Identification of Mycoplasma species and related organisms from ruminants in England and Wales during 2005-2019.

BACKGROUND: Mycoplasma species have been associated with economically important diseases affecting ruminants worldwide and include contagious bovine pleuropneumonia (CBPP), contagious caprine pleuropneumonia (CCPP) and contagious agalactia, listed by the World Organisation for Animal Health (OIE). The Mycoplasma Team at the Animal and Plant Health Agency provides an identification service for Mycoplasma and Ureaplasma species of veterinary importance to the United Kingdom (UK), supporting the detection of new and emerging pathogens, as well as contributing to the surveillance of endemic, and the OIE listed diseases exotic to the UK. Mycoplasma and other Mollicutes species were identified from diagnostic samples from farmed ruminants in England and Wales using a combination of culture and 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis, submitted between 2005 and 2019. RESULTS: A total of 5578 mollicutes identifications, which include mycoplasmas and the related acholeoplasmas and ureaplasmas, were made from farmed ruminant animals during the study period. Throughout the study period, the pathogen Mycoplasma bovis was consistently the most frequently identified species, accounting for 1411 (32%) of 4447 molecular identifications in cattle, primarily detected in the lungs of pneumonic calves, followed by joints and milk of cattle showing signs of arthritis and mastitis, respectively. M. bovirhinis, M. alkalescens, M. dispar, M. arginini and Ureaplasma diversum, were also common. Mixed species, principally M. bovis with M. alkalescens, M. arginini or M. bovirhinis were also prevalent, particularly from respiratory samples. The non-cultivable blood-borne haemoplasmas Candidatus 'Mycoplasma haemobos' and Mycoplasma wenyonii were identified from cattle, with the latter species most often associated with milk-drop. M. ovipneumoniae was the predominant species identified from sheep and goats experiencing respiratory disease, while M. conjunctivae preponderated in ocular samples. The UK remains free of the ruminant mycoplasmas listed by OIE. CONCLUSIONS: The continued high prevalence of M. bovis identifications confirms its ongoing dominance and importance as a significant pathogen of cattle in England and Wales, particularly in association with respiratory disease. M. ovipneumoniae has seen a general increase in prevalence in recent years, notably in coughing lambs and should therefore be considered as a primary differential diagnosis of respiratory disease in small ruminants.

Microbiome Analysis Reveals Diversity and Function of Mollicutes Associated with the Eastern Oyster, Crassostrea virginica.

Marine invertebrate microbiomes play important roles in diverse host and ecological processes. However, a mechanistic understanding of host-microbe interactions is currently available for a small number of model organisms. Here, an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica, was performed using 16S rRNA gene-based amplicon profiling, shotgun metagenomics, and genome-scale metabolic reconstruction. Relatively high variability of the microbiome was observed across individual oysters and among different tissue types. Specifically, a significantly higher alpha diversity was observed in the inner shell than in the gut, gill, mantle, and pallial fluid samples, and a distinct microbiome composition was revealed in the gut compared to other tissues examined in this study. Targeted metagenomic sequencing of the gut microbiota led to further characterization of a dominant bacterial taxon, the class Mollicutes, which was captured by the reconstruction of a metagenome-assembled genome (MAG). Genome-scale metabolic reconstruction of the oyster Mollicutes MAG revealed a reduced set of metabolic functions and a high reliance on the uptake of host-derived nutrients. A chitin degradation and an arginine deiminase pathway were unique to the MAG compared to closely related genomes of Mollicutes isolates, indicating distinct mechanisms of carbon and energy acquisition by the oyster-associated Mollicutes A systematic reanalysis of public eastern oyster-derived microbiome data revealed a high prevalence of the Mollicutes among adult oyster guts and a significantly lower relative abundance of the Mollicutes in oyster larvae and adult oyster biodeposits.IMPORTANCE Despite their biological and ecological significance, a mechanistic characterization of microbiome function is frequently missing from many nonmodel marine invertebrates. As an initial step toward filling this gap for the eastern oyster, Crassostrea virginica, this study provides an integrated taxonomic and functional analysis of the oyster microbiome using samples from a coastal salt pond in August 2017. The study identified high variability of the microbiome across tissue types and among individual oysters, with some dominant taxa showing higher relative abundance in specific tissues. A high prevalence of Mollicutes in the adult oyster gut was revealed by comparative analysis of the gut, biodeposit, and larva microbiomes. Phylogenomic analysis and metabolic reconstruction suggested the oyster-associated Mollicutes is closely related but functionally distinct from Mollicutes isolated from other marine invertebrates. To the best of our knowledge, this study represents the first metagenomics-derived functional inference of Mollicutes in the eastern oyster microbiome.

