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Applied and Environmental Microbiology, July 2005, p. 3872-3881, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3872-3881.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effects of Subtherapeutic Administration of Antimicrobial Agents to Beef Cattle on the Prevalence of Antimicrobial Resistance in Campylobacter jejuni and Campylobacter hyointestinalis{dagger}

G. D. Inglis,1* T. A. McAllister,1 H. W. Busz,1 L. J. Yanke,1 D. W. Morck,2 M. E. Olson,3 and R. R. Read3

Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta,1 Department of Biological Sciences, University of Calgary, Calgary, Alberta,2 Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada3

Received 2 September 2004/ Accepted 6 January 2005


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ABSTRACT
 
The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (≥94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.


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INTRODUCTION
 
Campylobacter species are recognized as one of the most frequent causes of acute diarrheal disease in humans in North America (Centers for Disease Control and Prevention, U.S. Department of Agriculture, and Food and Drug Administration Collaborating Sites Foodborne Disease Active Survey Network [Foodnet]; Health Canada website [http://dsol-smed.hc-sc.gc.ca/dsol-smed/ndis/c_dis_e.html]). Alberta, Canada, possesses a very large beef cattle population (approximately 6 million head), and approximately 2 million of these animals are in finished feedlots centered in the southern region of the province (Alberta government website [http://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/sdd1492?]). In the Chinook Health Region (CHR) of Alberta, the prevalence of Campylobacter infections in humans is higher than the national average and is increasing three times faster than population growth (Paul Hasselback, Canadian Laboratory Medicine Congress, Calgary, Alberta, Canada). Beef cattle production, including intensive feedlot operations, is predominant in the CHR, raising questions about the importance of beef cattle as a source of campylobacters infecting humans in this region. A diversity of Campylobacter species are shed in the feces of beef cattle in large numbers (23, 24, 25, 26, 35). Although the impact of cattle-borne campylobacters on human health has not been definitively determined, genotyping evidence points toward cattle as a significant source of human-pathogenic campylobacters (10, 38, 39, 45, 51). Furthermore, waterborne Campylobacter species from a bovine source were implicated in the infection of a large number of people in Walkerton, Ontario, Canada, in 2000 (8), and passive surveillance information for the CHR suggests that cattle production is linked to the transmission of Campylobacter to humans (Paul Hasselback, Canadian Laboratory Medicine Congress, Calgary, Alberta, Canada). Mounting evidence is implicating cattle as a significant source of Campylobacter strains that incite disease in humans (51).

In North America, antimicrobial agents have been used in feed for ca. 50 years for the prevention of disease and as growth promoters in beef cattle (12, 22, 56). Antimicrobials are typically administered in the diet, either continuously throughout the feeding period or at specific times of high disease risk. The subtherapeutic application of antimicrobial agents to cattle may contribute to the emergence of resistant pathogenic bacteria, and the continuous administration of antimicrobials at relatively low concentrations has been hypothesized to increase the likelihood of resistance development (27). Particular concerns include antimicrobial agents that are applied directly (e.g., tetracyclines) or that belong to the same chemical families as antimicrobials commonly used to treat bacterial infections in humans (e.g., the macrolides, tylosin, and erythromycin). Enteric disease incited by Campylobacter species is typically self-limiting (i.e., patients recover without intervention), with diarrhea persisting for approximately 3 to 7 days. However, it can result in the death of young, old, and immunocompromised individuals, with an overall mortality rate of 0.24% of culture-confirmed cases (49). The treatment of infected individuals with antimicrobial agents, such as erythromycin and ciprofloxacin, may decrease the severity of symptoms if the agents are administered soon after infection (48). However, the effectiveness of both of these antimicrobial agents has been severely compromised in some countries because of the development of antimicrobial resistance (AMR) (48). To our knowledge, no studies have examined the effect of antimicrobial use in beef cattle production on the development of antimicrobial resistance in Campylobacter species, despite the fact that more than 2 million kg of antimicrobial agents are administered to beef cattle in North America each year (33). Therefore, the objective of this study was to measure the extent to which antimicrobial agents (i.e., chlortetracycline alone, chlortetracycline and sulfamethazine in combination, virginiamycin, monensin, and tylosin) typically administered subtherapeutically to beef cattle affect the development of AMR and the carriage rates of Campylobacter species.


