MICROBIAL DRUG RESISTANCEVolume 12, Number 3, 2006© Mary Ann Liebert, Inc.

Molecular Characterization and Occurrence of Extended-Spectrum �-Lactamase Resistance Genes among Salmonella

enterica Serovar Corvallis from Thailand, Bulgaria, and Denmark

MARIE ARCHAMBAULT,1 PETAR PETROV,2 RENE SJØGREN HENDRIKSEN,3 G. ASSEVA,2

AROON BANGTRAKULNONTH,4 HENRIK HASMAN,3 and FRANK M. AARESTRUP3

ABSTRACT

Fifty nine Salmonella Corvallis isolates from humans and food products in Bulgaria, Denmark, and Thailandwere examined for antimicrobial susceptibility and characterized by pulsed-field gel electrophoresis (PFGE).Cephalosporin-resistant isolates were examined for the presence of genes encoding �-lactamases by PCR andsequencing. Ten different PFGE types were observed. One type (30 isolates) was recovered in all three coun-tries; three types were found only in Bulgaria, two only in Denmark, two only in Thailand, and two both inDenmark and Thailand. Ten isolates were susceptible to all antimicrobial agents tested, whereas 41 were re-sistant to three or more antimicrobials. Most resistance was observed among the isolates from Bulgaria. Ofthe 25 isolates from Bulgaria, 20 displayed resistance to ampicillin and the cephalosporins ceftiofur andcephalothin. All 20 isolates tested negative for blaCMY-1, blaCMY-2, and blaACC, but positive for blaSHV, of whichfive were sequenced to blaSHV-2. Plasmid profiling and hybridization revealed that the blaSHV gene was lo-cated on plasmids of approximately 70 kb. Five plasmid profiles were found among these 20 isolates. The plas-mid profiling confirmed the PFGE-type and was able to further subdivide the strains. Seventeen of these 20isolates contained also blaTEM, of which nine representatives were sequenced to blaTEM-1B, or blaTEM-1H. Oneisolate contained blaCTX-M-15, blaSHV-2, and blaTEM-1H, with the blaCTX-M-15, and blaTEM-1H genes located on a63-kb transferable plasmid. This study showed a high frequency of resistance among S. Corvallis isolatedfrom humans and food products in Bulgaria, with a lower frequency in Thailand and Denmark. The clonalrelatedness among the isolates from three countries could indicate a recent spread of this serovar.

INTRODUCTION

SALMONELLA ENTERICA is one of the most common causes ofhuman gastroenteritis worldwide. More than 2,500 differ-

ent serovars of S. enterica have been identified, and most ofthem have been described as the cause of human infections;however, most reports have mentioned S. enterica serovar Ty-phimurium and S. enterica serovar Enteritidis as the most com-mon causes of human salmonellosis worldwide.13,14,25 How-

ever, in some regions, other serovars have been reported to beof even greater importance,2 and changes in the importance ofdifferent serovars over time seem to take place.2,25 The occur-rence of different serovars in one country can be of global im-portance because of travel and trade of breeding animals andfood products worldwide. Knowledge regarding the occurrenceand molecular epidemiology of different serovars in differentcountries and geographic regions may assist in the recognitionand tracing of new emerging pathogens.

1Faculté de Médecine Vétérinaire, University of Montreal, Saint-Hyacinthe, Quebec, Canada.2National Center of Infectious and Parasitic Diseases, National Reference Laboratory for Enteric Pathogens,1504 Sofia, Bulgaria.3WHO Collaborating Centre for Antimicrobial Resistance in Foodborne Pathogens, Danish Institute for Food and Veterinary Research, DK-

1790 Copenhagen V, Denmark.4WHO International Salmonella and Shigella Centre, National Institute of Health, Department of Medical Sciences, Ministry of Public Health,

Bangkok, Thailand.

192

Salmonella resistant to multiple antimicrobial agents haveemerged worldwide in recent years. However, it is not resis-tance to all antimicrobial agents that is of equal importance.Fluoroquinolones are in many cases the drug of choice for treat-ment of gastrointestinal infections in humans. Thus, resistanceto this class of antimicrobial agents is associated with increasedmortality and morbidity1,12 and thus especially unwanted. Sal-monella isolates resistant to oxyiminocephalosporins due to theproduction of extended spectrum �-lactamases (ESBLs) haveemerged worldwide since 1992.3 This has also caused concernbecause cephalosporins are drugs of choice for the treatment ofsalmonellosis in children, to which fluoroquinolones must notbe administered.

