Studies on two bacterial resistance mechanisms to ß-lactam antibiot : extended spectrum ß-lactamases in Klebsiella oxytoca and methicillin resistance in staphylococci

University dissertation from Stockholm : Karolinska Institutet, Department of Laboratory Medicine

Abstract: Eleven clinical isolates of Klebsiella oxytoca from Stockholm hospitals were found to be resistant to aztreonam and cefuroxime (MIC > 16 mg/L). The resistance was caused by a ß-lactamase with pI 5.25 and designated KH. Assay of the substrate profiles revealed that the KH ß-lactarnase was similar to the chromosomally encoded OXY-2 ß-lactamase. The genetic determinant of the KH ß-lactamase (blaKH) was characterized by DNA sequencing to be one of the members in the OXY-2 ß-lactamase family of K. oxytoca. The same clinical isolates of K. oxytoca also contained a cryptic plasmid, pTKH11. Plasmid pTKH11 was observed after electroporation of K. oxytoca plasmid preparations into E. coli XAC. In the E. coli host, the plasmid expressed a high level of AmpC-like ß-lactamase designated KU and conferred the E. coli transformants to become resistant to ß-lactam antibiotics, such as aztreonam, cefuroxime, cefotaxime and ceftazidime. Determination of the nucleotide sequence of pTKH11 revealed that the 8199 bp plasmid encoded seven open reading frames including the KU ß-lactamase The blaKU product displayed sequence identity at amino acid level to the plasmidic CMY-2 ß-lactamase described in K. pneumoniae and to the chromosomal AmpC of Citrobacter freundii with 99.7% and 97.0%, respectively. The blaKU-Blc-SugE- cluster in pTKH11 exhibited an identical genetic organization as the ampC region on the chromosome of C. freundii. The mecA gene homologue identified in Staphylococcus sciuri isolate K1 shared overall similarity to the MRSA mecA by 88% in amino acid sequence, and by 96% in the transpeptidase domain. Furthermore, methicillin-resistant S. sciuri strains harbored a S. aureus mecA copy, and an open reading frame (UgpQ) conservatively located within the hypervariable region of MRSA was also identified downstream from the mecA homologue of strain K11 of S. sciuri subspecies carnaticum. These findings supported the hypothesis that the S. sciuri mecA is the evolutionary precursor of S. aureus mecA. The auxiliary gene mutant RUSA315 harbored an intact mecA, but showed a drop in methicillin MIC from 1600 to 1.5 mg/L. Cloning and DNA sequencing the insertion region of transposon Tn551 in S. aureus demonstrated that the inactivated gene femR315 encoded a polypeptide that showed 26% amino acid sequence identity and 57% similarity with the phosphoglucosamine mutase (GlmM) gene of E. coli. The biochemical function of femR315 product was characterized to be the GlmM of S. aureus. The DNA sequence of S. aureus transposon Tn551 was 5266 bp and differed from the enterococcal transposon Tn917 by eleven events of nucleotide alternative. The high degree of homology suggested that the two transposons should be regarded as identical transposable elements. Transcriptional analysis have determined the initiation start for the constitutive erm transcription and revealed that the erythromycin resistance conferred by Tn551 could also be enhanced by induction.

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