Full-length 16S rRNA Gene Sequences Research Articles

Characteristics of gut microbiome communities in the invasive African giant snail under urbanization gradient

To investigate the diversity and community structure of gut microbiome of the invasive species, Achatina fulica, along an urbanization gradient, we collected 30 A. fulica samples from five parks in the urban, suburban, and rural areas of Xiamen City. Using full-length 16S rRNA gene sequencing performed by the third generation PacBio sequencing platform, we analyzed the community characteristics of gut microbiome and soil microbiome in different habitats. We found a significant disparity between the composition of gut microbiome of A. fulica and that of the soil microbiome in their habitats. Furthermore, the gut microbiome of A. fulica were more sensitive to urbanization. The microbial α-diversity indices (Sobs, Chao, Shannon indices) in the soil of A. fulica habitats were consistently higher than those within their guts. Despite the similar β-diversity indices of microbial communities in urban, suburban, and rural soils, we found a significant discrepancy in gut microbiome composition. Urbanization significantly influenced A. fulica gut microbiome composition. Gut microbiome of A. fulica in urban and suburban regions primarily consisted of Enterobacteriaceae, Xanthomonadaceae, and Mycoplasmataceae, while that in rural areas chiefly composed of Streptococcaceae and Paenibacillaceae. The diversity and abundance of potential human pathogenic bacteria within the gut microbiome of A. fulica significantly increased in urban environments, suggesting that urbanization escalated the risk of A. fulica transmitting potential pathogens.

The Bactericidal Activity of a Novel Aneurinibacillus aneurinilyticus Isolate Effectively Controls Foodborne Pathogens Campylobacter jejuni and Listeria monocytogenes

Human zoonotic infection with Campylobacter is a major cause of gastroenteritis in the United States and worldwide. Listeria monocytogenes causes a potentially fatal infection in humans and is often attributed to contaminated food. Genetic typing has demonstrated that Campylobacter infection is often associated with the consumption of contaminated poultry products, with Campylobacter often colonizing the poultry gastrointestinal tract, while listeriosis is commonly associated with the consumption of contaminated ready-to-eat (RTE) foods. In this study, a strain of endospore-forming bacterium (strain NH) that is bactericidal towards the human food pathogens Campylobacter jejuni and Listeria monocytogenes was identified and characterized. Transwell experiments demonstrated that the bactericidal effect on both C. jejuni and L. monocytogenes is due to secretions from the spore former. These foodborne pathogens consistently exhibited 7 log reductions in growth when exposed to the NH cell-free culture filtrate. Sequencing of the 16s rRNA gene V4 variable region and analysis of the full-length 16s rRNA gene sequence from the WGS indicated that strain NH belongs to the species Aneurinibacillus aneurinilyticus. A microplate bioassay demonstrated that a bactericidal substance that is sensitive to protease could be collected from cell-free filtrates by salting out with ammonium sulfate. Gel filtration chromatography indicated a native molecular weight for the bactericidal protein of ca. 50 kDa, consistent with a class III bacteriocin. The active protein bound strongly to a cation-exchange resin and with an isoelectric point of ten, suggesting a positively charged protein. Both cation-exchange chromatography and isoelectric focusing indicated the enrichment of an 11 kDa protein on SDS-PAGE. This protein was identified through mass spectroscopy as the flgM protein, an anti-sigma factor. Analysis of whole genome sequencing (WGS) of the strain NH genome indicated the presence of a number of non-conservative amino acid substitutions in the flgM-gene-derived amino acid sequence of strain NH and A. aneurinilyticus compared to other members of the Aneurinibacillus genus. Further investigation is needed to determine whether these substitutions are correlated with the bactericidal activity. The identified strain may be useful as a feed additive for the pre-harvest control of Campylobacter jejuni in poultry.

SVep1, a temperate phage of human oral commensal Streptococcus vestibularis.

