Kevin HuginsBIOL 415Nevada State CollegeSpring 2014

Abstract

Bacterial antibiotic resistance is a topic that is causing increasing concern in the health community. Antibiotics are a necessary drug to help protect and heal us from pathogenic infections that our immune system is unable to successfully combat on its own. However, bacteria are very adept at utilizing evolutionary processes to develop antibiotic resistance in order to promote their own survival, reproduction and persistence. The development of antibiotic resistant bacteria is occurring at an alarming rate. Researchers are investigating the mechanisms that confer resistance on bacteria. With techniques for genomic sequencing now readily available, understanding of genetic mechanisms of resistance and evolution as a whole has been advancing rapidly. Researchers have found that bacteria are very adept at gene mutation and horizontal gene transfer. New insights regarding pleiotrophy and epistasis have been provided through these techniques. A possible result of this research will be the discovery of new antibiotic therapies. However, as the research is demonstrating, even if we develop new antibiotics, bacteria will develop resistance to them. Thus, important considerations to be taken from the research include finding ways to slow the development of resistance as we will most likely never be able to stop it entirely.

Introduction

Microorganisms are all around us. They play important roles in functions such as nutrient recycling, health, production of food, biodegradation, waste water treatment, and biosynthesis. However, microbes are also responsible for causing illness in animals and plants. Since their discovery in the 1920’s, antibiotics have played an important role in combating these pathogenic microorganisms. As production of antibiotics has become cheaper and more efficient, use has increased dramatically over time. Worldwide, over 50 million pounds of antibiotics are manufactured each year, with sales exceeding eight billion dollars annually. Only about half of the antibiotics produced are used for medical purposes. The other half is used in agriculture, either sprayed on crops or fed to livestock.(4) With so many antibiotics being produced and consumed, it would seem that both pathogenic and helpful bacteria would be on their way to extinction. In fact, the opposite is occurring. Microbes are adapting rapidly to develop antibiotic resistance, and some pathogenic strains show complete resistance to one or more commonly used antibiotics. The World Health Organization has warned that this is a serious global threat that may return mankind to the pre-antibiotic era.(10)

Antibiotic resistant bacteria have become an important health issue that has received a good deal of press lately. This is not a new problem however. The first documentation of antibiotic resistance was in the late 1930’s when bacterial resistance to sulfonamide was found. In 1940 researchers discovered a bacterial enzyme that conferred resistance to penicillin. By the mid 1940’s resistance to streptomycin had developed in Mycobacterium tuberculosis. In the 1950’s genetically transferred antibiotic resistance was reported in Japan.(1) In 1963 antibiotic resistance that resulted from systematic use of antibiotics in agriculture was documented.(2)

The antibiotics we use rely on the differences between eukaryotic and prokaryotic cells to make them effective. In order for an antibiotic to be useful it needs to be able to target microbial cells while not interacting with the cells of the plant or animal. There are hundreds of types of antibiotics produced but they all generally fall within the following classifications and modes of action. β-lactem antibiotics,

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which include penicillin and cephalosporin, interfere with the synthesis of the bacterial wall. Antibiotic classes such as aminoglycosides, tetracyclines, macrolides and others inhibit ribosomal translation within the bacteria. This action causes the bacteria to be unable to build the proteins required for life or replication. Common antibiotics within these classes include streptomycin, erythromycin, and azithromycin. Quinolones such as ciprofloxacin inhibit bacterial DNA replication.(1) With so many different antibiotic classes that attack different mechanisms within the bacteria, it would seem that humans should have the upper hand on these pesky little pathogens. However, if the alarm bells being sounded by governmental health organizations around the world are accurate, it appears that the microbes are the ones winning the war. On April 30, 2014, the WHO updated its Antimicrobial Resistance Fact sheet. The number of documented complete antibiotic resistant infections has increased significantly and has appeared in more countries since the last published report in May 2013.(10)

