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PubMed1990

A study of three blood culture media for isolating genital mycoplasmas from obstetrical and gynaecological patients.

Mycoplasma species are often found colonising the female genital tract. Their ability to become invasive and pathogenic, however, is often ignored, since attempts may not be made to culture these organisms from the bloodstream. We have investigated the ability of three types of blood culture media to support the growth of genital mycoplasmas. The media studied included brain-heart infusion broth, brain-heart infusion broth supplemented with 30% V/V sucrose and fastidious anaerobe broth. Genital mycoplasmas were cultured from the latter medium only. Since this was the sole medium which was liquoid-free, the inhibitory effects of liquoid on Mycoplasma spp. is discussed. This study comprised an investigation of 75 patients in obstetric and gynaecological wards with postpartum or post-operative fever. Genital mycoplasmas were isolated from five (6.7%) patients, four with Ureaplasma urealyticum and one with M. hominis. The value of considering these organisms in the differential diagnosis of fever in 'at risk' patients and of including appropriate media for their isolation is emphasised.

Adult↗

PubMed1984

Detection of mycoplasma contamination lymphoblastoid cell lines by monoclonal antibodies.

We have produced two monoclonal antibodies, SFR1-Myco 1 and SFR1-Myco 2, that detect Mycoplasma fermentans found to contaminate lymphoblastoid cell lines (LCL). The specificity of these monoclonal antibodies against the M. fermentans was determined by indirect immunofluorescence by demonstrating the reactivity of the monoclonal antibodies with known reference strains of mycoplasma grown on soft agar. The reactivity of these antibodies against LCL in a number of immunoassays correlates completely with the presence of mycoplasma in these cells as determined by a standard mycoplasma detection assay. Because of the potential for widespread contamination of B lymphoblastoid cell lines transformed with Epstein-Barr virus-containing supernatants obtained from marmoset cell lines contaminated with M. fermentans, these monoclonal antibodies have value as screening reagents for this mycoplasma species in LCL.

Antibodies, Bacterial↗

PubMed1992

Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction.

A fast and simple method to detect bacterial and especially mycoplasma contamination in tissue culture by means of polymerase chain reaction (PCR) amplification is described. In a first step the universal primer pairs P1/P2 (190-bp fragment) and P3/P4 (120-bp fragment) directed to different conserved parts of the prokaryotic 16S rRNA gene are used. A positive signal after amplification on cell culture DNA with these primers provides an indication of bacterial infection. Using the internal primers IP1, IP3 and IP'3 complementary to a part of the V4 and V8 variable regions of the 16S rRNA gene, in combination with a universal primer, cultures contaminated with mycoplasma could be identified. Six mycoplasma species, typical contaminants in tissue cultures, were investigated: Mycoplasma orale, M. fermentans, M. arginini, M. hyorhinis, M. hominis and Aeromonas laidlawii. This mycoplasma test is an easy, specific and sensitive assay which should be extremely useful in any tissue culture setting.

Bacteria↗

PubMed1995

Use of an enhanced Escherichia coli opal suppressor strain to screen a Mycoplasma hyopneumoniae library.

Many mycoplasma genes contain internal UGA (opal) codons because of their use as tryptophan coding codons. This results in a lack of expression of many cloned mycoplasma antigenic epitopes in Escherichia coli. It has been shown that opal suppressors can be used to enhance expression of defined mycoplasma gene sequences, but no studies have been published using E. coli suppressor strains to screen mycoplasma gene libraries for immunoreactive epitopes. The E. coli suppressor strain ISM612 was used to screen a Mycoplasma hyopneumoniae Lambda gene library. This strain contained an inducible opal suppressor, trpT, as well as the release factor 2 mutation prfB3. Strain ISM612 was shown to enhance antibody recognition of cloned mycoplasmal gene sequences.

Bacteriophage lambda↗

PubMed1982

Prevalence of Mycoplasma and Chlamydia in the nasal flora of dairy calves.

Nasal swabs from dairy calves in 20 selected herds were collected once in each of three sampling periods in one year and cultured for mycoplasmas and chlamydiae. Seventy-seven of 411 calves (19%) and 16 of 20 herds (80%) were positive for mycoplasmas at least once throughout the year, and all of 378 calves sampled from the 20 herds were negative for chlamydiae. Thirty-one percent of the isolates were Mycoplasma bovirhinis; other species isolated less frequently were Mycoplasma bovis, Mycoplasma arginini, and Acholeplasma laidlawaii. There was no evidence of respiratory tract disease at the time of sampling in calves studied.

Animals↗

PubMed1996

A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster.

