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J Clin Microbiol. 2010 March; 48(3): 883–888.
Published online 2009 December 23. doi:  10.1128/JCM.01792-09
PMCID: PMC2832443

Molecular Characterization of Extended-Spectrum Cephalosporinase-Producing Salmonella enterica Serovar Choleraesuis Isolates from Patients in Thailand and Denmark[down-pointing small open triangle]

Abstract

The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either blaCMY-2 containing incA/C or blaCTX-M-14 containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the blaCTX-M-14 gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A “travel-associated” spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.

Salmonella enterica is a common cause of human gastroenteritis and bacteremia worldwide (18, 31), and a wide variety of animals, particularly food animals, have been identified as reservoirs for non-Typhi Salmonella (11, 22, 23). Although approximately 2,600 serovars of Salmonella enterica have been identified, most human infections are caused by a limited number of serovars, and in general these infections are self-limiting. Some Salmonella serovars, including Salmonella Choleraesuis (swine) and Salmonella Dublin (cattle), which are adapted to a specific animal host, have a propensity to cause extraintestinal infections in humans. Compared to those with other serovars of non-Typhi Salmonella, infections with these serovars are associated with higher rates of bacteremia, meningitis, and mortality (4, 5, 24). For patients with severe salmonellosis, antimicrobial chemotherapy may be life-saving. Due to the increasing prevalence of fluoroquinolone resistance, extended-spectrum cephalosporins are increasingly used for the treatment of Salmonella infections in humans (17, 21, 25) and especially for children, for whom treatment of highly resistant Salmonella infections with fluoroquinolones is not advised, since such treatment has been associated with treatment failures (12, 13, 21). Therefore, these compounds have been designated critically important for human health by the World Health Organization (10).

We recently reported that the prevalence of human infections with Salmonella serovar Choleraesuis in Thailand increased from 1.5% (n = 87) in 1994 to 9.2% (n = 190) in 2006 (19). The group of people at highest risk for these infections was those between 6 and 40 years of age in the central region of Thailand (19). A 2007 study of Salmonella serovar Choleraesuis isolates from Thailand observed an increasing resistance to both extended-spectrum cephalosporins (ceftriaxone) and fluoroquinolones. Fifty-four isolates obtained between 2003 and 2005 were tested, of which 30% were found to be resistant to an extended-spectrum cephalosporin (ceftriaxone) (25).

To date, only two reports, both from Taiwan, have described mechanisms for extended-spectrum cephalosporin resistance in Salmonella serovar Choleraesuis. The first report was published in 2004 with the discovery of the blaCMY-2 AmpC β-lactamase gene located on a 140-kb F-like plasmid (6). The following year, the same authors detected blaCTX-M-3 in a Salmonella serovar Choleraesuis isolate from a patient admitted to a university hospital (30). In 2007, a massive increase of fluoroquinolone- and ceftriaxone-resistant Salmonella serovar Choleraesuis isolates was described in Thailand (25).

In Taiwan, the usage of antimicrobials in veterinary medicine and as growth promoters in animal feed may have promoted the emergence of resistance (5). Likewise, in Thailand, the extended-spectrum cephalosporin ceftiofur is used as a growth promoter in swine production (25). However, data on antimicrobial usage in disease prevention and as growth promoters are not accessible in both countries.

The objective of the present study was to characterize the mechanisms responsible for the emergence of resistance to extended-spectrum cephalosporins in isolates of Salmonella serovar Choleraesuis recovered from patients in Thailand and Denmark.

Additional objectives were to determine the clonality of the isolates resistant and susceptible to cephalosporins (ceftriaxone and cefoxitin) using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing and to find biological evidence of transmission through international travel.

MATERIALS AND METHODS

Bacterial isolates.

A total of 24 blood culture isolates from 22 patients were included in this study, originating from three different collections. The sets were collected by Aalborg Hospital, Aarhus University Hospital, Denmark (collection 1); the WHO National Salmonella and Shigella Center in Bangkok, Thailand (collection 2); and the Regional Medical Sciences Center in Samutsongkhram, Thailand (collection 3).

Twenty-three isolates were recovered in Thailand from 2003, 2007, and 2008, and one extended-spectrum cephalosporinase (ESC)-producing isolate (collection 1) was recovered in 2008 at Aalborg Hospital, Denmark.

