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Topic
What are the origins of antibiotic resistance in bacteria and how does it continue to evolve?

Annotated Bibliography
1. Cirz RT, Chin JK, Andes DR, de Crécy-Lagard V, Craig WA, et al. (2005) Inhibition of Mutation and Combating the Evolution of Antibiotic Resistance. PLoS Biol 3(6): e176. doi:10.1371/journal.pbio.0030176

This article suggests that mutation of chromosomal genes during therapy is one of the primary reasons for the rapid emergence of drug-resistance in bacteria. In this study, researchers interfered with the activity of the protease LexA in order to prevent induction of DNA repair in Escherichia coli. This rendered the pathogenic bacteria incapable of evolving resistance to antibiotics such as ciprofloxacin and rifampicin. Therefore, the results of this experiment indicate that the inhibition of mutations could serve as a therapeutic approach to combat the evolution of antibiotic resistance in bacteria.

2. GS Gray, WM Fitch. Evolution of antibiotic resistance genes: the DNA sequence of a kanamycin resistance gene from Staphylococcus aureus. Mol Biol Evol (1983) 1 (1): 57-66

This article looks at the evolutionary origins of antibiotic resistance in bacteria by comparing the kanamycin resistance gene in Staphylococcus aureus with similar genes in Streptomyces fradiae and two transposons isolated from Klebsiella pneumoniae and Salmonella typeimurium. The research finds that the genes are homologous but have undergone extensive divergence since their common ancestor. Therefore, it has been hypothesized that there have been multiple occurrences of gene transfer between these species.

3. BR Levin, V Perrot, Nina Walker. Compensatory Mutations, Antibiotic Resistance and the Population Genetics of Adaptive Evolution in Bacteria. Genetics March 1, 2000 vol. 154 no. 3 985-997.

This article points out that chromosomal mutations for resistance to antibiotics and other chemotherapeutic agents actually confers a cost to the fitness of the microorganisms that utilize them. The study specifically looks at antibiotic resistant strains of Escherichia coli, HIV, and Salmonella typhimurium and compares their fitness to the drug-sensitive revertants. The cost of resistance to the relative fitness of each of the species is determined by observing characteristics such as growth rate of the cultures and mutation rate in individuals.

4. SR Gill, DE Fouts, CM Fraser, et al. Insights on Evolution of Virulence and Resistance from the Complete Genome Analysis of an Early Methicillin-Resistant Staphylococcus aureus Strain and a Biofilm-Producing Methicillin-Resistant Staphylococcus epidermidis Strain. J Bacteriol. Apr 2005; 187(7): 2426–2438. doi:10.1128/JB.187.7.2426-2438.2005.

This article explores the evolution of virulence and resistance through comparative analysis between the sequenced genomes of Staphylococcus aureus, Staphylococcus epidermidis, and other staphylococcal genomes. The research determines that genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria also appears to have shaped their virulence and resistance profiles.

5. MTG Holden, EJ Feil, J Parkhill, et al. Complete genomes of two clinical Staphylococcus aureus strains: Evidence for the rapid evolution of virulence and drug resistance. Proc Natl Acad Sci U S A. Jun 29, 2004; 101(26): 9786–9791. Published online Jun 22, 2004. doi:10.1073/pnas.0402521101.

This articles describes general features found in the sequenced genomes of two clinical Staphylococcus aureus strains and highlights the significance of mobile genetic elements for the acquisition of traits of clinical importance, such as virulence and antibiotic resistance. Comparison between the genomes of the two clinal strains reveals extensive patterns of genomic diversification, which illustrates the relative speed with which resistance genes and virulence genes move between strains by lateral gene transfer.

Suggested Updates
https://en.wikipedia.org/wiki/Antibiotic_resistance

1. The article mentions the importance of the bacterial protein LexA in the accumulation of mutations that may confer antibiotic resistance, but it does not describe the mechanism by which this occurs. I would like to expand on this by pointing out that the common quinolone, ciprofloxacin, induces DNA damage by interfering with the two essential type II DNA topoisomerases in bacteria, gyrase and topoisomerase IV. The DNA damage caused by this interference induces the SOS gene repressor LexA to undergo autoproteolytic activity. This includes the transcription of genes encoding Pol II, Pol IV, and Pol V, which are three nonessential DNA polymerases that are required for mutation in response to DNA damage.

