Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics

<p>Abstract</p> <p>Background</p> <p>Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (<it>n </it>= 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of <it>tet</it>, <it>sul </it>and <it>erm </it>resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified <it>16S-rRNA.</it></p> <p>Results</p> <p>The concentrations of <it>16S-rRNA </it>in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of <it>16S-rRNA </it>differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of <it>tet</it>(B), <it>tet</it>(C), <it>sul1, sul2</it>, <it>erm</it>(A) tended to increase, and decline thereafter, whereas <it>tet</it>(M) and <it>tet</it>(W) gradually declined over 175 days. At day 7, the concentration of <it>erm</it>(X) was greatest in feces from cattle fed tylosin, compared to all other treatments.</p> <p>Conclusion</p> <p>The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.</p

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

<p>Abstract</p> <p>Background</p> <p>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 <it>Escherichia coli</it>. 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 <it>E. coli </it>as an indicator bacterium. <it>E. coli </it>isolates (<it>n </it>= 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 <it>E. coli </it>from cattle.</p> <p>Results</p> <p>Overall, patterns of antimicrobial resistance expressed by <it>E. coli </it>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 <it>E. coli </it>were readily transferred among steers within pens. Most M^Tisolates possessed the <it>tet</it>(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 <it>tem1</it>-like determinant was predominant (occurring in 50, 66.7, 80.3, and 100% of isolates from CON, T, TS, and V steers, respectively).</p> <p>Conclusions</p> <p>Factors other than, or in addition to subtherapeutic administration of antibiotics influence the establishment and transmission of AMR <it>E. coli </it>among feedlot cattle.</p

The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor

Background: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries. Results: We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively. Conclusion: Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins

Synergism of Cattle and Bison Inoculum on Ruminal Fermentation and Select Bacterial Communities in an Artificial Rumen (Rusitec) Fed a Barley Straw Based Diet

This study evaluated the effect of increasing the proportion of bison relative to cattle inoculum on fermentation and microbial populations within an artificial rumen (Rusitec). The experiment was a completely randomized design with a factorial treatment structure (proportion cattle:bison inoculum; 0:100, 33:67, 67:33 and 100:0) replicated in two Rusitec apparatuses (n=8 fermenters). The experiment was 15 d with 8 d of adaptation and 7 d of sampling. Fermenters were fed a diet of 70:30 barley straw:concentrate (DM basis). True digestibility of DM was determined after 48 h of incubation from d 13-15, and daily ammonia (NH3) and volatile fatty acid (VFA) production were measured on d 9-12. Protozoa counts were determined at d 9, 11, 13 and 15 and particle-associated bacteria (PAB) from d 13-15. Select bacterial populations in the PAB were measured using RT-qPCR. Fermenter was considered the experimental unit and day of sampling as a repeated measure. Increasing the proportion of bison inoculum resulted in a quadratic effect (P0.05). Increasing bison inoculum linearly increased (P<0.05) concentrate aNDF disappearance, total and concentrate N disappearance as well as total daily VFA and acetate production. A positive quadratic response (P<0.05) was observed for daily NH3-N, propionate, butyrate, valerate, isovalerate and isobutyrate production, as well as the acetate:propionate ratio. Increasing the proportion of bison inoculum linearly increased (P<0.05) total protozoa numbers. No effects were observed on pH, total gas and methane production, microbial N synthesis, or copies of 16S rRNA associated with total bacteria, Selenomonas ruminantium or Prevotella bryantii. Increasing bison inoculum had a quadratic effect (P<0.05) on Fibrobacter succinogenes, and tended to linearly (P<0.10) increase Ruminococcus flavefaciens and decrease (P<0.05) Ruminococcus albus copy numbers. In conclusion, bison inoculum increased the degradation of feed protein and fibre. A mixture of cattle and bison rumen inoculum acted synergistically, increasing the DM and aNDF disappearance of barley straw

