16S rRNA Gene Analysis Research Articles

Pseudomonas schmalbachii sp. nov., isolated from the gut of a millipede (Trigoniulus corallinus) from a coconut tree

During an investigation of microbes associated with arthropods living in decaying coconut trees, a Pseudomonas isolate, Milli4T, was cultured from the digestive tract of the common Asian millipede, Trigoniulus corallinus. Sequence analysis of 16S rRNA and rpoB genes found that Milli4T was closely related but not identical to Pseudomonas panipatensis Esp-1T, Pseudomonas knackmussi B13T and Pseudomonas humi CCA1T. Whole genome sequencing suggested that this isolate represents a new species, with average nucleotide identity (OrthoANIu) values of around 83.9-87.7% with its closest relatives. Genome-to-genome distance calculations between Milli4T and its closest relatives also suggested they are distinct species. The genomic DNA G+C content of Milli4T was approximately 65.0 mol%. Phenotypic and chemotaxonomic characterization and fatty acid methyl ester analysis was performed on Milli4T and its related type strains. Based on these data, the new species Pseudomonas schmalbachii sp. nov. is proposed, and the type strain is Milli4T (=BCRC 81294T=JCM 34414T=CIP 111980T).

The role of the microbiome and transcriptome in the development and progression of CIN

The article presents a systematic review of the results of modern clinical studies devoted to the problem of the microbiome and transcriptome in women with cervical intraepithelial neoplasia (CIN). Moderate to severe CIN (CIN II – III) can precede the development of cervical cancer (CC) by several years or even decades. Cervical cancer (CC) is an important global health problem. There is a year-on-year increase in the prevalence of CC. Currently, there are many known risk factors that contribute to the development of CIN and cervical cancer. However, the vaginal and cervical microbiome play an important role in the development and progression of CIN and CC, according to some authors. Thus, the timely detection and treatment of cervical intraepithelial lesion-associated genital infections is now especially important. From these point of view, bacterial vaginosis (BV) is considered an acute problem in gynecological practice, which affects the incidence of precancerous conditions of the cervix. The results of the studies have shown the importance of detailed analysis of the vaginal microbial community, which was performed by the method of next generation sequencing (NGS). These studies were conducted using the NGS method based on the analysis of bacterial 16S rRNA genes, which has a high diagnostic accuracy and allows to determine the verity of the microbial landscape. The study of the transcriptome in women with CIN showed a change in many microRNA molecules, which can become markers of the CIN and cervical cancer upon further study. The introduction of the NGS method into the laboratory diagnostics complex will improve the diagnosis and timely prevent the progression of CIN to cervical cancer.The study of the microbiome of the vaginal biotope and cervical canal will allow to identify the groups of patients at high risk for the progression of precancerous lesions of the cervix and cervical cancer. Transcriptome studies have shown changes in many microRNA molecules (SALL4, FOXO1, HBD-1, HBD-2, HBD-3, LL37, psoriasin and IL-8, etc.) in women with CIN and cervical cancer.

DNA Metabarcoding Revealed Interspecific Dietary Difference and Prey Selectivity in Juvenile Horseshoe Crabs Carcinoscorpius rotundicauda and Tachypleus tridentatus From Hong Kong

