Diabetes is associated with impaired immune function and increased susceptibility to severe bacterial infections, yet the pathogen-encoded mechanisms that exacerbate disease in this context remain poorly defined. Streptococcus pyogenes (group A Streptococcus [GAS]) causes invasive skin and soft tissue infections that are disproportionately severe in individuals with diabetes, often accompanied by delayed healing, excessive inflammation, and polymicrobial overgrowth. Here, we investigated how the GAS ClpX-dependent regulatory pathway (CDRP), a global virulence regulator, interacts with diabetic immune dysfunction to shape infection outcomes. Using two murine models of type I diabetes, we show that diabetic mice develop more severe and persistent GAS skin infections characterized by increased bacterial burden, exaggerated inflammatory responses, impaired neutrophil recruitment, excessive neutrophil extracellular trap (NET) accumulation, and frequent polymicrobial infections. Deletion of clpX significantly attenuated GAS virulence in both diabetic and non-diabetic hosts; however, the pathogenic consequences of CDRP were markedly amplified in the diabetic environment. In diabetic mice, CDRP promoted sustained inflammation, altered neutrophil behavior, impaired NET clearance, and enhanced tissue damage, leading to delayed resolution of infection. Mechanistically, ClpX-dependent virulence was associated with dysregulated protease-nuclease activity, excessive NET persistence, and defective neutrophil chemotaxis within diabetic lesions. Together, these findings indicate that while ClpX is a core regulator of GAS virulence, its downstream effects are disproportionately deleterious when host immune clearance is compromised. IMPORTANCE:Diabetic patients experience disproportionately severe bacterial infections, yet the microbial mechanisms that exacerbate disease in this immunocompromised context remain incompletely understood. This study demonstrates that the Streptococcus pyogenes ClpX-dependent regulatory pathway, a central regulator of virulence, amplifies tissue damage and inflammatory dysfunction during diabetic skin infection. ClpX-dependent regulation exacerbates disease by intensifying neutrophil dysregulation, excessive NET accumulation, and impaired resolution of infection in an already compromised host environment. These findings underscore the importance of host-pathogen interactions in shaping infection severity and suggest that targeting pathogen regulatory pathways may be particularly effective in settings of immune dysfunction such as diabetes.

Streptococcus pyogenes is a human pathogen that causes severe necrotizing soft tissue infections characterized by rapid and extensive tissue destruction. A key challenge in studying these infections is the lack of experimental models that allow independent assessment of bacterial replication and host-mediated tissue damage. This article presents a standardized and reproducible murine subcutaneous model of S. pyogenes necrotizing skin infection designed to overcome this limitation. The protocol describes the preparation of a log-phase bacterial inoculum, subcutaneous infection of mice, and longitudinal monitoring of lesion development. Key steps include sonication of bacterial cultures to standardize chain length, digital imaging of lesions for quantitative analysis of ulcer area, and determination of bacterial burden through tissue homogenization and plating. This versatile model can be readily adapted to evaluate the effects of specific bacterial genes, host factors, or therapeutic interventions on infection progression and tissue pathology. By enabling reproducible and quantitative assessment of both bacterial and host parameters, this method provides a robust platform for studying pathogenesis and testing strategies to limit tissue damage during invasive streptococcal infection.

