Molecular exploration for Mycoplasma amphoriforme, Mycoplasma fermentans and Ureaplasma spp. in patient samples previously investigated for Mycoplasma pneumoniae infection.

OBJECTIVES: To determine the presence and genotypic macrolide susceptibility of Mycoplasma amphoriforme, and the presence of Ureaplasma spp. and Mycoplasma fermentans among clinical samples from England previously investigated for Mycoplasma pneumoniae. METHODS: Quantitative and conventional PCR methods were used to retrospectively screen a collection of 160 clinical samples previously submitted to Public Health England (PHE) for the detection of M. pneumoniae between October 2016 and December 2017. Samples which were positive for M. amphoriforme DNA were further investigated for mutations associated with genotypic macrolide resistance by sequencing domain V of the 23s rRNA. RESULTS: M. amphoriforme was detected in 10/160 samples (6.3%), Ureaplasma parvum was detected in 4/160 samples (2.5%), and M. fermentans was not detected in any samples (0/160). Of the nine individuals (two samples were from the same patient) in which M. amphoriforme was detected, eight were male (age range 10-60 years) and one was female (age range 30-40 years). One individual with cystic fibrosis was positive for both M. amphoriforme and U. parvum. All M. amphoriforme DNA was genotypically susceptible to macrolides. CONCLUSIONS: Mycoplasma amphoriforme was found in clinical samples, including lower respiratory tract samples of patients with pneumonia. In the absence of other respiratory pathogens, these data suggest a potential role for this organism in human disease, with no evidence of acquired macrolide resistance. Ureaplasma parvum was detected in cerebrospinal fluid and respiratory tract samples. These data suggest that there is a need to consider these atypical respiratory pathogens in future diagnostic investigations.

Sulfur compounds block MCP-1 production by Mycoplasma fermentans-infected macrophages through NF-κB inhibition.

BACKGROUND AND AIMS: Hydrogen sulfide (H2S), together with nitric oxide (NO) and carbon monoxide (CO), belongs to a family of endogenous signaling mediators termed "gasotransmitters". Recent studies suggest that H2S modulates many cellular processes and it has been recognized to play a central role in inflammation, in the cardiovascular and nervous systems. By infecting monocytes/macrophages with Mycoplasma fermentans (M.F.), a well-known pro-inflammatory agent, we evaluated the effects of H2S. METHODS: M.F.-infected cells were analyzed by ELISA and real time RT-PCR to detect the M.F. effects on MCP-1 and on MMP-12 expression. The role of two different H2S donors (NaHS and GYY4137) on MF-infected cells was determined by treating infected cells with H2S and then testing the culture supernatants for MCP-1 and on MMP-12 production by ELISA assay. In order to identify the pathway/s mediating H2S- anti-inflammatory activity, cells were also treated with specific pharmaceutical inhibitors. Cytoplasmic and nuclear accumulation of NF-κB heterodimers was analyzed. RESULTS: We show that H2S was able to reduce the production of pro-inflammatory cytokine MCP-1, that was induced in monocytes/macrophages during M.F. infection. Moreover, MCP-1 was induced by M.F. through Toll-like receptor (TLR)-mediated nuclear factor-κB (NF-κB) activation, as demonstrated by the fact that TLR inhibitors TIRAP and MyD88 and NF-κB inhibitor IKK were able to block the cytokine production. In contrast H2S treatment of M.F. infected macrophages reduced nuclear accumulation of NF-κB heterodimer p65/p52. CONCLUSIONS: Our data demonstrate that under the present conditions H2S is effective in reducing Mycoplasma-induced inflammation by targeting the NF-κB pathway. This supports further studies for possible clinical applications.

[Antimycoplasmic Activity of Fermentation Broth of Trichoderma harzianum Rifai F-180, an Organism Producing L-Lysine-α-Oxidase, an Antitumor and Antiviral Enzyme].

A concentrate of the fermentation broth of Trichoderma harzianum Rifai F-180, an organism producing L-lysine-α-oxidase, an antitumor and antiviral enzyme, with the activity in the fermentation broth of 0.54-0.56 U/mI was recovered. The effect of the concentrate on the mycoplasmas growth was investigated for the first time. Two representatives of Mycoplasmafaceae, i.e. Mycoplasma hominis and Mycoplasma fermentans and one representative of Aholeplasmataceae. i. e. Aholeplasma laidlawii were used. It was shown that the fermentation broth inhibited the growth of Mycoplasma hominis after the preliminary exposure. The inhibition rate depended on the mycoplasma inoculation dose and the fermentation broth concentration.

