A phylogenomic and molecular markers based taxonomic framework for members of the order Entomoplasmatales: proposal for an emended order Mycoplasmatales containing the family Spiroplasmataceae and emended family Mycoplasmataceae comprised of six genera.

The "Spiroplasma cluster" is a taxonomically heterogeneous assemblage within the phylum Tenericutes encompassing different Entomoplasmatales species as well as the genus Mycoplasma, type genus of the order Mycoplasmatales. Within this cluster, the family Entomoplasmataceae contains two non-cohesive genera Entomoplasma and Mesoplasma with their members exhibiting extensive polyphyletic branching; additionally, the genus Mycoplasma is also embedded within this family. Genome sequences are now available for all 19 Entomoplasmataceae species with validly published names, as well as 6 of the 7 species from the genus Mycoplasma. With the aim of developing a reliable phylogenetic and taxonomic framework for the family Entomoplasmataceae, exhaustive phylogenetic and comparative genomic studies were carried out on these genome sequences. Phylogenetic trees were constructed based on concatenated sequences of 121 core proteins for this cluster, 67 conserved proteins shared with the phylum Firmicutes, 40 ribosomal proteins, three major subunits of RNA polymerase (RpoA, B and C) by different means and also for the 16S rRNA gene sequences. The interspecies relationships as well as different species groups observed in these trees were identical and robustly resolved. In all of these trees, members of the genera Mesoplasma and Entomoplasma formed three and two distinct clades, respectively, which were interspersed among the members of the other genus. The observed species groupings in the phylogenetic trees are independently strongly supported by our identification of 103 novel molecular markers or synapomorphies in the forms of conserved signature indels and conserved signature proteins, which are uniquely shared by the members of different observed species clades. To account for the different observed species clades, we are proposing a division of the genus Mesoplasma into an emended genus Mesoplasma and two new genera Tullyiplasma gen. nov. and Edwardiiplasma gen. nov. Likewise, to recognize the distinct species groupings of Entomoplasma, we are proposing its division into an emended genus Entomoplasma and a new genus Williamsoniiplasma gen. nov. Lastly, to rectify the long-existing taxonomic anomaly caused by the presence of genus Mycoplasma (order Mycoplasmatales) within the Entomoplasmatales, we are proposing an emendation of the family Mycoplasmataceae to include both Entomoplasmataceae plus Mycoplasma species and an emendation of the order Mycoplasmatales, which now comprises of the emended family Mycoplasmataceae and the family Spiroplasmataceae. The taxonomic reclassifications proposed here accurately reflect the species relationships within this group of Tenericutes and they should lead to a better understanding of their biological and pathogenic characteristics.

[Intestinal Microbiota in Migrating Barn Swallows around Osaka].

Migratory birds are considered as vectors of infectious diseases, owing to their potential for transmitting pathogens over large distances. The populations of barn swallow (Hirundo rustica) migrate from Southeast Asia to the Japanese mainland during spring and migrate back to Southeast Asia during autumn. This migratory population is estimated to comprise approximately hundreds to thousands of individuals per year. However, to date, not much is known about the gastrointestinal microbiota of the barn swallow. In this study, we characterized the fecal bacterial community in barn swallow. Using 16S rRNA gene metagenomic sequencing analysis, we examined the presence and composition of potentially pathogenic bacteria in the fecal samples, which were collected during spring season from Osaka. The number (±S.D.) of total bacteria was approximately 2.1(±3.4)×108 per gram of feces. In most samples, the bacterial community composition was dominated by families, such as Enterobacteriaceae, Pseudomonadaceae, Mycoplasmataceae, Enterococcaceae, Streptococcaceae, and Alcaligenaceae. However, no relationship was found between the bacterial community composition and geographical area in the fecal samples. Potentially pathogenic bacteria were detected at the rate of >0.1%, which included Pseudomonas spp., Escherichia/Shigella spp., Enterobacter spp., Yersinia spp., Mycoplasma spp., Enterococcus spp., Achromobacter spp., and Serratia spp. Our results suggested that barn swallow is instrumental in the transmission of these genera over large distances.

Identification of a novel Hemoplasma species from pigs in Zhejiang province, China.