Identification of contaminating mollicutes in mammalian cell culture as spiroplasma species.

Cell cultures have provided an ideal habitat for a wide variety of Mycoplasma and Acholeplasma species since the earliest days of in-vitro culture. The possibility of contamination with Spiroplasma species was addressed by Regulatory Authorities due to the increased commercial use of insect cells, recognising that Spiroplasmas have been isolated from many types of arthropod and also that insect cell cultures support Spiroplasma growth as they have been used for cultivation of fastidious species. In this study we re-examined two cell culture samples previously confirmed as contaminated with mollicutes by cultural methods. One isolate had undergone sequencing which had placed it in the S. citri phylogenetic group, whilst the other had not been identified. Using modern sequencing methods we were able to further identify both isolates to species level.

Metagenomic profiling of bacterial diversity and community structure in termite mounds and surrounding soils.

The study focuses on analysis of the compositional and diversity of bacteria in termite mound soils in comparison with the surrounding soils to verify the assertion that the high nutrient concentrations in termite mound soils influence a complex diversity of microorganisms. Here, whole DNA was extracted from soil samples collected from termite mounds and their surrounding soils which were 10 m apart and subsequently, sequenced using shotgun metagenomic approach. Our findings showed that both environments have several soil bacterial phyla in common. However, Proteobacteria and Actinobacteria significantly dominated the termite mound soils and the surrounding soils, respectively, with Tenericutes peculiar to only the termite mound soils. Furthermore, Bergeyella, Gloeothece, Thalassospira, and Glaciecola genera were exclusively identified in the termite mound soil samples. Diversity analysis showed that bacterial composition was different among the four sites (phyla level). This study also revealed a lot of unclassified groups of bacteria and this could point to the presence of potentially novel species. The differences observed in the bacterial structure and diversity from this study may be ascribed to variances in the physicochemical nature existing between the two environments. Mapping out schemes to culture these unclassified groups of bacteria discovered from this study would possibly set the platform for the discovery of novel bacteria for biotechnological applications.

Captivity affects diversity, abundance, and functional pathways of gut microbiota in the northern grass lizard Takydromus septentrionalis.

Animals in captivity undergo a range of environmental changes from wild animals. An increasing number of studies show that captivity significantly affects the abundance and community structure of gut microbiota. The northern grass lizard (Takydromus septentrionalis) is an extensively studied lacertid lizard and has a distributional range covering the central and southeastern parts of China. Nonetheless, little is known about the gut microbiota of this species, which may play a certain role in nutrient and energy metabolism as well as immune homeostasis. Here, we examined the differences in the gut microbiota between two groups (wild and captive) of lizards through 16S rRNA sequencing using the Illumina HiSeq platform. The results demonstrated that the dominant microbial components in both groups consisted of Proteobacteria, Firmicutes, and Tenericutes. The two groups did not differ in the abundance of these three phyla. Citrobacter was the most dominant genus in wild lizards, while Morganella was the most dominant genus in captive lizards. Moreover, gene function predictions showed that genes at the KEGG pathway levels2 were more abundant in wild lizards than in captive lizards but, at the KEGG pathway levels1, the differences in gene abundances between wild and captive lizards were not significant. In summary, captivity exerted a significant impact on the gut microbial community structure and diversity in T. septentrionalis, and future work could usefully investigate the causes of these changes using a comparative approach.

Phylogenomics of expanding uncultured environmental Tenericutes provides insights into their pathogenicity and evolutionary relationship with Bacilli.

