Susceptibilities of Mycoplasma hominis, M. pneumoniae, and Ureaplasma urealyticum to GAR-936, dalfopristin, dirithromycin, evernimicin, gatifloxacin, linezolid, moxifloxacin, quinupristin-dalfopristin, and telithromycin compared to their susceptibilities to reference macrolides, tetracyclines, and quinolones.

The susceptibilities of Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum to eight new antimicrobial agents were determined by agar dilution. M. pneumoniae was susceptible to the new glycylcycline GAR-936 at 0.12 microg/ml and evernimicin at 4 microg/ml, but it was resistant to linezolid. It was most susceptible to dirithromycin, quinupristin-dalfopristin, telithromycin, reference macrolides, and josamycin. M. hominis was susceptible to linezolid, evernimicin, and GAR-936. It was resistant to macrolides and the ketolide telithromycin but susceptible to quinupristin-dalfopristin and josamycin. U. urealyticum was susceptible to evernimicin (8 to 16 microg/ml) and resistant to linezolid. It was less susceptible to GAR-936 (4.0 microg/ml) than to tetracycline (0.5 microg/ml). Telithromycin and quinupristin-dalfopristin were the most active agents against ureaplasmas (0.06 microg/ml). The new quinolone gatifloxacin was active against M. pneumoniae and M. hominis at 0.12 to 0.25 microg/ml and active against ureaplasmas at 1.0 microg/ml. The MICs of macrolides were markedly affected by pH, with an 8- to 32-fold increase in the susceptibility of M. pneumoniae as the pH increased from 6.9 to 7.8. A similar increase in susceptibility with increasing pH was also observed with ureaplasmas. Tetracyclines showed a fourfold increase of activity as the pH decreased 1 U, whereas GAR-936 showed a fourfold decrease in activity with a decrease in pH.

Sparfloxacin selects gyrase mutations in first-step Mycoplasma hominis mutants, whereas ofloxacin selects topoisomerase IV mutations.

The role of mutations in the genes for GyrA and ParC in quinolone resistance in Mycoplasma hominis was studied. Selection with sparfloxacin gave mutations at GyrA83 (Ser-->Leu; Escherichia coli numbering) or GyrA87 (Glu-->Lys), and mutants had increased levels of resistance to sparfloxacin (8- to 16-fold) but not to ofloxacin. Selection with ofloxacin gave changes at ParC80 (Ser-->Ile) or ParC84 (Glu-->Lys), and mutants were four- to eightfold more resistant to ofloxacin but not to sparfloxacin. Selection of second-step mutants from strains with ParC mutations with either quinolone yielded double mutants with additional mutations at GyrA83 (Ser-->Trp or Ser-->Leu) or GyrA87 (Glu-->Lys). Second-step selection of GyrA mutants gave additional mutations at ParC80 (Ser-->Ile) or ParC84 (Glu-->Lys). Two-step mutants showed high levels of resistance to ofloxacin (MICs, 64 to 128 microg/ml) and moderate levels of resistance to sparfloxacin (MICs, 2 to 8 microg/ml). The primary target of ofloxacin in first-step mutants of Mycoplasma hominis was ParC, whereas that for sparfloxacin was GyrA.

Mycoplasma orale infection affects K+ and Cl- currents in the HSG salivary gland cell line.

The relations between K+ channel and Cl- channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K+ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K+ channels and K+ channel expression as estimated by ionomycin- or hypotonically induced K+ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl- channels, but only the latter decrease was statistically significant. Also, Cl- currents could be elicited more frequently than K+ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K+ channels relatively more than Cl- channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurred in vivo.

Susceptibilities of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum to a new quinolone, trovafloxacin (CP-99,219).