The WHO National Salmonella and Shigella Center in Bangkok receives all presumptive isolates of Salmonella spp. from all diagnostic laboratories throughout Thailand. In 2003, as part of another study, the National Food Institute, Technical University of Denmark (DTU-Food), received 82 isolates of Salmonella serovar Choleraesuis which were recovered from Thai patients (1). In 2008, this collection (collection 2) was screened for the presence of ESC-producing isolates and two ESC-producing strains, both isolated in 2003 from patients in Bangkok and Ratchaburi province, were identified and included.

In 2008, DTU-Food received a third collection of 12 Salmonella serovar Choleraesuis isolates from the Regional Medical Sciences Center in Samutsongkhram, Thailand. Ten of the isolates were ESC producers isolated in 2007 and 2008 from eight Thai patients from Ratchaburi province.

In addition, to assess the genetic diversity of Salmonella serovar Choleraesuis in Thailand, 11 Thai isolates from 2003, 2007, and 2008 (nine and two isolates from collections 2 and 3, respectively), which were susceptible to extended-spectrum cephalosporins, were included in the study. These non-ESC-producing isolates were all isolated from patients in Ratchaburi province and Bangkok and were randomly selected from the collection.

Serotyping.

The isolates were serotyped using slide agglutination in the country of origin.

O and H antigens were characterized by agglutination with hyperimmune sera (S & A Reagents Lab, Ltd, Bangkok, Thailand, and Statens Serum Institut, Copenhagen, Denmark), and serotype was assigned according to the Kauffmann-White scheme (16).

Antimicrobial susceptibility testing.

MIC testing of all 24 isolates was performed at the DTU-Food using previously described methods (20). Results were primarily interpreted using current European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.org) and European Food Safety Authority (EFSA) epidemiologic breakpoints (14). Due to the absence of some breakpoints in the EUCAST system, exceptions were made for the interpretation of cefepime and ceftriaxone, where Clinical and Laboratory Standards Institute guidelines and clinical breakpoints were utilized (7, 8, 9). Quality control using Escherichia coli ATCC 25922 was conducted according to CLSI guidelines.

Microarray.

DNA for the microarray analysis was prepared from bacterial cultures of the 13 ESC-producing isolates using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany). Detection of gene groups associated with the antimicrobial resistance phenotypes was carried out using miniaturized microarrays (Identibac Amr-ve array tubes; New Haw, Addlestone, Surrey, United Kingdom) containing probes for most relevant Gram-negative antimicrobial gene groups such as quinolone, sulfonamide, tetracycline, class 1/2 integrase, aminoglycoside, carbenicillinase, chloramphenicol exporter/acetyltransferase, florfenicol, trimethoprim, plasmidic AmpC, and beta-lactam groups. Analysis was performed as described by the manufacturer on the 13 ESC-producing isolates.

Detection of genes conferring resistance to extended-spectrum cephalosporins.

PCR amplification and sequencing of the blaCTX-M-9 group, blaTEM, and blaCMY-2 genes were performed on the 13 ESC-producing isolates using methods described previously (20, 28).

Amplicons produced were selected for sequencing. Prior to sequencing, the amplicons were purified using the GFX PCR DNA kit (GE Healthcare, Chalfont St. Giles, United Kingdom) following the protocol of the manufacturer. The DNA was shipped to Macrogen Inc., Seoul, South Korea, for sequencing using the same primers as those in the PCR analysis. Sequence analysis and alignment were performed using Vecton NTI suite 9 (InforMax Inc., Bethesda, MD) software. The resulting nucleotide sequences were compared to sequences obtained from the catalogue of unique beta-lactamases (http://www.lahey.org/studies/webt.html) referencing the sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/GenBank/index.html).

Plasmid characterization.

Plasmid DNA was extracted using the Qiagen plasmid minikit (Qiagen, Hilden, Germany). The plasmid DNA was transferred into electrocompetent E. coli DH10B cells and subjected to S1 nuclease PFGE as described below to ensure that only one plasmid had been transferred into the competent cells as well as to estimate the approximate sizes of the plasmids carrying the ESC phenotype. The electroporation was followed by selection of transformants on brain heart infusion (BHI) agar supplemented with cefotaxime (2 μg/ml). The presence of the plasmid in the transformants was confirmed by PCR detection of relevant bla genes as described above. Additionally, testing was performed to determine if any non-extended-spectrum cephalosporin resistance determinants cotransferred with the ESC plasmids. The 13 transformants were analyzed by PCR for all relevant resistance genes based on the results of the array tube analysis described above. Transformants were further subjected to replicon PCR and plasmid purification using Qiagen Tip-100 as described by the manufacturer (Qiagen, Hilden, Germany) followed by restriction fragment length polymorphism (RFLP).

