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1.  Diarylacylhydrazones: Clostridium-Selective Antibacterials with Activity Against Stationary-Phase Cells 
Current antibiotics for treating Clostridium difficile infections (CDI), i.e. metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This paper reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and C. perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure-activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.
doi:10.1016/j.bmcl.2013.12.015
PMCID: PMC3912389  PMID: 24360560
Clostridium difficile; antibacterial; protonophore; diarylacylhydrazone; CCCP; stationary phase cells
2.  In Vitro and In Vivo Activities of HPi1, a Selective Antimicrobial against Helicobacter pylori 
A high-throughput screen (HTS) was performed to identify molecules specifically active against Helicobacter pylori, the causative agent of peptic ulcer and gastric carcinoma. Currently, treatment of H. pylori infection is suboptimal, with failure rates approaching 25%, despite triple therapy with two broad-spectrum antibiotics and a proton pump inhibitor or quadruple therapy with added bismuth. The HTS was performed in 384-well plates, and reduction of the metabolic indicator resazurin was used as a reporter for cell growth. Diverse molecules from commercial sources were identified as hits, and in vitro validations included measurements of MIC and time-dependent killing as well as anaerobic susceptibility testing against a panel of gut microbes. In vivo validation included testing in the mouse model of H. pylori infection. The small molecule HPi1 (3-hydrazinoquinoxaline-2-thiol) had excellent potency, with an MIC of 0.08 to 0.16 μg/ml and good selectivity for H. pylori compared to a panel of commensal bacteria. HPi1 was also effective in a mouse model of H. pylori infection, reducing colony counts to below the limit of detection after oral dosing of 25 mg/kg/day for 3 days. HPi1 is a promising lead in the search for more effective and specific H. pylori therapeutics.
doi:10.1128/AAC.02573-13
PMCID: PMC4068456  PMID: 24687512
3.  Tackling antibiotic resistance 
Nature reviews. Microbiology  2011;9(12):894-896.
The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable, but can nevertheless be controlled and must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of thirty scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, May 16-18, 2011. From these discussions emerged a priority list of steps that need to be taken to resolve this global crisis.
doi:10.1038/nrmicro2693
PMCID: PMC4206945  PMID: 22048738
4.  A Screen for and Validation of Prodrug Antimicrobials 
The rise of resistant pathogens and chronic infections tolerant to antibiotics presents an unmet need for novel antimicrobial compounds. Identifying broad-spectrum leads is challenging due to the effective penetration barrier of Gram-negative bacteria, formed by an outer membrane restricting amphipathic compounds, and multidrug resistance (MDR) pumps. In chronic infections, pathogens are shielded from the immune system by biofilms or host cells, and dormant persisters tolerant to antibiotics are responsible for recalcitrance to chemotherapy with conventional antibiotics. We reasoned that the dual need for broad-spectrum and sterilizing compounds could be met by developing prodrugs that are activated by bacterium-specific enzymes and that these generally reactive compounds could kill persisters and accumulate over time due to irreversible binding to targets. We report the development of a screen for prodrugs, based on identifying compounds that nonspecifically inhibit reduction of the viability dye alamarBlue, and then eliminate generally toxic compounds by testing for cytotoxicity. A large pilot of 55,000 compounds against Escherichia coli produced 20 hits, 3 of which were further examined. One compound, ADC111, is an analog of a known nitrofuran prodrug nitrofurantoin, and its activity depends on the presence of activating enzymes nitroreductases. ADC112 is an analog of another known antimicrobial tilbroquinol with unknown mechanism of action, and ADC113 does not belong to an approved class. All three compounds had a good spectrum and showed good to excellent activity against persister cells in biofilm and stationary cultures. These results suggest that screening for overlooked prodrugs may present a viable platform for antimicrobial discovery.
