Prevalence of Mollicutes among men who have sex with men and transgender women aged 15 to 19 years in Salvador, North-eastern Brazil.

BACKGROUND: Some species of Mollicutes have been associated with different pathologies of the urogenital tract in humans, with a high prevalence among adult men who have sex with men (MSM) and transgender women (TGW). However, few studies have been performed to investigate its prevalence among adolescents. In this study, we estimated the initial prevalence of Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP); the rate of misdiagnosis at different anatomical sites; and the associated factors with positive tests for Mollicutes among MSM and TGW aged 15 to 19 years enrolled in the PrEP1519 study. METHODS: PrEP-1519 is the first study to investigate the effectiveness of pre-exposure prophylaxis for human immunodeficiency virus among adolescent MSM and TGW aged 15 to 19 in Latin America. Oral, anal, and urethral swabs were taken from 246 adolescents upon enrolment in the study to detect MG, MH, UU, and UP by quantitative polymerase chain reaction (qPCR). Bivariate and multivariate analyses were conducted by Poisson regression and 95% confidence intervals (95% CI) were estimated. RESULTS: The prevalence of Mollicutes was 32.1%. UU was the most prevalent species (20.7%), followed by MH (13.4%), MG (5.7%), and UP (3.2%); 67.3% of the positive samples would have been missed if only urethral samples had been taken. Receptive anal sex (prevalence ratio [PR] = 1.79; 95% CI = 1.07-3.01) and clinical suspicion of sexually transmitted infection (PR = 1.62; 95% CI = 1.01-2.61) were factors associated with the detection of Mollicutes in general. Group sex (PR = 1.98; 95% CI = 1.12-3.50) and receptive anal sex (PR = 2.36; 95% CI = 0.95-5.86) were associated with the detection of Mycoplasma spp. No sociodemographic, clinical, or behavioural variable was significantly associated with the detection of Ureaplasma spp. CONCLUSIONS: A high prevalence of Mollicutes was observed among adolescent MSM and TGW, especially at extragenital sites. Further research is required to understand the epidemiological profile of high-risk adolescents in different regions and contexts, and to investigate the pathogenesis of Mollicutes in the oral and anal mucosa before routine screening can be recommended in clinical practice.

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.

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.

Prevalence and antibiotic resistance of rectal Mollicutes in HIV-infected men who have sex with men at the University Hospital of Dresden, Germany.

BACKGROUND: Rectal sexually transmitted infections (STI) are common in men having sex with men (MSM). Mycoplasma genitalium is increasingly being reported in this localization, but due to frequent lack of symptoms at this site, clinical significance is still unclear. Rectal prevalence of Mycoplasma hominis and Ureaplasma species is not well studied so far. We aimed to investigate the prevalence and antibiotic sensitivity of rectal Mollicutes in our HIV-cohort. METHODS: In 227 MSM presenting for annual STI-screening, 317 anorectal swabs were collected from January 2017 to December 2018. PCR was performed for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, M. genitalium and also culture for M. hominis and Ureaplasma spec. RESULTS: Prevalence for M. genitalium, M. hominis, Ureaplasma spec., C. trachomatis and N. gonorrhoeae was 8.2%, 7.3%, 12.0%, 5.1% and 1.9%, respectively. Patients were asymptomatic with few exceptions. Seroprevalence of syphilis in 227 MSM was 41.9%. In 20 strains of M. genitalium, resistance-associated mutations to macrolides and quinolones were found in 60% and 30%, respectively; in five strains (25%) to both. M. hominis and Ureaplasma spec. frequently occurred combined, mostly in significant quantity consistent with infection. M. hominis and Ureaplasma spec. regularly showed sensitivity to tetracycline. CONCLUSION: At screening, rectal colonization with Mollicutes was common in our patients, but rarely caused symptoms. Due to rising antibiotic resistance of M. genitalium against quinolones, therapeutic options are increasingly limited. Treatment should be guided by antibiotic resistance testing including quinolones. In persisting anorectal symptoms, M. hominis and Ureaplasma spec. should also be taken into account.

Mycoplasma bovis and other Mollicutes in replacement dairy heifers from Mycoplasma bovis-infected and uninfected herds: A 2-year longitudinal study.

