Role of the Two Component Signal Transduction System CpxAR in Conferring Cefepime and Chloramphenicol Resistance in Klebsiella pneumoniae NTUH-K2044

Background: Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the c-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum b-lactamase producing strains. Twocomponent systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress. Principal Findings: In the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (DcpxAR) deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of DcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The DcpxAR was more susceptible to b-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxR KP to promoter region of ompC KP in a concentration dependent manner

Serine Protease Autotransporters of Enterobacteriaceae (SPATEs): Biogenesis and Function

Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins

Identification of Klebsiella pneumoniae Genes Involved in Intestinal Colonization and Adhesion Using Signature-Tagged Mutagenesis

Klebsiella pneumoniae is an opportunistic pathogen responsible for nosocomial infections that initially colonize the intestinal tract of patients. Signature-tagged mutagenesis was used to identify genes required for this function. A library of 2,200 mutants was analyzed for the inability of the mutants to survive in a murine model of intestinal colonization and to adhere to human intestinal cells (Int-407) in vitro. Twenty-nine attenuated mutants were selected for further analyses after competition assays against the wild-type strain. Whatever the screening model, most of the transposon insertions occurred in genes involved in metabolic pathways, membrane transport, DNA metabolism, transcriptional regulation, and unknown functions. Only one mutant was attenuated in both the murine colonization and the in vitro adhesion models, and the sequence disrupted by the transposon had homology to adhesin-encoding genes of Haemophilus sp

Effect of cumin (Cuminum cyminum) seed essential oil on biofilm formation and plasmid Integrity of Klebsiella pneumoniae

Seeds of the cumin plant (Cuminum cyminum L.) have been used since many years in Iranian traditional medicine. We assessed the effect of cumin seed essential oil on the biofilm-forming ability of Klebsiella pneumoniae strains and on the integrity of a native resistance plasmid DNA from K. pneumoniae isolates, treated with essential oil. Antibacterial coaction between the essential oil and selected antibiotic disks were determined for inhibiting K. pneumoniae. The essential oil of the cumin seeds was obtained by hydrodistillation in a Clavenger system. A simple method for the formation of biofilms on semiglass lamellas was established. The biofilms formed were observed by scanning electron microscopy (SEM). The effect of essential oil on plasmid integrity was studied through the induction of R-plasmid DNA degradation. The plasmid was incubated with essential oil, and agarose gel electrophoresis was performed. Disk diffusion assay was employed to determine the coaction. The essential oil decreased biofilm formation and enhanced the activity of the ciprofloxacin disk. The incubation of the R-plasmid DNA with essential oil could not induce plasmid DNA degradation. The results of this study suggest the potential use of cumin seed essential oil against K. pneumoniae in vitro, may contribute to the in vivo efficacy of this essential oil

Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of Uropathogenic Escherichia coli

The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P < 0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P < 0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S(256) as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S(256) is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside