Species Of Mollicutes Research Articles

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

BackgroundSome 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.MethodsPrEP-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.ResultsThe 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.ConclusionsA 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.

Microbial Sterility Testing for Mesenchymal Progenitor Cells Derived from Human Osteoarthritis and Rheumatoid Arthritis Cartilage.

The goal is to evaluate the microbial sterility of mesenchymal progenitor cells (MPCs)-derived from osteoarthritis (OA) and rheumatoid arthritis (RA) articular cartilages. Contaminants, including bacteria and fungi in MPC cultures were initially evaluated by inoculation culture methods and then affirmed through the amplification of 16S ribosomal DNA (rDNA) and internally transcribed spacer (ITS) regions, respectively, using polymerase chain reaction (PCR). Further, the mollicutes, if any, were identified by genus-specific 16S rDNA, and the positive samples were reamplified using species-specific primers. No bacteria or fungi were found to be compromising the sterility of MPCs (n = 20) assessed by both traditional culture methods and PCR. However, two in early passages and three in later passages of MPCs had the presence of mollicutes. Further rescreening for species of mollicutes indicated the presence of Mycoplasma hyorhinis, M. salivarium, and M. arginine. The PCR methods employed in this study could be beneficial as a rapid sterility testing of cell lines.

A nested-PCR strategy for molecular diagnosis of mollicutes in uncultured biological samples from cows with vulvovaginitis

Bacteria classified in Mycoplasma (M. bovis and M. bovigenitalium) and Ureaplasma (U. diversum) genera are associated with granular vulvovaginitis that affect heifers and cows at reproductive age. The traditional means for detection and speciation of mollicutes from clinical samples have been culture and serology. However, challenges experienced with these laboratory methods have hampered assessment of their impact in pathogenesis and epidemiology in cattle worldwide. The aim of this study was to develop a PCR strategy to detect and primarily discriminate between the main species of mollicutes associated with reproductive disorders of cattle in uncultured clinical samples. In order to amplify the 16S-23S rRNA internal transcribed spacer region of the genome, a consensual and species-specific nested-PCR assay was developed to identify and discriminate between main species of mollicutes. In addition, 31 vaginal swab samples from dairy and beef affected cows were investigated. This nested-PCR strategy was successfully employed in the diagnosis of single and mixed mollicute infections of diseased cows from cattle herds from Brazil. The developed system enabled the rapid and unambiguous identification of the main mollicute species known to be associated with this cattle reproductive disorder through differential amplification of partial fragments of the ITS region of mollicute genomes. The development of rapid and sensitive tools for mollicute detection and discrimination without the need for previous cultures or sequencing of PCR products is a high priority for accurate diagnosis in animal health. Therefore, the PCR strategy described herein may be helpful for diagnosis of this class of bacteria in genital swabs submitted to veterinary diagnostic laboratories, not demanding expertise in mycoplasma culture and identification.

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

BackgroundMollicutes 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.MethodsA 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.ResultsBoth 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.ConclusionsThese 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.

Loss and gain of GroEL in the MollicutesThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

GroEL is a chaperone thought of as essential for bacterial life. However, some species of Mollicutes are missing GroEL. We use phylogenetic analysis to show that the presence of GroEL is polyphyletic among the Mollicutes, and that there is evidence for lateral gene transfer of GroEL to Mycoplasma penetrans from the Proteobacteria. Furthermore, we propose that the presence of GroEL in Mycoplasma may be required for invasion of host tissue, suggesting that GroEL may act as an adhesin-invasin.

