Campylobacter spp are often identified as a cause of bacterial gastroenteritis in humans in the United States1,2 and outnumber other infectious causes of foodborne illness, such as Salmonella, Escherichia coli O157:h7, and Shigella.3 Each year, as many as 2 million cases of illness are estimated to be caused by Campylobacter jejuni.4 Most cases of enteritis are mild, self-limiting episodes of vomiting, cramping, and diarrhea5,6; however, a more serious form of campylobacteriosis can develop in infants, geriatric patients, and immunocompromised individuals. In those individuals, hematochezia, dehydration, septicemia, and long-term sequela can develop,7 including the demyelinating neurologic disorder Guillain-Barre syndrome or intermittent arthritis.8,9 Guillain-Barre syndrome develops subsequent to approximately 1 in 1,000 cases of enteritis caused by C jejuni and is usually transient; however, some individuals with Guillain-Barre syndrome continue to have neurologic deficits throughout life.8
In humans, many Campylobacter infections are associated with direct or indirect exposure to animals.10 Thermophilic Campylobacter spp can colonize the gastrointestinal tracts of mammals and birds without causing disease11; therefore, feces from apparently healthy animals may contaminate the environment with Campylobacter organisms. Research has been focused on Campylobacter-contaminated poultry in retail markets12,13; however, the dairy industry may also be a source of human exposure to Campylobacter organisms. Healthy adult cows and calves frequently shed this organism in their manure,14–15,a and a number of outbreaks of Campylobacter enteritis have been associated with consumption of raw milk,16–19 dairy farm visits,20 and water contamination.21–23 Consequently, the role of dairy cattle in the transmission of Campylobacter to humans should be examined in greater depth.
Another public health concern associated with Campylobacter is that this organism has developed resistance to antimicrobial agents. Campylobacter isolates from humans are becoming increasingly resistant to numerous classes of antimicrobial drugs with time and with the introduction of new pharmaceutical drugs.24 In developing countries, antimicrobial resistance may be associated with widespread availability of these drugs7 because easy access to antimicrobial agents often results in self-medicating to compensate for poor sanitary conditions.25,26 In developed countries, such as Denmark, domestically acquired Campylobacter spp isolated from humans in 2004 were resistant to nalidixic acid (31% of 107 isolates), ciprofloxacin (29%), tetracycline (24%), and erythromycin (5%).27
In countries with restricted availability of antimicrobial agents, including the United States, there is ongoing debate regarding the contribution of human medical, veterinary therapeutic, and animal husbandry practices to the decreased susceptibility of key bacteria to antimicrobials.28–30 Increased fluoroquinolone resistance has been detected in Campylobacter and other bacteria once these antimicrobials were approved in some food animal species,13,31 and there has been evidence of increased susceptibility in bacteria when certain antimicrobials are banned from use.32 However, most studies supporting the decrease in susceptibility are based on ecologic (aggregative) analysis of data (ie, which drugs are approved for veterinary use in a particular country) without ascertaining actual exposure to the drugs being studied, and the focus of much research on Campylobacter resistance has been on drug classes such as fluoroquinolones and macrolides. Studies33,34 of antimicrobial resistance to drugs used on dairy farms are limited; therefore, the role of dairy farm practices in the development of antimicrobial resistance in Campylobacter spp remains poorly defined.
To address the need for research on antimicrobial resistance and dairy cattle, the purpose of the study reported here was to describe antimicrobial susceptibility patterns of Campylobacter spp isolated from dairy cattle and farms managed organically and conventionally in the midwestern and northeastern United States.
Materials and Methods
The study reported here was part of a large longitudinal study investigating the prevalence and antimicrobial susceptibility patterns of Campylobacter spp and Salmonella spp isolated from dairy cattle from organic and conventionally managed farms in Michigan, Minnesota, New York, and Wisconsin. Results of the study associated with Salmonellaspp have been reported.35–38
Herds—One hundred thirty-two dairy farms were chosen from Michigan, Minnesota, New York, and Wisconsin, from which 128 farms had Campylobacter isolates available for antimicrobial susceptibility testing. Herds were enrolled according to farm type (organic vs conventional) and by farm size (No. of lactating and nonlactating cows). To be included in the study, a farm had to meet the following criteria: have at least 30 milking cows in the herd, have at least 90% of cows in the herd of Holstein breed, raise their own calves for replacement cattle, and ship milk all year. Organic farms had to be certified as organic by a recognized organic certification agency and may not have used antimicrobials in cattle > 1 year old for at least 3 years. Lists of farms were obtained from the respective state departments of agriculture, and herd owners within approximately 100 miles of the respective universities were randomly chosen to receive a mailing describing the study. Owners of farms were asked to indicate interest in participation in the study by returning a postcard. The final list of farms was obtained by randomly choosing names of respondents that had indicated willingness to participate in the study. To evaluate potential herd management practices as risk factors, a predetermined number of farms was enrolled within the following herd size categories: 30 to 49, 50 to 99, 100 to 199, and ≥ 200 cows. Because of the limited availability of organic farms, owners of all known organic farms within approximately 150 miles of the respective universities were contacted to determine eligibility on the basis of selection criteria and their desire to participate in the study. Farm visits for the collection of cattle and environmental samples took place every other month during a 12-month period.