BACKGROUND: The metabolic capacity, stress response and evolution of uncultured environmental Tenericutes have remained elusive, since previous studies have been largely focused on pathogenic species. In this study, we expanded analyses on Tenericutes lineages that inhabit various environments using a collection of 840 genomes. RESULTS: Several environmental lineages were discovered inhabiting the human gut, ground water, bioreactors and hypersaline lake and spanning the Haloplasmatales and Mycoplasmatales orders. A phylogenomics analysis of Bacilli and Tenericutes genomes revealed that some uncultured Tenericutes are affiliated with novel clades in Bacilli, such as RF39, RFN20 and ML615. Erysipelotrichales and two major gut lineages, RF39 and RFN20, were found to be neighboring clades of Mycoplasmatales. We detected habitat-specific functional patterns between the pathogenic, gut and the environmental Tenericutes, where genes involved in carbohydrate storage, carbon fixation, mutation repair, environmental response and amino acid cleavage are overrepresented in the genomes of environmental lineages, perhaps as a result of environmental adaptation. We hypothesize that the two major gut lineages, namely RF39 and RFN20, are probably acetate and hydrogen producers. Furthermore, deteriorating capacity of bactoprenol synthesis for cell wall peptidoglycan precursors secretion is a potential adaptive strategy employed by these lineages in response to the gut environment. CONCLUSIONS: This study uncovers the characteristic functions of environmental Tenericutes and their relationships with Bacilli, which sheds new light onto the pathogenicity and evolutionary processes of Mycoplasmatales.

A timetree for phytoplasmas (Mollicutes) with new insights on patterns of evolution and diversification.

The first comprehensive timetree is presented for phytoplasmas, a diverse group of obligate intracellular bacteria restricted to phloem sieve elements of vascular plants and tissues of their hemipteran insect vectors. Maximum likelihood-based phylogenetic analysis of DNA sequence data from the 16S rRNA and methionine aminopeptidase (map) genes yielded well resolved estimates of phylogenetic relationships among major phytoplasma lineages, 16Sr groups and known strains of phytoplasmas. Age estimates for divergences among two major lineages of Mollicutes based on a previous comprehensive bacterial timetree were used to calibrate an initial 16S timetree. A separate timetree was estimated based on the more rapidly-evolving map gene, with an internal calibration based on a recent divergence within two related 16Sr phytoplasma subgroups in group 16SrV thought to have been driven by the introduction of the North American leafhopper vector Scaphoideus titanus Ball into Europe during the early part of the 20th century. Combining the resulting divergence time estimates into a final 16S timetree suggests that evolutionary rates have remained relatively constant overall through the evolution of phytoplasmas and that the origin of this lineage, at ~641 million years ago (Ma), preceded the origin of land plants and hemipteran insects. Nevertheless, the crown group of phytoplasmas is estimated to have begun diversifying ~316 Ma, roughly coinciding with the origin of seed plants and Hemiptera. Some phytoplasma groups apparently associated with particular plant families or insect vector lineages generally arose more recently than their respective hosts and vectors, suggesting that vector-mediated host shifts have been an important mechanism in the evolutionary diversification of phytoplasmas. Further progress in understanding macroevolutionary patterns in phytoplasmas is hindered by large gaps in knowledge of the identity of competent vectors and lack of data on phytoplasma associations with non-economically important plants.

Necessity and rationale for the proposed name changes in the classification of Mollicutes species. Reply to: 'Recommended rejection of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceae fam. nov., Mycoplasmoidaceae fam. nov., Mycoplasmoidales ord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov. [Gupta, Sawnani, Adeolu, Alnajar and Oren 2018] and all proposed species comb. nov. placed therein', by M. Balish et al. (Int J Syst Evol Microbiol, 2019;69:3650-3653).

This response summarizes the highly disordered state of the Mollicutes taxonomy that existed until recently, where most Mollicutes taxa lacked proper circumscriptions and their names were not in accordance with the International Code of Nomenclature of Prokaryotes and illegitimate. We also summarize the comprehensive phylogenomic and comparative genomic studies forming the basis for the proposed changes in the classification of Mollicultes species. Our responses to the concerns raised by Balish et al., show that the proposed taxonomic changes do not violate any essential point of the Code. Instead the proposed name changes rectify numerous taxonomic anomalies that have long plagued the classification of Mollicutes species, leading to a better understanding of their evolutionary relationships and bringing their nomenclature in conformity with the Code.