Hemoplasmas belong to Mycoplasmataceae (Mollicutes: Mycoplasmatales) and are able to infect a broad range of mammalian species. We investigated prevalence of hemotropic mycoplasma species in pig farms in the region of Zhejiang by a PCR scheme using universal primers targeting 16S rRNA and RNase P RNA gene (rnpB). Representative positive samples from different farms were selected for sequencing of 16S rRNA and the 219bp rnpB gene fragments for phylogenetic analysis. Sequencing analysis of PCR products from first samples identified a novel hemoplasma species present in several pig farms in the region with highest nucleotide identity of 92% to Candidatus Mycoplasma turicensis. A duplex PCR assay was then designed for differential detection of the novel hemoplasma from Mycoplasma parvum/M. suis in field samples. Of 324 blood samples from clinically healthy pigs, 26.5% was positive for this novel hemoplasma species and 50% positive for M. suis/M. parvum, indicating that the novel hemotropic mycoplasma species were of considerably high prevalence in Zhejiang province, China.

Phylogenomics of "Candidatus Hepatoplasma crinochetorum," a lineage of mollicutes associated with noninsect arthropods.

Bacterial gut communities of arthropods are highly diverse and tightly related to host feeding habits. However, our understanding of the origin and role of the symbionts is often hindered by the lack of genetic information. "Candidatus Hepatoplasma crinochetorum" is a Mollicutes symbiont found in the midgut glands of terrestrial isopods. The only available nucleotide sequence for this symbiont is a partial 16S rRNA gene sequence. Here, we present the 657,101 bp assembled genome of Candidatus Hepatoplasma crinochetorum isolated from the terrestrial isopod Armadillidium vulgare. While previous 16S rRNA gene-based analyses have provided inconclusive results regarding the phylogenetic position of Candidatus Hepatoplasma crinochetorum within Mollicutes, we performed a phylogenomic analysis of 127 Mollicutes orthologous genes which confidently branches the species as a sister group to the Hominis group of Mycoplasma. Several genome properties of Candidatus Hepatoplasma crinochetorum are also highlighted compared with other Mollicutes genomes, including adjacent tryptophan tRNA genes, which further our understanding of the evolutionary dynamics of these genes in Mollicutes, and the presence of a probably inactivated CRISPR/Cas system, which constitutes a testimony of past interactions between Candidatus Hepatoplasma crinochetorum and mobile genetic elements, despite their current lack in this streamlined genome. Overall, the availability of the complete genome sequence of Candidatus Hepatoplasma crinochetorum paves the way for further investigation of its ecology and evolution.

Role of sexually-transmitted infections in the structural and functional reorganization of the prostate.

Intracellular reorganization of secretory epitheliocytes in the main, intermediate, and periurethral prostatic glands was studied in chronic prostatitis under conditions of sexually-transmitted infections. The destructive and autophagic processes in the secretory epitheliocytes were stimulated by persistence of microorganisms, Mycoplasmataceae (mainly mycoplasmas and ureaplasmas) and Chlamydia trachomatis, in the prostatic terminal compartments, epithelial layer, and epitheliocytes. Significant intracellular reorganization of smooth-muscle cells was found: focal destruction of ultrastructures (mainly in the perinuclear zone) and lythic changes in the myofilaments (focal and diffuse).

Mycoplasmateceae species are not found in Fallopian tubes of women with tubo-peritoneal infertility.

BACKGROUND: The role of mycoplasmas on the development and sequelae of pelvic inflammatory disease remains controversial. The objective of the present study is to correlate directly the presence of Mycoplasmateceae through polimerase chain reaction (PCR) determinations in cervix and Fallopian tubes of infertile patients with tubo-peritoneal factor diagnosed through laparoscopy. METHODS: Thirty patients with tubo-peritoneal infertility and 30 normal fertile patients were included in the study; cervical samples and tubal flushings were obtained during laparoscopy. PCR determinations for the detection of genetic material of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealiticum, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis in cervix and tubal flushings were performed. RESULTS: No Mycoplasmataceae species as "only" microorganisms were found in tubal flushings of tubo-peritoneal infertility patients, whereas three (10%) fertile patients with normal tubes were positive for mycoplasma presence. This difference was not significant (p = 0.237). Among the 30 patients suffering from tubal infertility diagnosed through laparoscopy, Mycoplasmatecae species were not detected in the Fallopian tubes by PCR determinations, while in normal tubes from fertile patients these and other microorganisms could be found without distorting tubal anatomy. CONCLUSION: Mycoplasmateceae species were not detected in Fallopian tubes of women with tubo-peritoneal infertility.

