Chryseobacterium oryctis sp. nov., isolated from the gut of the beetle Oryctes rhinoceros, and Chryseobacterium kimseyorum sp. nov., isolated from a stick insect rearing cage.

Two strains of Chryseobacterium identified from different experiments are proposed to represent new species. Strain WLa1L2M3T was isolated from the digestive tract of an Oryctes rhinoceros beetle larva. Strain 09-1422T was isolated from a cage housing the stick insect Eurycantha calcarata. Sequence analysis of the 16S rRNA and rpoB genes found both strains to be similar but not identical to other Chryseobacterium species. Whole-genome sequencing suggested the isolates represent new species, with average nucleotide identity values ranging from 74.6 to 80.5 %. Genome-to-genome distance calculations produced values below 25.3 %, and digital DNA-DNA hybridization values were 13.7-29.9 %, all suggesting they are distinct species. The genomic DNA G+C content of WLa1L2M3T is approximately 32.53 %, and of 09-1422T is approximately 35.89 %. The predominant cellular fatty acids of strain WLa1L2M3T are C15 : 0 iso, summed feature 9 (C16 : 0 10OH or C17 : 1 iso ω6c), C17 : 0 iso 3OH, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C15 : 0 iso 3OH, C15 : 0 anteiso and C13 : 0 iso, and those of strain 09-1422T are C15 : 0 iso, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C17 : 0 iso 3OH, C15 : 0 anteiso, C15 : 0 iso 3OH, C16 : 1 ω7c, C17 : 0 2OH and C18 : 0. In addition, physiological and biochemical tests revealed phenotypic differences from related Chryseobacterium type strains. These cumulative data indicate that the two strains represent novel species of the genus Chryseobacterium for which the names Chryseobacterium oryctis sp. nov. and Chryseobacterium kimseyorum sp. nov. are proposed with WLa1L2M3T (=BCRC 81350T=JCM 35215T=CIP 112035T) and 09-1422T (=UCDFST 09-1422T=BCRC 81359T=CIP 112165T), as type strains, respectively.

The QseEF Two-Component System-GlmY Small RNA Regulatory Pathway Controls Swarming in Uropathogenic Proteus mirabilis.

Bacterial sensing of environmental signals through the two-component system (TCS) plays a key role in modulating virulence. In the search for the host hormone-sensing TCS, we identified a conserved qseEGF locus following glmY, a small RNA (sRNA) gene in uropathogenic Proteus mirabilis. Genes of glmY-qseE-qseG-qseF constitute an operon, and QseF binding sites were found in the glmY promoter region. Deletion of glmY or qseF resulted in reduced swarming motility and swarming-related phenotypes relative to the wild-type and the respective complemented strains. The qseF mutant had decreased glmYqseEGF promoter activity. Both glmY and qseF mutants exhibited decreased flhDC promoter activity and mRNA level, while increased rcsB mRNA level was observed in both mutants. Prediction by TargetRNA2 revealed cheA as the target of GlmY. Then, construction of the translational fusions containing various lengths of cheA 5'UTR for reporter assay and site-directed mutagenesis were performed to investigate the cheA-GlmY interaction in cheA activation. Notably, loss of glmY reduced the cheA mRNA level, and urea could inhibit swarming in a QseF-dependent manner. Altogether, this is the first report elucidating the underlying mechanisms for modulation of swarming motility by a QseEF-regulated sRNA GlmY, involving expression of cheA, rcsB and flhDC in uropathogenic P. mirabilis.

Pseudomonas schmalbachii sp. nov., isolated from the gut of a millipede (Trigoniulus corallinus) from a coconut tree.

