Norio Okazaki

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, > or =256 microg/ml) and 1 was weakly resistant (MIC, 8 microg/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.

Some patients with Mycoplasma pneumoniae infection are clinically resistant to antibiotics such as erythromycin, clarithromycin, or clindamycin. We isolated M. pneumoniae from such patients and found that one of three isolates showed a point mutation in the 23S rRNA gene. Furthermore, 141 EM-sensitive clinical isolates of M. pneumoniae were cultured in broth medium containing 100 microg/ml of erythromycin (EM). Among 11 EM-resistant strains that grew in the medium, point mutations in the 23S rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM-resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.

Macrolide-resistant Mycoplasma pneumoniae (MR M. pneumoniae) has been isolated from clinical specimens in Japan since 2000. A comparative study was carried out to determine whether or not macrolides are effective in treating patients infected with MR M. pneumoniae. The clinical courses of 11 patients with MR M. pneumoniae infection (MR patients) treated with macrolides were compared with those of 26 patients with macrolide-susceptible M. pneumoniae infection (MS patients). The total febrile days and the number of febrile days during macrolide administration were longer in the MR patients than in the MS patients (median of 8 days versus median of 5 days [P = 0.019] and 3 days versus 1 day [P = 0.002], respectively). In addition, the MR patients were more likely than the MS patients to have had a change of the initially prescribed macrolide to another antimicrobial agent (63.6% versus 3.8%; odds ratio, 43.8; P < 0.001), which might reflect the pediatrician's judgment that the initially prescribed macrolide was not sufficiently effective in these patients. Despite the fact that the febrile period was prolonged in MR patients given macrolides, the fever resolved even when the initial prescription was not changed. These results show that macrolides are certainly less effective in MR patients.

Mycoplasma pneumoniae clinical isolates obtained between 1995 and 2005 were examined to determine the prevalent genotype. One hundred and twenty-seven strains isolated from bronchitis and pneumonia patients were genotyped by a PCR-RFLP method based on nucleotide sequence polymorphisms of the p1 gene, which encodes the major adhesin protein. The typing results established that 66 of the isolates were group I strains, 45 were group II strains and 16 were group II variants. Analysis of the annual occurrence of these isolates showed a predominance of group II strains between 1995 and 2001 (n=37). No group I strain was found during this period. However, group I strains appeared in the isolates from 2002 (2/5 isolates, 40 %) and increased in specimens taken after 2003, thereby constituting a large proportion of the isolates. In 2004 and 2005, no group II strains were found among the isolates (n=49), although there were nine group II variants. Throat swabs and sputum samples obtained from patients with respiratory infections between 1997 and 2005 were also analysed by PCR-RFLP or a new nested PCR to detect the p1 gene DNA. Typing analysis of these p1 gene DNAs also showed that the group I p1 gene was not present in specimens taken before 2000, but was present and dominant in specimens taken after 2001. These results indicate that, in Japan, the prevalent type of M. pneumoniae changed from a group II strain to a group I strain around 2002.

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.