Investigating horizontal gene transfer in untreated hospital wastewaters

SANNAZZARO, ANALIA INÉS; MILANI, STEFAN; RIBER, LEISE; SØRENSEN, SØREN

Copenhague

Simposio; Copenhagen Microbiology Center Symposium; 2012

Copenhagen Microbiology Center

Background: The role of horizontal gene transfer in evolution and emergence has always been considered very significant, but with the global emergence and rapid spread of multiple antibiotic resistance, it has become clear that the importance might be even larger than previously anticipated. Knowing that the hospital doesn?t treat the wastewater containing noumerous antibiotics it would be interesting to investigate the capability of wastewater being a hotspot for HGT in regards of the uptake of plasmids harbouring antibiotic resistance genes, as well as the taxonomical identity of the putative bacterial recipients. Aim: To investigate the plasmid uptake ability among non-treated hospital wastewater bacteria Methodology: Strains with three different plasmids (IncP, IncX and IncW) were used as donors in conjugation assays with wastewater bacteria. The donor strains were chromosomally marked with the gene encoding the repressor of the lacZ promotor, lacIq. Plasmids were tagged with the gfp reporter gene under control of the lacZ promotor. This leads to GFP expression in the plasmid receiving cells on a background of GFP-repressed donor cells. Filter mating assays were done and using flow cytometry wastewater transconjugants were detected and sorted for 16S sequencing, avoiding in this way, the drawbacks of culturability. Results: Transconjuganst were obtained with the three constructs, but only the IncP plasmid had sufficient high transfer rates to be used in the sorting experiments. DNA from transconjugants was used for 16S sequencing in order to identify the host range of this plasmid. This analysis is still in progress.