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PubMed2001

Re-evaluation of the classical Mycoplasma lipophilum cluster (Weisburg et al. 1989) and description of two new clusters in the hominis group based on 16S rDNA sequences.

The Mycoplasma lipophilum cluster (Weisburg et al. 1989) in the hominis group of the mollicutes is re-evaluated in this work to update the phylogenetic framework for classification of species within the genus Mycoplasma. Therefore, sequences of the 16S rRNA gene were determined from previously described species, and 11 were found to be closely related to the M. lipophilum cluster. A selection of members of the other hitherto defined clusters of the hominis group was included for phylogenetic analysis, revealing that the classical M. lipophilum cluster could be re-organized into two clusters, namely the M. lipophilum cluster and the Mycoplasma bovis cluster. The former was found to contain two species, while the latter contained 20 species. The two clusters were closely related, sharing an ancestral branch with the Mycoplasma synoviae cluster. Furthermore, the M. bovis cluster could be divided into subclusters. Interestingly, two species, Mycoplasma equigenitalium and Mycoplasma elephantis, formed a distinct and early branch of the M. lipophilum, M. bovis and M. synoviae clusters. This entity was termed the M. equigenitalium cluster. The clusters and subclusters could be verified by using neighbour-joining and maximum-likelihood analyses on a variety of data sets, bootstrap calculations, secondary structure analysis and signature nucleotides. Therefore, the new 16S rDNA data presented in this work were used to re-evaluate the M. lipophilum cluster, leading to the definition of two additional clusters. At present, the mollicutes belonging to the hominis group can be classified into ten evolutionary lineages.

Bacterial Typing Techniques↗

PubMed1989

Human cytomegalovirus and monocytes: limited infection and negligible immunosuppression in normal mononuclear cells infected in vitro with mycoplasma-free virus strains.

Human cytomegalovirus (HCMV) infection has previously been associated with the production of immunosuppression. The mechanism by which any such immunosuppressive effect might be mediated is unclear but previous work has implicated an effect of the virus on monocytes. We have attempted to characterize the immunosuppressive activity produced by in vitro infection of normal monocytes with HCMV strain AD169. We first examined the ability of HCMV AD169 and recent clinical isolates to infect normal peripheral blood mononuclear cells in vitro. We have found by immunofluorescence analysis that only a very limited number of peripheral blood mononuclear cells (0.2 to 0.5%) showed evidence of virus infection as demonstrated by expression of the major immediate early protein. We found that the inhibitory activity of supernatants of monocytes exposed to HCMV which suppressed mitogen-driven T cell responsiveness was associated with a protein of about 95K. Experiments to investigate the mechanism of action of this inhibitor suggested the possibility of mycoplasma contamination and we were subsequently able to isolate Mycoplasma hyorhinis from our AD169 virus stock. When a series of low passage clinical isolates of HCMV were examined for their ability to cause immunosuppression, there was a direct correlation between suppression and the presence of contaminating mycoplasmas. Using mycoplasma-free isolates of HCMV we could demonstrate no immunosuppressive effect on mitogen-mediated T cell proliferation of both unseparated human peripheral blood lymphocytes and nylon wool non-adherent T cells; these virus isolates also did not suppress accessory cell function or interleukin 1 production by monocytes infected in vitro. We conclude that the previously reported immunosuppressive effects of HCMV in vitro may be attributable to the presence of mycoplasmas and are unlikely to be due to expression of HCMV in monocytes. We suggest that mycoplasma contamination of isolates of HCMV may be a more extensive problem than is currently recognized.

Antibodies, Bacterial↗

PubMed1983

Synthesis of adenylate nucleotides by Mollicutes (mycoplasmas).

