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PubMed1996

Mechanisms and factors involved in Mycoplasma bovis adhesion to host cells.

Mycoplasma (M.) bovis cytadhesion was studied using permanent embryonic bovine lung (EBL) cells as host system. Adherence rates were found to be strongly dependent on temperature and the mycoplasma-to-EBL ratio near the point of saturation of the attachment isotherm was determined to be 225 : 1. Mild trypsinization of viable M. bovis cells caused a measurable decrease of adherence indicating that surface proteins, among them the P26 antigen, played a major part as adhesion factors. Neuraminidase treatment of mycoplasmas led to a drastic reduction of adherence rates, which emphasizes the importance of sialyl moieties in adhesive interactions. The ability of the P26 antigen, a hydrophilic 32-kDa protein, to function as an adhesin was confirmed using a competitive adherence assay, in which the HPLC-purified protein was shown to reduce mycoplasma adhesion. These data complement previous findings obtained with the corresponding monoclonal antibody (MAb) 4F6. In further inhibition experiments, it could be demonstrated that MAb 1E5, which is directed against a common epitope of at least three members of the Vsp (variable surface protein) family of M. bovis, was also capable of decreasing mycoplasma attachment to EBL cells. This is the first evidence of possible involvement of Vsps in cytadhesion. In an effort to identify more putative adhesion proteins of this organism, the reverse adherence screening assay was used, a procedure based on the specific binding of labelled mammalian tissue culture cells to Western-blotted mycoplasmal proteins.

Adhesins, Bacterial↗

PubMed2003

Mycoplasma pneumoniae: a reduced-genome intracellular bacterial pathogen.

Mycoplasma pneumoniae has classically been considered an extracellular (or membrane-associated) organism. Nevertheless, the recently elucidated genomic structure of this pathogen strongly suggest that this organism may have been subjected to the process of reductive genetic evolution which is characteristic of intracellular bacteria. We studied the Mycoplasma pneumoniae RYC15989 strain, recovered from a pericardial biopsy sample from a patient with atypical pneumonia and acute pericarditis. The interaction of this strain with human hepatocytes Hep-G2 and mouse neuroblastoma N2-A cell lines was investigated. Confocal laser scanning microscopy and electronic microscopy evidence is presented of the intracellular location of fluorochrome-labelled Mycoplasma pneumoniae in cell lines infected with the organism in vitro. This finding provides preliminary evidence of cellular invasive capacity of Mycoplasma pneumoniae and casts some new light on the pathogenic potential of Mycoplasma pneumoniae in host infection.

Animals↗

PubMed2000

A novel mechanism for control of antigenic variation in the haemagglutinin gene family of mycoplasma synoviae.

High-frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5' to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3' end of the gene, but conservation of the 5' end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5' coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single-copy 5' end of vlhA, but extending over one of four distinct overlapping regions of the 3' coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5' end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

Amino Acid Sequence↗

PubMed2003

Isolation of Mycoplasmas from nasal swabs of calves affected with respiratory diseases and antimicrobial susceptibility of their isolates.

Nasal swabs of 293 calves were examined for Mycoplasma. The samples were collected from calves affected with respiratory diseases on 71 farms in various parts of Japan between 1996 and 1997. Mycoplasma bovirhinis was isolated from 47 of 293 calves (16.0%). Mycoplasma alkalescens, M. bovis, M. arginini, M. bovigenitalium and Acholeplasma spp. were isolated from 19 (6.5%), seven (2.4%), four (1.4%), four (1.4%) and 18 (6.1%) calves, respectively. Pasteurella multocida and P. haemolytica were isolated from 60% of Mycoplasma-positive calves. However, other bacteria were not isolated from calves. To evaluate the antimicrobial susceptibility of their isolates, 68 M. bovirhinis, 21 M. alkalescens and 10 M. bovis strains were examined for 12 antimicrobial agents. All isolates showed higher susceptibility to tiamulin than to the other drugs used in the study. However, erythromycin had no effect on any of the Mycoplasma strains studied. The field isolates were less susceptible than the type strains to some drugs, such as spiramycin, oxytetracycline and tylosin.

Animals↗

PubMed1998

Mycoplasma contamination of Chlamydia pneumoniae isolates.

