Different Swine Production Systems Can Shape Slurry Resistome at Mechanism and Class Levels Based on Swine Manure Evaluation

Abstract

Antimicrobial resistance is a major threat to public health. Antimicrobial use in animal husbandry is a major concern since it can favor an increase in antimicrobial resistance among farms. Herein, we aim to better understand and characterize the main resistome profiles in microbial communities found in pig farms. Sampling of swine manure was performed in two different timepoints (October 2019 and January 2020) in each of the 14 different swine farms, located in the mesoregion of Western Santa Catarina state in Brazil, a pole of swine product production of worldwide importance. Samples were divided into three groups: farms with the opened regimen and no usage of antimicrobials (F1; n = 10), farms with the closed regimen and usage of antimicrobials (F2; n = 16), and farms with the closed regimen and no usage of antimicrobials (F3; n = 2). The metagenomic evaluation was performed to obtain and identify genetic elements related to antimicrobial resistance using nanopore sequencing. We used ResistoXplorer software to perform composition, alpha and beta diversity, and clustering analysis. In addition, PCR reactions were performed to confirm the presence or absence of seven different beta-lactamase family genes and five phosphoethanolamine transferase gene variants clinically relevant. Our findings based on the identification of resistance genes at the mechanism level showed a prevalence of alteration of the drug target (72.3%) profile, followed by drug inactivation (17.5%) and drug efflux (10.1%). We identified predominantly aminoglycosides (45.3%), tetracyclines (15.9%), and multiclass (11,2%) resistance genes. PCoA analysis indicates differences between F1 and F2 profiles. F2 samples showed increased diversity when compared to the F1 group. In addition, herein we first report the identification of mcr-4 in a slurry sample (C1F1.1) in Santa Catarina State. In general, our findings reinforce that many factors on the practices of animal husbandry are involved in the resistome profile at the mechanism and class levels. Further studies to better understand microbiome and mobilome aspects of these elements are necessary to elucidate transmission pathways between different bacteria and environments.

Keywords: MinION nanopore device®; NGS—next-generation sequencing; One Health (OH); Pigs (Sus domesticus); animal husbandry; antimicrobial resistance (AMR); metagenomics; swine manure (SM).

Figure 1

Venn diagram representing common and exclusive genes within groups.

Figure 2

Distribution of identified resistance gene abundance and composition classified by mechanism of action of each gene. (A) Stacked bar plot of relative abundance in each sample. (B) Stacked bar plot of actual abundance in number of identified genes in each sample. (C) Stacked bar plot of relative abundance in each farm considering both samplings. (D) Stacked bar plot of actual abundance in number of identified genes in each farm considering both samplings.

Figure 3

Distribution of identified resistance gene abundance and composition classified by the antimicrobial class that it confers resistance. (A) Stacked bar plot of relative abundance in each sample. (B) Stacked bar plot of actual abundance in number of identified genes in each sample. (C) Stacked bar plot of relative abundance in each farm considering both samplings. (D) Stacked bar plot of actual abundance in number of identified genes in each farm considering both samplings.

Figure 4

Alpha diversity within groups of the antimicrobial resistance genes classes measured by observed diversity at the (A) gene level, (B) mechanism of action level, and (C) class level. All samples are represented as a dot. Horizontal boxes represent interquartile range and median. Whiskers represent the extreme ranges.

Figure 5

Heatmaps represent the frequency that is classified by (A) the mechanisms of action and (B) the antimicrobial class that the genes confer resistance. The color spectrum represents the frequency of the class or mechanism as high (red) and low (blue).

Figure 6

Ordination analysis between groups at (A) genes level, (B) mechanism of action level and (C) class level. PCoA (Principal Coordinates Analysis) using Bray-Curtis Index as distance method. Each sample is represented by a dot. Ellipses were generated for each group, except for the F3 group due to its low n.