Final Report for GNC03-016
A comparison of antimicrobial usage in commercially-raised and organically-raised chickens and turkeys and the development of antimicrobial resistance in Campylobacter jejuni were investigated in this study. The results indicated that fluoroquinolone usage in commercial poultry operations could lead to the development of fluoroquinolone resistance in Campylobacter species. Although some commercial poultry farms did not directly get exposed to fluoroquinolones during the entire production period, the high fluoroquinolone resistance rates were also observed in these commercial poultry farms. This observation suggested that fluoroquinolone-resistant Campylobacter strains were stable and able to persist on the farms during several rotations.
Antimicrobial agents are widely used in agriculture especially in animal production to treat, prevent, and control of bacterial infections as well as to promote growth and enhance feed efficiency (1, 4, 6). There are direct evidences that the use of antibiotics for non-therapeutic purposes in animals can select for antimicrobial resistance in both pathogenic and non-pathogenic bacteria particularly the ones inhabit in the intestinal tracts of animals (1, 6, 12, 14). These antimicrobial-resistant organisms especially zoonotic foodborne bacterial pathogens may serve as reservoirs for antimicrobial resistance genes and infect and/or transfer these genes to humans via contaminated food of animal origin (6, 11, 13). Over the last decade the emergence of antimicrobial resistance in Campylobacter strains isolated from animals has increased dramatically in many countries around the world (2, 4, 7, 12, 14). The emergence of antimicrobial resistance in these foodborne pathogens is likely due to the widespread use of antimicrobial agents in food-producing animals (1, 4, 6, 12, 14).
As mentioned earlier antimicrobial agents can be used in commercial production practice, while the use of these antimicrobial substances has been restricted in organic production practice. Since no antimicrobials have been used in organically-raised chickens and turkeys and since the demand for organic animal produce has been increasing considerably over the last several years, the difference in antimicrobial resistance of Campylobacter isolates from commercial and organic poultry operations as well as the association between antimicrobial usage in animal production practice and the development of antimicrobial resistance in food-borne bacterial pathogens such as Campylobacter jejuni is of interest.
The objective of this project was to investigate the association between antimicrobial usage in chickens and turkeys in commercially-raised and organically-raised environment and the development of antimicrobial resistance in Campylobacter species.
Campylobacter jejuni isolates
A total of 694 Campylobacter isolates from commercial broilers, commercial turkeys, organic broilers, and organic turkeys were tested for antimicrobial susceptibility to quinolone and fluoroquinolone antibiotics.
During 2000 to 2002, the intestinal tracts of broilers and turkeys from 10 commercial broiler farms, 10 commercial turkey farms, 5 organic broiler farms, and 5 organic turkey farms were collected and cultured for Campylobacter. Briefly, the intestinal content from each intestine was directly plated onto Campy CVA agar (BBL Becton Dickinson Microbiology Systems, Cockeysville, MD) using sterile cotton-tipped swab and then incubated at 42 C for 48 hours under a microaerophilic environment (approximately 5% O2, 10% CO2, and 85% N2) that was created by CampyPak II anaerobic system jar with CampyPak gas generating system envelopes (BBL Becton Dickinson Microbiology Systems, Sparks, MD). Suspect Campylobacter colonies were confirmed by colony morphology and several biochemical tests including gram-stain, catalase test, oxidase test, hippurate hydrolysis test, and Campylobacter culture-plate latex agglutination confirmation test (INDX-Campy [jcl]; PanBio INDX, Inc., Baltimore, MD). Campylobacter isolates were stored in sterile microfuge tubes containing skim milk and glycerol at –85 C prior to antimicrobial susceptibility test.
Antimicrobial susceptibility testing
Antimicrobial susceptibility test to ciprofloxacin, norfloxacin, and nalidixic acid was performed by the agar dilution method as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (8). The concentrations of ciprofloxacin, norfloxacin, and nalidixic acid tested in this study were 0.008-128 microgram/ml, 0.06-128 microgram/ml, and 0.25-128 microgram/ml, respectively. Briefly, the suspension of Campylobacter grown in Mueller-Hinton broth was adjusted to turbidity equivalent to 0.5 McFarland standard. These suspensions were then applied onto Mueller-Hinton agar supplemented with 5% sheep blood by a multipoint inoculator. Campylobacter jejuni ATCC 33560 was inoculated on each plate to serve as a quality control organism. The inoculated plates were incubated at 42 C for 48 hours under a microaerophilic condition. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the antimicrobial agent producing no visible growth of colonies on the plates. Breakpoints for resistance of each antimicrobial agent were determined according to the NCCLS established guideline for veterinary pathogens, which were more than or equivalent to 4 µg/ml for ciprofloxacin, more than or equivalent to 16 µg/ml for norfloxacin, and more than or equivalent to 32 µg/ml for nalidixic acid (8).
Genomic DNA analysis
Pulsed-field gel electrophoresis (PFGE) was used for genotypic analysis of Campylobacter according to the previously published procedure (3). Briefly, fresh cultures of Campylobacter were embedded in chromosomal grade agarose (Bio-Rad) and treated with lysis buffer (0.25 M EDTA, 0.5% n-lauroylsarcosine, 1 mg of proteinase K per ml) at 50 C overnight. After being washed, the gel plugs were digested with KpnI restriction enzyme. DNA fragments were separated in pulsed-field-certified agarose (1%) with 0.5 X Tris-borate-EDTA (TBE) buffer at 14 C using a CHEF Mapper system (Bio-Rad). The electrophoresis conditions were optimized by using the embedded autoalgorithm in CHEF Mapper. After separation, the genomic DNA fingerprinting profiles were stained with ethidium bromide then visualized and photographed with a digital imaging system.