We examined 6 C. pneumonia isolates from The American Type Culture Collection (ATCC) and 2 Finnish isolates for Mycoplasma contamination. Three of the ATCC isolates and both of the Finnish isolates were Mycoplasma-contaminated. The contaminants were characterized by means of growth in BEa and BEg media, immunoblotting, polymerase chain reaction and pulsed field gel electrophoresis. Two of the 6 ATCC isolates [ATCC VR1355 (TWAR strain 2043) and ATCC VR1356 (TWAR strain 2023)] were infected with Mycoplasma hominis and 1 isolate [ATCC VR2282 (TWAR strain TW183)] was contaminated with both Mycoplasma hominis and Mycoplasma orale, whereas 3 of the ATCC isolates [ATCC VR1310, ATCC VR1360 (TWAR strain CM-1) and ATCC 53592 (TWAR strain AR39)] were not contaminated. The Finnish C. pneumoniae isolates Kajaani 6 and Parola were found to be contaminated with M. hominis and M. orale, respectively. The contamination of C. pneumoniae stock cultures, frequently used in the microimmunofluorescence test, with human pathogens, could pose a serious problem in C. pneumoniae serology.

Bacteriological Techniques↗

PubMed1966

The interaction of mycoplasmas with mammalian cells. I. HeLa cells, neutrophils, and eosinophils.

The ultrastructure of three mycoplasma species, Mycoplasma pneumoniae, Mycoplasma gallisepticum, and Mycoplasma neurolyticum, has been studied in isolated form as well as in HeLa cell cultures and following incubation with human peripheral blood leukocytes. During log growth phase, the organisms could be distinguished from each other on the basis of their fine structure. In mammalian cell cultures, PPLO's appeared to proliferate on the plasma membranes which had markedly increased their surface area by means of long cytoplasmic processes which extended toward and surrounded them. Some of the microorganisms affected in this way may well have lain, not in vacuoles, but at the bottom of crypts. It is suggested that the cytopathogenic effect exerted by PPLO's on some tissue cultures may be attributable to membrane damage. Mycoplasmas adhered to leukocyte plasma membranes in a similar manner. They were avidly phagocytosed by neutrophils and eosinophils with accompanying degranulation of the white cells. It is thus likely that the local inflammatory reaction induced by PPLO's does not differ in essence from that caused by bacteria.

Culture Techniques↗

PubMed1998

Immunologic cross-reaction between HIV type 1 p17 and Mycoplasma hyorhinis variable lipoprotein.

Monoclonal antibodies directed against the HIV-1 matrix protein p17 that react with a component present on the surface of HIV-1-infected cells have previously been described. In this study we show that one of these monoclonal antibodies binds to persistently HIV-1-infected cell lines that are coinfected with Mycoplasma hyorhinis, but not to cell lines that are uninfected with mycoplasma. Mycoplasma-infected cells secrete HIV-1 at a higher rate, have a slight increase in cell surface expression of gp120 and gp41, and are less sensitive to immunotoxins than uninfected cells. The anti-p17 antibody binds to a protein of M. hyorhinis grown in cell-free culture. The variable expression and size of the protein among strains is typical of the variable lipoprotein (Vlp) system of M. hyorhinis. Confirmation of the reactivity of the antibody with a Vlp was provided by demonstrating its specific binding to recombinant VlpF expressed in E. coli, and to a synthetic peptide representing the carboxy-terminal region of VlpF, but not to other recombinant Vlp products or peptides. This is a true cross-reaction because the antibody also binds to recombinant p17 expressed in E. coli and the binding is inhibited by the VlpF peptide. These analyses highlight the potential of mycoplasma contamination of tissue culture cell lines to cause anomalous results. With regard to HIV-1, mycoplasma infection of cells results in increased rates of virus secretion, and introduces a potential confounding immunologic cross-reaction as well. The existence of a cell surface form of p17 is unlikely.

Antibodies, Monoclonal↗

PubMed1988

Evaluation of three methods for curing hybridomas from mycoplasma contamination.

