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The proton-motive force (PMF), consisting of a pH gradient and a membrane potential (ΔΨ) underpins many processes essential to bacterial growth and/or survival. Yet bacteria often enter a bioenergetically diminished state characterized by a low PMF. Consequently, they have increased tolerance for diverse stressors, including clinical antibiotics. Despite the ubiquity of low metabolic rates in the environment, the extent to which bacteria have agency over entry into such a low-bioenergetic state has received relatively little attention. Here, we tested the hypothesis that production of redox-active metabolites (RAMs) could drive such a physiological transition. Pseudomonas aeruginosa is an opportunistic pathogen that produces phenazines, model RAMs that are highly toxic in the presence of molecular oxygen (O2). Under oxic conditions, the phenazines pyocyanin and phenazine-1- carboximide, as well as toxoflavin--a RAM produced by Burkholderia species--suppress the ΔΨ in distinct ways across distributions of single cells, reduce the efficiency of proton pumping, and lower cellular adenosine-triphosphate (ATP) levels. In planktonic culture, the degree and rate by which each RAM lowers the ΔΨ correlates with the protection it confers against antibiotics that strongly impact cellular energy flux. This bioenergetic suppression requires the RAM's presence and corresponds to its cellular reduction rate and abiotic oxidation rate by O2; it can be reversed by increasing the ΔΨ with nigericin. RAMs similarly impact the bioenergetic state of cells in (hyp)oxic biofilm aggregates. Collectively, these findings demonstrate that bacteria can suppress their bioenergetic state by the production of endogenous toxins in a manner that bolsters stress resilience. [ABSTRACT FROM AUTHOR]

Background: The interactions between bacterial pathogens and host cells are characterized by a multitude of complexities, leading to a wide range of heterogeneous outcomes. Despite extensive research, we still have a limited understanding of how bacterial motility in complex environments impacts their ability to tolerate antibiotics and adhere to mammalian cell surfaces. The challenge lies in unraveling the complexity of these interactions and developing quantitative microscopy approaches to predict the behavior of bacterial populations. Results: To address this challenge, we directed our efforts towards Pseudomonas aeruginosa, a pathogenic bacterium known for producing thick films in the lungs of cystic fibrosis patients, and Escherichia coli, used as a proof of concept to develop and demonstrate our single-cell tracking approaches. Our results revealed that P. aeruginosa exhibits diverse and complex interactions on mammalian cell surfaces, such as adhesion, rotational motion, and swimming, unlike the less interactive behavior of Escherichia coli. Our analysis indicated that P. aeruginosa demonstrated lower mean-squared displacement (MSD) values and greater adherence to mammalian cells compared to E. coli, which showed higher MSD slopes and less frequent adherence. Genetic mutations in membrane proteins of P. aeruginosa resulted in altered displacement patterns and reduced adhesion, with the ΔfliD mutant displaying a more Gaussian displacement distribution and significantly less adherence to mammalian cells. Adhesion and tolerance mechanisms are diverse and complex, potentially involving distinct pathways; however, our findings highlight the therapeutic potential of targeting the fliD gene (encoding a critical flagellum protein), as its deletion not only reduced adherence but also antibiotic tolerance. Conclusions: Overall, our findings underscore the importance of single cell tracking in accurately assessing bacterial behavior over short time periods and highlight its significant potential in guiding effective intervention strategies. [ABSTRACT FROM AUTHOR]

Introduction: Staphylococcus aureus is one of the chief pathogens that cause chronic and recurrent infections. Failure of the antibiotics to curb the infections contributes to relapse and is an important reason for the high mortality rate. Treatment failure may also be due to antibiotic tolerance. Accumulating evidence suggests that t the host immune environment plays an important role in inducing antibiotic tolerance of S. aureus, but research in this area has been limited. Methods: In this study, the minimum inhibitory concentration (MIC) of the antibiotics against S. aureus was determined using the standard broth microdilution method. The study evaluated whether itaconate induces antibiotic tolerance in S. aureus through an antibiotic bactericidal activity assay. The effect of itaconate on the growth of S. aureus was evaluated by monitoring the growth of S. aureus in medium supplemented with itaconate. Additionally, RNA sequencing and metabolomics analyses were used to determine transcriptional and metabolic changes in S. aureus when exposed to itaconate. Results and discussion: According to the study, we found that the immune metabolite itaconate can induce tolerance in both methicillin-resistant and -susceptible S. aureus to aminoglycosides. When S. aureus was exposed to itaconate, its growth slowed down and transcriptomic and metabolomic alterations associated with decreased energy metabolism, including the tricarboxylate cycle, glycolysis, pyruvate metabolism, and arginine biosynthesis, were observed. These changes are associated with aminoglycoside tolerance. This study highlights the role of immune signaling metabolites in bacterial antibiotic tolerance and suggests new strategies to improve antibiotic treatment by modulating the host immune response and stimulating the metabolism of bacteria. [ABSTRACT FROM AUTHOR]

Introduction: Helicobacter pylori causes chronic gastric diseases in nearly 50% of people around the world. It is suggested that biofilm formation has a pronounced effect on the dynamic resistance spread and recurrence of these infections. Methods: To mimic the scenario of therapeutic ineffectiveness, we investigated the impact of sub-minimal inhibitory concentrations (sub-MICs) of antibiotics on the development and parameters of biofilms produced by clinical H. pylori strains. Results: We observed that constant exposure of planktonic forms to metronidazole or levofloxacin stimulated the speed of autoaggregation and the amount of extracellular matrix, resulting in increased dimensions of the developed biofilms. Contrary to this, continuous exposure to clarithromycin negatively affected a number of biofilm-related reactions and led to the biofilm-weakening effect. Through assessing the membrane fatty acid profiles of antibiotic-exposed cells, we confirmed that metronidazole and levofloxacin induced a biofilm-like phenotype, while clarithromycin kept bacteria in a planktonic form. Discussion: Our results suggest that sub-MICs of antibiotics affect the biochemical and biophysical properties of the developing biofilm of H. pylori strains and may impact the effectiveness of antibiotic treatment. [ABSTRACT FROM AUTHOR]

The biofilm formation by various pathogens causes chronic infections and poses severe threats to industry, healthcare, and society. They can form biofilm on surfaces of medical implants, heart valves, pacemakers, contact lenses, vascular grafts, urinary catheters, dialysis catheters, etc. These biofilms play a central role in bacterial persistence and antibiotic tolerance. Biofilm formation occurs in a series of steps, and any interference in these steps can prevent its formation. Therefore, the hunt to explore and develop effective anti-biofilm strategies became necessary to decrease the rate of biofilm-related infections. In this review, we highlighted and discussed the current therapeutic approaches to eradicate biofilm formation and combat drug resistance by anti-biofilm drugs, phytocompounds, antimicrobial peptides (AMPs), antimicrobial lipids (AMLs), matrix-degrading enzymes, nanoparticles, phagebiotics, surface coatings, photodynamic therapy (PDT), riboswitches, vaccines, and antibodies. The clinical validation of these findings will provide novel preventive and therapeutic strategies for biofilm-associated infections to the medical world. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a bacterial species that is commonly found colonising healthy individuals but that presents a paradoxical nature: simultaneously, it can migrate within the body and cause a range of diseases. Many of these become chronic by resisting immune responses, antimicrobial treatment, and medical intervention. In part, this ability to persist can be attributed to the adoption of multiple cell types within a single cellular population. These dynamics in the S. aureus cell population could be the result of its interplay with host cells or other co-colonising bacteria—often coagulase-negative Staphylococcal (CoNS) species. Further understanding of the unique traits of S. aureus alternative cell types, the drivers for their selection or formation during disease, as well as their presence even during non-pathological colonisation could advance the development of diagnostic tools and drugs tailored to target specific cells that are eventually responsible for chronic infections. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both the exponential and stationary phases. Moreover, we could differentiate between triggered (induced using antibiotics such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation. [ABSTRACT FROM AUTHOR]

