16S rRNA Gene Research Articles

The first isolation of Clostridium massiliodielmoense from a dead beef cow in Japan and pitfalls to be aware of in identifying the Clostridium species.

Clostridium sp. was detected in the organs of a cow with black watery diarrhea in Japan. Results identifying this species were inconsistent; Clostridium novyi type A infection was suggested by PCR assay targeting Clostridium fliC region (fliC-multiplex PCR), while 16S rRNA gene sequencing identified the isolated bacteria as Clostridium massiliodielmoense. Sequencing of fliC-multiplex PCR products from the isolates revealed the presence of fliC region in C. massiliodielmoense, which had 92.7% nucleotide similarity to that of C. novyi type A JCM 1406T, leading to the false positive detection of C. novyi by the PCR. This is the first C. massiliodielmoense isolation from clinical specimens, suggesting the need for further research on its pathogenicity and improvement in fliC-multiplex PCR.

Microbial communities assembly in wastewater treatment plants in China

Community assembly processes determine community structure. Deterministic processes are essential for optimizing activated sludge (AS) bioreactor performance. However, the debate regarding the relative importance of determinism versus stochasticity remains contentious, and the influencing factors are indistinct. This study used large-scale 16S rRNA gene data derived from 252 AS samples collected from 28 cities across China to explore the mechanism of AS community assembly. Results showed that the northern communities possessed lower spatial turnover and more significant dispersal limitation than those in the south, whereas the latter had more substantial deterministic processes than the former (14.46 % v.s. 9.12 %). Meanwhile, the communities in the south exhibited lower network complexity and stability. We utilized a structural equation model to explore the drivers of deterministic processes. Results revealed that low network complexity (r = −0.56, P < 0.05) and high quorum sensing bacteria abundance (r = 0.25, P < 0.001) promoted deterministic assembly, which clarifies why determinism was stronger in southern communities than northern ones. Furthermore, total phosphorus and hydraulic retention time were found to be the primary abiotic drivers. These findings provide evidence to understand the community deterministic assembly, which is crucial for resolving community structure and improving bioreactor performance.

Fine-scale characterization of the soybean rhizosphere microbiome via synthetic long reads and avidity sequencing

BackgroundThe rhizosphere microbiome displays structural and functional dynamism driven by plant, microbial, and environmental factors. While such plasticity is a well-evidenced determinant of host health, individual and community-level microbial activity within the rhizosphere remain poorly understood, due in part to the insufficient taxonomic resolution achieved through traditional marker gene amplicon sequencing. This limitation necessitates more advanced approaches (e.g., long-read sequencing) to derive ecological inferences with practical application. To this end, the present study coupled synthetic long-read technology with avidity sequencing to investigate eukaryotic and prokaryotic microbiome dynamics within the soybean (Glycine max) rhizosphere under field conditions.ResultsSynthetic long-read sequencing permitted de novo reconstruction of the entire 18S-ITS1-ITS2 region of the eukaryotic rRNA operon as well as all nine hypervariable regions of the 16S rRNA gene. All full-length, mapped eukaryotic amplicon sequence variants displayed genus-level classification, and 44.77% achieved species-level classification. The resultant eukaryotic microbiome encompassed five kingdoms (19 genera) of protists in addition to fungi – a depth unattainable with conventional short-read methods. In the prokaryotic fraction, every full-length, mapped amplicon sequence variant was resolved at the species level, and 23.13% at the strain level. Thirteen species of Bradyrhizobium were thereby distinguished in the prokaryotic microbiome, with strain-level identification of the two Bradyrhizobium species most reported to nodulate soybean. Moreover, the applied methodology delineated structural and compositional dynamism in response to experimental parameters (i.e., growth stage, cultivar, and biostimulant application), unveiled a saprotroph-rich core microbiome, provided empirical evidence for host selection of mutualistic taxa, and identified key microbial co-occurrence network members likely associated with edaphic and agronomic properties.ConclusionsThis study is the first to combine synthetic long-read technology and avidity sequencing to profile both eukaryotic and prokaryotic fractions of a plant-associated microbiome. Findings herein provide an unparalleled taxonomic resolution of the soybean rhizosphere microbiota and represent significant biological and technological advancements in crop microbiome research.