Recommended rejection of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceae fam. nov., Mycoplasmoidaceae fam. nov., Mycoplasmoidales ord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov. [Gupta, Sawnani, Adeolu, Alnajar and Oren 2018] and all proposed species comb. nov. placed therein.

The consensus of the members of the International Committee on Systematics of Prokaryotes' Subcommittee on the taxonomy of Mollicutes is that recently proposed sweeping changes to nomenclature of members of the Mycoplasmatales, specifically involving introduction of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceaefam. nov., Mycoplasmoidaceaefam. nov., Mycoplasmoidalesord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov., and all proposed species or subspecies comb. nov. placed therein, should be rejected because they violate one or more essential points of the International Code of Nomenclature of Prokaryotes.

Effect of intestinal tapeworms on the gut microbiota of the common carp, Cyprinus carpio.

BACKGROUND: Parasitic protozoans, helminths, alter the gut microbiota in mammals, yet little is known about the influence of intestinal cestodes on gut microbiota in fish. In the present study, the composition and diversity of the hindgut microbiota were determined in the intestine of common carp (Cyprinus carpio) infected with two tapeworm species, Khawia japonensis and Atractolytocestus tenuicollis. RESULTS: The intestine contained a core microbiota composed of Proteobacteria, Fusobacteria and Tenericutes. Infection with the two cestode species had no significant effect on the microbial diversity and richness, but it altered the microbial composition at the genus level. PCoA analysis indicated that microbial communities in the infected and uninfected common carp could not be distinguished from each other. However, a Mantel test indicated that the abundance of K. japonensis was significantly correlated with the microbial composition (P = 0.015), while the abundance of A. tenuicollis was not (P = 0.954). According to Pearson's correlation analysis, the abundance of K. japonensis exhibited an extremely significant (P < 0.001) positive correlation with the following gut microbiota taxa: Epulopiscium, U114, Bacteroides, Clostridium and Peptostreptococcaceae (0.8< r < 0.9); and a significant (P < 0.05) correlation with Enterobacteriaceae, Micrococcaceae, Rummeliibacillus, Lysinibacillus boronitolerans, Veillonellaceae, Oxalobacteraceae, Aeromonadaceae (negative), Marinibacillus and Chitinilyticum (0.4< r < 0.7). CONCLUSIONS: These results suggest that the composition of gut microbiota was somewhat affected by the K. japonensis infection. Additionally, increased ratios of pathogenic bacteria (Lawsonia and Plesiomonas) were also associated with the K. japonensis infection, which may therefore increase the likelihood of disease.

Ureaplasma diversum protein interaction networks: evidence of horizontal gene transfer and evolution of reduced genomes among Mollicutes.

Ureaplasma diversum is a member of the Mollicutes class responsible for urogenital tract infection in cattle and small ruminants. Studies indicate that the process of horizontal gene transfer, the exchange of genetic material among different species, has a crucial role in mollicute evolution, affecting the group's characteristic genomic reduction process and simplification of metabolic pathways. Using bioinformatics tools and the STRING database of known and predicted protein interactions, we constructed the protein-protein interaction network of U. diversum and compared it with the networks of other members of the Mollicutes class. We also investigated horizontal gene transfer events in subnetworks of interest involved in purine and pyrimidine metabolism and urease function, chosen because of their intrinsic importance for host colonization and virulence. We identified horizontal gene transfer events among Mollicutes and from Ureaplasma to Staphylococcus aureus and Corynebacterium, bacterial groups that colonize the urogenital niche. The overall tendency of genome reduction and simplification in the Mollicutes is echoed in their protein interaction networks, which tend to be more generalized and less selective. Our data suggest that the process was permitted (or enabled) by an increase in host dependence and the available gene repertoire in the urogenital tract shared via horizontal gene transfer.

The time response of anaerobic digestion microbiome during an organic loading rate shock.

