Phenotypic characterization and PCR-ribotyping of Pseudomonas fluorescens isolates, in tracking contamination routes in the production line of pasteurized milk

Contamination sites of Pseudomonas fluorescens were traced in the production line for milk pasteurization in a large dairy plant in Shiraz, Iran. Samples of raw and pasteurized milk were collected at six sites along the line. All milk samples were incubated at 7°C until the aerobic plate count had reached 106-107 cfu mL-1. Colonies were picked randomly and identified. No growth of gram negative psychrotrophic bacteria (GNP) was detected in the immediately pasteurized milk samples (just after the pasteurization), during long incubation at 7°C. Recontamination most often occurred in the filling step. In this study 34.1 and 4.9% of the milk packages showed contamination with GNP and P. fluorescens, respectively. Twenty three P. fluorescens isolates were examined for phenotypic characteristics and 16S-23S PCR ribotyping. Phylogenetic analysis was conducted on the phenotypic and genotypic characteristics. The P. fluorescens isolates were shown to belong to 6 biotypes (B1-B6). The predominance of a particular ribotype was often observed for a given biotype, although there were two ribotypes in each of the B2 and B6 biotypes. The 16S-23S PCR-ribotyping technique allowed differentiation between the isolates. Based on this method, the isolates belonged to 5 subtypes. Phylogenetic analysis based on 16S-23S PCR-ribotyping and phenotypic characterization could be helpful in tracking contamination routes in the production line for milk pasteurization.