Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Pseudomonas chlororaphis subsp. piscium subsp. nov., isolated from freshwater fish

Gram-negative, aerobic, motile, rod-shaped bacteria were isolated from the intestines of freshwater fish on two separate occasions. Colonies of both strains, JF3835(T) and JF4413, produced non-diffusible green pigment following 4-5 days incubation on Luria-Bertani agar. The most abundant fatty acids were summed feature 3 (comprising C(16 : 1)ω7c and/or C(15 : 0) iso 2-OH), C(16 : 0) and C(18 : 1)ω7c. The DNA G+C content was 62.9 mol%. Sequence analysis of the 16S rRNA gene indicated 100 % sequence similarity between the two strains. In comparison with recognized species, the new strains exhibited the greatest degree of sequence similarity with members of the Pseudomonas chlororaphis subspecies: P. chlororaphis subsp. chlororaphis (99.84 %), P. chlororaphis subsp. aurantiaca (99.75 %) and P. chlororaphis subsp. aureofaciens (99.40 %). While DNA-DNA relatedness confirmed the placement of strains JF3835(T) and JF4413 as members of the species P. chlororaphis, multilocus sequencing indicated that the strains formed a distinct cluster within it. On the basis of genotypic and phenotypic evidence, strains JF3835(T) and JF4413 represent a novel subspecies of the species P. chlororaphis, for which the name Pseudomonas chlororaphis subsp. piscium subsp. nov. is proposed. The type strain is JF3835(T) (=NCIMB 14478(T)=DSM 21509(T)).

Uruburuella testudinis sp. nov., isolated from tortoise (Testudo)

A polyphasic taxonomic analysis was carried out on 11 uncommon Gram-stain-negative, non-motile, catalase- and oxidase-positive, but indole-negative, bacterial strains isolated from tortoises. Phenotypically and genetically they represented a homogeneous group of organisms most closely related to, but distinct from, Uruburuella suis. In a reconstructed 16S rRNA gene tree they clustered on a monophyletic branch next to U. suis with gene similarities between strains of 99.5-100%, and of up to 98.2% with U. suis . DNA-DNA hybridization indicated the organisms represented a novel species with only 40% DNA-DNA similarity with U. suis . Partial sequencing of rpoB resulted in two subclusters confirming the 16S rRNA gene phylogeny; both genes allowed clear separation and identification of the novel species. Furthermore, they could be unambiguously identified by matrix-assisted laser desorption ionization time-of-flight MS, where, again, they formed a highly homogeneous cluster separate from U. suis and other members of the family Neisseriaceae . The major fatty acids were C(16 : 0) and summed feature C(16 : 1)ω7c/iso-C(15 : 0) 2-OH. The DNA G+C content was 54.4 mol%. Based on phenotypic and genetic data we propose classifying these organisms as representatives of a novel species named Uruburuella testudinis sp. nov. The type strain is 07_OD624(T) ( = DSM 26510(T) = CCUG 63373(T)).

Classification of organisms previously reported as the SP and Stewart-Letscher groups, with descriptions of Necropsobacter gen. nov. and of Necropsobacter rosorum sp. nov. for organisms of the SP group