Knowledge of connections between operational conditions, process stability, and microbial community dynamics is essential to enhance anaerobic digestion (AD) process efficiency and management. In this study, the detailed temporal effects of a sudden glycerol-based organic overloading on the AD microbial community and process imbalance were investigated in two replicate anaerobic digesters by a time-intensive sampling scheme. The microbial community time response to the overloading event was shorter than the shifts of reactor performance parameters. An increase in bacterial community dynamics and in the abundances of several microbial taxa, mainly within the Firmicutes, Tenericutes, and Chloroflexi phyla and Methanoculleus genera, could be detected prior to any shift on the reactor operational parameters. Reactor acidification already started within the first 24 h of the shock and headed the AD process to total inhibition in 72 h alongside with the largest shifts on microbiome, mostly the increase of Anaerosinus sp. and hydrogenotrophic methanogenic Archaea. In sum, this work proved that AD microbial community reacts very quickly to an organic overloading and some shifts occur prior to alterations on the performance parameters. The latter is very interesting as it can be used to improve AD process management protocols.

Highly divergent Mollicutes symbionts coexist in the scorpion Androctonus australis.

Androctonus australis is one of the most ubiquitous and common scorpion species in desert and arid lands from North Africa to India and it has an important ecological role and social impact. The bacterial community associated to this arachnid is unknown and we aimed to dissect its species composition in the gut, gonads, and venom gland. A 16S rRNA gene culture-independent diversity analysis revealed, among six other taxonomic groups (Firmicutes, Betaproteobacteria, Gammaproteobacteria, Flavobacteria, Actinobacteria, and Cyanobacteria), a dominance of Mollicutes phylotypes recorded both in the digestive tract and the gonads. These related Mollicutes include two Spiroplasma phylotypes (12.5% of DGGE bands and 15% of clones), and a new Mycoplasma cluster (80% of clones) showing 16S rRNA sequence identities of 95 and 93% with Mollicutes detected in the Mexican scorpions Centruroides limpidus and Vaejovis smithi, respectively. Such scorpion-associated Mollicutes form a new lineage that share a distant ancestor with Mycoplasma hominis. The observed host specificity with the apparent phylogenetic divergence suggests a relatively long co-evolution of these symbionts with the scorpion hosts. From the ecological point of view, such association may play a beneficial role for the host fitness, especially during dormancy or molt periods.

Increased richness and diversity of the vaginal microbiota and spontaneous preterm birth.

BACKGROUND: The bacterial community present in the female lower genital tract plays an important role in maternal and neonatal health. Imbalances in this microbiota have been associated with negative reproductive outcomes, such as spontaneous preterm birth (sPTB), but the mechanisms underlying the association between a disturbed microbiota and sPTB remain poorly understood. An intrauterine infection ascending from the vagina is thought to be an important contributor to the onset of preterm labour. Our objective was to characterize the vaginal microbiota of pregnant women who had sPTB (n = 46) and compare to those of pregnant women who delivered at term (n = 170). Vaginal swabs were collected from women at 11-16 weeks of gestational age. Microbiota profiles were created by PCR amplification and pyrosequencing of the cpn60 universal target region. RESULTS: Profiles clustered into seven community state types: I (Lactobacillus crispatus dominated), II (Lactobacillus gasseri dominated), III (Lactobacillus iners dominated), IVA (Gardnerella vaginalis subgroup B or mix of species), IVC (G. vaginalis subgroup A dominated), IVD (G. vaginalis subgroup C dominated) and V (Lactobacillus jensenii dominated). The microbiota of women who experienced preterm birth (< 37 weeks gestation) had higher richness and diversity and higher Mollicutes prevalence when compared to those of women who delivered at term. The two groups did not cluster according to CST, likely because CST assignment is driven in most cases by the dominance of one particular species, overwhelming the contributions of more rare taxa. In conclusion, we did not identify a specific microbial community structure that predicts sPTB, but differences in microbiota richness, diversity and Mollicutes prevalence were observed between groups. CONCLUSIONS: Although a causal relationship remains to be determined, our results confirm previous reports of an association between Mollicutes and sPTB and further suggest that a more diverse microbiome may be important in the pathogenesis of some cases.

Horizontal Gene Transfers in Mycoplasmas (Mollicutes).

