The present study was initiated to evaluate the effect of sampling time and within-sample variability on the diversity in antimicrobial resistance patterns in fecal Escherichia coli from healthy pigs. Isolates were tested against 11 antimicrobials. A total of 25 different profiles were observed, involving resistance to ampicillin, streptomycin, tetracycline, sulfonamides, trimethoprim, and/or a trimethoprim/sulfonamide combination. No isolates were resistant to enrofloxacin, gentamicin, or chloramfenicol, whereas resistance against neomycin and nalidixic acid was sporadically detected in isolates from grower pigs. A model that clusters pigs within-sampling time as a repeated factor and clusters isolates within individual pigs as a random factor was used. For sows, the variance component ratio of sampling time to residuals was 0.28-0.56 for the different antimicrobials (except ampicillin) and 0.85-1.79 for grower pigs. The variance components for within-sample variation were zero or close to zero, except in isolates from sows where resistance to ampicillin explained 14.8 times more of the variation compared to residuals. Thus, the effect of an animal's status at a given sampling time was more influential on the variability in antimicrobial resistance than within-animal diversity. We conclude that repeated sampling and analysis of one isolate per animal each time may be preferable for screening general tendencies, whereas several isolates have to be tested when individual animals are focused.
"Our result supports the “single sample per farm” strategy that is currently applied in many countries , ,  and recommended by the EFSA. As demonstrated by previous studies targeting farm-level monitoring, the best strategies of resistance prevalence monitoring involve taking 1 sample per animal , . As an evaluation of national monitoring, our results agree with those of Regula et al. , who also demonstrated the advantage of a “single sample per farm” strategy using a bootstrap sampling procedure. "
[Show abstract][Hide abstract] ABSTRACT: Because antimicrobial resistance in food-producing animals is a major public health concern, many countries have implemented antimicrobial monitoring systems at a national level. When designing a sampling scheme for antimicrobial resistance monitoring, it is necessary to consider both cost effectiveness and statistical plausibility. In this study, we examined how sampling scheme precision and sensitivity can vary with the number of animals sampled from each farm, while keeping the overall sample size constant to avoid additional sampling costs. Five sampling strategies were investigated. These employed 1, 2, 3, 4 or 6 animal samples per farm, with a total of 12 animals sampled in each strategy. A total of 1,500 Escherichia coli isolates from 300 fattening pigs on 30 farms were tested for resistance against 12 antimicrobials. The performance of each sampling strategy was evaluated by bootstrap resampling from the observational data. In the bootstrapping procedure, farms, animals, and isolates were selected randomly with replacement, and a total of 10,000 replications were conducted. For each antimicrobial, we observed that the standard deviation and 2.5-97.5 percentile interval of resistance prevalence were smallest in the sampling strategy that employed 1 animal per farm. The proportion of bootstrap samples that included at least 1 isolate with resistance was also evaluated as an indicator of the sensitivity of the sampling strategy to previously unidentified antimicrobial resistance. The proportion was greatest with 1 sample per farm and decreased with larger samples per farm. We concluded that when the total number of samples is pre-specified, the most precise and sensitive sampling strategy involves collecting 1 sample per farm.
PLoS ONE 01/2014; 9(1):e87147. DOI:10.1371/journal.pone.0087147 · 3.23 Impact Factor
"Due to the volume of isolates collected and the laborious nature of PFGE, only one isolate was analysed per sample type which may have under-estimated the genetic diversity of E. coli O157:H7 compared to analysis of multiple isolates
, although analysis of a single isolate is generally indicative of the dominant strain
[21,22]. Consequently, the lack of congruity between simultaneously-collected fecal and PS isolates from individual super-shedders was surprising, as only 2 super-shedder steers had PS and fecal isolates in the same REPC (Table
[Show abstract][Hide abstract] ABSTRACT: Background
Cattle shedding at least 104 CFU Escherichia coli O157:H7/g feces are described as super-shedders and have been shown to increase transmission of E. coli O157:H7 to other cattle in feedlots. This study investigated relationships among fecal isolates from super-shedders (n = 162), perineal hide swab isolates (PS) from super-shedders (n = 137) and fecal isolates from low-shedder (< 104 CFU/g feces) pen-mates (n = 496) using pulsed-field gel electrophoresis (PFGE). A subsample of these fecal isolates (n = 474) was tested for antimicrobial resistance. Isolates of E. coli O157:H7 were obtained from cattle in pens (avg. 181 head) at 2 commercial feedlots in southern Alberta with each steer sampled at entry to the feedlot and prior to slaughter.