Replacement dairy heifers exposed to Mycoplasma bovis as calves may be at risk of future clinical disease and pathogen transmission, both within and between herds; however, little information is available about these risks. We conducted a 2-yr longitudinal (panel) study starting with 450 heifer calves reared to weaning in 8 herds (7 M. bovis infected with clinical disease, 1 uninfected) under the same ownership. After weaning, heifers were commingled and managed with non-study heifers at a single heifer rearing facility. Nose, conjunctival, and vaginal swabs were collected along with a blood sample at weaning, prebreeding, precalving, and approximately 1 mo postcalving. Additionally, a colostrum sample was collected upon calving and a composite milk sample was collected 1 mo postcalving. The swabs, colostrum, and milk samples were cultured for Mycoplasma spp., and serum from the blood was evaluated for serological evidence of exposure to M. bovis using an ELISA. Despite a high M. bovis ELISA seroprevalence at weaning in the heifers from the 7 M. bovis-infected herds with clinical disease [72% (289/400); range by herd: 28-98%], M. bovis was isolated from only 4% (16/400) of the same heifers at the same time. In heifers from the uninfected herd at weaning, M. bovis seroprevalence was 2% (1/50) and M. bovis was not detected by culture. Mycoplasma bovis was isolated from 0.5% (2/414) of heifers at prebreeding, 0% (0/374) of heifers at precalving, and 0.3% (1/356) of heifers 1 mo postcalving. The nose was the predominant anatomical site of M. bovis colonization (74%; 14/19 culture positives). A single heifer (from an M. bovis-infected herd with clinical disease) was repeatedly detected with M. bovis in its nose at weaning, prebreeding, and postcalving samplings. This demonstrates the possibility, albeit rare, of a long-term M. bovis carrier state in replacement heifers exposed to M. bovis as calves, up to at least 1 mo after entry into the milking herd. No M. bovis clinical disease was detected in any heifer from weaning through to the end of the study (approximately 1 mo after calving). Acholeplasma spp. were commonly isolated throughout the study. Mycoplasma bovigenitalium, Mycoplasma bovoculi, and Mycoplasma bovirhinis were isolated infrequently. Mycoplasma bovis seroprevalences at prebreeding, precalving, and postcalving samplings were 27% (112/414), 12% (46/374), and 18% (65/356), respectively. Overall, the results show that replacement heifers from groups exposed to M. bovis preweaning can become colonized with M. bovis and that colonization can, uncommonly, be present after their first calving. For groups of 50 or more heifers exposed to M. bovis preweaning, there is at least a nontrivial probability that the group will contain at least 1 shedding heifer postcalving.

Distribution of genital Mollicutes in the vaginal ecosystem of women with different clinical conditions.

Ureaplasma urealyticum (UU), Ureaplasma parvum (UP), Mycoplasma hominis (MH) and Mycoplasma genitalium (MG) are the most common Mollicutes of the female genital tract. Although many studies have addressed their possible role in the vaginal ecosystem, many aspects remain to be elucidated. The aim of this study was to evaluate the vaginal presence of ureaplasmas/mycoplasmas in women with different clinical conditions. By means of quantitative PCR assays, the prevalence and load of each Mollicute were assessed in different groups of pre-menopausal women: 'healthy' (n=29), women with bacterial vaginosis (BV) (n=21), patients with Chlamydia trachomatis (CT) infection (n=25) and subjects with vulvo-vaginal candidiasis (VVC) (n=23). Globally, UP was the most prevalent Mollicutes in the vagina (67.3%), followed by MH (14.3%), UU (9.2%) and MG (3.1%). The presence of UU and UP was almost never associated. MH showed a significantly higher prevalence and higher bacterial loads in BV-positive women (P<0.05), whereas patients with CT and VVC were characterized by a Mollicutes pattern similar to healthy women. Mollicutes can be frequently found in the vaginal ecosystem, even in asymptomatic 'healthy' women. Although its presence is not a strict requirement, MH displays a significant role in the pathogenesis of BV.

Screening for Mollicutes microorganisms in perinatal calf mortality cases in Polish dairy herds.

