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4 Antibiotic Resistance: Origins and Countermeasures OVERVIEW From Joshua Lederberg’s appreciation of the microbial world’s immense and fluid genetic resources came his recognition that humans, despite their domin- ion over “higher” forms of life, remain prey to microscopic predators. After a few decades in which it appeared that human ingenuity, in the form of anti- biotics, had outwitted the pathogens—but during which Lederberg and others warned of our distinct disadvantage in an escalating “arms race” with infectious microbes—antibiotic-resistant bacterial strains, or “superbugs,” have now become ubiquitous. As Stanley Cohen of Stanford University observes in his contribution to this chapter, “It seems quite remarkable that despite the enormous progress made in the treatment of infectious disease during Joshua Lederberg’s lifetime, the ominous microbial threat discussed by Lederberg on multiple occasions con- tinues.” The papers collected in this chapter explore the evolutionary origins of the antibiotic resistance phenomenon, take its measure as a present and future threat to public health, and propose scientific approaches to addressing it, includ- ing investigating environmental reservoirs of antibiotic resistance, identifying sources of novel antibiotics, and developing alternatives to conventional antibiotic therapies. In the chapter’s first paper, workshop speaker Julian Davies, of the Univer- sity of British Columbia, reviews the history of the development of antibiotic resistance, beginning in the early twentieth century. “Although we have gained considerable understanding of the biochemical and genetic bases of antibiotic resistance,” he writes, “we have failed dismally to control the development of antibiotic resistance, or to stop its transfer among bacterial strains.” This fail- 

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 ANTIBIOTIC RESISTANCE ure is perhaps understandable given the ubiquity of resistance genes in the environment—a vast collection of genes referred to by Davies and others as the “resistome”—and the many opportunities for genetic exchange that both nature and man have availed microbes. Thus, as Davies notes, “any drug usage, no mat- ter how well controlled, inevitably leads to the selection of multidrug-resistant pathogens.” Davies describes the spectrum of resistance mechanisms and places them in an evolutionary context, from their function in “virgin” (antibiotic-free) environ- ments to their potential for transmission and reassortment with other resistance elements in human-created environments, such as wastewater treatment systems. He also considers the function of the natural bioactive compounds from which many clinical antibiotics are derived and, in particular, the low-dose effects of natural antibiotics, which appear to differ significantly from the therapeutic effects of clinical antibiotics administered at high doses. In the next contribution to this chapter, Cohen contends that antibiotic resis- tance has become a global public health threat “largely as a consequence of the conventional approach used to treat infections, which is to attack the pathogen in the hope that the host will not be harmed by the drug.” He proposes a different approach to dealing with microbial pathogens: by interfering with the cooperative relationship that many of them have with their hosts. Host cells, he notes, furnish many invading pathogens with genes and gene products necessary for pathogen propagation and transmission. Cohen describes the strategy by which he and coworkers have identified several such pathogen-exploited host genes and discusses their prospects as therapeutic targets. “There is hope that devising antimicrobial therapies that target host cell genes exploited by pathogens, rather than—or in addition to—targeting the pathogens themselves may help in the effort to thwart the microbial threat,” he concludes. However, he also acknowledges that it remains to be determined whether such host-oriented therapies will, as expected, be less vulnerable to cir- cumvention by pathogen mutations. The final paper in this chapter, by Jo Handelsman of the University of Wisconsin, describes research in her laboratory on two topics relevant to antibi- otic resistance: the use of metagenomics to discover (potentially transferrable) resistance functions in soil bacteria; and the potential for manipulating endog- enous microbial communities so that they will defend their hosts against patho- gens, thus eliminating the need for antibiotic therapy. In order to understand the process of host invasion from the perspective of the commensal microbes involved in such events, Handelsman and coworkers are characterizing the composition, dynamics, and functions of model endogenous communities. Their studies of the microbial community of the gypsy moth gut have yielded intriguing evidence that commensal bacteria interact in ways that can influence host health, sometimes in surprising ways—including collaborating with invaders to kill their hosts. Handelsman’s group has also found that treat-

