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PubMed2005

Can mycoplasma-mediated oncogenesis be responsible for formation of conventional renal cell carcinoma?

OBJECTIVES: To investigate the association between Mycoplasma sp. infection and conventional renal cell carcinoma (RCC). METHODS: Normal kidney, renal intratubular neoplasia, and tumor tissue samples from 33 patients with RCC and 35 healthy controls were studied. Molecular DNA analysis was done after nested polymerase chain reaction performed in two steps with seven primers (four outer and three inner) that can recognize at least 15 different Mycoplasma sp. RESULTS: Mycoplasma sp. DNA was detected in normal kidney, renal intratubular neoplasia, and tumor tissue samples at a ratio of 36%, 67%, and 82%, respectively. In 6 of the 33 patients with RCC, no Mycoplasma sp. was detected from any of the three tissue samples. Mycoplasma sp. DNA was detected in only 5 (14%) of the 35 samples from the control group. CONCLUSIONS: The relationship between mycoplasma infection and conventional RCC has been investigated for the first time, and a significantly high existence of Mycoplasma sp. DNA was found in the tissues of patients with conventional RCC compared with that found in a healthy control group. This suggests that mycoplasma-mediated multistage carcinogenesis may play a role in the development of RCC.

Carcinoma, Renal Cell↗

PubMedDate not supplied

Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. International Research Programme on Comparative Mycoplasmology.

The absence of standardised procedures for minimum inhibitory concentration (MIC) testing of antimicrobial agents against veterinary mycoplasma and ureaplasma species (Mollicutes) has made it difficult to compare results originating from different laboratories. This report, prepared on behalf of the International Research Programme on Comparative Mycoplasmology (IRPCM), offers guidelines and recommendations for veterinary MIC testing of these organisms in an effort to rectify this problem. The subjects discussed include suitable media for broth and agar MIC assays, storage and preparation of antimicrobial agents, standardisation of mycoplasma inocula for MIC tests, validation of equipment, incubation conditions, and determination of MIC end points. A standard medium for all veterinary mycoplasma MIC tests cannot currently be recommended, owing to the diversity of nutritional requirements of different mycoplasma species. Instead mycoplasma broths or agars giving optimal growth of specific mycoplasmas or ureaplasmas are recommended, as suboptimal growth may lead to falsely low MIC results. The importance of using standardised mycoplasma inocula, for assays using either solid or liquid media is stressed. The growth phase may be less important as lag phase and logarithmic phase cultures of Mycoplasma gallisepticum, M. synoviae, M. bovis and M. hyopneumoniae have given very similar results in liquid MIC assays. The liquid method of Tanner and Wu and the agar method described by Hannan et al. are compared and described in detail. Methods for calculating MIC50s and MIC90s are described and the interpretation of results discussed. Methods for assessing mycoplasmacidal (MMC) activity of antimicrobial agents are also described. Adoption of these guidelines should lead to more consistent MIC results being obtained between laboratories.

Animals↗

PubMed2005

Mycoplasmas in semen of chronic prostatitis patients.

OBJECTIVE: To evaluate the occurrence of mycoplasmas in the semen of chronic prostatitis patients. MATERIAL AND METHODS: Genital mycoplasmas (Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Ureaplasma parvum) were sought in the semen of 121 chronic prostatitis patients [38 National Institutes of Health (NIH) category IIIa, 59 NIH category IIIb and 24 NIH category IV] and 40 controls. The commercially available kit Mycoplasma IST was applied to the semen samples of all 161 men, and polymerase chain reaction (PCR) to those of 60 randomly selected men. RESULTS: Ureaplasmas were found in all study groups (at frequencies ranging from 12% to 25%) using the Mycoplasma IST test, but M. hominis was found only in one NIH category IIIb patient. Using PCR, most of the ureaplasmas appeared to be U. parvum, which was found in all prostatitis groups (18% of NIH category IIIa, 15% of NIH category IIIb and 25% of NIH category IV patients) but not in the controls. M. genitalium was found in 18% of the NIH category IIIa patients. All of the mycoplasmas occurred significantly more frequently in prostatitis patients than in controls and in NIH category IIIa patients than in controls. CONCLUSION: Mycoplasmas occur more frequently in the semen of prostatitis patients than in that of healthy controls, with U. parvum being the most frequently occurring species.

Chronic Disease↗

PubMed1993

Interactions of mycoplasmas with B cells: antibody production and nonspecific effects.

Interactions between mycoplasmas and B cells consist primarily of the development of specific antibody and of nonspecific interactions with B lymphocytes or antibody. Antibody responses are important in the resistance to mycoplasmal disease in both humans and animals. However, the ability of mycoplasmas to survive in their host despite vigorous responses suggests that these play a limited role in the host's recovery from infection. Antibody also may prevent dissemination of mycoplasmal infections from mucosal sites and may account for the appearance of systemic mycoplasmal infections in immunocompromised patients. In some cases, antibody responses may contribute to disease pathogenesis through the development of hypersensitivity responses or the deposition of immune complexes. In addition, nonspecific interactions between mycoplasmas and B lymphocytes have been implicated in disease pathogenesis, possibly leading to autoimmune reactions, modulation of immunity, and/or promotion of lesion development. For example, several mycoplasmas, including Mycoplasma pneumoniae and Mycoplasma pulmonis, are able to activate B cells polyclonally in vitro and in vivo, but the mechanisms and consequences of these responses have yet to be defined. In addition to activating B lymphocytes, mycoplasmas are capable of producing chemotactic factors, Fc receptors, and immunoglobulin proteases that may also be involved in lesion development and/or survival of the organisms. Thus, both specific and nonspecific interactions of mycoplasmas with B cells can have important effects on disease progression, especially since many mycoplasmal infections are chronic and the cumulative effect of these interactions may be substantial.

Animals↗

PubMed2000

In vitro activity of gemifloxacin (SB 265805; LB20304a) against human mycoplasmas.

The in vitro activity of gemifloxacin, a new broad-spectrum fluoroquinolone, was compared with those of ciprofloxacin, erythromycin, azithromycin and doxycycline against 29 human respiratory or urogenital tract mycoplasmas. Gemifloxacin was highly active against all of the mycoplasma and ureaplasma species tested (MIC range 0.001-0.25 mg/L) and was 5- to 100-fold more active than ciprofloxacin. Doxycycline was less active than gemifloxacin against the mycoplasmas (MIC range 0.01-1 mg/L) but had similar activity against Ureaplasma urealyticum (MIC ranges 0.025-0.25 mg/L and 0.1-0. 25 mg/L, respectively). The macrolides, particularly azithromycin, were more active than gemifloxacin against Mycoplasma pneumoniae (MIC range 0.001-0.0025 mg/L) and Mycoplasma genitalium (0.0005-0. 001 mg/L) isolates but were less active against Mycoplasma fermentans and U. urealyticum and inactive against Mycoplasma hominis. Gemifloxacin may therefore be useful in the treatment of respiratory, urogenital or systemic mycoplasma infections in humans.

Anti-Bacterial Agents↗

PubMed1992

Relationships between members of the Mycoplasma mycoides cluster as shown by DNA probes and sequence analysis.

A gene probe, CAP-21, which demonstrated interrelationships between the members of the Mycoplasma mycoides cluster was developed. The probe easily differentiated mycoplasmas in this cluster by clear and predictable hybridization patterns in Southern blots and separated the cluster into four groups. Strains of M. mycoides subsp. mycoides which were capable of causing contagious bovine pleuropneumonia composed one group. Strains of M. mycoides subsp. mycoides which did not cause contagious bovine pleuropneumonia together with strains of M. mycoides subsp. capri composed the second group. Mycoplasma capricolum and the F38 mycoplasmas formed a third group, while the bovine group 7 mycoplasmas composed a separate, fourth group. Further support for the above grouping of the cluster was obtained when amplified DNA analogous to the probe from one representative strain of each of the cluster members was sequenced and these data were used to construct a phylogenic tree. Contagious caprine pleuropneumonia is recognized as an important disease, and the etiological agent of this disease is now known to be the F38 mycoplasma. The CAP-21 probe did not differentiate between M. capricolum and the closely related F38 mycoplasma. A second probe, F38-12, which was capable of distinguishing these two mycoplasmas was made.

Animals↗

PubMed1989

Interactions between mycoplasmas and the immune system.