Perinatal calf mortality in dairy herds has been reported worldwide. The etiology of stillbirth is multifactorial, and can be caused by various species of bacteria and environmental factors. Among them some potential pathogens from the Mollicutes class such as Mycoplasma (M.) spp. and Ureaplasma (U.) diversum can be isolated from the bovine genital tract and other organs of the suspected cattle. The aim of this study was to evaluate if the bacteria belonging to the Molli- cutes class i.e. M. bovis, M. bovigenitalium, M. canadense, M. canis, M. arginini, M. bovirhinis, M. dispar, M. alkalescens and U. diversum could have an impact on perinatal calf mortality in selected Polish dairy farms. The material was: 121 stillborn calves (SB), 21 live born calves (C) and 131 cows (dams) from 30 Polish Holstein-Friesian herds. Samples were examined from all the SB calves' and six control euthanized calves' abomasal contents and lung samples collected during necropsy, and from the dams' serum and placenta. In dams the serological ELISA, and in calves and placenta samples molecular PCR/denaturing gradient gel electrophoresis, methods were used. Screening of dams' sera for antibodies to M. bovis (ELISA) showed seven dams positive for M. bovis, whereas none of the nine examined Mollicutes microorganisms were detected in the placenta and calves.

'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.

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.

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.

Factors influencing ruminal bacterial community diversity and composition and microbial fibrolytic enzyme abundance in lactating dairy cows with a focus on the role of active dry yeast.

The objective of the current study was to employ a DNA-based sequencing technology to study the effect of active dry yeast (ADY) supplementation, diet type, and sample location within the rumen on rumen bacterial community diversity and composition, and to use an RNA-based method to study the effect of ADY supplementation on rumen microbial metabolism during high-grain feeding (HG). Our previous report demonstrated that the supplementation of lactating dairy cows with ADY attenuated the effect of subacute ruminal acidosis. Therefore, we used samples from that study, where 16 multiparous, rumen-cannulated lactating Holstein cows were randomly assigned to 1 of 2 dietary treatments: ADY (Saccharomyces cerevisiae strain Y1242, 80 billion cfu/animal per day) or control (carrier only). Cows received a high-forage diet (77:23, forage:concentrate), then were abruptly switched to HG (49:51, forage:concentrate). Rumen bacterial community diversity and structure were highly influenced by diet and sampling location (fluid, solids, epimural). The transition to HG reduced bacterial diversity, but epimural bacteria maintained a greater diversity than fluid and solids. Analysis of molecular variance indicated a significant separation due to diet × sampling location, but not due to treatment. Across all samples, the analysis yielded 6,254 nonsingleton operational taxonomic units (OTU), which were classified into several phyla: mainly Firmicutes, Bacteroidetes, Fibrobacteres, Tenericutes, and Proteobacteria. High forage and solids were dominated by OTU from Fibrobacter, whereas HG and fluid were dominated by OTU from Prevotella. Epimural samples, however, were dominated in part by Campylobacter. Active dry yeast had no effect on bacterial community diversity or structure. The phylum SR1 was more abundant in all ADY samples regardless of diet or sampling location. Furthermore, on HG, OTU2 and OTU3 (both classified into Fibrobacter succinogenes) were more abundant with ADY in fluid and solids than control samples. This increase with ADY was paralleled by a reduction in prominent Prevotella OTU. Metatranscriptomic profiling of rumen microbiome conducted on random samples from the HG phase showed that ADY increased the abundance of the cellulase endo-ß-1,4-glucanase and had a tendency to increase the hemicellulase α-glucuronidase. In conclusion, the shift from high forage to HG and sampling location had a more significant influence on ruminal bacterial community abundance and structure compared with ADY. However, evidence suggested that ADY can increase the abundance of some dominant anaerobic OTU belonging to F. succinogenes and phylum SR1. Further, microbial mRNA-based evidence suggested that ADY can increase the abundance of a specific microbial fibrolytic enzymes.

The short-chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis.

The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene (Ffar2) expression is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational ß-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of ß-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Together, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.

Frequency of different human mollicutes species in the mucosa of the oropharynx, conjunctiva, and genitalia of free-ranging and captive capuchin monkeys (Cebus spp.).

