Tracking the blue: A MLST approach to characterise the Pseudomonas fluorescens group
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, vegetation and water), with a wide range of animal and plant hosts (Anzai et al., 2000, Frapolli et al., 2007). The Pseudomonas fluorescens 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 characteristics of these bacteria, they can replicate at refrigeration temperatures 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 microflora 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, lipases and lecithinases, which are often heat resistant, responsible 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, rancidity and saponification when present in dairy products (Rajmohan et al., 2002, Arslan et al., 2011). Additionally, one of the most outstanding food-altering effects is the capacity of some Pseudomonas strains to produce coloured or fluorescent pigments, which causes food discolouration (Gennari and Dragotto, 1992). The interest in P. fluorescens as a spoiler of dairy products increased after the cases of “blue mozzarella” that occurred in 2010 in which some European consumers noted discolouration on some mozzarella products. The microbiological analyses on the cheese samples showed high concentrations of P. fluorescens, up to 106 CFU per gram of cheese (Bogdanova et al., 2010). Little information is available on the nature of the blue pigment that was observed in the mozzarella cheese, and the characteristics must be understood, in contrast with the thorough studies of other pigments, such as pyocyanine, pyoverdine, fluorescein, pyorubin and pyomelanin, that are typically produced by some P. fluorescens strains.
Thus, because the role of the P. fluorescens group in food spoilage has been established, the interest in methods for correctly identifying the different species of this group has grown. Concerning Pseudomonas taxonomy, several different phenotypic traits were chosen for species identification until molecular biology methods, such as DNA–DNA hybridisation (Wayne et al., 1987, Palleroni et al., 1973), RNA-DNA hybridisation (Palleroni et al., 1973, De Vos et al., 1985, De Vos et al., 1989), REP-PCR (Repetitive Extragenic Palindromic Sequence Polymerase Chain Reaction; Johnsen et al., 1996), 16S rRNA sequencing (Woese et al., 1984, Laguerre et al., 1994, Moore et al., 1996, Bennasar et al., 1998), 16S–23S rRNA intergenic spacer (ITS; Gurtler and Stanisich, 1996) and PFGE (Pulsed-Field Gel Electrophoresis, Nogarol et al., 2013) became more frequent. Because of the low level of variability in 16S rRNA and other housekeeping genes and past lateral gene transfer events, a single-gene-based identification approach may not be appropriate for strain typing (Frapolli et al., 2007). Since 1998, MLST has been proposed as a portable and universal method for characterising bacteria by sequence polymorphisms within internal fragments of housekeeping genes (Maiden, 2006). Different approaches have been developed to improve the characterisation of Pseudomonas spp.: Yamamoto et al. (2000) studied the genus Pseudomonas by analysing the gyrB and the rpoD genes, whereas Hilario and co-workers developed an approach based on atpD, carA, recA and 16S sequencing (Hilario et al., 2004). In 2010, Mulet and colleagues developed a MLST scheme to disclose the phylogenetic relationships within the Pseudomonas genus by 16S rRNA, gyrB, rpoB and rpoD sequencing of 107 Pseudomonas Type strains.
Many MLST schemes have been created as tracking instruments of pathogens (i.e., the first MLST scheme was created for Neisseria meningitidis by Maiden et al., 1998), but schemes to track food spoilers could be helpful to discover the causes of the spoilage reactions and to solve related problems.
Currently, no studies are available about the nature of the blue pigment involved in blue mozzarella events, whereas only one study has been conducted using PFGE analysis to highlight the phylogenetic relationships of the isolates involved in the case of the mozzarella contamination (Nogarol et al., 2013).
Here, five phenotypic tests were used to characterise strains for specific traits; furthermore, a MLST scheme based on the analysis of 7 loci was developed and applied to 18 reference strains, 117 food-borne strains and a human case of infection. The phenotypic and molecular results were compared to highlight a possible correlation between them, and the MLST data were used to reconstruct phylogenetic trees and conduct evolutionary analyses. Additionally, the first MLST online database specific to the P. fluorescens group was developed (http://pubmlst.org/pfluorescens).
Section snippets
Bacterial strains
A total of 18 reference and Type strains belonging to the P. fluorescens group were selected and included in the MLST scheme from previous studies conducted on the genus Pseudomonas (Mulet et al., 2010). Additionally, 118 field strains were isolated from the Istituto Zooprofilattico delle Venezie (IZSve; Legnaro, Italy), the Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana (IZStl; Pisa, Italy), the Department of Food Sciences (University of Udine), the School of Veterinary
Phenotypic characterisation
The results of the phenotypic characterisation are summarised in Table 3.
To standardise the data collection, a cut-off value was selected to verify the phenotypic traits. At 6 °C, the data were collected on the 10th day, when the traits were completely evident; at 22 °C and 31 °C, the cut-off was fixed on the 7th day, when the maximum expression of the traits was observed.
All strains grew on CFC PAB, whereas 9 (10.98%) strains did not grow on PDA at 31 °C. Several strains did not show any
Discussion
Pseudomonas spp. are among the most common bacteria involved in food spoilage (Martin et al., 2011), and the interest in their role in food grew after the “blue mozzarella” events that occurred in 2010. Health authorities and official laboratory analyses connected the outbreak to the contamination of processing water with strains of P. fluorescens (Marro et al., 2011). Specifically, in the Annual Report of RASFF, scientists confirmed the involvement of two species belonging to P. fluorescens
Acknowledgements
The study was supported by the University of Padova (Progetto di Ricerca di Ateneo 2011 to L.F. – Development of a molecular typing system) and evaluation of phenotype characters involved in food colouring and spoilage in Pseudomonas spp. strains. CPDA115333). Also supported by the PhD school of Veterinary Science of the University of Padova to support the education of N.A.A. The authors are grateful to the Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, to Dr. M. Marino
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Andreani N.A. and M.E. Martino contributed equally to this work.