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MATERIALS AND METHODS
 
Animals and treatments.
Three hundred crossbred steer calves (198 ± 20 kg of initial body weight) were housed in 30 pens at the Lethbridge Research Centre experimental feedlot. Calves originated from a common location and did not receive antimicrobial agents subtherapeutically before the initiation of the experiment. The calves were arbitrarily assigned to one of the following six treatments: (i) no antimicrobial agents (control treatment), (ii) 350 mg head–1 day–1 chlortetracycline and 350 mg head–1 day–1 sulfamethazine (CS treatment) (Aureo S-700 G; Alpharma Inc., N.J.), (iii) 11 ppm chlortetracycline (Ct treatment) (Aureomycin-100 G; Alpharma Inc.), (iv) 250 mg head–1 day–1 virginiamycin (Vi treatment) (V-Max; Pfizer Animal Health, N.Y.), (v) 25 ppm monensin (Mo treatment) (Rumensin; Elanco Animal Health, Alberta, Canada), and (vi) 11 ppm tylosin phosphate (Ty treatment) (Tylan; Elanco Animal Health). With the exception of virginiamycin, the antimicrobial agents were selected based on the commonality of their use in the Canadian feedlot industry and were fed at the concentrations recommended by the manufacturers. Virginiamycin is not registered for use in Canada, and as a result, neither calves nor their dams would have had prior exposure to this antimicrobial. Each treatment was replicated five times, and the treatment groups were arranged in a randomized complete block design; each block consisted of a separate pen containing 10 steers. Water troughs were shared between adjacent pens, but treatments were arranged in a manner so that only cattle that received the same antimicrobial agent could drink from the same water troughs.

All of the animals involved in this study were cared for according to the guidelines set out by the Canadian Council on Animal Care (7). Steers entering the feedlot were fed a forage-based diet, which consisted of 70% barley silage, 25% barley grain, and 5% (dry matter basis) supplementation with vitamins and minerals, for the first 115 days (i.e., backgrounding period) (Fig. 1). Cattle were subsequently switched from the forage-based diet to a grain-based diet (85% barley, 10% barley silage, 5% supplement) over a 21-day period, and then they were maintained on the grain-based diet for an additional 179 days (i.e., finishing period); this feeding regimen is typical for the Canadian feedlot industry. Cattle were fed once daily in a manner that ensured that all feed that was allotted to each pen was consumed. Antimicrobial agents were first introduced into the diets 18 days after the cattle arrived at the feedlot, and they were included in the forage-based diet for 56 days thereafter (Fig. 1). Antimicrobial agents were subsequently removed from the diet for 91 days and then reintroduced for an additional 42-day period when the grain-based diet was used. The antimicrobial administration periods were chosen to coincide with the two feeding periods. To avoid cross-contamination, we mixed the antimicrobial agents with 5 kg of a supplement containing minerals and vitamins and spread the mixture manually over the surface of feed within each of the appropriate pens during the morning feeding. All animals in the pen were capable of feeding at the feed trough at the same time. Cattle assigned to the control treatment were provided with a supplement that contained no antimicrobial agents.



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  		FIG. 1. Diagram showing the timing of antimicrobial administration to feedlot cattle during the periods that animals were fed a high-forage-content diet (backgrounding period) and a high-grain diet (finishing period). The experiment included 300 steers assigned to five pens (10 animals per pen), and the diagram shows the 11 sample times (A to K) at which fecal samples were obtained and campylobacters were isolated. The antimicrobial administration periods were chosen to coincide with the two feeding periods.

Sample collection and isolation of campylobacters.
All cattle were restrained 11 times during the course of the feedlot period (Fig. 1). Although animals were processed in groups according to pen assignments, all animals were processed in the same constraint device. Once each animal was constrained, deep fecal samples were obtained from the rectum by the use of rectal swabs (Starswap; Starplex Scientific, Ontario, Canada). To obtain each fecal sample, we inserted the swab approximately 4 to 5 cm into the rectum and rotated it until it was covered with feces. Within 1 h of collection, the samples were transported to the laboratory on ice. Each swab was aseptically placed in a tube containing 750 µl brain heart infusion broth (Difco, Sparks, Md.) containing 20% glycerol (vol/vol). Tubes were agitated with a vortexer (high setting). The resultant slurry was spread onto each of the following three media: (i) Campylobacter blood-free selective agar base (modified charcoal cefoperazone desoxycholate agar; Oxoid, Nepean, Ontario, Canada) containing the selective supplement SR155 (Oxoid) (mCCDA), (ii) mCCDA amended with 2.0 µg ml–1 of ciprofloxacin (Sigma-Genosys, Oakville, Ontario, Canada) (cCCDA), and (iii) mCCDA amended with 4.0 µg ml–1 erythromycin (Sigma-Genosys) (eCCDA). The concentrations of ciprofloxacin and erythromycin were one-half of the breakpoint values specified for other bacteria (National Committee for Clinical Laboratory Standards or best recommendation). For mCCDA, 20 µl of the slurry was spread over the surface of the medium, whereas 50 µl of the slurry was spread onto both cCCDA and eCCDA. Cultures were incubated at 42°C under microaerophilic conditions (10% CO2, 5% H2, and 85% nitrogen, with a trace of O2). At 48 and 72 h, one or two colonies per dish possessing the appropriate appearance (i.e., typical of Campylobacter) were streaked onto Trypticase soy agar. After incubation under microaerophilic conditions for 48 to 72 h, the biomass was transferred to brain heart infusion broth containing 20% glycerol. The isolates were stored at –80°C until processed.

