Molecular characterization of β-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A

Molecular characterization of b-lactamase genes blaA and blaB of Yersinia enterocolitica biovar1A Sachin Sharma, Shilpi Mittal, Sarita Mallik & Jugsharan S. Virdi Microbial Pathogenicity Laboratory, Department of Microbiology, University of Delhi South Campus, New Delhi, India

Correspondence: Jugsharan S. Virdi, Microbial Pathogenicity Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India. Tel.: 191 11 24110950; fax: 191 11 24115270; e-mail: [email protected]

Received 30 November 2005; revised 4 January 2006; accepted 6 February 2006. First published online March 2006. doi:10.1111/j.1574-6968.2006.00191.x

Abstract The b-lactamase genes blaA and blaB were detected by PCR amplification in strains of Yersinia enterocolitica biovar 1A isolated from India, Germany, France and the USA. Both genes were detected in all strains. Polymerase chain reactionrestriction fragment length polymorphism revealed genetic heterogeneity in blaA but not in blaB. Cluster analysis of blaA restriction profiles grouped the strains into three groups. The blaA gene of Y. enterocolitica biovar 1A showed a high degree of sequence homology to that of Y. enterocolitica 8081 (biovar 1B) and Y. enterocolitica Y-56 (biovar 4), whereas homology was low with class A b-lactamase genes of other members of the family Enterobacteriaceae. The pI 8.7 of enzyme Bla-A of Y. enterocolitica biovar 1A was similar to that of biovars 2, 3 and 4. The enzyme Bla-B focused at 6.8 and 7.1, indicating that biovar 1A strains produced a ‘B-like’ enzyme. This is the first study to have investigated the genetic heterogeneity of the b-lactamase genes of Y. enterocolitica.

Editor: Marco Soria Keywords biovar 1A; b-lactamase genes; Yersinia enterocolitica; RFLP; isoelectric focusing.

Introduction Yersinia enterocolitica, an important food- and water-borne enteropathogen, is known to cause a variety of gastrointestinal problems including acute diarrhea, terminal ileitis and mesenteric lymphadenitis. Long-term sequelae following infection include reactive arthritis and erythema nodosum (Bottone, 1999). Blood transfusion-associated septicemia because of Y. enterocolitica has been reported to have a high mortality rate (Leclercq et al., 2005). Yersinia enterocolitica is highly heterogeneous and is represented by six biovars (1A, 1B, 2, 3, 4 and 5) and more than 50 serovars. These biovars differ in their geographical distribution, ecological niches and pathogenic properties (Bottone, 1999). Strains belonging to different biovar/serovar combinations may exhibit different susceptibilities to b-lactams (Pham & Bell, 1993a; Pham et al., 2000; Stock et al., 2000), which may be due to the expression of two chromosomal blactamases. Bla-A is a class A constitutive broad spectrum penicillinase whereas Bla-B is a class C inducible cephalosporinase (AmpC) (Pham et al., 1991a, b). The distribution and expression of the two lactamases in different biovars of Y. enterocolitica has been well studied (Pham et al., 1999; Stock et al., 1999, 2000; Sharma et al., 2004). FEMS Microbiol Lett 257 (2006) 319–327

However, there have been few studies of bla genes of Y. enterocolitica strains, except those isolated in Europe. Biovar 2, 3, 4 and 5 strains of European origin were shown to possess both blaA and blaB genes (Stock et al., 1999, 2000). Biovar 1A strains unequivocally showed the presence of the blaB gene by PCR, but amplification of blaA gave multiple faint bands. This was speculated to be due to the genetic variability of the blaA gene (Stock et al., 2000). These observations clearly warrant further studies of the bla genes of Y. enterocolitica isolated in different parts of the world. Moreover, knowledge about the genetic heterogeneity of bla genes may be pivotal to understanding biovar-specific expression of b-lactamases in Y. enterocolitica. The present work reports molecular detection and heterogeneity in blaA and blaB genes of Y. enterocolitica biovar 1A isolated from India (Singh & Virdi, 1999; Sinha et al., 2000; Singh et al., 2003), France, Germany and the USA.