During an investigation of microbes associated with arthropods living in decaying coconut trees, a Pseudomonas isolate, Milli4T, was cultured from the digestive tract of the common Asian millipede, Trigoniulus corallinus. Sequence analysis of 16S rRNA and rpoB genes found that Milli4T was closely related but not identical to Pseudomonas panipatensis Esp-1T, Pseudomonas knackmussi B13T and Pseudomonas humi CCA1T. Whole genome sequencing suggested that this isolate represents a new species, with average nucleotide identity (OrthoANIu) values of around 83.9-87.7% with its closest relatives. Genome-to-genome distance calculations between Milli4T and its closest relatives also suggested they are distinct species. The genomic DNA G+C content of Milli4T was approximately 65.0 mol%. Phenotypic and chemotaxonomic characterization and fatty acid methyl ester analysis was performed on Milli4T and its related type strains. Based on these data, the new species Pseudomonas schmalbachii sp. nov. is proposed, and the type strain is Milli4T (=BCRC 81294T=JCM 34414T=CIP 111980T).

A CpxR-Regulated zapD Gene Involved in Biofilm Formation of Uropathogenic Proteus mirabilis.

Proteus mirabilis, a frequent uropathogen, forms extensive biofilms on catheters that are infamously difficult to treat. To explore the mechanisms of biofilm formation by P. mirabilis, we performed in vivo transposon mutagenesis. A mutant with impaired biofilm formation was isolated. The mutant was found to have Tn5 inserted in the zapD gene, encoding an outer membrane protein of the putative type 1 secretion system ZapBCD. zapBCD and its upstream zapA gene, encoding a protease, constitute an operon under the control of CpxR, a two-component regulator. The cpxR mutant and zapA mutant strains also had a biofilm-forming defect. CpxR positively regulates the promoter activities of zapABCD, cpxP, and cpxR An electrophoretic mobility shift assay revealed that CpxR binds zapA promoter DNA. The loss of zapD reduced CpxR-regulated gene expression of cpxR, zapA, cpxP, and mrpA, the mannose-resistant Proteus-like (MR/P) fimbrial major subunit gene. The restoration of biofilm formation in the zapD mutant with a CpxR-expressing plasmid reinforces the idea that CpxR-mediated gene expression contributes to zapD-involved biofilm formation. In trans expression of zapBCD from a zapBCD-expressing plasmid also reestablished the biofilm formation ability of the cpxR mutant to a certain level. The zapD and cpxR mutants had significantly lower protease activity, adhesion, and autoaggregation ability and production of exopolysaccharides and extracellular DNA (eDNA) than did the wild type. Finally, we identified copper as a signal for CpxR to increase biofilm formation. The loss of cpxR or zapD abolished the copper-mediated biofilm upshift. CpxR was required for copper-induced expression of zapA and cpxR Taken together, these data highlight the important role of CpxR-regulated zapD in biofilm formation and the underlying mechanisms in P. mirabilis.

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.

Serratia marcescens arn, a PhoP-regulated locus necessary for polymyxin B resistance.

Polymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly against Serratia marcescens. To investigate the underlying mechanisms, Tn5 mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5 inserted into the arnB and arnC genes. In other bacteria, arnB and arnC belong to the seven-gene arn operon, which is involved in lipopolysaccharide (LPS) modification. LPSs of arn mutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility in S. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression of phoP and arn in the wild-type strain but not in the phoP mutant. Complementation of the phoP mutant with the full-length phoP gene restored the PB MIC and induction by PB and low Mg2+ levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to the arn promoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+ levels protected S. marcescens from a PB challenge in the wild-type strain but not in the phoP mutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression of ugd, a gene required for LPS modification, in S. marcescens through a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression of arnA upon exposure to PB than did susceptible isolates. This is the first report to describe the role of S. marcescens arn in PB resistance and its modulation by PB and Mg2+ through the PhoP protein.

Copper affects virulence and diverse phenotypes of uropathogenic Proteus mirabilis.