Identification.
Isolates were identified to the species level by using colony PCRs specific for the Campylobacter genus (16S rRNA gene), Campylobacter coli (ceuE gene), C. fetus (23S rRNA gene), C. hyointestinalis (23S rRNA gene), C. jejuni (mapA gene), and C. lanienae (16S rRNA gene) (23). The conditions for amplification were 1 cycle at 95°C for 15 min; 25 cycles of 30 s at 94°C, 90 s at the annealing temperature, and 60 s at 72°C; and an extension cycle of 10 min at 72°C. The reaction mixtures consisted of a total volume of 20 µl containing 1x reaction buffer, a 0.2 mM concentration of each deoxynucleoside triphosphate, 2 mM MgCl2, a 0.5 µM concentration of each primer (Sigma-Genosys, Oakville, Ontario, Canada), 0.2 mg bovine serum albumin (Promega, Madison, Wis.), and 1 U HotStar Taq polymerase (QIAGEN Inc., Mississauga, Ontario, Canada). The DNA template consisted of 1 µl of a suspension containing 24- to 48-h-old cells of the isolate to be identified. For each isolate, cells from an individual colony were uniformly suspended in 100 µl of sterile Brucella broth in 96-well microtiter plates by the use of sterile pipette tips. Positive controls consisted of cells from reference strains, and negative controls consisted of Brucella broth alone. All PCR products (10 µl) were electrophoresed in a 2% Tris-borate-EDTA-agarose gel (Invitrogen Corp., Burlington, Ontario, Canada), visualized by staining with ethidium bromide, and viewed under UV light. A 100-bp ladder (Promega) was used to size the products.

In addition, partial 16S rRNA genes of five arbitrarily selected isolates per taxon were subsequently amplified and sequenced (23). PCR products were initially obtained with the C412F/C1228R primer set, using the same amplification conditions as those described above. The resulting PCR products (10 µl) were electrophoresed in a 2% Tris-acetate-EDTA-agarose gel, and a 100-bp ladder (Promega) was used to size the products. The PCR amplicons were purified with a QIAquick kit (QIAGEN Inc.) and then sequenced in forward (C412F) and reverse (C1228R) by use of an ABI PRISM Big Dye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, Calif.). Before sequencing, excess dye was removed with a DyeEx spin kit (QIAGEN Inc.). Contigs were constructed by using Staden (Medical Research Council, Laboratory of Molecular Biology, Cambridge, England), and all sequences were compared directly with the NCBI GenBank nonredundant nucleotide database using BLASTN. Representative isolates were also subjected to the following tests: microscopy for size, shape, and motility; tests for hippurate, nitrate, nitrite, and indoxyl acetate hydrolysis; tests for the production of catalase (3% H2O2) and urease; and a test of H2S production in triple sugar iron medium (23, 37). In total, 2,052 bacterial isolates were characterized.

Antimicrobial susceptibility testing.
The MICs of ampicillin (4, 8, 16, 32, and 64 µg ml–1), ciprofloxacin (0.5, 1, 2, 4, 8, and 16 µg ml–1), erythromycin (1, 2, 4, 8, 16, 32, and 64 µg ml–1), and tetracycline hydrochloride (2, 4, 8, 16, 32, and 64 µg ml–1) were determined by using the agar dilution methodology of the National Committee for Clinical Laboratory Standards (NCCLS); the medium used was Mueller-Hinton II agar (Difco, Sparks, Md.), but it did not contain blood. Cells were harvested from the surface of the medium after 24 h of growth under microaerophilic (5% O2, 10% CO2, 3% H2, and 82% N2) conditions at 42°C. The cells were suspended in sterile saline (0.075% NaCl), and the cell density was adjusted to a 0.5 McFarland turbidity standard. Aliquots (450 µl) of the saline suspension were pipetted into the seeding wells of a Cathra replicator (Oxoid, Inc.). Freshly prepared plates of Mueller-Hinton agar amended with antimicrobial agents were then inoculated by the use of 1-mm pins in the inoculating head of the replicator. Cultures were incubated microaerophilically at 42°C for 48 h, and the MIC was defined as the lowest concentration resulting in a complete inhibition of visible growth on the medium. Campylobacter jejuni (ATCC 33560), Klebsiella pneumoniae (ATCC 700603), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus aureus (ATCC 29213) were utilized as quality control strains. Since no breakpoints for Campylobacter have been defined by NCCLS, we used the following breakpoint values: for ciprofloxacin, 4 µg ml–1; for ampicillin, 8 µg ml–1 (NCCLS breakpoint for Enterobacteriaceae); and for tetracycline, 8 µg ml–1 (46). Although the British Society for Antimicrobial Chemotherapy indicates a breakpoint value of 2 µg ml–1 for Campylobacter, we used the breakpoint of 8 µg ml–1 for erythromycin as specified by the National Antimicrobial Resistance Monitoring System.

