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Antimicrobial Agents and Chemotherapy, June 2005, p. 2515-2518, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2515-2518.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Isolation and Expression of a Novel Molecular Class D ß-Lactamase, OXA-61, from Campylobacter jejuni

David A. Alfredson^* and Victoria Korolik

Microbial Glycobiology, Institute for Glycomics, Griffith University, Gold Coast, Southport, Queensland 4215, Australia

Received 22 October 2004/ Returned for modification 6 January 2005/ Accepted 8 February 2005

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A novel ß-lactamase gene, bla_OXA-61, from Campylobacter jejuni GC015 was cloned and its nucleotide sequence determined. bla_OXA-61 encodes a protein of 257 amino acids in which the active-site STFK tetrad and conserved class D ß-lactamase motifs YGN and KTG were identified. A conserved sequence upstream of bla_OXA-61 is required for expression in Campylobacter.

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The majority of thermotolerant Campylobacter species, including Campylobacter jejuni, the leading cause of bacterial food-borne diarrheal disease throughout the world (6), show various levels of resistance to the antimicrobial agents penicillin G, ampicillin, amoxicillin, ticarcillin, and piperacillin (9). Although ampicillin is not considered useful for the treatment of Campylobacter infections (5), 83 to 92% of strains of C. jejuni produce ß-lactamase (9, 16) and ß-lactamase-positive strains have been reported to be significantly more resistant to amoxicillin, ampicillin, and ticarcillin than ß-lactamase-negative strains (9).

Ambler molecular class D enzymes (oxacillinases) are active-site serine ß-lactamases (11), are poorly inhibited by clavulanic acid (7), and generally have a preference for penam substrates including oxacillin (10). Oxacillinases may be chromosomally encoded (4); however, most oxacillinase genes form part of gene cassettes in class 1 integrons (14) and the frequent location of OXA genes on mobile genetic elements (plasmids or integrons) facilitates their spread (8). In Campylobacter, ampicillin resistance and the associated ß-lactamase production are chromosomally encoded (17). This study describes the cloning and expression of a novel molecular class D ß-lactamase structural gene, bla_OXA-61, from a human clinical strain of C. jejuni, GC015, isolated in 2001.

Bacterial strains and plasmids used in this study are listed in Table 1. Plasmids from both Escherichia coli and C. jejuni were prepared using the Nucleospin Plasmid kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. ß-Lactam antibiotics used in this study were ampicillin, piperacillin, carbenicillin, potassium clavulanate, cephalothin, cefotaxime, meropenem, and imipenem. MICs of ß-lactam antibiotics, with the exception of imipenem, were determined by agar dilution according to the National Committee for Clinical Laboratory Standards method (12). Imipenem MIC testing was performed using Etest strips. For C. jejuni, MIC testing was performed as previously described (1). Potassium clavulanate was used at a concentration of 2 µg/ml.

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TABLE 1. Bacterial strains and plasmids used in this study

To amplify the putative C. jejuni ß-lactamase gene, primers DBla-F and DBla-R (Table 2) were designed utilizing the sequence of the putative periplasmic class D ß-lactamase gene reported in the C. jejuni ATCC 11168 genome sequence (GenBank accession no. NC_002163) and targeted the nucleotide sequences of the serine ß-lactamase STFK (serine-X-X-lysine) active-site tetrad and the class D ß-lactamase domain LKIS-X-X-EQ, respectively. A 310-bp PCR product, amplified from a crude lysate prepared from C. jejuni strain GC015, was ligated into pGU0202 (2), followed by CaCl₂-mediated transformation into E. coli HB101 and sequencing using primers pDA2-F and pDA2-R (Table 2). Sequencing of products was performed using the ABI 377 sequencer (Applied Biosystems, Foster City, CA) after dye terminator cycle sequencing (ABI Prism BigDye terminator sequencing kit; Applied Biosystems). To determine the precise sequence of the putative ß-lactamase gene, including its promoter region, inverse PCR was performed using ClaI-digested, self-ligated C. jejuni GC015 genomic DNA as a template. Pfu turbo proofreading DNA polymerase (Stratagene, La Jolla, CA) and inverse PCR primers Inv.D-F and Inv.D-R (Table 2), designed from the known sequence of the amplified segment of DNA, were used to amplify a 2.2-kb PCR product. Sequences were analyzed in silico to construct the entire GC015 putative ß-lactamase DNA sequence.