Materials and methods Bacterial strains Eighty one strains of Yersinia enterocolitica biovar 1A were examined. Among these, 65 strains were isolated from 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


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Table 1. Details of primers used in this study Primer

Sequence (5 0 –3 0 )

Gene

Expected amplicon size (bp)

Reference

blaA5 blaA3 A9-f A10-r blaB5 blaB3

AAATGCGCTACCGGCTTCAG AGTGGTGGTATCACGTGGGT GAGATTCAGGAATGAAGCACTCTTCG TCAGGATATTTGCGACAAAATTAT CCCACTTTATACCTTGGCACAAA GAACATATCTCCTGCCTGGGAAAT

blaA

439

Stock et al. (1999)

blaA

896

This study

blaB

827

Stock et al. (1999)

clinical and nonclinical sources viz. diarrheic human subjects (35 strains), wastewater (18 strains), pig throat (seven strains) and pork (five strains) in India. All these isolates have been authenticated by, and deposited with the Yersinia National Reference Laboratory and WHO Collaborating Center, Pasteur Institute, Paris (France). Of the remaining 16 isolates, 10 were kindly provided by Elisabeth Carniel (Yersinia National Reference Laboratory and WHO Collaborating Center, Pasteur Institute, Paris, France) and six were procured from J. Heesemann (Max von Pattenkofer Institute, Munich, Germany). The serovars, source of isolation, country of origin and reference laboratory accession numbers of these strains have been reported previously (Sachdeva & Virdi, 2004). All strains were maintained on trypticase soy agar (HiMedia, Mumbai, India) at 4 1C.

DNA extraction Genomic DNA was extracted from bacterial cultures grown overnight at 28 1C in trypticase soy broth. One milliliter of culture was pelleted by centrifugation (Sigma Loborzentrifugen GmbH, Osterode, Germany) at 7012 g for 10 min. Total genomic DNA was prepared from each strain using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) with modifications for gram-negative bacteria according to the manufacturer’s recommendations.

PCR amplification and sequencing of bla genes PCR amplification of blaA and blaB genes was performed with the primers (Microsynth GmbH, Balgach, Germany) listed in Table 1, using a PTC-100TM (MJ Research, Waltham, MA) thermal cycler. The PCR reaction mixture comprised 1  PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 1.5 mM KCl and 0.1% Triton X-100), 200 mM each of the four dNTPs (MBI Fermentas GmbH, St Leon-Rot, Germany), 10 pmol each of forward and reverse primers (Microsynth GmbH), 2 U of Taq DNA polymerase (DyNAzymeTM, Finnzymes, OY Espoo, Finland) and 50–100 ng of genomic DNA in a total volume of 25 mL. For blaA, PCR was performed at 95 1C for 5 min followed by 25 consecutive cycles of 30 s at 95 1C, 30 s at 56 1C, and 90 s at 72 1C and a final extension for 10 min at 72 1C. PCR for blaB was performed as described by Stock et al. (1999) using the 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


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primers indicated in Table 1. The PCR products were analyzed by electrophoresis in a 1% (weight in volume) agarose gel (GelroseTM LE, Life Technologies, New Delhi, India). The gels were stained with ethidium bromide (0.5 mg mL1) and visualized under UV transillumination. The PCR amplified products of bla genes of Y. enterocolitica biovar 1A were purified using the QIA Quick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction kit in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Frankfurt, Germany). The nucleotide sequences were analyzed by BLASTN, available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST). The nucleotide and the deduced amino-acid sequences of the blaA gene of Y. enterocolitica biovar 1A were aligned with class A b-lactamases of Y. enterocolitica 8081 (biovar 1B), Y. enterocolitica Y-56 (biovar 4) and other members of the family Enterobacteriaceae using ClustalW (http://www.ebi.ac.uk/clustalW).

Restriction fragment length polymorphism (RFLP) of bla genes The PCR amplified products of blaA and blaB genes were restricted with NciI and DraI, and with HaeIII and RsaI, respectively. Twenty-five microliters of PCR product was digested with 1 U of each enzyme separately by incubating at 37 1C for 12 h in the appropriate buffer as recommended by the supplier (New England Biolabs, Schwalbach, Germany). The digested DNA samples were resolved in a 2% agarose gel in 1  Tris-acetate-EDTA (TAE) buffer at 60 V for 6 h. The gels were stained with ethidium bromide and photographed using the Gel Doc 2000 (Bio-Rad, Hercules, CA). Genetic similarity was determined by cluster analysis of restriction profiles of blaA using NTSYSpc 2.02i software package and the dendrogram was constructed by the unweighted pair group method using arithmetic averages (UPGMA).