Hybridomas are as susceptible to mycoplasma contamination as animal cell cultures. Two in vitro methods and one in vivo passage into mice were compared for efficiency of curing hybridomas from mycoplasma contaminations. Four contaminated clones were treated with the combined action of 5-bromouracil (5-BrUra) and the Hoechst 33258 followed by photosensitization. The other in vitro method involved the use of BM-cycline. The success of overcoming the mycoplasma contamination was dependent on the level of the initial contamination of the individual hybridoma. BM-cycline was more efficient than the photosensitization method. For the most contaminated hybridomas, 10 successive treatments with 5-BrUra were necessary as compared to six treatments with BM-cycline. Moreover, the use of BM-cycline reduced the contamination by as much as 50% after the first treatment. After twenty passages following the curing of hybridomas with BM-cycline, cells were stable and retained their specificity and secretion of their respective immunoglobulins. Whereas, treatment with 5-bromouracil, recurrence of contamination was observed in one of the four hybridomas after 10 passages following treatment. Decontamination after one passage in peritoneal cavity in mice was not always sufficient since one of four hybridomas remained contaminated. BM-cycline appears to be the method of choice since it is more efficient, less time consuming, simpler and less expensive. Mycoplasma strains that could be identified were of bovine origin: Mycoplasma arginini and Acholeplasma laidlawii.

Animals↗

PubMed2005

Characterization of In Vivo acquired resistance of Mycoplasma hyopneumoniae to macrolides and lincosamides.

Macrolides and related antibiotics are used to control mycoplasma infections in the pig industry worldwide. Some porcine mycoplasmas, however, survive these treatments by acquiring resistance. The mechanism of acquired resistance to macrolides and lincosamides was studied in more detail for Mycoplasma hyopneumoniae by comparing both the phenotype and genotype of a resistant field isolate to five susceptible isolates. The MICs were significantly higher for the resistant strain for all antibiotics tested. The MICs for the 16-membered macrolide tylosin ranged from 8 to 16 microg for the resistant strain and from 0.03 to 0.125 microg/ml for the five susceptible strains. The MICs for the 15-membered macrolides and lincosamides were higher than 64 microg/ml for the resistant strain while only 0.06 to 0.5 microg/ml for the susceptible strains. Mycoplasma hyopneumoniae strains are intrinsically resistant to the 14-membered macrolides due to a G 2057 A transition (E. coli numbering) in their 23S rDNA. Therefore, high MICs were observed for all strains, although the MICs for the resistant strain were clearly increased. An additional, acquired A 2058 G point mutation was found in the 23S rRNA gene of the resistant strain. No differences linked to resistance were found in the ribosomal proteins L4 and L22. The present study showed that 23S rRNA mutations resulting in resistance to macrolides and lincosamides as described in other Mycoplasma spp. also occur under field conditions in M. hyopneumoniae.

Drug Resistance, Multiple, Bacterial↗

PubMed1993

Mycoplasma fermentans--HeLa cell interactions.

A survey of previous evidence for the intracellular localization of mycoplasmas within nonphagocytic cells indicated that it was insufficient to conclude unequivocally that such localization occurred. Illustrations of the seemingly intracellular existence of Mycoplasma fermentans in the tissues of patients with AIDS and other patients rekindled interest in the topic of mycoplasmal entrance into epithelial cells. Accordingly, the "incognitus" and PG18 strains of M. fermentans were sought and demonstrated within HeLa cells by electron microscopy following treatment of the cell cultures with gold-labeled antiserum specific to the mycoplasma, together with ruthenium red staining. The phenomenon is not unique to this mycoplasma, being demonstrated also with Mycoplasma hominis. In hindsight, some of the earlier investigators who hinted at intracellular localization were probably correct in doing so.

HeLa Cells↗

PubMed1993

Carbohydrates and the pathogenesis of Mycoplasma pneumoniae infection and AIDS--some observations and speculations.

Carbohydrate-mediated interactions are prominent in the pathobiology of human infection with Mycoplasma pneumoniae. The mycoplasma uses specific long-chain sialo-oligosaccharides at the host-cell surface as receptors. These sialo-oligosaccharides contain I antigen in their backbones, and they are richly expressed at the primary site of infection--the ciliated bronchial epithelium--and on erythrocytes. The autoantibodies of high titers directed to I antigen, cold hemagglutinins, frequently produced in this infection are therefore antibodies to receptors and may be triggered by an autoimmunogenic mycoplasma-receptor complex in which the lipid-rich microorganism serves as an adjuvant. Current interest is focused on the possibility that the course of infection with human immunodeficiency virus type 1 (HIV-1) may be exacerbated by coinfection with mycoplasmas. One mechanism for such potentiation would be specific interactions between the mycoplasmas and HIV-1 leading to a concentration of the virus at the mycoplasmal surface. The envelope glycoprotein gp120 of HIV-1 is richly decorated with an array of oligosaccharides whose biosynthesis is host cell directed. By an analogy with M. pneumoniae, I propose that recognition of carbohydrates by various mycoplasmal copathogens is a potential mechanism for interactions with HIV-1.