Mycoplasmas are a heterogenous group of prokaryotic organisms causing a wide variety of diseases, including autoimmune disorders. Thus, it is not surprising that various mycoplasmas strains, including Mycoplasma arginini, M. arthritidis, M. neurolyticum and M. pulmonis, are able to regulate the immune response. Though some of the studies of the immunomodulatory action of mycoplasmas have been done in vivo, the majority of the investigations have been conducted in vitro. This has led to the recognition that mycoplasmas are polyclonal activators of both B and T cells from several species, acting through MHC-restricted or -unrestricted pathways. Mycoplasma activation not only induces T-cell proliferation but also leads but to the formation of cytotoxic T cells. We, as well as others, have shown that mycoplasma-mediated B-cell activation induces proliferation as well as Ig secretion, and also that mycoplasma stimulation of lymphocytes may result in the production of cytokines. We communicate here our investigations into the effects of an M. arginini strain on the growth and maturation of preactivated B cells. After an initial biological characterization of the M. arginini effects in vitro, we established the protein nature of the growth-supporting activity and proceeded further on to isolate and identify the responsible proteins. The use of lipid- and lipoglycan-free extracts has allowed us to further extend our studies on the biological activities of the proteins from M. arginini and to compare these results with the effects obtained using live organisms. Furthermore, the study was extended to include a characterization of the in vivo-induced effects of live M. arginini. Altogether, the results from these experiments allow us to conclude that M. arginini is a T-cell independent polyclonal B-cell mitogen, mediated by five identified proteins, inducing growth and Ig secretion of both resting and preactivated B cells.

Animals↗

PubMed2004

A comparison of routine culture with polymerase chain reaction technology for the detection of Mycoplasma species in feline nasal samples.

Nasal flush samples were collected from 20 cats and submitted for Mycoplasma culture and polymerase chain reaction (PCR). Nasal biopsy samples were also obtained from each cat and simultaneously evaluated for Mycoplasma by standard culture and PCR. Concordance of the test results was determined through calculation of the kappa statistic. In 6 cats, nasal flush samples were culture positive for Mycoplasma. PCR was positive in each culture-positive cat and also positive in 1 flush sample that was culture negative. DNA sequencing of the PCR product from the culture negative flush sample identified the organism as Mycoplasma arginini. All other flush samples that were culture negative were also PCR negative (kappa = 0.89). Nasal biopsy samples from 7 cats were culture positive for Mycoplasma, and all were PCR positive. Biopsy samples that were culture negative for Mycoplasma were also PCR negative (kappa = 1.0). Results of culture and PCR for both nasal flush and biopsy were concordant in 19 of 20 cats, and PCR was able to identify an unusual Mycoplasma species that did not grow in culture. In most cats, organisms could be detected in either nasal flush or biopsy samples. In this study, PCR provided rapid and sensitive detection of Mycoplasma species in nasal samples from cats and detected 1 organism that did not grow in culture.

Animals↗

PubMedDate not supplied

Rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction.

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Animals↗

PubMed2006

The significance of a rapid cold hemagglutination test for detecting mycoplasma infections in children with asthma exacerbation.

BACKGROUND AND PURPOSE: Mycoplasma pneumoniae infection is a frequent cause of community-acquired respiratory infections in children and adults. However, standardized, rapid, specific methods for its diagnosis are lacking. The relationship between M. pneumoniae infection and asthma exacerbation has been recently discussed in the literature. We investigated the accuracy of rapid detection of mycoplasma infection by cold hemagglutination test compared to conventional enzyme immunoassays. The clinical characteristics of mycoplasma infection seen during emergent visits in asthmatic children were reviewed. METHODS: We retrospectively reviewed medical records of patients with asthma exacerbation visiting the Department of Pediatric Emergency, National Taiwan University Hospital, over a 12-month period. Subjects 2-18 years of age diagnosed with asthma at our outpatient clinic were included in this study. Patients with immunodeficiency, congenital anomalies, neurological diseases and irregular follow-up were excluded. RESULTS: A total of 269 children (174 males and 95 females) with a mean (+/- standard deviation) age of 6.15 +/- 3.08 years were included. The prevalence of asthma exacerbation in regular follow-up patients was 13.4%, and as many as 19.6% of cases (74/378 person-times) required hospitalization. Asthma attacks were most prevalent during December. 126 patients had both rapid cold hemagglutination testing and mycoplasma immunoglobulin M titers determined using acute blood samples drawn in the emergency room; 46 (36.5%) of these patients demonstrated mycoplasma infection. Sensitivity and specificity of the rapid cold hemagglutination test was 78.3% and 41.3%, respectively. The positive predictive value was 43.4%. Comparison of patients with or without mycoplasma infection revealed no differences in gender, age, chest X-ray findings, and most symptoms/signs and laboratory data, except that more signs of fever and auscultatory rales were seen in the non-mycoplasma infection group. CONCLUSIONS: Mycoplasma infections could be an exacerbating factor for asthma, and the rapid cold hemagglutination test should not be a guideline for prescribing macrolides in the emergency room.