Collection of samples—Approximately 10 g of fecal material was collected from the rectum of cattle and placed into plastic bags.b A separate glove was used to collect each sample. The number of samples collected per herd and the number of samples collected from specific cattle groups were determined by herd size. The total number of fecal samples from herds with 30 to 49, 50 to 99, 100 to 199, and ≥ 200 cows was 30, 40, 50, and 55 samples, respectively. Cattle management groups included preweaned heifer calves, cows to be culled within 14 days, periparturient cows (due to calve within 14 days and cows within 14 days in lactation after calving), sick cows as determined by farm personnel or the herd veterinarian, and healthy lactating cows. No effort was made to collect samples from the same individual cattle at subsequent herd visits.
Farm environmental samples—One sample from each location was collected at each sampling visit by wiping areas to be tested with sterile gauze pads soaked in double-strength skim milk (skim milk powderc reconstituted with 50% of the volume of water normally used and sterilized via autoclaving), which were placed into plastic bagsb for shipment. Sampling locations included areas in which cattle may be directly exposed to Campylobacter, including feed bunk of lactating cows; lagoon or manure pile; bird droppings in areas housing cows; and the walls, boards, or flooring of maternity pens and areas housing calves or sick cattle. In cows that were going to be culled, a sample was obtained by wiping the coat across the lower aspect of the flank and gluteal region with a swab soaked in double-strength skim milk. If a pen location was not used on a particular farm (eg, no sick pen), then no sample was collected for that location. Samples collected from pens used for more than 1 purpose, such as the sick cow pen and calving pen, were labeled according to the predominant use. A sample from a water source for cattle (eg, a water tank or a pooled swab specimen from 5 drinking cups), a bulk tank milk sample, and a milk line filter were also collected.
Shipment of samples—After collection, samples were shipped to a central laboratory at Michigan State University. Samples from Minnesota, New York, and Wisconsin were shipped via overnight delivery in insulated foam boxes with ice packs. Samples were shipped the same day as collection whenever possible; however, some samples were stored at 2° to 4°C for 2 to 36 hours until the next shipping opportunity.
Isolation and identification ofCampylobacter spp—Environmental swab specimens and milk filters were enriched in Bolton brothd containing 5% laked horse blood and selective antimicrobial agents (cefaperazone [20 mg/L], vancomycin [20 mg/L], trimethoprim [20 mg/L], and cycloheximide [50 mg/L]). Enriched samples were incubated at 42°C in 5% to 10% CO2 for 48 hours. Fecal and milk samples were suspended in phosphate buffer saline solution. Fecal and milk samples suspended in phosphate buffered saline solution and enriched samples were streaked on selective Campylobacter plates with 5 antimicrobial agents (amphotericin B, cephalothin, trimethoprim, vancomycin, and polymyxin B) and 10% defibrinated sheep bloode and incubated at 42°C in 5% to 10% CO2 for 48 hours. Typical colonies (small pinpoint gray colonies without hemolysis) were selected and streaked on sheep blood agar and incubated at 42°C in 5% to 10% CO2 for 48 hours. Identification of Campylobacterspp was performed from isolated colonies by gram staining, oxidase testing, and motility testing. Hippurate hydrolysis was used to speciate C jejuni by use of C jejuni ATCC 33560 as a positive control and Campylobacter coli ATCC 33559 as a negative control.
In vitro antimicrobial susceptibility testing—In vitro antimicrobial susceptibility testing was performed by use of a microbroth dilution method. At the time this study was performed in 2000 to 2001, the NCCLS had not approved standardized recommendations for in vitro antimicrobial susceptibility testing for Campylobacterspp.39 Because of the lack of a standard, in vitro antimicrobial susceptibility testing was performed by use of the microbroth dilution method, following the NCCLS guidelines available at that time for bacteria isolated from animals.39,40 In 2003, the NCCLS approved agar dilutions as the standardized method for antimicrobial susceptibility testing for Campylobacterspp39; therefore, a study41 was performed in our laboratory with a subset of those isolates to verify the performance of our microbroth dilution system with agar dilution. Briefly, results of that study indicated that there was no association in the classification of resistance by the testing methods used, and the quality control strain of C jejuni ATCC 33560 performed in a consistent manner for both agar dilution and microbroth dilution.41
Bacterial isolates from frozen stock were grown on BASB for 48 hours at 42°C in 5% to 10% CO2. Individual colonies from each plate were subcultured on BASB in similar conditions. Bacteria were swabbed from the BASB and suspended in 5 mL of water, and the turbidity was adjusted to a 0.5 McFarland standard. This suspension was used to make a dilution in a 1:10 ratio in Haemophilus testing medium,f resulting in a final bacterial inoculum concentration of approximately 8 × 105 colony forming units/mL.
Commercially prepared microbroth dilution plates were used for antimicrobial susceptibility testing. For all plates, C jejuni ATCC 33560 and C jejuni ATCC 81176 were used as quality control strains. Each plate was inoculated by adding 100 μL of bacterial suspension by use of an autoinoculator,g covered with a gas-permeable seal, and incubated at 42°C in microaerophilic conditions for 48 hours. The MIC, the minimum antimicrobial dilution at which no bacterial growth developed, was read manually from each plate for each isolate. The MIC50 and MIC90 from a given herd were calculated from the microbroth dilution plate with the largest range of MIC values for each drug.