RFLP was performed on purified plasmids digested with HincII (Promega, Madison, WI) and run on an 0.8% 20-cm agarose gel for 4 h at 100 V.

Plasmids within transformants were replicon typed as described previously (2).

Transferability of bla genes by conjugation.

Plate mating experiments were performed with transformants as donors and plasmid-free, rifampin- and nalidixic acid-resistant E. coli MT102RN cells as recipients (3, 28). The strains were grown to both late exponential and stationary phases, mixed (1:1), and incubated on solid blood agar at 37°C for 18 h. Transconjugants were selected on BHI medium supplemented with 50 μg/ml rifampin, 32 μg/ml nalidixic acid, and 2 μg/ml cefotaxime.

S1 digestion for plasmid size determination.

PFGE with S1 nuclease (Promega, Madison, WI) digestion of whole genomic DNA performed as described below was used to estimate sizes of larger plasmids. Following preincubation for 10 min in 1:10 diluted S1 buffer, 2-mm slices of PFGE plugs made from cultures with an optical density at 620 nm (OD620) of 0.6 were digested with 5 U of S1 (Promega, Madison, WI) for 45 min at 37°C. The slices were postincubated on ice for 10 min in 200 μl of ice-cold Tris-EDTA (TE) buffer (10:1), loaded on the gel, and run on a CHEF-DR III device (Bio-Rad, Hercules, CA) with a pulse time of 6.8 s to 38.4 s at 6 V/cm for 19 h. Salmonella serovar Braenderup H9812 digested with XbaI was used as a size marker.

Pulsed-field gel electrophoresis.

All 24 isolates included in this study were analyzed for genetic relatedness by PFGE using XbaI according to the CDC PulseNet protocol (29). The electrophoresis was performed with a CHEF-DR III System (Bio-Rad Laboratories, Hercules, CA) using 1% SeaKem agarose in 0.5× Tris-borate-EDTA (TBE) at 180 V. Running conditions consisted of one pulse time of 2.2 to 63.8 s for 22 h at 6 V/cm on a 120° angle in 14°C TBE buffer. Comparison of the PFGE profiles was performed by using Bionumerics software version 4.6 (Applied Maths, Sint-Martens-Latem, Belgium) and the Dice correlation for band matching with a 0.9% position tolerance and an optimization at 0.9%, both using XbaI.

RESULTS

Epidemiological data for the Thai patients.

Twenty-three Thai isolates were obtained from 21 Thai patients, with two patients each having two positive blood cultures. Thirteen of the isolates were obtained in Ratchaburi province, and 10 were from Bangkok. The 23 Thai strains were all equally isolated throughout the year whereas the 13 ESC-producing isolates were collected between April and May or between August and October. All of the 21 patients were unevenly distributed by gender, with 17 isolates being obtained from males and four from females. The ages of five patients were unknown. However, the ages of the remaining 16 patients ranged from 5 to 58 years with a median of 34 years (Fig. (Fig.1).1). Data on occupations of the patients were not collected.

FIG. 1.
Dendrographic analysis of PFGE (XbaI) data for Salmonella Choleraesuis isolates from Thailand and Denmark. Black and gray spots in the dendrogram represent the isolates as being either resistant or intermediate, respectively. Antimicrobials are abbreviated ...

Epidemiological investigations of the Danish patient.

A healthy 37-year-old Danish male was on assignment to an industrial company in Bangkok from 27 July to 14 August in 2008. He resided in a five-star international hotel at Sukhumvit Road in the center of Bangkok and worked full time at the Navanakorn Industrial Estate Zone 3 situated 45 km outside Bangkok. Meals were primarily served in the hotel and at the workplace. Typical Thai food (soup and rice dishes) was consumed daily and included either fish or minced pork.