Bacteriophages play a vital role in the human oral microbiome, yet their precise impact on bacterial physiology and microbial communities remains relatively understudied due to the limited isolation and characterization of oral phages. To address this gap, the current study aimed to isolate and characterize novel oral phages. To achieve this, oral bacteria were isolated using a culture-omics method from 30 samples collected from healthy individuals. These bacteria were then cultured in three different types of media under both aerobic and anaerobic conditions. The samples were subsequently subjected to full-length 16S rRNA gene sequencing for analysis. Subsequently, we performed the isolation of lytic and lysogenic phages targeting all these bacteria. In the initial step, a total of 75 bacterial strains were successfully isolated, representing 30 species and 9 genera. Among these strains, Streptococcus was found to have the highest number of species. Using a full-length 16S rRNA gene similarity threshold of 98.65%, 14 potential novel bacterial species were identified. In the subsequent phase, a temperate phage, which specifically targets the human oral commensal bacterium S. vestibularis strain SVE8, was isolated. The genome of S. vestibularis SVE8 consists of a 1.96-megabase chromosome, along with a 43,492-base pair prophage designated as SVep1. Annotation of SVep1 revealed the presence of 62 open reading frames (ORFs), with the majority of them associated with phage functions. However, it is worth noting that no plaque formation was observed in S. vestibularis SVE8 following lytic induction using mitomycin C. Phage particles were successfully isolated from the supernatant of mitomycin C-treated cultures of S. vestibularis SVE8, and examination using transmission electron microscopy confirmed that SVep1 is a siphovirus. Notably, phylogenetic analysis suggested a common ancestral origin between phage SVep1 and the cos-type phages found in S. thermophilus. The presence of SVep1 may confer immunity to S. vestibularis against infection by related phages and holds potential for being engineered as a genetic tool to regulate oral microbiome homeostasis and oral diseases.

Effect of biofilm thickness on the activity and community composition of phosphorus accumulating bacteria in a moving bed biofilm reactor

Can biofilms enhance the rates of phosphorus removal in wastewater treatment? In order to narrow the scientific gap on the effect of biofilm thickness on the activity and microbial community of phosphorus-accumulating bacteria, this study investigated biofilms of 30 to 1000 µm thickness in a moving bed biofilm reactor. Measurements on 5 different biofilm carriers showed that biomass-specific phosphorus release and uptake rates increased as a function of biofilm thickness for biofilms thinner than about 110 µm but were lower for thicker biofilms of about 550–1000 µm. The reduced phosphorus uptake and release rates in the thickest biofilms can result from substrate mass transfer limitations whereas the low activity in the thinnest biofilms can be related to a too high turnover rate in the biofilm due to heterotrophic growth. Additionally, the microbial ecology of the different biofilms confirms the observed phosphorus uptake and release rates. The results from the full-length 16S rRNA gene sequencing of the bacterial community showed that the thicker biofilms were characterized by higher relative abundance (40–58%) of potential phosphorus accumulating genera Zoogloea, Acinetobacter, Dechloromonas and Ca. Accumulibacter. In contrast, the thinner biofilms were dominated by the genus Ferribacterium (34–60%), which might be competing with phosphorus-accumulating bacteria as indicated by the relatively high acetate uptake rates in the thinner biofilms. It is concluded that there is an optimal biofilm thickness of 100–500 µm, at which the phosphorus accumulating bacteria have the highest activity.

Yeast strains do have an impact on the production of cured cocoa beans, as assessed with Costa Rican Trinitario cocoa fermentation processes and chocolates thereof.

The microbiological and metabolic outcomes of good cocoa fermentation practices can be standardized and influenced through the addition of starter culture mixtures composed of yeast and bacterial strains. The present study performed two spontaneous and 10 starter culture-initiated (SCI) cocoa fermentation processes (CFPs) in Costa Rica with local Trinitario cocoa. The yeast strains Saccharomyces cerevisiae IMDO 050523, Hanseniaspora opuntiae IMDO 020003, and Pichia kudriavzevii IMDO 060005 were used to compose starter culture mixtures in combination with the lactic acid bacterium strain Limosilactobacillus fermentum IMDO 0611222 and the acetic acid bacterium strain Acetobacter pasteurianus IMDO 0506386. The microbial community and metabolite dynamics of the cocoa pulp-bean mass fermentation, the metabolite dynamics of the drying cocoa beans, and the volatile organic compound (VOC) profiles of the chocolate production were assessed. An amplicon sequence variant approach based on full-length 16S rRNA gene sequencing instead of targeting the V4 region led to a highly accurate monitoring of the starter culture strains added, in particular the Liml. fermentum IMDO 0611222 strain. The latter strain always prevailed over the background lactic acid bacteria. A similar approach, based on the internal transcribed spacer (ITS1) region of the fungal rRNA transcribed unit, was used for yeast strain monitoring. The SCI CFPs evolved faster when compared to the spontaneous ones. Moreover, the yeast strains applied did have an impact. The presence of S. cerevisiae IMDO 050523 was necessary for successful fermentation of the cocoa pulp-bean mass, which was characterized by the production of higher alcohols and esters. In contrast, the inoculation of H. opuntiae IMDO 020003 as the sole yeast strain led to underfermentation and a poor VOC profile, mainly due to its low competitiveness. The P. kudriavzevii IMDO 060005 strain tested in the present study did not contribute to a richer VOC profile. Although differences in VOCs could be revealed in the cocoa liquors, no significant effect on the final chocolates could be obtained, mainly due to a great impact of cocoa liquor processing during chocolate-making. Hence, optimization of the starter culture mixture and cocoa liquor processing seem to be of pivotal importance.

