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Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is a frontline antibiotic for treating methicillin-resistant Staphylococcus aureus and other Gram-positive bacterial infections. However, vancomycin use has led to the increasing prevalence of bacterial strains with reduced susceptibility to vancomycin. Here, we show that unsaturated fatty acids act as potent vancomycin adjuvants to rapidly kill a range of Gram-positive bacteria, including vancomycin-tolerant and resistant populations. The synergistic bactericidal activity relies on the accumulation of membrane-bound cell wall intermediates that generate large fluid patches in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. Our findings provide a natural therapeutic option that enhances vancomycin activity against difficult-to-treat pathogens, and the underlying mechanism may be further exploited to develop antimicrobials that target recalcitrant infection.

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. Published version

Methicillin-resistant Staphylococcus aureus (MRSA) is highly prevalent in U.S. cystic fibrosis (CF) patients and is associated with worse clinical outcomes in CF. These infections often become chronic despite repeated antibiotic therapy. Here, we assessed whether bacterial phenotypes, including antibiotic tolerance, can predict the clinical outcomes of MRSA infections. MRSA isolates (

Background Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Many patients experience multiple episodes of GNB-BSI for unclear reasons. This study examines the genetic differences between initial and subsequent isolates of recurrent GNB-BSI. Methods We used a prospective cohort of patients with GNB-BSI at Duke Hospital to identify patients with >1 episode of GNB-BSI due to the same bacterial species. We performed pulsed field gel electrophoresis (PFGE) on paired isolates to determine if the events were Reinfection (paired isolates different) or Relapse (paired isolates genetically identical). We then used whole genome sequencing (WGS) to verify the PFGE findings and explore the genetic similarity between initial and relapsed GNB-BSI isolates. Results Among 1,423 unique patients with GNB-BSI, 60 (4%) experienced recurrent GNB-BSI with the same bacterial species. We performed genotyping (PFGE, followed by WGS) on the paired bacterial isolates from the index and recurrent bloodstream infections for the four most common species in our study population (Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, and Serratia marcescens) (n=48 pairs). We determined that 63% (30/48) of recurrent GNB-BSI episodes were due to relapse and 37% (18/48) were due to reinfection. PFGE correctly identified relapse versus reinfection in 98% (47/48) of cases. The WGS data illustrated the microevolution of relapsed GNB-BSI isolates within patients. When comparing initial to relapsed isolates, we calculated a median of 5 single-nucleotide polymorphisms (SNPs) in relapsed E. coli isolates and 2 SNPs in Klebsiella spp. isolates. Of these SNPs, 37% (E. coli) and 12% (Klebsiella) were non-synonymous changes in coding regions, most commonly genes associated with antimicrobial resistance (AMR), energy metabolism and stress response. Alterations in the AMR profile was common, with 20/30 relapsed isolates demonstrating either loss or acquisition of AMR. Figure 1:Time from initial to recurrent episode of gram-negative bloodstream infection. Relapse (i.e., same bacterial strain noted by green circle) versus reinfection (i.e., different bacterial strain noted by red circle) was determined by pulse field gel electrophoresis followed by WGS. Whole genome sequencing was performed on the initial and recurrent isolate from patients with relapsed GNB-BSI. We identified single nucleotide polymorphisms (SNPs) present in the recurrent isolate, which were not present in the initial isolate. We identified the function of genes containing SNPs in E. coli (n=28 SNPs) and Klebsiella spp. (n=13 SNPs). Synonymous mutations, insertions, deletions, and non-synonymous mutations in hypothetical proteins were excluded. Conclusion Most recurrent GNB-BSI with same species are due to relapse. PFGE has comparable accuracy to WGS to determine reinfection versus relapse. Multiple mutations in genes involved in stress response, AMR and metabolism were identified in relapsed isolates. Disclosures Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds.

Interactions between Staphylococcus aureus and the host immune system can have significant impacts on antibiotic efficacy, suggesting that targeting and modulating the immune response to S. aureus infection may improve antibiotic efficacy and improve infection outcome. As we've previously shown, high levels of reactive oxygen species (ROS), associated with an M1-like proinflammatory macrophage response, potently induce antibiotic tolerance in S. aureus. Although the proinflammatory immune response is critical for initial control of pathogen burden, recent studies demonstrate that modulation of the macrophage response to an anti-inflammatory, or M2-like, response facilitates resolution of established S. aureus skin and soft tissue infections, arthritis, and bacteremia. Here, we evaluated the impact of host-directed immunosuppressive chemotherapeutics and anti-inflammatory agents on antibiotic efficacy against S. aureus.

Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is of major clinical importance for the treatment of Gram-positive bacterial infections. Here we show that unsaturated fatty acids (UFAs) and vancomycin act synergistically to rapidly kill S. aureus, including vancomycin tolerant and resistant populations. Our results suggest that antibiotic-mediated accumulation of lipid II at the septum facilitates membrane invasion by antimicrobial UFAs. UFA-vancomycin dual treatment generates large fluid patches of flexible lipids in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. This mechanism of synergy may be exploited for the development of new antibiotic therapies that target lipid II to combat both antibiotic tolerance and resistance.

Staphylococcus aureus is a leading human pathogen that frequently causes relapsing infections. Host-pathogen interactions have been shown to have substantial impacts on antibiotic susceptibility and the formation of antibiotic tolerant cells. In this study, we interrogate how a major S. aureus virulence factor, α-toxin, interacts with macrophages to alter the microenvironment of the pathogen, thereby influencing its susceptibility to antibiotics. We find α-toxin-mediated activation of the NLRP3 inflammasome induces antibiotic tolerance in the host cell cytoplasm. Induction of antibiotic tolerance is driven by increased glycolysis in the host cells, resulting in glucose limitation and ATP depletion in S. aureus. Additionally, inhibition of NLRP3 activation improves antibiotic efficacy in vitro and in vivo. Our findings identify interactions between S. aureus and the host that result in metabolic crosstalk that can determine the outcome of antimicrobial therapy.

Methicillin-resistant Staphylococcus aureus (MRSA) is a devastating pathogen responsible for a variety of life-threatening infections. A distinctive characteristic of this pathogen is its ability to persist in the bloodstream for several days despite seemingly appropriate antibiotics. Persistent MRSA bacteremia is common and is associated with poor clinical outcomes. The etiology of persistent MRSA bacteremia is a result of the complex interplay between the host, the pathogen, and the antibiotic used to treat the infection. In this review, we explore the factors related to each component of the host–pathogen interaction and discuss the clinical relevance of each element. Next, we discuss the treatment options and diagnostic approaches for the management of persistent MRSA bacteremia.

Aminoglycosides are bactericidal drugs which require a proton motive force (PMF) for uptake into the bacterial cell. Low energy cells, such as persisters, maintain a PMF below the threshold for drug uptake and are tolerant to aminoglycosides. In this chapter, we discuss mechanisms to target the bacterial membrane and stimulate aminoglycoside uptake to kill Staphylococcus aureus persisters.

Antibiotic treatment failure of infection is common and frequently occurs in the absence of genetically encoded antibiotic resistance mechanisms. In such scenarios, the ability of bacteria to enter a phenotypic state that renders them tolerant to the killing activity of multiple antibiotic classes is thought to contribute to antibiotic failure. Phagocytic cells, which specialize in engulfing and destroying invading pathogens, may paradoxically contribute to antibiotic tolerance and treatment failure. Macrophages act as reservoirs for some pathogens and impede penetration of certain classes of antibiotics. In addition, increasing evidence suggests that subpopulations of bacteria can survive inside these cells and are coerced into an antibiotic-tolerant state by host cell activity. Uncovering the mechanisms that drive immune-mediated antibiotic tolerance may present novel strategies to improving antibiotic therapy.

Acyldepsipeptides are a unique class of antibiotics that act via allosterically dysregulated activation of the bacterial caseinolytic protease (ClpP). The ability of ClpP activators to kill nongrowing bacteria represents a new opportunity to combat deep-seated biofilm infections. However, the acyldepsipeptide scaffold is subject to rapid metabolism. Herein, we explore alteration of the potentially metabolically reactive α,β unsaturated acyl chain. Through targeted synthesis, a new class of phenyl urea substituted depsipeptide ClpP activators with improved metabolic stability is described. The ureadepsipeptides are potent activators of Staphylococcus aureus ClpP and show activity against Gram-positive bacteria, including S. aureus biofilms. These studies demonstrate that a phenyl urea motif can successfully mimic the double bond, maintaining potency equivalent to acyldepsipeptides but with decreased metabolic liability. Although removal of the double bond from acyldepsipeptides generally has a significant negative impact on potency, structural studies revealed that the phenyl ureadepsipeptides can retain potency through the formation of a third hydrogen bond between the urea and the key Tyr63 residue in the ClpP activation domain. Ureadepsipeptides represent a new class of ClpP activators with improved drug-like properties, potent antibacterial activity, and the tractability to be further optimized.

