The plasmidome of a Salmonella enterica serovar Derby isolated from pork meat.

The complete nucleotide sequences of four plasmids hosted by a Salmonella enterica serovar. Derby strain 6MK1 isolated from pork were determined by shotgun Sanger sequencing. A 107,637 base pairs (bp) conjugative plasmid pSD107 containing 150 putative coding sequences (CDS) could be assigned to the narrow host range incompatibility group IncI1. A detailed annotation of all CDS was carried out, revealing the presence of genes needed for plasmid replication, conjugal transfer, plasmid partitioning and stability as well as resistance to antimicrobials. The resistance determinants dhfrA1, aadA1, qacEΔ1, sul1 (supplied by a class 1 integron), blaTEM-1b (carried by a truncated Tn2 flanked by IS26), sul2 and strAB confer multidrug resistance to the host bacterium. In addition to pSD107, three small cryptic plasmids pSD4.0, pSD4.6 and pSD5.6 were identified, showing significant sequence similarities to already known replicons of Escherichia coli and S. enterica. In conjugation experiments performed on solid medium, pSD107 was successfully transferred to a nalidixic acid resistant E. coli DH5α, mobilizing pSD4.0 and, more infrequently, also pSD4.6. All transferred plasmids were stably propagated in the recipient strain without selective pressure for approximately 66 generations. The absolute plasmid copy numbers were determined in real time PCR experiments, revealing an approximate 1:1:1:1 ratio of the four replicons compared to the chromosome. The evolutionary position of pSD107 within the IncI1 family of plasmids was inferred from a maximum likelihood phylogenetic tree and by comparison of genetic key elements in a set of 17 IncI1 reference plasmids.

Psychroflexus halocasei sp. nov., isolated from a microbial consortium on a cheese.

A novel halophilic, Gram-reaction-negative, strictly aerobic, non-motile, rod-shaped and oxidase- and catalase-positive bacterial strain, designated WCC 4520(T), was isolated from a semi-hard, Raclette-type cheese. The colonies were yellow-orange; flexirubin-type pigments were not found. The strain hydrolysed gelatin, hippurate, tyrosine and Tweens 20 and 80. Optimal growth was observed with 6-8% (w/v) NaCl, at pH 7-8 and at 27-30 °C. The genomic DNA G+C content was 33.6 mol%. In phylogenetic analysis based on 16S rRNA gene sequences, strain WCC 4520(T) appeared to be a member of the family Flavobacteriaceae and the closest phylogenetic neighbours were identified as Psychroflexus gondwanensis DSM 5423(T) (94.0% 16S rRNA gene sequence similarity) and Psychroflexus salinarum CCUG 56752(T) (94.0%). The predominant cellular fatty acids were iso-C(15:0), anteiso-C(15:0), iso-C(15:1) G and iso-C(17:0) 3-OH. The only detected quinone was MK-6 and the major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid. Minor polar lipids and traces of polyamines were also detected. On the basis of the data presented, strain WCC 4520(T) represents a novel species of the genus Psychroflexus, for which the name Psychroflexus halocasei sp. nov. is proposed. The type strain is WCC 4520(T) ( = LMG 25857(T) = CCUG 59705(T)).

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese.

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.

Assessment of the anti-listerial activity of microfloras from the surface of smear-ripened cheeses.

The anti-listerial activity of microfloras from the surface of various smear-ripened cheeses was evaluated using four methods that were then compared. Method A measured the anti-listerial potential of supernatants from short-time liquid cultures, whereas in Method B, a model cheese was co-inoculated with the microflora and Listeria innocua test strains. Method C was based on successive propagations of the microfloras on this model cheese, and Method D on successive propagations of the microfloras together with Listeria test strains. Anti-listerial activity considerably depended on the microflora used. Significant correlations were obtained between Methods A and B and Methods C and D. With Methods C and D, the highest anti-listerial activity was obtained with the microflora from a Livarot-type cheese (FC12). To investigate the cause of the anti-listerial activity of FC12, Fourier Transform InfraRed (FTIR) analyses of microbial populations were performed on the original microflora as well as on the microflora after propagations on the model cheese. The composition of FC12 had changed considerably upon propagation, and in the propagated microflora, the population of yeasts was dominated by Yarrowia lipolytica strains, whereas the population of bacteria was dominated by Vagococcus species.

Vibrio casei sp. nov., isolated from the surfaces of two French red smear soft cheeses.

Three Gram-negative, rod-shaped, catalase- and oxidase-positive, facultatively anaerobic and motile bacteria, strains WS 4538, WS 4539T and WS 4540, were isolated from the surfaces of two fully ripened French red smear soft cheeses. Based on 16S rRNA gene sequence similarity, all three strains were shown to belong to the genus Vibrio. They are most closely related to Vibrio rumoiensis S-1T (96.3% similarity) and Vibrio litoralis MANO22DT (95.9%). DNA-DNA hybridization confirmed that all three isolates belong to the same species and clearly separated strain WS 4539T from V. rumoiensis DSM 19141T (38-42% relatedness) and V. litoralis DSM 17657(T) (28-37%). In contrast to their nearest relatives, the strains exhibited beta-galactosidase and aesculin hydrolase activities. A 14 bp insertion in the 16S rRNA gene sequence forms an elongated structure at helix 10 in the rRNA molecule and provides a tool for PCR-based identification of the novel species. Partial sequences of the housekeeping genes atpA, recA, rpoA and pyrH supported the conclusion that the three isolates constitute a separate species within the genus Vibrio. The name Vibrio casei sp. nov. is proposed for the novel taxon. Strain WS 4539T (=DSM 22364T =LMG 25240T; DNA G+C content 41.8 mol%) is the type strain and WS 4540 (=DSM 22378 =LMG 25241) is a reference strain.