Rapid and specific detection of organisms belonging to the Mycoplasma mycoides cluster, among them Mycoplasma (M.) mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia, is an important condition for effective control of the respective animal diseases. In an effort to improve diagnosis, a PCR identification scheme for these mycoplasmas was developed. A set of primer combinations derived from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides cluster was selected by means of which complete differentiation within the cluster can be accomplished. Nested PCR involving cluster-specific amplification at the first stage and group-specific amplification using internal primers at the second stage was shown to be applicable for identification of all six groups forming the cluster. For example, external primers P1 /P2 and internal primers P6/P7 were used to distinguish M. mycoides subsp. mycoides SC from LC strains. Using the present PCR procedure, identification of mycoplasmas could be considerably accelerated in comparison to conventional methods (5 h vs. one week) and specificity was also improved.

Animals↗

PubMed1990

Evaluation of different hybridization procedures for the detection of mycoplasma contamination in cell cultures.

Cell culture samples were analysed for mycoplasma contaminations with two different DNA probes which have been described earlier. One probe (the H900 probe), derived from the 23S rRNA gene of Mycoplasma hyorhinis, cross-hybridized with virtually all mycoplasmas (including the acholeplasmas). The other probe (the T2 probe), derived from a protein gene of Acholeplasma laidlawii, cross-hybridized with most acholeplasmas. The two probes were compared in three different direct filter hybridization procedures without previous isolation of DNA or RNA. One of the procedures, developed in the present study, gave the highest sensitivity in DNA-RNA hybridization but also worked satisfactorily in DNA-DNA hybridization. The sensitivity of the H900 probe in filter hybridization experiments was compared with the sensitivity of a commercial probe for detection of mycoplasma contaminations in cell cultures. The H900 probe was found to be at least 25 times more sensitive for all cell culture mycoplasmas except for A. laidlawii, for which they were equally sensitive.

Animals↗

PubMedDate not supplied

Mollicutes DNA polymerases: characterization of a single enzyme from Mycoplasma mycoides and Ureaplasma urealyticum and of three enzymes from Acholeplasma laidlawii.

The DNA polymerase activity of different members of Mollicutes was studied. A single DNA polymerase was found in Mycoplasma mycoides and Ureaplasma urealyticum, type species of the genera Mycoplasma and Ureaplasma, and was compared with the previously described Mycoplasma orale enzyme. Most of their properties were comparable; an immunological relationship was demonstrated between M. orale and M. mycoides enzymes by immunoblotting. In contrast to these results, three different DNA polymerases were purified in Acholeplasma laidlawii, type species of the genus Acholeplasma which, in this aspect, resembles the genus Spiroplasma. A 3'-5' exonuclease activity was found in the different purified preparations. In M. mycoides, M. orale and one of the three A. laidlawii preparations, the 3'-5' exonuclease could be separated from the DNA polymerase by non-denaturing PAGE. The presence of a single DNA polymerase seems to be a typical feature of the Mycoplasmataceae, which include the genera Mycoplasma and Ureaplasma, in contrast to the occurrence of three enzymes within the Acholeplasmataceae and Spiroplasmataceae. These results are in agreement with the phylogenetic tree of Mollicutes proposed from their 5 S and 16 S rRNA sequence comparisons, in which the evolution of Acholeplasma and Spiroplasma branches led, by genome reductions, to Mycoplasma and Ureaplasma species.

Acholeplasma laidlawii↗

PubMed2007

Tools for the genetic analysis of Mycoplasma.

The mycoplasmas have attracted much scientific attention since they contain the smallest known genomes of any independently viable bacterial species. However, the detailed genetic analysis of these bacteria has lagged behind the well-analyzed bacterial model organisms for a long time. This is due to the use of the UGA codon for tryptophan rather than as stop codon, which had often prevented the expression of full-length Mycoplasma proteins in heterologous hosts. Additionally, insufficient efficiency of homologous recombination prevented the targeted disruption of genes in some species such as M. pneumoniae whereas homologous recombination is operative in other mycoplasmas. Only recently, efficient screening systems for the use of transposon-based mutagenesis have been developed to circumvent this problem and to allow the targeted isolation of desired transposon insertion mutants. With the availability of several Mycoplasma genome sequences, artificial plasmids based on the chromosomal origin of replication were constructed that can now be used for complementation studies and for the stable introduction of foreign genetic material. In this review, we give an overview on recent developments in Mycoplasma genetics that facilitate the genetic manipulation of these interesting organisms.

Codon↗

PubMed2004

Production of prostaglandin E2 in monocytes stimulated in vitro by Chlamydia trachomatis, Chlamydophila pneumoniae, and Mycoplasma fermentans.