AIDS-Related Opportunistic Infections↗

PubMedDate not supplied

Cholesterol and phospholipid uptake by mycoplasmas.

Mycoplasma offer several unique advantages for investigating the mechanism controlling transfer and uptake of exogenous cholesterol and phospholipids by biomembranes, as their plasma membrane interacts directly with exogenous lipid donors and their endogenous lipid synthesis is restricted. Growing cells of five species of Mycoplasma were found to take up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three species of Acholeplasma failed to take up any of the exogenous phospholipids and incorporated only low amounts of free cholesterol and no esterified cholesterol. It is hypothesized that Mycoplasma species have receptors for serum lipoproteins and phospholipid-cholesterol vesicles that facilitate the transfer of cholesterol and phospholipids to the growing cell membrane. Our finding that gentle trypsin treatment of growing Mycoplasma capricolum cells decreased their cholesterol uptake ability by about 50% but did not affect cholesterol uptake by growing Acholeplasma laidlawii cells appears to support the existence of protein receptors for lipoproteins on the surface of Mycoplasma but not on Acholeplasma species. Digestion of membrane phospholipids by phospholipase A2 decreased the cholesterol-binding capacity of isolated A. laidlawii and M. capricolum membranes, roughly in proportion to the amount of phospholipid digested. The total removal of phosphatidylglycerol and diphosphatidylglycerol from A. laidlawii membranes by phospholipase A2 only decreased but did not abolish cholesterol uptake, an indication that glycolipids also participate in cholesterol uptake.

Acholeplasma↗

PubMed1975

T-mycoplasmas: Growth patterns and physical characteristics of some human strains.

Five T-mycoplasmas isolated from patients with nonspecific urethritis and five laboratory strains of T-mycoplasma were examined for differentiating biological properties. The strains differed by the presence or absence of a lag phase and the number of T-mycoplasmas constituting a colony-forming unit (cfu). Most T-mycoplasmas had no lag phase and a cfu consisting of single organisms. All had biphasic ultraviolet inactivation curves typical of suspensions containing both mononucleate and binucleate cells. Binucleate cells probably were in the process of division. They resisted sonication for 3 min. Sonication disrupted multicellular cfu in to single cells within 2 min. Stationary-phase organisms died more rapidly than exponential-phase cells. T-mycoplasmas replicated at 2 C; they grew more slowly in T-broth at 40 C and died in 2.5 min at 56 C. Inactivation curves at 45 C and 50 C differed but insufficiently to permit identification of individual strains. At pressures less than 5 psi, single-cell suspensions passed through filter membranes with 0.65-mum and 0.45-mum pores but were retained by membranes with 0.22-mum pores.

Cell Division↗

PubMed1997

A review of the in-vitro activity of quinupristin/dalfopristin against intracellular pathogens and mycoplasmas.

Quinupristin/dalfopristin displays in-vitro bacteriostatic activity against all Legionella spp. (MICs = 0.06-2 mg/L), with Legionella pneumophila usually being at least two-fold more sensitive to quinupristin/dalfopristin than Legionella bozemanii, Legionella dumoffii, Legionella gormanii and Legionella micdadei (MIC = 0.06-2 vs 1-2 mg/L, respectively). Against Legionella spp., quinupristin/dalfopristin was at least as active as erythromycin. Quinupristin/dalfopristin was active in vitro against all Mycoplasma spp. tested (MIC = 0.05-2 mg/L), with Mycoplasma hominis being less susceptible than other species. Quinupristin/dalfopristin was active against erythromycin-resistant strains of Mycoplasma fermentans and M. hominis (MIC90 = 0.5 and 2 mg/L, respectively), and doxycycline-resistant strains of Ureaplasma urealyticum (MIC90 = 1 mg/L). The in-vitro bacteriostatic activity against Mycoplasma pneumoniae and Mycoplasma genitalium (MIC90 = 0.1 and 0.05 mg/L, respectively) was similar to that of erythromycin and doxycycline. Quinupristin/dalfopristin was actively taken up by murine macrophages, and incubation of the drug (2.5 mg/L) with macrophages containing ingested Staphylococcus aureus resulted in the death of 70% of intracellular bacteria within 120 min. Intracellular concentrations of quinupristin/dalfopristin reached 50 and 30 times the extracellular concentration, respectively, showing that these compounds readily penetrate into cells. The intracellular activity of quinupristin/dalfopristin may make it suitable for use in some, presently difficult-to-treat, infections caused by intracellular organisms.

