<Emphasis Type="Italic">Ureaplasma urealyticum</Emphasis> and <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> Sensitivity to Bacteriocins Produced by Two Lactobacilli Strains

The purpose of the present study was to determine the inhibitory activities of two bacteriocins, produced by lactobacilli, against genital mycoplasmas. In this study, infections produced by genital mycoplasmas were studied; of these, 1.3% were caused by Mycoplasma hominis, 10.7% by Ureaplasma urealyticum and 5.6% by U. urealyticum + M. hominis. U. urealyticum was isolated from 75 out of 123 patients with genital mycoplasmas, while M. hominis was isolated from 9 patients (7.3%) and both U. urealyticum and M. hominis from 39 patients (31.7%). Bacteriocins, L23 and L60, produced by Lactobacillus fermentum and L. rhamnosus, respectively, appear to be two novel inhibitors of bacterial infection with potential antibacterial activity. Both bacteriocins proved to be active against 100% of strains tested; MICs of bacteriocin L23 ranged between 320 and 160 UA ml⁻¹ for 78% of the M. hominis strains and between 320 and 80 UA ml⁻¹ for 95% of the U. urealyticum strains. In addition, bacteriocin L60 was still active at 160 UA ml⁻¹ for a high percentage (56%) of M. hominis strains, and at 80 UA ml⁻¹ for 53% of the U. urealyticum strains. Interestingly, these antimicrobial substances produced by lactobacilli showed an inhibitory activity against genital mycoplasmas even when diluted. Altogether, our study indicates that the bacteriocins, L23 and L60, are good candidates for the treatment or prevention of genital infections in women.     

Attachment of Mycoplasma hominis and M. orale to Human Diploid Lung Fibroblasts

The process of attachment of Mycoplasma hominis and M. orale to HAIN-55 cells, derived from normal embryonic human lung, was investigated quantitatively. The attachment reached its maximum within about 2–4 hr at 37 C and increased linearly as a function of the number of organisms present in the system. The relative attachment efficiency of M. hominis was approximately 1% under our experimental conditions. Trypsin and EDTA were effective in detaching particles of M. hominis and M. orale from the surfaces of HAIN-55 cells. Therefore it was suggested that some proteinaceous substance and salt bridges might be involved in the attachment of these mycoplasmas to HAIN-55 cells.     

Life on Arginine for Mycoplasma hominis: Clues from Its Minimal Genome and Comparison with Other Human Urogenital Mycoplasmas

Mycoplasma hominis is an opportunistic human mycoplasma. Two other pathogenic human species, M. genitalium and Ureaplasma parvum, reside within the same natural niche as M. hominis: the urogenital tract. These three species have overlapping, but distinct, pathogenic roles. They have minimal genomes and, thus, reduced metabolic capabilities characterized by distinct energy-generating pathways. Analysis of the M. hominis PG21 genome sequence revealed that it is the second smallest genome among self-replicating free living organisms (665,445 bp, 537 coding sequences (CDSs)). Five clusters of genes were predicted to have undergone horizontal gene transfer (HGT) between M. hominis and the phylogenetically distant U. parvum species. We reconstructed M. hominis metabolic pathways from the predicted genes, with particular emphasis on energy-generating pathways. The Embden–Meyerhoff–Parnas pathway was incomplete, with a single enzyme absent. We identified the three proteins constituting the arginine dihydrolase pathway. This pathway was found essential to promote growth in vivo. The predicted presence of dimethylarginine dimethylaminohydrolase suggested that arginine catabolism is more complex than initially described. This enzyme may have been acquired by HGT from non-mollicute bacteria. Comparison of the three minimal mollicute genomes showed that 247 CDSs were common to all three genomes, whereas 220 CDSs were specific to M. hominis, 172 CDSs were specific to M. genitalium, and 280 CDSs were specific to U. parvum. Within these species-specific genes, two major sets of genes could be identified: one including genes involved in various energy-generating pathways, depending on the energy source used (glucose, urea, or arginine) and another involved in cytadherence and virulence. Therefore, a minimal mycoplasma cell, not including cytadherence and virulence-related genes, could be envisaged containing a core genome (247 genes), plus a set of genes required for providing energy. For M. hominis, this set would include 247+9 genes, resulting in a theoretical minimal genome of 256 genes.     

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell’s interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.     

