Abundance of Antibiotic Resistance Genes in Bacteriophage following Soil Fertilization with Dairy Manure or Municipal Biosolids, and Evidence for Potential Transduction

Abstract

Animal manures and municipal biosolids recycled onto crop production land carry antibiotic-resistant bacteria that can influence the antibiotic resistome of agricultural soils, but little is known about the contribution of bacteriophage to the dissemination of antibiotic resistance genes (ARGs) in this context. In this work, we quantified a set of ARGs in the bacterial and bacteriophage fractions of agricultural soil by quantitative PCR. All tested ARGs were present in both the bacterial and phage fractions. We demonstrate that fertilization of soil with dairy manure or human biosolids increases ARG abundance in the bacterial fraction but not the bacteriophage fraction and further show that pretreatment of dairy manure can impact ARG abundance in the bacterial fraction. Finally, we show that purified bacteriophage can confer increased antibiotic resistance to soil bacteria when combined with selective pressure. The results indicate that soilborne bacteriophage represents a substantial reservoir of antibiotic resistance and that bacteriophage could play a significant role in the horizontal transfer of resistance genes in the context of an agricultural soil microbiome. Overall, our work reinforces the advisability of composting or digesting fecal material prior to field application and suggests that application of some antibiotics at subclinical concentrations can promote bacteriophage-mediated horizontal transfer of ARGs in agricultural soil microbiomes. Overuse of antibiotics has contributed to the spread of antibiotic resistance due to the release of antibiotics, antibioticresistant bacteria, and antibiotic resistance genes (ARGs) into the environment (1). This phenomenon is mediated by horizontal gene transfer (HGT) of mobile genetic elements-such as plasmids, transposons, and integrons-between bacterial cells through conjugation and viral transduction (2-4).Transduction has been shown to occur in environmental matrices, including freshwater and wastewater; moreover, bacterial 16S ribosomal RNA sequences have been observed in the viral fraction of wastewater, confirming the ability of bacteriophage to carry bacterial genes (5, 6). In fact, while much attention has been paid to conjugation, more recent work has additionally implicated bacteriophage as a major vehicle for horizontal gene transfer and recombination (7-10).Using high-throughput sequencing of murine fecal phage populations, Modi et al. (11) demonstrated that antibiotic treatment leads to the enrichment of genes conferring resistance to the administered drug as well as to unrelated antibiotics in the phage genome. Furthermore, bacteriophage from drug-treated mice provided cultured naive microbiota with increased levels of resistance to the corresponding drug. Overall, this work concluded that antibiotic residues potentiate the transduction-mediated dissemination of ARGs.Antibiotic resistance genes have been found in the bacteriophage DNA fraction of various environmental matrices, such as activated sludge (12), urban sewage and river water (13), an...

Phages in the Human Body

“…The fact that phage capsids can mobilize bacterial DNA has multiple consequences, such as, for example, the fact that they can mobilize and transduce virulence genes (O’Brien et al, 1984; Griffiths et al, 2000; Allué-Guardia et al, 2011; Penades et al, 2015), antibiotic resistances (Muniesa et al, 2004; Colomer-Lluch et al, 2011b; Ross and Topp, 2015; Haaber et al, 2016) or genes related to fitness (Lindell et al, 2004; Müller et al, 2013) to new bacterial hosts. This causes horizontal genetic exchange and leads to the evolution of bacterial populations.…”

Genomics and the evolution of antibiotic resistance

“…37 More generally, metagenomics will be useful in understanding the dynamics of lateral transfer of antibiotic resistance in managed ecosystems. 166,167 It will be particularly important in tracking resistance in farm animals, 168,169 understanding the linkages between resistance elements, 170 and under-standing the modes by which resistance genes are shared between humans and their domestic animals. 171 New hotspots for generation of resistance elements will be identified, such as the potential for aquaculture facilities to be incubators of resistance.…”

Translational metagenomics and the human resistome: confronting the menace of the new millennium

“…The analysis of VLPs in human faeces from five individuals indicated levels of approximately 10 8 –10 9 VLPs per gramme faeces [56]. ARGs in bacteriophages can be detected by quantitative real-time PCR ([6, 9, 57]), as demonstrated in a study in which the presence of clinically relevant resistance genes was determined in the bacteriophage fraction of stool samples from 80 healthy individuals. In 77 % of the samples, at least one ARG could be detected.…”