Antibiotic Persistence Research Articles

Degradation of tetracycline, sulfamethazine, and tylosin in soil from prairie strips and row crops in Iowa

AbstractThe livestock industry in the United States relies on antibiotics for disease prevention and treatment. As a result, antibiotic‐laden manure is frequently applied to farmland to recycle nutrients. In the soil environment, antibiotics may accumulate and create selective pressure for antibiotic resistance in bacteria or travel to nearby water resources. This in vitro incubation study evaluated whether prairie buffer strips on farmland enhance degradation of three antibiotics—tetracycline, sulfamethazine, and tylosin—compared with degradation in soil from row crops adjacent to the strips. Soil from prairie strips of varying establishment ages and adjacent row crops were evaluated from three central Iowa sampling locations. Antibiotics mixed with swine manure slurry were added to soils at a starting concentration (10 μg kg–1) that reflects common veterinary antibiotic concentrations in soil and runoff after manure application. Antibiotic concentrations were quantified at six time points throughout a 72‐d incubation period and fit to a first‐order model to calculate decay rate constants and half‐lives. The mean half‐life for tetracycline was 0.54 d longer in prairie strip soil than row crop soil, whereas sulfamethazine and tylosin demonstrated no significant difference in persistence in strip or crop soil. Time since the establishment of the prairie strip did not affect antibiotic persistence. Concentrations of each antibiotic decreased to near‐background levels throughout the incubation period. This study suggests that prairie strips do not consistently enhance antibiotic degradation in farm fields, but that antibiotics are unlikely to persist throughout the growing season in soil under strip or crop management.

Tissue compartmentalization enables Salmonella persistence during chemotherapy

Antimicrobial chemotherapy can fail to eradicate the pathogen, even in the absence of antimicrobial resistance. Persisting pathogens can subsequently cause relapsing diseases. In vitro studies suggest various mechanisms of antibiotic persistence, but their invivo relevance remains unclear because of the difficulty of studying scarce pathogen survivors in complex host tissues. Here, we localized and characterized rare surviving Salmonella in mouse spleen using high-resolution whole-organ tomography. Chemotherapy cleared >99.5% of the Salmonella but was inefficient against a small Salmonella subset in the white pulp. Previous models could not explain these findings: drug exposure was adequate, Salmonella continued to replicate, and host stresses induced only limited Salmonella drug tolerance. Instead, antimicrobial clearance required support of Salmonella-killing neutrophils and monocytes, and the density of such cells was lower in the white pulp than in other spleen compartments containing higher Salmonella loads. Neutrophil densities declined further during treatment in response to receding Salmonella loads, resulting in insufficient support for Salmonella clearance from the white pulp and eradication failure. However, adjunctive therapies sustaining inflammatory support enabled effective clearance. These results identify uneven Salmonella tissue colonization and spatiotemporal inflammation dynamics as main causes of Salmonella persistence and establish a powerful approach to investigate scarce but impactful pathogen subsets in complex host environments.

Flagella-Mediated Antibiotic Persistence: A Scientific Enigma

Flagella-Mediated Antibiotic Persistence: A Scientific Enigma

The utilization of Blaptica dubia cockroaches as an in vivo model to test antibiotic efficacy

Insects are now well recognized as biologically relevant alternative hosts for dozens of mammalian pathogens and they are routinely used in microbial pathogenesis studies. Unfortunately, these models have yet to be incorporated into the drug development pipeline. The purpose of this work was to begin to evaluate the utility of orange spotted (Blaptica dubia) cockroaches in early antibiotic characterization. To determine whether these model hosts could exhibit mortality when infected with bacteria that are pathogenic to humans, we subjected B. dubia roaches to a range of infectious doses of Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii to identify the medial lethal dose. These results showed that lethal disease did not develop following infection of high doses of S. aureus, and A. baumannii. However, cockroaches infected with E. coli and K. pneumoniae succumbed to infection (LD50s of 5.82 × 106 and 2.58 × 106 respectively) suggesting that this model may have limitations based on pathogen specificity. However, because these cockroaches were susceptible to infection from E. coli and K. pneumoniae, we used these bacterial strains for subsequent antibiotic characterization studies. These studies suggested that β-lactam antibiotic persistence and dose was associated with reduction of hemolymph bacterial burden. Moreover, our data indicated that the reduction of bacterial CFU was directly due to the drug activity. Altogether, this work suggests that the orange-spotted cockroach infection model provides an alternative in vivo setting from which antibiotic efficacy can be evaluated.

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.

