The term broad-spectrum antibiotic refers to an antibiotic with activity against a wide range of disease-causing bacteria. It is also means that it acts against both Gram-positive and Gram-negative bacteria. This is in contrast to a narrow-spectrum antibiotic which is effective against only specific families of bacteria. A good example of a commonly used broad-spectrum antibiotic is levofloxacin.
Uses
Broad-spectrum antibiotics are properly used in the following medical situations:
* Empirically prior to identifying the causative bacteria when there is a wide differential and potentially serious illness would result in delay of treatment. This occurs, for example, in meningitis, where the patient can become so ill that he/she could die within hours if broad-spectrum antibiotics are not initiated.
* For drug resistant bacteria that do not respond to other, more narrow-spectrum antibiotics.
* In super-infections where there are multiple types of bacteria causing illness, thus warranting either a broad-spectrum antibiotic or combination antibiotic therapy.
Examples
In medicine:
* amoxycillin
* levofloxacin, gatifloxacin, moxifloxacin
In veterinary medicine, Co-amoxiclav, (in small animals); penicillin & streptomycin and oxytetracycline (in farm animals); penicillin and potentiated sulfonamides (in horses).
Others:
* streptomycin
* tetracycline
* chloramphenicol
Slightly-Broad:
* ampicillin
Thursday, December 17, 2009
Saturday, December 12, 2009
Drug resistance
Drug resistance is the reduction in effectiveness of a drug in curing a disease or improving a patient's symptoms. When the drug is not intended to kill or inhibit a pathogen, then the term is equivalent to dosage failure or drug tolerance. More commonly, the term is used in the context of diseases caused by pathogens.
Pathogens are said to be drug-resistant when drugs meant to neutralize them have reduced effect. When an organism is resistant to more than one drug, it is said to be multidrug resistant.
Drug resistance is an example of evolution in microorganisms. Individuals that are not susceptible to the drug effects are capable of surviving drug treatment, and therefore have greater fitness than susceptible individuals. By the process of natural selection, drug resistant traits are selected for in subsequent offspring, resulting in a population that is drug resistant.
Classification
Drug resistance occurs in several classes of pathogens:
* bacteria—antibiotic resistance
* endoparasites
* viruses—resistance to antiviral drugs
* fungi
* cancer cells
Mechanisms
Sometimes the target molecule of the drug evolves so the drugs won't bind as well. Sometimes the target cells or organisms evolve better enzymes to degrade the drug, or evolve better mechanisms to pump the drug out of the target cells.
Metabolic price
Biological cost or metabolic price is a measure of the increased energy metabolism required to achieve a function.
Drug resistance has a high metabolic price, in pathogens for which this concept is relevant (bacteria, endoparasites, and tumor cells.) In viruses, an equivalent "cost" is genomic complexity.
Other Problems
Drug resistance not only causes metabolic problems but also results in issues concerning what more can be done to help the infected people and what better and more effective ways can be used without any further drug resistance. Respiratory infections, HIV/AIDS, diarrhoeal diseases, tuberculosis and malaria are the leading killers among infectious diseases to this date. Resistance to first-line drugs has been observed in all of these diseases. In some cases, the level of resistance has forced a change to more expensive second or third-line agents. When resistance against these drugs also emerges, the world will run out of treatment options until other options emerge.
Pathogens are said to be drug-resistant when drugs meant to neutralize them have reduced effect. When an organism is resistant to more than one drug, it is said to be multidrug resistant.
Drug resistance is an example of evolution in microorganisms. Individuals that are not susceptible to the drug effects are capable of surviving drug treatment, and therefore have greater fitness than susceptible individuals. By the process of natural selection, drug resistant traits are selected for in subsequent offspring, resulting in a population that is drug resistant.
Classification
Drug resistance occurs in several classes of pathogens:
* bacteria—antibiotic resistance
* endoparasites
* viruses—resistance to antiviral drugs
* fungi
* cancer cells
Mechanisms
Sometimes the target molecule of the drug evolves so the drugs won't bind as well. Sometimes the target cells or organisms evolve better enzymes to degrade the drug, or evolve better mechanisms to pump the drug out of the target cells.
Metabolic price
Biological cost or metabolic price is a measure of the increased energy metabolism required to achieve a function.
Drug resistance has a high metabolic price, in pathogens for which this concept is relevant (bacteria, endoparasites, and tumor cells.) In viruses, an equivalent "cost" is genomic complexity.
Other Problems
Drug resistance not only causes metabolic problems but also results in issues concerning what more can be done to help the infected people and what better and more effective ways can be used without any further drug resistance. Respiratory infections, HIV/AIDS, diarrhoeal diseases, tuberculosis and malaria are the leading killers among infectious diseases to this date. Resistance to first-line drugs has been observed in all of these diseases. In some cases, the level of resistance has forced a change to more expensive second or third-line agents. When resistance against these drugs also emerges, the world will run out of treatment options until other options emerge.
Antibiotic misuse
Antibiotic misuse, (sometimes called antibiotic abuse or antibiotic overuse) refers to the misuse and overuse of antibiotics which has serious effects on public health. Antibiotic resistant bacteria is a growing threat and becoming increasingly common. This overuse creates multi-antibiotic resistant life threatening infections by "super bugs", sometimes out of relatively harmless bacteria. Antibiotic abuse also places the patient at unnecessary risk of adverse effects of antibiotics.
Epidemiology
Within a recent study concerning the proper use of this class in the emergency room it was revealed that 99% of these prescriptions were in error. Out of the one hundred total patients studied, eighty one received a fluoroquinolone for an inappropriate indication. Out of these cases, forty three (53%) were judged to be inappropriate because another agent was considered first line, twenty seven (33%) because there was no evidence of a bacterial infection to begin with (based on the documented evaluation) and eleven (14%) because of the need for such therapy was questionable. Out of the nineteen patients who received a fluoroquinolone for an appropriate indication, only one patient out of one hundred received both the correct dose and duration of therapy.
Within a 1994 study it was found that 75% of the fluoroquinolone prescription issued within a long term care setting were judged to be inappropriate by the authors. In more than fifty percent of the cases reviewed, the fluoroquinolone used were not considered to be a first line agent.
Social and economic impact
Increased hospitalizations attributed to adverse drug reactions alone account for billions of dollars each year within the US healthcare system. Severe reactions do occur with antibiotics and can add significantly to the cost of care. Antibacterial adverse effects account for nearly 25% of all adverse drug reactions amongst hospitalized patients. Adverse drug reactions to fluoroquinolones are easily and likely often misdiagnosed as seizure disorder or regular CNS or psychiatric symptoms and the diagnosis of quinolone toxicity or adverse reaction missed. Adverse event reporting in Italy by doctors showed fluoroquinolones among the top 3 prescribed drugs for adverse neurological and psychiatric adverse effects. These neuropsychiatric effects included tremor, confusion, anxiety, insomnia, agitation and in severe cases psychosis. Moxifloxacin came out worst amongst the quinolones for causing CNS toxicity. The central nervous system is an important target for fluoroquinolone mediated neurotoxicity.
Antibiotic resistance
Though antibiotics are considered to be a very important and necessary drugs required to treat severe and life threatening bacterial infections, the associated antibiotic abuse, has contributed to the problem of bacterial resistance. The overuse of antibiotics such as happens with children suffering from otitis media for example has given rise to a breed of super bacteria which are resistant to antibiotics entirely.
The overuse of fluoroquinolone and other antibiotics will eventually result in them becoming useless for treating antibiotic-resistant infections, for which broad-spectrum antibiotics are supposed to be reserved.
The over-prescribing and inappropriate use of antibiotics is fueling antibiotic resistance in bacteria. For example the inapprorpiate wide spread use of fluoroquinolones as first line antibiotics is leading to decreased bacterial sensitivity which has important implications for certain serious bacterial infections such as those associated with cystic fibrosis where quinolones are among the few available antibiotics.
Inappropriate use
Only about 5-10% of bronchitis cases are caused by a bacterial infection. Antibiotics have no effect upon viral infections such as the common head cold. Most cases of bronchitis are caused by a viral infection and are "self-limited" and resolve themselves in a few weeks. The use of antibiotics such as ofloxacin to treat bronchitis is to be considered unnecessary and as such exposes the patient to an unacceptable risk of suffering a severe adverse reaction. Nor does antibiotic treatment help sore throats. Prescribing antibiotics for sore throats encourages increased visits to the doctor. As most cases of sore throats are viral and are self limiting it has been recommended that antibiotic treatment is delayed in most cases. Nevertheless, for severe forms of community-acquired pneumonia the fluoroquinolones seem to be associated with improved treatment rates, but with no differences found in mortality between antibiotic regimens. In spite of this caveat, the use of the fluoroquinolone to treat community acquired pnuemonia (CAP) increased by >50%, from 25% to 39% of all prescriptions. This increase was at the expense of the macrolide class of antimicrobial drugs, the use of which declined 20% during the study period.
As with other fluoroquinolones their use as first line agents is not generally recommended. They are usually reserved for use in patients who are seriously ill and may soon require immediate hospitalization. Though considered to be a very important and necessary drug required to treat severe and life threatening bacterial infections, the associated overprescribing of fluoroquinolones remains unchecked, which has contributed to the problem of bacterial resistance. The overuse of antibiotics such as happens with children suffering from otitis media has given rise to a breed of super bacteria which are resistant to antibiotics entirely. “Fluoroquinolone resistance is an increasing problem not only in the U.S. but also worldwide, potentially due to the widespread misuse of this class of antimicrobials.” For example the use of the fuoroquinolones had increased three-fold in an emergency room environment in the United States between 1995 and 2002, while the use of safer alternatives such as macrolides declined significantly.
Chronic pelvic pain (category IIIB) is often misdiagnosed as chronic prostatitis and needlessly treated with a fluoroquinolone drug. Within a Bulgarian study, where by definition all patients had negative microbiological results, 65% of patients experienced an adverse drug reaction who were treated with a fluoroquinolone in comparison to a 9% rate for the placebo patients. This was combined with a higher cure rate (69% v 53%) found within the placebo group. The authors stated that “The results of our study show that antibiotics have an unacceptably high rate of adverse side effects as well as a statistically insignificant improvement over placebo...” Prostatitis has been termed "the waste basket of clinical ignorance" by prominent Stanford University Urologist Dr. Thomas Stamey. Campbell's Urology, the urologist's most authoritative reference text, identifies only about 5% of all patients with prostatitis as having bacterial prostatitis which can be "cured" at least in the short term by antibiotics. In other words, 95% of men with prostatitis have little hope for a cure with antibiotics alone since they don't actually have any identifiable bacterial infection.
There are limited indications for ciprofloxacin as a first-line therapy within Long Term Care Facilities. Within a 1994 study it was found that 75% of the prescriptions for fluoroquinolones issued within a long term care setting were judged to be inappropriate by the authors. In more than fifty percent of the cases reviewed fluoroquinolones were not considered to be a first line agent.
Epidemiology
Within a recent study concerning the proper use of this class in the emergency room it was revealed that 99% of these prescriptions were in error. Out of the one hundred total patients studied, eighty one received a fluoroquinolone for an inappropriate indication. Out of these cases, forty three (53%) were judged to be inappropriate because another agent was considered first line, twenty seven (33%) because there was no evidence of a bacterial infection to begin with (based on the documented evaluation) and eleven (14%) because of the need for such therapy was questionable. Out of the nineteen patients who received a fluoroquinolone for an appropriate indication, only one patient out of one hundred received both the correct dose and duration of therapy.
Within a 1994 study it was found that 75% of the fluoroquinolone prescription issued within a long term care setting were judged to be inappropriate by the authors. In more than fifty percent of the cases reviewed, the fluoroquinolone used were not considered to be a first line agent.
Social and economic impact
Increased hospitalizations attributed to adverse drug reactions alone account for billions of dollars each year within the US healthcare system. Severe reactions do occur with antibiotics and can add significantly to the cost of care. Antibacterial adverse effects account for nearly 25% of all adverse drug reactions amongst hospitalized patients. Adverse drug reactions to fluoroquinolones are easily and likely often misdiagnosed as seizure disorder or regular CNS or psychiatric symptoms and the diagnosis of quinolone toxicity or adverse reaction missed. Adverse event reporting in Italy by doctors showed fluoroquinolones among the top 3 prescribed drugs for adverse neurological and psychiatric adverse effects. These neuropsychiatric effects included tremor, confusion, anxiety, insomnia, agitation and in severe cases psychosis. Moxifloxacin came out worst amongst the quinolones for causing CNS toxicity. The central nervous system is an important target for fluoroquinolone mediated neurotoxicity.
Antibiotic resistance
Though antibiotics are considered to be a very important and necessary drugs required to treat severe and life threatening bacterial infections, the associated antibiotic abuse, has contributed to the problem of bacterial resistance. The overuse of antibiotics such as happens with children suffering from otitis media for example has given rise to a breed of super bacteria which are resistant to antibiotics entirely.
The overuse of fluoroquinolone and other antibiotics will eventually result in them becoming useless for treating antibiotic-resistant infections, for which broad-spectrum antibiotics are supposed to be reserved.
The over-prescribing and inappropriate use of antibiotics is fueling antibiotic resistance in bacteria. For example the inapprorpiate wide spread use of fluoroquinolones as first line antibiotics is leading to decreased bacterial sensitivity which has important implications for certain serious bacterial infections such as those associated with cystic fibrosis where quinolones are among the few available antibiotics.
Inappropriate use
Only about 5-10% of bronchitis cases are caused by a bacterial infection. Antibiotics have no effect upon viral infections such as the common head cold. Most cases of bronchitis are caused by a viral infection and are "self-limited" and resolve themselves in a few weeks. The use of antibiotics such as ofloxacin to treat bronchitis is to be considered unnecessary and as such exposes the patient to an unacceptable risk of suffering a severe adverse reaction. Nor does antibiotic treatment help sore throats. Prescribing antibiotics for sore throats encourages increased visits to the doctor. As most cases of sore throats are viral and are self limiting it has been recommended that antibiotic treatment is delayed in most cases. Nevertheless, for severe forms of community-acquired pneumonia the fluoroquinolones seem to be associated with improved treatment rates, but with no differences found in mortality between antibiotic regimens. In spite of this caveat, the use of the fluoroquinolone to treat community acquired pnuemonia (CAP) increased by >50%, from 25% to 39% of all prescriptions. This increase was at the expense of the macrolide class of antimicrobial drugs, the use of which declined 20% during the study period.
As with other fluoroquinolones their use as first line agents is not generally recommended. They are usually reserved for use in patients who are seriously ill and may soon require immediate hospitalization. Though considered to be a very important and necessary drug required to treat severe and life threatening bacterial infections, the associated overprescribing of fluoroquinolones remains unchecked, which has contributed to the problem of bacterial resistance. The overuse of antibiotics such as happens with children suffering from otitis media has given rise to a breed of super bacteria which are resistant to antibiotics entirely. “Fluoroquinolone resistance is an increasing problem not only in the U.S. but also worldwide, potentially due to the widespread misuse of this class of antimicrobials.” For example the use of the fuoroquinolones had increased three-fold in an emergency room environment in the United States between 1995 and 2002, while the use of safer alternatives such as macrolides declined significantly.
Chronic pelvic pain (category IIIB) is often misdiagnosed as chronic prostatitis and needlessly treated with a fluoroquinolone drug. Within a Bulgarian study, where by definition all patients had negative microbiological results, 65% of patients experienced an adverse drug reaction who were treated with a fluoroquinolone in comparison to a 9% rate for the placebo patients. This was combined with a higher cure rate (69% v 53%) found within the placebo group. The authors stated that “The results of our study show that antibiotics have an unacceptably high rate of adverse side effects as well as a statistically insignificant improvement over placebo...” Prostatitis has been termed "the waste basket of clinical ignorance" by prominent Stanford University Urologist Dr. Thomas Stamey. Campbell's Urology, the urologist's most authoritative reference text, identifies only about 5% of all patients with prostatitis as having bacterial prostatitis which can be "cured" at least in the short term by antibiotics. In other words, 95% of men with prostatitis have little hope for a cure with antibiotics alone since they don't actually have any identifiable bacterial infection.
There are limited indications for ciprofloxacin as a first-line therapy within Long Term Care Facilities. Within a 1994 study it was found that 75% of the prescriptions for fluoroquinolones issued within a long term care setting were judged to be inappropriate by the authors. In more than fifty percent of the cases reviewed fluoroquinolones were not considered to be a first line agent.
Monday, December 7, 2009
Xenobiotic metabolism
Xenobiotic metabolism is the set of metabolic pathways that modify the chemical structure of xenobiotics, which are compounds foreign to an organism's normal biochemistry, such as drugs and poisons. These pathways are a form of biotransformation present in all major groups of organisms, and are considered to be of ancient origin. These reactions often act to detoxify poisonous compounds; however, in some cases, the intermediates in xenobiotic metabolism can themselves be the cause of toxic effects.
Xenobiotic metabolism is divided into three phases. In phase I, enzymes such as cytochrome P450 oxidases introduce reactive or polar groups into xenobiotics. These modified compounds are then conjugated to polar compounds in phase II reactions. These reactions are catalysed by transferase enzymes such as glutathione S-transferases. Finally, in phase III, the conjugated xenobiotics may be further processed, before being recognised by efflux transporters and pumped out of cells.
The reactions in these pathways are of particular interest in medicine as part of drug metabolism and as a factor contributing to multidrug resistance in infectious diseases and cancer chemotherapy. The actions of some drugs as substrates or inhibitors of enzymes involved in xenobiotic metabolism are a common reason for hazardous drug interactions. These pathways are also important in environmental science, with the xenobiotic metabolism of microorganisms determining whether a pollutant will be broken down during bioremediation, or persist in the environment. The enzymes of xenobiotic metabolism, particularly the glutathione S-transferases are also important in agriculture, since they may produce resistance to pesticides and herbicides.
Permeability barriers and detoxification
That the exact compounds an organism is exposed to will be largely unpredictable, and may differ widely over time, is a major characteristic of xenobiotic toxic stress. The major challenge faced by xenobiotic detoxification systems is that they must be able to remove the almost-limitless number of xenobiotic compounds from the complex mixture of chemicals involved in normal metabolism. The solution that has evolved to address this problem is an elegant combination of physical barriers and low-specificity enzymatic systems.
