Prevalence and subtype analysis of Blastocystis in healthy Indian individuals.

There is a growing interest in subtype (ST) analysis of the intestinal parasite Blastocystis due to its extensive genetic diversity that might reflect differences in pathogenicity. Although essential for reference, few studies are available on Blastocystis in healthy individuals. Moreover, molecular epidemiology data on Blastocystis in India still remain to emerge. In the present study we identified the prevalence and ST distribution of Blastocystis in healthy Indian individuals. A total of 220 stool samples were obtained; four of 100 samples from 100 adults were chosen randomly for construction of small subunit (SSU) rRNA gene clone libraries in order to elucidate micro-eukaryotic diversity in the human gut. From the SSU rDNA library, 64 sequences annotated to Blastocystis were used for ST analysis along with sequences obtained by direct sequencing of SSU rDNA PCR products amplified from the remaining samples and generated using primers targeting Blastocystis. Of 220 stool samples collected, 120 samples from 30 infants (aged 1week to 1year) were PCR-negative. Of the remaining 100 samples from 100 adults, 27 resulted in specific amplification. Out of these 27, four samples were suspected of mixed ST infection and so these samples were further analyzed by construction of clone libraries. Analysis of cloned sequences revealed that indeed 2 samples had mixed ST infection (ST1 and ST3) while the remaining two showed infection with two separate ST3 strains. ST3 was the most common ST present in our study group (100%) followed by ST1 (7.4%); ST1 was seen only in mixed infections. SSU rDNA clone library sequences generated by processing of pooled samples were identified as ST3. The majority of ST3 sequences exhibited allele 34 commonly found in the European population.

Reclassification of Bacillus beijingensis Qiu et al. 2009 and Bacillus ginsengi Qiu et al. 2009 as Bhargavaea beijingensis comb. nov. and Bhargavaea ginsengi comb. nov. and emended description of the genus Bhargavaea.

We have carried out a polyphasic taxonomic characterization of Bacillus beijingensis DSM 19037(T) and Bacillus ginsengi DSM 19038(T), which are closely related phylogenetically to Bhargavaea cecembensis LMG 24411(T). All three strains are Gram-stain-positive, non-motile, moderately halotolerant and non-spore-forming. 16S rRNA gene sequence analyses showed that the strains constituted a coherent cluster, with sequence similarities between 99.7 and 98.7 %. The percentage similarity on the basis of amino acid sequences deduced from partial gyrB gene nucleotide sequences of these three type strains was 96.1-92.7 %. Phylogenetic trees based on the 16S rRNA gene and GyrB amino acid sequences, obtained by using three different algorithms, were consistent and showed that these three species constituted a deeply rooted cluster separated from the clades represented by the genera Bacillus, Planococcus, Planomicrobium, Sporosarcina, Lysinibacillus, Viridibacillus, Kurthia and Geobacillus, supporting their placement in the genus Bhargavaea. All three type strains have menaquinone MK-8 as the major respiratory quinone and showed similar fatty acid profiles. The main polar lipids present in the three type strains were diphosphatidylglycerol and phosphatidylglycerol, and the three strains showed peptidoglycan type A4α with L-lysine as the diagnostic diamino acid. The DNA G+C contents of Bacillus beijingensis DSM 19037(T), Bacillus ginsengi DSM 19038(T) and Bhargavaea cecembensis LMG 24411(T) were 53.1, 50.2 and 53.7 mol%, respectively. The level of DNA-DNA hybridization among the three strains was 57-39 %, indicating that they are members of different species of the genus Bhargavaea. The phenotypic data are consistent with the placement of these three species in a single genus and support their differentiation at the species level. On the basis of these data, we have emended the description of the genus Bhargavaea and propose the reclassification of Bacillus beijingensis and Bacillus ginsengi to the genus Bhargavaea, as Bhargavaea beijingensis comb. nov. (type strain ge10(T)  = DSM 19037(T)  = CGMCC 1.6762(T)) and Bhargavaea ginsengi comb. nov. (type strain ge14(T)  = DSM 19038(T)  = CGMCC 1.6763(T)).

