The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).Cocoa beans are the principal raw material for chocolate production (39,43,73,81). These seeds are derived from the fruit pods of the cocoa tree (Theobroma cacao L.), which is cultivated in plantations in the equatorial zone, with the Ivory Coast, Brazil, and Ghana as the major producers (2). The cocoa beans are embedded in a mucilaginous pulp inside the pods. Raw cocoa beans have an astringent, unpleasant taste and flavor and have to be fermented, dried, and roasted to obtain the desired characteristic cocoa flavor and taste (26, 73). The final chocolate flavor is influenced by the origin and cultivar of the cocoa beans, the on-the-farm fermentation and drying process, and the roasting and further processing performed by the cocoa and chocolate manufacturer (4,10,32,33,57,75).After removal of the beans from the pods, the first step in cocoa processing is a spontaneous 3-to 10-day fermentation of beans and pulp in heaps, boxes, baskets, or trays, of which spontaneous heap fermentation is the most widely used method in Ghana ...

The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S–23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA–DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii , the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain R 001T = LMG 10661T = DSM 7385T) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464T = LMG 27703T = DSM 27478T) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627T = LMG 27706T = DSM 27477T) for the BDB strains and Ralstonia pseudosolanacearum sp. nov. (type strain UQRS 461T = LMG 9673T = NCPPB 1029T) for the strains of R. solanacearum phylotypes I and III.

Species belonging to the genus of Pantoea are commonly isolated from plants, humans and the natural environment. The species of the genus are phenotypically closely related, making rapid identification of Pantoea strains to the species level difficult. Multilocus sequence analysis (MLSA) was evaluated as a means for rapid classification and identification of Pantoea strains. Four housekeeping genes, gyrB, rpoB, atpD and infB, were sequenced for strains assigned to the genus. Included in the study were (1) reference strains from the seven currently recognized species of Pantoea, (2) strains belonging to Brenner DNA groups II, IV and V, previously isolated from clinical samples and difficult to identify because of high phenotypic similarity to P. agglomerans or P. ananatis and (3) isolates from diseased Eucalyptus, maize and onion, assigned to the genus on the basis of phenotypic tests. Phylogenetic trees were constructed from the sequences of the four housekeeping genes. The ''core'' Pantoea species formed a cluster separate from the ''Japanese'' species which formed a tight cluster that included the genus Tatumella when the tree was based on concatenated sequences of the four genes. The MLSA data further suggested the existence of ten potential novel species, phylogenetically related to the currently recognized Pantoea species and the possible inclusion of Pectobacterium cypripedii in the genus Pantoea. When compared with DNA-DNA hybridization data, a good congruence was observed between both methods, with gyrB sequence data being the most consistent. In conclusion, MLSA of partial nucleotide sequences of the genes gyrB, rpoB, atpD and infB can be used for classification, identification and phylogenetic analyses of Pantoea strains. r 2008 Elsevier GmbH. All rights reserved. The GenBank/EMBL accession numbers for the sequences presented in this study are: EF988753-EF988838, EU145260-EU145275, EU344757-EU344760, FJ187830-FJ187834 (gyrB gene); EF988925-EF989010, EU145292-EU145307, EU344765-EU344768, FJ187840-FJ187844 (rpoB gene); EF988667-EF988752, EU145244-EU145259, EU344753-EU344756, FJ187825-FJ187829 (atpD gene); EF988839-EF988924, EU145276-EU145291, EU344761-EU344764, FJ187835-FJ187839 (infB gene) and EF688006-EF688012, EU216734-EU216737, EU344769-EU344770 (16S rRNA).Ã Corresponding author. Tel.: +2712 420 3934; fax: +2712 420 3960.E-mail address: teresa.coutinho@fabi.up.ac.za (T. Coutinho).Please cite this article as: C. Brady, et al., Phylogeny and identification of Pantoea species associated with plants, humans and the natural environment based on multilocus sequence analysis

Four staphylococcal isolates from clinical and necropsy specimens from a cat, a dog, a horse and a parrot (Psittacus erithacus timneh) were found to constitute a distinct taxon. 16S rRNA gene sequence analysis revealed that its closest phylogenetic relatives are Staphylococcus intermedius and Staphylococcus delphini. Growth characteristics, biochemical features and DNA-DNA hybridizations demonstrated that the strains differ from these and other known species and that they represent a single, novel Staphylococcus species for which the name Staphylococcus pseudintermedius sp. nov. is proposed. The novel species is commonly confused with S. intermedius in routine diagnostic veterinary bacteriology. Although the strains described were isolated from lesions and show several characteristics typical of pathogenic staphylococci, such as coagulase, DNase and b-haemolysin production, the pathogenic significance of the novel species remains unclear. The type strain, LMG 22219 T (=ON 86 T =CCUG 49543 T ), was isolated from lung tissue of a cat.

Background Enterobacter sakazakii is an opportunistic pathogen that can cause infections such as necrotizing enterocolitis, bacteraemia, meningitis and brain abscess/lesions. When the species was defined in 1980, 15 biogroups were described and it was suggested that these could represent multiple species. In this study the taxonomic relationship of strains described as E. sakazakii was further investigated. Results Strains identified as E. sakazakii were divided into separate groups on the basis of f-AFLP fingerprints, ribopatterns and full-length 16S rRNA gene sequences. DNA-DNA hybridizations revealed five genomospecies. The phenotypic profiles of the genomospecies were determined and biochemical markers identified. Conclusion This study clarifies the taxonomy of E. sakazakii and proposes a reclassification of these organisms.