Shiga Toxin-Producing Escherichia coli in Feces of Finisher Pigs: Isolation, Identification, and Public Health Implications of Major and Minor Serogroups†.

ABSTRACT: Shiga toxin-producing Escherichia coli (STEC) are major foodborne human pathogens that cause mild to hemorrhagic colitis, which could lead to complications of hemolytic uremic syndrome. Seven serogroups, O26, O45, O103, O111, O121, O145, and O157, account for the majority of the STEC illnesses in the United States. Shiga toxins 1 and 2, encoded by stx1 and stx2, respectively, and intimin, encoded by eae gene, are major virulence factors. Cattle are a major reservoir of STEC, but swine also harbor them in the hindgut and shed STEC in the feces. Our objectives were to use a culture method to isolate and identify major and minor serogroups of STEC in finisher pig feces. Shiga toxin genes were subtyped to assess public health implications of STEC. Fecal samples (n = 598) from finisher pigs, collected from 10 pig flows, were enriched in E. coli broth and tested for stx1, stx2, and eae by a multiplex PCR (mPCR) assay. Samples positive for stx1 or stx2 gene were subjected to culture methods, with or without immunomagnetic separation and plating on selective or nonselective media, for isolation and identification of stx-positive isolates. The culture method yielded a total of 178 isolates belonging to 23 serogroups. The three predominant serogroups were O8, O86, and O121. The 178 STEC strains included 26 strains with stx1a and 152 strains with stx2e subtypes. Strains with stx1a, particularly in association with eae (O26 and O103), have the potential to cause severe human infections. All stx2-positive isolates carried the subtype stx2e, a subtype that causes edema disease in swine, but is rarely involved in human infections. Several strains were also positive for genes that encode for enterotoxins, which are involved in neonatal and postweaning diarrhea in swine. In conclusion, our study showed that healthy finisher pigs harbored and shed several serogroups of E. coli carrying virulence genes involved in neonatal diarrhea, postweaning diarrhea, and edema disease, but prevalence of STEC of public health importance was low.

Genome Sequence of Classical Swine Fever Virus NIVEDI-165, Subtype 1.1, a Field Virus Strain Isolated from the Southern Part of India.

The whole-genome sequence of an Indian field isolate of classical swine fever virus, NIVEDI-165, was found to be subtype 1.1, and it showed 89 to 99% amino acid identity and 84 to 99% nucleotide identity with four and five Indian classical swine fever virus (CSFV) isolates, respectively. To the best of our knowledge, this is the first report on a full-genome sequence of CSFV from South India.

Effects of dietary supplementation of formaldehyde and crystalline amino acids on gut microbial composition of nursery pigs.

Formaldehyde-based feed additives are approved in the US for Salmonella control and reducing bacterial contamination in animal feed. However, we hypothesize formaldehyde inclusion in swine diets may influence gut microbial composition due to its antimicrobial properties which might negatively influence microbial populations and pig growth performance. Also, formaldehyde inclusion in diets is known to reduce the dietary availability of amino acids. Therefore, our study was conducted to characterize if the effects of feed formaldehyde-treatment are due to influences on microbial population or diet amino acid (AA) sources. Dietary treatments were arranged in a (2 × 2) + 1 factorial with formaldehyde treatment (none vs. 1000 ppm formaldehyde) and crystalline AA inclusion (low vs. high) with deficient AA content plus a positive control diet to contain adequate AA content without dietary formaldehyde. Treating diets with formaldehyde reduced growth rate (P = 0.001) while the AA inclusion had no evidence of impact. Formaldehyde reduced feed bacterial content and altered fecal microbial communities (P < 0.05). Therefore, we conclude that the negative influence on growth was due to the impact on the fecal microbial community. Implications are that strategies for feed pathogen control need to take into account potential negative impacts on the gut microbial community.

Effects of dietary chlortetracycline, Origanum essential oil, and pharmacological Cu and Zn on growth performance of nursery pigs.

Two 47-d experiments were conducted with 21-d-old weaned pigs (PIC 1050, initially 6.1 kg) to determine the effects of feeding low or high doses of chlortetracycline (CTC) and antibiotic alternatives (Cu, Zn, and essential oil [EO]), alone or in combination, on growth performance. On d 5 postweaning, pens of 5 pigs were allotted to diet treatments with 8 (exp. 1) or 7 (exp. 2) replicate pens per treatment. In exp. 1, treatments were fed from d 5 to 26 postweaning and arranged in a 2 × 3 factorial with main effects of added ZnO (0 vs. 2,500 ppm of Zn) and CTC (0, 55, or 441 ppm). In exp. 2, treatments were fed from d 5 to 33 and structured in a (2 × 2 × 2) + 2 factorial with main effects of added CuSO4 (0 vs. 125 ppm Cu), added ZnO (0 vs. 3,000 ppm Zn from d 5 to 12 and 2,000 ppm Zn from d 12 to 33), and Regano EX (0 vs. 0.1% Regano EX containing 5% Origanum oil). The 2 additional treatments were subtherapeutic (55 ppm) and therapeutic (441 ppm) levels of CTC. Following the treatment period, a common diet without antimicrobial was fed until d 47. All diets contained 16.5 ppm Cu and 110 ppm Zn from the trace mineral premix. In exp. 1, no ZnO × CTC interactions were observed. Feeding ZnO increased (P < 0.05) ADG, ADFI, and BW during the treatment period and increased (P < 0.05) ADG and ADFI overall (d 5 to 47). Pigs fed CTC had increased (linear, P < 0.05) ADG, ADFI, and BW during the treatment period and had marginally significant increases (linear, P < 0.10) in overall ADG and ADFI, but overall G:F tended (quadratic, P = 0.070) to increase then decrease as CTC increased. During the treatment period in exp. 2, EO did not affect ADG or ADFI, whereas pharmacological levels of Cu, Zn, and CTC increased (P < 0.05) ADG with coinciding increases (P = 0.055, 0.006, and linear 0.079, respectively) in ADFI. Copper, Zn, and CTC did not affect G:F. EO decreased (P = 0.009) G:F. Diet treatments had minimal carryover effects on subsequent nursery pig growth performance. Overall from d 5 to 47, Cu increased (P = 0.018) ADG, Zn increased (P < 0.05) ADG and ADFI, and EO tended to decrease (P = 0.086) G:F. In conclusion, increased dietary Cu, Zn, or CTC improved weanling pig performance while EO elicited no growth benefits. The benefits of added Zn from ZnO and CTC were additive and could be included together in diets to maximize growth performance of weaned pigs.

