Function and contribution of two putative Enterococcus faecalis glycosaminoglycan degrading enzymes to bacteremia and catheter-associated urinary tract infection.

Enterococcus faecalis is a common cause of healthcare-acquired bloodstream infections and catheter-associated urinary tract infections (CAUTIs) in both adults and children. Treatment of E. faecalis infection is frequently complicated by multi-drug resistance. Based on protein homology, E. faecalis encodes two putative hyaluronidases, EF3023 (HylA) and EF0818 (HylB). In other Gram-positive pathogens, hyaluronidases have been shown to contribute to tissue damage and immune evasion, but the function in E. faecalis has yet to be explored. Here, we show that both hylA and hylB contribute to E. faecalis pathogenesis. In a CAUTI model, ΔhylA exhibited defects in bladder colonization and dissemination to the bloodstream, and ΔhylB exhibited a defect in kidney colonization. Furthermore, a ΔhylAΔhylB double mutant exhibited a severe colonization defect in a model of bacteremia while the single mutants colonized to a similar level as the wild-type strain, suggesting potential functional redundancy within the bloodstream. We next examined enzymatic activity, and demonstrate that HylB is capable of digesting both hyaluronic acid (HA) and chondroitin sulfate in vitro, while HylA exhibits only a very modest activity against heparin. Importantly, HA degradation by HylB provided a modest increase in cell density during the stationary phase and also contributed to dampening of lipopolysaccharide-mediated NF-κB activation. Overall, these data demonstrate that glycosaminoglycan degradation is important for E. faecalis pathogenesis in the urinary tract and during bloodstream infection.

Preferential catabolism of l- vs d-serine by Proteus mirabilis contributes to pathogenesis and catheter-associated urinary tract infection.

Proteus mirabilis is a common cause of urinary tract infection, especially in catheterized individuals. Amino acids are the predominant nutrient for bacteria during growth in urine, and our prior studies identified several amino acid import and catabolism genes as fitness factors for P. mirabilis catheter-associated urinary tract infection (CAUTI), particularly those for d- and l-serine. In this study, we sought to determine the hierarchy of amino acid utilization by P. mirabilis and to examine the relative importance of d- vs l-serine catabolism for critical steps in CAUTI development and progression. Herein, we show that P. mirabilis preferentially catabolizes l-serine during growth in human urine, followed by d-serine, threonine, tyrosine, glutamine, tryptophan, and phenylalanine. Independently disrupting catabolism of either d- or l-serine has minimal impact on in vitro phenotypes while completely disrupting both pathways decreases motility, biofilm formation, and fitness due to perturbation of membrane potential and cell wall biosynthesis. In a mouse model of CAUTI, loss of either serine catabolism system decreased fitness, but disrupting l-serine catabolism caused a greater fitness defect than disrupting d-serine catabolism. We, therefore, conclude that the hierarchical utilization of amino acids may be a critical component of P. mirabilis colonization and pathogenesis within the urinary tract.

Catalase Activity is Critical for Proteus mirabilis Biofilm Development, Extracellular Polymeric Substance Composition, and Dissemination during Catheter-Associated Urinary Tract Infection.

Proteus mirabilis is a leading uropathogen of catheter-associated urinary tract infections (CAUTIs), which are among the most common health care-associated infections worldwide. A key factor that contributes to P. mirabilis pathogenesis and persistence during CAUTI is the formation of catheter biofilms, which provide increased resistance to antibiotic treatment and host defense mechanisms. Another factor that is important for bacterial persistence during CAUTI is the ability to resist reactive oxygen species (ROS), such as through the action of the catalase enzyme. Potent catalase activity is one of the defining biochemical characteristics of P. mirabilis, and the single catalase (katA) gene in strain HI4320 was recently identified as a candidate fitness factor for UTI, CAUTI, and bacteremia. Here, we show that disruption of katA results in increased ROS levels, increased sensitivity to peroxide, and decreased biofilm biomass. The biomass defect was due to a decrease in the production of extracellular polymeric substances (EPS) by the ΔkatA mutant and specifically due to reduced carbohydrate content. Importantly, the biofilm defect resulted in decreased antibiotic resistance in vitro and a colonization defect during experimental CAUTI. The ΔkatA mutant also exhibited decreased fitness in a bacteremia model, supporting a dual role for catalase in P. mirabilis biofilm development and immune evasion.

Polymicrobial interactions in the urinary tract: is the enemy of my enemy my friend?

