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PubMed1984

Inhibition of growth of mammalian cell cultures by extracts of arginine-utilizing mycoplasmas.

Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas. In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas. These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas. In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth. A highly positive correlation (r = 0.96, P less than 0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas.

Animals↗

PubMed2006

Symbiosis of Mycoplasma hominis in Trichomonas vaginalis may link metronidazole resistance in vitro.

Fourteen of 28 Trichomonas vaginalis isolates collected from patients in Guangzhou, China from 2003 to 2004 were found to be naturally infected with Mycoplasma hominis, as determined by PCR using specific primers. In vitro metronidazole sensitivity assay of the 28 isolates revealed four displaying low susceptibility [minimum lethal concentration (MLC)= approximately 13-25 microg/ml] and another four displaying high resistance (MLC=50-100 microg/ml). The overwhelming majority of these resistant isolates (7/8) were mycoplasma-infected. The mean of MLCs of mycoplasma-infected isolates is approximately 10-fold higher than the mean of noninfected isolates (p=0.029). Sequence analyses of PCR-amplified small subunit-large subunit rRNA interspacer regions (ITS1/5.8S/ITS2) revealed that 23 of the 28 samples are identical, the remaining five being separable into two groups, each with a single point mutation. These internal transcribed spacer sequence variants are associated neither with mycoplasma infection nor with drug resistance. In contrast, random amplified polymorphic DNA analyses of DNAs using 10 different primers showed that the drug-resistant isolates are clustered together in association with mycoplasma infection, albeit more loosely. Taken together, the results obtained from this study suggest that in vitro metronidazole resistance of T. vaginalis is related to mycoplasma infection of this protozoan.

Animals↗

PubMed1999

Mycoplasma pneumoniae-associated nephritis in children.

Mycoplasma pneumoniae infection is a rare cause of acute nephritis. Six children (2 girls) aged 5-10 years, admitted for nephritis, had serological tests showing recent Mycoplasma pneumoniae infection. The diagnosis of Mycoplasma pneumoniae infection was based on the presence of serum IgM, detected either by immunofluorescence (IF) (n = 1) or enzyme-linked immunosorbent assay (n = 5). Four children had a renal biopsy, with analysis of parenchymal Mycoplasma pneumoniae components by indirect IF and polymerase chain reaction. Extrarenal symptoms were: respiratory (n = 3), ear, nose and throat (n = 2), gastrointestinal (n = 3), hepatic (n = 1), neurological (n = 1), articular (n = 1), and hematological (n = 3). The patients presented with acute nephritis (1 had a nephrotic syndrome) or with acute renal failure and proteinuria. Pathological findings included type 1 membranoproliferative glomerulonephritis (MPGN, n = 1), proliferative endocapillary glomerulonephritis (n = 2) and minimal change disease (n = 1). The patient with type 1 MPGN progressed rapidly towards end-stage renal failure because of a congenital solitary kidney. Among the patients with endocapillary glomerulonephritis, 1 relapsed 6 months later and remained proteinuric, while the other recovered, as did the child with minimal change disease. The search for Mycoplasma pneumoniae antigens and nucleic acids in renal tissue was negative. However, the absence of the microorganism in the kidney is a common feature of post-streptococcal glomerulonephritis. We conclude that Mycoplasma pneumoniae is a rare yet potential cause of acute glomerulonephritis.

Acute Kidney Injury↗

PubMed1987

Rapid removal of mycoplasma from cell lines mediated by a direct effect of complement.

Mycoplasma can be removed from the surface of contaminated human and murine cell lines by incubation for 4 h with human, rabbit, guinea pig, or mouse sera. Several lines of evidence suggest the involvement of complement in this process: (1) The activity can be abrogated by heat treatment (56 degrees C for 45 min). (2) Using monoclonal antibodies directed against C3a and C3b, the deposition of C3b fragments on the surface of mycoplasma-positive cells can be demonstrated after 1 h incubation with human serum. (3) Ca2+ depletion ablates the ability of serum to remove the activity. (4) C2def' sera are inactive while addition of purified C2 reconstitutes the activity. The latter two findings implicate that activation of the classical pathway of complement is responsible for the effect. Antibody, however, is not required as demonstrated by the uncompromised activity of Ig-deficient sera from bursectomized chicken. Treatment with human serum or rabbit serum was used successfully to permanently cleanse 10/10 tumor cell lines of human and of murine origin. The complete removal of mycoplasma was monitored over at least 8 weeks by direct DNA staining and confirmed by agar culture and transfer of supernatants to mycoplasma-free Vero cells followed by DNA staining. Thus the direct interaction of mycoplasma and complement appears to be an effective and rapid means of curing cell lines from mycoplasma.

