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Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes The central clade of the genus Vibrio, also called the Vibrio core group, comprises six species that are tightly related (DNA-DNA reassociation values are very close to 70 % for most species pairs). Identification of novel strains to the species level within this group is troublesome and results are quite often dependent on the methodology employed. Therefore, this group represents an excellent framework to test the robustness of multilocus sequence analysis (MLSA) not only for inferring phylogeny but also as an identification tool without the need for DNA-DNA hybridization assays. The genes selected, 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR, were amplified by direct PCR from 44 Vibrio core-group strains. Subsequent analysis allowed us to recognize toxR and rpoD as the most resolving individual genes and showed that concatenated sequences of rpoD, rctB and toxR were more useful than concatenated sequences of all seven genes. To validate our conclusions, MLSA similarities have been correlated with DNA-DNA relatedness values obtained in this study and values taken from the literature. Although the seven concatenated genes gave the best correlation, the concatenated sequences of rpoD, rctB and toxR have the practical advantage of showing a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity recorded, meaning that they can be used quite conveniently for species identification of vibrios. INTRODUCTIONMLSA (multilocus sequence analysis) is a recently developed technique derived from multilocus sequence typing that has been used successfully in epidemiology studies. The use of MLSA as an alternative method for species delineation in bacteriology is currently being evaluated, following a recommendation of the ad hoc committee for the re-evaluation of the species definition in bacteriology (Stackebrandt et al., 2002). In MLSA studies, several housekeeping genes (more than five) are analysed and relationships between taxa are established. One important advantage of this kind of approach is the availability of genomic sequences from any laboratory, avoiding the problems of lack of comparability that are associated with DNA-DNA reassociation data. However, the usefulness of MLSA for describing bacterial species has to be demonstrated for each of the taxa or group of species studied and for the selected housekeeping genes. Authors should prove that there is a sufficient degree of congruence between the alternative technique (i.e. MLSA) and DNA-DNA reassociation data. To date, the technique has been applied to discrete bacterial taxa, mainly genera of lactic acid bacteria (Naser et al. et al., 2007, 2008) and the Vibrionaceae (Goarant et al., 2006;Urbanczyk et al., 2007;Thompson et al., 2008;Rameshkumar et al., 2008).In the family Vibrionaceae, sequence similarities for the 16S rRNA gene are ¢97.6 % among members of the so-called Vib...

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A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.

Aim:  To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum‐packed and refrigerated meat products. Methods and Results:  A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR‐restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, ‘morcilla’ and ‘fiambre de magro adobado’ obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. Conclusions:  Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum‐packed meat products. Significance and Impact of the Study:  Prevention of bloating spoilage in vacuum‐packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).