The class Mollicutes (trivial name "mycoplasma") is composed of wall-less bacteria with reduced genomes whose evolution was long thought to be only driven by gene losses. Recent evidences of massive horizontal gene transfer (HGT) within and across species provided a new frame to understand the successful adaptation of these minimal bacteria to a broad range of hosts. Mobile genetic elements are being identified in a growing number of mycoplasma species, but integrative and conjugative elements (ICEs) are emerging as pivotal in HGT. While sharing common traits with other bacterial ICEs, such as their chromosomal integration and the use of a type IV secretion system to mediate horizontal dissemination, mycoplasma ICEs (MICEs) revealed unique features: their chromosomal integration is totally random and driven by a DDE recombinase related to the Mutator-like superfamily. Mycoplasma conjugation is not restricted to ICE transmission, but also involves the transfer of large chromosomal fragments that generates progenies with mosaic genomes, nearly every position of chromosome being mobile. Mycoplasmas have thus developed efficient ways to gain access to a considerable reservoir of genetic resources distributed among a vast number of species expanding the concept of minimal cell to the broader context of flowing information.

Phylogenetic framework for the phylum Tenericutes based on genome sequence data: proposal for the creation of a new order Mycoplasmoidales ord. nov., containing two new families Mycoplasmoidaceae fam. nov. and Metamycoplasmataceae fam. nov. harbouring Eperythrozoon, Ureaplasma and five novel genera.

The genus Mycoplasma, including species earlier classified in the genera Eperythrozoon and Haemobartonella, contains ~ 120 species and constitutes an extensively polyphyletic assemblage of bacteria within the phylum Tenericutes. Due to their small genome sizes and lack of unique characteristics, the relationships among the mycoplasmas/Tenericutes are not reliably discerned. Using genome sequences for 140 Tenericutes, their evolutionary relationships were examined using multiple independent approaches. Phylogenomic trees were constructed for 63 conserved proteins, 45 ribosomal proteins, three main subunits of RNA polymerase and 16S rRNA gene sequences. In all of these trees, Tenericutes species reliably grouped into four main clades designated as the "Acholeplasma", "Spiroplasma", "Pneumoniae" and "Hominis" clusters. These clades are also distinguished based on a similarity matrix constructed based on 16S rRNA gene sequences. Mycoplasma species were dispersed across 3 of these 4 clades highlighting their extensive polyphyly. In parallel, our comparative genomic analyses have identified > 100 conserved signature indels (CSIs) and 14 conserved signature proteins (CSPs), which are uniquely shared by the members of four identified clades, strongly supporting their monophyly and identifying them in molecular terms. Mycoplasma mycoides, the type species of the genus Mycoplasma, and a small number of other Mycoplasma species, formed a strongly supported clade within the "Spiroplasma" cluster. Nine CSIs and 14 CSPs reliably distinguish this clade from all other Mycoplasmatales species. The remainder of the Mycoplasmatales species are part of the "Pneumoniae" and "Hominis" clusters, which group together in phylogenetic trees. Here we are proposing that the order Mycoplasmatales should be emended to encompass only the Mycoplasma species within the "Spiroplasma" cluster and that a new order, Mycoplasmoidales ord. nov., should be created to encompass the other Mycoplasma species. The "Pneumoniae" and the "Hominis" clusters are proposed as two new families, Mycoplasmoidaceae fam. nov., which includes the genera Eperythrozoon, Ureaplasma, and the newly proposed genera Malacoplasma and Mycoplasmoides, and Metamycoplasmataceae fam. nov. to contain the newly proposed genera Metamycoplasma, Mycoplasmopsis, and Mesomycoplasma. The results presented here allow reliable discernment, both in phylogenetic and molecular terms, of the members of the two proposed families as well as different described genera within these families including members of the genus Eperythrozoon, which is comprised of uncultivable organisms. The taxonomic reclassifications proposed here, which more accurately portray the genetic diversity among the Tenericutes/Mycoplasma species, provide a new framework for understanding the biological and clinical aspects of these important microbes.

The vaginal microbiome of pregnant women is less rich and diverse, with lower prevalence of Mollicutes, compared to non-pregnant women.

