Antimicrobial Drug Use and Antimicrobial Resistance in Enteric Bacteria Among Cattle from Alberta Feedlots

FOODBORNE PATHOGENS AND DISEASE Volume 7, Number 4, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=fpd.2009.0400

Sangeeta Rao,1 Joyce Van Donkersgoed,2 Valerie Bohaychuk,3 Thomas Besser,4 Xin-Ming Song,5 Bruce Wagner,6 Dale Hancock,4 David Renter,7 David Dargatz,6 and Paul S. Morley1

Abstract The purpose of this study was to determine whether antimicrobial resistance (AMR) in foodborne pathogens (Escherichia coli O157, Salmonella, and Campylobacter) and non–type-specific E. coli obtained from fecal samples of feedlot cattle was associated with antimicrobial drug (AMD) use. A secondary objective was to determine if AMR in non–type-specific E. coli could be used as a predictor of AMR in foodborne pathogens. Fecal samples were collected from pen floors in 21 Alberta feedlots during March through December 2004, and resistance prevalence was estimated by season (Spring, Fall) and cattle type (fewest days-on-feed and closest to slaughter). AMD exposures were obtained by calculating therapeutic animal daily doses for each drug before sampling from feedlot records. Generalized linear mixed models were used to investigate the relationship between each AMR and AMD use. Non–type-specific E. coli was commonly recovered from fecal samples (88.62%), and the highest prevalence of resistance was found toward tetracycline (53%), streptomycin (28%), and sulfadiazine (48%). Campylobacter jejuni was recovered from 55.3% of the fecal samples, and resistance was generally less for

the drugs that were evaluated (doxycycline 38.1%, ciprofloxacin 2.6%, nalidixic acid 1.64%, erythromycin 1.2%). E. coli O157 and Salmonella were recovered much less frequently (7% and 1% prevalence, respectively). The prevalence of recovery for the bacteria studied varied between seasons and cattle types, as did patterns of AMR. Among non–type-specific E. coli, resistance to tetracycline, streptomycin, and sulfadiazine was found to be positively associated with in-feed exposure as well as injectable tetracycline, but these differences were relatively small and of questionable practical relevance. Among C. jejuni isolates, cattle type was significantly associated with doxycycline resistance. Results suggested that resistance in non–type-specific E. coli to chloramphenicol, trimethoprim=sulfamethoxazole, and tetracycline might be used as predictors of resistance to these drugs in E. coli O157 recovered from the same fecal samples.

Introduction ronmental exposure (USDA, 1999b). Conscientious use of AMDs is among the ﬁrst-line control measures that are re- ntimicrobial resistance (AMR) among bacteria is a commended for combating AMR in feedlots. However, it re- Agrowing global concern for human and animal health. It mains unproven that increased use of AMDs in feedlot cattle is a complex issue that should not be oversimpliﬁed or over- creates meaningful increases in resistance among pathogenic generalized because there is not a simple, direct relationship or nonpathogenic bacteria recovered from these animals, or between antimicrobial drug (AMD) use and AMR (USDA, that such increased resistance increases health risks for hu- 1999b). There is a concern that AMD use in food animals can mans. lead to the selection of resistant enteric bacteria that may Based on the studies conducted by the U.S. Department of eventually be transferred to people by the consumption of Agriculture’s National Animal Health Monitoring System contaminated food or through direct animal contact or envi- between 1990 and 1997 in the United States, approximately

1Animal Population Health Institute, Colorado State University, Fort Collins, Colorado. 2Dr. Joyce Van Donkersgoed Veterinary Services, Inc., Coaldale, Alberta, Canada. 3Food Safety Division, Alberta Agriculture and Rural Development, Edmonton, Alberta, Canada. 4College of Veterinary Medicine, Washington State University, Pullman, Washington. 5Vaccine and Infectious Disease Organization, Saskatoon, Saskatchewan, Canada. 6Centers for Epidemiology and Animal Health, U.S. Department of Agriculture, Fort Collins, Colorado. 7Department of Diagnostic Medicine=Pathobiology, Kansas State University, Manhattan, Kansas.

449 450 RAO ET AL.