Comparative in vitro susceptibilities of human mycoplasmas and ureaplasmas to a new investigational ketolide, CEM-101.

MICs were determined for an investigational ketolide, CEM-101, and azithromycin, telithromycin, doxycycline, levofloxacin, clindamycin, and linezolid against 36 Mycoplasma pneumoniae, 5 Mycoplasma genitalium, 13 Mycoplasma hominis, 15 Mycoplasma fermentans, and 20 Ureaplasma isolates. All isolates, including two macrolide-resistant M. pneumoniae isolates, were inhibited by CEM-101 at < or = 0.5 microg/ml, making CEM-101 the most potent compound tested.

Comparative in vitro activities of the investigational fluoroquinolone DC-159a and other antimicrobial agents against human mycoplasmas and ureaplasmas.

The in vitro susceptibilities of 151 unique clinical isolates of Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium, and Ureaplasma species to DC-159a, an investigational fluoroquinolone, in comparison with those to other agents were determined. Macrolides were the most active agents against M. pneumoniae and M. genitalium, whereas clindamycin was most active against M. hominis. DC-159a MICs were or=99.9% of the inoculum at 24 h for 2 isolates. The excellent in vitro activity of DC-159a demonstrates its potential for use in the treatment of infections due to mycoplasmas and ureaplasmas.

Comparative in vitro activities of investigational peptide deformylase inhibitor NVP LBM-415 and other agents against human mycoplasmas and ureaplasmas.

Peptide deformylase inhibitor LBM-415 and seven other drugs were tested against Mycoplasma pneumoniae (100 isolates), Mycoplasma hominis (20 isolates), Mycoplasma fermentans (10 isolates), and Ureaplasma species (50 isolates). LBM-415 was active against M. pneumoniae (MICs,

Antimycoplasmal activity of hydroxytyrosol.

The aim of this study was to investigate the in vitro antimycoplasmal activity of hydroxytyrosol. Twenty strains of Mycoplasma hominis, three strains of Mycoplasma fermentans, and one strain of Mycoplasma pneumoniae were used. For M. pneumoniae, M. hominis, and M. fermentans, the MICs were 0.5, 0.03 (for 90% of the strains tested), and 0.25 microg/ml, respectively.

Comparative in vitro susceptibilities and bactericidal activities of investigational fluoroquinolone ABT-492 and other antimicrobial agents against human mycoplasmas and ureaplasmas.

We determined in vitro susceptibilities for ABT-492 and other antimicrobials against Mycoplasma pneumoniae, Mycoplasma fermentans, Mycoplasma hominis, and Ureaplasma species. ABT-492 MICs were < or =1 microg/ml, and the agent was bactericidal against selected isolates of M. pneumoniae and M. hominis. ABT-492 has potential for treatment of infections due to these microorganisms.

Mutations in 23S rRNA account for intrinsic resistance to macrolides in Mycoplasma hominis and Mycoplasma fermentans and for acquired resistance to macrolides in M. hominis.

The mechanisms of intrinsic resistance of Mycoplasma hominis to 14- and 15-membered macrolides were investigated in comparison with those of M. pneumoniae, which is naturally susceptible to macrolides. Radiolabeled erythromycin was not accumulated by M. hominis PG21, but addition of an ABC transporter inhibitor increased the level of erythromycin uptake more than two times, suggesting the existence of an active efflux process. The affinity of [(14)C]erythromycin to ribosomes isolated from M. hominis was dramatically reduced relative to that to ribosomes isolated from M. pneumoniae. The nucleotide sequences of 23S rRNA of both ribosomal operons rrnA and rrnB and ribosomal proteins L4 and L22 of M. hominis were obtained. Compared to the sequence of M. pneumoniae, M. hominis harbored a G2057A transition in its 23S rRNA sequence, as did M. fermentans, another mycoplasma that is erythromycin resistant. An additional C2610U change was also found in the sequence of M. hominis. Moreover, two M. hominis clinical isolates with acquired resistance to 16-membered macrolides were examined for mutations in domain II and domain V of 23S rRNA and in ribosomal proteins L4 and L22. Compared to the sequence of reference strain PG21, one isolate harbored a A2059G transition and a C2611U transition in one of the two rrn operons, while the other one was mutated only at position 2059, also on the same operon. No mutation was found in the two ribosomal protein sequences. Overall, the present study is an exhaustive characterization of the intrinsic resistance of M. hominis to 14- and 15-membered macrolides and the first description of mycoplasma clinical isolates resistant to macrolide, lincosamide, and streptogramin antibiotics harboring a mutation at position 2611 in the 23S rRNA.

Susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to membrane-active peptides and enrofloxacin in human tissue cell cultures.

Mycoplasmas, which are bacteria that are devoid of a cell wall and which belong to the class Mollicutes, are pathogenic for humans and animals and are frequent contaminants of tissue cell cultures. Although contamination of cultures with mycoplasma can easily be monitored with fluorescent dyes that stain DNA and/or with molecular probes, protection and decontamination of cultures remain serious challenges. In the present work, we investigated the susceptibilities of Mycoplasma fermentans and Mycoplasma hyorhinis to the membrane-active peptides alamethicin, dermaseptin B2, gramicidin S, and surfactin by growth inhibition and lethality assays. In the absence of serum, the four peptides killed mycoplasmas at minimal bactericidal concentrations that ranged from 12.5 to 100 microM, but in all cases the activities were decreased by the presence of serum. As a result, under standard culture conditions (10% serum) only alamethicin and gramicidin S were able to inhibit mycoplasma growth (MICs, 50 microM), while dermaseptin B2 and surfactin were ineffective. Furthermore, 8 days of treatment of HeLa cell cultures experimentally contaminated with either mycoplasma species with 70 microM enrofloxacin cured the cultures of infection, whereas treatment with alamethicin and gramicidin S alone was not reliable because the concentrations and treatment times required were toxic to the cells. However, combination of alamethicin or gramicidin S with 70 microM enrofloxacin allowed mycoplasma eradication after 30 min or 24 h of treatment, depending on the mycoplasma and peptide considered. HeLa cell cultures experimentally infected with mycoplasmas should prove to be a useful model for study of the antimycoplasma activities of antibiotics and membrane-active peptides under conditions close to those found in vivo.

Antisignature oligonucleotides and their analogs as inhibitors of mollicutes-cofactors of HIV.

Inhibition of mollicutes by synthetic oligonucleotides and their analogs complementary to specific "signature" regions of 16S rRNA and corresponding sequences of ribosomal operon DNA was studied. It was shown that antisignature oligonucleotides inhibited transcription in vitro for above 79% interacting specifically with ribosomal operon and non-specific with DNA-dependent RNA-polymerase. The inhibition efficiency depended on oligonucleotide sequence and type of modification. Translation in vitro was suppressed most efficiently (up to 60%) by oligonucleotides complementary to 3'-end region of 16S rRNA, also depending on their modification. Translation in vivo was inhibited most efficiently (up to 73%) by thiophosphate analogs of oligonucleotides complementary to sequences 499-507 and 523-532 of 16S rRNA responsible for binding of ribosomal "core" protein S4 starting the assembly of 30S ribosome subunit. With the simultaneous use of the last two oligonucleotides, the growth of mollicutes in SM IMV-72 medium rich in exogenous sources of nucleosides was suppressed for over 90%. It is supposed that under conditions where mollicutes have no free access to starting materials for their own synthesis of nucleic acid these nucleotides could suppress microorganisms completely. Antisignature oligonucleotides are considered as superspecific agents not leading to the development of resistance of mollicutes and believed to be the main future remedy against diseased caused by microorganisms lacking the system of nucleoside synthesis.

Observations on the possible origin of Mycoplasma fermentans incognitus strain based on antibiotic sensitivity tests.

