RESUMEN

Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.

Asunto(s)

Gardnerella vaginalis , Lactobacillus , Microbiota , Reacción en Cadena en Tiempo Real de la Polimerasa , Vagina , Vaginosis Bacteriana , Femenino , Vaginosis Bacteriana/diagnóstico , Vaginosis Bacteriana/microbiología , Humanos , Vagina/microbiología , Microbiota/genética , Lactobacillus/aislamiento & purificación , Lactobacillus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto , Gardnerella vaginalis/aislamiento & purificación , Gardnerella vaginalis/genética , Adulto Joven , Sensibilidad y Especificidad , Prevotella/aislamiento & purificación , Prevotella/genética , Megasphaera/aislamiento & purificación , Megasphaera/genética , Actinobacteria/aislamiento & purificación , Actinobacteria/genética , Actinobacteria/clasificación , Persona de Mediana Edad , Lactobacillus crispatus/aislamiento & purificación , Lactobacillus crispatus/genética , Adolescente , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/clasificación , Embarazo , ARN Ribosómico 16S/genética

RESUMEN

Human tumors harbor a plethora of microbiota. It has been shown that the composition and diversity of intratumor microbiome are significantly associated with the survival of patients with pancreatic ductal adenocarcinoma (PDAC). However, the association in Chinese patients as well as the effect of different microorganisms on inhibiting tumor growth are unclear. In this study, we collected tumor samples resected from long-term and short-term PDAC survivors and performed 16S rRNA amplicon sequencing. We found that the microbiome in samples with different survival time were significantly different, and the differential bacterial composition was associated with the metabolic pathways in the tumor microenvironment. Furthermore, administration of Megasphaera, one of the differential bacteria, induced a better tumor growth inhibition effect when combined with the immune checkpoint inhibitor anti-programmed cell death-1 (anti-PD-1) treatment in mice bearing 4T1 tumor. These results indicate that specific intratumor microbiome can enhance the anti-tumor effect in the host, laying a foundation for further clarifying the underlying detailed mechanism.

Asunto(s)

Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Megasphaera , Microbiota , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores , Carcinoma Ductal Pancreático/terapia , China , Citocinas/metabolismo , Modelos Animales de Enfermedad , Disbiosis , Femenino , Humanos , Inmunohistoquímica , Masculino , Megasphaera/clasificación , Megasphaera/genética , Redes y Vías Metabólicas , Metagenoma , Metagenómica/métodos , Ratones , Persona de Mediana Edad , Terapia Molecular Dirigida , Estadificación de Neoplasias , Neoplasias Pancreáticas/terapia , Pronóstico , Resultado del Tratamiento , Neoplasias Pancreáticas

RESUMEN

The composition of the human vaginal microbiome has been extensively studied and is known to influence reproductive health. However, the functional roles of individual taxa and their contributions to negative health outcomes have yet to be well characterized. Here, we examine two vaginal bacterial taxa grouped within the genus Megasphaera that have been previously associated with bacterial vaginosis (BV) and pregnancy complications. Phylogenetic analyses support the classification of these taxa as two distinct species. These two phylotypes, Megasphaera phylotype 1 (MP1) and Megasphaera phylotype 2 (MP2), differ in genomic structure and metabolic potential, suggestive of differential roles within the vaginal environment. Further, these vaginal taxa show evidence of genome reduction and changes in DNA base composition, which may be common features of host dependence and/or adaptation to the vaginal environment. In a cohort of 3870 women, we observed that MP1 has a stronger positive association with bacterial vaginosis whereas MP2 was positively associated with trichomoniasis. MP1, in contrast to MP2 and other common BV-associated organisms, was not significantly excluded in pregnancy. In a cohort of 52 pregnant women, MP1 was both present and transcriptionally active in 75.4 % of vaginal samples. Conversely, MP2 was largely absent in the pregnant cohort. This study provides insight into the evolutionary history, genomic potential and predicted functional role of two clinically relevant vaginal microbial taxa.

