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Institute of Medicine (US) Forum on Microbial Threats. The Science and Applications of Synthetic and Systems Biology: Workshop Summary. Washington (DC): National Academies Press (US); 2011.

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The Science and Applications of Synthetic and Systems Biology: Workshop Summary.

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A11NOVEL APPROACHES TO COMBAT BIOFILM DRUG TOLERANCE

60.

The Nature of the Threat

It is a given that new antibiotics are needed to combat drug-resistant pathogens. However, this is only part of the need—we actually never had antibiotics capable of eradicating an infection. Currently used antibiotics have been developed against rapidly growing bacteria, and most of them have no activity against stationary-state organisms, and none are effective against dormant persister cells. The relative effectiveness of antibiotics in treating disease is largely a result of a cooperation with the immune system, which mops up after antibiotics eliminate the bulk of a growing population. But the deficiency of existing antibiotics against supposedly drug-susceptible pathogens is becoming increasingly apparent with the rise of immunocompromised patients (HIV infected, undergoing chemotherapy) and the wide use of indwelling devices (catheters, prostheses, and heart valves), where the pathogen forms biofilms protecting cells from the components of the immune system. The ineffectiveness of the immune system leads to chronic diseases, which make up approximately half of all infectious disease cases in the developed world. The main culprit responsible for tolerance of pathogens to antibiotics are specialized survivors, persister cells (Lewis, 2007, 2010), which we examine in the following section.

Persisters

Persisters represent a small subpopulation of cells that spontaneously go into a dormant, nondividing state. When a population is treated with a bactericidal antibiotic, regular cells die, while persisters survive (Figure A11-1). In order to kill, antibiotics require active targets, which explains the tolerance of persisters. Taking samples and plating them for colony counts over time from a culture treated with antibiotic produces a biphasic pattern, with a distinct plateau of surviving persisters. By contrast, resistance mechanisms prevent antibiotics from binding to their targets (Figure A11-2).

A figure discussing persisters and bio films

FIGURE A11-1

Persisters and biofilms. Dose-dependent killing with a bactericidal antibiotic reveals a small subpopulation of tolerant cells, persisters, that are formed within a biofilm.

A diagram illustrating resistance and tolerance

FIGURE A11-2

Resistance and tolerance. Bactericidal antibiotics kill cells by forcing the active target to produce corrupted products. Persister proteins act by blocking the target, so no corrupted product can be produced. By contrast, all resistance mechanisms prevent (more...)

Infectious disease is often untreatable, even when caused by a pathogen that is not resistant to antibiotics. This is the essential paradox of chronic infections. In most cases, chronic infections are accompanied by the formation of biofilms, which seems to point to the source of the problem (Costerton et al., 1999; Del Pozo and Patel, 2007). Biofilms have been linked to dental disease, endocarditis, cystitis, urinary tract infection, deep-seated infections, indwelling device and catheter infections, and the incurable disease of cystic fibrosis. In the case of indwelling devices such as prostheses and heart valves, reoperation is the method of choice for treating the infection. Biofilms do not generally restrict penetration of antibiotics (Walters et al., 2003), but they do form a barrier for the larger components of the immune system (Jesaitis et al., 2003; Leid et al., 2002; Vuong et al., 2004). The presence of biofilm-specific resistance mechanisms was suggested to account for the recalcitrance of infectious diseases (Stewart and Costerton, 2001). However, the bulk of cells in the biofilm are actually highly susceptible to killing by antibiotics; only a small fraction of persisters remains alive (Spoering and Lewis, 2001). Based on these findings, we proposed a simple model of a relapsing chronic infection: antibiotics kill the majority of cells, and the immune system eliminates both regular cells and persisters from the bloodstream (Lewis, 2001) (Figure A11-3). The only remaining live cells are then persisters in the biofilm. Once the level of antibiotic drops, persisters repopulate the biofilm, and the infection relapses. While this is a plausible model, it is not the only one. A simpler possibility is that antibiotics fail to effectively reach at least some cells in vivo, resulting in a relapsing infection.

A model of relapsing bio film infection

FIGURE A11-3

A model of a relapsing biofilm infection. Regular cells (red) and persisters (blue) form in the biofilm and are shed off into surrounding tissue and the bloodstream. Antibiotics kill regular cells, and the immune system eliminates escaping persisters. (more...)