2. The article does not explain the evolutionary origins of any antibiotic resistance genes. I would like to add information on the evolutionary origin of genes that incur resistance to aminocyclitol antibiotics. This type of antibiotic resistance is found widely among bacteria, including Staphylococcus aureus, Streptococcus, Enterobacteriacae, Pseudomonadiaceae, Bacillus circulans, and actinomycetes. Research suggests that an aminocyclitol-producing bacterium was the source of genes for aminocyclitol phosphotransferases, which allow for resistance to the antibiotic aminocyclitol. The close structural relationship among the five phosphotransferase genes found in several different bacterial species suggests that the genes are homologous but have undergone extensive divergence since their common ancestor.

3. The article explains the advantages of adaptive antibiotic resistance in bacteria but does not mention the disadvantages it incurs. Research suggests that chromosomal mutations for resistance to antibiotics and other chemotherapeutic agents actually confers a cost to the fitness of the microorganisms that utilize them, such as Escherichia coli and Salmonella typhimurium. The cost of fitness is evident in the growth rate and mutation rate of antibiotic resistant bacterial colonies when compared to the drug-sensitive revertants.

Added Sentence
DNA damage induces the SOS gene repressor LexA to undergo autoproteolytic activity. This includes the transcription of genes encoding Pol II, Pol IV, and Pol V, which are three nonessential DNA polymerases that are required for mutation in response to DNA damage.

Reference: Cirz RT, Chin JK, Andes DR, de Crécy-Lagard V, Craig WA, et al. (2005) Inhibition of Mutation and Combating the Evolution of Antibiotic Resistance. PLoS Biol 3(6): e176. doi:10.1371/journal.pbio.0030176

Wikipedia Edits
These specialized pumps can be found within the cellular membrane of certain bacterial species and are used to pump antibiotics out of the cell before they are able to do any damage. These efflux pumps are often activated by a specific substrate associated with an antibiotic.

Another protective mechanism found among bacterial species is ribosomal protection proteins. These proteins protect the bacterial cell from antibiotics that target the cell’s ribosomes to inhibit protein synthesis. The mechanism involves the binding of the ribosomal protection proteins to the ribosomes of the bacterial cell, which in turn changes its conformational shape. This allows the ribosomes to continue synthesizing proteins essential to the cell while preventing antibiotics from binding to the ribosome to inhibit protein synthesis.

Most commonly, the protective enzymes produced by the bacterial cell will add an acetyl or phosphate group to a specific site on the antibiotic, which will reduce its ability to bind to the bacterial ribosomes and disrupt protein synthesis.

Mutations are rare but the fact that bacteria reproduce at such a high rate allows for the effect to be significant. A mutation may produce a change in the binding site of the antibiotic, which may allow the site to continue proper functioning in the presence of the antibiotic or prevent the binding of the antibiotic to the site altogether. Research has shown the bacterial protein LexA may play a key role in the acquisition of bacterial mutations giving resistance to quinolones and rifampicin. DNA damage induces the SOS gene repressor LexA to undergo autoproteolytic activity. This includes the transcription of genes encoding Pol II, Pol IV, and Pol V, which are three nonessential DNA polymerases that are required for mutation in response to DNA damage. Although these chromosomal mutations may seem to benefit the bacteria by providing antibiotic resistance, they also confer a cost of fitness. For example, a ribosomal mutation may protect a bacterial cell by changing the binding site of an antibiotic but it will also slow the process of protein synthesis. Additionally, a particular study specifically compared the overall fitness of antibiotic resistant strains of Escherichia coli and Salmonella typhimurium to their drug-sensitive revertants. They observed a reduced overall fitness in the antibiotic resistant strains, especially in growth rate.

