ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2004, p. 3179–3181 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.8.3179–3181.2004
Vol. 48, No. 8
Extended-Spectrum-Cephalosporin Resistance in Salmonella enterica Isolates of Animal Origin Jeffrey T. Gray,1* Laura L. Hungerford,2 Paula J. Fedorka-Cray,1 and Marcia L. Headrick3 Antimicrobial Resistance Research Unit, Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia 30604-56771; Center for Veterinary Medicine, Food and Drug Administration, Rockville, Maryland 208572; and Center for Veterinary Medicine, Food and Drug Administration, Athens, Georgia 306053 Received 24 October 2003/Returned for modification 24 January 2004/Accepted 27 April 2004
A total of 112 out of 5,709 Salmonella enterica isolates from domestic animal species exhibited decreased susceptibilities to ceftiofur and ceftriaxone, and each possessed the blaCMY gene. Ten Salmonella serotypes were significantly more likely to include resistant isolates. Isolates from turkeys, horses, cats, and dogs were significantly more likely to include resistant isolates. Salmonella enterica has been recovered from environmental samples, insects, and nearly all vertebrate species (11, 17). Extended-spectrum cephalosporins such as ceftriaxone and ceftiofur are important therapeutic agents and are often used for invasive Salmonella infections (4, 6). The emergence of Salmonella isolates that are resistant to extended-spectrum cephalosporins has been reported (7, 8, 14, 18). These isolates carry a plasmid-mediated AmpC-like beta-lactamase that hydrolyzes cephalosporins (CMY) (10), encoded by the blaCMY gene (3). Salmonella isolates carrying the blaCMY gene have been isolated from bovine, porcine, human, and foodstuff sources (8, 18, 19). In this study, isolates of Salmonella were obtained in 1997 and 1998 for antimicrobial susceptibility testing as part of the National Antimicrobial Resistance Monitoring System (http: //www.fda.gov/cvm/index/narms/narms_pg.html); these represent the general Salmonella population. A total of 5,709 isolates were tested from on-farm studies (n ⫽ 1,217), from diagnostic sources (n ⫽ 2,085), and from sampling carcasses from federally inspected slaughter establishments (n ⫽ 2,407). Susceptibility testing was performed (Sensititre; TREK Diagnostics, Inc., Westlake, Ohio) by using NCCLS (12, 13) standards and recommended quality control organisms. Isolates were examined for susceptibility to the following 17 antimicrobials: amikacin, amoxicillin-clavulanic acid, ampicillin, apramycin, cefoxitin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim-sulfamethoxazole. For a small proportion of the isolates (2.0%, 112 of 5,709), MICs of at least one of the extended-spectrum cephalosporins were found to be ⬎8 g/ml. This indicates that resistance to extended-spectrum cephalosporins among isolates originating from livestock and companion animals is a rare event in the United States. None of the 112 isolates were inhibited by the clavulanic acid-amoxicillin combination. The extended-spectrum-cephalosporin-resistant isolates were found
in 7 animal species, indicating that this resistance may be an emerging problem in a wide range of environments (Table 1). This expands the host species detected to carry this type of Salmonella to include live turkeys, horses, cats, and dogs. For animal species of origin and Salmonella serotype, an iterative latent variable technique (16) was used to determine the grouping that best divided isolates into those with higher and lower proportions of resistant isolates. This method compared all possible ways of partitioning categories into two groups to find the one that maximized the Cochran-MantelHaenszel (CMH) chi-square. A Monte Carlo simulation with 1,000 runs was used to determine the significance for this grouping (16). A significance level of 0.05 was used. The proportion of