Appendix A
Contributed Manuscripts

A1
THE CASE FOR PATHOGEN-SPECIFIC THERAPY1

Arturo Casadevall2

Albert Einstein College of Medicine


At the beginning of the twenty-first century, the treatment of microbial diseases is increasingly complicated by drug resistance, the emergence of new pathogenic microbes, the relatively inefficacy of antimicrobial therapy in immunocompromised hosts, and the reemergence of older diseases, often with drug-resistant microbes. Some of these problems can be traced to the switch between pathogen-specific antibacterial therapy and the nonspecific antibacterial therapy that followed the transition from serum therapy to modern antimicrobial chemotherapy. The widespread availability of cheap, effective, nontoxic wide-spectrum antibacterial therapy for almost 75 years fostered a culture of therapeutic empiricism that neglected diagnostic technologies. Despite unquestioned lifesaving efficacy for individuals with microbial diseases, the use of broad-spectrum antimicrobials was associated with fungal superinfections and antibiotic-associated

1

Reprinted from Casadevall, A. 2009. The case for pathogen-specific therapy. Expert Opinion in Pharmacotherapy 10(11):1699-1703 with permission from Taylor & Francis Ltd.

2

Affiliation: Arturo Casadevall, Albert Einstein College of Medicine, Division of Infectious Diseases of the Department of Medicine, Department of Microbiology and Immunology, 1300 Morris Park Avenue, Bronx, NY 10461, USATel: +1 781 430 3665; Fax: +1 718 430 8741



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Appendix A Contributed Manuscripts A1 THE CASE FOR PATHOGEN-SPECIFIC THERAPy1 Arturo Casadevall Albert Einstein College of Medicine At the beginning of the twenty-first century, the treatment of microbial diseases is increasingly complicated by drug resistance, the emergence of new pathogenic microbes, the relatively inefficacy of antimicrobial therapy in immu - nocompromised hosts, and the reemergence of older diseases, often with drug- resistant microbes. Some of these problems can be traced to the switch between pathogen-specific antibacterial therapy and the nonspecific antibacterial therapy that followed the transition from serum therapy to modern antimicrobial chemo- therapy. The widespread availability of cheap, effective, nontoxic wide-spectrum antibacterial therapy for almost 75 years fostered a culture of therapeutic empiri - cism that neglected diagnostic technologies. Despite unquestioned lifesaving efficacy for individuals with microbial diseases, the use of broad-spectrum anti- microbials was associated with fungal superinfections and antibiotic-associated 1 Reprinted from Casadevall, A. 2009. The case for pathogen-specific therapy. Expert Opinion in Pharmacotherapy 10(11):1699-1703 with permission from Taylor & Francis Ltd. 2 Affiliation: Arturo Casadevall, Albert Einstein College of Medicine, Division of Infectious Dis - eases of the Department of Medicine, Department of Microbiology and Immunology, 1300 Morris Park Avenue, Bronx, NY 10461, USATel: +1 781 430 3665; Fax: +1 718 430 8741 

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 ANTIBIOTIC RESISTANCE colitis, helped to catalyze the emergence of resistance, and is now tentatively asso- ciated in the pathogenesis of certain chronic diseases, including atopy, asthma and – perhaps – certain forms of cancer. This article briefly reviews these trends and suggests that the current strategy of nonspecific therapy is fundamentally unsound because it damages the microflora and – consequently – the human symbiont. The essay argues for the development of immunotherapy and pathogen-specific therapies, especially with regard to bacterial and fungal diseases, and suggests possible routes to that future. 1. The Problematic Status Quo Current antimicrobial therapy is largely pathogen-specific for viral diseases and nonpathogen-specific for bacterial, fungal, and parasitic diseases (Casadevall, 1996). Although some of the latter diseases are sometimes treated with pathogen- specific drugs, such as the use of isoniazid for tuberculosis, the overwhelming majority of compounds targeting bacteria, fungi, and parasitic diseases have activity against multiple microbes. Furthermore, these compounds target both pathogenic and nonpathogenic microbes. This current antimicrobial paradigm is currently in use at a time of significant upheaval in the therapy of microbial diseases, which is the only field of medicine in which one can argue that thera- peutic options have declined over time. For example, in the 1950s Jawetz noted that the then currently available antimicrobial drugs were satisfactory for the treatment of bacterial diseases (Jawetz, 1956). However, in recent years the field of infectious diseases has seen dramatic increases in antimicrobial resistance, an increasing prevalence of bacterial and fungal superinfections in treated individu - als, a relatively low therapeutic efficacy of antimicrobial therapy in individuals with impaired immunity, the emergence of new infectious diseases, and the reemergence of older microbial diseases, often with highly resistant microbes such as XDR-Tb. Given this status quo, it behooves us to ask the questions: How did we get here? What are the consequences of the choices made then and now? Can we do better and how do we get there? 2. How Did We Get Here? Effective antimicrobial therapy can be dated to the introduction of serum therapy in the 1890s, which, for the first time, provided physicians with the abil - ity to intervene and cause a favorable outcome for an infectious disease. Serum therapy was developed against numerous bacterial and viral diseases, including pneumococcal pneumonia, meningococcal meningitis, erysipelas, anthrax, and measles (for reviews, see refs Casadevall and Scharff, 1994; Casadevall and Scharff, 1995; Buchwald and Pirofski, 2003). The heyday of serum therapy was the 1930s, but the modality was rapidly abandoned because serum could not compete with small-molecule antimicrobial therapy, such as sulfonamides and