Horseshoe crabs provide important ecological services including bioturbation and linking of food web in the shallow waters, but their populations are declining globally, leading to major concerns on conservation of these iconic animals. Baseline information of horseshoe crab ecology, such as their trophic role and food source, is pre-requisite for habitat protection plan and captive restocking program. Trophic ecology of Asian horseshoe crabs is relatively poorly understood and previous studies on their juveniles have suggested that they are selective feeders rather than opportunistic generalists. This study demonstrates a non-invasive approach, using DNA metabarcoding analyses of the nuclear 18S rRNA gene on fecal samples to assess the dietary compositions of Carcinoscorpius rotundicauda and Tachypleus tridentatus juveniles to (1) determine their dietary compositions and trophic roles in their ecosystem, (2) determine any prey selectivity, and (3) distinguish the interspecific dietary differences with potential implications on the habitat requirement and ecological partitioning between these two horseshoe crab species. The results based on relative read abundance (RRA) suggested that oligochaetes were the major prey items for both C. rotundicauda (41.6%) and T. tridentatus (32.4%). Bivalves and crustaceans were second major prey groups for C. rotundicauda (8.6 and 8.4%, respectively). Surprisingly, anthozoans contributed a considerable portion of T. tridentatus’s diet (22.8%), which has never been reported. Furthermore, the major prey groups identified in the fecal samples were not the dominant benthic organisms in the studied area as revealed by environmental DNA (eDNA) analyses on the sediment samples, implying that both species are selective feeders rather than dietary generalists. Significant differences observed in the dietary compositions of the two species might be partially due to the difference in habitat preference between the two species. This study provides new insights into the trophic ecology of the two Asian horseshoe crab species in the estuarine habitat and establishes a new framework for future detailed molecular dietary analyses on all developmental stages of horseshoe crabs around the world, which will allow us to evaluate the food sources needed for the survival of horseshoe crabs and facilitate future conservation strategies without killing the animals.

Effects of Long-Term Triclosan Exposure on Microbiota in Zebrafish.

Background: Triclosan (TCS) is a widely used antibacterial agent in personal care products and is ubiquitous in the environment. We aimed to examine whether TCS exposure affects microbiota in the gastrointestinal tract of zebrafish.Methods: After exposure to TCS 0 (Dimethyl Sulphoxide, DMSO control), 0.03, 0.3, 3, 30, 100, and 300ng/ml, respectively, from day 0 to 120days post fertilization (dpf), or for 7days in adult 4-month zebrafish, the long- and short-term impact of TCS exposure on the microbiome in the gastrointestinal tract was evaluated by analyzing 16S rRNA gene V3-V4 region sequencing.Results: The top two most dominant microbiota phyla were Proteobacteria and Fusobacteria phylum in all zebrafish groups. In TCS exposure 0–120 dpf, compared with DMSO control, the mean number of microbial operational taxonomic units (OTUs) was 54.46 lower (p<0.0001), Chao indice 41.40 lower (p=0.0004), and Ace indice 34.10 lower (p=0.0044) in TCS 300ng/ml group, but no change was observed in most of the other TCS concentrations. PCoA diagram showed that the microbial community in the long-term TCS 300ng/ml exposure group clustered differently from those in the DMSO control and other TCS exposure groups. A shorter body length of the zebrafish was observed in the long-term TCS exposure at 0.03, 100, and 300ng/ml. For 7-day short-term exposure in adult zebrafish, no difference was observed in alpha or beta diversity of microbiota nor the relative abundance of Proteobacteria or Fusobacteria phylum among DMSO control and any TCS levels, but a minor difference in microbial composition was observed for TCS exposure.Conclusions: Long-term exposure to high TCS concentration in a window from early embryonic life to early adulthood may reduce diversity and alter the composition of microbiota in the gastrointestinal tract. The effect of short-term TCS exposure was not observed on the diversity of microbiota but there was a minor change of microbial composition in adult zebrafish with TCS exposure.

Cultivatable bacterial community in water from Lai Nullah contaminated with household sewage and industrial waste is more diverse and populated compared with nonpolluted water

