Complexity and Diversity of Klebsiella pneumoniae Strains with Extended-Spectrum β-Lactamases Isolated in 1994 and 1996 at a Teaching Hospital in Durban, South Africa

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Abstract
β-Lactamase production was investigated in cultures of 25 Klebsiella pneumoniae isolates isolated at a hospital in Durban, South Africa, in 1994 and 1996. Twenty of these isolates gave ceftazidime MIC/ceftazidime plus clavulanate MIC ratios of ≥8, implying production of extended-spectrum β-lactamases (ESBLs), and DNA sequencing identified an ESBL gene ( bla TEM-53 ) in a further two isolates. Pulsed-field gel electrophoresis (PFGE) defined 4 distinct strains among the 12 isolates collected in 1994 and 9 distinct strains among the 13 isolates collected in 1996. In three cases, multiple isolates from single patients varied in their PFGE profiles and antibiograms, implying mixed colonization or infection. Isoelectric focusing and DNA hybridization found both TEM and SHV enzymes and their genes in all 25 isolates. Many isolates had multiple identical or different β-lactamase gene variants, with at least 84 bla SHV and bla TEM gene copies among the 25 organisms. Sequencing identified the genes for the SHV-1, -2, and -5 enzymes and for four new SHV types (SHV-19, -20, -21, and -22). These new SHV variants had novel mutations remote from sites known to affect catalytic activity. Sequencing also found the genes for TEM-1, TEM-53, and one novel type, TEM-63. All the isolates had multiple and diverse plasmids. These complex and diverse patterns of ESBL production and strain epidemiology are far removed from the concept of an ESBL outbreak and suggest a situation in which ESBL production has become endemic and in which evolution is generating a wide range of enzyme combinations. This complexity and diversity complicates patient management and the design of antibiotic use policies.

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