To allow classification of bacteria previously reported as the SP group and the Stewart-Letscher group, 35 isolates from rodents (21), rabbits (eight), a dog and humans (five) were phenotypically and genotypically characterized. Comparison of partial rpoB sequences showed that 34 of the isolates were closely related, demonstrating at least 97.4 % similarity. 16S rRNA gene sequence comparison of 20 selected isolates confirmed the monophyly of the SP group and revealed 98.5 %-100 % similarity between isolates. A blast search using the 16S rRNA gene sequences showed that the highest similarity outside the SP group was 95.5 % to an unclassified rat isolate. The single strain, P625, representing the Stewart-Letscher group showed the highest 16S rRNA gene similarity (94.9-95.5 %) to members of the SP group. recN gene sequence analysis of 11 representative strains resulted in similarities of 97-100 % among the SP group strains, which showed 80 % sequence similarity to the Stewart-Letscher group strain. Sequence similarity values based on the recN gene, indicative for whole genome similarity, showed the SP group being clearly separated from established genera, whereas the Stewart-Letscher group strain was associated with the SP group. A new genus, Necropsobacter gen. nov., with only one species, Necropsobacter rosorum sp. nov., is proposed to include all members of the SP group. The new genus can be separated from existing genera of the family Pasteurellaceae by at least three phenotypic characters. The most characteristic properties of the new genus are that haemolysis is not observed on bovine blood agar, positive reactions are observed in the porphyrin test, acid is produced from (+)-L-arabinose, (+)-D-xylose, dulcitol, (+)-D-galactose, (+)-D-mannose, maltose and melibiose, and negative reactions are observed for symbiotic growth, urease, ornithine decarboxylase and indole. Previous publications have documented that both ubiquinones and demethylmenaquinone were produced by the proposed type strain of the new genus, Michel A/76(T), and that the major polyamine of representative strains (type strain not included) of the genus is 1,3-diaminopropane, spermidine is present in moderate amounts and putrescine and spermine are detectable only in minor amounts. The major fatty acids of strain Michel A/76(T) are C(14 : 0), C(16 : 0), C(16:1)omega7c and summed feature C(14 : 0) 3-OH/iso-C(16 : 1) I. This fatty acid profile is typical for members of the family Pasteurellaceae. The G+C content of DNA of strain Michel A/76(T) was estimated to be 52.5 mol% in a previous investigation. The type strain is P709(T) ( = Michel A/76(T) = CCUG 28028(T) = CIP 110147(T) = CCM 7802(T)).

Proposal of Bisgaardia hudsonensis gen. nov., sp. nov. and an additional genomospecies, isolated from seals, as new members of the family Pasteurellaceae

Phenotypic and phylogenetic studies were performed on eight Gram-negative-staining, rod-shaped bacteria isolated from seals. Biochemical and physiological studies showed identical profiles for all of the isolates and indicated that they were related to the family Pasteurellaceae. 16S rRNA gene sequencing demonstrated that the organism represented a distinct cluster with two sublines within the family Pasteurellaceae with <96% sequence similarity to any recognized species. Multilocus sequence analysis (MLSA) including rpoB, infB and recN genes further confirmed these findings with the eight isolates forming a genus-like cluster with two branches. Genome relatedness as deduced from recN gene sequences suggested that the isolates represented a new genus with two species. On the basis of the results of the phylogenetic analysis and phenotypic criteria, it is proposed that these bacteria from seals are classified as Bisgaardia hudsonensis gen. nov., sp. nov. (the type species) and Bisgaardia genomospecies 1. The G+C content of the DNA was 39.5 mol%. The type strain of Bisgaardia hudsonensis gen. nov., sp. nov. is M327/99/2(T) (=CCUG 43067(T)=NCTC 13475(T)=98-D-690B(T)) and the reference strain of Bisgaardia genomospecies 1 is M1765/96/5 (=CCUG 59551=NCTC 13474).

Staphylococcus rostri sp. nov., a haemolytic bacterium isolated from the noses of healthy pigs