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0 MICROBIAL EVOLUTION AND CO-ADAPTATION ment with antibiotics alters the response of their model microbial community to pathogens. These findings and observations have led to further inquiries into the nature of community robustness and the genetic attributes of invading microbes that permit them to overcome a robust community. ANTIBIOTIC RESISTANCE AND THE FUTURE OF ANTIBIOTICS Julian Davies, Ph.D.1 University of British Columbia Naturally occurring small molecules, and their synthetic and semisynthetic derivatives, have been used as the foundation of infectious disease therapy since the late 1930s. Following the introduction of the “wonder drugs” penicillin and streptomycin, dozens of novel and derived bioactive compounds have been developed and used for the treatment of microbial maladies in humans, animals, and plants. Figure 4-1 shows a brief history of antibiotic development from the pre-antibiotic era to the present. The use of these important therapeutic agents for nonhuman applications, such as animal feed additives, started in the early 1950s and has expanded enormously. More than half of the total annual production vol- umes of all antimicrobials today are employed for nontherapeutic use as growth promotants and prophylactics in the food animal and aquaculture industries and for many other agricultural purposes. In 2000, more than 25 million pounds of antibiotics were manufactured in the United States alone. Over a half century, this equates to about 1 million metric tons! Considering the fact that Russia, China, and India each currently produces more antibiotics than the United States, the amounts of these compounds made worldwide is very significant.2 From an ecological point of view, the dispersion of these bioactive compounds into the environment has created increasing pressure for the selection of antibiotic-resistant microbes throughout the entire biosphere. This selection pressure is clearly most crucial in specific therapeutic situations, such as hospitals and their associated intensive care units, but it is evident that antibiotic-resistant bacteria are now ubiquitous! The increasing use of antibiot- ics, since the 1950s, for both appropriate and inappropriate applications, has led almost simultaneously to increases in antibiotic-resistant bacteria, and the spec- trum of resistance determinants has grown correspondingly. Little wonder that antibiotic resistance is a continuing and major threat concomitant with efforts to cure human disease. A series of epidemics of resistant organisms have marked the antibiotic era: penicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus 1 Emeritus professor of microbiology and immunology. 2Accurate production figures are difficult to obtain.

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 ANTIBIOTIC RESISTANCE GRAMICIDIN (Peptide) PENICILLIN (b-lactam) CIPROFLOXACIN STREPTOMYCIN (aminoglycoside) (fluoroquinolone) CEPHALOSPORIN (b-lactam) LINEZOLID RIFAMYCIN SALVARSAN (oxazolidinone) (ansamycin) (arsenical) b-lactamase ? 1903 1932 1940 1950 1960 1962 1982 2000 2003 PRONTOSIL DAPTOMYCIN NALIDIXIC ACID (sulfonamide) (lipopeptide) (quinolone) THE GOLDEN AGE CHLORAMPHENICOL CHLORTETRACYCLINE POLYMIXIN (lipopeptide) ERYTHROMYCIN (macrolide) VANCOMYCIN (glycopeptide) VIRGINIAMYCIN (streptogramin) FIGURE 4-1 Major classes of antimicrobials and the year of their discovery. Figure 4-1.eps redrawn (MRSA),3 vancomycin-intermediate Staphylococcus aureus (VISA),4 aureus drug-resistant Vibrio cholerae, multidrug-resistant (MDR)5 and extensively drug- resistant (XDR)6 Mycobacterium tuberculosis (hereinafter, MDR- and XDR-TB), CTX-M resistant Escherichia coli and Klebsiella pneumoniae, Clostridium dif- 3 MRSA is a type of S. aureus that is resistant to antibiotics called β-lactams. β-lactam antibiotics include methicillin and other more common antibiotics such as oxacillin, penicillin, and amoxicillin (for more information, see http://www.cdc.gov/ncidod/dhqp/ar_MRSA_ca_public.html#2). 4VISA and vancomycin-resistant S. aureus (VRSA) are specific types of antimicrobial-resistant staphylococcal bacteria. Although most staphylococci are susceptible to the antimicrobial agent vancomycin, some have developed resistance to vancomycin. Infections caused by VISA and VRSA isolates cannot be successfully treated with vancomycin because these organisms are no longer responsive to vancomycin. However, to date, all VISA and VRSA isolates have been susceptible to other Food and Drug Administration (FDA)-approved drugs (for more information, see http://www. cdc.gov/ncidod/dhqp/ar_visavrsa_FAQ.html). 5 MDR-TB is tuberculosis that is resistant to at least two of the best anti-tuberculosis drugs, isonia- zid and rifampin. These drugs are considered first-line drugs and are used to treat all individuals with tuberculosis (for more information, see http://www.cdc.gov/tb/pubs/tbfactsheets/mdrtb.htm). 6 XDR-TB is a relatively rare type of MDR-TB. XDR-TB is defined an M. tuberculosis isolate that is resistant to isoniazid and rifampin plus any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin; for more information see http://www. cdc.gov/tb/pubs/tbfactsheets/mdrtb.htm).

