Detection of Salmonella in environmental water and sediment by a nested-multiplex polymerase chain reaction assay

From 1995 to 2002, 53 serovars of Salmonella were isolated in the Seine estuary (France). The 3 serovars most frequently found were S. enterica serovar Typhimurium, S. enterica serovar Infantis and S. enterica serovar Virchow. A nested multiplex PCR (nm-PCR) assay was developed to detect the presence of Salmonella in estuarine water and sediment samples. The target gene used was the phase 1 flagellin fliC chromosomal gene, present in all Salmonella serovars. A set of 4 primers was first used to amplify an 890-bp sequence of the fliC gene, and then a second set of 3 primers was used for the nested PCR. The nmPCR method has been successfully tested for 28 serovars, 13 of which are of epidemiological significance. The detection limit of the assay, without any pre-enrichment step, was estimated at 1 CFU in deionized water, and at 4-5 CFU in the reaction mixture when tested on estuarine water seeded with a Salmonella strain. When the nmPCR was used together with the classical culture method in environmental samples, it gave additional positive results for 11.3% of the sediment samples and 20% of the water samples despite a high background of other bacteria. Overall, the results demonstrated that this molecular approach informed us about the contamination by Salmonella of estuarine water and sediment samples. Positive amplifications suggested the presence of Salmonella DNA and could thus provide information about a recent (culturable) or past (non-culturable, released DNA) contamination of environmental samples by this pathogenic bacteria.