Molecular weight determinations of enzymes Bla-A and Bla-B Preparation of cell lysates containing Bla-A (uninduced) and Bla-B (induced with imipenem) was carried out by sonication of the washed cell pellet in cold buffer and FEMS Microbiol Lett 257 (2006) 319–327

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Molecular characterization of b-lactamase genes blaA and blaB

(a) M

1

2

3

4

5

6

(b) M

7

8

9

10

11

12

1000 bp Fig. 1. (a) Restriction profile of blaA with endonucleases NciI (Lanes 1–3) and DraI (Lanes 4–6). (b) Restriction profile of blaB with endonucleases RsaI (Lanes 7–9) and HaeIII (Lanes 10–12). PCR amplification and restriction analysis were carried out for 81 strains of Yersinia enterocolitica biovar 1A. M, 100 bp DNA ladder.

600 bp

200 bp 100 bp

sedimentation as described previously (Sharma et al., 2004). Cell lysates containing the enzymes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (1970). After PAGE, the gel was washed twice in renaturation buffer (100 mM Tris-HCl, pH 7.0 and 0.1% Triton-X-100) under mild shaking for 45 min each. b-lactamase activity was detected by overlaying the polyacrylamide gel with Whatman filter paper soaked in 0.5 mg mL1 nitrocefin (Oxoid, Basingstoke, England) for 2 min.

Isoelectric focusing (IEF) of Bla-A and Bla-B Isoelectric focusing of the cell extracts containing the crude enzymes was performed in a 6% polyacrylamide gel containing 2% ampholyte of pH 3–10 (Biolyte Ampholyte, BioRad). The extract containing 3 mg of protein was applied to the gel, and focused at 4 1C using a Mini IEF cell (Bio-Rad) according to the protocol specified by the manufacturer. Broad range IEF standard with pI ranging from 4.45 to 9.6 (Bio-Rad) was used as the pI marker. After focusing, the blactamase bands were visualized by overlaying the gel with nitrocefin as described above. Detection of Bla-A and Bla-B on the IEF gel was further confirmed by the selective inhibition of these enzymes by clavulanic acid (CLA) and aztreonam (ATM), respectively. For this, the gel was overlaid with filter paper soaked in 40 mM CLA or 20 mM ATM for 2 min before the application of nitrocefin.

Nucleotide sequence accession numbers The complete coding sequence of blaA and partial nucleotide sequence of blaB have been submitted to GenBankEMBL database under accession numbers AY954728 and DQ150252, respectively.

Results and discussion Primer design and PCR amplification of blaA When published primers blaA5 and blaA3 (Table 1) were used to amplify the blaA gene of Yersinia enterocolitica biovar 1A strains, amplicons of 1100 and 1200 bp were obtained, instead of the 479 bp as reported for biovar 2, 4 FEMS Microbiol Lett 257 (2006) 319–327

and 5 strains (Stock et al., 1999). These amplicons were obtained for all 81 strains of Y. enterocolitica biovar 1A studied. A similar observation was made by Stock et al. (2000), when they used these primers for amplification of blaA gene of biovar 1A strains isolated in Germany. They reported the appearance of multiple faint bands in the region of 1500 bp (Stock et al., 2000). Consequently, in order to amplify the blaA gene of Y. enterocolitica biovar 1A strains, consensus primers A9-f and A10-r (Table 1) were designed from the conserved regions of the blaA genes of Y. enterocolitica 8081, biovar 1B (http:// www.sanger.ac.uk/Projects/Y_enterocolitica), Y. enterocolitica Y-56, biovar 4 (Seoane & Garcia-Lobo, 1991a), and Klebsiella oxytoca (Arakawa et al., 1989). These primers amplified a 896 bp fragment unequivocally from all the 81 strains of Y. enterocolitica biovar 1A. Sequencing of one such amplicon confirmed it to be the complete coding sequence of the blaA gene of Y. enterocolitica.