Morphological and Sequence Analysis of Mycoplasma sp. Isolated from the Oral Cavity of a House Musk Shrew (Suncus murinus)

Mycoplasma sp. strain EDS-4 was isolated from the oral cavity of EDS line of a house musk shrew (Suncus murinus) originated from Bangladesh, and was distinguished from all previously described mollicutes. It lacks a cell wall; ferments glucose; does not produce film and spots; and does not hydrolyse arginine and urea. The strain could be distinguished from all previously described mollicutes by 16S rRNA gene sequence comparisons. The results suggest that the isolate is new species of mollicutes originated from the shrew. The strain EDS-4 has been deposited with Japan collection of Microorganisms, Bioresource Center, RIKEN in Japan (JCM15930). The 16S rRNA gene sequence of strain EDS-4 is available through the DDBJ under accession number (AB469852).

Ultrastructural findings in semen samples of infertile men infected with Chlamydia trachomatis and mycoplasmas

The aim of this study was to describe the ultrastructural features observed in semen samples of men with infertility and subfertility of unknown cause infected with Chlamydia trachomatis and mycoplasmas. The findings observed by ultrastructure included destruction or persistence of bacteria in leukocytes, phagocytosis of spermatozoa by leukocytes, and structural damage of spermatozoa.

Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes)

Minimal standards for novel species of the class Mollicutes (trivial term, mollicutes), last published in 1995, require revision. The International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Mollicutes proposes herein revised standards that reflect recent advances in molecular systematics and the species concept for prokaryotes. The mandatory requirements are: (i) deposition of the type strain into two recognized culture collections, preferably located in different countries; (ii) deposition of the 16S rRNA gene sequence into a public database, and a phylogenetic analysis of the relationships among the 16S rRNA gene sequences of the novel species and its neighbours; (iii) deposition of antiserum against the type strain into a recognized collection; (iv) demonstration, by using the combination of 16S rRNA gene sequence analyses, serological analyses and supplementary phenotypic data, that the type strain differs significantly from all previously named species; and (v) assignment to an order, a family and a genus in the class, with an appropriate specific epithet. The 16S rRNA gene sequence provides the primary basis for assignment to hierarchical rank, and may also constitute evidence of species novelty, but serological and supplementary phenotypic data must be presented to substantiate this. Serological methods have been documented to be congruent with DNA-DNA hybridization data and with 16S rRNA gene placements. The novel species must be tested serologically to the greatest extent that the investigators deem feasible against all neighbouring species whose 16S rRNA gene sequences show >0.94 similarity. The investigator is responsible for justifying which characters are most meaningful for assignment to the part of the mollicute phylogenetic tree in which a novel species is located, and for providing the means by which novel species can be identified by other investigators. The publication of the description should appear in a journal having wide circulation. If the journal is not the International Journal of Systematic and Evolutionary Microbiology, copies of the publication must be submitted to that journal so that the name may be considered for inclusion in a Validation List as required by the International Code of Bacteriological Nomenclature (the Bacteriological Code). Updated informal descriptions of the class Mollicutes and some of its constituent higher taxa are available as supplementary material in IJSEM Online.

Evolution and devolution of minimal standards for descriptions of species of the class Mollicutes: analysis of two Spiroplasma descriptions

Evolution and devolution of minimal standards for descriptions of species of the class Mollicutes: analysis of two Spiroplasma descriptions

Distinctive Repertoire of Contingency Genes Conferring Mutation- Based Phase Variation and Combinatorial Expression of Surface Lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides Phylogenetic Cluster

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.

Specific gene targeting in Spiroplasma citri: improved vectors and production of unmarked mutations using site-specific recombination

In Spiroplasma citri, where homologous recombination is inefficient, specific gene targeting could only be achieved by using replicative, oriC plasmids. To improve the probability of selecting rare recombination events without fastidious, extensive passaging of the transformants, a new targeting vector was constructed, which was used to inactivate the crr gene encoding the IIA component of the glucose phosphotransferase system (PTS) permease. Selection of recombinants was based on a two-step strategy using two distinct selection markers, one of which could only be expressed once recombination had occurred through one single crossover at the target gene. According to this strategy, spiroplasmal transformants were screened and multiplied in the presence of gentamicin before the crr recombinants were selected for their resistance to tetracycline. In contrast to the wild-type strain GII-3, the crr-disrupted mutant GII3-gt1 used neither glucose nor trehalose, indicating that in S. citri the glucose and trehalose PTS permeases function with a single IIA component. In addition, the feasibility of using the transposon gammadelta TnpR/res recombination system to produce unmarked mutations in S. citri was demonstrated. In an arginine deiminase (arcA-disrupted) mutant, the tetM gene flanked by the res sequences was efficiently excised from the chromosome through expression of the TnpR resolvase from a replicative oriC plasmid. Due to oriC incompatibility, plasmid loss occurred spontaneously when selection pressure was removed. This approach will be helpful for constructing unmarked mutations and generating multiple mutants with the same selection marker in S. citri. It should also be relevant to other species of mollicutes.

