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.     

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.     

Characterization of an IS Element in <Emphasis Type="Italic">Mycoplasma orale</Emphasis> That Is Highly Homologous to <Emphasis Type="Italic">M. fermentans</Emphasis> ISLE

In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L) structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two different mycoplasma species. Received: 17 April 2002 / Accepted: 9 July 2002     

Effects of Mycoplasmal LAMPs on Receptor Responses to Steroid Hormones in Mammalian Cells

Many individuals are chronically infected or parasitically colonized with mycoplasmas in their respiratory or urogenital tracts without apparent clinical significance. However, prolonged close interaction between prokaryotic agents and eukaryotic host cells may gradually and significantly alter normal biological or physiological properties of infected hosts. Steroid hormones are associated with rates of cancer formation in human. The purpose of this study is to establish a sensitive reporting system to examine whether mycoplasmal infections affect biological responses to steroid hormones in mammalian cells. We established pMTV-CAT stably transfected cell lines to test the effect of mycoplasmal lipid-associated membrane proteins (LAMPs). Results showed that LAMPs (1 μg/ml) from seven different species of human mycoplasmas—M. penetrans, M. fermentans, M. genitalium, M. salivarium, M. pneumoniae, M. orale, and M. hominis—had an inhibitory effect on androgen receptor (AR) response to 5α-dihydrotestosterone (DHT) in the E82 transfectants. The inhibitory effect of mycoplasmal LAMPs appeared to be dose dependent. LAMPs from M. penetrans, M. genitalium, M. salivarium, M. pneumoniae, and M. orale also had an inhibitory effect on glucocorticoid receptor (GR) response to hormone dexamethasone (Dex) in TSU transfectants. In contrast, LAMPs from M. fermentans and M. hominis showed a stimulatory effect on the GR response to Dex in these TSU cells. The results suggest that colonization or chronic infection by mycoplasmas may significantly affect the responses of mammalian host cells to various steroid hormones, potentially affecting rates of cancer formation. Received: 2 January 2001/Accepted: 26 January 2001     

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.     

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.     

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.     

Antibody response to MfGL-II, a phosphocholine-containing major lipid of Mycoplasma fermentans membranes

The choline-containing phosphoglycolipid, MfGL-II, is the major polar lipid of Mycoplasma fermentans PG18. Anti-MfGL-II antisera raised in rabbits using the purified MfGL-II as an immunogen were employed in immunogold electron microscopic and immunofluorescence studies showing that MfGL-II is uniformly distributed and exposed on the cell surface of M. fermentans cells. The specificity of the antibodies was determined by immunostaining of lipid extracts separated by thin layer chromatography. The antibodies recognize lipids specific to M. fermentans but did not cross-react with lipid extracts of M. penetrans, M. capricolum, M. gallisepticum or Acholeplasma laidlawii. As phosphocholine almost completely abolished antibody interaction with MfGL-II in an ELISA assay it is suggested that the anti-MfGL-II repertoire is composed primarily of anti-phosphocholine antibodies. The anti-MfGL-II antisera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGL-II plays a major role in M. fermentans-host cell interaction.     

Animal model of Mycoplasma fermentans respiratory infection

Background

Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.

Results

Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.

Conclusions

Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.     

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.     

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.     

Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines

Several antibiotics were examined for their potential to eliminate mycoplasmas from contaminated cell cultures. Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma hyorhinis and Mycoplasma orale were effectively eliminated from experimentally contaminated mouse fibroblasts and mink epithelial cells by the use of the antibiotics minocycline and tiamutin. An elimination procedure was established, which involved the consecutive treatment of the cultures over a period of 3 weeks, followed by cell cloning. This procedure was effective when applied to cell lines which had been contaminated with unidentified and partially non-cultivable strains of mycoplasmas.     

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.     

Preparation of reference strains for validation and comparison of mycoplasma testing methods

Aims: To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)‐based methods in comparison to the conventional microbiological methods. Methods and Results: A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co‐cultured with suspension of Chinese hamster ovary (CHO) cells. Conclusions: Tested mycoplasma strains harvested at the exponential‐early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. Significance and Impact of the Study: This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT‐based mycoplasma testing methods.     

Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas

Unexpected cytolysis was encountered when nonactivated murine peritoneal macrophages were cultured with [³H]TdR-prelabeled syngeneic or allogeneic tumor cells at a 10:1 ratio. The level of specific cytolysis reached 70% within 48 hr of cocultivation. Similar killing was observed whether the macrophages were derived from untreated, thioglycollate-treated, or germ-free mice. Cytolytic activity was also demonstrated when bone marrow-derived or peritoneal macrophages from 9- and 5-day in vitro cultures, respectively, were employed rather than freshly harvested peritoneal macrophages. Thus, the macrophage-mediated killing was neither the result of in vivo preactivation nor a consequence of the presence of lymphocytes in the assay. Moreover, macrophages derived from different strains caused similar effects. Our study revealed that the neoplastic target cell cultures susceptible to cytolysis by nonactivated macrophages were contaminated with mycoplasma. A mycoplasma was isolated from the supernatant of a culture of the A9HT fibrosarcoma line, identified as Mycoplasma orale, and cultivated. Addition of viable mycoplasma from that isolate to mixed cultures of thioglycollate-elicited macrophages and [³H]TdR-prelabeled mycoplasma-free target cells resulted in specific cytolysis of transformed A9 cells, but not of normal mouse fibroblasts. The level of macrophage-dependent cytolysis correlated with the number of viable mycoplasma cells added and was higher than that attained by activation with LPS at optimal concentration. Similar specific cytolysis was observed with heat-killed mycoplasmas. Our results demonstrate that mycoplasmas may cause selective macrophage-mediated cytolysis of neoplastic but not of normal target cells, perhaps via activation of the macrophages. It is suggested that undetected infection of experimental systems by mycoplasmas may account for some reports on lysis of neoplastic cells by nonactivated macrophages.     

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.     

Factors influencing microbiological assay of cell-culture mycoplasmas

Summary A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%).M. orale andA. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aerobic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains ofM. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas. These studies were supported in part by Contracts N01-AG-4-2865 and N01-AG-8-2117 from the National Institute on Aging and N01-GM-6-2119 from the National Institute of General Medical Sciences.     

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the “1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection” (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.     

O‐linked protein glycosylation in mycoplasma

Although mycoplasmas have a paucity of glycosyltransferases and nucleotidyltransferases recognizable by bioinformatics, these bacteria are known to produce polysaccharides and glycolipids. We show here that mycoplasmas also produce glycoproteins and hence have glycomes more complex than previously realized. Proteins from several species of Mycoplasma reacted with a glycoprotein stain, and the murine pathogen Mycoplasma arthritidis was chosen for further study. The presence of M. arthritidis glycoproteins was confirmed by high‐resolution mass spectrometry. O‐linked glycosylation was clearly identified at both serine and threonine residues. No consensus amino acid sequence was evident for the glycosylation sites of the glycoproteins. A single hexose was identified as the O‐linked modification, and glucose was inferred by ¹³C‐labelling to be the hexose at several of the glycosylation sites. This is the first study to conclusively identify sites of protein glycosylation in any of the mollicutes.     

Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

To understand the effects of the interaction between Mycoplasma and cells on the host cellular function, it is important to elucidate the influences of infection of cells with Mycoplasma on nuclear enzymes such as DNA Topoisomerase type I (Topo I). Human Topo I participates in DNA transaction processes and is the target of anti-cancer drugs, the camptothecins (CPTs). Here we investigated the mechanism by which infection of human tumor cells with Mycoplasma fermentans affects the activity and expression of cellular Topo I, and the anti-cancer efficacy of CPT. Human cancer cells were infected or treated with live or sonicated M. fermentans and the activity and expression of Topo I was determined. M. fermentans significantly reduced (by 80%) Topo I activity in the infected/treated tumor cells without affecting the level of Topo I protein. We demonstrate that this reduction in enzyme activity resulted from ADP-ribosylation of the Topo I protein by Poly-ADP-ribose polymerase (PARP-1). In addition, pERK was activated as a result of the induction of the MAPK signal transduction pathway by M. fermentans. Since PARP-1 was shown to be activated by pERK, we concluded that M. fermentans modified the cellular Topo I activity by activation of PARP-I via the induction of the MAPK signal transduction pathway. Moreover, the infection of tumor cells with M. fermentans diminished the inhibitory effect of CPT. The results of this study suggest that modification of Topo I activity by M. fermentans may alter cellular gene expression and the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capacity of Topo I antagonists.