Chlamydia trachomatis (CT) as well as Chlamydophila pneumoniae (CP) cause chronic inflammatory diseases in humans. Persistently infected monocytes are involved in the pathogenesis by inducing mediators of inflammation. An in vitro system of chlamydial persistence in human peripheral blood monocytes (HPBM) was used to investigate prostaglandin E(2) (PGE(2)) production and the expression of the key enzyme for prostaglandin production, cyclooxygenase-2 (COX-2). PGE(2) production was determined by PGE(2)-ELISA of HPBM-culture supernatants. Cox-2 mRNA expression was measured by real-time RT-PCR of total RNA isolated from HPBM. Both, CT and CP, stimulated PGE(2) production of HPBM in vitro. Equivalent numbers of CT per host cell induced a higher PGE(2)-response compared to CP. The amount of synthesized PGE(2) depended on the chlamydial multiplicity of infection (MOI). Even at an MOI of 10 the amount of CT- and CP-induced prostaglandin, respectively, was lower than the amount of prostaglandin induced by E. coli lipopolysaccharide (LPS) at a concentration of 10microg/ml. In contrast to stimulation with LPS, Chlamydia-induced PGE(2) production as well as cox-2 mRNA decreased after day 1 post infection (p.i.). These data indicate that Chlamydia stimulate PGE(2) production in human monocytes. Since Chlamydia are often contaminated by mycoplasma, the influence of mycoplasma on the prostaglandin production was investigated additionally. Mycoplasma fermentans (MF) also stimulated PGE(2) production. The co-infection of mycoplasma and Chlamydia resulted in an additive effect in the production of PGE(2). Thus it is important to use host cells and Chlamydia free of mycoplasma contamination for the analysis of Chlamydia-induced prostaglandin production.

Chlamydia trachomatis↗

PubMed1993

Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum.

Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum. The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M. capricolum cell suspension. The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M. capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells. The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur. Adaptation of M. capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein. The fusion activity of the adapted cells was very low or nonexistent. Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity. The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer. It is suggested that the role of cholesterol in mycoplasma-SUV fusion is not at the level of bulk bilayer viscosity but rather, affecting local lipid-lipid or lipid-protein interactions that are relevant to the fusion event.

Adaptation, Physiological↗

PubMed1983

The role of mycoplasmas in reproduction.

A study of the incidence of genital tract mycoplasmas in couples attending an infertility clinic and in a group of pregnant women showed that the frequency of isolation was significantly higher in the infertile group (P less than 0.001). In pregnancy, if mycoplasmas were isolated at the first prenatal visit, these organisms were harbored throughout pregnancy. Five percent of the women, however, developed the infection during pregnancy. No relationship was observed of female genital mycoplasmas with prematurity or babies small for gestational age. Treatment with doxycycline (Vibramycin, Pfizer Ltd., Sandwich, Kent, UK) was effective in eradicating mycoplasmas in 91% of couples, but the subsequent pregnancy rates in treated and untreated groups were similar. It appears that mycoplasmas are implicated neither in the etiology of reproductive failure nor in a poor pregnancy outcome.

Doxycycline↗

PubMed1996

A pathological and immunohistochemical study of caprine pleuropneumonia induced by subspecies of Mycoplasma mycoides.

In a population of 700 goats, 150 died; of these, 29 were necropsied. Ten of the 29 goats had pleuropneumonia. Mycoplasma mycoides subsp. mycoides (Large Colony) (MmmLC), Mycoplasma mycoides subsp. capri (Mmc), and Pasteurella multocida were isolated from five of the pleuropneumonic goats. Gross and microscopical lesions were typical of caprine pleuropneumonia (CPP), with bronchopneumonia, fibrinopurulent or fibrinonecrotic pleuropneumonia and dilatation of the interlobular septa and pleura. Immunohistochemical examination with antisera against MmmLC and Mmc showed mycoplasma antigens in all 10 goats with CPP. In all cases, both MmmLC and Mmc antigens were detected together. Mycoplasma antigens were present in the lumina of the airways and alveoli, mainly inside the cytoplasm of neutrophils and macrophages, but extracellular antigen was demonstrated in areas of necrosis. Pasteurella antigens were detected in four of the 10 animals with CPP. From the histological, immunohistochemical and microbiological results it was concluded that the two mycoplasmas, acting together, caused the pleuropneumonia, with P. multocida playing a subsidiary synergistic role.

Animals↗

PubMed1999

Failure of antibiotics gentamycin, tylosin, lincomycin and spectinomycin to eliminate Mycoplasma bovis in artificially infected frozen bovine semen.