RFLP, cluster analysis and sequence comparison of blaA Heterogeneity in blaA gene of Y. enterocolitica biovar 1A was studied by RFLP of the amplified 896 bp CDS, using NciI and DraI. Restriction digestion of blaA with NciI gave three different restriction profiles having 800 and 100 bp, 650 and 250 bp, and 550, 220 and 130 bp fragments, indicating limited heterogeneity (Fig. 1a). Digestion with DraI did not show any polymorphism in blaA gene and resulted in the consistent appearance of two fragments of sizes 550 and 350 bp. The profile obtained with NciI suggested that polymorphism observed in the blaA gene was due to the presence of four recognition sites for NciI. The heterogeneity in the blaA gene of Y. enterocolitica biovar 1A strains was also reflected in an earlier study, in which differential expression of enzyme Bla-A was reported by the double disc diffusion synergy test (Sharma et al., 2004). The genetic background of the antibiotic resistance genes has been shown to influence the minimal inhibitory concentrations of streptomycin in Escherichia coli (Sunde & Norstr¨om, 2005) and of penicillin in Streptococcus pneumoniae (Beall et al., 1998). A similarity analysis of restriction profiles of the blaA gene obtained with NciI, clustered the isolates into three 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


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Strain Serovar ID

Source

Country of origin

(a)

(b)

(c)

0.23

0.42

0.61 Coefficient

0.81

1.00

Fig. 2. Phylogenetic analysis of blaA gene of Yersinia enterocolitica biovar 1A based on restriction profiling. ND, not determined; NK, not known; NAG, nonagglutinable. (For more details of the strains refer to Sachdeva & Virdi, 2004.)

major groups – A, B and C (Fig. 2). Group A comprised primarily the clinical isolates, whereas there was preponderance of nonclinical isolates in the group B. The groups were related to each other at 49.5% similarity. Group C was represented by both clinical and nonclinical isolates in 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


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almost equal numbers. This group was related to the other two at approximately 23% similarity. All except one serogroup O:6,30-6,31 isolates of clinical origin clustered into group A, whereas those of non-clinical origin clustered into group B. The clinical serogroup O:6,30-6,31 isolates of FEMS Microbiol Lett 257 (2006) 319–327

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Fig. 3. Nucleotide sequence alignment of blaA genes. Dashes indicate gaps inserted in the alignment, and asterisks indicate identical nucleotides in the five genes. GenBank accession numbers of the genes are as follows: AY954728 (Yersinia enterocolitica biovar 1A, present study), 8081_biovar 1B (Y. enterocolitica 8081 biovar1B), X57074 (Y. enterocolitica Y-56 biovar 4), M27459 (Klebsiella oxytoca), X62610 (Citrobacter koseri).

Indian, European and American origin showed identical profiles. Previously, the BRO b-lactamases BRO-1 and BRO2 of Moraxella catarrhalis, which differ from each other in just five bases, have also been differentiated by restriction fragment length polymorphism (Plessis, 2001; Koseoglu et al., 2004). The blaCTX-M genes of K. pneumoniae were also differentiated using PCR-RFLP (Edelstein et al., 2003). The grouping of the strains of Y. enterocolitica biovar 1A based on restriction profiling of blaA is in agreement with an earlier study from our laboratory in which clustering of the same collection of Y. enterocolitica biovar 1A strains was performed using repetitive extragenic palindrome (REP)and enterobacterial repetitive intergenic consensus (ERIC)PCR based fingerprinting (Sachdeva & Virdi, 2004). A correlation between REP/ERIC types (Sachdeva & Virdi, 2004) and restriction profile of blaA was also discerned. For example, REP type 1 (R1) was predominantly associated with blaA group A, whereas ERIC type 4 (E4) was exclusively associated with blaA group C. Also, as with rep (REP/ERIC)PCR fingerprinting (Sachdeva & Virdi, 2004), the clinical and wastewater serogroup O:6,30-6,31 strains clustered into FEMS Microbiol Lett 257 (2006) 319–327

two separate groups. Another study reported that restriction profiles of b-lactamase genes (blaOXY-1, blaOXY-2) of K. oxytoca correlated very well with ERIC-PCR profiles (Granier et al., 2003). These observations suggest that restriction analysis of b-lactamase genes may be used to study epidemiology, or discern phylogenetic relationships. It would be of interest to explore further if restriction profiling of bla genes, such as blaA, may provide epidemiological/phylogenetic information about Y. enterocolitica and ‘Y. enterocolitica-like’ species (Virdi & Sachdeva, 2005) as obtained from other loci such as rrn (Kotetishvili et al., 2005) or gyrB (Demarta et al., 2004). Nucleotide sequence comparison of the blaA gene of Y. enterocolitica biovar 1A with that of Y. enterocolitica Y-56 (biovar 4) and Y. enterocolitica 8081 (biovar 1B) revealed a high degree of identity (93%). However, the homology of the blaA gene with that of chromosomal class A b-lactamase genes of other members of the family Enterobacteriaceae viz. K. oxytoca (Arakawa et al., 1989) and Citrobacter koseri (Perilli et al., 1991) was c. 50%, which was relatively low (Fig. 3). The low sequence similarities of blaA of 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


