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JAC Advance Access originally published online on May 24, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):398-401; doi:10.1093/jac/dkm168
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Rapid and widespread dissemination of multidrug-resistant blaCMY-2 Salmonella Typhimurium in Mexico

Mussaret B. Zaidi1,*, Verónica Leon1, Claudia Canche1, Carolina Perez1, Shaohua Zhao2, Susannah K. Hubert2, Jason Abbott2, Karen Blickenstaff2 and Patrick F. McDermott2

1 Depto. de Investigacion, Laboratorio de Investigación, Hospital General O'Horan, Av. Itzaes x Jacinto Canek, Mérida C.P. 97000, Yucatan, Mexico 2 Center for Veterinary Medicine, Food and Drug Administration, Laurel, MD, USA

* Corresponding author. Tel/Fax: +52-999-923-86-73; E-mail: mbzaidi{at}prodigy.net.mx or funsalud{at}yuc.quik.com

Received 29 January 2007; returned 27 February 2007; revised 12 April 2007; accepted 22 April 2007


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Objectives: We describe the emergence and dissemination of multidrug-resistant (MDR) Salmonella Typhimurium in humans, retail meat and food animals from Yucatan, Mexico.

Methods: Salmonella Typhimurium isolates were collected through an active surveillance system and tested for susceptibility to 12 antimicrobial agents. Isolates that were non-susceptible to ceftriaxone were tested with 10 additional antimicrobials and assayed by PCR for the presence of CMY, CTX-M, SHV, TEM and OXA ß-lactamase genes. Plasmid-borne phenotypes were identified by transfer to susceptible Escherichia coli. Isolates from humans, retail meat and food animals were compared by PFGE to determine genetic relatedness.

Results: MDR Salmonella Typhimurium containing a plasmid-mediated blaCMY-2 AmpC ß-lactamase rose from 0% (0/27) during 2000 and 2001 to 75% (63/84) in 2004 and 2005 (P < 0.0001). MDR blaCMY-2 Salmonella Typhimurium (n = 115) was most common in ill children (44.3%) and pork or swine intestine (36.5%). In several cities, MDR blaCMY-2 Salmonella Typhimurium from retail meat or swine intestine exhibited PFGE patterns and antibiograms indistinguishable from those in strains recovered from hospitalized children. The CMY gene was transferred to E. coli by electroporation, along with resistance to three to six other antimicrobials. Children with MDR blaCMY-2 Salmonella Typhimurium infection (n = 39) had a higher frequency of systemic infection (13% versus 0%), mortality (8% versus 0%) and hospital re-admission due to protracted diarrhoea (28% versus 17%) than children with non-MDR-Salmonella Typhimurium (n = 24), although the difference was not statistically significant.

Conclusions: The rapid and widespread dissemination of MDR blaCMY-2 Salmonella Typhimurium in Mexico calls for urgent interventions to contain this potentially fatal pathogen.

Keywords: swine , humans , PFGE , mortality , developing countries


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During the last two decades, Salmonella enterica subsp. enterica, in particular, serovar Typhimurium, has become increasingly resistant to antimicrobial compounds worldwide. During the 1980s, Salmonella Typhimurium definitive phage type (DT) 104 with resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline (ACSSuT resistance type) emerged in the UK and subsequently disseminated throughout Europe and North America. By the 1990s, Salmonella Typhimurium (both DT 104 and non-DT 104) had acquired additional resistance to trimethoprim–sulfamethoxazole, ciprofloxacin and extended-spectrum cephalosporins (ESCs).1 Although the ACSSuT phenotype, which is linked to a complex class I integron contained within Salmonella Genomic Island I, was the most prevalent multidrug-resistant (MDR) Salmonella phenotype during the 1980s and 1990s,1 the recent emergence of MDR Salmonella with AmpC-like ESC resistance represents a new challenge. In North America, this ESC resistance is mediated by a plasmid-borne CMY-2 ß-lactamase and has spread horizontally to different serotypes. These plasmids frequently harbour multiple resistance determinants to other antimicrobial classes.2,3

The emergence of MDR, ESC-resistant Salmonella is particularly worrisome, because clinicians are left with very few options for effectively treating severe infections. Furthermore, previous studies have shown that antibiotic-resistant Salmonella Typhimurium is associated with an increased frequency of bloodstream infection and mortality.4,5 This study is one of the few to describe the emergence and widespread dissemination of MDR blaCMY-2 Salmonella Typhimurium in a developing country such as Mexico, as well as the outcome of affected children.