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 MICROBIAL EVOLUTION AND CO-ADAPTATION ficile, and many others. Reports of new outbreaks of these so-called “superbugs” in the popular press are regular events. The CTX-M family of extended-spectrum β-lactamases is of particular interest and concern. These enzymes inactivate the extended-spectrum (third- generation) cephalosporins of the cefotaxime class that were first introduced for the treatment of infections caused by gram-negative organisms in the 1990s. The increasing use of these antibiotics led to the appearance of resistant strains in several countries. As indicated by the frequent reports of the increasing preva- lence of extended-spectrum β-lactamases, this resistance mechanism is now endemic in hospitals and the community throughout the world (Livermore et al., 2007; Queenan and Bush, 2007). Moreover, families of CTX-M enzymes that differ in amino acid sequence and catalytic activity have been reported in almost every country. Isolates of pathogenic Enterobacteriaceae carrying these resistance determinants, in particular K. pneumoniae, are essentially untreatable. In a number of hospitals and among certain populations, such as the military, the appearance of multidrug-resistant Acinetobacter baumannii isolates carrying a CTX-M enzyme has been of particular concern: this is an emerging pathogen capable of heightened mortality and morbidity (Peleg et al., 2008). Antibiotic-resistant infections are commonplace. Human and nonhuman use of antimicrobial agents has guaranteed this status quo; bacteria and other microbes evolve rapidly to adapt to diverse environments and novel stresses and remain alive. Nonetheless, in industrialized nations “old” antibiotics, penicillin and the sulfonamides, are effective most of the time treating in routine outpatient. Until recently the pharmaceutical industry has survived by developing novel agents (usually by synthetic chemical modification of existing compounds) in order to counter new types of infections; this continuing arms race has permitted modern medicine to keep up, in some measure, with bacterial genetics. Occa- sionally, structurally novel compounds have been isolated that offer a narrow window of therapeutic success, but this has become less common and there is no panacea yet. Given the seriousness of the current situation, a number of well-considered proposals have been mooted with the objective of controlling resistance develop- ment and restoring and maintaining the efficacy of treatments against infectious diseases (Norrby et al., 2005; Spellberg et al., 2008). But any drug usage, no matter how well controlled, inevitably leads to the selection of drug-resistant pathogens. Antibiotic Resistance in the Environment Since the first report of a penicillin-destroying enzyme (penicillinase) in a bacterial strain (Abraham and Chain, 1940), antibiotic resistance traits have been found in many environmental bacteria isolates (Table 4-1). It is generally believed that pristine environments represent the major reser-

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 ANTIBIOTIC RESISTANCE TABLE 4-1 Reports on Antibiotic Resistance Genes Isolated from the Environment Year Report Identification of β-lactamases in soil actinomycetes 1972 1974 Identification of aminoglycoside-modifying enzymes in soil bacteria 1988 Identification of Citrobacter spp. and Kluyvera spp. as origins of extended-spectrum β-lactamases 2001 Identification of gyrA allelism in soil isolates that provides such isolates with “natural” fluoroquinolone resistance 2004 Identification of resistance genes in the soil metagenome 2006 Identification of the environmental “resistome” that conveys multidrug resistance in soil isolates 2006 Identification of the “intrinsic” resistome of pathogens (gene knockouts) 2008 Identification of the environmental “subsistome”—a population of bacteria that degrades antibiotics voir of resistance genes to be acquired by bacterial pathogens. The identification of the “resistome” expanded the range of antibiotic resistance determinants and soil-derived actinomycete strains involved (D’Costa et al., 2006). More recently, the demonstration that a proportion of the microbes in the environment are capa- ble of metabolizing antibiotics provided additional evidence for the existence of enzymatic mechanisms for the biodegradation of antibiotics that enable bacteria to subsist on antibiotic substrates (the “subsistome”); this work has revealed the presence of a great diversity of antibiotic-modifying enzymes in nature, and these enzymes provide the basis for a wide range of antibiotic resistance mechanisms (Dantas et al., 2008). In addition, it has been shown that bacterial genomes pos- sess a significant number of genes that, if over-expressed in other hosts, would be candidate antibiotic resistance genes (intrinsic resistance; Tamae et al., 2008). In these latter cases, it has not yet been demonstrated that these genes are bona fide resistance genes; however, they have the potential to be. The existence of a universe of latent resistance genes in pathogens, commensals, and environmental organisms provides further evidence that resistance genes are common in nature; resistance is everywhere genotypically, if not phenotypically. In an ideal world the identification of a new antibiotic resistance phenotype might provide the basis for “early-warning” measures in clinical situations and could guide the development of preventive measures that should be taken to avoid dissemination of the determinant. However, this is difficult to achieve in practice, given that resistance genes may spread rapidly and become well established in the population before they are detected definitively. In addition, it is debatable how much of the environmental and intrinsic resistance is significant in clinical terms. The strategy certainly could have worked in the case of CTX-M determinants, since their putative source was first identified in environmental Kluyvera spp. and later in enteric pathogens.

