JAC Advance Access originally published online on March 21, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):1040-1042; doi:10.1093/jac/dkm075
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Correspondence |
Occurrence of CTX-M-1-producing Escherichia coli in pigs treated with ceftiofur
1 Department of Veterinary Pathobiology, Faculty of Life Science, University of Copenhagen, Frederiksberg C, Denmark 2 National Food Institute, Technical University of Denmark, Søborg, Denmark
* Corresponding author. Tel: +45-35282745; Fax: +45-35282755; E-mail: lg{at}life.ku.dk
Keywords: extended-spectrum ß-lactamases , cephalosporins , Enterobacteriaceae , antimicrobial resistance , swine
Resistance to cephalosporins due to extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is an increasing clinical and economical problem in human medicine.1 Various reports have recently documented the occurrence of ESBL-producing Enterobacteriaceae in food-producing animals,2–4 raising concern about the possible role played by food in the spread of ESBLs in the community. In this study, we investigated the possible impact of ceftiofur, a third-generation cephalosporin approved for therapeutic use in swine and cattle, on the occurrence of cephalosporin-resistant and ESBL-producing Escherichia coli in pigs. Twenty Danish farms were selected according to information on ceftiofur usage, including 10 farms using ceftiofur and 10 control farms without a history of ceftiofur usage in the 6 months prior to sampling (July–August 2006). Despite the fact that ceftiofur is registered for treatment of respiratory disease, the drug was used for other indications, i.e. systematic prophylaxis in 1-day-old piglets (eight farms) and treatment of diarrhoea (one farm) or arthritis (one farm). At each farm, faecal samples were taken from 10 piglets and 10 slaughter pigs, respectively. Following culture on MacConkey agar plates containing cefotaxime (2 mg/L), E. coli with reduced susceptibility to cefotaxime were obtained from 69 animals at five farms using ceftiofur and from 3 animals at one control farm. Statistical analysis showed a significant association (P = 0.02) between ceftiofur treatment in piglets and occurrence of E. coli with reduced susceptibility to cefotaxime. The analysis was carried out including data from both piglets and slaughter pigs (396 animals in total), and animal age was found not to be a statistically significant factor.
One colony with typical E. coli morphology was selected from each positive sample/animal. A total of 60 E. coli isolates were identified by biochemical tests (indole, methyl red, Voges–Proskauer, citrate) and analysed for ESBL production by the double disc method using cefotaxime and cefotaxime combined with clavulanic acid. ESBL production was demonstrated in 19 isolates originating from two farms (Table 1). All 19 isolates were resistant to cefotaxime and ceftiofur (MIC >64 mg/L) but susceptible to ceftazidime and cefoxitin. Sequencing of PCR products obtained using primers targeting TEM5 and CTX-M6 genes revealed that all 19 isolates carried CTX-M-1 and 5 of them additionally harboured TEM-1B. The CTX-M-1-positive isolates displayed distinct PFGE types and antimicrobial resistance patterns (Table 1), suggesting horizontal gene transfer between distinct E. coli strains rather than clonal spread within farms. The remaining 41 isolates had lower resistance to ceftiofur (MIC = 8–16 mg/L) and did not exhibit an ESBL phenotype by the disc diffusion test. Such isolates were not inhibited by clavulanic acid in combination with cefotaxime and were resistant to cefoxitin. Sequencing of the ampC promoter region of eight randomly selected isolates confirmed changes in the promoter region at positions –42, –18, –1 and +58, which have previously been shown to result in up-regulation of AmpC production.7
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The study demonstrated a statistically significant association between the use of ceftiofur and reduced susceptibility to cefotaxime due to production of either AmpC or CTX-M-1 ß-lactamase. Although the association between ceftiofur use and occurrence of CTX-M-1-producing E. coli was not statistically significant, such bacteria were only detected in farms using ceftiofur and the lack of statistical significance could be due to the relatively low number of farms that were included in the study. Furthermore, the actual occurrence of CTX-M-positive isolates could have been underestimated by only analysing a single isolate from each animal. The statistical analysis did not take into account factors that may have had a significant effect on the observed occurrence of resistance, namely the use of other antimicrobials that can cross- or co-select for cephalosporin resistance. E. coli-producing CTX-M-1 have been recently isolated from two infected pigs reared at Danish farms, with a history of cephalosporin usage.3 Associations between ceftiofur use and isolation of E. coli with reduced susceptibility to ceftriaxone have been reported in dairy cattle farms in the USA.4 These data indicate that usage of cephalosporins in animals selects for the occurrence of cephalosporin-resistant E. coli in animals. Further investigation is required to understand the magnitude of this selection and to assess the risk of zoonotic transmission via the food chain and by contact with animals. Preliminary results of a survey on the occurrence of ESBLs among E. coli isolates from Danish patients indicate that the majority of the ESBLs in human clinical strains belong to the CTX-M type (N. Frimodt-Møller, Statens Serum Institut, Denmark, personal communication). Thus, it is possible that CTX-M genes are exchanged between porcine and human E. coli populations, but the relative contribution of cephalosporin use in animals to the occurrence of these genes in strains causing human infections remains to be determined.
Off-label use of ceftiofur should be avoided in pig production to limit the spread of ESBL-producing bacteria and to preserve the efficacy of cephalosporins in both human and veterinary medicine. Prophylactic use of ceftiofur by a single injection in 1-day-old piglets and treatment of porcine bacterial infections other than respiratory disease should be discouraged because in addition to deleterious effects on selection for resistance, the clinical efficacy of this antimicrobial drug has not been documented for these indications. In Denmark, ceftiofur is only registered for treatment of respiratory diseases. Data from the Danish programme for surveillance of antimicrobial resistance in bacteria from livestock, foods and humans (DANMAP) show that consumption of ceftiofur in pig production increased markedly over the last 5 years and that 
80% of the total amount prescribed for pigs in 2005 was used in sows/piglets.8 The large amount prescribed for sows/piglets strongly indicates that off-label use is widespread since bacterial respiratory diseases are relatively uncommon in sows and piglets compared with slaughter pigs.
None to declare.
Acknowledgements
The work by Lina Cavaco was supported by a grant from the EU Marie Curie Early-Stage Training (EST) programme TRAINAU (MEST-CT-2004-007819).
References
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8 DANMAP. (2005) Use of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Bacteria From Food Animals, Food and Humans in Denmark ISSN 1600-2032. http://www.danmap.org 21 December 2006, date last accessed.
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