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Objective: To describe geographic, farm-type, and animal-type factors associated with multiple antimicrobial resistance (MAR) in fecal Escherichia coli isolates from cattle., Design: Cross-sectional field study., Sample Population: 1,736 fecal samples from cattle on 38 farms in California, Oregon, and Washington., Procedures: Fecal samples were collected from preweaned calves (2 to 4 weeks old) and cows that recently calved on dairy and beef cow-calf farms, preweaned calves on calf ranches, and 1-year-old steers on feedlots. One fecal E coli isolate per sample was isolated, and antimicrobial susceptibility was tested. Escherichia coli isolates were initially clustered by antimicrobial resistance patterns and categorized by number of antimicrobial resistances. A generalized estimating equations cumulative logistic regression model was used to identify factors associated with an increase in MAR in fecal E coli isolates from cattle., Results: MAR was higher in E coli isolates from cattle in California, compared with those from cattle in Washington or Oregon. Multiple antimicrobial resistance was highest in E coli isolates from calves on calf ranches and progressively lower in isolates from feedlot steers, dairy cattle, and beef cattle. Multiple antimicrobial resistance was higher in E coli isolates from calves than from adult cattle, in E coli isolates from cattle of conventional farms than of organic farms, and in isolates from beef cattle in intensive dairy farm regions than from beef cattle distant from dairy farm regions., Conclusions and Clinical Relevance: MAR in fecal E coli isolates from cattle was influenced by factors not directly associated with the use of antimicrobials, including geographic region, animal age, and purpose (beef vs dairy).

We examined how a dietary supplement affects the prevalence of antibiotic-resistant Escherichia coli on a dairy farm in Washington State. Between 2001 and 2004 the prevalence of fecal E. coli strains resistant to streptomycin, sulfadiazine, and tetracycline (SSuT strains) declined from 59.2% to 26.1% in the calf population. In 2003 the dairy discontinued use of a dietary supplement, and we hypothesized that the decline in prevalence of SSuT strains was related to this change in management. To test this we established three treatments in which calves received no supplement, the dietary supplement with oxytetracycline, or the dietary supplement without oxytetracycline. Calves receiving either dietary supplement had a significantly higher prevalence of SSuT E. coli than the no-supplement control group (approximately 37% versus 20%, respectively; P = 0.03). Importantly, there was no evidence that oxytetracycline contributed to an increased prevalence of fecal SSuT E. coli. We compared the growth characteristics of SSuT and non-SSuT E. coli in LB broth enriched with either the complete dietary supplement or its individual constituents. Both the complete dietary supplement and its vitamin D component supported a significantly higher cell density of SSuT strains (P = 0.003 and P = 0.001, respectively). The dry milk and vitamin A components of the dietary supplement did not support different cell densities. These results were consistent with selection and maintenance of SSuT E. coli due to environmental components independent of antibiotic selection.

Maintenance of antimicrobial drug resistance in bacteria can be influenced by factors unrelated to direct selection pressure such as close linkage to other selectively advantageous genes and secondary advantage conveyed by antimicrobial resistance genes in the absence of drug selection. Our previous trials at a dairy showed that the maintenance of the antimicrobial resistance genes is not influenced by specific antimicrobial selection and that the most prevalent antimicrobial resistance phenotype of Escherichia coli is specifically selected for in young calves. In this paper we examine the role of secondary advantages conveyed by antimicrobial resistance genes. We tested antimicrobial-susceptible null mutant strains for their ability to compete with their progenitor strains in vitro and in vivo. The null mutant strains were generated by selection for spontaneous loss of resistance genes in broth supplemented with fusaric acid or nickel chloride. On average, the null mutant strains were as competitive as the progenitor strains in vitro and in newborn calves (in vivo). Inoculation of newborn calves at the dairy with antimicrobial-susceptible strains of E. coli did not impact the prevalence of antimicrobial-resistant E. coli. Our results demonstrate that the antimicrobial resistance genes are not responsible for the greater fitness advantage of antimicrobial-resistant E. coli in calves, but the farm environment and the diet clearly exert critical selective pressures responsible for the maintenance of antimicrobial resistance genes. Our current hypothesis is that the antimicrobial resistance genes are linked to other genes responsible for differential fitness in dairy calves.