25% of the small cattle feedlot operations (feedlots with 1000 unique animal identification, animal weight, diagnosis, drug, to 7999 head count) and 70% of the large feedlot operations dose, route of injection, location of injection, person pulled (greater than 8000 head count) used antimicrobials at some animal, person treating animal, animal pen movements, and time during the feeding period (USDA, 1999a, edited 2007). drug meat withdrawal. A few operations only recorded the AMDs are administered to cattle in feedlots for a variety of number of animals that received a particular drug. reasons, including prevention or treatment of disease in in- To obtain fecal samples used in this study, each feedlot was dividuals and groups, control of liver abscesses, promotion of visited once in the Spring and once in the Fall during 2004 as more efficient feed conversion, and acceleration of weight previously described (Van Donkersgoed et al., 2009). Briefly, gain. Respiratory disease complex (e.g., pneumonia) is a at each visit, two types of cattle were randomly identified for major feedlot health problem and an important determinant sampling: one was the type of cattle (pen) that had most re- of antimicrobial use (USDA, 1999b). Pneumonia and neonatal cently arrived at the feedlot (newly arrived) and the other type diarrhea are major causes of calfhood mortality, and calves was identified from all the pens that were within 2 weeks of are often treated individually by injection or in groups slaughter (preslaughter). A total of 84 pens were sampled for through feed, water, or by injection (USDA, 1997; McEwen this project. Twenty-five fresh manure samples (25–50 g) were and Fedorka-Cray, 2002). collected from the floor of these selected pens in a represen- This study was designed to investigate associations be- tative systematic manner. The samples were placed on ice tween AMD use in feedlots and occurrence of AMR in enteric packs and sent by overnight courier to the laboratories where bacteria obtained from feces of cattle. This study also inves- they were cultured to isolate E. coli O157, Salmonella enterica, tigated whether AMR in non–type-specific Escherichia coli was C. jejuni, and non–type-specific E. coli. Non–type-specific E. predictive of AMR in foodborne pathogens recovered from coli, E. coli O157, Salmonella, and Campylobacter were isolated the same samples (E. coli O157, Campylobacter jejuni,orSal- as previously described (Van Donkersgoed et al., 2009). monella enterica). Antimicrobial susceptibility testing Materials and Methods The susceptibility of all non–type-specific E. coli isolates Study population and sample collection was characterized by agar dilution breakpoint assays according to the CLSI (2002) guidelines. The antimicrobials Twenty-one feedlots in Alberta with greater than 5000 head tested included amikacin, amoxicillin=clavulanic acid, ampi- capacity were randomly selected for participation in the study cillin, ceftiofur, cephalothin, chloramphenicol, gentamicin, (Van Donkersgoed et al., 2009). Cattle were managed in these nalidixic acid, streptomycin, sulfadiazine, sulfamethoxa- facilities using standard industry practices. zole=trimethoprim, and tetracycline. All Campylobacter iso-

Briefly, cattle were purchased from a variety of sources, lates were screened for resistance using agar dilution methods including individual ranches and livestock auction markets. with breakpoint concentrations of ciprofloxacin, doxycycline, Upon arrival, cattle were commonly treated with anti- erythromycin, and nalidixic acid (Ge et al., 2002). The control helmintic drugs, implanted with growth hormones, and strain C. jejuni ATCC 33560 was included on each agar dilu- vaccinated against respiratory disease agents and clostridial tion plate. E. coli O157 and Salmonella enterica were tested for agents. If individuals or groups were considered to be high antimicrobial susceptibility by a broth microdilution method risk for respiratory disease, they were treated with injectable (Sensititer, Trek Diagnostic Systems, West Sussex, UK) ac- AMDs as a prophylactic=metaphylactic disease control mea- cording to the manufacturer’s instructions, using E. coli ATCC sure. Incoming groups of cattle were assigned to specific pens 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus fae- for housing, and these cohorts generally remained together calis ATCC 29212, and Staphylococcus aureus ATCC 29213 as throughout the feeding period. Assignment to pens (i.e., controls. physical location) was made irrespective of the disease history and management strategies employed with cattle that had Exposure to AMDs previously been housed in the same pens. Thus, while not documented, cattle could have been housed in pens that Individual and mass antimicrobial treatment information previously housed cattle with a variety of AMD exposures was retrieved from records obtained from the 21 feedlots and disease histories. All cattle were fed nutritionally bal- enrolled in this study. A standard data capture form was used anced rations throughout the feeding periods. Trained feedlot to collect information from the feedlots on feed medications personnel observed cattle in all pens on a daily basis to and processing procedures. Copies of existing feedlot treat- identify animals that looked abnormal. These abnormal cattle ment records were requested for those pens sampled. The were moved from their pen to a hospital facility where trained amount of each drug administered to cattle was converted personnel worked under the direction of feedlot veterinarians into standardized animal daily doses (ADDs) according to to examine the animals and diagnose their condition. Cattle the licensing information regarding recommended doses thought to be clinically sick from conditions due to bacterial needed to provide therapeutic efficacy for the treatment of infections were treated with therapeutic doses of AMDs in respiratory disease in feedlot cattle (Animal drugs at Food accordance with treatment protocols prescribed by supervis- and Drug Administration: www.accessdata.fda.gov=scripts= ing veterinarians. Producers documented cattle treatment animaldrugsatfda=). Even though AMDs were sometimes information using a variety of methods ranging from com- administered for other purposes (e.g., in-feed medication for puterized animal health databases to paper records, and the liver abscess control), all ADDs were converted to ADDs that amount of information recorded varied among feedlots. were relevant to the treatment of respiratory disease. The Many feedlots maintained very detailed individual treatment AMD exposures (both in-feed and as injectables) used in an- records and recorded information such as the date treated, alyses were summarized at the group level as the total number ANTIMICROBIAL RESISTANCE IN BACTERIA FROM ALBERTA FEEDLOTS 451 of ADDs administered to a group of cattle before the sampling variables in the models. Random intercept mixed effect date. The ADDs for individual drugs were summed within models (generalized linear mixed model regression) were drug classes or subclasses (penicillins, cephalosporins, phe- used to control for the lack of independence in observations nicols, fluoroquinolones, macrolides, sulfonamides, and tet- due to the collection of multiple fecal samples from each pen racyclines). There was no reported administration of drugs (PROC GLIMMIX SAS v9.1; SAS Institute). Isolates were via drinking water at any of the feedlots. The therapeutic du- identified in models as repeated observations nested within ration of a single administration of long-acting tetracyclines, the feedlots, and feedlots were modeled as a random effect. macrolides, fluoroquinolones, phenicols, and long-acting Season (Spring=Fall) and type of cattle (newly arrived= penicillins was considered to be 3 days per administration as preslaughter) were analyzed as fixed effects. Exposure esti- per label recommendations for dosing (Animal drugs at Food mates for all drug classes were entered into all initial models, and Drug Administration). For estimating the number of and backward elimination was used to determine which ADDs associated with in-feed medication, all cattle were as- variables were retained in final models. Season (Spring=Fall) sumed to have an average body weight of 800 lbs; animals and type of cattle (newly arrived=preslaughter) were con- were arbitrarily assumed, based on the experience with trolled in all models as potential confounding variables. The feeding cattle, to have uniformly consumed feed at the rate of critical alpha for comparisons was set at 0.05. When inclusion 2.5% of its body weight per day, and the supplements were of variables in models created model instability (as evidenced assumed to have been added to the rations at 3% of the total by extremely high parameter estimates and extremely wide feed weight on a dry matter basis (unless otherwise specified confidence intervals [CIs]), these variables were removed in the treatment records). from models, and the model selection process continued until a stable model was obtained. Adjusted baseline resistance prevalence estimates were obtained from model intercepts, Statistical analysis and effects of AMD exposure were assessed by calculating ad- Data were entered into computer databases (Microsoft justed risk estimates using parameter estimates and standard Excel) and validated by comparison to paper records. Data error (SE) estimates obtained from the variance–covariance were evaluated graphically and by calculating unadjusted matrix. descriptive statistics (SAS v9.1; SAS Institute, Cary, NC). All To provide a practically relevant measure of effect from analyses regarding AMD exposures were performed by these analyses regarding injectable drugs, adjusted risk summarizing observations at the pen level. The average (probability) estimates for the occurrence were calculated for number of animals in each pen type was calculated and relative to AMD exposures that might be experienced by compared. Drug exposures were standardized by dividing cattle groups with low or high risk of respiratory disease.

pen-level exposures by the number of animals in the pen and Cattle from groups with a low theoretical risk of respiratory the amount of time cattle had been at risk of exposure before disease were assumed to have not received AMDs upon ar- sampling, yielding estimates of ADDs per head per day. Re- rival for prophylaxis=metaphylaxis and assumed to have gression analysis using generalized estimating equations approximately 0.5% crude mortality for the feeding period, (PROC GENMOD, SAS v9.1; SAS Institute) with normal with an accompanying 1% overall morbidity rate for diseases distribution function was used to compare the ADD expo- requiring injectable AMD treatment rate, that 25% of these sures (penicillins, cephalosporins, phenicols, fluor- requiring a second treatment (first relapse), and that 25% of oquinolones, macrolides, sulfonamides, and tetracyclines) the animals that were treated twice would require a third across sampling seasons (Spring, Fall) and type of ani- treatment (second relapse). Cattle in groups with a high the- mals=pens (newly arrived, preslaughter). To facilitate the oretical risk of respiratory disease were assumed to have ap- analysis of antimicrobial susceptibility, bacteria were cate- proximately 4% crude mortality rate for the feeding period. It gorized as resistant (included resistant and intermediates) or was also assumed that all high-risk cattle would receive an susceptible. Regression analysis using generalized estimating injectable AMD as prophylaxis=metaphylaxis for respiratory equations (PROC GENMOD, SAS v9.1; SAS Institute) with a disease, that there would be a 15% crude morbidity rate for binomial distribution function was used to evaluate whether diseases requiring initial injectable treatment, that 25% would season and type of cattle were associated with difference in experience a first relapse, and 25% would experience a second the likelihood of resistance in bacteria. The pens of newly relapse. Mortality, morbidity, and associated treatment rates arrived animals sampled during Spring season were consid- used in these assumptions were extrapolated from previously ered as the reference group to compare other categories of published literature (Perrett et al., 2008). All of these injectable animal=pen type and season. treatments were assumed to be with a long-acting AMD and Poisson regression was used to investigate associations that each treatment would therefore attribute three ADDs. between AMD exposure in pens and the occurrence of AMR To evaluate the AMR among non–type-specific E. coli as a at the group level. Counts of bacteria with AMR were the predictor of AMR in other bacteria within the same sample, outcome for these models, and drug exposure information logistic regression models using generalized estimating (ADDs per head per day) was the primary exposure variable equations (PROC GENMOD, SAS v9.1; SAS Institute) were of interest for these models. A separate model was con- constructed to separately compare resistance to each AMD. structed to investigate the influence of AMD use on suscep- Resistance (yes=no) among non–type-specific E. coli to a drug tibility to each AMD that was evaluated. Although each drug (chloramphenicol, sulfisoxazole, tetracycline, trimetho- resistance was analyzed in a different model, drug exposures prim=sulfamethoxazole, nalidixic acid) was included as an for all classes of drugs were evaluated in all models as po- independent predictor variable for resistance (yes=no) in a tential determinants of the likelihood of resistance. In-feed foodborne pathogen species. The antimicrobials that were and injectable drugs were analyzed as separate exposure evaluated in both non–type-specific E. coli and the other 452 RAO ET AL. bacteria in question were used in these models. A separate (26.0% from newly arrived pens and 25.9% from preslaughter analysis was performed to evaluate the association between pens both sampled in the Fall; 23.8% from newly arrived pens doxycycline resistance in Campylobacter and the resistance to and, 24.3% from preslaughter pens both sampled in the tetracycline in non–type-specific E. coli. The analyses were Spring). However, among all 84 pens sampled, antimicrobial performed at the level of the isolate. use data were available for only 75 pens. Among 1861 non–type-specific E. coli isolates, 74.4% Results showed resistance to at least one AMD (most commonly to tetracycline), 45.1% were resistant to at least two drugs, and Samples were collected from a total of 84 pens distributed 24.1% to at least three drugs (Table 3). Overall, the highest among the 21 participating feedlots. The mean numbers of resistance prevalence among season and pen type classifica- animals housed in newly arrived pens (mean ¼ 191, standard tions was seen for sulfadiazine during the Spring (>80% of deviation [SD] ¼ 90) and preslaughter pens (mean ¼ 171, isolates), followed by tetracycline resistance (45–48%), which SD ¼ 90) were significantly smaller in the Fall season was similar across all of these categories (Table 3). In fact, ( p < 0.0001) when compared with the newly arrived animals tetracycline resistance was the only resistance that did not in the Spring season. The mean number of animals in newly vary in prevalence among season and pen types. Resistance arrived (mean ¼ 220, SD ¼ 110) and preslaughter (mean ¼ among non–type-specific E. coli to amoxicillin=clauvulanic 208, SD ¼ 90) pens sampled in the Spring were not signifi- acid, cephalothin, streptomycin, and sulfadiazine was signif- cantly different ( p ¼ 0.62). The median number of days- icantly less frequent among isolates recovered from newly on-feed was 198 and 160 for preslaughter pens sampled in the arrived and preslaughter cattle during the Fall season when Spring and Fall, respectively, and the median number of days- compared with the newly arrived animals during the Spring on-feed for newly arrived pens was 15 and 12, respectively. season (Table 3). Resistance to chloramphenicol was signifi- Exposure to AMDs cantly higher among isolates recovered from newly arrived cattle and lower in those recovered from preslaughter cattle in Overall, differences in ADD exposures were significant the Fall when compared with the newly arrived animals between season and type of animals (Tables 1 and 2). As ex- sampled in the Spring; isolates recovered from preslaughter pected, the in-feed exposures to sulfonamides and macrolides animals sampled in the Spring were not different from the were greater in preslaughter animals during both seasons reference group. Resistance to ampicillin was lowest among when compared with the estimates for newly arrived cattle isolates from preslaughter animals in the Spring. Resistance to (Table 1). However, the average in-feed exposure to tetracy- ceftiofur was rare, but compared with the isolates recovered cline was lower in preslaughter animals during both seasons from newly arrived cattle in the Spring, resistance was sig- compared with the exposures accumulated by the newly ar- nificantly higher among isolates recovered from the other rived animals sampled during the Spring (Table 1). In con- groups. Nalidixic acid and trimethoprim=sulfamethoxazole trast, AMD exposures for newly arrived pens of cattle were resistance was quite rare and not significantly different greater for cattle sampled in the Fall when compared with the among different groups of cattle (Table 3). similar groups sampled during the Spring (Table 1). Average exposures to injectable AMDs were greatest for tetracycline AMR in E. coli O157. E. coli O157 was isolated from 148 and florfenicol, followed at some distance by exposures to fecal samples, representing 7.1% of the total samples. The macrolides (Table 2). However, average rates of exposure majority of isolates (54.7%) were recovered in the Fall from varied considerably among drug classes by season and type of newly arrived animals. In general, resistance was less common cattle group (Table 2). among E. coli O157 isolates compared with the non–type- specific E. coli isolates. Sixteen percent of the 148 samples Recovery of bacteria and AMR patterns showed resistance to at least two AMDs, 3% were resistant to AMR in non–type-specific E. coli. Non–type-specific three AMDs, and 2% to four drugs. Resistance to sulfisoxazole E. coli was recovered from 88.6% of fecal samples during the and tetracycline was identified in samples recovered from study (Table 3); thus, 1861 isolates were available for analysis newly arrived and preslaughter animals in both sampling