Mycoplasma fermentans (incognitus strain), isolated during transfection studies in NIH/3T3 cells with DNA extracted from Kaposi's sarcoma tissue from a patient suffering from AIDS, showed high levels of resistance to numerous aminoglycoside antibiotics (MICs > 250 to > 500 mg/L) and in this respect matched the aminoglycoside resistance patterns of M. fermentans strains isolated recently from tissue culture cells. Two M. fermentans strains isolated from urine deposits from AIDS patients, without the use of cell cultures, and six M. fermentans isolates from patients with acute respiratory infections differed markedly from the incognitus strain, in that they were sensitive to aminoglycosides (MIC range 0.25-25 mg/L). A much older strain (K7) isolated from leukaemic bone marrow tissue in the 1960s, with the aid of cell cultures, was resistant to streptomycin (MIC > 250 mg/L) but sensitive to other aminoglycosides (MIC range 0.625-6.25 mg/L). These results suggest that, although the aminoglycoside-resistance in M. fermentans incognitus could have developed during the isolation process or through treatment of the AIDS patient with aminoglycosides, in view of the unusual manner in which the strain was isolated, its multiple aminoglycoside resistance and the fact that other M. fermentans strains isolated from AIDS patients, without the use of tissue culture cells, were aminoglycoside-sensitive, it is more likely that it was a tissue culture contaminant.

[The validation of the possible use of monosugars and 6-azacytidine for the elimination of Mollicutes associated with HIV/AIDS from the human urogenital tract]. / Obhruntuvannia mozhlyvosti zastosuvannia monotsukriv i 6-azatsytydynu dlia eliminatsiï z urohenital'noho traktu liudyny molikutiv, iaki asotsiiuiut' z VIL/SNIDom.

A search for the methods new in principle which should block and eliminate AIDS-associated mycoplasmas was carried out. This work was conducted in two ways: 1) inhibition of vital activity of Mycoplasma fermentans PG-18 and Acholeplasma laidlawii PG-8 by 6-azacytidine; 2) establishment of carbohydrate composition of receptors for these mycoplasmas aimed at the competitive elimination of these microorganisms from urogenital tract of a man using carbohydrates. It is established that a 50%-inhibiting concentration of 6-azacytidine was 23.4 micrograms/ml for M. fermentans PG-18 and 62.5 micrograms/ml for A. laidlawii PG-8. alpha-D-glucose and N-acetylneuramine acid are two terminal carbohydrates that can serve as receptors for M. fermentans on human mucous membranes while D-mannose and N-acetyl-D-glucosamine for A. laidlawii PG-8. alpha-D-glucose in concentration 75 mM and N-acetylneuramine acid in concentration 150 mM competitively inhibit reception of M. fermentans on mucosae, while D-mannose in concentration 150 mM and N-acetyl-D-glucosamine in concentration 75 mM are antireceptor substances for A. laidlawii.

[The inhibition of translation in vivo in Mollicutes by thiophosphate analogs of oligodeoxyribonucleotides]. / Ingibirovanie transliatsii in vivo u mollikutov tiofosfatnymi analogami oligodezoksiribonukleotidov.

It is shown that the concentration of "antisignature" phosphorothioate analogs of oligodeoxynucleotides, complementary to the region of 165 rRNA Acholeplasma laidlawii PG-8 and Mycoplasma fermentans PG-18 responsible tor binding with ribosomal protein S4 being 0.5--1 microM synthesis of proteins in vivo decreases to 70%. A model of mechanisms is suggested to block oligonucleotides of the process of in vivo translation in mollicutes by "antisignature" phosphorothioate analogs. The advantages of the use of antisense oligonucleotides complementary to functionally significant plots of 16S rRNA to inhibit the in vivo translation are discussed in comparison with oligonucleotides, 5-nontranslated regions of mRNA serving a target for them.

Antibiotic susceptibility of Mycoplasma fermentans strains from various sources and the development of resistance to aminoglycosides in vitro.