Microbial-Transferred Metabolites of Black Tea Theaflavins by Human Gut Microbiota and Their Impact on Antioxidant Capacity.

Theaflavins (TFs), the primary bioactive components in black tea, are poorly absorbed in the small intestine. However, the biological activity of TFs does not match their low bioavailability, which suggests that the gut microbiota plays a crucial role in their biotransformation and activities. In this study, we aimed to investigate the biotransferred metabolites of TFs produced by the human gut microbiota and these metabolites' function. We profiled the microbial metabolites of TFs by in vitro anaerobic human gut microbiota fermentation using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. A total of 17 microbial metabolites were identified, and their corresponding metabolic pathways were proposed. Moreover, full-length 16S rRNA gene sequence analysis revealed that the TFs altered the gut microbiota diversity and increased the relative abundance of specific members of the microbiota involved in the catabolism of the TFs, including Flavonifractor_plautii, Bacteroides_uniformis, Eubacterium_ramulus, etc. Notably, the antioxidant capacity of the TF sample increased after fermentation compared to the initial sample. In conclusion, the results contribute to a more comprehensive understanding of the microbial metabolites and antioxidant capacity of TFs.

Exploring the bacterial diversity and composition with special emphasis on pathogens in ship ballast water and sediments using full-length 16S rRNA gene sequencing

Accurate detecting bacterial communities in ballast water and sediments supports risk management. This study uses full-length 16S rRNA gene sequencing to investigate the bacterial communities in ballast water and sediments, focusing on detecting pathogens. The results indicate that full-length sequencing more accurately reveals the species diversity. There is a significant difference (P < 0.05) in bacterial communities between ballast water and sediments, despite both being dominated by the Proteobacteria phylum. Thirty human and fish pathogens were identified by full-length sequencing, yet only five pathogens were detected from V3-V4 sequencing. Notably, emerging pathogens such as Citrobacter freundii and Nocardia nova are detected in samples, which are harmful to aquaculture and human health. Several opportunistic pathogens were also identified. In summary, this study provides important insights into the bacterial communities in ballast water and sediments, highlighting the need for strict management.

Defluviimonas sediminis sp. nov., isolated from mangrove sediment.

A Gram-stain-negative, facultatively aerobic, motile and ovoid- to rod-shaped bacterium, designated as FT324T, was isolated from a surface mangrove sediment sample from Shenzhen, PR China. The taxonomic position of strain FT324T was determined by a combination of phylogenetic, physiological, biochemical and chemotaxonomic analyses. Strain FT324T grew at 20-40 °C (optimum, 30-37 °C), pH 5.0-9.0 (optimum, pH 8.0) and in the presence of 0-3 % (w/v) NaCl (optimum, 1 %). Its full-length 16S rRNA gene sequence was the most similar to Frigidibacter oleivorans XJ4T (97.4 %), followed by Defluviimonas denitrificans DSM 18921T (96.62 %), Pseudothioclava arenosa CAU 1312T (96.54 %) and Defluviimonas nitratireducens DL5-4T (96.47 %). The major fatty acids (>10 %) of FT324T were C19 : 0 cyclo ω8c (51.9 %) and summed feature 8(29.6 %). The predominant respiratory quinone was Q-10. Its polar lipid profile contained phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified aminolipid, one unidentified lipid and two unidentified phospholipids. Its estimated genome size was 4 294 115 bp and the genomic DNA G+C content was 69.6 mol%. Based on its distinct phenotypic, chemotaxonomic and phylogenetic characteristics, strain FT324T should represent a novel species of the genus Defluviimonas, for which the name Defluviimonas sediminis sp. nov. is proposed (=MCCC 1K07685T=KCTC 92477T).