Catheter-associated urinary tract infections (CAUTIs) account for approximately 80% of urinary tract infections (UTI) and can lead to adverse outcomes. Most CAUTIs are polymicrobial with resilient communities maintaining a consistent composition of species over time. However, the mechanisms promoting persistence are poorly understood. Here, we examine how a chemical interaction between Enterococcus faecalis and Klebsiella pneumoniae can explain their high rate of co-occurrence on long-term indwelling catheters. Sequence analyses of longitudinal isolates from several patients coinfected with E. faecalis and K. pneumoniae revealed that despite frequent replacement, catheters became recolonized with the same or a nearly identical consortium of strains throughout the collection period. Using artificial urine medium (AUM), monoculture revealed that the K. pneumoniae isolates grew robustly and formed biofilm, while the E. faecalis isolates grew poorly and did not form biofilm. However, coculture of paired isolates resulted in enhanced E. faecalis growth and biofilm, which could be reproduced by supplementing E. faecalis with K. pneumoniae conditioned AUM supernatant (KpAUMSup). Analyses using comparative transcriptomics, mutant strains, and chemical inhibitors with cell culture and murine CAUTI models revealed that (i) KpAUMSup, but not AUM, stimulated expression of the E. faecalis Fsr quorum sensing system; (ii) Fsr was required for E. faecalis to respond to KpAUMSup; (iii) E. faecalis cultured in KpAUMSup was more efficient in initiating CAUTI; and (iv) disruption of Fsr inhibited initiation of CAUTI. This interspecies signaling may help explain the high rate of co-colonization of these CAUTI pathogens and highlights therapeutic strategies to treat polymicrobial CAUTI.

Building on our finding that Streptococcus pyogenes pyruvate dehydrogenase (PDH) suppresses host disease tolerance (DT) via short-chain fatty acid (SCFA)-mediated modulation of host acetyl-CoA and IL-10 levels, we characterize the global transcriptomic and epigenetic mechanisms underlying this immunometabolic manipulation. Combining new histological and ultrastructural analyses with an in-depth re-analysis of single-cell and bulk RNA-seq datasets to more comprehensively characterize the DT response, we show that PDH deficiency is associated with broad immunologic rewiring, characterized by intracellular bacterial containment within phagocytes, expansion of pro-resolving myeloid cells, and altered cell-cell communication. Metabolic analysis of ΔPdh-infected tissues revealed a shift away from acetyl-CoA metabolism towards glycolysis and the coordinated activation of a multi-faceted DT program, encompassing hypoxia signaling, iron handling, and the NRF2-mediated antioxidant response. Crucially, a focused re-analysis of existing transcriptome datasets from Histone Deacetylase (HDAC)-inhibited macrophages suggested that Trichostatin A (TSA) abrogates the protective transcriptome in ΔPdh infection, indicating that acetylation-dependent repression functions as a key regulator of the host DT response. By integrating new experimental data with advanced computational analyses, our work reveals a bacterial strategy of metabolic-epigenetic crosstalk, suggesting acetylation as a critical control point for mitigating infection-associated tissue damage.

Antimicrobial resistance (AMR) in common bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), is an increasingly dire public health threat, with MRSA accounting for up to 90% of S. aureus infections. To expand the treatment arsenal against MRSA infections, we developed a class of tunable three-dimensional tricyclic 2-pyridones, termed TriPcides, that can kill MRSA resistant to last-resort antibiotics and eliminate MRSA persister cells. No preexisting resistance was detected across hundreds of clinical isolates, and continuous exposure of MRSA to TriPcides did not elicit detectable resistance. Treatment with TriPcides causes a rapid decrease in membrane integrity and increased levels of reactive oxygen species. Last, TriPcides effectively reduce secretion of important virulence factors and result in reduced ulcer size and healing time in S. aureus murine skin and soft tissue infections but do not reduce bacterial burden.

Urinary catheterization, a frequent procedure in hospitals, nursing homes, and other healthcare facilities, is a primary driver of nosocomial infections. The most common of these are catheter-associated urinary tract infections (CAUTIs), which often progress to severe complication, sepsis, and ultimately death. The fungus Candida albicans is a primary causative agent of CAUTIs; yet, its tissue-specific pathogenesis remains poorly understood, which complicates development of efficient treatments. While Efg1 is a known virulence driver in CAUTI, its specific downstream targets within the unique bladder environment have not been defined. Here, we identify and validate the EFG1 regulon that is active during conditions that mimic the human catheterized bladder and, additionally, confirm the regulon by transcriptional profiling of catheters retrieved from patients with C. albicans infection. We found that this urine-specific signature is highly conserved in clinical samples, with Efg1-dependent genes being among the most robustly induced transcripts during active human infection. Furthermore, we characterized two of these key factors, ECE1 and EED1 , validating their roles in infection both in vitro in human urine and in vivo using a CAUTI mouse model. Elucidating this tissue-specific regulon offers a strategic roadmap for the development of targeted therapies to mitigate these ever-increasing life-threatening fungal infections.