All organisms use cell membranes as hydrophobic permeability barriers to control access to their internal environment. Polar compounds cannot diffuse across these cell membranes, and the uptake of useful molecules is mediated through transport proteins that specifically select substrates from the extracellular mixture. This selective uptake means that most hydrophilic molecules cannot enter cells, since they are not recognised by any specific transporters. In contrast, the diffusion of hydrophobic compounds across these barriers cannot be controlled, and organisms, therefore, cannot exclude lipid-soluble xenobiotics using membrane barriers.
However, the existence of a permeability barrier means that organisms were able to evolve detoxification systems that exploit the hydrophobicity common to membrane-permeable xenobiotics. These systems therefore solve the specificity problem by possessing such broad substrate specificities that they metabolise almost any non-polar compound. Useful metabolites are excluded since they are polar, and in general contain one or more charged groups.
The detoxification of the reactive by-products of normal metabolism cannot be achieved by the systems outlined above, because these species are derived from normal cellular constituents and usually share their polar characteristics. However, since these compounds are few in number, specific enzymes can recognize and remove them. Examples of these specific detoxification systems are the glyoxalase system, which removes the reactive aldehyde methylglyoxal, and the various antioxidant systems that eliminate reactive oxygen species.
Phases of detoxification
The metabolism of xenobiotics is often divided into three phases: modification, conjugation, and excretion. These reactions act in concert to detoxify xenobiotics and remove them from cells.
Phase I - modification
In phase I, a variety of enzymes acts to introduce reactive and polar groups into their substrates. One of the most common modifications is hydroxylation catalysed by the cytochrome P-450-dependent mixed-function oxidase system. These enzyme complexes act to incorporate an atom of oxygen into nonactivated hydrocarbons, which can result in either the introduction of hydroxyl groups or N-, O- and S-dealkylation of substrates. The reaction mechanism of the P-450 oxidases proceeds through the reduction of cytochrome-bound oxygen and the generation of a highly-reactive oxyferryl species, according to the following scheme:
\mbox{NADPH} + \mbox{H}^+ + \mbox{RH} \rightarrow \mbox{NADP}^+ + \mbox{H}_2\mbox{O} +\mbox{ROH} \,
Phase II - conjugation
In subsequent phase II reactions, these activated xenobiotic metabolites are conjugated with charged species such as glutathione (GSH), sulfate, glycine, or glucuronic acid. These reactions are catalysed by a large group of broad-specificity transferases, which in combination can metabolise almost any hydrophobic compound that contains nucleophilic or electrophilic groups. One of the most important of these groups are the glutathione S-transferases (GSTs). The addition of large anionic groups (such as GSH) detoxifies reactive electrophiles and produces more polar metabolites that cannot diffuse across membranes, and may, therefore, be actively transported.
Phase III - further modification and excretion
After phase II reactions, the xenobiotic conjugates may be further metabolised. A common example is the processing of glutathione conjugates to acetylcysteine (mercapturic acid) conjugates. Here, the γ-glutamate and glycine residues in the glutathione molecule are removed by Gamma-glutamyl transpeptidase and dipeptidases. In the final step, the cystine residue in the conjugate is acetylated.
Conjugates and their metabolites can be excreted from cells in phase III of their metabolism, with the anionic groups acting as affinity tags for a variety of membrane transporters of the multidrug resistance protein (MRP) family. These proteins are members of the family of ATP-binding cassette transporters and can catalyse the ATP-dependent transport of a huge variety of hydrophobic anions, and thus act to remove phase II products to the extracellular medium, where they may be further metabolised or excreted.
Endogenous toxins
The detoxification of endogenous reactive metabolites such as peroxides and reactive aldehydes often cannot be achieved by the system described above. This is the result of these species' being derived from normal cellular constituents and usually sharing their polar characteristics. However, since these compounds are few in number, it is possible for enzymatic systems to utilize specific molecular recognition to recognize and remove them. The similarity of these molecules to useful metabolites therefore means that different detoxification enzymes are usually required for the metabolism of each group of endogenous toxins. Examples of these specific detoxification systems are the glyoxalase system, which acts to dispose of the reactive aldehyde methylglyoxal, and the various antioxidant systems that remove reactive oxygen species.
History
Studies on how people transform the substances that they ingest began in the mid-nineteenth century, with chemists discovering that organic chemicals such as benzaldehyde could be oxidized and conjugated to amino acids in the human body. During the remainder of the nineteenth century, several other basic detoxification reactions were discovered, such as methylation, acetylation, and sulfonation.
In the early twentieth century, work moved on to the investigation of the enzymes and pathways that were responsible for the production of these metabolites. This field became defined as a separate area of study with the publication by Richard Williams of the book Detoxication mechanisms in 1947. This modern biochemical research resulted in the identification of glutathione S-transferases in 1961, followed by the discovery of cytochrome P450s in 1962, and the realization of their central role in xenobiotic metabolism in 1963
Xenobiotic metabolism is divided into three phases. In phase I, enzymes such as cytochrome P450 oxidases introduce reactive or polar groups into xenobiotics. These modified compounds are then conjugated to polar compounds in phase II reactions. These reactions are catalysed by transferase enzymes such as glutathione S-transferases. Finally, in phase III, the conjugated xenobiotics may be further processed, before being recognised by efflux transporters and pumped out of cells.
The reactions in these pathways are of particular interest in medicine as part of drug metabolism and as a factor contributing to multidrug resistance in infectious diseases and cancer chemotherapy. The actions of some drugs as substrates or inhibitors of enzymes involved in xenobiotic metabolism are a common reason for hazardous drug interactions. These pathways are also important in environmental science, with the xenobiotic metabolism of microorganisms determining whether a pollutant will be broken down during bioremediation, or persist in the environment. The enzymes of xenobiotic metabolism, particularly the glutathione S-transferases are also important in agriculture, since they may produce resistance to pesticides and herbicides.
Permeability barriers and detoxification
That the exact compounds an organism is exposed to will be largely unpredictable, and may differ widely over time, is a major characteristic of xenobiotic toxic stress. The major challenge faced by xenobiotic detoxification systems is that they must be able to remove the almost-limitless number of xenobiotic compounds from the complex mixture of chemicals involved in normal metabolism. The solution that has evolved to address this problem is an elegant combination of physical barriers and low-specificity enzymatic systems.
All organisms use cell membranes as hydrophobic permeability barriers to control access to their internal environment. Polar compounds cannot diffuse across these cell membranes, and the uptake of useful molecules is mediated through transport proteins that specifically select substrates from the extracellular mixture. This selective uptake means that most hydrophilic molecules cannot enter cells, since they are not recognised by any specific transporters. In contrast, the diffusion of hydrophobic compounds across these barriers cannot be controlled, and organisms, therefore, cannot exclude lipid-soluble xenobiotics using membrane barriers.
However, the existence of a permeability barrier means that organisms were able to evolve detoxification systems that exploit the hydrophobicity common to membrane-permeable xenobiotics. These systems therefore solve the specificity problem by possessing such broad substrate specificities that they metabolise almost any non-polar compound. Useful metabolites are excluded since they are polar, and in general contain one or more charged groups.
The detoxification of the reactive by-products of normal metabolism cannot be achieved by the systems outlined above, because these species are derived from normal cellular constituents and usually share their polar characteristics. However, since these compounds are few in number, specific enzymes can recognize and remove them. Examples of these specific detoxification systems are the glyoxalase system, which removes the reactive aldehyde methylglyoxal, and the various antioxidant systems that eliminate reactive oxygen species.
Phases of detoxification
The metabolism of xenobiotics is often divided into three phases: modification, conjugation, and excretion. These reactions act in concert to detoxify xenobiotics and remove them from cells.
Phase I - modification
In phase I, a variety of enzymes acts to introduce reactive and polar groups into their substrates. One of the most common modifications is hydroxylation catalysed by the cytochrome P-450-dependent mixed-function oxidase system. These enzyme complexes act to incorporate an atom of oxygen into nonactivated hydrocarbons, which can result in either the introduction of hydroxyl groups or N-, O- and S-dealkylation of substrates. The reaction mechanism of the P-450 oxidases proceeds through the reduction of cytochrome-bound oxygen and the generation of a highly-reactive oxyferryl species, according to the following scheme:
\mbox{NADPH} + \mbox{H}^+ + \mbox{RH} \rightarrow \mbox{NADP}^+ + \mbox{H}_2\mbox{O} +\mbox{ROH} \,
Phase II - conjugation
In subsequent phase II reactions, these activated xenobiotic metabolites are conjugated with charged species such as glutathione (GSH), sulfate, glycine, or glucuronic acid. These reactions are catalysed by a large group of broad-specificity transferases, which in combination can metabolise almost any hydrophobic compound that contains nucleophilic or electrophilic groups. One of the most important of these groups are the glutathione S-transferases (GSTs). The addition of large anionic groups (such as GSH) detoxifies reactive electrophiles and produces more polar metabolites that cannot diffuse across membranes, and may, therefore, be actively transported.
Phase III - further modification and excretion
After phase II reactions, the xenobiotic conjugates may be further metabolised. A common example is the processing of glutathione conjugates to acetylcysteine (mercapturic acid) conjugates. Here, the γ-glutamate and glycine residues in the glutathione molecule are removed by Gamma-glutamyl transpeptidase and dipeptidases. In the final step, the cystine residue in the conjugate is acetylated.
Conjugates and their metabolites can be excreted from cells in phase III of their metabolism, with the anionic groups acting as affinity tags for a variety of membrane transporters of the multidrug resistance protein (MRP) family. These proteins are members of the family of ATP-binding cassette transporters and can catalyse the ATP-dependent transport of a huge variety of hydrophobic anions, and thus act to remove phase II products to the extracellular medium, where they may be further metabolised or excreted.
Endogenous toxins
The detoxification of endogenous reactive metabolites such as peroxides and reactive aldehydes often cannot be achieved by the system described above. This is the result of these species' being derived from normal cellular constituents and usually sharing their polar characteristics. However, since these compounds are few in number, it is possible for enzymatic systems to utilize specific molecular recognition to recognize and remove them. The similarity of these molecules to useful metabolites therefore means that different detoxification enzymes are usually required for the metabolism of each group of endogenous toxins. Examples of these specific detoxification systems are the glyoxalase system, which acts to dispose of the reactive aldehyde methylglyoxal, and the various antioxidant systems that remove reactive oxygen species.
History
Studies on how people transform the substances that they ingest began in the mid-nineteenth century, with chemists discovering that organic chemicals such as benzaldehyde could be oxidized and conjugated to amino acids in the human body. During the remainder of the nineteenth century, several other basic detoxification reactions were discovered, such as methylation, acetylation, and sulfonation.
In the early twentieth century, work moved on to the investigation of the enzymes and pathways that were responsible for the production of these metabolites. This field became defined as a separate area of study with the publication by Richard Williams of the book Detoxication mechanisms in 1947. This modern biochemical research resulted in the identification of glutathione S-transferases in 1961, followed by the discovery of cytochrome P450s in 1962, and the realization of their central role in xenobiotic metabolism in 1963
Antibiotic resistance
Antibiotic resistance is a specific type of drug resistance when a microorganism has the ability of withstanding the effects of antibiotics. Antibiotic resistance evolves via natural selection acting upon random mutation, but it can also be engineered by applying an evolutionary stress on a population. Once such a gene is generated, bacteria can then transfer the genetic information in a horizontal fashion (between individuals) by plasmid exchange. If a bacterium carries several resistance genes, it is called multiresistant or, informally, a superbug. The term antimicrobial resistance is sometimes used to explicitly encompass organisms other than bacteria.
Antibiotic resistance can also be introduced artificially into a microorganism through transformation protocols. This can aid in implanting artificial genes into the microorganism. If the resistance gene is linked with the gene to be implanted, the antibiotic can be used to kill off organisms that lack the new gene.
Causes
The widespread use of antibiotics both inside and outside of medicine is playing a significant role in the emergence of resistant bacteria. They are often used in animals but also in other industries which at least in the case of agricultural use lead to the spread of resistant strains to human populations. In some countries antibiotics are sold over the counter without a prescription which compounds the problem. In human medicine the major problem of the emergence of resistant bacteria is due to misuse and overuse of antibiotics by doctors as well as patients. Other practices contributing towards resistance include the addition of antibiotics to the feed of livestock. Household use of antibacterials in soaps and other products, although not clearly contributing to resistance, is also discouraged (as not being effective at infection control). Also unsound practices in the pharmaceutical manufacturing industry can contribute towards the likelihood of creating antibiotic resistant strains.
Certain antibiotic classes are highly associated with colonisation with superbugs compared to other antibiotic classes. The risk for colonisation increases if there is a lack of sensitivity (resistance) of the superbugs to the antibiotic used and high tissue penetration as well as broad spectrum activity against "good bacteria". In the case of MRSA, increased rates of MRSA infections are seen with glycopeptides, cephalosporins and especially quinolones. In the case of colonisation with C difficile the high risk antibiotics include cephalosporins and in particular quinolones and clindamycin.
In medicine
The volume of antibiotic prescribed is the major factor in increasing rates or bacterial resistance rather than compliance with antibiotics. Inappropriate prescribing of antibiotics has been attributed to a number of causes including: people who insist on antibiotics, physicians simply prescribe them as they feel they do not have time to explain why they are not necessary, physicians who do not know when to prescribe antibiotics or else are overly cautious for medical legal reasons. A third of people for example believe that antibiotics are effective for the common cold and 22% of people do not finish a course of antibiotics primarily due to that fact that they feel better (varying from 10% to 44% depending on the country). Compliance with once daily antibiotics is better than with twice daily antibiotics. Sub optimum antibiotic concentrations in critically ill people increase the frequency of antibiotic resistance organisms. While taking antibiotics doses less than those recommended may increase rates of resistance, shortening the course of antibiotics may actually decrease rates of resistance.
Poor hand hygiene by hospital staff has been associated with the spread of resistant organisms and an increase in hand washing compliance results in decreased rates of these organisms.
Role of other animals
Drugs are used in animals that are used as human food, such as cows, pigs, chickens, fish, etc, and these drugs can affect the safety of the meat, milk, and eggs produced from those animals and can be the source of superbugs. For example, farm animals, particularly pigs, are believed to be able to infect people with MRSA. The resistant bacteria in animals due to antibiotic exposure can be transmitted to humans via three pathways, those being through the consumption of meat, from close or direct contact with animals, or through the environment.
The World Health Organization concluded that antibiotics as growth promoters in animal feeds should be prohibited (in the absence of risk assessments). In 1998, European Union health ministers voted to ban four antibiotics widely used to promote animal growth (despite their scientific panel's recommendations). Regulation banning the use of antibiotics in European feed, with the exception of two antibiotics in poultry feeds, became effective in 2006. In Scandinavia, there's evidence that the ban has led to a lower prevalence of antimicrobial resistance in (non-hazardous) animal bacterial populations. In the USA federal agencies do not collect data on antibiotic use in animals but animal to human spread of drug resistant organisms has been demonstrated in research studies. Antibiotics are still used in U.S. animal feed—along with other ingredients which have safety concerns.
Growing U.S. consumer concern about using antibiotics in animal feed has led to a niche market of "antibiotic-free" animal products, but this small market is unlikely to change entrenched industry-wide practices.
In 2001, the Union of Concerned Scientists estimated that greater than 70% of the antibiotics used in the US are given to food animals (e.g. chickens, pigs and cattle) in the absence of disease. In 2000 the US Food and Drug Administration (FDA) announced their intention to revoke approval of fluoroquinolone use in poultry production because of substantial evidence linking it to the emergence of fluoroquinolone resistant campylobacter infections in humans. The final decision to ban fluoroquinolones from use in poultry production was not made until five years later because of challenges from the food animal and pharmaceutical industries. Today, there are two federal bills (S. 549 and H.R. 962) aimed at phasing out "non-therapeutic" antibiotics in US food animal production.
Mechanisms
Schematic representation of how antibiotic resistance evolves via natural selection. The top section represents a population of bacteria before exposure to an antibiotic. The middle section shows the population directly after exposure, the phase in which selection took place. The last section shows the distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of individuals.
Researchers have recently demonstrated the bacterial protein LexA may play a key role in the acquisition of bacterial mutations.
Antibiotic resistance can be a result of horizontal gene transfer, and also of unlinked point mutations in the pathogen genome and a rate of about 1 in 108 per chromosomal replication. The antibiotic action against the pathogen can be seen as an environmental pressure; those bacteria which have a mutation allowing them to survive will live on to reproduce. They will then pass this trait to their offspring, which will result in a fully resistant colony.
The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:
1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases.
2. Alteration of target site: e.g. alteration of PBP—the binding target site of penicillins—in MRSA and other penicillin-resistant bacteria.
3. Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.
4. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.
There are three known mechanisms of fluoroquinolone resistance. Some types of efflux pumps can act to decrease intracellular quinolone concentration. In gram-negative bacteria, plasmid-mediated resistance genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Finally, mutations at key sites in DNA gyrase or Topoisomerase IV can decrease their binding affinity to quinolones, decreasing the drug's effectiveness.
Resistant pathogens
Staphylococcus aureus
Staphylococcus aureus (colloquially known as "Staph aureus" or a Staph infection) is one of the major resistant pathogens. Found on the mucous membranes and the skin of around a third of the population, it is extremely adaptable to antibiotic pressure. It was the first bacterium in which penicillin resistance was found—in 1947, just four years after the drug started being mass-produced. Methicillin was then the antibiotic of choice, but has since been replaced by oxacillin due to significant kidney toxicity. MRSA (methicillin-resistant Staphylococcus aureus) was first detected in Britain in 1961 and is now "quite common" in hospitals. MRSA was responsible for 37% of fatal cases of blood poisoning in the UK in 1999, up from 4% in 1991. Half of all S. aureus infections in the US are resistant to penicillin, methicillin, tetracycline and erythromycin.
This left vancomycin as the only effective agent available at the time. However, strains with intermediate (4-8 ug/ml) levels of resistance, termed GISA (glycopeptide intermediate Staphylococcus aureus) or VISA (vancomycin intermediate Staphylococcus aureus), began appearing in the late 1990s. The first identified case was in Japan in 1996, and strains have since been found in hospitals in England, France and the US. The first documented strain with complete (>16 ug/ml) resistance to vancomycin, termed VRSA (Vancomycin-resistant Staphylococcus aureus) appeared in the United States in 2002.
A new class of antibiotics, oxazolidinones, became available in the 1990s, and the first commercially available oxazolidinone, linezolid, is comparable to vancomycin in effectiveness against MRSA. Linezolid-resistance in Staphylococcus aureus was reported in 2003.