Antibiotic tolerance enables antibiotic-susceptible bacteria to withstand prolonged exposure to high concentrations of antibiotics. Although antibiotic tolerance presents a major challenge for public health, its underlying molecular mechanisms remain unclear. Previously, we have demonstrated that Campylobacter jejuni develops tolerance to clinically important antibiotics, including ciprofloxacin and tetracycline. To identify cellular responses associated with antibiotic tolerance, RNA-sequencing was conducted on C. jejuni after inducing antibiotic tolerance through exposure to ciprofloxacin or tetracycline. Additionally, knockout mutants were constructed for genes exhibiting significant changes in expression levels during antibiotic tolerance. The genes involved in protein chaperones, bacterial motility, DNA repair system, drug efflux pump, and iron homeostasis were significantly upregulated during antibiotic tolerance. These mutants displayed markedly reduced viability compared to the wild-type strain, indicating the critical role of these cellular responses in sustaining antibiotic tolerance. Notably, the protein chaperone mutants exhibited increased protein aggregation under antibiotic treatment, suggesting that protein chaperones play a critical role in managing protein disaggregation and facilitating survival during antibiotic tolerance. Our findings demonstrate that various cellular defense mechanisms collectively contribute to sustaining antibiotic tolerance in C. jejuni, providing novel insights into the molecular mechanisms underlying antibiotic tolerance.

IntroductionHelicobacter pylori causes chronic gastric diseases in nearly 50% of people around the world. It is suggested that biofilm formation has a pronounced effect on the dynamic resistance spread and recurrence of these infections.MethodsTo mimic the scenario of therapeutic ineffectiveness, we investigated the impact of sub-minimal inhibitory concentrations (sub-MICs) of antibiotics on the development and parameters of biofilms produced by clinical H. pylori strains.ResultsWe observed that constant exposure of planktonic forms to metronidazole or levofloxacin stimulated the speed of autoaggregation and the amount of extracellular matrix, resulting in increased dimensions of the developed biofilms. Contrary to this, continuous exposure to clarithromycin negatively affected a number of biofilm-related reactions and led to the biofilm-weakening effect. Through assessing the membrane fatty acid profiles of antibiotic-exposed cells, we confirmed that metronidazole and levofloxacin induced a biofilm-like phenotype, while clarithromycin kept bacteria in a planktonic form.DiscussionOur results suggest that sub-MICs of antibiotics affect the biochemical and biophysical properties of the developing biofilm of H. pylori strains and may impact the effectiveness of antibiotic treatment.

ABSTRACT: This study aimed to probe if xylooligosaccharide (XOS) could act as an antimetabolite to impact the cell cycle and antibiotic tolerance of avian pathogenic Escherichia coli (APEC). We firstly measured the bacteriostasis of XOS against APEC O78 and its effect on the growth of APEC O78 growing on different medium. Afterwards, the effects of XOS on xylose operon activation along with the cell cycle and antibiotic tolerance of APEC O78 were analyzed. The results showed that XOS caused no inhibitory circle against APEC O78 and did not affect (P > 0.05) the growth of APEC O78 growing on LB medium. Besides, APEC O78 was unable to grow on M9 medium (carbon-free) added with XOS. However, XOS exerted a similar role as xylose in increasing (P < 0.05) the expression of certain xylose operon genes including xylose isomerase (XylA)-encoding gene (xylA) and xylose-binding periplasmic protein (XylF)-encoding gene (xylF) in APEC O78. The molecular docking simulation revealed that the major monomer components (xylobiose, xylotriose and xylotetraose) of XOS had stable binding potentials to both XylA and XylF proteins of E. coli, as supported by the low binding free energy and the formation of considerable hydrogen bonds between them. The subsequent analysis showed that XOS altered certain cell cycle-related genes expression, especially elevated (P < 0.05) nrdB expression and decreased ihfB expression to a degree. Moreover, XOS played a similar role as 2-deoxy-glucose (a glucose analogue serving as a typical antimetabolite) in lowering (P < 0.05) the number of ampicillin-tolerant APEC O78. Collectively, XOS had no direct bacteriostasis against APEC and could not be metabolized/utilized by APEC O78. However, it might become an analogue of xylose and then activate xylose transport- and metabolism-related proteins in APEC O78, thus functioning as a potential antimetabolite and exerting antimetabolic actions. This could at least partially interpret the observed roles of XOS in interfering with the cell cycle and diminishing the antibiotic tolerance of APEC O78. The above findings expand the knowledges about the functions of XOS and provide a basis for exploring novel strategies to reduce the antibiotic tolerance of APEC.

For model bacteria, genetic drug resistance usually arises from antibiotic-tolerant subpopulations, but whether this is true for the globally important pathogen Mycobacterium tuberculosis-the cause of tuberculosis-is not known. Here, we discuss a recent article by Sebastian et al. (J. Sebastian, A. Thomas, C. Levine, R. Shrestha, et al., mBio 14:e0279522, 2023, 10.1128/mbio.02795-22) which leverages a robotic transwell microtiter experimental system coupled with deep sequencing of a barcoded library of M. tuberculosis to answer this question for rifampicin resistance. The authors investigate two distinct forms of antibiotic-tolerant subpopulations-classical tolerance, characterized by prolonged minimum duration of killing, and "differentially detectable" (DD) bacilli that are viable but can be recovered only in liquid medium as opposed to plating. They demonstrate that, indeed, resistance arises preferentially from both rifampicin-tolerant subpopulations, though earlier in the DD population. Use of barcoded libraries and parallel culture systems shows promise in investigating phenotypes mediated by minority subpopulations of bacteria such as development of antibiotic resistance.

