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Serratia marcescens swarms at 30[degrees]C but not at 37[degrees]C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis ors. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37[degrees]C but also earlier and faster than the parent strain swarms at 30[degrees]C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduetion sensor Mnase, upstream of which a potential response regulator gene (rssB) was located, rssA and rssB insertiondeletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37[degrees]C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30[degrees]C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-I strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. mareescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30[degrees]C or 37[degrees]C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.

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: 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]

Abstract: We describe a patient who received continuous ambulatory peritoneal dialysis and developed catheter-related peritonitis caused by Aspergillus sydowii. Identification of A. sydowii was confirmed by the sequence analysis of the ribosomal RNA genes. The patient remained well after removal of the peritoneal catheter without administration of any anti-fungal agent. [Copyright &y& Elsevier]

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]

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]

Two 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.

During an investigation of microbes associated with arthropods living in decaying coconut trees, a Pseudomonas isolate, Milli4 T , was cultured from the digestive tract of the common Asian millipede, Trigoniulus corallinus . Sequence analysis of 16S rRNA and rpoB genes found that Milli4 T was closely related but not identical to Pseudomonas panipatensis Esp-1 T , Pseudomonas knackmussi B13 T and Pseudomonas humi CCA1 T . 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 Milli4 T and its closest relatives also suggested they are distinct species. The genomic DNA G+C content of Milli4 T was approximately 65.0 mol%. Phenotypic and chemotaxonomic characterization and fatty acid methyl ester analysis was performed on Milli4 T and its related type strains. Based on these data, the new species Pseudomonas schmalbachii sp. nov. is proposed, and the type strain is Milli4 T (=BCRC 81294 T =JCM 34414 T =CIP 111980 T ).

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 Tn 5 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 ., (Copyright © 2020 American Society for Microbiology.)

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.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

In this study, we demonstrated that 10'(Z), 13'(E)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof Proteus mirabilis which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and rppA mutant but not in the rcsB mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the rcsB mutant. Moreover, the finding that HQ17-2 inhibits the expression of flhDC gene in the wild-type and rcsB-complemented strain but not in the rcsB mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and rcsB mutant but not in the rppA mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of rppA and pmrI, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the rppA mutant. The inhibition of rppA and pmrI expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications.

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 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.

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.

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.

We describe a case of empyema thoracis caused by Rhizopus oryzae diagnosed in an allogenic bone marrow transplant patient with acute lymphocytic leukemia. The isolate of R. oryzae was recovered from three pleural effusion specimens, which were black in color. It was identified on the basis of characteristic colonial appearance and microscopic findings, as well as the partial sequencing of rRNA genes. The patient died of uncontrolled R. oryzae empyema thoracis and concomitant nosocomial infection.

We present a case of fatal histoplasmosis in a patient with Addison's disease and long-term use of corticosteroid. The patient acquired Histoplasma capsulatum in China and initially presented with a laryngeal mass. He developed fulminant pneumonia and respiratory failure 9 years later. Nucleotide sequences of internal transcribed spacer regions of rRNA genes of the H. capsulatum isolate recovered from biopsied lung tissue and of the extracted fungal DNA from the laryngeal lesion were identical, indicating the recurrent nature of infection.

Escherichia coli strains are among the major bacterial causes of diarrheal illness. At least 5 categories of diarrheagenic E. coli (DEC) are recognized, namely enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and enterohemorrhagic E. coli (EHEC). Due to the need for costly and labor-intensive diagnostic procedures, identification of DEC is difficult at standard laboratories. Therefore, the epidemiology of DEC infections remains obscure in Taiwan. Recently, polymerase chain reaction (PCR) or dot blot has been used for genetic detection of DEC. In this study, we analyzed 150 E. coli isolates from diarrheal stools of children under 5 years old. The PCR tests detected 5 ETEC (3.3%), 6 EPEC (4%), 4 EIEC (2.7%), and 13 EAEC (8.7%) isolates. No EHEC was detected. Dot blot and sequence analysis were used to confirm the results of PCR. The cellular fatty acid (CFA) profiles from E. coli isolates were also analyzed. Comparison of CFA composition revealed minor variation in the percentage of each fatty acid detected among DEC isolates of ETEC, EPEC, EIEC and EAEC, but did not provide enough evidence for differentiating between categories of DEC by CFA profiles alone.

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.

Susceptibilities to 16 antimicrobial agents were determined by measurement of MICs for 344 isolates of anaerobic bacteria recovered from patients with significant infections. Resistance rates varied among antimicrobial agents and the species tested. The beta-lactams were more active in gram-positive than in gram-negative anaerobes. Resistance to meropenem was low (<1%). For beta-lactam-beta-lactamase inhibitors, piperacillin-tazobactam was most active for all species (resistance, <6%). The rates of resistance to cefoxitin (31 to 65%) and clindamycin (50 to 70%) for non-Bacteroides fragilis species of the B. fragilis group were higher than those for B. fragilis (4% resistant to cefoxitin and 33% resistant to clindamycin). Among members of B. fragilis group, Bacteroides thetaiotaomicron was the most resistant to clindamycin (70%) and cefoxitin (65%). Rates of susceptibility to imipenem and metronidazole for B. fragilis continue to be high compared to those from a previous study 10 years ago. However, resistance to metronidazole was found recently in five strains of B. fragilis. We analyzed the genetic relationships among the metronidazole-resistant B. fragilis strains by pulsed-field gel electrophoresis. The metronidazole-resistant B. fragilis strains showed genotypic heterogeneity, excluding the dissemination of a single clone.

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.