a Wyeth-Ayerst Research, 401 N. Middletown Rd, Pearl River, NY 10965 b Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND, USA
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Abstract |
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Introduction |
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Bovine calf scours is a severe form of diarrhoea that causes more financial loss to cow-calf producers than any other disease-related problem. Scours is a clinical sign of disease that may have many causes, although E. coli has been frequently implicated as the primary bacterial cause in calves.4 Antibiotics are frequently used by animal owners and veterinarians in the treatment and prevention of scours.
In recent years, bacterial resistance to ß-lactam antibiotics has risen dramatically in human 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 performed to collect data on the occurrence of antibiotic resistance and virulence factors among E. coli strains associated with bovine calf scours in the state of North Dakota, USA. This survey, revealed that about 13% of E. coli strains implicated in bovine calf scours were resistant to ceftiofur, an expanded-spectrum cephalosporin used in veterinary medicine. This study was performed to characterize the nature of bacterial resistance to expanded-spectrum cephalosporins.
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Materials and methods |
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Samples of E. coli obtained from individual bovine calf scours cases during 1996
were
examined for antimicrobial susceptibility. E. coli were selected from primary agar
cultures of
either bovine faeces from animals with diarrhoea, or intestinal tissue from septicaemic animals
taken
post mortem. All of the isolates represented cases that had failed antimicrobial therapy.
Thirty-two
strains that showed varying degrees of resistance to ceftiofur were chosen for further study. The
recipients for mating and transformation experiments were E. coli strains C600N and
DH5, respectively.
Susceptibility testing
Initial bacterial antibiotic susceptibilities were carried out using KirbyBauer disc diffusion assays performed by standard procedures.5 MICs were determined by microdilution using standard procedures.6 Antibiotics were obtained from their respective manufacturers.
Characterization of ß-lactamase
Isoelectric focusing (IEF) was performed by the method of Matthew et al.7 using an LKB Multiphor apparatus with prepared PAGplates, pH 3.59.5 or pH 4.06.5. (Pharmacia LKB, Piscataway, NJ, USA). The isoelectric point (pI) of each enzyme was determined by activity staining with nitrocefin (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) following IEF.
Nucleic acid techniques
DNA isolation and PCR were performed by standard techniques.8 Ribotyping was performed using the RiboPrinter system (Qualicon, Wilmington, DE). PCR was used to detect TEM ß-lactamase genes as well as genes for virulence factors including Shiga-like toxins I and II (STX-I, STX-II), E. coli attaching and effacing factor (EAE), cytotoxic necrotizing factor I (CNF-I), cytotoxic necrotizing factor II (CNF-II), heat labile enterotoxin (LT), K99 adhesion fimbriae (K99), heat stable enterotoxin a (STa) and heat stable enterotoxin b (STb), using specific oligonucleotide primers.
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Results |
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MICs of ß-lactam antibiotics for the clinical isolates are shown in Table I. All of the isolates tested were resistant to ampicillin and had reduced susceptibility to ticarcillin and piperacillin which suggested the presence of a ß-lactamase. The addition of a ß-lactamase inhibitor to these penicillins restored susceptibility to some, but not all of the isolates. In addition, 27 of the 32 isolates tested showed increased resistance to the expanded-spectrum cephalosporins, aztreonam and cefoxitin. None of the isolates were resistant to imipenem. Susceptibility to non-ß-lactam drugs is shown in Table II. All of the isolates were resistant to kanamycin, streptomycin, sulphisoxazole and tetracycline. In addition, many were resistant to chloramphenicol, gentamicin and trimethoprimsulphamethoxazole. Four isolates were nalidixic acid resistant but only two strains were ciprofloxacin resistant.
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IEF results (Table I) showed ß-lactamases with pIs of 5.4, 5.6 and 9.0 present in varying patterns among the isolates. The pI 9.0 ß-lactamase was a strong and prominent band which appeared rapidly after staining with nitrocefin. Five isolates produced only the pI 5.4 enzyme, 17 produced only the pI 9.0 enzyme and 10 produced the pI 9.0 ß-lactamase in combination with either the pI 5.4 or the pI 5.6 ß-lactamase. PCR with TEM-specific primers gave positive results, with plasmid DNA isolated from strains expressing a ß-lactamase of pI 5.4 or 5.6 (Table I). Pathogenic veterinary strains that produced only the TEM-type enzyme with a pI of 5.4 were all susceptible to extended-spectrum cephalosporins. Increased resistance to the expanded-spectrum cephalosporins, aztreonam and cefoxitin correlated with the presence of the pI 9.0 ß-lactamase and was presumed to be the AmpC of E. coli expressed at higher than usual levels.
Ribotyping
The ribogroups determined by ribotyping are shown in Table II. Ten of the isolates produced unique ribotype patterns. The remaining strains could be separated into seven distinct ribogroups. Of these, the most common was designated ribotype I and contained seven isolates. Groups IIVII consisted of two to four isolates. There was no correlation between ribotype and susceptibility, ß-lactamase type or carriage of virulence factors.
Characterization of virulence factors
Genes for STX-II, CNF-II, LT and STb were not detected among these isolates although eight strains were positive for the STa enterotoxin and K99 fimbriae genes, indicating that they were enterotoxigenic E. coli (ETEC). One isolate was positive for STX-I and EAE and one was positive for CNF-1. The remaining isolates were negative for the virulence factors assayed.
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Discussion |
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The ribotyping data indicated that there was not a clonal outbreak of a single multiply resistant strain, but that several strains arose following a number of independent mutational events. Several of the strains possessed virulence factors that are associated with human diarrhoeal disease. These genes are often encoded on plasmids and may possibly be co-transferred with antimicrobial resistance genes.
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 multidrug resistance may pose an increasing threat to successful treatment of E. coli-related veterinary diseases in the near future. The extensive use (and some misuse) of antimicrobials has led to the loss of efficacy through emergence of bacterial antibiotic resistance.10 Although only a few of the isolates in this study possessed virulence factors that are associated with human disease, they were resistant to multiple antibiotics. If these strains were to cross over into the human population, therapeutic options would be limited if antibiotic therapy were necessary.
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Acknowledgments |
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Notes |
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Present address: Rutgers University, Piscataway, NJ, USA.
Present address: Food and Drug Administration, Center for
Veterinary
Medicine, Laurel, MD, USA.
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References |
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Received 28 January 1999; returned 8 April 1999; revised 8 June 1999; accepted 29 June 1999