Within epithelial cells, Pseudomonas aeruginosa depends on its type III secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole-associated bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light-electron microscopy (CLEM), we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not their cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant (ΔexsA) unable to escape vacuoles phenocopied vacuole-associated subpopulations in wild-type PAO1-infected cells, with results revealing that epithelial cell death depended upon bacterial viability. Intravital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular subpopulations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic subpopulations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. IMPORTANCE Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative subpopulation of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this subpopulation, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing that it diversifies into multiple populations with T3SS-negative members enabling persistence while rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

Background: The commensal skin bacterium Cutibacterium acnes plays a role in the pathogenesis of acne vulgaris and also causes opportunistic infections of implanted medical devices due to its ability to form biofilms on biomaterial surfaces. Poly-β-(1→6)-N-acetyl-D-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm formation and biocide resistance in a wide range of bacterial pathogens. The objective of this study was to determine whether C. acnes produces PNAG, and whether PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. Methods: PNAG was detected on the surface of C. acnes cells by fluorescence confocal microscopy using the antigen-specific human IgG1 monoclonal antibody F598. PNAG was detected in C. acnes biofilms by measuring the ability of the PNAG-specific glycosidase dispersin B to inhibit biofilm formation and sensitize biofilms to biocide killing. Results: Monoclonal antibody F598 bound to the surface of C. acnes cells. Dispersin B inhibited attachment of C. acnes cells to polystyrene rods, inhibited biofilm formation by C. acnes in glass and polypropylene tubes, and sensitized C. acnes biofilms to killing by benzoyl peroxide and tetracycline. Conclusion: C. acnes produces PNAG, and PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. PNAG may play a role in C. acnes skin colonization, biocide resistance, and virulence in vivo. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug tolerance. Increased intracellular polyphosphate (polyP) in Mtb enhances tolerance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB. [ABSTRACT FROM AUTHOR]

Infections caused by Staphylococcus aureus, notably methicillin‐resistant S. aureus (MRSA), pose treatment challenges due to its ability to tolerate antibiotics and develop antibiotic resistance. The former, a mechanism independent of genetic changes, allows bacteria to withstand antibiotics by altering metabolic processes. Here, a potent methylazanediyl bisacetamide derivative, MB6, is described, which selectively targets MRSA membranes over mammalian membranes without observable resistance development. Although MB6 is effective against growing MRSA cells, its antimicrobial activity against MRSA persisters is limited. Nevertheless, MB6 significantly potentiates the bactericidal activity of gentamicin against MRSA persisters by facilitating gentamicin uptake. In addition, MB6 in combination with daptomycin exhibits enhanced anti‐persister activity through mutual reinforcement of their membrane‐disrupting activities. Crucially, the "triple" combination of MB6, gentamicin, and daptomycin exhibits a marked enhancement in the killing of MRSA persisters compared to individual components or any double combinations. These findings underscore the potential of MB6 to function as a potent and selective membrane‐active antimicrobial adjuvant to enhance the efficacy of existing antibiotics against persister cells. The molecular mechanisms of MB6 elucidated in this study provide valuable insights for designing anti‐persister adjuvants and for developing new antimicrobial combination strategies to overcome the current limitations of antibiotic treatments. [ABSTRACT FROM AUTHOR]

ABSTRACT Antibiotic therapy alters bacterial abundance and metabolism in the gut microbiome, leading to dysbiosis and opportunistic infections. Bacteroides thetaiotaomicron (Bth) is both a commensal in the gut and an opportunistic pathogen in other body sites. Past work has shown that Bth responds to β-lactam treatment differently depending on the metabolic environment both in vitro and in vivo. Studies of other bacteria show that an increase in respiratory metabolism independent of growth rate promotes susceptibility to bactericidal antibiotics. We propose that Bth enters a protected state linked to an increase in polysaccharide utilization and a decrease in the use of simple sugars. Here, we apply antibiotic susceptibility testing, transcriptomic analysis, and genetic manipulation to characterize this polysaccharide-mediated tolerance (PM tolerance) phenotype. We found that a variety of mono- and disaccharides increased the susceptibility of Bth to several different β-lactams compared to polysaccharides. Transcriptomics indicated a metabolic shift from reductive to oxidative branches of the tricarboxylic acid cycle on polysaccharides. Accordingly, supplementation with intermediates of central carbon metabolism had varying effects on PM tolerance. Transcriptional analysis also showed a decrease in the expression of the electron transport chain (ETC) protein NQR and an increase in the ETC protein NUO, when given fiber versus glucose. Deletion of NQR increased Bth susceptibility while deletion of NUO and a third ETC protein NDH2 had no effect. This work confirms that carbon source utilization modulates antibiotic susceptibility in Bth and that anaerobic respiratory metabolism and the ETC play an essential role.IMPORTANCEAntibiotics are indispensable medications that revolutionized modern medicine. However, their effectiveness is challenged by a large array of resistance and tolerance mechanisms. Treatment with antibiotics also disrupts the gut microbiome which can adversely affect health. Bacteroides are prevalent in the gut microbiome and yet are frequently involved in anaerobic infections. Thus, understanding how antibiotics affect these bacteria is necessary to implement proper treatment. Recent work has investigated the role of metabolism in antibiotic susceptibility in distantly related bacteria such as Escherichia coli. Using antibiotic susceptibility testing, transcriptomics, and genetic manipulation, we demonstrate that polysaccharides reduce β-lactam susceptibility when compared to monosaccharides. This finding underscores the profound impact of metabolic adaptation on the therapeutic efficacy of antibiotics. In the long term, this work indicates that modulation of metabolism could make Bacteroides more susceptible during infections or protect them in the context of the microbiome.

ABSTRACT Bacteria are involved in numerous interactions during infection and among host-associated microbial populations. Salmonella enterica serovar Typhimurium is a foodborne pathogen of great importance as well as a model organism to study interactions within a microbial community. In this study, we found that S. Typhimurium becomes tolerant to azithromycin when co-cultured with lactobacilli strains. Similarly, acidified media, from cell-free supernatant of lactobacilli cultures for instance, also induced the tolerance of S. Typhimurium to azithromycin. The addition of membrane disruptors restored the normal sensitivity to azithromycin in acidified media, but not when lactobacilli were present. These results suggested that the acidification of the media led to modification in envelope homeostasis, but that a different mechanism promoted the tolerance to azithromycin in the presence of lactobacilli strains. To further understand how lactobacilli strains modify the sensitivity of S. Typhimurium to azithromycin, a high-throughput assay was performed using the single-gene deletion collection of the S. Typhimurium (1) in co-culture with Lacticaseibacillus rhamnosus and (2) in sterile acidic conditions (pH 5.5 media only). As expected, both screens identified genes involved in envelope homeostasis and membrane permeability. Our results also suggest that changes in the metabolism of S. Typhimurium induce the tolerance observed in the presence of L. rhamnosus. Our results thus highlight two different mechanisms by which lactobacilli induce the tolerance of S. Typhimurium to azithromycin.IMPORTANCEThis study provides valuable insights into the intricate interactions between bacteria during infections and within host-associated microbial communities. Specifically, it sheds light on the significant role of lactobacilli in inducing antibiotic tolerance in Salmonella enterica serovar Typhimurium, a critical foodborne pathogen and model organism for microbial community studies. The findings not only uncover the mechanisms underlying this antibiotic tolerance but also reveal two distinct pathways through which strains of lactobacilli might influence Salmonella’s response to antibiotics. Understanding these mechanisms has the potential to enhance our knowledge of bacterial infections and may have implications for the development of strategies to combat antibiotic resistance in pathogens, such as Salmonella. Furthermore, our results underscore the necessity to explore beyond the direct antimicrobial effects of antibiotics, emphasizing the broader microbial community context.

