Elucidation of Proteus mirabilis as a Key Bacterium in Crohn's Disease Inflammation.

BACKGROUND & AIMS: Proteus spp, Gram-negative facultative anaerobic bacilli, have recently been associated with Crohn's disease (CD) recurrence after intestinal resection. We investigated the genomic and functional role of Proteus as a gut pathogen in CD. METHODS: Proteus spp abundance was assessed by ure gene-specific polymerase chain in 54 pairs of fecal samples and 101 intestinal biopsies from patients with CD and healthy controls. The adherence, invasion, and intracellular presence of 2 distinct isolates of Proteus mirabilis in epithelial cells were evaluated using immunofluorescence and electron microscopy. Intracellular gene expression profiles and regulated pathways were analyzed by RNA sequencing and KEGG pathway analysis. Biologic functions of 2 isolates of P mirabilis were determined by in vitro cell culture, and in vivo using conventional mice and germ-free mice. RESULTS: Proteus spp were significantly more prevalent and abundant in fecal samples and colonic tissue of patients with CD than controls. A greater abundance of the genus Fusobacterium and a lesser abundance of the genus Faecalibacterium were seen in patients with CD with a high Proteus spp abundance. All 24 Proteus monoclones isolated from patients with CD belonged to members of P mirabilis lineages and 2 isolates, recovered from stool or mucosa, were used in further studies. Mice gavaged with either P mirabilis strain had more severe colonic inflammation. Co-culture of the isolates with epithelial cell lines showed bacterial adherence, invasion, increased production of pro-inflammatory cytokines IL-18 and IL-1α, and cell necrosis. Both isolates induced key pro-inflammatory pathways, including NOD-like receptor signaling, Jak-STAT signaling, and MAPK signaling, and induced pro-inflammatory genes and activated inflammation-related pathways in gnotobiotic mice. CONCLUSIONS: P mirabilis in the gut is associated with CD and can induce inflammation in cells and animal models of colitis. P mirabilis can act as a pathobiont and play a crucial role in the pathogenesis of CD.

Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia.

Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

Peer pressure from a Proteus mirabilis self-recognition system controls participation in cooperative swarm motility.

Colonies of the opportunistic pathogen Proteus mirabilis can distinguish self from non-self: in swarming colonies of two different strains, one strain excludes the other from the expanding colony edge. Predominant models characterize bacterial kin discrimination as immediate antagonism towards non-kin cells, typically through delivery of toxin effector molecules from one cell into its neighbor. Upon effector delivery, receiving cells must either neutralize it by presenting a cognate anti-toxin as would a clonal sibling, or suffer cell death or irreversible growth inhibition as would a non-kin cell. Here we expand this paradigm to explain the non-lethal Ids self-recognition system, which stops access to a social behavior in P. mirabilis by selectively and transiently inducing non-self cells into a growth-arrested lifestyle incompatible with cooperative swarming. This state is characterized by reduced expression of genes associated with protein synthesis, virulence, and motility, and also causes non-self cells to tolerate previously lethal concentrations of antibiotics. We show that temporary activation of the stringent response is necessary for entry into this state, ultimately resulting in the iterative exclusion of non-self cells as a swarm colony migrates outwards. These data clarify the intricate connection between non-lethal recognition and the lifecycle of P. mirabilis swarm colonies.

Proteus mirabilis Urease: Unsuspected Non-Enzymatic Properties Relevant to Pathogenicity.

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.

Low antimicrobial resistance in general practice patients in Rotterdam, the city with the largest proportion of immigrants in the Netherlands.

Antimicrobial resistance (AMR) is an increasing problem. The prevalence of antimicrobial resistance in general practice patients is expected to be relatively high in Rotterdam, the Dutch city with the largest proportion non-Western immigrants. The aim of this study was to assess the prevalence of antibiotic-resistant uropathogens (Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis) in general practices in Rotterdam, and to find a possible association between the prevalence of antibiotic-resistant E. coli and age, gender, and socioeconomic status (SES). A retrospective analysis was performed of urine samples from general practice patients in 2016. The prevalence of AMR in uropathogens was compared with national resistance data, as was the prevalence of highly and multidrug resistant and extended spectrum ß-lactamase (ESBL) producing E. coli and K. pneumoniae. Univariate logistic regression was used to study associations between antibiotic-resistant E. coli and age, gender, and SES area score. No clinically relevant differences were observed in the prevalence of antibiotic-resistant uropathogens in Rotterdam compared with the national prevalence. For E. coli and K. pneumoniae, the prevalence was 3.6% for ESBL production (both pathogens together), while the prevalence ranged between 4.2%-5.0% for high resistance and between 1.2%-3.3% for multidrug resistance. Ciprofloxacin-resistant E. coli was significantly associated with higher age. Although Rotterdam has a high percentage of non-western immigrants and a low SES, AMR is low among general practice patients. This indicates that adherence to national guidelines in general practice enables maintenance of low AMR, even in high-risk populations.