Alpha-, gamma- and beta-proteobacteria detected in legume nodules in Latvia, using full-length 16S rRNA gene sequencing

ABSTRACT Rhizobia are soil bacteria that form nodules on legumes and fix atmospheric nitrogen, reducing the need for chemical fertilisers. In most studies rhizobia are described as alpha-proteobacteria and have been studied extensively for several decades. However, in recent years an increasing number of studies present beta- and gamma-proteobacteria strains as potential rhizobia-like bacteria. In this study we identify bacteria isolated from nodules of various legumes grown in Latvia, using a full-length 16S rRNA gene sequence, used for more precision, compared with a partial sequence of the gene. Bacterial strains in this study have been isolated from 1962–2019, part of which are rhizobia strains from a unique historical collection. This is the first time bacterial strains isolated from nodules in Latvian soils have been identified with molecular biology methods. A wide taxonomic diversity was detected – bacteria species from α-, β-, γ-proteobacteria classes and Paenibacillus polymyxa strains from the Bacilli class. P. polymyxa strains were detected only in the historical collection, while β- and γ-proteobacteria strains were obtained only from the newly isolated specimens, uncovering new potential strains for a commercial legume seed inoculum. Soil samples were collected, and phosphorus levels were determined. Several strains indicate phosphate solubilising properties.

Improved degradation of petroleum contaminants in hydraulic fracturing flowback and produced water using laccase immobilized on functionalized biochar

Large quantities of hydraulic fracturing flowback and produced water (HF-FPW) represent an important environmental issue owing to their inclusion of toxic petroleum contaminants. Enzymatic degradation is a promising approach for treating these pollutants. However, the low stability and poor reusability of free enzymes greatly inhibit their applications. This study investigated the effect of biochar-immobilized laccases on the biodegradation of petroleum contaminants in HF-FPW. Acid functionalization of corn straw biochar increased its surface area and pore volume, allowing it to adsorb additional laccase on the surface. Biochar-immobilized laccase exhibited a high tolerance to a low pH and high temperatures and retained >81.0% of its relative activity at 4 °C for 20 d. In addition, the immobilized laccase displayed reusability after six reaction cycles, retaining 40.9% of its activity. Supplementation with immobilized laccase substantially improved microbial growth and fluorescein diacetate hydrolase activity in HF-FPW, which were up to 25.5 times and 4.9 times higher than those of the free laccase treatment, respectively. The immobilized laccase also elevated the transcription levels of degradation genes such as rub and xylE, and greatly enhanced the biodegradation of n-alkanes (reduction from 112.4 mg/L to 41.9 mg/L) and polycyclic aromatic hydrocarbons (reduction from 2606.2μg/L to 1526.6μg/L). Full-length 16S rRNA gene sequencing revealed that Pseudomonas stutzeri, Pseudomonas balearica, and Stenotrophomonas maltophilia dominated the microbial community and were potential functional populations. This study represents the first attempt of utilizing biochar-immobilized laccase to treat HF-FPW and has important implications for efforts to develop strategies for HF-FPW remediation.

Symbiosis of Carpenter Bees with Uncharacterized Lactic Acid Bacteria Showing NAD Auxotrophy.