Establishing potential causality between persisters and therapy failure is not trivial, since these cells form a small subpopulation with a temporary phenotype, which precludes introducing them into an animal model of infection. We reasoned that causality can be tested based on what we know about selection for high persister (hip) mutants in vitro. Periodic application of high doses of bactericidal antibiotics leads to the selection of strains that produce increased levels of persisters (Moyed and Bertrand, 1983; Wolfson et al., 1990). This is precisely what happens in the course of treating chronic infections: the patient is periodically exposed to high doses of antibiotics, which may select for hip mutants. But hip mutants would only gain advantage if the drugs effectively reach, and kill, the regular cells of the pathogen.

Patients with cystic fibrosis (CF) are treated for decades for an incurable Pseudomonas aeruginosa infection to which they eventually succumb (Gibson et al., 2003). The periodic application of high doses of antibiotics provides some relief by decreasing the pathogen burden, but it does not clear the infection. If hip strains of pathogens were selected in vivo, they would most likely be present in a CF patient. We took advantage of a set of longitudinal P. aeruginosa isolates from a single patient, collected over the course of many years (Smith et al., 2006). Testing persister levels by monitoring survival after challenge with a high dose of ofloxacin showed a dramatic, 100-fold increase in surviving cells in the last four isolates (Mulcahy et al., 2010). Testing paired strains from additional patients showed that, in most cases, there was a considerable increase in persister levels in the late isolate from a patient. Interestingly, most of the hip isolates had no increase in minimum inhibitory concentration (MIC) compared to their clonal parent strain to ofloxacin, carbenicillin, and tobramycin, suggesting that classical acquired resistance plays little or no role in the recalcitrance of CF infection. These experiments directly link persisters to the clinical manifestation of the disease and suggest that persisters are responsible for the therapy failure of chronic CF infection. But why have the hip mutants with their striking survival phenotype evaded detection for such a long time?

The main focus of research in antimicrobials has been on drug resistance, and the basic starting experiment is to test a clinical isolate for its ability to grow in the presence of elevated levels of different antibiotics and to record any increases in the MIC. This is also the standard test employed by clinical microbiology laboratories. The hip mutants are of course missed by this test, which explains why they had remained undetected in spite of a major effort aimed at understanding pathogen survival to antimicrobial chemotherapy. Given that hip mutants are the likely main culprit responsible for morbidity and mortality of the CF infection, it makes sense to test for their presence. Testing for persister levels is not that much more difficult as compared to a MIC test.

Is selection for hip mutants a general feature of chronic infections? We recently examined patients with chronic oral thrush caused by Candida albicans (LaFleur et al., 2010). These were cancer patients undergoing chemotherapy, and suppression of the immune system caused the fungal infection. In patients where the disease did not resolve, the C. albicans isolates were almost invariably hip mutants, as compared to patients where the disease cleared within 3 weeks of treatment with chlorhexidine. The eukaryotic C. albicans forms persisters (Al-Dhaheri and Douglas, 2008; Harrison et al., 2007; LaFleur et al., 2006) through mechanisms that are probably analogous, rather than homologous, to that of their bacterial counterparts. Given the similar lifestyles of the unrelated P. aeruginosa and C. albicans, we may expect that the survival advantage of a hip mutation is universal. Just as multidrug resistance has become the prevalent danger in acute infections, multidrug tolerance of persisters and hip mutants may be the main, but largely overlooked, culprit of chronic infectious disease.

Biofilms apparently serve as a protective habitat for persisters (Harrison et al., 2005a,b, 2009; LaFleur et al., 2006; Spoering and Lewis, 2001), allowing them to evade the immune response. However, a more general paradigm is that persisters will be critical for pathogens to survive antimicrobial chemotherapy whenever the immune response is limited. Such cases would include disseminating infections in immunocompromised patients undergoing cancer chemotherapy or infected with HIV. Persisters are also likely to play an important role in immunocompetent individuals in cases where the pathogen is located at sites poorly accessible by components of the immune system. These include the central nervous system, where pathogens cause debilitating meningitis and brain abscesses (Honda and Warren, 2009), and the gastrointestinal tract, where hard-to-eradicate Helicobacter pylori causes gastroduodenal ulcers and gastric carcinoma (Peterson et al., 2000). Tuberculosis is perhaps the most prominent case of a chronic infection by a pathogen evading the immune system. The acute infection may resolve spontaneously or as a result of antimicrobial therapy, but the pathogen often remains in a “latent” form (Barry et al., 2009). It is estimated that one of every three people carry latent Mycobacterium tuberculosis, and 10 percent of carriers develop an acute infection at some stage in their lives. One simple possibility is that persisters are equivalent to the latent form of the pathogen. We recently isolated persisters from a growing culture of M. tuberculosis following lysis with d-cycloserine and found that these cells exhibit a general shutdown of biosynthetic processes, a hallmark of dormancy (Keren et al., 2011). The M. tuberculosis persisters also had several toxin-antitoxin modules overexpressed. These findings will enable the use of the “persister signature” to establish the equivalence of these cells and the dormant form of the pathogen in vivo.

