Comprehensive Identification of Drug-Resistant Bacteria in the Environment Using a Highly Sensitive Fluorescence In Situ Hybridization Technique and a Fluorescent Activated Cell Sorting

Abstract

 A comprehensive culture-independent analysis of drug-resistant bacteria linking drug-resistant genes derived from plasmid DNA with bacterial species has been developed using a combination of high-sensitivity fluorescence in situ hybridization (CARD-FISH) and fluorescence-activated cell sorting (FACS). A model experiment targeting the Class A β-lactamase TEM-1 gene employing E. coli cells and the pCR2.1 TOPO plasmid vector yielded fluorescent detection of E. coli cells harboring the TEM-1 gene. The application of this method to the activated sludge sample and gene quantification by means of real-time PCR led to a 6.2- to 45.3-fold enrichment of the TEM-1 gene in comparison to the pre-sort sample. The heightened prevalence of class Bacilli in the post-sorting samples suggested that the bacteria belonging to class Bacilli constituted the principal pool of TEM-1-harboring drug-resistant bacteria in the activated sludge.