doi:10.1128/AAC.02136-13
PMCID: PMC3957891  PMID: 24342644
5.  Potentiation of Azole Antifungals by 2-Adamantanamine 
Azoles are among the most successful classes of antifungals. They act by inhibiting α-14 lanosterol demethylase in the ergosterol biosynthesis pathway. Oropharyngeal candidiasis (OPC) occurs in about 90% of HIV-infected individuals, and 4 to 5% are refractory to current therapies, including azoles, due to the formation of resistant biofilms produced in the course of OPC. We reasoned that compounds affecting a different target may potentiate azoles to produce increased killing and an antibiofilm therapeutic. 2-Adamantanamine (AC17) was identified in a screen for compounds potentiating the action of miconazole against biofilms of Candida albicans. AC17, a close structural analog to the antiviral amantadine, did not affect the viability of C. albicans but caused the normally fungistatic azoles to become fungicidal. Transcriptome analysis of cells treated with AC17 revealed that the ergosterol and filamentation pathways were affected. Indeed, cells exposed to AC17 had decreased ergosterol contents and were unable to invade agar. In vivo, the combination of AC17 and fluconazole produced a significant reduction in fungal tissue burden in a guinea pig model of cutaneous candidiasis, while each treatment alone did not have a significant effect. The combination of fluconazole and AC17 also showed improved efficacy (P value of 0.018) compared to fluconazole alone when fungal lesions were evaluated. AC17 is a promising lead in the search for more effective antifungal therapeutics.
doi:10.1128/AAC.00294-13
PMCID: PMC3719723  PMID: 23689724
6.  Staphylococcus aureus persisters tolerant to bactericidal antibiotics 
Bacterial persister cells are non- or slow growing reversible phenotypic variants of the wild type, tolerant to bactericidal antibiotics. We here analyzed Staphylococcus aureus persister levels by monitoring colony forming unit (CFU) counts of planktonically grown cells treated with six different antimicrobials over time. Model laboratory strains HG001-HG003, SA113 and small colony variant (SCV) strains hemB and menD were challenged by the compounds at different logs of minimal inhibitory concentration (MIC) in exponential or stationary growth phase. Antibiotic tolerance was usually elevated in SCV strains compared to normally growing cells and in stationary vs. exponential phase cultures. Biphasic killing kinetics, typical for persister cell enrichment, were observed in both growth phases under different selective conditions. Treatment of exponential phase cultures of HG001-HG003 with 10-fold MIC of tobramycin resulted in the isolation of persisters which upon cultivation on plates formed either normal or phenotypically stable small colonies. Trajectories of different killing curves indicated physiological heterogeneity within persister subpopulations. Daptomycin added at 100-fold MIC to stationary phase SA113 cells rapidly isolated very robust persisters. Fractions of antibiotic tolerant cells were observed with all S. aureus strains and mutants tested. Our results refute the hypothesis that S. aureus stationary phase cells are equivalent to persisters, as not all of these cells showed antibiotic tolerance. Isolation of S. aureus persisters of different robustness seems to dependent on the kind and concentration of the antibiotic, as well as on the strain used.
doi:10.1159/000342449
PMCID: PMC3518770  PMID: 22986269
persister cell; multi drug tolerance; antibiotics; antimicrobials; Staphylococcus aureus
7.  Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers 
FEBS letters  2012;586(16):2529-2534.
We studied membrane activity of the bacterial peptide TisB involved in persister cell formation. TisB and its analogs form multi-state ion-conductive pores in planar lipid bilayers with all states of pores displaying similar anionic selectivity. TisB analogs differing by ±1 elementary charges show corresponding changes in selectivity. Probing TisB pores with poly-(ethylene glycol)s reveals only restricted partitioning even for the smallest polymers, suggesting that the pores are characterized by a relatively small diameter. These findings allow us to suggest that TisB forms clusters of narrow pores that are essential for its mechanism of action.
doi:10.1016/j.febslet.2012.06.021
PMCID: PMC3498054  PMID: 22728134
bacteria multi-drug tolerance; toxin/antitoxin system; multi-level conductance; cluster pore structure
8.  Role of Oxidative Stress in Persister Tolerance 
Persisters are dormant phenotypic variants of regular cells that are tolerant to antibiotics and play an important role in recalcitrance of chronic infections to therapy. Persisters can be produced stochastically in a population untreated with antibiotics. At the same time, a deterministic component of persister formation has also been documented in a population of cells with DNA damaged by fluoroquinolone treatment. Expression of the SOS response under these conditions induces formation of persisters by increasing expression of the TisB toxin. This suggests that other stress responses may also contribute to persister formation. Of particular interest is oxidative stress that pathogens encounter during infection. Activated macrophages produce reactive oxygen and nitrogen species which induce the SoxRS and OxyR regulons. Genes controlled by these regulons deactivate the oxidants and promote repair. We examined the ability of oxidative stress induced by paraquat (PQ) to affect persister formation. Preincubation of cells with PQ produced a dramatic increase in the number of persisters surviving challenge with fluoroquinolone antibiotics. PQ did not affect killing by kanamycin or ampicillin. Persisters in a culture treated with PQ that survived a challenge with a fluoroquinolone were also highly tolerant to other antibiotics. PQ induces SoxRS, which in turn induces expression of the AcrAB-TolC multidrug-resistant (MDR) pump. Fluoroquinolones are extruded by this MDR pump, and the effect of PQ on antibiotic tolerance was largely abolished in a mutant that was defective in the pump. It appears that PQ, acting through AcrAB-TolC, reduces the concentration of fluoroquinolones in the cells. This allows a larger fraction of cells to become persisters in the presence of a fluoroquinolone. Analysis of a lexA3 mutant indeed showed a dependence of persister induction under these conditions on SOS. These findings show that induction of a classical resistance mechanism, MDR efflux, by oxidative stress leads to an increase in multidrug-tolerant persister cells.
