by: Sarah Wu and Professor Yousif Shamoo
Abstract
Only recently has the U.S. public become aware of the dangers of antibiotic resistance. Overuse of antibiotics is chiefly responsible for the proliferation of super-resistant strains. An intimate understanding of resistance mechanisms will prove crucial for continued success against pathogenic bacteria. Traditionally, antibiotics have targeted processes such as cell wall synthesis and DNA replication. However, the decreasing effectiveness of optimizing existing antibiotics has prompted scientists to explore new strategies, such as using combinations of antibiotics, examining evolutionary pathways leading to resistance, and targeting metabolic processes.
Introduction
The problem of antibiotic resistance catapulted to the attention of the U.S. public when seventeen-year-old Ashton Bonds died in October 2007 from MRSA (methicillin-resistant Staphylococcus aureus), which he contracted from his high school locker room.1 Studies conducted by the Centers for Disease Control and Prevention estimate deaths in the U.S. due to MRSA in 2005 exceeded those of HIV-AIDS, Parkinson’s disease, emphysema and homicide.2 While traditionally associated with hospital ICUs and nursing homes, deadly bacterial infections emerged in schools, gyms, and day cares, alarming the nation.
Historically, antibiotics have demonstrated a remarkable effectiveness against bacteria. Many consider antibiotics as one of the most important discoveries in modern science. The discovery of penicillin by Alexander Fleming in 1928 prevented many deaths and amputations on the battlefields during WWII.3 Sulfa drugs not only accelerated research in the pharmaceutical industry but also were successful starting points for the creation of new drugs to treat a variety of diseases.4 Despite their amazing effectiveness, antibiotics are slowly losing their edge against pathogens, as seen by the recent rise of strains such as MRSA, which doubled from 127,000 infections in 1999 to 278,000 in 2005 and increased from 11,000 to more than 17,000 deaths.5
As medicine integrated antibiotics into common practice as a way to treat a variety of infectious diseases such as bronchitis, pneumonia, and syphilis, antibiotics have become the expected and preferred cure in the vocabulary of the general public for unpleasant symptoms. These drugs have come to be seen as the magic cure-all that doctors can easily dispense to their patients, who are increasingly expectant of instant treatments. According to rationalmedicine.org, physicians may over-prescribe antibiotics due to fear of lawsuits of negligence if drugs are not prescribed. In addition to facing pressure from drug companies to prescribe their medications, physicians risk losing their patients to other physicians who are less hesitant to prescribe antibiotics.6 Sore throat is usually caused by a viral infection (and is therefore unresponsive to antibiotics) with only 5-17% of cases caused by bacteria. However, a 2001 study by Linder and Stafford revealed that in the period from 1989 to 1999, there was a decrease in the use of recommended antibiotics like penicillin for treating sore throat and an increase in the use of nonrecommended broad spectrum antibiotics, such as extended-spectrum macrolides and fluroroquinolones.7 Another study showed that half of the antibiotic prescriptions written by emergency medicine physicians were for viral infections.8 Because of a variety of factors leading to faulty administration, antibiotics are not being used effectively.
Antibiotics are also widely used in agriculture. Frequently animals that are raised for consumption in the U.S. come from factory farms that maximize yield by placing as many animals as possible into a limited space. Close quarters such as these facilitate the spread of infectious disease, making it necessary to treat the animals regularly with antibiotics as a preventative measure. Antibiotics are also administered to make up for poor conditions like inadequate nutrition and imperfect caretaking.9 This is concerning as there have been reports of animal to human transmission of disease. A 2006 study found a case where a strain of MRSA from a family’s pig farm had infected a baby girl, demonstrating the ease with which resistant strains can arise in farm animal populations and spread to humans.10
Resistance against antibiotics is not a novel phenomenon in biology and has in fact occurred for millions of years. Streptomyces, a genus of bacteria that lives in soil and water, excretes a variety of anti-microbial substances into the nearby soil to prevent the growth of competing microorganisms.11 Penicillin mold has similar defenses. It is not surprising that we derive many of our antibiotics from nature.
If bacteria are quickly becoming resistant to antibiotics, why do pharmaceutical companies hesitate to develop replacement drugs? In short, drug development is very difficult, expensive, and unprofitable. It is estimated that the cost of developing one successful drug is around $802 million. Drugs take about 12 years to get approved by the FDA and only about one out of 5,000 substances are approved, a 0.02% success rate. The pharmaceutical company then has the rights to the drug patent for 20 years before the drug becomes generic, allowing anyone to produce it.12 In addition, the scope of bacteria against which most antibiotics are effective quickly narrows with increased usage. The drug company is under pressure to start selling as much of the drug as possible to recoup the cost of development, which means aggressive marketing to physicians. Physicians are subsequently faced with a dilemma, as they want to limit the use of antibiotics to maintain their effectiveness yet feel pressured into prescribing them. Currently, a large portion of research is dedicated to the mechanisms and selection of antibiotic resistance, which scientists attribute to evolutionary processes.