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.

Bacterial flora of liver abscesses in crossbred beef cattle and Holstein steers fed finishing diets with or without tylosin.

Holstein steers raised for beef production consistently have a higher prevalence and more severe form of liver abscesses than cattle of beef breeds. A study was conducted to compare bacterial flora of liver abscesses collected from multiple abattoirs from 4 groups of cattle, arranged in a 2 × 2 factorial design, consisting of crossbred cattle and Holstein steers, and each group fed a finishing diet supplemented with or without tylosin. A total of 383 liver abscess samples, consisting of 94 and 81 from crossbred cattle and 89 and 119 from Holstein steers fed finishing diets with or without tylosin, respectively, were subjected for anaerobic and aerobic bacterial isolations. The minimum inhibitory concentrations (MIC) of tylosin to the predominant bacterial species were determined. The likelihood chi-square test was performed to assess unadjusted differences in bacterial prevalence proportions between the 2 types of cattle (crossbred and Holstein steers) and feed type (tylosin or no tylosin). There was no interaction between cattle type and tylosin inclusion on the prevalence of any of the bacterial species isolated. Liver abscesses from Holstein steers yielded a higher total number of isolates compared to liver abscesses from crossbred cattle (1060 vs. 788). subsp. was isolated from all abscesses. The prevalence of subsp. was 19.1% and was not affected by the cattle type or tylosin. The prevalence of was higher ( < 0.01) in crossbred cattle (73.7%) compared to Holstein steers (29.8%). Also, the prevalence of was higher in abscesses from tylosin-fed (66.1%) cattle than no tylosin-fed cattle (35%). The overall prevalence of was 25.3% and was similar ( = 0.58) between cattle type, but the prevalence was lower ( < 0.01) in tylosin-fed (16.9%) compared to no tylosin-fed group (33%). Mean MIC of tylosin for and were similar across both cattle types and tylosin inclusion. Although bacterial flora of liver abscesses from Holstein steers appeared to be more diverse than that of crossbred cattle, there was no difference in the prevalence of the and and in fact, prevalence of was higher in crossbred than Holstein steers. Therefore, the difference in bacterial flora is not the likely reason for higher prevalence and severity of liver abscesses in Holstein steers than crossbred beef cattle.

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 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.

Liver abscesses in cattle: A review of incidence in Holsteins and of bacteriology and vaccine approaches to control in feedlot cattle.

Liver abscesses are the primary liver abnormality of feedlot cattle at slaughter. The incidence of liver abscesses is highly variable, but generally ranges from 10% to 20%. The incidence of total and the proportion of severely abscessed livers (A+) are greater in Holsteins fed for beef production and culled dairy cows than in beef breeds. The reason for the greater incidence of liver abscesses in Holstein steers is not known, but one of the reasons is likely because of increased days on feed. The high prevalence in cull cows is likely because no specific intervention, such as use of tylosin in the feed, is approved for use in dairy cows. Liver abscesses are generally a sequela to ruminal acidosis and rumenitis in cattle fed diets high in readily-fermentable carbohydrates and low in roughages; thus, the term "acidosis-rumenitis-liver abscess complex." Liver abscesses are almost always polymicrobial infections with Gram negative anaerobes constituting the predominant flora. Almost all studies have concluded that , a ruminal bacterium, is the primary causative agent and (formerly ) is the secondary pathogen. A limited number of studies have been done on the bacterial flora of liver abscesses of culled dairy cows and Holstein feedlot steers. A recent study has reported on isolation of from liver abscesses of Holstein cattle. The control of liver abscesses in feedlot cattle has depended on the use of antibiotics, particularly tylosin, in the feed combined with sound nutritional management to minimize occurrence of acidosis and subsequent rumenitis. Although there is no evidence of resistance development in , the future of tylosin use as a feed additive in feedlot cattle is uncertain. Regardless, beginning January 2017, the use of tylosin in feedlot cattle for the prevention of liver abscesses will be under veterinary oversight. Although tylosin is widely used in the feedlot industry, there is considerable interest in evaluating antibiotic alternatives, such as essential oils and vaccines, to control liver abscesses. Because liver abscess is a bacterial infection and the pathogenicity and virulence factors of have been studied widely, there have been considerable interest and efforts to develop an efficacious vaccine. The 2 antigens that have been targeted for vaccine production are leukotoxin and outer membrane proteins of .

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.

In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 µg/mL, and thymol, at 100 µg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal.

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.