Although considerable progress has been made in recent years regarding the classification of bacteria assigned to the phylum Bacteroidetes, there remains a need to further clarify taxonomic relationships within a diverse assemblage that includes organisms of clinical, piscicultural, and ecological importance. Bacteroidetes classification has proved to be difficult, not least when taxonomic decisions rested heavily on interpretation of poorly resolved 16S rRNA gene trees and a limited number of phenotypic features. Here, draft genome sequences of a greatly enlarged collection of genomes of more than 1,000 Bacteroidetes and outgroup type strains were used to infer phylogenetic trees from genome-scale data using the principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families and genera, including taxa proposed long ago such as Bacteroides, Cytophaga, and Flavobacterium but also quite recent taxa, as well as a few species were shown to be in need of revision. According proposals are made for the recognition of new orders, families and genera, as well as the transfer of a variety of species to other genera. In addition, emended descriptions are given for many species mainly involving information on DNA G+C content and (approximate) genome size, both of which can be considered valuable taxonomic markers. We detected many incongruities when comparing the results of the present study with existing classifications, which appear to be caused by insufficiently resolved 16S rRNA gene trees or incomplete taxon sampling. The few significant incongruities found between 16S rRNA gene and whole genome trees underline the pitfalls inherent in phylogenies based upon single gene sequences and the impediment in using ordinary bootstrapping in phylogenomic studies, particularly when combined with too narrow gene selections. While a significant degree of phylogenetic conservation was detected in all phenotypic characters investigated, the overall fit to the tree varied considerably, which is one of the probable causes of misclassifications in the past, much like the use of plesiomorphic character states as diagnostic features.

/pdf/analysis-of-1-000-type-strain-genomes-improves-taxonomic-345doatvi9.pdf

Analysis of 1,000 Type-Strain Genomes Improves Taxonomic Classification of Bacteroidetes .

The bacterial phylum Bacteroidetes, characterized by a distinct gliding motility, occurs in a broad variety of ecosystems, habitats, life styles, and physiologies. Accordingly, taxonomic classification of the phylum, based on a limited number of features, proved difficult and controversial in the past, for example, when decisions were based on unresolved phylogenetic trees of the 16S rRNA gene sequence. Here we use a large collection of type-strain genomes from Bacteroidetes and closely related phyla for assessing their taxonomy based on the principles of phylogenetic classification and trees inferred from genome-scale data. No significant conflict between 16S rRNA gene and whole-genome phylogenetic analysis is found, whereas many but not all of the involved taxa are supported as monophyletic groups, particularly in the genome-scale trees. Phenotypic and phylogenomic features support the separation of Balneolaceae as new phylum Balneolaeota from Rhodothermaeota and of Saprospiraceae as new class Saprospiria from Chitinophagia. Epilithonimonas is nested within the older genus Chryseobacterium and without significant phenotypic differences; thus merging the two genera is proposed. Similarly, Vitellibacter is proposed to be included in Aequorivita. Flexibacter is confirmed as being heterogeneous and dissected, yielding six distinct genera. Hallella seregens is a later heterotypic synonym of Prevotella dentalis. Compared to values directly calculated from genome sequences, the G+C content mentioned in many species descriptions is too imprecise; moreover, corrected G+C content values have a significantly better fit to the phylogeny. Corresponding emendations of species descriptions are provided where necessary. Whereas most observed conflict with the current classification of Bacteroidetes is already visible in 16S rRNA gene trees, as expected whole-genome phylogenies are much better resolved.

/pdf/genome-based-taxonomic-classification-of-bacteroidetes-5y671uumne.pdf

Genome-Based Taxonomic Classification of Bacteroidetes.

This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

Performance and Application of 16S rRNA Gene Cycle Sequencing for Routine Identification of Bacteria in the Clinical Microbiology Laboratory

Calcium precipitate induced aerobic granulation.

A Gram-stain-negative, rod-shaped, non-motile and yellow-pigmented bacterial strain, designated strain JJ013T, was isolated from an artificial lake in Jeollabuk-do, South Korea, and characterized using a polyphasic approach. The 16S rRNA gene sequence of strain JJ013T indicated that the isolate belonged to the family 
Flavobacteriaceae
and exhibited similarity levels of 96.6 % to the type strains of 
Flavobacterium cheonanense
and 
Flavobacterium koreense
and 96.5 % to the type strain of 
Flavobacterium chungnamense
. Growth was observed at 20–30 °C and pH 5.0–7.0. The major cellular fatty acids of the novel strain were iso-C15 : 0 (27.5 %), iso-C15 : 1 G (17.8 %), iso-C17 : 0 3-OH (9.4 %) and iso-C15 : 0 3-OH (9.2 %). Flexirubin-type pigments were present. The DNA G+C content of strain JJ013T was 33.9 mol%, the major respiratory quinone was menaquinone-6 (MK-6) and the major polyamine was sym-homospermidine. The polar lipid profile of the strain JJ013T consisted of a phosphatidylethanolamine (PE), two unknown aminolipids (AL1–2), three unidentified lipid (L1–3) and an unknown glycolipid (GL). On the basis of the morphological and physiological properties and biochemical evidence presented, it is concluded that strain JJ013T represents a novel species of the genus 
Flavobacterium
, for which the name Flavobacterium aciduliphilum sp. nov. is proposed; the type strain is JJ013T ( = KACC 16594T = JCM 18211T). Since C15 : 0, which is known as a predominant fatty acid of the genus 
Flavobacterium
was not detected in the novel strain and other reference strains, we propose an emended description of the genus 
Flavobacterium
.

