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Strength and regulation of the different promoters for chromosomal beta-lactamases of Klebsiella oxytoca.

ABSTRACT: The two groups of chromosomal beta-lactamases from Klebsiella oxytoca (OXY-1 and OXY-2) can be overproduced 73- to 223-fold, due to point mutations in the consensus sequences of their promoters. The different versions of promoters from blaOXY-1 and blaOXY-2 were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene of pKK232-8, and their relative strengths were determined in Escherichia coli and in K. oxytoca. The three different mutations in the OXY beta-lactamase promoters resulted in a 4- to 31-fold increase in CAT activity compared to that of the wild-type promoter. The G-->T transversion in the first base of the -10 consensus sequence caused a greater increase in the promoter strength of the wild-type promoter than the two other principal mutations (a G-to-A transition of the fifth base of the -10 consensus sequence and a T-to-A transversion of the fourth base of the -35 sequence). The strength of the promoter carrying a double mutation (transition in the Pribnow box and the transversion in the -35 hexamer) was increased 15- to 61-fold in comparison to that of the wild-type promoter. A change from 17 to 16 bp between the -35 and -10 consensus sequences resulted in a ninefold decrease of the promoter strength. The expression of the blaOXY promoter in E. coli differs from that in K. oxytoca, particularly for promoters carrying strong mutations. Furthermore, the blaOXY promoter appears not to be controlled by DNA supercoiling or an upstream curved DNA, but it is dependent on the gene copy number.

SUBMITTER: Fournier B

PROVIDER: S-EPMC89216 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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Strength and regulation of the different promoters for chromosomal beta-lactamases of Klebsiella oxytoca.

Fournier B B Gravel A A Hooper D C DC Roy P H PH

Antimicrobial agents and chemotherapy 19990401 4

The two groups of chromosomal beta-lactamases from Klebsiella oxytoca (OXY-1 and OXY-2) can be overproduced 73- to 223-fold, due to point mutations in the consensus sequences of their promoters. The different versions of promoters from blaOXY-1 and blaOXY-2 were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene of pKK232-8, and their relative strengths were determined in Escherichia coli and in K. oxytoca. The three different mutations in the OXY beta-lactamase promoters result ...[more]

PMID: 10103190

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Project description:We report the genetic characterization of 15 Klebsiella pneumoniae (KP) and 4 isolates of K. oxytoca (KO) from clinical cases in dogs and cats and showing extended-spectrum cephalosporin (ESC) resistance. Extended spectrum beta-lactamase (ESBL) and AmpC genes, plasmid-mediated quinolone resistance (PMQR) and co-resistances were investigated. Among KP isolates, ST101 clone was predominant (8/15, 53%), followed by ST15 (4/15, 27%). ST11 and ST340, belonging to Clonal Complex (CC)11, were detected in 2012 (3/15, 20%). MLST on KP isolates corresponded well with PFGE results, with 11 different PFGE patterns observed, including two clusters of two (ST340) and four (ST101) indistinguishable isolates, respectively. All isolates harbored at least one ESBL or AmpC gene, all carried on transferable plasmids (IncR, IncFII, IncI1, IncN), and 16/19 were positive for PMQR genes (qnr family or aac(6')-Ib-cr). The most frequent ESBL was CTX-M-15 (11/19, 58%), detected in all KP ST101, in one KP ST15 and in both KP ST340. blaCTX-M-15 was carried on IncR plasmids in all but one KP isolate. All KP ST15 isolates harbored different ESC resistance genes and different plasmids, and presented the non-transferable blaSHV-28 gene, in association with blaCTX-M-15, blaCTX-M-1 (on IncR, or on IncN), blaSHV-2a (on IncR) or blaCMY-2 genes (on IncI1). KO isolates were positive for blaCTX-M-9 gene (on IncHI2), or for the blaSHV-12 and blaDHA-1 genes (on IncL/M). They were all positive for qnr genes, and one also for the aac(6')-Ib-cr gene. All Klebsiella isolates showed multiresistance towards aminoglycosides, sulfonamides, tetracyclines, trimethoprim and amphenicols, mediated by strA/B, aadA2, aadB, ant (2")-Ia, aac(6')-Ib, sul, tet, dfr and cat genes in various combinations. The emergence in pets of multidrug-resistant Klebsiella with ESBL, AmpC and PMQR determinants, poses further and serious challenges in companion animal therapy and raise concerns for possible bi-directional transmission between pets and humans, especially at household level.

Project description:The aim of this study was to determine whether there is an association between serum resistance, O serotypes, and the production of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae. Ninety ESBL-producing and 178 non-ESBL-producing K. pneumoniae isolates gathered in five European countries were O serotyped and tested for sensitivity to the serum's bactericidal effect. The frequency of serum-resistant isolates was higher among ESBL-producing strains (30%; 27/90 isolates) than among non-ESBL-producing strains (17.9%; 32/178 isolates) (P = 0.037; odds ratio [OR] = 1.96; 95% confidence interval [95% CI] = 1.08 to 3.53). Although O1 was the most common O serotype in both Klebsiella groups, its frequency among ESBL-producing strains was significantly higher (59%; 53/90 isolates) than among non-ESBL producers (36%; 64/178 isolates) (P = 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29). Furthermore, the prevalence of the O1 serotype was higher among serum-resistant strains of both ESBL-producing (74%; 20/27isolates) and non-ESBL producers (75%; 24/32 isolates) than among serum-sensitive ESBL producers (52.4%; 33/63 isolates) and non-ESBL producers (27.4%; 40/146 isolates). Serum resistance among ESBL-producing strains (36%; 17/47 isolates) versus non-ESBL-producing strains (16%; 27/166 isolates) was also significantly higher after the exclusion of clonal strains (P = 0.0056; OR = 2.9; 95% CI = 1.41 to 6.01). Sixteen ESBL types were detected, among which the frequency of serum resistance was significantly lower among the SHV-producing strains (9/48 isolates) than among the TEM producers (16/35 isolates) (P = 0.016; OR = 3.65; CI = 1.3 to 9.7). Curing ESBL-coding plasmids did not influence the serum resistance of the bacteria; all six plasmid-cured derivatives maintained serum resistance. The present findings suggest that ESBL-producing strains have a greater pathogenic potential than non-ESBL-producing strains, but the linkage between O serotypes, serum resistance, and ESBL production remains unclear at this stage.

Dataset Information

Strength and regulation of the different promoters for chromosomal beta-lactamases of Klebsiella oxytoca.

Publications

Strength and regulation of the different promoters for chromosomal beta-lactamases of Klebsiella oxytoca.

Similar Datasets

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