The effect of environmental pollutants on living organisms can be assessed by studying the changes in the indigenous microbial community. Therefore, in this study, cultivatable bacterial community in nonpolluted as well as household sewage and industrially polluted water of Lai Nullah flowing through Islamabad and Rawalpindi, Pakistan was analyzed. Bacterial community composition and population present in the polluted water were significantly different from the nonpolluted water (P &lt; 0.05). Nonpolluted water had much fewer species and population of bacteria compared with polluted water. Sequence analysis of bacterial 16S rRNA gene revealed that Citrobacter freundii, Klebsiella pneumoniae, Escherichia coli, Lactobacillus plantarum, Geobacillus stearothermophilus, Enterococcus faecalis, Acinetobacter guillouiae, Ralstonia sp., Comamonas sp., and Stenotrophomonas maltophilia were specific to the polluted water. On the other hand, Aeromonas veronii, Exiguobacterium sp., and Lysinibacillus macroides were only found in the nonpolluted water. Among measured physicochemical parameters, higher colony count in the polluted water was best correlated with higher biological oxygen demand, phosphate, sodium, and chloride values (Spearman’s rho = 0.85). Concentration of heavy metals such as cadmium, chromium, copper, nickel, and lead were below 0.03 μg·mL−1 at all the study sites. During plate assay, bacterial strains found at polluted sites showed resistance to selected heavy metals with highest minimum inhibitory concentration for lead (8 mmol·L−1) followed by copper (5 mmol·L−1), nickel (3 mmol·L−1), and cadmium (1 mmol·L−1). All the bacterial isolates also showed various levels of resistance against antibiotics ampicillin, tetracycline, ciprofloxacin, and vancomycin using broth microdilution method. Current research provides new insight into the effect of household sewage and the industrially polluted water of Lai Nullah on the indigenous bacteria.

Metagenomic analysis of microbial communities across a transect from low to highly hydrocarbon-contaminated soils in King George Island, Maritime Antarctica.

Soil samples from a transect from low to highly hydrocarbon-contaminated soils were collected around the Brazilian Antarctic Station Comandante Ferraz (EACF), located at King George Island, Antarctica. Quantitative PCR (qPCR) analysis of bacterial 16S rRNA genes, 16S rRNA gene (iTag), and shotgun metagenomic sequencing were used to characterize microbial community structure and the potential for petroleum degradation by indigenous microbes. Hydrocarbon contamination did not affect bacterial abundance in EACF soils (bacterial 16S rRNA gene qPCR). However, analysis of 16S rRNA gene sequences revealed a successive change in the microbial community along the pollution gradient. Microbial richness and diversity decreased with the increase of hydrocarbon concentration in EACF soils. The abundance of Cytophaga, Methyloversatilis, Polaromonas, and Williamsia was positively correlated (p-value=<.05) with the concentration of total petroleum hydrocarbons (TPH) and/or polycyclic aromatic hydrocarbons (PAH). Annotation of metagenomic data revealed that the most abundant hydrocarbon degradation pathway in EACF soils was related to alkyl derivative-PAH degradation (mainly methylnaphthalenes) via the CYP450 enzyme family. The abundance of genes related to nitrogen fixation increased in EACF soils as the concentration of hydrocarbons increased. The results obtained here are valuable for the future of bioremediation of petroleum hydrocarbon-contaminated soils in polar environments.

Combination of rhizosphere bacteria isolated from resistant potato plants for biocontrol of potato late blight.

Potato late blight (PLB) caused by Phytophthora infestans is one of the most devastating plant diseases. The heavy use of chemical control agents is at odds with the development of sustainable and environmentally friendly agriculture practices. It is necessary to screen the antagonistic microorganisms of P. infestans and provide a new choice of PLB biocontrol. In vitro, eight bacterial strains (A, B, C, D, E, F, G, H) isolated from the rhizosphere of resistant potato plants had a significant inhibitory effect on the mycelium growth of P. infestans, and the inhibition rate was 35.02-79.20%. These isolates were assigned to Streptomyces, Pseudomonas, Saccharothrix and Nocardiopsis by phylogenetic analysis of 16S rRNA genes. Their physiological and biochemical characteristics suggested that they can produce cellulase and catalase, which may help to inhibit the infection of P. infestans. In vivo, each strain significantly inhibited the infection of P. infestans after individual inoculation into potato tubers, and no strains posed a pathogenic threat to tubers. In the field environment, multibacterial treatment significantly reduced the disease index. Compared with the control, multibacterial and single H treatment significantly increased the microbial species and abundance of the potato rhizosphere and enriched potential beneficial bacteria such as Rhizobiaceae. Meanwhile, multi-bacterial and single H treatment significantly reduced the abundance of Enterobacteriaceae and Bacillaceae. Our results provide some valuable native strains from the potato rhizosphere with the ability to inhibit P. infestans in vivo and in vitro, which may be a new option for PLB biocontrol. © 2021 Society of Chemical Industry.

