Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling.
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Penicillin G Acylas (PGA) of Providencia rettgeri (ATCC 25599) was evolved using a modified DNA family shuffling method. The identity of pga genes from Escherichia coli, Kluyvera citrophila and Providencia rettgeri ranges from 62.5% to 96.9%. The pga genes from above three species were recombined and shuffled to create interspecies pga gene fusion libraries. By substituting assembled chimaeras for corresponding region of pETPPGA, different recombinants were constructed and expressed in E. coli JM109(DE3). Mutants with obvious beta-lactam synthetic activity were selected from the plates and the ratios of synthesis to hydrolysis (S/H) were determined subsequently. It was shown that the primary structures of selected positives exhibited significant diversity among each library. The best mutant possessed 40% higher synthetic activity than the wild type enzyme of PrPGA. It was further proved in this study that the domain of alpha subunit contributed much more to improve the specific activity of synthesis. Results showed a recombinant PGA with higher synthetic activity was acquired by the method of DNA shuffling. (+info)
Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTX-M-1-derived cefotaximases.
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A gene identical to plasmid-borne bla(CTX-M-3) is present in the chromosome of one Kluyvera ascorbata strain. It is associated with a structure including an inverted repeat right and an open reading frame 477-like gene probably involved in the mobilization of bla(CTX-M-3). Two other K. ascorbata strains rendered the previously described bla(KLUA-9) gene. (+info)
Phylogenetic relationships of the genus Kluyvera: transfer of Enterobacter intermedius Izard et al. 1980 to the genus Kluyvera as Kluyvera intermedia comb. nov. and reclassification of Kluyvera cochleae as a later synonym of K. intermedia.
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In order to assess the relationship between the genus Kluyvera and other members of the family Enterobacteriaceae, the 16S rRNA genes of type strains of the recognized Kluyvera species, Kluyvera georgiana, Kluyvera cochleae, Kluyvera ascorbata and Kluyvera cryocrescens, were sequenced. A comparative phylogenetic analysis based on these 16S rRNA gene sequences and those available for strains belonging to several genera of the family Enterobacteriaceae showed that members of the genus Kluyvera form a cluster that contains all the known Kluyvera species. However, the type strain of Enterobacter intermedius (ATCC 33110T) was included within this cluster in a very close relationship with the type strain of K. cochleae (ATCC 51609T). In addition to the phylogenetic evidence, biochemical and DNA-DNA hybridization analyses of species within this cluster indicated that the type strain of E. intermedius is in fact a member of the genus Kluyvera and, within it, of the species Kluyvera cochleae. Therefore, following the current rules for bacterial nomenclature and classification, the transfer of E. intermedius to the genus Kluyvera as Kluyvera intermedia comb. nov. is proposed (type strain, ATCC 33110T=CIP 79.27T=LMG 2785T=CCUG 14183T). Biochemical analysis of four E. intermedius strains and one K. cochleae strain independent of the respective type strains further indicated that E. intermedius and K. cochleae represent the same species and are therefore heterotypic synonyms. Nomenclatural priority goes to the oldest legitimate epithet. Consequently, Kluyvera cochleae Muller et al. 1996 is a later synonym of Kluyvera intermedia (Izard et al. 1980) Pavan et al. 2005. (+info)
Clinically significant Kluyvera infections: a report of seven cases.
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To determine the clinical significance of Kluyvera isolates at our institution, we retrospectively analyzed clinical microbiology data from January 1999 to September 2003. We identified 11 isolates classified as Kluyvera ascorbata, 7 of which were considered clinically significant pathogens: 3 cases represented urinary tract infections; 2, bacteremia; 1, a soft tissue infection of the finger; and 1, acute appendicitis with a subsequent intra-abdominal abscess. The age distribution of patients was wide, ranging from 2 months to 73 years. Antimicrobial susceptibility studies of the clinically significant and non-clinically significant Kluyvera isolates showed susceptibility patterns similar to those reported in the medical literature, namely trends of resistance to ampicillin and first- and second-generation cephalosporins. Of the 4 non-clinically significant isolates in our study, 1 was resistant to ciprofloxacin, a finding reported in only 1 other isolate of Kluyvera in the medical literature. Patient outcome after treatment with third-generation cephalosporins and aminoglycosides in the 7 clinically significant cases was good, with no long-term sequelae. The potential virulence of K ascorbata highlights the need for heightened scrutiny of its antimicrobial susceptibility patterns for adequate clinical treatment. (+info)
Identification of a progenitor of the CTX-M-9 group of extended-spectrum beta-lactamases from Kluyvera georgiana isolated in Guyana.
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Chromosomal beta-lactamase genes (bla(KLUY)) from six Kluyvera georgiana strains isolated in Guyana were cloned and expressed in Escherichia coli. KLUY-1 exhibited 100% amino acid identity with the extended-spectrum beta-lactamase CTX-M-14. We also show that a 2.7-kb Kluyvera chromosomal region exhibits 99% nucleotide identity to a portion of In60 that includes bla(CTX-M-9). (+info)
In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring beta-lactamase gene blaCTX-M of Kluyvera ascorbata.
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ISEcp1B has been reported to be associated with and to mobilize the emerging expanded-spectrum beta-lactamase blaCTX-M genes in Enterobacteriaceae. Thus, the ability of this insertion sequence to mobilize the blaCTX-M-2 gene was tested from its progenitor, Kluyvera ascorbata. Insertions of ISEcp1B upstream of the blaCTX-M-2 gene in K. ascorbata reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 microg/ml). In those cases, ISEcp1B brought promoter sequences enhancing blaCTX-M-2 expression in K. ascorbata. Then, ISEcp1B-mediated mobilization of the blaCTX-M-2 gene from K. ascorbata to Escherichia coli J53 was attempted. The transposition frequency of ISEcp1B-blaCTX-M-2 occurred at (6.4+/-0.5)x10(-7) in E. coli. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40 degrees C. (+info)
Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains.
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Hickman Catheter-Related Bacteremia with Kluyvera cryocrescens: a Case Report.
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This report describes a 2-year-old child with neuroectodermal tumor presenting with febrile neutropenia. Blood cultures drawn from the peripheral vein and Hickman catheter revealed Kluyvera cryocrescens growth. The Hickman catheter was removed and the patient was successfully treated with cefepime and amikacin. Isolation of Kluyvera spp. from clinical specimens is rare. This saprophyte microorganism may cause serious central venous catheter infections, especially in immunosuppressed patients. Clinicians should be aware of its virulence and resistance to many antibiotics. (+info)