1 Department of Medical Microbiology (G4), Faculty of Health Sciences, University of the Free State, Bloemfontein; 2 Medical Research Council, Cape Town, South Africa
Received 23 January 2003; returned 7 March 2003; revised 22 April 2003; accepted 23 April 2003
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Abstract |
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Methods: Penicillin-binding protein (PBP) competition studies were conducted in Veillonella strains, with piperacillin MICs ranging from 0.5 to >128 mg/L and ampicillin MICs from 0.125 to 4 mg/L. Whole cell lysates were pre-incubated with piperacillin or ampicillin and post-labelled with [3H]benzylpenicillin.
Results: PBP competition studies showed that the PBP with greatest affinity for penicillin and ampicillin had a molecular weight of 66 kDa, and exhibited reduced binding of piperacillin in resistant strains.
Conclusions: This unusual focusing of different penicillins on one PBP may be the cause of selective mutants resulting from piperacillin MICs > 128 mg/L. In the absence of ß-lactamases, alterations in penicillin-binding were seen to be major contributors to high-level piperacillin resistance development.
Keywords: anaerobic bacteria, resistance, penicillins, Gram-negative bacteria, ß-lactams
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Introduction |
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Materials and methods |
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Thirty-one Veillonella spp. were isolated from clinically significant infections from 19961997 from the Universitas and Pelonomi Hospitals, Bloemfontein.9
MIC determination
MICs of ampicillin (8 mg/L), piperacillin (32 mg/L), cefoxitin (16 mg/L) and imipenem (4 mg/L) were determined by the NCCLS agar dilution methods. Susceptibility breakpoints used (indicated in parentheses) were those suggested by the NCCLS (mg/L).11 Wilkins Chalgren agar was supplemented with 5% lysed horse blood to enhance growth of fastidious bacteria, such as Veillonella. Eleven Veillonella spp. were selected for PBP analysis (Table 1).
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Cells were harvested from cultures grown overnight on BHI agar (Oxoid, Unipath, Basingstoke, UK) supplemented with vitamin K (10 mg/L), haemin (500 mg/L) and yeast extract (5000 mg/L) under an anaerobic atmosphere. The cells were suspended in Brucella broth (turbidity ±5 McFarland standard); 100 µL aliquots were centrifuged at 16 000g and the pellets stored at 20°C. Pellets were resuspended in 25 µL lysis buffer (0.02 M sodium phosphate buffer, pH 7 and 0.2% Triton X-100) to which 10 µL lysozyme (1 mg/mL) was added. For PBP competition studies, concentrations of piperacillin 10, 5, 2 and 1 mg/L, and ampicillin 5, 2, 1 and 0.5 mg/L were added to whole cell preparations of Veillonella spp. Cell preparations were incubated at 37°C for 10 min, and post-labelled with 2 µCi [3H]benzylpenicillin at 37°C for 10 min; 30 µL sample buffer (0.5 M Tris, pH 6.8, 10% glycerol, 2% SDS, 5% mercaptoethanol, 1% Bromophenol Blue) was added to each sample. PBPs were separated by SDSPAGE and visualized after fluorography (Amplify, Amersham). A protein molecular-weight marker (Rainbow, [14C]-labelled, Amersham) was included in each gel run.
ß-Lactamase production
To screen for ß-lactamase production, bacterial cells were mixed with nitrocefin (Oxoid) and observed extensively for 1 h for any change in colour. A change from red to yellow would indicate the production of ß-lactamase.
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Results and discussion |
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Footnotes |
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References |
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