Bioactivities of endophytic actinobacteria inhabiting Artemisia herba-alba emphasizing differences from free-living strains.

The endophytic actinobacteria associated with Artemisia herba-alba (synonym: Seriphidium herba-alba) are highly diverse. This study aimed to illustrate the extent of their differences from the free-living actinobacteria in the surrounding environment. A selection of eighteen actinobacteria inhabiting A. herba-alba were compared with twenty and ten actinobatceria isolates from the surrounding desert and groundwater, respectively, representing six genera. Antagonistic and enzymatic activities, plant growth-promoting traits, and the occurrence of biosynthetic genes were compared among the isolates. Data were analyzed statistically using principal component analysis (PCA) and were visualized using heat map. Endophytic strains showed higher antimicrobial activity and production of plant growth promoters compared to desert and groundwater strains. Polyketide synthase and non-ribosomal peptide synthetase gene clusters were detected at higher frequencies in the endophytic strains (8 and 11 strains, respectively) than the desert strains (1 and 2 strains, respectively). In contrast, both gene clusters were not detected in the groundwater strains. The PCA revealed unique metabolic characteristics of the endophytes. The heatmap clustered the endophytic strains apart from the free-living strains, indicating distinctive qualitative and quantitative bioactivities. Analysis of 16S rRNA genes confirmed the chemotaxonomic identity of all but two strains, with > 94.5% similarity. Six endophytes displayed < 99.5% similarity with their closest type strains, which might indicate species novelty. This study provides an evidence of functional differences and possible species novelty of the endophytic actinobacteria inhabiting A. herba-alba, compared with the free-living species.

Crosstalk among intestinal barrier, gut microbiota and serum metabolome after a polyphenol-rich diet in older subjects with “leaky gut”: The MaPLE trial

The MaPLE study was a randomized, controlled, crossover trial involving adults ≥60 y.o. (n=51) living in a residential care facility during an 8-week polyphenol-rich (PR)-diet. Results from the MaPLE trial showed that the PR-diet reduced the intestinal permeability (IP) in older adults by inducing changes to gut microbiota (GM). The present work aimed at studying the changes in serum metabolome in the MaPLE trial, as a further necessary step to depict the complex crosstalk between dietary polyphenols, GM, and intestinal barrier. Serum metabolome was monitored using a semi-targeted UHPLC-MS/MS analysis. Metataxonomic analysis (16S rRNA gene profiling) of GM was performed on faecal samples. Clinical characteristics and serum levels of the IP marker zonulin were linked to GM and metabolomics data in a multi-omics network. Compared to the control diet, the PR-diet increased serum metabolites related to polyphenols and methylxanthine intake. Theobromine and methylxanthines, derived from cocoa and/or green tea, were positively correlated with butyrate-producing bacteria (the order Clostridiales and the genera Roseburia, Butyricicoccus and Faecalibacterium) and inversely with zonulin. A direct correlation between polyphenol metabolites hydroxyphenylpropionic acid-sulfate, 2-methylpyrogallol-sulfate and catechol-sulfate with Butyricicoccus was also observed, while hydroxyphenylpropionic acid-sulfate and 2-methylpyrogallol-sulfate negatively correlated with Methanobrevibacter. The multi-omics network indicated that participant's age, baseline zonulin levels, and changes in Porphyromonadaceae abundance were the main factors driving the effects of a PR-diet on zonulin. Overall, these results reveal the complex relationships among polyphenols consumption, intestinal permeability, and GM composition in older adults, and they may be important when setting personalized dietary interventions for older adults. ISRCTN10214981.

