Characterisation of P-Nitrophenylglycerol-Resistant Proteus Mirabilis Super-Swarming Mutants

p-Nitrophenylglycerol (PNPG) inhibits the co-ordinately regulated activities of swarming behaviour and virulence factor expression in Proteus mirabilis. The inhibitory action of PNPG was investigated by the isolation of Tn5 insertion mutants that could swarm, albeit with much reduced ability, in the presence of PNPG. The mutants exhibited a super- swarming phenotype in the absence of PNPG; i.e., they migrated further in a given time than did the wild-type cells. Cloning and sequence analysis of the mutants indicated that Tn5 was inserted into the rsbA gene, which may encode a membrane sensor histidine kinase of the bacterial two- component signalling system. In the absence of PNPG, the mutants exhibited several swarming-related phenotypes that were different from those of the wild type; they initiated swarming earlier and had a less conspicuous consolidation phase, they differentiated earlier and maintained a differentiated state for longer, they started to express virulence factors earlier and maintained high expression levels of these factors for longer , and they had higher cell invasion ability than the wild type. These mutant phenotypes could be complemented by a plasmid-borne copy of rsbA. Together, these data suggest that RsbA may act as a repressor of swarming and virulence factor expression. In the presence of PNPG, these rsbA- mutated mutants could still swarm, differentiate and express virulence factors, whereas the wild type could not, suggesting that PNPG may target RsbA or RsbA-regulated pathways to exert its inhibitory effect. Together, these data reveal a novel mechanism through which bacteria may negatively regulate swarming differentiation and virulence factor expression and identify a potential target of PNPG action

In Vitro Activities of Antimicrobial Combinations against Clinical Isolates of Stenotrophomonas Maltophilia

Background and Purpose: Stenotrophomonas maltophilia, a major pathogen causing nosocomial infection, is inherently resistant to multiple antimicrobial agents. Evaluation of the effectiveness of recommended therapeutic options for S. maltophilia infections is crucial, particularly in areas with high antimicrobial resistance in this nosocomial pathogen. Methods: The in vitro activities of ceftazidime ( CAZ), ticarcillin- clavulanate (TIM), amikacin (AN), ciprofloxacin (CIP), and trimethoprim- sulfamethoxazole (TMP- SMZ) against 102 clinical isolates of S. maltophilia collected from January 1998 to December 1999 at a university hospital were evaluated. The disk diffusion and agar dilution susceptibilities of individual agents against these isolates were determined concomitantly. Errors between results obtained by the two methods were identified based on the guidelines for Acinetobacter species provided by the National Committee for Clinical Laboratory Standards. Activities of three two-drug combinations (AN+CIP, CAZ+CIP, and TIM+TMP- SMZ) against 32 of these isolates were analyzed using the checkerboard synergy test. Results. Among the agents tested, TMP-SMZ was the most active against S. maltophilia (83.3% susceptible), followed by CIP (63.7%) , CAZ (39.2%), TIM (36.2%), and AN (20.5%). Errors (very major and major) between the results obtained by the disk diffusion and agar dilution methods occurred at a high frequency for AN (15% and 3%), CAZ (8% and 6%), and CIP (3% and 3%). Synergy or partial synergy of antimicrobial agent combinations was detected predominantly with CAZ+CIP (81.3%) and TIM+TMP- SMZ (84.4%) but not with AN+CIP (37.5%). No antagonism was detected with any drug combinations. Conclusion: The dilution method is preferable to the disk diffusion method for susceptibility testing of S. maltophilia isolates, particularly for testing with AN, CAZ, and TIM, which have considerable error rates between the results obtained by the two methods. The findings from the synergy test suggest that TIM+TMP-SMZ and CAZ+CIP combinations are the treatments of choice for infections caused by S. maltophilia

Modulation of Swarming and Virulence by Fatty Acids through the Rsba Protein in Proteus Mirabilis