doi:10.1128/AAC.00921-12
PMCID: PMC3421885  PMID: 22777047
9.  Correction: Regulation of the Escherichia coli HipBA Toxin-Antitoxin System by Proteolysis 
PLoS ONE  2012;7(9):10.1371/annotation/e608601c-eadd-4c11-adb2-7b605aba9c44.
doi:10.1371/annotation/e608601c-eadd-4c11-adb2-7b605aba9c44
PMCID: PMC3465168
10.  Regulation of the Escherichia coli HipBA Toxin-Antitoxin System by Proteolysis 
PLoS ONE  2012;7(6):e39185.
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon- background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.
doi:10.1371/journal.pone.0039185
PMCID: PMC3376134  PMID: 22720069
11.  Novel high-throughput screen against Candida albicans identifies antifungal potentiators and agents effective against biofilms 
Objectives
Microbial adhesion and biofilms have important implications for human health and disease. Candida albicans is an opportunistic pathogen which forms drug-resistant biofilms that contribute to the recalcitrance of disease. We have developed a high-throughput screen for potentiators of clotrimazole, a common therapy for Candida infections, including vaginitis and thrush. The screen was performed against C. albicans biofilms grown in microtitre plates in order to target the most resilient forms of the pathogen.
Methods
Biofilm growth, in individual wells of 384-well plates, was measured using the metabolic indicator alamarBlue® and found to be very consistent and reproducible. This assay was used to test the effect of more than 120 000 small molecule compounds from the NIH Molecular Libraries Small Molecule Repository, and compounds that enhanced the activity of clotrimazole or acted on the biofilms alone were identified as hits.
Results
Nineteen compounds (0.016% hit rate) were identified and found to cause more than 30% metabolic inhibition of biofilms compared with clotrimazole alone, which had a modest effect on biofilm viability at the concentration tested. Hits were confirmed for activity against biofilms with dose–response measurements. Several compounds had increased activity in combination with clotrimazole, including a 1,3-benzothiazole scaffold that exhibited a >100-fold improvement against biofilms of three separate C. albicans isolates. Cytotoxicity experiments using human fibroblasts confirmed the presence of lead molecules with favourable antifungal activity relative to cytotoxicity.
Conclusions
We have validated a novel approach to identify antifungal potentiators and completed a high-throughput screen to identify small molecules with activity against C. albicans biofilms. These small molecules may specifically target the biofilm and make currently available antifungals more effective.
doi:10.1093/jac/dkq530
PMCID: PMC3058565  PMID: 21393183
clotrimazole; alamarBlue®; drug resistance
12.  Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters 
mBio  2011;2(3):e00100-11.
ABSTRACT
Tuberculosis continues to be a major public health problem in many parts of the world. Significant obstacles in controlling the epidemic are the length of treatment and the large reservoir of latently infected people. Bacteria form dormant, drug-tolerant persister cells, which may be responsible for the difficulty in treating both acute and latent infections. We find that in Mycobacterium  tuberculosis, low numbers of drug-tolerant persisters are present in lag and early exponential phases, increasing sharply at late exponential and stationary phases to make up ~1% of the population. This suggests that persister formation is governed by both stochastic and deterministic mechanisms. In order to isolate persisters, an exponentially growing population was treated with d-cycloserine, and cells surviving lysis were collected by centrifugation. A transcriptome of persisters was obtained by using hybridization to an Affymetrix array. The transcriptome shows downregulation of metabolic and biosynthetic pathways, consistent with a certain degree of dormancy. A set of genes was upregulated in persisters, and these are likely involved in persister formation and maintenance. A comparison of the persister transcriptome with transcriptomes obtained for several in vitro dormancy models identified a small number of genes upregulated in all cases, which may represent a core dormancy response.
