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.

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.

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.

Overcoming biological barriers to improve treatment of a Staphylococcus aureus wound infection.

Chronic wounds frequently become infected with bacterial biofilms which respond poorly to antibiotic therapy. Aminoglycoside antibiotics are ineffective at treating deep-seated wound infections due to poor drug penetration, poor drug uptake into persister cells, and widespread antibiotic resistance. In this study, we combat the two major barriers to successful aminoglycoside treatment against a biofilm-infected wound: limited antibiotic uptake and limited biofilm penetration. To combat the limited antibiotic uptake, we employ palmitoleic acid, a host-produced monounsaturated fatty acid that perturbs the membrane of gram-positive pathogens and induces gentamicin uptake. This novel drug combination overcomes gentamicin tolerance and resistance in multiple gram-positive wound pathogens. To combat biofilm penetration, we examined the ability of sonobactericide, a non-invasive ultrasound-mediated-drug delivery technology to improve antibiotic efficacy using an in vivo biofilm model. This dual approach dramatically improved antibiotic efficacy against a methicillin-resistant Staphylococcus aureus (MRSA) wound infection in diabetic mice.

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.

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.

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.

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.

Marine Actinomycetes, New Sources of Biotechnological Products.

The Actinomycetales order is one of great genetic and functional diversity, including diversity in the production of secondary metabolites which have uses in medical, environmental rehabilitation, and industrial applications. Secondary metabolites produced by actinomycete species are an abundant source of antibiotics, antitumor agents, anthelmintics, and antifungals. These actinomycete-derived medicines are in circulation as current treatments, but actinomycetes are also being explored as potential sources of new compounds to combat multidrug resistance in pathogenic bacteria. Actinomycetes as a potential to solve environmental concerns is another area of recent investigation, particularly their utility in the bioremediation of pesticides, toxic metals, radioactive wastes, and biofouling. Other applications include biofuels, detergents, and food preservatives/additives. Exploring other unique properties of actinomycetes will allow for a deeper understanding of this interesting taxonomic group. Combined with genetic engineering, microbial experimental evolution, and other enhancement techniques, it is reasonable to assume that the use of marine actinomycetes will continue to increase. Novel products will begin to be developed for diverse applied research purposes, including zymology and enology. This paper outlines the current knowledge of actinomycete usage in applied research, focusing on marine isolates and providing direction for future research.

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.

Harnessing ultrasound-stimulated phase change contrast agents to improve antibiotic efficacy against methicillin-resistant Staphylococcus aureus biofilms.

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.

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.

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.

Author Correction: Reactive oxygen species induce antibiotic tolerance during systemic Staphylococcus aureus infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reactive oxygen species induce antibiotic tolerance during systemic Staphylococcus aureus infection.

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 treatment1. Phagocytosis by macrophages and neutrophils is the primary mechanism through 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 generated by respiratory burst attack iron-sulfur cluster-containing proteins, including TCA-cycle enzymes, result in decreased respiration, lower ATP and increased antibiotic tolerance. We further show that respiratory burst induces antibiotic tolerance in the spleen during a murine systemic infection. These results suggest that a major component of the innate immune response is antagonistic to the bactericidal activities of antibiotics.

Chemical Induction of Aminoglycoside Uptake Overcomes Antibiotic Tolerance and Resistance in Staphylococcus aureus.

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.

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria.

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

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.

ATP-Dependent Persister Formation in Escherichia coli.

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.