CA-MRSA (Community-acquired MRSA) has now emerged as an epidemic that is responsible for rapidly progressive, fatal diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis. Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequently identified antimicrobial drug-resistant pathogen in US hospitals. The epidemiology of infections caused by MRSA is rapidly changing. In the past 10 years, infections caused by this organism have emerged in the community. The 2 MRSA clones in the United States most closely associated with community outbreaks, USA400 (MW2 strain, ST1 lineage) and USA300, often contain Panton-Valentine leukocidin (PVL) genes and, more frequently, have been associated with skin and soft tissue infections. Outbreaks of community-associated (CA)-MRSA infections have been reported in correctional facilities, among athletic teams, among military recruits, in newborn nurseries, and among men who engage in frequent homosexual activities. CA-MRSA infections now appear to be endemic in many urban regions and cause most CA-S. aureus infections.
Streptococcus and Enterococcus
Streptococcus pyogenes (Group A Streptococcus: GAS) infections can usually be treated with many different antibiotics. Early treatment may reduce the risk of death from invasive group A streptococcal disease. However, even the best medical care does not prevent death in every case. For those with very severe illness, supportive care in an intensive care unit may be needed. For persons with necrotizing fasciitis, surgery often is needed to remove damaged tissue. Strains of S. pyogenes resistant to macrolide antibiotics have emerged, however all strains remain uniformly sensitive to penicillin.
Resistance of Streptococcus pneumoniae to penicillin and other beta-lactams is increasing worldwide. The major mechanism of resistance involves the introduction of mutations in genes encoding penicillin-binding proteins. Selective pressure is thought to play an important role, and use of beta-lactam antibiotics has been implicated as a risk factor for infection and colonization. Streptococcus pneumoniae is responsible for pneumonia, bacteremia, otitis media, meningitis, sinusitis, peritonitis and arthritis.
Penicillin-resistant pneumonia caused by Streptococcus pneumoniae (commonly known as pneumococcus), was first detected in 1967, as was penicillin-resistant gonorrhea. Resistance to penicillin substitutes is also known as beyond S. aureus. By 1993 Escherichia coli was resistant to five fluoroquinolone variants. Mycobacterium tuberculosis is commonly resistant to isoniazid and rifampin and sometimes universally resistant to the common treatments. Other pathogens showing some resistance include Salmonella, Campylobacter, and Streptococci.
Enterococcus faecium is another superbug found in hospitals. Penicillin-Resistant Enterococcus was seen in 1983, vancomycin-resistant enterococcus (VRE) in 1987, and Linezolid-Resistant Enterococcus (LRE) in the late 1990s.
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a highly prevalent opportunistic pathogen. One of the most worrisome characteristics of P. aeruginosa consists in its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes (e.g. mexAB-oprM, mexXY etc) and the low permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily develop acquired resistance either by mutation in chromosomally-encoded genes, or by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events that include acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown that phenotypic resistance associated to biofilm formation or to the emergence of small-colony-variants may be important in the response of P. aeruginosa populations to antibiotics treatment.
Clostridium difficile
Clostridium difficile is a nosocomial pathogen that causes diarrheal disease in hospitals world wide. Clindamycin-resistant C. difficile was reported as the causative agent of large outbreaks of diarrheal disease in hospitals in New York, Arizona, Florida and Massachusetts between 1989 and 1992. Geographically dispersed outbreaks of C. difficile strains resistant to fluoroquinolone antibiotics, such as Cipro (ciprofloxacin) and Levaquin (levofloxacin), were also reported in North America in 2005.
Salmonella and E. coli
E. coli and Salmonella come directly from contaminated food. Of the meat that is contaminated with E. coli, eighty percent of the bacteria are resistant to one or more drugs made; it causes bladder infections that are resistant to antibiotics (“HSUS Fact Sheet”). Salmonella was first found in humans in the 1970s and in some cases is resistant to as many as nine different antibiotics (“HSUS Fact Sheet”). When both bacterium are spread, serious health conditions arise. Many people are hospitalized each year after becoming infected, and some die as a result.
Acinetobacter baumannii
On November 5, 2004, the Centers for Disease Control and Prevention (CDC) reported an increasing number of Acinetobacter baumannii bloodstream infections in patients at military medical facilities in which service members injured in the Iraq/Kuwait region during Operation Iraqi Freedom and in Afghanistan during Operation Enduring Freedom were treated. Most of these showed multidrug resistance (MRAB), with a few isolates resistant to all drugs tested.
Alternatives
Prevention
Rational use of antibiotics may reduce the chances of development of opportunistic infection by antibiotic-resistant bacteria due to dysbacteriosis. In one study the use of fluoroquinolones are clearly associated with Clostridium difficile infection, which is a leading cause of nosocomial diarrhea in the United States, and a major cause of death, worldwide.
There is clinical evidence that topical dermatological preparations containing tea tree oil and thyme oil may be effective in preventing transmittal of CA-MRSA.
Vaccines do not suffer the problem of resistance because a vaccine enhances the body's natural defenses, while an antibiotic operates separately from the body's normal defenses. Nevertheless, new strains may evolve that escape immunity induced by vaccines.
While theoretically promising, anti-staphylococcal vaccines have shown limited efficacy, because of immunological variation between Staphylococcus species, and the limited duration of effectiveness of the antibodies produced. Development and testing of more effective vaccines is under way.
The Australian Commonwealth Scientific and Industrial Research Organization (CSIRO), realizing the need for the reduction of antibiotic use, has been working on two alternatives. One alternative is to prevent diseases by adding cytokines instead of antibiotics to animal feed. These proteins are made in the animal body "naturally" after a disease and are not antibiotics so they do not contribute to the antibiotic resistance problem. Furthermore, studies on using cytokines have shown that they also enhance the growth of animals like the antibiotics now used, but without the drawbacks of non-therapeutic antibiotic use. Cytokines have the potential to achieve the animal growth rates traditionally sought by the use of antibiotics without the contribution of antibiotic resistance associated with the widespread non-therapeutic uses of antibiotics currently utilized in the food animal production industries. Additionally, CSIRO is working on vaccines for diseases.
Phage therapy
Phage therapy, an approach that has been extensively researched and utilized as a therapeutic agent for over 60 years, especially in the Soviet Union, is an alternative that might help with the problem of resistance. Phage therapy was widely used in the United States until the discovery of antibiotics, in the early 1940s. Bacteriophages or "phages" are viruses that invade bacterial cells and, in the case of lytic phages, disrupt bacterial metabolism and cause the bacterium to lyse. Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections.
Bacteriophage therapy is an important alternative to antibiotics in the current era of multidrug resistant pathogens. A review of studies that dealt with the therapeutic use of phages from 1966–1996 and few latest ongoing phage therapy projects via internet showed: phages were used topically, orally or systemically in Polish and Soviet studies. The success rate found in these studies was 80–95% with few gastrointestinal or allergic side effects. British studies also demonstrated significant efficacy of phages against Escherichia coli, Acinetobacter spp., Pseudomonas spp and Staphylococcus aureus. US studies dealt with improving the bioavailability of phage. Phage therapy may prove as an important alternative to antibiotics for treating multidrug resistant pathogens.
Research
New medications
Until recently, research and development (R&D) efforts have provided new drugs in time to treat bacteria that became resistant to older antibiotics. That is no longer the case. The potential crisis at hand is the result of a marked decrease in industry R&D, and the increasing prevalence of resistant bacteria. Infectious disease physicians are alarmed by the prospect that effective antibiotics may not be available to treat seriously ill patients in the near future.
The pipeline of new antibiotics is drying up. Major pharmaceutical companies are losing interest in the antibiotics market because these drugs may not be as profitable as drugs that treat chronic (long-term) conditions and lifestyle issues.
The resistance problem demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to current antibiotics. One of the possible strategies towards this objective is the rational localization of bioactive phytochemicals. Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives such as tannins. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total. In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Many of the herbs and spices used by humans to season food yield useful medicinal compounds including those having antibacterial activity.
Traditional healers have long used plants to prevent or cure infectious conditions. Many of these plants have been investigated scientifically for antimicrobial activity and a large number of plant products have been shown to inhibit growth of pathogenic bacteria. A number of these agents appear to have structures and modes of action that are distinct from those of the antibiotics in current use, suggesting that cross-resistance with agents already in use may be minimal. For example the combination of 5'-methoxyhydnocarpine and berberine in herbs like Hydrastis canadensis and Berberis vulgaris can block the MDR-pumps that cause multidrug resistance. This has been shown for Staphylococcus aureus.
Archaeocins is the name given to a new class of potentially useful antibiotics that are derived from the Archaea group of organisms. Eight archaeocins have been partially or fully characterized, but hundreds of archaeocins are believed to exist, especially within the haloarchaea. The prevalence of archaeocins is unknown simply because no one has looked for them. The discovery of new archaeocins hinges on recovery and cultivation of archaeal organisms from the environment. For example, samples from a novel hypersaline field site, Wilson Hot Springs, recovered 350 halophilic organisms; preliminary analysis of 75 isolates showed that 48 were archaeal and 27 were bacterial.
In research published on October 17, 2008 in Cell, a team of scientists pinpointed the place on bacteria where the antibiotic myxopyronin launches its attack, and why that attack is successful. The myxopyronin binds to and inhibits the crucial bacterial enzyme, RNA polymerase. The myxopyronin changes the structure of the switch-2 segment of the enzyme, inhibiting its function of reading and transmitting DNA code. This prevents RNA polymerase from delivering genetic information to the ribosomes, causing the bacteria to die.
One of the major causes of antibiotic resistance is the decrease of effective drug concentrations at their target place, due to the increased action of ABC transporters. Since ABC transporter blockers can be used in combination with current drugs to increase their effective intracellular concentration, the possible impact of ABC transporter inhibitors is of great clinical interest. ABC transporter blockers that may be useful to increase the efficacy of current drugs have entered clinical trials and are available to be used in therapeutic regimes.
Applications
Antibiotic resistance is an important tool for genetic engineering. By constructing a plasmid which contains an antibiotic resistance gene as well as the gene being engineered or expressed, a researcher can ensure that when bacteria replicate, only the copies which carry along the plasmid survive. This ensures that the gene being manipulated passes along when the bacteria replicates.
The most commonly used antibiotics in genetic engineering are generally "older" antibiotics which have largely fallen out of use in clinical practice. These include:
* ampicillin
* kanamycin
* tetracycline
* chloramphenicol
Industrially the use of antibiotic resistance is disfavored since maintaining bacterial cultures would require feeding them large quantities of antibiotics. Instead, the use of auxotrophic bacterial strains (and function-replacement plasmids) is preferred.
Antibiotic resistance can also be introduced artificially into a microorganism through transformation protocols. This can aid in implanting artificial genes into the microorganism. If the resistance gene is linked with the gene to be implanted, the antibiotic can be used to kill off organisms that lack the new gene.
Causes
The widespread use of antibiotics both inside and outside of medicine is playing a significant role in the emergence of resistant bacteria. They are often used in animals but also in other industries which at least in the case of agricultural use lead to the spread of resistant strains to human populations. In some countries antibiotics are sold over the counter without a prescription which compounds the problem. In human medicine the major problem of the emergence of resistant bacteria is due to misuse and overuse of antibiotics by doctors as well as patients. Other practices contributing towards resistance include the addition of antibiotics to the feed of livestock. Household use of antibacterials in soaps and other products, although not clearly contributing to resistance, is also discouraged (as not being effective at infection control). Also unsound practices in the pharmaceutical manufacturing industry can contribute towards the likelihood of creating antibiotic resistant strains.
Certain antibiotic classes are highly associated with colonisation with superbugs compared to other antibiotic classes. The risk for colonisation increases if there is a lack of sensitivity (resistance) of the superbugs to the antibiotic used and high tissue penetration as well as broad spectrum activity against "good bacteria". In the case of MRSA, increased rates of MRSA infections are seen with glycopeptides, cephalosporins and especially quinolones. In the case of colonisation with C difficile the high risk antibiotics include cephalosporins and in particular quinolones and clindamycin.
In medicine
The volume of antibiotic prescribed is the major factor in increasing rates or bacterial resistance rather than compliance with antibiotics. Inappropriate prescribing of antibiotics has been attributed to a number of causes including: people who insist on antibiotics, physicians simply prescribe them as they feel they do not have time to explain why they are not necessary, physicians who do not know when to prescribe antibiotics or else are overly cautious for medical legal reasons. A third of people for example believe that antibiotics are effective for the common cold and 22% of people do not finish a course of antibiotics primarily due to that fact that they feel better (varying from 10% to 44% depending on the country). Compliance with once daily antibiotics is better than with twice daily antibiotics. Sub optimum antibiotic concentrations in critically ill people increase the frequency of antibiotic resistance organisms. While taking antibiotics doses less than those recommended may increase rates of resistance, shortening the course of antibiotics may actually decrease rates of resistance.
Poor hand hygiene by hospital staff has been associated with the spread of resistant organisms and an increase in hand washing compliance results in decreased rates of these organisms.
Role of other animals
Drugs are used in animals that are used as human food, such as cows, pigs, chickens, fish, etc, and these drugs can affect the safety of the meat, milk, and eggs produced from those animals and can be the source of superbugs. For example, farm animals, particularly pigs, are believed to be able to infect people with MRSA. The resistant bacteria in animals due to antibiotic exposure can be transmitted to humans via three pathways, those being through the consumption of meat, from close or direct contact with animals, or through the environment.
The World Health Organization concluded that antibiotics as growth promoters in animal feeds should be prohibited (in the absence of risk assessments). In 1998, European Union health ministers voted to ban four antibiotics widely used to promote animal growth (despite their scientific panel's recommendations). Regulation banning the use of antibiotics in European feed, with the exception of two antibiotics in poultry feeds, became effective in 2006. In Scandinavia, there's evidence that the ban has led to a lower prevalence of antimicrobial resistance in (non-hazardous) animal bacterial populations. In the USA federal agencies do not collect data on antibiotic use in animals but animal to human spread of drug resistant organisms has been demonstrated in research studies. Antibiotics are still used in U.S. animal feed—along with other ingredients which have safety concerns.
Growing U.S. consumer concern about using antibiotics in animal feed has led to a niche market of "antibiotic-free" animal products, but this small market is unlikely to change entrenched industry-wide practices.
In 2001, the Union of Concerned Scientists estimated that greater than 70% of the antibiotics used in the US are given to food animals (e.g. chickens, pigs and cattle) in the absence of disease. In 2000 the US Food and Drug Administration (FDA) announced their intention to revoke approval of fluoroquinolone use in poultry production because of substantial evidence linking it to the emergence of fluoroquinolone resistant campylobacter infections in humans. The final decision to ban fluoroquinolones from use in poultry production was not made until five years later because of challenges from the food animal and pharmaceutical industries. Today, there are two federal bills (S. 549 and H.R. 962) aimed at phasing out "non-therapeutic" antibiotics in US food animal production.
Mechanisms
Schematic representation of how antibiotic resistance evolves via natural selection. The top section represents a population of bacteria before exposure to an antibiotic. The middle section shows the population directly after exposure, the phase in which selection took place. The last section shows the distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of individuals.
Researchers have recently demonstrated the bacterial protein LexA may play a key role in the acquisition of bacterial mutations.
Antibiotic resistance can be a result of horizontal gene transfer, and also of unlinked point mutations in the pathogen genome and a rate of about 1 in 108 per chromosomal replication. The antibiotic action against the pathogen can be seen as an environmental pressure; those bacteria which have a mutation allowing them to survive will live on to reproduce. They will then pass this trait to their offspring, which will result in a fully resistant colony.
The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:
1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases.
2. Alteration of target site: e.g. alteration of PBP—the binding target site of penicillins—in MRSA and other penicillin-resistant bacteria.
3. Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.
4. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.
There are three known mechanisms of fluoroquinolone resistance. Some types of efflux pumps can act to decrease intracellular quinolone concentration. In gram-negative bacteria, plasmid-mediated resistance genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Finally, mutations at key sites in DNA gyrase or Topoisomerase IV can decrease their binding affinity to quinolones, decreasing the drug's effectiveness.
Resistant pathogens
Staphylococcus aureus
Staphylococcus aureus (colloquially known as "Staph aureus" or a Staph infection) is one of the major resistant pathogens. Found on the mucous membranes and the skin of around a third of the population, it is extremely adaptable to antibiotic pressure. It was the first bacterium in which penicillin resistance was found—in 1947, just four years after the drug started being mass-produced. Methicillin was then the antibiotic of choice, but has since been replaced by oxacillin due to significant kidney toxicity. MRSA (methicillin-resistant Staphylococcus aureus) was first detected in Britain in 1961 and is now "quite common" in hospitals. MRSA was responsible for 37% of fatal cases of blood poisoning in the UK in 1999, up from 4% in 1991. Half of all S. aureus infections in the US are resistant to penicillin, methicillin, tetracycline and erythromycin.
This left vancomycin as the only effective agent available at the time. However, strains with intermediate (4-8 ug/ml) levels of resistance, termed GISA (glycopeptide intermediate Staphylococcus aureus) or VISA (vancomycin intermediate Staphylococcus aureus), began appearing in the late 1990s. The first identified case was in Japan in 1996, and strains have since been found in hospitals in England, France and the US. The first documented strain with complete (>16 ug/ml) resistance to vancomycin, termed VRSA (Vancomycin-resistant Staphylococcus aureus) appeared in the United States in 2002.
A new class of antibiotics, oxazolidinones, became available in the 1990s, and the first commercially available oxazolidinone, linezolid, is comparable to vancomycin in effectiveness against MRSA. Linezolid-resistance in Staphylococcus aureus was reported in 2003.
CA-MRSA (Community-acquired MRSA) has now emerged as an epidemic that is responsible for rapidly progressive, fatal diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis. Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequently identified antimicrobial drug-resistant pathogen in US hospitals. The epidemiology of infections caused by MRSA is rapidly changing. In the past 10 years, infections caused by this organism have emerged in the community. The 2 MRSA clones in the United States most closely associated with community outbreaks, USA400 (MW2 strain, ST1 lineage) and USA300, often contain Panton-Valentine leukocidin (PVL) genes and, more frequently, have been associated with skin and soft tissue infections. Outbreaks of community-associated (CA)-MRSA infections have been reported in correctional facilities, among athletic teams, among military recruits, in newborn nurseries, and among men who engage in frequent homosexual activities. CA-MRSA infections now appear to be endemic in many urban regions and cause most CA-S. aureus infections.