One week before his return to Denmark, the patient contracted diarrhea with an acute onset but without blood. The patient was febrile up to 38.8°C with dizziness, cephalgia, and mild muscle pain. Concurrently, the patient noticed flexor paresis of the interphalangeal joint of the left thumb accompanied by hypesthesia in the C6 dermatome (reduced sensibility to touch at the upper skin of the thumb). Except for paresis and hypesthesia, the symptoms abated in a few days and the diarrhea responded to loperamide. However, symptoms recurred several times and the patient was admitted to a Danish hospital on 10 September with a weight loss of 11 kg. A blood culture obtained by admission revealed Gram-negative rods on 12 September, and in response treatment with ciprofloxacin was commenced but was switched to pivmecillinam on the next day after notification of the finding of Salmonella spp. resistant to fluoroquinolone. A shift to meropenem was considered based on the multidrug-resistant phenotype of the isolate, but the patient felt well and he declined. Eight months later the patient was in good health, although the flexor paresis persisted.

Antimicrobial resistance.

All of the 13 ESC-producing isolates were multidrug resistant and exhibited resistance to at least 13 of the tested antimicrobials (eight antimicrobial drug classes) (Fig. (Fig.1).1). Resistance was not detected to apramycin (approved only for veterinary use), colistin, imipenem, meropenem, and trimethoprim. All 13 isolates were resistant to ampicillin, cefalothin, cefazolin, cefotaxime, cefpodoxime, ceftiofur, nalidixic acid, sulfamethoxazole, and tetracycline. Twelve isolates (92%) were resistant to chloramphenicol; four (31%) of these strains also were resistant to florfenicol (approved only for veterinary use). Resistance to ciprofloxacin was observed in 12 (92%) isolates; one isolate had a MIC just below the breakpoint of ciprofloxacin. Eleven (85%), four (31%), four (31%), and four (31%) of the isolates were resistant to ceftriaxone, ceftazidime, cefepime, and cefoxitin, respectively (Fig. (Fig.1).1). Four isolates (31%) were also resistant to amoxicillin-clavulanic acid, and another six isolates (46%) were resistant to gentamicin. Only two isolates (15%) were resistant to neomycin, whereas 11 (85%) and 10 (77%) isolates were resistant to spectinomycin and streptomycin, respectively.

In contrast, the remaining 11 non-ESC-producing isolates exhibited resistance to only nine different antimicrobials tested. All of the 11 isolates conferred resistance to streptomycin (100%) and sulfamethoxazole (100%). Resistance to ciprofloxacin and nalidixic acid was observed in seven (64%) isolates. Nine, six, three, two, and two isolates were resistant to tetracycline (82%), ampicillin (55%), neomycin (27%), streptomycin (18%), and gentamicin (18%), respectively (Fig. (Fig.1).1). Four isolates (36%) were resistant to chloramphenicol; one (9%) of these strains also was resistant to florfenicol. One of the non-ESC-producing isolates showed resistance to both trimethoprim (9%) and apramycin (9%) (Fig. (Fig.11).

Identification of resistance genes in the ESC-producing isolates.

Based on array tube analysis, 8 of the 13 ESC-producing isolates harbored the extended-spectrum β-lactamase gene group blaCTX-M-9, the chloramphenicol resistance acetyltransferase gene cmlA, the sulfonamide resistance gene sul3, the aminoglycoside resistance gene aadA1, and the tetracycline resistance gene tetB (Fig. (Fig.11).

Of the remaining five isolates, four contained the floR gene, conferring resistance to florfenicol; the sulfonamide resistance gene sul2; the aminoglycoside resistance genes strA and strB; the plasmidic ampC gene blaCMY; and the tetracycline resistance gene tetA. In addition, one (SH508/03) of the four isolates also harbored the tetB and sul1 genes. Furthermore, three (SH2870/08, SH2874/08, and SH508/03) of the four isolates contained also the aminoglycoside resistance gene aadA1 (Fig. (Fig.11).

One isolate (SH1208/03) was resistant to sulfamethoxazole and tetracycline, containing the sul1 and tetB genes, respectively. In addition, this isolate harbored the extended-spectrum β-lactamase gene group blaCTX-M-9 and the blaTEM gene (Fig. (Fig.11).

The isolates which harbored the blaCTX-M-9 group, blaCMY, and blaTEM genes according to the microarray results were confirmed by PCR and subsequently sequenced and showed 100% similarity to strains in GenBank carrying blaCTX-M-14, blaCMY-2, and blaTEM-1b, respectively.

Plasmid characterization of the 13 ESC-producing isolates.