The global issue of antimicrobial resistance poses significant challenges to public health. The World Health Organization (WHO) has highlighted it as a major global health threat, causing an estimated 700,000 deaths worldwide. Understanding the multifaceted nature of antibiotic resistance is crucial for developing effective strategies. Several physiological and biochemical mechanisms are involved in the development of antibiotic resistance. Bacterial cells may escape the bactericidal actions of the drugs by entering a physiologically dormant state known as bacterial persistence. Recent findings in this field suggest that bacterial persistence can be one of the main sources of chronic infections. The antibiotic tolerance developed by the persister cells could tolerate high levels of antibiotics and may give rise to persister offspring. These persister offspring could be attributed to antibiotic resistance mechanisms, especially in chronic infections. This review attempts to shed light on persister-induced antibiotic resistance and the current therapeutic strategies.

Aminoglycoside antibiotics target ribosomes and are effective against a wide range of bacteria. Here, we demonstrated that knockout strains related to energy metabolism in Escherichia coli showed increased tolerance to aminoglycosides during the mid-exponential growth phase. Contrary to expectations, these mutations did not reduce the proton motive force or aminoglycoside uptake, as there were no significant changes in metabolic indicators or intracellular gentamicin levels between wild-type and mutant strains. Our comprehensive proteomics analysis unveiled a noteworthy upregulation of proteins linked to the tricarboxylic acid (TCA) cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research provides valuable insights into the mechanisms of aminoglycoside tolerance, paving the way for novel strategies to combat such cells.

P. aeruginosa biofilms are aggregates of bacteria surrounded by a self-produced matrix which binds to some antibiotics such as aminoglycosides. P. aeruginosa biofilms are tolerant to antibiotics. The treatment of biofilm infections leads to a recurrence of symptoms after finishing antibiotic treatment, although the initial clinical response to the treatment is frequently favorable. There is a concentration gradient of oxygen and nutrients from the surface to the center of biofilms. Surface-located bacteria are multiplying and metabolizing, whereas deeper located bacteria are dormant and tolerant to most antibiotics. Colistin kills dormant bacteria, and combination therapy with colistin and antibiotics which kills multiplying bacteria is efficient in vitro. Some antibiotics such as imipenem induce additional production of the biofilm matrix and of chromosomal beta-lactamase in biofilms. Biofilms present a third Pharmacokinetic/Pharmacodynamic (PK/PD) micro-compartment (first: blood, second: tissue, third: biofilm) which must be taken into consideration when calculations try to predict the antibiotic concentrations in biofilms and thereby the probability of target attainment (PTA) for killing the biofilm. Treating biofilms with hyperbaric oxygen to wake up the dormant cells, destruction of the biofilm matrix, and the use of bacteriophage therapy in combination with antibiotics are promising possibilities which have shown proof of concept in in vitro experiments and in animal experiments.

Staphylococcus aureus is a bacterial species that is commonly found colonising healthy individuals but that presents a paradoxical nature: simultaneously, it can migrate within the body and cause a range of diseases. Many of these become chronic by resisting immune responses, antimicrobial treatment, and medical intervention. In part, this ability to persist can be attributed to the adoption of multiple cell types within a single cellular population. These dynamics in the S. aureus cell population could be the result of its interplay with host cells or other co-colonising bacteria—often coagulase-negative Staphylococcal (CoNS) species. Further understanding of the unique traits of S. aureus alternative cell types, the drivers for their selection or formation during disease, as well as their presence even during non-pathological colonisation could advance the development of diagnostic tools and drugs tailored to target specific cells that are eventually responsible for chronic infections.

The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy is the hallmark of bacterial persistence. In this study, we have screened a transposon library of Mycobacterium smegmatis mc2 155 strain using antibiotic tolerance, survival in mouse macrophages, and biofilm-forming ability of the mutants. Out of 10 thousand clones screened, we selected ten mutants defective in all the three phenotypes. Six mutants showed significantly lower persister abundance under different stress conditions. Insertions in three genes belonging to the pathways of oxidative phosphorylation msmeg_3233 (cydA), biotin metabolism msmeg_3194 (bioB), and oxidative metabolism msmeg_0719, a flavoprotein monooxygenase, significantly reduced the number of live cells, suggesting their role in pathways promoting long-term survival. Another group that displayed a moderate reduction in CFU included a glycosyltransferase, msmeg_0392, a hydrogenase subunit, msmeg_2263 (hybC), and a DNA binding protein, msmeg_2211. The study has revealed potential candidates likely to facilitate the long-term survival of M. smegmatis. The findings offer new targets to develop antibiotics against persisters. Further, investigating the corresponding genes in M. tuberculosis may provide valuable leads in improving the treatment of chronic and persistent tuberculosis infections. [ABSTRACT FROM AUTHOR]

Antibiotics are used to combat the ever-present threat of infectious diseases, but bacteria are continually evolving an assortment of defenses that enable their survival against even the most potent treatments. While the demand for novel antibiotic agents is high, the discovery of a new agent is exceedingly rare. We chose to focus on understanding how different signal transduction pathways in the gram-negative bacterium Escherichia coli (E. coli) influence the sensitivity of the organism to antibiotics from three different classes: tetracycline, chloramphenicol, and levofloxacin. Using the PHL628 strain of E. coli, we exogenously overexpressed two transcription factors, FliA and RpoH.I54N (a constitutively active mutant), to determine their influence on the minimum inhibitory concentration (MIC) and minimum duration of killing (MDK) concentration for each of the studied antibiotics. We hypothesized that activating these pathways, which upregulate genes that respond to specific stressors, could mitigate bacterial response to antibiotic treatment. We also compared the exogenous overexpression of the constitutively active RpoH mutant to thermal heat shock that has feedback loops maintained. While FliA overexpression had no impact on MIC or antibiotic tolerance, RpoH.I54N overexpression reduced the MIC for tetracycline and chloramphenicol but had no independent impact on antibiotic tolerance. Thermal heat shock alone also did not affect MIC or antibiotic tolerance. L-arabinose, the small molecule used to induce expression in our system, unexpectedly independently increased the MICs for tetracycline (>2-fold) and levofloxacin (3-fold). Additionally, the combination of thermal heat shock and arabinose provided a synergistic, 5-fold increase in MIC for chloramphenicol. Arabinose increased the tolerance, as assessed by MDK99, for chloramphenicol (2-fold) and levofloxacin (4-fold). These experiments highlight the potential of the RpoH pathway to modulate antibiotic sensitivity and the emerging implication of arabinose in enhanced MIC and antibiotic tolerance. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus, a human commensal, is also one of the most common and serious pathogens for humans. In recent years, its capacity to survive and replicate in phagocytic and non-phagocytic cells has been largely demonstrated. In these intracellular niches, bacteria are shielded from the immune response and antibiotics, turning host cells into long-term infectious reservoirs. Moreover, neutrophils carry intracellular bacteria in the bloodstream, leading to systemic spreading of the disease. Despite the serious threat posed by intracellular S. aureus to human health, the molecular mechanisms behind its intracellular survival and subsequent antibiotic treatment failure remain elusive. We give an overview of the killing mechanisms of phagocytes and of the impressive arsenal of virulence factors, toxins and stress responses deployed by S. aureus as a response. We then discuss the different barriers to antibiotic activity in this intracellular niche and finally describe innovative strategies to target intracellular persisting reservoirs. Intracellular niches represent a challenge in terms of diagnostic and treatment. Further research using ad-hoc in-vivo models and single cell approaches are needed to better understand the molecular mechanisms underlying intracellular survival and tolerance to antibiotics in order to identify strategies to eliminate these persistent bacteria. [ABSTRACT FROM AUTHOR]

ABSTRACTEscherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics.IMPORTANCEThe study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.

