Bloom of resident antibiotic-resistant bacteria in soil following manure fertilization

The increasing prevalence of antibiotic-resistant bacteria is a global threat to public health. Agricultural use of antibiotics is believed to contribute to the spread of antibiotic resistance, but the mechanisms by which many agricultural practices influence resistance remain obscure. Although manure from dairy farms is a common soil amendment in crop production, its impact on the soil microbiome and resistome is not known. To gain insight into this impact, we cultured bacteria from soil before and at 10 time points after application of manure from cows that had not received antibiotic treatment. Soil treated with manure contained a higher abundance of β-lactam–resistant bacteria than soil treated with inorganic fertilizer. Functional metagenomics identified β-lactam–resistance genes in treated and untreated soil, and indicated that the higher frequency of resistant bacteria in manure-amended soil was attributable to enrichment of resident soil bacteria that harbor β-lactamases. Quantitative PCR indicated that manure treatment enriched the bla_CEP-04 gene, which is highly similar (96%) to a gene found previously in a Pseudomonas sp. Analysis of 16S rRNA genes indicated that the abundance of Pseudomonas spp. increased in manure-amended soil. Populations of other soil bacteria that commonly harbor β-lactamases, including Janthinobacterium sp. and Psychrobacter pulmonis, also increased in response to manure treatment. These results indicate that manure amendment induced a bloom of certain antibiotic-resistant bacteria in soil that was independent of antibiotic exposure of the cows from which the manure was derived. Our data illustrate the unintended consequences that can result from agricultural practices, and demonstrate the need for empirical analysis of the agroecosystem.Agriculture affects human health through both the consumption and production of food for the human diet. Manure from pig and cattle farms is commonly used as a substitute for inorganic nitrogen and phosphorus fertilizers for agricultural crops worldwide, especially in organic farming practices (1–6). With the increasing consumer demand for organically produced food, the use of animal manure, which conforms to organic conventions, will likely increase in the future. According to the National Organic Program, raw manure may be used up to 90–120 d before harvest, depending on the crop, and composted manure may be applied at any time. There are no restrictions on the source of manure (1).Animal manure is an important reservoir of antibiotic-resistant bacteria, antibiotic-resistance genes (collectively known as the “resistome”), and pathogens (2, 7–12). Although antibiotic use increases antibiotic-resistance genes and resistant bacteria in manure (13–16), antibiotic-resistant bacteria are also abundant in manure from animals with no history of antibiotic treatment, indicating the natural presence of bacteria intrinsically resistant to antibiotics in animal gastrointestinal tracts (2, 17, 18).There is increasing concern about the use of manure as an agricultural amendment because of its possible contribution to the pool of resistance genes to resident soil bacteria and pathogens (2, 19). Antibiotic-resistance genes from the soil resistome can enter the food chain via contaminated crops or groundwater (5, 20), and have potential consequences for human health if transferred to human pathogens. Studies assessing the impact of fertilization with pig manure on the soil resistome have shown that excessive application of manure from farms with intensive sulfonamide use can lead to an increase of antibiotic-resistance genes in soil (2, 3); however, most studies have found that such increases are transient when the manure is applied at recommended rates (2, 21, 22). Cow manure from dairy farms, which use β-lactam antibiotics predominantly to prevent and treat diseases (23), is commonly used in crop production, but its impact on the soil resistome has yet to be investigated.Along with its impact on the soil resistome, the application of manure can affect the composition and functional properties of soil microbial communities, as has been demonstrated by community fingerprinting (21, 24). Recent advances in DNA-based analysis, such as metagenomics and quantitative PCR (qPCR), offer greater precision in such studies, enabling identification of affected community members (25) and their resistance genes (4).In the present study, we assessed the impact of cow manure on the composition and resistance profiles of bacterial communities in soil. Our results show that manure from cows that had not been treated with antibiotics increased the populations of resident soil bacteria harboring genes for resistance to β-lactam antibiotics, whereas inorganic fertilizers did not. These results demonstrate the complexity, and at times nonintuitive consequences, of agricultural practices.