Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. International Research Programme on Comparative Mycoplasmology.

The absence of standardised procedures for minimum inhibitory concentration (MIC) testing of antimicrobial agents against veterinary mycoplasma and ureaplasma species (Mollicutes) has made it difficult to compare results originating from different laboratories. This report, prepared on behalf of the International Research Programme on Comparative Mycoplasmology (IRPCM), offers guidelines and recommendations for veterinary MIC testing of these organisms in an effort to rectify this problem. The subjects discussed include suitable media for broth and agar MIC assays, storage and preparation of antimicrobial agents, standardisation of mycoplasma inocula for MIC tests, validation of equipment, incubation conditions, and determination of MIC end points. A standard medium for all veterinary mycoplasma MIC tests cannot currently be recommended, owing to the diversity of nutritional requirements of different mycoplasma species. Instead mycoplasma broths or agars giving optimal growth of specific mycoplasmas or ureaplasmas are recommended, as suboptimal growth may lead to falsely low MIC results. The importance of using standardised mycoplasma inocula, for assays using either solid or liquid media is stressed. The growth phase may be less important as lag phase and logarithmic phase cultures of Mycoplasma gallisepticum, M. synoviae, M. bovis and M. hyopneumoniae have given very similar results in liquid MIC assays. The liquid method of Tanner and Wu and the agar method described by Hannan et al. are compared and described in detail. Methods for calculating MIC50s and MIC90s are described and the interpretation of results discussed. Methods for assessing mycoplasmacidal (MMC) activity of antimicrobial agents are also described. Adoption of these guidelines should lead to more consistent MIC results being obtained between laboratories.

In vitro activity of gemifloxacin (SB 265805; LB20304a) against human mycoplasmas.

The in vitro activity of gemifloxacin, a new broad-spectrum fluoroquinolone, was compared with those of ciprofloxacin, erythromycin, azithromycin and doxycycline against 29 human respiratory or urogenital tract mycoplasmas. Gemifloxacin was highly active against all of the mycoplasma and ureaplasma species tested (MIC range 0.001-0.25 mg/L) and was 5- to 100-fold more active than ciprofloxacin. Doxycycline was less active than gemifloxacin against the mycoplasmas (MIC range 0.01-1 mg/L) but had similar activity against Ureaplasma urealyticum (MIC ranges 0.025-0.25 mg/L and 0.1-0. 25 mg/L, respectively). The macrolides, particularly azithromycin, were more active than gemifloxacin against Mycoplasma pneumoniae (MIC range 0.001-0.0025 mg/L) and Mycoplasma genitalium (0.0005-0. 001 mg/L) isolates but were less active against Mycoplasma fermentans and U. urealyticum and inactive against Mycoplasma hominis. Gemifloxacin may therefore be useful in the treatment of respiratory, urogenital or systemic mycoplasma infections in humans.

Comparative susceptibilities of various AIDS-associated and human urogenital tract mycoplasmas and strains of Mycoplasma pneumoniae to 10 classes of antimicrobial agent in vitro.

The susceptibilities of 40 strains of various Mycoplasma species to 10 classes of antimicrobial agents were compared in vitro by a broth microdilution method. The strains tested comprised 20 strains of four AIDS-associated species--M. penetrans (1 strain), M. fermentas (5 strains), M. pirum (6 strains) and M. genitalium (8 strains)--nine strains of the urogenital tract species M. hominis and 11 strains of M. pneumoniae. The results demonstrated wide variation in the susceptibilities of the different Mycoplasma spp. to different classes of antimicrobial agent. All the mycoplasmas were susceptible or highly susceptible to the fluoroquinolones, with sparfloxacin the most active, and to the diterpine antibiotic tiamulin. M. pneumoniae and M. genitalium strains were also highly susceptible to the macrolides, particularly azithromycin and had similar antibiotic susceptibility patterns to most other antimicrobial agents. However, all strains of M. genitalium were resistant to streptomycin (MIC 250->500 mg/L) whereas all M. pneumoniae isolates, except the MAC strain, were susceptible (MICs 1.25-12.5 mg/L). M. pirum isolates varied considerably in their susceptibility to macrolides (MIC range versus azithromycin 0.0025->100 mg/L). M. fermentans strains were susceptible to the tetracyclines, lincosamides and mupirocin, but varied in susceptibility to aminoglycosides. Most M. hominis strains were susceptible to the tetracyclines and all were susceptible to clindamycin and mupirocin. M. penetrans GTU 54 was susceptible to azithromycin, the tetracyclines and lincosamides as well as to the fluoroquinolones and tiamulin.