Observation of universal ageing dynamics in antibiotic persistence.

Stress responses allow cells to adapt to changes in external conditions by activating specific pathways1. Here we investigate the dynamics of single cells that were subjected to acute stress that is too strong for a regulated response but not lethal. We show that when the growth of bacteria is arrested by acute transient exposure to strong inhibitors, the statistics of their regrowth dynamics can be predicted by a model for the cellular network that ignores most of the details of the underlying molecular interactions. We observed that the same stress, applied eitherabruptly or gradually, can lead to totally different recovery dynamics. By measuring the regrowth dynamics after stress exposure on thousands of cells, we show that the model can predict the outcome of antibiotic persistence measurements. Our results may account for the ubiquitous antibiotic persistence phenotype2, as well as for the difficulty in attempts to link it to specific genes3. More generally, our approach suggests that two different cellular states can be observed under stress: a regulated state, which prepares cells for fast recovery, and a disrupted cellular state due to acute stress, with slow and heterogeneous recovery dynamics. The disrupted state may be described by general properties of large random networks rather than by specific pathway activation. Better understanding of the disrupted state could shed new light on the survival and evolution of cells under stress.

Many birds with one stone: targeting the (p)ppGpp signaling pathway of bacteria to improve antimicrobial therapy.

Winning the war against resistant bacteria will require a change of paradigm in antibiotic discovery. A promising new direction is the targeting of non-essential pathways required for successful infection, such as quorum-sensing, virulence, and biofilm formation. Similarly important will be strategies to prevent or revert antibiotic resistance. Here, we argue that the (p)ppGpp signaling pathway should be a prime target of this effort, since its inactivation could potentially achieve all these goals simultaneously. The hyperphosphorylated guanine nucleotide (p)ppGpp is an ancient and universal second messenger of bacteria that has pleotropic effects on the physiology of these organisms. Initially described as a stress signal-an alarmone-it is now clear that (p)ppGpp plays a more general and fundamental role in bacterial adaptation, by integrating multiple internal and environmental signals to establish the optimal balance between growth and maintenance functions at any given time. Given such centrality, perturbation of the (p)ppGpp pathway will affect bacteria in multiple ways, from the ability to adjust metabolism to the available nutrients to the capacity to differentiate into developmental forms adapted to colonize different niches. Here, we provide an overview of the (p)ppGpp pathway, how it affects bacterial growth, survival and virulence, and its connection with antibiotic tolerance and persistence. We will emphasize the dysfunctions of cells living without (p)ppGpp and finalize by reviewing the efforts and prospects of developing inhibitors of this pathway, and how these could be employed to improve current antibiotic therapy.

The vulnerable versatility of Salmonella antibiotic persisters during infection

Tolerance and persistence are superficially similar phenomena by which bacteria survive bactericidal antibiotics. It is assumed that the same physiology underlies survival of individual tolerant and persistent bacteria. However, by comparing tolerance and persistence during Salmonella Typhimurium infection, we reveal that these two phenomena are underpinned by different bacterial physiologies. Multidrug-tolerant mutant Salmonella enter a near-dormant state protected from immune-mediated genotoxic damages. However, the numerous tolerant cells, optimized for survival, lack the capabilities necessary to initiate infection relapse following antibiotic withdrawal. In contrast, persisters retain an active state. This leaves them vulnerable to accumulation of macrophage-induced dsDNA breaks but concurrently confers the versatility to initiate infection relapse if protected by RecA-mediated DNA repair. Accordingly, recurrent, invasive, non-typhoidal Salmonella clinical isolates display hallmarks of persistence rather than tolerance during antibiotic treatment. Our study highlights the complex trade-off that antibiotic-recalcitrant Salmonella balance to act as a reservoir for infection relapse.

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters.

Antibiotic persistence is a phenomenon in which rare cells of a clonal bacterial population can survive antibiotic doses that kill their kin, even though the entire population is genetically susceptible. With antibiotic treatment failure on the rise, there is growing interest in understanding the molecular mechanisms underlying bacterial phenotypic heterogeneity and antibiotic persistence. However, elucidating these rare cell states can be technically challenging. The advent of single-cell techniques has enabled us to observe and quantitatively investigate individual cells in complex, phenotypically heterogeneous populations. In this review, we will discuss current technologies for studying persister phenotypes, including fluorescent tags and biosensors used to elucidate cellular processes; advances in flow cytometry, mass spectrometry, Raman spectroscopy, and microfluidics that contribute high-throughput and high-content information; and next-generation sequencing for powerful insights into genetic and transcriptomic programs. We will further discuss existing knowledge gaps, cutting-edge technologies that can address them, and how advances in single-cell microbiology can potentially improve infectious disease treatment outcomes.