Animals↗

PubMed1985

Rapid and simple identification of mycoplasmas by immunobinding.

A simple and rapid method of species identification of mycoplasmas by immunobinding assay is described. Small amounts of antigen of supernatant from cell cultures, broth cultures or clinical specimens were spotted onto nitrocellulose paper. This was followed by application of specific anti-mycoplasma antisera. After incubation, an enzyme-conjugated antiserum against the first antiserum was applied. A positive reaction was indicated by the development of intense blue color reaction when substrate was added. This method identified mycoplasma species with monoclonal and polyclonal antibodies. It detected 9.3 X 10(3) - 7.5 X 10(4) CFU/ml of organisms depending on mycoplasma species. For identification of mycoplasma, ureaplasma, acholeplasma and spiroplasma species, this assay is useful and rapid compared with other serological methods. In limited studies, the method correlated with microbiological assay of clinical specimens for Mycoplasma pneumoniae.

Animals↗

PubMed1981

Cytoskeletal elements in mycoplasmas and other prokaryotes.

This paper reviews the relationship of mycoplasmas to eubacteria, the question of whether mycoplasmas and eubacteria have a cytoskeleton, and whether the unique ultrastructural features of certain mycoplasmas function as a mitotic-like apparatus. Although cytochalasins have inhibitory effects on some mycoplasmas and eubacteria, there are no data indicating that eubacteria have an actin-like protein or other cytoskeletal element. However, the situation for the mycoplasmas remain confusing. While mycoplasma may not contain actin, the data do suggest the presence of other cytoskeletal elements.

Bacteria↗

PubMed2005

The effect of Mycoplasma mycoides ssp. mycoides LC of bovine origin on in vitro fertilizing ability of bull spermatozoa and embryo development.

Several Mycoplasma species may adversely affect bovine spermatozoa viability and embryo development. Mycoplasma mycoides ssp. mycoides large-colony (LC) has been isolated from naturally aborted bovine fetuses and from bull semen. The objective of this study was to evaluate whether M. mycoides ssp. mycoides LC contaminated bovine ejaculates could (i) impair in vitro fertilizing ability of bull spermatozoa, (ii) impair embryo development, and (iii) evaluate potential spread by reproductive technologies. In the present study, spermatozoa of 10 fertile bulls were contaminated with M. mycoides ssp. mycoides LC, at a final concentration of 1.5 million CFU/ml and incubated for 60 min before evaluating spermatozoa motility and acrosome reaction inducibility with calcium ionophore. In addition, in vitro contaminated semen of a bull previously shown to have a good in vitro fertilizing ability, was used in an IVF procedure. Embryo development stage on Day-7 of culture was evaluated. Spermatozoa and embryos at morula and blastocyst stages were routinely processed for transmission electron microscopy observation. Both mean total and progressive motility decreased (P < 0.01 ) upon spermatozoa incubation with Mycoplasma. One-hour incubation with calcium ionophore increased the percentage of acrosome-reacted spermatozoa, although Mycoplasma contamination reduced calcium ionophore treatment efficacy (P < 0.05). Ultrastructurally, Mycoplasma microorganisms appeared as moderately electron-dense sphere-shaped particles, adhering to cell membranes. Sperm mid-piece sections showed numeric aberrations of the central singlets such as nine + zero or nine + one of the axonemal complex. Further morphological abnormalities included partial or total absence of dinein arms and radial fibers, with lack of the bridge and the central ring in 35.00 +/- 4.20% of contaminated cells, whereas these abnormalities were not observed in uninfected ones. The IVF trials showed that two-four cell blocks were higher (P < 0.05) in the infected group. Ultrastructure of Day-7 contaminated embryos showed Mycoplasma particles adhering and infiltrating the outer layer of the zona pellucida. Our investigations suggest that M. mycoides ssp. mycoides LC contaminating the bovine ejaculate induced adverse effects on in vitro spermatozoa-fertilizing ability and embryonic development. Some satisfactory quality transferable embryos could be produced in contaminated IVF systems. This could imply a potential transmission of this microorganism through reproductive technologies.

Acrosome Reaction↗

PubMed1978

On the etiologic role of ureaplasma urealyticum (T-mycoplasma) infection in infertility.

