In situ enumeration and localization of the probiotic Pediococcus sp strain Ab1 in the gut of abalone Haliotis gigantea

In this study, we established an optimized fluorescence in situ hybridization (FISH) protocol for detecting and enumerating host-derived probiotic Pediococcus sp. strain Ab1 cells in the gut of abalone. Samples were fixed with 4 % paraformaldehyde and hybridized at 40 A degrees C for 3 h using a TAMRA (5-carboxytetramethylrhodamine)-labeled probe (Rpt probe). Strain Ab1 could be clearly discriminated from other control bacteria (Enterococcus, Lactobacillus). Fluorescence signals of Rpt-positive bacteria were easily distinguished from any detectable background noise. Rpt-positive bacteria were detected in both probiotic-supplemented and control abalone; nevertheless, the number of Rpt-positive bacteria in probiotic-supplemented abalone was two orders of magnitude higher than in controls. Furthermore, the mean number of Rpt-positive bacteria (probiotic-supplemented abalone) in the gut-attached section was also two orders of magnitude higher than in the free-living section. Rpt-positive micro-colonies (MCs) were observed by thin-section FISH analysis only in probiotic-supplemented abalone, and these MCs were observed in contact with intestinal epithelial cells. These results indicate that the probiotic Pediococcus sp. strain Ab1 can colonize or adhere to the abalone digestive tract in numbers sufficient for therapeutic effects. The data obtained using our FISH method provide clear and direct evidence of the formation of probiotic colonies in the gut of supplemented abalone.

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