Inhibition of urease activity in the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis by dimethylsulfoxide (DMSO).

AIMS: Urease is a virulence factor for the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis. Dimethylsulfoxide (DMSO) is structurally similar to urea, used as a solvent for urease inhibitors, and an effective treatment for interstitial cystitis/bladder pain syndrome (IC/BPS). The aims of this study were to test DMSO as a urease inhibitor and determine its physiological effects on S. saprophyticus and P. mirabilis. METHODS AND RESULTS: Urease activity in extracts and whole cells was measured by the formation of ammonium ions. Urease was highly sensitive to noncompetitive inhibition by DMSO (Ki about 6 mmol l-1 ). DMSO inhibited urease activity in whole cells, limited bacterial growth in media containing urea, and slowed the increase in pH which occurred in artificial urine medium. CONCLUSIONS: DMSO should be used with caution as a solvent when testing plant extracts or other potential urease inhibitors. Because it can inhibit bacterial growth and delay an increase in pH, it may be an effective treatment for urinary tract infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study of the inhibition of urease by DMSO. Dimethylsulfoxide may be used to treat urinary tract infections that are resistant to antibiotics or herbal remedies.

Association among biofilm formation, virulence gene expression, and antibiotic resistance in Proteus mirabilis isolates from diarrhetic animals in Northeast China.

BACKGROUND: The aim of this study was to investigate the association among biofilm formation, virulence gene expression, and antibiotic resistance in P. mirabilis isolates collected from diarrhetic animals (n = 176) in northeast China between September 2014 and October 2016. RESULTS: Approximately 92.05% of the isolates were biofilm producers, whereas 7.95% of the isolates were non-producers. The prevalence of virulence genes in the biofilm producer group was significantly higher than that in the non-producer group. Biofilm production was significantly associated with the expression of ureC, zapA, rsmA, hmpA, mrpA, atfA, and pmfA (P < 0.05). The results of drug susceptibility tests revealed that approximately 76.7% of the isolates were multidrug-resistant (MDR) and extensively drug-resistant (XDR). Biofilm production was significantly associated with resistance to doxycycline, tetracycline, sulfamethoxazole, kanamycin, and cephalothin (P < 0.05). Although the pathogenicity of the biofilm producers was stronger than that of the non-producers, the biofilm-forming ability of the isolates was not significantly associated with morbidity and mortality in mice (P > 0.05). CONCLUSION: Our findings suggested that a high level of multidrug resistance in P. mirabilis isolates obtained from diarrhetic animals in northeast China. The results of this study indicated that the positive rates of the genes expressed by biofilm-producing P. mirabilis isolates were significantly higher than those expressed by non-producing isolates.

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.

Bacterial biofilm formation on indwelling urethral catheters.

Urethral catheters are the most commonly deployed medical devices and used to manage a wide range of conditions in both hospital and community care settings. The use of long-term catheterization, where the catheter remains in place for a period >28 days remains common, and the care of these patients is often undermined by the acquisition of infections and formation of biofilms on catheter surfaces. Particular problems arise from colonization with urease-producing species such as Proteus mirabilis, which form unusual crystalline biofilms that encrust catheter surfaces and block urine flow. Encrustation and blockage often lead to a range of serious clinical complications and emergency hospital referrals in long-term catheterized patients. Here we review current understanding of bacterial biofilm formation on urethral catheters, with a focus on crystalline biofilm formation by P. mirabilis, as well as approaches that may be used to control biofilm formation on these devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Urinary catheters are the most commonly used medical devices in many healthcare systems, but their use predisposes to infection and provide ideal conditions for bacterial biofilm formation. Patients managed by long-term urethral catheterization are particularly vulnerable to biofilm-related infections, with crystalline biofilm formation by urease producing species frequently leading to catheter blockage and other serious clinical complications. This review considers current knowledge regarding biofilm formation on urethral catheters, and possible strategies for their control.

Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation.

The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder.

