Development of a fixation-free fluorescence in situ hybridization for the detection of Salmonella species.

Abstract

Salmonella is one of the most important infectious bacteria causing severe gastroenteritis and deaths in humans and animals, and the prompt diagnosis is crucial for effective control and treatment. The detection of Salmonella still depends principally on culture-based methods, which is time-consuming and laborious. Recently, polyhexamethylene biguanide (PHMB) was discovered to have cellular delivery properties and its combination with the fluorescence in situ hybridization (FISH) method was exploited for oligomer delivery and the rapid detection of Salmonella spps in this study. Cell-associated fluorescence was quantified using Volocity® 3-D image analysis software (Volocity 6.3, PerkinElmer, Inc.). PHMB complexed with fluorophore—labelled species-specific oligomers permeabilized freshly grown viable strains of Salmonella cells and mediated strong cell-associated fluorescence signals. This strategy further enabled a fixation-free protocol and hybridization in a single reaction. Using the modified FISH method, monoculture Salmonella strains were validated as well as detected in artificially contaminated water and milk within a turnaround period of 5 h. The method was observed to be comparable with the standard FISH technique (sFISH; P > 0.05). The findings suggest that fixation-free delivery and hybridization of oligomers using PHMB can provide a simplified and prompt strategy for Salmonella detection at the species level, and promote early management responses to the disease and appropriate antimicrobial therapy.