Streptococcus and Enterococcus
Streptococcus pyogenes (Group A Streptococcus: GAS) infections can usually be treated with many different antibiotics. Early treatment may reduce the risk of death from invasive group A streptococcal disease. However, even the best medical care does not prevent death in every case. For those with very severe illness, supportive care in an intensive care unit may be needed. For persons with necrotizing fasciitis, surgery often is needed to remove damaged tissue. Strains of S. pyogenes resistant to macrolide antibiotics have emerged, however all strains remain uniformly sensitive to penicillin.
Resistance of Streptococcus pneumoniae to penicillin and other beta-lactams is increasing worldwide. The major mechanism of resistance involves the introduction of mutations in genes encoding penicillin-binding proteins. Selective pressure is thought to play an important role, and use of beta-lactam antibiotics has been implicated as a risk factor for infection and colonization. Streptococcus pneumoniae is responsible for pneumonia, bacteremia, otitis media, meningitis, sinusitis, peritonitis and arthritis.
Penicillin-resistant pneumonia caused by Streptococcus pneumoniae (commonly known as pneumococcus), was first detected in 1967, as was penicillin-resistant gonorrhea. Resistance to penicillin substitutes is also known as beyond S. aureus. By 1993 Escherichia coli was resistant to five fluoroquinolone variants. Mycobacterium tuberculosis is commonly resistant to isoniazid and rifampin and sometimes universally resistant to the common treatments. Other pathogens showing some resistance include Salmonella, Campylobacter, and Streptococci.
Enterococcus faecium is another superbug found in hospitals. Penicillin-Resistant Enterococcus was seen in 1983, vancomycin-resistant enterococcus (VRE) in 1987, and Linezolid-Resistant Enterococcus (LRE) in the late 1990s.
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a highly prevalent opportunistic pathogen. One of the most worrisome characteristics of P. aeruginosa consists in its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes (e.g. mexAB-oprM, mexXY etc) and the low permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily develop acquired resistance either by mutation in chromosomally-encoded genes, or by the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug resistance by P. aeruginosa isolates requires several different genetic events that include acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes. Hypermutation favours the selection of mutation-driven antibiotic resistance in P. aeruginosa strains producing chronic infections, whereas the clustering of several different antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance determinants. Some recent studies have shown that phenotypic resistance associated to biofilm formation or to the emergence of small-colony-variants may be important in the response of P. aeruginosa populations to antibiotics treatment.
Clostridium difficile
Clostridium difficile is a nosocomial pathogen that causes diarrheal disease in hospitals world wide. Clindamycin-resistant C. difficile was reported as the causative agent of large outbreaks of diarrheal disease in hospitals in New York, Arizona, Florida and Massachusetts between 1989 and 1992. Geographically dispersed outbreaks of C. difficile strains resistant to fluoroquinolone antibiotics, such as Cipro (ciprofloxacin) and Levaquin (levofloxacin), were also reported in North America in 2005.
Salmonella and E. coli
E. coli and Salmonella come directly from contaminated food. Of the meat that is contaminated with E. coli, eighty percent of the bacteria are resistant to one or more drugs made; it causes bladder infections that are resistant to antibiotics (“HSUS Fact Sheet”). Salmonella was first found in humans in the 1970s and in some cases is resistant to as many as nine different antibiotics (“HSUS Fact Sheet”). When both bacterium are spread, serious health conditions arise. Many people are hospitalized each year after becoming infected, and some die as a result.
Acinetobacter baumannii
On November 5, 2004, the Centers for Disease Control and Prevention (CDC) reported an increasing number of Acinetobacter baumannii bloodstream infections in patients at military medical facilities in which service members injured in the Iraq/Kuwait region during Operation Iraqi Freedom and in Afghanistan during Operation Enduring Freedom were treated. Most of these showed multidrug resistance (MRAB), with a few isolates resistant to all drugs tested.
Alternatives
Prevention
Rational use of antibiotics may reduce the chances of development of opportunistic infection by antibiotic-resistant bacteria due to dysbacteriosis. In one study the use of fluoroquinolones are clearly associated with Clostridium difficile infection, which is a leading cause of nosocomial diarrhea in the United States, and a major cause of death, worldwide.
There is clinical evidence that topical dermatological preparations containing tea tree oil and thyme oil may be effective in preventing transmittal of CA-MRSA.
Vaccines do not suffer the problem of resistance because a vaccine enhances the body's natural defenses, while an antibiotic operates separately from the body's normal defenses. Nevertheless, new strains may evolve that escape immunity induced by vaccines.
While theoretically promising, anti-staphylococcal vaccines have shown limited efficacy, because of immunological variation between Staphylococcus species, and the limited duration of effectiveness of the antibodies produced. Development and testing of more effective vaccines is under way.
The Australian Commonwealth Scientific and Industrial Research Organization (CSIRO), realizing the need for the reduction of antibiotic use, has been working on two alternatives. One alternative is to prevent diseases by adding cytokines instead of antibiotics to animal feed. These proteins are made in the animal body "naturally" after a disease and are not antibiotics so they do not contribute to the antibiotic resistance problem. Furthermore, studies on using cytokines have shown that they also enhance the growth of animals like the antibiotics now used, but without the drawbacks of non-therapeutic antibiotic use. Cytokines have the potential to achieve the animal growth rates traditionally sought by the use of antibiotics without the contribution of antibiotic resistance associated with the widespread non-therapeutic uses of antibiotics currently utilized in the food animal production industries. Additionally, CSIRO is working on vaccines for diseases.
Phage therapy
Phage therapy, an approach that has been extensively researched and utilized as a therapeutic agent for over 60 years, especially in the Soviet Union, is an alternative that might help with the problem of resistance. Phage therapy was widely used in the United States until the discovery of antibiotics, in the early 1940s. Bacteriophages or "phages" are viruses that invade bacterial cells and, in the case of lytic phages, disrupt bacterial metabolism and cause the bacterium to lyse. Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections.
Bacteriophage therapy is an important alternative to antibiotics in the current era of multidrug resistant pathogens. A review of studies that dealt with the therapeutic use of phages from 1966–1996 and few latest ongoing phage therapy projects via internet showed: phages were used topically, orally or systemically in Polish and Soviet studies. The success rate found in these studies was 80–95% with few gastrointestinal or allergic side effects. British studies also demonstrated significant efficacy of phages against Escherichia coli, Acinetobacter spp., Pseudomonas spp and Staphylococcus aureus. US studies dealt with improving the bioavailability of phage. Phage therapy may prove as an important alternative to antibiotics for treating multidrug resistant pathogens.
Research
New medications
Until recently, research and development (R&D) efforts have provided new drugs in time to treat bacteria that became resistant to older antibiotics. That is no longer the case. The potential crisis at hand is the result of a marked decrease in industry R&D, and the increasing prevalence of resistant bacteria. Infectious disease physicians are alarmed by the prospect that effective antibiotics may not be available to treat seriously ill patients in the near future.
The pipeline of new antibiotics is drying up. Major pharmaceutical companies are losing interest in the antibiotics market because these drugs may not be as profitable as drugs that treat chronic (long-term) conditions and lifestyle issues.
The resistance problem demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to current antibiotics. One of the possible strategies towards this objective is the rational localization of bioactive phytochemicals. Plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives such as tannins. Most are secondary metabolites, of which at least 12,000 have been isolated, a number estimated to be less than 10% of the total. In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Many of the herbs and spices used by humans to season food yield useful medicinal compounds including those having antibacterial activity.
Traditional healers have long used plants to prevent or cure infectious conditions. Many of these plants have been investigated scientifically for antimicrobial activity and a large number of plant products have been shown to inhibit growth of pathogenic bacteria. A number of these agents appear to have structures and modes of action that are distinct from those of the antibiotics in current use, suggesting that cross-resistance with agents already in use may be minimal. For example the combination of 5'-methoxyhydnocarpine and berberine in herbs like Hydrastis canadensis and Berberis vulgaris can block the MDR-pumps that cause multidrug resistance. This has been shown for Staphylococcus aureus.
Archaeocins is the name given to a new class of potentially useful antibiotics that are derived from the Archaea group of organisms. Eight archaeocins have been partially or fully characterized, but hundreds of archaeocins are believed to exist, especially within the haloarchaea. The prevalence of archaeocins is unknown simply because no one has looked for them. The discovery of new archaeocins hinges on recovery and cultivation of archaeal organisms from the environment. For example, samples from a novel hypersaline field site, Wilson Hot Springs, recovered 350 halophilic organisms; preliminary analysis of 75 isolates showed that 48 were archaeal and 27 were bacterial.
In research published on October 17, 2008 in Cell, a team of scientists pinpointed the place on bacteria where the antibiotic myxopyronin launches its attack, and why that attack is successful. The myxopyronin binds to and inhibits the crucial bacterial enzyme, RNA polymerase. The myxopyronin changes the structure of the switch-2 segment of the enzyme, inhibiting its function of reading and transmitting DNA code. This prevents RNA polymerase from delivering genetic information to the ribosomes, causing the bacteria to die.
One of the major causes of antibiotic resistance is the decrease of effective drug concentrations at their target place, due to the increased action of ABC transporters. Since ABC transporter blockers can be used in combination with current drugs to increase their effective intracellular concentration, the possible impact of ABC transporter inhibitors is of great clinical interest. ABC transporter blockers that may be useful to increase the efficacy of current drugs have entered clinical trials and are available to be used in therapeutic regimes.
Applications
Antibiotic resistance is an important tool for genetic engineering. By constructing a plasmid which contains an antibiotic resistance gene as well as the gene being engineered or expressed, a researcher can ensure that when bacteria replicate, only the copies which carry along the plasmid survive. This ensures that the gene being manipulated passes along when the bacteria replicates.
The most commonly used antibiotics in genetic engineering are generally "older" antibiotics which have largely fallen out of use in clinical practice. These include:
* ampicillin
* kanamycin
* tetracycline
* chloramphenicol
Industrially the use of antibiotic resistance is disfavored since maintaining bacterial cultures would require feeding them large quantities of antibiotics. Instead, the use of auxotrophic bacterial strains (and function-replacement plasmids) is preferred.
Sunday, November 1, 2009
Antibiotic
The term "antibiotic" was coined by Selman Waksman in 1942 to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. This original definition excluded naturally occurring substances that kill bacteria but are not produced by microorganisms (such as gastric juice and hydrogen peroxide) and also excluded synthetic antibacterial compounds such as the sulfonamides. Many antibiotics are relatively small molecules with a molecular weight less than 2000 Da.
With advances in medicinal chemistry, most antibiotics are now semisynthetic—modified chemically from original compounds found in nature, as is the case with beta-lactams (which include the penicillins, produced by fungi in the genus Penicillium, the cephalosporins, and the carbapenems). Some antibiotics are still produced and isolated from living organisms, such as the aminoglycosides, and others have been created through purely synthetic means: the sulfonamides, the quinolones, and the oxazolidinones. In addition to this origin-based classification into natural, semisynthetic, and synthetic, antibiotics may be divided into two broad groups according to their effect on microorganisms: those that kill bacteria are bactericidal agents, while those that only impair bacterial growth are known as bacteriostatic agents.
History of antibiotics
Many cures for infectious diseases prior to the beginning of the twentieth century were based on medicinal folklore. Cures for infection in ancient Chinese medicine using plants with antibiotic-like properties began to be described over 2,500 years ago. Many other ancient cultures, including the ancient Egyptians, ancient Greeks and medieval Arabs already used molds and plants to treat infections. Cinchona bark was a widely effective treatment of malaria in the 17th century, the disease caused by protozoan parasites of the genus Plasmodium. Scientific endeavours to understand the science behind what caused these diseases, the development of synthetic antibiotic chemotherapy, the isolation of the natural antibiotics marked milestones in antibiotic development.
Originally known as antiobiosis, antibiotics were drugs that had actions against bacteria. The term antibiosis which means ‘against life’ was introduced by the French bacteriologist Vuillemin as a descriptive name of the phenomenon exhibited by these drugs. (Antibiosis was first described in 1877 in bacteria when Louis Pasteur and Robert Koch observed that an airborne bacillus could inhibit the growth of Bacillus anthracis.). These drugs were later renamed antibiotics by Selman Wakeman, an American microbiologist in 1942.
Synthetic antibiotic chemotherapy as a science and the story of antibiotic development began in Germany with Paul Ehrlich, a German medical scientist in the late 1880s. Dr. Ehrlich noted that certain dyes would bind to and color human, animal or bacterial cells, while others did not. He then extended the idea that it might be possible to make certain dyes or chemicals that would act as a magic bullet or selective drug that would bind to and kill bacteria while not harming the human host. After much experimentation, screening hundreds of dyes against various organisms, he discovered the first medicinally useful drug, the man-made antibiotic, Salvarsan.However, the adverse side-effect profile of salvarsan, coupled with the later discovery of the antibiotic penicillin, superseded its use as an antibiotic. The work of Ehrlich, which marked the birth of the antibiotic revolution, was followed by the discovery of Prontosil by Domagk in 1932. Prontosil, the first commercially available antibacterial antibiotic was developed by a research team led by Gerhard Domagk (who received the 1939 Nobel Prize for Medicine for his efforts) at the Bayer Laboratories of the IG Farben conglomerate in Germany. Prontosil had a relatively broad effect against Gram-positive cocci but not against enterobacteria. The discovery and development of this first sulfonamide drug opened the era of antibiotics.
The discovery of natural antibiotics produced by microorganisms stemmed from earlier work on the observation of antibiosis between micro-organisms. Pasteur observed that "if we could intervene in the antagonism observed between some bacteria, it would offer ‘perhaps the greatest hopes for therapeutics’". Bacterial antagonism of Penicillium sp. were first described in England by John Tyndall in 1875. However, his work went by without much notice from the scientific community until Alexander Fleming's discovery of Penicillin in 1928. Even then the therapeutic potential of penicillin was not pursued. More than ten years later, Ernst Chain and Howard Florey became interested in Fleming's work, and came up with the purified form of penicillin. The purified antibiotic displayed antibacterial activity against a wide range of bacteria. It also had low toxicity and could be taken without causing adverse effects. Furthermore its activity was not inhibited by biological constituents such as pus, unlike the sulfonamides. At the time, no-one had discovered a compound equalling this activity. The discovery of penicillin led to renewed interest in the search for antibiotic compounds with similar capabilities. Because of their discovery of penicillin Ernst Chain, Howard Florey and Alexander Fleming shared the 1945 Nobel Prize in Medicine. In 1939, Rene Dubos isolated gramicidin, one of the first commercially manufactured antibiotics in use during World War II to prove highly effective in treating wounds and ulcers. Florey credited Dubos for reviving his research in penicillin.
Antimicrobial pharmacodynamics
The environment of individual antibiotics varies with the location of an infection, the ability of the antibiotic to reach the infection site, and the ability of the microbe to inactivate or excrete the antibiotic. At the highest level, antibiotics can be classified as either bactericidal or bacteriostatic. Bactericidals kill bacteria directly where bacteriostatics prevent cell division. However, these classifications are based on laboratory behavior; in practice, both of these are capable of ending a bacterial infection. The bactericidal activity of antibiotics may be growth phase dependent and in most but not all cases the action of many bactericidal antibiotics requires ongoing cell activity and cell division for the drugs' killing activity. The minimum inhibitory concentration and minimum bactericidal concentration are used to measure in vitro activity of an antimicrobial and are excellent indicators of antimicrobial potency. However, in clinical practice, these measurements alone are insufficient to predict clinical outcome. By combining the pharmacokinetic profile of an antibiotic with the antimicrobial activity, several pharmacological parameters appear to be significant markers of drug efficacy. The activity of antibiotics may be concentration-dependent and their characteristic antimicrobial activity increases with progressively higher antibiotic concentrations. They may also be time-dependent, where their antimicrobial activity does not increase with increasing antibiotic concentrations; however, it is critical that a minimum inhibitory serum concentration is maintained for a certain length of time.
Administration
Oral antibiotics are simply ingested, while intravenous antibiotics are used in more serious cases, such as deep-seated systemic infections. Antibiotics may also sometimes be administered topically, as with eye drops or ointments.
Antibiotic classes
Unlike many previous treatments for infections, which often consisted of administering chemical compounds such as strychnine and arsenic, which also have high toxicity against mammals, most antibiotics from microbes have fewer side-effects and high effective target activity. Most anti-bacterial antibiotics do not have activity against viruses, fungi, or other microbes. Anti-bacterial antibiotics can be categorized based on their target specificity: "narrow-spectrum" antibiotics target particular types of bacteria, such as Gram-negative or Gram-positive bacteria, while broad-spectrum antibiotics affect a wide range of bacteria.
Antibiotics which target the bacterial cell wall (penicillins, cephalosporins), or cell membrane (polymixins), or interfere with essential bacterial enzymes (quinolones, sulfonamides) usually are bactericidal in nature. Those which target protein synthesis, such as the aminoglycosides, macrolides and tetracyclines, are usually bacteriostatic.
In the last few years three new classes of antibiotics have been brought into clinical use. This follows a 40-year hiatus in discovering new classes of antibiotic compounds. These new antibiotics are of the following three classes: cyclic lipopeptides (daptomycin), glycylcyclines (tigecycline), and oxazolidinones (linezolid). Tigecycline is a broad-spectrum antibiotic, while the two others are used for Gram-positive infections. These developments show promise as a means to counteract the bacterial resistance to existing antibiotics.
Production
Since the first pioneering efforts of Florey and Chain in 1939, the importance of antibiotics to medicine has led to much research into discovering and producing them. The process of production usually involves the screening of wide ranges of microorganisms, and their testing and modification. Production is carried out using fermentation, usually in strongly aerobic form.
Side effects
Although antibiotics are generally considered safe and well tolerated, they have been associated with a wide range of adverse effects. Side effects are many, varied and can be very serious depending on the antibiotics used and the microbial organisms targeted. The safety profiles of newer medications may not be as well established as those that have been in use for many years. Adverse effects can range from fever and nausea to major allergic reactions including photodermatitis. One of the more common side effects is diarrhea, sometimes caused by the anaerobic bacterium Clostridium difficile, which results from the antibiotic disrupting the normal balance of the intestinal flora, Such overgrowth of pathogenic bacteria may be alleviated by ingesting probiotics during a course of antibiotics.. An antibiotic-induced disruption of the population of the bacteria normally present as constituents of the normal vaginal flora may also occur, and may lead to overgrowth of yeast species of the genus Candida in the vulvo-vaginal area. Other side effects can result from interaction with other drugs, such as elevated risk of tendon damage from administration of a quinolone antibiotic with a systemic corticosteroid.