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 APPENDIX A penicillin, with regard to price, stability, ease of use, and (low) toxicity. For some diseases such as meningococcal meningitis, small-molecule antimicrobial therapy was clearly more effective than serum therapy; however, for pneumococcal pneu- monia the difference in efficacy was less clear. In addition to serum therapy, the few other therapies available (e.g., quinine for malaria, salvarsan for syphilis, optochin for pneumococcus, and phage therapy) were all pathogen specific. In a prior essay (Casadevall, 2006), I argued that the time of serum therapy and the subsequent era of therapy with small molecules constituted the two first ages of antimicrobial therapy. When viewed through the prism of microbial specificity, the greatest difference in the therapeutic approach between the first and second ages of antimicrobial therapy was a switch from pathogen-specific to nonspecific therapy with regard to antibacterial therapeutics. In this essay, I argue that this change was to have enormous implications, which are root causes for some of the problems we face today. In evaluating the therapeutic paradigm for microbial diseases, it is worth- while contrasting it with the therapy of cancer. Like therapy for infectious dis - eases, the treatment of tumors has relieved [sic] heavily on antibiotics made by microorganisms; adryamicin, actinomycin D, bleomycin etc. are all microbial products. Like antimicrobial antibiotics, these antimetabolite antibiotics are each nonspecific in the sense that they are cytotoxic to multiple tumors. However, unlike most antimicrobial antibiotics, these agents have tremendous toxicity for the host and, consequently, are never used empirically. Hence, oncology practice has placed great emphasis on diagnosis and in exploiting subtle pharmacological differences between these agents to enhance their therapeutic index. In fairness to infectious diseases, it noteworthy that the temporal kinetics of microbial infections and tumorogenesis favored a more deliberate approach to diagnosis as tumors, which unlike microbes, seldom killed the host rapidly. Nevertheless, the analogy is relevant because it provides an inkling of how the practice of infectious diseases might have developed if early antimicrobials had more significant toxicity, as evidenced by the hesitant empiric use of amphoteri - cin b and Ara-C for fungal and herpetic diseases, respectively, Consistent with this notion, the development of the relatively nontoxic antiherpetic drug acyclo - vir as a replacement for Ara-C was followed with significantly greater empiric use, especially in neonates and cases of encephalitis. Similarly, the introduction of low-toxicity azoles and echinochandins as replacements for the highly toxic amphotericin b has promoted the empirical use of antifungal therapy. Hence, the advantage of low toxicity has the perverse effect of promoting empirical and inappropriate use. In comparing the ages of antimicrobial therapy, it is clear that the change in the specificity of therapeutic agents did not affect all types of antimicrobial therapy equally. Serum therapy for viral diseases was specific and current antivi- ral drugs remain largely pathogen-specific, with the caveat that some drugs like acyclovir have activity against multiple herperviruses [sic]. For mycobacterial

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 ANTIBIOTIC RESISTANCE diseases, there was no effective therapy in the preantibiotic era and most drugs that were subsequently developed (isoniazid, ethambutol, and others) were used primarily for the therapy of tuberculosis. For fungal diseases, there was no effec- tive therapy prior to the late 1950s when amphotericin B was introduced; a com - pound active against most fungal pathogens and antifungal therapy has always relied on nonpathogen-specific agents. For bacterial diseases, the change from serum to small-molecule therapeutics was a revolution, as therapeutic specificity was abandoned in favor of agents with increasingly greater spectrum of antimi- crobial activity. However, what made the switch from pathogen-specific to non- pathogen-specific therapy so significant with regard to antibacterial therapy is that the human host is a symbiont, with microflora consisting mostly of desired com- mensal bacteria. By contrast, there are no known desirable commensal viruses and the known fungal flora is limited to a few fungal species where Candida spp predominate. Unlike bacteria, a beneficial function has not been demonstrated for the host-associated fungal microflora. Hence, the use of nonspecific bacterial therapy carried an inherent potentially detrimental effect in damaging the associ- ated bacterial microflora, and thus the human symbiont. 3. The Consequences of Nonspecific Antimicrobial Therapy The nonspecificity of antibacterial, and to a lesser extent antifungal, thera - pies was to have profound consequences on the practice and outcome of infec- tious diseases that reverberate to current times. The availability of nonspecific antibacterial therapies with broad spectrum and low toxicity allowed physicians to rapidly treat many infectious diseases without a need for a microbial diagno - sis. For individuals with bacterial diseases, such therapy was often lifesaving. However, the ability to effectively treat many diseases safely without making a diagnosis deemphasized diagnostic clinical microbiology and fostered a culture of empiricism. For example, the diagnosis of pneumococcal pneumonia with the identification of the offending serotype took approximately 6 – 8 h in the 1930s and used the mouse peritoneal infection assay followed by typing with rabbit type-specific serum. This methodology was developed to rapidly ascertain the presence and serotype of pneumococcus in sputum because the efficacy of serum therapy depended on matching the bacterial serotype with the specificity of the antiserum. Despite the problems in unequivocally diagnosing pneumonia from sputum, this approach was successful for selecting therapeutic sera and sup- ported the use of serum therapy. However, the introduction of penicillin and later antimicrobial drugs made the test much less relevant and it was abandoned as a diagnostic tool. Currently, a definitive diagnosis of pneumococcal pneumonia is possible only when accompanied by bacteremia, information that requires 48 h. For fungal diseases, a full embrace of empiric therapy was checked by the toxicity of amphotericin b, but by the late 1990s, the availability of relatively nontoxic azole and echinocandin-type drugs had ushered greater empiric use. By contrast,