Eusocial bees (such as honey bees and bumble bees) harbor core gut microbiomes that are transmitted through social interaction between nestmates. Carpenter bees are not eusocial; however, recent microbiome analyses found that Xylocopa species harbor distinctive core gut microbiomes. In this study, we analyzed the gut microbiomes of three Xylocopa species in Japan between 2016 and 2021 by V1 to V2 region-based 16S rDNA amplicon sequencing, and 14 candidate novel species were detected based on the full-length 16S rRNA gene sequences. All Xylocopa species harbor core gut microbiomes consisting of primarily lactic acid bacteria (LAB) that were phylogenetically distant from known species. Although they were difficult to cultivate, two LAB species from two different Xylocopa species were isolated by supplementing bacterial culture supernatants. Both genomes exhibited an average LAB genome size with a large set of genes for carbohydrate utilization but lacked genes to synthesize an essential coenzyme NAD, which is unique among known insect symbionts. Our findings of phylogenetically distinct core LAB of NAD auxotrophy reflected the evolution of Xylocopa-restricted bacteria retention and maintenance through vertical transmission of microbes during solitary life. We propose five candidate novel species belonging to the families Lactobacillaceae and Bifidobacteriaceae, including a novel genus, and their potential functions in carbohydrate utilization. IMPORTANCE Recent investigations found unique microbiomes in carpenter bees, but the description of individual microbes, including isolation and genomics, remains largely unknown. Here, we found that the Japanese Xylocopa species also harbor core gut microbiomes. Although most of them were difficult to isolate a pure colony, we successfully isolated several strains. We performed whole-genome sequencing of the isolated candidate novel species and found that the two Lactobacillaceae strains belonging to the Xylocopa-specific novel LAB clade lack the genes for synthesizing NAD, a coenzyme central to metabolism in all living organisms. Here, we propose a novel genus for the two LAB species based on very low 16S rRNA gene sequence similarities and genotypic characters.

Altered Respiratory Microbiomes, Plasma Metabolites, and Immune Responses in Influenza A Virus and Methicillin-Resistant Staphylococcus aureus Coinfection.

Influenza A virus (IAV)-methicillin-resistant Staphylococcus aureus (MRSA) coinfection causes severe respiratory infections. The host microbiome plays an important role in respiratory tract infections. However, the relationships among the immune responses, metabolic characteristics, and respiratory microbial characteristics of IAV-MRSA coinfection have not been fully studied. We used specific-pathogen-free (SPF) C57BL/6N mice infected with IAV and MRSA to build a nonlethal model of IAV-MRSA coinfection and characterized the upper respiratory tract (URT) and lower respiratory tract (LRT) microbiomes at 4 and 13 days postinfection by full-length 16S rRNA gene sequencing. Immune response and plasma metabolism profile analyses were performed at 4 days postinfection by flow cytometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The relationships among the LRT microbiota, the immune response, and the plasma metabolism profile were analyzed by Spearman's correlation analysis. IAV-MRSA coinfection showed significant weight loss and lung injury and significantly increased loads of IAV and MRSA in bronchoalveolar lavage fluid (BALF). Microbiome data showed that coinfection significantly increased the relative abundances of Enterococcus faecalis, Enterobacter hormaechei, Citrobacter freundii, and Klebsiella pneumoniae and decreased the relative abundances of Lactobacillus reuteri and Lactobacillus murinus. The percentages of CD4+/CD8+ T cells and B cells in the spleen; the levels of interleukin-9 (IL-9), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-6, and IL-8 in the lung; and the level of mevalonolactone in plasma were increased in IAV-MRSA-coinfected mice. L. murinus was positively correlated with lung macrophages and natural killer (NK) cells, negatively correlated with spleen B cells and CD4+/CD8+ T cells, and correlated with multiple plasma metabolites. Future research is needed to clarify whether L. murinus mediates or alters the severity of IAV-MRSA coinfection. IMPORTANCE The respiratory microbiome plays an important role in respiratory tract infections. In this study, we characterized the URT and LRT microbiota, the host immune response, and plasma metabolic profiles during IAV-MRSA coinfection and evaluated their correlations. We observed that IAV-MRSA coinfection induced severe lung injury and dysregulated host immunity and plasma metabolic profiles, as evidenced by the aggravation of lung pathological damage, the reduction of innate immune cells, the strong adaptation of the immune response, and the upregulation of mevalonolactone in plasma. L. murinus was strongly correlated with immune cells and plasma metabolites. Our findings contribute to a better understanding of the role of the host microbiome in respiratory tract infections and identified a key bacterial species, L. murinus, that may provide important references for the development of probiotic therapies.

Identification of Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., two new members of the genus Nocardioides.