Comparative susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones.

The in vitro activities of six antimicrobial agents were tested against 162 mycoplasma strains of eight species isolated from poultry and livestock at different geographic sites. Tiamulin was most active (MICs at which 90% of the isolates were inhibited [MIC90s], 0.025 to 0.25 microg/ml); enrofloxacin and danofloxacin had near equivalent activities (MIC90s, 0.05 to 1.0 microg/ml), but were much more active than flumequine (MIC90s, 1 to 50 microg/ml). The MIC90s of tylosin and oxytetracycline were 0.25 to > 100 microg/ml and 0.25 to 100 microg/ml, respectively.

Differentiation of strains of Mycoplasma fermentans from various sources by pyrolysis mass spectrometry.

Mycoplasma fermentans has attracted much interest both as a cofactor for the progression of AIDS and as a pathogenic agent in non-AIDS related diseases. Previous studies with serological and genetic techniques suggest that M. fermentans represents a homogeneous group of organisms, with no significant differences identified among the strains examined. In this study, 25 cultures of M. fermentans, including isolates from human sources and tissue culture cells, were compared by pyrolysis mass spectrometry (PMS). It was possible to distinguish the 'type' strain PG-18 from an AIDS-associated M. fermentans strain 'incognitus' by this technique. PMS was also able to differentiate laboratory-induced aminoglycoside-resistant variants from their fully susceptible parents. Four AIDS-associated isolates were distinguished from each other, whilst five European cell culture isolates were shown to be closely related, as were six M. fermentans isolates from an outbreak of acute respiratory infection in Canada. PMS has proved useful in distinguishing isolates of M. fermentans, providing epidemiological data. In addition, PMS may help in determining the likely origin of a given isolate, and in the future may be of use in assessing the role of this micro-organism in human disease.

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.

In vitro susceptibilities of recent field isolates of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae to valnemulin (Econor), tiamulin and enrofloxacin and the in vitro development of resistance to certain antimicrobial agents in Mycoplasma hyopneumoniae.

The in vitro activities of valnemulin (Econor) and two other antimicrobial agents were determined against recent field strains of Mycoplasma hyopneumoniae and Mycoplasma hyosynoviae using a broth microdilution method. Valnemulin showed exceptional activity against M hyopneumoniae (MIC90 0.0005 microgram ml-1) and M hyosynoviae (MIC range 0.0001 microgram ml-1 to 0.00025 microgram ml-1) field strains. Tiamulin was 100-fold less active (MIC90 0.05 microgram ml-1) and enrofloxacin 20-fold less active (MIC90 0.01 microgram ml-1) than valnemulin against M hyopneumoniae field isolates and 20-fold to 25-fold less active (MIC range 0.0025 microgram ml-1 to 0.005 microgram ml-1) and 400-fold to 500-fold less active (MIC range 0.05 microgram ml-1 to 0.1 microgram ml-1) respectively against M hyosynoviae field isolates. No significant resistance developed to valnemulin or tiamulin in the type strain of M hyopneumoniae (strain J) or in a recent field isolate (MEVT G23) exposed to 10 in vitro passages in broths containing these antibiotics. Only slight resistance to oxytetracycline was observed. High resistance to tylosin developed in both M hyopneumoniae strains within five to seven in vitro passages in tylosin-containing broth. Providing that similar results are obtained in vivo under field conditions, valnemulin may well prove to be effective in the treatment of enzootic pneumonia and acute polyarthritis in pigs.

The chemistry of pseudomonic acid. 17. Dual-action C-1 oxazole derivatives of pseudomonic acid having an extended spectrum of antibacterial activity.