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 APPENDIX A for conditions that required specific therapy, such as viral and mycobacterial diseases, the practice ethos supported continued emphasis on diagnostic identifi - cation of the causative microbe. For bacterial and later fungal diseases, the availability of relatively nontoxic broad-spectrum therapy contributed to the emergence of resistance among both targeted and nontargeted microbes. Although specific therapy can also elicit resistance, as witnessed by the emergence of isoniazid-resistant Mycobacterium tuberculosis, only nonspecific therapy can elicit resistance among nontargeted microbes such as common inhabitants of the microflora. Furthermore, only non- specific therapy can damage the microflora to create alterations that foster the emergence of usually commensal microbes such as Candida and Enteroccocus spp, first as major pathogenic microbes and then as drug-resistant pathogenic microbes. Consequently, the discipline of infectious diseases may be the only specialty of medicine where previously effective therapeutic options have to be abandoned because of drug resistance creates [sic] obsolescence. Another consequence of nonspecific antibacterial and antifungal therapy was damage to the human symbiont. There is rapidly accumulating evidence that the human microflora is established early in life through complex steps and that there are individual differences in microbial species composition, a fact that could reflect differences in the timing of acquisition or modulation by the host immune system. The microbial flora is essential for development of the immune system, helps digestion, provides numerous nutrients including vitamins, and protects the human host by niche-denial to more pathogenic microbes. There is conclusive evidence that damage to the microflora by nonspecific antibacterial therapy can translate into antibiotic-associated colitis and fungal diseases such as oral thrush and candidal vaginitis. However, there are ominous signs that nonspecific anti - microbial use might translate into certain chronic diseases such as atopy (Kusel et al., 2008), asthma (Kozyrskyj et al., 2007), and even some types of cancer (Velicer et al., 2004), possibly by altering the development of the immune system in childhood and/or affecting metabolites produced by the microflora. In this regard, it is noteworthy that there is a temporal association between widespread antimicrobial use and the increase in immunoreactive diseases such as allergies and asthma, although it is premature to conclude causality as there may be con - founding variables (Wickens et al., 2008). Nevertheless, the available evidence does provide reason for concern. In summary, the development of effective, nontoxic, nonspecific antibacte- rial and antifungal therapy has had great consequences, some positive and some negative. Positive consequences include a significantly enhanced capacity to treat bacterial and fungal diseases early and effectively, which has translated to reduced mortality. Furthermore, the ability to treat early, safely, and without knowledge of the causative microbe has created a permissive environment for the development of complex surgeries, aggressive chemotherapy for tumors, and organ transplantation, procedures that would have unacceptable mortality without

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0 ANTIBIOTIC RESISTANCE such drugs. However, the same approach has also created a culture of empiricism that promoted antibiotic use, which in turn selected for resistance in targeted and nontargeted microbes, promoted the phenomenon of superinfection and damaged the symbiont with consequences that are only now beginning to be understood. In this regard, empiricism was a practice largely dictated by clinical findings and historical probability that essentially rejected causality in favor of associations. 4. Can We Do Better and How to Get There? Of course we can do better. Even for the short historical time that effec - tive antimicrobial therapy has been available it is clear that the effectiveness of therapy and diagnosis has fluctuated with time. In a previous essay (Casadevall, 2006), I argued that we are in the throes of a major paradigm shift that will usher in the third age of antimicrobial therapy. This age can be envisioned as an equi- lateral triangle with pathogen-specific therapy, greatly improved diagnostics, and immunotherapy at each apex. Nonspecific therapy will always have a role for the treatment of polymicrobial diseases and to insure proper coverage in individuals with fulminant disease but its use could be limited by the combination of rapid diagnostics and pathogen-specific drugs. Even for such polymicrobial diseases as abdominal sepsis originating from a ruptured viscus there is evidence that damage is caused by only a few microbial species and their identification would permit employment of pathogen-specific drugs. In this age, immunotherapy, whether with large molecules, such as antibodies or small-molecular-weight immuno - modulators, would have co-equal status with therapies designed to directly kill or inhibit the microbe. Although this author believes that third-age therapeutics will arrive in the twenty-first century, significant scientific, economic, and behavioral hurdles must be overcome for the realization of this vision. On the scientific front, drug discovery would have to move from trying to identify common therapeutic pathways among phylogenetically distant bacteria to exploiting differences in physiology and virulence mechanisms and/or to aug- menting host mechanisms that promote microbial clearance, which, interestingly, are nonspecific. This formidable task is made even more difficult by the econom- ics of antimicrobial drug discovery. As for other diseases, the economics of drug development is a function of the prevalence of the disease, which dictates market size. However, in antimicrobial drug discovery this formula is further modified by the fact that the market size is directly proportional to the width of the drug antimicrobial spectrum. Given the cost of drug development, the economics are stacked against pathogen-specific drugs in favor of broad-spectrum drugs. One caveat in this analysis is that drug resistance can disproportionately shorten the useful life of broad-spectrum drugs and that the emergence of resistant microbes can in itself create new market opportunities. For example, the emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) creates a niche such that a new staphylococcal-specific drug active against methicillin- and

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 APPENDIX A possibly vancomycin-resistant isolates would probably be developed clinically if available. The use of pathogen-specific drugs would necessitate advances in diagnostics to provide rapid and accurate information to support their use, and this would require new investments in research and laboratory assays. Finally, physicians would have to change their approach to patients with presumed infec - tious diseases, emphasizing the need for diagnosis to select appropriate therapy in an echo to the practices of physicians in the age of serum therapy. Perhaps the hurdles are so high that pathogen-specific therapy is only in the far horizon. If that is the case, there are concrete actions that can be taken in the present to slow the spread of drug resistance and damage to the human microbial flora. For example, educational campaigns aimed at physicians and the general public can promote more prudent use of antimicrobial drugs. At a political level, policy makers should be made aware of the economic and regulatory hurdles that slow the development of rapid diagnostic tests and pathogen-specific drugs. How- ever, perhaps things can change more rapidly that one can anticipate. Certainly, if future research was to associate disturbances in the microflora with such chronic diseases as asthma, atopy, and cancer, this would create tremendous medical and legal disincentives in the use of nonspecific microbial therapy. Another powerful force could be the categorization of such complications of broad-spectrum therapy as C. difficile colitis and candidiasis as medical errors, which would be followed by aversion of third-party payers for hospital and physician reimbursements. At the same time, economic incentives for the development of pathogen-specific therapy by industry could be created by linking the patent protection time of antimicrobial drugs to the width of the antimicrobial spectrum and inclusion of narrow-spectrum drugs as orphan drugs. For example, patent policy could be amended such that narrow-spectrum drugs with small markets enjoy much longer patent protection than broad-spectrum drugs. Although in 2009 a revolution in the antimicrobial therapeutic paradigm seems distant, it is worth noting that only a generation ago smoking was widely permitted and accepted in most public places. For smoking, it was the realization that second-hand smoke was dangerous that catalyzed the creation of smoke-free environments in most public places. Perhaps increased awareness of the consequences of long-term damage to the human flora will have a similar catalytic effect in promoting pathogen-specific antimicrobial therapies. The re-introduction of pathogen-specific therapy for bacterial diseases, and its extension to fungal diseases, would require a concerted effort and collaboration between intellectual leaders in the field, industry, and government to find mecha- nisms that would promote and encourage the development of such drugs. There are indications of movement in this direction. A recent report by the Institute of Medicine recommended ‘development of strategies that will selectively target pathogenic organisms while avoiding targeting the host and beneficial or benign organisms’, which in other words is pathogen-specific therapy.3 Several therapies 3 Available from http://www.nap.edu/catalog/11471.html.