The study of the means by which bacteria develop resistance to antibiotics has caught the attention of researchers in the areas of evolutionary biology, microbiology, genetics, chemistry, and other related sciences. This research has highlighted methods organisms use for adaptation and evolution, such as genetic mutation, homologous recombination (HR), and horizontal gene transfer (HGT).(6) Genetic mutation and HR occur when a bacterium’s DNA is altered within the specific bacterium. HGT occurs when DNA is transferred from one organism to another through processes outside of normal reproduction. This can occur with related bacteria or between different species. Not only can bacteria undergo mutation within its own genome, it can evolve using genes from other microbes leading to mosaic genes in which multiple organisms have contributed to the gene.(5) Researchers have also found that pleiotrophy and epistasis effect what mutations bacteria can undergo.(8) Pleiotrophy occurs when a single gene influences multiple phenotypic characteristics. Epistasis occurs when multiple genes influence a single phenotypic characteristic. This demonstrates that developing antibiotic resistance is an exceptionally complex evolutionary process.

Not all antibiotic resistance is the result of adaptation. Intrinsic antibiotic resistance occurs when the wild type of a species exhibits resistance to an antibiotic. The bacterial genus Mycoplasma lacks a cell wall so β-lactem antibiotics have no effect. Some bacteria such as Pseudomonas aeruginosa have several intrinsic mechanisms, making treatment of infection by them difficult from the start.(5)(1)

Bacteria that have not been endowed with intrinsic resistance must depend on evolutionary processes in order to persist in environments where antibiotics have been introduced. There are four general methods bacteria can use to develop antibiotic resistance. One method is to decrease uptake of the drug. This can occur through things such as the modification of porins. Increased export can also provide a mechanism of resistance and can be accomplished by upregulation of efflux pumps which pump the drug out of the cell. Some bacteria can inactivate or modify the target that the drug affects or make a new antibiotic resistant target. Bacteria can also hydrolyze or modify the drug using enzymes.(5)

Methods

The methods for studying antibiotic resistance in bacteria include in-vitro, such as in a petri dish, or in-vivo, within a living organism. Until recently, researchers were solely studying the evolution of phenotype in bacteria. With advancements in DNA sequencing, researchers can study changes in genotype arising from specific mutations. With bacterial genome information now widely available, this information can be correlated with phenotypic traits and computer simulations can be run. This speeds up study and provides some ability to predict adaptation.(6)(3)

Traditional laboratory techniques such as cloning, PCR and gene expression have been coupled with novel techniques as available technology has been advancing. The methods of studying antibiotic resistance in bacteria have advanced beyond culturing multiple petri dishes with varying concentrations of antibiotic. More sophisticated laboratory studies can now introduce antibiotics to a medium at

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different rates or gradients. Automatic feedback control can be implemented that will detect increasing levels of resistance and adjust the introduction of antibiotics accordingly. Such methods have provided insight into rates of resistance development with different microbes and antibiotics. This can better inform treatment guidelines that may slow resistance and increase effectiveness.(6)

Results

Previous theories have suggested that the biological costs associated with developing antibiotic resistance would decrease fitness of the bacteria. Wild type phenotypes would then be able to out-compete the mutated bacteria in environments that did not have the antibiotic present. Other theories suggested that when a population has adapted to a new environment those adaptations will disappear if the environment changes back to the original state.(3) These studies demonstrated that in the course of developing antibiotic resistance, the bacteria also underwent compensatory mutations that offset the biological cost of antibiotic resistance.(7)(3) These mutations often conferred increased fitness on the mutants compared to the wild type even when antibiotics were not present. Levin et al. specifically studied Salmonella typhimurium in mice. It was found that mutations in S. typhimurium, leading to resistance to various antibiotics, reduced the virulence of the bacteria. When the bacteria were transferred to antibiotic free mice, virulence was restored. When antibiotics were reintroduced, it was found that in most cases, S. typhimurium had maintained its resistance, and virulence was retained as a result of mutations at other loci. Additionally, they found that the mutation which makes Escherichia coli resistant to streptomycin reduces the E. coli ribosomes’ ability to translate proteins, a mutation that should significantly affect fitness. When E. coli was then transferred to an antibiotic free medium, compensatory mutations occurred which restored the ribosomes translational ability almost to wild type levels, bringing fitness back to almost equal with the wild type. The researchers then replaced the antibiotic resistant allele in the compensated strain with the antibiotic sensitive wild type allele from the wild type E. coli. This led to the lowest fitness of any of the experimental strains demonstrating that the new strain was unable to return to its original state of antibiotic sensitivity. Additionally, it was discovered that resistance persisted through subsequent generations. The genes providing antibiotic resistance may be repressed, but if the antibiotics were introduced into the environment of later generations, resistance was rapidly developed.(3)