Asunto(s)

Proteínas Bacterianas/genética , Megasphaera/clasificación , Análisis de Secuencia de ADN/métodos , Vagina/microbiología , Vaginosis Bacteriana/epidemiología , Composición de Base , Estudios de Casos y Controles , Evolución Molecular , Femenino , Regulación Bacteriana de la Expresión Génica , Tamaño del Genoma , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Megasphaera/genética , Megasphaera/aislamiento & purificación , Megasphaera/metabolismo , Filogenia , Embarazo , ARN Ribosómico 16S/genética , Salud Reproductiva , Vaginosis Bacteriana/microbiología

RESUMEN

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n = 21) and bacterial Keratitis patients (BK, n = 19). An increase in abundance of several antiinflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut-eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.

Asunto(s)

ADN Bacteriano/genética , ADN de Hongos/genética , Disbiosis/microbiología , Microbioma Gastrointestinal/fisiología , Queratitis/microbiología , Adulto , Aspergillus/clasificación , Aspergillus/genética , Aspergillus/aislamiento & purificación , Estudios de Casos y Controles , Clostridiales/clasificación , Clostridiales/genética , Clostridiales/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Disbiosis/diagnóstico , Disbiosis/patología , Faecalibacterium/clasificación , Faecalibacterium/genética , Faecalibacterium/aislamiento & purificación , Heces/microbiología , Femenino , Humanos , Queratitis/diagnóstico , Queratitis/patología , Kluyveromyces/clasificación , Kluyveromyces/genética , Kluyveromyces/aislamiento & purificación , Malassezia/clasificación , Malassezia/genética , Malassezia/aislamiento & purificación , Masculino , Megasphaera/clasificación , Megasphaera/genética , Megasphaera/aislamiento & purificación , Persona de Mediana Edad , Mortierella/clasificación , Mortierella/genética , Mortierella/aislamiento & purificación , Rhizopus/clasificación , Rhizopus/genética , Rhizopus/aislamiento & purificación , Ruminococcus/clasificación , Ruminococcus/genética , Ruminococcus/aislamiento & purificación , Veillonellaceae/clasificación , Veillonellaceae/genética , Veillonellaceae/aislamiento & purificación

RESUMEN

A novel mesophilic, anaerobic, Gram-stain-negative bacterium was isolated from the cecum of a healthy white leghorn chicken, and designated AJH120T. Cells were coccoid or diplococcoid with an average size of 0.8-1.8 µm and were non-motile with no evidence of spores. Phylogenetic analysis of 16S rRNA gene sequences revealed this organism to be a member of the genus Megasphaera, with the closest relatives being Megasphaera elsdenii (95 % sequence identity) and Megasphaera cerevisiae (95 % sequence identity). Growth was observed between 30 and 50 °C and between pH 5.0 and 9.0. AJH120T utilized a variety of carbon sources, including succinate, gluconate, fructose, ribose and pyruvate, as well as many individual amino acids. The DNA G+C content for the genome sequence of AJH120T was 52.1 mol%. Digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) and average amino acid identity (AAI) between AJH120T and close taxonomic relatives, indicated divergence consistent with the strain representing a novel species. The major fatty acid methyl esters of the organism were C12 : 0, C14 : 0 3-OH, C18 : 1ω9c, C16 : 0 and C16 : 1ω9c. AJH120T was able to produce several short chain fatty acids, including butyrate, acetate, propionate and isovalerate. Together, these data indicate that AJH120T represents a novel species within the genus Megasphaera. We propose the name Megasphaerastantonii sp. nov. for the species. The type strain of this species is AJH120T (=DSM 106750T=CCUG 71842T).