Gut microbiota alterations in response to sleep length among African-origin adults.

Sleep disorders are increasingly being characterized in modern society as contributing to a host of serious medical problems, including obesity and metabolic syndrome. Changes to the microbial community in the human gut have been reportedly associated with many of these cardiometabolic outcomes. In this study, we investigated the impact of sleep length on the gut microbiota in a large cohort of 655 participants of African descent, aged 25–45, from Ghana, South Africa (SA), Jamaica, and the United States (US). The sleep duration was self-reported via a questionnaire. Participants were classified into 3 sleep groups: short (<7hrs), normal (7-<9hrs), and long (≥9hrs). Forty-seven percent of US participants were classified as short sleepers and 88% of SA participants as long sleepers. Gut microbial composition analysis (16S rRNA gene sequencing) revealed that bacterial alpha diversity negatively correlated with sleep length (p<0.05). Furthermore, sleep length significantly contributed to the inter-individual beta diversity dissimilarity in gut microbial composition (p<0.01). Participants with both short and long-sleep durations exhibited significantly higher abundances of several taxonomic features, compared to normal sleep duration participants. The predicted relative proportion of two genes involved in the butyrate synthesis via lysine pathway were enriched in short sleep duration participants. Finally, co-occurrence relationships revealed by network analysis showed unique interactions among the short, normal and long duration sleepers. These results suggest that sleep length in humans may alter gut microbiota by driving population shifts of the whole microbiota and also specific changes in Exact Sequence Variants abundance, which may have implications for chronic inflammation associated diseases. The current findings suggest a possible relationship between disrupted sleep patterns and the composition of the gut microbiota. Prospective investigations in larger and more prolonged sleep researches and causally experimental studies are needed to confirm these findings, investigate the underlying mechanism and determine whether improving microbial homeostasis may buffer against sleep-related health decline in humans.

Proposal of Lactococcus garvieae subsp. bovis Varsha and Nampoothiri 2016 as a later heterotypic synonym of Lactococcus formosensis Chen et al. 2014 and Lactococcus formosensis subsp. bovis comb. nov.

Currently, Lactococcus garvieae contains two subspecies: L. garvieae subsp. bovis and L. garvieae subsp. garvieae. In a study by Varsha and Nampoothiri, high pheS (99.7 %) and rpoA (99.6 %) sequence similarities indicated that L. garvieae subsp. bovis and Lactococcus formosensis probably have a close taxonomic relationship; low pheS (92.2 %) and rpoA (97.8 %) sequence similarities and relatively low DNA-DNA hybridization value (75.8 %) indicated that L. garvieae subsp. bovis and L. garvieae subsp. garvieae probably represent two different species. In the present study, the taxonomic relationships between L. garvieae subsp. bovis, L. garvieae subsp. garvieae and L. formosensis were re-examined based on sequence analyses of 16S rRNA, pheS, recA, rpoA and rpoB genes, average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) values, average amino acid identity (AAI), fatty acid methyl ester analysis and phenotypic characterization. L. garvieae subsp. bovis LMG 30663T exhibited 97.3 % ANI, 78.3 % dDDH and 96.4 % AAI values to L. formosensis NBRC 109475T, higher than the threshold for species demarcation (95-96, 70 and 95-96 %, respectively), indicating that L. garvieae subsp. bovis LMG 30663T and L. formosensis NBRC 109475T belong to the same species. L. garvieae subsp. bovis LMG 30663T had 91.2 % ANI, 43.3 % dDDH and 92.9-93.0% AAI values with the type strain of L. garvieae subsp. garvieae, indicating that they represent two different species. Because L. formosensis has been proposed and validated before L. garvieae subsp. bovis, L. garvieae subsp. bovis is transferred to L. formosensis as L. formosensis subsp. bovis comb. nov. The type strain of L. formosensis subsp. bovis is BSN307T (=DSM 100577T=MCC 2824T=KCTC 21083T=LMG 30663T). The type strain of L. formosensis subsp. formosensis is 516T (=NBRC 109475T=BCRC 80576T).

