Macrolide-susceptible probiotic Enterococcus faecium ST296 exhibits faecal-environmental-oral microbial community cycling among beef cattle in feedlots.

Enterococci are included in the United States National Antimicrobial Resistance Monitoring System to track antibiotic resistance among commensal Gram-positive enteric bacteria, largely due to their high abundance in food animals and in retail meat. In the U.S. cattle industry, macrolides are used to prevent and control liver abscesses, which cause significant economic losses. Previous studies have suggested that feeding tylosin and the intensity of the pen environment, both expand and sustain respectively the prevalence of multidrug resistance among enterococci in feedlot cattle. This has led to research into alternative feed supplements and improved stewardship practices. In a randomized controlled trial, we measured the impact of a probiotic and an altered pen environment on antimicrobial resistance among faecal Enterococcus spp. in cattle fed tylosin. Supplementing cattle with an Enterococcus faecium and Saccharomyces cerevisiae-based probiotic yielded the isolation of E. faecium of the probiotic sequence type (ST296) from faecal and environmental samples in treatment groups, as well as from cattle and the manure pack in nearby pens. Of importance, the probiotic strain also was found in a desiccated and milled manure pack sample taken 120 days after the initial trial ended. Phylogenetic and SNP analyses revealed clonal survival and spread compatible with faecal-environmental-oral recycling of the probiotic strain within and among cattle and pens. The increase in prevalence of the ST296 strain occurred concomitant with a decrease in ST240, the dominant sequence type associated with ermB and tet(M) resistance genes in this trial. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrate that a macrolide-susceptible probiotic Enterococcus faecium ST296 strain fed to beef cattle becomes fully embedded in the microbial community cycling of bacteria via faecal-environmental-oral transmission within and among feedlot pens. An initial investment in feeding the probiotic is thereby leveraged into expanding numbers of susceptible bacteria in cattle and their environment, even among those cattle fed tylosin.

Interactive effects of supplemental Zn sulfate and ractopamine hydrochloride on growth performance, carcass traits, and plasma urea nitrogen in feedlot heifers.

Interactive effects of supplemental Zn and ractopamine hydrochloride (RH) were evaluated using 156 crossbred heifers (initial BW = 527 kg ± 6.61; gross BW × 0.96) to determine the impact on feedlot performance, plasma urea nitrogen (PUN), and carcass characteristics. The study was conducted as a randomized complete block design with a 2 × 2 factorial arrangement of treatments. Factors consisted of 1) 30 or 100 mg supplemental Zn/kg diet DM (30Zn or 100Zn) as Zn sulfate and 2) 0 or 200 mg RH/heifer daily. Heifers were blocked by BW and assigned randomly within block to treatments for a 43-d trial. Heifers were housed in partially covered feeding pens (3 heifers/pen; 13 pens/treatment) and provided ad libitum access to feed. Ractopamine hydrochloride was fed for 42 d and removed from the diet until cattle were harvested on d 43. Zinc treatments were fed until harvest. Plasma samples were collected on d 0 and 36 to assess changes in plasma Zn and PUN. On d 43, heifers were weighed, then transported to a commercial abattoir where HCW and incidence of liver abscesses were recorded. Carcass data were collected after 32 h of refrigeration. No Zn × RH interactions were observed for plasma Zn or PUN ( ≥ 0.58); however, there was a tendency for a RH × day interaction for PUN ( = 0.08). Supplementing 100Zn resulted in increased plasma Zn ( = 0.02) compared to 30Zn. No RH × Zn interactions were observed for feedlot performance ( ≥ 0.24). Final BW and ADG increased with RH supplementation ( ≤ 0.02), but DMI was not affected ( = 0.63); thus, feed efficiency improved ( < 0.01) when heifers were fed RH. Supplementing 100Zn tended to reduce ADG ( = 0.07) but did not affect other measures of feedlot performance ( ≥ 0.12). Zinc × RH interactions were observed for LM area and yield grade ( ≤ 0.01); LM area decreased and yield grade increased when heifers were supplemented 100Zn with no RH compared to other treatments. A tendency for a Zn × RH interaction was observed for dressed yield ( = 0.08), but no other interactions or effects of Zn were detected for carcass traits ( ≥ 0.11). Supplementing RH increased HCW ( = 0.03) but did not affect other carcass traits ( ≥ 0.13). In conclusion, supplemental Zn had little impact on feedlot performance or PUN concentration but may alter muscle and fat deposition when fed in conjunction with RH.