The vast majority of research pertaining to urinary tract infection has focused on a single pathogen in isolation, and predominantly Escherichia coli. However, polymicrobial urine colonization and infection are prevalent in several patient populations, including individuals with urinary catheters. The progression from asymptomatic colonization to symptomatic infection and severe disease is likely shaped by interactions between traditional pathogens as well as constituents of the normal urinary microbiota. Recent studies have begun to experimentally dissect the contribution of polymicrobial interactions to disease outcomes in the urinary tract, including their role in development of antimicrobial-resistant biofilm communities, modulating the innate immune response, tissue damage, and sepsis. This review aims to summarize the epidemiology of polymicrobial urine colonization, provide an overview of common urinary tract pathogens, and present key microbe-microbe and host-microbe interactions that influence infection progression, persistence, and severity.

Ynt is the primary nickel import system used by Proteus mirabilis and specifically contributes to fitness by supplying nickel for urease activity.

Proteus mirabilis is a Gram-negative uropathogen and frequent cause of catheter-associated urinary tract infection (CAUTI). One important virulence factor is its urease enzyme, which requires nickel to be catalytically active. It is, therefore, hypothesized that nickel import is critical for P. mirabilis urease activity and pathogenesis during infection. P. mirabilis strain HI4320 encodes two putative nickel import systems, designated Nik and Ynt. By disrupting the substrate-binding proteins from each import system (nikA and yntA), we show that Ynt is the primary nickel importer, while Nik only compensates for loss of Ynt at high nickel concentrations. We further demonstrate that these are the only binding proteins capable of importing nickel for incorporation into the urease enzyme. Loss of either nickel-binding protein results in a significant fitness defect in a murine model of CAUTI, but YntA is more crucial as the yntA mutant was significantly outcompeted by the nikA mutant. Furthermore, despite the importance of nickel transport for hydrogenase activity, the sole contribution of yntA and nikA to virulence is due to their role in urease activity, as neither mutant exhibited a fitness defect when disrupted in a urease-negative background.

Twin arginine translocation, ammonia incorporation, and polyamine biosynthesis are crucial for Proteus mirabilis fitness during bloodstream infection.

The Gram-negative bacterium Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTI), which can progress to secondary bacteremia. While numerous studies have investigated experimental infection with P. mirabilis in the urinary tract, little is known about pathogenesis in the bloodstream. This study identifies the genes that are important for survival in the bloodstream using a whole-genome transposon insertion-site sequencing (Tn-Seq) approach. A library of 50,000 transposon mutants was utilized to assess the relative contribution of each non-essential gene in the P. mirabilis HI4320 genome to fitness in the livers and spleens of mice at 24 hours following tail vein inoculation compared to growth in RPMI, heat-inactivated (HI) naïve serum, and HI acute phase serum. 138 genes were identified as ex vivo fitness factors in serum, which were primarily involved in amino acid transport and metabolism, and 143 genes were identified as infection-specific in vivo fitness factors for both spleen and liver colonization. Infection-specific fitness factors included genes involved in twin arginine translocation, ammonia incorporation, and polyamine biosynthesis. Mutants in sixteen genes were constructed to validate both the ex vivo and in vivo results of the transposon screen, and 12/16 (75%) exhibited the predicted phenotype. Our studies indicate a role for the twin arginine translocation (tatAC) system in motility, translocation of potential virulence factors, and fitness within the bloodstream. We also demonstrate the interplay between two nitrogen assimilation pathways in the bloodstream, providing evidence that the GS-GOGAT system may be preferentially utilized. Furthermore, we show that a dual-function arginine decarboxylase (speA) is important for fitness within the bloodstream due to its role in putrescine biosynthesis rather than its contribution to maintenance of membrane potential. This study therefore provides insight into pathways needed for fitness within the bloodstream, which may guide strategies to reduce bacteremia-associated mortality.

A Rare Opportunist, Morganella morganii, Decreases Severity of Polymicrobial Catheter-Associated Urinary Tract Infection.