Data analysis.
Analyses were conducted by using SAS software (43). For the prevalence of C. jejuni and C. hyointestinalis isolates (recovered on each of the three isolation media) associated with animals, the experiment was analyzed as a randomized complete block design, with 6 levels of antimicrobial treatments, 11 levels of time, and 5 levels of blocks (i.e., replicate pens), by using the mixed procedure of SAS. Since the same individuals were used for all sample times, the repeated-measurement statement was applied. The appropriate error structure was determined by using Akaike's information criterion and the Bayesian information criterion, and the Kenward-Roger degree of freedom feature was used to adjust the degrees of freedom of the error term. In conjunction with a significant F test, the LSMEAN statement of SAS was used to produce least-square means, and the DIFF option was applied to conduct the least significant difference test. The mean percent isolation rates of C. jejuni and C. hyointestinalis across sample times (i.e., data for animals and isolation events for combined isolation media) were also analyzed as a randomized complete block design by using the mixed procedure and LSMEAN statement of SAS. For carriage rates of resistant isolates, the same statistical model was used except that dependent variables were weighted to account for unequal observations and the analysis was unbalanced; replicates for which the frequency of isolation was ≤1 event were removed (i.e., two pens for each bacterium). In all instances, predicted values were plotted against residual values to determine whether residuals were randomly distributed, and the univariate procedure of SAS was applied to test for normality. Separate analyses were conducted for each species of Campylobacter and for each isolation medium.


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RESULTS
 
Frequency of isolation.
In total, 3,283 fecal samples were processed for the detection of campylobacters over the course of the experiment. Of the 2,052 isolates recovered from cattle feces on either mCCDA, cCCDA, or eCCDA, 1,898 isolates (92%) were confirmed as Campylobacter species. The majority (80.0%) of the isolates were identified as C. hyointestinalis (1,518 isolates), with the remaining 380 isolates identified as C. jejuni. An additional 276 isolates did not produce sufficient growth on Trypticase soy agar to be characterized.

(i) Campylobacter jejuni.
Two hundred forty-two of the 3,283 fecal samples were positive for C. jejuni (7.4%). The majority (98.4%) of the 380 isolates were recovered on mCCDA. No isolates were obtained on eCCDA, and only six isolates were recovered on cCCDA. Campylobacter jejuni was isolated from the feces of 130 of 300 steers (43.3%) over the course of the experiment and from 18.7% (n = 56) of the animals on more than one occasion; feces from three steers were positive for the bacterium at six or more sample times. The number of animals from which C. jejuni was isolated (combined media) did not differ (F = 2.6; df = 5, 20; P = 0.058) among the six antimicrobial treatment groups. In contrast, the times at which the bacterium was isolated (i.e., isolation events) differed (F = 3.2; df = 5, 20; P = 0.029) among the treatment groups. However, none of the treatments caused decreased (P ≥ 0.36) carriage rates relative to the control treatment (Table 1). A larger (P = 0.035) number of isolation events were recorded for animals administered Vi than for the control group.


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  		TABLE 1. Mean isolation rates of Campylobacter jejuni and Campylobacter hyointestinalis from beef steers administered subtherapeutic levels of antimicrobial agentsa

(ii) Campylobacter hyointestinalis.
Six hundred thirty-two, 148, and 738 isolates were recovered from cattle on mCCDA, cCCDA, and eCCDA, respectively, representing isolation frequencies of 19.3, 4.5, and 22.4%. Campylobacter hyointestinalis was isolated from 209 of the 300 steers (69.7%) at least once during the experiment, and overall, 565 of the 3,283 fecal samples (17.2%) were positive for the bacterium. One hundred forty-five of the cattle (48.3%) were positive for C. hyointestinalis on more than one occasion, and the bacterium was isolated from 18 animals (6.0%) at six or more sample times. The total number of animals from which C. hyointestinalis was isolated (combined media) (F = 5.2; df = 5, 20; P = 0.003) and the times at which the bacterium was isolated (i.e., isolation events) (F = 8.4; df = 5, 20; P < 0.001) differed significantly among the six antimicrobial treatment groups; relative to the control treatment group, a smaller (P = 0.002) number of animals administered CS carried C. hyointestinalis, and the number of times that the bacterium was isolated was lower for cattle treated with CS (P < 0.001) and Ct (P = 0.019) (Table 1). These findings are consistent with the results of analyses in which the sample time was included in the statistical model for each isolation medium. The frequency of isolation of C. hyointestinalis differed (F = 1.6 to 2.2; df = 50, 149 to 226; P ≤ 0.010) among the six treatment groups at the different collection times for all three media. Averaged over time, C. hyointestinalis was isolated on all three media from fewer (P ≤ 0.007) steers fed CS than from control animals. For the Ct treatment, the bacterium (averaged over time) was also isolated from fewer (P ≤ 0.021) steers, but only for mCCDA and cCCDA, not for eCCDA.