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We report the results obtained from 2000 to 2005 for an integrated surveillance system of the food chain in Yucatan, Mexico, that included samples from humans, retail meat and food-animal intestines.6 Sampling of meat and food-animal intestines was designed to reflect regional consumption of each meat product. Data on disease severity and outcome were obtained from the hospital records of children admitted for Salmonella Typhimurium infection. The study was approved by the Hospital General O'Horan Internal Review Board and written informed consent was obtained from all parents or guardians. Statistical testing of differences in proportions was conducted using the {chi}2 test; P values less than 0.05 were considered significant.

Methods for isolation, identification, susceptibility testing, serotyping and PFGE of Salmonella have been described previously.6 ESC-resistant Salmonella Typhimurium isolates were subjected to PFGE analysis and examined for the presence of CTX-M, CMY and the combination of SHV, TEM and OXA by PCR, as described by others.7,8 DNA sequences were determined and used to screen the GenBank database using the BLAST algorithm available at the National Center of Biotechnology Information's web site (http://www.ncbi.nlm.nih.gov/BLAST/). Transfer experiments were carried out on a subset of 4 isolates, which were selected from a total of 48 isolates on the basis of distinct plasmid RFLP patterns using PstI. Plasmid DNA was introduced by electroporation into Escherichia coli DH10B (EP-Max 10B) (Invitrogen, Carlsbad, CA, USA), and transformants were selected using ceftriaxone (4.0 mg/L). Transformed host cells were tested for antimicrobial susceptibility as before and assayed for the acquisition of CMY using PCR.


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The number of human, retail meat and food-animal intestine samples positive for Salmonella Typhimurium and MDR blaCMY-2 Salmonella Typhimurium is shown in Table 1. A total of 2431 Salmonella were recovered from 7206 samples, of which 177 (7.3%) isolates were serovar Typhimurium. Of the 177 Salmonella Typhimurium isolates, 115 (65%) were ESC-resistant. Most of these were recovered from ill children (44.3%) and from pork or swine intestine (36.5%). The prevalence of MDR blaCMY-2 Salmonella Typhimurium among all Salmonella Typhimurium isolates rose from 0% (0/27) during 2000 and 2001 to 75% (63/84) in 2004 and 2005 (P < 0.0001, {chi}2 for trend).


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  		Table 1. Prevalence of Salmonella Typhimurium and MDR blaCMY-2 Salmonella Typhimurium in humans, retail meat and food animals from Yucatan, Mexico, 2000–05

 
The ceftriaxone MIC50 and MIC90 for these isolates were 64 and 128 mg/L, respectively (range 16 to >128 mg/L). In addition to the ACSSuT phenotype, these isolates were non-susceptible to piperacillin, ticarcillin, cefoxitin, ceftazidime, cefotaxime, ceftiofur and aztreonam and did not show increased susceptibility in the presence of clavulanic acid. An important percentage of these strains was also non-susceptible to trimethoprim–sulfamethoxazole (87%), nalidixic acid (58%), gentamicin (44%) and kanamycin (23%) (Figure 1). All isolates were susceptible to cefepime, imipenem, ciprofloxacin and amikacin.


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  		Figure 1. PFGE patterns for MDR blaCMY-2 Salmonella Typhimurium from humans, retail meat and food animals in Mexico from 2002 to 2005. Isolates were collected from humans with systemic infection (HS), humans with symptomatic enteric infection (HE), humans with asymptomatic enteric infection (HA), chicken meat (CM), pork (PM), beef (BM) and swine intestine (SI) from 30 cities throughout the state of Yucatan. Non-susceptibility to antimicrobials, indicated by a black box, was present for ampicillin (AMP), cefoxitin (FOX), ceftriaxone (CRO), chloramphenicol (CHL), gentamicin (GEN), kanamycin (KAN), nalidixic acid (NAL), streptomycin (STR), sulfisoxazole (SSS), tetracycline (TET), trimethoprim/sulfamethoxazole (SXT) and ceftiofur (TIO). Clusters A–H, highlighted in boxes, indicate strains from humans and retail meat and/or swine intestine with indistinguishable PFGE patterns. Clusters B, E, F and G contain four of the five isolates from systemic infections. The fifth extraintestinal isolate (35 733) was closely related (97%) to strains from cluster C that included isolates from human enteric infection (32 090) and beef (35 423). Clusters 1–13, marked to the left of the dendrogram with numbered lines, indicate isolates of human or animal origin only. Most of the clusters contain isolates from identical or neighbouring cities. NA, not applicable.