MrpJ Directly Regulates Proteus mirabilis Virulence Factors, Including Fimbriae and Type VI Secretion, during Urinary Tract Infection.

Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs) and urolithiasis. The transcriptional regulator MrpJ inversely modulates two critical aspects of P. mirabilis UTI progression: fimbria-mediated attachment and flagellum-mediated motility. Transcriptome data indicated a network of virulence-associated genes under MrpJ's control. Here, we identify the direct gene regulon of MrpJ and its contribution to P. mirabilis pathogenesis, leading to the discovery of novel virulence targets. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was used for the first time in a CAUTI pathogen to probe for in vivo direct targets of MrpJ. Selected MrpJ-regulated genes were mutated and assessed for their contribution to UTI using a mouse model. ChIP-seq revealed a palindromic MrpJ binding sequence and 78 MrpJ-bound regions, including binding sites upstream of genes involved in motility, fimbriae, and a type VI secretion system (T6SS). A combinatorial mutation approach established the contribution of three fimbriae (fim8A, fim14A, and pmpA) to UTI and a new pathogenic role for the T6SS in UTI progression. In conclusion, this study (i) establishes the direct gene regulon and an MrpJ consensus binding site and (ii) led to the discovery of new virulence genes in P. mirabilis UTI, which could be targeted for therapeutic intervention of CAUTI.

Determination immunogenic property of truncated MrpH.FliC as a vaccine candidate against urinary tract infections caused by Proteus mirabilis.

Proteus mirabilis is common cause of urinary tract infections (UTIs) especially in complicated UTIs which are resistant to antibiotic therapy, Consequently, an ideal vaccine is inevitably required. The N-terminal domain of MrpH (Truncated form of MrpH) lies between the most critical antigens of P. mirabilis to consider as vaccine candidate. FliC of Salmonella typhimurium induces several pathways of immunity system, which leads to produce antibody and cytokines. In this study, adjuvant properties of FliC and efficacy of truncated MrpH as important antigen, in tMrpH.FliC were determined in in vitro and in vivo circumstances. Three proteins including: FliC, MrpH and tMrpH.FliC were injected to mice and subsequently sera and supernatant of cell culture were collected to evaluate different immune responses. According to our findings, tMrpH.FliC could stimulate both humoral and cellular immune responses, so that serum IgG, urine IgA, IL.4, IFN-γ and IL.17 were increased significantly in comparison to MrpH and FliC alone, this augmentation was considerable. Results showed significant decrease of bacterial load in all of the challenged groups compared to the control group, although this protective effect was the highest in mice vaccinated with tMrpH.FliC. Our results showed truncated MrpH, without an unwanted domain is an ideal vaccine target and FliC, as adjuvant, increases its immunogenic property. Thus, fusion protein tMrpH.FliC can be considered as promising vaccine against P. mirabilis.

Preferential use of central metabolism in vivo reveals a nutritional basis for polymicrobial infection.

The human genitourinary tract is a common anatomical niche for polymicrobial infection and a leading site for the development of bacteremia and sepsis. Most uncomplicated, community-acquired urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more often associated with complicated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent requirements for specific central pathways in vivo despite colonizing and occupying the same host environment. Using mutants of specific central metabolism enzymes, we determined glycolysis mutants lacking pgi, tpiA, pfkA, or pykA all have fitness defects in vivo for P. mirabilis but do not affect colonization of E. coli during UTI. Similarly, the oxidative pentose phosphate pathway is required only for P. mirabilis in vivo. In contrast, gluconeogenesis is required only for E. coli fitness in vivo. The remarkable difference in central pathway utilization between E. coli and P. mirabilis during experimental UTI was also observed for TCA cycle mutants in sdhB, fumC, and frdA. The distinct in vivo requirements between these pathogens suggest E. coli and P. mirabilis are not direct competitors within host urinary tract nutritional niche. In support of this, we found that co-infection with E. coli and P. mirabilis wild-type strains enhanced bacterial colonization and persistence of both pathogens during UTI. Our results reveal that complementary utilization of central carbon metabolism facilitates polymicrobial disease and suggests microbial activity in vivo alters the host urinary tract nutritional niche.

