Antibiotic-tolerant Persisters Research Articles

Stapled Phd Peptides Inhibit Doc Toxin Induced Growth Arrest in Salmonella.

Bacterial toxin inhibition is a promising approach to overcoming antibiotic failure. InSalmonella, knockout of the toxin Doc has been shown to significantly reduce the formation of antibiotic-tolerant persisters. Doc is a kinase that is inhibited in nontolerant cells by its cognate antitoxin, Phd. In this work, we have developed first-in-class stapled peptide antitoxin mimetics based on the Doc inhibitory sequence of Phd. After making a series of substitutions to improve bacterial uptake, we identified a lead stapled Phd peptide that is able to counteract Doc toxicity in Salmonella. This provides an exciting starting point for the further development of therapeutic peptides capable of reducing antibiotic persistence in pathogenic bacteria.

Decline in nitrosative stress drives antibiotic persister regrowth during infection

Internalization of pathogenic bacteria by macrophages results in formation of antibiotic-tolerant persisters. These cells are maintained in a non-growing state for extended periods of time, and it is assumed that their growth resumption causes infection relapse after cessation of antibiotic treatment. Despite this clinical relevance, the signals and conditions that drive persister regrowth during infection are not yet understood. Here, we found that after persister formation in macrophages, host reactive nitrogen species (RNS) produced in response to Salmonella infection lock persisters in growth arrest by intoxicating their TCA cycle, lowering cellular respiration and ATP production. Intracellular persisters resume growth when macrophage RNS production subsides and functionality of their TCA cycle is regained. Persister growth resumption within macrophages is slow and heterogeneous, dramatically extending the time the persister reservoir feeds infection relapse. Using an inhibitor of RNS production, we can force recalcitrant bacteria to regrow during antibiotic treatment, thereby facilitating their eradication.

Polymeric nano-system for macrophage reprogramming and intracellular MRSA eradication.

Intracellular Methicillin-Resistant Staphylococcus aureus (MRSA) remains a major factor of refractory and recurrent infections, which cannot be well addressed by antibiotic therapy. Here, we design a cellular infectious microenvironment-activatable polymeric nano-system to mediate targeted intracellular drug delivery for macrophage reprogramming and intracellular MRSA eradication. The polymeric nano-system is composed of a ferrocene-decorated polymeric nanovesicle formulated from poly(ferrocenemethyl methacrylate)-block-poly(2-methacryloyloxyethyl phosphorylcholine) (PFMMA-b-PMPC) copolymer with co-encapsulation of clofazimine (CFZ) and interferon-γ (IFN-γ). The cellular-targeting PMPC motifs render specific internalization by macrophages and allow efficient intracellular accumulation. Following the internalization, the ferrocene-derived polymer backbone sequentially undergoes hydrophobic-to-hydrophilic transition, charge reversal and Fe release in response to intracellular hydrogen peroxide over-produced upon infection, eventually triggering endosomal escape and on-site cytosolic drug delivery. The released IFN-γ reverses the immunosuppressive status of infected macrophages by reprogramming anti-inflammatory M2 to pro-inflammatory M1 phenotype. Meanwhile, intracellular Fe2+-mediated Fenton reaction together with antibiotic CFZ contributes to increased intracellular hydroxyl radical (•OH) generation. Ultimately, the nano-system achieves robust potency in ablating intracellular MRSA and antibiotic-tolerant persisters by synchronous immune modulation and efficient •OH killing, providing an innovative train of thought for intracellular MRSA control.

Antibiotic Tolerance Indicative of Persistence Is Pervasive among Clinical Streptococcus pneumoniae Isolates and Shows Strong Condition Dependence.