A polyphasic taxonomic characterization of two novel strain pairs (designated zg-579T/zg-578 and zg-536T/zg-ZUI104) isolated from the faeces of Marmota himalayana was conducted based on phylogenetic analysis of the nearly full-length 16S rRNA gene and genome, digital DNA-DNA hybridization, ortho-average nucleotide identity (Ortho-ANI), and phenotypic and chemotaxonomic traits. Comparative analysis of the nearly full-length 16S rRNA gene sequences showed that strain zg-579T was most closely related to Nocardioides dokdonensis FR1436T (97.57 %) and Nocardioides deserti SC8A-24T (97.36 %), whereas strain zg-536T had the highest similarity to Nocardioides caeni MN8T (98.33 %), Nocardioides convexus W2-2-3T (98.26 %) and Nocardioides daeguensis 2C1-5T (98.19 %). Low levels of DNA-DNA relatedness and Ortho-ANI values (19.8-31.0 %/78.6-88.2 %, zg-579T; 19.9-31.3 %/78.8-86.2 %, zg-536T) between the two new type strains and previously known species within the genus Nocardioides support the hypothesis that the four newly characterized strains could be considered to represent two novel species within this genus. The dominant cellular fatty acids found in strain pair zg-536T/zg-ZUI104 were iso-C16 : 0 and C18 : 1 ω9c, whereas C17 : 1 ω8c was major component in zg-579T/zg-578. Galactose and ribose were the main cell-wall sugars in these two new strain pairs. Diphosphatidylglycerol (DPG), phosphatidylcholine, phosphatidylglycerol (PG) and phosphatidylinositol (PI) were the major polar lipids in zg-579T, whereas DPG, PG and PI predominated in zg-536T. Both strain pairs had MK8(H4) as the major respiratory quinone and ll-diaminopimelic acid as the major cell-wall peptidoglycan. The optimal growth conditions for the two novel strain pairs were 30 °C, pH 7.0 and 0.5 % NaCl (w/v). Based on these polyphasic characterizations, two novel species within the genus Nocardioides are proposed, i.e. Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., with zg-579T (=CGMCC 4.7663T=JCM 33892T) and zg-536T (=CGMCC 4.7662T=JCM 33891T) as the type strains.

Cyanoseq: A database of cyanobacterial 16S rRNA gene sequences with curated taxonomy.

Cyanobacteria are photosynthetic bacteria that occupy various habitats across the globe, playing critical roles in many of Earth's biogeochemical cycles both in both aquatic and terrestrial systems. Despite their well-known significance, their taxonomy remains problematic and is the subject of much research. Taxonomic issues of Cyanobacteria have consequently led to inaccurate curation within known reference databases, ultimately leading to problematic taxonomic assignment during diversity studies. Recent advances in sequencing technologies have increased our ability to characterize and understand microbial communities, leading to the generation of thousands of sequences that require taxonomic assignment. We herein propose CyanoSeq (https://zenodo.org/record/7569105), a database of cyanobacterial 16S rRNA gene sequences with curated taxonomy. The taxonomy of CyanoSeq is based on the current state of cyanobacterial taxonomy, with ranks from the domain to genus level. Files are provided for use with common naive Bayes taxonomic classifiers, such as those included in DADA2 or the QIIME2 platform. Additionally, FASTA files are provided for creation of de novo phylogenetic trees with (near) full-length 16S rRNA gene sequences to determine the phylogenetic relationship of cyanobacterial strains and/or ASV/OTUs. The database currently consists of 5410 cyanobacterial 16S rRNA gene sequences along with 123 Chloroplast, Bacterial, and Vampirovibrionia (formally Melainabacteria) sequences.

Single-Molecule Approach to 16S rRNA for Vaginal Microbiome Signatures in Response to Metronidazole Treatment.