Cultures of the Mollicutes (mycoplasma) Acholeplasma laidlawii B, Acholeplasma morum, Mycoplasma bovis, Mycoplasma arginini, Mycoplasma fermentans and Mycoplasma gallisepticum, representing four metabolic groups, were sampled at intervals over a 40 to 50 h period and assayed for the numbers of c.f.u., changes in pH and glucose concentration, and concentrations of ATP, ADP, AMP, lactate and pyruvate. The adenylate energy charge (ECA), the mean generation time, and the number of nmol of ATP (mg dry weight)-1 were calculated for cultures in the mid-exponential growth phase. The maximum cell concentrations ranged from 0.2 X 10(10) to 5.0 X 10(10) c.f.u. ml-1. Doubling times ranged from 0.34 to 3.29 h. The fermentative, nonarginine-requiring A. laidlawii B, A. morum, and M. gallisepticum, as well as the fermentative, arginine-requiring M. fermentans, utilized glucose and produced lactate and pyruvate. The non-fermentative, non-arginine-requiring M. bovis neither utilized glucose nor produced lactate or pyruvate. The non-fermentative, arginine-requiring M. arginini utilized glucose, but did not produce lactate or pyruvate. At mid-exponential growth phase, the average ECA of A. laidlawii B was 0.90, a value similar to that reported for Spiroplasma citri and other bacteria. In contrast, the average ECA of A. morum and the four Mycoplasma species was 0.70. In A. laidlawii B at mid-exponential growth phase, ATP accounted for 97% of the total adenylate nucleotide pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Acholeplasma↗

PubMed1985

Nucleic acid relationships among the anaerobic mycoplasmas.

The genetic relatedness between twelve selected strains among four distinct serovars of anaerobic mycoplasmas was studied using [3H]DNA-DNA hybridization, and the results were compared with data obtained from biochemical and serological tests. Radiolabelled DNA probes were prepared from five strains representing four serovars. Based on the homology results, the anaerobic mycoplasmas can be divided into five distinct groups representing five distinct species and two distinct genera. There are two species in the Anaeroplasma bactoclasticum serovar 1 group represented by strains JR and A-2, one species in serovar 2, one species in A. abactoclasticum serovar 3 and one among the unclassified serovar 4 anaerobic mycoplasmas. The probe to nonsterol-requiring strain 161 of serovar 4 showed no homology with any of the established nonsterol-requiring Acholeplasma species DNAs, or with Mycoplasma hominis DNA, or with avian DNA which served as a negative control. There was good correlation between the phenotypic and genotypic properties of the five distinct anaerobic mycoplasma species but the results indicate that phenotypic properties are not always adequate for speciation of the anaerobic mycoplasmas.

Base Composition↗

PubMed1997

Widely used, commercially available Chlamydia pneumoniae antigen contaminated with mycoplasma.

Mycoplasma contamination was detected in a widely used commercially available Chlamydia pneumoniae antigen preparation. Contamination was studied with a mycoplasma group-specific 16S rRNA polymerase chain reaction (PCR) and sequence analysis. Several lots of the purified C. pneumoniae antigen from the Washington Research Foundation appeared to be contaminated with the same Mycoplasma species, which appeared to be closely related to M. arginini. Antigen slides prepared for the detection of chlamydia antibodies by MRL Diagnostics were contaminated with the same Mycoplasma sp. Chlamydia antigen slides from Labsystems OY and two chlamydia complement fixation reagents (Virion International Distribution Ltd and Behring Werke) were not contaminated. It is concluded that commercially available C. pneumoniae antigens may contain mycoplasma antigens as well. Although the impact of such mycoplasma contamination on the results of chlamydia serology may not be significant, routine screening of all antigen preparations obtained by tissue culture before their distribution and use is recommended.

Antibodies, Bacterial↗

PubMed1984

Increased sister chromatid exchange in human lymphocyte cultures infected with mycoplasma.

The effects of fermenting, poorly arginine-utilizing Mycoplasma fermentans and arginine-utilizing Mycoplasma salivarium on the frequency of sister chromatid exchange (SCE) in cultured human lymphocytes were examined. M. fermentans caused no apparent mitosis inhibition of lymphocytes and the increase in SCE frequency was dependent on the inoculum size of the mycoplasma. An evident increase in SCE frequency was observed in lymphocytes infected with smaller inoculum sizes of M. salivarium whereas there was mitosis inhibition of lymphocytes infected with larger inoculum sizes of the mycoplasma. In lymphocyte cultures infected with M. salivarium, the addition of arginine to the culture medium reduced mitosis inhibition but did not diminish the increase in SCE frequency, indicating that arginine depletion was not involved in causing the induction of SCEs in mycoplasma-infected lymphocytes. With regard to the genetic effectiveness of SCE, these results suggested that mycoplasmas are capable of inducing cytogenetic changes in infected host cells.

