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 g/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 g/ml), sulfadiazine (>512 g/ml), and tetracycline (>4 g/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.

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 (Ϸ37% 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.Antimicrobial drug-resistant bacteria present a significant risk to public health, and consequently there is great interest in reducing the prevalence of antimicrobial resistance (AR) genes in both commensal and pathogenic bacteria. One strategy to decrease the prevalence of AR bacteria is to discontinue using antimicrobial drugs for growth promotion and prophylaxis in food animals (4,11,22,43,44). Unfortunately, this strategy has produced mixed results that are dependent on the duration and amount of antimicrobial drug use (10,16,24,25,37,38,41). While pathogenic bacteria are the primary concern, commensal bacteria are an important reservoir for AR genes. It is hypothesized that, because AR genes can easily transfer among diverse bacterial species (6,29,32,35,39,45), then they can also move easily from commensal to pathogenic bacteria. Consequently abundance and ease of acquisition of the antimicrobial drug resistance genes ultimately complicate the orthodox treatment of diverse bacterial diseases (13,18).It is generally accepted that the prevalence of AR bacteria is directly correlated with antimicrobial drug use (19,20,40), but mechanisms unrelated to specific antimicrobial drug selection have also been implicated in the wide distribution and maintenance of AR genes. Some of these mechanisms include plasmid addiction (14,17) and close linkage...

Association of specific antimicrobial resistance patterns with unrelated selective traits has long been implicated in the maintenance of antimicrobial resistance in a population. Previously we demonstrated that Escherichia coli strains with a specific resistance pattern (resistant to streptomycin, sulfadiazine, and tetracycline [SSuT]) have a selective advantage in dairy calf intestinal environments and in the presence of a milk supplement commonly fed to the calves. In the present study we identified the sequence of the genetic element that confers the SSuT phenotype and show that this element is present in a genetically diverse group of E. coli isolates, as assessed by macrorestriction digestion and pulsed-field gel electrophoresis. This element was also found in E. coli isolates from 18 different cattle farms in Washington State. Using in vitro competition experiments we further demonstrated that SSuT strains from 17 of 18 farms were able to outcompete pansusceptible strains. In a separate set of experiments, we were able to transfer the antimicrobial resistance phenotype by electroporation to a laboratory strain of E. coli (DH10B), making that new strain more competitive during in vitro competition with the parental DH10B strain. These data indicate that a relatively large genetic element conferring the SSuT phenotype is widely distributed in E. coli from cattle in Washington State. Furthermore, our results indicate that this element is responsible for maintenance of these traits owing to linkage to genetic traits that confer a selective advantage in the intestinal lumens of dairy calves.

A 1-year-old, castrated male, mixed-breed dog was presented for sporadic episodes of kyphosis, tremors, and vocalizing. On neurologic examination, the lesion was localized to spinal cord segments T3-L3. Magnetic resonance imaging of the spine showed an expansile mass occupying most of the ventral aspect of the spinous process of T6. Fine-needle aspirates of the mass were examined cytologically. A moderately cellular population of pleomorphic spindle cells and abundant mucinous matrix were observed. The cytologic diagnosis was spindle cell neoplasia, with myxosarcoma and fibrosarcoma as the primary differential diagnoses. The dog was euthanized. Histopathologic evaluation of the mass and surrounding tissue confirmed a low-grade spindle cell sarcoma, with severe compressive myelopathy and mild neutrophilic inflammation. The neoplastic cells stained positive for mucopolysaccharides with Alcian blue, resulting in a final diagnosis of low-grade (grade 1) myxosarcoma. Fine-needle aspiration was useful in making a preliminary diagnosis of myxosarcoma in this case. Myxosarcoma should be included in the differential diagnosis for a vertebral mass in a young dog.