Catheter-associated urinary tract infections (CAUTIs) are common hospital-acquired infections and frequently polymicrobial, which complicates effective treatment. However, few studies experimentally address the consequences of polymicrobial interactions within the urinary tract, and the clinical significance of polymicrobial bacteriuria is not fully understood. Proteus mirabilis is one of the most common causes of monomicrobial and polymicrobial CAUTI and frequently cocolonizes with Enterococcus faecalis, Escherichia coli, Providencia stuartii, and Morganella morganiiP. mirabilis infections are particularly challenging due to its potent urease enzyme, which facilitates formation of struvite crystals, catheter encrustation, blockage, and formation of urinary stones. We previously determined that interactions between P. mirabilis and other uropathogens can enhance P. mirabilis urease activity, resulting in greater disease severity during experimental polymicrobial infection. Our present work reveals that M. morganii acts on P. mirabilis in a contact-independent manner to decrease urease activity. Furthermore, M. morganii actively prevents urease enhancement by E. faecalis, P. stuartii, and E. coli Importantly, these interactions translate to modulation of disease severity during experimental CAUTI, predominantly through a urease-dependent mechanism. Thus, products secreted by multiple bacterial species in the milieu of the catheterized urinary tract can directly impact prognosis.

Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements.

The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis CAUTI incidence and severity.

Autoinducer 2 (AI-2) Production by Nontypeable Haemophilus influenzae 86-028NP Promotes Expression of a Predicted Glycosyltransferase That Is a Determinant of Biofilm Maturation, Prevention of Dispersal, and Persistence In Vivo.

Nontypeable Haemophilus influenzae (NTHi) is an extremely common human pathobiont that persists on the airway mucosal surface within biofilm communities, and our previous work has shown that NTHi biofilm maturation is coordinated by the production and uptake of autoinducer 2 (AI-2) quorum signals. To directly test roles for AI-2 in maturation and maintenance of NTHi biofilms, we generated an NTHi 86-028NP mutant in which luxS transcription was under the control of the xylA promoter (NTHi 86-028NP luxS xylA::luxS), rendering AI-2 production inducible by xylose. Comparison of biofilms under inducing and noninducing conditions revealed a biofilm defect in the absence of xylose, whereas biofilm maturation increased following xylose induction. The removal of xylose resulted in the interruption of luxS expression and biofilm dispersal. Measurement of luxS transcript levels by real-time reverse transcription-PCR (RT-PCR) showed that luxS expression peaked as biofilms matured and waned before dispersal. Transcript profiling revealed significant changes following the induction of luxS, including increased transcript levels for a predicted family 8 glycosyltransferase (NTHI1750; designated gstA); this result was confirmed by real-time RT-PCR. An isogenic NTHi 86-028NP gstA mutant had a biofilm defect, including decreased levels of sialylated matrix and significantly altered biofilm structure. In experimental chinchilla infections, we observed a significant decrease in the number of bacteria in the biofilm population (but not in effusions) for NTHi 86-028NP gstA compared to the parental strain. Therefore, we conclude that AI-2 promotes NTHi biofilm maturation and the maintenance of biofilm integrity, due at least in part to the expression of a probable glycosyltransferase that is potentially involved in the synthesis of the biofilm matrix.

Urine Cytokine and Chemokine Levels Predict Urinary Tract Infection Severity Independent of Uropathogen, Urine Bacterial Burden, Host Genetics, and Host Age.

Urinary tract infections (UTIs) are among the most common infections worldwide. Diagnosing UTIs in older adults poses a significant challenge as asymptomatic colonization is common. Identification of a noninvasive profile that predicts likelihood of progressing from urine colonization to severe disease would provide a significant advantage in clinical practice. We monitored colonization susceptibility, disease severity, and immune response to two uropathogens in two mouse strains across three age groups to identify predictors of infection outcome. Proteus mirabilis caused more severe disease than Escherichia coli, regardless of mouse strain or age, and was associated with differences in interleukin-1ß (IL-1ß), beta interferon (IFN-ß), CXCL5 (LIX), CCL5 (RANTES), and CCL2 (MCP-1). In a comparison of responses to infection across age groups, mature adult mice were better able to control colonization and prevent progression to kidney colonization and bacteremia than young or aged mice, regardless of mouse strain or bacterial species, and this was associated with differences in IL-23, CXCL1, and CCL5. A bimodal distribution was noted for urine colonization, which was strongly associated with bladder CFU counts and the magnitude of the immune response but independent of age or disease severity. To determine the value of urine cytokine and chemokine levels for predicting severe disease, all infection data sets were combined and subjected to a series of logistic regressions. A multivariate model incorporating IL-1ß, CXCL1, and CCL2 had strong predictive value for identifying mice that did not develop kidney colonization or bacteremia, regardless of mouse genetic background, age, infecting bacterial species, or urine bacterial burden. In conclusion, urine cytokine profiles could potentially serve as a noninvasive decision support tool in clinical practice and contribute to antimicrobial stewardship.

The Pathogenic Potential of Proteus mirabilis Is Enhanced by Other Uropathogens during Polymicrobial Urinary Tract Infection.