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 ANTIBIOTIC RESISTANCE narrow-spectrum are currently in development, for example, the renewed interest in phage therapy, monoclonal antibody therapies, and drugs aimed primarily at targeting highly resistant bacteria. However, the task of refocusing anti-bacterial and antifungal therapy to pathogen specificity is too great for any individual party and cooperation from industry, government, and the medical community will be needed to effect change. There is an acute need for an economic model that would allow the development and use of pathogen-specific drugs. Despite these hurdles, it is clear that pathogen-specific therapy makes sense and, given that the current nonspecific strategies are increasingly bankrupt, it behooves all parties to begin a dialogue on how to get there, and get there sooner than later. Declaration of Interest The author states no conflict of interest and has received no payment in preparation of this manuscript. Bibliography Buchwald UK, Pirofski L. Immune therapy for infectious diseases at the dawn of the 21st century: the past, present and future role of antibody therapy, therapeutic vaccination and biological response modifiers. Curr Pharm Des 2003;9(12):945-68 Casadevall A. Crisis in Infectious Diseases: Time for a new paradigm? Clin Infect Dis 1996;23:790-4 Casadevall A, Scharff MD. “Serum Therapy” revisited: Animal models of infection and the develop - ment of passive antibody therapy. Antimicrob Agents Chemother 1994;38:1695-702 Casadevall A, Scharff MD. Return to the past: the case for antibody-based therapies in infectious diseases. Clin Infect Dis 1995;21:150-61 Casadevall A. The third age of antimicrobial therapy. Clin Infect Dis 2006;42(10):1414-6 Jawetz E. Antimicrobial therapy. Ann Rev Microbiol 1956;10:85-114 Kozyrskyj AL, Ernst P, Becker AB. Increased risk of childhood asthma from antibiotic use in early life. Chest 2007;131(6):1753-9 Kusel MM, de KN, Holt PG, Sly PD. Antibiotic use in the first year of life and risk of atopic disease in early childhood. Clin Exp Allergy 2008;38(12):1921-8 Velicer CM, Heckbert SR, Lampe JW, et al. Antibiotic use in relation to the risk of breast cancer. JAMA 2004;291(7):827-35 Wickens K, Ingham T, Epton M, et al. The association of early life exposure to antibiotics and the development of asthma, eczema and atopy in a birth cohort: confounding or causality? Clin Exp Allergy 2008;38(8):1318-24

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 APPENDIX A A2 WAVES OF RESISTANCE: STAPHyLoCoCCuS AuREuS IN THE ANTIBIOTIC ERA4 Henry F. Chambers and Frank R. DeLeo Abstract Staphylococcus aureus is notorious for its ability to become resistant to antibiotics. Infections that are caused by antibiotic-resistant strains often occur in epidemic waves that are initiated by one or a few successful clones. Methicillin-resistant S. aureus (MRSA) features prominently in these epi- demics. Historically associated with hospitals and other health care settings, MRSA has now emerged as a widespread cause of community infections. Community or community-associated MRSA (CA-MRSA) can spread rap - idly among healthy individuals. Outbreaks of CA-MRSA infections have been reported worldwide, and CA-MRSA strains are now epidemic in the United States. Here, we review the molecular epidemiology of the epidemic waves of penicillin- and methicillin-resistant strains of S. aureus that have occurred since 1940, with a focus on the clinical and molecular epidemiology of CA-MRSA. Staphylococcus aureus is naturally susceptible to virtually every antibiotic that has ever been developed. Resistance to antibiotics is often acquired by the horizontal transfer of genes from outside sources, although chromosomal muta - tion and antibiotic selection are also important. This exquisite susceptibility of S. aureus led to Alexander Fleming’s discovery of penicillin, which ushered in the ‘antibiotic era’. Penicillin was truly a miracle drug: uniformly fatal infections could now be cured. However, by the mid 1940s, only a few years after its intro - duction into clinical practice, penicillin resistance was encountered in hospitals, and within a decade it had become a notable problem in the community. A fundamental biological property of S. aureus is its ability to asymptomati- cally colonize healthy individuals. Approximately 30% of humans are asymptom- atic nasal carriers of S. aureus (Kluytmans and Verbaugh, 1997; Gorwitz et al., 2008) such that in these individuals S. aureus is part of the normal flora. S. aureus 4 Reprinted with permission from Nature Reviews Microbiology 7, 629-641 (September 2009). 5 Division of Infectious Diseases, Department of Medicine, San Francisco General Hospital, Uni - versity of California, San Francisco, California 94110, USA. 6 Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Insti - tute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Ham - ilton, Montana 59840, USA. Correspondence to H.F.C. e-mail: hchambers�medsfgh.ucsf.edu. doi:10.1038/nrmicro2200.