Drug-Drug interactions
Contraceptive pill
Hypothetically, interference of some antibiotics with the efficiency of birth control pills is thought to occur in two ways. Modification of the intestinal gut flora resulting in the reduced absorption of the estrogens and induction of hepatic liver enzymes which metabolise the pills active ingredients faster may affect the pill's usefulness. However, the majority of studies indicate that antibiotics do not interfere with contraception, even though a small percentage of women may experience decreased effectiveness of birth control pills while taking an antibiotic the failure rate is comparible to those taking the pill.Moreover, there have been no studies that have conclusively demonstrated that disruption of the gut flora affects contraception. Interaction with the combined oral contraceptive pill through induction of hepatic enzymes by the antifungal medication griseofulvin and the broad-spectrum antibiotic rifampicin has been shown to occur. It is recommended that extra contraceptive measures are applied during antimicrobial therapy using these antimicrobials.
Alcohol
Alcohol can interfere with the activity or metabolization of antibiotics. It may affect the activity of liver enzymes, which break down the antibiotics. Moreover, certain antibiotics, including metronidazole, tinidazole, co-trimoxazole, cephamandole, ketoconazole, latamoxef, cefoperazone, amoxicillin, cefmenoxime, and furazolidone, chemically react with alcohol, leading to serious side effects, which include severe vomiting, nausea, and shortness of breath. Alcohol consumption while taking such antibiotics is therefore not recommended. Additionally, serum levels of doxycycline and erythromycin succinate may, in certain circumstances, be significantly reduced by alcohol consumption.
Antibiotic resistance
The emergence of antibiotic resistance is an evolutionary process that is based on selection for organisms that have enhanced ability to survive doses of antibiotics that would have previously been lethal. Antibiotics like Penicillin and Erythromycin which used to be one-time miracle cures are now less effective because bacteria have become more resistant. Antibiotics themselves act as a selective pressure which allows the growth of resistant bacteria within a population and inhibits susceptible bacteria. Antibiotic selection of pre-existing antibiotic resistant mutants within bacterial populations was demonstrated in 1943 by the Luria-Delbrück experiment. Survival of bacteria often results from an inheritable resistance. Any antibiotic resistance may impose a biological cost and the spread of antibiotic resistant bacteria may be hampered by the reduced fitness associated with the resistance which proves disadvantageous for survival of the bacteria when antibiotic is not present. Additional mutations, however, may compensate for this fitness cost and aids the survival of these bacteria.
The underlying molecular mechanisms leading to antibiotic resistance can vary. Intrinsic resistance may naturally occur as a result of the bacteria's genetic makeup. The bacterial chromosome may fail to encode a protein which the antibiotic targets. Acquired resistance results from a mutation in the bacterial chromosome or the acquisition of extra-chromosomal DNA. Antibiotic-producing bacteria have evolved resistance mechanisms which have been shown to be similar to and may have been transferred to antibiotic resistant strains. The spread of antibiotic resistance mechanisms occurs through vertical transmission of inherited mutations from previous generations and genetic recombination of DNA by horizontal genetic exchange. Antibiotic resistance exchanged between different bacteria by plasmids that carry genes which encode antibiotic resistance which may result in co-resistance to multiple antibiotics. These plasmids can carry different genes with diverse resistance mechanisms to unrelated antibiotics but because they are located on the same plasmid multiple antibiotic resistance to more than one antibiotic is transferred. Alternatively, cross-resistance to other antibiotics within the bacteria results when the same resistance mechanism is responsible for resistance to more than one antibiotic is selected for.
Antibiotic misuse
The first rule of antibiotics is try not to use them, and the second rule is try not to use too many of them.
—Paul L. Marino, The ICU Book
Inappropriate antibiotic treatment and overuse of antibiotics have been a contributing factor to the emergence of resistant bacteria. The problem is further exacerbated by self-prescribing of antibiotics by individuals without the guidelines of a qualified clinician and the non-therapeutic use of antibiotics as growth promoters in agriculture. Antibiotics are frequently prescribed for indications in which their use is not warranted, an incorrect or sub-optimal antibiotic is prescribed or in some cases for infections likely to resolve without treatment.
Several organizations concerned with antimicrobial resistance are lobbying to improve the regulatory climate. Approaches to tackling the issues of misuse and overuse of antibiotics by the establishment of the U.S. Interagency Task Force on Antimicrobial Resistance which aims actively address the problem antimicrobial resistance are being organised and coordinated by the US Centers for Disease Control and Prevention, the Food and Drug Administration (FDA), and the National Institutes of Health (NIH) and also includes several other federal agencies. An NGO campaign group is Keep Antibiotics Working. In France, an "Antibiotics are not automatic" government campaign starting in 2002 led to a marked reduction of unnecessary antibiotic prescriptions, especially in children.
The overuse of antibiotics like penicillin and erythromycin which used to be one-time miracle cures were associated with emerging resistance since the 1950s. Therapeutic usage of antibiotics in hospitals has been seen to be associated with increases in multi-antibiotic resistant bacteria
Common forms of antibiotic misuse include failure to take into account the patient's weight and history of prior antibiotic use when prescribing, since both can strongly affect the efficacy of an antibiotic prescription, failure to take the entire prescribed course of the antibiotic, failure to prescribe or take the course of treatment at fairly precise correct daily intervals (e.g. "every 8 hours" rather than merely "3x per day"), or failure to rest for sufficient recovery to allow clearance of the infecting organism. These practices may facilitate the development of bacterial populations with antibiotic resistance. Inappropriate antibiotic treatment is another common form of antibiotic misuse. A common example is the prescription and use of antibiotics to treat viral infections such as the common cold that have no effect.
In agriculture, associated antibiotic resistance with the non-therapeutic use of antibiotics as growth promoters in animals resulted in their restricted use in the UK in the 1970 (Swann report 1969). Currently there is a EU wide ban on the non-therapeutic use of antibiotics as growth promoters. It is estimated that greater than 70% of the antibiotics used in U.S. are given to feed animals (e.g. chickens, pigs and cattle) in the absence of disease. Antibiotic use in food animal production has been associated with the emergence of antibiotic-resistant strains of bacteria including Salmonella spp., Campylobacter spp., Escherichia coli, and Enterococcus spp. Evidence from some US and European studies suggest that these resistant bacteria cause infections in humans that do not respond to commonly prescribed antibiotics. In response to these practices and attendant problems, several organizations (e.g. The American Society for Microbiology (ASM), American Public Health Association (APHA) and the American Medical Association (AMA)) have called for restrictions on antibiotic use in food animal production and an end to all non-therapeutic uses.However, delays in regulatory and legislative actions to limit the use of antibiotics are common, and may include resistance to these changes by industries using or selling antibiotics, as well as time spent on research to establish causal links between antibiotic use and emergence of untreatable bacterial diseases. Two federal bills (S.742 and H.R. 2562) aimed at phasing out non-therapeutic antibiotics in US food animal production were proposed but not passed. These bills were endorsed by public health and medical organizations including the American Holistic Nurses’ Association, the American Medical Association, and the American Public Health Association (APHA). The EU has banned the use of antibiotics as growth promotional agents since 2003.
One study on respiratory tract infections found "physicians were more likely to prescribe antibiotics to patients who they believed expected them, although they correctly identified only about 1 in 4 of those patients". Multifactorial interventions aimed at both physicians and patients can reduce inappropriate prescribing of antibiotics. Delaying antibiotics for 48 hours while observing for spontaneous resolution of respiratory tract infections may reduce antibiotic usage; however, this strategy may reduce patient satisfaction.
Excessive use of prophylactic antibiotics in travelers may also be classified as misuse.
In the United Kingdom, there are NHS posters in many doctors surgeries indicating that 'unfortunately, no amount of antibiotics will get rid of your cold', following on from many patients specifically requesting antibiotics from their doctor inappropriately, believing they will help treat viral infections.
Resistance modifying agents
One solution to combat resistance currently being researched is the development of pharmaceutical compounds that would revert multiple antibiotic resistance. These so called resistance modifying agents may target and inhibit MDR mechanisms, rendering the bacteria susceptible to antibiotics to which they were previously resistant. These compounds targets include among others
* Efflux inhibition(Phe-Arg-β-naphthylamide)
* Beta Lactamase inhibitors - Including Clavulanic acid and Sulbactam
Beyond antibiotics
The comparative ease of identifying compounds which safely cured bacterial infections was more difficult to duplicate in treatments of fungal and viral infections. Antibiotic research led to great strides in the knowledge of biochemistry, establishing large differences between the cellular and molecular physiology of the bacterial cell and that of the mammalian cell. This explained the observation that many compounds that are toxic to bacteria are non-toxic to human cells. In contrast, the basic biochemistries of the fungal cell and the mammalian cell are much more similar. This restricts the development and use of therapeutic compounds that attack a fungal cell, while not harming mammalian cells. Similar problems exist in antibiotic treatments of viral diseases. Human viral metabolic biochemistry is very closely similar to human biochemistry, and the possible targets of antiviral compounds are restricted to very few components unique to a mammalian virus.
Research into bacteriophages for use as antibiotics is presently ongoing. Several types of bacteriophage appear to exist that are specific for each bacterial taxonomic group or species. Research into bacteriophages for medicinal use is just beginning, but has led to advances in microscopic imaging. While bacteriophages provide a possible solution to the problem of antibiotic resistance, there is no clinical evidence yet that they can be deployed as therapeutic agents to cure disease.
Phage therapy, the use of particular viruses to attack bacteria, has been used in the past on humans in the US and Europe during the 1920s and 1930s, but these treatments had mixed results. With the discovery of penicillin in the 1940s, Europe and the US changed therapeutic strategies to using antibiotics. However, in the former Soviet Union phage therapies continued to be studied. In the Republic of Georgia, the Eliava Institute of Bacteriophage, Microbiology & Virology continues to research the use of phage therapy. Various companies and foundations in North America and Europe are currently researching phage therapies.However, phage are living and reproducing; concerns about genetic engineering in freely released viruses currently limit certain aspects of phage therapy.
Bacteriocins are also a growing alternative to the classic small-molecule antibiotics . Different classes of bacteriocins have different potential as therapeutic agents. Small molecule bacteriocins (microcins, for example, and lantibiotics) may be similar to the classic antibiotics; colicin-like bacteriocins are more likely to be narrow-spectrum, demanding new molecular diagnostics prior to therapy but also not raising the spectre of resistance to the same degree. One drawback to the large molecule antibiotics is that they will have relative difficulty crossing membranes and travelling systemically throughout the body. For this reason, they are most often proposed for application topically or gastrointestinally. Because bacteriocins are peptides, they are more readily engineered than small molecules. This may permit the generation of cocktails and dynamically improved antibiotics that are modified to overcome resistance.
Probiotics are another alternative that goes beyond traditional antibiotics by employing a live culture which may in theory establish itself as a symbiont, competing, inhibiting, or simply interfering with colonization by pathogens.
With advances in medicinal chemistry, most antibiotics are now semisynthetic—modified chemically from original compounds found in nature, as is the case with beta-lactams (which include the penicillins, produced by fungi in the genus Penicillium, the cephalosporins, and the carbapenems). Some antibiotics are still produced and isolated from living organisms, such as the aminoglycosides, and others have been created through purely synthetic means: the sulfonamides, the quinolones, and the oxazolidinones. In addition to this origin-based classification into natural, semisynthetic, and synthetic, antibiotics may be divided into two broad groups according to their effect on microorganisms: those that kill bacteria are bactericidal agents, while those that only impair bacterial growth are known as bacteriostatic agents.
History of antibiotics
Many cures for infectious diseases prior to the beginning of the twentieth century were based on medicinal folklore. Cures for infection in ancient Chinese medicine using plants with antibiotic-like properties began to be described over 2,500 years ago. Many other ancient cultures, including the ancient Egyptians, ancient Greeks and medieval Arabs already used molds and plants to treat infections. Cinchona bark was a widely effective treatment of malaria in the 17th century, the disease caused by protozoan parasites of the genus Plasmodium. Scientific endeavours to understand the science behind what caused these diseases, the development of synthetic antibiotic chemotherapy, the isolation of the natural antibiotics marked milestones in antibiotic development.
Originally known as antiobiosis, antibiotics were drugs that had actions against bacteria. The term antibiosis which means ‘against life’ was introduced by the French bacteriologist Vuillemin as a descriptive name of the phenomenon exhibited by these drugs. (Antibiosis was first described in 1877 in bacteria when Louis Pasteur and Robert Koch observed that an airborne bacillus could inhibit the growth of Bacillus anthracis.). These drugs were later renamed antibiotics by Selman Wakeman, an American microbiologist in 1942.
Synthetic antibiotic chemotherapy as a science and the story of antibiotic development began in Germany with Paul Ehrlich, a German medical scientist in the late 1880s. Dr. Ehrlich noted that certain dyes would bind to and color human, animal or bacterial cells, while others did not. He then extended the idea that it might be possible to make certain dyes or chemicals that would act as a magic bullet or selective drug that would bind to and kill bacteria while not harming the human host. After much experimentation, screening hundreds of dyes against various organisms, he discovered the first medicinally useful drug, the man-made antibiotic, Salvarsan.However, the adverse side-effect profile of salvarsan, coupled with the later discovery of the antibiotic penicillin, superseded its use as an antibiotic. The work of Ehrlich, which marked the birth of the antibiotic revolution, was followed by the discovery of Prontosil by Domagk in 1932. Prontosil, the first commercially available antibacterial antibiotic was developed by a research team led by Gerhard Domagk (who received the 1939 Nobel Prize for Medicine for his efforts) at the Bayer Laboratories of the IG Farben conglomerate in Germany. Prontosil had a relatively broad effect against Gram-positive cocci but not against enterobacteria. The discovery and development of this first sulfonamide drug opened the era of antibiotics.
The discovery of natural antibiotics produced by microorganisms stemmed from earlier work on the observation of antibiosis between micro-organisms. Pasteur observed that "if we could intervene in the antagonism observed between some bacteria, it would offer ‘perhaps the greatest hopes for therapeutics’". Bacterial antagonism of Penicillium sp. were first described in England by John Tyndall in 1875. However, his work went by without much notice from the scientific community until Alexander Fleming's discovery of Penicillin in 1928. Even then the therapeutic potential of penicillin was not pursued. More than ten years later, Ernst Chain and Howard Florey became interested in Fleming's work, and came up with the purified form of penicillin. The purified antibiotic displayed antibacterial activity against a wide range of bacteria. It also had low toxicity and could be taken without causing adverse effects. Furthermore its activity was not inhibited by biological constituents such as pus, unlike the sulfonamides. At the time, no-one had discovered a compound equalling this activity. The discovery of penicillin led to renewed interest in the search for antibiotic compounds with similar capabilities. Because of their discovery of penicillin Ernst Chain, Howard Florey and Alexander Fleming shared the 1945 Nobel Prize in Medicine. In 1939, Rene Dubos isolated gramicidin, one of the first commercially manufactured antibiotics in use during World War II to prove highly effective in treating wounds and ulcers. Florey credited Dubos for reviving his research in penicillin.
Antimicrobial pharmacodynamics
The environment of individual antibiotics varies with the location of an infection, the ability of the antibiotic to reach the infection site, and the ability of the microbe to inactivate or excrete the antibiotic. At the highest level, antibiotics can be classified as either bactericidal or bacteriostatic. Bactericidals kill bacteria directly where bacteriostatics prevent cell division. However, these classifications are based on laboratory behavior; in practice, both of these are capable of ending a bacterial infection. The bactericidal activity of antibiotics may be growth phase dependent and in most but not all cases the action of many bactericidal antibiotics requires ongoing cell activity and cell division for the drugs' killing activity. The minimum inhibitory concentration and minimum bactericidal concentration are used to measure in vitro activity of an antimicrobial and are excellent indicators of antimicrobial potency. However, in clinical practice, these measurements alone are insufficient to predict clinical outcome. By combining the pharmacokinetic profile of an antibiotic with the antimicrobial activity, several pharmacological parameters appear to be significant markers of drug efficacy. The activity of antibiotics may be concentration-dependent and their characteristic antimicrobial activity increases with progressively higher antibiotic concentrations. They may also be time-dependent, where their antimicrobial activity does not increase with increasing antibiotic concentrations; however, it is critical that a minimum inhibitory serum concentration is maintained for a certain length of time.
Administration
Oral antibiotics are simply ingested, while intravenous antibiotics are used in more serious cases, such as deep-seated systemic infections. Antibiotics may also sometimes be administered topically, as with eye drops or ointments.
Antibiotic classes
Unlike many previous treatments for infections, which often consisted of administering chemical compounds such as strychnine and arsenic, which also have high toxicity against mammals, most antibiotics from microbes have fewer side-effects and high effective target activity. Most anti-bacterial antibiotics do not have activity against viruses, fungi, or other microbes. Anti-bacterial antibiotics can be categorized based on their target specificity: "narrow-spectrum" antibiotics target particular types of bacteria, such as Gram-negative or Gram-positive bacteria, while broad-spectrum antibiotics affect a wide range of bacteria.
Antibiotics which target the bacterial cell wall (penicillins, cephalosporins), or cell membrane (polymixins), or interfere with essential bacterial enzymes (quinolones, sulfonamides) usually are bactericidal in nature. Those which target protein synthesis, such as the aminoglycosides, macrolides and tetracyclines, are usually bacteriostatic.
In the last few years three new classes of antibiotics have been brought into clinical use. This follows a 40-year hiatus in discovering new classes of antibiotic compounds. These new antibiotics are of the following three classes: cyclic lipopeptides (daptomycin), glycylcyclines (tigecycline), and oxazolidinones (linezolid). Tigecycline is a broad-spectrum antibiotic, while the two others are used for Gram-positive infections. These developments show promise as a means to counteract the bacterial resistance to existing antibiotics.
Production
Since the first pioneering efforts of Florey and Chain in 1939, the importance of antibiotics to medicine has led to much research into discovering and producing them. The process of production usually involves the screening of wide ranges of microorganisms, and their testing and modification. Production is carried out using fermentation, usually in strongly aerobic form.