Effects of inoculation of corn silage with Lactobacillus hilgardii and Lactobacillus buchneri on silage quality, aerobic stability, nutrient digestibility, and growth performance of growing beef cattle

This study evaluated the effects of inoculation of whole crop corn silage with a mixture of heterofermentative lactic acid bacteria (LAB) composed of Lactobacillus hilgardii and Lactobacillus buchneri on ensiling, aerobic stability, ruminal fermentation, total tract nutrient digestibility, and growth performance of beef cattle. Uninoculated control corn silage (CON) and silage inoculated with 3.0 × 105 cfu g-1 of LAB containing 1.5 × 105 cfu g-1 of L. hilgardii CNCM I-4785 and 1.5 × 105 cfu g-1 of L. buchneri NCIMB 40788 (INOC) were ensiled in silo bags. The pH did not differ (P > 0.05) between the two silages during ensiling but was greater (P 0.05) between heifers fed the two silages, while there was a tendency (P ≤ 0.07) for lower CP and starch digestibility for heifers fed INOC than CON. Total nitrogen (N) intake and N retention were lower (P ≤ 0.04) for heifers fed INOC than CON. Dry matter intake as a percentage of BW was lower (P < 0.04) and there was a tendency for improved feed efficieny (G:F; P = 0.07) in steers fed INOC vs. CON silage. The NEm and NEg contents were greater for INOC than CON diets. Results indicate that inoculation with a mixture of L. hilgardii and L. buchneri improved the aerobic stability of corn silage. Improvements in G:F of growing steers fed INOC silage even though the total tract digestibility of CP and starch tended to be lower for heifers fed INOC are likely because the difference in BW and growth requirements of these animals impacted the growth performance and nutrient utilization and a greater proportion of NDICP in INOC than CON.Fil: Nair, Jayakrishnan. Lethbridge Research Centre; CanadáFil: Huaxin, Niu. Inner Mongolia University for Nationals; ChinaFil: Andrada, Lidia Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Yang, Hee-Eun. Lethbridge Research Centre; CanadáFil: Chevaux, Eric. Lallemand SAS; FranciaFil: Drouin, Pascal. Lallemand Specialties Inc; Estados UnidosFil: McAllister, Tim A.. Lethbridge Research Centre; CanadáFil: Wang, Yuxi. Lethbridge Research Centre; Canad

Using condensed tannin containing forages to establish sustainable and productive forage-based cattle production systems

Legumes play a key role in ruminant production systems, with alfalfa being the most common legume forage in temperate climates. Alfalfa can maintain high production levels in grazing livestock, but its propensity to cause bloating discourages its inclusion at high planting densities. Alfalfa produces high molecular weight polyphenols known as condensed tannins, in their seed coat, while other legumes such as the corniculated lotus (Lotus corniculatus L.), wild chickpea (Astragalus cicer L.), purple prairie clover (Dalea purpurea Vent) and esparceta (Onobrychis vicifolia Scop.) produce condensed tannins in varying degrees. Condensed tannins (CT) are responsible for their bloat-safe nature, improving the use of ruminal N, controlling intestinal parasites, reducing Escherichia coli in faeces and possibly enteric methane emissions. Tympanism of grasses is almost always of the foamy type and arises as a result of complex interactions between the animal, the forage and the rumen microbiome. The risk that lucerne causes bloating is greater when it is in the vegetative stage and becomes considerably smaller as it enters the flowering stage. The risk of bloating in cattle pasturing alfalfa can be reduced through various management practices. Cutting and withering the alfalfa before allowing cattle access to the paddock can drastically reduce the incidence of bloat. The sainfoin is comparable to alfalfa in nutritional quality and results in average daily gains in cattle that are similar to alfalfa. The CTs they contain are of high molecular weight and show a greater affinity for Rubisco than most other CT scans, which possibly gives them a superior ability to prevent tympanism. Among TC forages, foam is perhaps the most adaptable for use in a high-performance grazing system where it is grown in mixed stands with alfalfa