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 MICROBIAL EVOLUTION AND CO-ADAPTATION TABLE 4-2 Mechanisms of Resistance, 2008a • • Decreased influxb Increased efflux • • Enzymatic inactivation Sequestration • • Target modificationb Target by-pass • • Target amplificationb Target repair/protection • • Biofilm formationb Intracellular localization aAll of the mechanisms are acquired by horizontal gene transfer. bAlso acquired by mutation. As has been known since the first use of antibiotics in research and the clinic, random mutation (spontaneous or induced) gives rise to antibiotic resis- tance. The mutations can occur in the genes encoding drug targets, drug export, or other mechanisms (Table 4-2). These mutations (usually point mutations) are pleiotropic;7 it could be argued, conversely, that resistant mutants are fre- quently the result of point mutations selected by nutritional or other pressures. Indeed, the pleiotropic effects may influence a wide variety of other functions. Even resistant strains that are detected in the environment (i.e., the resistome or subsistome) may, in reality, be the result of multifunctional activities, such as nutritional balances. Experiments performed in our laboratory demonstrate that antibiotic-resistant mutants may have multiple phenotypes that could act as means of resistance selection depending on the nutrients available in the environ- ment. Which came first? Cryptic resistance genes that are present in bacteria or bacterial popula- tions may be revealed by metagenomic cloning and expression (Riesenfeld et al., 2004), although it will be necessary to employ a wide range of expression hosts, in addition to E. coli, to identify functional resistance cloned from diverse bacterial genera. We do not yet know the mechanisms for gene “pick-up” from environmental sources, nor in what way such genes are “tailored” for efficient expression in heterologous hosts, such as bacterial pathogens. Environmental bacteria vary greatly in their G+C8 content and codon usage; converting the trans- ferred genes into functional genes in new hosts must require extensive “tailoring” by mutation and recombination. Native promoters need to be altered to permit transcription and translation in the new hosts. A good example of the way in which “new” genes may be acquired and expressed is provided by the omnipres- ent integron-mediated acquisition and expression system. In principle, any open reading frame can be inserted as a cassette into an attachment site associated with an integron recombinase and become a functional gene (Figure 4-2). The integron cassette promoter is strong and very effective in a variety of 7 Producing more than one effect; having multiple phenotypic expressions (see http://www.merriam- webster.com/dictionary/pleiotropic). 8 guanine-cytosine.

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 ANTIBIOTIC RESISTANCE Pant intI1 sul1 qacE ∆ integrase 3'- conserved segment attI attC R gene cassette Int Pant intI1 attI attC qacE ∆ R gene sul1 FIGURE 4-2 Integron mechanism of gene capture. Integron-mediated gene capture and the 4-2 COLOR.eps model for cassette exchange. Outline of the process by which circular antibiotic resistance gene cassettes (antR) are repeatedly inserted at the specific attI site in a class 1 integron downstream of the strong promoter Pc.intl, integrase-encoding gene; Int = integrase Intl1. SOURCE: Patrice Courvalin, Institut Pasteur, Paris, France. microbial hosts. However, the acquisition process is not well understood, and essentially all aspects of the origins and evolution of antibiotic resistance genes remain unsolved. Integron-encoded gene cassettes are widespread in all natural environments (marine and terrestrial), with the vast majority of such genes being of unknown function (Koenig et al., 2008). Although primarily of gram-negative bacterial origin, there have been reports of resistance integrons in gram-positive bacteria (Nandi et al., 2004). Gene exchange between bacterial genera is an evolutionary axiom, but it is still necessary to solve the dilemma of access to the resistance gene sources. Antibiotic Resistance in Urban Environments Urban areas, constantly exposed to the large variety of antibiotics that are commonly used in the hospital and the community, have considerable reservoirs of resistance. In large cities, thousands of people receive antibiotic treatment every day (e.g., in hospitals, in nursing homes, and at home), and accordingly,