Two different microbroth dilution plates were used throughout the study. Initially, to determine whether patterns of resistance detected in isolates from dairy cattle were comparable to those detected in isolates from humans, a customized antimicrobial panel (CMV1USDA),h with a prepared range of concentrations for azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid, and tetracycline, was purchased. After observing that isolates from dairy farms did not have resistance patterns similar to isolates from humans (decreased susceptibility to ciprofloxacin or azithromycin),13,24,29 another customized antimicrobial panel (CMV2DMSU)h was developed to address drug exposures that are common to dairy cattle management and to permit comparison with patterns of resistance detected in Salmonella spp.37,38 Antimicrobial panel CMV2DMSU included 17 drugs encompassing drug classes used on our study farms, such as β-lactams and cephalosporins,36 and its use replaced the CMV1USDA panel. Breakpoints used to classify isolates as resistant or not resistant were those recommended by the National Antimicrobial Resistance Monitoring System for Campylobacter spp for azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid, and tetracycline (Table 1).41 For the CMV2DMSU panel, general enteric breakpoints were used to classify isolates as resistant for the additional antimicrobials. Whereas selective media with antimicrobial agents (cefaperazone, vancomycin, trimethoprim, and cycloheximide) were used to isolate Campylobacter spp, any isolates detected would have been expected to be resistant to these agents. Consequently, analysis of resistance to ceftiofur, cephalothin, and trimethoprim-sulfamethoxazole was not pursued.
Dilution ranges for antimicrobial agents used, by panel, and interpretative breakpoints for in vitro antimicrobial susceptibility testing for Campylobacter isolates obtained from dairy cattle and farms managed conventionally and organically in the midwestern and northeastern United States.
| Antimicrobial | CMV1USDAh panel (μg/mL) | CMV2DMSUh panel (μg/mL) | Interpretative criteria for resistant strains (μg/mL) |
|---|---|---|---|
| Amoxicillin-clavulanic acid | |||
| Amoxicillin | NA | 2–64 | ≥32 |
| Clavulanic acid | NA | 1–32 | ≥16* |
| Ampicillin | NA | 2–64 | ≥32* |
| Azithromycin | 0.03–256 | 0.12–4 | ≥2† |
| Ceftiofur | NA | 1–16 | ≥8* |
| Ceftriaxone | NA | 4–128 | ≥64* |
| Cephalothin | NA | 4–64 | ≥32* |
| Chloramphenicol | 0.5–64 | 4–64 | ≥32* |
| Ciprofloxacin | 0.03–64 | 0.5–16 | ≥4† |
| Clindamycin | 0.06–256 | NA | ≥4† |
| Erythromycin | 0.12–256 | 0.25–16 | ≥8† |
| Florfenicol | NA | 2–32 | ≥16* |
| Gentamicin | 0.12–256 | 2–32 | ≥16† |
| Kanamycin | NA | 8–128 | ≥64* |
| Nalidixic acid | 0.12–128 | 4–128 | ≥32† |
| Streptomycin | NA | 16–128 | ≥64† |
| Sulfamethoxazole | NA | 64–512 | ≥512* |
| Tetracycline | 0.25–256 | 2–128 | ≥16† |
| Trimethoprim-sulfamethoxazole | |||
| Trimethoprim | NA | 1–8 | ≥4 |
| Sulfamethoxazole | NA | 16–512 | ≥64* |
Breakpointfrom NCCLS.41
NCCLS (Clinical and Laboratory Standards Institute) General Enteric breakpoints used by the National Antimicrobial Resistance Monitoring System.42NA = Not applicable.
Data analysis—To determine whether there was an association with the proportion of resistant isolates and farm type, descriptive breakpoints were used to classify isolates as resistant or susceptible for each antimicrobial agent. The proportion of resistant isolates by farm type (organic or conventional) was analyzed by use of χ2 tests via a computer software program.i
Results
A total of 2,030 Campylobacter isolates were available for antimicrobial susceptibility testing. Organic farms yielded 450 fecal, 8 environmental, and 2 milk and milk filter Campylobacter isolates for antimicrobial susceptibility testing, whereas conventional farms yielded 1,525 fecal, 33 environmental, and 12 milk and milk filter isolates (Table 2) . Greater than 97% of isolates were classified as C jejuni, and the rest were not further speciated.a
Distribution of Campylobacter isolates obtained from dairy cattle and farms managed conventionally and organically in the midwestern and northeastern United States used for antimicrobial susceptibility testing.
| Campylobacter isolates | Organic farm | Conventional farm |
|---|---|---|
| Farm environmental isolates | ||
| Feedbunk | 0 | 0 |
| Calf pen | 2 | 1 |
| Sick cow pen | 0 | 1 |
| Maternity pen | 2 | 3 |
| Water tank | 1 | 3 |
| Lagoon | 1 | 3 |
| Bulk tank milk | 0 | 3 |
| Milk filter | 2 | 9 |
| Bird droppings | 0 | 4 |
| Cull cow haircoat | 0 | 6 |
| Total | 8 | 33 |
| Cattle isolates | ||
| Preweaned calves | 132 | 427 |
| Healthy lactating cows | 238 | 683 |
| Cull cows | 3 | 32 |
| Cows due to calve in 14 days | 23 | 80 |
| Cows that calved within 14 days | 35 | 177 |
| Sick cows | 19 | 126 |
| Total | 450 | 1,525 |
Across farm type, conventional farms had slightly higher proportions of resistant isolates to most antimicrobial agents than organic farms. For fecal isolates, there were slightly higher proportions of reduced antimicrobial susceptibility in conventional farm isolates (9.9% resistant overall and 6.4% resistant excluding tetracycline), compared with organic farm isolates (8.9% resistant overall and 6.0% resistant excluding tetracycline; Table 3). The only significant (P = 0.007) difference detected was in resistance to tetracycline, in which the proportion of resistant isolates was higher for conventional farms (58.3%) than organic farms (49.3%). Similarly, there were no significant differences in MIC50 and MIC90 dilutions between conventional and organic isolates, with the exception of tetracycline. Conventional farm isolates required 4 times the antimicrobial concentration of tetracycline (32 μ g/mL) to inhibit growth of 50% of the isolates than required for organic farm isolates (8 μ g/mL). Higher proportions of reduced antimicrobial susceptibility were detected for antimicrobial agents in the CMV2DMSU panel (mean percentage resistant, 11.4%) than in the CMV1USDA panel (mean percentage resistant, 7.6%), and a higher proportion of multidrug resistance was detected in isolates from conventional farms (47%), compared with isolates from organic farms (40%), when the CMV2DMSU panel was used (Figure 1).