Final Paper
Origin and Evolution of Antibiotic Resistance in Bacteria The development and introduction of antibiotics in the twentieth century was a revolutionary period in human history. This so-called “antibiotic age” was initiated by the accidental discovery of penicillin by Alexander Fleming and its clinical introduction in 1943 (Bergstrom and Dugatkin 2012). Being the first modern antibiotic, penicillin was regarded as a “miracle” drug in that it therapeutically treated a variety of common infections that had plagued the human race for ages. In addition to penicillin, the “antibiotic age” witnessed the introduction of several other highly effective antibiotics such as tetracycline and kanamycin. Unfortunately, the success of these new drugs and their prominent impact on society would not last very long. Antibiotic resistance quickly spread among target bacteria rendering the aforementioned “miracle” drugs relatively useless. In fact, penicillin resistance was observed within a single year of its clinical introduction and had become common in numerous bacterial species within five years (Bergstrom and Dugatkin 2012). To further illustrate the speed of antibiotic resistance, it was estimated that 3-5% of Streptococcus pneumoniae were penicillin-resistant in 1980. This percentage grew to a hefty 34% by the year 1998. This substantial speed was also observed in normal human intestinal flora as tetracycline resistance increased from 2% in the 1950s to 80% in the 1990s (Criswell 2004). In effect, it did not take long to realize that an evolutionary war had broken out between humanity and bacteria. The next step was to find the underlying cause of this unparalleled antibiotic resistance growth and a way to stop it.

To understand the mechanisms of antibiotic resistance in bacteria, it is important to first understand the different modes of action antibiotics have on bacteria. The three main target sites for antibiotics are the cell wall, DNA, and ribosomes of the bacteria. The most common mode of action is inhibition of cell wall synthesis. This is usually a very effective mode of action because human cells do not contain a cell wall while bacterial cells do. This means that the antibiotic will be selectively effective against bacterial cells while eliminating the possibility of harming the human host. The cell wall is a protective barrier that is essential to the survival of most bacterial species. The antibiotics usually work by inhibiting peptidoglycan synthesis or disrupting peptidoglycan cross-linkage. Common antibiotics that utilize this mode of action include beta-lactams, vancomycin, and bacitracin. Another mode of action is inhibiting cell membrane function. Antibiotics that utilize this mode of action are usually less common in treatment since both bacterial and human cells contain cell membranes. Therefore, it is difficult to select against the bacteria without harming the human host. These antibiotics work by damaging the cell membrane, which results in reduced cell stability and leakage of solutes essential to the cell’s survival. Examples of this form of antibiotic include polymixin B and colistin. A third mode of action is inhibition of protein synthesis. Antibiotics in this class include macrolides, lincosamides, streptogramins and tetracycline. These antibiotics effectively disrupt protein synthesis of the bacterial cell by binding to the 30S or 50S subunits of the ribosome. These actions will inevitably lead to death of the bacterial cell and an inability to grow or reproduce. A fourth antibiotic mode of action is inhibition of nucleic acid synthesis. These antibiotics work by binding to components involved in the process of DNA or RNA synthesis. This effectively halts the multiplication of the bacteria and limits its likelihood to survive. Common antibiotics that utilize this mode of action include quinolones, metronidazole, and rifampin. One final mode of action commonly utilized by antibiotics is the inhibition of metabolic processes. Sulfonamides and trimethoprim utilize this mode of action by disrupting the folic acid pathway, which produces precursors necessary for DNA synthesis (Rollins and Joseph 2000).

With so many different antibiotics available that have different target sites and various modes of action it might be hard to understand how antibiotic resistance is so common among bacterial species. Well, the truth is that not all bacteria are susceptible to certain antibiotics. In fact, many species of bacteria have specialized cellular machinery or the ability to produce unique proteins to protect themselves from the antibiotic attack. One mechanism common among bacterial species is an efflux pump. These specialized pumps can be found within the cellular membrane of certain bacterial species and are used to pump antibiotics out of the cell before they are able to do any damage. These efflux pumps are often activated by a specific substrate associated with an antibiotic. Another protective mechanism found among bacterial species is ribosomal protection proteins. These proteins protect the bacterial cell from antibiotics that target the cell’s ribosomes to inhibit protein synthesis. The mechanism involves the binding of the ribosomal protection proteins to the ribosomes of the bacterial cell, which in turn changes its conformational shape. This allows the ribosomes to continue synthesizing proteins essential to the cell while preventing antibiotics from binding to the ribosome to inhibit protein synthesis (Aminov and Mackie 2007). One final mechanism utilized by certain species of bacteria is the production of enzymes that neutralize specific antibiotics. Most commonly, the protective enzymes produced by the bacterial cell will add an acetyl or phosphate group to a specific site on the antibiotic, which will reduce its ability to bind to the bacterial ribosomes and disrupt protein synthesis (Criswell 2004).