Streptococcus pneumoniae is an important human pathogen, being one of the most common causes of community-acquired pneumonia and otitis media. Antibiotic resistance in S. pneumoniae is an emerging problem, as it depletes our arsenal of effective drugs. In addition, persistence also contributes to the antibiotic crisis in many other pathogens, yet for S. pneumoniae, little is known about antibiotic-tolerant persisters and robust experimental means are lacking. Persister cells are phenotypic variants that exist as a subpopulation within a clonal culture. Being tolerant to lethal antibiotics, they underly the chronic nature of a variety of infections and even help in acquiring genetic resistance. In this study, we set out to identify and characterize persistence in S. pneumoniae. Specifically, we followed different strategies to overcome the self-limiting nature of S. pneumoniae as a confounding factor in the prolonged monitoring of antibiotic survival needed to study persistence. Under optimized conditions, we identified genuine persisters in various growth phases and for four relevant antibiotics through biphasic survival dynamics and heritability assays. Finally, we detected a high variety in antibiotic survival levels across a diverse collection of S. pneumoniae clinical isolates, which assumes that a high natural diversity in persistence is widely present in S. pneumoniae. Collectively, this proof of concept significantly progresses the understanding of the importance of antibiotic persistence in S. pneumoniae infections, which will set the stage for characterizing its relevance to clinical outcomes and advocates for increased attention to the phenotype in both fundamental and clinical research. IMPORTANCE S. pneumoniae is considered a serious threat by the Centers for Disease Control and Prevention because of rising antibiotic resistance. In addition to resistance, bacteria can also survive lethal antibiotic treatment by developing antibiotic tolerance, more specifically, antibiotic tolerance through persistence. This phenotypic variation seems omnipresent among bacterial life, is linked to therapy failure, and acts as a catalyst for resistance development. This study gives the first proof of the presence of persister cells in S. pneumoniae and shows a high variety in persistence levels among diverse strains, suggesting that persistence is a general trait in S. pneumoniae cultures. Our work advocates for higher interest for persistence in S. pneumoniae as a contributing factor for therapy failure and resistance development.

Arctic Psychrotolerant Pseudomonas sp. B14-6 Exhibits Temperature-Dependent Susceptibility to Aminoglycosides.

Bacteria can evade antibiotics by acquiring resistance genes, as well as switching to a non-growing dormant state without accompanying genetic modification. Bacteria in this quiescent state are called persisters, and this non-inheritable ability to withstand multiple antibiotics is referred to as antibiotic tolerance. Although all bacteria are considered to be able to form antibiotic-tolerant persisters, the antibiotic tolerance of extremophilic bacteria is poorly understood. Previously, we identified the psychrotolerant bacterium Pseudomonas sp. B14-6 from the glacier foreland of Midtre Lovénbreen in High Arctic Svalbard. Herein, we investigated the resistance and tolerance of Pseudomonas sp. B14-6 against aminoglycosides at various temperatures. This bacterium was resistant to streptomycin and susceptible to apramycin, gentamicin, kanamycin, and tobramycin. The two putative aminoglycoside phosphotransferase genes aph1 and aph2 were the most likely contributors to streptomycin resistance. Notably, unlike the mesophilic Pseudomonas aeruginosa PA14, this cold-adapted bacterium demonstrated reduced susceptibility to all tested aminoglycosides in a temperature-dependent manner. Pseudomonas sp. B14-6 at a lower temperature formed the persister cells that shows tolerance to the 100-fold minimum inhibitory concentration (MIC) of gentamicin, as well as the partially tolerant cells that withstand 25-fold MIC gentamicin. The temperature-dependent gentamicin tolerance appears to result from reduced metabolic activity. Lastly, the partially tolerant Pseudomonas sp. B14-6 cells could slowly proliferate under the bactericidal concentrations of aminoglycosides. Our results demonstrate that Pseudomonas sp. B14-6 has a characteristic ability to form cells with a range of tolerance, which appears to be inversely proportional to its growth rate.

Bugs on Drugs: A Drosophila melanogaster Gut Model to Study In Vivo Antibiotic Tolerance of E. coli.

With an antibiotic crisis upon us, we need to boost antibiotic development and improve antibiotics’ efficacy. Crucial is knowing how to efficiently kill bacteria, especially in more complex in vivo conditions. Indeed, many bacteria harbor antibiotic-tolerant persisters, variants that survive exposure to our most potent antibiotics and catalyze resistance development. However, persistence is often only studied in vitro as we lack flexible in vivo models. Here, I explored the potential of using Drosophila melanogaster as a model for antimicrobial research, combining methods in Drosophila with microbiology techniques: assessing fly development and feeding, generating germ-free or bacteria-associated Drosophila and in situ microscopy. Adult flies tolerate antibiotics at high doses, although germ-free larvae show impaired development. Orally presented E. coli associates with Drosophila and mostly resides in the crop. E. coli shows an overall high antibiotic tolerance in vivo potentially resulting from heterogeneity in growth rates. The hipA7 high-persistence mutant displays an increased antibiotic survival while the expected low persistence of ΔrelAΔspoT and ΔrpoS mutants cannot be confirmed in vivo. In conclusion, a Drosophila model for in vivo antibiotic tolerance research shows high potential and offers a flexible system to test findings from in vitro assays in a broader, more complex condition.

Membrane Transporters of the Major Facilitator Superfamily Are Essential for Long-Term Maintenance of Phenotypic Tolerance to Multiple Antibiotics in E. coli.