Antimicrobial resistance. (i) Campylobacter jejuni.
The MICs of ampicillin, ciprofloxacin, erythromycin, and tetracycline were determined for 375 isolates of C. jejuni (Table 2). Most isolates of C. jejuni (≥94%) were sensitive to the four antimicrobial agents tested. Only 22, 1, 3, and 20 isolates were resistant to ampicillin, ciprofloxacin, erythromycin, and tetracycline, respectively. For the animals from which at least one resistant C. jejuni isolate was recovered, an additional isolate sensitive to either ampicillin, erythromycin, or tetracycline was recovered from 35.2% (n = 6), 100% (n = 3), and 26.7% (n = 4) of the samples, respectively. Only 1 of the 375 C. jejuni isolates examined exhibited resistance to multiple antimicrobial agents (i.e., erythromycin and tetracycline).


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  		TABLE 2. MICs of ampicillin, ciprofloxacin, erythromycin, and tetracycline for C. jejuni and C. hyointestinalis isolates recovered from beef cattle across the subtherapeutic antimicrobial treatement groups

The administration of antimicrobial agents to cattle significantly affected the carriage rates of C. jejuni isolates resistant to ampicillin (F = 2.9 to 4.1; df = 5, 18; P ≤ 0.041) and tetracycline (F = 8.4; df = 5, 18; P < 0.001) but not to erythromycin (F = 0.4 to 0.9; df = 5, 18; P ≥ 0.53). The carriage rate of tetracycline-resistant isolates was increased (P < 0.001) only for cattle fed Ct (Table 3). In no instance did the administration of antimicrobial agents to cattle significantly increase (P ≥ 0.11) the carriage of C. jejuni isolates with resistance to ampicillin relative to the control treatment. However, the administration of Vi, Mo, and Ty did decrease (P ≤ 0.039) the carriage rates of ampicillin-resistant isolates (Table 3).


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  		TABLE 3. Recovery of resistant C. jejuni isolatesa

(ii) Campylobacter hyointestinalis.
The MICs of ampicillin, ciprofloxacin, erythromycin, and tetracycline were determined for 1,500 isolates. One hundred sixty-one (10.7%), 151 (10.1%), and 16 (1.1%) isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively (Table 2). No isolates resistant to ciprofloxacin (≥8 µg ml–1) were recovered. The relative frequencies of isolation of resistant isolates were comparable among the three media. For the animals from which at least one resistant C. hyointestinalis isolate was recovered, an additional isolate sensitive to either ampicillin, erythromycin, or tetracycline was recovered from 57.1% (n = 8), 44.2% (n = 53), and 58.9% (n = 14) of the samples, respectively. Only 12 of the 1,500 C. hyointestinalis isolates examined exhibited resistance to multiple antimicrobial agents.

The administration of antimicrobial agents to cattle did not affect (F = 1.2 to 1.4; df = 5, 18; P ≥ 0.27) the development of AMR to ampicillin but did influence resistance development to both erythromycin (F = 5.5 to 5.6; df = 5, 18; P = 0.003) and tetracycline (F = 5.1 to 8.7; df = 5, 18; P ≤ 0.004) (Table 4). An increased carriage rate of isolates with resistance to erythromycin was observed for cattle fed Ct (P ≤ 0.008) but not for cattle fed any other antimicrobial treatments (P ≥ 0.11), including the macrolide Ty. Very few C. hyointestinalis isolates with resistance to tetracycline were obtained from control steers (n = 1) and those fed Vi (n = 2) compared to animals treated with CS (n = 54), Ct (n = 34), Mo (n = 22), and Ty (n = 48). However, only the carriage rate of animals administered CS differed significantly (P < 0.001) from that of the control animals. Isolates of C. hyointestinalis with resistance to tetracycline were clearly aggregated in a relatively small number of pens (Fig. 2). In total, tetracycline-resistant isolates were obtained from animals in 12 of 30 pens, but the majority (91.3%; n = 147) of the resistant isolates recovered were obtained from steers in only 4 pens. They were CS-treated animals in pen 4 (n = 50), Ct-treated animals in pen 1 (n = 31), Mo-treated animals in pen 3 (n = 19), and Ty-treated animals in pen 2 (n = 47). Resistance to erythromycin in C. hyointestinalis isolates was detected in 26 of 30 pens, and no clear patterns of pen-related carriage were observed (data not shown).


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  		TABLE 4. Recovery of resistant C. hyointestinalis isolatesa



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  		FIG. 2. Animals positive for Campylobacter hyointestinalis isolated on mCCDA, cCCDA, and eCCDA. Ten animals per antimicrobial treatment group were assigned to each of five pens. Gray boxes represent animals from which the C. hyointestinalis isolate(s) did not exhibit antimicrobial resistance at the 11 sample times. Black boxes containing a "T" indicate isolates that were resistant to tetracycline hydrochloride (MIC, ≥8 µg ml–1). The subtherapeutic antimicrobial treatments were as follows: control, no antimicrobials; CS, chlortetracycline and sulfamethazine (Aureo S-700 G); Ct, chlortetracycline (Aureomycin-100 G); Vi, virginiamycin (V-Max); Mo, monensin (Rumensin); and Ty, tylosin phosphate (Tylan). In many instances, more than one isolate was recovered from each positive animal at each sample time.

