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

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.

Concentration and chemical form of dietary zinc shape the porcine colon microbiome, its functional capacity and antibiotic resistance gene repertoire

“…Genes such as aph(3”)-Ib , bla ROB , and arnA can be commonly found in enterobacteria [ 36 ]. In addition, aph(3”)-Ib , for example, has been recently shown to be associated with higher zinc tolerance in intestinal E. coli from weaned piglets [ 37 ]. This is supported by our findings here and by previous studies showing an increased abundance and diversity of enterobacteria with high dietary zinc levels in pigs [ 16 , 22 , 38 ].…”

High-Zinc Supplementation of Weaned Piglets Affects Frequencies of Virulence and Bacteriocin Associated Genes Among Intestinal Escherichia coli Populations

“…In total, 179 E. coli were collected as a stratified random sample (HZG = 99, CG = 80), with an average number of 11 E. coli isolates investigated per piglet (all three sampling sites). These isolates were previously studied with regard to their respective zinc tolerance levels and susceptibility patterns for antibiotics and biocides (22).…”

“…The E. coli isolates obtained from piglets fed with a high-zinc supplemented diet showed generally a lower VAG frequency. Based on this finding, we further investigated whether a specific pathotype is associated with a particular zinc tolerance level considering the previously determined ZnCl 2 MIC values of 128, 256, or 512 µg/ml (22,24) or not.…”

“…As mentioned before, the focus of our analysis was on the three different ZnCl 2 MIC values determined for the E. coli collection in a previous study (22).…”

“…While we have recently shown that high-zinc supplemented diets foster accumulation of E. coli associated with increased zinctolerance in weaned piglets (22), the effects of that particular feed additive on the occurrence of VAGs and bacteriocin associated genes (BAGs) among the intestinal E. coli population of these piglets were not investigated so far. In the current study, we analyzed the phylogenetic relationship and make up of a broad collection of E. coli obtained from a former piglet zinc-feeding trial together with strains isolated from pigs suffering from clinical disease in order to investigate the presence of a putative correlation between feeding group and the zinc tolerance level of the isolates with the occurrence of VAGs and/or BAGs.…”