Arginine↗

PubMed1992

Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas.

Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.

Biological Assay↗

PubMed1995

Metastasis-promoting activity of a novel molecule, Ag 243-5, derived from mycoplasma, and the complete nucleotide sequence.

We found that mycoplasma-infected cells have a higher ability to metastasize in vivo than non-mycoplasma-infected cells. To investigate this phenomenon, we obtained a monoclonal antibody, MAb 243-5, by immunization with Mycoplasma arginini-infected RPMI 4788 cells. This MAb recognized a mycoplasmal protein with an MW of 47 kDa and completely inhibited the experimental metastasis of M. arginini-infected RPMI 4788 cells using a nude mouse model. Using this MAb, we purified a molecule called Ag 243-5 and determined the N-terminal amino acid sequence and clarified the entire nucleotide sequence of the Ag 243-5 gene. PCR analysis showed the existence of a homologous gene in Mycoplasma hyorhinis. Four sequential injections of Ag 243-5 (30 micrograms/shot) promoted the experimental metastasis of non-mycoplasma-infected RPMI 4788 cells more than 10-fold using a nude mouse model. Ag 243-5 also promoted the experimental metastasis of the non-mycoplasma-infected mouse colon cancer cell line colon 26. This metastasis-promoting effect was neutralized by MAb 243-5.

Amino Acid Sequence↗

PubMed1995

Identification of mycoplasma membrane proteins by systematic Tn phoA mutagenesis of a recombinant library.

Wall-less prokaryotes in the genus Mycoplasma include over 90 species of infectious agents whose pathogenicity for humans and other animals is currently being assessed. Molecular characterization of surface proteins is critical in this regard but is hampered by the lack of genetic systems in these organisms. We used TnphoA transposition to systematically mutagenize, in Escherichia coli, a genomic plasmid library constructed from Mycoplasma fermentans, a potential human pathogen. The strategy circumvented problems of expressing mycoplasma genes containing UGA (Trp) codons and relied on the construction of the vector pG7ZCW, designed to reduce TnphoA transposition into vector sequences. Functional phoA gene fusions directly identified genes encoding 19 putative membrane-associated proteins of M. fermentans. Sequences of fusion constructs defined three types of export sequence: (1) non-cleavable, membrane-spanning sequences, (2) signal peptides with signal peptidase (SPase) I-like cleavage sites, and (3) signal peptides with SPase II-like lipoprotein-cleavage sites which, like most other mycoplasmal lipoprotein signals analysed to date, differed from those in several Gram-negative and Gram-positive eubacteria in their lack of a Leu residue at the -3 position. Antibodies to synthetic peptides that were deduced from two fusions to predicted lipoproteins, identified corresponding amphiphilic membrane proteins of 57 kDa and 78 kDa expressed in the mycoplasma. The P57 sequence contained a proline-rich N-terminal region analogous to an adhesin of Mycoplasma gallisepticum. The P78 protein was identical to a serologically defined phase-variant surface lipoprotein. TnphoA mutagenesis provides an efficient means of systematically characterizing functionally diverse lipoproteins and other exported proteins in mycoplasmas.

Alkaline Phosphatase↗

PubMed1994

Isolation of mycoplasmas from clinically healthy adult breeding geese in Germany.

Cloacal swabs from adult breeding geese of both sexes from six separate farms were culturally examined for mycoplasmas. Geese from these flocks did not show any clinical signs of illness, increased mortality or drop in egg production during the reproductive season. The results revealed the presence of mycoplasmas in all the flocks tested. Mycoplasma (M.) cloacale was found in 6 flocks, M. anseris in 4 flocks, Mycoplasma species 1220 in 3 flocks and non-identifiable Mycoplasmas in 2 flocks. More than one Mycoplasma species was simultaneously isolated from 14 out of 37 geese.

