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MSystems Oct 2022Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and...
Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and analysis methods, have made it possible to obtain complete bacterial genomes from metagenomic (i.e., multispecies) samples, including those from the human microbiome. In this study, methods are presented to obtain complete bacterial genomes from human saliva using complementary Oxford Nanopore (ONT) and Illumina sequencing. Applied to 3 human saliva samples, these methods resulted in 11 complete bacterial genomes: 3 clade G6 (also known as . Nanogingivalaceae HMT-870), 1 clade G1 HMT-348, 2 Rothia mucilaginosa, 2 Actinomyces graevenitzii, 1 Mogibacterium diversum, 1 HMT-096, and 1 ; and one circular chromosome of Ruminococcaceae HMT-075 (which likely has at least 2 chromosomes). The 4 genomes, as well as the genomes, represented the first complete genomes from their respective bacterial taxa. Aside from the complete genomes, the assemblies contained 147 contigs of over 500,000 bp each and thousands of smaller contigs, together representing a myriad of additional draft genomes including many which are likely nearly complete. The complete genomes enabled highly accurate pangenome analysis, which identified unique and missing features of each genome compared to its closest relatives with complete genomes available in public repositories. These features provide clues as to the lifestyle and ecological role of these bacteria within the human oral microbiota, which will be particularly useful in designing future studies of the taxa that have never been isolated or cultivated. Obtaining complete and accurate genomes is crucial to the study of any organism. Previously, obtaining complete genomes of bacteria, including those of the human microbiome, frequently required isolation of the organism, as well as low-throughput, manual sequencing methods to resolve repeat regions. Advancements in long-read sequencing technologies, including Oxford Nanopore (ONT), have made it possible to obtain complete, closed bacterial genomes from metagenomic samples. This study reports methods to obtain complete genomes from the human oral microbiome using complementary ONT and Illumina sequencing of saliva samples. Eleven complete genomes were obtained from 3 human saliva samples, with genomes of HMT-870, HMT-348, and being the first complete genomes from their respective taxa. Obtaining complete bacterial genomes in a high-throughput manner will help illuminate the metabolic and ecological roles of important members of the human microbiota, particularly those that have remained recalcitrant to isolation and cultivation.
Topics: Humans; Sequence Analysis, DNA; Nanopore Sequencing; Saliva; Genome, Bacterial; Microbiota; Bacteria
PubMed: 35993719
DOI: 10.1128/msystems.00491-22 -
Journal of Applied Microbiology Jan 2021To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM). (Comparative Study)
Comparative Study
AIMS
To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM).
METHODS AND RESULTS
Human milk DNA was extracted using the following methods: (i) Qiagen MagAttract Microbial DNA Isolation Kit (kit QM), (ii) Norgen Milk Bacterial DNA Isolation Kit (kit NM), (iii) Qiagen MagAttract Microbiome DNA/RNA Isolation Kit (kit MM) and (iv) TRIzol LS Reagent (method LS). The full-length 16S rRNA gene was sequenced. Kits MM and method LS were unable to extract detectable levels of DNA in 9/11 samples. Detectable levels of DNA were recovered from all samples using kits NM (mean = 0·68 ng μl ) and QM (mean = 0·55 ng μl ). For kits NM and QM, the greatest number of reads were associated with Staphylococcus epidermidis, Streptococcus vestibularis, Propionibacterium acnes, Veillonella dispar and Rothia mucilaginosa. Contamination profiles varied substantially between kits, with one bacterial species detected in negative extraction controls generated with kit QM and six with kit NM.
CONCLUSIONS
Kit QM is the most suitable of the kits tested for the extraction of bacterial DNA from human milk.
SIGNIFICANCE AND IMPACT OF THE STUDY
Choice of extraction method impacts the efficiency of bacterial DNA extraction from human milk and the resultant bacterial community profiles generated from these samples.
Topics: Bacteria; DNA, Bacterial; Humans; Microbiota; Milk, Human; RNA, Ribosomal, 16S; Reagent Kits, Diagnostic; Sequence Analysis, DNA
PubMed: 32654260
DOI: 10.1111/jam.14780 -
Frontiers in Microbiology 2021The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been recently demonstrated in the sputum or saliva, suggesting how the shedding of viral...