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AAX55643 8081_biovar1B

CAA40357 AAA25084 CAA44485

MKHSSLRRALLLAGITLPLVNFSLPTWAAAI--PGSLDKQLAALEHSANGRLGIAMINTG MKHSSLRRSLLLAGITLPLVNFALPTWAAAI--PGSLDKQLAALEHSANGRLGIAMINSG MKHSSLRRSLLLAGITLPLVSFALPAWANAL--PASVDKQLAELERNANGRLGVAMINTG MLKSSWRKTALMAAAAVPLLLASGSLWASAD----AIQQKLADLEKRSGGRLGVALINTA MFKKRGRQTVLIAAVLA-FFTASSPLLARTQGEPTQVQQKLAALEKQSGGRLGVALINTA * :. *:: *:*. .:. : . * : . ::::** **: :.****:*:**:. 70

AAX55643 8081_biovar1B

CAA40357 AAA25084 CAA44485

NGTKILYRGARRFPFCSTFKFMLAAAVLGQSQSQPNLLNKHINYHESDLLSYAPITRKNL AGTKILYRGAQRFPFCSTFKFMLAAAVLDQSQSQPNLLNKHINYHESDLLSYAPITRKNL NGTKILYRAAQRFPFCSTFKFMLAAAVLDQSQSQPNLLNKHINYHESDLLSYAPITRKNL DDSQTLYRGDERFAMCSTGKVMAAAAVLKQSESNPEVVNKRLEIKKSDLVVWSPITEKHL DRSQILYRGDERFAMCSTSKTMVAAAVLKQSETQHDILQQKMVIKKADLTNWNPVTEKYV :: ***. .**.:*** * * ***** **::: ::::::: :::** : *:*.* : 130

AAX55643 8081_biovar1B

CAA40357 AAA25084 CAA44485

AAX55643 8081_biovar1B

CAA40357 AAA25084 CAA44485

166

AHGMTVSELCAATIQYSDNTAANLLLKELGGLAAVNQFARSIGDQMFRLDRWEPDLN TAL ACGMTVSELCAATIQYSDNTAANLLIKELGGLAAVNQFARSIGDQMFRLDRWEPDLN TAL AHGMTVSELCAATIQYSDNTAANLLIKELGGLAAVNQFARSIGDQMFRLDRWEPDLN TAR QSGMTLAELSAAALQYSDNTAMNKMISYLGGPEKVTAFAQSIGDVTFRLDRTEPALN SAI DKEMTLAELSAATLQYSDNTAMNKLLEHLGGTSNVTAFARSIGDTTFRLDRKEPELN TAI **::**.**::******* * ::. *** *. **:**** ***** ** **:*

PNDPRDTTTPAAMAASINKLVLGDALHPAQRSQLTAWLKGNTTGDATIRAGAPTDWIVGD PNDPRDTTTPAAMAASMNKLVLGDALRPAQRSQLAAWLKGNTTGDATIRAGAPTDWIVGD PNDPRDTTTPAAMAASMNKLVLGDALRPAQRSQLAVWLKGNTTGDATIRAGAPTDWIVGD PGDKRDTTTPLAMAESLRKLTLGNALGEQQRAQLVTWLKGNTTGGQSIRAGLPASWAVGD PGDERDTTCPLAMAKSLHKLTLGDALAGAQRAQLVEWLKGNTTGGQSIRAGLPEGWVVGD *.* **** * *** *:.**.**:** **:**. ********. :**** * .* *** 234

AAX55643 8081_biovar1B

CAA40357 AAA25084 CAA44485

KTGSGDYGTTNDIAVLWPTKGAPIVLVVYFTQREKDAKPRRDVLASATKIILS--KTGSGDYGTTNDIAVLWPTKGAPIVLVVYFTQREKDAKPRRDVLASATQIILSQIS KTGSGDYGTTNDIAVLWPTKGAPIVLVVYFTQREKDAKPRRDVLASVTKIILS--KTGAGDYGTTNDIAVIWPENHAPLVLVTYFTQPQQDAKSRKEVLAAAAKIVTEGLKTGAGDYGTTNDIAVIWPEDRAPLILVTYFTQPQQDAKGRKDILAAAAKIVTEGL***:***********:** . **::**.**** ::*** *:::**:.::*: .