BACKGROUND: Copper plays a role in urinary tract infection (UTI) and urinary copper content is increased during Proteus mirabilis UTI. We therefore investigated the effect of copper on uropathogenic P. mirabilis and the underlying mechanisms, focusing on the virulence associated aspects. METHODS: Mouse colonization, swarming/swimming assays, measurement of cell length, flagellin level and urease activity, adhesion/invasion assay, biofilm formation, killing by macrophages, oxidative stress susceptibility, OMPs analysis, determination of MICs and persister cell formation, RT-PCR and transcriptional reporter assay were performed. RESULTS: We found that copper-supplemented mice were more resistant to be colonized in the urinary tract, together with decreased swarming/swimming, ureases activity, expression of type VI secretion system and adhesion/invasion to urothelial cells and increased killing by macrophages of P. mirabilis at a sublethal copper level. However, bacterial biofilm formation and resistance to oxidative stress were enhanced under the same copper level. Of note, the presence of copper led to increased ciprofloxacin MIC and more persister cell formation against ampicillin. In addition, the presence of copper altered the outer membrane protein profile and triggered expression of RcsB response regulator. For the first time, we unveiled the pleiotropic effects of copper on uropathogenic P. mirabilis, especially for induction of bacterial two-component signaling system regulating fitness and virulence. CONCLUSION: The finding of copper-mediated virulence and fitness reinforced the importance of copper for prevention and therapeutic interventions against P. mirabilis infections. As such, this study could facilitate the copper-based strategies against UTI by P. mirabilis.

Characterization of UDP-glucose dehydrogenase and UDP-glucose pyrophosphorylase mutants of Proteus mirabilis: defectiveness in polymyxin B resistance, swarming, and virulence.

Proteus mirabilis is known to be highly resistant to the action of polymyxin B (PB). However, the mechanism underlying PB resistance is not clear. In this study, we used Tn5 transposon mutagenesis to identify genes that may affect PB resistance in P. mirabilis. Two genes, ugd and galU, which may encode UDP-glucose dehydrogenase (Ugd) and UDP-glucose pyrophosphorylase (GalU), respectively, were identified. Knockout mutants of ugd and galU were found to be extremely sensitive to PB, presumably because of alterations in lipopolysaccharide (LPS) structure and cell surface architecture in these mutants. These mutants were defective in swarming, expressed lower levels of virulence factor hemolysin, and had lower cell invasion ability. Complementation of the ugd or galU mutant with the full-length ugd or galU gene, respectively, led to the restoration of wild-type phenotypic traits. Interestingly, we found that the expression of Ugd and GalU was induced by PB through RppA, a putative response regulator of the bacterial two-component system that we identified previously. Mutation in either ugd or galU led to activation of RpoE, an extracytoplasmic function sigma factor that has been shown to be activated by protein misfolding and alterations in cell surface structure in other bacteria. Activation of RpoE or RpoE overexpression was found to cause inhibition of FlhDC and hemolysin expression. To our knowledge, this is the first report describing the roles and regulation of Ugd and GalU in P. mirabilis.

Proteus mirabilis pmrI, an RppA-regulated gene necessary for polymyxin B resistance, biofilm formation, and urothelial cell invasion.

Proteus mirabilis is naturally resistant to polymyxin B (PB). To investigate the underlying mechanisms, Tn5 mutagenesis was performed, and a mutant exhibiting increased PB susceptibility was isolated. The mutant was found to have Tn5 inserted into the PpmrI (Proteus pmrI) gene, a gene which may encode a UDP-glucuronic acid decarboxylase. In other bacteria, pmrI belongs to the seven-gene pmrF operon, which is involved in lipopolysaccharide (LPS) modification. While the PpmrI knockout mutant had a wild-type LPS profile and produced amounts of LPS similar to those produced by the wild type, LPS of the knockout mutant had higher PB-binding activity than that of the wild type. PB could induce alterations of LPS in the wild type but not in the PpmrI knockout mutant. Moreover, the PpmrI knockout mutant exhibited decreased abilities in biofilm formation and urothelial cell invasion. Complementation of the PpmrI mutant with the full-length PpmrI gene led to restoration of the wild-type phenotypic traits. Previously we identified RppA, a response regulator of the bacterial two-component system, as a regulator of PB susceptibility and virulence factor expression in P. mirabilis. Here we showed that RppA could mediate the induction of PpmrI expression by PB. An electrophoretic mobility shift assay further demonstrated that RppA could bind directly to the putative PpmrI promoter. Together, these results provide a new insight into the regulatory mechanism underlying PB resistance and virulence expression in P. mirabilis.