ABSTRACTAntibiotic tolerance is not only the key underlying the cause of recurrent and chronic bacterial infections but it is also a factor linked to exacerbation of diseases, such as tuberculosis, cystic fibrosis-associated lung infection, and candidiasis. This phenomenon was previously attributed to a switch to physiological dormancy in a bacterial subpopulation triggered by environmental signals. However, we recently showed that expression of phenotypic antibiotic tolerance during nutrient starvation is highly dependent on robust production of proteins that actively maintain the bacterial transmembrane proton motive force (PMF), even under a nutrient-deficient environment. To investigate why PMF needs to be maintained for expression of phenotypic antibiotic tolerance, we tested the relative functional role of known transporters and efflux pumps in tolerance development by assessing the effect of deletion of specific efflux pump and transporter-encoding genes on long-term maintenance of antibiotic tolerance in an Escherichia coli population under starvation. We identified eight specific efflux pumps and transporters and two known efflux pump components, namely, ChaA, EmrK, EmrY, SsuA, NhaA, GadC, YdjK, YphD, TolC, and ChaB, that play a key role in tolerance development and maintenance. In particular, deletion of each of the nhaA and chaB genes is sufficient to totally abolish the tolerance phenotypes during prolonged antimicrobial treatment. These findings therefore depict active, efflux-mediated bacterial tolerance mechanisms and facilitate design of intervention strategies to prevent and treat chronic and recurrent infections due to persistence of antibiotic-tolerant subpopulations in the human body.IMPORTANCE We recently showed that the antibiotic-tolerant subpopulation of bacteria or persisters actively maintain the transmembrane proton motive force (PMF) to survive starvation stress for a prolonged period. This work further shows that the reason why antibiotic persisters need to maintain PMF is that PMF is required to support a range of efflux or transportation functions. Intriguingly, we found that tolerance-maintaining efflux activities were mainly encoded by 10 efflux or transporter genes. Because our study showed that deletion of even one of these genes could cause a significant reduction in tolerance level, we conclude that the products of these genes play an essential role in enhancing the survival fitness of bacteria during starvation or under other adverse environmental conditions. These gene products are therefore excellent targets for future design of antimicrobial agents that eradicate antibiotic tolerant persisters and prevent occurrence of chronic and recurrent human infections.

Population Bottlenecks Strongly Affect the Evolutionary Dynamics of Antibiotic Persistence.

Bacterial persistence is a potential cause of antibiotic therapy failure. Antibiotic-tolerant persisters originate from phenotypic differentiation within a susceptible population, occurring with a frequency that can be altered by mutations. Recent studies have proven that persistence is a highly evolvable trait and, consequently, an important evolutionary strategy of bacterial populations to adapt to high-dose antibiotic therapy. Yet, the factors that govern the evolutionary dynamics of persistence are currently poorly understood. Theoretical studies predict far-reaching effects of bottlenecking on the evolutionary adaption of bacterial populations, but these effects have never been investigated in the context of persistence. Bottlenecking events are frequently encountered by infecting pathogens during host-to-host transmission and antibiotic treatment. In this study, we used a combination of experimental evolution and barcoded knockout libraries to examine how population bottlenecking affects the evolutionary dynamics of persistence. In accordance with existing hypotheses, small bottlenecks were found to restrict the adaptive potential of populations and result in more heterogeneous evolutionary outcomes. Evolutionary trajectories followed in small-bottlenecking regimes additionally suggest that the fitness landscape associated with persistence has a rugged topography, with distinct trajectories toward increased persistence that are accessible to evolving populations. Furthermore, sequencing data of evolved populations and knockout libraries after selection reveal various genes that are potentially involved in persistence, including previously known as well as novel targets. Together, our results do not only provide experimental evidence for evolutionary theories, but also contribute to a better understanding of the environmental and genetic factors that guide bacterial adaptation to antibiotic treatment.

SC5005 dissipates the membrane potential to kill Staphylococcus aureus persisters without detectable resistance.