BackgroundThe commensal skin bacterium Cutibacterium acnes plays a role in the pathogenesis of acne vulgaris and also causes opportunistic infections of implanted medical devices due to its ability to form biofilms on biomaterial surfaces. Poly-β-(1→6)-N-acetyl-D-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm formation and biocide resistance in a wide range of bacterial pathogens. The objective of this study was to determine whether C. acnes produces PNAG, and whether PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro.MethodsPNAG was detected on the surface of C. acnes cells by fluorescence confocal microscopy using the antigen-specific human IgG1 monoclonal antibody F598. PNAG was detected in C. acnes biofilms by measuring the ability of the PNAG-specific glycosidase dispersin B to inhibit biofilm formation and sensitize biofilms to biocide killing.ResultsMonoclonal antibody F598 bound to the surface of C. acnes cells. Dispersin B inhibited attachment of C. acnes cells to polystyrene rods, inhibited biofilm formation by C. acnes in glass and polypropylene tubes, and sensitized C. acnes biofilms to killing by benzoyl peroxide and tetracycline.ConclusionC. acnes produces PNAG, and PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. PNAG may play a role in C. acnes skin colonization, biocide resistance, and virulence in vivo.

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug tolerance. Increased intracellular polyphosphate (polyP) in Mtb enhances tolerance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.

Abstract Infections caused by Staphylococcus aureus, notably methicillin‐resistant S. aureus (MRSA), pose treatment challenges due to its ability to tolerate antibiotics and develop antibiotic resistance. The former, a mechanism independent of genetic changes, allows bacteria to withstand antibiotics by altering metabolic processes. Here, a potent methylazanediyl bisacetamide derivative, MB6, is described, which selectively targets MRSA membranes over mammalian membranes without observable resistance development. Although MB6 is effective against growing MRSA cells, its antimicrobial activity against MRSA persisters is limited. Nevertheless, MB6 significantly potentiates the bactericidal activity of gentamicin against MRSA persisters by facilitating gentamicin uptake. In addition, MB6 in combination with daptomycin exhibits enhanced anti‐persister activity through mutual reinforcement of their membrane‐disrupting activities. Crucially, the “triple” combination of MB6, gentamicin, and daptomycin exhibits a marked enhancement in the killing of MRSA persisters compared to individual components or any double combinations. These findings underscore the potential of MB6 to function as a potent and selective membrane‐active antimicrobial adjuvant to enhance the efficacy of existing antibiotics against persister cells. The molecular mechanisms of MB6 elucidated in this study provide valuable insights for designing anti‐persister adjuvants and for developing new antimicrobial combination strategies to overcome the current limitations of antibiotic treatments.

Clostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both the exponential and stationary phases. Moreover, we could differentiate between triggered (induced using antibiotics such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation.

The prevalence of bacterial persisters is related to their phenotypic diversity and is responsible for the relapse of chronic infections. Tolerance to antibiotic therapy is the hallmark of bacterial persistence. In this study, we have screened a transposon library of Mycobacterium smegmatis mc2155 strain using antibiotic tolerance, survival in mouse macrophages, and biofilm-forming ability of the mutants. Out of 10 thousand clones screened, we selected ten mutants defective in all the three phenotypes. Six mutants showed significantly lower persister abundance under different stress conditions. Insertions in three genes belonging to the pathways of oxidative phosphorylation msmeg_3233 (cydA), biotin metabolism msmeg_3194 (bioB), and oxidative metabolism msmeg_0719, a flavoprotein monooxygenase, significantly reduced the number of live cells, suggesting their role in pathways promoting long-term survival. Another group that displayed a moderate reduction in CFU included a glycosyltransferase, msmeg_0392, a hydrogenase subunit, msmeg_2263 (hybC), and a DNA binding protein, msmeg_2211. The study has revealed potential candidates likely to facilitate the long-term survival of M. smegmatis. The findings offer new targets to develop antibiotics against persisters. Further, investigating the corresponding genes in M. tuberculosis may provide valuable leads in improving the treatment of chronic and persistent tuberculosis infections.

ABSTRACT The ubiquitous opportunistic pathogen Pseudomonas aeruginosa is highly adaptive and refractory to several different classes of antibiotics. However, we found in this study that stationary-phase P. aeruginosa cultures exhibit greater sensitivity to the fluoroquinolone Levofloxacin (Levo) than other bactericidal antibiotics, including an aminoglycoside (Tobramycin) and β-lactam (Aztreonam). To understand the basis of this sensitivity, we conducted time-lapse fluorescence microscopy experiments of cells during Levo treatment. We discovered that stationary-phase P. aeruginosa cells die rapidly during treatment and undergo heterogeneous morphological changes, including explosive lysis, filamentation, and gradual loss of membrane integrity as evidenced by propidium iodide uptake. These morphologies are reminiscent of how the model organism Escherichia coli appears when recovering from fluoroquinolone treatment, a period when activation of the DNA damage-induced SOS response is crucial. Accordingly, we monitored the morphologies and survival of P. aeruginosa ΔrecA mutants and found that the SOS response is not involved in P. aeruginosa Levo sensitivity like it is for E. coli. We hypothesized that Levo sensitivity may be due to P. aeruginosa maintaining active metabolism in stationary phase. We determined that stationary-phase P. aeruginosa cells transcribe, maintain reductase activity, and accumulate reactive metabolic species which contribute to Levo-mediated death. By elucidating how P. aeruginosa cells sustain metabolic activity during the stationary phase, we can design strategies to sensitize these persistent subpopulations to Levo and maintain the efficacy of this clinically important fluoroquinolone antibiotic. IMPORTANCE The bacterial pathogen Pseudomonas aeruginosa is responsible for a variety of chronic human infections. Even in the absence of identifiable resistance mutations, this pathogen can tolerate lethal antibiotic doses through phenotypic strategies like biofilm formation and metabolic quiescence. In this study, we determined that P. aeruginosa maintains greater metabolic activity in the stationary phase compared to the model organism, Escherichia coli, which has traditionally been used to study fluoroquinolone antibiotic tolerance. We demonstrate that hallmarks of E. coli fluoroquinolone tolerance are not conserved in P. aeruginosa, including the timing of cell death and necessity of the SOS DNA damage response for survival. The heightened sensitivity of stationary-phase P. aeruginosa to fluoroquinolones is attributed to maintained transcriptional and reductase activity. Our data suggest that perturbations that suppress transcription and respiration in P. aeruginosa may actually protect the pathogen against this important class of antibiotics.