The above analysis underscores the significance of drug tolerance as a barrier to effective antimicrobial chemotherapy. Given its significance—roughly half of all cases of infection (Figure A11-4)—the number of studies dedicated to tolerance is tiny compared to the number of publications on resistance. The difficulty in pinpointing the mechanism of biofilm recalcitrance and the formidable barriers to studying persister cells account for the lack of parity between these two comparably significant fields. Hopefully a better balance will be achieved, and the following discussion summarizes recent advances in understanding the mechanism of tolerance.

The two faces of recalcitrance

FIGURE A11-4

The two faces of recalcitrance. Drug resistance plays an important role in recalcitrance of acute infections, while drug tolerance is largely responsible for failures of chemotherapy in chronic infections. Tolerance allows the pathogen to survive for (more...)

Persisters were initially discovered in 1944, but the mechanism of their formation eluded us for a very long time. Only recently has the molecular mechanism of dormancy begun to emerge.

The most straightforward approach to finding an underlying mechanism of a complex function is by screening a library of transposon insertion mutants. This produces a set of candidate genes, and subsequent analysis leads to a pathway and a mechanism. This is indeed how the basic mechanisms of sporulation, flagellation, chemotaxis, virulence, and many other functions have been established. However, screening a Tn insertion library of Escherichia coli for an ability to tolerate high doses of antibiotics produced no mutants completely lacking persisters (Hu and Coates, 2005; Spoering, 2006). With the development of the complete, ordered E. coli gene knockout library by the Mori group (Baba et al., 2006) (the Keio collection), it seemed reasonable to revisit the screening approach. Indeed, there always remains a possibility that transposons missed a critical gene, or the library was not large enough. The use of the Keio collection largely resolves this uncertainty.

This advanced screen (Hansen et al., 2008), similarly to previous efforts, did not produce a single mutant lacking persisters, suggesting a high degree of redundancy. The screen did identify a number of interesting genes, with knockouts showing about a 10-fold decrease in persister formation. The majority of hits were in global regulators, DksA, DnaKJ, HupAB, and IhfAB. This is an independent indication of redundancy—a global regulator can affect expression of several persister genes simultaneously, resulting in a phenotype (Figure A11-5). The screen also produced two interesting candidate genes that may be more directly involved in persister formation: YgfA, which can inhibit nucleotide synthesis, and YigB, which may block metabolism by depleting the pool of flavin mononucleotide (FMN).

A diagram showing candidate persister genes

FIGURE A11-5

Candidate persister genes. Persisters are formed through parallel redundant pathways.

A similar screen of a P. aeruginosa mutant library was recently reported (De Groote et al., 2009). As in E. coli, no persisterless mutant was identified, pointing to the similar redundancy theme.

The main conclusion from the screens is that persister formation does not follow the main design theme of complex cellular functions—a single linear regulatory pathway controlling an execution mechanism. By contrast, persisters are apparently formed through a number of independent parallel mechanisms (Figure A11-5). There is a considerable adaptive advantage in this redundant design—no single compound will disable persister formation.

Screens for persister genes were useful in finding some possible candidate genes and pointing to redundancy of function. It seemed that a method better suited to uncover redundant elements would be transcriptome analysis. For this, persisters had to be isolated.

Persisters form a small and temporary population, making isolation challenging. The simplest approach is to lyse a population of growing cells with a β-lactam antibiotic and collect surviving persisters (Keren et al., 2004). This allows one to isolate enough E. coli cells to perform a transcriptome analysis. A more advanced method aimed at isolating native persisters was developed, based on a guess that these are dormant cells with diminished protein synthesis (Shah et al., 2006). If the strain expressed degradable green fluorescent protein (GFP), then cells that stochastically enter into dormancy will become dim. In a population of E. coli expressing degradable GFP under the control of a ribosomal promoter that is only active in dividing cells, a small number of cells indeed appeared to be dim. The difference in fluorescence allowed for the sorting of the two subpopulations. The dim cells were tolerant to ofloxacin, confirming that they are persisters.