Two hundred consecutive infertility patients and sixty-seven controls subjects were studied for the incidence of infection with Ureaplasma urealyticum (T-mycoplasma). On the basis of a complete infertility investigation, the infertility patients were subdivided into those with explained infertility and those with unexplained infertility. Of the patients with unexplained infertility, 55% had a positive culture for T-mycoplasma as compared with a 32% incidence of positive cultures in the control population. The differences were statistically significant. The 6-month pregnancy rate following successful antibiotic treatment in patients with unexplained infertility was 42%. The 6-month pregnancy rate in a comparable group of patients with unexplained infertility, seen during a 3-year period prior to mycoplasma culture and treatment, was 32%. The difference in pregnancy rates between the two groups was not statistically significant. No correlation was found between a poor postcoital test and the presence of T-mycoplasma infection nor between T-mycoplasma infection and poor cervical mucus. The role of T-mycoplasma infection in infertility was neither proven nor disproven by this study.

Cervix Uteri↗

PubMed2002

Mix-ups and mycoplasma: the enemies within.

Human leukemia-lymphoma (LL) cell lines represent important tools for experimental research. Among the various problems associated with cell lines, the two most common concern contaminations: (1) cross-contamination with unrelated cells and (2) contamination with microorganisms, in particular mycoplasma. The bad news is that about one-third of the cell lines are either cross-contaminated or mycoplasma-infected or both. The good news is that there are means to recognize and overcome these problems. In cases where, during attempts to establish new LL cell lines, primary LL cultures are cross-contaminated with continuous cell lines, intended new cell lines simply cannot be established ("early" cross-contamination). In cases of "late" cross-contamination of existing LL cell lines where the intrusive cells have a growth advantage, the original ("uncontaminated") cell lines may still be available elsewhere. DNA fingerprinting and cytogenetic analysis appear to be the most suitable approaches to detect cross-contaminations and to authenticate LL cell lines. A different but related aspect of "false" LL cell lines is the frequent misclassification of cell lines whereby the actual cell type of the cell line does not correspond to the purported model character of the cell line. Mycoplasma infection can have a multitude of effects on the eukaryotic cells which, due to the variety of infecting mycoplasma species and many other contributing parameters, cannot be predicted, rendering resulting data questionable at best. Practical procedures for the detection and elimination of mycoplasma contamination have been developed. Diagnostic and preventive strategies in order to hem the alarming increase in "false" and mycoplasma-positive LL cell lines are recommended.

Humans↗

PubMed1988

Survey of Mycoplasma infections in cell cultures and a comparison of detection methods.

A total of 1424 cell cultures was assayed for mycoplasmas by microbiological culture and fluorescent DNA staining. Of these cultures, 412 (29%) were infected with mycoplasmas. The most frequently occurring mycoplasma species were Mycoplasma orale (34%), M. hyorhinis (26%), M. arginini (21%), M. fermentans (13%) and Acholeplasma laidlawii (5%). A few isolates each of M. hominis, M. pulmonis and M. bovis were also detected. When detection methods were compared, microbiological culture produced false-negative results for 0.7% (3 of 412) of the infected cell cultures. DNA staining performed directly on the cells was falsely negative in 2.4% (5/207) of the mycoplasma-infected cultures that were compared, DNA staining performed on indicator cells was falsely negative in 3.1% (7/226). False positives appeared in direct DNA-staining in 1.8% (7/386) of the mycoplasma-free cultures and with DNA staining on indicator cells in 0.5% (3/620). For 11% of the cell cultures, the reading of the DNA staining was ambiguous. With DNA staining on indicator cells, 10% of the test results were ambiguous, but by further passage and staining on new indicator cells it was possible to get a definite diagnosis.

Animals↗

PubMed1996

cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product.