Antibiotic resistance is a major Public Health challenge worldwide. Mechanisms other than resistance are described as contributors to therapeutic failure. These include heteroresistance and tolerance, which escape the standardized procedures used for antibiotic treatment decision-making as they do not involve changes in minimal inhibitory concentration (MIC). Haemophilus influenzae causes chronic respiratory infection and is associated with exacerbations suffered by chronic obstructive pulmonary disease (COPD) patients. Although resistance to imipenem is rare in this bacterial species, heteroresistance has been reported, and antibiotic tolerance cannot be excluded. Moreover, development of antibiotic heteroresistance or tolerance during within-host H. influenzae pathoadaptive evolution is currently unknown. In this study, we assessed imipenem resistance, heteroresistance and tolerance in a previously sequenced longitudinal collection of H. influenzae COPD respiratory isolates. The use of Etest, disc diffusion, population analysis profiling, tolerance disc (TD)-test methods, and susceptibility breakpoint criteria when available, showed a significant proportion of imipenem heteroresistance with differences in terms of degree among strains, absence of imipenem tolerance, and no specific trends among serial and clonally related strains could be established. Analysis of allelic variation in the ftsI, acrA, acrB, and acrR genes rendered a panel of polymorphisms only found in heteroresistant strains, but gene expression and genome-wide analyses did not show clear genetic traits linked to heteroresistance. In summary, a significant proportion of imipenem heteroresistance was observed among H. influenzae strains isolated from COPD respiratory samples over time. These data should be useful for making more accurate clinical recommendations to COPD patients.

ABSTRACT RavA-ViaA were reported to play a role in aminoglycoside (AG) sensitivity, but the mechanisms remain elusive. Here, we performed competition and survival experiments to confirm that deletion of ravA-viaA increases tolerance of the Gram-negative pathogen Vibrio cholerae to low and high AG concentrations during aerobic growth. Using high-throughput strategies in this species, we identify Cpx and Zra2 two-component systems as new partners of RavA-ViaA. We show that the AG tolerance of ∆ravvia requires the presence of these membrane stress sensing two-component systems. We propose that deletion of the RavA-ViaA function facilitates the response AGs because of a pre-activated state of Cpx and Zra2 membrane stress response systems. We also find an impact of these genes on vancomycin resistance, and we show that simultaneous inactivation of ravvia function together with envelope stress response systems leads to outer membrane permeabilization. Vancomycin is mostly used for Gram-positive because of its low efficiency for crossing the Gram-negative outer membrane. Targeting of the ravA-viaA operon for inactivation could be a future strategy to allow uptake of vancomycin into multidrug-resistant Gram-negative bacteria. IMPORTANCE The RavA-ViaA complex was previously found to sensitize Escherichia coli to aminoglycosides (AGs) in anaerobic conditions, but the mechanism is unknown. AGs are antibiotics known for their high efficiency against Gram-negative bacteria. In order to elucidate how the expression of the ravA-viaA genes increases bacterial susceptibility to aminoglycosides, we aimed at identifying partner functions necessary for increased tolerance in the absence of RavA-ViaA, in Vibrio cholerae. We show that membrane stress response systems Cpx and Zra2 are required in the absence of RavA-ViaA, for the tolerance to AGs and for outer membrane integrity. In the absence of these systems, the ∆ravvia strain’s membrane becomes permeable to external agents such as the antibiotic vancomycin.

ABSTRACT Beta-lactam antibiotics are widely used to treat infections caused by the important human pathogen Staphylococcus aureus. Resistance to beta-lactams, as found in methicillin-resistant S. aureus, renders effective treatment difficult. The second messenger cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) promotes beta-lactam resistance in clinical S. aureus isolates. C-di-AMP plays a crucial role in the regulation of cellular processes such as virulence, cell wall homeostasis, and resistance to beta-lactams in many bacterial species. In S. aureus, c-di-AMP synthesis is mediated by the diadenylate cyclase DacA, while its degradation is carried out by the phosphodiesterases GdpP and Pde2. In this work, we assessed the effect of altered c-di-AMP levels due to mutations in dacA, gdpP, or gdpP/pde2 on virulence determinants. We report that a previously described growth defect in bacteria producing high c-di-AMP levels is mainly attributable to smaller cell size. High c-di-AMP levels also led to decreased survival upon oxidative stress, reduced production of the antioxidant staphyloxanthin, increased oxacillin and fosfomycin resistance, and increased cell wall thickness. While resistance to ceftaroline was not affected, high c-di-AMP levels promoted tolerance to this antibiotic. In response to cell wall stress induced by antibiotics, the three-component regulatory system VraTSR mediates an increase in cell wall synthesis via the cell wall stress stimulon (CWSS). Increased c-di-AMP levels led to an activation of the CWSS. Upon deletion of vraR, resistance to oxacillin and fosfomycin as well as cell wall thickness diminished in the ΔgdpP mutant, indicating a contribution of the VraTSR system to the cell wall related phenotypes. IMPORTANCE Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3′, 5′-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting.

For several decades, questions have been raised about the effects of endocrine disruptors (ED) on environment and health. In humans, EDs interferes with hormones that are responsible for the maintenance of homeostasis, reproduction and development and therefore can cause developmental, metabolic and reproductive disorders. Because of their ubiquity in the environment, EDs can adversely impact microbial communities and pathogens virulence. At a time when bacterial resistance is inevitably emerging, it is necessary to understand the effects of EDs on the behavior of pathogenic bacteria and to identify the resulting mechanisms. Increasing studies have shown that exposure to environmental EDs can affect bacteria physiology. This review aims to highlight current knowledge of the effect of EDs on the virulence of human bacterial pathogens and discuss the future directions to investigate bacteria/EDs interaction. Given the data presented here, extended studies are required to understand the mechanisms by which EDs could modulate bacterial phenotypes in order to understand the health risks. [ABSTRACT FROM AUTHOR]

Background: Toxin‐antitoxin systems are highly variable, even among strains of the same bacterial species. The MazEF toxin‐antitoxin system is found in many bacteria and plays important roles in various biological processes such as antibiotic tolerance and phage defense. However, no interplay of MazEF systems between different species was reported. Materials and Methods: MazEF toxin‐antitoxin system of Helicobacter macacae was examined in three Escherichia coli strains with and without endogenous MazEF knockout. In vivo toxicity, antibiotic tolerance, and live/dead staining followed by flowcytometry analysis were performed to evaluate the functionality and interplay of the toxin‐antitoxin system between the two species. Results: Controlled ectopic expression of MazF of H. macacae (MazFhm) in E. coli did not affect its growth. However, in endogenous MazEF knockout E. coli strains, MazFhm expression caused a sharp growth arrest. The toxicity of MazFhm could be neutralized by both the antitoxin of MazE homolog of H.macacae and the antitoxin of MazE of E. coli, indicating interplay of MazEF toxin‐antitoxin systems between the two species. Induced expression of MazFhm enhanced tolerance to a lethal dose of levofloxacin, suggesting enhanced persister formation, which was further confirmed by live/dead cell staining. Conclusions: The MazEF toxin‐antitoxin system of H. macace enhances persister formation and thus antibiotic tolerance in E. coli. Our findings reveal an interplay between the MazEF systems of H. macacae and E. coli, emphasizing the need to consider this interaction while evaluating the toxicity and functionality of MazF homologs from different species in future studies. [ABSTRACT FROM AUTHOR]

