Tracking the blue: A MLST approach to characterise the Pseudomonas fluorescens group N.A. Andreani a,1 , M.E. Martino a,1 , L. Fasolato a, * , L. Carraro a , F. Montemurro a , R. Mioni b , P. Bordin b , B. Cardazzo a a Department of Comparative Biomedicine and Food Science (University of Padova), Legnaro 35010, Padova, Italy b Istituto Zooprofilattico Sperimentale delle Venezie (IZSve), Legnaro 35020, Padova, Italy article info Article history: Received 10 July 2013 Received in revised form 18 November 2013 Accepted 20 November 2013 Available online 3 December 2013 Keywords: Pseudomonas fluorescens group Multilocus Sequence Typing Food spoilage Blue mozzarella abstract The Pseudomonas fluorescens group comprises several closely related species that are involved in food contamination and spoilage. Specifically, the interest in P. fluorescens as a spoiler of dairy products increased after the cases of “blue mozzarella” that occurred in Italy in 2010. A Multilocus Sequence Typing (MLST) scheme was developed and applied to characterise 136 isolates (reference strains and food borne isolates) at strain level, to reveal the genetic relationships among them and to disclose any possible genetic clustering of phenotypic markers involved in food spoilage (protease, lipase, lecithinase activities and pigmented or fluorescent molecule production). The production of dark blue diffusible pigment was evaluated on several bacterial culture media and directly on mozzarella cheese. The MLST scheme provided precise genotyping at the strain level, and the population analyses of the concatenated sequences allowed major taxa to be defined. This approach was revealed to be suitable for tracking the strains according to their origin, such as dairy plants or food matrices. The genetic analysis revealed the presence of a connection between the blue pigment production and a specific phylogenetic cluster. The development of the online database specific to the P. fluorescens group (http://pubmlst.org/ pfluorescens) will facilitate the application of the scheme and the sharing of the data. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The strains ascribed to the genus Pseudomonas are Gram- negative, rod-shaped, ubiquitous bacteria, characterised by poor nutritional needs and present in various environments (soil, organic material under decomposition, atmospheric dust, vegeta- tion and water), with a wide range of animal and plant hosts (Anzai et al., 2000; Frapolli et al., 2007). The Pseudomonas fluo- rescens group is strictly connected to food spoilage, and its importance is clearly related to the food trade and hygienic standards. Because of the psychrotrophic and mesophilic charac- teristics of these bacteria, they can replicate at refrigeration tem- peratures and are easily destroyed by heat treatments; however, long periods of shelf life can easily increase the Pseudomonas concentration in foods (Marchand et al., 2009; Munsch-Alatossava and Alatossava, 2005). Strains belonging to the P. fluorescens group are found in a wide range of foods, such as ready-prepared fresh vegetables, raw fish (especially sushi or sashimi), meat and dairy products (Arnaut-Rollier et al., 1999; Franzetti and Scarpellini, 2007). Specifically, Pseudomonas represents part of the main micro- flora of raw milk and its products, which can be contaminated via defiled water and soil, inadequately sanitised milking surfaces, storage and transporting equipment (Munsch-Alatossava and Alatossava, 2005). Dairy products are a particularly favourable substrate to grow different bacteria, including Pseudomonas, because of their nutritional value, water content and a neutral pH (Marchand et al., 2009). As previously reported, psychrotrophic bacteria are not resistant to heat treatments, but they are able to produce extracellular enzymes, such as different proteases, li- pases and lecithinases, which are often heat resistant, respon- sible for spoilage and instability problems in food, and their production increases in suboptimal storage conditions (Marchand et al., 2009; De Jonghe et al., 2011). Their activity is more remarkable at refrigeration temperatures, and they induce grey colouration, unpleasant bitter off-flavours, gelation, a decrease of cheese-making performance, variation in pH, * Corresponding author. viale dell’Università, 16, Legnaro, 350210 Padova, Italy. Tel.: þ39 049 8272965. E-mail address: luca.fasolato@unipd.it (L. Fasolato). 1 Andreani N.A. and M.E. Martino contributed equally to this work. Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.11.012 Food Microbiology 39 (2014) 116e126