Comparison of Ciliostasis by Mycoplasmas in Mouse and Chicken Tracheal Organ Cultures

The cilia-stopping effect of mycoplasmas of human and various animal origin in mouse and chicken tracheal organ cultures was studied. From the results in mouse tracheal organ cultures, the mycoplasma strains tested were divided into three groups: Mycoplasma pulmonis m53, M. pulmonis JB, M. pulmonis OK, M. mycoides subsp. mycoides PG1 and M. gallisepticum S6 showed a strong cilia-stopping effect; M. pulmonis PG22, M. mycoides subsp. capri PG3, M. meleagridis 19729, M. neurolyticum Type A and M. arthritidis PG6 showed a mild effect; and M. pneumoniae FH, M. salivarium Hup, M. hominis type 1-C and M. orale N-C of human origin and Acholeplasma laidlawii PG8 showed a weak effect. On the other hand, in chicken tracheal organ cultures, only M. gallisepticum S6 showed a strong effect, M. meleagridis 19729 was affected to a lesser degree, and other mycoplasma strains showed a weak or no effect. The results indicate that some murine and poultry mycoplasmas showed a cilia-stopping tendency in mouse and chicken tracheal organ cultures, respectively, while human mycoplasmas showed weak or little effect in both organ cultures. In mouse tracheal organ cultures, M. pulmonis m53 treated with heat, trypsin or formaldehyde, and the sterile filtrate of an m53 broth culture showed no cilia-stopping effect. The relationship of the pathogenicity of mycoplasmas for their natural hosts to that for cultured respiratory cells is discussed.     

Development of real-time PCR for detection of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>

Background  

Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target.     

Localization of division protein FtsZ in Mycoplasma hominis

The localization of FtsZ protein in M. hominis cells was studied by immunoelectron microscopy with polyclonal antibodies to this protein. Cell polymorphism typical for mycoplasmas was seen on electron microscopic pictures. Among the diversity of cell shapes, we distinguished dumbbell-shaped dividing cells and cells connected with each other by membrane tubules (former constrictions). The label was predominantly observed in the constriction area of dividing M. hominis cells and on thin membrane tubules. A septum and the gold labeling of this structure have not been described before in mycoplasma cells. For the first time, in some rounded and oval cells, colloidal gold particles labeled the entire plasma membrane, probably marking a submembranous contractile ring (Z ring). These observations confirm the implication of FtsZ protein in M. hominis cytokinesis. In some cells, the spiral-like distribution of gold particles was observed. Most likely, FtsZ protofilaments in M. hominis cells form spiral structures similar to Z spirals in Bacillus subtilis and Escherichia coli. Their presence in mycoplasma cells may be considered to be an important argument in favor of Z ring assembly through the reorganization of Z spirals. FtsZ as a bacterial cytoskeleton protein binding with membrane directly or through intermediates may be engaged in maintenance of M. hominis cell shape.     

Experimental Infections with Mycoplasma agalactiae Identify Key Factors Involved in Host-Colonization

Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.     

Proteomic characterization of <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis> nanoforming

The goal of this work was to create a model for the long persistence of Mycoplasma gallisepticum in depleted medium and under low growth temperature followed by proteomic study of the model. Nanoforms and revertants for M. gallisepticum were obtained. Proteomic maps were produced for different stages of the formation of nanoforms and revertants. It is shown that proteins responsible for essential cellular processes of glycolysis, translation elongation, and DnaK chaperone involved in the stabilization of newly synthesized proteins are crucial for the reversion of M. gallisepticum to a vegetative form. Based on the current data, it is assumed that changes in the metabolism of M. gallisepticum during nanoforming are not post-mortal, thus M. gallisepticum does not transform to uncultivable form, but remains in a reversible dormant state during prolonged unfavorable conditions.     

Adaptation of <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis> to unfavorable growth conditions: Changes in morphological and physiological characteristics

Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions results in altered morphological and physiological characteristics of the cells. M. gallisepticum populations in a complete nutrient medium contain pear-shaped vegetative cells ( d ～ 0.3 μm; l ～ 0.8 μm) with pronounced polar and cytoskeletonlike structures. Such mycoplasma cells are able to induce damage in a bacterial genome, causing an SOS response of the test strain (Escherichia coli PQ37). In a starvation medium, M. gallisepticum produces nanoforms, small coccoid cells (d ～ 0.15–0.2 μm) without either polar or cytoskeleton-like structures. Unlike vegetative cells, nanoforms do not induce genome damage. Alleviation of unfavorable growth conditions results in a reversion of nanoforms to vegetative cells.     