Table 1. In-Feed Administration of Antimicrobial Drugs to Cattle Groups Before Sampling (per 1000 Head per Day)

Sulfonamides Macrolides Tetracyclines

ADDs ADDs ADDs

Season Type of group Mean SD p-Value Mean SD p-Value Mean SD p-Value

Spring Shortest on feed 0 0 Reference 14 50 Reference 1437.2 2261.3 Reference Spring Preslaughter 42.8 94.9 <0.0001 205.2 493.1 <0.0001 657.3 1301.9 <0.0001 Fall Shortest on feed 0 0 1 0.19 0.7 0.57 3398.6 4586.7 <0.0001 Fall Preslaughter 13.5 52.4 0.0003 167.8 340.3 <0.0001 998.5 1742.2 0.01

p-Values were obtained from generalized estimating equations (PROC GENMOD, SAS v9.1; SAS Institute) with normal distribution function to compare the ADD exposures across sampling seasons and groups of animals. Sulfonamide class of antimicrobial in feed included sulfamethazine; macrolide class included tylosin; tetracycline class included tetracyline. ADDs, animal daily doses, SD, standard deviation. Table 2. Injectable Antimicrobial Drugs Administered to Cattle Before Sampling (per 1000 Head per Day)

Penicillins Cephalosporins Phenicols Fluoroquinolones

ADDs ADDs ADDs ADDs

Season Pen type Mean SD p-Value Mean SD p-Value Mean SD p-Value Mean SD p-Value

Spring SOF 0.05 0.2 Reference 0.1 0.4 Reference 16.5 52.5 Reference 0 0 Reference Spring PS 0.2 0.4 0.02 0.4 0.6 <0.0001 2.2 5.8 <0.0001 0 0 1 Fall SOF 0 0 0.01 0.3 0.6 <0.0001 15.2 35.5 0.1 0.3 0.98 <0.0001 Fall PS 0.1 0.3 <0.0001 0.3 0.4 <0.0001 0.5 1.5 <0.0001 0.002 0.01 0.9