Shewanella indica sp. nov., isolated from sediment of the Arabian Sea.

A Gram-negative, facultatively anaerobic, rod-shaped, catalase- and oxidase-positive bacterium, motile by means of a single polar flagellum and designated strain KJW27(T), was isolated from the marine sediment of Karwar jetty, west coast of India. The strain was ß-haemolytic and grew with 0-10 % (w/v) NaCl, at 10-45 °C and at pH 6.5-10, with optimum growth with 2 % (w/v) NaCl, at 37 °C and at pH 7.5. The major fatty acids were iso-C15:0 (22.2 %), C17:1ω8c (21 %), summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c; 10.2 %), C16:0 (7.1 %), iso-C13:0 (5.6 %) and C17:0 (4.4 %). The DNA G+C content was 51.2 mol%. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences showed that strain KJW27(T) forms a lineage within the genus Shewanella and is closely related to Shewanella algae ATCC 51192(T) (98.8 %), Shewanella haliotis DW01(T) (98.8 %) and Shewanella chilikensis JC5(T) (98.2 %). Sequence identity with other members of this genus ranges from 92.2 to 96.4 %. The DNA-DNA relatedness of strain KJW27(T) with S. algae ATCC 51192(T), S. haliotis DW01(T) and S. chilikensis JC5(T) was 52, 44 and 33 %, respectively. The phenotypic, genotypic and DNA-DNA relatedness data indicate that strain KJW27(T) should be distinguished from S. algae ATCC 51192(T), S. haliotis DW01(T) and S. chilikensis JC5(T). On the basis of the data presented in this study, strain KJW27(T) represents a novel species, for which the name Shewanella indica sp. nov. is proposed. The type strain is KJW27(T) ( = KCTC 23171(T)  = BCC 41031(T)  = NCIM 5388(T)).

Ignatzschineria indica sp. nov. and Ignatzschineria ureiclastica sp. nov., isolated from adult flesh flies (Diptera: Sarcophagidae).

Two Gram-negative-staining, aerobic, non-motile, rod-shaped bacteria, designated strains FFA1(T) and FFA3(T), and belonging to the class Gammaproteobacteria were isolated from the gastrointestinal tract of adult flesh flies (Diptera: Sarcophagidae). Phylogenetic analysis of 16S rRNA gene sequence data placed these two strains within the genus Ignatzschineria with similarities of 98.6 % (FFA1(T)) and 99.35 % (FFA3(T)) to Ignatzschineria larvae L1/68(T). The level of gene sequence similarity between strains FFA1(T) and FFA3(T) was 99 %, 97.15 % and 78.1 % based on the 16S rRNA, 23S rRNA and gyrB gene sequences, respectively. Strains FFA1(T) and FFA3(T) shared 24 % DNA-DNA relatedness. DNA-DNA hybridization revealed a very low level of relatedness between the novel strains (22 % for strain FFA1(T) and 44 % for strain FFA3(T)) and I. larvae L1/68(T) genomic DNA. The respiratory quinone was Q-8 in both novel strains. The DNA G+C contents were 41.1 mol% and 40.1 mol% for strains FFA1(T) and FFA3(T), respectively. The cell membrane of both strains consisted of phosphatidylglycerol, phosphatidylethanolamine, phospholipids and aminophospholipid. The major fatty acids for both strains were C(16 : 0), summed feature 8 (C(18 : 1)ω7c and/or C(18 : 1)ω6c), CyC(19 : 0)ω8c and C(14 : 0). The results of DNA-DNA hybridization between the two new strains and I. larvae L1/68(T), in combination with phylogenetic, chemotaxonomic, biochemical and electron microscopic data, demonstrated that strains FFA1(T) and FFA3(T) represented two novel species of the genus Ignatzschineria for which the names Ignatzschineria indica sp. nov. (type strain FFA1(T) = DSM 22309(T) = KCTC 22643(T) = NCIM 5325(T)) and Ignatzschineria ureiclastica sp. nov. (type strain FFA3(T) = DSM 22310(T) = KCTC 22644(T) = NCIM 5326(T)) are proposed.