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

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Effects of Live Yeast and Yeast Extracts with and without Pharmacological Levels Effects of Live Yeast and Yeast Extracts with and without Pharmacological Levels of Zinc on Antimicrobial Susceptibilities of Fecal Escherichia coli in Nursery Pigs of Zinc on Antimicrobial Susceptibilities of Fecal Escherichia coli in Nursery Pigs
Summary 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% Nucle-oSaf 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.

Introduction
Feeding pharmacological levels of Zn (2,000 to 3,000 ppm) has become a concern for AMR to antimicrobials of importance to human and animal medicine. One potential replacement strategy for pharmacological levels of Zn in the early nursery is the use of pre-and probiotics. This report is a companion to our previous paper in which we evaluated the effects of pharmacological levels of Zn with or without the addition of the live yeast Saccharomyces cerevisiae strain NCYC Sc 47 and yeast-based prebiotics derived from Saccharomyces cerevisiae on weanling pig growth performance. 6 This paper reports the effects of live yeast and yeast-based prebiotics on AMR patterns for E. coli isolated from nursery pig feces.

General
The Kansas State University Institutional Animal Care and Use Committee approved the protocol used in this experiment. The study was conducted at the Kansas State University Segregated Early Weaning Facility in Manhattan, KS. The facility has two identical barns that are completely enclosed, environmentally controlled, and mechanically ventilated. Treatments were equally represented in each barn. Each pen contained a 4-hole, dry self-feeder and a cup waterer to provide ad libitum access to feed and water. Pens (4 × 4 ft) had metal tri-bar floors and allowed approximately 2.7 ft 2 /pig.

Animals and treatment structure
A total of 360 barrows (Line 200 × 400, DNA Genetics; initially 12.4 ± 0.05 lb BW) were used in a 42-d study with 5 pigs per pen and 18 pens per treatment (9 pens per barn). Details as to pig allotment, experimental design, and diet preparation and analysis can be found in Chance et al. (2021). 6 Briefly, dietary treatments were arranged in a 2 × 2 factorial with main effects of yeastbased 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 added yeast products or pharmacological levels of Zn. The live yeast Saccharomyces cerevisiae strain NCYC Sc 47 (ActiSaf Sc 47 HR+; Phileo by Lesaffre, Milwaukee, WI) served as the yeast-based probiotic. The yeast-based prebiotics included a yeast cell wall fraction with concentrated mannan-oligosaccharides and β-glucans from Saccharomyces cerevisiae (SafMannan; Phileo by Lesaffre, Milwaukee, WI) and a yeast extract containing ≥6% unbound nucleotides from Saccharomyces cerevisiae (NucleoSaf; Phileo by Lesaffre, Milwaukee, WI).

Fecal collection
Fecal samples were collected on d 4, 21, and 42 of the experiment to isolate E. coli and determine AMR. Fecal samples were collected directly from the rectum of the same three randomly selected pigs from each pen and pooled by pen to form one composite sample. Fecal samples were collected using a sterile, single-use cotton tipped applicator (Fisher Healthcare, Pittsburgh, PA), stored in a clean, single-use zipper storage bag placed on ice and delivered the same day to the laboratory of Dr. Raghavendra Amachawadi at the Kansas State University College of Veterinary Medicine for bacterial isolation and further characterization.

E. coli isolation
Approximately 1 g of fecal sample was suspended in 9 mL of phosphate-buffered saline.

Statistical analysis
For each of the 14 antimicrobials, minimum inhibitory concentration (MIC) data were summarized with appropriate descriptive statistics by treatment group at each sampling day. Due to the lack of variability, MICs of tetracycline were excluded from the statistical analysis since all isolates were resistant. The MIC data of the remaining antimicrobials were analyzed using the linear mixed model. To better achieve model assumptions, data underwent natural log transformation before statistical modeling. Statistical analysis was performed using the MIXED procedure of SAS (v. 9.4, SAS Inst., Inc., Cary, NC) with option DDFM=KR in the MODEL statement. Fixed effects of the model included Zn, yeast, time, and their second-and third-order interactions. Random effects included block and pen. Treatment effect was assessed via back-transformed least squares means, i.e., geometric means of the MIC values. The variance-covariance structure of pen was taken as compound symmetry, first-order autoregressive, or unstructured according to the model fitting criteria. Differences between treatments were considered significant at P ≤ 0.05 and marginally significant at 0.05 < P ≤ 0.10.

Results and Discussion
From fecal samples collected and E. coli isolated, there were no two-way or three-way interactions observed for any of the antibiotics tested. Thus, MIC values of fecal E. coli isolates in response to the inclusion of yeast-based pre-and probiotics, pharmacological levels of Zn, and sampling day were further explored (Table2). There was evidence for increased (P < 0.05) MIC values over time for ampicillin, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, sulfisoxazole, and trimethoprim/sulfamethoxazole with a tendency (P < 0.10) for increased MIC values of chloramphenicol and gentamicin. Azithromycin was the only antibiotic that had evidence for decreased (P < 0.001) MIC values over time.
Of the fecal samples collected and E. coli isolated, MICs were not influenced by the presence of dietary addition of live yeast and yeast extracts. Only fecal E. coli isolated from pigs fed pharmacological levels of Zn from d 0 to 21 had a marginally significant effect (P = 0.051) where the AMR to ciprofloxacin was higher compared to those that were not fed added Zn. However, all median MICs were still well under the CLSI 5 antimicrobial breakpoint for ciprofloxacin.
In conclusion, there was minimal impact on the AMR of fecal E. coli of pigs fed diets with pharmacological levels of Zn and/or yeast-based pre-and probiotics. However, feeding high levels of Zn tended to increase the possibility of fecal E. coli's AMR to ciprofloxacin, but based on the breakpoint the isolates were considered susceptible.

Kansas State University Agricultural Experiment Station and Cooperative Extension Service
Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. Persons using such products assume responsibility for their use in accordance with current label directions of the manufacturer.  239 ± 10 b 0.010 Trimethoprim/Sulfamethoxazole 1:19 ratio 5 0.25 ± 0.025 0.24 ± 0.025 0.848 0.23 ± 0.023 0.27 ± 0.027 0.210 0.20 ± 0.020 b 0.15 ± 0.013 a 0.51 ± 0.089 c <0.001 1 A total of 360 barrows (initially12.4 ± 0.05lb) were used in a 42-d study with 5 pigs per pen and 18 pens per treatment. Data reported as geometric mean of minimal inhibitory concentration (MIC) ± standard error of the mean. (Clinical and Laboratory Standards Institute (CLSI). 2018. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Approved standard, 5th ed. CLSI supplement VET08. CLSI, Wayne, PA.) 2 Fecal samples from the same 3 pigs/pen were collected on d 4, 21, and 42 for E. coli isolation and further characterization. 3 Yeast pre-and probiotics included ActiSaf Sc 47 HR+ at 0.1%, SafMannan at 0.05% and NucleoSaf at 0.05% in phase 1 diets, and then concentrations were lowered by 50% in phase 2 diets (Phileo by Lesaffre, Milwaukee, WI). 4 Zinc oxide was added to supply 3,000 ppm of Zn for the duration of phase 1, and 2,000 ppm of Zn for the duration of phase 2. 5 The MIC numerator of the ratio was reported for the antimicrobial's amoxicillin:clavulanic acid 2:1 ratio and trimethoprim/sulfamethoxazole 1:19 ratio. a,b,c Superscripts signify statistical difference among sampling days at P < 0.05.