Antibodies, Bacterial↗

PubMed1990

[Basic and clinical studies on a new method for detecting anti-Mycoplasma pneumoniae antibody, using high density particles].

We developed a new method for detecting anti-Mycoplasma pneumoniae antibody, using high density particles (HDP). We characterized the HDP, and also studied the clinical applicability of the HDP agglutination method (HDPA). HDP were uniform, spherical particles, having a specific gravity of 2.0 and a mean particle volume of 18 fl. Adsorption of IgG by HDP was approximately 2 times that by polystyrene latex. The sensitized antigen to the HDP, extracted from the Mycoplasma pneumoniae (Mac strain) bound specifically to the anti-Mycoplasma pneumoniae antibody. With HDPA, the antibody was determined within 30 minutes, which was more rapid than with other routine methods such as complement fixation test, passive hemagglutination test and particle agglutination test (PA). HDPA showed excellent correlation with PA. However, the titers with HDPA were generally 2 to 5 tubes higher than that with PA. A follow-up study on 7 cases of mycoplasma pneumonia revealed remarkably high titers of anti-Mycoplasma pneumoniae antibodies in the acute phase of mycoplasma pneumonia by HDPA, compared to that by PA. HDPA is highly sensitive and specific test for detection of anti-Mycoplasma pneumoniae antibody, and useful for rapid diagnosis of mycoplasma pneumonia.

Agglutination Tests↗

PubMed1990

[Examination of cattle with respiratory diseases for Mycoplasma and bacterial bronchopneumonia agents].

A total of 247 mycoplasma strains was isolated from 435 lungs, tracheobronchial secretions and nasal swabs originating from cattle with symptoms of bronchopneumonia. Mycoplasma (M.) bovis was found 89 times (36%) and was the most common mycoplasma species in the lungs. M. bovirhinis, M. bovigenitalium, M. spec. and Acholeplasma (A.) laidlawii were isolated 158 times (64%). Among these mycoplasmas M. bovirhinis was the most widespread species (114 isolations). In 55 cases (62%) M. bovis was associated with Pasteurella or Actinomyces (A.) pyogenes. The other mycoplasma species were found in 67 cases (42%) together with these bacteria. Without mycoplasmas Pasteurella and A. pyogenes occurred in 33 of the probes investigated (21%). Beside mycoplasmas Haemophilus (H.) somnus was isolated from 16 of 162 tracheobronchial secretions investigated. The results confirm earlier suppositions that in most of the cases bronchopneumonia of cattle is a multifactorial event, frequently associated with mycoplasmas--especially M. bovis.

Actinomyces↗

PubMed2005

[Detection and classification of Mycoplasma in cell lines].

OBJECTIVE: To detect the rate of Mycoplasma infection in cell lines and further determine its types. METHODS: We performed nest PCR amplification of Mycoplasma's conserved regions (16S-23S) and sequenced the spacer with different length between conserved regions. RESULTS: Within the tested 22 cell lines, 17 (77.3%) showed Mycoplasma infections, of which 5 had two or more types of Mycoplasma. M. fermentans and hyorhinis were more frequently detected within the types of infected Mycoplasma. CONCLUSION: The high rate of Mycoplasma infection in cell lines makes it necessary for researchers to pay more attention to its influence on research data when using cell lines as models. Establishment of detection and classifying techniques make it possible to further study the pathogenesis of different types of Mycoplasma.

Base Sequence↗

PubMedDate not supplied

Mycoplasmas and arthritis.

At least 13 mycoplasma species cause arthritis in animals. In some cases it remains chronic, and in particular Mycoplasma hyorhinis in swine and Mycoplasma synoviae in poultry clinically and histologically resemble rheumatoid arthritis in man. Of patients with serologically verified Mycoplasma pneumoniae infections 0.9% had arthritis. Usually it is mild, but may sometimes be the dominant clinical feature. This mycoplasma has twice been cultured from the synovial fluid, as has Ureaplasma urealyticum. A few scientists have succeeded in isolating fastidious mycoplasmas from joint fluid specimens taken from human subjects with acute and chronic arthritis. Their subcultivation has often been difficult or unsuccessful. With the new methods available, renewed attempts to enlighten the possible role of mycoplasmas in human joint disease could now usefully be made.

Animals↗

PubMed2000

Intracellular DNA replication and long-term survival of pathogenic mycoplasmas.