Effects of "brain-gut coherence" method of acupuncture on motor function and intestinal microflora in the patients with cerebral ischemic stroke

To observe the clinical effect of "brain-gut coherence" method of acupuncture on cerebral ischemic stroke (CIS) and explore its action mechanism. A total of 82 patients with CIS were randomly divided into an observation group (41 cases, 3 cases dropped out, 2 cases discontinued) and a control group (41 cases, 4 cases dropped out, 2 cases excluded). The conventional basic treatment was administered in the two groups. Additionally, in the observation group, "brain-gut coherence" method of acupuncture was delivered. The stimulating points included the parietal and temporal anterior oblique line on the affected side, Zhongwan (CV 12), Guanyuan (CV 4), and bilateral Tianshu (ST 25), Zusanli (ST 36), Shangjuxu (ST 37) and Xiajuxu (ST 39). In the control group, the routine acupuncture was operated at Baihui (GV 20), Yintang (GV 24+), bilateral Fengchi (GB 20) and Zusanli (ST 36), and Hegu (LI 4), Jianyu (LI 15), Quchi (LI 11), Waiguan (TE 5), Futu (ST 32), Sanyinjiao (SP 6) and Taichong (LR 3) on the affected side. Acupuncture stimulation lasted 30 min each time, once daily, and for 5 days a week. The intervention for 4 weeks was required. The scores of Fugl-Meyer assessment scale (FMA), Berg balance scale (BBS) and the modified Barthel index (MBI), as well as the score of gastrointestinal symptoms were compared before and after treatment in the two groups. The neutrophil count (NUE) and the content of the serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) were detected before and after treatment in the two groups. Using 16S rRNA gene sequencing, the structure and relative abundance of intestinal microflora was detected before and after treatment; and with the enzyme linked immunosorbent assay (ELISA) adopted, the levels of intestinal fatty acid-binding protein (iFABP), D-lactate (D-LA), lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP), tumor necrosis factor-α(TNF-α), interleukin (IL)-1β and IL-6 in the serum were detected before and after treatment in the two groups. After treatment, the scores of FMA, BBS and MBI were increased (P<0.05), and the scores of gastrointestinal symptoms were decreased (P<0.05) compared with those before treatment in the two groups. Compared with the control group, the scores of FMA, BBS and MBI were higher (P<0.05) and the score of gastrointestinal symptoms was lower (P<0.05) in the observation group after treatment. NEU and the content of serum NT-proBNP were reduced in the two groups (P<0.05), and the content of serum NT-proBNP in the observation group was lower than that of the control group (P<0.05) after treatment. Chao1, Ace, Sobs and Shannon indexes were increased after treatment compared with those before treatment in the two groups (P<0.05); and these indexes in the observation group were higher when compared with the control group (P<0.05). After treatment, the relative abundance of Bacteroidaceae, Enterobacteriaceae, Oscillospiraceae, Streptococcaceae and Sutterellaceae was reduced in comparison with that before treatment in the two groups (P<0.05); and the relative abundance of these microflora was lower in the observation group when compared with the control group (P<0.05). After treatment, the relative abundance of Lachnospiraceae, Ruminococcaceae, Bifidobacteriaceae and Coriobacteriaceae was increased in comparison with that before treatment in the two groups (P<0.05); and the relative abundance of these microflora was elevated in the observation group when compared with the control group (P<0.05). After treatment, the levels of iFABP, D-LA, LPS, LBP, TNF-α, IL-1β and IL-6 were reduced when compared with those before treatment in the two groups (P<0.05), and these levels of the observation group were lower than those of the control group (P<0.05). "Brain-gut coherence" method of acupuncture can improve the motor function and gastrointestinal function of the patients with cerebral ischemic stroke, which may be related to modulating the structure of intestinal microflora, alleviating inflammatory reactions and accelerating the intestinal barrier repair.

Integrative analysis of microbiome and metabolome revealed the effect of microbial inoculant on microbial community diversity and function in rhizospheric soil under tobacco monoculture.