Molecular detection of Babesia spp. in European bison (Bison bonasus) and their ticks

Babesia spp. are tick-borne haemoparasites that infect a wide range of domestic and wild mammals. Free-ranging ungulates are considered to be important reservoir hosts of Babesia parasites. The European bison (Bison bonasus) is a large and rare ungulate species, reintroduced into the forests of Central Europe after an absence of several decades. Owing to their protected status, studies of tick-borne pathogens in European bison have so far been rare and fragmented. The aim of this study was to investigate the presence of Babesia infection in free-ranging and captive herds of European bison and their ticks. Tissue samples obtained from 37 European bison individuals and 242 ticks belonging to two species, Ixodes ricinus and Dermacentor reticulatus, collected from bison were subjected to PCR analysis of the 18S rRNA gene followed by sequencing. Babesia spp. were detected in 8% of the samples from European bison and in 11% of the ticks. Sequence analysis of partial 18S rRNA gene indicated the presence of B. divergens and B. capreoli in European bison, while B. divergens, B. microti and B. venatorum were detected in ixodid ticks. To the best of authors’ knowledge, this is the first molecular detection and characterization of Babesia spp. in European bison and their ticks.

P–632 Examination of temporal changes in phenotype and gut microbiome during the process of growth in polycystic ovary syndrome (PCOS) model induced by prenatal androgen exposure

Abstract Study question From when do abnormality in gut microbiome and phenotypes of PCOS appear during the process of growth? Summary answer Reproductive phenotypes of PCOS appear from 6 weeks and metabolic phenotypes from 12 weeks onward. Alteration in gut microbiome appears as early as 4 weeks. What is known already The etiology of PCOS remains largely unknown, however PCOS is considered as a complex multigenic disorder with strong epigenetic and environmental influence. Previous studies have suggested that fetal over-exposure to androgens could be the main factor of the development of PCOS after birth. On the other hands, recent studies on both human and PCOS rodent models have demonstrated the association between PCOS and alteration of gut microbiome in adulthood. Furthermore, it was recently reported that gut microbiome in obese adolescent with PCOS is different from obese adolescent without PCOS. Study design, size, duration A rodent PCOS model induced by prenatal dehydroepiandrosterone (DHT) exposure was applied to this study. Phenotypes and gut microbiome were compared between PCOS model mice (n = 12/group) and control mice (n = 10/group) at each stage of growth; 4 weeks (prepuberty), 6 weeks (puberty), 8 weeks (adolescent), 12 weeks (young adult), and 16 weeks (adult). The determinants for PCOS phenotypes are onset of puberty, estrous cycle, morphology of ovaries, serum testosterone level, body weight, and insulin resistance. Participants/materials, setting, methods Pregnant dams were subcutaneously injected on days of 16, 17, and 18 of gestation with either sesame oil for control groups or sesame oil containing 250µg of DHT for prenatal DHT groups. The evaluation of PCOS phenotypes and gut microbiome in female offspring were performed at each stage of growth. For examination of gut microbiota, next generation sequencing and bioinformatics analysis of 16S rRNA genes were performed on DNA extracted from mouse fecal samples. Main results and the role of chance Prenatal DHT mice exhibited delayed puberty onset, disrupted estrous cycle, and significantly increased testosterone levels from 6 weeks onward. Significantly increased atretic antral follicles were observed in prenatal DHT mice at 6, 12, and 16 weeks. Prenatal DHT mice showed significantly decreased body weight at 4, 6, 8 weeks and increased body weight from 12 weeks onward. As for gut microbiome, alpha-diversity was significantly different between control and prenatal DHT mice from 8 weeks onward and beta-diversity was significantly different at 6 and 8 weeks. Altered composition of gut microbiota was observed as early as 4 weeks. At phylum level, Firmicutes are significantly increased in prenatal DHT mice at 4 and 8 weeks and decreased at 16 weeks. Actinobacteria phylum showed significant decrease at 6 and 8 weeks in prenatal DHT mice. At genus level, relative abundance of several bacterial taxa significantly differed between control and prenatal DHT mice; some taxa, such as Allobaculum, Adlercreutzia, Bilophila, Clostridium, Gemella, Gemmiger, Roseburia, Ruminococcus, Staphylococcus, and Sutterella, exhibited constant increase or decrease in prenatal DHT mice during the process of growth. Interestingly, Roseburia was never detected in prenatal DHT mice, while approximately half of control mice harbored Roseburia at 12 and 16 weeks. Limitations, reasons for caution It is not clearly determined whether alteration in gut microbiome is cause or result of PCOS development, although the changes in gut microbiome seemed to precede the appearance of typical PCOS phenotypes in the present study. Mouse model does not completely recapitulate human PCOS. Wider implications of the findings: Our findings suggest that prenatal androgen exposure causes alteration of gut microbiome from pre-puberty onward, even before PCOS phenotypes become apparent. Intervention for girls at risk of PCOS with pre/pro-biotics may prevent them from developing PCOS in future. Trial registration number Not applicable