A series of C-1 oxazole isosteres of pseudomonic acid A (mupirocin) bearing a nitroheterocycle have been synthesized, and significant differences in both spectrum of activity and potency were found between these derivatives and mupirocin. Additionally, the antibacterial potency of two members of this class of compounds against mupirocin-resistant staphylococci could not be accounted for solely by inhibition of the target enzyme isoleucyl-tRNA synthetase (IRS), indicating an additional mode of action. The most potent compound, the nitrofuran 3f (SB 205952), was the most electron affinic derivative prepared and was transformed by NAD(P)H-dependent bacterial reductases at a rate similar to that for nitrofurantoin. The second mode of action of this compound may therefore arise from its reduction to a species with cellular targets other than IRS. In in vivo studies, 3f was shown to be a very effective agent by both the subcutaneous and oral routes of administration.

Chemistry of pseudomonic acid. Part 16. Aryl and heteroaryl ketone derivatives of monic acid.

The synthesis, antibacterial activities, murine pharmacokinetic and infection model data for a range of aryl and heteroaryl ketone derivatives of monic acid (2a) are reported. The best results were found for the 3-furyl and 2-methoxy thiazol-5-yl analogues.

The chemistry of pseudomonic acid. Part 14. Synthesis and in vivo biological activity of heterocyclyl substituted oxazole derivatives.

Semisynthetic analogues of pseudomonic acid A have been prepared containing a heterocyclyl substituted oxazole. Derivatives in which the heterocycle was thiophene, furan, pyridine, or isoxazole showed good antibacterial potency and were further evaluated in vivo. Both pharmacokinetic parameters and oral activity against an experimental intraperitoneal sepsis were superior to results obtained from previously described pseudomonic acid A derivatives.

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.

Treatment of experimental enzootic pneumonia of the pig by norfloxacin or its 6-chloro analogue.

The 6-chloro analogue of norfloxacin (compound A) administered continuously in the feed at 400 ppm for 21 days markedly reduced the extent and activity of pneumonic lesions in pigs with pneumonia induced experimentally with an homogenate of pneumonic lung and broth cultures of Mycoplasma hyopneumoniae. Norfloxacin at 100 ppm or compound A at 200 ppm in the feed did not reduce the extent of lung lesions, although half the pigs treated with norfloxacin had lesions which appeared histologically to be healing. M hyopneumoniae was detected either by culture or immunofluorescence in the lungs of 60 per cent of the pigs treated with compound A at 400 ppm compared with all the pigs in the other groups. These results were related to the amount of drug in the lungs and body fluids during therapy. Only compound A at 400 ppm produced concentrations in the lungs and bronchial secretions exceeding the minimum inhibitory concentration against M hyopneumoniae. Mycoplasmacidal concentrations were not reached either in the lungs or bronchial secretions which might account partly for the frequent detection of M hyopneumoniae in the lungs after treatment. Drug resistance did not appear to be responsible for the persistence of M hyopneumoniae in vivo since the M hyopneumoniae isolates from the pigs after therapy were sensitive in vitro to both quinolones. As daily weight gain and feed-conversion efficiency improved in all groups of treated pigs compared with the controls, these effects were probably unrelated to the antimycoplasmal activities of the two quinolones.

Biological properties of ER 42859, a novel erythromycin derivative.

The antimicrobial activity of a new semi-synthetic oral erythromycin derivative, ER 42859, was evaluated in vitro and in vivo in comparison with erythromycin, spiramycin, josamycin, oleandomycin and the newer semi-synthetic derivatives flurithromycin, roxithromycin and A-56268. MIC values of ER 42859 were superior to those of roxithromycin, oleandomycin, josamycin and spiramycin but generally 2-fold poorer than those of erythromycin. The activity equalled that of erythromycin against Haemophilus influenzae and was superior to that of roxithromycin and A-56268 against this organism. MIC values of the compound were greatly influenced by pH due to the dibasic nature of the molecule. ER 42859 had markedly superior activity to erythromycin, spiramycin, josamycin, oleandomycin and flurithromycin against experimental infections in mice and similar activity to roxithromycin and A-56268. Blood and tissue levels were high and prolonged in rodents. In volunteers, blood levels were prolonged but inferior to those of erythromycin.

