Abundance dynamics and sequence variation of neomycin phosphotransferase gene (nptII) homologs in river water

Abstract

Monitoring the abundance and dynamics of antibiotic resistance genes is essential for assessing their potential effects on environmental and human health. The objectives of the present study were to investigate: (1) the abundance of neomycin phosphotransferase (nptII) gene homologs in water samples collected monthly from the South Saskatchewan River (Canada) for 24 mo, and (2) sequence variation of the cloned nptII gene homologs retrieved from the river. DNA from river micro- bial communities was used to transform a natural competent Pseudomonas stutzeri strain containing a truncated nptII gene on a plasmid (P. stutzeri pMR7). Of the 24 water samples, the recovery of kanamycin resistant (Km R ) transformants from DNA of 4 samples indicated the presence of nptII gene homologous sequences. However, the natural transformation process required ~3.2 × 10 4 copies of the nptII gene to recover a single Km R transformant. To overcome the limitation of detection, a real- time PCR assay using SYBR Green I was developed to quantify the abundance of nptII gene homolo- gous sequences in river microbial communities. The results showed that nptII gene homologous sequences were present in the river, and their abundance varied over the course of this study, rang- ing from undetectable levels to 4.36 × 10 6 copies l -1 water. Furthermore, nptII gene homologous sequences amplified from river microbial community DNA were cloned, and unique clones were sequenced. Comparison of the nucleotide and deduced amino acid sequences of the cloned frag- ments to those of the nptII gene on transposon Tn5 showed that they had over 96% homology.