Urinary catheter use is prevalent in health care settings, and polymicrobial colonization by urease-positive organisms, such as Proteus mirabilis and Providencia stuartii, commonly occurs with long-term catheterization. We previously demonstrated that coinfection with P. mirabilis and P. stuartii increased overall urease activity in vitro and disease severity in a model of urinary tract infection (UTI). In this study, we expanded these findings to a murine model of catheter-associated UTI (CAUTI), delineated the contribution of enhanced urease activity to coinfection pathogenesis, and screened for enhanced urease activity with other common CAUTI pathogens. In the UTI model, mice coinfected with the two species exhibited higher urine pH values, urolithiasis, bacteremia, and more pronounced tissue damage and inflammation compared to the findings for mice infected with a single species, despite having a similar bacterial burden within the urinary tract. The presence of P. stuartii, regardless of urease production by this organism, was sufficient to enhance P. mirabilis urease activity and increase disease severity, and enhanced urease activity was the predominant factor driving tissue damage and the dissemination of both organisms to the bloodstream during coinfection. These findings were largely recapitulated in the CAUTI model. Other uropathogens also enhanced P. mirabilis urease activity in vitro, including recent clinical isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa We therefore conclude that the underlying mechanism of enhanced urease activity may represent a widespread target for limiting the detrimental consequences of polymicrobial catheter colonization, particularly by P. mirabilis and other urease-positive bacteria.

Peroxiredoxin-glutaredoxin and catalase promote resistance of nontypeable Haemophilus influenzae 86-028NP to oxidants and survival within neutrophil extracellular traps.

Nontypeable Haemophilus influenzae (NTHI) is a common commensal and opportunistic pathogen of the human airways. For example, NTHI is a leading cause of otitis media and is the most common cause of airway infections associated with chronic obstructive pulmonary disease (COPD). These infections are often chronic/recurrent in nature and involve bacterial persistence within biofilm communities that are highly resistant to host clearance. Our previous work has shown that NTHI within biofilms has increased expression of factors associated with oxidative stress responses. The goal of this study was to define the roles of catalase (encoded by hktE) and a bifunctional peroxiredoxin-glutaredoxin (encoded by pdgX) in resistance of NTHI to oxidants and persistence in vivo. Isogenic NTHI strain 86-028NP mutants lacking hktE and pdgX had increased susceptibility to peroxide. Moreover, these strains had persistence defects in the chinchilla infection model for otitis media, as well as in a murine model for COPD. Additional work showed that pdgX and hktE were important determinants of NTHI survival within neutrophil extracellular traps (NETs), which we have shown to be an integral part of NTHI biofilms in vivo. Based on these data, we conclude that catalase and peroxiredoxin-glutaredoxin are determinants of bacterial persistence during chronic/recurrent NTHI infections that promote bacterial survival within NETs.

Increased incidence of urolithiasis and bacteremia during Proteus mirabilis and Providencia stuartii coinfection due to synergistic induction of urease activity.

BACKGROUND: Catheter-associated urinary tract infections (CaUTIs) are the most common hospital-acquired infections worldwide and are frequently polymicrobial. The urease-positive species Proteus mirabilis and Providencia stuartii are two of the leading causes of CaUTIs and commonly co-colonize catheters. These species can also cause urolithiasis and bacteremia. However, the impact of coinfection on these complications has never been addressed experimentally. METHODS: A mouse model of ascending UTI was utilized to determine the impact of coinfection on colonization, urolithiasis, and bacteremia. Mice were infected with P. mirabilis or a urease mutant, P. stuartii, or a combination of these organisms. In vitro experiments were conducted to assess growth dynamics and impact of co-culture on urease activity. RESULTS: Coinfection resulted in a bacterial load similar to monospecies infection but with increased incidence of urolithiasis and bacteremia. These complications were urease-dependent as they were not observed during coinfection with a P. mirabilis urease mutant. Furthermore, total urease activity was increased during co-culture. CONCLUSIONS: We conclude that P. mirabilis and P. stuartii coinfection promotes urolithiasis and bacteremia in a urease-dependent manner, at least in part through synergistic induction of urease activity. These data provide a possible explanation for the high incidence of bacteremia resulting from polymicrobial CaUTI.

Inhibition of urease-mediated ammonia production by 2-octynohydroxamic acid in hepatic encephalopathy.

