In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, >256 g/ml) and 1 was weakly resistant (MIC, 8 g/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan.Mycoplasma pneumoniae is a pathogen causing human respiratory infections such as atypical pneumonia, mainly in children and younger adults. In the chemotherapy of M. pneumoniae infection in children, erythromycin (ERY) and clarithromycin (CLR) among 14-membered macrolides and the 15-membered macrolide azithromycin (AZM) are usually considered the first-choice agents in Japan. Although there was no report on the isolation of ERY-resistant M. pneumoniae before 2000 in Japan, we found that ca. 20% of M. pneumoniae strains isolated from patients from 2000 to 2003 were ERY resistant. These results are consistent with pediatricians' impression that antibiotics such as ERY, CLR, and clindamycin (CLI) are not effective for some patients with M. pneumoniae infection.It is well known that the macrolide-lincosamide-streptogramin B (MLS) antibiotics inhibit protein synthesis by binding to domain II and/or domain V of 23S rRNA (3, 26). Lucier et al. (10) and Okazaki et al. (17) found that an A-to-G transition or A-to-C transversion at position 2063 (corresponding to 2058 in Escherichia coli numbering) or 2064 of the 23S rRNA gene resulted in high resistance to macrolide antibiotics. No point mutation was found in domain II of 23S rRNA of the ERYresistant M. pneumoniae strains used in the present study.We report here the prevalence of macrolide-resistant M. pneumoniae infection in Japan. By using 13 ERY-resistant M. pneumoniae strains, we investigated the mechanisms of resistance to MLS antibiotics. Furthermore, we established restriction fragment length polymorphism (RFLP) techniques to detect point mutations in domain V of 23S rRNA...

Two hundred fifty strains of Mycoplasma pneumoniae isolated during the past 20 years in Japan were classified into two groups (I and II) based upon different PCR-restriction fragment length polymorphism patterns of their P1 cytadhesin genes. Clear shifts between the M. pneumoniae groups were observed but did not appear to be correlated with M. pneumoniae epidemic cycles. Patients' sera showed relatively higher levels of antiadhesin antibodies to M. pneumoniae strains homologous with the infecting strain.

Chicken egg yolk broth medium containing 10% (vol/vol) phosphate-buffered saline extract of egg yolk instead of horse serum showed better growth-promoting activity for Mycoplasma pneumoniae than did Chanock medium and was effective in promoting the growth of small numbers of M. pneumoniae. Moreover, the phosphate-buffered saline extract of egg yolk proved superior to horse serum in the following respects: (i) it was consistent in growth-promoting activity for M. pneumoniae among different batches; (ii) it could be preserved at 4 degrees C for at least 2 years; (iii) it is inexpensive and easy to obtain; and (iv) it contains large amounts of lipoproteins, cholesterol, and phospholipids.

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Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas. In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas. These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas. In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth. A highly positive correlation (r = 0.96, P less than 0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas.