scholarly journals Identification of Novel phoP-phoQ Regulated Genes that Contribute to Polymyxin B Tolerance in Pseudomonas aeruginosa

2021 ◽  
Vol 9 (2) ◽  
pp. 344
Author(s):  
Baopeng Yang ◽  
Chang Liu ◽  
Xiaolei Pan ◽  
Weixin Fu ◽  
Zheng Fan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Survival Rates ◽  
Sodium Dodecyl ◽  
Membrane Integrity ◽  
Regulatory System ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Bacterial Survival ◽  
Bacterial Binding ◽  
Chromatin Immunoprecipitation Sequencing

Polymyxin B and E (colistin) are the last resorts to treat multidrug-resistant Gram-negative pathogens. Pseudomonas aeruginosa is intrinsically resistant to a variety of antibiotics. The PhoP-PhoQ two-component regulatory system contributes to the resistance to polymyxins by regulating an arnBCADTEF-pmrE operon that encodes lipopolysaccharide modification enzymes. To identify additional PhoP-regulated genes that contribute to the tolerance to polymyxin B, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) assay and found novel PhoP binding sites on the chromosome. We further verified that PhoP directly controls the expression of PA14_46900, PA14_50740 and PA14_52340, and the operons of PA14_11970-PA14_11960 and PA14_52350-PA14_52370. Our results demonstrated that mutation of PA14_46900 increased the bacterial binding and susceptibility to polymyxin B. Meanwhile, mutation of PA14_11960 (papP), PA14_11970 (mpl), PA14_50740 (slyB), PA14_52350 (ppgS), and PA14_52370 (ppgH) reduced the bacterial survival rates and increased ethidium bromide influx under polymyxin B or Sodium dodecyl sulfate (SDS) treatment, indicating roles of these genes in maintaining membrane integrity in response to the stresses. By 1-N-phenylnaphthylamine (NPN) and propidium iodide (PI) staining assay, we found that papP and slyB are involved in maintaining outer membrane integrity, and mpl and ppgS-ppgH are involved in maintaining inner membrane integrity. Overall, our results reveal novel PhoP-PhoQ regulated genes that contribute to polymyxin B tolerance.

scholarly journals Clinically Relevant Concentrations of Polymyxin B and Meropenem Synergistically Kill Multidrug-Resistant Pseudomonas aeruginosa and Minimize Biofilm Formation

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 405
Author(s):  
Hasini Wickremasinghe ◽  
Heidi H. Yu ◽  
Mohammad A. K. Azad ◽  
Jinxin Zhao ◽  
Phillip J. Bergen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Respiratory Infections ◽  
Population Analysis ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Bacterial Killing

The emergence of antibiotic resistance has severely impaired the treatment of chronic respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. Since the reintroduction of polymyxins as a last-line therapy against MDR Gram-negative bacteria, resistance to its monotherapy and recurrent infections continue to be reported and synergistic antibiotic combinations have been investigated. In this study, comprehensive in vitro microbiological evaluations including synergy panel screening, population analysis profiling, time-kill kinetics, anti-biofilm formation and membrane damage analysis studies were conducted to evaluate the combination of polymyxin B and meropenem against biofilm-producing, polymyxin-resistant MDR P. aeruginosa. Two phylogenetically unrelated MDR P. aeruginosa strains, FADDI-PA060 (MIC of polymyxin B [MICpolymyxin B], 64 mg/L; MICmeropenem, 64 mg/L) and FADDI-PA107 (MICpolymyxin B, 32 mg/L; MICmeropenem, 4 mg/L) were investigated. Genome sequencing identified 57 (FADDI-PA060) and 50 (FADDI-PA107) genes predicted to confer resistance to a variety of antimicrobials, as well as multiple virulence factors in each strain. The presence of resistance genes to a particular antibiotic class generally aligned with MIC results. For both strains, all monotherapies of polymyxin B failed with substantial regrowth and biofilm formation. The combination of polymyxin B (16 mg/L)/meropenem (16 mg/L) was most effective, enhancing initial bacterial killing of FADDI-PA060 by ~3 log10 CFU/mL, followed by a prolonged inhibition of regrowth for up to 24 h with a significant reduction in biofilm formation (* p < 0.05). Membrane integrity studies revealed a substantial increase in membrane depolarization and membrane permeability in the surviving cells. Against FADDI-PA107, planktonic and biofilm bacteria were completely eradicated. In summary, the combination of polymyxin B and meropenem demonstrated synergistic bacterial killing while reinstating the efficacy of two previously ineffective antibiotics against difficult-to-treat polymyxin-resistant MDR P. aeruginosa.

Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

2022 ◽  
Author(s):  
Baopeng Yang ◽  
Yujun Jiang ◽  
Yongxin Jin ◽  
Fang Bai ◽  
Zhihui Cheng ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Defense Mechanism ◽  
Extracellular Polysaccharide ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Guanosine Monophosphate ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Bacterial Survival

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .

scholarly journals Evolution under low antibiotic concentrations: a risk for the selection of Pseudomonas aeruginosa multidrug resistant mutants in nature

2021 ◽  
Author(s):  
Fernando Sanz-García ◽  
Sara Hernando-Amado ◽  
José Luis Martínez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Published Data ◽  
Natural Ecosystems ◽  
Clinical Value ◽  
Drug Concentrations ◽  
Resistant Mutants

ABSTRACTBACKGROUNDAntibiotic pollution of non-clinical environments might have a relevant impact on human health if resistant pathogens are selected. However, this potential risk is often overlooked, since drug concentrations in nature are usually below their minimal inhibitory concentrations (MICs). Albeit, antibiotic resistant bacteria can be selected even at sub-MIC concentrations, in a range that is dubbed the sub-MIC selective window, which depends on both the antibiotic and the pathogen.OBJECTIVESDetermine the sub-MIC selective windows of seven antibiotics of clinical relevance in the opportunistic pathogen Pseudomonas aeruginosa and evaluate the risk for selecting resistant mutants in nature, based on published data about the amount of antimicrobials detected in natural environments.METHODSWe conducted evolution experiments of P. aeruginosa PA14 in presence of sub-MIC concentrations of ceftazidime, amikacin, levofloxacin, ciprofloxacin, tetracycline, polymyxin B or imipenem, and measured drug susceptibility of the evolved populations.RESULTSSub-MIC selective window of quinolones was the largest, and the ones of polymyxin B and imipenem, the narrowest. Clinically relevant multidrug resistant (MDR) mutants (presenting MICs above EUCAST clinical breakpoints) arose within the sub-MIC selective windows of the majority of antibiotics tested, being these phenotypes probably mediated by efflux pumps′ activity.DISCUSSIONOur data show that the concentration of antibiotics reported in aquatic ecosystems -colonizable by P. aeruginosa- are, in occasions, higher than the ones able to select MDR mutants. This finding has implications for understanding the role of different ecosystems and conditions in the emergence of antibiotic resistance from a One-Health point of view. Further, it highlights the importance of delineating the sub-MIC selective windows for drugs of clinical value in pathogens with environmental niches, in order to evaluate the health risks due to antibiotic pollution of natural ecosystems and ultimately tackle antibiotic resistance.

scholarly journals Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Anna Olsson ◽  
Pikkei Wistrand-Yuen ◽  
Elisabet I. Nielsen ◽  
Lena E. Friberg ◽  
Linus Sandegren ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Time Lapse ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Positive Interactions ◽  
Synergistic Activity ◽  
Content Type ◽  
Time Lapse Microscopy ◽  
Time Kill ◽  
Antibiotic Combination

ABSTRACT Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa. We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (≤106 CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal β-lactams, ciprofloxacin, and amikacin. Genes encoding β-lactamases (e.g., blaPAO and blaOXA-50) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations.

scholarly journals Analysis of Susceptibility Patterns of Pseudomonas aeruginosa and Isolation, Characterization of Lytic Bacteriophages Targeting Multi Drug Resistant Pseudomonas aeruginosa

2018 ◽  
Vol 11 (2) ◽  
pp. 1105-1117 ◽  
Author(s):  
Shri Natrajan Arumugam ◽  
Akarsh Chickamagalur Rudraradhya ◽  
Sathish Sadagopan ◽  
Sunilkumar Sukumaran ◽  
Ganesh Sambasivam ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Host Range ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Dilution Method ◽  
Drug Resistant ◽  
Susceptibility Pattern ◽  
Infectivity Rate

Pseudomonas aeruginosa is known to be a major cause of Hospital Acquired Infections leading to high mortality in immune-compromised patients. Due to precipitous rise in antibiotic resistance, bacteriophages are significant alternative therapeutic approach for treatment and to combat resistance development. Objective of the current study was to identify MDR Pseudomonas aeruginosa from clinical isolates and to isolate bacteriophages from sewage samples against these MDR Pseudomonas aeruginosa strains. One hundred and forty-four Pseudomonas isolates were tested for their susceptibility pattern with 13 different antibiotics by micro-broth dilution method. Frequency of multidrug resistant (MDR) and Extensive Drug resistant (XDR) of Pseudomonas aeruginosa were found to be 35.5% and 23.6%, respectively. 7.61% isolates were identified as Pan drug resistant (PDR). Rate of susceptibility pattern were Piperacillin/Tazobactam 75%, Polymyxin B 74.6%, Meropenem 73.6%, Colistin 69.2%, Cefepime 54.9%, Ciprofloxacin 54.2%, Gentamicin 54.2%, Aztreonam 53.5%, Tobramycin 47.9%, Ticarcillin/Clavulanic acid 46.9%, Ertapenem 45.8%, Ceftazidime 40.3% and Imipenem 39.2%. Ninety-four bacteriophages were isolated from sewage samples against Pseudomonas aeruginosa PAO1/ATCC9027/clinical strains and host range testing study was carried out with all MDR clinical isolates. Among 51 MDR strains 34 strains were infected by phages. Phage infectivity rate were calculated for individual phages based on their host range infectivity results. AP025 and AP006 phages exhibited good infectivity rate of 39% and 30% respectively against MDR strains. Combination of 5 phages (AP002, AP006, AP011, AP025 and AP067) lysed 62.7% of the strains. Based on the obtained results, phages could be employed for treatment of infections caused by MDR strains with substantiated in-vivo experiments.

scholarly journals 2151. Biofilm and Metallo-β-Lactamase (MBL) Production Among Imipenem-Resistant Pseudomonas aeruginosa and Acinetobacter spp.

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S729-S730
Author(s):  
Yang Metok ◽  
Supram Hosuru Subramanya ◽  
Upendra Thapa Shrestha ◽  
Leandro Reus Rodrigues Perez ◽  
Nabaraj Adhikari ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Hospital Setting ◽  
Polymyxin B ◽  
Microtiter Plate ◽  
Multidrug Resistant ◽  
High Rate ◽  
Bacterial Strains ◽  
Microbiological Techniques ◽  
Acinetobacter Spp ◽  
Imipenem Resistance

Abstract Background Pseudomonas aeruginosa and Acinetobacter spp. head the list of hospital-acquired infections. Resistance to carbapenem as reserve drug is under threat with the emergence of Metallo-β-lactamase (MBL) and biofilm producing bacterial strains. This study was thus undertaken to determine the rate of MBL and biofilm production among imipenem-resistant P. aeruginosa (IRPA) and imipenem-resistant Acinetobacter spp. (IRAS) isolates. Methods A total of 79 P. aeruginosa and 117 Acinetobacter spp. were isolated from different clinical specimens of patients visiting Manipal Teaching Hospital, Pokhara Nepal from July 2016 to January 2017. Isolation, identification and antibiotic susceptibility testing of the isolates were performed by standard microbiological techniques. Combined disc test and Epsilometer test (E-test) were employed to detect MBL in IRPA and IRAS isolates. Microtiter plate using crystal violet method was employed for detection of biofilm in imipenem-resistant isolates. Results 9 (11.4%) of P. aeruginosa and 49 (41.9%) of Acinetobacter spp. were Multidrug Resistant (MDR). Similarly, 22 (27.8%) of P. aeruginosa and 23 (19.7%) of Acinetobacter spp. were Extensively Drug Resistant (XDR). Imipenem resistance was detected among 15 (19%) P. aeruginosa and 57 (48.7%) Acinetobacter spp. isolates. 8 (53.3%) of IRPA and 22 (38.6%) of IRAS isolates were MBL producers while all (100%) of IRPA and 47 (82.5%) of IRAS were biofilm producers. All the biofilm producer IRPA isolates were XDR and 62.5% of XDR IRAS strains were moderate biofilm producers. However, 80% of IRPA, 49.1% of IRAS and 63% of both MBL producer isolates were weak biofilm formers. Polymyxin B and ampicillin-sulbactam showed a better degree of susceptibility against MBL cum biofilm producer IRPA and IRAS isolates respectively. Conclusion The study showed high propensity of IRPA and IRAS to form biofilm, which is strongly associated with higher drug resistance. Such high rate of MBL and biofilm producing P. aeruginosa and Acinetobacter spp. alarms the rapid spread of such strains in our hospital setting. Disclosures All authors: No reported disclosures.

scholarly journals Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Fatma Ben Jeddou ◽  
Léna Falconnet ◽  
Alexandre Luscher ◽  
Thissa Siriwardena ◽  
Jean-Louis Reymond ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Sensor Kinase ◽  
Loss Of Function ◽  
Adaptive Responses ◽  
Kinase Gene ◽  
Polyamine Biosynthesis ◽  
Content Type

ABSTRACT Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

scholarly journals Comparative Metabolomics and Transcriptomics Reveal Multiple Pathways Associated with Polymyxin Killing in Pseudomonas aeruginosa

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Mei-Ling Han ◽  
Yan Zhu ◽  
Darren J. Creek ◽  
Yu-Wei Lin ◽  
Alina D. Gutu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Therapeutic Option ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Central Carbon Metabolism ◽  
Dynamic Interactions ◽  
Content Type ◽  
Comparative Metabolomics

ABSTRACT Polymyxins are a last-line therapy against multidrug-resistant Pseudomonas aeruginosa; however, resistance to polymyxins has been increasingly reported. Therefore, understanding the mechanisms of polymyxin activity and resistance is crucial for preserving their clinical usefulness. This study employed comparative metabolomics and transcriptomics to investigate the responses of polymyxin-susceptible P. aeruginosa PAK (polymyxin B MIC, 1 mg/liter) and its polymyxin-resistant pmrB mutant PAKpmrB6 (MIC, 16 mg/liter) to polymyxin B (4, 8, and 128 mg/liter) at 1, 4, and 24 h, respectively. Our results revealed that polymyxin B at 4 mg/liter induced different metabolic and transcriptomic responses between polymyxin-susceptible and -resistant P. aeruginosa. In strain PAK, polymyxin B significantly activated PmrAB and the mediated arn operon, leading to increased 4-amino-4-deoxy-L-arabinose (L-Ara4N) synthesis and the addition to lipid A. In contrast, polymyxin B did not increase lipid A modification in strain PAKpmrB6. Moreover, the syntheses of lipopolysaccharide and peptidoglycan were significantly decreased in strain PAK but increased in strain PAKpmrB6 due to polymyxin B treatment. In addition, 4 mg/liter polymyxin B significantly perturbed phospholipid and fatty acid levels and induced oxidative stress in strain PAK, but not in PAKpmrB6. Notably, the increased trehalose-6-phosphate levels indicate that polymyxin B potentially caused osmotic imbalance in both strains. Furthermore, 8 and 128 mg/liter polymyxin B significantly elevated lipoamino acid levels and decreased phospholipid levels but without dramatic changes in lipid A modification in wild-type and mutant strains, respectively. Overall, this systems study is the first to elucidate the complex and dynamic interactions of multiple cellular pathways associated with the polymyxin mode of action against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has been highlighted by the recent WHO Global Priority Pathogen List due to multidrug resistance. Without new antibiotics, polymyxins remain a last-line therapeutic option for this difficult-to-treat pathogen. The emergence of polymyxin resistance highlights the growing threat to our already very limited antibiotic armamentarium and the urgency to understand the exact mechanisms of polymyxin activity and resistance. Integration of the correlative metabolomics and transcriptomics results in the present study discovered that polymyxin treatment caused significant perturbations in the biosynthesis of lipids, lipopolysaccharide, and peptidoglycan, central carbon metabolism, and oxidative stress. Importantly, lipid A modifications were surprisingly rapid in response to polymyxin treatment at clinically relevant concentrations. This is the first study to reveal the dynamics of polymyxin-induced cellular responses at the systems level, which highlights that combination therapy should be considered to minimize resistance to the last-line polymyxins. The results also provide much-needed mechanistic information which potentially benefits the discovery of new-generation polymyxins.

Multidrug-resistant Pseudomonas aeruginosa in Brooklyn, New York: molecular epidemiology and in vitro activity of polymyxin B

2005 ◽  
Vol 24 (3) ◽  
pp. 196-201 ◽  
Author(s):  
S. Bratu ◽  
J. Quale ◽  
S. Cebular ◽  
R. Heddurshetti ◽  
D. Landman
Keyword(s):  
New York ◽  
Pseudomonas Aeruginosa ◽  
Molecular Epidemiology ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Vitro Activity ◽  
In Vitro Activity

scholarly journals The Three RelE Homologs of Mycobacterium tuberculosis Have Individual, Drug-Specific Effects on Bacterial Antibiotic Tolerance

2010 ◽  
Vol 192 (5) ◽  
pp. 1279-1291 ◽  
Author(s):  
Ramandeep Singh ◽  
Clifton E. Barry ◽  
Helena I. M. Boshoff
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Survival Rates ◽  
Specific Effect ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Bacterial Survival ◽  
E Coli ◽  
The Face

ABSTRACT In Escherichia coli, expression of the RelE and HipA toxins in the absence of their cognate antitoxins has been associated with generating multidrug-tolerant “persisters.” Here we show that unlike persisters of E. coli, persisters of Mycobacterium tuberculosis selected with one drug do not acquire cross-resistance to other classes of drugs. M. tuberculosis has three homologs of RelE arranged in operons with their apparent antitoxins. Each toxin individually arrests growth of both M. tuberculosis and E. coli, an effect that is neutralized by coexpression of the cognate antitoxin. Overexpression or deletion of each of the RelE toxins had a toxin- and drug-specific effect on the proportion of bacilli surviving antibiotic killing. All three toxins were upregulated in vivo, but none of the deletions affected survival during murine infection. RelE2 overexpression increased bacterial survival rates in the presence of rifampin in vitro, while deletion significantly decreased survival rates. Strikingly, deletion of this toxin had no discernible effect on the level of persisters seen in rifampin-treated mice. Our results suggest that, in vivo, RelE-generated persisters are unlikely to play a significant role in the generation of bacilli that survive in the face of multidrug therapy or in the generation of multidrug-resistant M. tuberculosis.

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