Resistance to tetracycline was detected at a low level at the first sample time. For the CS treatment group, resistance to tetracycline was first detected in isolates obtained on day 36 and increased rapidly thereafter (Fig. 3A); this period corresponded to the period when cattle were consuming a forage-based diet (Fig. 1). For the Ct, Vi, Mo, and Ty treatment groups, limited tetracycline resistance was observed when cattle were fed the forage-based diet compared to that when cattle were fed the grain-based diet (Fig. 3A). Thereafter, a larger number of isolation events including tetracycline-resistant C. hyointestinalis isolates were observed for the Ct, Mo, and Ty treatment groups, but not the Vi treatment group, than for the control group. With the exception of the CS treatment group, from which only one resistant isolate was recovered over the 315-day duration of the study, the rates of resistance development to erythromycin were similar among the treatment groups, including animals administered the macrolide Ty (Fig. 3B).



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  		FIG. 3. Cumulative carriage rates of Campylobacter hyointestinalis isolates (number of animals [n = 50] from which resistant isolates were obtained per treatment at each of 11 sample times). (A) Resistance to tetracycline; (B) resistance to erythromycin. The subtherapeutic antimicrobial treatments were as follows: control, no antimicrobials; CS, chlortetracycline and sulfamethazine (Aureo S-700 G); Ct, chlortetracycline (Aureomycin-100 G); Vi, virginiamycin (V-Max); Mo, monensin (Rumensin); and Ty, tylosin phosphate (Tylan). Arrow 1 indicates the start of the high-forage-content diet period; arrows 2 and 3 indicate the start of the transition period and the start of the high-grain diet period, respectively.


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DISCUSSION
 
An examination of the effect of subtherapeutic antimicrobial use in beef cattle on the development of AMR in campylobacters from beef cattle was undertaken in the present study. Beef cattle were maintained in an experimental feedlot for 315 days and were administered one of six antimicrobial treatments for two periods. During the course of the experiment, we isolated two Campylobacter species from fecal material removed from the rectums of beef cattle. These were C. hyointestinalis (17% of samples) and C. jejuni (7% of samples). Others have previously isolated these two campylobacters from cattle at various frequencies (e.g., see references 3, 6, 16, 19, and 55). All media used to isolate Campylobacter species select for specific taxa and even genotypes (3, 5, 13, 28, 47). As a result, microbiological methods that employ selective media do not necessarily provide a true measure of the frequency and diversity of Campylobacter species associated with livestock and their feces. However, the isolation frequency that we observed is consistent with the findings of a PCR-based survey (26) in which 8% and 13% of fecal samples obtained from beef steers contained DNAs of C. hyointestinalis and C. jejuni, respectively. Interestingly, we did not isolate C. coli in the present study, but we have previously shown that this bacterium is shed at low frequencies from beef cattle (25, 26).

Campylobacter jejuni is recognized as the primary cause of gastroenteritis in humans (32), but the significance of strains originating from cattle on human health has not been fully ascertained. However, evidence is now pointing toward C. jejuni from cattle as an important source of infectious strains (e.g., see references 8, 10, 38, 39, 45, and 51). The pathogenic role of C. hyointestinalis in animals and humans is not well understood, but numerous reports of infections in humans (11, 14, 29, 34) and animals (9, 18) have been reported. Furthermore, evidence for the transmission of C. hyointestinalis from swine to humans has been reported, demonstrating the zoonotic potential of this bacterium (20). Given these observations, along with the high frequency at which this bacterium is shed from livestock such as cattle, the clinical significance of C. hyointestinalis as a zoonotic pathogen of humans should not be discounted.

The antimicrobial agents included in the present study were selected because they are typically used in beef production in North America (NebGuide, University of Nebraska, Lincoln [http://ianrpubs.unl.edu/beef/g761.htm]). Chlortetracycline, alone and in combination with sulfamethazine, is typically used to improve weight gain and feed efficiency, aid in the prevention of liver abscesses, reduce bacterial diarrhea, prevent foot rot, and reduce the incidence of bovine respiratory disease. Monensin is an ionophore that inhibits the growth of gram-positive bacteria in the rumen and has been shown to improve the feed efficiency of cattle fed a high-grain diet. The macrolide tylosin is primarily included in the diet to reduce the incidence of liver abscesses in beef cattle. Virginiamycin is not currently registered for use in Canada, but it is used as a feed additive for beef cattle in the United States. This antimicrobial has been shown to increase daily weight gain, improve feed efficiency, and reduce the incidence of liver abscesses (42). We observed that the administration of chlortetracycline and sulfamethazine (i.e., CS treatment) had the greatest impact on the frequency of isolation of C. hyointestinalis. However, chlortetracycline alone also decreased the isolation rates for C. hyointestinalis. Although we did not quantify the numbers of Campylobacter cells in feces or in the gastrointestinal (GI) tract, our observations of decreased isolation rates suggest that antimicrobial agents can affect the colonization of the GI tract and/or the shedding of campylobacters.