Flavobacterium aciduliphilum sp. nov., isolated from freshwater, and emended description of the genus Flavobacterium

A Gram-negative, strictly aerobic, yellow-pigmented rod, designated DK69T, was isolated from soil collected from the waste liquid treatment facility of Bafeng Pharmaceutical Company in the city of Enshi, Hubei Province, China. Phylogenetic analysis based on 16S rRNA gene sequences placed strain DK69T in the genus 
Flavobacterium
of the family 
Flavobacteriaceae
. The highest 16S rRNA gene sequence similarities were found with 
Flavobacterium cauense
R2A-7T (96.9 %), 
Flavobacterium saliperosum
AS 1.3801T (96.3 %) and 
Flavobacterium suncheonense
GH29-5T (95.7 %). The major fatty acids (≥5 %) were iso-C15 : 0, iso-C17 : 1ω9c, C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid. The major respiratory quinone was menaquinone-6. The genomic DNA G+C content was 34.4 mol%. Strain DK69T represents a novel species of the genus 
Flavobacterium
, for which the name Flavobacterium enshiense sp. nov. is proposed. The type strain is DK69T ( = CCTCC AB 2011144T  = KCTC 23775T). Emended descriptions of the genus 
Flavobacterium
and 
Flavobacterium cauense
, 
Flavobacterium saliperosum
and 
Flavobacterium suncheonense
are also proposed.

Flavobacterium enshiense sp. nov., isolated from soil, and emended descriptions of the genus Flavobacterium and Flavobacterium cauense, Flavobacterium saliperosum and Flavobacterium suncheonense

A bacterial isolate, designated strain TS3(T), was isolated from soil collected from a metal mine in Tieshan District, Daye City, Hubei Province, in central China. Cells of this strain were Gram-negative, motile and rod-shaped. The strain had ubiquinone Q-8 as the predominant respiratory quinone, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as the major polar lipids and summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(16:0) and C(18:1)ω7c as the major fatty acids. The G+C content was 65.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain TS3(T) was most closely related to Massilia niastensis 5516S-1(T) (98.5%), Massilia consociata CCUG 58010(T) (97.6%), Massilia aerilata 5516S-11(T) (97.4%) and Massilia varians CCUG 35299(T) (97.2%). DNA-DNA hybridization revealed low relatedness between strain TS3(T) and M. niastensis KACC 12599(T) (36.5%), M. consociata CCUG 58010(T) (27.1%), M. aerilata KACC 12505(T) (22.7%) and M. varians CCUG 35299(T) (46.5%). On the basis of phenotypic and phylogenetic characteristics, strain TS3(T) belongs to the genus Massilia, but is clearly differentiated from other members of the genus. The strain represents a novel species, for which the name Massilia tieshanensis sp. nov. is proposed. The type strain is TS3(T) ( = CCTCC AB 2010202(T) = KACC 14940(T)).

/pdf/massilia-tieshanensis-sp-nov-isolated-from-mining-soil-4dsz8in4mt.pdf

Massilia tieshanensis sp. nov., isolated from mining soil.

A Gram-positive, aerobic, non-spore-forming actinobacterial strain, designated strain TL1T, was isolated from pig manure in Wuhan, China. The cell wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids were phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol. The predominant menaquinone was MK-8(H4). The major fatty acids (>10 %) were iso-C16 : 0, iso-C15 : 0 and C17 : 1ω8c. The genomic DNA G+C content was 70.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain TL1T was most closely related to the type strains of Knoellia sinensis (98.5 %), Knoellia subterranea (98.2 %) and Knoellia aerolata (96.9 %). DNA–DNA relatedness values of strain TL1T with the type strains of K. sinensis and K. subterranea were 27.3 and 34.0 %, respectively. Comparison of phenotypic, chemotaxonomic and phylogenetic characteristics among strain TL1T and related organisms revealed that the isolate represents a novel species of the genus Knoellia, for which the name Knoellia flava sp. nov. is proposed; the type strain is TL1T ( = CGMCC 1.10749T = KCTC 19810T).

/pdf/knoellia-flava-sp-nov-isolated-from-pig-manure-4dtspp84mb.pdf

Knoellia flava sp. nov., isolated from pig manure.

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Flavobacterium enshiense sp. nov., isolated from soil, and emended descriptions of the genus Flavobacterium and Flavobacterium cauense, Flavobacterium saliperosum and Flavobacterium suncheonense

Massilia tieshanensis sp. nov., isolated from mining soil.

Knoellia flava sp. nov., isolated from pig manure.