In vitro antimicrobial susceptibility of Campylobacter isolates from feces of dairy cattle on farms managed conventionally or organically in the midwestern and northeastern United States.
| Antimicrobial | Conventional farm isolates | Organic farm isolates | ||||||
|---|---|---|---|---|---|---|---|---|
| No. of cattle | MIC50 | MIC90 | Resistant (%) | No. of cattle | MIC50 | MIC90 | Resistant (%) | |
| Amoxicillin-clavulanic acid* | 686 | 2 | 2 | 0.1 | 168 | 2 | 2 | 0 |
| Ampicillin | 686 | 4 | 8 | 8.6 | 168 | 8 | 16 | 7.1 |
| Azithromycin† | 1,525 | 0.12 | 0.12 | 1.3 | 450 | 0.06 | 0.12 | 1.1 |
| Ceftriaxone* | 686 | 16 | 16 | 1.4 | 168 | 16 | 32 | 2.3 |
| Chloramphenicol† | 1,525 | 2 | 4 | 1.1 | 450 | 2 | 4 | 0 |
| Ciprofloxacin† | 1,525 | 0.12 | 0.5 | 1.1 | 450 | 0.12 | 0.5 | 0.9 |
| Clindamycin† | 840 | 0.12 | 0.5 | 1.3 | 282 | 0.12 | 0.25 | 1.0 |
| Erythromycin† | 1,525 | 0.5 | 1.0 | 1.2 | 450 | 0.5 | 1.0 | 1.1 |
| Florfenicol* | 686 | 2 | 2 | 0.3 | 168 | 2 | 2 | 0 |
| Gentamicin† | 1,525 | 2 | 2 | 0.1 | 450 | 1 | 2 | 0 |
| Kanamycin* | 686 | 8 | >128 | 32.4 | 168 | 8 | >128 | 30.0 |
| Nalidixic acid† | 1,525 | 4 | 8 | 1.9 | 450 | 4 | 8 | 1.3 |
| Streptomycin* | 686 | 16 | 16 | 1.6 | 168 | 16 | 16 | 0.6 |
| Sulfamethoxazole* | 686 | 256 | 512 | 37.2 | 168 | 256 | 256 | 38.7 |
| Tetracycline† | 1,525 | 32 | 128 | 58.3 | 450 | 8 | 128 | 49.3 |
Sample numbers change because not all antimicrobials were present on all panels used to test for antimicrobial susceptibility.
CMV2DMSU.h
CMV1USDA.h
Percentage of Campylobacterisolates with multidrug resistance as determined by in vitro antimicrobial susceptibility testing (customized panel with 17 antimicrobials [CMV2DMSU]) of samples obtained from dairy cattle and farms managed conventionally (black bars; n = 696) or organically (stippled bars;169) in the midwestern and northeastern United States.
Citation: Journal of the American Veterinary Medical Association 228, 7; 10.2460/javma.228.7.1074
Percentage of Campylobacterisolates with multidrug resistance as determined by in vitro antimicrobial susceptibility testing (customized panel with 17 antimicrobials [CMV2DMSU]) of samples obtained from dairy cattle and farms managed conventionally (black bars; n = 696) or organically (stippled bars;169) in the midwestern and northeastern United States.
Citation: Journal of the American Veterinary Medical Association 228, 7; 10.2460/javma.228.7.1074
Percentage of Campylobacterisolates with multidrug resistance as determined by in vitro antimicrobial susceptibility testing (customized panel with 17 antimicrobials [CMV2DMSU]) of samples obtained from dairy cattle and farms managed conventionally (black bars; n = 696) or organically (stippled bars;169) in the midwestern and northeastern United States.
Citation: Journal of the American Veterinary Medical Association 228, 7; 10.2460/javma.228.7.1074
Given the low numbers of isolates from farm environmental samples and milk and milk filters, MIC50 and MIC90 were not calculated. Resistance in isolates from farm environmental samples was low, with no resistant isolates detected for amoxicillin-clavulanic acid, ceftriaxone, chloramphenicol, ciprofloxacin, florfenicol, gentamicin, kanamycin, and streptomycin (Table 4) . Resistance to sulfamethoxazole was detected in 50% of isolates from conventional farms. Decreased antimicrobial susceptibility to tetracycline was detected in 5 of 12 Campylobacter isolates from milk and milk filters from conventional farms.