Side effects
Although antibiotics are generally considered safe and well tolerated, they have been associated with a wide range of adverse effects. Side effects are many, varied and can be very serious depending on the antibiotics used and the microbial organisms targeted. The safety profiles of newer medications may not be as well established as those that have been in use for many years. Adverse effects can range from fever and nausea to major allergic reactions including photodermatitis. One of the more common side effects is diarrhea, sometimes caused by the anaerobic bacterium Clostridium difficile, which results from the antibiotic disrupting the normal balance of the intestinal flora, Such overgrowth of pathogenic bacteria may be alleviated by ingesting probiotics during a course of antibiotics.. An antibiotic-induced disruption of the population of the bacteria normally present as constituents of the normal vaginal flora may also occur, and may lead to overgrowth of yeast species of the genus Candida in the vulvo-vaginal area. Other side effects can result from interaction with other drugs, such as elevated risk of tendon damage from administration of a quinolone antibiotic with a systemic corticosteroid.
Drug-Drug interactions
Contraceptive pill
Hypothetically, interference of some antibiotics with the efficiency of birth control pills is thought to occur in two ways. Modification of the intestinal gut flora resulting in the reduced absorption of the estrogens and induction of hepatic liver enzymes which metabolise the pills active ingredients faster may affect the pill's usefulness. However, the majority of studies indicate that antibiotics do not interfere with contraception, even though a small percentage of women may experience decreased effectiveness of birth control pills while taking an antibiotic the failure rate is comparible to those taking the pill.Moreover, there have been no studies that have conclusively demonstrated that disruption of the gut flora affects contraception. Interaction with the combined oral contraceptive pill through induction of hepatic enzymes by the antifungal medication griseofulvin and the broad-spectrum antibiotic rifampicin has been shown to occur. It is recommended that extra contraceptive measures are applied during antimicrobial therapy using these antimicrobials.
Alcohol
Alcohol can interfere with the activity or metabolization of antibiotics. It may affect the activity of liver enzymes, which break down the antibiotics. Moreover, certain antibiotics, including metronidazole, tinidazole, co-trimoxazole, cephamandole, ketoconazole, latamoxef, cefoperazone, amoxicillin, cefmenoxime, and furazolidone, chemically react with alcohol, leading to serious side effects, which include severe vomiting, nausea, and shortness of breath. Alcohol consumption while taking such antibiotics is therefore not recommended. Additionally, serum levels of doxycycline and erythromycin succinate may, in certain circumstances, be significantly reduced by alcohol consumption.
Antibiotic resistance
The emergence of antibiotic resistance is an evolutionary process that is based on selection for organisms that have enhanced ability to survive doses of antibiotics that would have previously been lethal. Antibiotics like Penicillin and Erythromycin which used to be one-time miracle cures are now less effective because bacteria have become more resistant. Antibiotics themselves act as a selective pressure which allows the growth of resistant bacteria within a population and inhibits susceptible bacteria. Antibiotic selection of pre-existing antibiotic resistant mutants within bacterial populations was demonstrated in 1943 by the Luria-Delbrück experiment. Survival of bacteria often results from an inheritable resistance. Any antibiotic resistance may impose a biological cost and the spread of antibiotic resistant bacteria may be hampered by the reduced fitness associated with the resistance which proves disadvantageous for survival of the bacteria when antibiotic is not present. Additional mutations, however, may compensate for this fitness cost and aids the survival of these bacteria.
The underlying molecular mechanisms leading to antibiotic resistance can vary. Intrinsic resistance may naturally occur as a result of the bacteria's genetic makeup. The bacterial chromosome may fail to encode a protein which the antibiotic targets. Acquired resistance results from a mutation in the bacterial chromosome or the acquisition of extra-chromosomal DNA. Antibiotic-producing bacteria have evolved resistance mechanisms which have been shown to be similar to and may have been transferred to antibiotic resistant strains. The spread of antibiotic resistance mechanisms occurs through vertical transmission of inherited mutations from previous generations and genetic recombination of DNA by horizontal genetic exchange. Antibiotic resistance exchanged between different bacteria by plasmids that carry genes which encode antibiotic resistance which may result in co-resistance to multiple antibiotics. These plasmids can carry different genes with diverse resistance mechanisms to unrelated antibiotics but because they are located on the same plasmid multiple antibiotic resistance to more than one antibiotic is transferred. Alternatively, cross-resistance to other antibiotics within the bacteria results when the same resistance mechanism is responsible for resistance to more than one antibiotic is selected for.
Antibiotic misuse
The first rule of antibiotics is try not to use them, and the second rule is try not to use too many of them.
—Paul L. Marino, The ICU Book
Inappropriate antibiotic treatment and overuse of antibiotics have been a contributing factor to the emergence of resistant bacteria. The problem is further exacerbated by self-prescribing of antibiotics by individuals without the guidelines of a qualified clinician and the non-therapeutic use of antibiotics as growth promoters in agriculture. Antibiotics are frequently prescribed for indications in which their use is not warranted, an incorrect or sub-optimal antibiotic is prescribed or in some cases for infections likely to resolve without treatment.
Several organizations concerned with antimicrobial resistance are lobbying to improve the regulatory climate. Approaches to tackling the issues of misuse and overuse of antibiotics by the establishment of the U.S. Interagency Task Force on Antimicrobial Resistance which aims actively address the problem antimicrobial resistance are being organised and coordinated by the US Centers for Disease Control and Prevention, the Food and Drug Administration (FDA), and the National Institutes of Health (NIH) and also includes several other federal agencies. An NGO campaign group is Keep Antibiotics Working. In France, an "Antibiotics are not automatic" government campaign starting in 2002 led to a marked reduction of unnecessary antibiotic prescriptions, especially in children.
The overuse of antibiotics like penicillin and erythromycin which used to be one-time miracle cures were associated with emerging resistance since the 1950s. Therapeutic usage of antibiotics in hospitals has been seen to be associated with increases in multi-antibiotic resistant bacteria
Common forms of antibiotic misuse include failure to take into account the patient's weight and history of prior antibiotic use when prescribing, since both can strongly affect the efficacy of an antibiotic prescription, failure to take the entire prescribed course of the antibiotic, failure to prescribe or take the course of treatment at fairly precise correct daily intervals (e.g. "every 8 hours" rather than merely "3x per day"), or failure to rest for sufficient recovery to allow clearance of the infecting organism. These practices may facilitate the development of bacterial populations with antibiotic resistance. Inappropriate antibiotic treatment is another common form of antibiotic misuse. A common example is the prescription and use of antibiotics to treat viral infections such as the common cold that have no effect.
In agriculture, associated antibiotic resistance with the non-therapeutic use of antibiotics as growth promoters in animals resulted in their restricted use in the UK in the 1970 (Swann report 1969). Currently there is a EU wide ban on the non-therapeutic use of antibiotics as growth promoters. It is estimated that greater than 70% of the antibiotics used in U.S. are given to feed animals (e.g. chickens, pigs and cattle) in the absence of disease. Antibiotic use in food animal production has been associated with the emergence of antibiotic-resistant strains of bacteria including Salmonella spp., Campylobacter spp., Escherichia coli, and Enterococcus spp. Evidence from some US and European studies suggest that these resistant bacteria cause infections in humans that do not respond to commonly prescribed antibiotics. In response to these practices and attendant problems, several organizations (e.g. The American Society for Microbiology (ASM), American Public Health Association (APHA) and the American Medical Association (AMA)) have called for restrictions on antibiotic use in food animal production and an end to all non-therapeutic uses.However, delays in regulatory and legislative actions to limit the use of antibiotics are common, and may include resistance to these changes by industries using or selling antibiotics, as well as time spent on research to establish causal links between antibiotic use and emergence of untreatable bacterial diseases. Two federal bills (S.742 and H.R. 2562) aimed at phasing out non-therapeutic antibiotics in US food animal production were proposed but not passed. These bills were endorsed by public health and medical organizations including the American Holistic Nurses’ Association, the American Medical Association, and the American Public Health Association (APHA). The EU has banned the use of antibiotics as growth promotional agents since 2003.
One study on respiratory tract infections found "physicians were more likely to prescribe antibiotics to patients who they believed expected them, although they correctly identified only about 1 in 4 of those patients". Multifactorial interventions aimed at both physicians and patients can reduce inappropriate prescribing of antibiotics. Delaying antibiotics for 48 hours while observing for spontaneous resolution of respiratory tract infections may reduce antibiotic usage; however, this strategy may reduce patient satisfaction.
Excessive use of prophylactic antibiotics in travelers may also be classified as misuse.
In the United Kingdom, there are NHS posters in many doctors surgeries indicating that 'unfortunately, no amount of antibiotics will get rid of your cold', following on from many patients specifically requesting antibiotics from their doctor inappropriately, believing they will help treat viral infections.
Resistance modifying agents
One solution to combat resistance currently being researched is the development of pharmaceutical compounds that would revert multiple antibiotic resistance. These so called resistance modifying agents may target and inhibit MDR mechanisms, rendering the bacteria susceptible to antibiotics to which they were previously resistant. These compounds targets include among others
* Efflux inhibition(Phe-Arg-β-naphthylamide)
* Beta Lactamase inhibitors - Including Clavulanic acid and Sulbactam
Beyond antibiotics
The comparative ease of identifying compounds which safely cured bacterial infections was more difficult to duplicate in treatments of fungal and viral infections. Antibiotic research led to great strides in the knowledge of biochemistry, establishing large differences between the cellular and molecular physiology of the bacterial cell and that of the mammalian cell. This explained the observation that many compounds that are toxic to bacteria are non-toxic to human cells. In contrast, the basic biochemistries of the fungal cell and the mammalian cell are much more similar. This restricts the development and use of therapeutic compounds that attack a fungal cell, while not harming mammalian cells. Similar problems exist in antibiotic treatments of viral diseases. Human viral metabolic biochemistry is very closely similar to human biochemistry, and the possible targets of antiviral compounds are restricted to very few components unique to a mammalian virus.
Research into bacteriophages for use as antibiotics is presently ongoing. Several types of bacteriophage appear to exist that are specific for each bacterial taxonomic group or species. Research into bacteriophages for medicinal use is just beginning, but has led to advances in microscopic imaging. While bacteriophages provide a possible solution to the problem of antibiotic resistance, there is no clinical evidence yet that they can be deployed as therapeutic agents to cure disease.
Phage therapy, the use of particular viruses to attack bacteria, has been used in the past on humans in the US and Europe during the 1920s and 1930s, but these treatments had mixed results. With the discovery of penicillin in the 1940s, Europe and the US changed therapeutic strategies to using antibiotics. However, in the former Soviet Union phage therapies continued to be studied. In the Republic of Georgia, the Eliava Institute of Bacteriophage, Microbiology & Virology continues to research the use of phage therapy. Various companies and foundations in North America and Europe are currently researching phage therapies.However, phage are living and reproducing; concerns about genetic engineering in freely released viruses currently limit certain aspects of phage therapy.
Bacteriocins are also a growing alternative to the classic small-molecule antibiotics . Different classes of bacteriocins have different potential as therapeutic agents. Small molecule bacteriocins (microcins, for example, and lantibiotics) may be similar to the classic antibiotics; colicin-like bacteriocins are more likely to be narrow-spectrum, demanding new molecular diagnostics prior to therapy but also not raising the spectre of resistance to the same degree. One drawback to the large molecule antibiotics is that they will have relative difficulty crossing membranes and travelling systemically throughout the body. For this reason, they are most often proposed for application topically or gastrointestinally. Because bacteriocins are peptides, they are more readily engineered than small molecules. This may permit the generation of cocktails and dynamically improved antibiotics that are modified to overcome resistance.
Probiotics are another alternative that goes beyond traditional antibiotics by employing a live culture which may in theory establish itself as a symbiont, competing, inhibiting, or simply interfering with colonization by pathogens.
Friday, October 23, 2009
SARS Virus Genetically Engineered
The SARS epidemic started in the weeks that the ‘allied forces’ were waging war on Iraq to hunt down Saddam Hussein and his still elusive ‘weapons of mass destruction’.
SARS – Severe Acute Respiratory Syndrome – is a completely new infectious disease spread by human contact. By 20 June 2003, World Health Organisation figures registered 8461 cases in 31 countries worldwide, and 804 deaths. The overall death rate is nearly 10% and could be 20% or higher.
Although there are signs that the disease is under control, there are also fears that it may return.
Mystery of the SARS virus
The World Health Organisation, which played the key role in coordinating the research of a dozen laboratories, formally announced on 16 April that a new pathogen, a member of the coronavirus family never before seen in humans, is the cause of SARS, though lingering doubt has remained. The virus cannot be identified all patients diagnosed with SARS, and it can only be isolated from cultured green monkey kidney cells.
Known coronaviruses are placed in three groups based on similarities in their genomes. Group 1 contains the porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV) and human coronovirus 229E (HuCV229E); Group 2 contains the avian infectious bronchitis virus (AIBV) and turkey coronavirus; while Group 3 contains the murine hepatitis virus (MHV) bovine coronavirus (BCV), human coronavirus (HuOC43) and others.
The molecular phylogenies published 10 April in the New England Journal of Medicine, based on small fragments of the polymerase gene, have placed the SARS virus in a separate group somewhere between groups 2 and 3.
More detailed analysis, subsequently published in the New England Journal of Medicine, Science and the Lancet indicate that the new virus is not closely related to any known virus at all, human, mouse, bovine, cat, pig, bird, notwithstanding. It is neither a mutant that switched host, nor a recombinant from existing coronaviruses. It is more complicated than that.
SARS virus - a product of genetic engineering?
Two scientists who have genetic engineered coronaviruses in their laboratories, Holmes and Enjuanes, suggested in a commentary in the journal Science that the SARS virus probably "evolved separately from an ancestor of the known coronavirus, and infected an unidentified animal, bird, or reptile host for a very long time before infecting humans and starting the SARS epidemic." (p.1377)
Following soon afterwards, there was a claim that the SARS virus came from the masked civet cat in south China. But that claim could not be substantiated. An alternative hypothesis entertained in the mainstream journals was that the virus came from outerspace.
There are very unusual features to the SARS virus. Its sequence most closely matches that of mouse hepatitis virus (MHV) and Bovine corona virus (BCV), both in group 3. The match is quite good in the middle third of the genome that’s nearly 30 000nt long, and not good at all for the first third or last third of the sequence.
But, antibodies to the SARS virus cross react with FIPV, HuCV229E and TGEV, all in Group 1. And the SARS virus can grow in Vero green monkey kidney cells, which no other coronavirus can, with the exception of PEDV, another virus in Group 1.
Could the SARS virus have come from genetic engineering? This is a question that Ho and Cummins have put to the scientific community. So far, we have not had a proper reply.
Holmes and Enjuanes stated in their commentary, "SARS-CoV is also unlikely to have been created from known coronaviruses by genetic engineering, because at present it would be impossible to modify 50% of a coronavirus genome without abrogating viral infectivity."
This is a quite a feeble response. The whole point to genetic engineering is that it greatly increases the scope of recombination, and provides selective tools to find the most unlikely recombinants that are still infectious.
Coronaviruses have been subjected to increasing genetic manipulation since the latter half of the 1990s, when P.S. Masters in Wadworth Center, New York State Department of Health and New York State University at Albany, used RNA recombination to introduce extensive changes into the genome of mouse hepatitis virus (MHV). In a review published in 1999, he wrote, "targeted recombination could be used to create extensive substitutions to the cornavirus genome, generating recombinants that could not be made otherwise between two viruses separated by a species barrier." (p.254)
‘Defective interfering RNAs’ – sequences of the viral genome with large deletions as well as mutations and substitutions or insertions - were used as donor sequences to introduce major substitutions and point mutations into the genome of the viruses by RNA recombination.
In the course of such work, researchers have even isolated a recombinant of cororanvirus with the green fluorescent protein (GFP) gene, presumably from cells in which coronaviruses have been cultured, which has become inserted into the spike protein gene. The GFP gene, originally from a jelly-fish, is extensively used in genetic engineering as a marker gene because it makes the cells that have taken up the foreign genes give off a green glow under uv light. The GFP-coronavirus recombinant could only have come about as an unintended by-product of genetic engineering.
In the same review, P.S. Masters showed that both point mutations and large substitutions can readily be transferred to the last third of the genome of MHV and other coronaviruses. He further indicated that similar strategies could be used to mutate and substitute the first third of the genome, though not for the middle third. "A comprehensive genetic study of the highly complex gene for the RNA polymerase and all of its associated activities [encoded by the middle third of the genome] will likely await either the construction of an infectious full-length clone or the development of an innovative scheme for mutant selection." (p.259)
Is that why the middle third of SARS virus genome has retained good homology to MHV and BCV, which were the first coronaviruses to be engineered in this manner, while the other parts are much more different?
Another feature of the SARS virus is that the spike protein, which determines host range, is unlike the spike protein of any known coronavirus. Instead, it appears to have homologies to segments of the human chromosome 7, according to sequence analysis performed by Howard Urnovitz.
Urnovitz believes that the spike protein of the SARS virus is the result of genetic rearrangements provoked by environmental genotoxic agents, much like those he and his colleagues have detected in Gulf War I veterans suffering from Gulf War Syndrome.
But how did the virus get to south China? A possible answer was provided by Urnovitz: Migratory birds that frequent gene-swapping hot spots like southeast China could have carried the SARS virus there.
Urnovitz himself doesn’t think the SARS virus is the real cause of SARS. Instead, it is the piece of reshuffled human chromosome 7 that others are referring to as the spike protein gene of the SARS virus. That alone is sufficient to trigger serious autoimmune responses in people.
Hence, to create vaccines against that ‘spike’ protein is also tantamount to vaccinating people against their own genes (see "Dynamic genomics")
SARS – Severe Acute Respiratory Syndrome – is a completely new infectious disease spread by human contact. By 20 June 2003, World Health Organisation figures registered 8461 cases in 31 countries worldwide, and 804 deaths. The overall death rate is nearly 10% and could be 20% or higher.
Although there are signs that the disease is under control, there are also fears that it may return.
Mystery of the SARS virus
The World Health Organisation, which played the key role in coordinating the research of a dozen laboratories, formally announced on 16 April that a new pathogen, a member of the coronavirus family never before seen in humans, is the cause of SARS, though lingering doubt has remained. The virus cannot be identified all patients diagnosed with SARS, and it can only be isolated from cultured green monkey kidney cells.
Known coronaviruses are placed in three groups based on similarities in their genomes. Group 1 contains the porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV) and human coronovirus 229E (HuCV229E); Group 2 contains the avian infectious bronchitis virus (AIBV) and turkey coronavirus; while Group 3 contains the murine hepatitis virus (MHV) bovine coronavirus (BCV), human coronavirus (HuOC43) and others.