Only 1 steer maintained super-shedder status at both samplings, although approximately 30% of super-shedders in sampling 1 had low-shedder status at sampling 2. A total of 85 restriction endonuclease digestion clusters (REPC; 90% or greater similarity) and 86 unique isolates (< 90% similarity) were detected, with the predominant REPC (30% of isolates) being isolated from cattle in all feedlot pens, although it was not associated with shedding status (super- or low-shedder; P = 0.94). Only 2/21 super-shedders had fecal isolates in the same REPC at both samplings. Fecal and PS isolates from individual super-shedders generally belonged to different REPCs, although fecal isolates of E. coli O157:H7 from super- and low-shedders showed greater similarity (P < 0.001) than those from PS. For 77% of super-shedders, PFGE profiles of super-shedder fecal and PS isolates were distinct from all low-shedder fecal isolates collected in the same pen. A low level of antimicrobial resistance (3.7%) was detected and prevalence of antimicrobial resistance did not differ among super- and low-shedder isolates (P = 0.69), although all super-shedder isolates with antimicrobial resistance (n = 3) were resistant to multiple antimicrobials.
Super-shedders did not have increased antimicrobial resistance compared to low-shedder pen mates. Our data demonstrated that PFGE profiles of individual super-shedders varied over time and that only 1/162 steers remained a super-shedder at 2 samplings. In these two commercial feedlots, PFGE subtypes of E. coli O157:H7 from fecal isolates of super- and low-shedders were frequently different as were subtypes of fecal and perineal hide isolates from super-shedders.
BMC Veterinary Research 09/2012; 8(1):178. DOI:10.1186/1746-6148-8-178 · 1.78 Impact Factor
"Ensuring representative sampling is always a challenge considering the voluminous nature of digesta within the bovine intestinal tract and the number of cattle that are typically housed within a feedlot. Others have reported that examining single vs multiple isolates did not compromise interpretation of the temporal trends or the nature of diversity of E. coli within cohorts [50,51]. In early samples, where we did select two isolates, PFGE frequently identified both isolates as clones. "
[Show abstract][Hide abstract] ABSTRACT: Feedlot cattle in North America are routinely fed subtherapeutic levels of antimicrobials to prevent disease and improve the efficiency of growth. This practice has been shown to promote antimicrobial resistance (AMR) in subpopulations of intestinal microflora including Escherichia coli. To date, studies of AMR in feedlot production settings have rarely employed selective isolation, therefore yielding too few AMR isolates to enable characterization of the emergence and nature of AMR in E. coli as an indicator bacterium. E. coli isolates (n = 531) were recovered from 140 cattle that were housed (10 animals/pen) in 14 pens and received no dietary antimicrobials (control--5 pens, CON), or were intermittently administered subtherapeutic levels of chlortetracycline (5 pens-T), chlortetracycline + sulfamethazine (4 pens-TS), or virginiamycin (5 pens-V) for two separate periods over a 9-month feeding period. Phenotype and genotype of the isolates were determined by susceptibility testing and pulsed field gel electrophoresis and distribution of characterized isolates among housed cattle reported. It was hypothesized that the feeding of subtherapeutic antibiotics would increase the isolation of distinct genotypes of AMR E. coli from cattle.
Overall, patterns of antimicrobial resistance expressed by E. coli isolates did not change among diet groups (CON vs. antibiotic treatments), however; isolates obtained on selective plates (i.e., M(A),M(T)), exhibited multi-resistance to sulfamethoxazole and chloramphenicol more frequently when obtained from TS-fed steers than from other treatments. Antibiograms and PFGE patterns suggested that AMR E. coli were readily transferred among steers within pens. Most M(T) isolates possessed the tet(B) efflux gene (58.2, 53.5, 40.8, and 50.6% of isolates from CON, T, TS, and V steers, respectively) whereas among the M(A) (ampicillin-resistant) isolates, the tem1-like determinant was predominant (occurring in 50, 66.7, 80.3, and 100% of isolates from CON, T, TS, and V steers, respectively).
Factors other than, or in addition to subtherapeutic administration of antibiotics influence the establishment and transmission of AMR E. coli among feedlot cattle.
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