Investigation on the presence of blue pigment-producing Pseudomonas strains along a production line of fresh mozzarella cheese

Abstract

The aim of this study was to investigate on the presence, dissemination and persistence of blue pigmenting Pseudomonas strains in a cheese factory previously involved in cases of blue spoilage of mozzarella cheese. The production of the blue pigment is strain or species specific, and an effective identification of Pseudomonas spp. involved is important to know its microbial ecology and to trace the origin of the contamination in the cheese processing environments. To know the incidence, the genetic diversity and the species distribution of Pseudomonas spp., a survey on milk, cheese and processing fluid samples collected during a period of 5 months from a production line of mozzarella cheese was carried out. Pseudomonas spp. were detected in 50% of the 74 samples of milk, cheese and processing fluid analyzed. In particular, Pseudomonas were more frequently detected (66.7%) in processing fluid samples, like processing water, brine and preserving liquid. Ninety-four strains were isolated and typed by RAPD-PCR. They were grouped into 32 different genotypic clusters and, by partial sequencing of the 16S rRNA gene, were identified as belonging to P. fluorescens group (n = 40), P. putida group (n = 10), P. aeruginosa group (n = 6), Pseudomonas oleovorans group (n = 3), and Pseudomonas anguilliseptica group (n = 1). Three blue-pigmenting strains all belonging to P. fluorescens group were isolated from cooling and hardening water (n = 2) and preserving liquid (n = 1) sampled on three different occasions, confirming the persistency in the environment. The MLST analysis of the concatenated sequences of seven housekeeping genes (glnS, gyrB, ileS, nuoD, recA, rpoB and rpoD) displayed a new MLST allelic profile for the blue-producing strains. The comparison with a database specific to the P. fluorescens group (http://pubmlst.org/pfluorescens) confirmed the connection between the blue pigment production and a specific phylogenetic cluster within the group. The wide phylogenetic and genotypic variability of Pseudomonas observed in this work emphasized the importance of an effective identification of the blue-pigment producing strains to trace and control their presence in the dairy environments and prevent their possible occurrence in cheese spoilage.