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Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H2O2 and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H2O2 in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Gemella morbillorum, a low G+C content Gram-positive bacterium, is considered to be a commensal organism in humans but occasionally causes endocarditis or other diseases. We determined the sequences of groESL, dnaK and their flanking regions in G. morbillorum. Sequence analysis revealed the presence of putative CtsR binding sites in both groE and dnaK operons, but the lack of CIRCE in groE and the presence of CIRCE in dnaK. This finding suggests in addition to the known regulatory systems for the class I heat shock protein genes, there may be another model in G. morbillorum. Furthermore, an unusual organization of the groE operon as groES-groEL-trxA was found. Genome sequence on GenBank database and southern blot indicate that there is only one copy of trxA in G. morbillorum. Sequencing of the groE locus from other Gemella species and clinical isolates revealed the same genetic structure, suggesting the conservation of the structure in Gemella species. Northern hybridization revealed that there were two transcripts, a large transcript, groES-groEL-trxA and a small transcript, trxA, in groE operon. Treatment of heat or diamide increased the transcription level of groES-groEL-trxA, whereas these two stresses did not affect the small trxA transcript. Thus, this study reveals that the trxA is co-transcribed with the groE operon, and most possibly under the control of the CtsR. [Copyright &y& Elsevier]