Urinary catheterization, a common procedure in hospitals and nursing home facilities, is a primary driver of hospital-acquired infections (HAI). These devices frequently lead to catheter-associated urinary tract infections (CAUTIs), which often progress to severe complication, sepsis, and ultimately death. The fungus Candida albicans has emerged as the second most common causative agent of CAUTIs; yet, its pathogenesis is poorly understood, which complicates development of efficient treatments. Previously, we identified the transcription factor Efg1 as a critical virulence driver in C. albicans CAUTIs. However, its specific downstream targets within the unique bladder microenvironment remained unknown. This study identifies, for the first time, the complete Efg1 regulon that is active during growth in human urine. We confirmed the clinical relevance of this discovery, finding that many of these Efg1-regulated factors are present and significantly upregulated in catheter samples from patients with C. albicans infections. Furthermore, we characterized two of these key factors, ECE1 and EED1, validating their roles both in vitro in urine conditions and in vivo using a CAUTI mouse model. Identifying the tissue-specific downstream targets of Efg1 elucidates the precise mechanism of fungal CAUTI. This knowledge provides a new roadmap for developing targeted therapeutics, offering vital antimicrobial-sparing strategies to combat these life-threatening infections.

Disease tolerance is a host response to infection that limits collateral damage to host tissues while having a neutral effect on pathogen fitness. Previously, we found that the pathogenic lactic acid bacterium Streptococcus pyogenes manipulates disease tolerance using its aerobic mixed-acid fermentation pathway via the enzyme pyruvate dehydrogenase, but the microbe-derived molecules that mediate communication with the host's disease tolerance pathways remain elusive. Here we show in a murine model that aerobic mixed-acid fermentation inhibits the accumulation of inflammatory cells including neutrophils and macrophages, reduces the immunosuppressive cytokine interleukin-10, and delays bacterial clearance and wound healing. In infected macrophages, the aerobic mixed-acid fermentation end-products acetate and formate from streptococcal upregulate host acetyl-CoA metabolism and reduce interleukin-10 expression. Inhibiting aerobic mixed-acid fermentation using a bacterial-specific pyruvate dehydrogenase inhibitor reduces tissue damage during murine infection, correlating with increased interleukin-10 expression. Our results thus suggest that reprogramming carbon flow provides a therapeutic strategy to mitigate tissue damage during infection.

