Attachment of Mycoplasma hominis and M. orale to Human Diploid Lung Fibroblasts

The process of attachment of Mycoplasma hominis and M. orale to HAIN-55 cells, derived from normal embryonic human lung, was investigated quantitatively. The attachment reached its maximum within about 2–4 hr at 37 C and increased linearly as a function of the number of organisms present in the system. The relative attachment efficiency of M. hominis was approximately 1% under our experimental conditions. Trypsin and EDTA were effective in detaching particles of M. hominis and M. orale from the surfaces of HAIN-55 cells. Therefore it was suggested that some proteinaceous substance and salt bridges might be involved in the attachment of these mycoplasmas to HAIN-55 cells.     

Effect of Mycoplasmas on Apoptosis of 32D Cells Is Species-Dependent

We previously showed that mycoplasmal infection effectively prevented apoptosis of infected cells, whereas other researchers have indicated that mycoplasmal infection promoted apoptosis. To understand the mechanism underlying this discrepancy, five different species of mycoplasmas were investigated for their effects on apoptosis of interleukin (IL)-3–dependent 32D cells. Results revealed that Mycoplasma fermentans and M. penetrans effectively supported continuous growth of 32D cells after IL-3 withdrawal. M. fermentans was more potent than M. penetrans. This effect was achieved by way of preventing apoptosis and stimulating cell proliferation. On the contrary, M. hominis and M. salivarium accelerated apoptosis of 32D cells. M. genitalium had no significant effect on apoptosis. The RNase protection assay indicated that the proapoptotic and antiapoptotic mycoplasmas altered the expression of major apoptosis regulatory genes differently. The difference in apoptosis regulatory gene expression induced by different species of mycoplasmas might be accountable for their effects on host cell apoptosis.     

Cultivation of Mycoplasmas on Glass

Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.     

<Emphasis Type="Italic">Ureaplasma urealyticum</Emphasis> and <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> Sensitivity to Bacteriocins Produced by Two Lactobacilli Strains

The purpose of the present study was to determine the inhibitory activities of two bacteriocins, produced by lactobacilli, against genital mycoplasmas. In this study, infections produced by genital mycoplasmas were studied; of these, 1.3% were caused by Mycoplasma hominis, 10.7% by Ureaplasma urealyticum and 5.6% by U. urealyticum + M. hominis. U. urealyticum was isolated from 75 out of 123 patients with genital mycoplasmas, while M. hominis was isolated from 9 patients (7.3%) and both U. urealyticum and M. hominis from 39 patients (31.7%). Bacteriocins, L23 and L60, produced by Lactobacillus fermentum and L. rhamnosus, respectively, appear to be two novel inhibitors of bacterial infection with potential antibacterial activity. Both bacteriocins proved to be active against 100% of strains tested; MICs of bacteriocin L23 ranged between 320 and 160 UA ml⁻¹ for 78% of the M. hominis strains and between 320 and 80 UA ml⁻¹ for 95% of the U. urealyticum strains. In addition, bacteriocin L60 was still active at 160 UA ml⁻¹ for a high percentage (56%) of M. hominis strains, and at 80 UA ml⁻¹ for 53% of the U. urealyticum strains. Interestingly, these antimicrobial substances produced by lactobacilli showed an inhibitory activity against genital mycoplasmas even when diluted. Altogether, our study indicates that the bacteriocins, L23 and L60, are good candidates for the treatment or prevention of genital infections in women.     

Mycoplasma penetrans infections and seroconversion in patients with AIDS: identification of major mycoplasmal antigens targeted by host antibody response

We examined Mycoplasma penetrans-specific antibodies in sera of five male homosexual AIDS patients from whom M. penetrans was isolated during the disease process. No consistent immune reaction pattern could be recognized in Western blot using whole cell proteins. Serum samples obtained prior to M. penetrans isolation reacted with a number of M. penetrans proteins, most likely due to non-specific cross-reactions. Further analysis revealed that patients produced prominent antibody reaction to lipid-associated membrane proteins (LAMPs) of M. penetrans at the time of mycoplasma isolation, which could not be observed for serum samples obtained prior to M. penetrans isolation. The positive antibody reaction was mainly directed against two major LAMPs of M. penetrans with molecular mass of 35 and 38 kDa and produced a distinctive pattern of positive immunoreaction bands. Our observation suggested that, comparing with whole mycoplasmal proteins, LAMPs were more specific target antigens in serological assays for M. penetrans infection.     

Differential Responses of Single Cells and Aggregates of Mycoplasmas to Ultraviolet Irradiation

Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.     