Mycoplasma fermentans strains reputedly from human infections or tissue culture cells were much more susceptible to azithromycin than to clarithromycin or erythromycin. Lincomycin, clindamycin and several tetracyclines also exhibited good mycoplasmastatic activity but mycoplasmacidal concentrations were substantially greater than the MICs. Ciprofloxacin was the most active of three fluoroquinolones tested and was mycoplasmacidal at concentrations close to the MIC. Tiamulin and mupirocin were also very active. Synergy with specific M. fermentans antiserum plus guinea-pig complement was not observed with any class of antibiotic although the number of viable mycoplasmas was markedly reduced by the combined immunological components. Marked differences in susceptibility to various aminoglycosides were observed. Human strains isolated in cell-free media up to 1967 were aminoglycoside susceptible (MIC range 0.5-25 mg/L) but recent human isolates and strains isolated from tissue culture cells often showed either single or multiple aminoglycoside resistance (MIC > 500 mg/L). Two aminoglycoside-susceptible strains developed resistance to streptomycin or neomycin (> 500 mg/L) within five passages in broth containing streptomycin or neomycin, respectively. Resistance to tobramycin, kanamycin or gentamicin emerged after seven, eight and 14 cycles of exposure to the respective antibiotic. Streptomycin resistance was associated with a five-fold increase in resistance to tobramycin. Neomycin-, kanamycin-, gentamicin- and tobramycin-resistant variants showed mutual cross-resistance but remained susceptible to streptomycin. Induced resistance persisted for at least 17 passages in aminoglycoside-free broth. The use of aminoglycosides in human medicine and the frequent inclusion of some of these drugs in tissue cell cultures to combat bacterial and mycoplasmal contamination might account for the aminoglycoside resistance of recent M. fermentans isolates.

Identification of phosphocholine-containing glycoglycerolipids purified from Mycoplasma fermentans-infected human helper T-cell culture as components of M. fermentans.

Previously, we have reported the occurrence of novel phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) in human helper T-cell (MT-4 cell line) (Mustuda et al, Glycoconjugate J. 10:340). However, the GGPLs disappeared from the MT-4 after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent-untreated Mt-4 cell culture produced mycoplasma-like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated Mt-4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL-I and GGPL-III. Thin-layer chromatography-mass spectrometry and immunological analyses showed that these glycoglycerolipid which were derived from the isolated M. fermentans were identical with GGPL-I and GGPL-III previously obtained. This is the first report that shows mycoplasma has phosphocholine-containing glycoglycerolipids.

Antibiotic susceptibilities of AIDS-associated mycoplasmas.

Because mycoplasmas may be a cofactor in the progression of human immunodeficiency virus infection to AIDS, their susceptibilities to antibiotics need to be known in the event that appropriate therapy is required. The mycoplasmas studied were a stock culture strain of Mycoplasma fermentans, two strains of M. fermentans isolated from patients with AIDS, M. fermentans var. incognitus, Mycoplasma penetrans, and Mycoplasma pirum. The antibiotics tested were doxycycline, tetracycline, clindamycin, ofloxacin, erythromycin, azithromycin, and clarithromycin at levels consistent with the attainable levels in serum. By the macrodilution metabolic inhibition method, all six mycoplasma strains were susceptible to doxycycline, tetracycline, clindamycin, ofloxacin, azithromycin, and clarithromycin. M. penetrans was susceptible to erythromycin. The M. fermentans strains and M. pirum were resistant to erythromycin. The macrodilution metabolic inhibition method results showed agreement with the Sensititre Gram Positive MIC Panel results for tetracycline, clindamycin, and erythromycin. MICs of clarithromycin for all six mycoplasma isolates tested were low, indicating susceptibility.

In vitro antibiotic susceptibility testing of different strains of Mycoplasma fermentans isolated from a variety of sources.

The in vitro susceptibilities to antibiotics of 24 strains of Mycoplasma fermentans (isolated from human immunodeficiency virus type 1-infected AIDS patients, non-AIDS patients with acute respiratory disease, and tissue culture) were determined. MICs for 90% of the strains tested (micrograms per milliliter) were obtained for chloramphenicol (1.25), ciprofloxacin (0.078), clindamycin (0.078), doxycycline (0.625), erythromycin (> 10), gentamicin (> 10), levofloxacin (0.078), lincomycin (0.156), streptomycin (> 10), and tetracycline (0.625).

Susceptibility of mycoplasmas to antimicrobial agents: clinical implications.

Mycoplasmas are susceptible to antimicrobial agents that affect DNA, RNA, protein synthesis, or the integrity of the cell membrane. Mycoplasmas are not susceptible to agents that interfere with synthesis of folic acid or that act on the cell wall. Tetracyclines, erythromycin, clindamycin, chloramphenicol, aminoglycosides, and fluoroquinolones have been shown to have activity against one or more mycoplasmal species. Tetracycline-resistant isolates of Mycoplasma hominis and Ureaplasma urealyticum contain DNA sequences homologous to the streptococcal determinant tetM. Resistance to tetracycline in vitro is associated with failure of tetracycline treatment to eradicate M. hominis and U. urealyticum from the human urogenital tract.