S. enterica serovar Corvallis resistant to oxyimino-cephalosporins due to the production of SHV-2 was recentlyreported in Bulgaria.16 This serovar has also in 2004 and 2005entered into the top 40 as a cause of salmonella infections inhumans in Denmark (http://www.germ.dk). However, there isonly limited information on the occurrence of antimicrobial re-sistance and molecular diversity of S. Corvallis.

This study was conducted to determine the occurrence of an-timicrobial resistance and molecular variation of S. Corvallisfrom humans, food products, and animals in Denmark, Thai-land, and Bulgaria. The implication of the findings in relationto global spread of new serovars is discussed.

MATERIALS AND METHODS

Occurrence of Salmonella Corvallis in Bulgaria,Denmark, and Thailand

Data on the prevalence of S. Corvallis were obtained fromthe diagnostic laboratory authorities of Bulgaria, Denmark, andThailand.

Bacterial isolates

A total of 59 isolates of S. Corvallis were examined fromBulgaria (25), Thailand (22), and Denmark (12). The isolateswere collected during 2001–2004. The isolates originatedfrom humans (45) and from food products (13); one isolatewas from an unknown source. Human isolates were from fe-cal samples of infants in Bulgaria (23), Denmark (1), and Thai-land (10), ranging in age from 3 days to 30 months, and adultsin Bulgaria (1) and Denmark (10) from 17 to 65 years of age.Among the isolates obtained from Danish patients, 2 adult pa-tients had a history of foreign travel to Thailand. The 13 iso-lates from food products were from minced meat (1) in Bul-garia and beef (2), chicken meat (6), and pork (4) in Thailand.The isolates were initially identified and serotyped in the orig-inating countries. The isolates were shipped to The Danish In-stitute for Food and Veterinary Research where the serotypewas confirmed.

Serotyping

Serotyping of Salmonella sp. isolates was performed on thebasis of somatic O and phase 1 and phase 2 flagellar antigensby agglutination tests with antisera according to the Kauf-mann–White scheme.28

Antimicrobial susceptibility testing

Susceptibility to antimicrobial agents was performed as MICdeterminations using a commercially prepared, dehydrated panel(Sensititre®). The following antimicrobial agents were used:amoxicillin-clavulanic acid, ampicillin, apramycin, ceftiofur,cephalothin, chloramphenicol, ciprofloxacin, colistin, florfeni-col, gentamicin, nalidixic acid, neomycin, spectinomycin, strep-tomycin, sulfamethoxazole, tetracycline, and trimethoprim. Allplates were inoculated following the guidelines recommendedby the Clinical and Laboratory Standards Institute (CLSI), andCLSI breakpoints were used for interpretation of the MIC results.22

Detection of resistance genes

Twenty ampicillin- and ceftiofur-resistant isolates were analyzed by PCR to detect the presence of blaTEM, blaCTX, blaCMY-1, blaCMY-2, blaSHV, and blaACC. PCR amplification wasperformed using primer pairs 5�-ATGAGTATTCAACATTTC-CG-3� and 5�-ACCAATGCTTAATCAGTGAG-3� (blaTEM),23

5� ATGTGCAGYACCAGTAARGTKATGGC-3�, and 5�TGGGTRAARTARGTSACCAGAAYCAGCGG-3� (blaCTX),18

5�-TGGTGGATGCCAGCATCCA-3� and 5�-GGTCGAGCCG-GTCTTGTTGAA-3�, (blaCMY-1)11, 5�-GCACTTAGCCACC-TATACGGCAG-3� and 5�-GCTTTTCAAGAATGCGCCA-GG-3� (blaCMY-2),11 and 5�-AGCCTCAGCAGCCGGTTAC-3�and 5�-GAAGCCGTTAGTTGATCCGG-3� (blaACC-1),11 and5�-TTATCTCCCTGTTAGCCACC-3� and 5�-GATTTGCTGA-TTTCGCTCGG-3� (blaSHV).11 The amplicon sizes of blaTEM,blaCTX, blaCMY-1, blaCMY-2, blaSHV, and blaACC were 964 bp,593 bp, 915 bp, 758 bp, 854 bp, and 818 bp, respectively. Pos-itive controls for the blaTEM, blaCTX, blaCMY-2, blaSHV, andblaACC, PCR reactions were S. Blockley 34.60, S. Virchow7522438-1, Klebsiella pneumoniae MISC339, S. Heidelberg7512893-1, S. Concord 38.47, and S. Bareilly 50.32, respec-tively.11