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 MICROBIAL EVOLUTION AND CO-ADAPTATION many antibiotics are released as part of the waste stream of the sanitary sewer system into wastewater. The “hotspots” are generally considered to be hospital- associated, but given that the main disposal route is through sewers, it is very likely that wastewater treatment plants (WWTPs) are also hotspots, with signifi- cant concentrations of antibiotic-resistant microbes containing multiple resistance genes from a wide variety of antibiotics and myriad potential vectors. That WWTPs provide ideal environments for gene exchange and gene acqui- sition has been confirmed by studies of antibiotic resistance plasmids isolated from WWTP bacterial cultures (Tennstedt et al., 2005). Sequencing of purified plasmid DNAs demonstrated that very complex genomes are formed, as evi- denced by the combinations of resistance genes, transfer factors, transposases, integrons and their associated integrases, bacteriophage remnants, and other potential mobile elements. Thus, a considerable level of genetic mixing and matching occurs, with the consequence that novel combinations of antibiotic resistance determinants may be discharged into WTTP effluents (Schlüter et al., 2007). It is also possible that virulence genes in the form of pathogenicity islands are acquired by plasmids. While the roles of such newly formed resistance elements may be a matter of speculation there is absolutely no doubt that they are produced, and could contribute to, the gene pool responsible for increasing antibiotic resistance in the human community. Antibiotic Hormesis Antibiotics have long been known to have multiple effects on target cells. For many years, clinicians and a growing number of microbiologists and biochemists have reported that sub-inhibitory concentrations of commonly used antibiotics may affect microbial cell growth, morphology, structure, adhesion, virulence, and a variety of other phenotypes (Davies et al., 2006). In the majority of studies the changes induced were advantageous to the microbes. For example, a number of so-called antibiotics induce the formation of biofilms that permit microbial communities to survive under adverse conditions; others may promote swarm- ing and motility, perhaps enhancing nutrient accessibility; even protein synthe- sis inhibitors may cause changes in cell-wall structure and function. Recently, detailed studies of transcriptional and proteomic changes (rather than phenotypic ones) have confirmed the wide range of responses of microbes to bioactive small molecules. Almost all antibiotics have side effects that in many cases require careful dosage monitoring; these effects often occur at sub-inhibitory concentrations. The recent demonstration that these compounds exert the phenomenon of hormesis 9 9 Hormesis defines a dose-response activity of antibiotics (and other agents). It usually refers to a positive (beneficial) effect at low concentrations and a negative (toxic or inhibitory) effect at higher concentrations (Yim et al., 2006).

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 ANTIBIOTIC RESISTANCE provides an explanation for the dual activities of microbially-derived natural products (Davies, 2006). The differences are due to transcriptional effects that are concentration-dependent. At low concentrations, antibiotics cause strong stimula- tion of transcription from specific groups of promoters. At higher concentrations (near-inhibitory), the transcription patterns change, as illustrated in Figure 4-3. Such dose-response dependence has been demonstrated by the use of promoter- reporter constructs or microarray analyses in the laboratory. We have proposed that these hormetic effects of antibiotics account for the differences in activity of low-molecular-weight compounds in their natural environment (soils, etc.) compared to their role in the treatment of infectious diseases. In pristine environments, complex microbial populations can be assumed to exist in some form of homeostatic equilibrium that change as a result of fluctua- tions in nutritional resources. Stability in the population is probably maintained by cell-to-cell signaling that modulates their metabolic activity. When soil iso- lates of bacteria producing useful bioactive compounds (such as antibiotics) are Figure 4-3.eps FIGURE 4-3 Concentration dependence of transcription modulation by antibiotics. MIC = minimal inhibitory concentrations; SMs = small molecules. bitmap image SOURCE: Adapted from Yim et al. (2006).

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 MICROBIAL EVOLUTION AND CO-ADAPTATION transplanted to a completely different environment (a laboratory), and exposed to potent mutagens and fermentation on unusual substrates to very high densi- ties under artificial conditions, the yields of small molecules are often amplified considerably, generating sufficiently large amounts of the desired compounds to permit their testing as antibiotics at elevated (inhibitory) concentrations. Antibiotics and Other Anthropomorphisms The microbiological literature is spiced with comments such as “the bacteria have to make a decision” or “the microbe has made a choice.” These statements are patently ridiculous. Microbes are genetically programmed to respond to differ- ent external environments; they do not make choices. Perhaps the most pervasive of such comments refers to the activity of microbially-produced small molecules. Selman Waksman’s seminal work on the discovery of potent compounds such as neomycin and streptomycin led him to define them as antibiotics. He later real- ized that this definition was based on laboratory studies of soil microbes, grown in media containing exotic substrates and tested for their activities against human pathogens, not environmental bacteria. Waksman (1961) subsequently made a different judgment: The existence of microbes that have the capacity to produce antibiotics in artificial culture cannot be interpreted as signifying that such phenomena are important in controlling microbial populations in nature. . . . Unless one accepts the argument that laboratory environments are natural, one is forced to conclude that antibiotics play no part in modifying or influencing living processes that normally occur in nature. We may disagree with his conclusion, in the light of current knowledge, but must concur with the distinction between the laboratory and complex natural environments. Another “misinterpretation” is that all of these compounds kill bacteria. On the contrary, it is well known that most of the antimicrobial agents used thera- peutically do not kill other bacteria—they only inhibit the growth of the target organism. This is an important aspect of antimicrobial therapy: the drug retards or inhibits bacterial growth and virulence and allows the human immune system to eliminate the weakened pathogens.10 The Global Microbiome Bacteria are the most abundant living organisms on this planet and, given that they are essential to the maintenance of all other living organisms, they are 10 For this reason, treatment with compounds with a cidal action is favored for patients who are immunosuppressed or in cases of critical infections caused by highly virulent organisms.

