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U.S. Department of Health and Human Services

Animal & Veterinary

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Expanded-Spectrum Cephalosporin Resistance in E. coli Isolates Associated with Bovine Calf Diarrheal Disease

FDA Veterinarian Newsletter January/February 2000 Volume XV, No I

by David G. White, Ph.D. (FDA/CVM, Office of Research) and Patricia A. Bradford, Ph.D. (Wyeth-Ayerst Research, Pearl River, NY)

Pathogenic Escherichia coli are one of the most important groups of bacteria causing diarrhea and extraintestinal infections in humans and animals.1 There have been many recent reports of morbidity and mortality due to outbreaks of disease-causing E. coli which have been attributed to foods of bovine origin or other foods cross-contaminated by beef products or cow manure.2 The occurrence of toxigenic E. coli strains in humans and calves with diarrhea is well documented and cattle have been considered an important reservoir of Shiga-like toxin (SLT) producing E. coli strains involved in human disease.3Toxigenic E. coli are also frequently implicated as the primary bacterial cause of scours in neonatal calves.4 Bovine calf scours is a severe form of diarrhea which is thought to cause more financial loss to cow-calf producers than any other disease-related problem. The most important aspect in treatment of calf scours is directed at correcting the accompanying electrolyte losses, dehydration, and acidosis. Antimicrobial therapy is often implemented at the same time in an attempt to treat and prevent further occurrences. However, strains of E. coli (animal and human origin) have become increasingly resistant to most frontline antimicrobials, including third generation cephalosporins, aminoglycosides, and even fluoroquinolones. Infections caused by antibiotic resistant bacteria are a major and costly animal health problem. These infections can lead to prolonged illness and if not treated in time with more expensive, alternate antimicrobial agents, can lead to increased mortality. This potential problem will continue to have a large impact on the animal industry in terms of both animal health and food safety if not investigated.

In recent years, bacterial resistance to ß-lactam antimicrobials has risen dramatically among human bacterial pathogens. The use of expanded-spectrum cephalosporins in health institutions contributes to the emergence of such resistance. However, there is limited knowledge concerning the incidence and prevalence of extended-spectrum ß -lactamases (ESBLs) among pathogenic veterinary strains of E. coli incriminated in animal disease. Therefore, a survey was initiated to collect data on the occurrence of multiple antibiotic resistance and virulence factors among E. coli strains associated with bovine calf scours in the state of North Dakota, USA. As a result of this survey, it was noted that about 13% of E. coli strains implicated in bovine calf scours displayed decreased susceptibilities to ceftiofur, an expanded-spectrum cephalosporin used in veterinary medicine. Ceftiofur, a veterinary expanded-spectrum cephalosporin, is active against a variety of animal pathogens, including microorganisms associated with bovine and swine respiratory disease.5 Although this antimicrobial is not approved for treatment of bovine calf scours, ceftiofur is sometimes used in an extralabel manner to treat bacterial diarrheal diseases when these pathogenic strains are resistant to other less efficacious veterinary antimicrobials. This study was performed to characterize the nature of bacterial resistance to expanded-spectrum cephalosporins and to determine if ESBLs were present among these isolates.

E. coli isolates were selected from primary agar cultures of either bovine feces from diarrheic animals, or intestinal tissue from septicemic animals taken post mortem from scours cases submitted to the North Dakota State University Veterinary Diagnostic Laboratory during 1996 and 1997. Isolates were further examined for antimicrobial susceptibility according to NCCLS methodologies.6,7

Thirty-two strains exhibiting varying degrees of resistance to ceftiofur were chosen for further study. All of the isolates tested were resistant to ampicillin and had reduced susceptibility to ticarcillin and piperacillin which suggested the presence of a $ –lactamase. The addition of a ß-lactamase inhibitor to these penicillins restored susceptibility to some, but not all of the strains. In addition, 27 of the 32 strains tested displayed increased resistance to the expanded-spectrum cephalosporins, aztreonam and cefoxitin. None of the strains were resistant to imipenem. All of the isolates were resistant to kanamycin, streptomycin, sulfisoxazole and tetracycline. In addition, most of the strains were also resistant to trimethoprim-sulfamethoxazole (84%), chloramphenicol (59%), and gentamicin (53%). Four strains were nalidixic acid resistant, and two strains were ciprofloxacin-resistant.