All PCRs were conducted with the buffer supplied by themanufacturers using 20 pmol of each primer and 0.5 U of Su-per Taq® (HT Biotechnology, UK). All reaction mixtures (50�l) were amplified using the following programs: 3 min at94°C; 25 cycles of 1 min at 94°C, 1 min. at 50°C, and 1 minat 72°C; 10 min at 72°C (blaTEM), 5 min at 94°C; 30 cycles of45 sec at 94°C, 45 sec at 45°C, and 45 sec at 72°C; 10 min at72°C (blaCTX), 5 min at 94°C, 25 cycles of 1 min at 94°C, 1min at 58°C, and 1 min at 72°C; 10 min at 72°C (blaCMY-1), 5min at 94°C; 25 cycles of 45 sec at 94°C, 45 sec at 58°C and1 min at 72°C; 10 min at 72°C (blaCMY-2) and 3 min at 95°C;35 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C;10 min at 72°C (blaSHV) and 5 min at 94°C; 30 cycles of 45sec at 94°C, 45 sec at 55°C, and 1.5 min at 72°C; 10 min at72°C (blaACC).

Nine out of 17 PCR amplicons positive for blaTEM, five outof 20 amplicons positive for blaSHV, and one amplicon againstblaCTX were selected for sequencing. Prior to sequencing, theamplicons were purified using the GFX™ PCR DNA kit (Amer-sham Biosciences). Sequencing and sequence analysis of allgenes were performed on an ABI 377 DNA sequencer (PerkinElmer, Applied Biosystems) using the same primers as in thePCR analysis. The resulting nucleotide sequences were com-pared to sequences obtained from the GenBank database and

CHARACTERIZATION OF SALMONELLA CORVALLIS 193

http://www.lahey.org/studies/webt.html. The software VektorNTI suite 8 (InforMax, Inc.) was used for alignment.

To distinguish between the genes blaCTX-M-15, blaCTX-M-28,and blaUOE1 in the Bulgarian isolate 489 (No. 58 in Table 3,below), another amplification of blaCTX was performed and sequenced using two other primer pairs 5-�CCATGGT-TAAAAAATCACTGCG-3�(blaCTX-M-15 front P1) and 5�-TG-AAGTGGTATCACGCGGATC -3� (blaCTX-M-15 front P2) and5�-CCGTTTCCSCTATTACAAACCG-3�(blaCTX-M-15 end P2)and 5�-ATGTGCAGYACCAGTAARGTKATGGC-3� (blaCTX

M U1). The following program was used for both PCR amplifi-cations: 3 min at 94°C; 30 cycles of 1 min at 94°C, 1 min at57°C, and 1 min at 72°C; 10 min at 72°C with amplicon sizesof 557 bp and 680 bp, respectively. The nucleotide sequencesof the genes detected in the study were matched to known genesin the GenBank database; the accession numbers are AAT48025(blaCTX-M-15), AY293072 (blaTEM-1B), AAA25526 (blaSHV-2),and AY394610 (blaTEM-1H).

Plasmid analysis and conjugation

Plasmid DNA of the 20 Bulgarian isolates showing a posi-tive reaction against blaSHV, including the one also showing apositive reaction against blaCTX, was purified by an alkaline ly-sis method, using Nucleobond® AX (Macherey-Nagel Inc., Eas-ton, PA). The plasmid DNA was detected on an 0.8% agarosegel electrophoresis for 2 hr at 120 V. The molecular sizes ofthe bands were determined using the software Quantity One4.2.2 and the Gel Doc 2000 (Bio Rad®) by reference to plas-mids of known band sizes (E. coli 39R: 7, 36, 63, 147 kbp).