Over-application of chemical fertilizers and continuous cropping obstacles seriously restrict the sustainable development of tobacco production. Localized fertilization of beneficial microbes has potential advantages in achieving higher productivity, but the underlying biological mechanisms of interactions between rhizospheric microorganisms and the related metabolic cycle remain poorly characterized. Here, an integrative analysis of microbiomes with non-targeted metabolomics was performed on 30 soil samples of rhizosphere, root surrounding, and bulk soils from flue-cured tobacco under continuous and non-continuous monocropping systems. The analysis was conducted using UPLC-MS/MS platforms and high-throughput amplicon sequencing targeting the bacterial 16S rRNA gene and fungal ITS gene. The microbial inoculant consisted of Bacillus subtilis, B. velezensis, and B. licheniformis at the ratio of 1:1:1 in effective microbial counts, improved the cured leaf yield and disease resistance of tobacco, and enhanced nicotine and nitrogen contents of tobacco leaves. The bacterial taxa Rhizobium, Pseudomonas, Sphingomonadaceae, and Burkholderiaceae of the phylum Proteobacteria accumulated in high relative abundance and were identified as biomarkers following the application of the microbial inoculant. Under continuous monocropping, metabolomics demonstrated that the application of the microbial inoculant significantly affected the soil metabolite spectrum, and the differential metabolites were significantly enriched to the synthesis and degradation of nicotine (nicotinate and nicotinamide metabolism and biosynthesis of alkaloids derived from nicotinic acid). In addition, microbes were closely related to the accumulation of metabolites through correlation analysis. The interactions between plant roots and rhizospheric microorganisms provide valuable information for understanding how these beneficial microbes affect complex biological processes and the adaption capacity of plants to environments.IMPORTANCEThis study elaborated on how the microbial fertilizer significantly changed overall community structures and metabolite spectrum of rhizospheric microbes, which provide insights into the process of rhizosphere microbial remolding in response to continuous monocropping. we verified the hypothesis that the application of the microbial inoculant in continuous cropping would lead to the selection of distinct microbiota communities by establishing models to correlate biomarkers. Through correlation analysis of the microbiome and metabolome, we proved that rhizospheric microbes were closely related to the accumulation of metabolites, including the synthesis and degradation of nicotine. The interactions between plant roots and rhizospheric microorganisms provide valuable information for understanding how these beneficial microbes affect complex biological processes and the adaption capacity of plants to environments.

Plant roots affect free-living diazotroph communities in temperate grassland soils despite decades of fertilization

Fixation of atmospheric N2 by free-living diazotrophs accounts for an important proportion of nitrogen naturally introduced to temperate grasslands. The effect of plants or fertilization on the general microbial community has been extensively studied, yet an understanding of the potential combinatorial effects on the community structure and activity of free-living diazotrophs is lacking. In this study we provide a multilevel assessment of the single and interactive effects of different long-term fertilization treatments, plant species and vicinity to roots on the free-living diazotroph community in relation to the general microbial community in grassland soils. We sequenced the dinitrogenase reductase (nifH) and the 16S rRNA genes of bulk soil and root-associated compartments (rhizosphere soil, rhizoplane and root) of two grass species (Arrhenatherum elatius and Anthoxanthum odoratum) and two herb species (Galium album and Plantago lanceolata) growing in Austrian grassland soils treated with different fertilizers (N, P, NPK) since 1960. Overall, fertilization has the strongest effect on the diazotroph and general microbial community structure, however with vicinity to the root, the plant effect increases. Despite the long-term fertilization, plants strongly influence the diazotroph communities emphasizing the complexity of soil microbial communities’ responses to changing nutrient conditions in temperate grasslands.

Gut Microbiome and colorectal cancer: discovery of bacterial changes with metagenomics application in Turkısh population.

Colorectal cancer (CRC) is the 3rd most common cancer in the world and colonic carcinogenesis is a multifactorial disease that involves environmental and genetic factors. Gut microbiota plays a critical role in the regulation of intestinal homeostasis. Increasing evidence shows that the gut microbiome plays a role in CRC development and may be a biomarker for early diagnosis. This study aimed to determine the clinical prognostic significance of gut microbiota in CRC patients in the Turkish population by metagenomic analysis and to determine the microbial composition in tumor tissue biopsy samples. Tissue biopsies were taken from the participants with sterile forceps during colonoscopy and stored at -80°C. Then, DNA isolation was performed from the tissue samples and the V3-V4 region of the 16S rRNA gene was sequenced on the Illumina MiSeq platform. Quality control of the obtained sequence data was performed. Operational taxonomic units (OTUs) were classified according to the Greengenes database. Alpha diversity (Shannon index) and beta diversity (Bray-Curtis distance) analyses were performed. The most common bacterial species in CRC patients and healthy controls were determined and whether there were statistically significant differences between the groups was tested. A total of 40 individuals, 13 CRC patients and 20 healthy control individuals were included in our metagenomic study. The mean age of the patients was 64.83 and BMI was 25.85. In CRC patients, the level of Bacteroidetes at the phylum taxonomy was significantly increased (p = 0.04), the level of Clostridia at the class taxonomy was increased (p = 0.23), and the level of Enterococcus at the genus taxonomy was significantly increased (p = 0.01). When CRC patients were compared with the control group, significant increases were detected in the species of Gemmiger formicilis (p = 0.15), Prevotella copri (p = 0.02) and Ruminococcus bromii (p = 0.001) at the species taxonomy. Metagenomic analysis of intestinal microbiota composition in CRC patients provides important data for determining the treatment options for these patients. The results of this study suggest that it may be beneficial in terms of early diagnosis, poor prognosis and survival rates in CRC patients. In addition, this metagenomic study is the first study on the colon microbiome associated with CRC mucosa in the Turkish population.