P48 is a 48 kDa monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukaemia cell line. It induces growth arrest and differentiation of HL-60 human promyelocytic leukaemia cells along the monocytic pathway and the production of the cytokines interleukin 1, tumour necrosis factor-alpha and interleukin 6 in human monocytes and monocytic cell lines. The cDNA for P48 was cloned from Reh cellular RNA using 3' reverse amplification of cDNA ends. Southern blot probing with P48 cDNA revealed hybridization with DNA from Reh and Molt-4 cells, but not with DNA from human peripheral blood mononuclear cells. Subsequent studies using PCR and Southern analysis revealed P48 sequences in DNA isolated from Mycoplasma fermentans but not M. hominis, M.iowae, M.synoviae or M.lypophilum. Although initial studies using Mycoplasma culture and hybridization techniques had failed to reveal Mycoplasma infection in our Reh and Molt-4 cell lines, subsequent PCR studies using Mycoplasma genus-specific rRNA primrs revealed Mycoplasma sequences in these cell lines. Using the P48 cDNA probe, we isolated a genomic clone from M. fermentans DNA which was found to be 98.5% identical with the P48 cDNA clone, and the deduced amino acid sequence agreed with N-terminal microsequencing data for P48 protein purified from the Reh cell line conditioned medium. The 5' end of the gene has a number of consensus sequences characteristic of prokaryotic genes, and the deduced amino acid sequence has a number of features suggesting that P48 is a lipoprotein. The P48 cDNA was expressed in pMAL in Escherichia coli, and the 60 kDa expressed fusion protein was found to react with anti-P48 antibodies on Western blots. This is consistent with a pMAL fusion protein representing the sum of the 42 kDa maltose-binding protein and 18 kDa of P48 recombinant protein, suggesting that native P48 has significant post-translational modification. Consistent with this, Northern blot studies revealed a single 1 kb transcript. The recombinant fusion protein was found to possess anti-proliferative activity against HL-60 cells, and antibodies against recombinant P48 were found to block the biological activity of native P48 isolated from conditioned medium. These studies demonstrate that P48, a molecule with immunomodulatory and haematopoietic differentiation activities, is derived from M. fermentans or a closely related species. P48 may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian haematopoietic cell differentiation, immune function and cytokine biosynthesis.

Amino Acid Sequence↗

PubMed2006

Introduction of a validation concept for a PCR-based Mycoplasma detection assay.

BACKGROUND: Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS: The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS: The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION: The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.

DNA, Bacterial↗

PubMed1966

Studies of PPLO infection. IV. The neurotoxicity of intact mycoplasmas, and their production of toxin in vivo and in vitro.

Concentrated suspensions of washed Mycoplasma neurolyticum produce rolling disease in mice and rats, with neurological manifestations and pathological lesions similar to those seen with the exotoxin of this organism. Pretreatment of animals with tetracycline protects completely against the toxic effects of washed suspensions of mycoplasmas, while tetracycline affords no protection against the exotoxin. Freeze-thawing disruption of mycoplasma suspensions eliminates their neurotoxicity, while the same treatment does not affect exotoxin. The toxicity of intact organisms is not affected by exposure to the sedimentable component of brain, nor to ganglioside. These observations are interpreted to indicate that the neurotoxicity of living mycoplasmas must be due to their production of toxin after they have been injected into the animal. Resting mycoplasmas, suspended in Ringer's solution in dialysis sacs submerged in PPLO broth) produce considerable amounts of toxin within 15 min of incubation at 37 degrees C. Toxin is also produced, although in somewhat less amount, by washed organisms suspended in phosphate buffer containing glucose. The formation of toxin is prevented by the presence of puromycin, but not by the aminonucleoside analogue of puromycin, indicating that active protein synthesis is involved in the elaboration of toxin. The similarities between the neurotoxicity of the intact organisms of M. neurolyticum and Mycoplasma gallisepticum are discussed.

Animals↗

PubMed2002

Suspected utility of enzymes with multiple activities in the small genome Mycoplasma species: the replacement of the missing "household" nucleoside diphosphate kinase gene and activity by glycolytic kinases.

The small genome Mollicutes whose DNAs are completely sequenced (Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pulmonis, and Ureaplasma urealyticum [parvum]) lack a gene (ndk) for the presumably essential nucleoside diphosphate kinase (NDPK). We hypothesized that other activities might replace NDPK activity. We found in M. genitalium G37(T), Mycoplasma pneumoniae FH(T), Mycoplasma fermentans PG18(T), and Mycoplasma capricolum subsp. capricolum Kid(T) that their 6-phosphofructokinases (6-PFKs), phosphoglycerate kinases (PGKs), pyruvate kinases (PKs), and acetate kinases (AKs), besides reactant ADP/ATP, could use other ribo- and deoxyribo-purine and pyrimidine NDPs and NTPs. These activities could compensate for the absence of an orthologous ndk gene in the Mycoplasmataceae. They suggest a metabolically varied and consequential role for unrelated and perhaps unsuspected "replacement" or compensatory enzymes that may confound metabolic prediction. We partially purified and biochemically characterized the PKs, 6-PFKs, PGKs, and AKs from M. capricolum subsp. capricolum Kid(T) and M. fermentans PG18(T).