Evolution’s role in antibiotic resistance
Evolution is the continual adaptation of a population to its environment. Mutations at the nucleotide level influence the structure of the proteins to produce a phenotype that could confer increased resistance to antibiotics. When a naive population of bacteria is exposed to an antibiotic, the vast majority is eliminated. However, the surviving bacteria are resistant and will give rise to antibiotic-resistant progeny. Thus, repeated administration of the same antibiotic results in the proliferation of resistant phenotypes and decrease in antibiotic efficacy. Because bacteria are unicellular organisms that divide roughly every thirty minutes, they quickly adapt to environmental challenges such as antibiotics. The combined effect of many divisions and mutations gives rise to numerous resistance mechanisms.
Antibiotics: Mechanism and Response
Bacterial cell walls serve the critical function of maintaining osmotic stability, making their synthesis a favorite target of bactericidal antibiotics. Bacteria are classified into two categories based on the type of cell walls that they have: gram-positive or gram-negative (Fig. 1). Gram refers to the staining protocol developed by Hans Christian Gram in 1884.13 The crystal violet stain used in this procedure will color the outer peptidoglycan wall of gram-positive bacteria purple but leaves the gram-negative bacteria pink, as the peptidoglycan net is contained inside its dual membranes. This peptidoglycan net is made up of alternating units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) which are cross-linked by the enzyme transpeptidase, also known as penicillin-binding proteins (PBP). The peptidoglycan net is a critical component to the bacteria as it maintains the high osmotic pressure inside the bacteria.
β-lactam drugs (Fig. 2), such as penicillin and ampicillin, are the most widely used antibiotics. β-lactams are a class of drugs that inhibit bacterial cell wall synthesis. Because their structure mimics the penultimate D-Ala-D-Ala pentapeptide chain of NAM, they function by being mistakenly used by the transpeptidases as a substrate, resulting in the acylation of the enzyme. The acylated transpeptidase is unable to hydrolyze the β-lactam and is thus not functional, which hinders cell wall synthesis. The cell wall becomes susceptible to the autolytic enzymes that degrade the cell wall and eventually becomes permeable to the environment, leading to bacterial lysis.14
Bacteria have responded in three main ways to β-lactam drugs. The active site of the transpeptidase can be changed to have a lower affinity for β-lactams. In gram-negative bacteria, the cell can also restrict the influx of drugs into the periplasm by altering the expression of outer membrane proteins to restrict drug entry or by the expression of antibiotic efflux pumps such as MexA.15 The most common mechanism of β-lactam resistance uses an enzyme to inactivate the drug before it can bind transpeptidase. This enzyme, known as β-lactamase (Fig. 3), is very similar in structure to the PBP and uses a strategically placed water molecule to hydrolyze the β-lactam ring of the drug, rendering it inactive. However, as new forms of β-lactam drugs have been introduced, bacteria have evolved more efficient forms of this enzyme.
Cell wall synthesis, however, is not the only common target of antibiotics. Fluoroquinolones (Fig. 2) inhibit DNA replication by stabilizing the enzyme-DNA complex created by the enzymes topoisomerase II (DNA gyrase) or topoisomerase IV, blocking DNA synthesis. Bacterial mechanisms for resistance to fluoroquinolones are very similar to those for β-lactam resistance. Mutations to the target sites of DNA gyrase and topoisomerase IV decrease drug binding, increasing expression of efflux pumps to remove the drug. Alternatively, losing porins in the membrane restrict entry of the drug into the cytoplasm.16
Current Research in the Field
For many years, the main response to developed resistance was to introduce a new form of the antibiotic. Unfortunately, little progress has been made recently in developing new effective compounds, eliminating the possibility of a quick score in terms of discovering a bacterial growth inhibitor.17 This necessitates the study of novel approaches to eradicating pathogenic bacteria.
Drug cocktails
Some researchers have pursued the idea of combining antibiotics in hope of a synergistic effect. The combination of two drugs is expected to lead to a more effective therapy, as is the case with the cocktail of drugs given to HIV patients. However, predicting the efficacy of drug combinations has proven to be difficult, making direct experimentation necessary for verifying efficacy. Cefotaxime and minocycline were found to be an effective combination against Vibrio vulnificus18, although the treatment of sepsis with the addition of aminoglycosides to β-lactams was found to have no advantage and actually increased risk of nephrotoxicity.19 One study characterized combinations of drugs as synergistic, additive, or antagonistic (depending on if the cumulative effect of the drugs is greater than, equal to, or less than the effect of their individual activity) and demonstrated how certain combinations of drugs could select against drug resistance. By conducting a competition assay between doxycycline-resistant and wild type E. coli at certain concentrations of doxycycline and ciprofloxacin, the wild type strain was found to have a growth advantage over the doxycycline-resistant strain.20 While this study was limited to sublethal concentrations of antibiotics, it suggests the potential to forestall drug resistance through combinations that select against the development of resistance.