Taxonomic diversity and environmental tolerance of cultivable extremophilic bacteria from a high-altitude meltwater pond on Ojos del Salado (Chile).

Earth harbors unique environments where only microorganisms adapted to extreme conditions, known as extremophiles, can survive. This study focused on a high-altitude meltwater pond, located in the Puna de Atacama, Dry Andes. The extremophilic bacteria of this habitat must adapt to a range of extremities, including cold and dry climate, high UV radiation, high daily temperature fluctuations, low-nutrient availability, and negative water balance. This study aimed to explore the taxonomic diversity of cultivable extremophilic bacteria from sediment samples of a desiccated, high-altitude, meltwater pond using media with different organic matter contents and different incubation temperatures. Based on the 16S rRNA gene sequence analysis, the isolates were identified as members of the phyla Actinobacteria, Proteobacteria, and Firmicutes. The most abundant genera were Arthrobacter and Pseudoarthrobacter. The isolates had oligocarbophilic and psychrotrophic properties, suggesting that they have adapted to the extreme environmental parameters of their natural habitats. The results indicate a positive correlation between nutrient concentration and temperature tolerance.

Bocconia frutescens little leaf, a new plant disease associated to a ´Candidatus Phytoplasma pruni´ related strain in Costa Rica

Objective/Background. Bocconia frutescens (Papaveraceae) is a small tree distributed naturally from Mexico to Argentina and the Caribbean Bassin. Bocconia trees showing symptoms resembling phytoplasmas infection, such as little leaves and witches´-broom, were found in Cartago province, Costa Rica. Detection and identification of the potential phytoplasmas associated with B. frutescens little leaf symptoms was the objective out of this study. Materials and Methods. Evaluation of leaves tissue using transmission electron microscopy (TEM), nested PCR using universal and specific primers to amplify phytoplasmas 16S rRNA and secA genes. Nucleotidic sequences (Sanger method) were obtained from amplicons, and used for BLASTn, phylogenetic analyses, and in silico RFLP’s. Results. Presence of phytoplasmas into phloem tissue, only in symptomatic trees, was evidenced by TEM. Comparison of partial sequences (16Sr and secA genes) by BLASTn, in silico RFLP´s and phylogenetic analyses, showed the occurrence of a ´Candidatus Phytoplasma pruni´ related strain in the samples evaluated. Conclusion. Phytoplasmas were found only in the symptomatic B. frutescens trees evaluated. The phytoplasmas were identified as a ´Ca. Phytoplasma pruni´ related strain. This is the first report of B. frutescens as a natural host of ´Ca. Phytoplasma pruni´.

Divergent marine anaerobic ciliates harbor closely related Methanocorpusculum endosymbionts.

Ciliates are a diverse group of protists known for their ability to establish various partnerships and thrive in a wide variety of oxygen-depleted environments. Most anaerobic ciliates harbor methanogens, one of the few known archaea living intracellularly. These methanogens increase the metabolic efficiency of host fermentation via syntrophic use of host end-product in methanogenesis. Despite the ubiquity of these symbioses in anoxic habitats, patterns of symbiont specificity and fidelity are not well known. We surveyed two unrelated, commonly found groups of anaerobic ciliates, the Plagiopylea and Metopida, isolated from anoxic marine sediments. We sequenced host 18S rRNA and symbiont 16S rRNA marker genes as well as the symbiont ITS region from our cultured ciliates to identify hosts and their associated methanogenic symbionts. We found that marine ciliates from both of these co-occurring, divergent groups harbor closely related yet distinct intracellular archaea within the Methanocorpusculum genus. The symbionts appear to be stable at the host species level, but at higher taxonomic levels, there is evidence that symbiont replacements have occurred. Gaining insight into this unique association will deepen our understanding of the complex transmission modes of marine microbial symbionts, and the mutualistic microbial interactions occurring across domains of life.