Asunto(s)

Ciego/microbiología , Pollos/microbiología , Megasphaera/clasificación , Filogenia , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Megasphaera/genética , Megasphaera/aislamiento & purificación , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: Data evaluating the impact of contraceptives on the vaginal microbiome are limited and inconsistent. OBJECTIVE: We hypothesized that women initiating copper intrauterine device use would have increased bacterial vaginosis and bacterial vaginosis-associated microbes with use compared to women initiating and using hormonal contraceptive methods. STUDY DESIGN: Vaginal swabs (N = 1047 from 266 participants seeking contraception) for Nugent score determination of bacterial vaginosis and quantitative polymerase chain reaction analyses for assessment of specific microbiota were collected from asymptomatic, healthy women aged 18-35 years in Harare, Zimbabwe, who were confirmed to be free of nonstudy hormones by mass spectrometry at each visit. Contraception was initiated with an injectable (depot medroxyprogesterone acetate [n = 41], norethisterone enanthate [n = 44], or medroxyprogesterone acetate and ethinyl estradiol [n = 40]), implant (levonorgestrel [n = 45] or etonogestrel [n = 48]), or copper intrauterine device (n = 48) and repeat vaginal swabs were collected after 30, 90, and 180 days of continuous use. Self-reported condom use was similar across all arms at baseline. Quantitative polymerase chain reaction was used to detect Lactobacillus crispatus, L jensenii, L gasseri/johnsonii group, L vaginalis, L iners, Gardnerella vaginalis, Atopobium vaginae, and Megasphaera-like bacterium phylotype I from swabs. Modified Poisson regression and mixed effects linear models were used to compare marginal prevalence and mean difference in quantity (expressed as gene copies/swab) prior to and during contraceptive use. RESULTS: Bacterial vaginosis prevalence increased in women initiating copper intrauterine devices from 27% at baseline, 35% at 30 days, 40% at 90 days, and 49% at 180 days (P = .005 compared to marginal prevalence at enrollment). Women initiating hormonal methods had no change in bacterial vaginosis prevalence over 180 days. The mean increase in Nugent score was 1.2 (95% confidence interval, 0.5-2.0; P = .001) in women using copper intrauterine devices. Although the frequency and density of beneficial lactobacilli did not change among intrauterine device users over 6 months, there was an increase in the log concentration of G vaginalis (4.7, 5.2, 5.8, 5.9; P = .046) and A vaginae (3.0, 3.8, 4.6, 5.1; P = .002) between baseline and 30, 90, and 180 days after initiation. Among other contraceptive groups, women using depot medroxyprogesterone acetate had decreased L iners (mean decrease log concentration = 0.8; 95% confidence interval, 0.3-1.5; P = .004) and there were no significant changes in beneficial Lactobacillus species over 180 days regardless of contraceptive method used. CONCLUSION: Copper intrauterine device use may increase colonization by bacterial vaginosis-associated microbiota, resulting in increased prevalence of bacterial vaginosis. Use of most hormonal contraception does not alter vaginal microbiota.

Asunto(s)

Anticonceptivos Femeninos/uso terapéutico , Dispositivos Intrauterinos de Cobre , Microbiota/genética , Vagina/microbiología , Vaginosis Bacteriana/epidemiología , Adulto , ADN Bacteriano/genética , Desogestrel/uso terapéutico , Implantes de Medicamentos , Etinilestradiol/uso terapéutico , Femenino , Gardnerella vaginalis/genética , Gardnerella vaginalis/aislamiento & purificación , Humanos , Lactobacillus crispatus/genética , Lactobacillus crispatus/aislamiento & purificación , Lactobacillus gasseri/genética , Lactobacillus gasseri/aislamiento & purificación , Levonorgestrel/uso terapéutico , Acetato de Medroxiprogesterona/uso terapéutico , Megasphaera/genética , Megasphaera/aislamiento & purificación , Noretindrona/análogos & derivados , Noretindrona/uso terapéutico , Reacción en Cadena de la Polimerasa , Factores Protectores , Factores de Riesgo , Vaginosis Bacteriana/microbiología , Adulto Joven

RESUMEN

The development and evaluation of a 6-hours laboratory class, based on capillary electrophoresis (CE) and the detection of microbial contaminants, is described. It can be easily scaled up or down, to suit class sizes up to 188 and completed in a shorter time scale. CE uses narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. A laboratory exercise has been devised to illustrate how CE-based genetic analysis system processes DNA fragment analysis to detect three microbial contaminants. The protocol is relatively inexpensive and uses standard molecular biology reagents and equipment. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):279-284, 2018.