After sensing external signals, Proteus mirabilis undergoes a multicellular behavior called swarming which is coordinately regulated with the expression of virulence factors. Here we report that exogenously added fatty acids could act as signals to regulate swarming in P. mirabilis. Specifically, while oleic acid enhanced swarming, some saturated fatty acids, such as lauric acid, myristic acid, palmitic acid, and stearic acid, inhibited swarming. We also found that expression of hemolysin, which has been shown to be coordinately regulated with swarming , was also inhibited by the above saturated fatty acids. Previously we identified a gene, rsbA, which may encode a histidine- containing phosphotransmitter of the bacterial two-component signaling system and act as a repressor of swarming and virulence factor expression in P. mirabilis. We found that while myristic acid, lauric acid, and palmitic acid exerted their inhibitory effect on swarming and hemolysin expression through an RsbA-dependent pathway, the inhibition by stearic acid was mediated through an RsbA-independent pathway. Biofilm formation and extracellular polysaccharide (EPS) production play an important role in P. mirabilis infection. We found that RsbA may act as a positive regulator of biofilm formation and EPS production. Myristic acid was found to slightly stimulate biofilm formation and EPS production, and this stimulation was mediated through an RsbA-dependent pathway. Together, these data suggest that fatty acids may act as environmental cues to regulate swarming and virulence in P. mirabilis and that RsbA may play an important role in this process

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, Haernophilus influenzae and Bacillus subtilis and could complement an Escherichia coli csrA mutant in glycogen synthesis. A low-copy-number plasmid carrying rsmApm 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

Modulation of Swarming and Virulence by Fatty Acids through the RsbA Protein in Proteus mirabilis

After sensing external signals, Proteus mirabilis undergoes a multicellular behavior called swarming which is coordinately regulated with the expression of virulence factors. Here we report that exogenously added fatty acids could act as signals to regulate swarming in P. mirabilis. Specifically, while oleic acid enhanced swarming, some saturated fatty acids, such as lauric acid, myristic acid, palmitic acid, and stearic acid, inhibited swarming. We also found that expression of hemolysin, which has been shown to be coordinately regulated with swarming, was also inhibited by the above saturated fatty acids. Previously we identified a gene, rsbA, which may encode a histidine-containing phosphotransmitter of the bacterial two-component signaling system and act as a repressor of swarming and virulence factor expression in P. mirabilis. We found that while myristic acid, lauric acid, and palmitic acid exerted their inhibitory effect on swarming and hemolysin expression through an RsbA-dependent pathway, the inhibition by stearic acid was mediated through an RsbA-independent pathway. Biofilm formation and extracellular polysaccharide (EPS) production play an important role in P. mirabilis infection. We found that RsbA may act as a positive regulator of biofilm formation and EPS production. Myristic acid was found to slightly stimulate biofilm formation and EPS production, and this stimulation was mediated through an RsbA-dependent pathway. Together, these data suggest that fatty acids may act as environmental cues to regulate swarming and virulence in P. mirabilis and that RsbA may play an important role in this process

The RNA chaperone Hfq is involved in stress tolerance and virulence in uropathogenic Proteus mirabilis.

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

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

International audienceTwo strains of Chryseobacterium identified from different experiments are proposed to represent new species. Strain WLa1L2M3 T was isolated from the digestive tract of an Oryctes rhinoceros beetle larva. Strain 09-1422 T 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 WLa1L2M3 T is approximately 32.53 %, and of 09-1422 T is approximately 35.89 %. The predominant cellular fatty acids of strain WLa1L2M3 T are C 15 : 0 iso, summed feature 9 (C 16 : 0 10OH or C 17 : 1 iso ω6 c ), C 17 : 0 iso 3OH, summed feature 3 (C 16 : 1 ω7 c and/or C 16 : 1 ω6c), C 15 : 0 iso 3OH, C 15 : 0 anteiso and C 13 : 0 iso, and those of strain 09-1422 T are C 15 : 0 iso, summed feature 3 (C 16 : 1 ω7c and/or C 16 : 1 ω6 c ), C 17 : 0 iso 3OH, C 15 : 0 anteiso, C 15 : 0 iso 3OH, C 16 : 1 ω7 c , C 17 : 0 2OH and C 18 : 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 WLa1L2M3 T (=BCRC 81350 T =JCM 35215 T =CIP 112035 T ) and 09-1422 T (=UCDFST 09-1422 T =BCRC 81359 T =CIP 112165 T ), as type strains, respectively