Bacterial biofilms can be found in most environments on our planet, and the human body is no exception. Consisting of microbial cells encased in a matrix of extracellular polymers, biofilms enable bacteria to sequester themselves in favorable niches, while also increasing their ability to resist numerous stresses and survive under hostile circumstances. In recent decades, biofilms have increasingly been recognized as a major contributor to the pathogenesis of chronic infections. However, biofilms also occur in or on certain tissues in healthy individuals, and their constituent species are not restricted to canonical pathogens. In this review, we discuss the evidence for where, when, and what types of biofilms occur in the human body, as well as the diverse ways in which they can impact host health under homeostatic and dysbiotic states. [ABSTRACT FROM AUTHOR]

Abstract The antibiotic treatment of periprosthetic joint infections (PJI) is complicated by the presence of biofilm produced by bacteria on the abiotic surface of the implant. Bacteria within the deeper layers of the biofilm become metabolically less active, resulting in antibiotic tolerance due to several mechanisms. This review describes the basic principles of antibiotic treatment in PJI in relation to the behavior of bacteria within the biofilm. The concept of biofilm-active antibiotics will be explained from an in vitro as well as in vivo perspective. Evidence from clinical studies on biofilm-active antibiotics in PJI will be highlighted, mainly focusing on the role of rifampicin for Gram-positive microorganisms and fluoroquinolones for Gram-negative microorganisms. The optimal treatment duration will be discussed as the timing of switching to oral antibiotic therapy.

For several decades, questions have been raised about the effects of endocrine disruptors (ED) on environment and health. In humans, EDs interferes with hormones that are responsible for the maintenance of homeostasis, reproduction and development and therefore can cause developmental, metabolic and reproductive disorders. Because of their ubiquity in the environment, EDs can adversely impact microbial communities and pathogens virulence. At a time when bacterial resistance is inevitably emerging, it is necessary to understand the effects of EDs on the behavior of pathogenic bacteria and to identify the resulting mechanisms. Increasing studies have shown that exposure to environmental EDs can affect bacteria physiology. This review aims to highlight current knowledge of the effect of EDs on the virulence of human bacterial pathogens and discuss the future directions to investigate bacteria/EDs interaction. Given the data presented here, extended studies are required to understand the mechanisms by which EDs could modulate bacterial phenotypes in order to understand the health risks.

ABSTRACT Bacterial antibiotic tolerance, a phenomenon first observed in 1944, is known to be responsible for both onset and exacerbation of recurrent and chronic bacterial infections. The development of antibiotic tolerance was previously thought to be due to a switch to physiological dormancy when bacteria encounter adverse growth conditions. Our recent laboratory findings, however, showed that a set of genes related to the maintenance of proton motive force (PMF) are up-regulated under starvation, indicating that the tolerant sub-population, which are commonly known as persisters, can actively maintain their tolerance phenotypes. In this study, we investigated the relative functional roles of proteins involved in the maintenance and active generation of PMF in mediating tolerance formation in bacteria and found that the PspA and RcsB proteins play a key role in PMF maintenance in persisters, as deletion of genes encoding these two proteins resulted in significantly lower tolerance levels. Consistently, expression of the OsmC and Bdm proteins, which is under regulation by RcsB, is required to maintain PMF and the antibiotic tolerance phenotypes. On the other hand, the NuoL, Ndh, AppC, CyoB, and NuoF proteins, which are electron transport chain (ETC) components, were also found to be actively expressed in persisters in order to generate PMF to support functioning of various tolerance mechanisms such as efflux activities. Our data show that active generation of PMF is even more important than the PMF maintenance functions of PspA and RcsB in the expression of antibiotic tolerance phenotypes in persisters. Assessment of double- and triple-gene knockout strains, in which the PMF maintenance genes and those encoding ETC components were simultaneously deleted, confirms that these two groups of genes are both required for the expression of antibiotic tolerance phenotypes and that a lack of these functions would result in complete PMF dissipation and accumulation of antibiotics in the intracellular compartment of persisters and eventually cell death. Products of these genes are, therefore, ideal targets for future development of anti-tolerance agents. IMPORTANCE In this work, bacteria were found to undergo active generation and maintenance of proton motive force (PMF) under adverse conditions, such as starvation so as to support a range of physiological functions in order to survive under such conditions for a prolonged period. The ability to maintain a substantial level of PMF was found to be directly linked to that exhibiting phenotypic antibiotic tolerance under nutrient starvation or other adverse conditions. These findings infer that bacteria do not simply become physiologically dormant when they become antibiotic tolerant, instead they need to produce a wide range of proteins including those which help prevent PMF dissipation, such as PspA and RcsB, and the electron transport chain components, such as NuoL and Ndh, that actively generate PMF even during long-term starvation. As antibiotic tolerant sub-population is known to play a role in eliciting recurrent and chronic infections, especially among patients with a weakened immune system, the PMF maintenance mechanisms identified in this work are potential targets for the development of new strategies to control recurrent and chronic infections.

ABSTRACT Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population called persister cells to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically repressed state. We occasionally noticed a high variation in persister levels, and to explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbrück fluctuation test. Although we kept the conditions same, the diversity in persistence level among clones was relatively consistent: varying from ~60- to 100- and ~40- to 70-fold for ampicillin and apramycin, respectively. Then, we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for approximately five generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>seven generations). Our result demonstrates (1) the existence of a small population prepared for stress (“primed cells”) resulting in higher persister numbers; (2) the primed memory state is reproducible and transient, passed down for generations but eventually lost; and (3) a heterogeneous persister population is a result of a transiently primed reversible cell state and not due to a pre-existing genetic mutation. IMPORTANCE Antibiotics have been highly effective in treating lethal infectious diseases for almost a century. However, the increasing threat of antibiotic resistance is again causing these diseases to become life-threatening. The longer a bacteria can survive antibiotics, the more likely it is to develop resistance. Complicating matters is that non-genetic factors can allow bacterial cells with identical DNA to gain transient resistance (also known as persistence). Here, we show that a small fraction of the bacterial population called primed cells can pass down non-genetic information (“memory”) to their offspring, enabling them to survive lethal antibiotics for a long time. However, this memory is eventually lost. These results demonstrate how bacteria can leverage differences among genetically identical cells formed through non-genetic factors to form primed cells with a selective advantage to survive antibiotics.

Depletion attraction is a common entropy force observed in colloidal systems. As a common phenomenon in colloidal and bacterial systems, studying the mechanism and application of depletion attraction is of great value for controlling the state of colloidal solutions, regulating the morphology of colloidal particles, disease treatment, and water pollution treatment. Based on the current research status, we briefly introduce the calculation and measurement methods of depletion attractions. And we review the application of depletion attractions in colloidal systems, and summarize the different phenomena and aggregation mechanisms caused by depletion attraction in active colloidal particle-bacterial systems. Understanding the specific role of depletion aggregation in colloidal and bacterial systems provides more possibilities for further exploring depletion aggregation mechanisms and utilizing depletion aggregation phenomena in nature.

