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Two bacterial strains, DSM 20253T and DSM 20254, isolated from rennin and regarded as causing cheese spoilage, were deposited in the DSMZ as Lactobacillus sp. by J. Stadhouders. The strains show 99·9 % 16S rRNA gene sequence similarity and have less than 94·3 % similarity with any other species of the genus. Lactobacillus coryniformis is their closest phylogenetic neighbour. DNA–DNA hybridization experiments confirmed that the two strains are members of the same species with separate status within the genus Lactobacillus. The strains are homofermentative lactic acid bacteria and can be phenotypically and genotypically distinguished from their closest relatives. 16S rRNA gene-targeted specific primers were designed to enable PCR detection of the novel species. The name Lactobacillus rennini sp. nov. is proposed for the novel isolates, with strain CECT 5922T (=DSM 20253T) as the type strain.

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus , 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease ( nuc ) and enterotoxin ( sea to see ) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea , 22 (17%) for sec , one (0.8%) for sed , and three (2.2%) for sea and sec . No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans . The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.

M.C. MACIAN, E. CHENOLL AND R. AZNAR. 2004. Aims: To develop a multiplex PCR approach for simultaneous detection of Leuconostoc and Carnobacterium and its validation in meat products. Methods and Results: Two multiplex PCR assays were developed using newly designed 16S rDNA-directed primers adapted to the current taxonomic situation of genera Leuconostoc and Carnobacterium that allow: (i) simultaneous detection of both genera, and members of the nonmotile species of genus Carnobacterium and (ii) identification in a single assay of the nonmotile species C. divergens, C. maltaromicum and C. gallinarum. Sensitivity values of 10 3 and 10 4 CFU g )1 were determined for multiplex PCR detection of Carnobacterium and Leuconostoc, respectively, following artificially inoculated meat trials. In addition, both multiplex PCR assays were validated in 14 naturally contaminated samples covering nine types of meat products. Results obtained by colony identification were confirmed by PCR detection. Conclusions: The methods described in this study provide a rapid and reliable tool for PCR detection of Carnobacterium and Leuconostoc, in meat products, and for colony identification. Significance and Impact of the Study: This multiplex PCR approach will help in the analysis of the spoilage microbiota of refrigerated vacuum-packaged meat product in order to determine the appropriate preservation method.

IntroductionVery little is known about the impact of n-3 long-chain fatty acids (n-3 LCFAs) on the microbiota of sows and their piglets. The aim of this study was to evaluate the effect of n-3 LCFA in sow diets on the microbiota composition of sows’ feces, colostrum, and milk as well as that of piglets’ feces.MethodsTwenty-two sows were randomly assigned to either a control or an n-3 LCFA diet from service to weaning. Sows’ and piglets’ performance was monitored. The gestating and lactating sows’ microbiomes in feces, colostrum, and milk were characterized by 16s ribosomal RNA gene sequencing. The fecal microbiome from the two lowest (>800 g) and the two highest birth weight piglets per litter was also characterized, and the LPS levels in plasma were analyzed at weaning.Results and Discussionn-3 LCFA increased microbiota alpha diversity in suckling piglets’ and gestating sows’ feces. However, no effects were observed in colostrum, milk, or lactating sows’ feces. Dietary n-3 LCFA modified the microbiota composition of gestating sows’ feces, milk, and suckling piglets’ feces, without affecting lactating sows’ feces or colostrum. In gestating sows’ feces and milk, the decrease in genus Succinivibrio and the increase of Proteobacteria phylum, due to the increased genera Brenneria and Escherichia, respectively, stand out. In the feces of suckling piglets, the higher abundance of the beneficial genus Akkermansia and Bacteroides, and different species of Lactobacillus are highlighted. In addition, positive correlations for families and genera were found between lactating sows’ feces and milk, milk and suckling piglets’ feces, and lactating sows’ feces and suckling piglets’ feces. To conclude, dietary n-3 LCFA had a positive impact on the microbiome of suckling piglet’s feces by increasing microbial diversity and some beneficial bacteria populations, had a few minor modifications on the microbiome of milk and gestating sows’ feces and did not change the microbiome in lactating sows’ feces or colostrum. Therefore, this study shows the effect of dietary n-3 LCFA on the microbiota of sows, colostrum, milk, and suckling piglets during the lactation period providing crucial information on the microbiota status at the early stages of life, which have an impact on the post-weaning.

In this work, a complete analytical procedure is investigated to differentiate several food-spoilage lactic acid bacteria. To do that, a method involving multiplex Polymerase Chain Reaction (PCR), capillary gel electrophoresis (CGE), and laser-induced fluorescence (LIF) is developed. The PCR-CGE-LIF protocol allows the simultaneous detection and differentiation of the genera Leuconostoc and Carnobacterium, the nonmotile group of species within the genus Carnobacterium, and the three species of the group individually (C. divergens, C. gallinarum, and C. maltaromicum). The capability of this approach is clearly illustrated through the sensitive and efficient analysis of the two closest amplicons, with sizes equal to 397 and 412 bp, showing very different yields in all of the amplification reactions tested. These two fragments, which could not be resolved by agarose gel electrophoresis (AGE), are clearly distinguishable by CGE-LIF even when very different areas for both peaks are obtained. The PCR-CGE-LIF method also allows the sensitive detection of these bacteria, demonstrating both a significant resolution improvement compared with traditional AGE and the usefulness of this approach to solve real-life analytical challenges. Good reproducibility of the CGE-LIF procedure is shown for the analysis of multiplex PCR samples with percent relative standard deviation values for migration times and corrected peak areas as low as 0.80 and 6.50 for the same sample and three different days (n = 12), respectively.

The intraspecific diversity of Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, and Lactobacillus plantarum was analyzed by randomly amplified polymorphic DNA (RAPD) PCR with universal primers M13 and T3. The study included 100 reference strains and 210 isolates recovered from two vacuum-packed Spanish meat products, fiambre de magro adobado and morcilla, previously identified by rDNA-restriction fragment length polymorphism profiles. The RAPD-M13 profiles identified isolates at species level in L. plantarum and L. mesenteroides, while RAPD-T3 provided profiles in L. sakei. The combination of RAPD-M13 and RAPD-T3 fingerprints revealed a total of 17 profiles in L. mesenteroides, 6 in L. sakei, 12 in L. plantarum, and 6 in L. curvatus. Of these, six profiles corresponding to L. mesenteroides and one corresponding to L. sakei were found in both products. The Shannon-Weaver diversity index (H'), calculated according to RAPD-M13 and RAPD-T3 profiles during storage, revealed that most profiles appeared only in single samplings in both products, indicating a high strain substitution rate during chilled storage of vacuum-packed meat products. When bloating appeared, only one profile corresponding to L. mesenteroides subsp. dextranicum was present throughout the storage period.