1 Division of Infectious Disease, Department of Medicine, 2 Wayne State University School of Medicine, William Beaumont Hospital, 3811 West 13 Mile Road, Royal Oak, MI 48073; 3 Michigan State College of Veterinary Medicine, East Lansing, MI; 4 Wayne State University, School of Medicine, Detroit, MI, USA
Received 8 June 2004; returned 15 July 2004; revised 20 October 2004; accepted 23 October 2004
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Abstract |
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Methods: Enterococci from faecal samples from 18 beef cattle, 18 dairy cattle, 18 swine, 13 chicken, and eight turkey farms were prospectively evaluated over a 6 year period from 1998 to 2003.
Results: We evaluated 1256 isolates of Enterococcus faecium and 656 isolates of Enterococcus faecalis. None was vancomycin resistant. Quinupristin/dalfopristin, gentamicin and ciprofloxacin resistance rates in E. faecium were 2%, 0% and 55% in beef cattle, 8%, 7% and 47% in dairy cattle, 21%, 1% and 47% in swine, 85%, 12% and 23% in chicken, and 52%, 13% and 24% in turkey isolates, respectively. For E. faecalis, gentamicin resistance rates were 0% in beef cattle, 24% in dairy cattle, 37% in swine, 32% in chicken, and 29% in turkey isolates, whereas 12%, 9%, 21%, 64% and none of isolates from beef, dairy, swine, chicken, and turkey farms, respectively, were resistant to ciprofloxacin. Quinupristin/dalfopristin resistance in E. faecium was more common on chicken and turkey farms using virginiamycin (P<0.0001 for both) compared with farms not using a streptogramin, gentamicin resistance was more common on dairy farms using gentamicin (P<0.0001) compared with farms not using this antibiotic, and ciprofloxacin resistance was more common on turkey and dairy farms using enrofloxacin compared with those with no enrofloxacin use (P=0.02 and P=0.04, respectively). For E. faecalis, gentamicin resistance was more frequently detected on dairy and swine farms using gentamicin (P<0.0001 and P=0.0052, respectively) and ciprofloxacin resistance was more common on beef farms using enrofloxacin (P<0.0001) compared with farms not using these antimicrobials. PFGE showed multiple strain types with some clones common between animals of the same animal species.
Conclusions: This study shows the presence of a significant reservoir of antibiotic-resistant enterococci among farm animals. Resistance was more common on farms using antimicrobial agents.
Keywords: farms , antimicrobial use , molecular analysis
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Introduction |
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Recent studies in the United States have provided evidence for spread of gentamicin-resistant2 and in Europe for spread of vancomycin-resistant3 enterococci through the food chain from animals to humans. Quinupristin/dalfopristin, a combination of streptogramin A and B antimicrobial agents was approved in the United States for therapy of vancomycin-resistant enterococci (VRE) in late 1999. Quinupristin/dalfopristin resistance has been reported in human surveillance stool cultures and clinical isolates of Enterococcus faecium.4 Importantly, these findings were reported before quinupristin/dalfopristin was approved for human use.
The role that non-human sources and reservoirs other than hospitalized patients may play in the spread of Enterococcus is controversial and poorly understood.5 Although much has been learned over the last 10 years about the epidemiology of nosocomial enterococci, there is little information on the prevalence and epidemiology of antimicrobial resistance in enterococci outside of the hospital setting. We therefore prospectively studied enterococci from animal farms in three mid-western states in the United States (Michigan, Wisconsin and Indiana) over a 6 year period to evaluate the epidemiology of antimicrobial resistance and the potential relation of resistance to antimicrobial use.
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Materials and methods |
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We prospectively evaluated faecal samples from animals from 18 beef cattle, 18 dairy cattle, 18 swine, 13 chicken and eight turkey farms in Michigan, Wisconsin and Indiana from 1998 to 2003. A random sample of various sizes and location of cattle, poultry and swine farms was selected. Animals of different species were geographically separated. There were no animal transfers between farms. Farms ranged in size from 60 to 3000 cattle and swine and up to 30 000 turkeys and 200 000 to 400 000 chickens per year, and included three organic dairy farms. The identity of the farms was anonymous to the investigators during the laboratory analysis of samples.
