A culture-independent approach, supervised machine learning, and the characterization of the microbial community composition of coastal areas across the Bay of Bengal and the Arabian Sea.

BACKGROUND: Coastal areas are subject to various anthropogenic and natural influences. In this study, we investigated and compared the characteristics of two coastal regions, Andhra Pradesh (AP) and Goa (GA), focusing on pollution, anthropogenic activities, and recreational impacts. We explored three main factors influencing the differences between these coastlines: The Bay of Bengal's shallower depth and lower salinity; upwelling phenomena due to the thermocline in the Arabian Sea; and high tides that can cause strong currents that transport pollutants and debris. RESULTS: The microbial diversity in GA was significantly higher than that in AP, which might be attributed to differences in temperature, soil type, and vegetation cover. 16S rRNA amplicon sequencing and bioinformatics analysis indicated the presence of diverse microbial phyla, including candidate phyla radiation (CPR). Statistical analysis, random forest regression, and supervised machine learning models classification confirm the diversity of the microbiome accurately. Furthermore, we have identified 450 cultures of heterotrophic, biotechnologically important bacteria. Some strains were identified as novel taxa based on 16S rRNA gene sequencing, showing promising potential for further study. CONCLUSION: Thus, our study provides valuable insights into the microbial diversity and pollution levels of coastal areas in AP and GA. These findings contribute to a better understanding of the impact of anthropogenic activities and climate variations on biology of coastal ecosystems and biodiversity.

Clostridium punense sp. nov., an obligate anaerobe isolated from healthy human faeces.

An obligately anaerobic, rod-shaped (0.5-1.0 × 2.0-10.0 µm), Gram-stain-positive bacterium, occurring mainly singly or in pairs, and designated BLPYG-8T, was isolated from faeces of a healthy human volunteer aged 56 years. Cells were non-motile. Oval, terminal spores were formed that swell the cells. The strain was affiliated with the genus Clostridium sensu stricto (Clostridium rRNA cluster I) as revealed by 16S rRNA gene sequence analysis. Strain BLPYG-8T showed 97.3 to 97.4 % 16S rRNA gene sequence similarity with Clostridium sulfidigenes DSM 18982T, Clostridium subterminale DSM 6970T and Clostridium thiosulfatireducens DSM 13105T. DNA-DNA hybridization and phenotypic analysis showed that the strain was distinct from its closest relatives, C. sulfidigenes DSM 18982T, C. subterminale DSM 6970T, C. thiosulfatireducens DSM 13105T with 54.2, 53.9 and 53.3 % DNA-DNA relatedness, respectively. Strain BLPYG-8T grew in PYG broth at temperatures between 20 and 40 °C (optimum 37 °C). The strain utilized a range of amino acids as well as carbohydrates as a source of carbon and energy. Glucose fermentation resulted in the formation of volatile fatty acids mainly acetic acid, n-butyric acid and organic acids such as succinic and lactic acid. The DNA G+C content of strain BLPYG-8T was 44.1 mol%. The major fatty acids (>10 %) were C14 : 0, iso-C15 : 0, C16 : 1ω7c and C16 : 0. Phylogenetic analysis and specific phenotypic characteristics and/or DNA G+C content differentiated the strain from its closest relatives. On the basis of these data, strain BLPYG-8T represents a novel species of the genus Clostridium, for which the name Clostridium punense sp. nov. is proposed. The type strain is BLPYG-8T ( = DSM 28650T = CCUG 64195T = MCC 2737T).

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.

Bhargavaea indica sp. nov., a member of the phylum Firmicutes, isolated from Arabian Sea sediment.

A Gram-positive, aerobic, coccoid-rod shaped, non-motile, catalase- and oxidase-positive bacterium, designated strain KJW98(T), was isolated from the marine sediment of Karwar jetty, west coast of India. The strain was ß-haemolytic, non-endospore-forming and grew with 0-8.5% (w/v) NaCl, at 15-48°C and at pH 6.5-9.0, with optimum growth with 0.5% (w/v) NaCl, at 42°C and at pH 7.0-8.0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences showed that strain KJW98(T) forms a lineage within the genus Bhargavaea. The G+C content of the genomic DNA was 55 mol%. The DNA-DNA relatedness values of strain KJW98(T) with B. beijingensis DSM 19037(T), B. cecembensis LMG 24411(T) and B. ginsengi DSM 19038(T) were 43.2, 39 and 26.5%, respectively. The major fatty acids were anteiso-C15:0 (37.7%), iso-C15:0 (19.7%), anteiso-C17:0 (17.0%) and iso-C16:0 (11.1%). The predominant menaquinone was MK-8 and the cell-wall peptidoglycan was of A4α type with L-lysine as the diagnostic diamino acid. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The phenotypic, genotypic and DNA-DNA relatedness data indicate that strain KJW98(T) should be distinguished from the members of the genus Bhargavaea, for which the name Bhargavaea indica sp. nov. is proposed with the type strain KJW98(T) (=KCTC 13583(T) =LMG 25219(T)).

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.