Downloaded from www.microbiologyresearch.org by IP: 93.91.26.97 On: Fri, 06 Nov 2015 10:32:14 Genetic diversity in Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequence typing and pulsed-field gel electrophoresis analyses Claudia Picozzi, Gaia Bonacina, Ileana Vigentini and Roberto Foschino Correspondence Roberto Foschino roberto.foschino@unimi.it Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita ` degli Studi di Milano, via Celoria 2, 20133 Milano, Italy Received 21 December 2009 Revised 4 March 2010 Accepted 29 March 2010 Lactobacillus sanfranciscensis is a lactic acid bacterium that characterizes the sourdough environment. The genetic differences of 24 strains isolated in different years from sourdoughs, mostly collected in Italy, were examined and compared by PFGE and multilocus sequence typing (MLST). The MLST scheme, based on the analysis of six housekeeping genes (gdh, gyrA, mapA, nox, pgmA and pta) was developed for this study. PFGE with the restriction enzyme ApaI proved to have higher discriminatory power, since it revealed 22 different pulsotypes, while 19 sequence types were recognized through MLST analysis. Notably, restriction profiles generated from three isolates collected from the same firm but in three consecutive years clustered in a single pulsotype and showed the same sequence type, emphasizing the fact that the main factors affecting the dominance of a strain are correlated with processing conditions and the manufacturing environment rather than the geographical area. All results indicated a limited recombination among genes and the presence of a clonal population in L. sanfranciscensis. The MLST scheme proposed in this work can be considered a useful tool for characterization of isolates and for in- depth examination of the strain diversity and evolution of this species. INTRODUCTION Sourdough technology is widespread in Europe, and is traditionally used in bread making for a variety of baked products, such as rye and wheat bread, crackers, brioches, Italian panettone and similar cakes, some pizzas and numerous local baked goods (De Vuyst & Neysens, 2005). The type of cereal, recipe formulation, starter method (back- slopping or use of selected cultures), dough yield, redox potential, fermentation time and temperature are the environmental factors that affect the dominant microbiota, which is mainly constituted of lactic acid bacteria and yeasts (Vogel et al., 1996, 1999; De Vuyst et al., 2009). In particular, the metabolic activities of heterofermentative lactobacilli involved in most sourdough production provide beneficial effects on flavour (Thiele et al., 2002; Hansen & Schieberle, 2005), nutritional properties (Liljeberg et al., 1995; Liljeberg & Bjo ¨rck, 1996; Di Cagno et al., 2002, 2004), texture (Korakli et al. 2001; Tieking & Ga ¨nzle, 2005; Arendt et al., 2007) and shelf-life of the end products (Corsetti et al., 1998a; Schnu ¨rer & Magnusson, 2005). Lactobacillus sanfranciscensis is considered a key micro- organism (Gobbetti et al., 1996), being the predominant bacterial species in type I sourdoughs for San Francisco bread and many traditional Italian and German baked products (Corsetti et al., 2001; Foschino et al., 2001; Meroth et al., 2003). The increasing knowledge of sourdough ecology is leading to the improvement of the quality of the end product through the use of starter cultures with enhanced technological capabilities, such as exopolysaccharide pro- duction (Korakli et al., 2003; Lacaze et al., 2007), antifungal activity (Corsetti et al., 1998b; Schnu ¨rer & Magnusson, 2005), antistaling properties (Corsetti et al., 2000; Moore et al., 2007) and decreased gluten intolerance (Di Cagno et al., 2005; Gobbetti et al., 2007). The identification of L. sanfranciscensis at strain level proves to be crucial as a consequence of the rising interest in the above-mentioned strain-related functional traits and for monitoring patented technologies in sourdough production. Moreover, the accurate detection of a bacterial strain could be a valuable criterion for traceability systems, in order to support the typicality of the traditional fermented foods by providing information for consumers and safeguarding the autoch- thonous microbiota. In fact, the possibility of extracting Abbreviations: MLST, multilocus sequence typing; ST, sequence type; UPGMA, unweighted pair group method with arithmetic means. The GenBank/EMBL/DDBJ accession numbers for the gdh, gyrA, mapA, nox, pgmA and pta sequences of the Lactobacillus sanfrancis- censis strains analysed in this study are GU454588–GU454611, GU454612–GU454635, GU454636–GU454659, GU454660– GU454683, GU454684–GU454707 and GU454708–GU454731, respectively. Microbiology (2010), 156, 2035–2045 DOI 10.1099/mic.0.037341-0 037341 G 2010 SGM Printed in Great Britain 2035