Conjugation was performed by the filter mating method30

using a mutated plasmid-free, amoxicillin-sensitive andnalidixid acid- and rifampicin-resistant Salmonella DublinJEO6624 as recipient for conjugation of the ceftiofur-resistantBulgarian isolate 489 showing a positive amplicon against

blaCTX and 10 other isolates showing positive amplicons againstblaSHV. Transconjugates were isolated on brain heart infusion(BHI) agar containing cephalothin (32 �g/ml), nalidixic acid(50 �g/ml), and rifampicin (50 �g/ml). The transconjugate489AxJEO66 was investigated by PCR, plasmid profiling, andpulsed-field gel electrophoresis (PFGE) to confirm the presenceof the original plasmid, the blaTEM-1H and the blaCTX genes ob-tained from strain 489, and to match the PFGE pattern with theone of the recipients.

Pulsed-field gel electrophoresis

PFGE was performed using XbaI according to CDC PulseNetprotocols.29 To perform the phylogenetic analysis of the types,TIFF files were analyzed with BioNumerics software (AppliedMaths, Sint-Martens-Latem, Belgium). Cluster analysis usingthe Dice correlation for band matching with a 0.7% positiontolerance was used to generate a dendrogram describing the re-lationship among the different types and subtypes.

RESULTS

Occurrence of S. Corvallis

A total of 1,046 Salmonella were isolated from patients in Bul-garia in 2003, of which 1,027 were serotyped. S. Enteritidis(82.9%), S. Typhimurium (8.1%), and S. Corvallis (3.1%) rankedfirst, second, and third in prevalence, respectively. In Thailand,S. Corvallis was isolated from 3.8% of the salmonella-positivesamples from humans in 2003 and 3.88% in 2004 (data until Sep-tember) and accounted for 3.4% of the Salmonella isolates fromfood products. In Denmark, this serovar ranked 11th in the over-all Salmonella serovar list for 2005. Only one veterinary isolatewas recorded in 1997 with an unknown origin.

ARCHAMBAULT ET AL.194

TABLE 1. OCCURRENCE OF ANTIMICROBIAL RESISTANCE AND PFGE TYPES AMONG 59 SALMONELLA CORVALLIS ISOLATES FROM BULGARIA, DENMARK, AND THAILAND

Number of PFGECountry isolates types AMP CEF CHL GEN NAL STR SUL TET

Bulgaria 4 I 3 3 3 3 3 3 3 0(25) 6 II 6 6 6 6 0 6 6 0

14 V 12 11 12 12 1 11 9 01 VI 0 0 0 0 0 0 0 0

Denmark 5 V 0 0 0 0 4 3 3 3(12) 1 VIII 0 0 0 0 0 0 0 0

4 IX 1 0 0 0 3 3 3 41 X 0 0 0 0 0 1 1 11 XI 0 0 1 0 0 1 1 1

Thailand 11 V 0 0 0 0 9 6 6 6(22) 2 VII 0 0 0 0 1 0 0 0

4 VII 0 0 0 0 1 3 3 34 IX 0 0 0 0 2 3 3 31 XII 0 0 0 0 0 0 0 0

Total 59 10 22 20 22 21 24 40 38 21(37.3) (33.9) (37.3) (35.6) (40.7) (67.8) (64.4) (35.6)

aAMP, Ampicillin; CEF, ceftiofur; CHL, chloramphenicol; GEN, gentamicin; NAL, nalidixic acid; STR, streptomycin; SUL,sulfamethoxazole; TET, tetracycline.

Number of isolates with antimicrobial resistancea (%)

Antimicrobial resistance and PFGE typing

Only 10 (16.9%) of the 59 isolates were susceptible to allantimicrobial agents tested, whereas 41 (69.5%) were resistantto three or more antimicrobial agents. All isolates were sus-ceptible to apramycin, ciprofloxacin, florfenicol, colistin,neomycin, and spectinomycin. A higher frequency of resistancewas found toward streptomycin (67.8%) and sulfonamides(64.4%) (Table 1). Resistance was also observed to ampicillin(37.3%), ceftiofur (33.9%), chloramphenicol (37.3%), gentam-icin (35.6%), nalidixic acid (40.7%), and tetracyclines (35.6%).In Denmark, no ceftiofur, cephalothin, or gentamicin resistancewere found. In Thailand, no resistance to ampicillin, ceftiofur,cephalothin, chloramphenicol, and gentamicin were observed.Of the 24 human isolates from Bulgaria, 20 displayed resistanceto ampicillin, cephalothin, and ceftiofur. The isolate fromminced meat in Bulgaria was susceptible to all antimicrobialstested. No difference in susceptibility between the isolates fromfood products and patients in Thailand was observed.