Staphylococcus aureus is a leading human pathogen that frequently causes chronic and relapsing infections. Antibiotic tolerant persister cells contribute to frequent antibiotic failure in patients. Macrophages represent an important niche during S. aureus bacteremia and recent work has identified a role for oxidative burst in the formation of antibiotic tolerant S. aureus. We find that host-derived peroxynitrite, the reaction product of superoxide and nitric oxide, is the main mediator of antibiotic tolerance in macrophages. Using a collection of S. aureus clinical isolates, we find that, despite significant variation in persister formation in pure culture, all strains were similarly enriched for antibiotic tolerance following internalization by activated macrophages. Our findings suggest that host interaction strongly induces antibiotic tolerance and may negate bacterial mechanisms of persister formation, established in pure culture. These findings emphasize the importance of studying antibiotic tolerance in the context of bacterial interaction with the host suggest that modulation of the host response may represent a viable therapeutic strategy to sensitize S. aureus to antibiotics.

Antibiotic treatment failure of Staphylococcus aureus infections is very common. In addition to genetically encoded mechanisms of antibiotic resistance, numerous additional factors limit the efficacy of antibiotics in vivo. Identifying and removing the barriers to antibiotic efficacy are of major importance, as even if new antibiotics become available, they will likely face the same barriers to efficacy as their predecessors. One major obstacle to antibiotic efficacy is the proficiency of S. aureus to enter a physiological state that is incompatible with antibiotic killing. Multiple pathways leading to antibiotic tolerance and the formation of tolerant subpopulations called persister cells have been described for S. aureus. Additionally, S. aureus is a versatile pathogen that can infect numerous tissues and invade a variety of cell types, of which some are poorly penetrable to antibiotics. It is therefore unlikely that there will be a single solution to the problem of recalcitrant S. aureus infection. Instead, specific approaches may be required for targeting tolerant cells within different niches, be it through direct targeting of persister cells, sensitization of persisters to conventional antibiotics, improved penetration of antibiotics to particular niches, or any combination thereof. Here, we examine two well-described reservoirs of antibiotic-tolerant S. aureus, the biofilm and the macrophage, the barriers these environments present to antibiotic efficacy, and potential solutions to the problem.

Aminoglycosides are bactericidal drugs which require a proton motive force (PMF) for uptake into the bacterial cell. Low energy cells, such as persisters, maintain a PMF below the threshold for drug uptake and are tolerant to aminoglycosides. In this chapter, we discuss mechanisms to target the bacterial membrane and stimulate aminoglycoside uptake to kill Staphylococcus aureus persisters.

Biofilms are associated with chronic infection and frequently require surgical intervention even after prolonged antibiotic therapy. Bactericidal antibiotics are often ineffective at eradicating genetically susceptible cells within a biofilm because: 1) most conventional antibiotics target ATP-dependent processes and so work poorly on metabolically indolent persister cells within a biofilm; and 2) the biofilm matrix can act as a physical barrier to drug diffusion of certain classes of antibiotics. Antibiotic therapy that fails to completely eradicate the pathogen leads to chronic and relapsing infections, major financial healthcare burdens and significant mortality. Numerous approaches have been taken to improve biofilm killing but these often fail to achieve eradication. We address this problem with a novel two-pronged strategy with the aim of eradicating biofilm infection using 1) antibiotics which target persister cells as well as 2) improving drug penetration using ultrasound-stimulated phase-change contrast agents (US-PCCA). We previously demonstrated that rhamnolipids, biosurfactant molecules produced by Pseudomonas aeruginosa, significantly potentiated aminoglycoside efficacy against S. aureus biofilm. We have also shown that US-PCCA can transiently disrupt biological barriers to therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could eradicate methicillin resistant S. aureus (MRSA) biofilms. To investigate this, we treated MRSA biofilms with a range of conventional and anti-persister antibiotics and/or US-PCCA. We found that vancomycin was significantly potentiated by US-PCCA, but a fraction of cells remained viable after the combination treatment. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically reduced biofilm viability, frequently culminating in complete eradication of the biofilm. These data demonstrate that biofilm eradication can be achieved using a combined approach improving drug penetration and therapeutics that target persister cells.