The bacterium Enterococcus faecalis is a leading cause of catheter-associated urinary tract infection (CAUTI), whose treatment is increasingly challenged by antibiotic resistance. In examining alternative therapies, we previously found that the cysteine protease inhibitor E64 dramatically reduced bladder inflammation and bacterial dissemination in a murine model of E. faecalis CAUTI. However, the role of cysteine proteases in pathogenesis and the target(s) of E64 are unknown. Here, we found that while it did not affect E. faecalis growth in vitro, in a murine CAUTI model, E64 reduced host caspase-1-dependent cellular apoptosis and necrosis, levels of several pro-inflammatory cytokines, bladder epithelial damage, and formation of renal abscesses. Analysis of host cell transcription and inflammatory cell populations revealed that E64 did not affect neutrophil numbers but did enhance the expression of C-C chemokine receptor type 3 (CCR3), a receptor for eosinophil-specific chemokines, with a concomitant increase in eosinophil numbers. Treatments that reduced or increased the eosinophil response (anti-interleukin 5 antibody or eotaxin, respectively) confirmed a role for eosinophils in controlling bacterial burdens. Analysis of a panel of host cysteine proteases in vivo demonstrated that E64 decreased the activation of cathepsin L; subsequently, we found that the infection of cathepsin L-deficient mice yielded lower catheter and bladder colonization compared to wild-type mice. In total, we have shown that host cysteine proteases exacerbate CAUTI pathogenesis, suggesting that cysteine protease modulation may represent a novel approach for the treatment of persistent CAUTI.IMPORTANCECatheter-associated urinary tract infections (CAUTIs) are the most prevalent healthcare-associated infection globally, with Enterococcus faecalis posing a significant threat due to widespread antibiotic resistance. This study identifies host cysteine proteases-particularly cathepsin L and caspase-1-as unrecognized drivers of CAUTI pathogenesis and renal fibrosis. Pharmacologic inhibition of these proteases using E64 reduces bladder inflammation, epithelial disruption, and kidney abscesses, while restoring fibrinogen and collagen homeostasis. Strikingly, E64 treatment unmasks a protective eosinophil response via CCR3 signaling that enhances bacterial clearance. Genetic deletion of cathepsin L recapitulates these protective effects, establishing it as a key host factor in E. faecalis persistence. These findings reveal host cysteine proteases as viable therapeutic targets for CAUTI and provide proof-of-concept for host-directed strategies that bypass antibiotic resistance.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis , a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Catheter-associated urinary tract infections (CAUTIs), a common cause of healthcare-associated infections, are caused by a diverse array of pathogens that are increasingly becoming antibiotic resistant. We analyze the microbial occurrences in catheter and urine samples from 55 human long-term catheterized patients collected over one year. Although most of these patients were prescribed antibiotics over several collection periods, their catheter samples remain colonized by one or more bacterial species. Examination of a total of 366 catheter and urine samples identify 13 positive and 13 negative genus co-occurrences over 12 collection periods, representing associations that occur more or less frequently than expected by chance. We find that for many patients, the microbial species composition between collection periods is similar. In a subset of patients, we find that the most frequently sampled bacteria, Escherichia coli and Enterococcus faecalis , co-localize on catheter samples. Further, co-culture of paired isolates recovered from the same patients reveals that E. coli significantly augments E. faecalis growth in an artificial urine medium, where E. faecalis monoculture grows poorly. These findings suggest novel strategies to collapse polymicrobial CAUTI in long-term catheterized patients by targeting mechanisms that promote positive co-associations.

We have developed GmPcides from a peptidomimetic dihydrothiazolo ring-fused 2-pyridone scaffold that has antimicrobial activities against a broad spectrum of Gram-positive pathogens. Here, we examine the treatment efficacy of GmPcides using skin and soft tissue infection (SSTI) and biofilm formation models by Streptococcus pyogenes . Screening our compound library for minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations identified GmPcide PS757 as highly active against S. pyogenes . Treatment of S. pyogenes biofilm with PS757 revealed robust efficacy against all phases of biofilm formation by preventing initial biofilm development, ceasing biofilm maturation and eradicating mature biofilm. In a murine model of S. pyogenes SSTI, subcutaneous delivery of PS757 resulted in reduced levels of tissue damage, decreased bacterial burdens, and accelerated rates of wound healing, which were associated with down-regulation of key virulence factors, including M protein and the SpeB cysteine protease. These data demonstrate that GmPcides show considerable promise for treating S. pyogenes infections.

Microbial pathogens balance growth against tissue damage to achieve maximum fitness. Central carbon metabolism is connected to growth, but how it influences growth/damage balance is largely unknown. Here we examined how carbon flux through the exclusively fermentative metabolism of the pathogenic lactic acid bacterium Streptococcus pyogenes impacts patterns of growth and tissue damage. Using a murine model of soft tissue infection, we systematically examined single and pair-wise mutants that constrained carbon flux through the three major pathways that S . pyogenes employs for reduction of the glycolytic intermediate pyruvate, revealing distinct disease outcomes. Its canonical lactic acid pathway (via lactate dehydrogenase) made a minimal contribution to virulence. In contrast, its two parallel pathways for mixed-acid fermentation played important, but non-overlapping roles. Anaerobic mixed acid fermentation (via pyruvate formate lyase) was required for growth in tissue, while aerobic mixed-acid pathway (via pyruvate dehydrogenase) was not required for growth, but instead regulated levels of tissue damage. Infection of macrophages in vitro revealed that pyruvate dehydrogenase was required to prevent phagolysosomal acidification, which altered expression of the immunosuppressive cytokine IL-10. Infection of IL-10 deficient mice confirmed that the ability of aerobic metabolism to regulate levels of IL-10 plays a key role in the ability of S . pyogenes to modulate levels of tissue damage. Taken together, these results show critical non-overlapping roles for anaerobic and aerobic metabolism in soft tissue infection and provide a mechanism for how oxygen and carbon flux act coordinately to regulate growth/damage balance. Therapies targeting carbon flux could be developed to mitigate tissue damage during severe S . pyogenes infection.