Fig. 4. Amino-acid sequence alignment of b-lactamase (Bla-A) from Yersinia enterocolitica biovar 1A (strain ID 1) with its nearest b-lactamase class A neighbors. Asterisks indicate identical amino acids. The conserved motifs (70SXXK73, 130SDN132 and 234KTG236) typical of class A b-lactamases are in boldface. The motif 166EXXLN170 responsible for O loop formation is in boldface and italics. Arrows indicate the putative O loop region. GenBank accession numbers of the b-lactamases are as follows: AAX55643 (present study), CAA40357 (Y-56), AAA25084 (Klebsiella oxytoca) CAA44485 (Citrobacter koseri).

Y. enterocolitica biovar 1A with class A b-lactamase genes of K. oxytoca and C. koseri suggested separate lineages of these genes. The percent amino-acid sequence identity was high with Y. enterocolitica Y-56, biovar 4 and Y. enterocolitica 8081, biovar 1B (93–94%) compared with those of K. oxytoca (55.7%) and C. koseri (53.4%). The amino-acid sequence alignment of chromosomal class A b-lactamases is shown in Fig. 4. Multiple sequence alignment showed the presence of four highly conserved motifs viz., a serinethreonine-phenylalanine-lysine (STFK) at position 70–74, serine-aspartic acid-asparagine (SDN) at position 130–132, glutamate-X-X-leucine-asparagine (EXXLN) at position 166–170 and KTG at position 234–236. STFK, SDN and 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


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KTG are characteristic of class A b-lactamases possessing a serine active site and are known to be involved in catalytic mechanism and substrate binding. Another motif, EXXLN, is characteristic of O loop formation (Cheung et al., 2002). The presence of an O loop in Bla-A of Y. enterocolitica biovar 1A suggests that the enzyme has a globular shape (Fetrow, 1995).

PCR amplification, RFLP and sequence comparison of blaB The primers blaB5 and blaB3, described by Stock et al. (1999), consistently amplified the 871 bp fragment of the blaB for all the isolates of Y. enterocolitica biovar 1A. RFLP FEMS Microbiol Lett 257 (2006) 319–327

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Table 2. Isoelectric points (pI) of b-lactamases (Bla-A and Bla-B) of Yersinia enterocolitica biovar 1A pI Strains

n

Bla-A

Bla-B

Clinical Indian European American Nonclinical

35 15 1 30

8.7 8.7 8.7 8.7

Both 6.8 and 7.1 Either 6.8 or 7.1; rarely both Both 6.8 and 7.1 Both 6.8 and 7.1

Isolated from India; includes wastewater (18 strains), pig throat (seven strains) and pork (five strains).

n, number of the strains studied.

with two endonucleases viz. RsaI and HaeIII resulted in the consistent appearance of two fragments of 550 and 324 bp for RsaI, and 650 and 110 bp for HaeIII in all the strains, suggesting that there was no polymorphism in this gene (Fig. 1b). The lack of heterogeneity in this gene was also suggested by the fact that the blaB gene of Y. enterocolitica biovar 1A strains could be amplified unequivocally with primers designed for amplification of blaB of biovars 2, 3 and 5 (Stock et al., 1999). A previous investigation of Y. enterocolitica biovar 1A strains also suggested a lack of heterogeneity in Bla-B as identical expression of the enzyme was seen in all the strains by the double disc-diffusion test (Sharma et al., 2004). Sequence analysis of the blaB gene of Y. enterocolitica biovar 1A strain showed 96% nucleotide sequence identity with that of Y. enterocolitica 8081, biovar 1B and Y. enterocolitica IP97, biovar 2 (Seoane et al., 1992), and 88% with Y. bercovieri ATCC 43970 ampC gene (Schiefer et al., 2005), whereas sequence homology with other members of the genus Yersinia (Y. aldovae and Y. ruckeri) was relatively low (56–61%) (Schiefer et al., 2005).