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TABLE 2. Oligonuleotides used in this study

An open reading frame of 774 bp was identified which encoded a putative 257-amino-acid protein, designated OXA-61. The serine ß-lactamase active-site STFK tetrad (positions 58 to 61) and conserved motifs characteristic of molecular class D ß-lactamase structural elements, YGN (positions 133 to 135) and KTG (positions 196 to 198), were identified (Fig. 1). The overall GC content of bla_oxa-61 is 27.4%, which is similar to the overall GC content (30.6%) of the C. jejuni ATCC 11168 chromosome (15). Database searches carried out through the National Center for Biotechnology Information using the BLAST program (3) showed that the putative product of bla_OXA-61 had only one amino acid substitution, Glu Gly (position 202), compared to a putative class D ß-lactamase from C. jejuni ATCC 11168 (GenBank accession no. NC_002163). A 122-bp region identified directly upstream of bla_OXA-61 showed 99% identity with a region directly upstream of the C. jejuni ATCC 11168 putative class D ß-lactamase gene. OXA-61 showed the highest identity to the oxacillinase FUS-1 (45%) from Fusobacterium nucleatum (GenBank accession no. AAP69916) and also shares identity with OXA-10 (37%), OXA-23 (35%), and OXA-27 (34%) (GenBank accession no. AAB60534, AAS59523, and AAG35609, respectively).

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FIG. 1. Nucleotide sequence of the blaOXA-61(pGU0401) gene. The deduced amino acid sequence of OXA-61 is aligned with the respective codons. The ß-lactamase active-site STFK tetrad and the conserved motifs YGN and KTG are shown in underlined boldface type. A 16-amino-acid signal peptide was deduced (using the program SignalP V1.1. at the Center for Biological Sequence Analysis [http://www.cbs.dtu.dk/services/SignalP/] [13]) and is indicated in italics; the deduced cleavage site is indicated by a slash. The 122-bp conserved region directly upstream of the oxa-61 initiation codon is shaded. Putative C. jejuni promoter sequences (–10, –35) and the putative ribosome-binding site (RBS) (18) are underlined. The bracketed broken line indicates the nucleotide sequence deleted in recombinant plasmid pGU0403.

Three plasmid constructs were generated utilizing the previously described shuttle vector pGU0202 (2) (Table 1) to determine whether bla_OXA-61 was expressed using its own promoter sequences and whether the entire 122-bp sequence upstream of the ß-lactamase gene was required for expression. Thus generated recombinant plasmids pGU0401, pGU0402, and pGU0403 were used to transform ß-lactamase-negative C. jejuni strain 428, which does not harbor a homologue of bla_OXA-61 (data not shown), and E. coli HB101. ß-Lactam MICs for E. coli HB101 and C. jejuni 428 were then compared to those for E. coli and C. jejuni 428 carrying bla_OXA-61 (Table 3).

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TABLE 3. MICs of ß-lactam antibiotics for E. coli HB101, C. jejuni GC015, C. jejuni 428, and OXA-61-producing transformants

Recombinant plasmids pGU0401 and pGU0402 conferred a 32-fold rise in the MICs of ampicillin, piperacillin, and carbenicillin in C. jejuni 428 cells, indicating a functional ß-lactamase being expressed from its own promoter regions; resistance to cefotaxime and imipenem was not conferred. Consistent with the class D ß-lactamases, clavulanic acid, at a concentration of 2 µg/ml, did not significantly inhibit hydrolysis of the penicillins by bla_OXA-61. The meropenem MIC increased fivefold; however, pGU0202 is a multicopy plasmid (>50 copies/cell) and weak carbapenemase activity has been reported previously from recombinant strains expressing cloned oxacillinase genes (14). Truncation of the sequence upstream of the bla_OXA-61 putative –35 promoter region in recombinant plasmid pGU0403 resulted in reduction of ß-lactam MICs to the same level as the ß-lactam-susceptible, wild-type C. jejuni strain 428. RNA primer extension analysis is required to determine the transcription start point for bla_OXA-61 in C. jejuni; the exact function of the 122-bp sequence upstream of bla_OXA-61 remains to be determined.

In E. coli, the bla_OXA-61 promoter region is only poorly recognized, resulting in weak transcription of the ß-lactamase gene. A four- to eightfold rise in the carbenicillin MIC and a two- to fourfold rise in the ampicillin MIC were still conferred by bla_OXA-61, indicating a low level of transcription of bla_OXA-61 from its promoter region in E. coli.

This study indicates that OXA-61 may contribute to resistance to ß-lactams in C. jejuni. Future biochemical analyses will determine its functional classification according to the scheme of Bush et al. (7).

Nucleotide sequence accession number. The nucleotide sequence of bla_OXA-61 has been deposited in GenBank under accession number AY587956.

 
The scientific community considers that George Jacoby and Karen Bush have made a considerable work with their website "Amino Acid Sequences for TEM, SHV, and OXA Extended-Spectrum and Inhibitor Resistant ß-Lactamases," and we thank George Jacoby for assistance with assigning the designation of OXA-61 to the novel C. jejuni ß-lactamase.

 
* Corresponding author. Mailing address: Microbiology Department, Queensland Health Pathology Services, Gold Coast Hospital, 108 Nerang St., Southport, Queensland, Australia 4215. Phone: 61 7 5519 8497. Fax: 61 7 5519 8496. E-mail: David_Alfredson{at}health.qld.gov.au.

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Antimicrobial Agents and Chemotherapy, June 2005, p. 2515-2518, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2515-2518.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alfredson, D. A. Articles by Korolik, V. Search for Related Content PubMed PubMed Citation Articles by Alfredson, D. A. Articles by Korolik, V.