Deacidification by FhlA-dependent hydrogenase is involved in urease activity and urinary stone formation in uropathogenic Proteus mirabilis.

Proteus mirabilis is an important uropathogen, featured with urinary stone formation. Formate hydrogenlyase (FHL), consisting of formate dehydrogenase H and hydrogenase for converting proton to hydrogen, has been implicated in virulence. In this study, we investigated the role of P. mirabilis FHL hydrogenase and the FHL activator, FhlA. fhlA and hyfG (encoding hydrogenase large subunit) displayed a defect in acid resistance. fhlA and hyfG mutants displayed a delay in medium deacidification compared to wild-type and ureC mutant failed to deacidify the medium. In addition, loss of fhlA or hyfG decreased urease activity in the pH range of 5-8. The reduction of urease activities in fhlA and hyfG mutants subsided gradually over the pH range and disappeared at pH 9. Furthermore, mutation of fhlA or hyfG resulted in a decrease in urinary stone formation in synthetic urine. These indicate fhlA- and hyf-mediated deacidification affected urease activity and stone formation. Finally, fhlA and hyfG mutants exhibited attenuated colonization in mice. Altogether, we found expression of fhlA and hyf confers medium deacidification via facilitating urease activity, thereby urinary stone formation and mouse colonization. The link of acid resistance to urease activity provides a potential strategy for counteracting urinary tract infections by P. mirabilis.

Role of RppA in the regulation of polymyxin b susceptibility, swarming, and virulence factor expression in Proteus mirabilis.

Proteus mirabilis, a human pathogen that frequently causes urinary tract infections, is intrinsically highly resistant to cationic antimicrobial peptides, such as polymyxin B (PB). To explore the mechanisms underlying P. mirabilis resistance to PB, a mutant which displayed increased (> 160-fold) sensitivity to PB was identified by transposon mutagenesis. This mutant was found to have Tn5 inserted into a novel gene, rppA. Sequence analysis indicated that rppA may encode a response regulator of the two-component system and is located upstream of the rppB gene, which may encode a membrane sensor kinase. An rppA knockout mutant of P. mirabilis had an altered lipopolysaccharide (LPS) profile. The LPS purified from the rppA knockout mutant could bind more PB than the LPS purified from the wild type. These properties of the rppA knockout mutant may contribute to its PB-sensitive phenotype. The rppA knockout mutant exhibited greater swarming motility and cytotoxic activity and expressed higher levels of flagellin and hemolysin than the wild type, suggesting that RppA negatively regulates swarming, hemolysin expression, and cytotoxic activity in P. mirabilis. PB could modulate LPS synthesis and modification, swarming, hemolysin expression, and cytotoxic activity in P. mirabilis through an RppA-dependent pathway, suggesting that PB could serve as a signal to regulate RppA activity. Finally, we demonstrated that the expression of rppA was up-regulated by a low concentration of PB and down-regulated by a high concentration of Mg2+. Together, these data highlight the essential role of RppA in regulating PB susceptibility and virulence functions in P. mirabilis.

Extensively drug-resistant Stenotrophomonas maltophilia in a tertiary care hospital in Taiwan: microbiologic characteristics, clinical features, and outcomes.