Among bovine fecal and recto-anal mucosal swab samples cultured in our laboratory for Escherichia coli O157:H7, we frequently isolated E. coli organisms that were phenotypically similar to the O157:H7 serotype as non-sorbitol fermenting and negative for beta-glucuronidase activity but serotyped O nontypeable:H25 (ONT:H25). This study determined the prevalence and virulence properties of the E. coli ONT:H25 isolates. Among dairy and feedlot cattle (n = 170) sampled in Washington, Idaho, and Alberta, Canada, the percentage of animals culture positive for E. coli ONT:H25 ranged from 7.5% to 22.5%, compared to the prevalence of E. coli O157:H7 that ranged from 0% to 15%. A longitudinal 8-month study of dairy heifers (n = 40) showed that 0 to 15% of the heifers were culture positive for E. coli O157:H7, while 15 to 22.5% of the animals were culture positive for E. coli ONT:H25. As determined by a multiplex PCR, the E. coli ONT:H25 isolates carried a combination of virulence genes characteristic of the enterohemorrhagic E. coli, including intimin, translocated intimin receptor, Stx2, and hemolysin (eae-beta, tir, stx(2vh-a), and hly). E. coli ONT:H25 isolates from diverse geographic locations and over time were fingerprinted by separating XbaI-restricted chromosomal DNA by pulsed-field gel electrophoresis (PFGE) separation. Two strains of E. coli ONT:H25 were highly similar by PFGE pattern. Experimental inoculation of cattle showed that E. coli ONT:H25, like E. coli O157:H7, colonized the bovine recto-anal junction mucosa for more than 4 weeks following a single rectal application of bacteria.

The objective of this study was to characterize Escherichia coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. The rationale of the study was that, isolates of the same serotypes that were obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Multiplex polymerase chain reaction (PCR) was used to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) in E. coli isolates (n = 400). Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2 and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle, and food) isolates were stx+. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx+ . Five of 6 stx+ O26 and O111 produced Stx1, however, stx+ O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-O157 stx+ strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n = 105), and pigs, dogs, parrot and iguanas (n = 48) were stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between humans and cattle.

Shiga-toxigenic Escherichia coli (STEC) strains were isolated from 7.4% of 1,440 fecal and farm environmental samples. Shiga toxin gene and STEC prevalences were significantly associated with animal production type and season. A range of serogroups were identified. Nine percent of isolates possessed all three principal virulence markers: stx(2), eae, and ehx.

The prevalence of antimicrobial drug-resistant bacteria is typically highest in younger animals, and prevalence is not necessarily related to recent use of antimicrobial drugs. In dairy cattle, we hypothesize that antimicrobial drug-resistant, neonate-adapted bacteria are responsible for the observed high frequencies of resistant Escherichia coli in calves. To explore this issue, we examined the age distribution of antimicrobial drug-resistant E. coli from Holstein cattle at a local dairy and conducted an experiment to determine if low doses of oxytetracycline affected the prevalence of antimicrobial drug-resistant E. coli. Isolates resistant to tetracycline (>4 microg/ml) were more prevalent in <3-month-old calves (79%) compared with lactating cows (14%). In an experimental trial where calves received diets supplemented with or without oxytetracycline, the prevalence of tetracycline-resistant E. coli was slightly higher for the latter group (P = 0.039), indicating that drug use was not required to maintain a high prevalence of resistant E. coli. The most common resistance pattern among calf E. coli isolates included resistance to streptomycin (>12 microg/ml), sulfadiazine (>512 microg/ml), and tetracycline (>4 microg/ml) (SSuT), and this resistance pattern was most prevalent during the period when calves were on milk diets. To determine if prevalence was a function of differential fitness, we orally inoculated animals with nalidixic acid-resistant strains of SSuT E. coli and susceptible E. coli. Shedding of SSuT E. coli was significantly greater than that of susceptible strains in neonatal calves (P < 0.001), whereas there was no difference in older animals (P = 0.5). These data support the hypothesis that active selection for traits linked to the SSuT phenotype are responsible for maintaining drug-resistant E. coli in this population of dairy calves.

The use of antimicrobial drugs in livestock is suspected to contribute to bacterial antimicrobial resistance (AR) development. Dairy farms experiencing recent outbreaks of salmonellosis involving multi-resistant (MR) Salmonella strains were compared to control farms with respect to AR among bovine commensal E. coli isolates. For most antimicrobials tested, the percentage of AR E. coli isolated from salmonellosis-affected farms was significantly higher than that from control farms. Calf E. coli from both case and control farms had greater levels of AR than cow isolates. Commensal E. coli isolates from case farms and calves tended to more frequently be MR. These data are consistent with the existence of higher antimicrobial selection pressure on farms with recent salmonellosis outbreaks, however, the directionality of the relationship remains to be elucidated. An improved understanding of the epidemiology of AR bacteria in livestock production, both at the herd and molecular level, is essential to mitigate risk to public health and food safety.