The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been recently demonstrated in the sputum or saliva, suggesting how the shedding of viral RNA outlasts the end of symptoms. Recent data from transcriptome analysis show that the oral cavity mucosa harbors high levels of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), highlighting its role as a double-edged sword for SARS-CoV-2 body entrance or interpersonal transmission. Here, we studied the oral microbiota structure and inflammatory profile of 26 naive severe coronavirus disease 2019 (COVID-19) patients and 15 controls by 16S rRNA V2 automated targeted sequencing and magnetic bead-based multiplex immunoassays, respectively. A significant diminution in species richness was observed in COVID-19 patients, along with a marked difference in beta-diversity. Species such as and were distinctive for COVID-19 patients, while and were predominant in controls. Interestingly, these two groups of oral species oppositely clustered within the bacterial network, defining two distinct Species Interacting Groups (SIGs). COVID-19-related pro-inflammatory cytokines were found in both oral and serum samples, along with a specific bacterial consortium able to counteract them. We introduced a new parameter, named CytoCOV, able to predict COVID-19 susceptibility for an unknown subject at 71% of power with an Area Under Curve (AUC) equal to 0.995. This pilot study evidenced a distinctive oral microbiota composition in COVID-19 subjects, with a definite structural network in relation to secreted cytokines. Our results would be usable in clinics against COVID-19, using bacterial consortia as biomarkers or to reduce local inflammation.
PubMed: 34394024
DOI: 10.3389/fmicb.2021.671813 -
Frontiers in Cellular and Infection... 2021Oral microbiota is constantly changing with the host state, whereas the oral microbiome of chronic erythematous candidiasis remains poorly understood. The aim of this...
Oral microbiota is constantly changing with the host state, whereas the oral microbiome of chronic erythematous candidiasis remains poorly understood. The aim of this study was to compare oral microbial signatures and functional profiling between chronic erythematous candidiasis and healthy subjects. Using shotgun metagenomic sequencing, we analyzed the microbiome in 12 chronic erythematous candidiasis, 12 healthy subjects, and 2 chronic erythematous candidiasis cured by antifungal therapy. We found that the salivary microbiota of chronic erythematous candidiasis was significantly different from that of healthy subjects. Among them, and were the most abundant disease-enriched species (Mann-Whitney U-test, < 0.05). In addition, co-occurrence network analysis showed that formed densely connected modules with oral bacterial species and was mainly positive connected to species. Furthermore, we investigated the functional potentials of the microbiome and identified a set of microbial marker genes associated with chronic erythematous candidiasis. Some of these genes enriching in chronic erythematous candidiasis are involved in eukaryotic ribosome, putative glutamine transport system, and cytochrome bc1 complex respiratory unit. Altogether, this study revealed the changes of oral microbial composition, the co-occurrence between and oral bacteria, as well as the changes of microbial marker genes during chronic erythematous candidiasis, which provides evidence of oral microbiome as a target for the treatment and prevention of chronic erythematous candidiasis.
Topics: Candidiasis, Oral; Humans; Metagenomics; Microbiota; Micrococcaceae
PubMed: 34490138
DOI: 10.3389/fcimb.2021.691092 -
Frontiers in Microbiology 2022There are two main types of echinococcosis, namely alveolar echinococcosis (AE) and cystic echinococcosis (CE). They are zoonotic parasitic diseases caused by the...
There are two main types of echinococcosis, namely alveolar echinococcosis (AE) and cystic echinococcosis (CE). They are zoonotic parasitic diseases caused by the metacestodes of and . In order to explore the gut microbiome composition of patients with echinococcosis, we analyzed fecal samples of seven patients with AE, six patients with CE, and 13 healthy individuals from the Qinghai-Tibetan Plateau, China. Using metagenomic next-generation sequencing, we identified fecal bacteria in the patients with AE and CE. The gut microbiota was analyzed by next-generation metagenomic sequencing (mNGS) to compare patients with either AE or CE against healthy individuals. We found there were some differences between them in abundant bacteria. Our results led to five findings: (1) Between patients with echinococcosis and healthy individuals, the differential bacteria were from four phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria. (2) , , , , and were abundant in the feces of patients with AE. (3) sp_E4742, and were abundant in the feces of the patients with CE. (4) At the phylum and class level, compared to the AE group, the healthy group was characterized by higher numbers of Actinobacteria. (5) At the family level, Lachnospiraceae and Eubacteriaceae were more abundant in the feces of healthy individuals than in AE patients. The genera , , and were more abundant in the healthy group, while the genus was more abundant in the AE group. The results of this study enrich our understanding of the gut microbiome composition of patients with AE and CE in the Qinghai-Tibetan Plateau.
PubMed: 35615499
DOI: 10.3389/fmicb.2022.860909 -
Frontiers in Microbiology 2022Dysbacteriosis is thought to play an important role in the pathogenesis of necrotizing enterocolitis (NEC). We aimed to identify new biomarkers among gut microbiota and...
BACKGROUND
Dysbacteriosis is thought to play an important role in the pathogenesis of necrotizing enterocolitis (NEC). We aimed to identify new biomarkers among gut microbiota and short-chain fatty acids (SCFAs) for the early prediction of NEC.