The vaginal microbiome plays an important role in maternal and neonatal health. Imbalances in this microbiota (dysbiosis) during pregnancy are associated with negative reproductive outcomes, such as pregnancy loss and preterm birth, but the underlying mechanisms remain poorly understood. Consequently a comprehensive understanding of the baseline microbiome in healthy pregnancy is needed. We characterized the vaginal microbiomes of healthy pregnant women at 11-16 weeks of gestational age (n = 182) and compared them to those of non-pregnant women (n = 310). Profiles were created by pyrosequencing of the cpn60 universal target region. Microbiome profiles of pregnant women clustered into six Community State Types: I, II, III, IVC, IVD and V. Overall microbiome profiles could not be distinguished based on pregnancy status. However, the vaginal microbiomes of women with healthy ongoing pregnancies had lower richness and diversity, lower prevalence of Mycoplasma and Ureaplasma and higher bacterial load when compared to non-pregnant women. Lactobacillus abundance was also greater in the microbiomes of pregnant women with Lactobacillus-dominated CSTs in comparison with non-pregnant women. This study provides further information regarding characteristics of the vaginal microbiome of low-risk pregnant women, providing a baseline for forthcoming studies investigating the diagnostic potential of the microbiome for prediction of adverse pregnancy outcomes.

Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes.

BACKGROUND: Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes. METHODS: A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis. RESULTS: Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10-4 and 10-5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well. CONCLUSIONS: These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult.

Description of two novel members of the family Erysipelotrichaceae: Ileibacterium valens gen. nov., sp. nov. and Dubosiella newyorkensis, gen. nov., sp. nov., from the murine intestine, and emendation to the description of Faecalibaculum rodentium.

To better characterize murine intestinal microbiota, a large number (187) of Gram-positive-staining, rod- and coccoid-shaped, and facultatively or strictly anaerobic bacteria were isolated from small and large intestinal contents from mice. Based on 16S rRNA gene sequencing, a total 115 isolates formed three phylogenetically distinct clusters located within the family Erysipelotrichaceae. Group 1, as represented by strain NYU-BL-A3T, was most closely related to Allobaculum stercoricanis, with 16S rRNA gene sequence similarity values of 87.7 %. A second group, represented by NYU-BL-A4T, was most closely related to Faecalibaculum rodentium, with 86.6 % 16S rRNA gene sequence similarity. A third group had a nearly identical 16S rRNA gene sequence (99.9 %) compared with the recently described Faecalibaculum rodentium, also recovered from a laboratory mouse; however, this strain had a few differences in biochemical characteristics, which are detailed in an emended description. The predominant (>10 %) cellular fatty acids of strain NYU-BL-A3T were C16 : 0 and C18 : 0, and those of strain NYU-BL-A4T were C10 : 0, C16 : 0, C18 : 0 and C18 : 1ω9c. The two groups could also be distinguished by multiple biochemical reactions, with the group represented by NYU-BL-A4T being considerably more active. Based on phylogenetic, biochemical and chemotaxonomic criteria, two novel genera are proposed, Ileibacterium valens gen. nov., sp. nov. with NYU-BL-A3T (=ATCC TSD-63T=DSM 103668T) as the type strain and Dubosiella newyorkensis gen. nov., sp. nov. with NYU-BL-A4T (=ATCC TSD-64T=DSM 103457T) as the type strain.

'Candidatus Moeniiplasma glomeromycotorum', an endobacterium of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF, subphylum Glomeromycotina) are symbionts of most terrestrial plants. They commonly harbour endobacteria of a largely unknown biology, referred to as MRE (Mollicutes/mycoplasma-related endobacteria). Here, we propose to accommodate MRE in the novel genus 'Candidatus Moeniiplasma.' Phylogeny reconstructions based on the 16S rRNA gene sequences cluster 'Ca.Moeniiplasma' with representatives of the class Mollicutes, whereas phylogenies derived from amino acid sequences of 19 genes indicate that it is a discrete lineage sharing ancestry with the members of the family Mycoplasmataceae. Cells of 'Ca.Moeniiplasma' reside directly in the host cytoplasm and have not yet been cultivated. They are coccoid, ~500 nm in diameter, with an electron-dense layer outside the plasma membrane. However, the draft genomes of 'Ca.Moeniiplasma' suggest that this structure is not a Gram-positive cell wall. The evolution of 'Ca.Moeniiplasma' appears to be driven by an ultrarapid rate of mutation accumulation related to the loss of DNA repair mechanisms. Moreover, molecular evolution patterns suggest that, in addition to vertical transmission, 'Ca.Moeniiplasma' is able to transmit horizontally among distinct Glomeromycotina host lineages and exchange genes. On the basis of these unique lifestyle features, the new species 'Candidatus Moeniiplasma glomeromycotorum' is proposed.