Mycoplasmateceae species are not found in fallopian tubes of women with tubo-peritoneal infertility

BACKGROUND: The role of mycoplasmas on the development and sequelae of pelvic inflammatory disease remains controversial. The objective of the present study is to correlate directly the presence of Mycoplasmateceae through polimerase chain reaction (PCR) determinations in cervix and Fallopian tubes of infertile patients with tubo-peritoneal factor diagnosed through laparoscopy. METHODS: Thirty patients with tubo-peritoneal infertility and 30 normal fertile patients were included in the study; cervical samples and tubal flushings were obtained during laparoscopy. PCR determinations for the detection of genetic material of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealiticum, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis in cervix and tubal flushings were performed. RESULTS: No Mycoplasmataceae species as "only" microorganisms were found in tubal flushings of tubo-peritoneal infertility patients, whereas three (10%) fertile patients with normal tubes were positive for mycoplasma presence. This difference was not significant (p = 0.237). Among the 30 patients suffering from tubal infertility diagnosed through laparoscopy, Mycoplasmatecae species were not detected in the Fallopian tubes by PCR determinations, while in normal tubes from fertile patients these and other microorganisms could be found without distorting tubal anatomy. CONCLUSION: Mycoplasmateceae species were not detected in Fallopian tubes of women with tubo-peritoneal infertility.

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections.

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex(®) STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO™) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex(®) STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.

Prevalence of genital mycoplasmas, ureaplasmas and chlamydia in pregnancy.

The study was designed to determine the prevalence of genital mycoplasmas, ureaplasmas and Chlamydia on women attending their first prenatal visit, in conjunction with pre-term labour or HIV status. For pre-term labour (2003), 199 women were monitored for pre-term delivery (<37 weeks); for colonisation and HIV (2005), 219 women were screened. Microbial detection was performed on DNA extracted from endocervical swabs employing PCR techniques. Colonisation was seen to be highest in the 14-20 year age group from 2003. In women aged > or = 21 years, co-colonisation was 13%, although there was a shift from co-colonisation with Mycoplasma hominis and Ureaplasma urealyticum in 2003, to other dual/triple combinations in 2005. Overall, major trends from both collection periods were that the prevalence of U. urealyticum tended to be higher in women > or = 26 years, while the prevalence of Chlamydia trachomatis and M. hominis lower. No association was evident between colonisation with M. hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, U. urealyticum or C. trachomatis. The importance of genital mycoplasmas, ureaplasmas and C. trachomatis in long-term aetiologies requires further investigations, certainly in relation to syndromic management regimens that fail to reduce colonisation rates.

Azithromycin treatment for nongonococcal urethritis negative for Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum.

Some patients with nongonococcal urethritis (NGU) are negative for Chlamydia trachomatis, mycoplasmas, and ureaplasmas. The optimal antimicrobial chemotherapy for such NGU has not fully been clarified. We assessed the efficacy of azithromycin for treatment of nonmycoplasmal, nonureaplasmal, nonchlamydial NGU (NMNUNCNGU). Thirty-eight men whose first-pass urine was negative for Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were treated with a single dose of 1 g azithromycin. Urethritis symptoms and polymorphonuclear leukocytes in urethral smears or in first-pass urine were assessed before and after treatment with azithromycin. Thirty-two (84.2%) of the 38 men with NMNUNCNGU showed no signs of urethral inflammation after treatment. The efficacy of this azithromycin regimen was comparable to that of the 7-day regimen of levofloxacin, gatifloxacin, minocycline, or clarithromycin reported previously. A single dose of 1 g azithromycin, which is effective not only for NGU due to specific pathogens but also for NMNUNCNGU, is an appropriate treatment for NGU.

Host-specificity of environmentally transmitted Mycoplasma-like isopod symbionts.

Mycoplasms are known as pathogens of economic and medical interest in plants, animals and man. Here, we show a positive correlation between the presence of Mycoplasma-like symbionts in their isopod hosts and survivorship on low-quality food. Most isopods that survived feeding on a cellulose-based low-quality diet for 90 days harboured 'Candidatus Hepatoplasma' in their midgut glands, while those that died within 90 days mostly either harboured no or other bacterial symbionts. We detected 'Candidatus Hepatoplasma' in all but one of the examined species of terrestrial isopods from different habitats and locations, suggesting an evolutionarily ancient association between terrestrial isopods and their Mycoplasma-like symbionts. Phylogenetic analyses clustered symbionts from different populations of the same isopod species together, and clearly distinguished between symbionts of different isopod species, indicating host-specificity of 'Candidatus Hepatoplasma', although a previous study provided evidence for environmental symbiont transmission. Nonetheless, horizontal exchange of symbionts between species may have been possible in evolutionary earlier stages, as suggested by only limited congruency of phylogenetic trees of hosts and symbionts. Another symbiont, 'Candidatus Hepatincola porcellionum', was only detected in midgut glands of the most terrestrial tribe of isopods (Crinocheta), suggesting an evolutionarily younger host-symbiont association. This symbiont proved to be negatively correlated with host longevity, even on high-quality food.