Using established iso-electric focusing (IEF) techniques, the ß -lactamases of the pathogenic bovine E. coli isolates were further characterized.8 Five isolates produced only the plasmid mediated TEM-1 $ –lactamase, 17 isolates produced only the chromosomal AmpC ß-lactamase and 10 isolates produced the AmpC ß -lactamase in combination with either the plasmid mediated TEM-1 or TEM-2 ß -lactamase. Pathogenic veterinary E. coli isolates that produced only the TEM-type enzymes were all susceptible to expanded-spectrum ß -lactams. Increased resistance to the expanded-spectrum cephalosporins, aztreonam, cefoxitin and ceftiofur correlated with overexpression of the E. coli chromosomal AmpC ß -lactamase.

In this study, many of the E. coli strains associated with bovine calf scours were found to be resistant to several antimicrobial classes including expanded-spectrum ß-lactam antimicrobials. Resistance to the expanded-spectrum ß-lactam antimicrobials was most likely due to the hyperproduction of the chromosomally-encoded AmpC ß -lactamase naturally found in E. coli, but usually not expressed at high levels. Although there is no direct correlation between the occurrence of the cephalosporin-resistance with the use of ß -lactams in veterinary medicine, these results suggest that the use of these antimicrobials may have contributed to the in vivo selection of E. coli strains which hyperproduce this enzyme. No extended-spectrum ß -lactamases were found among this population of isolates which suggests that extended-spectrum ß -lactamases do not routinely play a role in expanded-spectrum cephalosporin resistance among veterinary isolates of E. coli. However, identification of several TEM-type ß -lactamases among these isolates suggests that the emergence of ESBLs in this population of pathogens is possibly only a matter of time. The somewhat surprising finding of so many E. coli isolates that over express their ampC gene constitutes a serious threat to veterinary ß -lactam therapy of E. coli related diseases, as well as serving as a potential reservoir for drug resistant human and veterinary pathogens.

The results from this study demonstrated that resistance to front line antimicrobials is present among E. coli strains incriminated in bovine calf scours. This combination of virulence coupled with multi-drug resistance is an increasing threat to successful treatment of E. coli related veterinary diseases in the near future.

This work was published in the Journal of Antimicrobial Chemotherapy, 1999, Vol. 44:607-610.

References

1. Levine, M. M. (1987). Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J. Infect. Dis. 155, 377-389.

2. Griffin, P. M. and Tauxe, R. V. (1991). The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13, 60-68.

3. Saridakis, H. O., Gared, S. A. E., Vidotto, M. C. and Guth, B. E. C. (1997). Virulence properties of Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups isolated from calves with diarrhea. Vet. Microbiol. 54, 145-153.

4. Yamamoto, T. and Nakazawa, M. (1997). Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea. J. Clin. Microbiol. 35, 223-227.

5. Burton, P. J., Thornsberry, C., Cheung, Y. Y., Watts, J. L. and Jr., R. J. Y. (1996). Interpretive criteria for antimicrobial susceptibility testing of ceftiofur against bacteria associated with swine respiratory disease. J. Vet. Diag. Invest. 8, 464-468.

6. National Committee for Clinical Laboratory Standards.(1997). Performance standards for antimicrobial disk susceptibility tests. Approved Standard M2-A4. National Committee for Clinical Laboratory Standards, Wayne, PA. .

7. National Committee for Clinical Laboratory Standards. (1997). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A2. National Committee for Clinical Laboratory Standards, Wayne, PA.

8. Matthew, M. A., Harris, A. M., Marshall, M. J. and Ross, G. W. (1975). The use of isoelectric focusing for detection and identification of beta-lactamases. J. Gen. Microbiol. 88, 169-178.