DIPLOTRIAENA OBTUSA (NEMATODA: DIPLOTRIAENIDAE) FROM BARN SWALLOWS (HIRUNDO RUSTICA) AND CLIFF SWALLOWS (PETROCHELIDON PYRRHONOTA) COLLECTED DURING MORTALITY EVENTS IN THE UPPER MIDWEST, USA.

Several mortality events involving barn swallows (Hirundo rustica) and cliff swallows (Petrochelidon pyrrhonota) were reported in the Upper Midwestern states in 2017 and 2018. Barn swallow mortality followed unseasonal cold snaps, with the primary cause of death being emaciation with concurrent air sac nematodiasis. Lesions in cliff swallows were consistent with blunt force trauma from suspected car impacts. Examination of air sac nematodes from both bird species revealed morphological characters consistent with Diplotriaena obtusa. Sequence analysis of the partial 18S rRNA gene indicated the samples clustered with other species in the genus Diplotriaena. These nematodes provide a link between morphological specimens and DNA sequence data for D. obtusa.

Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period.

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3–20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

CLONING AND in silico STUDIES OF LIPASE GENE OF A THERMOPHILIC BACTERIA ISOLATED FROM THE HYDROTHERMAL VENT AREA OF KAWIO, NORTH SULAWESI

Underwater volcano is an example of a natural habitat of thermophilic bacteria, such as hydrothermal vent Kawio in North Sulawesi. The focus of this research was to screen the bacterial isolates originally from hydrothermal vent Kawio for the production of thermostable lipase. In this study, the lipase gene from the selected isolate was cloned, sequenced, and in silico analysis was performed to predict the characteristics of the lipase protein. One of three isolates examined, showed a positive reaction. Phylogenetic analysis of 16S rRNA gene showed that the isolate was a novel bacteria closely related to Bacillus safensis strain FO-26b with 96,87% homology. Amino acid sequence analysis of the lipase showed similarity between lipase of Bacillus pumilus strain B106 with 98% homology. Further analysis at the protein level indicated that the lipase isolated from marine water was a member of family I.4 of ‘true lipase’ group.

Isolation and Identification of Cellulolytic Bacteria Symbiont from Various Termites on Different Nest Type in Bukit Baka Bukit Raya National Park, West Kalimantan, Indonesia