Antibiotic resistance is already present in certain species of bacteria to some extent. However, that still does not explain how certain species of bacteria, which were previously susceptible to a certain antibiotic, can become resistant to it. There are two possible ways this can happen. The first one to be discussed will be lateral gene transfer. This process, which is unique to bacteria, allows for the transfer of genetic information between bacterial cells. Most antibiotic resistant genes are located on a circular strand of DNA known as the R-plasmid. During the process of conjugation, a bacterial cell has the ability to transfer genes from its R-plasmid to a neighboring bacterial cell. If the genes transferred from one bacterial cell to the other happen to contain the genetic code for a certain type of antibiotic resistance, the bacterial cell receiving those genes will display that respective antibiotic resistance (Criswell 2004). In studies focused on determining the evolutionary origin of the kanamycin resistance gene, researchers found that the gene is homologous in multiple bacterial species including Staphylococcus aureus, Streptomyces fradiae, Klebsiella pneumoniae, and Salmonella typeimurium but has undergone extensive divergence since their common ancestor. Therefore, it has been hypothesized that there have been multiple occurrences of gene transfer between these species (Gray and Fitch 1983). Another research study compared the sequenced genomes of Staphylococcus aureus, Staphylococcus epidermidis, and other staphylococcal species and found that the primary source of variations in pathogenicity and resistance were located on specific genome islands in nonsyntenic regions (Gill et al. 2005). They hypothesized that their virulence and resistance profiles where shaped through gene transfer with low-GC-content gram-positive bacteria (Enright et al. 2002). One final research study pertaining to lateral gene transfer comparatively analyzed the sequenced genomes of two clinical strains of Staphylococcus aureus. They noted that the acquisition of traits of clinical importance, such as virulence and antibiotic resistance, were mostly due to mobile genetic elements gained through lateral gene transfer. The extensive genomic diversification between the two clinical strains illustrates the incredible speed at which divergence can occur through lateral gene transfer (Holden et al. 2004).

The second way that a species of bacteria once susceptible to a certain antibiotic can become resistance to it is through chromosomal mutation and proliferation of that mutation through asexual reproduction. Mutations are rare but the fact that bacteria reproduce at such a high rate allows for the effect to be significant. A mutation may produce a change in the binding site of the antibiotic, which may allow the site to continue proper functioning in the presence of the antibiotic or prevent the binding of the antibiotic to the site altogether. Research has shown the bacterial protein LexA may play a key role in the acquisition of bacterial mutations giving resistance to quinolones and rifampicin. DNA damage induces the SOS gene repressor LexA to undergo autoproteolytic activity. This includes the transcription of genes encoding Pol II, Pol IV, and Pol V, which are three nonessential DNA polymerases that are required for mutation in response to DNA damage (Cirz et al. 2005). Although these chromosomal mutations may seem to benefit the bacteria by providing antibiotic resistance, they also confer a cost of fitness. For example, a ribosomal mutation may protect a bacterial cell by changing the binding site of an antibiotic but it will also slow the process of protein synthesis (Criswell 2004). Additionally, a particular study specifically compared the overall fitness of antibiotic resistant strains of Escherichia coli and Salmonella typhimurium to their drug-sensitive revertants. They observed a reduced overall fitness in the antibiotic resistant strains, especially in growth rate (Levin et al. 2000). Ultimately, chromosomal mutations causing antibiotic resistance give the bacterial cells a selective advantage in the presence of the antibiotic but a reduced ability to compete with bacterial cells without mutation in the absence of the antibiotic (Perron et al. 2007).

The last question left to ask is how antibiotic resistance proliferates so quickly among a species of bacteria after being exposed to the antibiotic. This question can simply be answered by the concept of natural selection. The use of antibiotics imposes a strong environmental pressure on a population of bacteria. It will kill off most of the bacteria but there is a good chance that at least one or a few will survive in a large population due to antibiotic resistance conferred by lateral gene transmission or chromosomal mutation. These survivors will then proliferate asexually, passing their genes on to the next generations, producing a population of antibiotic resistant bacteria. In effect, extensive antibiotic use essentially produces a bottleneck as it greatly reduces the number of less fit bacteria allowing the more fit, antibiotic resistant bacteria to proliferate and possibly pass their antibiotic resistance on to bacterial cells without it through lateral gene transfer.