To study the effect of antibiotics upon Mycoplasma bovis in fresh bovine semen just before freezing, specimens of bovine semen were artificially infected with 1 of 9 different strains of M. bovis. Inocula of each strain were prepared to contain 10(5) to 10(6)/mL colony-forming units of M. bovis at 3 different stages of the growth phase. The infected semen was diluted with a Tris extender by a 3-step procedure using an antibiotic mixture of gentamicin, tylosin, lincomycin and spectinomycin (GTLS). This semen-antibiotic mixture was placed into French straws that were stored at -196 degrees C. The control semen specimens contained no antibiotics Mycoplasmas were counted after 8 d of storage in 3 decimal dilutions of the frozen semen. No evident effect was noticed upon the 9 tested strains of mycoplasmas in the semen frozen with the antibiotics, compared with that of the untreated control samples. It was further shown that this lack of effect was irrespective of the stage of the growth phase of the mycoplasmas. It was concluded that the antibiotic mixture (GTLS) in semen specimens is not capable of total elimination of mycoplasmas in frozen bovine semen.

Animals↗

PubMed1988

The effects of Mycoplasma mobile 163 K on the ciliary epithelium of tracheal organ cultures.

Mycoplasma (M.) mobile 163 K, isolated from the gills of a tench (Tinca tinca L.), was examined for cytotoxic capacities using tracheal organ cultures from gnotobiotic rats and gnotobiotic piglets in pH-controlled experiments (pH 7.2-7.6). The mycoplasmas caused an inhibition of the ciliary activity at incubation temperatures of 20, 25 and 30 degrees C. The strongest cilia stopping effect was observed at an incubation temperature of 25 degrees C, the optimal growth temperature of the mycoplasmas. No ciliostasis occurred at 37 degrees C. The number of the organisms did clearly affect the severity of ciliostasis in the range from 4.0 x 10(4) to 7.7 x 10(8) c.f.u. in rat as well as in porcine tracheal organ cultures. Toxic substances, secreted by the mycoplasmas into the culture medium, could not be detected. In histological investigations cytopathological changes were observed in the epithelial cells, apparent in the destruction and loss of cilia, cytoplasmatic vacuolization, swelling of mitochondria, peripheral orientation of the nuclear chromatin and detachment of epithelial cells from each other and the basal membrane. The final stage of the infection was characterized by complete exfoliation of the epithelial cells and the complete destruction of the multi-layer epithelium. The localization of the mycoplasmas attached to the ciliary epithelium was shown by scanning and transmission electron microscopy.

Animals↗

PubMedDate not supplied

Adenosine phosphorylase activity as a technique for detection of mycoplasmas in biological media.

The importance of cell culture contamination by mycoplasmas is well recognized, but the means used to detect such contamination still need improvement. Most mycoplasmas possess an enzyme, adenosine phosphorylase, which is not found in cell lines. We used the ultramicromethod of Uitendaal et al. to detect the presence of mycoplasmas in sera and in tissue culture medium. The absence of adenosine phosphorylase activity seems to be the best guarantee that a serum is not contaminated by mycoplasmas. This test is also most efficient for the detection of mycoplasmas in tissue or cell cultures in vitro.

Animals↗

PubMedDate not supplied

Isolation and pathogenicity of mycoplasmas from geese.

Several mycoplasma isolation trials were performed on infertile goose eggs and goose embryos which died during incubation, as well as on geese of different ages. A total of 43 out of 110 goose eggs proved to be contaminated by mycoplasmas. Upon autopsy of birds which laid positive eggs, lesions were observed in the airsacs. Mycoplasmas could be isolated from their air sacs and oviduct. Four out of 15 strains examined biochemically and serologically with antisera prepared against all known avian mycoplasma species were identified as Acholeplasma laidlawii and A. axanthum, respectively. Two strains proved to be glucose-positive and arginine-negative and 9 were glucose-negative but arginine-positive. Some strains caused 50-80% mortality among embryos inoculated intra-yolk-sac at 12 days. In goslings inoculated at the age of 3 days with these strains, we observed fibrinous airsacculitis and peritonitis. By inoculating laying geese with one of the strains, we demonstrated decreasing egg production, increasing early-embryo mortality and egg transmission of mycoplasmas.

Acholeplasma↗

PubMed1992

Protein and antigen variability among strains of Mycoplasma arthritidis.

Six strains of Mycoplasma arthritidis isolated from different host tissues were examined for differences in their proteins and antigens by using one- and two-dimensional electrophoretic techniques as well as immunoblotting. One-dimensional electrophoresis revealed differences in concentrations of individual bands, but not differences in the overall banding pattern. By two-dimensional electrophoretic analysis, 25 proteins were identified as strain-variable whereas the majority of protein spots was strain-constant (about 195 after IEF-2D-PAGE and 145 after NEPHGE-2D-PAGE). Immunoblot analysis using an antiserum against the type-strain of Mycoplasma arthritidis (PG 6) revealed size-heterogeneity of antigens of all six strains. An epitopic relationship between these size-variant antigens could be demonstrated by using monospecific antibodies produced against some of these antigens of Mycoplasma arthritidis. Furthermore, we describe a highly variable antigen of Mycoplasma arthritidis similar to that shown previously in Mycoplasma pulmonis.

Animals↗