Macrolides Sulfonamides Tetracyclines

ADDs ADDs ADDs

Season Pen type Mean SD p-Value Mean SD p-Value Mean SD p-Value

Spring SOF 0.8 1.5 Reference 0.2 0.5 Reference 29.0 106.9 Reference Spring PS 1.7 3.4 0.02 0.3 0.7 0.0005 7.0 11.2 <0.0001 Fall SOF 3.4 8.6 <0.0001 0.1 0.3 0.0004 122.9 164.7 <0.0001 Fall PS 0.06 0.1 0.02 0.2 0.3 0.02 3.1 6.0 <0.0001

‘‘Season’’ indicates two sampling periods in 2004 (March to July—designated as Spring—and September to December—designated as Fall). ‘‘Pen type’’ indicates two groups of cattle (pen): one that had most recently arrived at the feedlot (shortest on feed=newly arrived) and the other group were within 2 weeks of slaughter (preslaughter). p-Values were obtained from generalized estimating equations (PROC GENMOD, SAS v9.1; SAS Institute) with normal distribution function to compare the ADD exposures across sampling seasons and groups of animals. Penicillin class of injectable antimicrobial included penicillin, long-acting penicillin, and procaine penicillin; cephalosporins included ceftiofur sodium; phenicols included florfenicol; fluoroquinolones included enrofloxacin; macrolides included tilmicosin; sulfonamides included trimethoprim=sulfadoxine; tetracycline class included biomycin, chlortetracycline, liquamycin, oxytetracyline, long-acting oxymsycine, and tetracycline.

Table 3. Unadjusted Prevalence (%) of Antimicrobial Resistance Among Non–Type-Speciﬁc Escherichia coli Recovered from the Feces of Feedlot Cattle

Spring Fall

Newly arrived Preslaughter Newly arrived Preslaughter Antimicrobial (%) n ¼ 443 n ¼ 454 n ¼ 483 n ¼ 481

Amikacin % Prevalence 0 0 0 0 95% CI 0 0 0 0 Ampicillin % Prevalence 6.3 2.4a 6.6 4.6 95% CI 4.32–9.11 1.28–4.42 4.64–9.32 2.96–6.95 Amoxicillin–clavulanate % Prevalence 13.8 11.0 0.4a 0.4a 95% CI 10.77–17.41 8.36–14.35 0.072–1.66 0.072–1.66 Ceftiofur % Prevalence 0 0.7b 0.8b 0.6b 95% CI 0 0.17–2.09 0.27–2.26 0.16–1.97 Cephalothin % Prevalence 14.2 11.9 5.2a 5.4a 95% CI 11.17–17.9 9.13–15.32 3.45–7.65 3.63–7.92 Chloramphenicol % Prevalence 3.2 2.6 6.2b 1.0a 95% CI 1.81–5.37 1.44–4.7 4.3–8.85 0.38–2.55 Gentamicin % Prevalence 0 0 0.2 0 95% CI 0 0 0.011–1.33 0 Nalidixic acid % Prevalence 0.5 0.2 0.4 0.6 95% CI 0.078–1.8 0.012–1.42 0.072–1.66 0.16–1.97 Streptomycin % Prevalence 35.7 39.2 19.9a 19.3a 95% CI 31.24–40.35 34.72–43.88 16.46–23.78 15.96–23.21 Sulfadiazine % Prevalence 86.7 83.3 19.0a 10.8a 95% CI 83.08–89.63 79.43–86.51 15.7–22.9 8.25–14.02 Tetracycline % Prevalence 51.5 57.5 45.8 55.3 95% CI 46.71–56.2 52.79–62.06 41.26–50.32 50.73–59.79 Trimethoprim–sulfamethoxazole % Prevalence 1.8 0.4 3.7 1.2 95% CI 0.84–3.67 0.076–1.76 2.29–5.94 0.51–2.83

