Molecular characterization of Î²-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A

Molecular characterizationof b-lactamasegenesblaA andblaBofYersinia enterocolitica biovar1ASachin 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

Editor: Marco Soria

Keywords

biovar 1A; b-lactamase genes; Yersinia

enterocolitica; RFLP; isoelectric focusing.

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 reaction-

restriction 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. enterocoli-

tica 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.

Introduction

Yersinia enterocolitica, an important food- and water-borne

enteropathogen, is known to cause a variety of gastrointest-

inal 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 repre-

sented by six biovars (1A, 1B, 2, 3, 4 and 5) and more than 50

serovars. These biovars differ in their geographical distribu-

tion, ecological niches and pathogenic properties (Bottone,

1999). Strains belonging to different biovar/serovar combi-

nations 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 b-

lactamases. Bla-A is a class A constitutive broad spectrum

penicillinase whereas Bla-B is a class C inducible cephalos-

porinase (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).

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

FEMS Microbiol Lett 257 (2006) 319–327 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

clinical and nonclinical sources viz. diarrheic human sub-

jects (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 Colla-

borating Center, Pasteur Institute, Paris, France) and six

were procured from J. Heesemann (Max von Pattenkofer

Institute, Munich, Germany). The serovars, source of isola-

tion, 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 Loborzentri-

fugen 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 mod-

ifications for gram-negative bacteria according to the man-

ufacturer’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, Wal-

tham, 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 (DyNA-

zymeTM, 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

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 mL�1) and visualized under UV transillumination.

The PCR amplified products of bla genes of Y. enteroco-

litica 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-lacta-

mases of Y. enterocolitica 8081 (biovar 1B), Y. enterocolitica

Y-56 (biovar 4) and other members of the family Enterobac-

teriaceae 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 enzymesBla-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

Table 1. Details of primers used in this study

Primer Sequence (50–30) Gene Expected amplicon size (bp) Reference

blaA5 AAATGCGCTACCGGCTTCAG blaA 439 Stock et al. (1999)

blaA3 AGTGGTGGTATCACGTGGGT

A9-f GAGATTCAGGAATGAAGCACTCTTCG blaA 896 This study

A10-r TCAGGATATTTGCGACAAAATTAT

blaB5 CCCACTTTATACCTTGGCACAAA blaB 827 Stock et al. (1999)

blaB3 GAACATATCTCCTGCCTGGGAAAT

FEMS Microbiol Lett 257 (2006) 319–327c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

320 S. Sharma et al.

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 mL�1 nitrocefin (Oxoid, Basing-

stoke, 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 contain-

ing 2% ampholyte of pH 3–10 (Biolyte Ampholyte, Bio-

Rad). 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 b-

lactamase 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 40mM 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 nucleo-

tide sequence of blaB have been submitted to GenBank-

EMBL 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

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. enterocoliti-

ca 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 comparisonof 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 poly-

morphism 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 influ-

ence the minimal inhibitory concentrations of streptomycin

in Escherichia coli (Sunde & Norstrom, 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

M 1 2 3 4 5 6 M 7 8 9 10 11 121000 bp

600 bp

200 bp

100 bp

(a) (b)

Fig. 1. (a) Restriction profile of blaA with en-

donucleases NciI (Lanes 1–3) and DraI (Lanes

4–6). (b) Restriction profile of blaB with endo-

nucleases RsaI (Lanes 7–9) and HaeIII (Lanes

10–12). PCR amplification and restriction analy-

sis were carried out for 81 strains of Yersinia

enterocolitica biovar 1A. M, 100 bp DNA ladder.


321Molecular characterization of b-lactamase genes blaA and blaB

major groups – A, B and C (Fig. 2). Group A comprised

primarily the clinical isolates, whereas there was preponder-

ance 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

almost equal numbers. This group was related to the other

two at approximately 23% similarity. All except one ser-

ogroup 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

StrainID

Serovar Source Countryof origin

0.23 0.42 0.61 0.81 1.00Coefficient

(a)

(b)

(c)

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.)



Indian, European and American origin showed identical

profiles. Previously, the BRO b-lactamases BRO-1 and BRO-

2 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

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 (Gran-

ier et al., 2003). These observations suggest that restriction

analysis of b-lactamase genes may be used to study epide-

miology, or discern phylogenetic relationships. It would be

of interest to explore further if restriction profiling of bla

genes, such as blaA, may provide epidemiological/phyloge-

netic information about Y. enterocolitica and ‘Y. enterocoliti-

ca-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

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).



