Intensified livestock farming increases antibiotic resistance genotypes and phenotypes in animal feces

Animal feces, a significant reservoir of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB), pose a considerable threat to environmental health. The One Health approach underscores the interconnectedness of human, animal, and environmental health, highlighting the urgency of addressing antibiotic resistance in farm waste. Intensive farming practices, driven by growing populations, lead to increased antibiotic use in livestock, resulting in substantial ARG excretion via feces. This study focuses on the impact of farming intensification on the fecal resistome, considering both genotypic (ARGs and MGEs) and phenotypic resistance. The study area is the southwestern rim of the Qinghai-Tibet Plateau in China, a region experiencing a shift from traditional free-range farming to more intensive systems. This transition necessitates understanding its influence on antibiotic resistance development.

Prior research has demonstrated the high abundance of ARGs in feces from intensive livestock operations, significantly exceeding background levels in soil. Studies have identified hundreds of ARGs in feces from various animals, conferring resistance to multiple antibiotic classes. However, a clear gap exists in understanding how fecal resistance changes across different farming intensities. While the impact of intensive pig and dairy farming has been studied, the effect on various livestock types across an intensification gradient has received less attention. Existing studies have compared conventional, antibiotic-free, and organic farming practices, yielding varying results on ARG abundance. Some studies show a lower prevalence of resistance in antibiotic-free systems, while others challenge the effectiveness of such practices. There is a need for comparative studies that utilize both culture-dependent and culture-independent methods to fully capture the resistome, particularly in developing countries where intensified farming is rapidly expanding.

This study compared antibiotic resistance genotypes and phenotypes in fecal samples from eight Chinese farms. Farms represented three farming intensities (free-range, small-scale, and intensive) and four animal types (yak, sheep, pig, and horse). High-throughput qPCR was used to profile the fecal resistomes (317 ARGs and 57 MGEs), with relative and absolute abundances determined. 16S rRNA gene sequencing characterized bacterial communities, identifying potential pathogens using a bacterial pathogen database and predicting OTU phenotypes using BugBase. Shotgun metagenome sequencing further analyzed ARGs and MGEs, examining ARG-MGE co-location on contigs and identifying potential bacterial hosts. The resistance phenotypes of 1324 bacterial isolates were assessed using a disc diffusion assay, determining minimum inhibitory concentration (MIC) values and multidrug resistance indices. Statistical analyses included variation partitioning analysis (VPA), random forest algorithms to predict ARG-bacteria associations, co-occurrence analysis of ARGs and resistance phenotypes, and phylogenetic signal analysis using Blomberg's K. Soil samples served as a control for phenotypic resistance comparisons.

Intensive farms had higher levels of micro- and meso-elements (calcium, magnesium, sulfur) and heavy metals (copper, cadmium, zinc) in feces compared to free-range farms. Higher concentrations of various antibiotics were detected in feces from intensive farms. Fecal samples from intensive farms harbored significantly more ARGs and MGEs than free-range samples. Intensive pig farms showed the highest number of ARGs (137 on average), significantly higher than free-range yaks (34 on average). The relative abundance of ARGs conferring resistance to aminoglycosides, macrolide-lincosamide-streptogramin B (MLSB), multidrug, sulfonamides, and tetracyclines increased with farming intensification. MGE abundance also increased with intensification, with a strong positive correlation between ARG and MGE abundance (R=0.826, p<0.001). Bacterial community composition differed significantly across farming intensities, with intensive farms exhibiting a higher abundance of bacteria carrying MGEs. Variation partitioning analysis revealed that farming intensity, followed by animal species, explained the most variation in ARG abundance. Machine-learning analysis showed that pathogens, rather than dominant genera, were stronger predictors of ARG presence, particularly multidrug resistance genes. Metagenomic analysis confirmed the higher proportions of ARGs and MGEs in intensive farms, with ARGs more frequently co-located with MGEs on the same contigs (27.38% in captive farms vs. 17.52% in free-range farms). Intensive pig farms showed the highest co-occurrence of ARGs and MGEs. Resistance phenotype profiling showed higher resistance in isolates from captive animals than in free-range animals or soil samples, particularly for sulfonamides, tetracyclines, and aminoglycosides. Isolates from intensive farms showed higher levels of multidrug resistance. Phylogenetic analysis showed a reduced dependence of resistance phenotypes on bacterial phylogeny in intensive systems, suggesting increased horizontal gene transfer.

The findings strongly support the hypothesis that intensified livestock farming significantly increases both genotypic and phenotypic antibiotic resistance in animal feces. The increased abundance of ARGs and MGEs, coupled with higher antibiotic concentrations in feces from intensive farms, points to the role of antibiotic selection pressure and horizontal gene transfer. The greater prevalence of multidrug resistance, particularly associated with pathogenic bacteria, highlights the potential public health implications. The decoupling of resistance phenotypes from bacterial phylogeny in intensive systems further supports the significance of horizontal gene transfer in shaping antibiotic resistance. This study underscores the need for interventions to mitigate antibiotic resistance associated with intensified livestock farming, considering both waste management and antibiotic use practices. The findings highlight the interconnectedness of antibiotic use, environmental contamination, and the emergence of multidrug-resistant pathogens.

Intensified livestock farming significantly increases antibiotic resistance in animal feces, both genotypically and phenotypically, primarily affecting resistance to aminoglycosides, tetracyclines, and sulfonamides. This increase is strongly linked to elevated MGE abundance and co-location with ARGs, facilitating horizontal gene transfer and multidrug resistance, especially within pathogenic bacteria. Future research should focus on developing sustainable farming practices to reduce antibiotic use and improve waste management, minimizing the environmental dissemination of antibiotic resistance. Further investigations into the fitness costs associated with resistance development and the specific mechanisms of horizontal gene transfer in these systems are warranted.

The study was conducted in a specific geographic region in China, and findings may not be directly generalizable to other regions or farming systems. The number of farms sampled per category was relatively small, potentially limiting statistical power. While soil samples provided a control for phenotypic resistance, environmental factors beyond those directly measured could influence the observed patterns. The culturing method might not capture the entire spectrum of bacterial diversity and resistance, potentially leading to an underestimation of the total resistance present.