In-patient evolution of a high-persister Escherichia coli strain with reduced in vivo antibiotic susceptibility.

Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Despite appropriate antibiotic treatment, relapse of GNB-BSI with the same bacterial strain is common and associated with poor clinical outcomes and high healthcare costs. The role of persister cells, which are sub-populations of bacteria that survive for prolonged periods in the presence of bactericidal antibiotics, in relapse of GNB-BSI is unclear. Using a cohort of patients with relapsed GNB-BSI, we aimed to determine how the pathogen evolves within the patient between the initial and subsequent episodes of GNB-BSI and how these changes impact persistence. Using Escherichia coli clinical bloodstream isolate pairs (initial and relapse isolates) from patients with relapsed GNB-BSI, we found that 4/11 (36%) of the relapse isolates displayed a significant increase in persisters cells relative to the initial bloodstream infection isolate. In the relapsed E. coli strain with the greatest increase in persisters (100-fold relative to initial isolate), we determined that the increase was due to a loss-of-function mutation in the ptsI gene encoding Enzyme I of the phosphoenolpyruvate phosphotransferase system. The ptsI mutant was equally virulent in a murine bacteremia infection model but exhibited 10-fold increased survival to antibiotic treatment. This work addresses the controversy regarding the clinical relevance of persister formation by providing compelling data that not only do high-persister mutations arise during bloodstream infection in humans but also that these mutants display increased survival to antibiotic challenge in vivo.

Fibrin(ogen) engagement of S. aureus promotes the host antimicrobial response and suppression of microbe dissemination following peritoneal infection.

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) ß2-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.

Shooting yourself in the foot: How immune cells induce antibiotic tolerance in microbial pathogens.

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.

Recalcitrant Staphylococcus aureus Infections: Obstacles and Solutions.

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.

Macrophage-Produced Peroxynitrite Induces Antibiotic Tolerance and Supersedes Intrinsic Mechanisms of Persister Formation.

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 and suggest that modulation of the host response may represent a viable therapeutic strategy to sensitize S. aureus to antibiotics.

A new antibiotic kills pathogens without detectable resistance.

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.

Pseudomonas aeruginosa exoproducts determine antibiotic efficacy against Staphylococcus aureus.

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.

Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis.

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.

Staphylococcus aureus chronic and relapsing infections: Evidence of a role for persister cells: An investigation of persister cells, their formation and their role in S. aureus disease.

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.

Role for the A domain of unprocessed accumulation-associated protein (Aap) in the attachment phase of the Staphylococcus epidermidis biofilm phenotype.

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.

Host stress drives tolerance and persistence: The bane of anti-microbial therapeutics.

Antibiotic resistance, typically associated with genetic changes within a bacterial population, is a frequent contributor to antibiotic treatment failures. Antibiotic persistence and tolerance, which we collectively term recalcitrance, represent transient phenotypic changes in the bacterial population that prolong survival in the presence of typically lethal concentrations of antibiotics. Antibiotic recalcitrance is challenging to detect and investigate-traditionally studied under in vitro conditions, our understanding during infection and its contribution to antibiotic failure is limited. Recently, significant progress has been made in the study of antibiotic-recalcitrant populations in pathogenic species, including Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica, and Yersiniae, in the context of the host environment. Despite the diversity of these pathogens and infection models, shared signals and responses promote recalcitrance, and common features and vulnerabilities of persisters and tolerant bacteria have emerged. These will be discussed here, along with progress toward developing therapeutic interventions to better treat recalcitrant pathogens.

Persistent Methicillin-Resistant Staphylococcus aureus Bacteremia: Host, Pathogen, and Treatment.

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.

Antibiotic Tolerance and Treatment Outcomes in Cystic Fibrosis Methicillin-Resistant Staphylococcus aureus Infections.

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 (n = 90) collected at the incident (i.e., acute) and early infection states from 57 patients were characterized for growth rates, biofilm formation, hemolysis, pigmentation, and vancomycin tolerance. The resistance profiles were consistent with those in prior studies. Isolates from the early stage of infection were found to produce biofilms, and 70% of the isolates exhibited delta-hemolysis, an indicator of agr activity. Strong vancomycin tolerance was present in 24% of the isolates but was not associated with intermediate vancomycin susceptibility. There were no associations between these phenotypic measures, antibiotic tolerance, and MRSA clearance. Our research suggests that additional factors may be relevant for predicting the clearance of MRSA. IMPORTANCE Chronic MRSA infections remain challenging to treat in patients with cystic fibrosis (CF). The ability of the bacterial population to survive high concentrations of bactericidal antibiotics, including vancomycin, despite lacking resistance is considered one of the main reasons for treatment failures. The connection between antibiotic tolerance and treatment outcomes remains unexplored and can be crucial for prognosis and regimen design toward eradication. In this study, we measured the capacity of 90 MRSA isolates from CF patients to form vancomycin-tolerant persister cells and evaluated their correlation with the clinical outcomes. Additionally, various traits that could reflect the metabolism and/or virulence of those MRSA isolates were systematically phenotyped and included for their predictive power. Our research highlights that despite the importance of antibiotic tolerance, additional factors need to be considered for predicting the clearance of MRSA.

Inflammasome-mediated glucose limitation induces antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus is a leading human pathogen that frequently causes relapsing infections. The failure of antibiotics to eradicate infection contributes to infection relapse. Host-pathogen interactions have a substantial impact 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. Induction of 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, suggesting that this strategy has potential as a host-directed therapeutic to improve outcomes. Our findings identify interactions between S. aureus and the host that result in metabolic crosstalk that can determine the outcome of antimicrobial therapy.

Antibiotic-induced accumulation of lipid II synergizes with antimicrobial fatty acids to eradicate bacterial populations.

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 Fail to Eradicate Staphylococcus aureus Biofilm from Infected Implant Materials.

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.

The Use of Acute Immunosuppressive Therapy to Improve Antibiotic Efficacy against Intracellular Staphylococcus aureus.

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. IMPORTANCE Staphylococcus aureus is the leading cause of hospital-acquired infections in the United States with high rates of antibiotic treatment failure. Macrophages represent an important intracellular niche in experimental models of S. aureus bacteremia. Although a proinflammatory macrophage response is critical for controlling infection, previous studies have identified an antagonistic relationship between antibiotic treatment and the proinflammatory macrophage response. Reactive oxygen species, produced by macrophages during respiratory burst, coerce S. aureus into an antibiotic tolerant state, leading to poor treatment outcome. Here, we aimed to determine the potential of host-directed immunomodulators that reduce the production of reactive oxygen species to improve antibiotic efficacy against intracellular S. aureus.

Stimulating Aminoglycoside Uptake to Kill Staphylococcus aureus Persisters.

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.