Development of Flouroquinolone (Ciprofloxacin) Resistance in Mycoplasma hominis in the Presence of HeLa Cells

The effect of cocultivation of eukaryotic HeLa cells and Mycoplasma hominis mycoplasma on the resistance of the latter to fluoroquinolones (ciprofloxacin) was examined. It was shown that cocultivation of the M. homonisand HeLa cells during 24 h with subsequent addition of ciprofloxacin resulted in an increase of the micoplasma resistance to this antimicrobial agent. In the M. hominis cells cultivated in the presence of HeLa cells and the increasing concentration of ciprofloxacin mutations in the parC gene were observed only at low concentrations of the antimicrobial agent, while mutations in the gyrA gene were never detected. A gradual elevation of ciprofloxacin concentration up to 10 g/ml resulted in the reversion of the parC mutations in mycoplasmas. Mycoplasma cells resistant to high flouroquinolone concentrations and isolated after cocultivation with the HeLa cells were characterized by the wild-type genotype in respect of the gyrA and parC genes. It was shown that infection of HeLa cells resulted in the appearance of genome rearrangements in M. hominis cells.     

Mycoplasma adaptation to stress factors: Nucleotide sequences absent in vegetative forms of <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis> S6 cells are found in nonculturable forms

The adaptation of Mycoplasma gallisepticum S6 to adverse environmental conditions is associated with the transformation of vegetative cell forms to viable but nonculturable (VBNC) forms. The vegetative and VBNC forms proved to differ in the spectrum of PCR products amplified from pvpA-gene, which codes for the phase-variable cytoadhesion protein. The vegetative forms displayed only one amplicon, which contained one open reading frame (1086 bp) with a high homology (97%) to pvpA-gene of M. gallisepticum R and Pendik. The VBNC forms of M. gallisepticum S6 had additional amplicons, whose open reading frames were absent from the complete database sequence of the mycoplasma genome. A high nucleotide sequence homology (54–55%) between pvpA-gene and the additional pvpA-gene amplicons made it possible to suggest that pvpA-gene provided a source for the formation of new regions within the mycoplasma genome during adaptation to adverse environmental conditions.     

Allelic Polymorphism of the tet(M) Determinant in Mycoplasma hominis and Ureaplasma urealyticum Clinical Isolates Resistant to Tetracyclines

Of the 130 clinical isolates of Mycoplasma hominisfrom patients with nonspecific inflammatory diseases of the urogenital tract (UGT), approximately 10% contained the tet(M) gene after the course of treatment with tetracyclines. This gene was found in nine (25%) of the 36 Ureaplasma urealyticum clinical isolates. The nucleotide sequence of 13 tet(M) genes in Tc^R clinical isolates ofM. hominis and five genes in U. urealyticum Tc^R clinical isolates was determined. A comparison of nucleotide sequences of eight tetM genes of different origin and tet(M) genes ofGardnerella vaginalis and M. hominis and U. urealyticumclinical isolates showed that the mosaic structure of thetet(M) gene is completely identical in 11 of 13 M. hominis Tc^Risolates but belongs to an unidentified allele different from those described earlier. Another new allelic variant oftet(M) was found in two isolates. In three of five Tc^R clinical isolates of U. urealyticum, a tet(M) gene, whose mosaic structure was identical to that of tet(M) reported previously for ureaplasmas, and also two new allelic variants, which have not been described so far, were found.     

Electrophoretic analysis of proteins from Mycoplasma capricolum and related serotypes using extracts from intact cells and from minicells containing cloned mycoplasma DNA

The acidic proteins of six different mycoplasma serotypes causing bovine or caprine pleuropneumonia were compared by two-dimensional gel electrophoresis of extracts of 35S-labelled cells. The organisms investigated were Mycoplasma mycoides subsp. mycoides (PG1), M. mycoides subsp. mycoides (Y-goat), M. mycoides subsp. capri (PG3), M. capricolum (California kid), the unclassified bovine serogroup 7 of Leach (PG50) and the F38-like group (F38). The results suggested a close relationship between M. capricolum and F38 and a similarly close relationship between the different M. mycoides subspecies, whereas the two M. mycoides subspecies appeared to be quite distant from M. capricolum and F38. The representative strain of the bovine serogroup 7 of Leach was equally distant from F38, M. capricolum and the three strains of M. mycoides. Strikingly, all six mycoplasma strains apparently shared six proteins in the two-dimensional gels. In Escherichia coli minicells, DNA from strain PG50 cloned in the vector pBR325 gave rise to incorporation of radioactive label into proteins which were identified as mycoplasma proteins by two-dimensional electrophoresis and immunoprecipitation.     

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.     

Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.