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 MICROBIAL EVOLUTION AND CO-ADAPTATION resistance genes from cultured organisms; we think these genes may confer resis- tance through a regulatory function. One of the most interesting clones that we obtained from the Alaskan soil encodes the first known example of a hybrid β-lactamase: two different kinds of β-lactamases fused into a single protein. One end confers resistance to the penicillin-like compounds, and the other end confers resistance to the cephalosporin-like compounds; together, they confer very broad resistance to β-lactam antibiotics (Allen et al., 2009). This is a gene that we hope to never see in clinically important pathogens, but based on what we know about the transfer of antibiotic resistance determinants, this is entirely possible. We should, there- fore, continue to examine and anticipate the broad range of antibiotic resistance genes in our environment. Essential in this exploration is that we are mindful of antibiotic resistance in communities and that we account for both culturable and nonculturable members of those communities since either type of organism could provide an important source of resistance to human pathogens. Phalanx or Traitors? The second part of this essay concerns the nature of the microbial com- munities that comprise the normal gastrointestinal flora of animals. Do these commensal communities protect their hosts from disease, or do they encourage the disease process? My group is interested in commensal gut communities for a variety of rea- sons. We know from many studies18 in mammals, as well as in invertebrates, that the normal gut flora influences host physiology. There is evidence that endogenous microbial communities contribute to obesity, diabetes, and high cholesterol (Ley et al., 2006); there is also evidence that the microbiota is essential to the health of the host. We also know that most pathogens live as commensals and then—under the right conditions—they become invasive, and thereby pathogenic. We are very interested in that switch, because if we want to use microbial communities to protect us from disease (e.g., through the use of probiotics or through the delib- erate manipulation of microbial community dynamics), we need to understand how the opposite sometimes happens. We study the role of microorganisms in communities that inhabit the midguts of gypsy moth and cabbage white butterfly larvae. These are good model systems, as the larvae are easy to rear and dissect and their gut microbial communities can be readily and naturally manipulated via host feeding. Moreover, the intestinal epithelium of these insects is surprisingly similar to that of humans and other mammals, in terms of both its anatomy and 18 See Bregman and Kirsner (1965), Fleming et al. (1986), Hirayama and Rafter (1999), Hooper (2004), Hooper and Gordon (2001), Hooper et al. (2001, 2002), Johannsen et al. (1990), Krueger et al. (1982), Ley et al. (2006), and Rath et al. (1996).

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 ANTIBIOTIC RESISTANCE immunological function. Using the larval model systems, we have explored the role of microbial communities in health and disease of the host. Who Is There? We use both culture-based and culture-independent methods to examine the composition of communities. We find uncultured organisms that are readily recognizable as members of phylogenetic groups that have many culturable mem- bers, but these particular organisms cannot themselves be cultured, for largely unknown reasons. Table 4-3 shows the diversity of bacterial phylotypes that we have identified in the gypsy moth system. Most of these are members of two phyla: the Proteo- bacteria and the Firmicutes. Over time and under different feeding regimes, we have observed changes in community composition at the species level, but rarely at the phylum level. Are Signals Exchanged in the Gut Community? Many disease processes induced by bacteria are dependent on microbial com- munication through small molecules. Some bacterial species use a mechanism, called quorum sensing, to gauge the density of their populations (Engebrecht and Silverman, 1984; Fuqua et al., 1994; Miller and Bassler, 2001). Our first question was: Does quorum sensing occur in the larval midgut? Quorum sensing is mediated by small molecules called homoserine lac- tones. Some thought it was impossible that these molecules—and therefore quo- TABLE 4-3 Phylogeny of Cultured and Uncultured Bacteria from Third Instar Gypsy Moth Midguts Feeding on an Artificial Diet Phylotype Division Genus Species 1a low G+C gram positive Enterococcus spp. E. faecalis 2a low G+C gram positive Staphylococcus spp. S. lentus 3a low G+C gram positive Staphylococcus spp. S. cohnii 4a low G+C gram positive Staphylococcus spp. S. xylosus γ-Proteobacterium 5a Enterobacter spp. γ-Proteobacterium 6a Pseudomonas P. putida γ-Proteobacterium 7a Pantoea spp. P. agglomerans 8b low G+C gram positive Enterococcus spp. γ-Proteobacterium 9b Enterobacter spp. a-Proteobacterium 10b Agrobacterium spp. aCultured. bUncultured. SOURCE: Adapted from Broderick et al. (2004).