Transcriptomes obtained by both methods pointed to downregulation of biosynthesis genes and indicated increased expression of several toxin-antitoxin (TA) modules (RelBE, MazEF, DinJYafQ, YgiU). TA modules are found on plasmids, where they constitute a maintenance mechanism (Gerdes et al., 1986b; Hayes, 2003). Typically, the toxin is a protein that inhibits an important cellular function, such as translation or replication, and forms an inactive complex with the antitoxin. The toxin is stable, while the antitoxin is degradable. If a daughter cell does not receive a plasmid after segregation, the antitoxin level decreases due to proteolysis, leaving a toxin that either kills the cell or inhibits propagation. TA modules are also commonly found on bacterial chromosomes, but their role is largely unknown. In E. coli, MazF and an unrelated toxin RelE induce stasis by cleaving mRNA, which of course inhibits translation, a condition that can be reversed by expression of corresponding antitoxins (Christensen and Gerdes, 2003; Pedersen et al., 2002). This property of toxins makes them excellent candidates for persister genes.

Ectopic expression of RelE (Keren et al., 2004) or MazF (Vazquez-Laslop et al., 2006) strongly increased tolerance to antibiotics. The first gene linked to persisters, hipA (Moyed and Bertrand, 1983), is also a toxin, and its ectopic expression causes multidrug tolerance as well (Correia et al., 2006; Falla and Chopra, 1998; Korch and Hill, 2006; Vazquez-Laslop et al., 2006). Interestingly, a bioinformatics analysis indicates that HipA is a member of the TOR family of kinases, which have been extensively studied in eukaryotes (Schmelzle and Hall, 2000) but have not been previously identified in bacteria. HipA is indeed a kinase; it autophosphorylates on ser150, and site-directed mutagenesis replaces it, or other conserved amino acids in the catalytic and Mg2+-binding sites abolish its ability to stop cell growth and confer drug tolerance (Correia et al., 2006). The crystal structure of HipA in complex with its antitoxin HipB was recently resolved, and a pull-down experiment showed that the substrate of HipA is elongation factor EF-Tu (Schumacher et al., 2009). Phosphorylated EF-Tu is inactive, which leads to a block in translation and dormancy (Figure A11-6).

An illustration of how the HipA toxin causes dormancy in E. coli by phosphorylating elongation factor Tu, which inhibits protein synthesis

FIGURE A11-6

The HipA toxin causes dormancy in E. coli by phosphorylating elongation factor Tu, which inhibits protein synthesis.

The deletion of potential candidates of persister genes noted above does not produce a discernible phenotype affecting persister production, possibly due to the high degree of redundancy of these elements. In E. coli, there are at least 15 TA modules (Alix and Blanc-Potard, 2009; Pandey and Gerdes, 2005; Pedersen and Gerdes, 1999) and there are more than 80 in M. tuberculosis (Ramage et al., 2009).

High redundancy of TA genes would explain the lack of a multidrug-tolerance phenotype in knockout mutants, and therefore it seemed useful to search for conditions where a particular toxin would be highly expressed in a wild-type strain and then examine a possible link to persister formation.

Several TA modules contain the Lex box and are induced by the SOS response. These are symER, hokE, yafN/yafO, and tisAB/istr1 (Courcelle et al., 2001; Fernandez De Henestrosa et al., 2000; Kawano et al., 2007; McKenzie et al., 2003; Motiejunaite et al., 2007; Pedersen and Gerdes, 1999; Singletary et al., 2009; Vogel et al., 2004). Fluoroquinolones induce the SOS response (Phillips et al., 1987), and we tested the ability of ciprofloxacin to induce persister formation (Dorr et al., 2009).

Examination of deletion strains showed that the level of persisters dropped dramatically, 10- to 100-fold, in a ΔtisAB mutant. This suggests that TisB was responsible for the formation of the majority of persisters under conditions of SOS induction. The level of persisters was unaffected in strains deleted in the other Lex box containing TA modules. Persister levels observed in time-dependent killing experiments with ampicillin or streptomycin that do not cause DNA damage were unchanged in the ΔtisAB strain. TisB only had a phenotype in the presence of a functional RecA protein, confirming the dependence on the SOS pathway.

Ectopic overexpression of tisB sharply increased the level of persisters. The drop in persisters in a deletion strain and the increase upon overexpression gives reasonable confidence in functionality of a persister gene. The dependence of TisB-induced persisters on a particular regulatory pathway, the SOS response, further strengthens the case for TisB as a specialized persister protein (Figure A11-7). Incidentally, a tisB mutant is not present in the otherwise fairly complete Keio knockout library, and the small open reading frame might have been easily missed by Tn mutagenesis as well, evading detection by the generalized screens for persister genes.

A diagram showing persister induction by antibiotic

FIGURE A11-7

Persister induction by antibiotic. The common antibiotic ciprofloxacin causes DNA damage by converting its targets, DNA gyrase and topoisomerase, into endonucleases. This activates the canonical SOS response, leading to increased expression of DNA repair (more...)

