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On-Farm Anaerobic Digestion of Dairy Manure Reduces the Abundance of Antibiotic Resistance-Associated Gene Targets and the Potential for Plasmid Transfer.

ABSTRACT: The present study investigated the impact of on-farm anaerobic digestion on the abundance of enteric bacteria, antibiotic resistance-associated gene targets, and the horizontal transfer potential of extended-spectrum β-lactamase (ESBL) genes. Samples of raw and digested manure were obtained from six commercial dairy farms in Ontario, Canada. Digestion significantly abated populations of viable coliforms in all six farms. Conjugative transfer of plasmids carrying β-lactamase genes from manure bacteria enriched overnight with buffered peptone containing 4 mg/liter cefotaxime into a β-lactam-sensitive green fluorescent protein (GFP)-labeled Escherichia coli recipient strain was evaluated in patch matings. Digestion significantly decreased the frequency of the horizontal transfer of ESBL genes. Twenty-five transconjugants were sequenced, revealing six distinct plasmids, ranging in size from 40 to 180 kb. A variety of ESBL genes were identified: bla_CTX-M-1, bla_CTX-M-14, bla_CTX-M-15, bla_CTX-M-27, bla_CTX-M-55, and bla_PER-1. bla_CTX-M-15 was the most prevalent ESBL gene detected on plasmids harbored by transconjugants. Various mobile genetic elements were found located proximal to resistance genes. Ten gene targets, including sul1, str(A), str(B), erm(B), erm(F), intI1, aadA, incW, bla_PSE, and bla_OXA-20, were quantified by quantitative PCR on a subset of 18 raw and 18 digested samples. Most targets were significantly more abundant in raw manure; however, erm(B) and erm(F) targets were more abundant in digested samples. Overall, on-farm digestion of dairy manure abated coliform bacteria, a number of antibiotic resistance-associated gene targets, and the potential for in vitro conjugation of plasmids conferring resistance to extended-spectrum β-lactams and other classes of antibiotics into E. coli CV601. IMPORTANCE Using livestock manure for fertilization can entrain antibiotic-resistant bacteria into soil. Manure on some dairy farms is anaerobically digested before being land applied. Recommending the widespread implementation of the practice should be founded on understanding the impact of this treatment on various endpoints of human health concern. Although lab-scale anaerobic treatments have shown potential for reducing the abundance of antibiotic resistance genes, there are very few data from commercial farms. Anaerobic digestion of manure on six dairy farms efficiently abated coliform bacteria, E. coli, and a majority of antibiotic resistance-associated gene targets. In addition, the conjugation potential of plasmids carrying ESBL genes into introduced E. coli strain CV601 was reduced. Overall, anaerobic digestion abated coliform bacteria, the genes that they carry, and the potential for ESBL-carrying plasmid transfer.

SUBMITTER: Tran TT

PROVIDER: S-EPMC8231723 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study employed high-throughput quantitative PCR and 16S rRNA sequencing to evaluate the effect of temperature and residual antibiotics on the dynamics of antibiotic resistance genes (ARGs) and microbial communities during anaerobic digestion of swine manure. The abundances of total ARGs and 16S rRNA genes significantly decreased in all of four treatments (25°C, 37°C, and 37°C with 50 mg of wet weight antibiotics of body weight, and 55°C). The abundances of most ARG types were significantly correlated with those of the 16S rRNA gene and transposase gene (P < 0.01). However, the abundances of total ARGs at 55°C were much higher than those of other treatments. Meanwhile, the microbial communities at 55°C, where the Streptococcus pathogen remained at a relatively high abundance and cellulose degraders and hydrogen producers, such as Ethanoligenens and Coprococcus bacteria, increased, were markedly different from those of other treatments. Redundancy analysis indicates that temperature, pH, and the genus Streptococcus had the highest explanation for ARG variation among experimental factors, chemical properties, and representative genera, respectively. Network analysis further showed that the genus Streptococcus contributed greatly to the higher ARG abundance at 55°C. The moderate antibiotic residue only caused a slight and transitory inhibition for microbially diverse populations and promotion for ARG abundance, probably due to the degradation of antibiotics and microbial adaptability. Our results clarify the cooperativity of gene transfer-related items on ARG variation and intensively prove that higher temperature cannot always achieve better ARG removal in anaerobic digestion unless pathogens and gene transfer elements are more efficiently inhibited.IMPORTANCE Antibiotic resistance genes (ARGs) are frequently detected with high abundance in manure-applied soils. Anaerobic digestion is one of widely used processes for animal waste treatment. Thus, it is critical to understand the potential of anaerobic digestion to attenuate ARGs. Although some previous studies recommended thermophilic digestion for ARG removal, they did not get sufficient evidence to support this view. The antibiotics applied to animals are mostly excreted through feces and urine because of incomplete metabolism. It is indispensable to know whether residual antibiotics in manure will hinder ARG attenuation in anaerobic digesters. The significance of our research is in comprehensively understanding the evolution and mechanism of ARGs in anaerobic digestion of swine manure affected by temperature and residual antibiotics, which will allow the development of an ARG elimination strategy before their release into the environment.

Project description:Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.

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On-Farm Anaerobic Digestion of Dairy Manure Reduces the Abundance of Antibiotic Resistance-Associated Gene Targets and the Potential for Plasmid Transfer.

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