"Candidatus hepatoplasma crinochetorum," a new, stalk-forming lineage of Mollicutes colonizing the midgut glands of a terrestrial isopod.

Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales. Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name "Candidatus Hepatoplasma crinochetorum" is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P. scaber.

Comparison of multiplex PCR assay with culture for detection of genital mycoplasmas.

Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.

[Genital mycoplasma colonization in the female genital tract].

Cervical or vaginal specimens obtained from 1,178 females at different physiologic periods of life were cultured for ureaplasma urealyticum and Mycoplasma hominis. Low recovery rates of U. urealyticum were obtained in the neonates (20/72, 27.8%) and puerperal (22/83, 26.5%) and postmenopausal (21/70, 30.0%) women. The highest incidence (114/157, 72.6%) was observed in pregnant women. It is suggested that the colonization of genital mycoplasma is associated with the hormonal status of the women.

[Evaluation of the Mycoscreen technique for the detection and quantification of genital mycoplasmas]. / Evaluación del método Mycoscreen para la detección y cuantificación de micoplasmas genitales.

A total of 161 samples from genital sources were evaluated using two different methods for genital Mycoplasma isolation: inoculation to urea broth, arginine broth and A7 solid media (standard method) and a new enzymatic method (Mycoscreen Oxoid). The isolation rates for Ureaplasma urealitycum were 42% (standard method) and 49% (Mycoscreen) (sensitivity: 100%; specificity: 88%). The isolation rates for Mycoplasma hominis were 13% (standard method) and 14% (Mycoscreen) (sensitivity: 80%; specificity: 95%). Using the enzymatic method, manufacturer's positive result is defined as a count greater than 10,000 UCC-ml at 24 hours. When we compare the 24 hours results, the concordance rates was 84% (Ureaplasma urealyticum) and 62% (Mycoplasma hominis). Mycoscreen method is a valid and fast technique for the isolation and identification of genital Mycoplasma. It also allows a fast screening of pathologic counts of Ureaplasma urealyticum in genital exudates.

Colonization with genital mycoplasmas in pregnant women and their neonates and birth weight.

The colonization by genital mycoplasmas of mothers and their newborn infants was examined in 114 pregnant women and their 84 neonates. Urine and cervical swabs were taken from the pregnant women in the last trimester, and urine from the neonates within six days after birth. Ureaplasma urealyticum was found in 73.7% of the pregnant women and in 17.9% of the neonates. Mycoplasma hominis was isolated in 8.8% of the material from the pregnant women and in 1.2% of that from the neonates. The isolation rate of U. urealyticum from newborn girls was significantly higher than that from newborn boys (p less than 0.01). There was no correlation between the germ density in the urine of the pregnant women and that of the neonates. The urine of the neonates harboured as many U. urealyticum as that of the adults. The frequency of colonization by mycoplasmas in the pregnant women or the neonates was not related to the duration of gestation or the babies' birth weight.

Mycoplasmas recovered from bovine genitalia, aborted foetuses and placentas in the Republic of South Africa.

A total of 917 Mycoplasma isolations were made from 4 092 specimens originating from 2 874 cattle in private herds and at AI stations. The percentages of isolations from the different sources were: cervico-vaginal mucus 14,6%, semen 43%, preputial wash 25%, foetuses 3,3% and placentas 15%. Mycoplasma bovigenitalium, the most common isolate, was recovered from 39% of males, 47% of females, 25% of foetuses and 11% of placentas. A wide spectrum of mycoplasmas was present, and varying combinations were common. The possible pathogenic significance of the isolates is discussed.

Urine as a specimen for diagnosis of sexually transmitted diseases.

The case of specimen collection has led to the suggestion that urine might be a useful specimen for the isolation of sexually transmitted disease agents. It would only be an appropriate specimen for agents that infect the urethra, such as Neisseria gonorrhoeae or Chlamydia trachomatis. Comparative tests have shown that culture of urine for chlamydiae (from men with urethritis) or for gonococci from women is an insensitive procedure. Gonococci can be isolated from urine from men at rates essentially equivalent to culture of urethral swabs. If specimens can be processed promptly (to avoid bactericidal effects of urine), culture of urine can likely be useful for screening asymptomatic men for gonococcal infection.