Effects of feeding elevated concentration of copper on prevalence and selection of fecal enterococci positive for transferable copper resistance gene in piglets

Master of ScienceDepartment of Diagnostic Medicine/PathobiologyTiruvoor G. NagarajaCopper, as copper sulfate, is often supplemented at elevated concentrations in swine diets, particularly in piglets, to promote growth. Growth promotional effects of copper are believed to be similar to that of antibiotics in that gut microbial flora is altered to reduce loss of nutrients and suppress pathogens. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and E. faecalis, resistance is conferred by a plasmid-borne transferable copper resistance (tcrB) gene. The plasmid also carries macrolide [erm(B)] and glycopeptide (vanA) antibiotics resistance genes. The objectives of the research were to 1) determine the prevalence of tcrB gene in fecal enterococci of piglets in relation to normal (16.5 ppm) and elevated level (125 ppm) of copper supplementation, 2) determine the relationship of tcrB gene and susceptibilities to copper, erythromycin, and vancomycin, and 3) determine the transferability of tcrB gene in enterococci by conjugation. Weaned piglets, housed in pens, fed normal (16.5 ppm; control) or elevated level of copper (125 ppm) were used. Fecal samples were collected weekly for isolation of enterococci. Isolates were speciated by multiplex PCR and sodA gene sequence analysis. The prevalence of tcrB-positive enterococcal isolates was higher (P < 0.05) in the copper supplemented group than the control group. The prevalence of tcrB was affected by sampling days (P < 0.05) with a significant treatment and sampling time interaction (P < 0.05). The tcrB positive isolates were either E. faecium or E. faecalis, and majority of isolates was E. faecium. The mean MIC of copper for tcrB-positive isolates (21.1 mM) was higher (P < 0.001) compared to tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin, tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains suggests the possibility that copper supplementation may exert selection pressure for antibiotic resistance. The positive association between copper supplementation and prevalence of tcrB gene has important implications for antimicrobial resistance and food safety, which warrants further investigation

Antimicrobial Susceptibility of Enteric Gram Negative Facultative Anaerobe Bacilli in Aerobic versus Anaerobic Conditions

Citation: DeMars, Z., Biswas, S., Amachawadi, R. G., Renter, D. G., & Volkova, V. V. (2016). Antimicrobial Susceptibility of Enteric Gram Negative Facultative Anaerobe Bacilli in Aerobic versus Anaerobic Conditions. Plos One, 11(5). doi:10.1371/journal.pone.0155599Antimicrobial treatments result in the host's enteric bacteria being exposed to the antimicrobials. Pharmacodynamic models can describe how this exposure affects the enteric bacteria and their antimicrobial resistance. The models utilize measurements of bacterial antimicrobial susceptibility traditionally obtained in vitro in aerobic conditions. However, in vivo enteric bacteria are exposed to antimicrobials in anaerobic conditions of the lower intestine. Some of enteric bacteria of food animals are potential foodborne pathogens, e.g., Gram-negative bacilli Escherichia coli and Salmonella enterica. These are facultative anaerobes; their physiology and growth rates change in anaerobic conditions. We hypothesized that their antimicrobial susceptibility also changes, and evaluated differences in the susceptibility in aerobic vs. anaerobic conditions of generic E. coli and Salmonella enterica of diverse serovars isolated from cattle feces. Susceptibility of an isolate was evaluated as its minimum inhibitory concentration (MIC) measured by E-TestR following 24 hours of adaptation to the conditions on Mueller-Hinton agar, and on a more complex tryptic soy agar with 5% sheep blood (BAP) media. We considered all major antimicrobial drug classes used in the U.S. to treat cattle: ?-lactams (specifically, ampicillin and ceftriaxone E-Test1 ), aminoglycosides (gentamicin and kanamycin), fluoroquinolones (enrofloxacin), classical macrolides (erythromycin), azalides (azithromycin), sulfanomides (sulfamethoxazole/ trimethoprim), and tetracyclines (tetracycline). Statistical analyses were conducted for the isolates (n?30) interpreted as susceptible to the antimicrobials based on the clinical breakpoint interpretation for human infection. Bacterial susceptibility to every antimicrobial tested was statistically significantly different in anaerobic vs. aerobic conditions on both media, except for no difference in susceptibility to ceftriaxone on BAP agar. A satellite experiment suggested that during first days in anaerobic conditions the susceptibility changes with time. The results demonstrate that assessing effects of antimicrobial treatments on resistance in the host's enteric bacteria that are Gram negative facultative Anaerobe Bacilli requires data on the bacterial antimicrobial susceptibility in the conditions resembling those in the intestine. © 2016 DeMars et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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