Ten different PFGE types of S. Corvallis (I, II, V, VI, VII,VIII, IX, X, XI, and XII) were observed among the isolates(Table 1) and their phylogeny is shown in Fig. 1. Types I, II,and VI were found only in Bulgaria whereas types X and XIwere found only in Denmark. Types VII and XII were foundonly in Thailand, whereas types VIII and IX were common toboth Denmark and Thailand. Type V was the only commontype recovered in all three countries.

Detection and identification of blaTEM, blaCTX-M, blaCMY-1,blaCMY-2, blaSHV, and blaACC

All 20 ampicillin- and ceftiofur-resistant isolates gave neg-ative reactions for blaCMY-1, blaCMY-2, and blaACC genes butwere positive for blaSHV. Five of them were sequenced toblaSHV-2. Seventeen of the 20 isolates also gave positive reac-tions for blaTEM, and nine representative isolates were se-quenced. A comparison with GenBank revealed 100% identityto the sequence of blaTEM-1B for eight isolates and to blaTEM-1H for one isolate. The blaTEM-1H-positive isolate of S.Corvallis was also positive for blaCTX-M-15. The blaCTX-M-15

gene was located on a 63-kb transferable plasmid, which alsocarried the blaTEM-1H gene. This is the first blaSHV-2 and blaCTX-

M-15 containing isolates of S. Corvallis reported to date.

Plasmid analysis and conjugation

Five different plasmid profiles were found among the 20 iso-lates investigated (Fig. 2). The plasmid profiles confirmed inmost cases the PFGE-type (Table 2). However, the six isolatesof PFGE type II were assigned to two different plasmid pro-files (four of plasmid profile II and two of profile III). This was

CHARACTERIZATION OF SALMONELLA CORVALLIS 195

FIG. 1. Phylogeny of the PFGE types of Salmonella Corvallis isolates from Bulgaria, Denmark, and Thailand.

FIG. 2. Plasmid profiles of undigested plasmid DNA foundamong 20 blaSHV-positive Salmonella Corvallis isolates fromBulgaria. Lanes M1, plasmid marker V517 invisible on blot;lanes 1, plasmid profile I (#37 / 14670: PFGE type I); lanes 2,plasmid profile II (#39 / 15048: PFGE type II); lanes 3, plas-mid profile III (#61 / 391: PFGE type II); lanes 4, plasmid pro-file IV (#48 / 557: PFGE type V); lanes 5, plasmid profile V(#58 / 489: PFGE type V); lanes M2, plasmid marker 39R861invisible on blot; lanes M3, � DNA HindIII marker.

consistent with the fact that the four isolates were obtained in2002 and believed to be part of a minor outbreak where a 40-year-old female had infected infants, whereas the two remain-ing isolates were isolated more than a year later. The blaCTX-M-15 gene was located on a plasmid of approximately63 kb, giving rise to a new plasmid profile. The plasmid withblaCTX-M-15 was transferable by conjugation, whereas notransconjugants were obtained from the isolates only contain-ing blaSHV. Hybridization showed that the blaSHV gene was pre-sent on plasmids of different sizes of approximately 70 kb.

DISCUSSION

In Japan, S. Corvallis was recently involved in a hospitalfood poisoning outbreak where the isolates recovered were allsusceptible to the antimicrobial tested.10 Peek et al.27 recentlyreported the isolation of S. Corvallis from the environment ofone dairy without any history of clinical salmonellosis. Thisisolate was also susceptible to all antimicrobial agents tested.S. Corvallis was reported in the top five serovars isolated froma U.S. monitoring study of chicken houses and an egg-pro-cessing facility in a laying farm between 1994 and 1998.21 InTunis, S. Corvallis was one of the most frequent serovars iso-lated from food products of animal origin between 1989 and1993; the bacteria was mainly recovered in turkey meat.9 Fi-nally, a study on Salmonella sp. contamination of restaurantmeat products collected over a period of 1 year found that S.Corvallis was detected in red meat and fowl.15 Although isola-tion of S. Corvallis seems to be a rare event in most countries,our study suggests the contrary for Bulgaria, Denmark, andThailand. Thus, this serovar might have the potential for caus-ing local or global outbreaks.