Diatomaceous earth/zinc oxide micro-composite assisted antibiotics in fungal therapy

As the second wave of COVID-19 hits South Asia, an increasing deadly complication ‘fungal infections (such as Mycosis, Candida and Aspergillus) outbreak’ has been raised concern about the insufficient technologies and medicals for its diagnosis and therapy. Biosilica based nano-therapy can be used for therapeutic efficacy, yet their direct role as antibiotic agent with biocompatibility and stability remains unclear. Here, we report that a diatomaceous earth (DE) framework semiconductor composite conjugated DE and in-house synthesized zinc oxide (DE-ZnO), as an antibiotic agent for the enhancement of antibiotic efficacy and persistence. We found that the DE-ZnO composite had enhanced antibiotic activity against fungi (A. fumigatus) and Gram-negative bacteria (E. coli, S. enterica). The DE-ZnO composite provides enhancing large surface areas for enhancement of target pathogen binding affinity, as well as produces active ions including reactive oxygen species and metal ion for breaking the cellular network of fungi and Gram-negative bacteria. Additionally, the toxicity of DE-ZnO with 3 time less amount of dosage is 6 times lower than the commercial SiO2-ZnO. Finally, a synergistic effect of DE-ZnO and existing antifungal agents (Itraconazole and Amphotericin B) showed a better antifungal activity, which could be reduced the side effects due to the antifungal agents overdose, than a single antibiotic agent use. We envision that this DE-ZnO composite can be used to enhance antibiotic activity and its persistence, with less-toxicity, biocompatibility and high stability against fungi and Gram-negative bacteria which could be a valuable candidate in medical science and industrial engineering.

Scan4CFU: Low-cost, open-source bacterial colony tracking over large areas and extended incubation times

A hallmark of bacterial populations cultured in vitro is their homogeneity of growth, where the majority of cells display identical growth rate, cell size and content. Recent insights, however, have revealed that even cells growing in exponential growth phase can be heterogeneous with respect to variables typically used to measure cell growth. Bacterial heterogeneity has important implications for how bacteria respond to environmental stresses, such as antibiotics. The phenomenon of antimicrobial persistence, for example, has been linked to a small subpopulation of cells that have entered into a state of dormancy where antibiotics are no longer effective. While methods have been developed for identifying individual non-growing cells in bacterial cultures, there has been less attention paid to how these cells may influence growth in colonies on a solid surface. In response, we have developed a low-cost, open-source platform to perform automated image capture and image analysis of bacterial colony growth on multiple nutrient agar plates simultaneously. The descriptions of the hardware and software are included, along with details about the temperature-controlled growth chamber, high-resolution scanner, and graphical interface to extract and plot the colony lag time and growth kinetics. Experiments were conducted using a wild type strain of Escherichia coli K12 to demonstrate the feasibility and operation of our setup. By automated tracking of bacterial growth kinetics in colonies, the system holds the potential to reveal new insights into understanding the impact of microbial heterogeneity on antibiotic resistance and persistence.

Effects of Partial Organic Substitution for Chemical Fertilizer on Antibiotic Residues in Peri-Urban Agricultural Soil in China.

Recycling of organic wastes in agricultural ecosystems to partially substitute chemical fertilizer is recommended to improve soil productivity and alleviate environmental degradation. However, livestock manure- and sewage sludge-derived amendments are widely known to potentially carry antibiotic residues. The aim of this study is to investigate how substituting organic fertilizer for chemical fertilizer affects soil quality and antibiotic residues in agricultural soil, as well as their tradeoffs. A field experiment was conducted with the different treatments of pig manure and sewage sludge as typical organic fertilizers at equal total nitrogen application rates. The analysis of variance showed that the increments on the levels of residual antibiotics in the agricultural soils due to organic substitution for chemical fertilizer by pig manure and sewage sludge were observed. The antibiotic residues ranged from 13.73 to 76.83 ng/g for all treatments. Partial organic substitution significantly increased the sequestration of antibiotics in agricultural soil by 138.1~332.5%. Organic substitution will also significantly improve soil quality, especially for nutrient availability. Based on principal component analysis, organic substitution will strongly affected soil quality and antibiotic contamination. Pearson’s correlation showed that soil physicochemical properties had significant correlations with concentrations of antibiotics in soil, indicating organic fertilizers can promote the persistence of antibiotics in soil by modifying soil quality. To balance the benefits and risks, appropriate management practices of organic fertilizers should be adopted.

Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments.

Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections; however, none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial persisters when inoculated on abiotic surfaces to form a film of bacterial agglomerates (biofilm-like environment). Our data suggest that efflux pumps were possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persisters arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The persister cells showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis revealed that susceptible cells express higher levels of ribosome proteins. It is remarkable that previous studies have reported the recovery of S. pyogenes viable cells from tissue biopsies of patients presented with GAS invasive infections and submitted to therapy with antibiotics. The persistence phenomenon described herein brings new insights into the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving protein synthesis inhibition, cell growth impairment and efflux pumps seem to play roles in the formation of antimicrobial persisters in S. pyogenes.

Histone-like nucleoid-structuring protein (H-NS) regulatory role in antibiotic resistance in Acinetobacter baumannii

In the multidrug resistant (MDR) pathogen Acinetobacter baumannii the global repressor H-NS was shown to modulate the expression of genes involved in pathogenesis and stress response. In addition, H-NS inactivation results in an increased resistance to colistin, and in a hypermotile phenotype an altered stress response. To further contribute to the knowledge of this key transcriptional regulator in A. baumannii behavior, we studied the role of H-NS in antimicrobial resistance. Using two well characterized A. baumannii model strains with distinctive resistance profile and pathogenicity traits (AB5075 and A118), complementary transcriptomic and phenotypic approaches were used to study the role of H-NS in antimicrobial resistance, biofilm and quorum sensing gene expression. An increased expression of genes associated with β-lactam resistance, aminoglycosides, quinolones, chloramphenicol, trimethoprim and sulfonamides resistance in the Δhns mutant background was observed. Genes codifying for efflux pumps were also up-regulated, with the exception of adeFGH. The wild-type transcriptional level was restored in the complemented strain. In addition, the expression of biofilm related genes and biofilm production was lowered when the transcriptional repressor was absent. The quorum network genes aidA, abaI, kar and fadD were up-regulated in Δhns mutant strains. Overall, our results showed the complexity and scope of the regulatory network control by H-NS (genes involved in antibiotic resistance and persistence). These observations brings us one step closer to understanding the regulatory role of hns to combat A. baumannii infections.

Proteome Dynamics during Antibiotic Persistence and Resuscitation

ABSTRACTDuring antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here, we used sustained addition of stable isotope-labeled lysine to selectively label the proteome during hipA-induced persistence and hipB-induced resuscitation of Escherichia coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-h measurement of label incorporation allowed detection of over 500 newly synthesized proteins in viable cells, demonstrating low but widespread protein synthesis during persistence. Many essential proteins were newly synthesized, and several ribosome-associated proteins such as RaiA and Sra showed high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized the ribosome-splitting GTPase HflX and various ABC transporters, restored translation machinery, and resumed metabolism by inducing glycolysis and biosynthesis of amino acids.IMPORTANCE While bactericidal antibiotics typically require actively growing cells to exploit their function, persister cells are slowly replicating which makes them tolerant to the lethal action of antimicrobials. Here, we used an established in vitro model of bacterial persistence based on overexpression of the paradigm toxin-antitoxin (TA) system hipA/hipB to devise a generic method for temporal analysis of protein synthesis during toxin-induced persistence and antitoxin-mediated resuscitation. Our time-resolved, 24-h measurement of label incorporation demonstrated low but widespread protein synthesis during persistence. At the onset of resuscitation, cells restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. Our study provides the first global analysis of protein synthesis in persisting and resuscitating bacterial cells, and as such, presents an unprecedented resource to study the processes governing antibiotic persistence.

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens.

Bacterial gene expression is under the control of a large set of molecules acting at multiple levels. In addition to the transcription factors (TFs) already known to be involved in global regulation of gene expression, small regulatory RNAs (sRNAs) are emerging as major players in gene regulatory networks, where they allow environmental adaptation and fitness. Developments in high-throughput screening have enabled their detection in the entire bacterial kingdom. These sRNAs influence a plethora of biological processes, including but not limited to outer membrane synthesis, metabolism, TF regulation, transcription termination, virulence, and antibiotic resistance and persistence. Almost always noncoding, they regulate target genes at the post-transcriptional level, usually through base-pair interactions with mRNAs, alone or with the help of dedicated chaperones. There is growing evidence that sRNA-mediated mechanisms of actions are far more diverse than initially thought, and that they go beyond the so-called cis- and trans-encoded classifications. These molecules can be derived and processed from 5' untranslated regions (UTRs), coding or non-coding sequences, and even from 3' UTRs. They usually act within the bacterial cytoplasm, but recent studies showed sRNAs in extracellular vesicles, where they influence host cell interactions. In this review, we highlight the various functions of sRNAs in bacterial pathogens, and focus on the increasing examples of widely diverse regulatory mechanisms that might compel us to reconsider what constitute the sRNA.