Effect of preemptive flunixin meglumine and lidocaine on behavioral and physiological indicators of pain post-band and knife castration in 6-mo-old beef calves

One hundred and seventy-four Angus bull calves (248 ± 27.1 kg of body weight (BW), 6-mo-old) were used in a 71 d study to assess the efficacy of the combination of flunixin meglumine and lidocaine in mitigating pain associated with band and knife castration. The experiment consisted of a 3 × 2 factorial design that included castration method -sham (C), band (B) or knife (K); and medication – lidocaine (scrotal ring block 30 mL, 2% HCl lidocaine) and flunixin meglumine (single s.c. dose of 2.2 mg/kg BW) (M), or saline solution (NM). Animals were weighed on d 0 and weekly until d 71 (final BW) post-castration to obtain ADG. Physiological indicators included salivary cortisol collected on d 0 (30, 60, 120 and 240 min), d 2, 8, and weekly until d 48 post-castration; scrotal and eye temperature assessed on d 1, 2, 6, 8, and weekly until d 36 post-castration; fecal samples for E. coli collected on d 0, 2, 6, 8, and 22 post-castration. Behavioral measures included stride length on d 0, 8, and weekly until d 36, visual analog scale (VAS) evaluated during castration, and feeding behavior collected daily from d 0 to d 71 post-castration. Final BW and ADG were greater (P  0.10) were observed for stride length. The VAS scores were greater (P = 0.01) in K than C and B calves, while NM had greater scores (P < 0.01) than M calves. Dry matter intake and meal size were greater (P = 0.05) in M than NM calves. Meal duration was greater (P = 0.01) in B and C than K calves on d 0, while K calves had greater (P < 0.01) meal duration than C calves 1 and 2-wk post-castration. Overall, the combination of flunixin meglumine and lidocaine reduced physiological and behavioral indicators of pain, suggesting that their combined use was effective at mitigating pain associated with band and knife castration.info:eu-repo/semantics/acceptedVersio

Comparative analysis of macroalgae supplementation on the rumen microbial community: Asparagopsis taxiformis inhibits major ruminal methanogenic, fibrolytic, and volatile fatty acid-producing microbes in vitro

Seaweeds have received a great deal of attention recently for their potential as methane-suppressing feed additives in ruminants. To date, Asparagopsis taxiformis has proven a potent enteric methane inhibitor, but it is a priority to identify local seaweed varieties that hold similar properties. It is essential that any methane inhibitor does not compromise the function of the rumen microbiome. In this study, we conducted an in vitro experiment using the RUSITEC system to evaluate the impact of three red seaweeds, A. taxiformis, Palmaria mollis, and Mazzaella japonica, on rumen prokaryotic communities. 16S rRNA sequencing showed that A. taxiformis had a profound effect on the microbiome, particularly on methanogens. Weighted Unifrac distances showed significant separation of A. taxiformis samples from the control and other seaweeds (p &lt; 0.05). Neither P. mollis nor M. japonica had a substantial effect on the microbiome (p &gt; 0.05). A. taxiformis reduced the abundance of all major archaeal species (p &lt; 0.05), leading to an almost total disappearance of the methanogens. Prominent fiber-degrading and volatile fatty acid (VFA)-producing bacteria including Fibrobacter and Ruminococcus were also inhibited by A. taxiformis (p &lt; 0.05), as were other genera involved in propionate production. The relative abundance of several other bacteria including Prevotella, Bifidobacterium, Succinivibrio, Ruminobacter, and unclassified Lachnospiraceae were increased by A. taxiformis suggesting that the rumen microbiome adapted to an initial perturbation. Our study provides baseline knowledge of microbial dynamics in response to seaweed feeding over an extended period and suggests that feeding A. taxiformis to cattle to reduce methane may directly, or indirectly, inhibit important fiber-degrading and VFA-producing bacteria