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 ANTIBIOTIC RESISTANCE carriers are at higher risk of infection and they are presumed to be an important source of the S. aureus strains that spread among individuals. The primary mode of transmission of S. aureus is by direct contact, usually skin-to-skin contact with a colonized or infected individual, although contact with contaminated objects and surfaces might also have a role (Miller and Diep, 2008; Kazakova et al., 2005; Lowy, 1998; Muto et al., 2003). Various host factors can predispose individuals to infection, including the loss of the normal skin barrier, the presence of underlying diseases such as diabetes or AIDS and defects in neutrophil function. Infections that are caused by antibiotic-resistant strains of S. aureus have reached epidemic proportions globally (Tiemersma, 2006). The overall burden of staphylococcal disease, particularly disease caused by methicillin-resistant S. aureus (MRSA) strains, is increasing in many countries in both health care and community settings (Kaplan et al., 2005; Hersh et al., 2008; Klevens et al., 2007; Hope et al., 2008; Laupland et al., 2008; European Antimicrobial resistance Surveillance System, 2008). In the United States, the emergence of community associated MRSA (CA-MRSA) strains accounts for much of this increase, as it is a major cause of skin and soft-tissue infections (Moran et al., 2006; Fridkin et al., 2005). The rapidity and extent of the spread of CA-MRSA strains has been remarkable. In addition to the United States, CA-MRSA strains have been reported in Canada, Asia, South America and Australia as well as throughout Europe, including in countries that historically have a low prevalence of MRSA, such as Norway, the Netherlands, Denmark and Finland (Laupland et al., 2008; Larsen et al., 2007; Larsen et al., 2008; Wannet et al., 2005; Deurenberg et al., 2009; Vandenesch et al., 2003; Stam-Bolink et al., 2007; Huang et al., 2007; Nimmo and Coombs, 2008; Kanerva et al., 2009; Park et al., 2009; Gardella et al., 2008; Francois et al., 2008; Fang et al., 2008; Conly and Johnston, 2003). Globally, CA-MRSA strains have shown considerable diversity in the number of different clones that have been identified. In addition to their increasing prevalence and incidence, CA-MRSA strains seem to be particularly virulent. Overwhelming and tissue-destructive infections, such as necrotizing fasciitis and fulminant, necrotizing pneumonia (Francis et al., 2005; Gonzalez et al., 2005; Kallen et al., 2009), were rarely seen before the emergence of CA-MRSA strains. The factor (or factors) that is responsible for this hypervirulent behaviour is not known, but Panton–Valentine leukocidin (PVL), which has been epidemiologically associated with severe skin infections and pneumonia that are caused by methicillin-susceptible S. aureus (MSSA) strains (Lina et al., 1999), is a leading candidate. Antibiotics arguably constitute the most concentrated selective pressure on S. aureus in its long coevolutionary history with mankind. The consequences of this selective pressure, in conjunction with horizontal and vertical gene trans - fer, are discussed in this Review. Given their crucial importance as therapeutic agents, we focus on resistance to penicillins and the structurally related β-lactam antibiotics.

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 APPENDIX A Epidemic Waves of Resistance The emergence of antibiotic resistance in S. aureus can be visualized as a series of waves (Figure A2-1). The first wave began in the mid 1940s as the pro- portion of infections caused by penicillin-resistant strains of S. aureus increased in hospitals (Kirby, 1944; Barber and Rozwadowska-Dowzenko, 1948). These strains produced a plasmid-encoded penicillinase, which hydrolyses the β-lactam ring of penicillin that is essential for its antimicrobial activity. Penicillin-resistant strains soon began to cause community infections, and by the early 1950s they had become pandemic (Roundtree and Freeman, 1956). These infections, both in hospitals and in the community, were frequently caused by an S. aureus clone known as phage type 80/81 (Roundtree and Freeman, 1956; Blair and Carr, 1960; Bynoe et al., 1956; Roundtree and Beard, 1958). Pandemic phage type 80/81 S. aureus infections largely disappeared after the introduction of methicillin (Jevons and Parker, 1964), but the prevalence of penicillinase-producing strains from other S. aureus lineages has remained high. The introduction of methicillin marks the onset of the second wave of resis - tance (Figure A2-1). The first reports of a S. aureus strain that was resistant to methicillin were published in 1961 (Barber, 1961; Jevons, 1961). Although the specific gene responsible for methicillin resistance (mecA, which encodes the low-affinity penicillin-binding protein PbP2a (also known as PbP2′)) was not identified until over 20 years later, it was appreciated early on that the resistance mechanism involved was different from penicillinase-mediated resistance because drug inactivation did not occur. Unlike penicillinase-mediated resistance, which is narrow in its spectrum of activity, methicillin resistance is broad, conferring resistance to the entire β-lactam class of antibiotics, which include penicillins, cephalosporins and carbapenems. Among the earliest MRSA clinical isolates was the archetypal MRSA strain COL, a member of the ‘archaic’ clone of MRSA and perhaps the most studied MRSA strain, which was isolated from a patient in Colindale, UK, in 1960 (Jevons, 1961). COL is a member of the most successful MRSA lineage, which includes both hospital and community-associated strains. Archaic MRSA strains circulated in hospitals throughout Europe until the 1970s (Crisostomo et al., 2001). There were also isolated reports of MRSA in hospitals in the United States (Barrett et al., 1968; Bran et al., 1972), but the rest of the world was largely unaffected, and these early MRSA strains never gained a foothold in the community. By the 1980s, for reasons that remain unclear, the archaic MRSA clone had largely disappeared from European hospitals, marking the end of the second and the beginning of the third wave of antibiotic resistance. Descendants of the archaic MRSA clone (for example, the Iberian and Rome clones (Mato et al., 2004) and other, highly successful MRSA lineages emerged (Enright et al., 2002; Robinson and Enright, 2003; Deurenberg and Stobberingh, 2008) (Table A2-1). Outbreaks of infections caused by MRSA strains were reported in hospitals in the United States in the late 1970s, and by the mid 1980s

