Development of a Multiplex PCR Assay for Detection of Pseudomonas fluorescens with Biofilm Formation Ability

Under the cold storage and processing conditions of raw milk, the psychrotrophic Pseudomonas fluorescens is usually found as predominant bacteria causing its spoilage. In this study, a multiplex PCR assay was developed for rapid and selective detection of P. fluorescens with biofilm formation ability. The target sequences were 2 genes (adnA and fliC) related to biofilm formation and flagella biosynthesis of P. fluorescens. The specificity of the mPCR assay was evaluated with 7 reference strains, isolated from raw milk, belonging to P. fluorescens, Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas putida, Pseudomonas monteilii, and 2 unclassified Pseudomonas species (Pseudomonas sp1 and Pseudomonas sp8). The detection limit for the target strain was 10² CFU/mL. Seventy-three strains were evaluated by the mPCR assay. The adnA gene was detected in 23 strains while fliC gene was detected in only 3 strains. However, both target genes (adnA and fliC) were detected by amplification in 12 strains belonging to P. fluorescens species. The biofilm formation ability of P. fluorescens following cultivation in 10% UHT milk at 30 °C or 4 °C were evaluated by the microtiter plate assay. The result showed that all the P. fluorescens strains with the target gene (adnA or fliC, or both 2 genes) had the biofilm-forming ability. The phylogenetic analysis showed that adnA gene tree had a higher resolution than rpoB tree, and the strains in adnA phylogenetic dendrogram could be divided into 4 different groups according with the matrix of their biofilm-forming ability. The results indicated a promising use of adnA gene as a taxonomic marker for subdividing P. fluorescens.

A mPCR assay targeting adnA and fliC genes showed rapid and reliable detection of P. fluorescens with biofilm formation ability, which could be useful to detect this contamination in milk samples.