In vitro evaluation of various quinolone antibacterial agents against veterinary mycoplasmas and porcine respiratory bacterial pathogens.

The in vitro activities of 12 quinolones and four antibiotics were determined against 15 veterinary mycoplasmal species and four species of bacteria commonly involved in respiratory infections in pigs. The newer quinolones were markedly more active in vitro against a wide range of mycoplasmas than nalidixic acid and the earlier quinolones. Against Mycoplasma hyopneumoniae ciprofloxacin was the most active quinolone with a geometric mean minimal inhibitory concentration (MIC) against 16 strains of 0.01 microgram ml-1 compared with 0.04 microgram ml-1 for tiamulin, 0.06 microgram ml-1 for tylosin, 0.17 microgram ml-1 for oxytetracycline and 0.23 microgram ml-1 for gentamicin. M hyosynoviae was less sensitive to the quinolones with mean MICs of 0.6 microgram ml-1 for ofloxacin and 0.7 microgram ml-1 for ciprofloxacin compared with 0.034 microgram ml-1, or less, for tiamulin. Norfloxacin and its 6-chloro analogue were both mycoplasmacidal in vitro at five or 10 times their MICs against M hyopneumoniae UCD4. Tiamulin was mycoplasmastatic. The quinolones were also active against porcine Bordetella bronchiseptica and Pasteurella multocida strains and Haemophilus species. Ciprofloxacin was the most active quinolone with mean MICs of 0.58 microgram ml-1 against B bronchiseptica (nine strains), 0.026 microgram ml-1 against P multocida (five strains) and 0.01 microgram ml-1, or less, against Haemophilus pleuropneumoniae (nine strains) and H parasuis (two strains) compared with mean MICs of from 0.5 microgram ml-1 to 64 micrograms ml-1, or more, for the antibiotics. This combination of excellent mycoplasmacidal activity against M hyopneumoniae and good antibacterial activity, suggests that the quinolones have great potential for treating respiratory infections in pigs, including enzootic pneumonia.

Antimycoplasmal activities of the pseudomonic acids and structure-activity relationships of monic acid A derivatives.

The antimycoplasmal activities of the pseudomonic acids isolated from Pseudomonas fluorescens NCIB 10586 are reported. Structure-activity relationships of a variety of ester, amide and thiol ester derivatives of the nucleus, monic acid A, are described. Enhanced antimycoplasmal activity is reported for a number of monic acid A esters and the most potent derivative, m-nitrobenzyl monate A, is a 100-fold more active against Mycoplasma hyopneumoniae than pseudomonic acid A.

Production of mycoplasma-specific antisera in rabbits immunologically tolerized at birth to mycoplasma medium constituents.

Neonatal rabbits were injected intraperitoneally (i.p.) within 12 h of birth followed by similar injections every day for 10 consecutive days and then every second day for a further 8 weeks, with mycoplasma broth medium (tolerogen), to induce immune tolerance. The rabbits were then immunized with the porcine mycoplasmas, M. hyopneumoniae or M. hyorhinis at 9 weeks of age. Immune sera obtained from these rabbits and from normal control rabbits were tested for antibodies against both mycoplasma antigens and for antibodies to medium components by double immunodiffusion in agarose and by enzyme-linked immunosorbent assay (ELISA). Antisera obtained from the tolerized rabbits contained no antibodies to medium components as evidenced by lack of reactivity in both assays. In immunofluorescence tests the antisera obtained from tolerized rabbits permitted specific staining of colonies of the homologous mycoplasma grown on mycoplasma agarose medium. In contrast the antisera obtained from normal rabbits produced strong reactions in all of the tests and non-specific background fluorescence due to reactions with components of the culture medium.

Reproducible pneumonia in gnotobiotic piglets induced with broth cultures of Mycoplasma hyopneumoniae and the effect of animal passage on virulence.