The link between Proteus mirabilis, environmental factors and autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a relatively common and potentially disabling immune-mediated inflammatory systemic disease, predominantly affecting women and characterised by multiple small joint arthritis. Extensive data supports the roles of genetic, environmental and microbial factors in the triggering and development of this disease. Proteus mirabilis is considered as the main microbial culprit in the causation of RA. The evidence for the role of these microbes in RA and their links with commonly associated autoantibodies such as rheumatoid factors and anti-citrullinated peptide antibodies have been elucidated together with their relations with some of the non-microbial environmental factors which have been implicated in the aetiopathogenesis of RA. The most likely mechanism in the development of RA is "molecular mimicry" where Proteus antigens were found to share homologous sequences, which cross-react with certain self-antigens present in synovial tissues. This could raise possibilities for implementing a new therapeutic strategy in the treatment of RA.

The impact of production of extended-spectrum ß-lactamases on the 28-day mortality rate of patients with Proteus mirabilis bacteremia in Korea.

BACKGROUND: The incidence of Proteus mirabilis antimicrobial resistance, especially that mediated by extended-spectrum ß-lactamases (ESBLs), has increased. We investigated the impact of ESBL production on the mortality of patients with P. mirabilis bacteremia in Korea. METHODS: Patients diagnosed with P. mirabilis bacteremia between November 2005 and December 2013 at a 2000-bed tertiary care center in South Korea were included in this study. Phenotypic and molecular analyses were performed to assess ESBL expression. Characteristics and treatment outcomes were investigated among ESBL-producing and non-ESBL-producing P. mirabilis bacteremia groups. A multivariate analysis of 28-day mortality rates was performed to evaluate the independent impact of ESBLs. RESULTS: Among 62 P. mirabilis isolates from 62 patients, 14 expressed ESBLs (CTX-M, 2; TEM, 5; both, 6; other, 1), and the 28-day mortality rate of the 62 patients was 17.74%. No clinical factor was significantly associated with ESBL production. The 28-day mortality rate in the ESBL-producing group was significantly higher than that in the non-ESBL-producing group (50% vs. 8.3%, p = 0.001). A multivariate analysis showed that ESBL production (odds ratio [OR], 11.53, 95% confidence interval [CI], 2.11-63.05, p = 0.005) was independently associated with the 28-day mortality rate in patients with P. mirabilis bacteremia. CONCLUSIONS: ESBL production is significantly associated with mortality in patients with bacteremia caused by P. mirabilis. Rapid detection of ESBL expression and prompt appropriate antimicrobial therapy are required to reduce mortality caused by P. mirabilis bacteremia.

Method for Bacterial Growth and Ammonia Production and Effect of Inhibitory Substances in Disposable Absorbent Hygiene Products.

PURPOSE: The purpose of this study was to evaluate a pragmatic laboratory method to provide a technique for developing incontinence products better able to reduce malodor when used in the clinical setting. METHODS: Bacterial growth and bacterially formed ammonia in disposable absorbent incontinence products was measured by adding synthetic urine inoculated with bacteria to test samples cut from the crotch area of the product. The inhibitory effect's of low pH (4.5 and 4.9) and 3 antimicrobial substances-chlorhexidine, polyhexamethylene biguanide (PHMB), and thymol-at 2 concentrations each, were studied. RESULTS: From the initial inocula of 3.3 log colony-forming units per milliliter (cfu/mL) at baseline, the bacterial growth of the references increased to 5.0 to 6.0 log cfu/mL at 6 hours for Escherichia coli, Proteus mirabilis, and Enterococcus faecalis. At 12 hours there was a further increase to 7.0 to 8.9 log cfu/mL. Adjusting the pH of the superabsorbent in the incontinence product from 6.0 to pH 4.5 and pH 4.9 significantly (P < .05) inhibited the bacterial growth rates, in most cases, both at 6 and 12 hours. The effect was most pronounced at pH 4.5. Chlorhexidine had significant (P < .05) inhibitory effect on E. coli and E. faecalis, and at 12 hours also on P. mirabilis. For PHMB and thymol the results varied. At 6 hours, the ammonia concentration in the references (pH 6.0) was 200 to 300 ppm and it was 1500 to 1600 ppm at 8 hours. At pH 4.5, no or little ammonia production was measured at 6 and 8 hours. At pH 4.9, there was a significant reduction (P < .01). Chlorhexidine and PHMB exerted a significant (P < .01 or P < .001) inhibitory effect on ammonia production at both concentrations and at 6 and 8 hours. Thymol 0.003% and 0.03% showed inhibitory effect at both 6 hours (P < .01 or P < .001) and at 8 hours (P < .05 or P < .001). CONCLUSION: The method described in this study can be used to compare the ability of various disposable absorbent products to inhibit bacterial growth and ammonia production. This technique, we describe, provides a pragmatic method for assessing the odor-inhibiting capacity of specific incontinence products.

