Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed mil k were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases f or each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) ( cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E), Isolates within each cluster also displayed similar enzyme activities, Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isola tes in cluster B were predominantly positive for all three enzyme activitie s; and isolates in cluster E were negative for lecithinase but predominantl y positive for protease and lipase activities. Thus, only isolates from clu sters B and E produced enzyme activities associated with dairy product flav or defects. Thirty-eight ribogroups were differentiated among the 70 isolat es. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, wi th a Simpson's index of discrimination of 0.955. Isolates of the same ribot ype were never classified into different clusters, and ribotypes within a g iven cluster generally showed similar ribotype patterns; thus, specific rib otype fragments may be useful markers for tracking the sources of pseudomon ads in dairy production systems. Our results suggest that ribogroups are ge nerally homogeneous with respect to nomenspecies and biovars, confirming th e identification potential of ribotyping for Pseudomonas spp.