Distribution of MICs for farm environmental Campylobacter isolates obtained from dairy farms managed organically and con ventionally in the midwestern and northeastern United States.
| Antimicrobial | MIC (μg/mL) | No. of isolates | Antimicrobial | MIC (μg/mL) | No. of isolates | ||
|---|---|---|---|---|---|---|---|
| Conventional | Organic | Conventional | Organic | ||||
| Amoxicillin-clavulanic acid* (MIC resistance breakpoint ≥32/16) | 2 | 9 | 1 | Erythromycin*† (MIC resistance breakpoint ≥8) | 0.12 | 1 | 0 |
| 4 | 1 | 0 | 0.25 | 10 | 0 | ||
| 8 | 0 | 0 | 0.5 | 11 | 3 | ||
| 16 | 0 | 0 | 1 | 8 | 3 | ||
| 2 | 2 | 2 | |||||
| 4 | 0 | 0 | |||||
| Ampicillin* (MIC resistance breakpoint ≥32) | 2 | 3 | 1 | 8 | 0 | 0 | |
| 4 | 1 | 0 | 16 | 0 | 0 | ||
| 8 | 4 | 0 | 32 | 1 | 0 | ||
| 16 | 1 | 0 | 64 | 0 | 0 | ||
| 32 | 0 | 0 | 128 | 0 | 0 | ||
| 64 | 1 | 0 | 256 | 0 | 0 | ||
| Azithromycin*† (MIC resistance breakpoint ≥2) | 0.03 | 10 | 3 | Florfenicol* (MIC resistance breakpoint ≥16) | 2 | 10 | 1 |
| 0.06 | 4 | 3 | 4 | 0 | 0 | ||
| 0.12 | 15 | 2 | 8 | 0 | 0 | ||
| 0.25 | 3 | 0 | 16 | 0 | 0 | ||
| 0.5 | 0 | 0 | 32 | 0 | 0 | ||
| 1 | 0 | 0 | |||||
| 2 | 0 | 0 | Gentamicin*† (MIC resistance breakpoint ≥16) | 0.12 | 0 | 0 | |
| 4 | 0 | 0 | 0.25 | 0 | 1 | ||
| 8 | 1 | 0 | 0.5 | 0 | 1 | ||
| 16 | 0 | 0 | 1 | 18 | 3 | ||
| 32 | 0 | 0 | 2 | 14 | 3 | ||
| 64 | 0 | 0 | 4 | 0 | 0 | ||
| 128 | 0 | 0 | 8 | 1 | 0 | ||
| 256 | 0 | 0 | 16 | 0 | 0 | ||
| 32 | 0 | 0 | |||||
| Ceftriaxone* (MIC resistance breakpoint ≥64) | 4 | 1 | 0 | 64 | 0 | 0 | |
| 8 | 1 | 0 | 128 | 0 | 0 | ||
| 16 | 6 | 1 | 256 | 0 | 0 | ||
| 32 | 2 | 0 | |||||
| 64 | 0 | 0 | Kanamycin* (MIC resistance breakpoint ≥64) | 8 | 10 | 1 | |
| 128 | 0 | 0 | 16 | 0 | 0 | ||
| 32 | 0 | 0 | |||||
| 64 | 0 | 0 | |||||
| Chloramphenicol*† (MIC resistance breakpoint 32) | 0.5 | 1 | 1 | 128 | 0 | 0 | |
| 1 | 8 | 3 | |||||
| 2 | 13 | 3 | Nalidixic acid*† (MIC resistance | 0.12 | 0 | 0 | |
| 4 | 10 | 1 | 0.25 | 0 | 0 | ||
| 8 | 1 | 0 | 0.5 | 0 | 0 | ||
| 16 | 0 | 0 | 1 | 0 | 0 | ||
| 32 | 0 | 0 | 2 | 2 | 1 | ||
| 64 | 0 | 0 | 4 | 25 | 3 | ||
| 8 | 5 | 3 | |||||
| 16 | 0 | 0 | |||||
| Ciprofloxacin*† (MIC resistance breakpoint ≥4) | 0.03 | 1 | 0 | 32 | 1 | 1 | |
| 0.06 | 6 | 3 | 64 | 0 | 0 | ||
| 0.12 | 13 | 3 | 128 | 0 | 0 | ||
| 0.25 | 1 | 1 | |||||
| 0.5 | 11 | 1 | Streptomycin* (MIC resistance breakpoint ≥64) | 16 | 10 | 1 | |
| 1 | 0 | 0 | 32 | 0 | 0 | ||
| 2 | 1 | 0 | 64 | 0 | 0 | ||
| 4 | 0 | 0 | 128 | 0 | 0 | ||
| 8 | 0 | 0 | |||||
| 16 | 0 | 0 | Sulfamethoxazole* (MIC resistance breakpoint ≥512) | 64 | 1 | 0 | |
| 32 | 0 | 0 | 128 | 1 | 0 | ||
| 64 | 0 | 0 | 256 | 3 | 0 | ||
| 512 | 3 | 1 | |||||
| Clindamycin† (MIC resistance breakpoint ≥4) | 0.06 | 4 | 4 | >512 | 2 | 0 | |
| 0.12 | 3 | 2 | |||||
| 0.25 | 9 | 1 | Tetracycline*† (MIC resistance breakpoint ≥16) | 0.25 | 12 | 5 | |
| 0.5 | 6 | 1 | 0.5 | 0 | 1 | ||
| 1 | 0 | 0 | 1 | 0 | 0 | ||
| 2 | 0 | 0 | 2 | 6 | 1 | ||
| 4 | 0 | 0 | 4 | 0 | 0 | ||
| 8 | 0 | 0 | 8 | 1 | 0 | ||
| 16 | 0 | 0 | 16 | 3 | 0 | ||
| 32 | 0 | 0 | 32 | 2 | 0 | ||
| 64 | 1 | 0 | 64 | 2 | 1 | ||
| 128 | 0 | 0 | 128 | 4 | 0 | ||
| 256 | 0 | 0 | 256 | 3 | 0 | ||
CMV2DMSU (10 conventional isolates and 1 organic isolate)
†CMV1USDA (23 conventional isolates and 8 organic isolates).