To our knowledge, our study is the first to empirically investigate the impact of subtherapeutic antimicrobial use in beef cattle on the development of AMR in campylobacters. Several surveys have previously demonstrated the occurrence of resistance to antimicrobial agents in thermophilic campylobacters (primarily C. jejuni and C. coli) originating from cattle (1, 2, 40, 41, 54). Although such studies documented the occurrence of AMR and the possible risk associated with the contamination of foods by AMR strains, they provide limited information on the degree to which antimicrobial use in beef production induces the development of resistance. It has been established with other animals (e.g., poultry) that the administration of antimicrobials during production can select for resistant bacteria which are subsequently transferred to humans via contaminated food or water (50). Our findings indicate that antimicrobial administration to beef cattle does indeed select for AMR campylobacters. In particular, we observed a substantial development of resistance to tetracycline but only limited resistance to erythromycin, ampicillin, and ciprofloxacin. Similarly, Sato et al. (44) observed no or very limited resistance to ciprofloxacin, gentamicin, and erythromycin in 332 Campylobacter isolates (primarily C. jejuni) obtained from dairy cows reared on conventional or organic farms. However, they observed relatively high levels of resistance to tetracycline (45%), but there was no difference between the two farm types, suggesting that resistance to tetracycline occurred naturally in the populations of Campylobacter and/or that non-antimicrobial agent factors are involved in the selection of resistance to tetracycline. This contrasts with the findings of our study, which showed significant levels of resistance to tetracycline for isolates obtained from animals fed subtherapeutic doses of chlortetracycline relative to those from animals given the control treatment. One possible reason for the difference between the two studies may be the differential selection pressure encountered in beef versus dairy production, since dairy cows are not administered antimicrobial agents subtherapeutically.

Tetracycline has been suggested as a treatment for humans infected with C. jejuni and C. coli (36). However, the extensive development of resistance to tetracyclines in countries including Canada has led to a decrease in their clinical use (53). For example, Gaudreau and Gilbert (17) observed a significant increase in the number of C. jejuni isolates with resistance to tetracycline in Quebec; the resistance rates were 19% in 1985-1986 and 56% in 1995-1997. We observed that most of the C. jejuni isolates that developed resistance to tetracycline were obtained from animals treated with chlortetracycline (i.e., Ct treatment). For C. hyointestinalis, the majority of tetracycline-resistant isolates were isolated from animals treated with chlortetracycline and sulfamethazine (i.e., CS treatment) and, to a lesser extent, those treated with chlortetracycline alone. The reasons for the lack of activity of CS against C. jejuni are uncertain. The long-term application of subtherapeutic quantities of chlortetracycline would be expected to exert selection pressure for the development of resistance, which we have confirmed for Escherichia coli isolated from beef cattle fed CS (31).

Although it was not significant, a considerable amount of resistance to tetracycline was observed in C. hyointestinalis isolates obtained from animals fed monensin and tylosin phosphate. The reasons for this finding are currently unknown. One possibility is that tetracycline resistance developed initially in animals fed chlortetracycline and that AMR isolates were transmitted to other animals (e.g., during sampling). We observed that the occurrence of resistance to tetracycline did not occur randomly in the pens, and the vast majority of isolates with resistance to tetracycline were obtained from animals housed in only four pens. Resistance to tetracycline was primarily restricted to one pen for the monensin and tylosin phosphate treatments. The transmission of campylobacters from water to dairy cattle has been shown (21), and Minihan et al. (35) observed that the prevalence of campylobacters was higher among penmates, suggesting that transmission may readily occur among animals within pens. Using similar rearing settings, we have also shown that AMR strains of E. coli are readily transferred among cattle within a pen (31) but that the movement of resistant strains between cattle in different pens occurs far less frequently (52). Furthermore, rapid transfer of the tetO gene between C. jejuni strains occurs even in the absence of selection pressure within the GI tracts of chickens (4). In the present study, we did not evaluate the genetic diversity of the isolates that we recovered, and this information is necessary to elucidate the mechanisms by which the aggregation and transmission of AMR strains occur. For example, do the isolates belong to one or a limited number of genotypes? These data are necessary to determine whether a single strain may have evolved in a single animal and been subsequently transmitted to cohorts within the same pen and/or transferred to animals maintained in separate pens. Such transmission may occur independently of the type of antibiotic that is administered in the diet.