Interaction between supplemental zinc oxide and zilpaterol hydrochloride on growth performance, carcass traits, and blood metabolites in feedlot steers.

Interactive effects of supplemental Zn and zilpaterol hydrochloride (ZH) were evaluated in feedlot steers ( = 40; 652 kg ± 14 initial BW) to determine their impact on feedlot performance, blood constituents, and carcass traits. The study was conducted as a randomized complete block design with a 2 × 2 factorial treatment arrangement. Steers were blocked by BW and randomly assigned to treatments. Factors consisted of supplemental Zn (60 or 300 mg/kg diet DM) and ZH (0 or 8.33 mg/kg) in the diets. For diets supplemented with 300 mg Zn/kg DM, 60 mg Zn/kg was supplemented as zinc sulfate and 240 mg Zn/kg was supplemented as zinc oxide, and the diet was fed for 24 d. Zilpaterol hydrochloride was fed for 21 d followed by a 3-d withdrawal. Cattle were housed in partially covered individual feeding pens equipped with automatic waterers and fence-line feed bunks and were fed once daily for ad libitum intake. Plasma samples were collected on d 0 and 21 to assess changes in Zn, plasma urea nitrogen (PUN), glucose, and lactate concentrations, and serum samples were collected on d 21 to assess IGF-1 concentration. On d 25, cattle were weighed and transported 450 km to a commercial abattoir for harvest; HCW and incidence of liver abscesses were recorded. Carcass data were collected after 36 h of refrigeration. Data were analyzed as a mixed model with Zn, ZH, and Zn × ZH as fixed effects; block as a random effect; and steer as the experimental unit. No interaction or effects of Zn or ZH were observed for IGF-1 concentration, plasma glucose, or lactate concentrations ( ≥ 0.25). No interaction between Zn and ZH was observed for PUN concentration, but PUN decreased with ZH ( < 0.01). There were no effects of ZH or Zn on ADG, DMI, final BW, feed efficiency, HCW, back fat, KPH, quality grade, or incidence of liver abscesses ( > 0.05). Zinc supplementation tended ( = 0.08) to improve the proportion of carcasses grading USDA Choice. Feeding ZH decreased yield grade ( = 0.05) and tended to increase LM area ( = 0.07). In conclusion, increasing dietary concentrations of Zn does not impact response to ZH, but feeding ZH altered circulating concentrations of PUN.

Effects of Menthol Supplementation in Feedlot Cattle Diets on the Fecal Prevalence of Antimicrobial-Resistant Escherichia coli.

The pool of antimicrobial resistance determinants in the environment and in the gut flora of cattle is a serious public health concern. In addition to being a source of human exposure, these bacteria can transfer antibiotic resistance determinants to pathogenic bacteria and endanger the future of antimicrobial therapy. The occurrence of antimicrobial resistance genes on mobile genetic elements, such as plasmids, facilitates spread of resistance. Recent work has shown in vitro anti-plasmid activity of menthol, a plant-based compound with the potential to be used as a feed additive to beneficially alter ruminal fermentation. The present study aimed to determine if menthol supplementation in diets of feedlot cattle decreases the prevalence of multidrug-resistant bacteria in feces. Menthol was included in diets of steers at 0.3% of diet dry matter. Fecal samples were collected weekly for 4 weeks and analyzed for total coliforms counts, antimicrobial susceptibilities, and the prevalence of tet genes in E. coli isolates. Results revealed no effect of menthol supplementation on total coliforms counts or prevalence of E. coli resistant to amoxicillin, ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, and sulfamethoxazole; however, 30 days of menthol addition to steer diets increased the prevalence of tetracycline-resistant E. coli (P < 0.02). Although the mechanism by which menthol exerts its effects remains unclear, results of our study suggest that menthol may have an impact on antimicrobial resistance in gut bacteria.