Bacterial vaginosis (BV) is the most common infection of the lower reproductive tract among women of reproductive age, characterized by a depletion of health-associated Lactobacillus and an overgrowth of anaerobes. Metronidazole has been recommended as a first-line therapy for treating BV for decades. Although most cases are cured by the treatment, recurrent infections of BV seriously affect women's reproductive health. Until now, limited information on the vaginal microbiota has been explored at the species level. Here, we adopted a single molecular sequencing approach for the 16S rRNA gene, named FLAST (full-length assembly sequencing technology), to analyze the human vaginal microbiota that improved species-level resolution for taxonomy and identified microbiota alterations in the vaginal tract in response to treatment with metronidazole. Appling high-throughput sequencing, we identified 96 and 189 novel full-length 16S rRNA gene sequences in Lactobacillus and Prevotella, respectively, which had not previously been reported in vaginal samples. Moreover, we found that Lactobacillus iners was significantly enriched in the cured group before metronidazole treatment, and that was maintained in a high frequency after the treatment, suggesting an important role for this species in response to metronidazole treatment. Our research also highlights the importance of the single-molecule paradigm for progressing the field of microbiology and applying these insights to better understand the dynamic microbiota during BV treatment. Subsequent novel treatment approaches should be proposed to improve BV treatment outcomes, optimize the vaginal microbiome, and reduce gynecological and obstetric sequelae. IMPORTANCE Bacterial vaginosis (BV) is a common infectious disease of the reproductive tract. Metronidazole treatment, as the first line of treatment, frequently fails at recovery of the microbiome. However, the precise types of Lactobacillus and other bacteria involved in BV remain unclear, and this has resulted in a failure to identify potential markers to predict clinic outcomes. In this study, we adopted a 16S rRNA gene full-length assembly sequencing technology for the taxonomy analysis and evaluation of vaginal microbiota before and after treatment with metronidazole. We additionally identified 96 and 189 novel 16S rRNA gene sequences in Lactobacillus and Prevotella species, respectively, in vaginal samples, which improves our understanding of the vaginal microbiota. Moreover, we found that the abundance of Lactobacillus iners and Prevotella bivia before treatment was associated with a lack of cure. These potential biomarkers will help to facilitate future studies aimed at improving BV treatment outcomes, optimize the vaginal microbiome, and reduce adverse sexual and reproductive outcomes.

Succession of bacterial biofilm communities following removal of chloramine from a full-scale drinking water distribution system

Monochloramine is used to regulate microbial regrowth in drinking water distribution systems (DWDS) but produces carcinogenic disinfection byproducts and constitutes a source of energy for nitrifying bacteria. This study followed biofilm-dispersed microbial communities of a full-scale DWDS distributing ultrafiltered water over three years, before and after removal of monochloramine. Communities were described using flow cytometry and amplicon sequencing, including full-length 16S rRNA gene sequencing. Removal of monochloramine increased total cell counts by up to 440%. Increased abundance of heterotrophic bacteria was followed by emergence of the predatory bacteria Bdellovibrio, and a community potentially metabolizing small organic compounds replaced the nitrifying core community. No increased abundance of Mycobacterium or Legionella was observed. Co-occurrence analysis identified a network of Nitrosomonas, Nitrospira, Sphingomonas and Hyphomicrobium, suggesting that monochloramine supported this biofilm community. While some species expanded into the changed niche, no immediate biological risk to consumers was indicated within the DWDS.

Bacterial diversity of middle ear cholesteatoma by 16S rRNA gene sequencing in China

In this study, the bacterial diversity of acquired middle ear cholesteatoma (MEC) was evaluated to reveal its pathogenesis and provides a guide for the use of antibiotics. Twenty-nine cases of acquired MEC and eight cases of healthy middle ears undergoing cochlear implantation (CI) were evaluated. Full-length 16S rRNA gene sequencing was performed to profile the bacterial communities in lesions and healthy tissues of the middle ear. ACE (P = 0.043) and Chao1 (P = 0.039) indices showed significant differences in alpha diversity (P < 0.05). Analysis of PERMANOVA/Anosim using the Bray–Curtis distance matrix results suggested that the between-group differences were greater than the within-group differences (R = 0.238, P < 0.05, R2 = 0.066, P < 0.05). Bacterial community analysis revealed that Alphaproteobacteria at the class level and Caulobacterales and Sphingomonadales at the order level were significantly different (P < 0.05). In the LefSe (Linear discriminant analysis effect size) analysis, Porphyromonas bennonis was elevated, and Bryum argenteum and unclassified Cyanobacteriales were reduced at the species level in MEC (P < 0.05). Fifteen metabolic pathways were found to be significantly different between the two groups by analysing the abundance of metabolic pathways in level 2 of the Kyoto Encyclopaedia of Genes and Genomes (KEGG). Seven and eight metabolic pathways were significantly elevated in the MEC and control groups, respectively (P < 0.05). The role of bacteria in the pathogenesis of acquired MEC was further refined through analysis of metabolic pathways. These findings indicate that the acquired MEC and healthy middle ear contain more diverse microbial communities than previously thought.