The susceptibilities of mycoplasmas to a new quinolone, trovafloxacin (CP-99,219), were compared with those to sparfloxacin and ofloxacin. Mycoplasma pneumoniae was as susceptible to trovafloxacin (MIC = 0.25 microgram/ml) as to sparfloxacin and fourfold less susceptible to ofloxacin. Mycoplasma hominis was highly susceptible to trovafloxacin (MIC = 0.06 microgram/ml) and sparfloxacin (MIC = 0.03 microgram/ml) and less susceptible to ofloxacin (MIC = 0.5 microgram/ml). Ureaplasma urealyticum was most susceptible to trovafloxacin, with susceptibilities ranging from 0.06 to 0.5 microgram/ml compared with 0.25 to 1.0 microgram of sparfloxacin per ml and 1 to 4 micrograms of ofloxacin per ml.

Susceptibilities of Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum to new glycylcyclines in comparison with those to older tetracyclines.

The glycylcyclines are new tetracycline derivatives that include the N,N-dimethylglycylamido derivative of minocycline (DMG-MINO) and the N,N-dimethylglycylamido derivative of 6-demethyl-6-deoxytetracycline (DMG-DMDOT). The susceptibilities of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum to DMG-MINO, DMG-DMDOT, tetracycline, doxycycline, and minocycline were determined by the agar dilution method. The glycylcyclines with MICs at which 50% of the isolates are inhibited of 0.25 to 0.5 micrograms/ml for M. pneumoniae were two- to fourfold more active than tetracycline and had the same activity as minocycline and doxycycline. Tetracycline-susceptible M. hominis strains were four- to eightfold more susceptible to the glycylcyclines (0.12 to 0.25 micrograms/ml) than to tetracycline. Strains of M. hominis known to be resistant to tetracycline, doxycycline, and minocycline because of the tet(M) determinant were as susceptible to the glycylcyclines as the tetracycline-susceptible strains. For tetracycline-susceptible U. urealyticum strains, the glycylcyclines showed the same activity as tetracycline (MICs at which 50% of the isolates are inhibited of 1 to 2 micrograms/ml). Tetracycline-resistant strains of U. urealyticum were resistant to doxycycline and minocycline and showed variable susceptibility to the glycylcyclines (range, 0.5 to 32 micrograms/ml). In view of the increasing resistance of M. hominis and U. urealyticum strains to tetracyclines, the glycylcyclines have promise, pending assessment of their pharmacokinetic and safety profiles.

Susceptibilities of Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma urealyticum to a new quinolone, OPC 17116.

The susceptibilities of human mycoplasmas to OPC 17116 (Otsuka America Pharmaceutical, Inc., Rockville, Md.) and temafloxacin (Abbott Laboratories, Chicago, Ill.) were determined by the agar dilution method and were compared with those to sparfloxacin and ofloxacin. The MICs of OPC 17116 for 90% of Mycoplasma pneumoniae (0.25 microgram/ml) and Mycoplasma hominis (0.125 micrograms/ml) isolates tested were closely similar to those of sparfloxacin and were four- to eightfold greater than those of ofloxacin. Temafloxacin was two- to fourfold more active than ofloxacin. Ureaplasma urealyticum was less susceptible; the MICs of OPC 17116 and temafloxacin for 90% of isolates tested were 2 and 4.0 micrograms/ml, respectively.

Effect of pH, inoculum size, and incubation time on the susceptibility of Ureaplasma urealyticum to erythromycin in vitro.

Determinations of the susceptibility of Ureaplasma urealyticum to erythromycin in vitro as measured by the broth dilution method have shown wide variations with minimal inhibitory concentrations (MICs) from 0.04 to > or = 8 micrograms/mL, indicating a need for standardization. The effects of pH, inoculum size, and incubation were studied. In the broth dilution test, pH had an important effect. The apparent MIC was 4- to 16-fold higher at pH 6.0 than at pH 7.0, with the MIC falling progressively from values of 8 to > or = 8 micrograms/mL to values of 0.25 to 1 microgram/mL as the pH of the medium was increased in steps to pH 7.0. Large inocula also inflated the MICs 2- to 4-fold. In addition, the time of incubation influenced the apparent MIC, with increases of 4- to 16-fold between days 1 and 5. In agar dilution assays, MICs decreased from 2 micrograms/mL at pH 6.2 to 0.5 microgram/mL at pH 6.6. Since pH, inoculum level, and incubation time appear to be responsible for most of the variation in results, we propose that susceptibility testing for ureaplasmas can be improved by using medium with a more neutral pH than that usually used (pH 6) and by standardizing the inoculum size and incubation period.