Faecal samples were collected by faecal extraction for adult cattle and swine, by rectal swab for calves and piglets, and by a drag sample for each turkey and chicken pen. Drag samples were collected by fastening sterile booties soaked in sterile water to the end of a string, and dragging the swabs through the turkey or chicken pen. All samples (338 from chicken, 487 from swine, 212 from beef, 13 from chicken, and eight from turkey farms) were immediately placed on ice and kept at refrigerator temperature during transport. A questionnaire was used to collect information from the farmers regarding usual use of antibiotics in the animals within the year before collection of specimens. Cultures from farmers or other clinical information on the farmers themselves were not obtained.
Isolation, identification and susceptibility testing of enterococci
Enterococcosel broth (Becton Dickinson, Cockeysville, MD, USA) was used to select for enterococci. For each faecal culture sample, 5 mL of Enterococcosel broth containing 16 mg/L vancomycin, 100 mg/L gentamicin, 4 mg/L ciprofloxacin, or 4 mg/L quinupristin/dalfopristin was inoculated with 0.5 g of faeces. After 48 h of incubation at 37°C, broth was sub-cultured (10 µL) to Enterococcosel agar plate containing the same antibiotic as the broth. Plates were then incubated for 48 h at 37°C. A representative of each colony type from each plate was isolated for evaluation. All E. faecium and Enterococcus faecalis isolates were screened on MuellerHinton II agar plates containing each of the antibiotics at the same concentrations as in the broth. Previously described methods were used for identification of enterococci.6 Only E. faecium and E. faecalis isolates were evaluated for antimicrobial susceptibility and all quinupristin/dalfopristin-resistant and gentamicin-resistant isolates were analysed by PFGE. Antimicrobial susceptibility tests were carried out using NCCLS guidelines.
PFGE
Genomic DNA was prepared using previously described methods.7,8 Strains were considered related if their SmaI restriction patterns differed by less than six bands. Duplicate isolates from the same culture were excluded using the results of PFGE.
Statistical analysis
Two-Tailed Fisher's Exact Test was used for analysis. P values less than an alpha of 0.05 (Probability of Type I Error) were considered significant throughout this study. Statistical analysis was carried out using the SAS System for Windows version 8.02.
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Results |
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Mean gentamicin resistance rates in E. faecium were 0% in beef cattle, 7% in dairy cattle, 1% in swine, 12% in chicken and 13% in turkey farms. Rates of resistance to gentamicin in E. faecalis were 0% in beef cattle, 24% in dairy cattle, 37% in swine, 32% in chicken and 29% in turkey farms. Dairy and swine farms using aminoglycosides had higher gentamicin resistance rates (39 of 301 versus 3 of 323 E. faecium isolates, P < 0.0001, and 55 of 158 versus 10 of 112 E. faecalis isolates from dairy farms, and 26 of 48 versus 39 of 127 E. faecalis isolates from swine farms, P=0.005) than farms with no aminoglycoside use. Gentamicin use was reported more frequently in calves compared with cows and 90% of the gentamicin-resistant isolates were isolated from calves on the farms.
Ciprofloxacin resistance rates in E. faecium were 55% in beef cattle, 47% in dairy cattle and swine, 23% in chickens, and 24% in turkey farms, whereas 12%, 9%, 21%, 64%, and none of E. faecalis isolates from beef, dairy, swine, chicken, and turkey farms, respectively, were resistant to ciprofloxacin. Use of enrofloxacin was associated with higher ciprofloxacin resistance in E. faecium on turkey and dairy farms (9 of 20 versus 25 of 122 isolates, P=0.02, and 21 of 34 versus 275 of 590 isolates, P=0.04, respectively) as well as higher ciprofloxacin resistance in E. faecalis on beef farms (15 of 15 versus 1 of 114 isolates, P < 0.0001).