Bacterial persisters are a transient subpopulation of non-growing, antibiotic-tolerant cells. There is increasing evidence that bacterial persisters play an important role in treatment failure leading to recurring infections and promoting the development of antibiotic resistance. Current research reveals that recurring legionellosis is often the result of relapse rather than reinfection and suggests that the mechanism of bacterial persistence may play a role. The development of single-cell techniques such as the Timerbac system allows us to identify potential persister cells and investigate their physiology. Here, we tested the persister forming capacity of 7 pairs of Legionella pneumophila (Lp) clinical isolates, with isolate pairs corresponding to two episodes of legionellosis in the same patient. We distinguished non-growing subpopulations from their replicating counterparts during infection in an amoeba model. Imaging flow cytometry allowed us to identify single non-growing bacteria within amoeba cells 17 h post-infection, thus corresponding to this subpopulation of potential persister cells. Interestingly the magnitude of this subpopulation varies between the 7 pairs of Lp clinical isolates. Biphasic killing kinetics using ofloxacin stress confirmed the persister development capacity of ST1 clinical isolates, highlighting enhanced persister formation during the host cell infection. Thus, persister formation appears to be strain or ST (sequence type) dependent. Genome sequence analysis was carried out between ST1 clinical isolates and ST1 Paris. No genetic microevolution (SNP) linked to possible increase of persistence capacity was revealed among all the clones tested, even in clones issued from two persistence cycle experiments, confirming the transient reversible phenotypic status of persistence. Treatment failure in legionellosis is a serious issue as infections have a 5-10% mortality rate, and investigations into persistence in a clinical context and the mechanisms involved may allow us to combat this issue. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is a nosocomial pathogen of serious healthcare concern that is becoming increasingly difficult to treat due to antibiotic treatment failure. Recent studies have revealed that clinically defined antibiotic-susceptible strains upregulate the expression of a repertoire of putative drug efflux pumps during their growth under biologically relevant conditions, e.g., in human serum, resulting in efflux-associated resistance to physiologically achievable antibiotic levels within a patient. This phenomenon, termed Adaptive Efflux Mediated Resistance (AEMR), has been hypothesized to account for one mechanism by which antibiotic-susceptible A. baumannii fails to respond to antibiotic treatment. In the current study, we sought to identify genetic determinants that contribute to A. baumannii serum-associated AEMR by screening a transposon mutant library for members that display a loss of the AEMR phenotype. Results revealed that mutation of a putative pirin-like protein, YhaK, results in a loss of AEMR, a phenotype that could be complemented by a wild-type copy of the yhaK gene and was verified in a second strain background. Ethidium bromide efflux assays confirmed that the loss of AEMR phenotype due to pirin-like protein mutation correlated with reduced overarching efflux capacity. Further, flow cytometry and confocal microscopy measures of a fluorophore 7-(dimethylamino)-coumarin-4-acetic acid (DMACA)-tagged levofloxacin isomer, ofloxacin, further verified that YhaK mutation reduces AEMR-mediated antibiotic efflux. RNA-sequencing studies revealed that YhaK may be required for the expression of multiple efflux-associated systems, including MATE and ABC families of efflux pumps. Collectively, the data indicate that the A. baumannii YhaK pirin-like protein plays a role in modulating the organism's adaptive efflux-mediated resistance phenotype. [ABSTRACT FROM AUTHOR]

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide, povidone-iodine, and chlorhexidine are antiseptics that are commonly added to irrigants to either prevent or treat infection. There are little clinical data available that demonstrate efficacy of adding antiseptics to irrigants in the treatment of periprosthetic joint infection after biofilm establishment. The objective of the study was to assess the bactericidal activity of the antiseptics on S. aureus planktonic and biofilm. For planktonic irrigation, S. aureus was exposed to different concentrations of antiseptics. S. aureus biofilm was developed by submerging a Kirschner wire into normalized bacteria and allowing it to grow for forty-eight hours. The Kirschner wire was then treated with irrigation solutions and plated for CFU analysis. Hydrogen peroxide, povidone-iodine, and chlorhexidine were bactericidal against planktonic bacteria with over a 3 log reduction (p < 0.0001). Unlike cefazolin, the antiseptics were not bactericidal (less than 3 log reduction) against biofilm bacteria but did have a statistical reduction in biofilm as compared to the initial time point (p < 0.0001). As compared to cefazolin treatment alone, the addition of hydrogen peroxide or povidone-iodine to cefazolin treatment only additionally reduced the biofilm burden by less than 1 log. The antiseptics demonstrated bactericidal properties with planktonic S. aureus; however, when used to irrigate S. aureus biofilms, these antiseptics were unable to decrease biofilm mass below a 3 log reduction, suggesting that S. aureus biofilm has a tolerance to antiseptics. This information should be considered when considering antibiotic tolerance in established S. aureus biofilm treatment. [ABSTRACT FROM AUTHOR]

Helicobacter pylori, a WHO class I carcinogen, is one of the most successful human pathogens colonizing the stomach of over 4.4 billion of the world's population. Antibiotic therapy represents the best solution but poor response rates have hampered the elimination of H. pylori. A growing body of evidence suggests that H. pylori forms biofilms, but the role of this growth mode in infection remains elusive. Here, we demonstrate that H. pylori cells within a biofilm are tolerant to multiple antibiotics in a manner that depends partially on extracellular proteins. Biofilm-forming cells were tolerant to multiple antibiotics that target distinct pathways, including amoxicillin, clarithromycin, and tetracycline. Furthermore, this tolerance was significantly dampened following proteinase K treatment. These data suggest that H. pylori adapts its phenotype during biofilm growth resulting in decreased antibiotic susceptibility but this tolerance can be partially ameliorated by extracellular protease treatment.

Abstract Streptococcus suis is a zoonotic agent that causes sepsis and meningitis in pigs and humans. S. suis infections are responsible for large economic losses in pig production. The lack of effective vaccines to prevent the disease has promoted the extensive use of antibiotics worldwide. This has been followed by the emergence of resistance against different classes of antibiotics. The rates of resistance to tetracyclines, lincosamides, and macrolides are extremely high, and resistance has spread worldwide. The genetic origin of S. suis resistance is multiple and includes the production of target-modifying and antibiotic-inactivating enzymes and mutations in antibiotic targets. S. suis genomes contain traits of horizontal gene transfer. Many mobile genetic elements carry a variety of genes that confer resistance to antibiotics as well as genes for autonomous DNA transfer and, thus, S. suis can rapidly acquire multiresistance. In addition, S. suis forms microcolonies on host tissues, which are associations of microorganisms that generate tolerance to antibiotics through a variety of mechanisms and favor the exchange of genetic material. Thus, alternatives to currently used antibiotics are highly demanded. A deep understanding of the mechanisms by which S. suis becomes resistant or tolerant to antibiotics may help to develop novel molecules or combinations of antimicrobials to fight these infections. Meanwhile, phage therapy and vaccination are promising alternative strategies, which could alleviate disease pressure and, thereby, antibiotic use.