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

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.

Recent Advances in Understanding the Influence of Zinc, Copper, and Manganese on the Gastrointestinal Environment of Pigs and Poultry

“…Various (mainly) negative, as well as positive, correlations between free ions or protein-bound zinc and numbers of different bacterial taxa or groups in the GIT were reported by [ 18 ], while seemingly similar Zn fractions between groups have been associated with different microbial profiles [ 16 ] or apparent fractional differences between groups associated with relatively similar gut microbiological parameters [ 20 ]. Excess Zn has been reported to be negatively correlated with the occurrence of enteric E. coli pathotypes, which could at least partially explain the associated reduction in diarrhea, but seemingly increases the carriage of virulence-associated genes within the intestinal E. coli populations [ 23 ], and has been proposed to slow the decline in fecal shedding of an administered Salmonella serovar [ 24 ].…”

“…In weaned pigs, Højberg et al [ 23 ] reported that 175 mg/kg dietary Cu (from CuSO 4 ) reduced lactic acid bacteria counts in the stomach, and coliforms in the cecum and the colon, while Namkung et al [ 40 ] found the GI microbiota diversity to be reduced with 250 mg/kg additional Cu. A lower microbiota diversity is often considered undesirable, although often a feature of antimicrobial supplementation, but the reduction in the putative beneficial lactic acid bacteria was surprising.…”

Role played by the environment in the emergence and spread of antimicrobial resistance (AMR) through the food chain

“…These studies are complicated by the fact that many pathogens of concern are opportunists and do not necessarily cause infection at the time of exposure or transmission. Therefore, colonisation, virulence potential linked to AMR and heavy metal resistance and potential for gene transfer, as well as relevance to human infection should also be considered as an end point for epidemiological studies (in addition to infection) as part of overarching One Health‐based studies (Hernando‐Amado et al., 2019; Findlay et al., 2020; Johanns et al., 2020).…”

Gut Microbiota as a Mediator of Essential and Toxic Effects of Zinc in the Intestines and Other Tissues

“…A detailed analysis of 179 Escherichia coli genomes obtained from piglets after completion of a Zn oxide feeding trial demonstrated that genes and operons associated with virulence and bacteriocin production, as well as enterotoxigenic, enteropathogenic, and Shiga toxin-producing pathotypes were less abundant in high Zn-supplemented animals [ 66 ]. In enteropathogenic Escherichia coli , exposure to Zn was shown to reduce expression of virulence factors and reduced bacterial adhesion to the cells.…”