The molecular phylogenies published 10 April in the New England Journal of Medicine, based on small fragments of the polymerase gene, have placed the SARS virus in a separate group somewhere between groups 2 and 3.
More detailed analysis, subsequently published in the New England Journal of Medicine, Science and the Lancet indicate that the new virus is not closely related to any known virus at all, human, mouse, bovine, cat, pig, bird, notwithstanding. It is neither a mutant that switched host, nor a recombinant from existing coronaviruses. It is more complicated than that.
SARS virus - a product of genetic engineering?
Two scientists who have genetic engineered coronaviruses in their laboratories, Holmes and Enjuanes, suggested in a commentary in the journal Science that the SARS virus probably "evolved separately from an ancestor of the known coronavirus, and infected an unidentified animal, bird, or reptile host for a very long time before infecting humans and starting the SARS epidemic." (p.1377)
Following soon afterwards, there was a claim that the SARS virus came from the masked civet cat in south China. But that claim could not be substantiated. An alternative hypothesis entertained in the mainstream journals was that the virus came from outerspace.
There are very unusual features to the SARS virus. Its sequence most closely matches that of mouse hepatitis virus (MHV) and Bovine corona virus (BCV), both in group 3. The match is quite good in the middle third of the genome that’s nearly 30 000nt long, and not good at all for the first third or last third of the sequence.
But, antibodies to the SARS virus cross react with FIPV, HuCV229E and TGEV, all in Group 1. And the SARS virus can grow in Vero green monkey kidney cells, which no other coronavirus can, with the exception of PEDV, another virus in Group 1.
Could the SARS virus have come from genetic engineering? This is a question that Ho and Cummins have put to the scientific community. So far, we have not had a proper reply.
Holmes and Enjuanes stated in their commentary, "SARS-CoV is also unlikely to have been created from known coronaviruses by genetic engineering, because at present it would be impossible to modify 50% of a coronavirus genome without abrogating viral infectivity."
This is a quite a feeble response. The whole point to genetic engineering is that it greatly increases the scope of recombination, and provides selective tools to find the most unlikely recombinants that are still infectious.
Coronaviruses have been subjected to increasing genetic manipulation since the latter half of the 1990s, when P.S. Masters in Wadworth Center, New York State Department of Health and New York State University at Albany, used RNA recombination to introduce extensive changes into the genome of mouse hepatitis virus (MHV). In a review published in 1999, he wrote, "targeted recombination could be used to create extensive substitutions to the cornavirus genome, generating recombinants that could not be made otherwise between two viruses separated by a species barrier." (p.254)
‘Defective interfering RNAs’ – sequences of the viral genome with large deletions as well as mutations and substitutions or insertions - were used as donor sequences to introduce major substitutions and point mutations into the genome of the viruses by RNA recombination.
In the course of such work, researchers have even isolated a recombinant of cororanvirus with the green fluorescent protein (GFP) gene, presumably from cells in which coronaviruses have been cultured, which has become inserted into the spike protein gene. The GFP gene, originally from a jelly-fish, is extensively used in genetic engineering as a marker gene because it makes the cells that have taken up the foreign genes give off a green glow under uv light. The GFP-coronavirus recombinant could only have come about as an unintended by-product of genetic engineering.
In the same review, P.S. Masters showed that both point mutations and large substitutions can readily be transferred to the last third of the genome of MHV and other coronaviruses. He further indicated that similar strategies could be used to mutate and substitute the first third of the genome, though not for the middle third. "A comprehensive genetic study of the highly complex gene for the RNA polymerase and all of its associated activities [encoded by the middle third of the genome] will likely await either the construction of an infectious full-length clone or the development of an innovative scheme for mutant selection." (p.259)
Is that why the middle third of SARS virus genome has retained good homology to MHV and BCV, which were the first coronaviruses to be engineered in this manner, while the other parts are much more different?
Another feature of the SARS virus is that the spike protein, which determines host range, is unlike the spike protein of any known coronavirus. Instead, it appears to have homologies to segments of the human chromosome 7, according to sequence analysis performed by Howard Urnovitz.
Urnovitz believes that the spike protein of the SARS virus is the result of genetic rearrangements provoked by environmental genotoxic agents, much like those he and his colleagues have detected in Gulf War I veterans suffering from Gulf War Syndrome.
But how did the virus get to south China? A possible answer was provided by Urnovitz: Migratory birds that frequent gene-swapping hot spots like southeast China could have carried the SARS virus there.
Urnovitz himself doesn’t think the SARS virus is the real cause of SARS. Instead, it is the piece of reshuffled human chromosome 7 that others are referring to as the spike protein gene of the SARS virus. That alone is sufficient to trigger serious autoimmune responses in people.
Hence, to create vaccines against that ‘spike’ protein is also tantamount to vaccinating people against their own genes (see "Dynamic genomics")
Adult Bone Marrow Cells Mend Heart without Transplant
Sudden blockages of a major artery to the heart cuts off blood supply and lead to rapid death of the muscle cells and blood vessels in the heart. This condition, myocardial infarction, is a common form of heart disease. Despite the demonstration that some of the heart muscle cells can multiply and new vessels formed, regeneration is restricted to the living part of the heart wall. The ‘infarcted’ or dead area is irreversible, and in time, scar tissue is formed. Attempts to replace the dead tissue by transplanting heart muscle cells or skeletal muscle cells have failed to mend the damaged part properly.
In previous experiments on mice, researchers in New York Medical College and the National Institute of Health injected bone marrow cells along the border of the damaged area of the heart, and found that the cells did differentiate into muscle and blood vessels. But this surgical intervention killed a high number of the mice and the grafting success was only 40%. This prompted them to consider a ‘non-invasive’ method, which involved stimulating the mice to overproduce bone marrow cells before and after myocardial infarction was induced .
For the purpose, the mice were given daily injections of two cytokines (small molecules that influence the activities of cells), stem cell factor (SCF) and granulocyte-colony-stimulating factor (G-CSF), which increased the number of circulating stem cells two to three hundred fold.
Mice given cytokines had a survival rate of 73% after the operation, compared with 20% in controls not given cytokines. There were clear signs of repair in the damaged area of the heart in the cytokine-injected group, both new heart muscle and blood vessels were formed, whereas only scar-tissue was found in controls. The hearts of the cytokine-injected group also performed significantly better than the controls.
The experimental results looked impressive enough even though the protocol of inducing myocardial infarction in such large numbers of animals is debatable. In addition, there was an unaccountably small number of experimental animals, only 15 compared to 52 in the group of controls. This may be because the researchers excluded mice that died within 48h of the operation, "to minimize the influence of the surgical trauma". But could it be that the mice died from stress of overproduction of bone marrow cells caused by the cytokines injected? There are certainly more ways to be invasive; and much more effort should be devoted to reducing unnecessary and stressful interventions, both physical and chemical.
In previous experiments on mice, researchers in New York Medical College and the National Institute of Health injected bone marrow cells along the border of the damaged area of the heart, and found that the cells did differentiate into muscle and blood vessels. But this surgical intervention killed a high number of the mice and the grafting success was only 40%. This prompted them to consider a ‘non-invasive’ method, which involved stimulating the mice to overproduce bone marrow cells before and after myocardial infarction was induced .
For the purpose, the mice were given daily injections of two cytokines (small molecules that influence the activities of cells), stem cell factor (SCF) and granulocyte-colony-stimulating factor (G-CSF), which increased the number of circulating stem cells two to three hundred fold.
Mice given cytokines had a survival rate of 73% after the operation, compared with 20% in controls not given cytokines. There were clear signs of repair in the damaged area of the heart in the cytokine-injected group, both new heart muscle and blood vessels were formed, whereas only scar-tissue was found in controls. The hearts of the cytokine-injected group also performed significantly better than the controls.
The experimental results looked impressive enough even though the protocol of inducing myocardial infarction in such large numbers of animals is debatable. In addition, there was an unaccountably small number of experimental animals, only 15 compared to 52 in the group of controls. This may be because the researchers excluded mice that died within 48h of the operation, "to minimize the influence of the surgical trauma". But could it be that the mice died from stress of overproduction of bone marrow cells caused by the cytokines injected? There are certainly more ways to be invasive; and much more effort should be devoted to reducing unnecessary and stressful interventions, both physical and chemical.
Thursday, October 22, 2009
Scrambled Genomes in Human Gene Therapy and Transgenic Plants
Human gene therapy is usually considered separate and distinct from genetic modification (GM) of crops, but this is misleading.
Adeno-associated virus vectors (AAV) are most commonly used in clinical gene-therapy trials. The wild-type adenovirus has terminal repeats that enable it to integrate into human chromosome 19 at a specific site. The AAV vector, however, has no specific integration site. It often integrates into chromosome 19, though not at the integration site of the wild-type virus, and it may also integrate into any of the other human chromosomes. AAV vector is preferred for most gene therapy experiments because the chromosome insertion is more stable and the AAV vector transforms both dividing and non-dividing cells.
Crop GM is achieved using Agrobacterium transformation or direct plasmid transfer using biolistic transformation (gene gun) methods. The Agrobacterium T –DNA vector is flanked by 25 base-pair direct repeats that facilitate integration of plasmid sequences into the plant chromosome.
The common features of gene therapy in human cells and crop GM is the presence of integrating vector sequences flanking transgene(s) each equipped with a promoter to drive expression.
While many studies have been carried out on transgenic DNA in plants, there have been relatively few that analyze host genome at the site of insertion. In a recent issue of Nature Genetics, researchers in the Department of Medicine, University of Washington Seattle, report that integrated AAV are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).
The researchers analysed the chromosomal DNA flanking the site of vector insertion. By searching the human genome sequence databases, the junctions were located to 6 different chromosomes. Four integrated into genes. Four of nine inserts went into a relatively large, 22-Mb (millions of base pairs) region of chromosome 10, but not the wild type site, which only spans 1kb.
Chromosomal deletions and additions were found, as well as translocations of parts of one chromosome to another. There were also unexpected vector sequences at integration sites.
The study is reminiscent of the recent finding of unexpected sequences and genome scrambling associated with transgenes in GM crops such as soybean (see "Scrambled genome of Roundup Ready Soya", ISIS News 9/10 www.i-sis.org.uk). Further analysis is likely to show that scrambled and unexpected sequences are commonplace in GM crops. We have questioned the legality and safety of approving crops that contain unknown, uncharacterised DNA sequences. If scrambled and unexpected sequences are found even in the most widely distributed and established commercial GM crop, the problems are likely to be worse with newer transgenic crops of corn, cotton or canola, which have yet to be analysed. Certainly, government regulators and their academic satellites seem passive and submissive in dealing with important findings that question the safety of GM crops.
The observation that gene scrambling occurs in both human gene therapy and in GM crops suggest that there is a fundamental flaw in both genetic engineering technology and in the auditing of molecular properties of the modified humans or crops.
The corporate audit of molecular characteristics in gene therapy vectors and those of GM crops may be analogous to the Enron audits of the Arthur Anderson Accounting Firm. Those outside of direct involvement in gene therapy and crop genetic modification should be well enough informed to require full and truthful molecular audits of gene therapy vectors and GM crops.
Adeno-associated virus vectors (AAV) are most commonly used in clinical gene-therapy trials. The wild-type adenovirus has terminal repeats that enable it to integrate into human chromosome 19 at a specific site. The AAV vector, however, has no specific integration site. It often integrates into chromosome 19, though not at the integration site of the wild-type virus, and it may also integrate into any of the other human chromosomes. AAV vector is preferred for most gene therapy experiments because the chromosome insertion is more stable and the AAV vector transforms both dividing and non-dividing cells.
Crop GM is achieved using Agrobacterium transformation or direct plasmid transfer using biolistic transformation (gene gun) methods. The Agrobacterium T –DNA vector is flanked by 25 base-pair direct repeats that facilitate integration of plasmid sequences into the plant chromosome.
The common features of gene therapy in human cells and crop GM is the presence of integrating vector sequences flanking transgene(s) each equipped with a promoter to drive expression.
While many studies have been carried out on transgenic DNA in plants, there have been relatively few that analyze host genome at the site of insertion. In a recent issue of Nature Genetics, researchers in the Department of Medicine, University of Washington Seattle, report that integrated AAV are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).
The researchers analysed the chromosomal DNA flanking the site of vector insertion. By searching the human genome sequence databases, the junctions were located to 6 different chromosomes. Four integrated into genes. Four of nine inserts went into a relatively large, 22-Mb (millions of base pairs) region of chromosome 10, but not the wild type site, which only spans 1kb.
Chromosomal deletions and additions were found, as well as translocations of parts of one chromosome to another. There were also unexpected vector sequences at integration sites.
The study is reminiscent of the recent finding of unexpected sequences and genome scrambling associated with transgenes in GM crops such as soybean (see "Scrambled genome of Roundup Ready Soya", ISIS News 9/10 www.i-sis.org.uk). Further analysis is likely to show that scrambled and unexpected sequences are commonplace in GM crops. We have questioned the legality and safety of approving crops that contain unknown, uncharacterised DNA sequences. If scrambled and unexpected sequences are found even in the most widely distributed and established commercial GM crop, the problems are likely to be worse with newer transgenic crops of corn, cotton or canola, which have yet to be analysed. Certainly, government regulators and their academic satellites seem passive and submissive in dealing with important findings that question the safety of GM crops.
The observation that gene scrambling occurs in both human gene therapy and in GM crops suggest that there is a fundamental flaw in both genetic engineering technology and in the auditing of molecular properties of the modified humans or crops.
The corporate audit of molecular characteristics in gene therapy vectors and those of GM crops may be analogous to the Enron audits of the Arthur Anderson Accounting Firm. Those outside of direct involvement in gene therapy and crop genetic modification should be well enough informed to require full and truthful molecular audits of gene therapy vectors and GM crops.
Saturday, October 3, 2009
Stem cell
Stem cells are cells found in most, if not all, multi-cellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. Research in the stem cell field grew out of findings by Canadian scientists Ernest A. McCulloch and James E. Till in the 1960s. The two broad types of mammalian stem cells are: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.
Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies.
Properties
The classical definition of a stem cell requires that it possess two properties:
* Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
* Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent - to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.
Potency definitions
Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. The stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta.
Human embryonic stem cells
A: Cell colonies that are not yet differentiated.
B: Nerve cell
Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.
* Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
* Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
* Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.
* Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
* Unipotent cells can produce only one cell type, their own, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).
Identification
The practical definition of a stem cell is the functional definition - a cell that has the potential to regenerate tissue over a lifetime. For example, the gold standard test for a bone marrow or hematopoietic stem cell (HSC) is the ability to transplant one cell and save an individual without HSCs. In this case, a stem cell must be able to produce new blood cells and immune cells over a long term, demonstrating potency. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.
Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew. As well, stem cells can be isolated based on a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells.
Embryonic
Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos. A blastocyst is an early stage embryo—approximately four to five days old in humans and consisting of 50–150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.
Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal culture conditions or genetic manipulation, embryonic stem cells will rapidly differentiate.
A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.
After nearly ten years of research, there are no approved treatments using embryonic stem cells. The first human trial was approved by the US Food & Drug Administration in January 2009. ES cells, being pluripotent cells, require specific signals for correct differentiation - if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face. Many nations currently have moratoria on either ES cell research or the production of new ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.
Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies.
Properties
The classical definition of a stem cell requires that it possess two properties:
* Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
* Potency - the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent - to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.
Potency definitions
Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. The stem cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta.
Human embryonic stem cells
A: Cell colonies that are not yet differentiated.
B: Nerve cell
Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.
* Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable, organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.
* Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.
* Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.
* Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
* Unipotent cells can produce only one cell type, their own, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).
Identification
The practical definition of a stem cell is the functional definition - a cell that has the potential to regenerate tissue over a lifetime. For example, the gold standard test for a bone marrow or hematopoietic stem cell (HSC) is the ability to transplant one cell and save an individual without HSCs. In this case, a stem cell must be able to produce new blood cells and immune cells over a long term, demonstrating potency. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.
Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, where single cells are characterized by their ability to differentiate and self-renew. As well, stem cells can be isolated based on a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. Considerable debate exists whether some proposed adult cell populations are truly stem cells.
Embryonic
Embryonic stem cell lines (ES cell lines) are cultures of cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos. A blastocyst is an early stage embryo—approximately four to five days old in humans and consisting of 50–150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.
Nearly all research to date has taken place using mouse embryonic stem cells (mES) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of Leukemia Inhibitory Factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal culture conditions or genetic manipulation, embryonic stem cells will rapidly differentiate.
A human embryonic stem cell is also defined by the presence of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency. The cell surface antigens most commonly used to identify hES cells are the glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.
After nearly ten years of research, there are no approved treatments using embryonic stem cells. The first human trial was approved by the US Food & Drug Administration in January 2009. ES cells, being pluripotent cells, require specific signals for correct differentiation - if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face. Many nations currently have moratoria on either ES cell research or the production of new ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.
Friday, October 2, 2009
Molecular Genetic Engineers in Junk DNA
Perhaps only 1% of the human genome codes for genes, and that’s what the human genome map contains. The rest is mainly repetitive DNA, commonly known as ‘junk DNA’.
However, evidence has been emerging that lurking within junk DNA are armies of transposons (mobile genetic elements) that play an indispensable role in ‘natural genetic engineering’ the genome. They make up nearly half of the human genome, and serve as ‘recombination hotspots’ for cutting and splicing, and hence reshuffling the genome. They are also a source of ready to use motifs for gene expression, as well as new protein-coding sequences.
These important transposons are scattered throughout the genome. There are two main categories: Long Interspersed Elements (LINEs) about 6.7 kilobasepairs in length and Short Interspersed Elements (SINEs) of several hundred basepairs.
The most abundant SINEs are Alu elements, of which 1.4 million copies exist, comprising 10% of the human genome, and are apparently only found in primates.
Most LINEs are dormant, except for LINE1. But both LINE 1 and other LINEs are abundant in areas of the genome where the bases A and T predominate, which do not have many genes. Alu elements, however, are more common in ‘GC-rich’regions that are also gene-rich. This is quite baffling as Alu itself cannot move, but depends on enzymes encoded by LINE1 in order to insert itself.
Alu elements, like LINE1 are retrotransposons that move and multiply by being transcribed into RNA, then reverse transcribed into DNA copies that jump into new sites.
New research is suggesting that Alu elements may help create new proteins from existing ones. The reasons the human genome contains so few genes – the latest count is just under 25 000, is that more than half the genes are interrupted and subject to alternative splicing.