The macrolide erythromycin was the first antimicrobial agent used to treat Campylobacter infections in humans, and it remains the treatment of choice for patients with uncomplicated enteritis in many countries (48). Resistance to erythromycin has remained consistently low (<5%) in most regions of the world, including Canada (36), but considerable resistance (up to 50%) has been reported for some countries, such as Thailand (48). We observed a significant development of resistance to erythromycin in C. hyointestinalis, but not C. jejuni, isolates. We used a medium amended with one-half the breakpoint value of erythromycin (i.e., 4 µg ml–1), but we did not observe a conspicuous shift in the frequency distribution of MICs relative to that for the medium that did not contain erythromycin. The possibility that the actual breakpoint for erythromycin may actually be lower than that suggested by the National Antimicrobial Resistance Monitoring System may account for the lack of C. jejuni isolates on the medium amended with erythromycin at a concentration of 4 µg ml–1. Erythromycin resistance in C. coli is relatively common relative to that in C. jejuni (15), but little is known about AMR development in C. hyointestinalis. It is well documented that erythromycin resistance is readily selected for in bacteria occurring in the GI tracts of animals ingesting feed supplemented with the macrolide tylosin (27). Surprisingly, we did not observe substantial levels of erythromycin resistance in C. hyointestinalis isolates originating from cattle fed subtherapeutic levels of tylosin. In contrast, significant levels of erythromycin resistance were observed for the chlortetracycline treatment group. The reasons for this occurrence are unknown.

One problem encountered when studying the mechanism(s) of resistance development in campylobacters associated with cattle is the relatively low frequency at which they are shed in feces; C. jejuni and C. coli do not typically colonize the GI tracts of many cattle (e.g., see references 25 and 26). We observed that the numbers of resistant isolates recovered were too small for us to conduct adequate risk assessment analyses, and the use of indicator taxa that are more commonly associated with cattle may prove useful. In the present study, C. hyointestinalis was far more prevalent (1,518 isolates) than C. jejuni (380 isolates), and the increased prevalence of this bacterium facilitated analyses of AMR development. Whether C. hyointestinalis will prove to be a good indicator taxon for resistance in C. jejuni and C. coli remains to be determined but will depend on the mechanisms of AMR and possibly on aspects of its ecology in the GI tracts of cattle. Another possible indicator taxon is C. lanienae. Although fastidious for isolation and culture, C. lanienae is frequently shed in the feces of beef cattle (25, 26). Campylobacter lanienae is typically not isolated on mCCDA (23), which explains why we did not observe this bacterium in the present study. Currently, its human pathogenicity is not certain (30), but its high abundance relative to C. jejuni may make it a good indicator taxon in subsequent studies of AMR development in campylobacters associated with beef cattle.

In conclusion, isolates of C. hyointestinalis and C. jejuni were recovered from beef cattle maintained in feedlots. The administration of chlortetracycline alone and in combination with sulfamethazine significantly reduced the numbers of animals from which C. hyointestinalis was isolated. Minimal or no resistance to ampicillin or ciprofloxacin was observed. The exposure to subtherapeutic levels of antimicrobial agents during the feedlot period resulted in the development of resistance to tetracyclines and, to a lesser extent, erythromycin. Furthermore, we observed that resistance to tetracycline was aggregated in cattle maintained in a relatively small number of pens. Erythromycin resistance developed in C. hyointestinalis strains isolated from animals treated with chlortetracycline but not in those isolated from animals fed the macrolide tylosin. Large quantities of in-feed tetracyclines and other antimicrobial agents administered continuously at relatively low concentrations are typically used in beef production in North America. The findings of this study show that this practice selects for AMR campylobacters. However, the impact that AMR campylobacters originating from beef cattle have on human health remains to be determined.


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ACKNOWLEDGMENTS
 
We are most grateful for the financial support received from the Canada-Alberta Beef Industry Development Fund for this project.

This research could not have been completed without the dedication and excellent work of several individuals at the Agriculture and Agri-Food Canada Research Center at Lethbridge, and we thank the following people: Kathaleen House for sequencing the 16S rRNA genes of Campylobacter isolates; Toby Entz for his advice on statistical analyses; Aaron Minkley, Ken Wright, Wendi Smart, and Brant Baker for collecting fecal samples, conducting microbiological isolations, and storing Campylobacter isolates; and Merlin Anderson and his staff at the feedlot for the care of the cattle. Deseret Ranches of Alberta (Raymond) provided the cattle used for the study. We also thank the anonymous reviewers for their suggestions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Agriculture & Agri-Food Canada Research Centre, 5403 1st Avenue S, Lethbridge, AB T1J 4B1, Canada. Phone: (403) 317-3355. Fax: (403) 382-3156. E-mail: inglisd{at}agr.gc.ca. Back

{dagger} Contribution 04044 from the Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, Canada. Back


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Applied and Environmental Microbiology, July 2005, p. 3872-3881, Vol. 71, No. 7
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  • Alexander, T. W., Yanke, L. J., Topp, E., Olson, M. E., Read, R. R., Morck, D. W., McAllister, T. A. (2008). Effect of Subtherapeutic Administration of Antibiotics on the Prevalence of Antibiotic-Resistant Escherichia coli Bacteria in Feedlot Cattle. Appl. Environ. Microbiol. 74: 4405-4416 [Abstract] [Full Text]  
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