A Chi-square test at a significance level of P < 0.05 (two-tailed) with Yates correction for continuity was used for statistical calculation.
Antimicrobial resistance rates
The difference in quinolone and fluoroquinolone resistance rates between Campylobacter strains isolated from commercially-raised and organically-raised broilers and turkeys was observed in this study. Approximately 46% of Campylobacter strains isolated from commercially-raised broilers and 67% of Campylobacter strains isolated from commercially-raised turkeys were resistant to ciprofloxacin, norfloxacin, and nalidixic acid. In contrast, none of Campylobacter strains isolated from organically-raised broilers and less than 2% of Campylobacter strains isolated from organically-raised turkeys were resistant to these antibiotics. Since quinolones and fluoroquinolones have never been used in these organic poultry operations, it is not surprising that little or no fluoroquinolone resistance was observed among Campylobacter strains isolated from organic poultry farms. A high prevalence of fluoroquinolone resistance observed in Campylobacter isolates from commercial broiler farms is interesting. Although no fluoroquinolones were used in commercial broiler flocks from which the samples were collected, it should be noted that these antimicrobial agents were used in the previous flocks of these commercially-raised broilers. Because certain quinolone-resistant clones were stable and able to persist on the farms during several rotations even there had been no selective pressure on that farm for a long period of time (9, 10) and because fluoroquinolone-resistant Campylobacter strains could out compete fluoroquinolone-susceptible Campylobacter strains in the absence of antimicrobial usage (5), this may be the explanation of a high fluoroquinolone resistance rate observed among Campylobacter isolates from commercially-raised broilers. In contrast, since fluoroquinolones were used in commercial turkey operation, a high prevalence of fluoroquinolone resistance observed in Campylobacter strains isolated from commercially-raised turkeys is not unexpected.
In summary, significant difference (P < 0.05) in quinolone and fluoroquinolone resistance rates between Campylobacter strains isolated from commercial poultry operations and organic poultry operations observed in this study suggests that the practice of fluoroquinolone usage in commercial poultry production systems can lead to the development of fluoroquinolone resistance in Campylobacter species.
The genetic diversity of representative Campylobacter isolates from different poultry operation types was analyzed by PFGE. The PFGE patterns generated by KpnI digestion showed great differences among the strains isolated from different farms, while the isolates from a single poultry farm had indistinguishable PFGE patterns. This result reveals a high level of diversity among Campylobacter species isolated from these commercial and organic poultry operations.
Educational & Outreach Activities
These findings were presented at the 141st annual meeting of the American Veterinary Medical Association, which is a national meeting of the veterinary profession that was held in Philadelphia, Pennsylvania during July 24-28, 2004. In addition, this study was also presented at the Advances in Veterinary Medicine Research Day, which was held at The Ohio State University on April 14, 2005 as well as at the Annual Meeting Ohio Branch of the American Society for Microbiology, which was held in Delaware, Ohio during April 15-16, 2005. In terms of publication, the manuscript of this study has been accepted for publication in the Public Health Microbiology section of the Applied and Environmental Microbiology journal, a peer review journal published by the American Society for Microbiology.
Recently, organic poultry are becoming an increasingly important sector of the retail chicken market in many industrialized countries and most of organic poultry productions are small-scale sustainable farming operations. Since the results of our study have shown that Campylobacter isolates from organically-raised broilers and turkeys were significantly less resistant to fluoroquinolones than the isolates from commercially-raised poultry, this finding will undoubtedly help promote organic and/or antibiotic-free poultry production systems, which normally are small-scale sustainable farms.
In addition, because C. jejuni is one of the most important causes of foodborne illness in humans and because it can be transferred from food animals especially poultry to humans via contaminated or improperly handled/cooked foods, the results of this study are directly affected the quality of life of humans (both farmers and consumers) as well as society in the North Central region. Finally, although this study alone may not be able to stop the inappropriate use of antimicrobial agents in animal agriculture, it at least reveals the consequences of antimicrobial usage in animal production systems and it therefore should lead to the development of innovative methods to limit the inappropriate use of antimicrobial agents in animal agriculture in order to prevent the spreading of antibiotic-resistant bacteria from animals to humans.
No economic analysis conducted for this project.
Farmers may adopt the results of this study by reducing the inappropriate use of antibiotics on the farms since it is clearly shown from this study that antimicrobial usage in animal production systems such as in the case of fluoroquinolone usage in commercial poultry operations can lead to the development of antimicrobial resistance in foodborne bacterial pathogens.
Areas needing additional study
Since this study is focused only on C. jejuni, more studies on the prevalence and antimicrobial resistance of other foodborne pathogens in these organically-raised chickens and turkeys will be useful. Also, the information on quality and safety of organically-raised poultry such as the residues of antibiotics or other chemical substances in these organically-raised chicken and turkey meats should be investigated or compared to those of commercially-raised poultry meats. This type of information will be very useful and will also help support organic poultry production systems especially if the results show that organically-raised chickens and turkeys are safer than commercially-raised poultry. However, since the production cycle of organic chickens takes longer time before the birds reach the market age than that of commercial chickens, an economic analysis of these organic poultry production systems should also be determined.