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0 APPENDIX A Acinetobacter, Burkholderia and others are intrinsically highly drug resistant. In fact, these organisms are often cited as among the most concerning to infectious disease specialists looking for new antibiotics to treat disease (Spellberg et al., 2008; Livermore, 2009). Antibiotic resistance in these opportunistic pathogens of environmental origin is often combinatorial; dominated by multiple efflux pumps, but also including specific enzymes that inactivate particular classes of antibiotics. The genome sequence of Pseudomonas aeruginosa PA01, for example, reveals > 20 efflux pump associated genes and several genes encoding enzymes for resis- tance to chloramphenicol, aminoglycoside and β-lactam antibiotics (Stover et al., 2000). This arsenal of resistance elements no doubt reflects the evolutionary history of these bacteria within their (often broad) environmental niches, where the ability to evade the activities of toxic molecules produced by a myriad of organisms offers a selective advantage. Such an advantage is perhaps not neces- sary for common human pathogens such as Staphylococci and Streptococci that have a more restricted host range, and in fact are often human commensals, and as a result are much more intrinsically drug-sensitive. Another hallmark of many bacteria is the ability to acquire genes via HGT (Barlow, 2009). Microbial genome sequences have revealed the remarkable extent of this phenomenon where the scars of HGT are recognizable in the presence of genes (and pseudogenes) encoding elements required for HGT (transposases, resolvases, etc.), which are often distributed throughout microbial genomes. Fre- quently, these are physically adjacent in the chromosome to resistance elements (reviewed in D’Costa et al., 2007). HGT can be a recent event as evidenced by the sequencing of the genome of Acinetobacter baumannii strain AYE that showed the acquisition of an 86 kb multi- resistance island containing 45 resistance genes that was absent in the wild-type A. baumannii strain SDF (Fournier et al., 2006). Environmental bacteria produce many antibiotics and other cytotoxic small molecules. The important question of whether these molecules are synthesized exclusively for their cytotoxic activity, that is, as chemical warfare, is receiv- ing increased scrutiny (Yim et al., 2006, 2007; Linares et al., 2006). Sub-lethal concentrations of many antibiotics have been found to have myriad effects on cellular processes (Yim et al., 2006; Davies et al., 2006;Tsui et al., 2004) and the actual roles of the so-called ‘antibiotics’ that are produced by bacteria are probably much more complex. Whatever their real functions in the ecology of producing bacteria, small molecules with cytotoxic bioactivities are intimately associated with resistance. For example, members of the order Actinomycetales are especially prolific in their ability to synthesize antibiotics. Of course, the capacity to produce antibiotics must co-evolve with resistance for producing organisms to avoid suicide (Cundliffe, 1989). As early as 1973, Benveniste and Davies recognized that resistance mechanisms in aminoglycoside antibiotic pro - ducers shared similarities with those found in pathogens. These similarities were extended in later work to include other antibiotics including glycopeptides such as vancomycin.

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0 ANTIBIOTIC RESISTANCE Vancomycin resistance emerged in enterococci in the late 1980s and was found to be the product of a five-gene cassette that included two regulatory genes and three enzyme encoding genes (Leclerq et al., 1988; Courvalin, 2006). This cassette results in antibiotic inducible reprogramming of intrinsic bacterial peptidoglycan biosynthesis to enrich cell walls with peptidoglycan terminating in D-Alanyl-D-Lactate in place of the canonical D-Alanyl-D-Alanine (Kahne et al., 2005). The latter is the recognition site for the glycopeptide antibiotic such as vancomycin while antibiotics do not bind to the former (Bugg et al., 1991). This complex resistance mechanism and gene cassette is found in glycopeptide antibiotic producers (Marshall et al., 1997, 1998) and non-producing environ - mental organisms (Hing et al., 2004). Recently, a variant of the van gene cassette has been characterized from the environmental anaerobe Desulfitobacterium halfniense demonstrating genetic variability of glycopeptide resistance (Kalan et al., 2009). The growing anecdotal evidence that actinomycetes were possible sources of antibiotic resistance elements that share similarity with those found in clinical pathogens prompted us to conduct a systematic study of these organisms from various soils and a survey of their antibiotic resistance profiles (D’Costa et al., 2006). A collection of 480 wild-type actinomycetes was screened against a panel of 21 antibiotics. These included natural products, their semi-synthetic derivatives and completely synthetic molecules. Resistance to all antibiotics was observed and on average each bacterial strain was resistant to seven to eight antibiotics. Analysis of the molecular basis of resistance identified modes of resistance that are shared with clinical pathogens as well as novel mechanisms that so far have not been detected in the clinic. For example, the van gene cluster was found in all five vancomycin resistant strains in the collection, demonstrating that this gene cassette is readily identifiable in the environment. On the other hand, a new mechanism of resistance to the semi-synthetic ketolide antibiotic, telithromycin, was characterized in one strain. Analysis of the inactive drug revealed that anti - biotic glucosylation was the mode of resistance (D’Costa et al., 2006). A subsequent study by Dantas et al. showed that in addition to resistance, it was possible to select environmental organisms that subsist on antibiotics (Dantas et al., 2008). The authors identified bacteria from diverse orders (not just acti - nomycetes) that used natural products antibiotics such as penicillin G and van - comycin or synthetic antibiotics including ciprofloxacin as sole carbon sources. A subsequent investigation by this group on resistance genes in the human gut and oral microbiomes revealed resistance genes in cultured bacteria and in the metagenome (Sommer et al., 2009). Resistance to a number of classes of drugs was widespread in the bacteria described in this study that looked at two unrelated healthy human subjects. Functional metagenomic analyses of soils by the Handelsman group has identified a number of resistance genes even in the so-called ‘pristine’ environ - ments not expected to have been exposed to antibiotics of human or agricultural

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 APPENDIX A origin (Allen et al., 2009; Handelsman, 2004). Another metagenomic study of activated sludge identified several bleomycin resistance genes (Mori et al., 2008). Other studies on microbes associated with insects (Allen et al., 2009; Kadavy et al., 2000), animals (Cloud-Hansen et al., 2007; Poeta et al., 2009, 2007; Gilliver et al., 1999) and birds (Bonnedahl et al., 2009; Sjolund et al., 2008; Poeta et al., 2008) demonstrate the wide distribution of microorganisms harboring resistance genes associated with wild animals. There is substantial variation in the frequency of resistance elements in wild animals, however, and there are increased numbers of resistant bacteria in animals that have contact with humans than those with little exposure (Thaller et al., 2010; Osterblad et al., 2001). Not surprisingly then, there is an even larger literature concerning antibiotic resistant organisms asso - ciated with farmed animals (Aarestrup et al., 2008a) including poultry (Gyles, 2008), swine (Aarestrup et al., 2008b), cattle (Call et al., 2008) and fish (Cabello, 2006) and often increases in resistant bacteria can be directly correlated with agricultural and aquacultural antibiotic use. Antibiotic exposure then can select low abundance resistant strains in the environment or select for HGT of resistance genes (Baquero et al., 2009). These studies and our growing understanding of natural product biosynthesis demonstrate that there is nothing particularly special about antibiotics as chemi - cals. These small molecules, like other primary and secondary metabolites, are part of the natural chemical ecology of the Earth. As such, numerous resistance mechanisms have evolved across microbial genera to either deal specifically with selected antibiotics or classes of antibiotics (for example, inactivating enzymes), or more generally respond to the presence of toxic small molecules, for example, through the expression of broad spectrum efflux proteins. This is analogous to acquired and innate immunity in higher organisms where the innate immune pro - cesses are deployed in response to general threats. This contrasts with acquired immunity through antibody production, which provides highly specific and robust immunity. The analogy breaks down, however, in that bacteria can often readily acquire high-level resistance through HGT. The fact that the vast majority of sequenced microbial genomes show evidence of HGT in addition to the presence of resistance genes concretely demonstrates the density of resistance elements in the environment. When considered in the context of a global bacterial population of 5 × 1030, the probability of the emergence of antibiotic resistance in clinically important pathogens becomes a virtual certainty. 5. The Clinical Resistome Most clinicians and medical microbiologists restrict their study of antibiotic resistance to clinically important pathogens for good reason. Understanding the mechanisms, dissemination and epidemiology of resistance in pathogenic bacteria is vital to drug use, management and discovery. As suggested above, unlike opportunistic pathogens of environmental origin the bacteria that often