Significance was based on generalized estimating equations (PROC GENMOD, SAS v9.1; SAS Institute) with a binomial distribution function to evaluate whether season and type of cattle were associated with difference in the likelihood of resistance in bacteria. aSignificantly lower than Spring newly arrived. bSignificantly higher than Spring newly arrived. CI, confidence interval. 454 RAO ET AL. seasons. Resistance to each of the two drugs was less common Associations between AMD use and AMR during Spring (5.9%) and Fall (8%) in preslaughter and fall Non–type-specific E. coli. In-feed medication with tetra- newly arrived animals (14.8%) when compared with newly cyclines was found to be significantly associated with resis- arrived animals during Spring (36%). Resistance to chloram- tance among non–type-specific E. coli to three drugs phenicol was highest in isolates obtained from newly arrived (streptomycin, sulfadiazine, and tetracycline) (Table 5). animals during the Spring (12%) and moderate in isolates ob- Comparing estimates for cattle that did not receive any in- tained from cattle sampled in the Fall (3.7%). Resistance to feed tetracycline treatment to estimates for groups in which trimethoprim=sulfamethoxazole (12%) was detected among every animal received one therapeutic ADD (resistance isolates recovered from the newly arrived cattle sampled in the prevalence in cattle with AMD exposure in Table 5) of tetra- Spring animals but not detected in isolates recovered from cycline in-feed every day before sampling, the predicted dif- other cattle. ferences in resistance prevalence were modest (1.9%, 6.0%, and 1.95% absolute differences for resistance to streptomycin, AMR in Salmonella. Salmonella enterica representing a sulfadiazine, and tetracycline, respectively; Table 5). Com- variety of serotypes (Rubislaw, Saintpaul, Enteritidis, Mban- paring untreated cattle to groups of cattle with an assumed daka, 4,5,12:i:- and Typhimurium) were isolated from only low-risk AMD exposure, the differences in resistance preva- 0.95% of the fecal samples (n ¼ 20). Twenty-five percent of the lence were either absent or quite small (0.0%, 0.01%, and isolates represented serotype Saintpaul that exhibited a 0.01% absolute differences for resistance to streptomycin, common pentadrug resistance pattern (ampicillin, chloram- sulfadiazine, and tetracycline, respectively). In-feed medica- phenicol, streptomycin, sulfisoxazole, and tetracycline) in tion with macrolides (i.e., tylosan) was not associated with addition to amoxicillin–clavulanate, cefoxitin, and ceftiofur any detectable differences in resistance prevalence among resistance. Five percent (n ¼ 1) of the isolates represented se- non–type-specific E. coli. For injectable AMDs, only exposure rotype Mbandaka and another 5% (n ¼ 1) represented sero- to tetracycline injections was associated with differences in type 4,5,12:i:-, both showed pentadrug resistance pattern in AMR; injectable tetracycline exposure was associated with addition to amoxicillin–clavulanate resistance. Another 5% detectable differences in resistance to streptomycin, sulfadi- (n ¼ 1) of the isolates belonged to serotype Typhimurium that azine, and tetracycline (Table 5). However, comparing un- showed pentadrug resistance in addition to kanamycin and treated cattle to groups of cattle with an assumed low-risk trimethoprim–sulfamethoxazole resistance. AMD exposure, the differences in resistance prevalence were quite small (0.01%, 0.02%, and 0.01% absolute differences for AMR in Campylobacter. A total of 1183 Campylobacter resistance to streptomycin, sulfadiazine, and tetracycline, re- isolates were isolated; 98.2% (n ¼ 1162) were C. jejuni and spectively; Table 5). Comparing untreated cattle to groups of

1.8% (n ¼ 21) were Campylobacter coli. Seventy-one percent (15 cattle with an assumed high-risk AMD exposure, the differ- out of 21) of the C. coli isolates showed resistance to doxycy- ences in resistance prevalence were small also (0.87%, 2.16%, cline. However, C. coli were excluded from further analysis and 0.68% absolute differences for resistance to streptomycin, because there were so few isolates. Overall, C. jejuni were sulfadiazine, and tetracycline, respectively; Table 5). Although isolated from 55.3% of the fecal samples collected. About 38% the variable ‘‘type of cattle’’ (newly arrived=preslaughter) was were resistant to at least one AMD, 2% to two AMDs, and included in all models, it was only found to be signiﬁcantly 0.8% to three AMDs (Table 4). In general, there was moderate associated with differences in tetracycline resistance preva- resistance to doxycycline, and resistance to other drugs was lence; adjusting for other variables in the model, the average rare. Resistance to doxycycline was highest among isolates resistance prevalence was lower in isolates obtained from recovered from preslaughter animals (sampled from both cattle in newly arrived pens (46%, 95% CI ¼ 32.6–52.8%) than seasons) when compared with the isolates from the newly in isolates obtained from cattle in preslaughter pens (54%, arrived animals (Table 4). 95% CI ¼ 53.5–63.3%).

Table 4. Unadjusted Prevalence (%) of Antimicrobial Resistance in Campylobacter Jejuni in the Manure of Feedlot Cattle by Sampling Season and Type of Cattle

Spring Fall

Newly arrived Preslaughter Newly arrived Preslaughter Antimicrobial (%) n ¼ 220 n ¼ 309 n ¼ 206 n ¼ 427

Ciproﬂoxacin % Prevalence 1.4 1.9 6.3a 1.9 95% CI 0–2.9 0.4–3.5 3.0–9.7 0.6–3.2 Doxycycline % Prevalence 27.7 42.1a 25.7 46.6a 95% CI 21.8–33.7 36.5–47.6 19.7–31.7 41.9–51.4 Erythromycin % Prevalence 0.5 1.9 3.4 0 95% CI 0–1.4 0.4–3.5 0.9–5.9 0 Nalidixic Acid % Prevalence 0.5 1.3 3.9a 1.4 95% CI 0–1.4 0.03–2.6 1.2–6.5 0.3–2.5

Table 5. Prevalence Estimates for Antimicrobial Resistance Predicted from Multivariable Models Relative to Varying Levels of Exposures to Drugs That Were Signiﬁcantly Associated with Differences in Antimicrobial Resistance Among Non–Type-Speciﬁc Escherichia coli Recovered from the Feces of Feedlot Cattle

Antimicrobial drug Unexposed Low High exposure Resistance (%) 95% CI exposure (%) 95% CI exposure (%) 95% CI