Citation: Feldpausch, J. A., Amachawadi, R. G., Tokach, M. D., Scott, H. M., Nagaraja, T. G., Dritz, S. S., . . . DeRouchey, J. M. (2016). Effects of dietary copper, zinc, and ractopamine hydrochloride on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria. Journal of Animal Science, 94(8), 3278-3293. doi:10.2527/jas2016-0340A 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 (CuSO4; 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, P = 0.057, P = 0.068, and P = 0.064 for G:F and caloric efficiency on a ME and NE basis, respectively). Ractopamine improved (P &lt; 0.001) overall ADG, G:F, and caloric efficiency, thereby increasing final BW by 3% with no change in ADFI. Ractopamine also increased (P &lt; 0.001) HCW, percentage carcass yield, G:F, loin depth, and percent fat-free lean and decreased (P = 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, P = 0.011 and P = 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. Enterococcus spp. and Escherichia coli isolates displayed varying levels of resistance to certain antibiotics prior to initiation of treatments on d 0. Resistance to most antibiotics decreased (P &lt; 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. © 2016 American Society of Animal Science. All rights reserved

COVID-19 PANDEMIC: A SYSTEMATIC REVIEW ON THE CORONAVIRUSES OF ANIMALS AND SARS-CoV-2

Coronaviruses (CoVs), classified into four genera, viz., alpha-, beta-, gamma-, and Delta- CoV, represent an important group of diverse transboundary pathogens that can infect a variety of mammalian and avian species including humans, animals, poultry, and non-poultry birds. CoVs primarily infect lung and gut epithelial cells, besides monocytes and macrophages. CoVs have high mutation rates causing changes in host specificity, tissue tropism, and mode of virus excretion and transmissions. The recent CoV zoonoses are SARS, MERS, and COVID-19 that are caused by the transmission of beta-CoVs of bats to humans.&nbsp; Recently, reverse zoonoses of the COVID-19 virus have been detected in dogs, tigers, and minks. Beta-CoV strains also infect bovine (BCoV) and canine species (CRCoV); both these beta-CoVs might have originated from a common ancestor. Despite the high genetic similarity between BCoV, CRCoV, and HCoV-OC43, these differ in species specificity. Alpha-CoV strains infect canine (CCoV), feline (FIPV), swine (TGEV and PEDV), and humans (HCoV229E and NL63). Six coronavirus species are known to infect and cause disease in pigs, seven in human beings, and two in dogs. The high mutation rate in CoVs is attributed to error-prone 3′-5′ exoribonuclease (NSP 14), and genetic recombination to template shift by the polymerase. The present compilation describes the important features of the CoVs and diseases caused in humans, animals, and birds that are essential in surveillance of diverse pool of CoVs circulating in nature, and monitoring interspecies transmission, zoonoses, and reverse zoonoses

Effects of dietary zinc oxide and chlortetracycline on nursery pig growth performance