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 MICROBIAL EVOLUTION AND CO-ADAPTATION rum sensing—could be viable in the lepidopteran (moth and butterfly) midgut, because it is an extremely alkaline environment, typically reaching pH 10 to 12, a pH expected to destroy lactones. Nevertheless, we found that quorum sensing did indeed occur in the gut. To determine whether a molecular signal was being exchanged between cells in the bacterial community, we used a simple luminescence-based reporter system, described in Figure 4-6. This reporter system enabled us to visualize the impact of homoserine lactone signal exchange among cells within the larval gut. We transformed bacterial cells in the midgut with biosensors that emit light only if they receive quorum-sensing signals sent by another cell. We detected lumines- cence in whole, living, larvae: clear evidence that the members of the gut bacterial community are communicating with each other in the highly alkaline gut. What is the biological role of these signals in the insect midgut? We asked whether quorum-sensing signals that are required for Pseudomonas aeruginosa infection in other systems are also required in the cabbage white larval gut system; as Figure 4-7 illustrates, the answer was a resounding “yes.” We then demonstrated that we could chemically inhibit the larval gut quorum-sensing system using a known quorum-sensing inhibitor synthesized in the laboratory of our colleague at the University of Wisconsin, Helen Blackwell. Both approaches indicated that quorum-sensing is required for the virulence of P. aeruginosa in the larval gut. Furthermore, these results suggest that the gut microbes are, in fact, acting as a community and are not merely coexisting. Image Min = −1.8561e+05 Max = 84991 p/sec/cm 2 /sr 10,000 9,000 8,000 7,000 6,000 5,000 4,000 FIGURE 4-6 Detection of quorum-sensing activity and signal exchange in the guts of Figure 4-6 COLOR.eps cabbage white butterfly larvae. Bioluminescence was detected in the individual guts of larvae fed Pantoea pSB401image w/ antoea mixed with Pantoea panI::Tn pSB401 bitmap (top row), P vector type elements (middle row) and Pantoea panlI::TN pSB401 (bottom row). SOURCE: Borlee et al. (2008).

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 ANTIBIOTIC RESISTANCE 100 90 P. aeruginosa P. aeruginosa + Indole 80 P. aeruginosa signal No treatment 70 Mortality (%) 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 Days After Inoculation FIGURE 4-7 Mortality of cabbage white butterfly larvae fed P. aeruginosa strains and the Figure 4-7.eps quorum-sensing analog indole inhibitor. Treatments include P. aeruginosa PAO1, P. aeru- ginosa PAO1 and indole inhibitor, P. aeruginosa PAO1-JP2 (lasI rhlI N-acyl-L-lomoserine lactone-deficient mutant), and no P. aeruginosa PAO1 control. Values represent the mean mortality as a percentage of 18 larvae per treatment replicated in five independent experi- ments. Error bars are the standard errors. SOURCE: Borlee et al. (2008). Does the Community Affect the Health of the Host? Bacillus thuringiensis (Bt) is a well-characterized pathogen of gypsy moth and, in fact, of all members of the order Lepidoptera. The bacterium produces rhomboid protein crystals that are highly toxic only to lepidopterans. The Bt toxin is known to resemble bacterial pore-forming toxins that affect mammals; thus, we are interested in exploring the interaction of Bt, its toxin, and the microbial community in which they function as a model for mammalian gut disease. Bt has been used widely for the last 50 years to control insect pests that can- not be deterred or eradicated by other means, and in organic agriculture in lieu of synthetic pesticides. Interestingly, despite this broad usage, resistance to Bt has not developed in the field. Over the last decade, crop plants have been genetically engineered to express the Bt toxin gene and cultivated according to strict guide- lines designed to prevent the development of resistance to the toxin. Although Bt toxin-expressing crops are now grown on a massive scale in the United States, resistance has not become a problem. Why this is the case, even though antibiotic resistance is rampant, is not known.

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 MICROBIAL EVOLUTION AND CO-ADAPTATION Bt was discovered nearly 100 years ago, and both the bacterium and the interaction between its toxin and the cells of the lepidopteran gut epithelium— where the toxin forms pores—have been very well studied. However, the pathway by which pore formation (which damages the intestinal epithelium) leads to the animal’s death is not known. It has been assumed that either bacteremia caused by Bt or starvation (because pore formation is associated with reduced feeding) is the proximal cause of death. Neither of those putative mechanisms satisfied one of my graduate students, Nichole Broderick, whose research had shown that some compounds known to enhance insects’ sensitivity to Bt toxin also affect the bacterial community of the gut. As a result, she developed the hypothesis that the gut microbial community acts as a protection—a phalanx—against infection by Bt, and she predicted that eliminating the gut bacteria would enhance Bt activity. To test this hypothesis, we treated insects with increasing concentrations of an antibiotic cocktail that we knew would kill all of the bacteria that we could detect in the gut (Broderick et al., 2006). However, antibiotic treatment did not enhance Bt killing; instead, the more antibiotic the larvae received in their diet, the greater the larval survival following exposure to Bt (Figure 4-8). That result FIGURE 4-8 Gypsy moth larvae reared on antibiotics are not susceptible to Bt. The vertical line indicates the concentration of antibiotic at which no bacteria are detected in the midgut. IU = internationalFigure 4-8 COLOR.eps units. SOURCE: Broderick et al. (2006). bitmap image