Molecular weight determination and IEF analysis of Bla-A and Bla-B Two distinct bands having molecular weights of c. 35 and 29 kDa were obtained on SDS-PAGE representing the enzyme activities of Bla-A and Bla-B, respectively. A molecular weight of 30 kDa has, however, been reported for class A b-lactamases of Klebsiella oxytoca (Arakawa et al., 1989), Burkholderia pseudomallei (Cheung et al., 2002), Y. frederiksenii (Schiefer et al., 2005) and Citrobacter sedlakii (Petrella et al., 2001). Bla-B, the inducible cephalosporinase (AmpC), was consistently found to be of 29 kDa. This was further confirmed as induction by imipenem lead to an increase in intensity of the band. This is in agreement with previous reports that showed AmpC b-lactamases have molecular weights in the range 29–39 kDa (Seoane & Garcia-Lobo, 1991b; Weng et al., 2004; Schiefer et al., 2005). FEMS Microbiol Lett 257 (2006) 319–327

The two b-lactamases, Bla-A and Bla-B produced by Y. enterocolitica have been distinguished by their distinct isoelectric points (Pham et al., 1991a, 1995a, 1999, 2000; Pham & Bell, 1993b). Table 2 shows IEF data of 81 strains of Y. enterocolitica biovar 1A, for uninduced (Bla-A) and induced (Bla-B) enzymes separately. Bla-A was visualized as a single faint band in the alkaline region of the gel in all isolates and exhibited a pI of 8.7. The intensity of the Bla-A band was similar in both uninduced and induced enzyme preparations. The pI 8.7 for the enzyme Bla-A produced by Y. enterocolitica biovar 1A strains was similar to that produced by strains of biovars 2, 3 and 4 (Pham & Bell, 1993b; Pham et al., 1995b, 2000), or biovar 1A strains isolated in other parts of the world (Pham & Bell, 1993b; Pham et al., 2000). Bla-B, on the other hand, was observed as multiple bands on the IEF gel. Major bands were seen at pIs 6.8 and 7.1 for all the isolates. This was in contrast to the reported pIs 5.3 and 5.7 of enzyme B, of other biovars (Pham et al., 1995a). These results suggested that Y. enterocolitica biovar 1A produces a ‘B-like’ enzyme instead of Bla-B. That the two bands represented the enzyme ‘B-like’ was confirmed by thickening and increase in the intensity of these bands following induction with imipenem (Pham et al., 1991a). Interestingly, although the majority of the Indian strains and the sole American strain studied showed both bands, the majority of the European strains showed either of the two and rarely both. This may be attributed to differential expression of enzymes in strains isolated in different parts of the world as suggested by Pham et al. (2000). Minor bands with pI 6.0 or 7.8 were also seen in a small percentage of the strains. This however indicated the range of pI of ‘Blike’ enzyme that may be elaborated by biovar 1A strains. In the majority of the isolates, Bla-B was seen to express even without induction. The isolates which did not show such basal level of Bla-B activity, nevertheless expressed it after induction with imipenem. Of the 35 clinical strains isolated in India, two however failed to express the enzyme B even after induction. The clinical and the nonclinical biovar 1A strains did not show any difference in respect of the isoelectric points of their enzymes. 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved


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In conclusion, both blaA and blaB genes were detected in biovar 1A strains of Y. enterocolitica by PCR amplification. PCR-RFLP revealed heterogeneity in blaA, which was represented by three restriction profiles. The blaB failed to reveal any such genetic heterogeneity. The cluster analysis of the restriction profiles of blaA provided information broadly similar to that inferred previously from rep (REP/ERIC)PCR fingerprinting of the isolates. To the best of our knowledge, this is the first study in which the heterogeneity of the chromosomal b-lactamase genes of Y. enterocolitica has been investigated.

Acknowledgements This work was supported by a grant to J. S. V. from Defense Research and Development Organization, Govt. of India, Senior Research Fellowship to S. S. from Indian Council of Medical Research and postdoctoral fellowship to S. M. from Department of Biotechnology. We thank Dr P. K. Burma for help in primer design. Sachin Sharma and Shilpi Mittal contributed equally to this work.

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