Understanding of the phylogeny and interrelationships of the genera within the order ‘ Enterobacteriales ’ has proven difficult using the 16S rRNA gene and other single-gene or limited multi-gene approaches. In this work, we have completed comprehensive comparative genomic analyses of the members of the order ‘ Enterobacteriales ’ which includes phylogenetic reconstructions based on 1548 core proteins, 53 ribosomal proteins and four multilocus sequence analysis proteins, as well as examining the overall genome similarity amongst the members of this order. The results of these analyses all support the existence of seven distinct monophyletic groups of genera within the order ‘ Enterobacteriales ’. In parallel, our analyses of protein sequences from the ‘ Enterobacteriales ’ genomes have identified numerous molecular characteristics in the forms of conserved signature insertions/deletions, which are specifically shared by the members of the identified clades and independently support their monophyly and distinctness. Many of these groupings, either in part or in whole, have been recognized in previous evolutionary studies, but have not been consistently resolved as monophyletic entities in 16S rRNA gene trees. The work presented here represents the first comprehensive, genome-scale taxonomic analysis of the entirety of the order ‘ Enterobacteriales ’. On the basis of phylogenetic analyses and the numerous identified conserved molecular characteristics, which clearly distinguish members of the order ‘ Enterobacteriales ’ and the seven reported clades within this order, a proposal is made here for the order Enterobacterales ord. nov. which consists of seven families: Enterobacteriaceae , Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov.
A polyphasic taxonomic approach was applied to strains of the species Staphylococcus sciuri in order to clarify the taxonomic legitimacy of the delineation of S. sciuri into S. sciuri subsp. sciuri , S. sciuri subsp. carnaticus and S. sciuri subsp. rodentium . A group of 81 S. sciuri isolates obtained from human (n=62) and veterinary (n=17) clinical materials and foods (n=2) and ten reference and type strains obtained from the Czech Collection of Microorganisms were characterized by extensive biotyping using conventional tests and commercial identification kits (ID 32 Staph, STAPHYtest, Biolog Microbial ID System), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, automated ribotyping with EcoRI restriction enzyme, 16S–23S rRNA gene intergenic transcribed spacer PCR fingerprinting and repetitive sequence-based PCR fingerprinting with the (GTG)5 primer. Selected strains representing different ribotypes were further characterized using sequencing of the β-subunit of RNA polymerase (rpoB) gene. Individual techniques revealed high heterogeneity within the analysed S. sciuri strains but differentiation of the investigated strains into groups corresponding to the aforementioned S. sciuri subspecies and supported by these techniques was not clearly revealed. Based on obtained results and data retrieved from literature we propose rejecting the separation of S. sciuri species into S. sciuri subsp. sciuri , S. sciuri subsp. carnaticus and S. sciuri subsp. rodentium and we suggest reclassification these subspecies as S. sciuri with the type strain W.E. Kloos SC 116T (=ATCC 29062T=BCRC 12927T=CCM 3473T=CCUG 15598T=CNCTC 5683T=DSM 20345T=JCM 2425T=NCTC 12103T).
Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.
Two novel species names, Acinetobacter bohemicus and Acinetobacter pakistanensis , appeared on validation list no. 161 (January 2015) under priority numbers 26 and 28, respectively. As the published data suggested a high similarity of the organisms associated with these names, we aimed to define their taxonomic relationship. The study set included all strains used in the original nomenclatural proposals, i.e. 25 strains of A. bohemicus and one strain of A. pakistanensis . The average nucleotide identity values (95.9 and 96.1 % based on blast and MUMmer, respectively) between the whole-genome sequences of A. bohemicus ANC 3994T and A. pakistanensis KCTC 42081T supported the identity of these type strains at the species level. Based on the genus-wide comparative analyses of the rpoB sequences and whole-cell fingerprints generated by matrix-assisted laser desorption/ionization-time-of-flight MS, A. pakistanensis KCTC 42081T fell within the respective clusters formed by the 25 A . bohemicus strains. The same picture was obtained on the basis of comparative analysis of 16S rRNA gene sequences of KCTC 42081T and three A. bohemicus strains. Finally, the metabolic and physiological features of KCTC 42081T were found to be congruent with those of A. bohemicus . Based on these results, we conclude that Acinetobacter pakistanensis is a later heterotypic synonym of Acinetobacter bohemicus.