The role of TisB in persister formation is unexpected based on what we know about this type of protein. TisB is a small, 29 amino acid hydrophobic peptide that binds to the membrane and disrupts the proton motive force (pmf), which leads to a drop in ATP levels (Unoson and Wagner, 2008). Bacteria, plants, and animals all produce antimicrobial membrane-acting peptides (Garcia-Olmedo et al., 1998; Sahl and Bierbaum, 1998; Zasloff, 2002). Toxins of many TA loci found on plasmids belong to this type as well. If a daughter cell does not inherit a plasmid, the concentration of a labile antitoxin decreases, and the toxin such as the membrane-acting hok kills the cell (Gerdes et al., 1986a). High-level artificial overexpression of TisB also causes cell death (Unoson and Wagner, 2008). It is remarkable from this perspective that the membrane-acting TisB under conditions of natural (mild) expression has the exact opposite effect of protecting the cell from antibiotics.

Fluoroquinolones such as ciprofloxacin are widely used broad-spectrum antibiotics, and their ability to induce multidrug-tolerant cells is unexpected and a cause of considerable concern. The induction of persister formation by fluoroquinolones may contribute to the ineffectiveness of antibiotics in eradicating infections. Indeed, preexposure with a low dose of ciprofloxacin drastically increased tolerance to subsequent exposure with a high dose, and TisB persisters are multidrug tolerant.

The finding of the role of TisB in tolerance opens an intriguing possibility of a wider link between other stress responses and persister formation. Pathogens are exposed to many stress factors in the host environment apart from DNA damaging agents: oxidants, high temperature, low pH, and membrane-acting agents. It is possible that all stress responses induce the formation of surviving persisters.

While resistance and tolerance are mechanistically distinct, there is sufficient reason to believe that tolerance may be a major cause for developing resistance. Indeed, the probability of resistance development is proportional to the size of the pathogen population, and a lingering chronic infection that cannot be eradicated due to tolerance will go on to produce resistant mutants and strains acquiring resistant determinants by transmission from other bacteria (Levin and Rozen, 2006). Combating tolerance then becomes a major component in preventing resistance.

The Discovery Challenge

Source Compounds

The discovery of penicillin was an isolated event, but the development of screening for antimicrobial activity from soil actinomycetes by Salman Waxman produced the first, and also the only known, effective platform technology for antibiotic discovery (Schatz et al., 1944). Cultivable actinomycetes, however, are a limited resource; ~99 percent of microbes do not readily grow in the lab and are known as “uncultured” (Lewis et al., 2010). Overmining of actinomycetes by the early 1960s replaced discovery of novel compounds with rediscovery of knowns.

In response to the dwindling returns in natural product antibiotic discovery, the industry responded by focusing on synthetics. Indeed, a number of antimicrobials are synthetic (metronidazole, trimethoprim, isoniazid, ethionamide, pyrazinamide, and ethambutol), and there is one highly effective class of synthetic broad-spectrum antibiotics, the fluoroquinolones. Encouraged by these examples, and by dramatic advances in synthetic and combinatorial chemistry, high-throughput robotics, genomics, and proteomics, a new discovery platform emerged (Figure A11-8). Combinatorial chemistry provided a large number of test compounds, which were screened in high-throughput format against isolated essential target proteins determined by genomics. This platform, however, failed to produce a new class of broad-spectrum antibiotics, leading to the closure of anti-infectives divisions in many of the Big Pharma companies. The main reasons for failure is well understood: high-throughput screening hits were literally running into the penetration barrier of Gram-negative bacteria, which is made of transenvelope multidrug resistance (MDR) pumps that extrude amphipathic compounds across the outer membrane barrier (Lomovskaya et al., 2008). Drugs have to be amphipathic in order to penetrate across the hydrophobic inner membrane, but this is precisely the feature that the outer membrane restricts and the MDRs recognize. There are few compounds that pass this seemingly impenetrable barrier rather effectively: the broad-spectrum aminoglycosides, tetracyclines, fluoroquinolones, some β-lactams, chloramphenicol, and azithromycin. Fluoroquinolones are the only synthetics on this list, and they were discovered 50 years ago.

The high-tech platform

FIGURE A11-8

The high-tech platform. Early leads from screening compound libraries are extruded by transenvelope MDR pumps of Gram-negative species.

But what about less challenging narrow-spectrums, with good activity against at least Gram-positive species? Seventy high-throughput screens performed by GlaxoSmithKline, for example, against a large number of targets produced no viable leads (Payne et al., 2007). Glaxo scientists realized that penetration is a serious problem, and therefore also performed in vivo screens against E. coli, but only obtained “nuisance” hits, such as membrane-acting compounds. One obvious conclusion from this negative experience is that the libraries do not carry good starting compounds.

