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, >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...

Molecular mechanism of macrolide–lincosamide resistance in Moraxella catarrhalis

“…In previous studies, a single mutation, A2058 or A2059 (E. coli numbering), in one of the 23S rRNA copies has been shown to be sufficient to confer high-level resistance to 14-and 15-membered macrolides in bacteria that possess either one [Mycobacterium smegmatis (Li et al, 2011)] or two [Helicobacter pylori (Versalovic et al, 1996) and Mycoplasma pneumonia (Matsuoka et al, 2004)] copies of this gene. On the other hand, in a Gram-negative diplococcus, Neisseria gonorrhoeae, which is similar to M. catarrhalis in that it also contains four copies of the rrn operon (Chisholm et al, 2010), a single mutation at position 2059 in at least three of the 23S rRNA alleles confers high-level resistance to azithromycin (MIC.256 mg l 21 ) (Chisholm et al, 2010).…”

“…In general, mechanisms underlying resistance against these antimicrobials are known to involve active efflux of the antibiotics and modification of the ribosomal target by enzymes such as rRNA methylase, enzymic inactivation or mutations in the 23S rRNA gene, as described previously in various organisms by Chisholm et al (2010), Li et al (2011) and Versalovic et al (1996). Another mechanism, involving point mutations in genes encoding the ribosomal proteins L4 (rplD) and L22 (rplV), has been reported in some clinical isolates (Matsuoka et al, 2004;Tait-Kamradt et al, 2000).…”

In Vitro Activities of 11 Antimicrobial Agents against Macrolide-Resistant Mycoplasma pneumoniae Isolates from Pediatric Patients: Results from a Multicenter Surveillance Study

“…pneumonia isolates had mutations in domain V of the 23S rRNA gene (1,2,10,11). Several studies indicated that macrolide resistance in M. pneumoniae may have clinical significance in terms of diminished response to treatment with drugs in this class (2,12,13).…”

“…We performed full-length sequencing of the 23S rRNA gene in M. pneumoniae isolates showing macrolide resistance, as reported previously (11).…”

Mycoplasma Removal from Cell Culture Using Antimicrobial Photodynamic Therapy

“…In the case of mycoplasma infection, Mycoplasma pneumoniae strains have been reported to be resistant to antibiotics. 12 Therefore, it is valuable to establish a method to remove bacteria and mycoplasmas from infected patients or cell culture without using antibiotics. As described, aPDT is expected to be a potent method for bacterial removal without using antibiotics.…”