Asunto(s)

ADN Bacteriano/análisis , Laboratorios , Megasphaera/aislamiento & purificación , Biología Molecular/educación , Pectinatus/aislamiento & purificación , Pediococcus pentosaceus/aislamiento & purificación , ADN Bacteriano/genética , Electroforesis Capilar , Megasphaera/genética , Pectinatus/genética , Pediococcus pentosaceus/genética , Reacción en Cadena de la Polimerasa

RESUMEN

Strain MHT, a strictly anaerobic, Gram-stain-negative, non-spore-forming, spherical coccus or coccoid-shaped microorganism, was isolated from a cow rumen during a screen for hexanoic acid-producing bacteria. The microorganism grew at 30-40 °C and pH 5.5-7.5 and exhibited production of various short- and medium-chain carboxylic acids (acetic acid, butyric acid, pentanoic acid, isobutyric acid, isovaleric acid, hexanoic acid, heptanoic acid and octanoic acid), as well as H2 and CO2 as biogas. Phylogenetic analysis based on 16S rRNA gene sequencing demonstrated that MHT represents a member of the genus Megasphaera, with the closest relatives being Megapsphaera indica NMBHI-10T (94.1 % 16S rRNA sequence similarity), Megasphaera elsdenii DSM 20460T (93.8 %) and Megasphaera paucivorans DSM 16981T (93.8 %). The major cellular fatty acids produced by MHT included C12 : 0, C16 : 0, C18 : 1cis 9, and C18 : 0, and the DNA G+C content of the MHT genome is 51.8 mol%. Together, the distinctive phenotypic and phylogenetic characteristics of MHT indicate that this microorganism represents a novel species of the genus Megasphaera, for which the name Megasphaera hexanoica sp. nov. is herein proposed. The type strain of this species is MHT (=KCCM 43214T=JCM 31403T).

Asunto(s)

Ácidos Carboxílicos/metabolismo , Bovinos/microbiología , Megasphaera/clasificación , Filogenia , Rumen/microbiología , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Femenino , Megasphaera/genética , Megasphaera/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN

RESUMEN

Much of the work in periodontal microbiology in recent years has focused on identifying and understanding periodontal pathogens. As the majority of oral microbes have not yet been isolated in pure form, it is essential to understand the phenotypic characteristics of microbes to decipher their role in oral environment. In this study, strain DISK18 was isolated from gingival sulcus and identified as a Megasphaera species. Although metagenomics studies revealed Megasphaera species as a major group within the oral habitat, they have never been isolated in cultivable form to date. Therefore, we have characterized the DISK18 strain to better understand its role in the periodontal ecosystem. Strain Megasphaera sp. DISK18 displayed the ability to adhere and self-aggregate, which are essential requisite features for inhabiting and persisting in oral cavity. It also coaggregated with other pioneer oral colonizers like Streptococcus and Lactobacillus species but not with Veillonella. This behaviour points towards its role in the ecologic succession of a multispecies biofilm as an early colonizer. The absence of virulence determining genes as observed in whole genome sequence analysis coupled with an inability to degrade collagen reveals that Megasphaera sp. strain DISK18 is likely not a pathogenic species and emphasizes its commensal lifestyle.

Asunto(s)

Placa Dental/microbiología , Megasphaera/química , Megasphaera/genética , Metagenoma , Metagenómica , Algoritmos , Adhesión Bacteriana , Biopelículas , Femenino , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Megasphaera/clasificación , Megasphaera/aislamiento & purificación , Metagenómica/métodos , Filogenia , Azúcares/metabolismo , Azufre/química , Temperatura , Compuestos Orgánicos Volátiles/química , Secuenciación Completa del Genoma , Adulto Joven