In part this is due to the fact that libraries are constructed based on Lipinski rules (Lipinski, 2003), which are good for predicting druglike properties for compounds acting against mammalian cell targets but do not work well for bacteria because of peculiarities of permeation (O’Shea and Moser, 2008; Silver, 2008). Another important consideration is the probability of resistance development. Pathogen populations produce 109 cells in an infected patient, which means that the probability of resistance development should be <109. This is readily achieved with most of the antibiotic classes currently in use, since they hit more than one target (fluoroquinolones attack DNA gyrase and topoisomerase, β-lactams inhibit several penicillin-binding proteins, and ribosomal inhibitors bind to rRNA which is coded by multiple genes) (Silver, 2007). This requirement severely limits the number of realistic targets for antimicrobial drug discovery.

The above analysis presents an extremely bleak picture; if we cannot even discover compounds acting against rapidly growing Gram-positive bacteria, what are the prospects of finding broad-spectrum antimicrobials acting against non-growing stationary cells and persisters?

Opportunities

There are many steps in the drug discovery pipeline, but if there are no viable leads, there is no pipeline. Indeed, at the last Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) meeting (2009), there was not a single broad-spectrum lead presented. This means that the number of realistic broad-spectrum leads in the global antimicrobial drug discovery pipeline is zero. This is where the process needs to be restarted, and this is where allocation of resources will make a tangible impact.

A Fresh Look at Potential Sources of Compounds

Natural products There are two largely untapped and potentially enormous new sources of natural products: uncultured microorganisms and silent operons coding for secondary metabolites.

A recent resurgence in cultivation efforts aimed at gaining access to uncultured microorganisms has been sparked by the vast diversity of uncultured bacterial groups revealed by environmental surveys of 16S rRNA (Aoi et al., 2009; Bollmann et al., 2007; Bruns et al., 2002; Connon and Giovannoni, 2002; Davis et al., 2005; Ferrari et al., 2005; Gavrish et al., 2008; Kaeberlein et al., 2002; Nichols et al., 2008; Rappe et al., 2002; Stevenson et al., 2004; Zengler et al., 2002). While some novel bacterial species were successfully cultured by varying media and growth conditions (Joseph et al., 2003), significant departures from conventional techniques were clearly in order, and indeed the new technologies substantially diverged from traditional cultivation methods by adopting single-cell and high-throughput strategies (Connon and Giovannoni, 2002; Nichols et al., 2008; Rappe et al., 2002; Zengler et al., 2002), better mimicking the natural milieu (Aoi et al., 2009; Bruns et al., 2002; Ferrari et al., 2005; Stevenson et al., 2004), increasing the length of incubation, and lowering the concentration of nutrients (Davis et al., 2005). High-throughput extinction culturing is based on the dilution of natural communities of bacteria to 1 to 10 cells per well in low-nutrient, filtered marine water. This strategy resulted in the cultivation of the first member of the ubiquitous, previously uncultured clade, SAR11 (Rappe et al., 2002). Our research group contributed to the effort by developing three cultivation methodologies (Gavrish et al., 2008; Kaeberlein et al., 2002; Nichols et al., 2008). All three strategies aim to provide microorganisms with their natural growth conditions by incubating them in simulated natural environments.

The diffusion chamber is designed to essentially “trick” cells into thinking they are growing in their natural environment by creating an incubation strategy that very closely mimics their natural habitat (Kaeberlein et al., 2002). The diffusion chamber consists of a stainless steel washer and 0.03-μm pore-size membranes (Figure A11-9). After gluing a membrane to one side of the washer, the inoculum (a mix of environmental cells and warm agar) is introduced, and the second membrane seals the chamber. Nutrients from the environment can diffuse into the chamber; therefore, it is not necessary to add them to the medium. Once inoculated and assembled, the chamber can be returned to the original location of sampling or in a simulated natural environment such as a block of sediment kept in an aquarium in lab. Microcolonies grow in the chamber during such incubation. A recovery rate of 22 percent on average was observed in the diffusion chambers. In this study and follow-up research (Bollmann et al., 2007; Nichols et al., 2008) we isolated numerous species that did not grow in Petri dishes inoculated with environmental samples but were successfully grown in the diffusion chambers.

A diffusion chamber for growing bacteria in situ

FIGURE A11-9

A diffusion chamber for growing bacteria in situ. A sample from marine sediment is diluted, mixed with agar, and sandwiched between the two semipermeable membranes of the diffusion chamber, which is returned to the environment.