The S1 nuclease assay revealed the sizes of the plasmids responsible for ESC production to range from approximately 75 kb to 200 kb (Fig. (Fig.22).

FIG. 2.
Dendrographic analysis of plasmid replicons from Salmonella Choleraesuis transformants originating from Thai and Danish patients and subjected to RFLP. Plasmid sizes are in kb. Data with the same numbers of asterisks are from the same patient.

Plasmid profiling by RFLP separated the plasmids into three distinct clusters (I, II, and III in Fig. Fig.2).2). Furthermore, one plasmid profile (from strain SH1208/03) was not associated with any of the three main groups. The plasmid with blaCMY-2 genes was associated with cluster I, while plasmids harboring the blaCTX-M-14 gene belonged to the two remaining clusters as well as the plasmid out of line with the three clusters (Fig. (Fig.2).2). Replicon typing identified the incA/C replicon in RFLP cluster I, incFrepB in cluster II, and incFIIA in cluster III, while the plasmid from SH1208/03 was untypeable by the PCR method.

PCR results revealed that only one (SH2862/08) of the eight ESC-producing isolates which harbored the extended-spectrum β-lactamase gene group blaCTX-M-14 successfully cotransferred antimicrobial resistance determinants other than ESC determinants. In addition to blaCTX-M-14, the transformant contained the sul3, aadA1, and tetB genes.

The four transformants containing plasmid ampC gene blaCMY-2 seemed to harbor many of the other antimicrobial resistance determinants. All four transformants contained the floR, sul2, strA/strB, and tetA genes. In addition, one (SH508/03) of the four transformants also contained the sul1 gene, and three transformants (SH2870/08, SH2874/08, and SH508/03) contained the aadA1 gene.

The transformant of one isolate (SH1208/03) which harbored blaCTX-M-14 also contained the blaTEM gene.

Conjugation experiments on the 13 ESC-producing isolates.

By conjugation experiments we found the ESC phenotype to be readily transferable from wild-type strains carrying plasmids belonging to RFLP cluster I and cluster II as well as from isolate SH1208/03. However, the four strains carrying the incFIIA-type plasmids of RFLP cluster III could not be transferred by conjugation.

PFGE typing.

The 24 Salmonella serovar Choleraesuis isolates from 22 patients were subtyped by PFGE. Sixteen unique XbaI PFGE patterns were observed (Fig. (Fig.1).1). There were five distinct PFGE clusters with ≥2 indistinguishable isolates. Three clusters contained ESC-producing isolates, of which two included two indistinguishable isolates from different Thai patients (SH2858/08 and SH2867/08; SH2870/08 and SH2874/08). The third cluster of ESC-producing isolates contained three isolates with indistinguishable patterns, two isolates from one Thai patient (SH2871-08 and SH2872-08) (different susceptibility profiles) and the isolate (08-120226) from the Danish traveler to Thailand. The two remaining clusters contained only isolates susceptible to extended-spectrum cephalosporins, of which one included isolates from both 2003 and 2008.

DISCUSSION

This study provides the first description of blaCTX-M-14 in Salmonella serovar Choleraesuis isolates and is the first reported isolation of an ESC-producing Salmonella serovar Choleraesuis isolate in an international traveler who recovered with sequelae from his infection. In addition, these data provide evidence that ESC-producing isolates have emerged in Thailand on several plasmids and in several clusters of Salmonella Choleraesuis.

Characterization of the antimicrobial resistance genes indicates some similarity among isolates harboring either blaCTX-M-14 or blaCMY-2. The data from the plasmid characterization, conjugation, replicon typing, and RFLP also suggested that these are not highly clonal strains and further grouped the isolates into four distinct replicon clusters. Based on the data of the unknown replicon, one could speculate if the plasmid of isolate SH1208/03 was the ancestor to the other isolates harboring the blaCTX-M-14 gene and simply evolved rather than spread to other strains. All of the analyses indicate that multiple clones and multiple plasmids are responsible for extended-spectrum cephalosporin resistance among Salmonella serovar Choleraesuis isolates obtained from patients in Thailand and Denmark. All of the analyses indicate multiple clones and multiple plasmids being responsible for the resistance to extended-spectrum cephalosporins found in Salmonella serovar Choleraesuis isolates obtained from patients in Thailand and Denmark.