Discussion
Although infections and outbreaks in humans caused by Campylobacter spp have been associated with or linked to dairy cattle sources,20,21,43 little critical evaluation of the antimicrobial susceptibility of those isolates has been performed. Although multidrug-resistant Salmonella infections in humans have been traced to dairy farms through meat or milk consumption,44 evaluation of this association would also seem prudent for Campylobacter spp. An additional concern is that the current consumer interest in organic and alternative food sources has resulted in some consumers bypassing such food safety measures as pasteurization.45 In light of increased consumer interest in raw milk and minimally processed food products, it is noteworthy that decreased susceptibility was observed in some isolates from raw milk and milk filter samples to the 8 antimicrobials of interest in treating infections in humans. The practice of drinking raw milk has lead to infections in humans with Campylobacter and Salmonella spp.43,44,46 Therefore, unprocessed dairy products may be capable of transmitting not only foodborne pathogens but also antimicrobial resistance determinants through the exchange of mobile genetic elements such as plasmids or integrons.
In addressing the primary purpose of evaluating the susceptibility of Campylobacter spp by farm type in the United States, results of our study indicated that Campylobacter isolates from both types of dairy farms are generally susceptible to most antimicrobials. This finding agrees with results of studies47–50 on farming systems in countries in which antimicrobial use is regulated more than it is in the United States. With the exception of tetracycline, no significant association between decreases in antimicrobial resistance and organic farming practices was detected, which agrees with results of a study51 indicating that no clear associations between on-farm antimicrobial use and susceptibility patterns in Campylobacter isolates to tetracycline, kanamycin, ciprofloxacin, erythromycin, or nalidixic acid were detected. Increased susceptibility to tetracycline in isolates from organic dairies was detected, which agrees with results of other studies52,53 indicating that antimicrobial susceptibility among organic farming systems increased, compared with isolates from conventional farms.
In addition to differences in selection pressures from antimicrobial use, organic farms are often small and use different animal management practices, such as pasture grazing or exposure of cattle to free-living bird environments,36,54 compared with large, conventionally managed dairies. Resistance of Campylobacter spp in free-living wild birds has also been detected, suggesting that wildlife may play a role in the ecology of antimicrobial resistance.55 Consequently, various management practices must be considered when evaluating the ecology of antimicrobial resistance in a farm environment.
There were 2 drugs, kanamycin and tetracycline, for which resistance was common to both farm types. Antimicrobial susceptibility to tetracycline, which has a much wider spectrum of use in cattle than kanamycin, was significantly decreased on conventional farms, compared with organic farms. In other species, resistance to tetracycline has been found to be associated with use of this drug in broiler chicken flocks and in birds that had been exposed to a coccidiostat only.56 Coccidostats are frequently used in dairy heifer rations on conventional farms36; however, this was not a common practice in organic herds used in our study. Reportedly, genetic determinants for kanamycin and tetracycline resistance (KanR and tetO, respectively) are carried on plasmids in C jejuni,57 and it is possible that these mobile genetic elements are continually exchanged between other bacteria and C jejuni, despite a lack of selective pressure in the animal host from which it was isolated. Genetic markers for tetracycline resistance have been detected in farming environments,58 making environmental contamination a viable source for an animal to acquire resistance factors regardless of whether an individual animal was treated with antimicrobial drugs.
Results of our study indicated that compared with conventional farms, Campylobacter isolates obtained from organic farms were not more susceptible to all classes of antimicrobials studied. However, differences observed in tetracycline resistance between various farm management types and results of other studies32,47-50 suggested that the issue of antimicrobial resistance in food animals warrants investigation of modifiable herd and individual animal risk factors to aid in the planning and implementation of interventions that will promote food safety and a healthy livestock population.
ABBREVIATIONS
| ATCC | American Type Culture Collection |
| BASB | Brucellaagar with supplemental 5% defibrinated sheep blood |
| MIC | Minimum inhibitory concentration |
| MIC50 | MIC inhibiting growth of 50% of isolates |
| MIC90 | MIC inhibiting growth of 90% of isolates |
Green AM. Patterns of occurrence of Campylobacter in organic and conventional diary farms in midwestern and northeastern United States. MS thesis, Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, Mich, 2002.
Whirl-Pak, Nasco, Fort Atkinson, Wis.
Becton-Dickinson Microbiology Systems (formerly Difco), Sparks, Md.
Bolton broth, Oxoid USA (Remel Inc), Lenexa, Kan.
Campylobacter agar with 5 antimicrobics and 10% sheep blood (Blaser), BD Diagnostics, Franklin Lakes, NJ.
Haemophilus testing medium, TREK Diagnostics Systems Inc, Cleveland, Ohio.
SensiTitre, TREK Diagnostics Systems Inc, Cleveland, Ohio.
TREK Diagnostic Systems Inc, Cleveland, Ohio.
SAS, version 8.2, SAS Institute Inc, Cary, NC.
References
- 1.
Acheson DWK. Foodborne diseases update: current trends in foodborne disease. Medscape Infect Dis 2001;3:1–9.
- 2.
Altekruse SF, Tollefson LK. Human campylobacteriosis: a challenge for the veterinary profession. J Am Vet Med Assoc 2003;223:445–452.
- 3.↑
Mead PS, Slutsker L, Dietz V, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999;5:607–625.
- 4.↑
Allos MB. Campylobacter jejuni infections: update on emerging issues and trends. Clin Infect Dis 2001;32:1201–1206.
- 5.