 
Of the 115 ESC-resistant isolates, 113 were available for molecular testing. The blaCMY-2 gene was identified in all of these. With the exception of a single isolate (CVM34474) that also carried TEM-1, none of the other assayed bla genes was detected. The CMY ß-lactam resistance phenotype was transferred by electrotransformation of purified plasmid DNA to E. coli DH10 recipient, along with different patterns of resistance to sulfamethoxazole, chloramphenicol, tetracycline, gentamicin, trimethoprim–sulfamethoxazole and kanamycin. For transformant 35417-T, the MIC of ceftriaxone increased 8-fold relative to the wild-type donor strains. Plasmids from this strain type were also distinct in that trimethoprim–sulfamethoxazole, tetracycline and chloramphenicol resistances failed to transfer to the E. coli recipient.

PFGE analysis of the 113 MDR blaCMY-2 Salmonella Typhimurium isolates revealed 21 clusters, each of which contained at least two strains with indistinguishable banding patterns. Most of the clusters contained isolates from identical or neighbouring cities. In 8 of the 21 clusters (clusters A–H), human isolates were found to be closely associated with those from swine intestine or retail meat. (Figure 1).

Clinical charts were available for 63 children with Salmonella Typhimurium infection. Children with MDR blaCMY-2 Salmonella Typhimurium infection (n = 39) had a higher frequency of systemic infection (12.8% versus 0%, P = 0.15), mortality (7.7% versus 0%, P = 0.28) and hospital re-admission due to protracted diarrhoea (28% versus 16.6%, P = 0.46) than children with non-MDR blaCMY-2 Salmonella Typhimurium (n = 24); however, we could not demonstrate statistical significance because of small sample size. Of the children with MDR blaCMY-2 Salmonella Typhimurium infection, two presented severe thrombocytopenia (2000 and 18 000 platelets/mm3, respectively), one of whom died from a cerebral haemorrhage; two other infants died from bloodstream infection.


   Discussion
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This study documents the emergence and rapid dissemination of MDR blaCMY-2 Salmonella Typhimurium in food animals and humans from Yucatan, Mexico. MDR blaCMY-2 Salmonella Typhimurium has become the predominant phenotype of serovar Typhimurium in the state, currently accounting for 75% of all Salmonella Typhimurium isolates; it is resistant to at least eight antimicrobials and has caused severe enteric and systemic infections in children, three of whom died. Molecular analysis enabled us to presumptively identify swine as the major source of these strains and to determine that ESC-resistance is mediated by a blaCMY-2 gene harboured on different plasmids.

Among food animals, the blaCMY-2 phenotype was only found in swine intestines, suggesting that the main selection pressure for ESC-resistant Salmonella Typhimurium in Yucatan originated and persists in swine production. Its presence in pork, beef and chicken meat is likely due to cross-contamination during slaughter or at retail. Although we made no effort to mobilize them by conjugation, our transformation experiments show that the blaCMY-2 gene resides on plasmids with differing resistance gene content and that they are able to replicate in E. coli. The extent to which CMY dissemination has been mediated by other genera is not known.

In North America, MDR blaCMY-2 salmonellae first emerged in Salmonella Typhimurium and later as part of the multiple resistance genotype in food animal and food-borne isolates of Salmonella Newport. In the USA, blaCMY-2 Salmonella has been primarily associated with cattle and beef, and in Canada, with turkey.2,3,9 Differences in animal reservoirs could be related to food-animal production practices, including the amount and type of antimicrobials used in each country. In both Mexico and the USA, blaCMY-2 Salmonella appears to have established itself in an animal reservoir before spreading to humans.

blaCMY-2 Salmonella is infrequent in countries outside North America, where ESC resistance is more commonly mediated by molecular class A ß-lactamases such as TEM, SHV, OXA and CTX.10 Possible explanations for this low frequency could be differences in the prevalence of specific serovars among countries, the lack of systematic screening for AmpC-type ß-lactamase genes in the veterinary sector and cephalosporin usage. Some investigators attribute the emergence of CMY-2 to the use of ceftiofur, a third-generation cephalosporin used exclusively in food animals.2,3 Although there are no reliable data in Mexico or the USA on the quantity of third-generation cephalosporins used, these compounds are readily available in veterinary and medical pharmacies throughout Yucatan and can be purchased without prescription (M. Zaidi, unpublished results).

