Rapid identification and enumeration of antibiotic resistant bacteria in urban canals by microcolony-fluorescence in situ hybridization

The abundance and phylogenetic composition of antibiotic resistant bacteria in canals of metropolitan Bangkok, Thailand, were investigated using a microcolony method and fluorescence in situ hybridization (FISH). Cells were directly trapped from aquatic samples onto polycarbonate membranes and incubated for 24 hr on selective agar containing antibiotics. Individual antibiotic resistant bacterial microcolonies samples were classified on the filter using FISH with rRNA-targeted probes. The numbers of microcolony forming units (mCFU) on selective medium containing antibiotics were 0.5 to 8.1-fold (average, 3.4-fold) higher than those of colony forming units (CFU) in all samples, and mCFU and CFU closely correlated in all samples (r² = 0.89). Estimates of Escherichia coli (E. coli) determined by FISH with rRNA-targeted probe accounted for approximately 1% of bacteria detectable by probe EUB338 among microcolony-forming bacteria on nonselective medium. However, they accounted for approximately 10% of bacteria detectable by probe EUB338 among microcolony-forming norfloxacin/tetracycline-resistant bacteria. Microcolony-FISH on selective medium containing antibiotics would be a valuable tool that could help in obtaining information about the numbers and phylogenetic affiliations of yet-to be-cultured antibiotic-resistant bacteria in aquatic environments.