Comparisons of identical experiments, with the only difference being that one is in-vivo and the other in-vitro, have resulted in very different compensatory mutations, indicating that environment will effect the mutations that occur.(5) The bacterial evolution is contingent on the overall environment it is in. Thus, the development of resistance is not a static process but can be adapted to bring about the highest possible fitness for the organism.

All of the studies investigated the presence and function of R-factors. R-factors are specific plasmids found in enteric bacteria. The plasmid provides coding both for resistance and also enhancement of the transfer of those genes in related populations. This is an example of HGT that promotes rapidly developing resistance.(5) R-genes have been detected in bacterial libraries and soil samples where the bacteria have not had exposure to antibiotics.(1) This discovery is important for two reasons. First, it demonstrates that in many bacteria the ability to develop resistance is intrinsic and not the result of direct exposure to antibiotics. Also, by cataloging potential R-factors, researchers can better predict resistance that may be transferred in the future.(6)

Some bacteria are classified as “promiscuous.” When these microbes obtain resistant plasmids they can transfer these not only within their own species but to other species as well. This transfer will occur in both the absence and the presence of the antibiotic.

Some bacteria can also move between different species of hosts. For example, antibiotic resistant Salmonella that develop in chickens being given feed containing antibiotics can lead to

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antibiotic resistant infections in humans who eat the eggs or meat. It is even possible for the bacteria to be passed to pets eating tainted pet food, who can then transmit it to humans.(2)

A study by Rodríguez-Verdugo et al. demonstrated pleiotrophy and epistasis within bacterial adaptation. In the study, 114 populations of E. coli were evolved for 2,000 generations in an increased temperature of 42.2 oC. Analysis of the genomes of the clones showed mutations that conferred resistance to rifampicin.(8) This demonstrated how adaptive mutations for a specific phenotype can cause a change in a phenotypic trait that is not related to the conditions being adapted to.

Some in-vitro studies of multi drug resistant bacteria have demonstrated that these mutations can lead to low fitness due to increased energy costs and ultimately lead to low long term survival rates of the adapted bacteria. In-vivo studies have demonstrated different results. These studies have shown that mutations can occur repeatedly over time with continued persistence of the microbe. In one study a patient hospitalized with an S. aureus infection was treated with vancomycin. Isolates of the bacteria were taken and the genome was sequenced at regular intervals over the three month period it took for recovery. Before the bacterium was finally killed, 35 genetic mutations were detected over the course of treatment. In similar studies of M. tuberculosis, from 29 to 35 different independent mutations were detected.(1)

Automatic feedback mechanisms have demonstrated that when exposed to some drugs the genotype can be variable yet is fixed and predictable when exposed to others. Resistance to some drugs can increase 1,000 fold in 20 days and other drugs only 10 fold.(6)

Discussion and Conclusions

Current research in the field of antibiotic resistant bacteria is helping researchers, health organizations and governments chart a path and make public health recommendations to slow the progression of this problem. It has also had the consequence of bringing new insights and understanding in the area of evolutionary biology. As studies of bacterial genomes continue, researchers may be able to identify new targets within bacteria that can be exploited through novel means. As similarities are found between different types of pathogens, antibiotics may be developed that effect wider ranges of microbes with a single drug. However, as has been demonstrated, even if we are able to develop new antibiotics it is only a matter of time before the bacteria will be able to develop resistance.

