L. Jay Yanke

Selected Antimicrobial Resistance during Composting of Manure from Cattle Administered Sub-Therapeutic Antimicrobials

Composting is being increasingly employed for the recycling of nutrients in manure from the livestock industry. However, composting manure from animals fed antimicrobials has not been well characterized. In this study, compost windrows were prepared using manure collected from cattle (Bos Taurus L.) fed tylosin (TY), chlortetracycline-sulphamethazine (TS), and control cattle (no antimicrobials). The objectives of the 18-wk trial were to quantitatively assess the survival of total E. coli, E. coli resistant to ampicillin (Amp(r)) and tetracycline (Tet(r)), and select tetracycline (tet) and erythromycin resistance methylase (erm) genes. We found that while compost windrows did not reach the recommended temperature of 55 degrees C for 15 d, composting reduced high initial levels of total, Amp(r), and Tet(r) E. coli as early as Week 2. A significant antimicrobial effect on total (P = 0.04) and Amp(r) (P = 0.03) E. coli was observed. Significant antimicrobial x time interactions were observed from Week 0 to Week 3 (Total E. coli: P = 0.04; Amp(r): P = 0.02; Tet(r): P = <0.001). Low absolute abundance of tet and erm genes (<10(6) copies g(-1)) was found and the resistance genes displayed different dynamics; tet(A,C) and erm(A) increased marginally at Week 11 relative to Week 0 and 5 and the remaining genes (tet(G), RPP tet, erm(B), erm(C), erm(F), erm(T), and erm(X)) decreased for most time points and treatments. These results indicate that even though composting reduces antimicrobial resistant E. coli, tet and erm genes could still be detected. Our experiments reiterate advantages of polymerase chain reaction (PCR)-based quantitative assays over cultivation-based methods for the rapid identification of composting effectiveness in eliminating resistance genes before land application.

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis.

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.

Distribution and characterization of ampicillin- and tetracycline-resistant Escherichia coli from feedlot cattle fed subtherapeutic antimicrobials

BackgroundFeedlot 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.ResultsOverall, 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., MA,MT), 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 MT 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 MA (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).ConclusionsFactors other than, or in addition to subtherapeutic administration of antibiotics influence the establishment and transmission of AMR E. coli among feedlot cattle.

Antibiotics conspicuously affect community profiles and richness, but not the density of bacterial cells associated with mucosa in the large and small intestines of mice

The influence of three antibiotics (bacitracin, enrofloxacin, and neomycin sulfate) on the mucosa-associated enteric microbiota and the intestines of mice was examined. Antibiotics caused conspicuous enlargement of ceca and an increase in overall length of the intestine. However, there were no pathologic changes associated with increased cecal size or length of the intestine. Conspicuous reductions in the richness of mucosa-associated bacteria and changes to community profiles within the small (duodenum, proximal jejunum, middle jejunum, distal jejunum, and ileum) and large (cecum, ascending colon, and descending colon) intestine occurred in mice administered antibiotics. Communities in antibiotic-treated mice were dominated by a limited number of Clostridium-like (i.e. clostridial cluster XIVa) and Bacteroides species. The richness of mucosa-associated communities within the small and large intestine increased during the 14-day recovery period. However, community profiles within the large intestine did not return to baseline (i.e. relative to the control). Although antibiotic administration greatly reduced bacterial richness, densities of mucosa-associated bacteria were not reduced correspondingly. These data showed that the antibiotics, bacitracin, enrofloxacin, and neomycin sulfate, administered for 21 days to mice did not sterilize the intestine, but did impart a tremendous and prolonged impact on mucosa-associated bacterial communities throughout the small and large intestine.

High-throughput species identification of enterococci using pyrosequencing

Here we report the development and validation of an automated high-throughput pyrosequencing-based method for the reliable identification of isolated Enterococcus species. This method exploits the discrete species-specificity of hypervariable groES-EL spacer region and utilizes a universal dispensation order optimized for a wide range of Enterococcus species.

Impact of β2-1 fructan on faecal community change: results from a placebo-controlled, randomised, double-blinded, cross-over study in healthy adults

Healthy adults (n 30) participated in a placebo-controlled, randomised, double-blinded, cross-over study consisting of two 28 d treatments (β2-1 fructan or maltodextrin; 3×5 g/d) separated by a 14-d washout. Subjects provided 1 d faecal collections at days 0 and 28 of each treatment. The ability of faecal bacteria to metabolise β2-1 fructan was common; eighty-seven species (thirty genera, and four phyla) were isolated using anaerobic medium containing β2-1 fructan as the sole carbohydrate source. β2-1 fructan altered the faecal community as determined through analysis of terminal restriction fragment length polymorphisms and 16S rRNA genes. Supplementation with β2-1 fructan reduced faecal community richness, and two patterns of community change were observed. In most subjects, β2-1 fructan reduced the content of phylotypes aligning within the Bacteroides, whereas increasing those aligning within bifidobacteria, Faecalibacterium and the family Lachnospiraceae. In the remaining subjects, supplementation increased the abundance of Bacteroidetes and to a lesser extent bifidobacteria, accompanied by decreases within the Faecalibacterium and family Lachnospiraceae. β2-1 Fructan had no impact on the metagenome or glycoside hydrolase profiles in faeces from four subjects. Few relationships were found between the faecal bacterial community and various host parameters; Bacteroidetes content correlated with faecal propionate, subjects whose faecal community contained higher Bacteroidetes produced more caproic acid independent of treatment, and subjects having lower faecal Bacteroidetes exhibited increased concentrations of serum lipopolysaccharide and lipopolysaccharide binding protein independent of treatment. We found no evidence to support a defined health benefit for the use of β2-1 fructans in healthy subjects.