We examined intracellular survival and growth of pathogenic mycoplasmas (Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma genitalium) in cultured human cells. By using the eukaryotic nuclear DNA synthesis inhibitor, aphidicolin, we detected the selective synthesis of mycoplasma (My) and mitochondria (Mt) DNA, which could be further differentiated by restriction enzyme analyses. Also, intracellular M. pneumoniae and M. penetrans infectivity of human cells was detected over 6 months using subfractionation of infected cells and determination of mycoIplasma colony forming units (cfu). For M. genitalium, which we failed to re-grow from infected cells, species-specific PCR primers were used to implicate long-term mycoplasma survivability. Data indicated that pathogenic mycoplasmas reside and replicate intracellularly over extended periods in human cells, consistent with the ability of mycoplasmas to circumvent antibiotic therapy and immune surveillance and establish chronic infections.

Aphidicolin↗

PubMed1984

Detection of mycoplasmas infecting cell cultures by DNA hybridization.

Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of the Eco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes of Mycoplasma capricolum. The probe does not hybridize with eukaryotic DNA. The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants, Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, and Acholeplasma laidlawii. The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas.

Animals↗

PubMed1989

The effect of mycoplasma on the autocrine stimulation of human small cell lung cancer in vitro by bombesin and beta-endorphin.

The tumor stem cell clonogenic assay was utilized to investigate the autocrine growth response of small cell lung cancer (SCLC) to bombesin (BN) and beta-endorphin (beta-E). Mycoplasma contamination was detected in the human SCLC cell line NCl-H345 by a nucleic acid hybridization assay which detects mycoplasma ribosomal RNA. Clonogenic assays of mycoplasma (+) cells were compared to assays of the same cell line following treatment for mycoplasma. Concentrations of beta-E ranging from 0.1nM to 25nM or BN (0.1nM-100nM) were added to cells, media and agarose and applied to prepared base layers. Following incubation for 12-14 days at 37 degrees C, the degree of clonal growth stimulation was determined by colony counts greater than or equal to 42 mu. The non-infected cell population grew in the presence of 25nM BN up to 69% over control growth. The infected cells, however, did not grow more than 27% above control. In the presence of 10nM beta-E, colony counts of non-infected cells exceeded the control values by up to 187% whereas the mycoplasma (+) colonies did not grow more than 20% over the control values. These results indicate a marked reduction in the response of SCLC cell lines to the peptides BN and beta-E when infected with mycoplasma. Since infecting mycoplasma typically adhere to cellular membranes, these adherent mycoplasma may interfere with membrane receptors or alter signal transduction, thus, inhibiting the development of the autocrine response.

Bombesin↗

PubMed2004

Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins.

Chinese hamster ovary (CHO) cell cultures used to produce biopharmaceuticals are tested for mycoplasma contamination as part of the ensurance of a safe and pure product. The current U.S. Food and Drug Administration (FDA) regulatory guideline recommends using two procedures: broth/agar cultures and DNA staining of indicator cell cultures. Although these culture methods are relatively sensitive to most species, theoretically capable of detecting as few as 1-10 cfu/ml of most species, the overall procedure is lengthy (28 d), costly and less sensitive to noncultivable species. The detection of mycoplasma using the polymerase chain reaction (PCR) method has been considered an alternative method because it is relatively fast (1-2 d), inexpensive, and independent of culture conditions, however, limitations in sensitivity (limit of detection >/=1000 cfu/ml) and the risk of false positive and false negative results have prevented PCR from replacing the traditional culture methods in the industrial setting. In this report, we describe a new PCR assay for mycoplasma detection that appears to resolve these issues while being sufficiently simple and inexpensive for routine use. This assay applies readily available techniques in DNA extraction together with a modified single-step PCR using a previously characterized primer pair that is homologous to a broad spectrum of mycoplasma species known to infect mammalian cell cultures. Analysis is made easy by the detection of only a single amplification product within a narrow size range, 438-470 bp. A high sensitivity and specificity for mycoplasma detection in CHO cell production cultures is made possible through the combination of three key techniques: 8-methoxypsoralen and UV light treatment to decontaminate PCR reagents of DNA; hot-start Taq DNA polymerase to reduce nonspecific priming events; and touchdown- (TD-) PCR to increase sensitivity while also reducing nonspecific priming events. In extracts of mycoplasma DNA, the limit of detection for eight different mycoplasma species is 10 genomic copies. In CHO cell production cultures containing gentamicin, the limit of detection for a model organism, gentamicin-resistant M. hyorhinis, is 1 cfu/ml. The sensitivity and specificity of this PCR assay for mycoplasma detection in CHO cell production cultures appear similar to the currently used culture methods and thus should be considered as an alternative method by the biopharmaceutical industry.

Animals↗