Predicting the evolutionary trajectories of antibiotic resistance
Other research concentrates on the evolutionary processes of resistance. One study identified five key β-lactamase mutations that led to a 100,000-fold increase in resistance against cefotaxime. While in principle there are 120 (5!) possible pathways (also known as trajectories) to reach the final state of accumulating all five mutations, the study demonstrated that a large number of these pathways were already inaccessible as a beneficial mutation has a much higher probability of fixation in the population as opposed to a deleterious or neutral one. Other combinations of those 5 mutations did not increase resistance unless certain mutations preceded others sequentially. In the end, they found only 18 probable trajectories leading towards the super effective enzyme with five mutations.21 This study shows the surprisingly limited nature of evolutionary pathways and the possibly predictable nature of evolution. While this study was strictly theoretical in its execution, similar investigations into the evolutionary trajectories of antibiotic resistance at the molecular level are being conducted using in vivo models with the hope of understanding the mutational landscape of the development of drug resistance.
Targeting Bacterial Metabolism
Other studies have looked beyond conventional drug targets and in bacterial metabolism, suggesting that all antibiotics kill bacteria in the same way by causing them to produce hydroxyl radicals, which damage proteins, membrane lipids, and DNA. A methodical sequence of experiments probes this idea, starting with experiments that show the increased production of hydroxyl radicals in bacteria when treated with bactericidal antibiotics. The Fenton reaction, which produces hydroxyl radicals by the reduction of hydrogen peroxide by ferrous iron, is considered the most significant contributor of hydroxyl radicals. Bacteria were found to live longer when exposed to thiourea, a hydroxyl radical scavenger, as well as 2,2’-dipyridyl, an iron chelator that inhibits the Fenton reaction. To determine the source of the ferrous iron as either extracellular or intracellular, a knockout strain (∆tonB) was created with disabled iron import as well as a knockout (∆iscS) with impaired iron-sulfur cluster synthesis abilities. While ∆tonB exhibited no advantage against antibiotics, ∆iscS showed reduced hydroxyl radical formation and cell death, pointing to intracellular ferrous iron as the source of hydroxyl radicals. It is established that ferrous iron is released when superoxide damages the iron-sulfur clusters and the most superoxide formation comes from electron transport chain oxidation and conversion of NADH to NAD+. Gene expression microarrays revealed that upon exposure to antibiotics, NADH dehydrogenase I was a key upregulated pathway. Bacteria respond to hydroxyl radicals by activating RecA, which stimulates SOS response genes to initiate DNA repair mechanisms. RecA knockout strains had a significant increase in cell death compared to wild type. The study proposes the following mechanism: antibiotics stimulate oxidation of NADH, which induces hyperactivation of the electron transport chain, thus stimulating superoxide formation that damages iron-sulfur clusters in the cell. These clusters release ferrous iron, which is oxidized by the Fenton reaction, producing the hydroxyl radicals. Bacteria have SOS response mechanisms that are activated by the RecA gene, leading to the stimulation of SOS response genes. By knocking out this gene, they found the bacteria had a significant increase in sensitivity to antibiotics. The study demonstrated the possibilities of targeting the TCA cycle or respiratory chain when developing new antibiotics.22
Complementary strategies
While a large burden rests in the hands of the scientists and drug companies, there are steps the public can take to decrease the rate of proliferation of resistant strains. The federal Center for Disease Control and Prevention recommends not pressuring one’s health care provider to prescribe antibiotics and to follow directions when taking medication by only taking antibiotics for bacterial infections as well as completing the prescribed dose of antibiotics completely to avoid sparing bacteria that could potentially become drug resistant.23 Improving infection control would prevent the spread of resistant strains as rapid methods of identifying pathogens would lead to faster isolation of colonized patients, making it harder for disease to spread.24 The simple practice of washing hands can limit the spread of disease. Citizens can also advocate legislation that limits the use of antibiotics in the agricultural industry.
As observed in the laboratory and daily life, carelessness with the application of antibiotics results in the disappearance of our painstakingly acquired advantage against bacteria. But even with cautious usage, antibiotics will still select for resistant strains. Continuous ongoing research is critical to discovering successful novel treatment strategies.
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