Abstract

Spoilage induced by Pseudomonas strains is commonly found in a wide range of food products as a result of the ubiquitous presence of these strains and their ability to induce alteration through different mechanisms. Particular attention has been recently paid on those P. fluorescens strains able to induce a blue discolouration on several food matrices (e.g. dairy or meat products). Actually, poor data are available about this curious event that draw the attention of European consumer from 2010.
In the present manuscript a step-by-step investigation of the spoilage potential of Pseudomonas fluorescens species complex strains is reported, focusing in particular on the ability to produce an unpleasant blue pigment in food.
Firstly, some general information is given to the reader to understand the P. fluorescens group as food spoiler. Then, the application of a polyphasic approach is described with the aim to investigate 136 Pseudomonas fluorescens group strains. Additionally, the achievement and the analyses of draft genomes and transcriptomes for 4 P. fluorescens strains are described to investigate the biosynthetic pathways involved in the blue pigment production.
The attempt to chemically characterise the blue molecule using MALDI-TOF mass spectrometry is also reported. Finally, the execution of a transposon-mediated mutagenesis is described to confirm previously obtained genomic data and to highlight further genes involved in the blue-pigment production.
The phenotypic and genotypic characterisation, based on the combination of classical microbiological tests and a MLST scheme, allowed the reconstruction of phylogenetic relationships among the isolates and the identification of a monophyletic group (named “the blue branch”) grouping all the blue-pigmenting and few uncoloured strains. The real
involvement of these strains in the blue mozzarella event was confirmed by their ability to induce a blue discolouration on mozzarella cheese during a challenge test. The genomic investigation confirmed the strict phylogenetic relationship between the strains belonging to the “blue branch”. Additionally, comparative genomic tools revealed the presence of a genetic cluster unique to the blue pigmenting strains containing a second copy of five trp genes, clearly involved in the blue pigment production. The biochemical characterisation of the pigment, hampered by strong issues of solubility, led to the conclusion that the molecule is an indigo-derivative.
Transposon-induced mutants confirmed the involvement of the previously identified unique cluster and the association of several genes affecting directly or indirectly the blue molecule production. Furthermore, the phenotypic
characterisation of the mutants revealed a key role of iron in the production of the pigment, such as absence of any advantage of the wild-type strain in co-culture with a non-pigmenting mutant.
To conclude, the present work represents an exhaustive investigation of the spoilage potential of the blue-pigmenting P. fluorescens strains, giving to food industry reliable approaches to identify, track and prevent spoilage related to the growth of these interesting bacteria.