Publications

4359

ABSTRACT “ Candidatus Neoehrlichia mikurensis” is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of “ Ca . Neoehrlichia” has been studied only by comparing 16S rRNA genes and groEL operon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical “ Ca . Neoehrlichia” strains in Europe and their relatedness to other species within the Anaplasmataceae family. Six genes were selected: ftsZ , clpB , gatB , lipA , groEL , and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed “ Ca . Neoehrlichia” infection from Sweden ( n = 9), the Czech Republic ( n = 2), and Germany ( n = 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identical lipA sequences, while the lipA sequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in the clpB sequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA, ftsZ , gatB , and groEL . According to the MLSA, among the Anaplasmataceae , “ Ca . Neoehrlichia” is most closely related to Ehrlichia ruminantium , less so to Anaplasma phagocytophilum , and least to Wolbachia endosymbionts. To conclude, three sequence types of infectious “ Ca . Neoehrlichia” were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

The phenotypic and genotypic characteristics of four Bordetella hinzii-like strains from human respiratory specimens and representing nrdA gene sequence based genogroups 3, 14 and 15 were examined. In a 16S rRNA gene sequence based phylogenetic tree, the four strains consistently formed a single coherent lineage but their assignment to the genus Bordetella was equivocal. The respiratory quinone, polar lipid and fatty acid profiles generally conformed to those of species of the genus Bordetella and were characterized by the presence of ubiquinone 8, of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several aminolipids, and of high percentages of C16 : 0, cyclo-C17 : 0 and summed feature 2, as major chemotaxonomic marker molecules, respectively. The DNA G+C content was about 66 mol%, which corresponded with that of the high-percentage DNA G+C content genera of the family Alcaligenaceae including the genus Bordetella. DNA–DNA hybridization experiments revealed the presence of three distinct genomospecies and thus confirmed phenotypic differences as revealed by means of extensive biochemical characterization. We therefore propose to formally classify Bordetella genogroups 3, 14 and 15 as Bordetella bronchialis sp. nov. (type strain LMG 28640T = AU3182T = CCUG 56828T), Bordetella sputigena sp. nov. (type strain LMG 28641T = CCUG 56478T) and Bordetella flabilis sp. nov. (type strain LMG 28642T = AU10664T = CCUG 56827T). In addition, we propose to reclassify Achromobacter sediminum into the novel genus Verticia, as Verticia sediminum, gen. nov., comb. nov., on the basis of its unique phylogenetic position, its marine origin and its distinctive phenotypic, fatty acid and polar lipid profile.

rinjal is an important vegetable crop grown worldwide. A field survey conducted during 2012 in Kerala state of South India, the little leaf disease affected brinjal plant was observed in one of the home gardens located at Nemmara (Palakkad district). The diseased and healthy samples were collected; genomic DNA was isolated and 16SrRNA gene amplified by performing nested PCR using universal primers pairs P1/P7 and R16F2n/R2 respectively. The association of Phytoplasma was detected in all the three symptomatic brinjal samples and in Phytoplasma affected Periwinkle used as positive control. An expected amplicon size of 1250bp from nested PCR was purified and sequenced. The nucleotide sequence analysis (nBLAST) of 1250bp fragment of sequence KL-50(KP027530) revealed 97% similarity with Candidatus phytoplasma trifolii (JX104336). Phylogenetic analysis and study of virtual RFLP gel pattern of the sequence also revealed the association of Candidatus phytoplasma trifolii.