ABSTRACT Sore throat is one of the most common complaints encountered in the ambulatory clinical setting. Rapid, culture-independent diagnostic techniques that do not rely on pharyngeal swabs would be highly valuable as a point-of-care strategy to guide outpatient antibiotic treatment. Despite the promise of this approach, efforts to detect volatiles during oropharyngeal infection have yet been limited. In our research study, we sought to evaluate for specific bacterial volatile organic compounds (VOC) biomarkers in isolated cultures in vitro , in order to establish proof-of-concept prior to initial clinical studies of breath biomarkers. A particular challenge for the diagnosis of pharyngitis due to Streptococcus pyogenes is the likelihood that many metabolites may be shared by S. pyogenes and other related oropharyngeal colonizing bacterial species. Therefore, we evaluated whether sufficient metabolic differences are present, which distinguish the volatile metabolome of Group A streptococci from other streptococcal species that also colonize the respiratory mucosa, such as Streptococcus pneumoniae and Streptococcus intermedius . In this work, we identified 27 discriminatory VOCs ( q -values < 0.05), composed of aldehydes, alcohols, nitrogen-containing compounds, hydrocarbons, ketones, aromatic compounds, esters, ethers, and carboxylic acid. From this group of volatiles, we identify candidate biomarkers that distinguish S. pyogenes from other species and establish highly produced VOCs that indicate the presence of S. pyogenes in vitro , supporting future breath-based diagnostic testing for streptococcal pharyngitis. IMPORTANCE Acute pharyngitis accounts for approximately 15 million ambulatory care visits in the United States. The most common and important bacterial cause of pharyngitis is Streptococcus pyogenesis , accounting for 15%–30% of pediatric pharyngitis. Distinguishing between bacterial and viral pharyngitis is key to management in US practice. The culture of a specimen obtained by a throat swab is the standard laboratory procedure for the microbiologic confirmation of pharyngitis; however, this method is time-consuming, which delays appropriate treatment. If left untreated, S. pyogenes pharyngitis may lead to local and distant complications. In this study, we characterized the volatile metabolomes of S. pyogenes and other related oropharyngeal colonizing bacterial species. We identify candidate biomarkers that distinguish S. pyogenes from other species and provide evidence to support future breath-based diagnostic testing for streptococcal pharyngitis.