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 ANTIBIOTIC RESISTANCE are associated with infection are (or at least were) largely antibiotic-sensitive. There is good evidence that the emergence of resistance in pathogens in many cases is the direct result of natural selection in the clinic. For example, a survey of the Murray collection of enterobacteria (433 stains) collected between 1917 and 1952 showed that while pre-antibiotic use bacterial isolates harbored conju - gatitive plasmids capable of HGT, none of them carried resistance genes (Hughes and Datta, 2003). In contrast, following the introduction of antibiotics in a single patient, in a clinical care setting, or across populations, increased prevalence of resistance is the norm and is predictable (e.g., Livermore, 2009; Grayson et al., 1991; Hawkey and Jones, 2009). Clinical resistance can be the result of selection for single mutants, for exam- ple, point mutations in the RNA polymerase gene rpoB that result in rifampin resistance, DNA gyrA and toposiomerase parC that confer fluroquinolone resis- tance, rpsL and streptomycin resistance. Such mutations usually diminish the pro- ductive binding of drug to target. Mutations that result in upregulation of genes can confer resistance and this is not uncommon with efflux mechanisms; alterna - tively, downregulation of transport proteins such as porins can result in resistance by blocking entry of the antibiotic into the cell. Acquisition of genes and their stable integration into the bacterial chromosome is another form of mutation that leads to resistance. The acquisition of the SCCmec cassette in MRSA is an example of this mechanism (deLencestre et al., 2007). Clonal dissemination of such strains results in resistant populations that can have geographic limits, for example, to distinct healthcare institutions or wards. In such cases, it is the founder strains that can dominate as a result of selective pressures from drug use. Genotyping of bacteria can identify lineages and help to map the natural history of the clinical resistome linked to a specific outbreak, for example. Alternatively, resistance elements can migrate between strains by HGT resulting in relatively rapid adaptation and radial dissemination of genes into several strains, each of which has the potential to be founders. Genotype analysis in this case can be difficult to interpret. HGT can occur through transformation, transduction or conjugation. Plasmid-mediated HGT has the potential to move resistance genes through microbial populations. It is clear that since the introduc- tion of antibiotics in the 1940s, plasmids have accumulated a greater number of resistance genes and often times these are on transposable elements that facilitate movement of genes into the chromosome (Barlow, 2009). The collection of resis- tance genes on mobile genetic elements such as a plasmid or transposon means that selection for resistance to one class of antibiotic can inadvertently result in co-selection for genes that confer resistance to other structurally distinct drugs. This mobility and co-selection make the clinical resistome a challenge to map and model. The source of the resistance genes found on these genetic elements is not known, but the growing understanding of the extent of the environmental resistome as discussed above suggests a strong link. The vast numbers of resis -

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 APPENDIX A tance genes and organisms in the environment are consistent with identifying it as the wellspring of much of the clinical resistome. The similarity of the vancomycin resistance gene cassette in environmental and pathogenic organisms is one exam- ple of a likely connection. Another is the link of the CTX-M extended spectrum β-lactamases that are prevalent in clinics across the globe and a reservoir in the environmental bacterium Kluyvera ascorbata (Humeniuk et al., 2002). 6. The Intrinsic Resistome Bacteria are not uniformly sensitive or resistant to antibiotics. The presence or absence of genes that contribute to resistance confer species- (and even strain)- specific built-in resistance to drugs. These genes comprise the intrinsic resistome (Breidenstein et al., 2008; Fajardo et al., 2008; Tamae et al., 2008). In some cases, these genes are readily recognizable as members of well-known resistance gene families. For example, Enterococcus faecalis are uniformly insensitive to lincos- amide and streptogramin antibiotics as a result of the presence of Lsa efflux pro - tein, which is characteristic of this species (Singh et al., 2002). As noted above, opportunistic pathogens such as P. aeruginosa encode a number of resistance genes, particularly efflux proteins that can provide broad drug insensitivity. In addition to such well-characterized elements, several systematic studies have revealed a network of genes that contribute to intrinsic resistance. Chemical– synthetic interaction studies have been particularly enlightening. In such experi - ments, a library of mutants is screened for sensitivity or resistance to an antibiotic at sub-lethal concentrations. Mutants that confer sensitivity to the antibiotic are candidates for the intrinsic resistome. These have potential as targets for new drugs that could potentiate the action of antibiotics. One caveat is that the nature of these studies excludes essential genes from being sampled and, therefore, could underestimate the extent of the intrinsic resistome. A screen of a P. aeruginosa PA14 transposon mutant library against sub- lethal concentrations of the fluroquinolone antibiotic ciprofloxacin identified 35 mutants with increased sensitivity to that antibiotic and 79 mutants with decreased sensitivity (Breidenstein et al., 2008). Genes linked to intrinsic resis - tance included expected efflux systems, but also non-obvious genes such as those involved in DNA repair and replication and the ClpX and ClpP proteases. The former has recently been identified as the target for the acyldepsipeptide antibiotics (Brotz-Oesterhelt et al., 2005) and this offers the possibility of syner- gistic combinations with fluoroquinolones. In a similar study, two P. aeruginosa transposon mutant libraries were screened against a panel of six antibiotics rep - resentative of distinct antibiotic classes (Fajardo et al., 2008). This study found that several mutants increased sensitivity to more than one antibiotic, suggesting a significant lack of discrimination by the genetic networks that protect the cell from toxic molecules. An analogous screen was reported in a transposon mutant library of Acinetobacter baylyi (Gomez and Neyfakh, 2006). This work studied