Phenotypic heterogeneity in persisters: a novel 'hunker' theory of persistence.

ABSTRACTPersistence has been linked to treatment failure since its discovery over 70 years ago and understanding formation, nature and survival of this key antibiotic refractory subpopulation is crucial to enhancing treatment success and combatting the threat of antimicrobial resistance (AMR). The term ‘persistence’ is often used interchangeably with other terms such as tolerance or dormancy. In this review we focus on ‘antibiotic persistence’ which we broadly define as a feature of a subpopulation of bacterial cells that possesses the non-heritable character of surviving exposure to one or more antibiotics; and persisters as cells that possess this characteristic. We discuss novel molecular mechanisms involved in persister cell formation, as well as environmental factors which can contribute to increased antibiotic persistence in vivo, highlighting recent developments advanced by single-cell studies. We also aim to provide a comprehensive model of persistence, the ‘hunker’ theory which is grounded in intrinsic heterogeneity of bacterial populations and a myriad of ‘hunkering down’ mechanisms which can contribute to antibiotic survival of the persister subpopulation. Finally, we discuss antibiotic persistence as a ‘stepping-stone’ to AMR and stress the urgent need to develop effective anti-persister treatment regimes to treat this highly clinically relevant bacterial sub-population.

Bacterial toxin-antitoxin modules: classification, functions, and association with persistence.

Toxin-antitoxin (TA) modules are ubiquitous gene loci among bacteria and are comprised of a toxin part and its cognate antitoxin part. Under normal physiological conditions, antitoxin counteracts the toxicity of the toxin whereas, during stress conditions, TA modules play a crucial role in bacterial physiology through involvement in the post-segregational killing, abortive infection, biofilms, and persister cell formation. Most of the toxins are proteinaceous that affect translation or DNA replication, although some other intracellular molecular targets have also been described. While antitoxins may be a protein or RNA, that generally neutralizes its cognate toxin by direct interaction or with the help of other signaling elements and thus helps in the TA module regulation. In this review, we have discussed the current state of the multifaceted TA (type I-VIII) modules by highlighting their classification and specific targets. We have also discussed the presence of TA modules in the various pathogens and their role in antibiotic persistence development as well as biofilm formation, by influencing the different cellular processes. In the end, assembling knowledge about ubiquitous TA systems from pathogenic bacteria facilitated us to propose multiple novel antibacterial strategies involving artificial activation of TA modules.

The RNA Chaperone Hfq Participates in Persistence to Multiple Antibiotics in the Fish Pathogen Yersinia ruckeri.

Yersinia ruckeri causes outbreaks of enteric redmouth disease in salmon aquaculture all over the world. The transient antibiotic tolerance exhibited by bacterial persisters is commonly thought to be responsible for outbreaks; however, the molecular factors underlying this behavior have not been explored in Y. ruckeri. In this study, we investigated the participation of the RNA chaperone Hfq from Y. ruckeri in antibiotic persistence. Cultures of the hfq-knockout mutant (Δhfq) exhibited faster replication, increased ATP levels and a more reductive environment than the wild type. The growth curves of bacteria exposed to sublethal concentrations of ampicillin, oxolinic acid, ciprofloxacin and polymyxin B revealed a greater susceptibility for the Δhfq strain. The time-kill curves of bacteria treated with the antibiotics mentioned above and florfenicol, using inoculums from exponential, stationary and biofilm cultures, demonstrated that the Δhfq strain has significant defects in persister cells production. To shed more light on the role of Hfq in antibiotic persistence, we analyzed its dependence on the (p)ppGpp synthetase RelA by determining the persister cells production in the absence of the relA gene. The ΔrelA and ΔrelAΔhfq strains displayed similar defects in persister cells formation, but higher than Δhfq strain. Similarly, stationary cultures of the ΔrelA and ΔrelAΔhfq strains exhibited comparable levels of ATP but higher than that of the Δhfq strain, indicating that relA is epistatic over hfq. Taken together, our findings provide valuable information on antibiotic persistence in Y. ruckeri, shedding light on the participation of Hfq in the persistence phenomenon.

Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response.

ABSTRACTBacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNAPhe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence.