Sequence analysis of cfxA2-like ß-lactamases in Prevotella species

Chantal Giraud-Morin1, Isabelle Madinier1,2 and Thierry Fosse1,*

1 Laboratoire de Bactériologie, Hôpital l’Archet 2, Centre Hospitalier Universitaire, 151 route de Saint Antoine Ginestière, 06202 Nice Cedex 3; 2 Laboratoire Surfaces–Interfaces en Odontologie, Faculté de Chirurgie Dentaire, Nice, France

Received 13 August 2002; returned 9 October 2002; revised 19 February 2003; accepted 20 February 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sixty-two strains of oral (32) and non-oral (30) Prevotella producing ß-lactamases were screened for cfxA by PCR, using an intragenic primer pair. All 62 were cfxA/cfxA2 positive. Fourteen of these strains, representing seven pigmented and seven non-pigmented Prevotella species were submitted to further PCR with specific primers that amplified the whole ß-lactamase structural gene (966 bp). After cloning and sequencing, the deduced amino acid sequences were compared with that of Bacteroides vulgatus CfxA ß-lactamase. All 14 sequences possessed the E272K substitution characteristic of CfxA2. CfxA sensu stricto was not observed in the present series. G83D, F/V189L, W193L and D239Y substitutions were observed more than once, without species specificity. This sequence analysis indicates that most oral and non-oral ß-lactamase-producing Prevotella isolates from French patients produce variants of the CfxA enzyme.

Keywords: anaerobes, antibiotic resistance, ß-lactam, Prevotella


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prevotella are the main ß-lactamase-producing species in odontogenic infections.1 These Gram-negative strict anaerobes belong to Bacteroidaceae. In the Bacteroides fragilis group, >90% of B. fragilis strains produce endogenous chromosomal ß-lactamases. Three enzymes have been described in B. fragilis: (i) CepA, a cephalosporinase inactive against cefoxitin and imipenem (class A/group 2e);24 (ii) CfxA, a cephalosporinase initially described in Bacteroides vulgatus (class A/group 2e);5 and (iii) CfiA, an extended-spectrum metallo-ß-lactamase (class B/group 3).3 CblA is another class A/group 2e cephalosporinase described in a Bacteroides uniformis isolate.6 CfxA2 (class A/group 2e) has recently been cloned and characterized from a periodontal strain of Prevotella intermedia. cfxA2 shares >98% identity with cfxA, and the deduced amino acid sequence has a K272E substitution.7 In a previous study, PCR screening revealed a high prevalence of cfxA/cfxA2 versus cepA/cblA in oral Prevotella isolated from adult outpatients suffering from periodontitis, but did not identify the cfxA type.8 In the present study, we aimed to determine cfxA type by gene sequencing, both in oral and non-oral isolates of Prevotella species.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and susceptibility determination

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-l’Étoile, 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 Wilkins–Chalgren 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 Tris–HCl (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 (INV{alpha}F'), using the manufacturer’s 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thirty-two oral and 30 non-oral ß-lactamase-producing strains of Prevotella were screened for cfxA: Prevotella bivia (10), Prevotella buccae (10), Prevotella denticola (10), Prevotella disiens (five), Prevotella intermedia (20), Prevotella melaninogenica (five) and Prevotella oralis (two). All appeared to produce class A/group 2e ß-lactamases and were resistant to amoxicillin, ticarcillin, cefalothin, cefuroxime and cefazolin, intermediate to cefoxitin, cefpirome and cefotaxime, and susceptible to co-amoxiclav, ceftazidime, imipenem and aztreonam. All 62 were cfxA/cfxA2 positive. Fourteen strains, representing seven Prevotella species, were selected for cloning and sequencing of the entire ß-lactamase gene. Three sequences were identical to cfxA2 GenBank accession no. AF118110. The 11 remaining sequences were submitted to GenBank (accession no. AF504909 to AF504919). Nucleic sequence analysis revealed a 764–783 region with a higher rate of substitution. Silent nucleic acid substitutions were observed in 8/14 sequences. All the deduced amino acid sequences possessed the E272K substitution specific to CfxA2. G83D, F/V189L, W193L and D239Y substitutions were observed more than once, without species specificity.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, cfxA-type ß-lactamases were identified in all of the 62 strains of ß-lactamase-producing Prevotella investigated. Further identification by sequencing of amplification products from 14 Prevotella strains did not identify cfxA sensu stricto, initially described by Parker & Smith5 in Northern America; the E272K substitution characteristic of CfxA2 was always observed.7 This CfxA/CfxA2 type partition could be genus related (Bacteroides versus Prevotella), but we have previously identified cfxA2 in a strain of B. vulgatus (NI-2869).8 It could also be related to the geographic origin of the strains, with CfxA type predominant in Northern America and CfxA2 type in France.5,7 A CfxA2-like ß-lactamase has been sequenced from a French oral strain of Capnocytophaga ochracea (CfxA3: D239Y/E272K) (GenBank accession no. AF472622). No correlation was observed between the clinical origin of the strains and the amino acid sequence of the CfxA ß-lactamase they produced (Table Go). Indeed, it must be noted that the two strains P. buccae NI-104 (G83D/D133N/F189L/E272K) and P. oralis NI-114 (F155L/R159S/R170S/E272K) were periodontal strains isolated from the same patient.


