Background: Following the ban on antimicrobial usage for growth promotion in animal husbandry in the EU, nonantimicrobial agents including heavy metal ions (e.g. zinc and copper), prebiotics or probiotics have been suggested as alternatives. Zinc has extensively been used in pig farming, particularly during weaning of piglets to improve animal health and growth rates. Recent studies, however, have suggested that high dietary zinc feeding during weaning of piglets increases the proportion of multi-drug resistant E. coli in the gut, contraindicating the appropriateness of zinc as an alternative. The underlying mechanisms of zinc effects on resistant bacteria remains unclear, but coselection processes could be involved. In this study, we determined whether E. coli isolates from intestinal contents of piglets that had been supplemented with high concentrations of zinc acquired a higher tolerance towards zinc, and whether multi-drug resistant isolates tolerated higher zinc concentrations. In addition, we compared phenotypic zinc and copper resistance of E. coli isolates for possible correlation between phenotypic resistance/tolerance to different bivalent ionic metals. Results: We screened phenotypic zinc/copper tolerance of 210 isolates (including antimicrobial resistant, multi-drug resistant, and non-resistant E. coli) selected from two, independent zinc-feeding animal trials by determining a zinc/ copper minimal inhibitory concentration (Merlin, Bornheim-Hersel, Germany). In both trials, groups of piglets were supplemented either with high dietary zinc (> 2000 ppm) or control (50-70 ppm, background) concentrations. Our observations showed that high concentration zinc exposure did not have an effect on either zinc or copper phenotypic tolerance of E. coli isolates from the animals. No significant association was found between antimicrobial resistance and phenotypic zinc/copper tolerance of the same isolates. Conclusion: Our findings argue against a co-selection mechanism of antimicrobial drug-resistance and zinc tolerance after dietary zinc supplementation in weaning piglets. An explanation for an increase in multi-drug resistant isolates from piglets with high zinc dietary feeding could be that resistant bacteria to antimicrobial agents are more persistent to stresses such as zinc or copper exposure.

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.

Background Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. Results We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. Conclusions This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

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.

Escherichia coli is an opportunistic pathogen that can colonize or infect various host species. There is a significant gap in our understanding to what extent genetic lineages of E. coli are adapted or restricted to specific hosts. In addition, genomic determinants underlying such host specificity are unknown. By analyzing a randomly sampled collection of 1198 whole-genome sequenced E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, we demonstrate that certain lineages of E. coli are frequently detected in specific hosts. We report a novel nan gene cluster, designated nan-9, putatively encoding acetylesterases and determinants of uptake and metabolism of sialic acid, to be associated with the human host as identified through genome wide association studies. In silico characterization predicts nan-9 to be involved in sialic acid (Sia) metabolism. In vitro growth experiments with a representative Δnan E. coli mutant strain, using sialic acids 5-N-acetyl neuraminic acid (Neu5Ac) and N-glycolyl neuraminic acid (Neu5Gc) as the sole carbon source, indicate an impaired growth behaviour compared to the wild-type. In addition, we identified several additional E. coli genes that are potentially associated with adaptation to human, cattle and chicken hosts, but not for the pig host. Collectively, this study provides an extensive overview of genetic determinants which may mediate host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.