IMPORTANCE
It is estimated that every third person on the planet is infected with Mycobacterium tuberculosis. The two major problems in controlling M. tuberculosis are the length of the treatment and the large reservoir of latently infected people. Dormant persister cells may be responsible for both problems. We find that M. tuberculosis produces persisters in vitro in a growth phase-dependent manner. Persisters were isolated from an exponentially growing population, and their transcriptome shows a distinct pattern of dormancy. These results give the first insight into M. tuberculosis persisters and point to possible mechanisms responsible for their formation.
doi:10.1128/mBio.00100-11
PMCID: PMC3119538  PMID: 21673191
13.  Emergence of Pseudomonas aeruginosa Strains Producing High Levels of Persister Cells in Patients with Cystic Fibrosis▿  
Journal of Bacteriology  2010;192(23):6191-6199.
The majority of cystic fibrosis (CF) patients succumb to a chronic infection of the airway with Pseudomonas aeruginosa. Paradoxically, pathogenic strains are often susceptible to antibiotics, but the infection cannot be eradicated with antimicrobial therapy. We find that in a majority of patients with airway infections, late isolates of P. aeruginosa produce increased levels of drug-tolerant persister cells. The genomes of a clonal pair of early/late isolates from a single patient have been previously sequenced, and the late isolate (obtained at age 96 months) showed a 100-fold increase in persister levels. The 96-month isolate carries a large number of mutations, including a mutation in mutS that confers a hypermutator phenotype. There is also a mutation in the mexZ repressor controlling the expression of the MexXY-OprM multidrug pump, which results in a moderate increase in the ofloxacin, carbenicillin, and tobramycin MICs. Knocking out the mexXY locus restored the resistance to that of the parent strain but did not affect the high levels of persisters formed by the 96-month isolate. This suggests that the late isolate is a high-persister (hip) mutant. Increased persister formation was observed in exponential phase, stationary phase, and biofilm populations of the 96-month isolate. Analysis of late isolates from 14 additional patients indicated that 10 of them are hip mutants. Most of these hip mutants did not have higher drug resistance. Increased persister formation appears to be their sole mechanism for surviving chemotherapy. Taken together, these findings suggest a link between persisters and recalcitrance of CF infection and identify an overlooked culprit—high-persister mutants producing elevated levels of drug-tolerant cells. Persisters may play a similarly critical role in the recalcitrance of other chronic infections.
doi:10.1128/JB.01651-09
PMCID: PMC2981199  PMID: 20935098
14.  Isolation and Physiology of Bacteria from Contaminated Subsurface Sediments▿ †  
Applied and Environmental Microbiology  2010;76(22):7413-7419.
The majority of environmental microorganisms cannot be grown by traditional techniques. Here we employed, and contrasted with conventional plating, an alternative approach based on cultivation of microorganisms inside diffusion chambers incubated within natural samples, followed by subculturing in petri dishes. Using this approach, we isolated microorganisms from subsurface sediments from the Field Research Center (FRC) in Oak Ridge, TN. The sediments were acidic and highly contaminated with uranium, heavy metals, nitrate, and organic pollutants. Phylogenetic analysis of 16S rRNA gene sequences revealed clear differences between diversity of isolates obtained by the diffusion chamber approach and those obtained by conventional plating. The latter approach led to isolation of members of the Alpha- and Gammaproteobacteria, Actinobacteria, and Verrucomicrobia. Isolates obtained via the diffusion chamber approach represented the Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Notably, one-third of the isolates obtained by the new method were closely related to species known from previous molecular surveys conducted in the FRC area. Since the initial growth of microorganisms inside diffusion chambers occurred in the presence of the environmental stress factors, we expected the isolates we obtained to be tolerant of these factors. We investigated the physiologies of selected isolates and discovered that the majority were indeed capable of growth under low pH and/or high concentrations of heavy metals and nitrate. This indicated that in contrast to conventional isolation, the diffusion chamber-based approach leads to isolation of species that are novel, exhibit tolerance to extant environmental conditions, and match some of the species previously discovered by molecular methods.
doi:10.1128/AEM.00376-10
PMCID: PMC2976195  PMID: 20870785
15.  Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria 
PLoS ONE  2011;6(4):e18127.