Effects of dietary copper, zinc, and ractopamine hydrochloride on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria.

A total of 480 pigs (PIC 327 × 1050; initially 48.7 ± 2.3 kg) were used to determine the interactive effects of supplemental Cu, Zn, and ractopamine HCl (RAC) on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria. Treatments were arranged in a 2 × 2 × 2 factorial with the main effects of added Cu (CuSO; 0 vs. 125 mg/kg Cu), Zn (ZnO; 0 vs. 150 mg/kg Zn), and RAC (0 vs. 10 mg/kg during the last 28 d prior to marketing). All diets contained 11 mg/kg Cu and 73 mg/kg Zn from the trace mineral premix. Pens of pigs were balanced and blocked on initial BW and then randomly allotted to 1 of the 4 mineral treatment diets. At 28 d prior to marketing, pens within each block and mineral treatment were randomly assigned to receive either 0 or 10 mg/kg RAC in addition to the mineral treatment. Adding either Cu or Zn alone did not improve ADG or ADFI yet resulted in numerical improvements in overall G:F and caloric efficiencies, but improvements were not additive (Cu × Zn, = 0.057, = 0.068, and = 0.064 for G:F and caloric efficiency on a ME and NE basis, respectively). Ractopamine improved ( < 0.001) overall ADG, G:F, and caloric efficiency, thereby increasing final BW by 3% with no change in ADFI. Ractopamine also increased ( < 0.001) HCW, percentage carcass yield, G:F, loin depth, and percent fat-free lean and decreased ( = 0.014) backfat. Adding Zn or Cu alone to diets containing RAC numerically improved percent yield and HCW G:F, but this effect was absent when the Cu or Zn was added to the control diet or when Cu and Zn were fed in combination in RAC diets (Cu × Zn × RAC, = 0.011 and = 0.018 for yield and HCW G:F, respectively). Fecal samples were collected on d 0 and at the conclusion of the finishing period (d 90) for bacterial isolation and antimicrobial susceptibility determinations according to Clinical and Laboratory Standards Institute minimal inhibitory concentrations breakpoints. spp. and isolates displayed varying levels of resistance to certain antibiotics prior to initiation of treatments on d 0. Resistance to most antibiotics decreased ( < 0.05) over time or was stable for those that had a low baseline percentage of resistance. Neither Zn nor RAC adversely affected antimicrobial resistance. However, extended feeding of 125 mg/kg Cu throughout the finishing period seems to decrease enterococcal susceptability to tetracycline, tylosin, and quinupristin/dalfopristin.

Effects of in-feed copper and tylosin supplementations on copper and antimicrobial resistance in faecal enterococci of feedlot cattle.

AIMS: The objective was to investigate whether in-feed supplementation of copper, at elevated level, co-selects for macrolide resistance in faecal enterococci. METHODS AND RESULTS: The study was conducted in cattle (n = 80) with a 2 × 2 factorial design of copper (10 or 100 mg kg(-1) of feed) and tylosin (0 or 10 mg kg(-1) of feed). Thirty-seven isolates (4·6%; 37/800) of faecal enterococci were positive for the tcrB and all were Enterococcus faecium. The prevalence was higher among cattle fed diets with copper and tylosin (8·5%) compared to control (2·0%), copper (4·5%) and tylosin (3·5%) alone. All tcrB-positive isolates were positive for erm(B) and tet(M) genes. Median copper minimum inhibitory concentrations (MICs) for tcrB-positive and tcrB-negative enterococci were 20 and 4 mmol l(-1) , respectively. CONCLUSIONS: Feeding of elevated dietary copper and tylosin alone or in combination resulted in an increased prevalence of tcrB and erm(B)-mediated copper and tylosin-resistant faecal enterococci in feedlot cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: In-feed supplementation of elevated dietary copper has the potential to co-select for macrolide resistance. Further studies are warranted to investigate the factors involved in maintenance and dissemination of the resistance determinants and their co-selection mechanism in relation to feed-grade antimicrobials' usage in feedlot cattle.