Swine Industry Day, 2014 is known as Swine Day, 2014A total of 240 weaned pigs (PIC 1050; initially 13.4 lb) were used in a 47-d study to compare the effects of added Zn from zinc oxide (ZnO), alone or in combination with a low or high dose of chlortetracycline (CTC), on nursery pig performance. Pigs were allotted to pens at weaning (d 0) and fed a common starter diet with no antimicrobial for 5 d before the start of the experiment. On d 5, pens of 5 pigs were allotted to 1 of 6 dietary treatments in a randomized complete block design with 8 replications per treatment. Dietary treatments were arranged in a 2 × 3 factorial with main effects of added ZnO (0 vs. 2,500 ppm of Zn) and CTC (0, 50, or 400 g/ton). Pigs were fed experimental diets from d 5 to 26 after weaning followed by a common corn-soybean meal–based diet without antimicrobial from d 26 to 47. Pigs on the 50 g/ton treatment received CTC continuously from d 5 to 26; however, to comply with FDA guidelines, CTC was removed on d 15 from the diets of pigs fed 400 g/ton CTC, then added again from d 16 to 26. All diets contained 110 ppm of Zn from ZnO in the trace mineral premix. No ZnO × CTC interactions were observed. Pigs fed added ZnO had increased (P = 0.001) ADG, ADFI, and ending BW during the treatment period but increased F/G (P = 0.03) from d 26 to 47 when a common diet was fed. Pigs fed CTC had increased (linear, P < 0.05) ADG, ADFI, and ending BW during the treatment period as well as a tendency (quadratic, P = 0.08) for improved F/G. Overall (d 5 to 47), pigs fed added ZnO had increased (P < 0.05) ADG and ADFI. Overall, pigs fed CTC tended to have increased (linear, P = 0.06) ADG and ADFI, but F/G tended (quadratic, P = 0.07) to decrease then increase as CTC increased. In summary, when ZnO or CTC were added to the diets, increased ADG and ADFI were observed, but additional carryover benefits were not evident after these feed additives were removed from the diets. The benefits of added Zn from ZnO and CTC are additive and could be included together in diets to get the maximum benefit in growth performance of weaned pigs

Effects of dietary Cu, Zn, and ractopamine-HCl on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria

Citation: Feldpausch, J. A., Amachawadi, R., Tokach, M. D., Scott, H. M., Dritz, S. S., Nagaraja, T. G., . . . DeRouchey, J. M. (2016). Effects of dietary Cu, Zn, and ractopamine-HCl on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria. Journal of Animal Science, 94, 73-74. doi:10.2527/msasas2016-157A total of 480 pigs (PIC 327 × 1050; initially 48.7 kg) were used to determine the interactive effects of supplemental Cu, Zn, and ractopamine HCl on finishing pig growth, carcass characteristics, and antimicrobial susceptibility of enteric bacteria. Treatments were arranged in a 2 × 2 × 2 factorial with main effects of added copper (CuSO4; 0 vs. 125 ppm Cu), added zinc (ZnO; 0 vs. 150 ppm Zn) and ractopamine HCl (0 vs. 10 ppm during the last 28 d before marketing; Paylean®; Elanco Animal Health, Greenfield, IN). All diets contained 11 ppm Cu and 73 ppm Zn from the trace mineral premix. Pens of pigs were balanced and blocked on initial BW then randomly allotted to 1 of the 4 mineral treatment diets. Twenty-eight d before marketing, pens within each block and mineral treatment were randomly assigned to receive either 0 or 10 ppm ractopamine 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, P = 0.057, 0.068 and 0.064 for G:F and caloric efficiency on a ME and NE basis, respectively). Ractopamine improved (P < 0.001) overall ADG, G:F, and caloric efficiency thereby increasing final BW by 3% with no change in ADFI. Ractopamine increased (P < 0.001) HCW, percent carcass yield, HCW G:F, loin depth, and percent fat-free lean and decreased (P = 0.014) backfat. An interaction existed whereby adding Zn or Cu alone to diets containing ractopamine numerically improved percent carcass yield and HCW G:F, but no improvement was observed when the Cu or Zn was added to the control diet or when Cu and Zn were fed in combination in the ractopamine diets (Cu × Zn × ractopamine, P = 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 minimal inhibitory concentration breakpoints. Escherichia coli and Enterococcus spp. isolates displayed varying levels of resistance to certain antibiotics before initiation of treatments on d 0. Resistance to most antibiotics decreased (P < 0.05) over time or was stable for those that had a low base-line percentage of resistance. Ractopamine and Zn did not adversely affect antimicrobial resistance but extended feeding of 125 ppm Cu throughout the finishing period appeared to antagonize any time-associated decrease in enterococcal resistance to tetracycline, tylosin, and quinupristin/dalfopristin

Comparative effects of dietary copper, zinc, essential oils, and chlortetracycline on nursery pig growth performance

