Abstract
Plasmid conjugation is one of the dominant mechanisms of horizontal gene transfer, playing a noticeable role in the rapid spread of antibiotic resistance genes (ARGs). Broad host range plasmids are known to transfer to diverse bacteria in extracted soil bacterial communities when evaluated by filter mating incubation. However, the persistence and dissemination of broad range plasmid in natural soil has not been well studied. In this study, Pseudomonas putida with a conjugative antibiotic resistance plasmid RP4 was inoculated into a soil microcosm, the fate and persistence of P. putida and RP4 were monitored by quantitative PCR. The concentrations of P. putida and RP4 both rapidly decreased within 15-day incubation. P. putida then decayed at a significantly lower rate during subsequent incubation, however, no further decay of RP4 was observed, resulting in an elevated RP4/P. putida ratio (up to 10) after 75-day incubation, which implied potential transfer of RP4 to soil microbiota. We further sorted RP4 recipient bacteria from the soil microcosms by fluorescence-activated cell sorting. Spread of RP4 increased during 75-day microcosm operation and was estimated at around 10-4 transconjugants per recipient at the end of incubation. Analysis of 16S rRNA gene sequences of transconjugants showed that host bacteria of RP4 were affiliated to more than 15 phyla, with increased diversity and shift in the composition of host bacteria. Proteobacteria was the most dominant phylum in the transconjugant pools. Transient transfer of RP4 to some host bacteria was observed. These results emphasize the prolonged persistence of P. putida and RP4 in natural soil microcosms, and highlight the potential risks of increased spread potential of plasmid and broader range of host bacteria in disseminating ARGs in soil.
Highlights
The global increased antimicrobial resistance level in human pathogens has posed significant threat to human health (UNEP, 2017)
The massive use of antibiotics has resulted in high concentration of antibiotic residues in the environments, which may serve as a selection pressure that promote the dissemination of antibiotic resistance genes (ARGs) among environmental bacteria, including pathogenic species (Pruden et al, 2012; Guo et al, 2018)
The copy numbers of 16S rRNA gene in soil with KT2442/RP4 inoculation and control soil were at similar levels (3.3–4.9 × 109 copies g−1 dry soil), and they both remained relatively stable during the incubation
Summary
The global increased antimicrobial resistance level in human pathogens has posed significant threat to human health (UNEP, 2017). Plasmid conjugation is one of the most important mechanisms of HGT (Sorensen et al, 2005; Thomas and Nielsen, 2005), contributing to the wide spread of ARGs among environmental bacteria and human pathogens. Conjugative plasmids are often implicated in the rapid propagation and proliferation of ARGs (Dodd, 2012) These plasmids contain genes for the formation of pilus to transfer copies of themselves to other bacteria, enabling horizontal transfer of complete set of genes on the plasmid, including ARGs (Willetts and Wilkins, 1984; Willey et al, 2008). The massive use of antibiotics has resulted in high concentration of antibiotic residues in the environments, which may serve as a selection pressure that promote the dissemination of ARGs among environmental bacteria, including pathogenic species (Pruden et al, 2012; Guo et al, 2018). Conjugative plasmids selected with antibiotics have been found to persist among bacterial populations or invade new strains even without antibiotics pressure (Dionisio et al, 2005)
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