In the past few decades, multiple-antibiotic-resistant Staphylococcus aureus has emerged and quickly spread in hospitals and communities worldwide. Additionally, the formation of antibiotic-tolerant persisters and biofilms further reduces treatment efficacy. Previously, we identified a sorafenib derivative, SC5005, with bactericidal activity against MRSA in vitro and in vivo. Here, we sought to elucidate the resistance status, mode of action and anti-persister activity of this compound. The propensity of S. aureus to develop SC5005 resistance was evaluated by assessment of spontaneous resistance and by multi-passage selection. The mode of action of SC5005 was investigated using macromolecular synthesis, LIVE/DEAD and ATPlite assays and DiOC2(3) staining. The effect of SC5005 on the mammalian cytoplasmic membrane was measured using haemolytic and lactate dehydrogenase (LDH) assays and flow cytometry. SC5005 depolarized and permeabilized the bacterial cytoplasmic membrane, leading to reduced ATP production. Because of this mode of action, no resistance of S. aureus to SC5005 was observed after constant exposure to sub-lethal concentrations for 200 passages. The membrane-perturbing activity of SC5005 was specific to bacteria, as no significant haemolysis or release of LDH from human HT-29 cells was detected. Additionally, compared with other bactericidal antibiotics, SC5005 exhibited superior activity in eradicating both planktonic and biofilm-embedded S. aureus persisters. Because of its low propensity for resistance development and potent persister-eradicating activity, SC5005 is a promising lead compound for developing new therapies for biofilm-related infections caused by S. aureus.

Inside Cover: Poly(2‐Oxazoline)‐Based Functional Peptide Mimics: Eradicating MRSA Infections and Persisters while Alleviating Antimicrobial Resistance (Angew. Chem. Int. Ed. 16/2020)

Poly(2-oxazoline)s (POXs) were employed as a new class of host defense peptide mimics. As reported by R. Liu and co-workers in their Research Article on page 6412, these functional peptide mimics show potent in vitro and in vivo antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) while alleviating antibiotic resistance and killing antibiotic-tolerant MRSA persisters through a reactive oxygen species (ROS)-dependent mechanism.

The Functional and Molecular Effects of Doxycycline Treatment on Borrelia burgdorferi Phenotype.

Recent studies have shown that Borrelia burgdorferi can form antibiotic-tolerant persisters in the presence of microbiostatic drugs such as doxycycline. Precisely how this occurs is yet unknown. Our goal was to examine gene transcription by B. burgdorferi following doxycycline treatment in an effort to identify both persister-associated genes and possible targets for antimicrobial intervention. To do so, we performed next-generation RNA sequencing on doxycycline-treated spirochetes and treated spirochetes following regrowth, comparing them to untreated B. burgdorferi. A number of genes were perturbed and most of those which were statistically significant were down-regulated in the treated versus the untreated or treated/re-grown. Genes upregulated in the treated B. burgdorferi included a number of Erp genes and rplU, a 50S ribosomal protein. Among those genes associated with post-treatment regrowth were bba74 (Oms28), bba03, several peptide ABC transporters, ospA, ospB, ospC, dbpA and bba62. Studies are underway to determine if these same genes are perturbed in B. burgdorferi treated with doxycycline in a host environment.

Salmonella persisters undermine host immune defenses during antibiotic treatment.

Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of Salmonella species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that Salmonella persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the Salmonella pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing Salmonella cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.

Prophages and Growth Dynamics Confound Experimental Results with Antibiotic-Tolerant Persister Cells.

ABSTRACTBacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ϕ80, a notorious laboratory contaminant, apparently caused several of the phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon but no longer support a role of TA modules in E. coli persister formation under unstressed conditions. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.

Novel Antibacterial Compounds and their Drug Targets - Successes and Challenges.

Infectious diseases are one of the most important and urgent health problems in the world. According to the World Health Organization (WHO) statistics, infectious and parasitic diseases are a cause of about 16% of all deaths worldwide and over 40% of deaths in Africa. A considerable progress that has been made during last hundred years in the fight against infectious diseases, in particular bacterial infections, can be attributed mainly to three factors: (1) the general improvement of living conditions, in particular sanitation; (2) development of vaccines and (3) development of efficient antibacterial drugs. Although considerable progress in reduction of the number of cases of bacterial infections, especially in lethal cases, has been made, continued cases and outbreaks of these diseases persist, which is caused by different contributing factors. Indeed, during last sixty years antibacterial drugs were used against various infectious diseases caused by bacterial pathogens with an undoubtable success. The most fruitful period for antibiotic development lasted from 40's to 60's of the last century and resulted in the majority of antibiotics currently on the market, which were obtained by screening actinomycetes derived from soil. Although the market for antibacterial drugs is nowadays greater than 25 billion US dollars per year, novel antibacterial drugs are still demanded due to developed resistance of many pathogenic bacteria against current antibiotics. In the last five years, one can observe a dramatic increase in cases of resistant bacteria strains (e.g. Klebsiella pneumoniae and E. coli) which are responsible for difficult to treat pneumonia and infections of urinary tract. The development of resistant bacteria strains is a side effect of antibiotic application for treatment: the infections become untreatable as a result of the existence of antibiotic-tolerant persisters. In this review, we discuss the challenges in antibacterial drug discovery, including the molecular basis of drug resistance, drug targets for novel antibacterial drugs, and new compounds (since year 2010) from different chemical classes with antibacterial activity, focusing on structure-activity relationships.

