Application of miniaturized most probable number method for bacterial detection in water samples: detection of multi-drug-resistant Ralstonia insidiosa in drinking water

Abstract

ABSTRACT The detection of bacterial contamination in drinking water is essential for monitoring the spread of foodborne diseases. We developed a simple, portable, and low-cost method of mini most probable number (mini MPN) to semi-enumerate bacterial suspension in water as a drinking water analogue. In this study, there is no significant difference between mini MPN and the standard method, technique plate count (TPC), at 10 and 100 CFU/ml Klebsiella pneumoniae suspension with a P-value of 0.28. For the ease-of-use aspect of this method, we tested several variables to prove it can be mass-applied in society. The usage of a sterile-plastic pipette, sample inoculation conducted in a biosafety cabinet (BSC), the usage of a 3-month storage medium, and incubation temperature conducted at room temperature compared to aseptic standard laboratory technique showed P-value > 0.05. In a trial for this method, we used commercialized drinking water for bacterial enumeration and characterization. We found multi-drug resistant (MDR) Ralstonia insidiosa which was resistant to at least four antimicrobial classes, including aminoglycosides, penicillins, cephalosporin, and carbapenem. Vitek 2 Compact was used for bacterial identification and antimicrobial susceptibility testing. A virulence test in Omphisa fuscidentalis larvae showed R. insidiosa strain D had a low virulence.