Background
Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria.
Methodology/Principal Findings
In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology.
Conclusions/Significance
These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4′,5′-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.
doi:10.1371/journal.pone.0018127
PMCID: PMC3070693  PMID: 21483731
16.  Siderophores from Neighboring Organisms Promote the Growth of Uncultured Bacteria 
Chemistry & biology  2010;17(3):254-264.
Summary
The majority of bacterial species do not grow on synthetic media. Many non-growers require growth factors from other bacteria, but the nature of these compounds is largely unknown. We show here that previously uncultured isolates from marine sediment biofilm grow on a Petri dish in the presence of cultured organisms from the same environment. The growth factors produced by one cultured helper strain were identified as new acyl-desferrioxamine siderophores. A panel of previously uncultured isolates exhibited a range of siderophore promiscuity for growth promotion. This siderophore-based approach has enabled the culturing of organisms only distantly related to previously cultured microbes. The lack of growth in the lab for many strains from this habitat stems from an inability to autonomously produce siderophores, and the resulting chemical dependence on other microorganisms regulates community establishment in the environment.
doi:10.1016/j.chembiol.2010.02.010
PMCID: PMC2895992  PMID: 20338517
17.  Berberine-INF55 (5-Nitro-2-Phenylindole) Hybrid Antimicrobials: Effects of Varying the Relative Orientation of the Berberine and INF55 Components ▿  
Hybrid antimicrobials containing an antibacterial linked to a multidrug resistance (MDR) pump inhibitor make up a promising new class of agents for countering efflux-mediated bacterial drug resistance. This study explores the effects of varying the relative orientation of the antibacterial and efflux pump inhibitor components in three isomeric hybrids (SS14, SS14-M, and SS14-P) which link the antibacterial alkaloid and known substrate for the NorA MDR pump berberine to different positions on INF55 (5-nitro-2-phenylindole), an inhibitor of NorA. The MICs for all three hybrids against wild-type, NorA-knockout, and NorA-overexpressing Staphylococcus aureus cells were found to be similar (9.4 to 40.2 μM), indicating that these compounds are not effectively effluxed by NorA. The three hybrids were also found to have similar curing effects in a Caenorhabditis elegans live infection model. Each hybrid was shown to accumulate in S. aureus cells to a greater extent than either berberine or berberine in the presence of INF55, and the uptake kinetics of SS14 were found to differ from those of SS14-M and SS14-P. The effects on the uptake and efflux of the NorA substrate ethidium bromide into S. aureus cells in the presence or absence of the hybrids were used to confirm MDR inhibition by the hybrids. MDR-inhibitory activity was confirmed for SS14-M and SS14-P but not for SS14. Molecular dynamics simulations revealed that SS14 prefers to adopt a conformation that is not prevalent in either SS14-M or SS14-P, which may explain why some properties of SS14 diverge from those of its two isomers. In summary, subtle repositioning of the pump-blocking INF55 moiety in berberine-INF55 hybrids was found to have a minimal effect on their antibacterial activities but to significantly alter their effects on MDR pumps.
doi:10.1128/AAC.01715-09
PMCID: PMC2916310  PMID: 20498327
18.  High Throughput Screen for Novel Antimicrobials using a Whole Animal Infection Model 
ACS chemical biology  2009;4(7):527-533.
The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen with a mortality rate of up to 37% that is increasingly acquiring resistance to antibiotics. Here, we describe an automated, high throughput screen of 37,200 compounds and natural product extracts for those that enhance survival of C. elegans infected with E. faecalis. The screen uses a robot to accurately dispense live, infected animals into 384-well plates, and automated microscopy and image analysis to generate quantitative, high content data. We identified 28 compounds and extracts that were not previously reported to have antimicrobial properties, including 6 structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use. Our versatile and robust screening system can be easily adapted for other whole animal assays to probe a broad range of biological processes.
