Unravelling mechanisms by which Zinc influences diversity and multi-resistance of porcine intestinal Escherichia coli populations

• Applicant: Professor Dr. Lothar H. Wieler
• Subject Area: Veterinary Medical Science
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 292118477

Despite the ban of antimicrobial growth promoters the prevalence of MDR bacteria, especially MRSA and ESBL-producing Escherichia (E.) coli, raised tremendously in livestock. Instead alternative feed additives - like the cationic trace element zinc - are increasingly used. Genes conferring resistance against cationic trace elements are often co-localized with genes conferring antimicrobial resistance. Therefore we raised the hypothesis that zinc feeding is one cause of the increase of MDR bacteria. As E. coli is both a major commensal and pathogenic bacterial species of the intestinal microbiota, in two feeding trials of piglets with zinc-oxid (ZnO) of particle sizes between 100-200 µm within the SFB852, we molecularly analyzed nearly 1,500 intestinal E. coli to the clonal level. Indeed, zinc feeding had a strong impact on E. coli, as strains from zinc fed piglets showed (i) a greater genetic diversity, and even more importantly (ii) an increased rate of MDR E. coli. Unexpectedly, antimicrobial resistance of E. coli was not significantly associated with zinc tolerance. The analyzed intestinal habitats did also not contain an increased E. coli density, so that increased physical contact does not explain a possible higher conjugation rate. In conclusion, co-selection of zinc tolerance and drug resistance is not the sole explanation for our findings.To understand the mechanisms underlying our observations of a higher diversity and an increase in MDR associated with zinc feeding, the presented project aims at:(i) Identifying genetic differences between E. coli strains isolated from the feeding and the control group of the mentioned feeding trials by whole genome sequence analysis. (ii) Characterizing and functionally proving the mechanisms responsible for the higher rate of MDR E. coli by initial whole genome sequence analysis and in vitro assays.As basis for these experiments we utilize E. coli strains that were isolated from the mentioned feeding trials and which have been extensively characterized by macrorestriction analysis (defining clones), by Multiplex-PCR (defining pathotypes) and by MLST) defining phylotypes). We thereby circumvent biases of previous studies which either performed functional investigations on E. coli K-12 lab strains only or judged on the intestinal E. coli population by random colony picking. Taking into consideration the growing knowledge on the genetic diversity of E. coli genomes, ranging in size from 4.5 to 5.5 Mbp and in gene contents from ~4,100 to ~5,800 genes, our aand in gene contents from ~4,100 to ~5,800 genes, our approach will tremendously increase the knowledge on the influence of zinc on natural E. coli populations in piglets and beyond.This proposal is a straightforward continuation of previous work performed in the institute. Most importantly, unravelling the mechanisms by which zinc increases the occurrence of MDR E. coli will have fundamental impact on feeding procedures in livestock.

• DFG Programme: Research Grants