RESUMEN

Recent studies showing clear differences in the airway microbiota between healthy and diseased individuals shed light on the importance of the airway microbiota in health. Here, we report the associations of host genetics and lifestyles such as smoking, alcohol consumption, and physical activity with the composition of the sputum microbiota using 16S rRNA gene sequence data generated from 257 sputum samples of Korean twin-family cohort. By estimating the heritability of each microbial taxon, we found that several taxa, including Providencia and Bacteroides, were significantly influenced by host genetic factors. Smoking had the strongest effect on the overall microbial community structure among the tested lifestyle factors. The abundances of Veillonella and Megasphaera were higher in current-smokers, and increased with the pack-year value and the Fagerstrom Test of Nicotine Dependence (FTND) score. In contrast, Haemophilus decreased with the pack-year of smoking and the FTND score. Co-occurrence network analysis showed that the taxa were clustered according to the direction of associations with smoking, and that the taxa influenced by host genetics were found together. These results demonstrate that the relationships among sputum microbial taxa are closely associated with not only smoking but also host genetics.

Asunto(s)

Consumo de Bebidas Alcohólicas/genética , Microbiota/genética , Fumar/genética , Esputo/microbiología , Tabaquismo/genética , Adulto , Bacteroides/clasificación , Bacteroides/genética , Ejercicio Físico/fisiología , Femenino , Interacción Gen-Ambiente , Haemophilus/clasificación , Haemophilus/genética , Humanos , Masculino , Megasphaera/clasificación , Megasphaera/genética , Persona de Mediana Edad , ARN Ribosómico 16S/genética , Tabaquismo/microbiología , Veillonella/clasificación , Veillonella/genética

RESUMEN

Megasphaera elsdenii is a lactate-utilizing bacterium whose ruminal abundance has been shown to be greatly elevated during milk fat depression (MFD). To further examine this association, a total of 23 cannulated multiparous Holstein cows were examined in 3 experiments in which strains of M. elsdenii were directly dosed into the rumen (~2 × 10(12) cells/dose); control cows were dosed with sterile lactate-free culture medium. Cows were fed a total mixed ration (292 g of starch/kg of dry matter) that contained primarily corn silage, alfalfa silage, finely ground high-moisture corn, supplemental protein, and corn oil (3 g/kg of dry matter). Experiments differed in stage of lactation of the cows (early or late), dosing events (single dose, or 4 doses over a 5-d period), timing of dose (prefeed or 4 h postfeed), and M. elsdenii strain (laboratory strain YI9 or 3 strains isolated from cows in the same herd). Dry matter intake and milk yield and composition were measured from 5 to 0 d before dosing and 1 to 7d after first dosing, plus later time points that varied by experiment. Milk yield and composition were not affected by dosing. Megasphaera elsdenii was quantified in the liquid phase of ruminal contents by automated ribosomal intergenic spacer analysis, or by PCR with relative quantification (M. elsdenii 16S rRNA gene copy number as a percentage of total bacterial 16S rRNA gene copies). Neither the M. elsdenii-dosed or control cows displayed MFD after dosing, and in almost all cases M. elsdenii populations returned to low baseline levels (<0.02% of 16S rRNA gene copy number) within 24 h of dosing. This rapid decline in M. elsdenii also occurred in several cows that were dosed with a strain of M. elsdenii that had been isolated from that particular cow during a previous bout of MFD. Ruminal pH and total millimolar volatile fatty acids and lactate did not differ between dosed and control cows, although acetate-to-propionate ratio declined in both groups and butyrate increased after dosing with M. elsdenii. The results confirm that establishing exogenously added bacterial strains in the rumen is difficult, even for strains previously isolated from the recipient cow. The potential role of M. elsdenii as an agent of MFD remains unclear in the absence of successful establishment of the dosed strains.

Asunto(s)

Bovinos/microbiología , Megasphaera/fisiología , Leche/química , Rumen/química , Acetatos/análisis , Animales , Bovinos/fisiología , ADN Bacteriano/genética , Industria Lechera , Dieta/veterinaria , Ácidos Grasos Volátiles/análisis , Femenino , Lactancia , Medicago sativa , Megasphaera/genética , Propionatos/análisis , ARN Ribosómico/genética , ARN Ribosómico 16S/genética , Rumen/microbiología , Ensilaje/análisis , Zea mays

RESUMEN

Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421-432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2 O2 , whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2 O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles' heel of anaerobes when exposed to air.