ABSTRACT Sore throat is one of the most common complaints encountered in the ambulatory clinical setting. Rapid, culture-independent diagnostic techniques that do not rely on pharyngeal swabs would be highly valuable as a point-of-care strategy to guide outpatient antibiotic treatment. Despite the promise of this approach, efforts to detect volatiles during oropharyngeal infection have yet been limited. In our research study, we sought to evaluate for specific bacterial volatile organic compounds (VOC) biomarkers in isolated cultures in vitro , in order to establish proof-of-concept prior to initial clinical studies of breath biomarkers. A particular challenge for diagnosis of pharyngitis due to Streptococcus pyogenes is the likelihood that many metabolites may be shared by S. pyogenes and other related oropharyngeal colonizing bacterial species. Therefore, we evaluated whether sufficient metabolic differences are present that distinguish the volatile metabolome of Group A streptococci from other streptococcal species that also colonize the respiratory mucosa, such as S. pneumoniae and S. intermedius . In this work, we identify candidate biomarkers that distinguish S. pyogenes from other species, and establish highly produced VOCs that indicate presence of S. pyogenes in vitro , supporting future breath-based diagnostic testing for streptococcal pharyngitis. IMPORTANCE Acute pharyngitis accounts for approximately 15 million ambulatory care visits in the USA. The most common and important bacterial cause of pharyngitis is Streptococcus pyogenesis , accounting for 15% to 30% of pediatric pharyngitis. Distinguishing between bacterial and viral pharyngitis is key to management in US practice. Culture of a specimen obtained by throat swab is the standard laboratory procedure for the microbiologic confirmation of pharyngitis, however this method is time consuming which delays appropriate treatment. If left untreated, S. pyogenes pharyngitis may lead to local and distant complications. In this study, we characterized the volatile metabolomes of S. pyogenes and other related oropharyngeal colonizing bacterial species. We identify candidate biomarkers that distinguish S. pyogenes from other species and provides evidence to support future breath-based diagnostic testing for streptococcal pharyngitis.

MRSA-1369 is a uropathogenic methicillin-resistant Staphylococcus aureus (MRSA) strain. Here, we present the complete genome sequence of MRSA-1369, which consists of one chromosome (2.87 Mb) and two plasmids (16.68 kb and 3.13 kb). This will serve as a reference genome for future Staphylococcus aureus pathogenesis and multiomic studies.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Antibiotic-resistant bacteria in the genus Enterococcus are a major cause of nosocomial infections and are an emergent public health concern. Similar to a number of bacterial species, resistance to the antibiotic rifampicin (RifR) in enterococci is associated with mutations in the gene encoding the β subunit of RNA polymerase (rpoB). In Mycobacterium tuberculosis, RifRrpoB mutations alter mycobacterial surface lipid expression and are associated with an altered IL-1 cytokine response in macrophages upon infection. However, it is not clear if RifR mutations modulate host cytokine responses by other bacteria. To address this question, we utilized Enterococcus faecalis (E. faecalis). Here, we treated human monocyte-derived macrophages with heat-inactivated wild type or RifRrpoB mutants of E. faecalis and found that RifR mutations reduced IL-1β cytokine production. However, RifR mutations elicited other potent pro- and anti-inflammatory responses, indicating that they can impact other immune pathways beyond IL-1R1 signaling. Our findings suggest that immunomodulation by mutations in rpoB may be conserved across diverse bacterial species and that subversion of IL-1R1 pathway is shared by RifR bacteria.

Purpose Catheter-associated urinary tract infections (CAUTIs) are a significant cause of morbidity worldwide, as they account for 40% of all hospital-associated infections. Microbial biofilm formation on urinary catheters (UCs) limits antibiotic efficacy, making CAUTI extremely difficult to treat. To gain insight into the spatiotemporal microbe interactions on the catheter surface we sought to determine how the presence or absence of bacteriuria prior to catheterization affects the organism that ultimately forms a biofilm on the UC and how long after catheterization they emerge. Methods Thirty UCs were collected from patients who received a urine culture prior to catheterization, a UC, and antibiotics as part of standard of care. Immunofluorescence imaging and scanning electron microscopy were used to visualize patient UCs. Results Most patients did not have bacteria in their urine (based on standard urinalysis) prior to catheterization, yet microbes were detected on the majority of UCs, even with dwell times of < 3 days. The most frequently identified microbes were Staphylococcus epidermidis, Enterococcus faecalis, and Escherichia coli. Conclusions This study indicates that despite patients having negative urine cultures and receiving antibiotics prior to catheter placement, microbes, including uropathogens associated with causing CAUTI, could be readily detected on UCs with short dwell times. This suggests that a potential microbial catheter reservoir can form soon after placement, even in the presence of antibiotics, which may serve to facilitate the development of CAUTI. Thus, removing and/or replacing UCs as soon as possible is of critical importance to reduce the risk of developing CAUTI.

Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease. IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae. However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.