Twenty coagulase-negative Staphylococcus strains displaying alpha-haemolysis (delta-haemolysin) on sheep-blood agar were isolated from the noses of different pigs in Switzerland. The strains were Gram-stain-positive, non-motile cocci, catalase-positive and coagulase-negative. Sequence analysis of the 16S rRNA gene, sodA, rpoB, dnaJ and hsp60 and phylogenetic characteristics revealed that the strains showed the closest relatedness to Staphylococcus microti CCM 4903(T) and Staphylococcus muscae DSM 7068(T). The strains can be differentiated from S. microti by the absence of mannose fermentation and arginine arylamidase and from S. muscae by the absence of beta-glucuronidase activity and production of alkaline phosphatase. The chosen type strain ARI 262(T) shared 20.1 and 31.9 % DNA relatedness with S. microti DSM 22147(T) and S. muscae CCM 4903(T), respectively, by DNA-DNA hybridization. iso-C(15 : 0), anteiso-C(15 : 0) and iso-C(17 : 0) were the most common fatty acids. Cell-wall structure analysis revealed the peptidoglycan type A3alpha l-Lys-Gly(2)-l-Ser-Gly (type A11.3). The presence of teichoic acid was determined by sequencing the N-acetyl-beta-d-mannosaminyltransferase gene tarA, which is involved in biosynthesis of ribitol teichoic acid. Menaquinone 7 (MK-7) was the predominant respiratory quinone. The G+C content of ARI 262(T) was 38.8 mol%. The isolated strains represent a novel species of the genus Staphylococcus, for which we propose the name Staphylococcus rostri sp. nov. The type strain is ARI 262(T) (=DSM 21968(T) =CCUG 57266(T)) and strain ARI 602 (=DSM 21969 =CCUG 57267) is a reference strain.

Arsenicicoccus dermatophilus sp. nov., a hypha-forming bacterium isolated from the skin of greater flamingos (Phoenicopterus roseus) with pododermatitis

Dermatophilus-like bacteria were observed in histological examinations of samples of diseased foot skin from greater flamingos (Phoenicopterus roseus) living in zoological gardens in Switzerland. When grown on TSA-SB containing polymyxin B, the bacteria isolated from these skin samples formed hyphae, as is typical for Dermatophilus congolensis, but these bacteria were non-haemolytic. The closest relatives based on 16S rRNA gene sequences were the two members of the genus Arsenicicoccus, Arsenicicoccus bolidensis and Arsenicicoccus piscis. A representative of the isolated strains shared 34.3 % DNA-DNA relatedness with the type strain of A. bolidensis, 32.3 % with the type strain of A. piscis and 34.5 % with the type strain of D. congolensis, demonstrating that these strains do not belong to any of these species. The phenotypic characteristics differed from those of members of the genus Arsenicicoccus as well as from those of D. congolensis. The G+C content of strain KM 894/11(T) was 71.6 mol%. The most abundant fatty acids were iso-C15 : 0, summed feature 3 (including C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω9c. MK-8(H4) was the predominant menaquinone. Cell-wall structure analysis revealed that the peptidoglycan type was A3γ ll-Dpm-Gly (type A41.1). Based on genotypic and chemotaxonomic characteristics, the isolated strains represent a novel species within the genus Arsenicicoccus, for which the name Arsenicicoccus dermatophilus sp. nov. is proposed. The type strain is KM 894/11(T) ( = DSM 25571(T) = CCUG 62181(T) = CCOS 690(T)), and strain KM 1/12 ( = DSM 25572 = CCUG 62182 = CCOS 691) is a reference strain.

Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data

BACKGROUND The best-known cause of intolerance to fluoropyrimidines is dihydropyrimidine dehydrogenase (DPD) deficiency, which can result from deleterious polymorphisms in the gene encoding DPD (DPYD), including DPYD*2A and c.2846A>T. Three other variants-DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A-have been associated with DPD deficiency, but no definitive evidence for the clinical validity of these variants is available. The primary objective of this systematic review and meta-analysis was to assess the clinical validity of c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity. METHODS We did a systematic review of the literature published before Dec 17, 2014, to identify cohort studies investigating associations between DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A and severe (grade ≥3) fluoropyrimidine-associated toxicity in patients treated with fluoropyrimidines (fluorouracil, capecitabine, or tegafur-uracil as single agents, in combination with other anticancer drugs, or with radiotherapy). Individual patient data were retrieved and analysed in a multivariable analysis to obtain an adjusted relative risk (RR). Effect estimates were pooled by use of a random-effects meta-analysis. The threshold for significance was set at a p value of less than 0·0167 (Bonferroni correction). FINDINGS 7365 patients from eight studies were included in the meta-analysis. DPYD c.1679T>G was significantly associated with fluoropyrimidine-associated toxicity (adjusted RR 4·40, 95% CI 2·08-9·30, p<0·0001), as was c.1236G>A/HapB3 (1·59, 1·29-1·97, p<0·0001). The association between c.1601G>A and fluoropyrimidine-associated toxicity was not significant (adjusted RR 1·52, 95% CI 0·86-2·70, p=0·15). Analysis of individual types of toxicity showed consistent associations of c.1679T>G and c.1236G>A/HapB3 with gastrointestinal toxicity (adjusted RR 5·72, 95% CI 1·40-23·33, p=0·015; and 2·04, 1·49-2·78, p<0·0001, respectively) and haematological toxicity (adjusted RR 9·76, 95% CI 3·03-31·48, p=0·00014; and 2·07, 1·17-3·68, p=0·013, respectively), but not with hand-foot syndrome. DPYD*2A and c.2846A>T were also significantly associated with severe fluoropyrimidine-associated toxicity (adjusted RR 2·85, 95% CI 1·75-4·62, p<0·0001; and 3·02, 2·22-4·10, p<0·0001, respectively). INTERPRETATION DPYD variants c.1679T>G and c.1236G>A/HapB3 are clinically relevant predictors of fluoropyrimidine-associated toxicity. Upfront screening for these variants, in addition to the established variants DPYD*2A and c.2846A>T, is recommended to improve the safety of patients with cancer treated with fluoropyrimidines. FUNDING None.

Draft Genome Sequence of the Virulent Avibacterium paragallinarum Serotype A Strain JF4211 and Identification of Two Toxins

Avibacterium paragallinarum is an important pathogen of chicken livestock causing infectious coryza. Here, we report the draft genome sequence of the virulent A. paragallinarum serotype A strain JF4211 (2.8 Mbp and G+C content of 41%) and the two toxin operons discovered from the annotation of the genome.

Draft Genome Sequence of the Virulent Clostridium chauvoei Reference Strain JF4335

Clostridium chauvoei is the etiological agent of blackleg, a disease of cattle and sheep with high mortality rates, causing severe economic losses in livestock production. Here, we report the draft genome sequence of the virulent C. chauvoei strain JF4335 (2.8 Mbp and 28% G+C content) and the annotation of the genome.

Complete Genome Sequences of Virulent Mycoplasma capricolum subsp. capripneumoniae Strains F38 and ILRI181

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae is a severe epidemic affecting mainly domestic Caprinae species but also affects wild Caprinae species. M. capricolum subsp. capripneumoniae belongs to the "Mycoplasma mycoides cluster." The disease features prominently in East Africa, in particular Kenya, Tanzania, and Ethiopia. CCPP also endangers wildlife and thus affects not only basic nutritional resources of large populations but also expensively built-up game resorts in affected countries. Here, we report the complete sequences of two M. capricolum subsp. capripneumoniae strains: the type strain F38 and strain ILRI181 isolated druing a recent outbreak in Kenya. Both genomes have a G+C content of 24% with sizes of 1,016,760 bp and 1,017,183 bp for strains F38 and ILRI181, respectively.

Alternative DNA base-pairs: from efforts to expand the genetic code to potential material applications

The complementary Watson-Crick base-pairs, A:T and G:C, have long been recognized as pivotal to both the stability of the DNA double helix and replication/transcription. Recently, the replacement of the Watson-Crick base-pairs with other molecular entities has received considerable attention. In this tutorial review we highlight different approaches used to replace natural base-pairs and equip them with novel function. We also discuss the advantages that non-natural base-pairs convey with respect to practical applications.