Growth resumption from stationary phase reveals memory in Escherichia coli cultures.

Frequent changes in nutrient availability often result in repeated cycles of bacterial growth and dormancy. The timing of growth resumption can differ among isogenic cells and delayed growth resumption can lead to antibiotic tolerant persisters. Here we describe a correlation between the timing of entry into stationary phase and resuming growth in the next period of cell proliferation. E. coli cells can follow a last in first out rule: the last ones to shut down their metabolism in the beginning of stationary phase are the first to recover in response to nutrients. This memory effect can last for several days in stationary phase and is not influenced by environmental changes. We observe that the speed and heterogeneity of growth resumption depends on the carbon source. A good carbon source (glucose) can promote rapid growth resumption even at low concentrations, and is seen to act more like a signal than a growth substrate. Heterogeneous growth resumption can protect the population from adverse effect of stress, investigated here using heat-shock, because the stress-resilient dormant cells are always present.

Antibacterial Property of Graphene Quantum Dots (Both Source Material and Bacterial Shape Matter).

Whereas diverse graphene quantum dots (GQDs) with basal planes similar to those of graphene oxide sheets (i.e., GO-GQDs) lack antibacterial property, that prepared by rupturing C60 cage (i.e., C60-GQD) effectively kills Staphylococcus aureus, including its antibiotic-tolerant persisters, but not Bacillus subtilis, Escherichia coli, or Pseudomonas aeruginosa. The observed activity may correlate with a GQD's ability to disrupt bacterial cell envelop. Surface-Gaussian-curvature match between a GQD and a target bacterium may play critical role in the association of the GQD with bacterial cell surface, the initial step for cell envelope disruption, suggesting the importance of both GQDs' source materials and bacterial shape.

Surface Disinfection Enabled by a Layer-by-Layer Thin Film of Polyelectrolyte-Stabilized Reduced Graphene Oxide upon Solar Near-Infrared Irradiation.

We report an antibacterial surface that kills airborne bacteria on contact upon minutes of solar near-infrared (NIR) irradiation. This antibacterial surface employs reduced graphene oxide (rGO), a well-known near-infrared photothermal conversion agent, as the photosensitizer and is prepared by assembling oppositely charged polyelectrolyte-stabilized rGO sheets (PEL-rGO) on a quartz substrate with the layer-by-layer (LBL) technique. Upon solar irradiation, the resulting PEL-rGO LBL multilayer efficiently generates rapid localized heating and, within minutes, kills >90% airborne bacteria, including antibiotic-tolerant persisters, on contact, likely by permeabilizing their cellular membranes. The observed activity is retained even when the PEL-rGO LBL multilayer is placed underneath a piece of 3 mm thick pork tissue, indicating that solar light in the near-infrared region plays dominant roles in the observed activity. This work may pave the way toward NIR-light-activated antibacterial surfaces, and our PEL-rGO LBL multilayer may be a novel surface coating material for conveniently disinfecting biomedical implants and common objects touched by people in daily life in the looming postantibiotic era with only minutes of solar exposure.

Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4–6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.

Energy production genessucBandubiF  are involved in persister survival and tolerance to multiple antibiotics and stresses inEscherichia coli

Persisters are a small population of slowly growing or nongrowing bacteria that are phenotypically resistant to antibiotics, but the mechanisms involved are not well understood. The aim of this study is to determine new mechanisms underlying antibiotic-tolerant persisters. The Escherichia coli deletion mutant library was screened to identify mutants that had a defect in persister survival after exposure to ampicillin for 24 h or 5 days. The identified mutants and the parent strain were subjected to minimum inhibitory concentration (MIC) and minimum bactericidal tests and antibiotic or stress conditions in exposure assays. sucB and ubiF mutants deficient in energy production were identified from the mutant screens to have defective persister survival as demonstrated by higher susceptibility to various antibiotics, including ampicillin, norfloxacin, tetracycline and gentamicin, and different stresses such as oxidative stress, acid pH and weak acid compared with the parent strain. In addition, both sucB and ubiF had a twofold lower MIC than the parent strain. The above sucB and ubiF mutant phenotypes could be complemented by their respective functional genes. Defective energy production through mutations in sucB and ubiF affects persister survival and could serve as new drug targets for persister bacteria.