Animals↗

PubMed1997

Systematic detection of mycoplasmas by culture and polymerase chain reaction (PCR) procedures in 209 synovial fluid samples.

The objective was to investigate the presence of mycoplasmas in rheumatoid arthritis (RA) and other chronic arthritides. Samples of synovial fluid (SF) were systematically collected from all patients presenting with an articular effusion. Each sample was divided into three parts. The first was kept for cytological count and culture on standard media for pyogens and mycobacteria, the second was cultivated on specific media for mycoplasmas and the third frozen for subsequent study by polymerase chain reaction (PCR). A total of 209 samples were studied. Half of the patients had inflammatory rheumatic diseases: RA (27), spondyloarthropathy (28), connective tissue disease (5), unclassified arthritis (45). The remaining suffered from other conditions, including osteoarthritis (60), gouty arthritis (19), haemarthrosis (5), post-traumatic effusion (2). Eight samples were positive by culture, two for Mycoplasma hominis; three for M. fermentans, one for M. salivarium, one for M. orale and one for Ureaplasma urealyticum. All the patients concerned had an inflammatory rheumatic disease: five had RA, one had psoriatic arthritis and two had unclassified arthritis. These results were confirmed by PCR in two cases (one M. fermentans, one U. urealyticum). The lack of sensitivity of the conventional PCR assay on SF is discussed. Mycoplasmas were mainly detected in SF of RA patients. These results raise the question of the possible role of mycoplasmas in the triggering and maintenance of inflammatory rheumatic diseases, especially RA.

Adult↗

PubMed1992

Mycoplasma penetrans sp. nov., from the urogenital tract of patients with AIDS.

An unusual mycoplasma, which was isolated from the urine of a human immunodeficiency virus-positive male homosexual patient, has an elongated flask shape and two unique sharply divided internal compartments. The tiplike compartment is densely packed with fine granules, and the body compartment is loosely filled with coarse granules consistent with ribosomal structures. The organism has properties of adherence, hemadsorption, and cytadsorption and invades many different types of mammalian cells. Adhesion and penetration apparently involve the terminally located tiplike structure. Cholesterol is required for growth, and the mycoplasma ferments glucose and hydrolyzes arginine, but does not hydrolyze urea. The results of DNA homology studies revealed that this organism is not genetically related to previously described mycoplasma species that have the same biochemical properties. The results of serologic studies demonstrated that this organism is antigenically distinct from all previously described mycoplasmas. We propose that this new mollicute species should be named Mycoplasma penetrans sp. nov. The type strain is strain GTU-54-6A1 (= ATCC 55252).

Acquired Immunodeficiency Syndrome↗

PubMed1995

Mycoplasma adleri sp. nov., an isolate from a goat.

Mycoplasma sp. strain G145T (T = type strain) was isolated from a goat's abscessed ankle. Strain G145T required cholesterol or serum for growth and possessed characteristics similar to those of other members of the genus Mycoplasma. This strain was serologically distinct from previously described Mycoplasma species and from a group of currently unnamed strains thought to belong to the genus Mycoplasma. Strain G145T hydrolyzed arginine, but did not hydrolyze urea or ferment glucose. The guanine-plus-cytosine content of the DNA was 29.6 mol%. We propose that strain G145 (= ATCC 27948) is the type strain of a new species, for which we propose the name Mycoplasma adleri.

Animals↗

PubMed1980

Partial nucleotide sequence similarity within species of Mycoplasma and Acholeplasma.

The nucleotide sequence relationships between 18 species of Mycoplasma and 3 species of Acholeplasma were examined by solution DNA hybridization. Radiolabeled DNAs from strains representing 13 Mycoplasma and 2 Acholeplasma species were used as hybridization probes. The mycoplasmas were heterogeneous but displayed a low degree of conserved information of the order of 3 to 5% of the genome. However, several species within each genus showed 13 to 15% homology. There was no detectable homology between species from the two genera, and M. pneumoniae and M. neurolyticum appeared to be unrelated to any of the other Mycoplasma species or to each other. These results suggest that it may be possible to isolate genes common to most, if not all, Mycoplasma and Acholeplasma species.

Acholeplasma↗