Divergent marine anaerobic ciliates harbor closely related Methanocorpusculum endosymbionts.

Ciliates are a diverse group of protists known for their ability to establish various partnerships and thrive in a wide variety of oxygen-depleted environments. Most anaerobic ciliates harbor methanogens, one of the few known archaea living intracellularly. These methanogens increase the metabolic efficiency of host fermentation via syntrophic use of host end-product in methanogenesis. Despite the ubiquity of these symbioses in anoxic habitats, patterns of symbiont specificity and fidelity are not well known. We surveyed two unrelated, commonly found groups of anaerobic ciliates, the Plagiopylea and Metopida, isolated from anoxic marine sediments. We sequenced host 18S rRNA and symbiont 16S rRNA marker genes as well as the symbiont ITS region from our cultured ciliates to identify hosts and their associated methanogenic symbionts. We found that marine ciliates from both of these co-occurring, divergent groups harbor closely related yet distinct intracellular archaea within the Methanocorpusculum genus. The symbionts appear to be stable at the host species level, but at higher taxonomic levels, there is evidence that symbiont replacements have occurred. Gaining insight into this unique association will deepen our understanding of the complex transmission modes of marine microbial symbionts, and the mutualistic microbial interactions occurring across domains of life.

Exploring the occurrence of Listeria in biofilms and deciphering the bacterial community in a frozen vegetable producing environment.

The establishment of Listeria (L.) monocytogenes within food processing environments constitutes a significant public health concern. This versatile bacterium demonstrates an exceptional capacity to endure challenging environmental conditions in the food processing environment, where contamination of food products regularly occurs. The diverse repertoire of stress resistance genes, the potential to colonize biofilms, and the support of a co-existing microbiota have been proposed as root causes for the survival of L. monocytogenes in food processing environments. In this study, 71 sites were sampled after cleaning and disinfection in a European frozen vegetable processing facility, where L. monocytogenes in-house clones persisted for years. L. monocytogenes and L. innocua were detected by a culture-dependent method at 14 sampling sites, primarily on conveyor belts and associated parts. The presence of biofilms, as determined by the quantification of bacterial load and the analysis of extracellular matrix components (carbohydrates, proteins, extracellular DNA) was confirmed at nine sites (12.7%). In two cases, L. innocua was detected in a biofilm. Furthermore, we explored the resident microbial community in the processing environment and on biofilm-positive sites, as well as the co-occurrence of bacterial taxa with Listeria by 16S rRNA gene sequencing. Pseudomonas, Acinetobacter, and Exiguobacterium dominated the microbial community of the processing environment. Using differential abundance analysis, amplicon sequence variants (ASVs) assigned to Enterobacterales (Enterobacter, Serratia, unclassified Enterobacteriaceae) and Carnobacterium were found to be significantly higher abundant in Listeria-positive samples. Several Pseudomonas ASVs were less abundant in Listeria-positive compared to Listeria-negative samples. Acinetobacter, Pseudomonas, Janthinobacterium, Brevundimonas, and Exiguobacterium were key players in the microbial community in biofilms, and Exiguobacterium and Janthinobacterium were more relatively abundant in biofilms. Further, the microbial composition varied between the different areas and the surface materials.

Successful operation of nitrifying granules at low pH in a continuous-flow reactor: Nitrification performance, granule stability, and microbial community

Acidic nitrification, as a novel process for treating wastewater without sufficient alkalinity, has received increasing attention over the years. In this study, a continuous-flow reactor with aerobic granular sludge was successful operated at low pH (<6.5) performing high-rate acidic nitrification. Volumetric ammonium oxidation rate of 0.4–1.2 kg/(m3·d) were achieved with the specific biomass activities of 5.8–13.9 mg N/(gVSS·h). Stable partial nitritation with nitrite accumulation efficiency over 85% could be maintained at pH above 6 with the aid of residual ammonium, whereas the nitrite accumulation disappeared when pH was below 6. Interestingly, the granule morphology significantly improved during the acidic operation. The increased secretion of extracellular polymeric substances (especially polysaccharides) suggested a self-protective behavior of microbes in the aerobic granules against acidic stress. 16S rRNA gene sequencing analyses indicated that Candidatus Nitrospira defluvii was always the dominant nitrite-oxidizing bacteria, while the dominant ammonia-oxidizing bacteria shifted from Nitrosomonas europaea to Nitrosomonas mobilis. This study, for the first time, demonstrated the improved stability of aerobic granules under acidic conditions, and also highlighted aerobic granules as a useful solution to achieve high-rate acidic nitrification.

Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses

BackgroundCurrently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region.ResultsThe largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities.ConclusionsFrozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.

First Report of bacterial wilt disease of banana caused by Klebsiella variicola in Malaysia.

In Malaysia, bananas (Musa spp.) are the second most cultivated fruit and the fourth most cultivated fruit in terms of export revenue. In October 2018, about 5.0 out of 6.6 hectares of a banana plantation located in Teluk Intan, Malaysia, was impacted by an outbreak of banana disease. The onset of bacterial wilt symptoms is characterized by initial leaf wilting, followed by the subsequent withering of the entire plant during later stages, fruit stalk and fruit pulp discoloration, fruit rotting, and pseudostem necrosis. The diseased banana's symptomatic pseudostems and fruit pulps were surface-sterilised in 70% ethanol for 30 s, followed by 2% NaClO for 3 min, rinsed three times in sterilised water, and cut into small pieces approximately 5 mm2 in size. The tissues were macerated in a sterilised 0.85% NaCl solution for 5 min, and the resulting suspension was streaked onto nutrient agar, followed by incubation at 28°C for 2 days. After incubation, bacterial colonies with five unique morphological characteristics were observed. Two colonies of each unique morphological type were randomly chosen and subjected to preliminary bacterial identification by 16S rRNA gene sequencing. Based on BLASTn analysis, the five unique morphological types of bacteria were preliminarily identified as Enterobacter cloacae, Citrobacter farmeri, Klebsiella variicola, Kosakonia radicincitans, and Phytobacter ursingii. Previous reports identified K. variicola and K. radicincitans as banana pathogens, but Malaysia has yet to report the former. The amplified partial 16S rDNA sequences of both K. variicola isolates (designated as UTAR-BC1 and UTAR-BC2; GenBank accession numbers: PP531448 and PP531460, respectively), which were chosen to be the focus of this study, exhibited complete similarity to each other and were 100% identical (1426/1426 identity and 1420/1420 identity, respectively) to K. variicola (CP026013.1). To verify the identity of the bacterial isolate, three housekeeping genes, namely, infB(PP538994), rpoB (PP538995), and gyrB (PP538996) of UTAR-BC1, were amplified, sequenced, and subjected to multilocus phylogenetic analysis via the neighbour-joining method (1,000 bootstrap values). Phylogenetic analysis revealed that UTAR-BC1 belongs to the K. variicola clade. A pathogenicity assay of UTAR-BC1 was conducted on 4-month-old healthy banana plantlets (cv. Nangka) using the pseudostem injection method (Tripathi et al., 2008). First, UTAR-BC1 was grown overnight in nutrient broth and then adjusted to 108 CFU/ml in a sterile 10 mM MgCl2 solution. A total volume of 100 µL of the bacterial suspension was injected into the pseudostem of five healthy banana plantlets via a syringe with a needle. Control plants were mock-inoculated with a sterile 10 mM MgCl2 solution. The experiments were replicated thrice and inoculated plants were maintained at room temperature with natural sunlight and humidity, which resembled the field conditions. Two months after inoculation, all of the UTAR-BC1 inoculated spots of banana plantlets showed severe necrosis, while the banana leaves showed symptoms of wilted appearance, whereas the control plants remained symptomless. The reisolated pathogen from 90% of the symptomatic pseudostems and leaf blades shares the same morphological and molecular features as UTAR-BC1, thus fulfilling Koch's postulates. Previously, K. variicola has been reported to be a banana pathogen causing rhizome rot in India (Loganathan et al., 2021), plantain soft rot in Haiti (Fulton et al. 2020), and sheath rot and bulb rot in China (Sun et al., 2023; Jiang et al., 2024). To the best of our knowledge, this is the first report of bacterial wilt disease in bananas attributed to K. variicola in Malaysia. This finding will facilitate the surveillance of K. variicola as an emerging pathogen in banana plants in this region, thereby safeguarding the country's food security and promoting socio-economic growth.