Animals↗

PubMed1992

Peculiar properties of mycoplasmas: the smallest self-replicating prokaryotes.

Mycoplasmas are the smallest and simplest prokaryotes capable of self-replication, with information provided by a genome which may be as small as 600 kb, estimated to carry less than 500 genes. Keeping the number of structural elements, metabolic pathways and components of the protein synthesizing machinery to an essential minimum places mycoplasmas closest to the concept of 'minimum cells'. Mycoplasmas are, therefore, most adequate candidates for the complete deciphering of the machinery of a self-replicating organism, and studies towards this goal are already underway. Living as 'minimum cells' was made possible by adopting a parasitic mode of life, securing from the host the many nutrients which cannot be synthesized by the mycoplasmas themselves. When pathogenic, infections by mycoplasmas usually follow a chronic course, with host immune reactions playing an important role in symptom production. Recent studies on the possible association of mycoplasmas with rheumatoid arthritis and AIDS are reviewed.

Acquired Immunodeficiency Syndrome↗

PubMed1980

Mycoplasma pneumoniae antigens demonstrated by crossed immunoelectrophoresis.

Soluble Mycoplasma pneumoniae antigens were analysed by crossed immunoelectrophoresis. At least 15 precipitation arcs were developed between the soluble antigens and rabbit anti-Mycoplasma pneumoniae hyperimmune sera. Sera from Mycoplasma pneumoniae infected patients formed precipitation arcs which were identified with those formed by a chloroform-methanol extract of Mycoplasma pneumoniae. Low concentrations of human antibodies, not detectable in a complement fixation test, were found to react with these antigens. The antigens were shown to cross-react with antigens of Mycoplasma neurolyticum. Emphasis was placed on the technical development of the crossed immunoelectrophoresis for the analysis of immune-precipitable antigens of Mycoplasma pneumoniae.

Animals↗

PubMed1987

Transposition of gram-positive transposon Tn916 in Acholeplasma laidlawii and Mycoplasma pulmonis.

Mycoplasma genetics has been limited by a lack of genetic tools such as selectable markers, methods to transfer DNA, and suitable vectors for cloning. Studies were undertaken to examine the potential of using the streptococcal transposon Tn916 as a mycoplasma genetic tool. The Escherichia coli plasmid pAM120, which contains Tn916, was transformed into Acholeplasma laidlawii and Mycoplasma pulmonis. Transposition of Tn916 into the mycoplasma chromosome apparently occurred by an excision-insertion mechanism. This example shows that newly introduced DNA from other bacteria can be successfully expressed in mycoplasma and that Tn916 should serve as a powerful genetic tool for the study of mycoplasmas.

Acholeplasma laidlawii↗

PubMed2004

In vitro activities of garenoxacin and levofloxacin against Chlamydia pneumoniae are not affected by presence of Mycoplasma DNA.

We studied 20 Chlamydia pneumoniae isolates obtained from respiratory sites and atheroma tissue of patients from various geographic areas to determine the susceptibilities of these isolates to a new des-fluoroquinolone, garenoxacin, and to levofloxacin. In addition, we assessed the cultures with these isolates by PCR for the presence or absence of Mycoplasma sp. DNA. Both the MIC at which 90% of isolates are inhibited (MIC(90)) and the minimal bactericidal concentration at which 90% of isolates are killed (MBC(90)) for garenoxacin were 0.06 microg/ml, and both the MIC(90) and the MBC(90) for levofloxacin were 2.0 microg/ml. The activity of garenoxacin against C. pneumoniae was 32-fold greater than that of levofloxacin. Mycoplasma sp. DNA was detected by PCR in 17 of 20 cultures. Mycoplasma amplicons from five Mycoplasma DNA-positive C. pneumoniae cultures were sequenced and found to represent four Mycoplasma species. Our data demonstrate that C. pneumoniae cultures frequently contain Mycoplasma DNA and that its presence in C. pneumoniae cultures does not appear to affect the susceptibility results for the two fluoroquinolones that we tested.

Anti-Infective Agents↗

PubMed1974

Induction of interferon in mice by mycoplasmas.