Mollicutes

Mollicutes

Detection and differentiation of ruminant mycoplasmas.

More than 20 different species of mollicutes, most of them belonging to the genus Mycoplasma, have been identified from ruminant hosts to date. While a considerable part of this group is conceived to be of minor epidemiological relevance, it contains some important pathogenic agents that have specific host ranges. Due to several peculiar properties of mycoplasmas, which include the absence of a cell wall and the capability of surface antigen variation, diseases caused by mycoplasmas are difficult to control in the conventional fashion by chemotherapy or immunoprophylaxis. Another general feature of mycoplasma infections is their protracted and occasionally chronic course. Five mycoplasmas of economic and welfare importance are mentioned here and serve as examples for the variety of clinical and diagnostic circumstances that can be resolved using polymerase chain reaction (PCR).

Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family

Dermaseptins are a family of cationic (Lys-rich) antimicrobial peptides that are abundant in the skin secretions of the arboreal frogs Phyllomedusa bicolor and P. sauvagii. In vitro, these peptides are microbicidal against a wide variety of microorganisms including Gram-positive and Gram-negative bacteria, yeasts, protozoa and fungi. To date, 6 dermaseptin B mature peptides, 24–34 residues long, 2 dermaseptin B cDNAs and 2 gene sequences have been identified in P. bicolor. To assess dermaseptin related genes further, we screened a P. bicolor genomic library with 32P -labeled cDNAs coding either for prepro-dermaseptins B1 or B2 (adenoregulin). A gene sequence was identified that coded a novel dermaseptin B, termed Drg3, which exhibits 23–42% amino acids identities with other members of the family. Analysis of the cDNAs coding precursors for several opioid and antimicrobial peptides originating from the skin of various amphibian species revealed that the 25-residue preproregion of these preproforms are all encoded by conserved nucleotides encompassed by the first coding exon of the Drg3 gene. Synthetic dermaseptin Drg3 exhibited a bactericidal activity towards several species of mollicutes (wall-less eubacteria), firmicutes (Gram-positive eubacteria), and gracilicutes (Gram-negative eubacteria), with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 μM. Experiments performed on Acholeplasma laidlawii cells revealed that this peptide is membranotropic and that it efficiently depolarizes the plasma membrane.

Identification of mycoplasmas by immunofluorescence.

Immunofluorescence has been used as a diagnostic tool for identification of mycoplasmas cultured on artificial medium and also for detection of the organisms in situ in infected hosts and for detection of contaminated cell cultures. The technique has been used in research investigations to locate mycoplasmas in pathogenicity studies in both animals and in organ cultures, and it is also one of the recommended serological tests for the characterization of new species of Mollicutes (). Another diagnostic application of immunofluorescence is the detection of mycoplasma antibodies (), but the topic falls outside the scope of this chapter.