A small number of isolates demonstrated resistance to more than one class of antimicrobials. Two percent of E. faecium isolates from dairy, 11% from chicken, and 1.4% from turkey farms were resistant to both quinupristin/dalfopristin and gentamicin; 0.5% isolates from dairy, 1.4% from chicken, and 0.7% from turkey farms were resistant to both gentamicin and ciprofloxacin; and 1.4% isolates from dairy, 2.5% from swine, 11% from chicken, and 12.7% from turkey farms demonstrated resistance to both quinupristin/dalfopristin and ciprofloxacin. Among E. faecalis isolates, there were 5.1% from swine farms and 17% from chicken farms that were resistant to both gentamicin and ciprofloxacin. None of the E. faecium or E. faecalis isolates were resistant to all three classes of antimicrobials.
Cephalosporins (ceftiofur), penicillins (procaine penicillin G), macrolides (tylosin, tilmicosin, and erythromycin), lincosamides (lincomycin), florfenicol, tetracyclines (chlortetracycline, oxytetracycline, and chlortetracycline/sulfamethazine) and sulfonamides (sulfadimethoxine) were other antimicrobials used on the various farms.
PFGE results are summarized in Table 2.
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Discussion |
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Resistant strains of enterococci were isolated from farms not using antimicrobial agents, however lower rates of resistance were observed. We did not collect information regarding transfer of farm workers between farms, but previous studies showing colonization of farm workers with resistant enterococci,9 raise the possibility of farm workers playing a role in potential transfer of resistance from farm to farm.
Avoparcin is not approved for use in animal husbandry in the United States and we did not recover any vancomycin resistance among all E. faecalis and E. faecium isolates we evaluated over the study period. These results are different from European studies where avoparcin was used commonly in animals and high prevalence of vancomycin-resistant enterococci was discovered not only among animal farms, but related strains also from healthy humans.
Quinupristin/dalfopristin resistance was seen in E. faecium isolates from all types of farms; however, farms that used virginiamycin as a growth-promoting agent had higher rates of quinupristin/dalfopristin resistance compared with farms that did not use virginiamycin. The results of this study show a high prevalence of resistance to quinupristin/dalfopristin among E. faecium isolated from farm animals. Further work is necessary to determine the molecular mechanism for quinupristin/dalfopristin-resistant E. faecium in animal isolates and possible transferability to humans.
We did not specifically evaluate feed productivity and animal health; however, there were no reports of concerns on the farms not using antimicrobial agents (data not shown). Antibiotics other than aminoglycosides, streptogramins, or fluoroquinolones, which are important in management of infections in humans, were used commonly in some of the animal farms. Although we did not evaluate the rate of resistance to these other agents, the effect of their utilization and how it contributes to bacterial population and antimicrobial resistance rates requires further assessment.
This study provides clear evidence that resistance in enterococci to quinupristin/dalfopristin, gentamicin and fluoroquinolones is common in various food animals and that the increase in consumption of antibiotics by animals has been accompanied by an increase in antibiotic resistance. We also found considerable diversity in the strains isolated from animals, suggesting a single strain or limited number of strains is not responsible for this resistance. However, the potential role that antibiotic use in veterinary medicine and animal husbandry plays in the transfer of antibiotic-resistant bacteria to humans remains unresolved. Considering the effect antimicrobial resistance has on human health and also its economic impact, measures to preserve these agents and delay the development of resistance are urgently needed.5,10 This includes judicious use of antibiotics for human infections, control measures to prevent spread of resistant pathogens in healthcare facilities and decrease in resistance in reservoirs on farms and in the environment.
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Acknowledgements |
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Footnotes |
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References |
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2
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van den Bogaard, A. E., Willems, R., London, N. et al. (2002). Antibiotic resistance of faecal enterococci in poultry, poultry farmers and poultry slaughterers. Journal of Antimicrobial Chemotherapy 49, 497505.
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