In other words, the coding sequence of the gene is broken up into segments (exons) interrupted by non-coding segments (introns) that are spliced out from the RNA transcript before it is translated into protein. But there are alternative splicing pathways that join different exons together, with the result that many different proteins can be made from a single gene.
It appears that about 5% of alternatively spliced internal exons in the human genome originate in an Alu sequence. It suggests that Alu elements can actually jump into genes and, instead of destroying that gene, actually contributes a new coding sequence to it.
There are two ways in which this could be done, either by jumping into an existing gene so that the gene gains a new exon and increases its repertoire of possible proteins by alternative splicing, or else, the gene can become duplicated first, with one copy remaining intact, while the other is crafted by the Alu element. Currently, it is estimated that 238 000 Alu elements are located within introns of protein-coding genes and each one can actually or potentially become an exon.
Gil Ast, head of a group in Tel Aviv University, Israel, which has made some of the most significant discoveries about Alu, is understandably pleased. "We believe that Alus allowed the shuffling of genetic information that may have led to the evolution of primates," said Ast. "They may contribute to a lot of disorders we don’t even know about yet. But they have also created genetic diversity."
Alternative splicing is quite precisely regulated, and all other things being equal, depends on the spacing of potential splice sites consisting of the dinucleotide, AG, as well as neighbouring nucleotides. A single base change can destroy alternative splicing, and this can cause a disease, as in the case of Alport syndrome.
Also, gaining an exon is not always a good thing. There are numerous ‘insertion mutations’ associated with the inappropriate insertion of transposons into genes. And even Alu sequences that are already in introns can cause problems. More than a decade ago, it was discovered that a point mutation in an Alu element residing in the third intron of the ornithine aminotransferase gene activated a cryptic splice site, and led to part of the Alu element becoming incorporated into the coding sequence. Unfortunately, it carried a stop codon, which cut the protein short, leading to ornithine aminotransferase deficiency.
There is increasing evidence that physical and chemical stresses to the cell, such as heat shock, chemical poisons and viral infections, tend to activate Alu elements. The resultant gene reshuffling may be responsible for a variety of chronic diseases.
source:- i-sis.org.uk
However, evidence has been emerging that lurking within junk DNA are armies of transposons (mobile genetic elements) that play an indispensable role in ‘natural genetic engineering’ the genome. They make up nearly half of the human genome, and serve as ‘recombination hotspots’ for cutting and splicing, and hence reshuffling the genome. They are also a source of ready to use motifs for gene expression, as well as new protein-coding sequences.
These important transposons are scattered throughout the genome. There are two main categories: Long Interspersed Elements (LINEs) about 6.7 kilobasepairs in length and Short Interspersed Elements (SINEs) of several hundred basepairs.
The most abundant SINEs are Alu elements, of which 1.4 million copies exist, comprising 10% of the human genome, and are apparently only found in primates.
Most LINEs are dormant, except for LINE1. But both LINE 1 and other LINEs are abundant in areas of the genome where the bases A and T predominate, which do not have many genes. Alu elements, however, are more common in ‘GC-rich’regions that are also gene-rich. This is quite baffling as Alu itself cannot move, but depends on enzymes encoded by LINE1 in order to insert itself.
Alu elements, like LINE1 are retrotransposons that move and multiply by being transcribed into RNA, then reverse transcribed into DNA copies that jump into new sites.
New research is suggesting that Alu elements may help create new proteins from existing ones. The reasons the human genome contains so few genes – the latest count is just under 25 000, is that more than half the genes are interrupted and subject to alternative splicing.
In other words, the coding sequence of the gene is broken up into segments (exons) interrupted by non-coding segments (introns) that are spliced out from the RNA transcript before it is translated into protein. But there are alternative splicing pathways that join different exons together, with the result that many different proteins can be made from a single gene.
It appears that about 5% of alternatively spliced internal exons in the human genome originate in an Alu sequence. It suggests that Alu elements can actually jump into genes and, instead of destroying that gene, actually contributes a new coding sequence to it.
There are two ways in which this could be done, either by jumping into an existing gene so that the gene gains a new exon and increases its repertoire of possible proteins by alternative splicing, or else, the gene can become duplicated first, with one copy remaining intact, while the other is crafted by the Alu element. Currently, it is estimated that 238 000 Alu elements are located within introns of protein-coding genes and each one can actually or potentially become an exon.
Gil Ast, head of a group in Tel Aviv University, Israel, which has made some of the most significant discoveries about Alu, is understandably pleased. "We believe that Alus allowed the shuffling of genetic information that may have led to the evolution of primates," said Ast. "They may contribute to a lot of disorders we don’t even know about yet. But they have also created genetic diversity."
Alternative splicing is quite precisely regulated, and all other things being equal, depends on the spacing of potential splice sites consisting of the dinucleotide, AG, as well as neighbouring nucleotides. A single base change can destroy alternative splicing, and this can cause a disease, as in the case of Alport syndrome.
Also, gaining an exon is not always a good thing. There are numerous ‘insertion mutations’ associated with the inappropriate insertion of transposons into genes. And even Alu sequences that are already in introns can cause problems. More than a decade ago, it was discovered that a point mutation in an Alu element residing in the third intron of the ornithine aminotransferase gene activated a cryptic splice site, and led to part of the Alu element becoming incorporated into the coding sequence. Unfortunately, it carried a stop codon, which cut the protein short, leading to ornithine aminotransferase deficiency.
There is increasing evidence that physical and chemical stresses to the cell, such as heat shock, chemical poisons and viral infections, tend to activate Alu elements. The resultant gene reshuffling may be responsible for a variety of chronic diseases.
source:- i-sis.org.uk
Endogenous Viruses and Chronic Disease
Retroviruses are RNA viruses found in all vertebrates. The retrovirus reproduces by reverse transcribing (copying) its RNA into DNA that is then integrated into the host-cell genome to be transcribed into viruses. Many retroviral sequences have become permanently integrated into the human genome as human endogenous retroviruses, or HERVs. The human genome (indeed all genomes) also contains retrovirus-like retrotransposons, mobile elements that multiply by making RNA copies that are reverse transcribed into DNA and integrated into new sites in the genome. The main difference between a retrovirus and a retrotransposon is that the latter lacks an envelope.
Most HERVs and retrotransposons are defective, having lost one or more gene functions; but can nevertheless multiply and move with the help of other elements or infecting viruses.
Which came first, retrovirus or retrotransposon? As viruses depend on cells to reproduce, it seems reasonable to suppose that retroviruses are simply retrotransposons that have gained an envelop, which helps in entering other cells. That idea was first put forward by Howard Temin, who discovered reverse transcriptase, the enzyme that reverse transcribes, encoded by the retrovirus and retrotransposon.
Retrotransposons play an important role in turning genes on or off, and in rearranging and amplifying genes during development and cell differentiation. And evidence has been accumulating that they are the natural molecular genetic engineers of the fluid genome, which are necessary for the survival of the organism .
HERVs are flanked by ‘long terminal repeats’ that contain strong promoters for gene expression. Promoters are stretches of DNA with binding sites for transcription factors of the host cell that boosts transcription, effectively saying to the cell, "make many copies of the message following". HERVs and retrotransposons are regulated by the cell, and ultimately, by the organism as a whole, which stops most of them from being expressed; though they can be activated under a variety of conditions (see below).
Many endogenous viruses show xenotropism, i.e., they are not active in the host cell but can become infectious to cells of non-host species. Xenotropism is one of the major health hazards of xenotransplantation, the transplant of organs and tissues between species, as from pig to humans.
Defective or dormant HERVs, like defective retrotransposons, can become expressed when missing gene-functions are provided by a ‘helper’ virus that happens to infect the cell, and that includes ‘attenuated’ viruses in vaccines. Like retrotransposons, HERVs can also be induced: by X-rays or various chemical agents and drugs, such as inhibitors of protein synthesis, organophosphates and other pesticides, inflammatory cytokines (hormone-like factors that influence cells of the immune system) or steroid hormones, and retinoic acid.
In a comprehensive review published in 1996, virologists Howard Urnovitz and William Murphy raised the possibility that many chronic debilitating diseases may be linked to HERVs. These include leukaemia and other cancers, B-cell immunoglobulin diseases, inflammatory diseases of the nervous system, autoimmune rheumatic and connective tissue disease and chronic fatigue syndrome.
There are several mechanisms linking HERVs with chronic diseases, though it is not at all clear which mechanism comes into effect under different circumstances.
One way in which endogenous viruses can cause disease is for them to move and insert itself next to certain genes, that, when over-expressed, results in uncontrolled cell division, or cancer. This mechanism may be involved in mouse and human leukaemia, breast cancer and teratocarcinoma. This is also the mechanism that causes cancer in gene therapy, when viral vectors integrate next to these same genes.
Another possibility is for an HERV to recombine with an infecting virus to generate new viruses. One theory for the origin of the AIDS virus it that it may have come from early preparations of polioviruses used for vaccines that were propagated in rhesus and African green monkey kidney cells. At least 26 monkey viruses, including adenoviruses, cosackievirus, herpesvirus, echovirus, and possibly other groups of viruses were found as contaminants in such preparations. Current vaccines are presumably free of such contamination, though that does not necessarily make them safe (see below).
Urnovitz and Murphy suggested that human immunodeficiency virus type 1 (HIV-1) may be a chimera between one of the simian (monkey) viruses (simian immunodeficiency virus) and HERV sequences. Simian immunodeficiency virus capable of causing simian AIDS, appears to occur exclusively in African monkeys, particularly in the African green monkey. And DNA sequences related to the highly conserved domain of the HIV reverse transcriptase and glycoprotein gp41 (part of the gp160 polyprotein of the HIV that’s cleaved into gp120 and gp41) have been found in the human genome.
Another way in which disease may arise is when HERV encoded proteins are expressed. This provokes antibodies against the body’s own cells, giving rise to autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, Sjögren’s syndrome, mixed connective tissue diseases and inflammatory neurologic disease. The inflammatory response could be the most important trigger for the development of autoimmune disease, as infecting viruses can strongly activate HERVs to express, resulting in production of HERV protein antigens.
In this context, vaccines came in for special criticism in Unovitz’ testimony to the United States House of Representatives in 1999.
In his view, "there appears to be a limit on how much foreign material to which the human body can be exposed before some level of genetic damage occurs and a chronic disease initiates". Gulf War I veterans (GWIVs) were given large numbers of vaccines (see "Gulf War Syndrome and vaccinations", this series), and vaccine overload is a significant factor in Gulf War Syndrome.
Urnovitz described a case of a woman who died from a mysterious case of AIDS. Over several years, laboratory tests failed to reveal HIV-1 antibodies in her blood. However, she was subsequently tested HIV-1 positive in her urine. The virus was eventually isolated and sequenced; and came to be known as HIV-1 Group O. Analyses of the viral genetic material suggest that the virus originated, in part, from genetic reshuffling of human chromosomal material.
Vaccination against HIV-1 is particularly dangerous, if as Urnovitz and Murphy have suggested, HIV arose from recombination between the simian aids virus and HERV sequences (see also "AIDS vaccines worse than useless?" this series).
"To put it simply, are we embarking on a course that will vaccinate people against their own genes?" Dr. Urnovitz asked.
A second example is the intensive effort directed to creating a vaccine for the hepatitis C virus. Unfortunately, there is no hard evidence in the scientific literature that hepatitis C virus exists. Urnovitz continued, "As a scientist I am compelled to ask, how can we vaccinate people against a disease-causing agent that has not been fully characterised?"
Finally, he drew attention to the contaminated polio vaccines that is now being increasingly implicated in cancer,
"Had my mother and father known that the poliovirus vaccines of the 1950s were heavily contaminated with more than 26 monkey viruses, including the cancer virus SV40, I can say with certainty that they would not have allowed their children and themselves to take those vaccines. Both of my parents might not have developed cancers suspected of being vaccine-related, and might even be alive today."
But even uncontaminated Polio vaccines are of doubtful efficacy in protecting against viral infections and chronic disease. Enteroviruses have been shown to be a major factor in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome by John Richardson who studied more than 4000 patients over a 50 year period. Vaccination against polio provides protection against only 3 strains of polio leaving no protection against the other 70 or so enteroviruses- coxsackie, echo and others. These viruses have a range of pathological effects on the central nervous system, the cardiovascular system, and endocrine and exocrine glands.
source:- i-sis.org.uk
Most HERVs and retrotransposons are defective, having lost one or more gene functions; but can nevertheless multiply and move with the help of other elements or infecting viruses.
Which came first, retrovirus or retrotransposon? As viruses depend on cells to reproduce, it seems reasonable to suppose that retroviruses are simply retrotransposons that have gained an envelop, which helps in entering other cells. That idea was first put forward by Howard Temin, who discovered reverse transcriptase, the enzyme that reverse transcribes, encoded by the retrovirus and retrotransposon.
Retrotransposons play an important role in turning genes on or off, and in rearranging and amplifying genes during development and cell differentiation. And evidence has been accumulating that they are the natural molecular genetic engineers of the fluid genome, which are necessary for the survival of the organism .
HERVs are flanked by ‘long terminal repeats’ that contain strong promoters for gene expression. Promoters are stretches of DNA with binding sites for transcription factors of the host cell that boosts transcription, effectively saying to the cell, "make many copies of the message following". HERVs and retrotransposons are regulated by the cell, and ultimately, by the organism as a whole, which stops most of them from being expressed; though they can be activated under a variety of conditions (see below).
Many endogenous viruses show xenotropism, i.e., they are not active in the host cell but can become infectious to cells of non-host species. Xenotropism is one of the major health hazards of xenotransplantation, the transplant of organs and tissues between species, as from pig to humans.
Defective or dormant HERVs, like defective retrotransposons, can become expressed when missing gene-functions are provided by a ‘helper’ virus that happens to infect the cell, and that includes ‘attenuated’ viruses in vaccines. Like retrotransposons, HERVs can also be induced: by X-rays or various chemical agents and drugs, such as inhibitors of protein synthesis, organophosphates and other pesticides, inflammatory cytokines (hormone-like factors that influence cells of the immune system) or steroid hormones, and retinoic acid.
In a comprehensive review published in 1996, virologists Howard Urnovitz and William Murphy raised the possibility that many chronic debilitating diseases may be linked to HERVs. These include leukaemia and other cancers, B-cell immunoglobulin diseases, inflammatory diseases of the nervous system, autoimmune rheumatic and connective tissue disease and chronic fatigue syndrome.
There are several mechanisms linking HERVs with chronic diseases, though it is not at all clear which mechanism comes into effect under different circumstances.
One way in which endogenous viruses can cause disease is for them to move and insert itself next to certain genes, that, when over-expressed, results in uncontrolled cell division, or cancer. This mechanism may be involved in mouse and human leukaemia, breast cancer and teratocarcinoma. This is also the mechanism that causes cancer in gene therapy, when viral vectors integrate next to these same genes.
Another possibility is for an HERV to recombine with an infecting virus to generate new viruses. One theory for the origin of the AIDS virus it that it may have come from early preparations of polioviruses used for vaccines that were propagated in rhesus and African green monkey kidney cells. At least 26 monkey viruses, including adenoviruses, cosackievirus, herpesvirus, echovirus, and possibly other groups of viruses were found as contaminants in such preparations. Current vaccines are presumably free of such contamination, though that does not necessarily make them safe (see below).
Urnovitz and Murphy suggested that human immunodeficiency virus type 1 (HIV-1) may be a chimera between one of the simian (monkey) viruses (simian immunodeficiency virus) and HERV sequences. Simian immunodeficiency virus capable of causing simian AIDS, appears to occur exclusively in African monkeys, particularly in the African green monkey. And DNA sequences related to the highly conserved domain of the HIV reverse transcriptase and glycoprotein gp41 (part of the gp160 polyprotein of the HIV that’s cleaved into gp120 and gp41) have been found in the human genome.
Another way in which disease may arise is when HERV encoded proteins are expressed. This provokes antibodies against the body’s own cells, giving rise to autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, Sjögren’s syndrome, mixed connective tissue diseases and inflammatory neurologic disease. The inflammatory response could be the most important trigger for the development of autoimmune disease, as infecting viruses can strongly activate HERVs to express, resulting in production of HERV protein antigens.
In this context, vaccines came in for special criticism in Unovitz’ testimony to the United States House of Representatives in 1999.
In his view, "there appears to be a limit on how much foreign material to which the human body can be exposed before some level of genetic damage occurs and a chronic disease initiates". Gulf War I veterans (GWIVs) were given large numbers of vaccines (see "Gulf War Syndrome and vaccinations", this series), and vaccine overload is a significant factor in Gulf War Syndrome.
Urnovitz described a case of a woman who died from a mysterious case of AIDS. Over several years, laboratory tests failed to reveal HIV-1 antibodies in her blood. However, she was subsequently tested HIV-1 positive in her urine. The virus was eventually isolated and sequenced; and came to be known as HIV-1 Group O. Analyses of the viral genetic material suggest that the virus originated, in part, from genetic reshuffling of human chromosomal material.
Vaccination against HIV-1 is particularly dangerous, if as Urnovitz and Murphy have suggested, HIV arose from recombination between the simian aids virus and HERV sequences (see also "AIDS vaccines worse than useless?" this series).
"To put it simply, are we embarking on a course that will vaccinate people against their own genes?" Dr. Urnovitz asked.
A second example is the intensive effort directed to creating a vaccine for the hepatitis C virus. Unfortunately, there is no hard evidence in the scientific literature that hepatitis C virus exists. Urnovitz continued, "As a scientist I am compelled to ask, how can we vaccinate people against a disease-causing agent that has not been fully characterised?"
Finally, he drew attention to the contaminated polio vaccines that is now being increasingly implicated in cancer,
"Had my mother and father known that the poliovirus vaccines of the 1950s were heavily contaminated with more than 26 monkey viruses, including the cancer virus SV40, I can say with certainty that they would not have allowed their children and themselves to take those vaccines. Both of my parents might not have developed cancers suspected of being vaccine-related, and might even be alive today."
But even uncontaminated Polio vaccines are of doubtful efficacy in protecting against viral infections and chronic disease. Enteroviruses have been shown to be a major factor in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome by John Richardson who studied more than 4000 patients over a 50 year period. Vaccination against polio provides protection against only 3 strains of polio leaving no protection against the other 70 or so enteroviruses- coxsackie, echo and others. These viruses have a range of pathological effects on the central nervous system, the cardiovascular system, and endocrine and exocrine glands.
source:- i-sis.org.uk
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