extended-spectrum-cephalosporin-resistant isolates differed significantly between host animal species (P ⬍ 0.0001). Among all isolates from a particular host, the most significant grouping placed turkey, horse, cat, and dog isolates together as the set with the highest proportion of resistant isolates compared to cattle, chicken, swine, exotics, and others (P ⬍ 0.0001). In general, the proportion of resistant isolates was higher among samples from clinical cases than among those from healthy animals. Clinical isolates from turkeys, horses, and cats had significantly higher proportions of resistant isolates than did those from cattle, chickens, dogs, swine, exotics, and others. Serotyping and phage typing were performed by the National Veterinary Services Laboratory, Ames, Iowa. None of the isolates were phage type DT104. Twenty-one serotypes were identified among the extended-spectrum-cephalosporinresistant isolates (Table 2). This distribution was significantly different than that of the general Salmonella population, which included 111 different serotypes (among 4,510 that were serotypeable). When the 20 cephalosporin-resistant serotypes were partitioned, based on the proportion of resistant isolates for each serotype, those with the highest proportion of resistant isolates were S. enterica serotype Bredeney (27.0% resistant), S. enterica serotype Infantis (5.2%), S. enterica serotype Newport (8.3%), S. enterica serotype Ohio (8.1%), S. enterica serotype Stanley (100%), S. enterica serotype Typhimurium (4.2%), S. enterica serotype Typhimurium-Copenhagen (5.5%), S. enterica serotype Uganda (7.1%), S. enterica sero-
* Corresponding author. Present address: University of Guelph, Dept. of Pathobiology, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 54642. Fax: (519) 824-5930. E-mail:
[email protected]. 3179
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Host animal species origin of extended-spectrumcephalosporin-resistant Salmonella isolates Host animal group
Total no. of Salmonella isolates
No. (%) of resistant isolatesa
No. of resistant isolatesa/no. of clinical isolatesb (%)
Cattlec Turkeysd Horsesd Chickensc Swinec Catsd Dogsd Exoticsc Othersc
1,751 677 169 1,121 1,580 55 95 140 91
34 (1.9) 32 (4.7) 16 (9.5) 11 (1.0) 10 (0.6) 5 (8.3) 4 (4.0) 0 0
6.0 11.0 9.5 3.0 2.3 8.3 4.0 0 0
Total
5,709
112 (2.0)
a
Extended-spectrum-cephalosporin-resistant isolates. Total number of clinical isolates. Species in group with lowest proportions of resistant isolates, based on partitioning to maximize the CMH chi-square (16). d Species in group with highest proportions of resistant isolates, based on partitioning to maximize the CMH chi-square (16). b c
type Holcomb (50%), and untypeable isolates (8.0%). Four different cephalosporin-resistant Salmonella serotypes have been described for humans (7), and only two human isolate serotypes overlapped with the animal isolate serotypes described here, serotype Typhimurium and serotype Newport. PCR analysis was performed on total DNA prepared by boiling or purified plasmid DNA. DNA amplification was performed with consensus primers for the bla gene encoding CMY. Primers were F (5⬘-GACAGCCTCTTTCTCCACA-3⬘) and R (5⬘-TGGAACGAAGGCTACGTA-3⬘), based on the blaCMY-2 gene sequence of Klebsiella pneumoniae (GenBank
TABLE 2. Serotypes of extended-spectrum-cephalosporin-resistant Salmonella isolates S. enterica serotypea
Serogroupb
No. of isolates
Typhimurium-Copenhagen Bredeney Typhimurium Heidelberg Agona Infantis Newport Reading Ohio Montevideo Meleagridis None (untypeable) Schwarzengrund Thompson Stanley Uganda Holcomb Dublin Hadar Braenderup Seftenberg
B B B B B C1 C2 B C1 C1 E1 B C1 B E1 C2 D1 C2 C1 E4
26 20 17 9 8 5 5 4 3 2 2 2 1 1 1 1 1 1 1 1 1
Total
20b
112
a
Serotypes and serogroups were defined by using the Kaufmann-White antigenic scheme. b Twenty serotypes and 2 untypeable isolates were identified.