The blood-clotting protein fibrin(ogen) plays a critical role in host defense against invading pathogens, particularly against peritoneal infection by the Gram-positive microbe Staphylococcus aureus. Here, we tested the hypothesis that direct binding between fibrin(ogen) and S. aureus is a component of the primary host antimicrobial response mechanism and prevention of secondary microbe dissemination from the peritoneal cavity. To establish a model system, we showed that fibrinogen isolated from FibγΔ5 mice, which express a mutant form lacking the final 5 amino acids of the fibrinogen γ chain (termed fibrinogenγΔ5), did not support S. aureus adherence when immobilized and clumping when in suspension. In contrast, purified wildtype fibrinogen supported robust adhesion and clumping that was largely dependent on S. aureus expression of the receptor clumping factor A (ClfA). Following peritoneal infection with S. aureus USA300, FibγΔ5 mice displayed worse survival compared to WT mice coupled to reduced bacterial killing within the peritoneal cavity and increased dissemination of the microbes into circulation and distant organs. The failure of acute bacterial killing, but not enhanced dissemination, was partially recapitulated by mice infected with S. aureus USA300 lacking ClfA. Fibrin polymer formation and coagulation transglutaminase Factor XIII each contributed to killing of the microbes within the peritoneal cavity, but only elimination of polymer formation enhanced systemic dissemination. Host macrophage depletion or selective elimination of the fibrin(ogen) β2-integrin binding motif both compromised local bacterial killing and enhanced S. aureus systemic dissemination, suggesting fibrin polymer formation in and of itself was not sufficient to retain S. aureus within the peritoneal cavity. Collectively, these findings suggest that following peritoneal infection, the binding of S. aureus to stabilized fibrin matrices promotes a local, macrophage-mediated antimicrobial response essential for prevention of microbe dissemination and downstream host mortality., Author summary The Gram-positive bacterium Staphylococcus aureus (S. aureus) produces a number of soluble and surface-associated proteins that bind the host coagulation protein fibrinogen. The contribution of fibrinogen-S. aureus binding through the fibrinogen receptor clumping factor A (ClfA) in peritoneal infection has not been defined. Elimination of the binding motif on fibrinogen for ClfA or deletion of ClfA from S. aureus significantly reduced S. aureus-fibrinogen binding and bacterial clumping in solution. In a mouse model of peritonitis, loss of these activities resulted in diminished bacterial killing, increased bacterial dissemination, and worsened host survival. Although fibrin polymer formation and fibrin(ogen)-macrophage binding are mechanistically linked to the local antimicrobial response, fibrin formation in and of itself is not sufficient to suppress microbe dissemination. These discoveries have identified important components of the fibrin(ogen)-dependent host antimicrobial response against S. aureus, providing further understanding of this physiological response to infection which could uncover potential therapeutic strategies for peritonitis patients.

Accurate prediction of antimicrobial efficacy is essential for successful treatment of bacterial infection. Beyond genetically encoded mechanisms of antibiotic resistance, the determinants of antibiotic susceptibility during infection remain poorly understood, and treatment failure is common. Traditional antibiotic susceptibility testing fails to account for extrinsic determinants of antibiotic susceptibility present in the complex infection environment and is therefore a poor predictor of antibiotic treatment outcome. Here we discuss how host-pathogen interaction, microbial interspecies interaction, and metabolic heterogeneity contribute to the success or failure of antibiotic therapy. Consideration of these factors during the treatment of disease will improve our ability to successfully resolve recalcitrant bacterial infection and improve patient health.

Bacterial biofilms, often associated with chronic infections, respond poorly to antibiotic therapy and frequently require surgical intervention. Biofilms harbor persister cells, metabolically indolent cells, which are tolerant to most conventional antibiotics. In addition, the biofilm matrix can act as a physical barrier, impeding diffusion of antibiotics. Novel therapeutic approaches frequently improve biofilm killing, but usually fail to achieve eradication. Failure to eradicate the biofilm leads to chronic and relapsing infection, is associated with major financial healthcare costs and significant morbidity and mortality. We address this problem with a two-pronged strategy using 1) antibiotics that target persister cells and 2) ultrasound-stimulated phase-change contrast agents (US-PCCA), which improve antibiotic penetration. We previously demonstrated that rhamnolipids, produced by Pseudomonas aeruginosa, could induce aminoglycoside uptake in gram-positive organisms, leading to persister cell death. We have also shown that US-PCCA can transiently disrupt biological barriers to improve penetration of therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could improve treatment of methicillin resistant S. aureus (MRSA) biofilms. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically improved biofilm killing. This novel treatment strategy has the potential for rapid clinical translation as the PCCA formulation is a variant of FDA-approved ultrasound contrast agents that are already in clinical practice and the low-pressure ultrasound settings used in our study can be achieved with existing ultrasound hardware at pressures below the FDA set limits for diagnostic imaging.