The virulence of a laboratory adapted culture of Mycoplasma hyopneumoniae strain NB12 was determined in three- to five-day-old gnotobiotic piglets. Intranasal inoculation or exposure to an aerosol of the culture caused low incidences of pneumonia in the piglets. Passage of M hyopneumoniae strain NB12 in gnotobiotic piglets resulted in a rapid increase in virulence. After only three in vivo passages, severe pneumonia involving most lobes of the lung developed in all inoculated piglets within three and a half weeks. All 49 piglets inoculated with the piglet-passaged NB12 strain in nine subsequent experiments developed pneumonia but the extent of the pneumonic lesions varied considerably from piglet to piglet. The histopathology of the lung lesions was similar to that reported as being induced by other strains of M hyopneumoniae in gnotobiotic piglets and resembled that seen previously in conventionally reared neonatal piglets inoculated with homogenised lung from pigs with enzootic pneumonia. Aspiration pneumonia caused by milk inhalation occurred in some piglets. The pneumonia induced with the piglet-passaged NB12 strain was judged to be suitable for the study of porcine enzootic pneumonia or for the evaluation of chemotherapeutic agents.

Tylosin tartrate and tiamutilin effects on experimental piglet pneumonia induced with pneumonic pig lung homogenate containing mycoplasmas, bacteria and viruses.

The effects of tylosin tartrate and tiamutilin were examined in pneumonias induced experimentally in neonatal piglets with a homogenate of pneumonic pig lung, obtained from pigs with naturally acquired enzootic pneumonia. The homogenate contained mycoplasmas, including Mycoplasma hyopneumoniae (M suipneumoniae) and M hyorhinis, and certain bacteria and viruses. The experimental pneumonias generally resembled mycoplasmal pneumonia histologically but were complicated by aspiration pneumonia in some animals. both tylosin tartrate (50 mg/kg) and tiamutilin (10 mg/kg) administered orally twice daily for 10 days, beginning 14 days after intranasal infection, significantly reduced the incidence and severity of macroscopical pneumonic lung lesions. M hyopneumoniae could be isolated from the lungs of the unmedicated piglets, but not from drug-treated piglets. The numbers of M hyorhinis, Acholeplasma granularum, Haemophilus parasuis, Pasteurella multocida and P haemolytica in the lung tissue of the infected piglets were significantly reduced by drug therapy. The role of bacterial in the experimental infection appeared to be that of secondary invaders.

Sodium aurothiomalate, gold keratinate, and various tetracyclines in mycoplasma-induced arthritis of rodents.

Sodium aurothiomalate (ATM), gold keratinate and five different tetracyclines were investigated for activity against M. arthritidis strain ATCC 14124 and M. pulmonis strain JB, both in vitro and in rodents with arthritis caused by these mycoplasmas. In vitro, ATM had only slight activity against M. arthritidis and M. pulmonis, while gold keratinate was virtually inactive against M. pulmonis. In contrast, the tetracyclines were highly active against both mycoplasmas. The tetracyclines and the gold salts were both predominantly mycoplasmastatic. In both rats and mice, parenteral administration of ATM, begun shortly before or after infection of rodents with mycoplasmas, prevented the development of arthritis. ATM or gold keratinate, given subcutaneously to mice already arthritic from infection with M. pulmonis, reduced the severity of the arthritis, even although gold keratinate was inactive aganist this mycoplasma in vitro. Moreover, direct testing of serum, collected from mice treated with gold keratinate, failed to demonstrate antimycoplasmal activity in vitro. These results suggest that the action of gold-containing drugs in mycoplasmal arthritis is due to biological properties of gold other than antimycoplasmal activity. Tetracyclines were also found to be effective in preventing arthritis in rats and mice when given subcutaneously. With high doses, subcutaneous, but not oral, therapy significantly reduced the severity of established arthritis in mice infected with M. pulmonis. The blood levels achieved with the different tetracyclines, when related to their therapeutic activity, indicated that good antimycoplasmal activity and adequate absorption from the gut were not the only properties needed for optimal effectiveness. The results are discussed in relation to treatment of rheumatoid patients with tetracycline HCl.