Hepatic encephalopathy is a neuropsychiatric complication of liver disease which is partly associated with elevated ammonemia. Urea hydrolysis by urease-producing bacteria in the colon is often mentioned as one of the main routes of ammonia production in the body, yet research on treatments targeting bacterial ureases in hepatic encephalopathy is limited. Herein we report a hydroxamate-based urease inhibitor, 2-octynohydroxamic acid, exhibiting improved in vitro potency compared to hydroxamic acids that were previously investigated for hepatic encephalopathy. 2-octynohydroxamic acid shows low cytotoxic and mutagenic potential within a micromolar concentration range as well as reduces ammonemia in rodent models of liver disease. Furthermore, 2-octynohydroxamic acid treatment decreases cerebellar glutamine, a product of ammonia metabolism, in male bile duct ligated rats. A prototype colonic formulation enables reduced systemic exposure to 2-octynohydroxamic acid in male dogs. Overall, this work suggests that urease inhibitors delivered to the colon by means of colonic formulations represent a prospective approach for the treatment of hepatic encephalopathy.

Function and contribution of two putative Enterococcus faecalis glycosaminoglycan degrading enzymes to bacteremia and catheter-associated urinary tract infection.

Enterococcus faecalis is a common cause of healthcare acquired bloodstream infections and catheter associated urinary tract infections (CAUTI) in both adults and children. Treatment of E. faecalis infection is frequently complicated by multi-drug resistance. Based on protein homology, E. faecalis encodes two putative hyaluronidases, EF3023 (HylA) and EF0818 (HylB). In other Gram-positive pathogens, hyaluronidases have been shown to contribute to tissue damage and immune evasion, but function in E. faecalis has yet to be explored. Here, we show that both hylA and hylB contribute to E. faecalis pathogenesis. In a CAUTI model, Δ hylA exhibited defects in bladder colonization and dissemination to the bloodstream, and Δ hylB exhibited a defect in kidney colonization. Furthermore, a Δ hylA Δ hylB double mutant exhibited a severe colonization defect in a model of bacteremia while the single mutants colonized to a similar level as the wild-type strain, suggesting potential functional redundancy within the bloodstream. We next examined enzymatic activity, and demonstrate that HylB is capable of digesting both HA and CS in vitro while HylA exhibits only a very modest activity against heparin. Importantly, HA degradation by HylB provided a modest increase in cell density during stationary phase and also contributed to dampening of LPS-mediated NF-Bκ activation. Overall, these data demonstrate that glycosaminoglycan degradation is important for E. faecalis pathogenesis in the urinary tract and during bloodstream infection.

Initiation of swarming motility by Proteus mirabilis occurs in response to specific cues present in urine and requires excess L-glutamine.

Proteus mirabilis, a leading cause of catheter-associated urinary tract infection (CaUTI), differentiates into swarm cells that migrate across catheter surfaces and medium solidified with 1.5% agar. While many genes and nutrient requirements involved in the swarming process have been identified, few studies have addressed the signals that promote initiation of swarming following initial contact with a surface. In this study, we show that P. mirabilis CaUTI isolates initiate swarming in response to specific nutrients and environmental cues. Thirty-three compounds, including amino acids, polyamines, fatty acids, and tricarboxylic acid (TCA) cycle intermediates, were tested for the ability to promote swarming when added to normally nonpermissive media. L-Arginine, L-glutamine, DL-histidine, malate, and DL-ornithine promoted swarming on several types of media without enhancing swimming motility or growth rate. Testing of isogenic mutants revealed that swarming in response to the cues required putrescine biosynthesis and pathways involved in amino acid metabolism. Furthermore, excess glutamine was found to be a strict requirement for swarming on normal swarm agar in addition to being a swarming cue under normally nonpermissive conditions. We thus conclude that initiation of swarming occurs in response to specific cues and that manipulating concentrations of key nutrient cues can signal whether or not a particular environment is permissive for swarming.

Differential Contribution of Hydrogen Metabolism to Proteus mirabilis Fitness during Single-Species and Polymicrobial Catheterized Urinary Tract Infection.