Staphylococcus aureus has an incredible ability to survive, either by adapting to environmental conditions or defending against exogenous stress. Although there are certainly important genetic traits, in part this ability is provided by the breadth of modes of growth S. aureus can adopt. It has been proposed that while within their host, S. aureus survives host-generated and therapeutic antimicrobial stress via alternative lifestyles: a persister sub-population, through biofilm growth on host tissue or by growing as small colony variants (SCVs). Key to an understanding of chronic and relapsing S. aureus infections is determining the molecular basis for its switch to these quasi-dormant lifestyles. In a multicellular biofilm, the metabolically quiescent bacterial community additionally produces a highly protective extracellular polymeric substance (EPS). Furthermore, there are bacteria within a biofilm community that have an altered physiology potentially equivalent to persister cells. Recent studies have directly linked the cellular ATP production by persister cells as their key feature and the basis for their tolerance of a range of antibiotics. In clinical settings, SCVs of S. aureus have been observed for many years; when cultured, these cells form non-pigmented colonies and are approximately ten times smaller than their counterparts. Various genotypic factors have been identified in attempts to characterize S. aureus SCVs and different environmental stresses have been implicated as important inducers.

Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now., Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation.

Bacterial invasion of synovial joints, as in infectious or septic arthritis, can be difficult to treat in both veterinary and human clinical practice. Biofilms, in the form of free-floating clumps or aggregates, are involved with the pathogenesis of infectious arthritis and periprosthetic joint infection (PJI). Infection of a joint containing an orthopedic implant can additionally complicate these infections due to the presence of adherent biofilms. Because of these biofilm phenotypes, bacteria within these infected joints show increased antimicrobial tolerance even at high antibiotic concentrations. To date, animal models of PJI or infectious arthritis have been limited to small animals such as rodents or rabbits. Small animal models, however, yield limited quantities of synovial fluid making them impractical for in vitro research. Herein, we describe the use of ex vivo equine and porcine models for the study of synovial fluid induced biofilm aggregate formation and antimicrobial tolerance. We observed Staphylococcus aureus and other bacterial pathogens adapt the same biofilm aggregate phenotype with significant antimicrobial tolerance in both equine and porcine synovial fluid, analogous to human synovial fluid. We also demonstrate that enzymatic dispersal of synovial fluid aggregates restores the activity of antimicrobials. Future studies investigating the interaction of bacterial cell surface proteins with host synovial fluid proteins can be readily carried out in equine or porcine ex vivo models to identify novel drug targets for treatment of prevention of these difficult to treat infectious diseases.

Staphylococcus aureus is a major human pathogen that causes an array of infections ranging from minor skin infections to more serious infections including osteomyelitis, endocarditis, necrotizing pneumonia and sepsis1. These more serious infections usually arise from an initial bloodstream infection and are frequently recalcitrant to antibiotic treatment 1. Phagocytosis by macrophages and neutrophils is the primary mechanism by which S. aureus infection is controlled by the immune system2. Macrophages have been shown to be a major reservoir of S. aureus in vivo3 but the role of macrophages in the induction of antibiotic tolerance has not been explored. Here we show that macrophages not only fail to efficiently kill phagocytosed S. aureus but also induce tolerance to multiple antibiotics. Reactive oxygen species (ROS) generated by respiratory burst attack iron-sulfur (Fe-S) cluster containing proteins, including TCA cycle enzymes, resulting in decreased respiration, lower ATP and increased antibiotic tolerance. We further show that during a murine systemic infection, respiratory burst induces antibiotic tolerance in the spleen. These results suggest that a major component of the innate immune response is antagonistic to the bactericidal activities of antibiotics.