doi:10.1021/cb900084v
PMCID: PMC2745594  PMID: 19572548
19.  Patients with Long-Term Oral Carriage Harbor High-Persister Mutants of Candida albicans▿  
Fungal biofilms produce a small number of persister cells which can tolerate high concentrations of fungicidal agents. Persisters form upon attachment to a surface, an important step in the pathogenesis of Candida strains. The periodic application of antimicrobial agents may select for strains with increased levels of persister cells. In order to test this possibility, 150 isolates of Candida albicans and C. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day. Persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of amphotericin B and plating for survivors. The persister levels of the isolates varied from 0.2 to 9%, and strains isolated from patients with long-term carriage had high levels of persisters. High-persister strains were isolated from every patient with Candida carriage of more than 8 consecutive weeks but from no patients with transient carriage. All of the high-persister isolates had an amphotericin B MIC that was the same as that for the wild type, indicating that these strains were drug-tolerant rather than drug-resistant mutants. Biofilms of the majority of high-persister strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. This study suggests that persister cells are clinically relevant, and antimicrobial therapy selects for high-persister strains in vivo. The drug tolerance of persisters may be a critical but overlooked component responsible for antimicrobial drug failure and relapsing infections.
doi:10.1128/AAC.00860-09
PMCID: PMC2798516  PMID: 19841146
20.  Antibacterial Activity of Berberine-NorA Pump Inhibitor Hybrids with a Methylene Ether Linking Group 
Bioorganic & medicinal chemistry  2009;17(11):3866-3872.
Conjugation of the NorA substrate berberine and the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group gave the 13-substituted berberine-NorA inhibitor hybrid, 3. A series of simpler arylmethyl ether hybrid structures were also synthesized. The hybrid 3 showed excellent antibacterial activity (MIC Staphylococcus aureus, 1.7 μM), which was over 382-fold more active than the parent antibacterial berberine, against this bacterium. This compound was also shown to block the NorA efflux pump in S. aureus.
doi:10.1016/j.bmc.2009.04.028
PMCID: PMC2759347  PMID: 19419877
13-Substituted berberine; Antibacterial; NorA efflux pump inhibitors; Dual action; Hybrid
21.  Ciprofloxacin Causes Persister Formation by Inducing the TisB toxin in Escherichia coli 
PLoS Biology  2010;8(2):e1000317.
Persisters are specialized survivor cells that arise in populations of E. coli after antibiotic-mediated DNA damage induces the production of a small membrane-acting peptide TisB, which causes reversible dormancy. The TisB-dependent persisters are tolerant to multiple antibiotics.
Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage–induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.
Author Summary
Bacterial populations contain a small number of dormant cells (persisters) that are tolerant to antibiotics. Persisters are not mutants, but rather phenotypic variants of regular cells. Persisters play a major role in resistance of bacterial biofilms to death, and are likely to be responsible for recalcitrance of chronic infections to antibiotics. A lead into the mechanism by which these specialized survivor cells arise comes from the fact that DNA damage induces the SOS response in bacteria, a signaling pathway that up-regulates DNA repair functions. SOS response induction also leads to expression in E. coli of a tisB “toxin” gene encoding a small membrane-acting peptide that leads to a decrease in ATP and can kill cells if artificially overexpressed. We reasoned that tisB may actually be a persister gene and its product induces reversible dormancy by shutting down cell metabolism. We show that a knockout of tisB resulted in a sharply decreased frequency of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage, whereas mild overproduction of the peptide induced persister formation. TisB-dependent persisters also were highly tolerant to unrelated antibiotics. It appears that production of persisters tolerant to all antimicrobials is a “side-effect” of fluoroquinolone antibiotics. Our results suggest that induction of TisB by the SOS response controls production of multidrug-tolerant cells and represents, to our knowledge, the first mechanism of persister formation.
doi:10.1371/journal.pbio.1000317
PMCID: PMC2826370  PMID: 20186264
22.  SOS Response Induces Persistence to Fluoroquinolones in Escherichia coli 
PLoS Genetics  2009;5(12):e1000760.
Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance. We investigated persistence to the fluoroquinolone ciprofloxacin in Escherichia coli. Our data show that a majority of persisters to ciprofloxacin were formed upon exposure to the antibiotic, in a manner dependent on the SOS gene network. These findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means. SOS-induced persistence is a novel mechanism by which cells can counteract DNA damage and promote survival to fluoroquinolones. This unique survival mechanism may be an important factor influencing the outcome of antibiotic therapy in vivo.