The microbial symbiotic community in the digestive tract of termites is reportedly influenced by the taxonomy and feeding habit of the host. Both factors are strongly correlated with the nest type. This study aimed to isolate the cellulolytic bacteria from termite’s digestive tract on different nest types and characterize and identify the potential isolates. The research methods included termite sampling conducted in Bukit Baka Bukit Raya National Park (BBBRNP), Melawi, West Kalimantan, isolation of cellulolytic bacteria from termites’ gut, endoglucanase activity test, biochemical characterization, and DNA analysis based on the amplification of 16S rRNA gene. Thirty isolates from 6 different species of termites on three different nest types were successfully isolated. Sixteen potential endoglucanase bacterial isolates were tested in terms of their endoglucanase activity. The cellulolytic index measured from those isolates ranged from 1.162 - 4.894. Three isolates (MRH.13.S, MRH.13.AF, and MRH.13.O2) with the highest cellulolytic index on each nest type were identified. The analysis of 16S rRNA gene using BLAST (Basic Local Alignment Search Tool for Nucleotides) revealed that isolate MRH.13.S had the closest relationship with Bacillus tequilensis (99 % homology). Based on biochemical characterization, MRH.13.AF and MRH.13.O2 isolates were related to Bacillus spp. HIGHLIGHTS Potential cellulolytic bacteria from termite intestinal tract from different nests (i.e., soil, wood, and arboreal) were isolated and compared Termites were obtained from a lowland dipterocarp primary forest ecosystem in Bukit Baka Bukit Raya National Park, West Kalimantan Province, Indonesia Termite species collected were Termes comis, Dicuspiditermes garthwaitei, Synhamitermes quadriceps, Havilanditermes proatripennis, Bulbitermes borneensis, and Bulbitermes parapusillus Potential cellulolytic bacteria acquired were closely related with Bacillus tequilensis and Bacillus spp GRAPHICAL ABSTRACT

Comparative Analysis of 16S rRNA Gene and Metagenome Sequencing in Pediatric Gut Microbiomes

The colonization of the human gut microbiome begins at birth, and over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon vs. shotgun metagenomic sequencing techniques in 338 fecal samples; younger than 15, 15–30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

Drivers and ecological consequences of arsenite detoxification in aged semi-aerobic landfill

Microbial populations responsible for arsenite [As(III)] detoxification were examined in aged refuse treated with 75 μM As(III) under semi-aerobic conditions. As(III) was rapidly oxidized to As(V) via microbial activity, and substantial As was fixed in the solid phase. The abundance of arsenite oxidase genes (aioA) was about four times higher in the moderate As(III) stressed treatment than in the untreated control. Network analysis of microbial community 16S rRNA genes based on MRT (random matrix theory) further illuminated details about microbe-microbe interactions, and showed six ecological clusters. A total of 166 “core” taxa were identified by within-module connectivity and among-module connectivity values. When compared with the control treatment without As(III), 12 putative keystone operational taxonomic units were positively correlated with As(III) oxidation, of which 10 of these were annotated to genera level. Eight genera were associated with As(III) detoxification: Pseudomonas, Paenalcaligenes, Proteiniphilum, Moheibacter, Mobilitalea, Anaerosporobacter, Syntrophomonas and Pusillimonas. Most of those putative keystone taxa were rare species in landfill, which suggests that low-abundance taxa might significantly contribute to As(III) oxidation.

Pinocchia daecheonga sp. nov. (Synechococcales, Cyanobacteria) isolated from a Daecheong Lake in Geum River, Republic of Korea

The collection of Pinocchia daecheonga sp. nov. was performed at the Daecheong Lake in Korea, five strains of which with four clones were investigated through light microscopy, transmission electron microscopy and molecular data comprising the 16S rRNA to 23S rRNA gene. P. daecheonga differed from type species P. polymorpha by absence of the polar aerotopes. Investigated stains and four clones of P. daecheonga turned out to be a sister clade to the P. polymorpha according to the phylogenetic analysis of 16S rRNA gene. In addition, the Pinocchia was clustered with the family Leptolyngbyaceae members, genera Leptothoe and Leptoelongatus. 16S–23S rRNA intergenic transcribed spacer (ITS) region of P. daecheonga was found to be substantially distinct to P. polymorpha in terms of the secondary structure and nucleotide sequence composition, which concludes that the Pinocchia daecheonga isolated from the Daecheong Lake is a unique species due to differences in morphology and genetic traits compared to the relative P. polymorpha.