Proteus mirabilis is a common uropathogen and a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Through a genome-wide screen, we previously identified two [NiFe] hydrogenases as candidate fitness factors for P. mirabilis CAUTI: a Hyb-type Group 1c H2-uptake hydrogenase and a Hyf-type Group 4a H2-producing hydrogenase. In this study, we disrupted one gene of each system (hyfE and hybC) and also generated a double mutant to examine the contribution of flexible H2 metabolism to P. mirabilis growth and fitness in vitro and during experimental CAUTI. Since P. mirabilis is typically present as part of a polymicrobial community in the urinary tract, we also examined the impact of two common co-colonization partners, Providencia stuartii and Enterococcus faecalis, on the expression and contribution of each hydrogenase to fitness. Our data demonstrate that neither system alone is critical for P. mirabilis growth in vitro or fitness during experimental CAUTI. However, perturbation of flexible H2 metabolism in the ∆hybC∆hyfE double mutant decreased P. mirabilis fitness in vitro and during infection. The Hyf system alone contributed to the generation of proton motive force and swarming motility, but only during anaerobic conditions. Unexpectedly, both systems contributed to benzyl viologen reduction in TYET medium, and disruption of either system increased expression of the other. We further demonstrate that polymicrobial interactions with P. stuartii and E. faecalis alter the expression of Hyb and Hyf in vitro as well as the contribution of each system to P. mirabilis fitness during CAUTI.

Role of Bacterial Surface Components in the Pathogenicity of Proteus mirabilis in a Murine Model of Catheter-Associated Urinary Tract Infection.

Proteus mirabilis (PM) is a Gram-negative, rod-shaped bacterium that causes catheter-associated urinary tract infections (CAUTIs). The specific roles of bacterial surface components (BSCs) in PM pathogenicity and CAUTIs remain unknown. To address this knowledge gap, we utilized relevant in vitro adhesion/invasion models and a well-established murine model of CAUTI to assess the ability of wildtype (WT) and seven mutant strains (MSs) of PM with deficiencies in various genes encoding BSCs to undergo the infectious process (including adhesion to catheters) in both model systems. Overall, MSs adhesion to catheters and the different cell types tested was significantly reduced compared to WT, while no invasion of cells was evident at 24 h. In vivo, WT showed a greater number of planktonic (urine) bacteria, bacteria adherent to catheters, and bacteria adherent to/invading bladder tissue when compared to the MSs. Bacterial counts in urine for PMI3191 and waaE mutants were lower than that for WT and other MSs. The complementation of mutated BSC genes resulting in the biggest defects restored the invasion phenotype both in vitro and in vivo. BSCs play a critical role at various steps in the pathogenicity of PM including adhesion to indwelling medical devices and adhesion/invasion of urinary tissue in vivo.

Metabolic interplay between Proteus mirabilis and Enterococcus faecalis facilitates polymicrobial biofilm formation and invasive disease.

Polymicrobial biofilms play an important role in the development and pathogenesis of CAUTI. Proteus mirabilis and Enterococcus faecalis are common CAUTI pathogens that persistently co-colonize the catheterized urinary tract and form biofilms with increased biomass and antibiotic resistance. In this study, we uncover the metabolic interplay that drives biofilm enhancement and examine the contribution to CAUTI severity. Through compositional and proteomic biofilm analyses, we determined that the increase in biofilm biomass stems from an increase in the protein fraction of the polymicrobial biofilm matrix. We further observed an enrichment in proteins associated with ornithine and arginine metabolism in polymicrobial biofilms compared to single-species biofilms. We show that L-ornithine secretion by E. faecalis promotes arginine biosynthesis in P. mirabilis, and that disruption of this metabolic interplay abrogates the biofilm enhancement we see in vitro and leads to significant decreases in infection severity and dissemination in a murine CAUTI model.

RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP.

Nontypeable Haemophilus influenzae (NTHI) is a respiratory commensal and opportunistic pathogen, which persists within biofilms on airway mucosal surfaces. For many species, biofilm formation is impacted by quorum signalling. Our prior work shows that production of autoinducer-2 (AI-2) promotes biofilm development and persistence for NTHI 86-028NP. NTHI 86-028NP encodes an ABC transporter annotated as a ribose transport system that includes a protein (RbsB) with similarity to the Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2. In this study, inactivation of rbsB significantly reduced uptake of AI-2 and the AI-2 precursor dihydroxypentanedione (DPD) by NTHI 86-028NP. Moreover, DPD uptake was not competitively inhibited by ribose or other pentose sugars. Transcript levels of rbsB increased in response to DPD and as bacteria approached stationary-phase growth. The NTHI 86-028NP rbsB mutant also formed biofilms with significantly reduced thickness and total biomass and reduced surface phosphorylcholine, similar to a luxS mutant. Infection studies revealed that loss of rbsB impaired bacterial persistence in the chinchilla middle ear, similar to our previous results with luxS mutants. Based on these data, we conclude that in NTHI 86-028NP, RbsB is a LuxS/AI-2 regulated protein that is required for uptake of and response to AI-2.