Effects of Heat-Treated Lactobacillus helveticus CP790-Fermented Milk on Gastrointestinal Health in Healthy Adults: A Randomized Double-Blind Placebo-Controlled Trial.

Probiotic-fermented milk is commonly used to maintain intestinal health. However, the effects of heat-treated fermented milk, which does not contain live microorganisms, on intestinal function are not yet fully understood. This study aimed to investigate whether heat-treated Lactobacillus helveticus CP790-fermented milk affects fecal microbiota and gut health as a "postbiotic". A randomized, double-blind, placebo-controlled trial was conducted in healthy Japanese individuals aged 20-59 years with a tendency toward constipation. Participants consumed 100 mL of either the test beverage (n = 60) or placebo beverage (n = 60) for four weeks. The test beverages were prepared with heat-treated CP790-fermented milk, while the placebo beverages were prepared with nonfermented milk flavored with lactic acid. Fecal samples were analyzed using 16S rRNA gene sequencing. Constipation symptoms were assessed using defecation logs and the Patient Assessment of Constipation Symptoms (PAC-SYM) questionnaire. Mood state was also assessed using the Profile of Mood States 2 (POMS2) questionnaire to explore its potential as a "psychobiotic". Desulfobacterota were significantly decreased by CP790-fermented milk intake. PICRUSt2 analysis predicted a decrease in the proportion of genes involved in the sulfate reduction pathway following the consumption of CP790-fermented milk. The CP790-fermented milk intervention significantly improved stool consistency and straining during defecation. These improvements were correlated with a decrease in Desulfobacterota. After the intervention, overall mood, expressed as total mood disturbance, and depression-dejection were significantly better in the CP790 group than in the placebo group. These results suggest that the intake of CP790-fermented milk could be effective in modulating gut microbiota and improving constipation symptoms and mood states.

Undibacterium cyanobacteriorum sp. nov., an auxin-producing bacterium isolated from fresh water during cyanobacterial bloom period.

A novel Gram-negative, white-pigmented, and auxin-producing strain, 20NA77.5T, was isolated from fresh water during cyanobacterial bloom period. Pairwise comparison of the 16S rRNA gene sequences showed that strain 20NA77.5T belonged to the genus Undibacterium and exhibited the highest sequence similarity to the type strains of Undibacterium danionis (98.00%), Undibacterium baiyunense (97.93%), Undibacterium macrobrachii (97.92%), and Undibacterium fentianense (97.71%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain 20NA77.5T and its related type strains were below 79.93 and 23.80%, respectively. The predominant fatty acids (> 10% of the total fatty acids) were C16:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The genomic DNA G + C content of strain 20NA77.5T was found to be 48.61%. Based on the phylogenetic distinctness, chemotaxonomic features, and phenotypic features, strain 20NA77.5T is considered to represent a novel species of the genus Undibacterium, for which the name Undibacterium cyanobacteriorum sp. nov is proposed. The type strain is 20NA77.5T (= KCTC 8005T = LMG 33136T).

Gut Microbiota and Clinical Manifestations in Thai Pediatric Patients with Attention-Deficit Hyperactivity Disorder.

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder potentially linked to gut dysbiosis. This comparative cross-sectional study profiled the gut microbiota in 24 treatment-naïve Thai children diagnosed with ADHD and 24 healthy ones matched by age and gender (median age: 7 years). Fecal microbial compositions were genetically analyzed using 16s rRNA gene amplicon sequencing. The study findings indicated no statistically significant differences in microbial diversity between groups, although Firmicutes and Actinobacteria appeared dominant in both groups. Moreover, ADHD patients exhibited enrichment in Alloprevotella, CAG-352, Succinivibrio, and Acidaminococcus genera, while healthy controls had higher levels of Megamonas, Enterobacter, Eubacterium hallii, and Negativibacillus genera. Spearman correlation analysis demonstrated a significant positive association between CAG-352 and inattention and hyperactivity/impulsivity scores, whereas the Eubacterium hallii group and Megamonas exhibited negative correlations with these symptomatology domains. Beta-carotene intake was associated with the Eubacterium hallii group and Succinivibrio: likewise, vitamin B2 intake was associated with Alloprevotella. Additional research should aim to elucidate the underlying mechanisms influencing clinical biomarkers that signify alterations in specific gut microbiome profiles linked to ADHD.