MATERIALS AND METHODS
Thirty-four preterm infants with gestational ages of ≤ 34 weeks who developed gastrointestinal symptoms were divided into the NEC group ( = 17) and non-NEC group ( = 17). In the NEC group, the gut microbiota and SCFAs in feces were assessed when the infants were enrolled (Group P) and when they were diagnosed with NEC (Group N). In the non-NEC group, samples were assessed when the infants were enrolled (Group C).
RESULTS
The Ace and Chao1 indices were higher in Group P than in Group C ( < 0.05), and there was no difference between Groups C and N or between Groups P and N ( > 0.05). There was no significant difference in the Simpson and Shannon indices among Groups C, P and N ( > 0.05). The four main phyla showed no differences ( > 0.05) in composition, while at the genus level, compared with Group C, in Group P, , and were increased, while and were decreased ( < 0.05). At the species level, and increased, while decreased ( < 0.05). In Group N, at the genus level, , and increased ( < 0.05). Compared with those in Group C, the levels of acetic acid, propanoic acid and butyric acid decreased significantly in Groups P and N ( < 0.05), and the areas under the curves (AUCs) of these three SCFAs between groups C and P were 0.73, 0.70, and 0.68, respectively.
CONCLUSION
The increase in and and decrease in , as well as the decrease in acetic, propionic and butyric acids, may help in the early prediction of NEC.
PubMed: 36060739
DOI: 10.3389/fmicb.2022.969656 -
Life (Basel, Switzerland) Mar 2023The beneficial effects of balneotherapy have been proven by numerous clinical studies on locomotor disorders. To date, there is only scant data on changes in the...
The beneficial effects of balneotherapy have been proven by numerous clinical studies on locomotor disorders. To date, there is only scant data on changes in the microbiome system of the skin during balneotherapy. The aim of this study was to compare the effects of thermal water and tap water on the skin's microbiome in healthy volunteers. 30 healthy female volunteers participated in the study. The experimental group (of 15 women) spent 30-min 10 times, in Gabriella Spring's thermal baths (i.e., mineral water containing sodium hydrogen carbonate).The controlled group (15 women) had the same, but in tap water. The results of this study have proven that there is a difference in the influencing effects of tap water and medicinal water on the microbiome of the skin. After bathing in the thermal water of Lakitelek, increased significantly at the genus level, and the tendency for bacteria also increased. At the species level, increased significantly, while and the tendency for decreased. When the values of the two trial groups after bathing at the genus level were compared, increased significantly, while tended to increase, tended to decrease, tended to increase significantly, and tended to decrease. At the species level, decreased significantly, and the tendency for decreased. The growth of and the decrease in the tendency of , and confirm the beneficial effect of balneotherapy. In this study, trends are represented by the uncorrected value. The main result was that the thermal water changed certain bacteria of the skin, both on the genus and species levels, but there were no significant changes in the tap water used, either at the genus or species level. We first compared the worlds of thermal water and tap water's microbiome systems. The thermal water decreased the number of certain inflammatory infectious agents and could enhance some of their positive effects, which have been proven at the molecular level. Our results can provide an important clue in the treatment of certain skin diseases. The research of the skin microbiome during balneotherapy can be one of the most intriguing and exciting topics of the future and can bring us closer to understanding the mechanism of action of balneotherapy.
PubMed: 36983902
DOI: 10.3390/life13030746 -
Frontiers in Microbiology 2023Cardiac surgery-associated acute kidney injury (CSA-AKI) is a common hospital-acquired AKI that carries a grave disease burden. Recently, gut-kidney crosstalk has...
INTRODUCTION
Cardiac surgery-associated acute kidney injury (CSA-AKI) is a common hospital-acquired AKI that carries a grave disease burden. Recently, gut-kidney crosstalk has greatly changed our understanding of the pathogenesis of kidney diseases. However, the relationship between gut microbial dysbiosis and CSA-AKI remains unclear. The purpose of this study was to investigate the possible contributions of gut microbiota alterations in CSA-AKI patients.
METHODS
Patients undergoing cardiac surgery were enrolled and divided into acute kidney injury (AKI) and Non_AKI groups. Faecal samples were collected before the operation. Shotgun metagenomic sequencing was performed to identify the taxonomic composition of the intestinal microbiome. All groups were statistically compared with alpha- and beta-diversity analysis, and linear discriminant analysis effect size (LEfSe) analysis was performed.
RESULTS
A total of 70 individuals comprising 35 AKI and 35 Non_AKI were enrolled in the study. There was no significant difference between the AKI and Non_AKI groups with respect to the alpha-and beta-diversity of the Shannon index, Simpson or Chao1 index values except with respect to functional pathways ( < 0.05). However, the relative abundance of top 10 gut microbiota in CSA-AKI was different from the Non_AKI group. Interestingly, both LEfSe and multivariate analysis confirmed that the species , , and were associated with CSA-AKI. Moreover, correlation heat map indicated that altered pathways and disrupted function could be attributed to disturbances of gut microbiota involving .