We found major differences in the occurrence of antimicro-bial resistance between countries and origin. There was highfrequency of resistance among S. Corvallis isolated from hu-mans and food in Bulgaria, with a lower frequency in Thailandand Denmark. Twenty ampicillin- and ceftiofur-resistant iso-lates were recovered from Bulgaria where the dominant resis-tance profile was ampicillin, ceftiofur, chloramphenicol, gen-tamicin, streptomycin, and sulfamethoxazole. This resistanceprofile was not seen in Denmark or Thailand. Resistance totetracyclines was observed among isolates from Thailand andDenmark, but not from Bulgaria. A similar predominant pat-tern of resistance to nalidixic acid, streptomycin, sulfamethox-azole, and tetracycline were found in isolates from Denmarkand Thailand.

This high frequency of resistance might be because thisserovar easily acquires resistance, or because the natural reser-voirs were exposed to large amounts of antimicrobial agents. Itis tempting to speculate that the first hypothesis might be morerelevant for this serovar when we look at the age of the infantwhose isolates showed the higher resistance (Table 2). Also,knowing that infants in Bulgaria between 7 days and 9 monthshave generally not been exposed to large amounts of antimi-crobial agents, it is logical to think that transmission of thisserovar from adults to infants is a possibility that should be se-riously considered.

All S. Corvallis isolates were related, as determined byPFGE. Thus, isolates belonging to this serovar might havespread recently between these three countries. However, ourresults also showed that the PFGE clone V has evolved dif-ferently in Bulgaria, Thailand, and Denmark from a perspec-tive of antimicrobial resistance. In Thailand and Denmark, thisclone showed a resistance pattern to nalidixic acid, strepto-mycin, sulfamethoxazole, and tetracycline, whereas in Bul-

ARCHAMBAULT ET AL.196

TABLE 2. ORIGIN, DATE OF ISOLATION, PFGE, AND PLASMID PROFILES OF THE

20 blaSHV-POSITIVE SALMONELLA CORVALLIS ISOLATES FROM BULGARIA

PlasmidNumber Date Province Age of patient �-Lactamase genes PFGE type profile

36 Jan. 9, 2001 Sofia 18 months blaSHV, blaTEM I I37 Jan. 8, 2002 Stara Zagora 12 months blaSHV, blaTEM I I38 Jan. 8, 2003 Stara Zagora 3 months blaSHV, blaTEM I I39 Feb. 7, 2002 Stara Zagora 40 year blaSHV, blaTEM II II40 Jan. 24, 2002 Stara Zagora 25 days blaSHV, blaTEM II II41 Jan. 24, 2002 Stara Zagora 23 days blaSHV, blaTEM II II42 Jan. 24, 2002 Stara Zagora 17 days blaSHV, blaTEM II II60 April 21, 2003 Stara Zagora 7 days blaSHV, blaTEM II III61 April 21, 2003 Stara Zagora 22 days blaSHV II III46 Feb. 20, 2004 Gabrovo 18 months blaSHV, blaTEM V IV48 Sep. 16, 2003 Stara Zagora 4 months blaSHV, blaTEM V IV50 Sep. 16, 2003 Stara Zagora 9 days blaSHV, blaTEM V IV51 Sep. 16, 2003 Stara Zagora 3 months blaSHV, blaTEM V IV52 Sep. 16, 2003 Stara Zagora 9 months blaSHV, blaTEM V IV53 Sep. 16, 2003 Stara Zagora 19 months blaSHV, blaTEM V IV54 Sep. 16, 2003 Stara Zagora 6 months blaSHV, blaTEM V IV56 Sep. 16, 2003 Stara Zagora 5 months blaSHV, blaTEM V IV57 Sep. 16, 2003 Stara Zagora 15 months blaSHV V IV58 Aug. 29, 2003 Gabrovo 30 months blaSHV, blaTEM, blaCTX V V59 Aug. 29, 2003 Gabrovo 1 month blaSHV V IV

garia the pattern was ampicillin, ceftiofur, chloramphenicol,gentamicin, streptomycin, and sulfamethoxazole. The resis-tance to streptomycin and sulfamethoxazole is the only com-mon feature in their antimicrobial resistance profile. Sometype V isolates, which were susceptible to all antimicrobialtested, were also recovered from these countries. Therefore,mobile elements are suspected as a mean of antimicrobial re-sistance transfer.