In-feeda STR 28.20 21.9–36.3 28.20 21.91–36.3 30.11 22.5–40.3 Tetracycline SUL 74.10 63.0–87.1 74.11 63.05–87.12 80.15 65.9–97.5 TET 43.50 34.9–54.2 43.51 34.93–54.2 45.46 35.5–58.3 STR 28.20 21.9–36.3 28.21 21.9–36.3 29.07 22.2–38.1 Injectableb SUL 74.11 63.1–87.1 74.13 63.1–87.2 76.27 64.0–91.0 Tetracycline TET 43.51 34.9–54.2 43.52 34.9–54.2 44.19 35.0–55.8

aAssuming one therapeutic ADD was administered in feed per head on every day before sampling. bTheoretically unexposed groups were assumed to have no morbidity requiring parenteral treatment. Estimates for low exposures to injectable drugs were based on an assumption that 1% of the group received an initial injectable treatment, 25% of these received a second treatement (first relapse), and 25% of those received a third treatment (second relapse) with no metaphylaxis. Estimates for high-risk exposures were based on the assumption that 100% of the group received metaphylactic treatment at arrival, 15% received an initial injectable treatment, 25% of these received a second treatment (first relapse), and 25% of those received a third treatment (second relapse). All injectable treatments were assumed to be with long-acting drugs that had three ADDs per treatment. STR, streptomycin; SUL, sulfisoxazole; TET, tetracycline.

Campylobacter. Exposure to AMDs was not associated specific E. coli was not significantly associated with resistance with detectable differences in resistance among C. jejuni iso- in Campylobacter. However, resistance to tetracycline in non– lates. Although there was a detectable difference among type-specific E. coli was significantly associated with the re- C. jejuni in doxycycline resistance relative to the type of cattle sistance to doxycycline in Campylobacter (OR ¼ 1.6, 95% and season (Table 4) that were sampled, this difference was CI ¼ 1.1–2.3). Predictors of resistance in Salmonella were not small and of questionable practical relevance: isolates ob- analyzed because of the low numbers of Salmonella isolates tained from newly arrived cattle had an average resistance (n ¼ 20). prevalence of 21.1% (95% CI ¼ 14.0–31.7%) compared with

the isolates from preslaughter cattle, which had an average Discussion resistance prevalence of 21.4% (95% CI ¼ 11.8–38.9%). This study showed that the prevalence of recovery for the bacteria varied between seasons and among the cattle types, AMR in non–type-specific E. coli as a predictor as did patterns of AMR (Van Donkersgoed et al., 2009). There to determine AMR in other bacteria were some significant associations detected among the AMD Only predictors for chloramphenicol, streptomycin, sulfi- exposures and AMR in bacteria. Although some associations soxazole, tetracycline, and trimethoprim=sulfamethoxazole were identified, the size of the effect was moderate for in-feed could be analyzed because of the lack of detected resistance to exposures and small for injectable drugs. Further, the signif- other drugs among E. coli O157. Similarly, only nalidixic acid icance of these differences relative to animal or public health is resistance was analyzed regarding predictors for Campylo- not clear. For example, although resistance among non–type- bacter phenotype as this was the only drug resistance evalu- specific E. coli isolates was most commonly detected against ated in both non–type-specific E. coli and Campylobacter. The tetracycline and in-feed exposure was associated with a 2% odds ratios (ORs) suggested that resistance to chloramphen- difference in the resistance prevalence between the two icol, trimethoprim=sulfamethoxazole, and tetracycline in groups (nonexposed vs. exposed tetracycline in-feed group), non–type-specific E. coli could be predictors for resistance to this class of drugs continues to be efficacious and useful for a the same drugs among E. coli O157 recovered from the same variety of purposes in feedlot cattle, including treatment of fecal samples (respectively, OR ¼ 22.6, 95% CI ¼ 1.2–416.1; respiratory disease (O’Connor et al., 2002), control of liver OR ¼ 57.5, 95% CI ¼ 4.3–773.6; OR ¼ 4.5, 95% CI ¼ 1.2–16.1) abscesses, and growth promotion (McEwen and Fedorka- (Table 6). Models for sulfisoxazole and streptomycin were not Cray, 2002; Schunicht 2002a, 2002b). Tetracycline treatment significant. Resistance to nalidixic acid among non–type- was also associated with tetracycline and sulfizoxazole

Table 6. Antimicrobial Resistance in Non–Type-Speciﬁc Escherichia coli as a Predictor of Antimicrobial Resistance in Escherichia coli O157 in the Manure of Feedlot Cattle