Reinoculation of material both from marine and soil environments from chamber to chamber produces “domesticated” variants that grow on regular media on a Petri dish and can be exploited for secondary metabolite production (Bollmann et al., 2007; Nichols et al., 2008).

The diffusion chamber typically produces a mixed culture, which requires considerable time to isolate, purify, and reinoculate individual colonies. In order to streamline this process into a high-throughput system, we developed a variant of the diffusion chamber for massively parallel microbial isolation. The Isolation Chip, or ichip for short (Nichols et al., 2010), consists of hundreds of miniature diffusion chambers that can be loaded with an average of one cell per chamber. The ichip enables microbial growth and isolation in a single step with hundreds of individual cultures incubating on a single chip.

Microorganisms that are particularly important for drug discovery, microscopic fungi and actinomycetes, grow by forming filaments capable of penetrating soft substrates. Since actinomycetes can pass through 0.2-μm pores, we reasoned this could be used to design a trap for the specific capture of these organisms (Gavrish et al., 2008). The trap is similar in design to the diffusion chamber, except the membranes have larger pores and the agar inside the trap is initially sterile when placed in the environment. Any growth observed afterward inside the trap is due to the movement of cells into the trap during incubation. The majority of organisms grown in the traps proved to be actinomycetes, some of which represented rare and unusual species from the genera Dactylosporangium, Catellatospora, Catenulispora, Lentzea, and Streptacidiphilus.

We noticed that some organisms forming colonies in the diffusion chamber can grow on a Petri dish, but only in the presence of other species from the same environment (Kaeberlein et al., 2002; Nichols et al., 2008) and suggested that uncultured bacteria only commit to division in a familiar environment, which they recognize by the presence of growth factors released by their neighbors. In order to assess the commonality of the growth dependence of uncultured organisms on neighboring species and pick good models for study, we chose an environment where bacteria live in a tightly packed community (D’Onofrio et al., 2010). This is a biofilm that envelopes sand particles of a tidal ocean beach (Figure A11-10). There were disproportionately more colonies appearing on densely inoculated plates compared with more dilute plates. This indicated that some of the cells that grew on the densely seeded plates were receiving growth factors from neighboring colonies. To test the possible growth dependence of microorganisms on neighboring species, pairs of colonies growing within a short distance of each other were restreaked in close proximity to each other. Potential uncultured isolates were identified by their diminishing growth with increasing distance from the cultivable “helper” strain on the cross-streak plates. Colonies of the culturable organism Micrococcus luteus KLE1011 (a marine sand sediment isolate 99.5 percent identical to M. luteus DSM 200030T according to 16S rRNA gene sequence) grew larger as their distance from other colonies increased (Figure A11-10). Approximately 100 randomly picked pairs of colonies were re-streaked from the high-density plates, and 10 percent of these pairs showed this pattern of growth induction on cross-streaked plates.

A series of images for understanding the mechanism of uncultivability

FIGURE A11-10

Understanding the mechanism of uncultivability. Marine sand particles are covered by a multispecies biofilm (top). Cells from the biofilm form colonies on a densely seeded plate, and pairing them together reveals that some of them are uncultured bacteria (more...)

In order to isolate growth factors, spent medium from the helper M. luteus KLE1011 was tested and shown to induce growth of the uncultured M. polysiphoniae KLE1104. An assay-guided fractionation led to isolation and structure determination of five different siderophores and each of them was able to induce growth of M. polysiphoniae KLE1104. This demonstrated that siderophores represent the growth factors responsible for the helping activity. The siderophores consisted of a central core with alternating N-hydroxycadaverine and succinic acid units and were of the desferrioxamine class (Challis, 2005). Both close relatives of known microorganisms and novel species were isolated by this approach. This study identified the first class of growth factors for uncultured bacteria and suggests that additional ones will come from analyzing organisms growing in co-culture.

Silent operons Whole-genome sequencing of several actinomycetes showed that there are many more potential biosynthetic pathways for production of secondary metabolites than there are known antibiotics made by these organisms (Ikeda et al., 2003). Ecopia used fermentation in 40 different media to entice production of additional compounds and discovered a novel type of enediyne with anticancer activity (Zazopoulos et al., 2003). No novel antimicrobials emerged from this effort. However, in order to be effective, one needs to develop a high-throughput approach to induce production of such compounds. This is entirely doable.

Synthetics Are existing libraries, both commercially available and proprietary collections in Big Pharma, useless for antibiotic discovery? It does seem so, since they have obviously already been screened for actives, including nonbiased screens for growth inhibition of whole cells, and produced no viable leads. But does it not seem strange that a screen of a collection of 600 dyes by Domagk produced the first viable antibiotic, while a screen of the total global library of ~107 compounds produced nothing at all? As the libraries grew, a number of innovations were introduced aimed at improving the screening outcome; thus we have in vitro screening, targeted screens, Lipinski rules, and specificity validation. My feeling is that each time we tried to improve things, the result was to discard valuable compounds. I think that the existing libraries do harbor useful molecules; the question is how to identify them.

