Abstract
A total of 195 Mycoplasma pneumoniae strains were isolated from 2,462 clinical specimens collected between April 2002 and March 2004 from pediatric outpatients with respiratory tract infections. Susceptibilities to six macrolide antibiotics (ML), telithromycin, minocycline, levofloxacin, and sitafloxacin were determined by the microdilution method using PPLO broth. A total of 183 M. pneumoniae isolates were susceptible to all agents and had excellent MIC90s in the following order: 0.00195 μg/ml for azithromycin and telithromycin, 0.0078 μg/ml for clarithromycin, 0.0156 μg/ml for erythromycin, 0.0625 μg/ml for sitafloxacin, 0.5 μg/ml for minocycline, and 1 μg/ml for levofloxacin. Notably, 12 ML-resistant M. pneumoniae strains were isolated from patients with pneumonia (10 strains) or acute bronchitis (2 strains). These strains showed resistance to ML with MICs of ≥1 μg/ml, except to rokitamycin. Transition mutations of A2063G or A2064G, which correspond to A2058 and A2059 in Escherichia coli, in domain V on the 23S rRNA gene in 11 ML-resistant strains were identified. By pulsed-field gel electrophoresis typing, these strains were classified into groups I and Vb, as described previously (A. Cousin-Allery, A. Charron, B. D. Barbeyrac, G. Fremy, J. S. Jensen, H. Renaudin, and C. Bebear, Epidemiol. Infect. 124:103-111, 2000). These findings suggest that excessive usage of MLs acts as a trigger to select mutations on the corresponding 23S rRNA gene with the resultant occurrence of ML-resistant M. pneumoniae. Monitoring ML susceptibilities for M. pneumoniae is necessary in the future.
Mycoplasma pneumoniae is one of the main pathogens in respiratory tract infections (RTI) acquired in the community. In school-aged children and young adults with community-acquired pneumonia (CAP), M. pneumoniae accounts for as many as 10 to 30% of cases (4, 6, 13).
In recent years, the clinical diagnosis for M. pneumoniae infection has relied on serological methods such as passive agglutination (Serodia-Myco II kit, Fujirebio, Tokyo, Japan) and complement fixation, even though a PCR method was partially applied (3, 22, 26). Accordingly, culture methods for this pathogen that require up to 1 week or more are rarely carried out in the laboratory. Thus, the current status of susceptibility to macrolide antibiotics (ML) used as the first-choice agent for M. pneumoniae is unclear.
In Japan, in parallel with the increase in usage of oral 14-membered ring ML (14-ML) and azithromycin for RTI, M. pneumoniae showing resistance to 14-ML has been isolated from clinical samples from pediatric outpatients with CAP (12, 16). We suspected an increase in cases with ML-resistant M. pneumoniae infection from prolonged clinical symptoms. In such cases, PCR positivity with rapid identification of M. pneumoniae persisted after the administration of clarithromycin or azithromycin for several days.
We isolated causative M. pneumoniae by cultivation from clinical specimens collected from pediatric outpatients with RTI to determine susceptibilities to 10 oral antibiotics and identified a transition mutation on the 23S rRNA gene in ML-resistant isolates.
MATERIALS AND METHODS
Microorganisms.
M. pneumoniae was isolated from clinical specimens from patients with RTI. The specimens were collected from pediatric outpatients who visited 10 medical Japanese institutions participating in the study group on acute respiratory diseases (ARD). A total of 2,462 samples were sent to our laboratory (Kitasato Institute for Life Sciences, Kitasato University) between April 2002 and March 2004.
Rapid detection by PCR for M. pneumoniae was performed initially using a primer set constructed on the 16S rRNA gene and methods described previously (14).
Cultivation of M. pneumoniae was carried out for 262 PCR-positive specimens with PPLO broth and agar plates. Composition of the broth was as follows: 70 ml PPLO broth (Difco, Inc., Detroit, Mich.) supplemented with 20 ml horse serum, 5 ml 50% yeast extract, 2.5 ml 20% glucose, 200 μl 1% phenol red, 1 ml 2.5% thallium acetate, 0.5 ml 200,000 units/ml potassium penicillin G, and 0.5 ml 20,000 μg/ml cefotaxime. M. pneumoniae was identified by a change in the color of the broth from red to yellow, by the resulting utilization of glucose, and by hemadsorption when colonies were overlaid with a 5% suspension of guinea pig erythrocytes. Incubation of PPLO broth and agar plates was performed according to previously described methods (25). M. pneumoniae strains isolated after purification procedures were stored at −80°C until use.