Swine Industry Day, 2014 is known as Swine Day, 2014A total of 350 weaned pigs (PIC 1050; initially 13.3 lb) were used in a 47-d study to compare the effects of feeding antibiotic alternatives (copper, zinc, and essential oils), alone or in combination, on nursery pig performance. Pigs were allotted to pens at weaning (d 0) and fed a common starter diet with no antimicrobial for 5 d before the start of the experiment. On d 5, pens of 5 pigs were allotted to 1 of 10 dietary treatments in a randomized complete block design with 7 replications per treatment. Dietary treatments were arranged in a 2 × 2 × 2 + 2 factorial with main effects of added copper sulfate (CuSO4; 0 vs. 125 ppm Cu), added zinc oxide (ZnO; none 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. 45 g/ton essential oils blend; Ralco Animal Nutrition, Marshall, MN). The 2 additional treatments were growth-promoting and therapeutic levels of chlortetracycline (CTC at 50 or 400 g/ton). Pigs were fed experimental diets from d 5 to 33 followed by a common corn-soybean meal–based diet without any antimicrobial, essential oils, or pharmacological levels of Cu or Zn from d 33 to 47. To comply with FDA guidelines, CTC was removed on d 19 from the diet of pigs fed 400 g/ton CTC, then added again from d 20 to 33. All diets contained 16.5 ppm Cu and 165 ppm of Zn from the trace mineral premix. Essential oils had no effect on daily gain, but feeding CTC or pharmacological levels of Cu or Zn improved the growth rate of nursery pigs. Carryover effects from any of these dietary treatments on subsequent nursery growth performance were minimal. Although there were no improvements in feed efficiency due to Cu or Zn, the inclusion of an essential oils blend worsened feed and caloric efficiencies

Isolation and characterization of lactic acid bacteria with potential probiotic activity and further investigation of their activity by α-amylase and α-glucosidase inhibitions of fermented batters

Probiotic microbiota plays a vital role in gastrointestinal health and possesses other beneficial attributes such as antimicrobial and antibiotic agents along with a significant role in the management of diabetes. The present study identifies the probiotic potential of Lactobacillus spp. isolated from three traditionally fermented foods namely, jalebi, medhu vada, and kallappam batters at biochemical, physiological, and molecular levels. By 16S rRNA gene amplification and sequencing, the isolates were identified. A similarity of &gt;98% to Lacticaseibacillus rhamnosus RAMULAB13, Lactiplantibacillus plantarum RAMULAB14, Lactiplantibacillus pentosus RAMULAB15, Lacticaseibacillus paracasei RAMULAB16, Lacticaseibacillus casei RAMULAB17, Lacticaseibacillus casei RAMULAB20, and Lacticaseibacillus paracasei RAMULAB21 was suggested when searched for homology using NCBI database. Utilizing the cell-free supernatant (CS), intact cells (IC), and cell-free extract (CE) of the isolates, inhibitory potential activity against the carbohydrate hydrolyzing enzymes α-glucosidase and α-amylase was assessed. CS, CE, and IC of the isolates had a varying capability of inhibition against α-glucosidase (15.08 to 59.55%) and α-amylase (18.79 to 63.42%) enzymes. To assess the probiotic potential of seven isolates, various preliminary characteristics were examined. All the isolates exhibited substantial tolerance toward gastrointestinal conditions and also demonstrated the highest survival rate (&gt; 99%), hydrophobicity (&gt; 65%), aggregation (&gt; 76%), adherence to HT-29 cells (&gt; 84%), and chicken crop epithelial cells suggesting that the isolates had a high probiotic attribute. Additionally, the strains showed remarkable results in safety assessment assays (DNase and hemolytic), and antibacterial and antibiotic evaluations. The study concludes that the lactic acid bacteria (LAB) characterized possesses outstanding probiotic properties and has antidiabetic effects. In order to obtain various health advantages, LAB can be utilized as probiotic supplements

Evaluation of Clay-Based Binders and In-Feed Antimicrobials on Growth Performance and Biological Measurements in Nursery Pigs

