JAC Advance Access originally published online on June 12, 2003
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Journal of Antimicrobial Chemotherapy (2003) 52, 120-122
© 2003 The British Society for Antimicrobial Chemotherapy
Penicillin-binding proteins involved in high-level piperacillin resistance in Veillonella spp.
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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Objectives: To investigate high-level piperacillin resistance in Veillonella spp. in the absence of ß-lactamase activity.
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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Veillonella spp. are Gram-negative anaerobic cocci and are part of the normal flora of the mouth, gastrointestinal tract and vagina of humans. Although clinically isolated, Veillonella spp. are often regarded as contaminants; they are often associated with oral infections, bite wounds, head, neck and various soft tissue infections, and have recently been implicated as pathogens in infections of the sinuses, lungs, heart, bone and central nervous system.1,2 Recent reports have also indicated their isolation in pure culture in septic arthritis and meningitis.2,3 ß-Lactam antibiotics are used frequently, and for many years have been the first choice of treatment and prophylaxis for anaerobic infections.4,5 However, susceptibility varies depending on the ß-lactam and bacterial species to be targeted.5 Piperacillin has maintained efficacy, although it may be inactivated by chromosomal class A ß-lactamases produced by anaerobes, and is invariably given in combination with a ß-lactamase inhibitor, tazobactam.6,7 Resistance to piperacillin/tazobactam has been reported in Veillonella spp., and the occurrence of high-level resistance to piperacillin in ß-lactamase-negative Veillonella spp. has been noted.8–10 The current study investigates alterations in penicillin-binding protein (PBP) involved in piperacillin resistance development in Veillonella spp.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Veillonella isolates
Thirty-one Veillonella spp. were isolated from clinically significant infections from 1996–1997 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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PBP labelling of whole cells
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 SDS–PAGE 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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Selective resistance to piperacillin (MIC ≥ 64 mg/L) was found in the absence of ß-lactamase activity in 21/31 (68%) Veillonella isolates.9 Alteration of PBP prompted investigations into resistance development. Results from PBP ampicillin/piperacillin competition studies performed on Veillonella strains with piperacillin MICs ranging from 0.5 to >128 mg/L are illustrated in Figure 1(a–c). Ampicillin was seen to bind strongly (as with penicillin) to the high-molecular-weight PBP (
66 kDa), as only a faint band was noted in lanes 8 and 9 of Figure 1(b and c). In contrast, affinity of piperacillin for the 66 kDa PBP was considerably lower in piperacillin-resistant strain V18 (MIC > 128 mg/L), as reduced binding to this PBP was noted, indicated by the strong binding of [3H]benzylpenicillin in lanes 2–5 (Figure 1c). Other resistant strains that showed the same results were V1, V4 and V28 (MICs > 128 mg/L). The PBP competition profile of strain V31 (Figure 1b) with intermediate resistance to piperacillin (MIC 64 mg/L), showed a reduction in piperacillin binding to this PBP similar to that found with V18 (Figure 1c), when compared with the binding to piperacillin in susceptible strain V2 (MIC 0.5 mg/L) (Figure 1a). Two other susceptible strains, V11 and V19 (MIC 16 mg/L), showed PBP binding similar to that of V2. It was evident that the PBP (66 kDa) revealing the greatest affinity for penicillin and ampicillin was seen to exhibit the lowest affinity for piperacillin in piperacillin-resistant strains (Figure 1a–c).
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For many bacterial genera, it is unusual for the affinities of different penicillins to be focused on one high affinity PBP, yet for piperacillin, specific mutants exhibiting high-level resistance to piperacillin appear to have evolved. In 1996, Wren12 found that some strains of Veillonella were susceptible to carboxypenicillins and ureidopenicillins, and in 1997 Mendes, Gordon & Mitchell8 reported strains of Veillonella from Australia that were resistant to piperacillin/tazobactam. It is also known that a target PBP can alter affinity selectively with respect to one particular ß-lactam.4 The findings of the present study and those of other researchers demonstrate the importance of further investigations into the involvement of PBPs in piperacillin resistance development in Veillonella spp.
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Footnotes |
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* Corresponding author. Tel: +27-51-4053648; Fax: +27-51-4443437; E-mail: gnmbml{at}med.uovs.ac.za
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References |
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