Hitoshi Kotani

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

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

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.

Detection of cell culture mycoplasmas by a genetic probe

The utility of a genetic probe prepared against mycoplasmal ribosomal RNA was evaluated. The probe consisted of a tritiated DNA probe prepared with reverse transcriptase against Mycoplasma and Acholeplasma. Using a batch process without Southern blots and autoradiography, the probe assay detected mycoplasma infection in 27 of 52 cell cultures, an incidence of 51.9%. No false-positives were detected. Two false-negatives occurred; these were unusual mycoplasma infections of cell cultures grown in serum-free media. The sensitivity of the probe varied with mycoplasma species, from 9.3 X 10(3) for M. arginini to 2.6 X 10(6) for M. orale. The assay is rapid, taking only 2-3 h.

Microbiological cultivation ofMycoplasma hyorhinis from cell cultures

SummaryThe failure of many cell culture isolates ofMycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly prepared yeast extract in mycoplasmal media together with incubation in 5% CO2/air successfully detectedM. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures.

Comparative studies to determine the efficiency of 6 methylpurine deoxyriboside to detect cell culture mycoplasmas

SummaryStudies were performed to compare three methods to detect mycoplasmal infection of cell cultures. The methods included microbiological assay by inoculation into broth and onto agar with anaerobic incubation, fluorescent DNA staining by Hoechst 33258, and mycoplasmal mediated cytotoxicity by 6 methylpurine deoxyriboside (6MPDR). Fluorescent DNA staining and 6MPDR assays were performed in an indicator cell culture system. A total of 2589 cell cultures were assayed. Mycoplasmas were detected in 174, an incidence of 6.7%. Species isolated were:Acholeplasma laidlawii, Mycoplasma orale, M. arginini, M. hyorhinis, M. fermentans, M. pirum, and M. pneumoniae. In separate studies, 6MPDR also detected infection withSpiroplasma mirum when this organisms was deliberately inoculated into cell cultures. The efficiencies of microbiological testing, fluorescent DNA assays, and 6MPDR were 43.1, 98. 8, and 97.1%, respectively.

Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma,Spiroplasma mirum, strain SMCA

SummaryA helical mycoplasma,Spiroplasma mirum strain SMCA, produced malignant transformation in mouse NIH 3T3 cells and monkey kidney CV-1 cells. The transformed cells exhibited morphological changes consistent with the transformed phenotype, grew in soft agar and produced tumors in athymic and BALB/c mice. Transmission electron microscopy revealed structures morphologically similar to mycoplasmas present in the cytoplasm of transformed but not untransformed 3T cells. The time of inoculation ofS. mirum SMCA to 3T3 cells and the passage level of 3T3 cells affected transformation.

Elimination of mycoplasmas from cell cultures by a novel soft agar technique

SummaryMycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12–21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1–3 days. In separate studies, it was shown that certainMycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas.

Uridine phosphorylase activity among the class mollicutes

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of14C-uracil from14C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.

Ureaplasma-eukaryotic cell interactions in vitro.

Ureaplasmas from six animal species were inoculated into cell cultures derived from human, simian and murine hosts. All ureaplasmas infected at least two of the cell cultures tested. Supplemental medium urea was not a requirement for cell culture infection, and Ureaplasma growth in cell cultures occurred in as little urea as 4 micrograms/ml, similar to findings in broth-propagated organisms. These findings are of potential significance due to an observation (HM Jernigan, Jr: Exp Eye Res 37:551, 1983) that certain tissues produce urea locally. This may explain why ureaplasmas can be isolated from tissues outside the urinary tract where large amounts of urea are not routinely encountered. Ureaplasmas generally increased from titers of approximately 10(2) to 10(4) to ones of 10(5) to 10(6), but occasionally 10(7), color changing-units/ml within 2 days. Apparently a cell culture product is required for Ureaplasma growth. In several systems, Ureaplasma infection persisted and Ureaplasma-infected cell cultures could be passaged.

Mycoplasma-derived leukocyte mitogens

It is well established that many species of mycoplasmas are potent mitogens for leukocytes from a variety of mammals including humans, rodents, and rabbits. The complexity of mycoplasma-leukocyte interactions necessitates the use of purified mycoplasma mitogens to understand the mechanisms and consequences of mycoplasma-induced leukocyte mitogenesis. This has been the objective of several groups and is the subject of this review. Mitogenic activity has been described for 13 of the 80 known species of mycoplasmas. This phenomenon has been recently extensively reviewed (5). Mycoplasmas are the etiologic agents of primary atypical pneumonia (PAP) in humans, atypical pneumonia and arthritis in rodents, rabbits, swine, ruminants, and fowl, and genitourinary and neurologic infections in humans and rodents. Although the consequences of mycoplasma-induced leukocyte mitogenesis in vivo are poorly understood, it is likely that it plays an important role in the etiology of atypical pneumonia (8), but not arthritis (4) in rodents. The mitogenic membrane fraction of Mycoplasma pulmonis, an etiologic agent of pneumonia in rats, and Concanavalin A both induced pneumonia in rats. These pneumonias were characterized by peribronchial mononuclear cell infiltrates typical of human disease induced by M. pneumoniae. This suggests that mitogenesis may be an important factor in PAP. Mycoplasma infection can be devastating in immunologic studies in vitro. Mycoplasmas are common cell culture contaminants; as many as 10-15% of cell lines in the United States, and up to 25% in Europe are infected. All of the most common cell culture contaminants, M. hyorhinis, M. orale, M. arginini, M. fermentans, and A. laidlawii, are leukocyte mitogens. In addition to being mitogenic, several species of mycoplasmas can induce the secretion of soluble immune-response mediators, including granulocyte/monocyte colony-stimulating factor and alpha interferon. Thus, the presence of mycoplasmas can lead to misinterpretation of immunologic data. M. hyorhinis, the most common cell culture contaminant, is highly mitogenic for lymphocytes from many species including human, mouse, and hamster. Mitogenic stimulation of leukocytes by intact infectious mycoplasmas or mycoplasma products is a complex process involving binding and direct stimulation of T cells, B cells, and macrophages, as well as indirect stimulation of lymphocytes by macrophages. Mycoplasmas bound to the plasma membranes of lymphoid and nonlymphoid tumor cell lines can also activate macrophages (5). Evaluation of the pathophysiologic consequences of mycoplasma induced leukocyte mitogenesis is further complicated by the ability of some species of mycoplasmas to stimulate multiple leukocyte types, such as T cells, B cells, macrophages, and NK cells. Since individual species of mycoplasmas can be mitogenic for multiple leukocyte types, they may possess either individual polyspecific mitogens able to stimulate many different target cell types or, alternatively, multiple monospecific mitogens, or both. Purification of mycoplasma mitogens is crucially needed to understand the nature and consequences of mycoplasmainduced mitogenesis. The data presented below describe our efforts to isolate rodent leukocyte mitogens from M. hyorhinis. Although mycoplasmas are capable of free living, they are often associated with host cells as parasites on the outer plasma membrane surface. The adsorption of mycoplasmas to host plasma membranes is a complex phenomenon that may be mediated by association with membrane glycoproteins, by hydrophobic interactions, or both. The association of mycoplasmas or mycoplasma products with host membranes is a necessary first step in mitogenesis. Many species of mycoplasmas are potent mammalian lymphocyte mitogens. These include mycoplasmas that are mitogenic for both human and rodent lymphocytes (M. hyorhinis, M. pneumoniae, M. fermentans, S. mirum, and A. laidlawii), as well as a variety of other mycoplasma species known to be mitogenic only for rodent lymphocytes (i.e., M. arthritidis and M. pulmonis).