Susceptibilities of Mycoplasma hominis and Ureaplasma urealyticum to two new quinolones, sparfloxacin and WIN 57273.

Mycoplasma hominis was highly susceptible to two new quinolones, with MICs for 90% of isolates tested of 0.004 micrograms/ml for WIN 57273 and 0.063 micrograms/ml for sparfloxacin, which were activities much greater than the 1 microgram/ml found for ofloxacin and tetracycline. Although Ureaplasma urealyticum was less susceptible, the MICs for 90% of isolates tested of 0.25 micrograms/ml for WIN 57273 and 0.5 micrograms/ml for sparfloxacin were four- to eightfold greater than those found for ofloxacin (2 micrograms/ml) and tetracycline (2 micrograms/ml). The finding that U. urealyticum and M. hominis are more susceptible to WIN 57273 and sparfloxacin than they are to other quinolones suggests that these quinolones may be therapeutically useful.

Susceptibility of Mycoplasma pneumoniae to several new quinolones, tetracycline, and erythromycin.

Mycoplasma pneumoniae (39 strains) was most susceptible to two quinolones, WIN 57273 and sparfloxacin, with MICs for 90% of the strains (MIC90S) of 0.125 and 0.25 micrograms/ml, respectively. It was susceptible to ofloxacin and ciprofloxacin at 2 micrograms/ml and to lomefloxacin and fleroxacin at 4 micrograms/ml. The MIC90 of erythromycin was 0.062 microgram/ml, and that of tetracycline was 1 microgram/ml.

Susceptibilities of genital mycoplasmas to the newer quinolones as determined by the agar dilution method.

The increasing resistance of genital mycoplasmas to tetracycline poses a problem because tetracycline is one of the few antimicrobial agents active against Mycoplasma hominis, Ureaplasma urealyticum, chlamydiae, gonococci, and other agents of genitourinary-tract disease. Since the quinolones are a promising group of antimicrobial agents, the susceptibilities of M. hominis and U. urealyticum to the newer 6-fluoroquinolones were determined by the agar dilution method. Ciprofloxacin, difloxacin, and ofloxacin had good activity against M. hominis, with the MIC for 50% of isolates tested (MIC50) being 1 microgram/ml. Fleroxacin, lomefloxacin, pefloxacin, and rosoxacin had MIC50s of 2 micrograms/ml. Enoxacin, norfloxacin, and amifloxacin had MIC50s of 8 to 16 micrograms/ml, and cinoxacin and nalidixic acid were inactive (MIC50, greater than or equal to 256 micrograms/ml). Overall, the activities of 6-fluoroquinolones for ureaplasmas were similar to those for M. hominis, with MICs being the same or twofold greater. The most active 6-fluoroquinolones against ureaplasmas were difloxacin, ofloxacin, and pefloxacin, with MIC50s of 1 to 2 micrograms/ml. Ciprofloxacin was unusual in that the MIC50 for M. hominis was 1 microgram/ml, whereas the MIC50 for ureaplasmas was 8 micrograms/ml. Since the MIC50s for the most active quinolones approximate achievable concentrations in blood and urine, quinolones have promise in treating mycoplasmal infections.

The principal protein antigens of isolates of Mycoplasma pneumoniae as measured by levels of immunoglobulin G in human serum are stable in strains collected over a 10-year period.