Asunto(s)

Proteínas Bacterianas/metabolismo , Flavoproteínas Transportadoras de Electrones/metabolismo , Ferredoxinas/metabolismo , Megasphaera/metabolismo , Acidaminococcus/genética , Acidaminococcus/metabolismo , Anaerobiosis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Butiril-CoA Deshidrogenasa/química , Butiril-CoA Deshidrogenasa/genética , Butiril-CoA Deshidrogenasa/metabolismo , Transporte de Electrón , Flavoproteínas Transportadoras de Electrones/química , Flavoproteínas Transportadoras de Electrones/genética , Megasphaera/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría

RESUMEN

OBJECTIVE: The objective of the study was to evaluate the upper genital tract (UGT) presence of vaginal bacterial species using sensitive molecular methods capable of detecting fastidious bacterial vaginosis (BV)-associated bacteria. STUDY DESIGN: Vaginal swabs were collected prior to hysterectomy. The excised uterus was sterilely opened and swabs collected from the endometrium and upper endocervix. DNA was tested in 11 quantitative polymerase chain reaction (PCR) assays for 12 bacterial species: Lactobacillus iners, L crispatus, L jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp, Prevotella spp, Leptotrichia/Sneathia, BVAB1, BVAB2, BVAB3, and a broad-range16S ribosomal ribonucleic acid gene assay. Endometrial fluid was tested with Luminex and an enzyme-linked immunosorbent assay for cytokines and defensins and tissue for gene expression of defensins and cathelicidin. RESULTS: We enrolled 58 women: mean aged 43±7 years, mostly white (n=46; 79%) and BV negative (n=43; 74%). By species-specific quantitative PCR, 55 (95%) had UGT colonization with at least 1 species (n=52) or were positive by 16S PCR (n=3). The most common species were L iners (45% UGT, 61% vagina), Prevotella spp (33% UGT, 76% vagina) and L crispatus (33% UGT, 56% vagina). Median quantities of bacteria in the UGT were lower than vaginal levels by 2-4 log10 ribosomal ribonucleic acid gene copies per swab. There were no differences in the endometrial inflammatory markers between women with no bacteria, Lactobacillus only, or any BV-associated species in the UGT. CONCLUSION: Our data suggest that the endometrial cavity is not sterile in most women undergoing hysterectomy and that the presence of low levels of bacteria in the uterus is not associated with significant inflammation.

Asunto(s)

Portador Sano/epidemiología , Cuello del Útero/microbiología , Endometrio/microbiología , Vagina/microbiología , Actinobacteria/genética , Actinobacteria/aislamiento & purificación , Adulto , Portador Sano/microbiología , Femenino , Gardnerella vaginalis/genética , Gardnerella vaginalis/aislamiento & purificación , Humanos , Histerectomía , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Leptotrichia/genética , Leptotrichia/aislamiento & purificación , Megasphaera/genética , Megasphaera/aislamiento & purificación , Microbiota , Persona de Mediana Edad , Tipificación Molecular , Prevotella/genética , Prevotella/aislamiento & purificación , ARN Ribosómico 16S/genética , Enfermedades Uterinas/cirugía