Author Summary
The frequent failure of antibiotic treatments is an acute public health problem. Bacteria can escape the lethal action of antibiotics by a mutation in the cell's DNA, leading to antibiotic resistance. Alternatively, they can enter a physiological state in which the antibiotics do not affect them. This phenomenon, referred to as persistence, is different from resistance because there is no genetic modification and because it is transient. Persisters are believed to form stochastically prior to antibiotic treatment. The presence of persister cells in bacterial biofilms contributes to the difficulty in treating biofilm-related infections. We investigated the persistence of Escherichia coli to one of the most widely used antibiotics, ciprofloxacin. We show that the majority of persister cells are formed in response to this antibiotic, contrary to the prevailing view of persister formation. Ciprofloxacin kills bacteria by damaging their DNA. DNA damage activates a SOS gene network, the result of which is the production of various repair proteins. We uncovered a novel part of this network that leads to the formation of tolerant persister cells. The induced tolerance as a side effect of antibiotic treatment is an effective bacterial survival strategy and is likely to contribute to recalcitrance of infections.
doi:10.1371/journal.pgen.1000760
PMCID: PMC2780357  PMID: 20011100
23.  Molecular Mechanisms of HipA Mediated Multidrug Tolerance and its Neutralization by HipB 
Science (New York, N.Y.)  2009;323(5912):396-401.
Summary
Bacterial multidrug tolerance is largely responsible for the inability of antibiotics to eradicate infections and is caused by a small population of dormant bacteria called persisters. HipA is a critical Escherichia coli persistence factor that is normally neutralized by HipB, a transcription repressor, which also regulates hipBA expression. Here we report multiple structures of HipA and a HipA-HipB-DNA complex. HipA has a eukaryotic Ser/Thr kinase-like fold and can phosphorylate the translation factor, EF-Tu, suggesting a persistence mechanism via cell stasis. The HipA-HipB-DNA structure reveals the HipB-operator binding mechanism, ~70° DNA bending and unexpected HipA-DNA contacts. Dimeric HipB interacts with two HipA molecules to inhibit its kinase activity through sequestration and conformational inactivation. Combined, these studies suggest mechanisms for HipA-mediated persistence and its neutralization by HipB.
doi:10.1126/science.1163806
PMCID: PMC2764309  PMID: 19150849
24.  Structure–Activity Relationships of 2-Aryl-1H-indole Inhibitors of the NorA Efflux Pump in Staphylococcus aureus 
The synthesis of 22 2-aryl-1H-indoles, including 12 new compounds, has been achieved via Pd- or Rh- mediated methodologies, or selective electrophilic substitution. All three methods were based on elaborations from simple indole precursors. SAR studies on these indoles and 2-phenyl-1H-indole in S. aureus as NorA efflux pump inhibitors indicated 5-nitro-2-(3-methoxycarbonyl)phenyl-1H-indole was a slightly more potent inhibitor than the lead INF55. A promising new antibacterial lead compound against S. aureus, (2-phenyl-1H-indol-5-yl)-methanol, was also found.
doi:10.1016/j.bmcl.2008.06.093
PMCID: PMC2590755  PMID: 18632270
2-Arylindoles; NorA efflux pump inhibitors; Antibacterial
25.  A trap for in situ cultivation of filamentous actinobacteria 
The approach of growing microorganisms in situ, or in a simulated natural environment is appealing, and different versions of it have been described by several groups. The major difficulties with these approaches are that they are not selective for actinomycetes – a group of gram-positive bacteria well known as a rich source of antibiotics. In order to efficiently access actinomycetes, a trap for specifically capturing and cultivating these microorganisms in situ has been developed, based on the ability of these bacteria to form hyphae and penetrate solid environments. The trap is formed by two semi-permeable membranes (0.2 – 0.6 μm pore-size bottom membrane and 0.03 μm pore-size top membrane) glued to a plastic washer with sterile agar or gellan gum inside. The trap is placed on top of soil, and filamentous microorganisms selectively penetrate into the device and form colonies. Decreasing the size of the pores of the lower membrane to 0.2 μm restricted penetration of fungi. The trap produced more filamentous actinobacteria, and a higher variety of them, as compared to a conventional Petri dish cultivation from the same soil sample. Importantly, the trap cultivation resulted in the isolation of unusual and rare actinomycetes.
doi:10.1016/j.mimet.2007.12.009
PMCID: PMC2293972  PMID: 18255181

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