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 ANTIBIOTIC RESISTANCE the impact of 12 antibiotics at subinhibitory concentrations and identified 11 genes with chemical synthetic lethal phenotypes. Several genes were associated with efflux systems and cell wall metabolism; however, like the Pseudomonas screens described above, many genes were unrelated to known antibiotic targets of resistance elements. Finally, a systematic analysis of the susceptibility of ~4000 single-gene deletion strains of Escherichia coli (the Keio collection; Baba et al., 2006) ver- sus seven antibiotics identified 140 novel synthetic chemical lethal interactions (Tamae et al., 2008). This work was recently updated to cover 22 antibiotics further supporting the complexity of the intrinsic resistome genetic network and at the same time generating a distinctive sensitivity profile that is predictive of antibiotic classes (Liu et al., 2010). This cellular ‘bar code’ has the potential to be applied in antibiotic typing during drug discovery. 7. Expert Opinion The concept of the antibiotic resistome is a framework to understand the evolution, origins and genetic complexity of resistance. The majority of the past research on antibiotic resistance focused narrowly on the emergence of resistance in clinical pathogens and its epidemiology. The growing understanding of the molecular mechanisms of resistance along with knowledge of the 3D-structure of these elements, and the fact that many similar resistance genes are found in non-pathogenic organisms help to understand why resistance is so prevalent and emerges so rapidly after antibiotic deployment in the clinic. Furthermore, the resistome concept which reveals the remarkable depth of the gene pool to source resistance and the ease of HGT in bacterial populations explain why resistance-proof antibiotics are a fiction. What is lacking in the field is a thorough understanding of the precise mechanisms of HGT in the environment and more examples of unimpeachable evidence of recent HGT from environmental organ- ism to pathogens. A response to the pervasiveness of the resistome and inevitability of anti- biotic resistance can be despair. Certainly, the challenges of new antibiotic drug discovery are significant (Payne et al., 2007) and the fact that the resistome is so broad is a contributing factor. Nevertheless, antibiotics have proven to be miracle drugs and immensely profitable to the pharmaceutical sector over the past 6 decades despite the fact that the resistome was in existence during this time. There is a lot of room for optimism and understanding the resistome provides new opportunities for drug discovery field. First, traditional antibiotic discovery from natural and synthetic sources has proven to be successful and should continue. Screening of environmental organ - isms for resistance to candidate drugs early in the discovery process could help to identify protoresistance and bonafide resistance elements that may eventually emerge in the clinic. This can be used to make strategic decisions in lead opti -

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 APPENDIX A mization or between competing candidates in the preclinical discovery phase. Furthermore, it could identify resistance elements that may emerge in pathogens and thus provide opportunities for diagnostic tests to be used during clinical trials or post approval. Second, the resistome concept leads naturally to the consideration of combina- tions of drugs in antibacterial treatment. Combinations of antibiotics are common in infectious disease practice; however, there are relatively few formulated combina- tions (Synercid® [dalfopristin and quinupristin] and co-trimoxazole are exceptions). Combinations of antibiotics and inhibitors of antibiotic resistance enzymes are thus far limited to three β-lactamase inhibitors (clavulanic acid, sulbactam, tazobactam) formulated with a few penicillins. There is great opportunity to expand this rep- ertoire. In combination with molecular diagnostics that can identify the presence of specific resistance genes, these combinations could be a powerful anti-infective strategy. The revelation of the intrinsic resistome of bacteria presents a number of potential targets for inhibitors of non-essential gene products that could be used in combination with known antibiotics. Such combination strategies could extend the lifetimes of our existing collection of antibiotics for which we have ample under- standing of toxicology, pharmacology and so on. There are significant challenges to this approach including matching of pharmacological profiles of bioactive compounds in any formulated combination drug, not to mention regulatory hurdles and clinical trial design of any combi - nation. However, as the frequency of multidrug resistant bacterial pathogens increases in the healthcare sector and in the community, leveraging our growing understanding of the resistome through the use of drug combinations will become more attractive. Declaration of Interest Research in the author’s lab on antibiotic resistance is supported by a Canada Research Chair and the Canadian Institutes Health of Research and the Natural Sciences and Engineering Research Council. References Aarestrup FM, Oliver Duran C, Burch DG. Antimicrobial resistance in swine production. Anim Health Res Rev 2008b;9(2):135-48 Aarestrup FM, Wegener HC, Collignon P. Resistance in bacteria of the food chain: epidemiology and control strategies. Expert Rev Anti Infect Ther 2008a;6(5):733-50 Allen HK, Moe LA, Rodbumrer J, et al. Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil. ISME J 2009;3(2):243-51 Allen HK, Cloud-Hansen KA, Wolinski JM, et al. Resident microbiota of the gypsy moth midgut harbors antibiotic resistance determinants. DNA Cell Biol 2009;28:109-17 Baba T, Ara T, Hasegawa M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2006;2:2006-8

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 APPENDIX A Yim G, Wang HH, Davies J. Antibiotics as signalling molecules. Philos Trans R Soc Lond B Biol Sci 2007;362(1483):1195-200 Yim G, de la Cruz F, Spiegelman GB, Davies J. Transcription modulation of Salmonella enterica serovar Typhimurium promoters by sub-MIC levels of rifampin. J Bacteriol 2006;188(22):7988-91 Young PG, Walanj R, Lakshmi V, et al. The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2´´-phosphotransferase-IIa [APH(2´´)-IIa] provide in- sights into substrate selectivity in the APH(2´´) subfamily. J Bacteriol 2009;191(13):4133-43