ABSTRACT
A nationwide surveillance for antimicrobial susceptibility in Escherichia coli strains isolated from food-producing animals in Japan was conducted from 1999 to 2002. Eighteen cefazolin-resistant E. coli strains were isolated from broilers. Six were CTX-M-type producing, and eight were CMY-2 producing, while eight had mutations at the ampC promoter region.
Recently, the relationship between the use of antimicrobials in food-producing animals and the emergence of resistant bacteria in the food chain has become of great concern and has been the subject of numerous international meetings (6, 11, 12). However, until recently there was a lack of nationwide information available on antimicrobial resistance of bacteria isolated from animal origins. Consequently, we established the Japanese Veterinary Antimicrobial Resistance Monitoring program in 1999 (9).
In Japan, CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae are important in nosocomial infections. Yagi et al. reported that Toho-1-like ESBLs were the most prevalent type of ESBL in clinical isolates of Escherichia coli (13, 14). The aim of this study was to characterize cephalosporin-resistant E. coli strains recovered from healthy animals and especially to investigate isolates resistant to ceftiofur, an expanded-spectrum cephalosporin used in animals.
Fresh fecal samples were collected from healthy farm animals. In principle, one fecal sample per farm was collected and two E. coli isolates from each sample were kept using desoxycholate-hydrogen sulfate-lactose agar. Overall, a total of 2,747 isolates (872 isolates from 453 cattle farms, 793 isolates from 417 pig farms, 406 isolates from 219 layer farms, and 676 isolates from 354 broiler farms) were collected during 4 years (1999 to 2002).
MICs were determined by the agar dilution method (4, 5). The cefazolin MIC for 18 isolates from 12 broiler farms was ≥32 μg/ml, and these isolates were further investigated in this study. The MICs of 19 antibiotics for the 18 cefazolin-resistant isolates are shown in Table 1. The resistance profiles of isolates collected from the same farm were always identical to each other, suggesting that those isolates were likely replicates. Six isolates from four farms were also resistant to ceftiofur, cefpodoxime, cefotaxime, and cefepime while retaining susceptibility to cefoxitin. A double-disk synergy test for detection of ESBLs, carried out as described previously (3), revealed synergy between clavulanate and cefotaxime, ceftadizime, cefpodoxime, or aztreonam disks (Nissui Pharmaceutical, Co., Ltd, Tokyo, Japan) with these six isolates, suggesting production of an ESBL (Table 2). The remaining 12 isolates exhibited increased cefoxitin MICs while retaining very low cefepime MICs, suggesting the production of a class C β-lactamase. Double-disk synergy testing yielded negative results with these isolates (Table 2).
Detection of several β-lactamase genes, including blaTEM, blaSHV, blaPSE-1, blaCTX-M-2, blaCTX-M-9, blaCMY-1, blaCMY-2, and blaFOX, and amplification of the promoter region of the ampC gene were carried out by PCR (94°C for 3 min; 30 cycles of amplification at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and 72°C for 7 min) using primers listed in Table 3. Nucleotide sequences were determined on both strands, directly on PCR products. The DNA alignments and deduced amino acid sequences were examined using the BLAST program (1). Mutations in the ampC promoter region were defined in comparison with E. coli K-12 strain LA5 (7).
The results of β-lactamase gene detection and analysis of the ampC promoter region in the cefazolin-resistant isolates are reported in Table 2. Acquired β-lactamase genes were detected in most isolates. CTX-M-2 or CTX-M-18 β-lactamase genes were detected in the six ceftiofur-resistant isolates, in agreement with the resistance phenotype. The four CTX-M-2-producing E. coli strains were isolated from three different farms (farm F is 500 km away from farm G and 400 km away from farm K; farm G is 100 km away from farm K) in different years. A CMY-2 β-lactamase gene, alone or in combination with blaTEM-1 or blaPSE-1, was detected in eight of the other isolates, in agreement with the resistance phenotype. In the remaining four isolates, either a blaTEM-1 gene or none of the acquired β-lactamase genes searched in this work was detected. In these isolates, however, mutations at positions −42 (C→T), −18 (G→A), −1 (C→T), and +58 (C→T) were detected. Though we did not perform enzyme expression experiments, mutations at these points could be associated with AmpC hyperproduction (2) and thus explain the resistance phenotype.
