1 Laboratoire de Bactériologie, Hôpital lArchet 2, Centre Hospitalier Universitaire, 151 route de Saint Antoine Ginestière, 06202 Nice Cedex 3; 2 Laboratoire SurfacesInterfaces en Odontologie, Faculté de Chirurgie Dentaire, Nice, France
Received 13 August 2002; returned 9 October 2002; revised 19 February 2003; accepted 20 February 2003
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Abstract |
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Keywords: anaerobes, antibiotic resistance, ß-lactam, Prevotella
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Introduction |
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Materials and methods |
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Sixty-two ß-lactamase-producing Prevotella were retrieved from a laboratory collection (Bacteriology Laboratory, Nice University Hospital, France). These clinical samples were sent by medical and dental hospital staff for routine diagnosis. Thirty-two oral strains were isolated from outpatients with periodontitis or acute dentoalveolar abscesses. All these strains were previously shown, by PCR, to carry a cfxA-type gene.1 Thirty non-oral strains were obtained from hospitalized patients suffering from intestinal, respiratory, gynaecological or maxillary bone infections. After standard identification (Api 32A, bioMérieux, Marcy-lÉtoile, France) and susceptibility testing as previously described,1 all the strains were stored at 70°C before PCR protocols. Briefly, susceptibility profiles were carried out by a standard disc diffusion method on WilkinsChalgren agar supplemented with 5% sterile defibrinated sheep blood with amoxicillin, co-amoxiclav, ticarcillin, cefalothin, cefuroxime, cefazolin, cefoxitin, cefpirome, cefotaxime, ceftazidime, imipenem and aztreonam. Concordance charts between diameters of inhibition and susceptibility breakpoints according to NCCLS criteria were supplied by the manufacturer (Bio-Rad, Marnes-La-Coquette, France). This standard method does not allow MIC determination. ß-Lactamase production was confirmed using nitrocefin, a chromogenic cephalosporin (cefinase; AB Biodisk, Solna, Sweden).
Primer design and specificity controls
For screening purposes, an intragenic primer pair was designed to amplify an 802 bp conserved region within cfxA and cfxA2 (5'-CGTAGTTTTGAGTATAGCTTT-3' and 5'-GATGTTGCCTATATATGTC-3'). For cloning experiments, we used a primer pair flanking cfxA/cfxA2 and designed to amplify the whole ß-lactamase gene (966 bp) (5'-GAAAAAAACAGAAAAAAACAAATC-3' and 5'-TTAAGATTTTACTGAAGTTTG-3'). These two primer pairs were designed from cfxA and cfxA2 gene sequences [GenBank accession nos U38243 (cfxA) and AF118110 (cfxA2)]. Three control strains were used in preliminary experiments: (i) B. vulgatus CLA 341, the wild-type strain from which cfxA was initially cloned and sequenced; (ii) an Escherichia coli EC 351 transformant harbouring cfxA (pFD351);5 and (iii) an E. coli NI-141 transformant harbouring cfxA2 (pNCE-3).7 Reference strains of E. coli producing TEM-1, SHV-1 and OXA-3 enzymes were tested as negative controls.
PCR protocol
For PCR experiments, whole-cell DNA was obtained from bacterial suspensions (100 µL, 1.5 McFarland standard density) disrupted for 10 min at 100°C, cooled on ice, and centrifuged for 5 min at 48 000g. The supernatant was used as a template. Positive and negative controls were used in each run. Amplification was carried out in 100 µL volumes containing 10 mM TrisHCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 0.4 mM dNTP, 20 pmol of each primer and 0.5 U of Taq DNA polymerase (Perkin-Elmer PCR reagents; Roche Molecular Systems, Branchburg, NJ, USA), to which 5 µL of bacterial DNA supernatant was added. DNA was amplified in a PCR system (PCR Sprint; Hybaid, Ashford, UK), programmed for 25 cycles of 1 min at 94°C, 1 min at 58°C and 1 min 30 s at 72°C. Initial and final steps of 5 min at 94°C and 10 min at 72°C, respectively, were added. Amplification products were detected by electrophoresis in agarose gels (0.8%).
Cloning experiments and sequence analysis
After initial screening of cfxA/cfxA2 in 62 Prevotella, we selected 14 strains representative of Prevotella species, either pigmented or not pigmented, for cloning experiments and further gene sequence analysis. Briefly, the entire ß-lactamase genes were amplified (966 bp), cloned in the plasmid vector pCR 2.1 and expressed in E. coli (INVF'), using the manufacturers media and compounds (TA Cloning Kit; Invitrogen, Carlsbad, CA, USA). Sequences were determined from both strands of DNA with an Applied Biosystems sequencer (Eurogentec, Herstal, Belgium). Relatedness of the deduced protein sequences (321 amino acids) was investigated by comparison with the GenBank/EMBL databases.
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Results |
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Discussion |
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CfxA and CfxA2 have the same kinetic parameters,5,7 and in routine susceptibility testing the 62 Prevotella strains tested appeared as regular class A/group 2e ß-lactamase producers. Nonetheless, detailed susceptibility profile and biochemical characterization are needed in order to determine whether the additional G83D, F/V189L, W193L and D239Y substitutions affect enzyme properties. Mutations in the genes encoding ß-lactamases may extend their substrate specificity from aminopenicillins and older cephalosporins to newer molecules, as has been well documented in Gram-negative bacilli producing TEM and SHV. Additional studies are needed to investigate the distribution of cfiA, cepA, cblA, cfxA and cfxA2 in a larger series of Bacteroidaceae.4,8 Also, cfxA genes are carried on transposons and their combination with tetQ and ermF also deserves investigation.9 A better knowledge of the distribution of ß-lactamase genes within the Bacteroidaceae could help to standardize PCR detection of resistance genes in clinical isolates. Until now, ß-lactamase detection in these strict anaerobes has relied upon phenotypic methods.10
In conclusion, this sequence analysis suggests that most oral and non-oral ß-lactamase-producing Prevotella isolates from French patients produce the CfxA2 enzyme or a variant thereof.
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Acknowledgements |
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Footnotes |
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References |
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3 . Podglajen, I., Breuil, J., Casin, I. & Collatz, E. (1995). Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content. Journal of Bacteriology 177, 52705.[Abstract]
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