Several studies have described plasmids carrying blaCMY-2 containing the incA/C replicon along with other resistance genes. A recent Canadian study investigated 38 E. coli isolates where all of the isolates harbored a plasmid carrying blaCMY-2 containing the incA/C replicon (27). A similar association between blaCTX-M-14 and incFII has been described previously. Marcadé et al. found that the great majority of genes carrying blaCTX-M-14 and blaCTX-M-15 were carried by incF replicons. Of 15 E. coli isolates harboring blaCTX-M-14, eight of them contained the incFII replicon (26).

PFGE and antimicrobial resistance patterns revealed a high degree of clonal diversity among the 24 isolates. ESC-producing and non-ESC-producing isolates were generally interspersed, although some rare clusters comprised solely resistant or susceptible isolates. This indicates that the emergence of resistance is not a recent spread of a single clone.

Salmonella serovar Choleraesuis has been eradicated from the primary production of swine in Denmark and many other industrialized countries. The isolate from the Danish traveler shared an identical PFGE pattern with an isolate from a Thai patient infected in the autumn of 2008. The isolates were resistant to the same antimicrobials and harbored the same resistance genes with the exception of two additional resistance traits in the isolate from the Danish patient, namely, resistance to amoxicillin-clavulanic acid and gentamicin. We have no explanation for this discrepancy because the Danish patient received only symptom therapy prior to admission to the hospital in Denmark.

The Danish case was remarkable for neurologic symptoms localized to the left hand which coincided with diarrheal illness and persisted for at least 9 months. There was no evidence of focal infection, but the similarity with mononeuropathy in association with typhoid fever may indicate a common pathogenesis (15).

Thailand is a popular tourist destination for Europeans. Thus, in 2008 149,570 Danes visited Thailand. During the same year, 3,022 confirmed cases of Salmonella infections in humans were reported to the Statens Serum Institut, Denmark. Of these, 706 (23.3%) were confirmed as travel associated and 95 (13.4%) cases were linked to traveling to Thailand (data not published). Overall, 0.06% of the Danes visiting Thailand might bring back a Salmonella infection based on these data, but the number is believed to be underestimated. In Denmark, the level of underestimation of gastrointestinal diseases has not been investigated. However, British data on an underestimation of 10 to 20 times are often referred to in Denmark (32).

The infections with Salmonella serovar Choleraesuis have recently increased in Thailand, and the emergence of ESC-producing Salmonella serovar Choleraesuis isolates makes this problem even more serious (19). We therefore urge the Thai authorities to take action in order to prevent and control the spread of this serovar among animals and the human population. Targeted interventions can benefit swine farmers by reducing losses and possible export restrictions. These interventions can also reduce the high costs of hospitalization associated with treatment of invasive Salmonella serovar Choleraesuis infections, which include the necessity of using carbapenems, which are antibiotics of last resort. We recommend enforcing a strict policy on the usage of antimicrobials in food animals and a ban on the usage of extended-spectrum cephalosporin (ceftiofur) as a growth promoter in Thailand.

This study provides for the first time a description of blaCTX-M-14 found in Salmonella serovar Choleraesuis isolates and documents a case of bacteremia with an ESC-producing Salmonella serovar Choleraesuis isolate acquired by a Danish traveler during a stay in Bangkok. The data suggest that ESC-producing isolates have emerged in Thailand on several plasmids and in multiple clones of Salmonella serovar Choleraesuis.

We found two genes and four replicons responsible for the resistance to extended-spectrum cephalosporins present in the isolates from both 2003 and 2008. In addition, the isolates exhibit a huge diversity among the molecular patterns, indicating a variable population despite similar resistance patterns and genes.

The Thai authorities should initiate immediate actions to control and prevent infections with this invasive serovar for the benefit of the Thai people and tourists traveling to Thailand. The first step could be specific serovar-targeted intervention and limitations on antimicrobial usage in the production of food animals.

Acknowledgments

Permission to include clinical information was obtained from the Danish patient and the Department of Infectious Diseases, Aalborg Hospital.

We are grateful to Christina Aaby Svendsen and Lisbeth Andersen (National Food Institute) for outstanding technical assistance, to Matthew Mikoleit (Centers for Disease Control and Prevention) for reviewing the manuscript and improving the English, and to Steen Ethelberg (Statens Serum Institut, Denmark) for providing data on travel-associated infections in Danish patients.

This work was supported by the World Health Organization Global Salm-Surv (www.who.int/salmsurv).

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 December 2009.

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