Effects of chlortetracycline and copper supplementation on antimicrobial resistance of fecal Escherichia coli from weaned pigs.

Feed-grade chlortetracycline (CTC) and copper are both widely utilized in U.S. pig production. Cluster randomized experiment was conducted to evaluate the effects of CTC and copper supplementation in weaned pigs on antimicrobial resistance (AMR) among fecal Escherichia coli. Four treatment groups: control, copper, CTC, or copper plus CTC were randomly allocated to 32 pens with five pigs per pen. Fecal samples were collected weekly from three pigs per pen for six weeks. Two E. coli isolates per fecal sample were tested for phenotypic and genotypic resistance against antibiotics and copper. Data were analyzed with multilevel mixed effects logistic regression, multivariate probit analysis and discrete time survival analysis. CTC-supplementation was significantly (99% [95% CI=98-100%]) associated with increased tetracycline resistance compared to the control group (95% [95% CI=94-97%]). Copper supplementation was associated with decreased resistance to most of the antibiotics tested, including cephalosporins, over the treatment period. Overall, 91% of the E. coli isolates were multidrug resistant (MDR) (resistant to ≥3 antimicrobial classes). tetA and blaCMY-2 genes were positively associated (P<0.05) with MDR categorization, while tetB and pcoD were negatively associated with MDR. tetA and blaCMY-2 were positively associated with each other and in turn, these were negatively associated with both tetB and pcoD genes; which were also positively associated with one another. Copper minimum inhibitory concentration was not affected by copper supplementation or by pcoD gene carriage. CTC supplementation was significantly associated with increased susceptibilities of E. coli to copper (HR=7 [95% CI=2.5-19.5]) during treatment period. In conclusion, E. coli isolates from the nursery pigs exhibited high levels of antibiotic resistance, with diverse multi-resistant phenotypic profiles. The roles of copper supplementation in pig production, and pco-mediated copper resistance among E. coli in particular, need to be further explored since a strong negative association of pco with both tetA and blaCMY-2 points to opportunities for selecting a more innocuous resistance profile.

Occurrence of the transferable copper resistance gene tcrB among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets.

Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2×2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut.

Selection of fecal enterococci exhibiting tcrB-mediated copper resistance in pigs fed diets supplemented with copper.

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ∼194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance.

Cloning and characterization of a novel insulin-regulated membrane aminopeptidase from Glut4 vesicles.

The insulin-regulated glucose transporter isotype GlutT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle.

Starvation and nutrient resuscitation of Klebsiella pneumoniae isolated from oil well waters.

Klebsiella pneumoniae isolated from oil well waters reduced in size in response to nutrient starvation. The cells remained viable during starvation and later were able to grow rapidly when stimulated by nutrients. The heterotrophic potential, culture absorbance and extracellular polysaccharide production decreased during cell starvation whereas an initial increase in colony-forming units was observed on agar plates. Transmission electron microscopy (TEM) after 24 d revealed that the cells had changed to small rods or cocci between 0.5 by 0.25 micron and 0.87 by 0.55 micron. When transferred to half-strength brain heart infusion medium, TEM showed cell division and rod-shaped cells after 45 min and full resuscitation within 4 h. Cell response was much slower in sodium citrate medium and resuscitation took 8 h.