ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2004, p. 1066–1067 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.3.1066–1067.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 3
Antibiotic Resistance and Distribution of Tetracycline Resistance Genes in Escherichia coli O157:H7 Isolates from Humans and Bovines racycline (30 g), and trimethoprim-sulfamethoxazole (1.25 and 23.75 g, respectively). Antibiotic disks were supplied by Becton Dickinson Microbiology Systems (Franklin Lakes, N.J.). Forty-four (6.6%) bovine isolates and 29 (12.2%) human isolates were resistant to one or more antibiotics. Tetracycline resistance (Tcr) was the most common resistance found, with 43 of 44 (98%) resistant bovine isolates and 15 of 29 (52%) resistant human isolates being tetracycline resistant. Streptomycin resistance was found in 29 (66%) resistant bovine isolates and 13 (45%) resistant human isolates. Ampicillin was the third most common resistance phenotype with four (9%) of the resistant bovine isolates and eight (27%) of the resistant human isolates being ampicillin resistant. Thirty (68%) of the resistant bovine isolates and 15 (52%) of the resistant human isolates were multidrug resistant, defined as resistance to two or more different classes of antibiotics such as tetracycline and ampicillin. These results are similar to those of Kim et al. (4), who reported 13 of 176 (7.4%) isolates collected between 1989 and 1991 to be resistant to streptomycin, sulfisoxazole, and tetracycline. Schroeder et al. (10) also found similar results with 5% of 85 human isolates being ampicillin resistant and 11% of 93 cow isolates being tetracycline resistant among isolates collected between 1985 and 2002. In contrast, Galland et al. (3) found 10 (38.5%) isolates resistant to tetracycline and 2 (7.7%) resistant to ampicillin from 26 cattle isolates of E. coli O157:H7. However, this group tested very few isolates, which may account for the differences found. The level of antibiotic resistance within E. coli O157:H7 from humans and bovines between 1997 and 2000 was lower than that traditionally found with other E. coli isolates; however the level of resistance appears stable compared to those found in other studies of resistance within O157:H7 (3, 4, 10).
Escherichia coli O157:H7 is rare yet widely distributed within bovine populations, where it is a benign commensal of the gastrointestinal tract (3). Most E. coli O157:H7-related human illness has been the result of contamination of meat, other foodstuffs, and/or drinking water with bovine wastes containing this organism (Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Escherichia coli O157:H7 [http://www.cdc.gov/ncidod/dbmd/diseaseinfo /escherichiacoli_g.htm]). In humans, E. coli O157:H7 may cause no symptoms, mild to severe diarrhea, hemorrhagic colitis, or hemolytic-uremic syndrome (11; http://www.cdc.gov/ncidod/dbmd /diseaseinfo/escherichiacoli_g.htm). As previously reported, an increase in antibiotic resistance has been noted within E. coli O157:H7 populations over the last 20 years (3, 4, 10). Testing of susceptibility to 12 different antibiotics and antibiotic combinations was performed on 901 randomly selected E. coli O157:H7 isolates collected between 1997 and 2000 from our collection. This included 663 bovine isolates from feedlots in the midwestern United States and 238 human isolates, which represented both outbreaks and sporadic cases, from public health departments of Washington, Oregon, Nevada, Wisconsin, Georgia, and Illinois. Isolates were verified as E. coli O157:H7 by being plated on sorbitol MacConkey agar (Becton Dickinson, Sparks, Md.), followed by a latex agglutination test (Oxoid, Basingstoke, Hampshire, United Kingdom). Disk diffusion assays were done using the National Committee for Laboratory Standards protocol and the control E. coli strain ATCC 25922 (7). The following antibiotic disks were used: ampicillin (10 g), ampicillin-sulbactam (10 and 10 g, respectively), ceftriaxone (30 g), nalidixic acid (30 g), ciprofloxacin (5 g), kanamycin (30 g), gentamicin (10 g), amikacin (30 g), streptomycin (10 g), tet-
TABLE 1. Primers used for DNA-DNA hybridization of tetracycline resistance determinants and distribution of tetracycline genes by host Gene
Primer designation
No. (%) of isolatesb Sequencea (5⬘ to 3⬘)
Bovine (n ⫽ 42)
Human (n ⫽ 15)
4 (10)
3 (20) 9 (60)
tet(A)
A1 A3
CGA GCC ATT CGC GAG AGC GCC TCC TGC GCG ATC TGG
tet(B)
BF BR
CAG TGC TGT TGT TGT CAT TAA GCT TGG AAT ACT GAG TGT AA
25 (60)
tet(C)
CI CR
CTT GAG AGC CTT CAA CCC AG TGG TCG TCA TCT ACC TGC C
3 (7)
0
tet(D)
DF DR
GGA TAT CTC ACC GCA TCT GC CAT CCA TCC GGA AGT GAT AGC
0
0
tet(E)
EF ER
TCC ATA CGC GAG ATG ATC TCC CGA TTA CAG CTG TCA GCT GGG
0
0
tet(G)
G
AAC AAT GTC AGC AGT AAC AAG
1 (2)
3 (20)
13 (31)
3 (20)
None a b
All sequences are from reference 6. Five isolates carried two tet genes, and one isolate carried three tet genes.