Effect of natural amphipathic peptides on viability, membrane potential, cell shape and motility of mollicutes

The antibiotic activity of ten amphipathic peptides was investigated in six species of mollicutes belonging to the genera Acholeplasma, Mycoplasma and Spiroplasma. A. laidlawii was the most sensitive and M. mycoides subsp. mycoides SC the most resistant. Animal defence peptides (cecropins A and P1, and magainin 2) proved to be less potent than bee-venom mellitin and most of the peptides produced by bacteria (globomycin, gramicidin S, surfactin and valinomycin) or fungi (alamethicin). Gramicidin S was by far the most active peptide, with minimal inhibitory concentrations ranging from 2 to 50 nM. Alamethicin, gramicidin S, mellitin and surfactin had a cidal effect, whilst cecropins, globomycin, magainin 2, polymyxin B and valinomycin proved to be static. The peptides altered the membrane potential of spiroplasma cells with a potency independent of their linear or cyclic structure. However, globomycin depolarized the plasma membrane only weakly, whilst polymyxin B, in order to be active, required prior hyperpolarization of the membrane. The peptides also induced the loss of cell motility and helicity in spiroplasmas, suggesting that motility and cell shape in these bacteria are coupled to the transmembrane electrochemical gradient. Globomycin, an inhibitor of signal-peptidase II, prevented the growth of spiroplasmas, M. gallisepticum, and M. genitalium, but not that of A. laidlawii and M. mycoides subsp. mycoides SC, although the latter also synthesized membrane lipoproteins. Inhibition of spiralin processing by globomycin was demonstrated in S. citri and S. melliferum, with a more pronounced effect in the second species.

Revised Minimum Standards for Description of New Species of the Class Mollicutes (Division Tenericutes)

In this paper the Subcommittee on the Taxonomy of Mollicutes proposes minimum standards for descriptions of new cultivable species of the class Mollicutes (trivial term, mollicutes) to replace the proposals published in 1972 and 1979. The major class characteristics of these organisms are the lack of a cell wall, the tendency to form fried-egg-type colonies on solid media, the passage of cells through 450- and 220-nm-pore-size membrane filters, the presence of small A-T-rich genomes, and the failure of the wall-less strains to revert to walled bacteria under appropriate conditions. Placement in orders, families, and genera is based on morphology, host origin, optimum growth temperature, and cultural and biochemical properties. Demonstration that an organism differs from previously described species requires a detailed serological analysis and further definition of some cultural and biochemical characteristics. The precautions that need to be taken in the application of these tests are defined. The subcommittee recommends the following basic requirements, most of which are derived from the International Code of Nomenclature of Bacteria, for naming a new species: (i) designation of a type strain; (ii) assignment to an order, a family, and a genus in the class, with selection of an appropriate specific epithet; (iii) demonstration that the type strain and related strains differ significantly from members of all previously named species; and (iv) deposition of the type strain in a recognized culture collection, such as the American Type Culture Collection or the National Collection of Type Cultures. The publication of the description should appear in a journal having wide circulation. If the journal is not the International Journal of Systematic Bacteriology, a reprint must be submitted to that journal so that the name may be considered for inclusion in a validation list as required by the International Code of Nomenclature of Bacteria.

Current status of the mollicute flora of humans.

Of the more than 20 species of mollicutes reported to be present in human tissues, 15 have been isolated more than once and are currently thought to typify mollicutes of human origin. In the past decade a number of new and potentially significant mollicutes have been added to the list of species inhabiting humans. As our understanding of the human mollicute flora increases, diagnostic and clinical advances should permit the identification and control of those species that are significant pathogens in human disease.

Development of a PCR-based method for diagnosing Mycoplasma pneumoniae infections.

The polymerase chain reaction was used to detect clinical samples of Mycoplasma pneumoniae. A 245-bp region of the cytoadhesin P1 gene was shown to be specifically amplified in Myc. pneumoniae, but not in other species of Mollicutes. Picogram amounts of Myc. pneumoniae. DNA could be detected per ml blood serum by use of a simple and reliable protocol for sample preparation and a PCR reaction involving two rounds of amplification. Application of the PCR-based method for the detection of Myc. pneumoniae in serum samples and throat swabs from patients with atypical pneumonia showed that it could be used in clinical diagnosis.

Identification and characterization of proteinase K-resistant proteins in members of the class Mollicutes

Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.