It is important that as studies of resistance continue we use the knowledge we gain about how resistance is developed to find ways to slow that resistance. Finding ways to prolong the usefulness of current antibiotics is as important as developing new ones. Group A Streptococcus (GAS) provides a poignant example of our need to be vigilant. GAS is a very common bacterium that leads to diseases such as strep throat and impetigo. However, if GAS finds its way into muscles, blood, or lungs, it can cause necrotizing fasciitis or streptococcal toxic shock syndrome (STSS). Over 50% of STSS cases lead to death as does approximately 20% of cases of necrotizing fasciitis.(9) GAS is still very sensitive to penicillin, which makes treatment of the common infections easy and thus reduces the incidents of the deadly infections. If GAS were to make the jump to penicillin resistance, the global consequences could be severe.(5)

Though some mutations that confer antibiotic resistance happen in a single step, it is often a multi-step process that occurs gradually over time. Remember that time can be a relative consideration. If bacteria divide every 20 minutes, seven days provides ample opportunity to undergo a multi-step mutation. One recommendation to slow antibiotic resistance is to give the highest dose possible of an antibiotic for a shorter treatment period to try to cut that process short.(5)

Understanding cross resistance can lead to better prescribing practices.(6) These studies are helping inform recommendations for more effective prescribing of antibiotics. When dealing with

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infections that are known to be developing resistance, doctors now prescribe two different antibiotics that the bacteria is most sensitive to at the same time. This tactic also helps arrest multi-step evolution of resistance.

Cycling the use of antibiotics has been suggested by some researchers in the past.(1) However as these studies have demonstrated, resistance is not lost over time in the absence of the antibiotic. Once a strain has developed resistance to an antibiotic, that resistance can be re-expressed in future generations.

Three recommendations that the World Health Organization has issued for patients should be noted for all of us to consider and act upon. Only use antibiotics when they are prescribed by a certified health professional. Always complete the full course of treatment, even if you feel better. Never share antibiotics with others or use leftover prescriptions.(10)

In order to combat and slow the threat of antibiotic resistant bacteria, coordinated action is needed from everyone involved. Patients, health workers, pharmacists, policy makers, scientists, and industries such as agriculture and pharmaceuticals, must all do their part in order to slow or reverse this dangerous trend.

References

1. Davies, J., & Davies, D. (2010). Origins and Evolution of Antibiotic Resistance. Microbiology and Molecular Biology Reviews, 74(3), 417-433. doi: 10.1128/MMBR.00016-10

2. Khachatourians, G. G. (1998, November 03). Agricultural Use of Antibiotics and the Evolution and Transfer of Antibiotic-resistant Bacteria. Canadian Medical Association Journal, 159(9), 1129-1136. Retrieved April 30, 2014, from http://www.cmaj.ca/content/159/9/1129.abstract

3. Levin, B. R., Perrot, V., & Walker, N. (2000, March 01). Compensatory Mutations, Antibiotic Resistance and the Population Genetics of Adaptive Evolution in Bacteria. Genetics, 154(3), 985-997. Retrieved April 30, 2014, from http://www.genetics.org/content/154/3/985.abstract

4. Meade-Callahan, M. (2001, January). Microbes: What They Do & How Antibiotics Change Them. Retrieved March 15, 2014, from http://www.actionbioscience.org/evolution/meade_callahan.html

5. Normark, B. H., & Normark, S. (2002). Evolution and Spread of Antibiotic Resistance. Journal of Internal Medicine, 252(2), 91-106. doi: 10.1046/j.1365-2796.2002.01026.x

6. Palmer, A. C., & Kishony, R. (2013). Understanding, Predicting and Manipulating the Genotypic Evolution of Antibiotic Resistance. Nature Reviews Genetics, 14(4), 243-248. doi:10.1038/nrg3351

7. Perron, G., Hall, A., & Buckling, A. (2010). Hypermutability and Compensatory Adaptation in Antibiotic Resistant Bacteria.‐ The American Naturalist, 176(3), 303-311. doi: 10.1086/655217

8. Schenk, M. F., & Visser, J. A. (2013). Predicting the Evolution of Antibiotic Resistance. BMC Biology, 11(1), 14. doi: 10.1186/1741-7007-11-14

9. USA, Department of Health and Human Services, Centers for Disease Control and Prevention. (2008, April 3). Group A Streptococcal (GAS) Disease. Retrieved April 30, 2014, from http://www.cdc.gov/ncidod/dbmd/diseaseinfo/groupastreptococcal_g.htm

10. World Health Organization. (2014, April). Antimicrobial Resistance. Retrieved May 1, 2014, from http://www.who.int/mediacentre/factsheets/fs194/en/

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