Urinary Concentrations and Antibacterial Activity of BAL30072, a Novel Siderophore Monosulfactam, against Uropathogens after Intravenous Administration in Healthy Subjects.

This annex study to a phase 1 study aimed to correlate urinary concentrations and bactericidal titers (UBTs) of BAL30072, a novel siderophore monosulfactam, in healthy subjects in order to evaluate which dosage of BAL30072 should be investigated in a clinical study on complicated urinary tract infection (UTI). Three cohorts of a total of 19 healthy male subjects were included in the add-on study and received the following BAL30072 dosages. The 1st cohort received 1 g once a day (q.d.) intravenously (i.v.) (1 h) on day 1 and 1 g thrice daily (t.i.d.) on day 2, the 2nd cohort received 2 g q.d. i.v. (1 h) on day 1 and 2 g t.i.d. on day 2, and the 3rd cohort received 1 g q.d. i.v. (4-h infusion) on day 8. Urine was collected up to 24 h after drug administration. UBTs were determined for seven Escherichia coli isolates (three wild type [WT], CTX-M-15, TEM-3, TEM-5, NDM-1), two Klebsiella pneumoniae isolates (WT, KPC), one Proteus mirabilis isolate (WT), and two Pseudomonas aeruginosa isolates (WT, VIM-1 plus AmpC). Urine drug concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The median urinary excretions of BAL30072 ranged between 38% and 46% (3 cohorts). The median UBTs after i.v. administration of 1 or 2 g q.d. and after 1 or 2 g t.i.d. showed positive UBTs for 24 h after the lowest dosage (1 g q.d.) for 5 of 7 of the Enterobacteriaceae strains and after the higher dosage of 2 g administered i.v. t.i.d. for all strains tested. After i.v. infusion of 1 g over 4 h, positive UBTs were demonstrated for three E. coli strains for up to 12 h, for the K. pneumoniae (KPC) strain for up to 8 h, and for the P. aeruginosa (VIM-1 plus AmpC) strain for up to only 4 h. The minimal bactericidal concentrations (MBCs) of the E. coli (NDM-1) strain and the K. pneumoniae (WT) strain correlated well between broth and urine but did not correlate well for the two P. aeruginosa strains. BAL30072 exhibits positive UBTs for 24 h even after a dosage of 1 g administered i.v. q.d. for 5 of 7 Enterobacteriaceae strains and after 2 g administered i.v. t.i.d. for all strains except one P. aeruginosa strain (50% of the time). In general, the UBTs correlated well with the MICs of the Enterobacteriaceae but were lower for P. aeruginosa The clinical efficacy with a dosage regimen of BAL30072 of 2 g administered i.v. t.i.d. should be evaluated in the treatment of complicated UTI.

The Rcs regulon in Proteus mirabilis: implications for motility, biofilm formation, and virulence.

The overall role of the Rcs phosphorelay in Proteus mirabilis is largely unknown. Previous work had demonstrated that the Rcs phosphorelay represses the flhDC operon and activates the minCDE cell division inhibition system. To identify additional cellular functions regulated by the Rcs phosphorelay, an analysis of RNA-seq data was undertaken. In this report, the results of the RNA-sequencing are discussed with an emphasis on the predicted roles of the Rcs phosphorelay in swarmer cell differentiation, motility, biofilm formation, and virulence. RcsB is shown to activate genes important for differentiation and fimbriae formation, while repressing the expression of genes important for motility and virulence. Additionally, to follow up on the RNA-Seq data, we demonstrate that an rcsB mutant is deficient in its ability to form biofilm and exhibits enhanced virulence in a Galleria mellonella waxworm model. Overall, these results indicate the Rcs regulon in P. mirabilis extends beyond flagellar genes to include those involved in biofilm formation and virulence. Furthermore, the information presented in this study may provide clues to additional roles of the Rcs phosphorelay in other members of the Enterobacteriaceae.

Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence.

New aspects of RpoE in uropathogenic Proteus mirabilis.

Proteus mirabilis is a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms for P. mirabilis to establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulating mrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted from rpoE mutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness of P. mirabilis in the host, including the avoidance of immune attacks. Accordingly, rpoE mutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and the rpoE mutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression of rpoE by the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness of P. mirabilis, to build up a UTI.