1066
VOL. 48, 2004
LETTERS TO THE EDITOR
Previously the tet(A), tet(B), tet(C), tet(D), tet(E), and tet(G) genes have been identified in tetracycline-resistant E. coli strains (1, 2, 3, 4, 5, 6, 9). However, little is known about distribution among E. coli O157:H7 isolates from either humans or animals. Whole-bacterium dot blots were used to screen for the presence of the six genes as previously described (6, 8). The oligonucleotide sequence for each probe and the number of isolates carrying each gene are listed in Table 1. Sixty percent of the Tcr strains carried the tet(B) gene, which was the most prevalent tet gene found (Table 1). In a previous study, of non-O157 E. coli strains, tet(B) was also the dominant gene and found in 80% of Tcr E. coli isolates (1). The tet(A) gene was found in four (10%) of the Tcr bovine isolates and three (20%) of the Tcr human isolates. The tet(C) gene was found in three (7%) of the Tcr bovine isolates and no human isolates. The tet(G) gene was found in one (2%) Tcr bovine isolate and three (20%) Tcr human isolates. The tet(D) and tet(E) genes were not found in any of the isolates. One Tcr bovine isolates and four Tcr human isolates carried two different tetracycline resistance genes, and one Tcr bovine isolate carried three different tet genes. Thirteen (31%) Tcr bovine isolates and three (20%) Tcr human isolates did not hybridize with any of the known genes examined. In preliminary analysis, none of the 16 isolates hybridized with the recently identified tet(Y) gene (9), suggesting that these strains either may carry one of the other known tet genes not previously identified in E. coli or may have a novel gene. To distinguish between these hypotheses, the other known efflux tet genes, the only type currently identified in E. coli, will need to be screened. The finding that 31% of the Tcr bovine isolates carried unidentified tetracycline resistance genes, in this study, is very similar to a recent report that 40% of the Tcr isolates from Chilean salmon farms (6) carried unidentified tetracycline resistance genes. The data from these two studies suggest that Tcr gram-negative bacteria taken from nonhuman animals and/or the environment carry a more diverse group of tetracycline resistance genes than are usually found when characterizing Tcr gramnegative bacteria from humans. REFERENCES 1. Blake, D., R. Humphry, K. Scott, K. Hillman, D. Fenlon, and J. Low. 2003. Influence of tetracycline on tetracycline resistance and the carriage of tet-
2. 3.
4.
5. 6. 7.
8. 9. 10.
11.
1067
racycline resistance genes within commensal Escherichia coli populations. J. Appl. Microbiol. 94:1087–1097. Chopra, I., and M. C. Roberts. 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 65:232–260. Galland, J., D. Hyatt, S. Crupper, and D. Acheson. 2001. Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots. Appl. Environ. Microbiol. 67: 1619–1627. Kim, H., M. Samadpour, L. Grimm, C. Clausen, T. Besser, M. Baylor, J. Kobayashi, M. L. Neill, F. Schoenknecht, and P. Tarr. 1994. Characteristics of antibiotic-resistant Escherichia coli O157:H7 in Washington State, 1984– 1991. J. Infect. Dis. 170:1606–1609. Lanz, R., P. Kuhner, and P. Boerlin. 2003. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet. Microbiol. 91:73–84. Miranda, C. D., C. Kehrenberg, C. Ulep, S. Schwarz, and M. C. Roberts. 2003. Diversity of tetracycline resistance genes from bacteria isolated from Chilean salmon farms. Antimicrob. Agents Chemother. 47:883–888. National Committee for Clinical Laboratory Standards. 1998. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved standard M7-A5, M100-S10. National Committee for Clinical Laboratory Standards, Wayne, Pa. Pang, Y., T. Bosch, and M. C. Roberts. 1994. Single polymerase chain reaction for the detection of tetracycline resistant determinants Tet K and Tet L. Mol. Cell. Probes 8:417–422. Roberts, M. C. 2002. Resistance to tetracycline, macrolide-lincosamidestreptogramin, trimethoprim and sulfonamide drug classes. Mol. Biotechnol. 20:261–283. Schroeder, C., C. Zhao, C. Debroy, J. Torcolini, S. Zhao, D. White, D. Wagner, P. McDermott, R. Walker, and J. Meng. 2002. Antimicrobial resistance of Escherichia coli O157 isolated from humans, cattle, swine, and food. Appl. Environ. Microbiol. 68:576–581. Wong, C., S. Jelacic, R. Habeeb, S. Watkins, and P. Tarr. 2000. The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N. Engl. J. Med. 342:1930–1936.
Chris Wilkerson Mansour Samadpour Departments of Environmental and Occupational Health Sciences Nicole van Kirk Marilyn C. Roberts* Department of Pathobiology University of Washington Seattle, WA 98195 *Phone: (206) 543-8001 Fax: (206) 543-3873 E-mail:
[email protected]