A retrospective study was performed in a university-affiliated referral center from year 1999 to year 2004 to understand the trend of resistance in Stenotrophomonas maltophilia isolates associated with nosocomial infections and describe the emergence of an extensively drug-resistant S. maltophilia (XDRSM). A 49% increase in all types of nosocomial infections and an 85% increase in nosocomial bloodstream infections caused by S. maltophilia were noted over the 5-year period. Trends of resistance to 7 antimicrobial agents against S. maltophilia remained stable. Seventeen patients had positive cultures of XDRSM in the year 2004, including 6 with pneumonia and 1 with biliary tract infection, and the other 10 patients had XDRSM colonization. Twelve patients (70.6%) had positive cultures of non-XDRSM prior to the acquisition of XDRSM. The mortality rate of patients with XDRSM infection was higher than those with colonization (85.7% versus 10%, P = 0.004). XDRSM infection, presence of shock, and the number of dysfunctional organ systems at the time of XDRSM isolation were significantly associated with in-hospital mortality (P < 0.05). Genotyping of the 17 XDRSM isolates revealed 16 different clones. All the isolates tested possessed class 1 integron and formed biofilms and melanin. The high genotypic heterogeneity among XDRSM isolates was indicative of the polyclonal nature of emergence of these resistant bacteria. Majority of patients had positive cultures of non-XDRSM prior to the acquisition of XDRSM, indicating that the use of antibiotics might have resulted in selection of XDRSM.

Invasive pulmonary aspergillosis: high incidence of disseminated intravascular coagulation in fatal cases.

BACKGROUND AND PURPOSE: Disseminated intravascular coagulation (DIC) is a rarely described finding in invasive pulmonary aspergillosis (IPA) with unclear impact on mortality. METHODS: This study included patients with positive cultures of Aspergillus spp. from respiratory specimens, serological evidence of aspergillosis, or lung biopsy findings supporting aspergillosis treated at National Taiwan University Hospital from January 1999 to June 2005. IPA was defined based on the consensus of the European Organization for Research and Treatment of Cancer, and the Mycosis Study Group of the National Institute of Allergy and Infectious Diseases. Univariate logistic regression analysis was used to evaluate the factors associated with mortality. RESULTS: Proven or probable IPA was diagnosed in 26 patients. Hematological malignancy was found in 11 patients (42%) and immunosuppressive agents had been administered to 17 patients (65%). Among 20 culture-proven infections (77%), the most frequently encountered fungi were Aspergillus fumigatus (46%) and Aspergillus flavus (23%). The overall mortality rate was 62%. Univariate and multivariate analyses revealed that DIC was the only factor that was significantly associated with death attributable to IPA (p<0.01). CONCLUSIONS: IPA is associated with a high mortality rate, particularly for patients with DIC.

cAMP receptor protein regulates mouse colonization, motility, fimbria-mediated adhesion, and stress tolerance in uropathogenic Proteus mirabilis.

Cyclic AMP receptor protein (Crp) is a major transcriptional regulator in bacteria. This study demonstrated that Crp affects numerous virulence-related phenotypes, including colonization of mice, motility, fimbria-mediated adhesion, and glucose stress tolerance in uropathogenic Proteus mirabilis. Diabetic mice were more susceptible to kidney colonization by wild-type strain than nondiabetic mice, in which the crp mutant exhibited increased kidney colonization. Loss of crp or addition of 10% glucose increased the P. mirabilis adhesion to kidney cells. Direct negative regulation of pmpA (which encodes the major subunit of P-like fimbriae) expression by Crp was demonstrated using a reporter assay and DNase I footprinting. Moreover, the pmpA/crp double mutant exhibited reduced kidney adhesion comparable to that of the pmpA mutant, and mouse kidney colonization by the pmpA mutant was significantly attenuated. Hence, the upregulation of P-like fimbriae in the crp mutant substantially enhanced kidney colonization. Moreover, increased survival in macrophages, increased stress tolerance, RpoS upregulation, and flagellum deficiency leading to immune evasion may promote kidney colonization by the crp mutant. This is the first study to elucidate the role of Crp in the virulence of uropathogenic P. mirabilis, underlying mechanisms, and related therapeutic potential.

Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol.

Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a phytoalexin compound with anti-inflammatory and antioxidant activities. The effect of resveratrol on swarming and virulence factor expression of Proteus mirabilis, an important pathogen infecting the urinary tract, was determined on swarming agar plates with and without the compound. Bacteria harvested at different times were assayed for cell length and the production of flagella, haemolysin and urease. Resveratrol inhibited P. mirabilis swarming and virulence factor expression in a dose-dependent manner. Resveratrol significantly inhibited swarming at 15 microg ml(-1), and completely inhibited swarming at 60 microg ml(-1). Inhibition of swarming and virulence factor expression was mediated through RsbA, a His-containing phosphotransmitter of the bacterial two-component signalling system possibly involved in quorum sensing. Complementation of an rsbA-defective mutant with the rsbA gene restored its responsiveness to resveratrol. The compound also inhibited the ability of P. mirabilis to invade human urothelial cells. These findings suggest that resveratrol has potential to be developed as an antimicrobial agent against P. mirabilis infection.

Stenotrophomonas maltophilia PhoP, a Two-Component Response Regulator, Involved in Antimicrobial Susceptibilities.

Stenotrophomonas maltophilia, a gram-negative bacterium, has increasingly emerged as an important nosocomial pathogen. It is well-known for resistance to a variety of antimicrobial agents including cationic antimicrobial polypeptides (CAPs). Resistance to polymyxin B, a kind of CAPs, is known to be controlled by the two-component system PhoPQ. To unravel the role of PhoPQ in polymyxin B resistance of S. maltophilia, a phoP mutant was constructed. We found MICs of polymyxin B, chloramphenicol, ampicillin, gentamicin, kanamycin, streptomycin and spectinomycin decreased 2-64 fold in the phoP mutant. Complementation of the phoP mutant by the wild-type phoP gene restored all of the MICs to the wild type levels. Expression of PhoP was shown to be autoregulated and responsive to Mg2+ levels. The polymyxin B and gentamicin killing tests indicated that pretreatment of low Mg2+ can protect the wild-type S. maltophilia from killing but not phoP mutant. Interestingly, we found phoP mutant had a decrease in expression of SmeZ, an efflux transporter protein for aminoglycosides in S. maltophilia. Moreover, phoP mutant showed increased permeability in the cell membrane relative to the wild-type. In summary, we demonstrated the two-component regulator PhoP of S. maltophilia is involved in antimicrobial susceptibilities and low Mg2+ serves as a signal for triggering the pathway. Both the alteration in membrane permeability and downregulation of SmeZ efflux transporter in the phoP mutant contributed to the increased drug susceptibilities of S. maltophilia, in particular for aminoglycosides. This is the first report to describe the role of the Mg2+-sensing PhoP signaling pathway of S. maltophilia in regulation of the SmeZ efflux transporter and in antimicrobial susceptibilities. This study suggests PhoPQ TCS may serve as a target for development of antimicrobial agents against multidrug-resistant S. maltophilia.

Overexpression of an outer membrane protein associated with decreased susceptibility to carbapenems in Proteus mirabilis.