RESUMEN

Two coccoid, non-motile, obligately anaerobic, Gram-stain-negative bacteria, occurring singly or in pairs, or as short chains, with a mean size of 1.4-2.5 µm were isolated from the faeces of two healthy human volunteers, aged 26 and 56 years, and were designated NMBHI-10(T) and BLPYG-7, respectively. Both the strains were affiliated to the sub-branch Sporomusa of the class Clostridia as revealed by 16S rRNA gene sequence analysis. The isolates NMBHI-10(T) and BLPYG-7 showed 99.1 and 99.2% 16S rRNA gene sequence similarity, respectively, with Megasphaera elsdenii JCM 1772(T). DNA-DNA hybridization and phenotypic analysis showed that both the strains were distinct from their closest relative, M. elsdenii JCM 1772(T) (42 and 53% DNA-DNA relatedness with NMBHI-10(T) and BLPYG-7, respectively), but belong to the same species (DNA-DNA relatedness of 80.9 % between the isolates). According to DNA-DNA hybridization results, the coccoid strains belong to the same genospecies, and neither is related to any of the recognized species of the genus Megasphaera. Strains NMBHI-10(T) and BLPYG-7 grew in PYG broth at temperatures of between 15 and 40 °C (optimum 37 °C), but not at 45 °C. The strains utilized a range of carbohydrates as sources of carbon and energy including glucose, lactose, cellobiose, rhamnose, galactose and sucrose. Glucose fermentation resulted in the formation of volatile fatty acids, mainly caproic acid and organic acids such as succinic acid. Phylogenetic analysis, specific phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The DNA G+C contents of strains NMBHI-10(T) and BLPYG-7 are 57.7 and 54.9 mol%, respectively. The major fatty acids were 12 : 0 FAME and 17 : 0 CYC FAME. On the basis of these data, we conclude that strains NMBHI-10(T) and BLPYG-7 should be classified as representing a novel species of the genus Megasphaera, for which the name Megsphaera indica sp. nov. is proposed. The type strain is NMBHI-10(T) ( = DSM 25563(T) = MCC 2481(T)).

Asunto(s)

Heces/microbiología , Megasphaera/clasificación , Filogenia , Adulto , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Fermentación , Humanos , India , Masculino , Megasphaera/genética , Megasphaera/aislamiento & purificación , Persona de Mediana Edad , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN

RESUMEN

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

Asunto(s)

Cerveza/microbiología , Sondas de ADN , Microbiología de Alimentos/métodos , Megasphaera/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Pectinatus/aislamiento & purificación , Sondas de ADN/química , Sondas de ADN/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , Megasphaera/genética , Hibridación de Ácido Nucleico , Pectinatus/genética , Sensibilidad y Especificidad

RESUMEN

With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563) and BL7 (DSM25562), isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460) highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the "glycobiome" based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes) amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate), vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the human host.

Asunto(s)

Tracto Gastrointestinal/microbiología , Genoma Bacteriano/genética , Megasphaera/genética , Aminoácidos/metabolismo , Ácidos Grasos Volátiles/metabolismo , Humanos , Megasphaera/clasificación , Megasphaera/metabolismo , Megasphaera/fisiología , Filogenia , Vitaminas/metabolismo

RESUMEN

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.

Asunto(s)

Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Metagenoma , ARN Bacteriano/análisis , ARN/análisis , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de Varianza , Bacillus cereus/genética , Bacillus cereus/metabolismo , Gardnerella vaginalis/genética , Gardnerella vaginalis/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Riñón/citología , Riñón/metabolismo , Lactobacillus/genética , Lactobacillus/metabolismo , Hígado/citología , Hígado/metabolismo , Megasphaera/genética , Megasphaera/metabolismo , Prevotella/genética , Prevotella/metabolismo , Reproducibilidad de los Resultados , Tamaño de la Muestra

RESUMEN

The class Clostridia in the phylum Firmicutes (formerly low-G+C Gram-positive bacteria) includes diverse bacteria of medical, environmental and biotechnological importance. The Selenomonas-Megasphaera-Sporomusa branch, which unifies members of the Firmicutes with Gram-negative-type cell envelopes, was recently moved from Clostridia to a separate class Negativicutes. However, draft genome sequences of the spore-forming members of the Negativicutes revealed typically clostridial sets of sporulation genes. To address this and other questions in clostridial phylogeny, we have compared a phylogenetic tree for a concatenated set of 50 widespread ribosomal proteins with the trees for beta subunits of the RNA polymerase (RpoB) and DNA gyrase (GyrB) and with the 16S rRNA-based phylogeny. The results obtained by these methods showed remarkable consistency, suggesting that they reflect the true evolutionary history of these bacteria. These data put the Selenomonas-Megasphaera-Sporomusa group back within the Clostridia. They also support placement of Clostridium difficile and its close relatives within the family Peptostreptococcaceae; we suggest resolving the long-standing naming conundrum by renaming it Peptoclostridium difficile. These data also indicate the existence of a group of cellulolytic clostridia that belong to the family Ruminococcaceae. As a tentative solution to resolve the current taxonomical problems, we propose assigning 78 validly described Clostridium species that clearly fall outside the family Clostridiaceae to six new genera: Peptoclostridium, Lachnoclostridium, Ruminiclostridium, Erysipelatoclostridium, Gottschalkia and Tyzzerella. This work reaffirms that 16S rRNA and ribosomal protein sequences are better indicators of evolutionary proximity than phenotypic traits, even such key ones as the structure of the cell envelope and Gram-staining pattern.