To determine whether antigenic variation in protein antigens of Mycoplasma pneumoniae occurred over time, 12 isolates obtained from pneumonia patients over a 10-year period (1964 to 1974) were compared by immunoblotting (Western blotting) against acute and convalescent human serum samples obtained from the same patients. The strains selected were isolated from patients who had low anti-lipid complement-fixing antibody titers in their acute-phase serum samples and high titers in their convalescent-phase serum samples. The polypeptide composition of the strains was closely similar by protein staining even when compared with prototype FH-Liu. On immunoblotting, all strains showed five bands (170, 130, 90, 45, and 35 kilodaltons [kDa]) which were stained more intensely by convalescent-phase than by acute-phase specimens. A sixth band (62 kDa) was detected by the conjugate alone. In FH-Liu, one band (110 kDa) was prominently stained by convalescent-phase specimens; this band was much less apparent in all of the clinical isolates. Two isolates possessed an additional band (92 kDa) which was stained more prominently by some but not all convalescent-phase specimens. Because of its known antigenic relationships and culture similarities, Mycoplasma genitalium was used for comparison. More polypeptides of M. genitalium than of M. pneumoniae were recognized by acute-phase serum samples, and 4 of 12 convalescent-phase serum samples showed increases in antibodies to certain M. genitalium polypeptides. However, these reactive polypeptides did not correspond in molecular mass to polypeptides recognized in M. pneumoniae; thus the signature profile of human convalescent-phase specimens with M. pneumoniae was distinct. These five polypeptides, individually or in combination, are especially promising for use in detection of human serum antibodies by enzyme-linked immunosorbent assay because they were found in all M. pneumoniae isolates tested.

Agar dilution method for determination of antibiotic susceptibility of Ureaplasma urealyticum.

The agar dilution method was adapted for use in determining antibiotic sensitivities of clinical isolates of Ureaplasma urealyticum. The reproducibility of end points (minimal inhibitory concentrations) for tetracycline was excellent within one dilution; however, for spectinomycin and erythromycin end points increased 2-fold between 3 and 4 days of incubation. When incubation times were held constant for these antibiotics the method was reproducible within two dilutions (4-fold). The use of a Steers' replicator permitted many strains to be tested. Although it is recognized that ureaplasmas present unique problems among mycoplasmas because of their small colony size, suitable developmental studies will make agar dilution an important method for determining antibiotic susceptibilities of ureaplasmas.

Immunoblotting for determination of the antigenic specificities of antibodies to the Mycoplasmatales.

Determination of the nature of antigens towards which specific antibodies are directed has caused great difficulties in studies of the antigenic structure of the Mycoplasmatales. In immunoblotting, polypeptides are separated first by SDS polyacrylamide gel electrophoresis and transferred to cellulose nitrate electrophoretically. The resultant pattern is stained by enzyme-linked staining techniques. This permits direct detection of the antigenic specificities recognized by human and animal immune serum. For example, human convalescent sera from patients with Mycoplasma pneumoniae pneumonia recognize 2 to 7 polypeptides in M. pneumoniae, whereas human sera from patients with postpartum fever from whom Ureaplasma urealyticum has been isolated from the bloodstream detect 15 to 25 polypeptides. A comparison of M. pneumoniae with M. genitalium using rabbit antisera demonstrated that these two organisms show strong cross-reactions, although the organisms can be distinguished. Although certain antigens (epitopes) are destroyed in the procedure, it appears that about two-thirds of the polypeptides retain antigenicity. Immunoblotting provides a powerful means for identifying and subsequently fractionating antigens important to the human immune response.

Effect of urea concentration on growth of Ureaplasma urealyticum (T-strain mycoplasma).

The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 x 10(4) colony-forming units per ml. The maximum yield of organisms, 8.0 x 10(7) colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 x 10(10) total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K(s) value for growth was 2.0 x 10(-4) M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.