FIG. 1. Southern blot analysis of plasmid hybridization with PCRamplified blacmy gene probes. Lane 1, supercoiled ladder; lane 2, positive control, S. enterica serotype Typhimurium, extended-spectrumcephalosporin-resistant isolates; lane 3, S. enterica serotype Typhimurium 42/98; lane 4, S. enterica serotype Typhimurium-Copenhagen 4/97; lane 5, S. enterica serotype Typhimurium-Copenhagen 36/98; lane 6, S. enterica serotype Bredeney 72/98; lane 7, S. enterica serotype Heidelberg 61/98; lane 8, S. enterica serotype Newport 11/98; lane 9, S. enterica serotype Reading 57/98; lane 10, negative control, S. enterica serotype Typhimurium 798; lane 11, labeled lambda marker (23, 9.4, 6.5, 4.3, 2.3, and 2.0 kb). The arrow indicates native plasmid hybridization.
accession no. Y16784). Amplifications were carried out as described previously (2), with an expected 1,143-kb product size. Amplification of the blaCMY gene sequence yielded positive results for all 112 cephalosporin-resistant isolates and was not amplified in 550 random comparison strains that were susceptible to ceftiofur. Confirmatory sequence homology to blaCMY-2 (97%) was observed for purified PCR-generated DNA fragments from four PCR-positive isolates, as previously described (19). Plasmid DNA isolations were performed by using an isolation procedure modified from that of Kado and Liu (9). Plasmid DNA was digested with PlasmidSafe DNase (Epicentre Technologies, Madison, Wis.) at a concentration of 0.2 U/l and then electrophoresed on a 0.8% agarose gel in Tris-acetate EDTA buffer with 90 V of constant voltage. A subset of the cephalosporin-resistant isolates was analyzed by Southern blot analysis (1, 15). Probes for the blacmy gene were prepared by PCR amplification with a digoxigenin labeling kit (Roche, Indianapolis, Ind.). Subsequently, the membrane was hybridized at 37°C, washed, and probed with antidigoxigenin antibody. The bands were visualized with the CDP star system (Roche). One large plasmid, ranging in size from 60 to 75 kb, was observed for all 112 isolates. Additionally, 26% of the isolates carried smaller plasmids between the sizes of 3 and 14 kb (data
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NOTES
not shown). Southern blot analysis of the native plasmid preparation indicated that the CMY probe consistently hybridized to a large plasmid near the origin of migration for each of the isolates (Fig. 1). Multiple antimicrobial resistances and plasmid transfer have been demonstrated with the large blacmycontaining plasmid in Salmonella isolates from the United States (5, 18, 19). In this study, the plasmid was found in all major domestic animal species and in 20 different Salmonella serotypes. The Salmonella isolates used in this study were widely distributed among serotypes and host species. This complicates deductions about possible mechanisms promoting selection of these pathogens. The combination of ubiquity and potential to acquire or transfer a multiple antibiotic resistance plasmid suggests that these food-borne, zoonotic pathogens create a serious public health concern. It will take creative strategies to identify and control these Salmonella isolates and/or the plasmid that carries the blacmy gene. REFERENCES 1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. More, J. G. Seidman, J. A. Smith, and K. Stzubl. 1993. Current protocols in molecular biology. John Wiley and Sons, Inc., New York, N.Y. 2. Barnaud, G., G. Arlet, C. Verdet, O. Gaillot, P. H. Lagrange, and A. Philippon. 1998. Salmonella enteritidis: AmpC plasmid-mediated inducible -lactamase (DHA-1) with an ampR gene from Morganella morganii. Antimicrob. Agents Chemother. 42:2352–2358. 3. Bauernfeind, A., I. Stemplinger, R. Jungwirth, and H. Giamarllou. 1996. Characterization of the plasmidic -lactamase CMY-2, which is responsible for cephamycin resistance. Antimicrob. Agents Chemother. 40:221–224. 4. Bryan, J. P., and W. M. Scheld. 1992. Therapy of experimental meningitis due to Salmonella enteritidis. Antimicrob. Agents Chemother. 36:949–954. 5. Carattoli, A., F. Tosini, W. P. Giles, M. E. Rupp, S. H. Hinrichs, F. J. Angulo T. J. Barrett, P. D. Fey. 2002. Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella strains isolated in the United States between 1996 and 1998. Antimicrob. Agents Chemother. 46:1269–1272.
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