Asunto(s)

Clostridium/clasificación , Genoma Bacteriano , Bacterias Gramnegativas/clasificación , Filogenia , Secuencia de Bases , Pared Celular/metabolismo , Clostridium/genética , Girasa de ADN/genética , Bacterias Gramnegativas/genética , Megasphaera/clasificación , Megasphaera/genética , ARN Polimerasa II/genética , ARN Ribosómico 16S/genética , Selenomonas/clasificación , Selenomonas/genética , Especificidad de la Especie , Esporas

RESUMEN

BACKGROUND AND OBJECTIVE: Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis. MATERIALS AND METHODS: Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3-V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated. RESULTS: Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor. CONCLUSIONS: Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.

Asunto(s)

ADN Bacteriano/genética , Metagenoma/genética , ARN Ribosómico 16S/genética , Vagina/microbiología , Vaginosis Bacteriana/diagnóstico , Vaginosis Bacteriana/microbiología , Actinobacteria/genética , Actinobacteria/aislamiento & purificación , Adolescente , Adulto , Estudios de Casos y Controles , ADN Bacteriano/aislamiento & purificación , Femenino , Gardnerella vaginalis/genética , Gardnerella vaginalis/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Megasphaera/genética , Megasphaera/aislamiento & purificación , Persona de Mediana Edad , ARN Ribosómico 16S/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

BACKGROUND: Bacterial vaginosis (BV) represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. Although antibiotics relieve symptoms and temporarily eradicate BV-associated bacteria (BVAB), BV usually recurs. We investigated the role of extravaginal BVAB reservoirs in recurrence. METHODS: Risks for BV acquisition over the course of 1 year were defined. DNA in vaginal, anal, and oral swab samples from enrollment was subjected to quantitative polymerase chain reaction assays targeting 16S ribosomal RNA genes of Gardnerella vaginalis, Lactobacillus crispatus, BVAB1, BVAB2, BVAB3, Megasphaera spp., Lactobacillus jensenii, and Leptotrichia/Sneathia spp. A case-control approach analyzed BVAB detection at enrollment for case patients (BV acquisition) versus controls (none). RESULTS: Of 239 women enrolled without BV, 199 were seen in follow-up, and 40 experienced BV; 15 had all samples for analysis. Detection of G. vaginalis in oral cavity or anal samples and Leptotrichia/Sneathia spp. in anal samples was more common at enrollment among case patients, who also had higher concentrations of these bacteria and Megasphaera relative to 30 controls at each site. In contrast, L. crispatus was detected more frequently in anal samples among controls. CONCLUSIONS: Women who acquire BV are more likely have previous colonization of extravaginal reservoirs with some BVAB, and less likely to have L. crispatus, suggesting that BVAB may be acquired vaginally from extravaginal reservoirs.

Asunto(s)

Canal Anal/microbiología , Bacterias/aislamiento & purificación , Reservorios de Enfermedades/microbiología , Boca/microbiología , Vaginosis Bacteriana/microbiología , Adolescente , Adulto , Bacterias/genética , Estudios de Casos y Controles , Estudios de Cohortes , ADN Bacteriano/genética , Femenino , Estudios de Seguimiento , Gardnerella vaginalis/genética , Gardnerella vaginalis/aislamiento & purificación , Humanos , Lactobacillus/genética , Lactobacillus/aislamiento & purificación , Leptotrichia/genética , Leptotrichia/aislamiento & purificación , Megasphaera/genética , Megasphaera/aislamiento & purificación , Metagenoma , ARN Ribosómico 16S/genética , Factores de Riesgo , Vaginosis Bacteriana/epidemiología , Adulto Joven