CONCLUSION
Dysbiosis of the intestinal microbiota in preoperative stool affects susceptibility to CSA-AKI, indicating the crucial role of key microbial players in the development of CSA-AKI. This work provides valuable knowledge for further study of the contribution of gut microbiota in CSA-AKI.
PubMed: 37065117
DOI: 10.3389/fmicb.2023.1119959 -
Indian Journal of Pathology &... 2022Adenocarcinoma is a more common type of Non-small cell lung cancer (NSCLC). Lung cancer showed a statistically significant increment in the Kamrup Urban district of...
BACKGROUND
Adenocarcinoma is a more common type of Non-small cell lung cancer (NSCLC). Lung cancer showed a statistically significant increment in the Kamrup Urban district of Assam, Tripura, Sikkim, and Manipur of India. The goal of our pilot study is to identify non-invasive microbial biomarkers to detect lung adenocarcinoma (LAC).
MATERIAL AND METHODS
DNA extraction from saliva samples of five LAC patients and five healthy controls was performed by Qiagen DNeasy blood and tissue kit using Lysozyme (3mg/ml) treatment. 16S rRNA genes of distinct regions (V3-V4) were amplified from saliva DNA by PCR. Paired-end sequencing targeting the V3-V4 region of the 16S rRNA gene has been performed on the Illumina MiSeq platform. Raw sequences were analyzed using the QIIME(Quantitative Insights Into Microbial Ecology) software package.
RESULTS
Our preliminary results showed that Rothia mucilaginosa, Veillonella dispar, Prevotella melaninogenica, Prevotella pallens, Prevotella copri, Haemophilus parainfluenzae, Neisseria bacilliformis and Aggregatibacter segnis were significantly elevated in saliva of LAC which may serve as potential non-invasive biomarkers for LAC detection. Functional prediction analysis showed that bacterial genes involved in glycosyltransferase, peptidases, amino sugar, and nucleotide sugar metabolism, starch and sucrose metabolism were significantly enriched in LAC.
CONCLUSION
These salivary bacteria may contribute to the development of LAC by increasing expression of glycosyltransferase and peptidases. However to understand their role in pathobiology, studies are required to perform in large cohort.
Topics: Adenocarcinoma of Lung; Bacteria; Biomarkers, Tumor; DNA, Bacterial; Dysbiosis; Female; Humans; India; Male; Microbiota; Middle Aged; Pilot Projects; RNA, Ribosomal, 16S; Saliva
PubMed: 35074976
DOI: 10.4103/IJPM.IJPM_1111_20 -
Japanese Journal of Infectious Diseases Sep 2021To improve our current understanding of normal flora in children, we investigated bacterial isolates from the pharynx and nasopharynx of 173 and 233 healthy children,...
To improve our current understanding of normal flora in children, we investigated bacterial isolates from the pharynx and nasopharynx of 173 and 233 healthy children, respectively. The bacterial isolation rates were compared among three age groups: infants (<1 year), toddlers (1-5 years), and school-aged children (6-15 years). Gram-positive cocci were the predominant bacteria in the pharynx (Streptococcus mitis/oralis, 87.3%; Streptococcus salivarius, 54.3%; Rothia mucilaginosa, 41.6%; Staphylococcus aureus, 39.3%). Among infants, S. salivarius and Neisseria subflava, which are related to the development of teeth, were significantly lower than in the other age groups (P <0.0001, S. salivarius; P <0.01, N. subflava). With the exception of Corynebacterium pseudodiphtheriticum (44.2%, gram-positive rods), gram-negative rods largely predominated the nasopharynx (Moraxella catarrhalis, 32.1%; Moraxella nonliquefaciens, 28.3%). Among toddlers, M. catarrhalis and Streptococcus pneumoniae, which are the most common pathogens in acute otitis media, were significantly higher than in the infant group (P <0.05). Among the bacterial species implicated in pediatric respiratory infections, Streptococcus pyogenes was isolated in 3.5% of the pharyngeal samples. S. pneumoniae and Haemophilus influenzae were isolated in 22.3% and 17.2% of the nasopharyngeal samples, respectively. In conclusion, the normal flora of the respiratory tract differs not only by the sampling site but also by the age group.
Topics: Adolescent; Bacterial Infections; Child; Child, Preschool; Female; Gram-Negative Aerobic Rods and Cocci; Gram-Positive Cocci; Haemophilus influenzae; Humans; Infant; Male; Moraxella; Moraxella catarrhalis; Nasopharynx; Pharynx; Pneumococcal Infections; Streptococcus pneumoniae; Streptococcus pyogenes
PubMed: 33642434
DOI: 10.7883/yoken.JJID.2020.824