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 ANTIBIOTIC RESISTANCE FIGURE 4-9 Restoration of B. thuringiensis toxicity by an Enterobacter spp. after elimi- nation of the detectable gut flora and B. thuringiensis activity by antibiotics. Lymantria Figure 4-9.eps dispar larvae were reared until the third instar on a sterile artificial diet amended with bitmap image 500 μg each of penicillin, gentamicin, rifampin, and streptomycin per milliliter (antibiotic cocktail). Each bar represents the mean mortality ± the standard error of the mean for 48 larvae (four replications with 12 larvae each). Values at the bottom represent the sizes of the populations of the Enterobacter spp. as detected by culture. nd = not detected. SOURCE: Broderick et al. (2006). led us to the traitor hypothesis: if Bt is inactive in the absence of the endogenous microbial community, then perhaps community members actually collaborate with Bt in a multispecies infection. If so, one would predict that if we introduced members of the gut bacterial community into antibiotic-treated larvae that lacked a gut flora and were resistant to Bt, sensitivity to Bt would be restored. We tested this hypothesis by rearing larvae on high levels of the antibiotic cocktail (Figure 4-9), letting the antibiotic clear, then feeding the larvae with Enterobacter, a gram-negative bacterium that we have found consistently in the gypsy moth gut; we then assessed the susceptibility of these larvae to Bt

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 MICROBIAL EVOLUTION AND CO-ADAPTATION (Broderick et al., 2004). In control experiments with larvae raised without anti - biotics, Bt and Bt plus Enterobacter both kill nearly 100 percent of the larvae (Broderick et al., 2006). Antibiotics reduced killing by Bt, and the addition of Enterobacter prior to Bt exposure restored the killing. This suggests that Entero- bacter and Bt work together to kill the insects. Conclusion Our work with the lepidopteran gut system is at an early stage. We have learned that the microbial assemblage it contains is relatively simple, compris- ing two phyla that are also found among the human gut microbiota. Chemical signals are exchanged between bacterial cells in the gut, and when this signaling process is inhibited chemically or genetically, the pathogenesis of P. aeruginosa is attenuated. The microbial community affects host health. When the pathogen Bt is intro- duced, the normally benign gut microbiota mediate pathogenesis. In the absence of the normal gut microbiota, Bt does not induce killing and reintroduction of normal gut residents restores killing. This system might provide a model for studying the common phenomenon that commensal microbes can act as patho- gens under the right conditions. The insect gut system and the soil metagenomic analysis of antibiotic resis- tance genes both reveal the importance of accounting for communities in the analysis of microbial behavior and genetic potential in biological systems. REFERENCES Davies References Abraham, E. P., and E. Chain. 1940. An enzyme from bacteria able to destroy penicillin. Nature 146(3713):837. Bäckhed, F., R. E. Ley, J. L. Sonnenburg, D. A. Peterson, and J. I. Gordon. 2005. Host-bacterial mutualism in the human intestine. Science 307(5717):1915-1920. Bartoloni, A., L. Pallecchi, H. Rodriguez, C. Fernandez, A. Mantella, F. Bartalesi, M. Strohmeyer, C. Kristiansson, E. Gotuzzo, F. Paradisi, and G. M. Rossolini. 2009. Antibiotic resistance in a very remote Amazonas community. International Journal of Antimicrobial Agents 33(2):125-129. Dantas, G., M. O. A. Sommer, R. D. Oluwasegun, and G. M. Church. 2008. Bacteria subsisting on antibiotics. Science 320(5872):100-103. Davies, J. 2006. Are antibiotics naturally antibiotics? Journal of Industrial Microbiology and Bio- technology 33(7):496-499. Davies, J., G. B. Spiegelman, and G. Yim. 2006. The world of subinhibitory antibiotic concentrations. Current Opinion in Microbiology 9(5):1-9. D’Costa, V. M., K. M. McGrann, D. W. Hughes, and G. D. Wright. 2006. Sampling the antibiotic resistome. Science 311(5759):374-377. Gardner, P., D. H. Smith, H. Beer, and R. C. Moellering, Jr. 1969. Recovery of resistance (R) factors from a drug-free community. Lancet 2(7624):774-776.

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