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.
Highlights
Enterotoxigenic Escherichia coli (ETEC) are commonly associated with post-weaning diarrhea (PWD) in piglets, a disease causing serious losses in the pig industry worldwide (Fairbrother et al, 2005; Rhouma et al, 2017a)
The aim of this work is to study the effects of zinc-rich diets on a representatively selected collection of intestinal E. coli obtained from post-weaning piglets, considering a putative association of a nutritive zinc oxide excess and (i) phenotypic zinc tolerance levels, (ii) antibiotic- and biocide susceptibility profiles, and (iii) genes involved in antimicrobial resistance, zincand biocide tolerance
Since co-selection of antibiotic- and metal resistance is an issue of utmost importance and metal resistance genes are often co-located on mobile genetic elements (MGEs) alongside antibiotic resistance genes (ARGs) (Baker-Austin et al, 2006; Fard et al, 2011; Holzel et al, 2012; Fang et al, 2016; Song et al, 2017; Argudín et al, 2019), we further investigated the occurrence of genes known to be associated with either antibioticor metal resistance on mobile genetic elements among our isolate collection (Supplementary Table S1)
Summary
Enterotoxigenic Escherichia coli (ETEC) are commonly associated with post-weaning diarrhea (PWD) in piglets, a disease causing serious losses in the pig industry worldwide (Fairbrother et al, 2005; Rhouma et al, 2017a). Zinc is the second most abundant transition metal in most phyla and generally considered as essential for life. Together with copper, it is an important trace element required for hormone function, reproduction, vitamin synthesis, enzyme formation and it promotes a strong immune system function (Yu et al, 2017). Both metals are usually added to animal feed in amounts necessary for physiological body function (Yazdankhah et al, 2014; Yu et al, 2017)
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