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 serine-

threonine-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

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 sequencecomparison 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

AAX55643 MKHSSLRRALLLAGITLPLVNFSLPTWAAAI--PGSLDKQLAALEHSANGRLGIAMINTG8081_biovar1B MKHSSLRRSLLLAGITLPLVNFALPTWAAAI--PGSLDKQLAALEHSANGRLGIAMINSGCAA40357 MKHSSLRRSLLLAGITLPLVSFALPAWANAL--PASVDKQLAELERNANGRLGVAMINTGAAA25084 MLKSSWRKTALMAAAAVPLLLASGSLWASAD----AIQQKLADLEKRSGGRLGVALINTACAA44485 MFKKRGRQTVLIAAVLA-FFTASSPLLARTQGEPTQVQQKLAALEKQSGGRLGVALINTA * :. *:: *:*. .:. : . * : . ::::** **: :.****:*:**:.

AAX55643 NGTKILYRGARRFPFCSTFKFMLAAAVLGQSQSQPNLLNKHINYHESDLLSYAPITRKNL8081_biovar1B AGTKILYRGAQRFPFCSTFKFMLAAAVLDQSQSQPNLLNKHINYHESDLLSYAPITRKNLCAA40357 NGTKILYRAAQRFPFCSTFKFMLAAAVLDQSQSQPNLLNKHINYHESDLLSYAPITRKNLAAA25084 DDSQTLYRGDERFAMCSTGKVMAAAAVLKQSESNPEVVNKRLEIKKSDLVVWSPITEKHLCAA44485 DRSQILYRGDERFAMCSTSKTMVAAAVLKQSETQHDILQQKMVIKKADLTNWNPVTEKYV :: ***. .**.:*** * * ***** **::: ::::::: :::** : *:*.* :

AAX55643 AHGMTVSELCAATIQYSDNTAANLLLKELGGLAAVNQFARSIGDQMFRLDRWEPDLNTAL8081_biovar1B ACGMTVSELCAATIQYSDNTAANLLIKELGGLAAVNQFARSIGDQMFRLDRWEPDLNTALCAA40357 AHGMTVSELCAATIQYSDNTAANLLIKELGGLAAVNQFARSIGDQMFRLDRWEPDLNTARAAA25084 QSGMTLAELSAAALQYSDNTAMNKMISYLGGPEKVTAFAQSIGDVTFRLDRTEPALNSAICAA44485 DKEMTLAELSAATLQYSDNTAMNKLLEHLGGTSNVTAFARSIGDTTFRLDRKEPELNTAI **::**.**::******* * ::. *** *. **:**** ***** ** **:*

AAX55643 PNDPRDTTTPAAMAASINKLVLGDALHPAQRSQLTAWLKGNTTGDATIRAGAPTDWIVGD8081_biovar1B PNDPRDTTTPAAMAASMNKLVLGDALRPAQRSQLAAWLKGNTTGDATIRAGAPTDWIVGDCAA40357 PNDPRDTTTPAAMAASMNKLVLGDALRPAQRSQLAVWLKGNTTGDATIRAGAPTDWIVGDAAA25084 PGDKRDTTTPLAMAESLRKLTLGNALGEQQRAQLVTWLKGNTTGGQSIRAGLPASWAVGDCAA44485 PGDERDTTCPLAMAKSLHKLTLGDALAGAQRAQLVEWLKGNTTGGQSIRAGLPEGWVVGD *.* **** * *** *:.**.**:** **:**. ********. :**** * .* ***

AAX55643 KTGSGDYGTTNDIAVLWPTKGAPIVLVVYFTQREKDAKPRRDVLASATKIILS---8081_biovar1B KTGSGDYGTTNDIAVLWPTKGAPIVLVVYFTQREKDAKPRRDVLASATQIILSQISCAA40357 KTGSGDYGTTNDIAVLWPTKGAPIVLVVYFTQREKDAKPRRDVLASVTKIILS---AAA25084 KTGAGDYGTTNDIAVIWPENHAPLVLVTYFTQPQQDAKSRKEVLAAAAKIVTEGL-CAA44485 KTGAGDYGTTNDIAVIWPEDRAPLILVTYFTQPQQDAKGRKDILAAAAKIVTEGL- ***:***********:** . **::**.**** ::*** *:::**:.::*: .

234

130 166

70

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).



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. enter-

ocolitica 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 IEFanalysis 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 mole-

cular 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 cephalos-

porinase (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).

The two b-lactamases, Bla-A and Bla-B produced by Y.

enterocolitica have been distinguished by their distinct iso-

electric 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 prepara-

tions. 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 ‘B-

like’ 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 iso-

electric points of their enzymes.

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

Strains n

pI

Bla-A Bla-B

Clinical

Indian 35 8.7 Both 6.8 and 7.1

European 15 8.7 Either 6.8 or 7.1; rarely both

American 1 8.7 Both 6.8 and 7.1

Nonclinical� 30 8.7 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.



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 repre-

sented 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 knowl-

edge, 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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Molecular characterization of Î²-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A