Interferon was induced in mice after intraperitoneal inoculation with four different mycoplasmas. Peak levels of between 100 and 300 U of interferon per ml were attained by 6 h postinfection with each of the mycoplasmas except Mycoplasma arthritidis, which induced higher titers (400 to 11,800 U/ml) by this time. A fifth mycoplasma, M. pulmonis, induced interferon inconsistently and at a later (72 to 96 h) time. Mycoplasmatales virus MVL51 and sterile mycoplasmal broth did not stimulate interferon production in vivo. All of the mycoplasmas and MVL51 failed to induce interferon in murine spleen cell, peritoneal exudate cell, or peripheral blood leukocyte cultures. Preinfecting the mycoplasmas with MVL51 or treating the organisms with trypsin or dilutions of specific antisera did not enhance their ability to induce interferon in vitro.

Acholeplasma laidlawii↗

PubMed1979

Interaction of Mycoplasma pneumoniae with human lung fibroblasts: characterization of the in vitro model.

The interaction of pathogenic Mycoplasma pneumoniae and host cells was studied in cell cultures of MRC-5 human lung fibroblasts. A comparison of results obtained with fibroblasts in a monolayer format and with hamster tracheal explant cultures indicated that the former can bind significantly larger numbers of mycoplasmas. In addition, the attachment was 96% specific, that is, mediated through a neuraminidase-sensitive receptor on the host cell. Uptake of mycoplasmas was directly related to the number of mycoplasma cells present in the inoculum, and attachment was virtually complete within a 30-min period at 37 degrees C. High doses of M. pneumoniae induced a marked cytopathic effect, whereas doses of less than or equal to 10(6) colony-forming units per ml produced grossly observable cell damage that was moderate and variable. Transmission electron microscopy studies indicated that attachment of M. pneumoniae to the surface of lung fibroblasts occurred with the specialized terminal structure or binding site oriented closest to the epithelial cell surface. The filamentous mycoplasma cells were spatially arranged in several configurations and were not limited to a vertical orientation. The advantages and disadvantages of human lung fibroblast monolayer cultures, in reference to other in vitro models are discussed. A new mycoplasma agar medium (G-200 agar) with a defined tissue culture base and 10% horse serum is also described.

Binding Sites↗

PubMed1980

Antibody-mediated neutralization of virus is abrogated by mycoplasma.

The ability of a mouse mammary tumor cell line to abrogate antibody neutralization of vesicular stomatitis virus was shown to be due to the presence of mycoplasma. The mycoplasma was isolated from the cell line and typed as Mycoplasma orale. Colonies of this mycoplasma were used to deliberately infect cell cultures which then gained the capacity to reactivate antibody-neutralized virus. The extent of the reactivation depended on the source of neutralizing antiserum. Other species of mycoplasma were tested and were found to reactivate neutralized virus, indicating that this may be a general phenomenon of mycoplasma contamination.

Animals↗

PubMed1986

Expression of Mycoplasma pneumoniae antigens in Escherichia coli.

A genomic library of Mycoplasma pneumoniae was generated by using bacteriophage lambda EMBL3 as the vector. Screening of the library for the expression of M. pneumoniae protein antigens with adsorbed anti-M. pneumoniae serum revealed strong reactivity from a third of those clones which contained mycoplasma DNA inserts. Three of the most highly reactive clones were analyzed in detail and found to synthesize discrete mycoplasma proteins. Two carried overlapping fragments of mycoplasma DNA which encoded a protein that was readily detected in Escherichia coli after infection with recombinant bacteriophage. The third clone contained a novel mycoplasma DNA fragment which directed the synthesis of two additional mycoplasma proteins. Further screening of the library with antiserum raised against the major M. pneumoniae adhesin protein P1 (165 kilodaltons [kDa]) yielded one clone which produced an immunologically reactive protein of 140 kDa. Adsorption of anti-P1 serum by this clone selected a population of antibodies that were reactive with M. pneumoniae adhesin P1 (165 kDa). These results demonstrate that immunologically active M. pneumoniae proteins are synthesized in E. coli.

Adhesiveness↗