Characterization and molecular analysis of macrolide-resistant Mycoplasma pneumoniae clinical isolates obtained in Japan

Abstract

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.

FIG. 1.

Multiple alignment of 23S rRNA gene of ERY-resistant M. pneumoniae strains and M. pneumoniae M129, FH, and Mac. Partial sequences of the peptidyltransferase (domain V) from positions 2051 to 2081 and 2601 to 2630 are presented. The nucleotides are numbered on the basis of M. pneumoniae. The nucleotide sequence of M. pneumoniae M129 was according to GenBank accession no. X68422. Identical nucleotides are indicated by dashes. The positions of 2063, 2064, and 2617 are underlined.

FIG. 2.

Secondary structure of the peptidyltransferase loop in domain V of M. pneumoniae 23S rRNA. Positions of the newly found mutations (A2063C and C2617G), as well as previously reported in vitro mutations (A2063G, A2064G, and A2064C), in clinical isolates are indicated by using the numbering for M. pneumoniae 23S rRNA (accession no. X68422). The numbers in parentheses indicate E. coli numbering.

FIG. 3.

Nucleotide sequence of the 927-bp amplicon from positions 1758 to 2684 of the 23S rRNA gene from M. pneumoniae M129. A long arrow indicates a primer sequence with direction. A short arrow indicates a site of mutation with a substituted base, i.e., A2063G, A2063C, A2064G, or C2617A. A newly constructed restriction site and the responsible base change with underline is shown in parentheses with the corresponding restriction enzyme.

FIG. 4.

Restriction analysis of 210-bp (A) and 108-bp (B) amplicons from the peptidyltransferase region (domain V) in 23S rRNA of M. pneumoniae. (A) Restriction profile for detection of the A2063G, A2063C, and A2064G mutations. Lanes: 1, DNA size marker (25-bp DNA step ladder; Promega); 2, 4, and 6, M. pneumoniae M129 (susceptible strain) treated with BbsI (lane 2, 124-, and 86-bp products) and BceAI and BsaI (lanes 4 and 6, respectively; uncut 210-bp product); 3, strain 375 (A2063G) treated with BbsI (124-, 57-, and 52-bp products); 5, strain 376 (A2063C) treated with BceAI (158- and 52-bp products); 7, strain 1020 (A2064G) treated with BsaI (141- and 69-bp products). (B) Restriction profile for detection of C2617 mutation with BsmFI digestion. Although M. pneumoniae M129 and strain 375 (A2063G) produced two fragments of 81 and 27 bp (lanes 1 and 2), the 108-bp fragment remained uncut in strains 377 and 1020-EMR3 (C2617G) as a result of loss of the restriction site for BsmF1 (lanes 3 and 4). Lane 5, DNA size marker (25-bp DNA step ladder; Promega).