Tauxe RV, Hargrett-Bean N, Patton CM. Campylobacter isolates in the United States, 1982–1986. MMWR CDC Surveill Summ 1988;37:1–13.
- 6.
Altekruse SF, Swerdlow DL, Stern NJ. Microbial food borne pathogens. Campylobacter jejuni. Vet Clin North Am Food Anim Pract 1998;14:31–40.
- 7.↑
Blaser MJ. Epidemiologic and clinical features of Campylobacter jejuni infections. J Infect Dis 1997;176 (suppl 2):S103–S105.
- 8.↑
Rees JH, Soudain SE, Gregson NA, et al. Campylobacter jejuni infection and Guillain-Barre syndrome. N Engl J Med 1995;333:1374–1379.
- 9.
Nachamkin I, Allos BM, Ho T. Campylobacter species and Guillain-Barre syndrome. Clin Microbiol Rev 1998;11:555–567.
- 10.↑
Deming MS, Tauxe RV, Blake PA, et al. Campylobacter enteritis at a university: transmission from eating chicken and from cats. Am J Epidemiol 1987;126:526–534.
- 12.
Harris NV, Thompson D, Martin DC, et al. A survey of Campylobacter and other bacterial contaminants of pre-market chicken and retail poultry and meats, King County, Washington. Am J Public Health 1986;76:401–406.
- 13.
Smith KE, Besser JM, Hedberg CW, et al. Quinolone-resistant Campylobacter jejuni infections in Minnesota, 1993–1998. Investigation team. N Engl J Med 1999;340:1525–1532.
- 14.
Wesley IV, Wells SJ, Harmon KM, et al. Fecal shedding of Campylobacter and Arcobacter spp in dairy cattle. Appl Environ Microbiol 2000;66:1994–2000.
- 15.
Nielsen EM. Occurrence and strain diversity of thermophilic campylobacters in cattle of different age groups in dairy herds. Lett Appl Microbiol 2002;35:85–89.
- 16.
Warner DP, Bryner JH, Beran GW. Epidemiologic study of campylobacteriosis in Iowa cattle and the possible role of unpasteurized milk as a vehicle of infection. Am J Vet Res 1986;47:254–258.
- 17.
Dilworth CR, Lior H, Belliveau MA. Campylobacter enteritis acquired from cattle. Can J Public Health 1988;79:60–62.
- 18.
Kalman M, Szollosi E, Czermann B, et al. Milkborne Campylobacter infection in Hungary. J Food Prot 2000;63:1426–1429.
- 19.
Lehner A, Schneck C, Feierl G, et al. Epidemiologic application of pulsed-field gel electrophoresis to an outbreak of Campylobacter jejuni in an Austrian youth centre. Epidemiol Infect 2000;125:13–16.
- 20.↑
Evans MR, Roberts RJ, Ribeiro CD, et al. A milk-borne Campylobacter outbreak following an educational farm visit. Epidemiol Infect 1996;117:457–462.
- 21.
Duke LA, Breathnach AS, Jenkins DR, et al. A mixed outbreak of Cryptosporidium and Campylobacter infection associated with a private water supply. Epidemiol Infect 1996;116:303–308.
- 22.
Melby KK, Svendby JG, Eggebo T, et al. Outbreak of Campylobacter infection in a subartic community. Eur J Clin Microbiol Infect Dis 2000;19:542–544.
- 23.
Frost JA, Gillespie IA, O'Brien SJ. Public health implications of Campylobacter outbreaks in England and Wales, 1995–9: epidemiological and microbiological investigations. Epidemiol Infect 2002;128:111–118.
- 24.↑
Engberg J, Aarestrup FM, Taylor DE, et al. Quinolone and macrolide resistance in Campylobacter jejuni and C coli: resistance mechanisms and trends in human isolates. Emerg Infect Dis 2001;7:24–34.
- 25.
Oberhelman RA, Taylor DN. Campylobacter infections in the developing world. In: Nachamkin I, Blaser M, eds. Campylobacter. 2nd ed. Washington, DC: ASM Press, 2000;139–153.
- 26.
Padungton P, Kaneene JB. Campylobacter spp in human, chickens, pigs and their antimicrobial resistance. J Vet Med Sci 2003;65:161–170.
- 27.↑
Emborg HD, Heuer HO, Larsen PB. DANMAP 2004—Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark. Copenhagen, Denmark: Statens Serum Institut, Danish Veterinary and Food Administration, Danish Medicines Agency, Danish Veterinary Institute, 2004.
- 28.
Threlfall EJ, Ward LR, Frost JA, et al. The emergence and spread of antibiotic resistance in food-borne bacteria. Int J Food Microbiol 2000;62:1–5.
- 29.
Smith KE, Bender JB, Osterholm MT. Antimicrobial resistance in animals and relevance to human infections. In: Nachamkin I, Blaser M, eds. Campylobacter. 2nd ed. Washington, DC: ASM Press, 2000;483–495.
- 30.
Wagner J, Jabbusch M, Eisenblatter M, et al. Susceptibilities of Campylobacter jejuni isolates from Germany to ciprofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline. Antimicrob Agents Chemother 2003;47:2358–2361.
- 31.
McDermott PF, Bodeis SM, English LL, et al. Ciprofloxacin resistance in Campylobacter jejuni evolves rapidly in chickens treated with fluoroquinolones. J Infect Dis 2002;185:837–840.
- 32.↑
Aarestrup FM, Seyfarth AM, Emborg HD, et al. Effect of abolishment of the use of antimicrobial agents for growth promotion on the occurrence of antimicrobial resistance in fecal enterococci from food animals in Denmark. Antimicrob Agents Chemother 2001;45:2054–2059.