Good Compounds from Bad Libraries

Back to Domagk The first screen was also perfect: Domagk tested compounds against mice infected with streptococci. The result was the discovery of prontosil, a sulfa drug that has no activity in vitro. The compound is cleaved in the intestine by gut bacteria, releasing the active sulfonamide moiety, which inhibits dehydropteroate synthase in the folate pathway. An in vitro test would have missed prontosil. There are obvious advantages to testing compounds in situ—this automatically eliminates the significant burden of toxic molecules and demonstrates efficacy, again automatically eliminating substances with problems of action in an animal, such as serum binding, instability, or poor tissue distribution. In addition, different types of compounds may be uniquely uncovered, such as those requiring activation in situ and those hitting targets that are only important in an infection but not in vitro. While this would theoretically be the perfect way to go, testing in 107 mice is not an option for a variety of reasons, including ethical considerations and the large amounts of required test compound. We therefore considered a useful intermediate between in vitro and a mammal—an animal that, unlike mice, can be dispersed in microtiter wells. Caenorhabditis elegans can be infected with human pathogens by simply ingesting them, and we found that the worm can be cured by common antibiotics such as tetracycline and vancomycin, and at concentrations typically achieved in human plasma (Moy et al., 2006). Worms infected with a pathogen such as Enterococcus feacalis die, stop moving, their shape changes from curved to straight, and they can be detected by typical eukaryotic vital dyes (Figure A11-11). Using these parameters, an automated approach was developed, and a large pilot screen of compound libraries uncovered hits, some of which had no activity in vitro (Moy et al., 2006, 2009). This approach shows that C. elegans points us in the right direction—back to Domagk, but with larger libraries.

A high-throughput screen for antimicrobials in an animal model

FIGURE A11-11

A high-throughput screen for antimicrobials in an animal model. C. elegans are infected with E. faecalis, and cured with ampicillin. This provides for an assay of compounds that cure the worm in situ.

Better libraries and rules of penetration Of course it would be great to have a better library, constructed based not on Lipinski rules but on “rules of penetration.” We have a small number of broad-spectrum compounds that are able to largely bypass the MDRs and get across the impermeable barrier of Gram-negative membranes—tetracycline, chloramphenicol, aminoglycosides, trimethoprim, β-lactams (these only need to traverse the outer membrane), fluoroquinolones, and metronidazole. This set is too small to enable us to discern rules for penetration. But testing a large number of unbiased compounds from a library for their ability to enter into the cytoplasm of Gram-negative bacteria should allow us to deduce general rules that favor penetration. Once these are available, this would drive the synthesis and combinatorial chemistry of new compound libraries specifically geared toward antimicrobial discovery.

Prodrugs It is useful to consider the theoretically perfect antibiotic from first principles and then decide whether it is realistic. Approaches we discussed so far do not address the daunting challenge of killing persister cells while at the same time showing broad-spectrum activity. It is useful to start with the end result: a highly reactive compound will kill all cells, including persisters. In order to spare the host, the compound must be delivered as a prodrug, and then a bacteria-specific enzyme will activate it into a generally reactive molecule which will covalently bind to unrelated targets. Importantly, this mechanism creates an irreversible sink, largely resolving the issue of MDR efflux, so the antimicrobial is automatically broad spectrum. Is this realistic? Several existing antimicrobials closely match the properties of this idealized prodrug antibiotic. These are isoniazid, pyrazinamide, ethionamide, and metronidazole. The first three are anti-Mtb drugs, while metronidazole is a broad-spectrum compound acting against anaerobic bacteria. All four compounds convert into active antiseptic-type molecules inside the cell that covalently bind to their targets. It seems to be no accident that prodrug antibiotics make up the core of the anti-Mtb drug arsenal, since an ability to kill the pathogen is critical for treating the disease. Preferred targets have been identified for isoniazid and ethionamide (Vilcheze et al., 2005), suggesting a relatively limited reactivity of these compounds. The existence of preferred targets indicates that the prodrug products are not that reactive, and there is considerable room for developing better sterilizing antibiotics based on the same principle.

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60

Antimicrobial Discovery Center and the Department of Biology, Northeastern University, Boston, MA, USA. E-mail: ude.uen@siwel.k, http://www​.biology.neu​.edu/faculty03/lewis03.html.

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