Ampicillin- and ceftiofur-resistant isolates were examinedfor the presence of selected extended �-lactamase resistancegenes by PCR. A total of 17 isolates contained blaTEM

(blaTEM-1B or blaTEM-1H). blaSHV-2 was found in all 20 ceftio-fur-resistant isolates from Bulgaria. The gene could not be trans-ferred by conjugation from the 10 isolates where it was at-tempted, but hybridization to plasmid profiles indicated that thegene was located on plasmids of approximately 70 kb. Genesof the SHV family are commonly reported from Enterobacte-riaceae worldwide, but seem especially to be commonly foundin Klebsiella spp.6,17,26 blaSHV-2 has previously been reportedfrom Salmonella Wien in Tunisia,14 Salmonella Typhimuriumfrom France,8 and Salmonella Virchow from The Nether-lands.11 blaSHV-2a has recently been reported to be commonamong Enterobacteriaceae in Hungary, whereas blaSHV-2 werenot detected.31

The blaCTX-M-15 gene was present in one isolate also con-taining blaSHV-2 and blaTEM-1H. The blaCTX-M-15 gene was lo-cated on a 63-kb transferable plasmid, which also carried theblaTEM-1H gene. The blaCTX-M-15 gene has previously been as-sociated with mobile elements, most commonly insertion se-quences, such as ISEcp1, or integron-associated blaCTX-M.7,19

Co-carriage of ESBL genes is not a new finding, but it is re-ported as uncommon.19,20 It has been reported that blaCTX-M-14

and blaCTX-M-13 were carried together with either blaSHV-12 orblaSHV-11.5 In Canada, an isolate containing the blaCTX-M-14

gene was found to also contain blaSHV-12.17 In The Netherlands,co-carriage of blaCTX-M2 and blaSHV-2 was observed in a S. Vir-chow isolate.11 Co-carriage of blaCTX-M-2 and blaSHV-5 geneshas also been reported in Klebsiella spp.4,26 We believe this tobe the first report of co-carriage of blaCTX-M-15, blaSHV-2, andblaTEM-1H.

Our investigation in Bulgaria indicated that some of these S.Corvallis isolates were certainly acquired in the same hospitalin Stara Zagora, among three infants of 17, 23, and 25 days ofage and 1 female care giver of 40 years of age, who was inclose contact. They all had the same PFGE type II with thesame resistance and plasmid profile and contained both blaSHV

and blaTEM. The minced meat isolate of S. Corvallis from Bul-garia was a type V and showed no antimicrobial resistance toany of the antibiotics tested. These findings stress the urgentneed to determine the origin of a contamination and to intro-duce adequate antibiotic regimen into hospitals and nurseriesto prevent further transmission and dissemination of such strain.

In conclusion, this study showed a high frequency of resis-tance among S. Corvallis isolated from humans, adults, and in-fants, and other sources in Bulgaria, and a lower frequency inThailand and Denmark. PFGE typing suggests that all of theisolates from the different countries are clonally related, whichcould indicate that isolates belonging to this serovar have spreadrecently and that resistance has evolved locally among the iso-lates in Bulgaria.

ACKNOWLEDGMENTS

We are grateful to the following persons at The Danish In-stitute for Food and Veterinary Research (DFVF): ChristinaAaby Svendsen and Jane Nygaard Larsen for their technical sup-port, Gitte Sørensen for providing data regarding the veterinaryisolate and prevalence data from Denmark. Furthermore, wewant to thank Ingrid Bøcker Jensen at Statens Serum Institut(SSI) for providing strains from humans in Denmark and DikMevius at the Central Institute for Animal Disease Control,Lelystad, The Netherlands for providing the strains S. Blockley34.60 and S. Bareilly 50.32. We wish to acknowledge the sup-port of the WHO Global Salmonella Survey Program and theFaculté de Médecine Vétérinaire, University of Montreal.

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Address reprint requests to:Dr. Frank M. Aarestrup

WHO Collaborating Centre for Antimicrobial Resistance inFoodborne Pathogens

Danish Institute for Food and Veterinary ResearchBulowsvej 27

DK-1790 Copenhagen V, Denmark

E-mail: faa@dfvf.dk

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