Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

Vanessa C Johanns; Fereshteh Ghazisaeedi; Lennard Epping; Torsten Semmler; Antina Lübke-Becker; Yvonne Pfeifer; Astrid Bethe; Inga Eichhorn; Roswitha Merle; Birgit Walther; Lothar H Wieler

doi:10.3389/fmicb.2019.02734

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

Front Microbiol. 2019 Nov 28:10:2734. doi: 10.3389/fmicb.2019.02734. eCollection 2019.

Affiliations

¹ Advanced Light and Electron Microscopy (ZBS-4), Robert Koch Institute, Berlin, Germany.
² Institute of Microbiology and Epizootics, Centre for Infection Medicine, Freie Universität Berlin, Berlin, Germany.
³ Microbial Genomics (NG1), Robert Koch Institute, Berlin, Germany.
⁴ Nosocomial Pathogens and Antibiotic Resistances, Robert Koch Institute, Wernigerode, Germany.
⁵ Institute for Veterinary Epidemiology and Biostatistics, Freie Universität Berlin, Berlin, Germany.
⁶ Robert Koch Institute, Berlin, Germany.

Abstract

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from "pigs fed with high zinc concentrations" (high zinc group, [HZG]: n = 99) and a corresponding "control group" ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl₂ MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl₂ (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between "higher" ZnCl₂ MICs and isolates representing the HZG as well as "lower ZnCl₂ MICs" with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and - detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes - a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems.

Keywords: Escherichia coli; antimicrobial resistance; heavy metal tolerance; pig; zinc.