- 33.
Hady PJ, Lloyd JW, Kaneene JB. Antibacterial use in lactating dairy cattle. J Am Vet Med Assoc 1993;203:210–220.
- 34.
Sundlof SF, Kaneene JB, Miller R. National survey on veterinarian-initiated drug use in lactating dairy cattle. J Am Vet Med Assoc 1995;207:347–352.
- 35.
Warnick LD, Kaneene JB, Ruegg PL, et al. Evaluation of herd sampling for Salmonella isolation on midwest and northeast US dairy farms. Prev Vet Med 2003;60:195–206.
- 36.↑
Zwald A, Ruegg P, Kaneene J, et al. Management practices and reported antimicrobial usage on conventional and organic dairy farms. J Dairy Sci 2004;87:191–201.
- 37.
Fossler CP, Wells SJ, Kaneene JB, et al. Prevalence of Salmonella spp on conventional and organic dairy farms. J Am Vet Med Assoc 2004;225:567–573.
- 38.
Fossler CP, Wells SJ, Kaneene JB, et al. Cattle and environmental sample-level factors associated with presence of Salmonella in a multi-state study of conventional and organic dairy farms. Prev Vet Med 2005;67:39–53.
- 39.↑
National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard 23. Wayne, Pa: NCCLS, 2003.
- 40.
National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard 19—first edition (M31-A). Wayne, Pa: NCCLS, 1999.
- 41.↑
Halbert LW, Kaneene JB, Mansfield LS, et al. Comparison of automated microbroth dilution and agar dilution for antimicrobial susceptibility of Campylobacter jejuni isolated from dairy sources. J Antimicrob Chemother 2005;56:686–691.
- 42.
National Antimicrobial Resistance Monitoring System (NARMS). National Antimicrobial Resistance Monitoring System: enteric bacteria—2000 annual report. Atlanta, Ga: CDC, 2001.
- 43.
CDC. Outbreak of Campylobacter jejuni infections associated with drinking unpasteurized milk procured through a cow-leasing program—Wisconsin 2001. MMWR Morb Mortal Wkly Rep 2002;51:548–549.
- 44.↑
Villar RG, Macek MD, Simons S, et al. Investigation of multidrug-resistant Salmonella serotype Typhimurium DT 104 infections linked to raw-milk cheese in Washington State. JAMA 1999;281:1811–1816.
- 45.↑
Potter ME, Kaufmann AF, Blake PA, et al. Unpasteurized milk. The hazards of a health fetish. JAMA 1984;252:2048–2052.
- 46.
CDC. Multistate outbreak of Salmonella serotype Typhimurium infections associated with drinking unpasteurized milk—Illinois, Indiana, Ohio, Tennessee, 2002–2003. MMWR Morb Mortal Wkly Rep 2003;52:613–615.
- 47.
Aarestrup FM, Nielsen EM, Madsen M, et al. Antimicrobial susceptibility patterns of thermophilic Campylobacter spp from humans, pigs, cattle, and broilers in Denmark. Antimicrob Agents Chemother 1997;41:2244–2250.
- 48.
Bager F, Aarestrup FM, Jensen NE, et al. Design of a system for monitoring antimicrobial resistance in pathogenic, zoonotic and indicator bacteria from food animals. Acta Vet Scand 1999;92:77–86.
- 49.
Aarestrup FM, Jensen NE, Jorsal SE, et al. Emergence of resistance to fluoroquinolones among bacteria causing infections in food animals in Denmark. Vet Rec 2000;146:76–78.
- 50.
Emborg HD, Heuer HO. DANMAP 2002—Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark. Copenhagen, Denmark: Statens Serum Institut, Danish Veterinary and Food Administration, Danish Medicines Agency, Danish Veterinary Institute, 2002.
- 51.↑
Piddock LJ, Ricci V, Stanley K, et al. Activity of antibiotics used in human medicine for Campylobacter jejuni isolated from farm animals and their environment in Lancashire, UK. J Antimicrob Chemother 2000;46:303–306.
- 52.
Blake DP, Humphry RW, Scott KP, et al. Influence of tetracycline exposure on tetracycline resistance and the carriage of tetracycline resistance genes within commensal Escherichia coli populations. J Appl Microbiol 2003;94:1087–1097.
- 53.
Mathew AG, Beckmann MA, Saxton AM. A comparison of antibiotic resistance in bacteria isolated from swine herds in which antibiotics were used or excluded. J Swine Health Prod 2001;9:125–129.
- 54.
Regula G, Stephan R, Danuser J, et al. Reduced antibiotic resistance to fluoroquinolones and streptomycin in “animal-friendly” pig fattening farms in Switzerland. Vet Rec 2003;152:80–81.
- 55.↑
Stanley KN, Jones K. High frequency of metronidazole resistance among strains of Campylobacter jejuni isolated from birds. Lett Appl Microbiol 1998;27:247–250.
- 56.↑
Avrain L, Humbert F, L'Hospitalier R, et al. Antimicrobial resistance in Campylobacter from broilers: association with production type and antimicrobial use. Vet Microbiol 2003;96:267–276.
- 57.↑
Tenover FC, Fennell CL, Lee L, et al. Characterization of two plasmids from Campylobacter jejuni isolates that carry the aphA-7 kanamycin resistance determinant. Antimicrob Agents Chemother 1992;36:712–716.
- 58.↑
Aminov RI, Garrigues-Jeanjean JN, Mackie RI. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl Environ Microbiol 2001;67:22–32.