It is noteworthy that systemic Salmonella Typhimurium infection and mortality were only seen in children with MDR blaCMY-2 strains. Moreover, the occurrence of severe thrombocytopenia with haemorrhagic syndrome, a very uncommon presentation of non-typhoidal salmonellosis, should be alerted to all physicians. Although we were unable to demonstrate a statistically significant association between MDR and increased severity of disease, our findings show a clinically significant trend that supports the growing body of literature that associates MDR Salmonella with increased morbidity.4,5

In conclusion, our findings highlight the rapid dissemination of MDR CMY-2-producing Salmonella in Mexico, a developing country that currently lacks the infrastructure for effective containment measures. Policy makers need to implement a series of urgent interventions to prevent and control selection of, and environmental contamination with, MDR Salmonella throughout the food chain. Foremost measures include tighter regulations for antimicrobial use and the establishment of an effective surveillance programme to monitor the public health impact of this emerging pathogen.


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Financial support was provided by the United States Food and Drug Administration (grant number FD-U-001934).


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None to declare.


   Acknowledgements
 
We wish to thank Dr Maria Teresa Estrada for critical review of the manuscript. This research was presented in part at the Forty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004 (Abstract C2-1717) and the 107th General Meeting of the American Society for Microbiology, Toronto, Canada, 2007 (Poster A-042).


   References
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1 Threlfall EJ. Epidemic Salmonella typhimurium DT 104-a truly international multiresistant clone. J Antimicrob Chemother (2000) 46:7–10.[Free Full Text]

2 Winokur PL, Brueggemann A, DeSalvo DL, et al. Animal and human multidrug-resistant, cephalosporin-resistant Salmonella isolates expressing a plasmid-mediated CMY-2 AmpC ß-lactamase. Antimicrob Agents Chemother (2000) 44:2777–83.[Abstract/Free Full Text]

3 Gupta A, Fontana J, Crowe C, et al. Emergence of multidrug-resistant Salmonella enterica serotype Newport infections resistant to expanded-spectrum cephalosporins in the United States. J Infect Dis (2003) 188:1707–16.[CrossRef][Web of Science][Medline]

4 Martin LJ, Fyfe M, Dore K, et al. Increased burden of illness associated with antimicrobial-resistant Salmonella enterica serotype typhimurium infections. J Infect Dis (2004) 189:377–84.[CrossRef][Web of Science][Medline]

5 Varma JK, Molbak K, Barrett TJ, et al. Antimicrobial-resistant nontyphoidal Salmonella is associated with excess bloodstream infections and hospitalizations. J Infect Dis (2005) 191:554–61.[CrossRef][Web of Science][Medline]

6 Zaidi MB, McDermott PF, Fedorka-Cray P, et al. Nontyphoidal Salmonella from human clinical cases, asymptomatic children, and raw retail meats in Yucatan, Mexico. Clin Infect Dis (2006) 42:21–8.[CrossRef][Web of Science][Medline]

7 Zhao S, White DG, McDermott PF, et al. Identification and expression of cephamycinase blaCMY genes in Escherichia coli and Salmonella isolates from food animals and ground meat. Antimicrob Agents Chemother (2001) 45:3647–50.[Abstract/Free Full Text]

8 Brinas L, Zarazaga M, Saenz Y, et al. ß-Lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob Agents Chemother (2002) 46:3156–63.[Abstract/Free Full Text]

9 Allen KJ, Poppe C. Occurrence and characterization of resistance to extended-spectrum cephalosporins mediated by ß-lactamase CMY-2 in Salmonella isolated from food-producing animals in Canada. Can J Vet Res (2002) 66:137–44.[Web of Science][Medline]

10 Miriagou V, Tassios PT, Legakis NJ, et al. Expanded-spectrum cephalosporin resistance in non-typhoid Salmonella. Int J Antimicrob Agents (2004) 23:547–55.[CrossRef][Web of Science][Medline]


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