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Table 1.  Amino acid sequence analysis of CfxA2-like ß-lactamases produced by Prevotella species compared with CfxA/B. vulgatus CLA 341:5 all 14 sequences have the E272K substitution specific to CfxA2
 
This work demonstrates that oral and non-oral strains of Prevotella share a common pool of cfxA2-like resistance genes towards ß-lactam antibiotics, probably selected by antibiotic treatments. These findings are consistent with the detection by Arzese et al.9 of tetQ (tetracycline) and ermF (erythromycin) resistance genes in Prevotella and Porphyromonas isolates from either sites of infection or various body sites in healthy subjects.

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.


    Acknowledgements
 
We are grateful to C. J. Smith, who kindly provided B. vulgatus CLA 341 and E. coli EC 351 strains and also to F. La Louze and S. Ourang for technical assistance. This work was supported by a grant from Ministère de la Santé, Projet Hospitalier de Recherche Clinique 2001 PHRC 01-01 UF 807.


    Footnotes
 
* Corresponding author. Tel: +33-4-92-03-62-14; Fax: +33-4-92-03-65-49; E-mail: fosse{at}unice.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Fosse, T., Madinier, I., Hitzig, C. & Charbit, Y. (1999). Prevalence of ß-lactamase-producing strains among 149 anaerobic gram-negative rods isolated from periodontal pockets. Oral Microbiology and Immunology 14, 352–7.[CrossRef][ISI][Medline]

2 . Rogers, M. B., Parker, A. C. & Smith, C. J. (1993). Cloning and characterization of the endogenous cephalosporinase gene, cepA, from Bacteroides fragilis reveals a new subgroup of Ambler class A ß-lactamases. Antimicrobial Agents and Chemotherapy 37, 2391–400.[Abstract]

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, 5270–5.[Abstract]

4 . Gutacker, M., Valsangiacomo, C. & Piffaretti, J.-C. (2000). Identification of two genetic groups in Bacteroides fragilis by multilocus enzyme electrophoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin (bft) encoding genes. Microbiology 146, 1241–54.[Abstract/Free Full Text]

5 . Parker, A. C. & Smith, C. J. (1993). Genetic and biochemical analysis of a novel Ambler class A ß-lactamase responsible for cefoxitin resistance in Bacteroides species. Antimicrobial Agents and Chemotherapy 37, 1028–36.[Abstract]

6 . Smith, C. J., Bennett, T. K. & Parker, A. C. (1994). Molecular and genetic analysis of the Bacteroides uniformis cephalosporinase gene, cblA, encoding the species-specific beta-lactamase. Antimicrobial Agents and Chemotherapy 38, 1711–5.[Abstract]

7 . Madinier, I., Fosse, T., Giudicelli, J. & Labia, R. (2001). Cloning and biochemical characterization of a class A ß-lactamase from Prevotella intermedia. Antimicrobial Agents and Chemotherapy 45, 2386–9.[Abstract/Free Full Text]

8 . Fosse, T., Madinier, I., Hannoun, L., Giraud-Morin, C., Hitzig, C., Charbit, Y. et al. (2002). High prevalence of cfxA ß-lactamase in aminopenicillin-resistant Prevotella strains isolated from periodontal pockets. Oral Microbiology and Immunology 17, 85–8.[CrossRef][ISI][Medline]

9 . Arzese, A. R., Tomasetig, L. & Botta, G. A. (2000). Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans. Journal of Antimicrobial Chemotherapy 45, 577–82.[Abstract/Free Full Text]

10 . Lewis, M. A., Pankhurst, C. L., Douglas, C. W., Martin, M. V., Absi, E. G., Bishop, P. B. et al. (2000). Assessment of the Etest method for detection of penicillin resistance in acute suppurative oral infection. Journal of Antimicrobial Chemotherapy 46, 323–42.[Free Full Text]