This study is the first to evaluate the occurrence of several Mollicutes species in Brazilian capuchin monkeys (Cebus spp.). Mollicutes were detected by culture and polymerase chain reaction (PCR) in samples of the oropharyngeal, conjuctiva, and genital mucosae of 58 monkeys. In the oropharynx, Mollicutes in general (generic PCR to the Class), and those of the genus Ureaplasma (genus PCR), were detected in 72.4% and 43.0% of the samples, respectively. The identified species in this site included: Mycoplasma arginini (43.1%), M. salivarium (41.4%), and M. pneumoniae (19.0%). Both Ureaplasma and Mycoplasma are genera of the order Mycoplasmatales. In the preputial/vaginal mucosa, PCR detected Mollicutes in general in 27.58% of the samples, the genus Ureaplasma in 32.7%, the species M. arginini in 8.6%, and Acholeplasma laidlawii of the order Acholeplasmatales in 1.7% In the conjunctiva, Mollicutes in general were detected in 29.3% of the samples, with 1.7% being identified as A. laidlawii. Culturing was difficult due to contamination, but two isolates were successfully obtained. The Mollicutes species of this study provided new insights into these bacteria in Brazilian Cebus. Studies are lacking of the actual risk of Mollicutes infection or the frequency at which primates serve as permanent or temporary reservoirs for Mollicutes. In the present study, the samples were collected from monkeys without clinical signs of infection. The mere presence of Mollicutes, particularly those also found in humans, nevertheless signals a need for studies to evaluate the impact of these microorganisms on the health of non-human primates (NHPs) and the possibility of cross-species transmission between NHPs and humans.

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines.

BACKGROUND AIMS: The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. METHODS: We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. RESULTS: Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. CONCLUSIONS: We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

Mycoplasma hominis in Cuban Trichomonas vaginalis isolates: association with parasite genetic polymorphism.

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival.

Army ants harbor a host-specific clade of Entomoplasmatales bacteria.

In this article, we describe the distributions of Entomoplasmatales bacteria across the ants, identifying a novel lineage of gut bacteria that is unique to the army ants. While our findings indicate that the Entomoplasmatales are not essential for growth or development, molecular analyses suggest that this relationship is host specific and potentially ancient. The documented trends add to a growing body of literature that hints at a diversity of undiscovered associations between ants and bacterial symbionts.

Detection of Mollicutes in bioreactor samples by real-time transcription-mediated amplification.

AIM: Contamination by Mollicutes is a significant challenge for research laboratories and biopharmaceutical industry. It leads to alteration of results or production quality as well as loss of time, materials and revenue. These organisms can czoriginate from mammalian, avian, insect, plant or fish cells. Culture-based methods may require 28 days to detect Mollicutes. Traditional microbiology could advantageously be replaced by nucleic acid testing for earlier detection. METHODS AND RESULTS: A membrane filtration-based concentration of the Mollicutes has been coupled to real-time transcription-mediated amplification (real-time TMA) to demonstrate these advantages. The eight species required by European Pharmacopoeia have been tested and were detected with sensitivity below 100 CFU per 20-ml sample. Co-culture experiments, in which Mollicutes are grown with CHO-S (suspension) or HEK 293 (adherent) cells, were also performed to respectively mimic a bioreactor or flask contamination. Despite the fact that Mollicutes can attach to or invade mammalian cells, they were consistently detected over multiple days. CONCLUSIONS: the sample preparation and amplification method used in this study increases sensitivity and reduces time-to-result for detection of Mollicutes. SIGNIFICANCE AND IMPACT OF THE STUDY: the described system allows real-time monitoring for microbial contamination of cell-based processes and products for the biopharmaceutical industry.

Mollicutes species and Mycoplasma bovis prevalence and association with health outcomes in beef feeder calves at arrival and initial treatment for bovine respiratory disease.

Mollicutes nasal swab culture status and potential associations with health outcomes were determined in beef feeder calves. Mollicutes culture was positive in 7.6% (22/291) of calves at arrival and in 26.2% (34/130) of calves at first disease treatment. Positive Mollicutes culture at first treatment was associated with increased odds for subsequent retreatment or death.