Proteus mirabilis isolates commonly have decreased susceptibility to imipenem. Previously, we found P. mirabilis hfq mutant was more resistant to imipenem and an outer membrane protein (OMP) could be involved. Therefore, we investigated the role of this OMP in carbapenem susceptibility. By SDS-PAGE we found this OMP (named ImpR) was increased in hfq mutant and LC-MS/MS revealed it to be the homologue of Salmonella YbfM, which is a porin for chitobiose and subject to MicM (a small RNA) regulation. We demonstrated that ImpR overexpression resulted in increased carbapenem MICs in the laboratory strain and clinical isolates. Chitobiose induced expression of chb (a chitobiose utilization operon). Real-time RT-PCR and SDS-PAGE were performed to elucidate the relationship of hfq, impR, chb and MicM in P. mirabilis. We found MicM RNA was decreased in hfq mutant and chbBC-intergenic region (chbBC-IGR) overexpression strain (chbIGRov), while impR mRNA was increased in hfq mutant, micM mutant and chbIGRov strain. In addition, mutation of hfq or micM and overexpression of chbBC-IGR increased ImpR protein level. Accordingly, chitobiose made wild-type have higher levels of ImpR protein and are more resistant to carbapenems. Hfq- and MicM-complemented strains restored wild-type MICs. Mutation of both impR and hfq eliminated the increase in carbapenem MICs observed in hfq mutant and ImpR-complementation of hfq/impR double mutant resulted in MICs as hfq mutant, indicating that the ImpR-dependent decreased carbapenem susceptibility of hfq mutant. These indicate MicM was antisense to impR mRNA and was negatively-regulated by chbBC-IGR. Together, overexpression of ImpR contributed to the decreased carbapenem susceptibility in P. mirabilis.

Inhibition of virulence factor expression and swarming differentiation in Proteus mirabilis by p-nitrophenylglycerol.

Proteus mirabilis is a common cause of upper urinary tract infections that can involve invasion of host urothelial cells. The ability to invade urothelial cells is coupled closely to swarming, a form of multicellular behaviour in which vegetative bacteria differentiate into hyperflagellate, filamentous swarming cells capable of co-ordinated and rapid population migration. Co-ordinate expression of virulence factors including urease, protease, haemolysin and flagellin during swarm-cell differentiation in P. mirabilis has been reported. To investigate the effects of p-nitrophenylglycerol (PNPG), a potent anti-swarming agent, on the various swarming-associated traits of P. mirabilis and to elucidate the relationships among them, P. mirabilis growth rate, swarming/swimming activity, cell invasion ability and the ability to express various virulence factors were monitored in the presence or absence of PNPG. It was found that PNPG could inhibit the growth rate, swarming differentiation and swarming/swimming activities of P. mirabilis. The expression of virulence factors such as protease, urease, haemolysin and flagellin in P. mirabilis was also inhibited by PNPG. The ability of P. mirabilis to invade human urothelial cells was reduced dramatically in the presence of PNPG. These results suggest that PNPG has the potential to be developed as an agent active against the effects of P. mirabilis infection.

Role of RsmA in the regulation of swarming motility and virulence factor expression in Proteus mirabilis.

Swarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper-flagellated swarm cells that undergo cycles of rapid and co-ordinated population migration across surfaces and exhibit high levels of virulence gene expression. RsmA (repressor of secondary metabolites) and CsrA, its homologue in Escherichia coli, control many phenotypic traits, such as motility and pathogenesis in Erwinia species, glycogen biosynthesis, cell size and biofilm formation in Escherichia coli and swarming motility in Serratia marcescens. To investigate the role of RsmA in Proteus mirabilis, the rsmA gene from Proteus mirabilis (hereafter referred to as rsmA(Pm)) was cloned. RsmA(Pm) showed high sequence similarity to Escherichia coli CsrA and RsmA cloned from Erwinia carotovora subsp. carotovora, Serratia marcescens, Haemophilus influenzae and Bacillus subtilis and could complement an Escherichia coli csrA mutant in glycogen synthesis. A low-copy-number plasmid carrying rsmA(Pm) expressed from its native promoter caused suppression of swarming motility and expression of virulence factors in Proteus mirabilis. mRNA stability assays suggested that RsmA(Pm) inhibited virulence factor expression through promoting mRNA degradation. RsmA homologues cloned from Serratia marcescens and Erwinia carotovora subsp. carotovora could also inhibit swarming and virulence factor expression in Proteus mirabilis.