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

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

doi:10.3389/fvets.2020.614513

Vanessa C Johanns, Astrid Bethe + Show 9 more

Open Access

https://doi.org/10.3389/fvets.2020.614513

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Abstract

To prevent economic losses due to post-weaning diarrhea (PWD) in industrial pig production, zinc (Zn) feed additives have been widely used, especially since awareness has risen that the regular application of antibiotics promotes buildup of antimicrobial resistance in both commensal and pathogenic bacteria. In a previous study on 179 Escherichia coli collected from piglets sacrificed at the end of a Zn feeding trial, including isolates obtained from animals of a high-zinc fed group (HZG) and a corresponding control group (CG), we found that the isolate collection exhibited three different levels of tolerance toward zinc, i.e., the minimal inhibitory concentration (MIC) detected was 128, followed by 256 and 512 μg/ml ZnCl2. We further provided evidence that enhanced zinc tolerance in porcine intestinal E. coli populations is clearly linked to excessive zinc feeding. Here we provide insights about the genomic make-up and phylogenetic background of these 179 E. coli genomes. Bayesian analysis of the population structure (BAPS) revealed a lack of association between the actual zinc tolerance level and a particular phylogenetic E. coli cluster or even branch for both, isolates belonging to the HZG and CG. In addition, detection rates for genes and operons associated with virulence (VAG) and bacteriocins (BAG) were lower in isolates originating from the HZG (41 vs. 65% and 22 vs. 35%, p < 0.001 and p = 0.002, resp.). Strikingly, E. coli harboring genes defining distinct pathotypes associated with intestinal disease, i.e., enterotoxigenic, enteropathogenic, and Shiga toxin-producing E. coli (ETEC, EPEC, and STEC) constituted 1% of the isolates belonging to the HZG but 14% of those from the CG. Notably, these pathotypes were positively associated with enhanced zinc tolerance (512 μg/ml ZnCl2 MIC, p < 0.001). Taken together, zinc excess seems to influence carriage rates of VAGs and BAGs in porcine intestinal E. coli populations, and high-zinc feeding is negatively correlated with enteral pathotype occurrences, which might explain earlier observations concerning the relative increase of Enterobacterales considering the overall intestinal microbiota of piglets during zinc feeding trials while PWD rates have decreased.

Highlights

The gastrointestinal microbiota of pigs is a large, multifaceted and complex microbial community which has been estimated to comprise of 1010-1011 bacteria per gram of gut content [1]
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 virulence associated genes (VAGs) and bacteriocin associated genes (BAGs) among the intestinal E. coli population of these piglets were not investigated so far
Considering the overall phylogenetic diversity, we identified 2,804 orthologous genes representing the “maximum common genome” (MCG) of 186 genomic sequences representing isolates from both feeding groups (HZG = 99, control group (CG) = 80), six isolates previously collected from diagnostic samples of severely ill piglets and one further isolate of human origin [RKI6122 [24]] which exhibited a MIC value of 1,024 μg/ml ZnCl2

Summary

Introduction

The gastrointestinal microbiota of pigs is a large, multifaceted and complex microbial community which has been estimated to comprise of 1010-1011 bacteria per gram of gut content [1]. Complex mechanisms of interaction between commensal and pathogenic bacteria have evolved within the gut, including competition for nutrients, shielding from the activated enteral immune response and induction of competitoreliminating bacteriocin production [7]. These bacteriocins are polypeptide toxins comprising colicins and microcins which exhibit a broad range of different cytotoxic mechanisms [8,9,10]. Both colicins and microcins are capable of killing a narrow spectrum of competing Enterobacterales, including other E. coli lineages [9]. Porcine pathogenic E. coli have been shown to produce predominantly colicins, especially colicin BM and Ib [11]

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