Behavior of Mycoplasma hominis in a Human Diploid Cell Culture System

The behavior of Mycoplasma hominis in normal human embryonic lung fibroblast (HAIN-55) cell cultures was investigated. Multiplication patterns of cell-associated mycoplasmas and of extracellular mycoplasmas in the HAIN-55 cultures depended upon the size of the inoculum. This relationship did not vary with the number of days the cells had been cultured, nor with the number of HAIN-55 cell passages. The maximum mycoplasmal growth was obtained with inoculum sizes of 10⁵ to 10⁶ colony-forming units (CFU)/ml. The recovery of mycoplasmas decreased rapidly with inoculum size beyond 10⁷ CFU/ml, and growth of the HAIN-55 cells was inhibited. Growth of the cells was also inhibited by the addition of the cytoplasmic fraction of Mycoplasma hominis.     

Comparison of Ciliostasis by Mycoplasmas in Mouse and Chicken Tracheal Organ Cultures

The cilia-stopping effect of mycoplasmas of human and various animal origin in mouse and chicken tracheal organ cultures was studied. From the results in mouse tracheal organ cultures, the mycoplasma strains tested were divided into three groups: Mycoplasma pulmonis m53, M. pulmonis JB, M. pulmonis OK, M. mycoides subsp. mycoides PG1 and M. gallisepticum S6 showed a strong cilia-stopping effect; M. pulmonis PG22, M. mycoides subsp. capri PG3, M. meleagridis 19729, M. neurolyticum Type A and M. arthritidis PG6 showed a mild effect; and M. pneumoniae FH, M. salivarium Hup, M. hominis type 1-C and M. orale N-C of human origin and Acholeplasma laidlawii PG8 showed a weak effect. On the other hand, in chicken tracheal organ cultures, only M. gallisepticum S6 showed a strong effect, M. meleagridis 19729 was affected to a lesser degree, and other mycoplasma strains showed a weak or no effect. The results indicate that some murine and poultry mycoplasmas showed a cilia-stopping tendency in mouse and chicken tracheal organ cultures, respectively, while human mycoplasmas showed weak or little effect in both organ cultures. In mouse tracheal organ cultures, M. pulmonis m53 treated with heat, trypsin or formaldehyde, and the sterile filtrate of an m53 broth culture showed no cilia-stopping effect. The relationship of the pathogenicity of mycoplasmas for their natural hosts to that for cultured respiratory cells is discussed.     

Spread and control of mycoplasmal infection of cell cultures

Summary Environmental sampling was performed during trypsinization and passage of 3T-6 cell cultures that contained a mean of 4.3×10⁷ colony forming units (CFU) per ml supernatant ofA. laidlawii. The lip of the culture flask and the outside of the used pipet were always heavily contaminated. The outside of the culture flask (3/7), the work surface (8/12) and the outside of a pan of disinfectant (4/5) were regularly contaminated with mycoplasmas. Airborne mycoplasmas were detected eight of 32 times (25%) by settling plates; simultaneous forced-air samplers by two different methods were always negative. The technician’s hands were contaminated two of 15 samples. When hands were contaminated, more contamination was detected in the environment. Droplets ofA. laidlawii andM. orale inoculated onto work surfaces survived drying for a minimum of 3 days, even in laminar airflow cabinets. Twenty-five of 31 (80.6%) cell culture technicians carriedM. salivarium in their throats; only two carriedM. orale. It is concluded that mycoplasma-infected cultures are the most common source of further infection. Recommendations for prevention and control of mycoplasmal infection are listed. These studies were supported in part by Contract No. 1-GM-2112 from the National Institute of General Medical Sciences, Contract No. 1-CB-23868 from the National Cancer Institute, General Research Support Grant 5-S01-RRO5582 from Research Resources, National Institutes of Health, and by a Grant-in-Aid from the State of New Jersey.     

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell’s interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.     

Effect of mycoplasma infection on pyruvate dehydrogenase complex activity of normal and pyruvate dehydrogenase complex-deficient fibroblasts

The fermentative mycoplasmas A. laidlawii JS, M. hyorhinis DBS-50, M. hyorhinis GDL and M. pneumoniae FH have very high apparent activities of pyruvate dehydrogenase (PDH) (EC 1.2.4.1) and pyruvate dehydrogenase complex (PDHC). Infection of normal and PDHC-deficient fibroblasts with these mycoplasma species resulted in a marked increase of the specific activity of these two enzymes, and under certain conditions could conceal the enzymatic defect. The non-fermentative mycoplasmas M. salivarium VV and M. arthritidis PG-6 have very low apparent activities of these two enzymes. Normal fibroblasts infected with non-fermentative mycoplasmas could appear as deficient in these two enzymes. The degree of interference depends on the number of mycoplasmas associated with the harvested cells. Besides the mycoplasma species, this depends (1) on the duration of infection which determines mycoplasmal titers and also can have a killing effect on both host cells and/or mycoplasmas; (2) harvest of the cells by scraping or trypsinization; (3) centrifugal force used in the collection of the cells; (4) washing and the inherent mechanical treatment; and (5) other possibilities.     

生殖支原体脂质相关膜蛋白激活核因子κB诱导人单核细胞表达前炎症细胞因子及凋亡

为了解生殖支原体(Mg)潜在的致病性及其脂质相关膜蛋白(LAMPs)诱导人单核细胞(THP-1)凋亡及表达前炎症细胞因子(CKs)的分子机制,用Mg提取的LAMPs刺激THP-1细胞,以ELISA法和RT-PCR方法分析CKs产生和其mRNA的表达。不同试实验组的细胞经AnnexinV联合PI染色后通过流式细胞仪检测细胞凋亡。采用EMSA方法检测LAMPs处理的THP-1细胞中核转录因子kappaB(NF-κB)的激活,并分析NF-κB抑制剂二硫代氨基甲酸吡咯烷(pyrrolidine dithiocoarbamate,PDTC)对LAMPs处理的THP-1细胞产生CKs的量和其mRNA表达及细胞凋亡的影响。LAMPs能以时间和剂量依赖方式刺激THP-1细胞产生TNF-α、IL-1β和IL-6,且能激活NF-κB诱导THP-1细胞表达CKs的mRNA及发生凋亡,PDTC能显著抑制CKs的mRNA表达水平和细胞凋亡。由于LAMPs能激活NF-κB诱导THP-1细胞表达CKs及产生细胞凋亡,因而可能是一个重要的致病因素。     

Detection and identification of mycoplasmas by amplification of rDNA

Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR primers, several mycoplasmas including the four human parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale were detected. This DNA amplification was restricted to species of the genus Mycoplasma while no cross-reaction was observed with DNA from other bacteria and eukaryotic cells. Subsequent analysis of amplified products by either specific oligonucleotide hybridization or dideoxy sequencing specified the identity of the detected mycoplasmas. This method offers a highly discriminating and sensitive assay for the direct detection and identification of these microorganisms without the need for prior cultivation.     

Immunologic reactions of rabbit anti-Mycoplasma arthritidis serum with in vitro cultivated rat synovial cells

Summary The pathogenesis of the intra-articular, arthritic-inflammatory reaction caused byMycoplasma arthritidis in susceptible rats and mice is poorly understood. To investigate this problem, synovial cells from normal Sprague-Dawley rats were cultured and studied in vitro. These cells continued to produce hyaluronic acid as measured by viscosity and chemical assays. Normal synovial cells were treated with rabbit serum specimens taken before and after immunization withM. arthritidis. Cytotoxicity assays indicated that the cells were killed in the presence of rabbit anti-M. arthritidis serum but not with preimmunization serum specimens. The anti-M. arthritidis serum was not cytotoxic to monolayer cultures of HEp-2, Vero, or L-cells. Antiserum produced in response toM. fermentans, M. hominis, andM. pulmonis did not produce a cytotoxic effect on the cultured synovial cells. From immunofluorescence studies it was demonstrated that the interactions occurred between the rabbit anti-M. arthritidis serum and synovial cell surface antigens. Extreme precautions were taken to prevent mycoplasmal contamination of rats and the synovial cells in culture. These observations would appear to support previous reports implicating mycoplasmas as biological triggering mechanisms of autoimmune reactions. This research was supported in part by funds from the National Science Foundation, Grant DPP72-05787, and the U.S. Veterans Administration.     

Development of Flouroquinolone (Ciprofloxacin) Resistance in Mycoplasma hominis in the Presence of HeLa Cells

The effect of cocultivation of eukaryotic HeLa cells and Mycoplasma hominis mycoplasma on the resistance of the latter to fluoroquinolones (ciprofloxacin) was examined. It was shown that cocultivation of the M. homonisand HeLa cells during 24 h with subsequent addition of ciprofloxacin resulted in an increase of the micoplasma resistance to this antimicrobial agent. In the M. hominis cells cultivated in the presence of HeLa cells and the increasing concentration of ciprofloxacin mutations in the parC gene were observed only at low concentrations of the antimicrobial agent, while mutations in the gyrA gene were never detected. A gradual elevation of ciprofloxacin concentration up to 10 g/ml resulted in the reversion of the parC mutations in mycoplasmas. Mycoplasma cells resistant to high flouroquinolone concentrations and isolated after cocultivation with the HeLa cells were characterized by the wild-type genotype in respect of the gyrA and parC genes. It was shown that infection of HeLa cells resulted in the appearance of genome rearrangements in M. hominis cells.     

Membrane-associated hemolysin activities in mycoplasmas

Abstract Mycoplasmas are cell wall-less organisms that require membrane precursors for growth. Activities involved in the acquisition of these materials have been hypothesized as mycoplasmal virulence factors because of the effects these activities might have on host cells. Twenty-nine species or strains of mycoplasmas were examined for membrane-associated hemolysis activity similar to that previously identified in Mycoplasma pulmonis . Membrane-associated hemolytic activity was found in most mycoplasma species, but the amount of activity varied between and within the species. All of the arginine-utilizing mycoplasmal species, one M. pulmonis strain, one Acholeplasma species, and the intracellular human pathogens M. penetrans and M. fermentans ssp. incognitus were devoid of activity. The wide distribution of the membrane-associated hemolysis activity suggests that it may be important to the survival of the organism.