Acetate Kinase↗

PubMed1995

Membrane-associated and cytosolic species-specific antigens of Mycoplasma bovis recognized by monoclonal antibodies.

Mycoplasma bovis is a causative agent of bovine mastitis, arthritis, and pneumonia. Six monoclonal antibodies (MAbs) against M. bovis were prepared and used to characterize specific antigens of the mycoplasma. Reactivity of the MAbs to six M. bovis strains was tested by IFA, ELISA, and immunoblotting. The specificity of these MAbs was tested by the same methods against 8 other species of bovine mycoplasmas and 1 mycoplasma species of sheep and goats (Mycoplasma agalactiae) that is highly cross-reactive with M. bovis. Three of the MAbs were used on Western blots of trypsin-treated whole organisms to determine if the antigens were exposed on the surface of M. bovis By isotyping, MAbs were identified as kappa chain IgG1 (3 MAbs), and IgM (3MAbs). The MAbs reacted with all six M. bovis strains in IFA, ELISA, and Western blots. Four of the antigens recognized were highly specific for M. bovis in ELISA, and 3 were cross-reactive with M. agalactiae or other bovine mycoplasmas in Western blots. One MAb reacted with multiple bands with all M. bovis strains, indicating recognition of a size-variant antigen. The size-variant antigen and one of the M. bovis-specific antigens were recognized as surface proteins. A large M. bovis-specific antigen was a conserved cytosolic protein. The M. bovis antigens discovered may be used for specific detection of the organism or measurement of antibody responses, particularly if used in tests with nondenatured alkali-treated antigen, such as ELISA.

Animals↗

PubMed1993

Identification of mycoplasmas in urine from persons infected with human immunodeficiency virus.

Voided urine samples from persons with and without human immunodeficiency virus (HIV) infection were examined for mycoplasmas. Mycoplasma hominis organisms were identified in cultures of urine from 32 (18%) of 180 HIV-positive individuals and from 8 (21%) of 38 HIV-negative individuals. In contrast, glucose-utilizing mycoplasmas were identified in the urine of 30 (17%) of the HIV-positive individuals and in none of those who were HIV-negative. Assays of growth inhibition around disks containing specific antisera identified 14 of the 30 glucose-utilizing mycoplasmas as Mycoplasma fermentans. Four isolates were presumptively identified as Mycoplasma pirum. Growth on solid media was insufficient to permit the identification of the species of the other 12 isolates by growth inhibition.

Adult↗

PubMed1999

Isolation pattern and clinical outcome of genital mycoplasma in neonates from a tertiary care neonatal unit.

The role of genital mycoplasma in perinatal mortality and morbidity has been debated. This study was undertaken to determine the frequency of isolation of genital mycoplasma and evaluate its association with clinical outcome. Sixty-six cerebrospinal fluid (CSF) and 49 tracheal aspirates taken from 100 low birthweight infants who had suspected meningitis and/or respiratory distress respectively were cultured for genital mycoplasma. Ureaplasma urealyticum was isolated from 9 per cent of CSF and 14 per cent of tracheal aspirates. Mycoplasma hominis was isolated from CSF in one case and none at the tracheal aspirates. Three out of seven mycoplasma-infected CNS cases showed CSF pleocytosis while three out of seven patients whose tracheal aspirates grew mycoplasma had congenital pneumonia. None of the patients were treated with antimycoplasmal therapy and none developed chronic lung disease.

Female↗

PubMed1994

Eosinophil cationic protein in the sera of patients with Mycoplasma pneumonia.

In 1985 we reported that eosinophilia was found in many children with Mycoplasma pneumonia. We measured the serum concentrations of eosinophil cationic protein (ECP) produced by eosinophils in 25 children with Mycoplasma pneumonia, 25 with asthma and 11 with no disease (normal controls). The mean concentrations (+/- SD) of serum ECP in children with Mycoplasma pneumonia, with asthma and the normal controls were 18.7 +/- 12.6, 23.7 +/- 12.2 and 6.5 +/- 1.4 micrograms/ml, respectively. When compared with those of normal controls, these higher serum concentrations of ECP in children with Mycoplasma pneumonia and asthma were statistically significant (P < 0.001). The data were directly correlated with the presence of eosinophilia (r = 0.349). There was no relationship between the amount of ECP and the age of children with Mycoplasma pneumonia. These results suggest that ECP may work as a factor causing a persistent cough similar to asthma in children with Mycoplasma pneumonia.

Adolescent↗