Probable airborne transmission of Burkholderia pseudomallei causing an urban outbreak of melioidosis during typhoon season in Hong Kong, China

ABSTRACT Between January 2015 and October 2022, 38 patients with culture-confirmed melioidosis were identified in the Kowloon West (KW) Region, Hong Kong. Notably, 30 of them were clustered in the Sham Shui Po (SSP) district, which covers an estimated area of 2.5 km2. Between August and October 2022, 18 patients were identified in this district after heavy rainfall and typhoons. The sudden upsurge in cases prompted an environmental investigation, which involved collecting 20 air samples and 72 soil samples from residential areas near the patients. A viable isolate of Burkholderia pseudomallei was obtained from an air sample collected at a building site five days after a typhoon. B. pseudomallei DNA was also detected in 21 soil samples collected from the building site and adjacent gardening areas using full-length 16S rRNA gene sequencing, suggesting that B. psuedomallei is widely distributed in the soil environment surrounding the district. Core genome-multilocus sequence typing showed that the air sample isolate was phylogenetically clustered with the outbreak isolates in KW Region. Multispectral satellite imagery revealed a continuous reduction in vegetation region in SSP district by 162,255 m2 from 2016 to 2022, supporting the hypothesis of inhalation of aerosols from the contaminated soil as the transmission route of melioidosis during extreme weather events. This is because the bacteria in unvegetated soil are more easily spread by winds. In consistent with inhalational melioidosis, 24 (63.2%) patients had pneumonia. Clinicians should be aware of melioidosis during typhoon season and initiate appropriate investigation and treatment for patients with compatible symptoms.

Maternal Psychosocial Stress Is Associated with Reduced Diversity in the Early Infant Gut Microbiome.

The developing infant gut microbiome is highly sensitive to environmental exposures, enabling its evolution into an organ that supports the immune system, confers protection from infection, and facilitates optimal gut and central nervous system function. In this study, we focus on the impact of maternal psychosocial stress on the infant gut microbiome. Forty-seven mother-infant dyads were recruited at the HEAL Africa Hospital in Goma, Democratic Republic of Congo. Extensive medical, demographic, and psychosocial stress data were collected at birth, and infant stool samples were collected at six weeks, three months, and six months. A composite maternal psychosocial stress score was created, based on eight questionnaires to capture a diverse range of stress exposures. Full-length 16S rRNA gene sequences were generated. Infants of mothers with high composite stress scores showed lower levels of gut microbiome beta diversity at six weeks and three months, as well as higher levels of alpha diversity at six months compared to infants of low stress mothers. Longitudinal analyses showed that infants of high stress mothers had lower levels of health-promoting Lactobacillus gasseri and Bifidobacterium pseudocatenulatum at six weeks compared to infants of low stress mothers, but the differences largely disappeared by three to six months. Previous research has shown that L. gasseri can be used as a probiotic to reduce inflammation, stress, and fatigue, as well as to improve mental state, while B. pseudocatenulatum is important in modulating the gut-brain axis in early development and in preventing mood disorders. Our finding of reduced levels of these health-promoting bacteria in infants of high stress mothers suggests that the infant gut microbiome may help mediate the effect of maternal stress on infant health and development.

Rare Bacteria Can Be Used as Ecological Indicators of Grassland Degradation

Grassland degradation is a major ecological problem at present, leading to changes in the grassland environment and the soil microbial community. Here, based on full-length 16S rRNA gene sequencing, we highlight the importance of small-scale environmental changes on the Qinghai-Tibet Plateau grassland for the composition and assembly processes of abundant and rare bacterial taxa. The results showed that grassland vegetation coverage affected the taxonomic and phylogenetic composition of rare bacterial taxa more than abundant bacterial taxa. The taxonomic composition and phylogenetic composition of rare bacterial taxa were also affected by soil nutrients. The relative contribution of deterministic processes (variable selection and homogeneous selection) to rare bacterial taxa was higher than that of the abundant bacterial taxa. The competitive potential within rare bacterial taxa was lower than that of the competitive potential between rare and non-rare bacterial taxa or within non-rare bacterial taxa. The assembly of rare bacterial taxa was more susceptible to environmental changes caused by grassland degradation than the abundant bacterial taxa. Furthermore, the distribution of rare bacterial taxa in the different degraded grassland soil was more local than that of abundant bacterial taxa. Thus, rare bacterial taxa could be considered an ecological indicator of grassland degradation. These findings help to improve our understanding of the composition and assembly mechanism of the bacterial communities in degraded grassland and provide a basis for the establishment of the grassland degradation management strategy.