Aminoglycoside antibiotics require proton motive force (PMF) for bacterial internalization. In non-respiring populations, PMF drops below the level required for drug influx, limiting the utility of aminoglycosides against strict and facultative anaerobes. We recently demonstrated that rhamnolipids (RLs), biosurfactant molecules produced by Pseudomonas aeruginosa, potentiate aminoglycoside activity against Staphylococcus aureus. Here, we demonstrate that RLs induce PMF-independent aminoglycoside uptake to restore sensitivity to otherwise tolerant persister, biofilm, small colony variant, and anaerobic populations of S. aureus. Furthermore, we show that this approach prevents the rise of resistance, restores sensitivity to highly resistant clinical isolates, and is effective against other gram-positive pathogens. Finally, while other membrane-acting agents can synergize with aminoglycosides, induction of PMF-independent uptake is uncommon, and distinct to RLs among several compounds tested. In all, small molecule induction of PMF-independent aminoglycoside uptake circumvents phenotypic tolerance, overcomes genotypic resistance, and expands the utility of aminoglycosides against intrinsically recalcitrant bacterial populations.

Persisters are dormant variants that form a subpopulation of cells tolerant to antibiotics. Persisters are largely responsible for the recalcitrance of chronic infections to therapy. In Escherichia coli, one widely accepted model of persister formation holds that stochastic accumulation of ppGpp causes activation of the Lon protease that degrades antitoxins; active toxins then inhibit translation, resulting in dormant, drug-tolerant persisters. We found that various stresses induce toxin-antitoxin (TA) expression but that induction of TAs does not necessarily increase persisters. The 16S rRNA promoter rrnB P1 was proposed to be a persister reporter and an indicator of toxin activation regulated by ppGpp. Using fluorescence-activated cell sorting (FACS), we confirmed the enrichment for persisters in the fraction of rrnB P1-gfp dim cells; however, this is independent of toxin-antitoxins. rrnB P1 is coregulated by ppGpp and ATP. We show that rrnB P1 can report persisters in a relA/spoT deletion background, suggesting that rrnB P1 is a persister marker responding to ATP. Consistent with this finding, decreasing the level of ATP by arsenate treatment causes drug tolerance. Lowering ATP slows translation and prevents the formation of DNA double-strand breaks upon fluoroquinolone treatment. We conclude that variation in ATP levels leads to persister formation by decreasing the activity of antibiotic targets., IMPORTANCE Persisters are a subpopulation of antibiotic-tolerant cells responsible for the recalcitrance of chronic infections. Our current understanding of persister formation is primarily based on studies of E. coli. The activation of toxin-antitoxin systems by ppGpp has become a widely accepted model for persister formation. In this study, we found that stress-induced activation of mRNA interferase-type toxins does not necessarily cause persister formation. We also found that the persister marker rrnB P1 reports persister cells because it detects a drop in cellular ATP levels. Consistent with this, lowering the ATP level decreases antibiotic target activity and, thus, leads to persister formation. We conclude that stochastic variation in ATP is the main mechanism of persister formation. A decrease in ATP provides a satisfactory explanation for the drug tolerance of persisters, since bactericidal antibiotics act by corrupting energy-dependent targets.

Staphylococcus aureus is an opportunistic pathogen capable of causing a variety of diseases including osteomyelitis, endocarditis, infections of indwelling devices and wound infections. These infections are often chronic and highly recalcitrant to antibiotic treatment. Persister cells appear to be central to this recalcitrance. A multitude of factors contribute to S. aureus virulence and high levels of treatment failure. These include its ability to colonize the skin and nares of the host, its ability to evade the host immune system and its development of resistance to a variety of antibiotics. Less understood is the phenomenon of persister cells and their role in S. aureus infections and treatment outcome. Persister cells occur as a sub-population of phenotypic variants that are tolerant to antibiotic treatment. This review examines the importance of persisters in chronic and relapsing S. aureus infections and proposes methods for their eradication.

Summary Aminoglycoside antibiotics require proton motive force (PMF) for bacterial internalization. In non-respiring populations, PMF drops below the level required for drug influx, limiting the utility of aminoglycosides against strict and facultative anaerobes. We recently demonstrated that rhamnolipids (RLs), biosurfactant molecules produced by Pseudomonas aeruginosa, potentiate aminoglycoside activity against Staphylococcus aureus. Here, we demonstrate that RLs induce PMF-independent aminoglycoside uptake to restore sensitivity to otherwise tolerant persister, biofilm, small colony variant, and anaerobic populations of S. aureus. Furthermore, we show that this approach represses the rise of resistance, restores sensitivity to highly resistant clinical isolates, and is effective against other Gram-positive pathogens. Finally, while other membrane-acting agents can synergize with aminoglycosides, induction of PMF-independent uptake is uncommon, and distinct to RLs among several compounds tested. In all, small-molecule induction of PMF-independent aminoglycoside uptake circumvents phenotypic tolerance, overcomes genotypic resistance, and expands the utility of aminoglycosides against intrinsically recalcitrant bacterial populations.