The effect of vaccination with a commercially available R-mutant coliform mastitis vaccine on the survival of comingled dairy calves on a farm with endemic salmonellosis was examined. A total of 864 calves were randomly assigned to either vaccine (n = 435) or control (n = 429) groups. Passive transfer status of each calf was determined using refractometer determination of serum total protein concentration. Logistic models were developed to determine the effects of vaccine group and passive transfer status on calf survival to 100 days of age. In a model in which serum protein concentration was treated as a categorical variable, increasing serum total protein concentrations were associated with decreased mortality until these concentrations exceeded 6.0 g/dL. Calves with serum protein concentrations > 6.0 g/dL had increased risk for mortality compared with calves with serum protein concentrations > 5.5 g/dL but < or = 6.0 g/dL. This increased risk for mortality was supported by the results of a logistic model in which serum protein concentration was treated as a continuous variable. The increased risks associated with high serum protein concentration probably reflect the effect of dehydration in calves with occult disease. Neither model demonstrated any significant association between vaccination status and survival to 100 days of age. Based on these results, the routine immunization of calves cannot be recommended as a strategy to prevent mortality on farms with endemic salmonellosis.

Sixty-three of 209 (30.1%) samples of cattle feed that were collected from multiple commercial sources and from farms were found to contain Escherichia coli. However, none of the feed samples examined were culture-positive for E. coli O157. Replication of fecal E. coli, including E. coli O157, was demonstrated in a variety of feeds at temperatures that were similar to those found on farms in summer months. Fresh mixed rations containing corn silage were sampled from 16 dairies. Rations from 12 of these dairies were found to contain E. coli, and the rations from 5 dairies had concentrations of E. coli that were greater than 1000 cfu/g. The ability of experimental mixed rations to support the replication of E. coli was correlated with the concentration of organic acids in the corn silage that was used in the ration. Widespread contamination of cattle feeds with E. coli and the ability of E. coli to replicate in feeds suggest that feeds are a potentially important factor in the ecology of organisms that can be transmitted from feces to mouth, such as E. coli O157.

The sensitivities of several plating and broth enrichment methods for the detection of Escherichia coli O157:H7 in (i) bovine fecal samples directly inoculated with E. coli O157:H7, (ii) fecal samples from cattle in herds previously positive for E. coli O157:H7, and (iii) fecal samples from calves shedding E. coli O157:H7 after experimental oral inoculation were compared. Three enrichment protocols and three plating protocols were evaluated with directly inoculated fecal samples. All broth enrichment methods were superior to direct plating when they were combined with subsequent plating on sorbitol-MacConkey with cefixime and tellurite (SMACct). SMACct was the most sensitive plating medium, and the three alternative broth enrichment methods gave similar improvements in sensitivity. Of 351 fecal samples from known positive herds, 24 samples (6.8%) were positive by one or more methods. By the most sensitive plating method, cultures of 10-g samples were slightly more sensitive (19 of 351 [5.4%]) than cotton-tipped swab fecal samples (14 of 351 [4.0%]); however, this difference was not significant. For samples from calves orally inoculated with E. coli O157:H7, separation by immunomagnetic beads was slightly more sensitive (79%) than broth enrichment followed by plating at two dilutions (10(-3) and 10(-4)) (71%); however, this difference was not significant. The combination of overnight enrichment of swab fecal samples (0.1 g) and plating on SMACct at two dilutions (10(-3) and 10(-4)) appears to be a sensitive method for detection in large-scale studies involving hundreds of samples per week.

A case-control study was conducted to determine risk factors for fecal shedding of Escherichia coli O157:H7 (ECO) in dairy calves. Three herds previously found to lack calves that shed ECO in their feces were selected for each herd previously found to have calves that shed ECO. Fecal samples from 965 calves on 64 farms were tested for ECO by microbial culture. Sample prevalence of ECO in calves less than 8 weeks old was 1.4% and in calves 8 weeks or older was 4.8%. Calves were 3 times more likely to shed ECO after weaning than before weaning. Shedding of ECO was associated with grouping calves before weaning; feeding whole cottonseed was negatively associated with ECO shedding. The change in results between testing periods illustrated that an individual herd's status cannot be defined by a single testing of a small sample of cattle.

Foods of bovine origin have been linked to human disease outbreaks caused by Escherichia coli O157:H7 and may be linked to the more common sporadic cases as well. In this study, E. coli O157:H7 from the bovine reservoir (22 isolates: 12 from dairy and 10 from beef breed cows) and from human patients (50 isolates from sporadic human infections) were compared using Shiga-like toxin genotypes, plasmid profiles, and DNA restriction fragment length polymorphisms identified with a bacteriophage lambda probe (lambda-RFLP). Twenty-three lambda-RFLP profiles, 4 Shiga-like toxin genotypes, and 8 plasmid profiles were identified among the isolates tested. Together the typing methods distinguished 43 strains, of which 3 were isolated from both humans (5 isolates) and cattle (6 isolates; 5 from dairy herds). These data demonstrate the value of lambda-RFLP as a means of strain identification for E. coli O157:H7.