A total of 360 barrows (DNA 200 × 400; initially 14.2 ± 0.08 lb) were used in a 42-d growth study to evaluate clay-based binders or an in-feed antimicrobial on growth performance and biological measurements including fecal and blood analysis in nursery pigs. Pigs were weaned at approximately 21 d of age and randomly allotted to 1 of 4 dietary treatments in a completely randomized design. There were 5 pigs per pen and 18 replications per treatment. Dietary treatments were corn-soybean meal-based and fed in two phases from d 0 to 9 (phase 1) and 9 to 21 (phase 2) after weaning. Either Protek (0.40% of the diet; Nutriquest, Mason City, IA); Protect-8 Plus (0.10% of the diet; Essential Ag Solutions, Sioux Falls, SD); or Kavault (0.04% of the diet; Avilamycin; Elanco, Greenfield, IN) were added to the control diet to create the experimental treatments. A common phase 3 diet was fed to all pigs from d 21 to 42. Overall (d 0 to 42), pigs fed Kavault had increased (P \u3c 0.05) final BW, ADG, and ADFI compared to all other treatments. There was evidence that frequency of fecal scores with softer feces increased over time (P \u3c 0.001), with d 21 having the greatest frequency of diarrhea and soft feces. Fecal Escherichia coli colony count was lower (P \u3c 0.001) on d 21 compared to d 9. For fecal myeloperoxidase (MPO), concentrations were lower (P \u3c 0.05) on d 21 compared to d 6 and 9. For fecal DM, pigs fed Kavault had decreased (P \u3c 0.05) DM percentage compared to all other treatments. Fecal DM percentage was higher (P \u3c 0.05) on d 6 and 9 compared to d 21. No differences (P \u3e 0.10) were observed across treatments for fecal scores, fecal E. coli colony count, fecal MPO or virulence genes associated with E. coli. Similarly, no differences (P \u3e 0.10) were observed across treatments for TNF-alpha and IL-6 blood assays. For IL-6, concentrations were greater (P \u3c 0.05) on d 9 compared to d 21. In summary, pigs fed Kavault had increased BW, ADG, and ADFI, compared to those fed the 2 clay-based additives or the control diet. There were no treatment effects on fecal score, fecal MPO, or blood measurements. However, we observed a day effect indicating that feces were softer and had less DM on d 21 compared to d 6 and 9. Additionally, fecal E. coli colony count and MPO had lower concentrations of d 21 compared to d 9. There was a strong negative correlation between fecal DM and score (P \u3c 0.001) on d 6, 9, and 21 indicating that as fecal DM increased, the score became closer to 0, representing a firmer fecal sample. Fecal DM on d 6 and fecal DM on d 9 were negatively correlated with ADG from d 0 to 9 meaning that as growth rate increased, fecal DM decreased

Effects of Live Yeast and Yeast Extracts with and without Pharmacological Levels of Zinc on Antimicrobial Susceptibilities of Fecal Escherichia coli in Nursery Pigs

A total of 360 weanling barrows (Line 200 × 400, DNA Genetics; initial BW 12.4 ± 0.05 lb) were used in a 42-d study to evaluate yeast-based pre- and probiotics (Phileo by Lesaffre, Milwaukee, WI) in diets with or without pharmacological levels of Zn on antimicrobial resistance (AMR) patterns of fecal Escherichia coli. Pens were assigned to 1 of 4 dietary treatments with 5 pigs per pen and 18 pens per treatment. Dietary treatments were arranged in a 2 × 2 factorial with main effects of live yeast-based pre- and probiotics (none vs. 0.10% ActiSafSc 47 HR+, 0.05% SafMannan, and 0.05% NucleoSaf from d 0 to 7, then concentrations were lowered by 50% from day 7 to 21) and pharmacological levels of Zn (110 vs. 3,000 ppm from d 0 to 7, and 2,000 ppm from d 7 to 21 provided by ZnO). All pigs were fed a common diet from d 21 to 42 post-weaning without live yeast-based pre- and probiotics or pharmacological Zn. Fecal samples were collected on d 4, 21, and 42 from the same three pigs per pen for fecal E. coli isolation. The identification of E. coli was by PCR detection of uidA and clpB genes. The AMR patterns of E. coli were determined by microbroth dilution method using Sensititre CMV3AGNF panel containing 14 different antimicrobials. The addition of pharmacological levels of Zn had a marginally significant effect (P = 0.051) to increase the minimum inhibitory concentration (MIC) values of ciprofloxacin; however, median MIC values were still under the Clinical and Laboratory Standards Institute (2018) classified resistant breakpoint for ciprofloxacin. There was no evidence for differences (P \u3e 0.05) for yeast additives or Zn for AMR of fecal E. coli isolates to any of the remaining antibiotics. In conclusion, pharmacological levels of Zn tended to increase the AMR of fecal E. coli to ciprofloxacin while the medians were below a resistant breakpoint. There was no influence of live yeast and yeast extracts on AMR patterns of fecal E. coli against tested antimicrobials