Conjugation experiments were carried out as described previously (15) using a rifampin-resistant mutant of E. coli INVαF′ (Invitrogen Corp. Carlsbad, CA) generated in our laboratory. Transconjugants were selected on LB agar (Difco Laboratories, Detroit, MI) containing rifampin (50 μg/ml) and cefazolin (50 μg/ml). Cefazolin-resistant transconjugants were obtained from 14 isolates, including those producing CTX-M-type and CMY-2 enzymes. Resistance profiles of the transconjugants were consistent with transfer of a CTX-M-type or CMY-2 β-lactamase gene, respectively (Table 4). The presence of the respective β-lactamase genes was confirmed in all transconjugants by PCR analysis with primers encoding CTX-M types or CMY-2. The transfer of resistance traits to non-β-lactam agents was also observed in most cases (Table 4), suggesting that additional resistance genes were cotransferable with the β-lactamase genes.
Plasmid restriction profiles of the six CTX-M-producing transconjugants are shown in Fig. 1. Restriction profiles of plasmids carrying the same type of blaCTX-M gene were identical or similar to each other, suggesting a common origin. On the other hand, restriction profiles of plasmids carrying different types of CTX-M determinants were remarkably different from each other.
In our survey, cefazolin-resistant E. coli strains were isolated only from broilers. In Japan, six cephalosporins are approved for parenteral use, but in cattle and pigs only. Some reports discuss the relationship between the use of ceftiofur and the appearance of resistant strains in cattle and/or pigs (8, 10). However, our results suggest that the use of the expanded-spectrum cephalosporins in healthy animals at the farm level does not directly influence the appearance of resistant strains. For some reason, ESBL-producing E. coli strains were only isolated from broilers and not layers, which suggests there might be some other factor, possibly in their specific environment, that introduces the plasmids encoding CTX-M-type ESBLs into E. coli during the husbandry of broilers.
In conclusion, we report on the emergence of extended-spectrum class A and class C β-lactamases in E. coli strains from healthy broilers. Even if at present there is a low level of isolation in food-producing animals, it is necessary to monitor the spread of expanded-spectrum cephalosporin-resistant bacteria and further research including animals and humans and their environments should be carried out.
Restriction profiles of plasmids from CTX-M-producing transconjugants digested with ClaI (A), EcoRI (B), and SphI (C). The plasmids shown in lanes 1 (plasmid pC5-4; 65.1 kbp), 2 (pC6-8; 68.1 kbp), 3 (pC33-13; 66.8 kbp), 4 (pC20-5; 67.4 kbp), and 5 and 6 (pC79-6 and pC80-12; 97.3 kbp) were derived from field isolates 13-C-005, 13-C-006, 13-C-033, 14-C-020, 14-C-079, and 14-C-080, respectively. M1 and M2, lambda DNA digested with HindIII marker and 1-kb DNA ladder marker, respectively (Takara Bio Inc., Shiga, Japan).
MICs of various antimicrobial agents for 18 cefazolin-resistant field strains isolated from 1999 to 2002
Phenotypes and β-lactamase genotypes of 18 cefazolin-resistant E. coli field isolates
Primers used for PCR and DNA sequencing
MICs of various antimicrobial agents for transconjugants and recipient strain
ACKNOWLEDGMENTS
We thank the staff of the Livestock Hygiene Service Centers across Japan for sampling, collection of drug usage data of individual animals, and isolation and identification of E. coli strains and K. S. Thomson, Creighton University School of Medicine, for useful advice.
FOOTNOTES
- Received 14 August 2004.
- Returned for modification 8 November 2004.
- Accepted 1 May 2005.
- Copyright © 2005 American Society for Microbiology
