Metagenomic Analysis of Acquired Antibiotic Resistance Determinants in the Gut Microbiota of Wild Boars (Sus Scrofa) - Preliminary Results

Abstract

Introduction: Land application of manure that contains antibiotics and resistant bacteria may facilitate the establishment of an environmental reservoir of antibiotic-resistant microbes, promoting their dissemination into agricultural and natural habitats. The main objective of this study was to search for acquired antibiotic resistance determinants in the gut microbiota of wild boar populations living in natural habitats.

Material and methods: Gastrointestinal samples of free-living wild boars were collected in the Zemplén Mountains in Hungary and were characterised by culture-based, metagenomic, and molecular microbiological methods. Bioinformatic analysis of the faecal microbiome of a hunted wild boar from Japan was used for comparative studies. Also, shotgun metagenomic sequencing data of two untreated sewage wastewater samples from North Pest (Hungary) from 2016 were analysed by bioinformatic methods. Minimum spanning tree diagrams for seven-gene MLST profiles of 104 E. coli strains isolated in Europe from wild boars and domestic pigs were generated in Enterobase.

Results: In the ileum of a diarrhoeic boar, a dominant E. coli O112ab:H2 strain with intermediate resistance to gentamicin, tobramycin, and amikacin was identified, displaying sequence type ST388 and harbouring the EAST1 toxin astA gene. Metagenomic analyses of the colon and rectum digesta revealed the presence of the tetQ, tetW, tetO, and mefA antibiotic resistance genes that were also detected in the gut microbiome of four other wild boars from the mountains. Furthermore, the tetQ and cfxA genes were identified in the faecal microbiome of a hunted wild boar from Japan.

Conclusion: The gastrointestinal microbiota of the free-living wild boars examined in this study carried acquired antibiotic resistance determinants that are highly prevalent among domestic livestock populations.

Keywords: antibiotic resistance; intestinal microbiota; natural habitat; tetracycline; wild boar.

Fig. 1

Phylum-level bacterial composition of the microbiota in three sections of the gastrointestinal tract of the diarrhoeic wild boar (WB) from Hungary analysed in this work in comparison with those established earlier for the domestic pig (DP) by a meta-analysis (13)

Fig. 2

GrapeTree diagram generated in EnteroBase based on seven-gene MLST profiles of 104 E. coli strains isolated in Europe from wild boars and domestic pigs (2, 28, 29). Countries of origin (and number of strains): Denmark (48), Hungary (8), Czech Republic (7), Spain (7), Ireland (6), Luxembourg (5), Italy (4), Switzerland (3), United Kingdom (3), Croatia (2), France (2), Germany (2), Portugal (2), Austria (1), Belgium (1), Bulgaria (1), Netherlands (1), and Sweden (1). Dark blue, light blue, and orange indicate E. coli isolates of domestic pig, wild boar, and food origin, respectively. Numbers stand for sequence types by the Achtman seven-gene MLST scheme (28). The node size in the GrapeTree diagram is proportional to the number of bacterial strains with the same sequence type (ST), and the length of edges are proportional to inter-node genetic distances (29). ST388 determined for the E. coli O112ab:H2 strain characterised in this work is marked by an asterisk (*)

Fig. 3

Immediate genetic environment of the tetracycline resistance determinants identified from the gut microbiome of free-living wild boars in Hungary. Schematic diagrams of metagenomic contigs for the tetQ (A), tetW (B), and tetO genes (C), respectively. Abbreviations for selected genes: traG – conjugal transfer protein TraG; tnp – transposase; mobC – mobilisation protein MobC; tnpV – transposon-encoded protein TnpV; and ctp – conjugal transfer protein

Fig. 4

Proportions of the sales of various veterinary antimicrobial agents in three countries of the European Union in 2015 (9) compared to those in Japan in 2013 for food producing animals (4)