Teixobactin represents the first member of a newly discovered class of antibiotics that act through inhibition of cell wall synthesis. Teixobactin binds multiple bactoprenol-coupled cell wall precursors, inhibiting both peptidoglycan and teichoic acid synthesis. Here, we show that the impressive bactericidal activity of teixobactin is due to the synergistic inhibition of both targets, resulting in cell wall damage, delocalization of autolysins, and subsequent cell lysis. We also find that teixobactin does not bind mature peptidoglycan, further increasing its activity at high cell densities and against vancomycin-intermediate Staphylococcus aureus (VISA) isolates with thickened peptidoglycan layers. These findings add to the attractiveness of teixobactin as a potential therapeutic agent for the treatment of infection caused by antibiotic-resistant Gram-positive pathogens.

TheicaADBC-encoded polysaccharide intercellular adhesin (PIA) and wall teichoic acids (WTA) are structural components ofStaphylococcus epidermidisbiofilms. Deletion oftagO,which encodes the first enzymic step in WTA biosynthesis, had pleiotropic effects, including enhanced intercellular aggregation and autolytic activity, and impaired biofilm production. The biofilm-negative phenotype of thetagOmutant, named TAGO1, was associated with increased cell surface hydrophobicity, lower rates of primary attachment to polystyrene, and reducedicaADBCoperon and PIA expression. Mild acid stress induced by growth in BHI glucose media reduced rates of stationary phase autolysis and enhanced aggregation by TAGO1, leading to formation of a pellicle, which unlike a biofilm was only loosely attached to the polystyrene surface. TAGO1 pellicles were dispersed by proteinase K and DNase I but not sodium metaperiodate, implicating protein and extracellular DNA (eDNA) and not PIA in this phenotype. Substantially increased levels of eDNA were recovered from TAGO1 culture supernatants compared with the wild-type. These data indicate that WTA are essential for the primary attachment and accumulation phases of theS. epidermidisbiofilm phenotype. Furthermore, in the absence of WTA, proteins and eDNA can promote cell aggregation and pellicle formation, which also appear to limit interactions with artificial surfaces.

Persister cells are phenotypic variants surviving a lethal dose of antibiotic, sufficient to kill the bulk of an exponential phase population. In this chapter we summarize current techniques to isolate persisters and discuss limitations associated with identifying mechanisms of persister formation.

Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics(1). Persisters are associated with chronic infections and antibiotic treatment failure(1-3). In Escherichia coli, toxin-antitoxin modules have been linked to persister formation(4-6). The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting toxin-antitoxin modules in S. aureus did not affect the level of persisters. Here, we show that S. aureus persisters are produced due to a stochastic entrance into the stationary phase accompanied by a drop in intracellular adenosine triphosphate. Cells expressing stationary-state markers are present throughout the growth phase, and increase in frequency with cell density. Cell sorting revealed that the expression of stationary markers is associated with a 100-1,000-fold increase in the likelihood of survival to antibiotic challenge. The adenosine triphosphate level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.