CHL FIS TET SXT

E. coli O157 p-Value 0.036 0.22 0.023 0.002 OR 22.6 2.06 4.45 57.5 95% CI 1.23–416.07 0.64–6.62 1.23–16.07 4.27–773.56

OR, odds ratio; CHL, chloramphenicol; FIS, sulfisoxazole; TET, tetracycline; SXT, trimethoprim=sulfamethoxazole. 456 RAO ET AL. resistance. It was interesting that resistance to streptomycin, Conclusions sulfisoxazole, and tetracycline was associated with both in- Although there were some associations detected between feed and injectable treatment with tetracycline, which also the in-feed exposure of AMD and AMR in these feedlots in had the highest prevalence of resistance among non–type- this study, differences associated with treatment were small specific E. coli, which may suggest that a co-selection method and of unknown relevance. More such in-depth studies would was responsible. be helpful in answering questions on drug use in cattle and Other studies in feedlot cattle have identified varied re- the development of AMR in cattle, as well as what risk this sponses between AMR and AMD exposures. Studies on fecal poses to animal and human health. non–type-specific E. coli have suggested that a single administration of the long-acting medications can have detectable, Acknowledgments short-term impacts on the prevalence of resistant isolates, but that measureable differences between treated and untreated We would like to thank the 21 Alberta feedlot producers cattle disappear after a short period of time (Stabler et al., 1982; who participated in this study. We are grateful to the technical Berge et al., 2005; Lowrance et al., 2007). A study in feedlot support by Robin Downen, Darren Malchow, and Larry Sus- steers in Canada that received subtherapeutic levels of tetra- helnitski in the field collection of samples. Laboratory support cycline in combination with sulfamethazine showed an in- was given by Scott Nelson, Washington State University. We creased prevalence of tetracycline- and ampicillin-resistant are grateful for the technical assistance of staff at the Agri-Food E. coli (Alexander et al., 2008). In contrast, another study Laboratories Branch, Alberta Agriculture and Rural Devel- conducted in a western Canadian feedlot found no associa- opment. CANFAX is thanked for helping us randomly select tions between antimicrobial use and AMR (Checkley et al., participating feedlots for the study. Funding was provided by 2008). Another study in feedlot cattle found an association Alberta Livestock Industry Development Fund, Alberta between injectable oxytetracycline and an increase in the Agricultural Research Institute, and Alberta Beef Producers. prevalence of resistance to sulfisoxazole and chloramphenicol among E. coli beyond those due to the in-feed chlortetracy- Disclosure Statement cline, and these changes were detectable a substantial time after treatment with injectable oxytetracycline (O’Connor No competing financial interests exist. et al., 2002). The reason proposed was that the in-feed chlortetracycline increased the level of resistance to tetracycline to References ‘‘saturation,’’ and therefore injectable oxytetracycline could Alexander TW, Yanke LJ, Topp E, Olson ME, Read RR, Morck have no detectable additional impact on the prevalence of DW, and McAllister TA. Effect of subtherapeutic administra- resistance. Thus, further work is needed to clarify whether tion of antibiotics on the prevalence of antibiotic-resistant these differences are consistently found in other feedlot pop- Escherichia coli bacteria in feedlot cattle. Appl Environ Micro ulations and whether they are associated with significant 2008;74:4405–4416. health risks to cattle or people. Berge AC, Epperson WB, and Pritchard RH. Assessing the effect Resistance to certain drugs in non–type-specific E. coli and of a single dose florfenicol treatment in feedlot cattle on the C. jejuni was more prevalent during the Fall season in the antimicrobial resistance patterns in faecal Escherichia coli. Vet newly arrived animals compared to other groups. This dif- Res 2005;36:723–734. ference may have occurred because fall placed calves are the Checkley SL, Campbell JR, Chirino-Trejo M, Janzen ED, and highest risk cattle for bovine respiratory disease and generally McKinnon JJ. Antimicrobial resistance in generic fecal Es- tend to be treated more commonly than backgrounded Spring cherichia coli obtained from beef cattle on arrival at the feedlot placed calves. However, it is possible that this association is and prior to slaughter, and associations with volume of total specific to some unique aspect of this dataset rather than being individual cattle antimicrobial treatments in one western Ca- a common finding relative to all feedlot cattle in Alberta. The nadian feedlot. Can J Vet Res 2008;72:101–108. findings of AMR in C. jejuni showed high resistance to [CLSI] Clinical and Laboratory Standards Institute, National doxycycline (belonging to tetracycline group) and low resis- Committee for Clinical Laboratory Standards. Performance tance to erythromycin, ciprofloxacin, and nalidixic acid. Si- Standards for Antimicrobial Susceptibility Tests for Bacteria Iso- nd milar observations were made in a study in Canada which lated from Animals,2 edition, volume 22. Approved standard showed that antimicrobial administration to beef cattle selects M31-A2. Wayne, PA: National Committee for Clinical La- for AMR campylobacters, developing substantial resistance to boratory Standards, 2002, p. 86. tetracycline and only limited resistance to erythromycin, Ge B, Bodeis S, Walker RD, White DG, Zhao S, McDermott PF, and Meng J. Comparison of the Etest and agar dilution for ampicillin, and ciprofloxacin (Inglis et al., 2005). in vitro antimicrobial susceptibility testing of Campylobacter. When investigating the use of AMR in E. coli as a predictor J Antimicrob Chemother 2002;50:487–494. of resistance in other bacteria, strong associations were found Inglis GD, McAllister TA, Busz HW, Yanke LJ, Morck DW, Olson between resistance in non–type-specific E. coli and resis- ME, and Read RR. Effects of subtherapeutic administration of tance in E. coli O157 for chloramphenicol, trimethoprim= antimicrobial agents to beef cattle on the prevalence of anti- sulfamethoxazole, and tetracycline. A strong association was microbial resistance in Campylobacter jejuni and Campylobacter also detected between the resistance to tetracycline in non– hyointestinalis. Appl Environ Micro 2005;71:3872–3881. type-specific E. coli and resistance to doxycycline in Campy- Lowrance TC, Loneragan GH, Kunze DJ, Platt TM, Ives SE, Scott lobacter; both of these drugs belong to the same antimicrobial HM, Norby B, Echeverry A, and Brashears MM. Changes in class. These findings may have useful implications, but fur- antimicrobial susceptibility in a population of Escherichia coli ther work is needed to corroborate these findings, especially isolated from feedlot cattle administered ceftiofur crystalline- given the relatively rare isolation of E. coli O157. free acid. Am J Vet Res 2007;68:501–507. ANTIMICROBIAL RESISTANCE IN BACTERIA FROM ALBERTA FEEDLOTS 457

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