Multi-omics surveillance of antimicrobial resistance in the pig gut microbiome

Abstract

Background: High-throughput sequencing technologies play an increasingly active role in the surveillance of major global health challenges, such as the emergence of antimicrobial resistance. The post-weaning period is of critical importance for the swine industry and antimicrobials are still required when infection occurs during this period. Here, two sequencing approaches, shotgun metagenomics and metatranscriptomics, have been applied to decipher the effect of different treatments used in post-weaning diarrhea on the transcriptome and resistome of pig gut microbiome. With this objective, a metagenome-assembled genome (MAG) catalogue was generated to use as a reference database for transcript mapping obtained from a total of 140 pig fecal samples in a cross-sectional and longitudinal design to study differential gene expression. The different treatments included antimicrobials trimethoprim/sulfamethoxazole, colistin, gentamicin, and amoxicillin, and an oral commercial vaccine, a control with water acidification, and an untreated control. For metatranscriptomics, fecal samples from pigs were selected before weaning, three days and four weeks post-treatment.

Results: The final non-redundant MAGs collection comprised a total of 1396 genomes obtained from single assemblies and co-assemblies per treatment group and sampling time from the metagenomics data. Analysis of antimicrobial resistance genes (ARGs) at this assembly level considerably reduced the total number of ARGs identified in comparison to those found at the reads level. Besides, from the metatranscriptomics data, half of those ARGs were detected transcriptionally active in all treatment groups. Differential gene expression between sampling times after treatment found major number of differential expressed genes (DEGs) against the group treated continuously with amoxicillin, with DEGs being correlated with antimicrobial resistance. Moreover, at three days post-treatment, a high number of significantly downregulated genes was detected in the group treated with gentamicin. At this sampling time, this group showed an altered expression of ribosomal-related genes, demonstrating the rapid effect of gentamicin to inhibit bacterial protein synthesis.

Conclusions: Different antimicrobial treatments can impact differently the transcriptome and resistome of microbial communities, highlighting the relevance of novel sequencing approaches to monitor the resistome and contribute to a more efficient antimicrobial stewardship.

Keywords: Antimicrobial resistance; Metagenome-assembled genomes; Metatranscriptomics; Post-weaning; Swine.

Fig. 1

Experimental design describing the seven different treatment groups (G1–G6, GG) sampled at four sampling times (ST1–ST4). Metagenomics sequencing was performed from DNA extracted from the faeces of ten animals per group at all sampling times, while metatranscriptomics sequencing was performed from RNA extracted from the faeces of seven animals per group at three sampling times

Fig. 2

A. Percentage of completeness and contamination means per treatment group and sampling time for both single assembly (SA) bins and final refined bins from single assemblies and co-assemblies (SCOA). B. Taxonomic classification at the phylum level of SCOA per treatment group and sampling times. C. Percentage of completeness and contamination means of the final bins included in the metagenome-assembled genome (MAG) collection refined from 280 pig faecal samples. D. Taxonomic classification at the phylum level of the final bins included in the MAG collection. E. Phylogenetic reconstruction of the collection of MAGs. Different colours (same as panel D) indicate different phyla and layers indicate phyla, presence (purple) or absence (white) of resistance genes identified in each MAG, completeness (grey gradient scale), contamination (red gradient scale), and genome size (grey bars). For Bacillota, s.s. are sensu stricto. G1: trimethoprim/sulfamethoxazole, G2: colistin, G3: oral vaccination, G4: gentamicin, G5: untreated control with water acidification, G6: untreated control, GG: amoxicillin (farm of origin). ST1: one day before weaning, ST2: three days post-treatment, ST3: two weeks post-treatment, ST4: four weeks post-treatment

Fig. 3

A. Principal component analysis (PCA) of metatranscriptomic expression profiles from different treatment groups and sampling times. B. Functional traits of the differential expressed genes (DEGs) against the gentamicin-treated group (G4) at three days post-treatment (ST2) at a 1% significance level (P-adj < 0.01). G1: trimethoprim/sulfamethoxazole, G2: colistin, G3: oral vaccination, G4: gentamicin, G5: untreated control with water acidification, G6: untreated control, GG: amoxicillin (farm of origin). ST1: one day before weaning, ST2: three days post-treatment, ST4: four weeks post-treatment

Fig. 4

A. Significant differentially expressed genes (P-adj < 0.01) associated with resistance specific terms at three days post-treatment (ST2) in comparison to before weaning (ST1) for all treatment groups. G1: trimethoprim/sulfamethoxazole, G2: colistin, G3: oral vaccination, G4: gentamicin, G5: untreated control with water acidification, G6: untreated control, GG: amoxicillin (farm of origin). B. Significant differentially expressed genes (P-adj < 0.01) associated with resistance specific terms between sampling times for the group that remained at the farm of origin treated with amoxicillin (GG). ST1: one day before weaning, ST2: three days post-treatment, ST4: four weeks post-treatment

Fig. 5

Expression levels of ARGs to the different antibiotic classes represented in a logarithmic scale of transcripts per million (TPM) of transcriptomic reads by treatment group and sampling times. Only ARGs found in at least 2% of the samples are represented. G1: trimethoprim/sulfamethoxazole, G2: colistin, G3: oral vaccination, G4: gentamicin, G5: untreated control with water acidification, G6: untreated control, GG: amoxicillin (farm of origin). ST1: one day before weaning, ST2: three days post-treatment, ST4: four weeks post-treatment