Genetic diversity and prevalence of emerging Rickettsiales in Yunnan Province: a large-scale study

BackgroundRickettsia and related diseases have been identified as significant global public health threats. This study involved comprehensive field and systematic investigations of various rickettsial organisms in Yunnan Province.MethodsBetween May 18, 2011 and November 23, 2020, field investigations were conducted across 42 counties in Yunnan Province, China, encompassing small mammals, livestock, and ticks. Preliminary screenings for Rickettsiales involved amplifying the 16S rRNA genes, along with additional genus- or species-specific genes, which were subsequently confirmed through sequencing results. Sequence comparisons were carried out using the Basic Local Alignment Search Tool (BLAST). Phylogenetic relationships were analyzed using the default parameters in the Molecular Evolutionary Genetics Analysis (MEGA) program. The chi-squared test was used to assess the diversities and component ratios of rickettsial agents across various parameters.ResultsA total of 7964 samples were collected from small mammals, livestock, and ticks through Yunnan Province and submitted for screening for rickettsial organisms. Sixteen rickettsial species from the genera Rickettsia, Anaplasma, Ehrlichia, Neoehrlichia, and Wolbachia were detected, with an overall prevalence of 14.72%. Among these, 11 species were identified as pathogens or potential pathogens to humans and livestock. Specifically, 10 rickettsial organisms were widely found in 42.11% (24 out of 57) of small mammal species. High prevalence was observed in Dremomys samples at 5.60%, in samples from regions with latitudes above 4000 m or alpine meadows, and in those obtained from Yuanmou County. Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis were broadly infecting multiple genera of animal hosts. In contrast, the small mammal genera Neodon, Dremomys, Ochotona, Anourosorex, and Mus were carrying individually specific rickettsial agents, indicating host tropism. There were 13 rickettsial species detected in 57.14% (8 out of 14) of tick species, with the highest prevalence (37.07%) observed in the genus Rhipicephalus. Eight rickettsial species were identified in 2375 livestock samples. Notably, six new Rickettsiales variants/strains were discovered, and Candidatus Rickettsia longicornii was unambiguously identified.ConclusionsThis large-scale survey provided further insight into the high genetic diversity and overall prevalence of emerging Rickettsiales within endemic hotspots in Yunnan Province. The potential threats posed by these emerging tick-borne Rickettsiales to public health warrant attention, underscoring the need for effective strategies to guide the prevention and control of emerging zoonotic diseases in China.Graphical

Revealing microbial community characteristics in healthy human, cat and canine salivas and looking for species-specific microbes.

As two kinds of increasingly popular pets, the saliva of cat or canine is most likely to be left at the crime scene compared with the common types of body fluids in forensics. Accurately identifying the species of saliva samples found at the crime scene involving pets will help the investigators find available testing materials, reduce the consumption of reagents and save the investigative time of the case. Therefore, it is necessary to explore the characteristics and differences of saliva microbiomes of cat, canine and human. In this study, 16S rRNA gene amplicon sequencing technology was used to reveal microbial communities of saliva samples of healthy human, cat, and canine. Alpha diversity analyses indicated that canine saliva demonstrated the highest microbial diversity, followed by cat saliva, whereas human saliva microbial diversity was the lowest. The saliva samples of the three species all had their own unique microbial community compositions, and the dominant phyla of canine and cat salivas were Proteobacteria and Bacteroidete, while the dominant phyla of human saliva were Firmicutes and Proteobacteria. There was no significant statistical difference in the salivary microbiota obtained by the two collection methods (cotton swab and liquid saliva). The gender of cats and canines might have no effect on the salivary microbiota, but the different breeds had an impact on their saliva microbiomes. Principal coordinates analysis, non-metric multidimensional scaling analysis and random forest analysis all indicated significant differences in microbial community structures among the three species, allowing inference on the species sources of saliva samples by microbiome method. Differential microbial biomarkers for the salivas of three species were screened out using a variety of bioinformatics analyses, and the results demonstrated that Prevotella melaninogenica, Veillonella parvula, and Haemophilus parainfluenzae could be used as species-specific microbial biomarkers of human saliva. The detections of human species-specific microbes provide a potential method for determining human saliva.