In conclusion, it is obvious that humans are now fully engaged in an evolutionary war with bacteria. However, rather than fight this war with force, it would be wise to fight it with intelligence. There are numerous hypothetical ways to slow the evolution of antibiotic resistance in bacteria and it is our responsibility to put forth a concerted effort to do so. One way is through drug overkill with the use of drug cocktails. Attacking the bacteria through multiple modes of action with the use of multi-drug therapy should reduce the risk of leaving behind any survivors. Another solution is direct observation therapy. This would entail bringing drug doses to patients individually to make sure that they are being compliant. Patients that do not take the proper dose recommended by the doctor are increasing the chances of antibiotic resistance development. Finally, it is important to withhold the most powerful drug. Less powerful drugs exert less pressure on the bacteria to evolve, which effectively increases the life span of the drugs (Palumbi 2001). Utilization of these simple methods will surely curtail the immense growth of antibiotic resistance that the world has seen since the dawn of the “antibiotic age.” Therefore, it is important that we develop strict standards for the widespread practice of drug therapy and continue exploring other methods of antibiotic resistance control through further research.

References

Bergstrom, Carl T., and Lee A. Dugatkin. “An Overview of Evolutionary Biology.” Evolution. New York: W.W. Norton, 2012. 8-11. Print.

BR Levin, V Perrot, Nina Walker. Compensatory Mutations, Antibiotic Resistance and the Population Genetics of Adaptive Evolution in Bacteria. Genetics March 1, 2000 vol. 154 no. 3 985-997.

Cirz RT, Chin JK, Andes DR, de Crécy-Lagard V, Craig WA, et al. (2005) Inhibition of Mutation and Combating the Evolution of Antibiotic Resistance. PLoS Biol 3(6): e176. doi:10.1371/journal.pbio.0030176

Criswell, Daniel. "The "Evolution" of Antibiotic Resistance." Institute for Creation Research. N.p., 2004. Web. 28 Oct. 2014.

GG Perron, A Gonzalez, A Buckling. Source–sink dynamics shape the evolution of antibiotic 	resistance and its pleiotropic fitness cost. The Royal Society. 22 September 2007 doi: 	10.1098/rspb.2007.0640. Vol. 274 no. 1623 2351-2356

GS Gray, WM Fitch. Evolution of antibiotic resistance genes: the DNA sequence of a kanamycin resistance gene from Staphylococcus aureus. Mol Biol Evol (1983) 1 (1): 57-66

MC Enright, DA Robinson, G Randle, et al. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). PNAS. 28 May 2002. Vol. 99 No. 11.

MTG Holden, EJ Feil, J Parkhill, et al. Complete genomes of two clinical Staphylococcus aureus strains: Evidence for the rapid evolution of virulence and drug resistance. Proc Natl Acad 	Sci U S A. Jun 29, 2004; 101(26): 9786–9791. Published online Jun 22, 2004. doi:10.1073/pnas.0402521101.

Rollins, D. M., and S. W. Joseph. "Basic Mechanisms of Antibiotic Action and Resistance." BSCI 424 Pathogenic Microbiology. University of Maryland, 2000. Web. 29 Oct. 2014.

RI Aminov, RI Mackie. Evolution and ecology of antibiotic resistance genes. Microbiology Letters. 8 May 2007. DOI: 10.1111/j.1574-6968.2007.00757.x

SR Gill, DE Fouts, CM Fraser, et al. Insights on Evolution of Virulence and Resistance from the Complete Genome Analysis of an Early Methicillin-Resistant Staphylococcus aureus Strain and a Biofilm-Producing Methicillin-Resistant Staphylococcus epidermidis Strain. J Bacteriol. Apr 2005; 187(7): 2426–2438. doi:10.1128/JB.187.7.2426-2438.2005.

SR Palumbi. Humans as the World’s Greatest Evolutionary Force. Science 293, 1786 (2001); DOI 10.1126/science.293.5536.1786.