Chronic coinfections of Staphylococcus aureus and Pseudomonas aeruginosa frequently fail to respond to antibiotic treatment, leading to significant patient morbidity and mortality. Currently, the impact of interspecies interaction on S. aureus antibiotic susceptibility remains poorly understood. In this study, we utilize a panel of P. aeruginosa burn wound and cystic fibrosis (CF) lung isolates to demonstrate that P. aeruginosa alters S. aureus susceptibility to bactericidal antibiotics in a variable, strain-dependent manner and further identify 3 independent interactions responsible for antagonizing or potentiating antibiotic activity against S. aureus. We find that P. aeruginosa LasA endopeptidase potentiates lysis of S. aureus by vancomycin, rhamnolipids facilitate proton-motive force-independent tobramycin uptake, and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) induces multidrug tolerance in S. aureus through respiratory inhibition and reduction of cellular ATP. We find that the production of each of these factors varies between clinical isolates and corresponds to the capacity of each isolate to alter S. aureus antibiotic susceptibility. Furthermore, we demonstrate that vancomycin treatment of a S. aureus mouse burn infection is potentiated by the presence of a LasA-producing P. aeruginosa population. These findings demonstrate that antibiotic susceptibility is complex and dependent not only upon the genotype of the pathogen being targeted, but also on interactions with other microorganisms in the infection environment. Consideration of these interactions will improve the treatment of polymicrobial infections., Author summary Accurate prediction of antimicrobial efficacy is essential for successful treatment of a bacterial infection. While many studies have considered the impacts of genetically encoded mechanisms of resistance, nongenetic determinants of antibiotic susceptibility during infection remain poorly understood. Here we show that a single interspecies interaction between 2 human pathogens, S. aureus and P. aeruginosa, can completely transform the antibiotic susceptibility profile of S. aureus. Through multiple distinct mechanisms, P. aeruginosa can antagonize or potentiate the efficacy of multiple classes of antibiotics against S. aureus. We identify the exoproducts responsible for altering S. aureus susceptibility to antibiotic killing, and furthermore demonstrate that these compounds are produced at varying levels in P. aeruginosa clinical isolates, with dramatic repercussions for S. aureus antibiotic susceptibility. Finally, we use a mouse model of P. aeruginosa–S. aureus coinfection to demonstrate that the presence of P. aeruginosa significantly alters the outcome of S. aureus antibiotic therapy in a host. These findings indicate that the efficacy of antibiotic treatment in polymicrobial infection is determined at the community level, with interspecies interaction playing an important and previously unappreciated role.

The polysaccharide intercellular adhesin or the cell wall-anchored accumulation-associated protein (Aap) mediates cellular accumulation during Staphylococcus epidermidis biofilm maturation. Mutation of sortase, which anchors up to 11 proteins (including Aap) to the cell wall, blocked biofilm development by the cerebrospinal fluid isolate CSF41498. Aap was implicated in this phenotype when Western blots and two-dimensional (2D) electrophoresis revealed increased levels of the protein in culture supernatants. Unexpectedly, reduced levels of primary attachment were associated with impaired biofilm formation by CSF41498 srtA and aap mutants. In contrast to previous studies, which implicated Aap proteolytic cleavage and, specifically, the Aap B domains in biofilm accumulation, the CSF41498 Aap protein was unprocessed. Furthermore, aap appeared to play a less important role in the biofilm phenotype of S. epidermidis 1457, in which the Aap protein is processed. Anti-Aap A-domain IgG inhibited primary attachment and biofilm formation in strain CSF41498 but not in strain 1457. The nucleotide sequences of the aap gene A-domain region and cleavage site in strains CSF41498 and 1457 were identical, implicating altered protease activity in the differential Aap processing results in the two strains. These data reveal a new role for the A domain of unprocessed Aap in the attachment phase of biofilm formation and suggest that extracellular protease activity can influence whether Aap contributes to the attachment or accumulation phases of the S. epidermidis biofilm phenotype.

Persister cells are phenotypic variants of a bacterial population that display tolerance to killing by bactericidal antibiotics. In this chapter we discuss the formation of persisters and their role in biofilm associated infections.

Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing dormant cells-activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections.

An initially quiescent quarter‐plane of fluid is set into motion by the action of a wall jet, i.e., a jet parallel and adjacent to a wall. The circumstances for which the jet separates from the wall and forms a dipole at the upstream head of the jet are studied numerically. The streamfunction‐vorticity formulation is used to track the time evolution of vorticity over a range of Reynolds numbers, 50≤Re≤1000, based on jet velocity and width. By implementing both a no‐slip and a slip boundary condition and comparing the results for the time evolution of vorticity, it is found that the boundary condition has a negligible effect on dipole development. By contrast, the relative magnitude of positive ω+ and negative ω− vorticity present in the inlet jet flow controls whether dipole formation occurs. In particular, dipole formation requires a sufficiently large magnitude of negative vorticity ω−/ω+≳0.65. The results refine a conjecture by Yushina (‘‘Evolution of the near‐wall jet,’’ in General Circulations of the...

Nature 517, 455–459 (2015); doi:10.1038/nature14098 In Fig. 3d of this Article, the ‘2:1’ and ‘1:1’ labels at the bottom of the panel were inadvertently switched during the production process; this figure has now been corrected in the online versions of the paper.