Antibiotic susceptibility testing.
MICs of 10 agents for M. pneumoniae strains were determined by microdilution methods with PPLO broth (17, 21). The following agents were provided by the indicated manufacturer: erythromycin (Shionogi Pharmaceutical Co., Ltd., Osaka, Japan), clarithromycin (Taisho Pharmaceutical Co., Ltd., Tokyo, Japan), josamycin (Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan), midecamycin (Meiji Seika Kaisha, Ltd., Tokyo, Japan), rokitamycin (Asahi Kasei Co., Tokyo, Japan), azithromycin (Pfizer Japan, Inc., Tokyo, Japan), telithromycin (Aventis Pharma, Ltd., Bridgewater, N.J.), minocycline (Wyeth Japan, Tokyo, Japan), and levofloxacin and sitafloxacin (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan). M. pneumoniae strain M129 was used as a control.
After approximately 30 μl of each M. pneumoniae suspension was inoculated into 1.5 ml of PPLO broth, and the inoculates were subcultured for 4 to 5 days at 37°C until they changed to a yellow color. Each 10 μl of the culture, estimated to be 105 CFU/ml of M. pneumoniae, was inoculated into 96-well microplates with 90 μl of PPLO broth, which contained serially diluted antibiotics. The microplate was sealed and incubated aerobically for 14 days at 37°C until color change in the antibiotic-free growth control was confirmed. The MIC was defined as the lowest concentration of each antibiotic without color change.
Sequencing.
The total length of the 23S rRNA gene in 12 M. pneumoniae strains showing ML resistance was sequenced by methods described previously (24). Two primer sets shown in Table 1 were used for amplification of the entire 23S rRNA gene. The sequencing reaction was conducted with a DYEnamic ET Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech, Piscataway, N.J.). The nucleotide sequences were determined with an ABI PRISM 377 DNA sequencer, and the results were compared to the genome sequence (8) and to ML-resistant strains (12, 16).
TABLE 1.
Primers used in this study
| Primer target | Primer name | Sequence | Nucleotide position | Product size (bp) |
|---|---|---|---|---|
| 16S rRNA | Mpn-S | 5′-GTAATACTTTAGAGGCGAACG-3′ | 77-97 | 225 |
| Mpn-R | 5′-TACTTCTCAGCATAGCTACAC-3′ | 281-301 | ||
| 23S rRNA | Mp-1S | 5′-GTTACTAAGGGCTTATGGTG-3′ | 7-26 | 1,583 |
| Mp-1R | 5′-GATTAATACCACCTTCGCTAC-3′ | 1569-1589 | ||
| Mp-2S | 5′-GGACAACAGGTTAATATTCCTG-3′ | 1406-1427 | 1,498 | |
| Mp-2R | 5′-CAATAAGTCCTCGAGCAATTAG-3′ | 2882-2903 |
Genes encoding ribosomal proteins L4 and L22, respectively, were sequenced using primers described by Pereyre et al. (17).
PFGE typing.
Pulsed-field gel electrophoresis (PFGE) typing was carried out by a modification of the methods of Hasegawa et al. (7). M. pneumoniae grown in 5 ml PPLO broth for 5 to 7 days was harvested by centrifugation at 12,000 rpm at 4°C for 30 min, followed by three washes with saline-EDTA solution. After cells were resuspended in Pet IV solution (1 M NaCl, 10 mM EDTA), 1.6× this volume of melted 2.0% low-melting-point agarose was added (InCert agarose; FMC Bioproducts, Rockland, Maine). The mixture was poured into an insert mold and chilled at 4°C for 20 min. Plugs removed from the mold were treated with 0.2 mg proteinase K (Sigma Chemical Co., St. Louis, Mo.) per ml in ES solution (0.25 M EDTA [pH 8.0], 1% Sarkosyl) at 50°C for 17 h. The ES solution was decanted, and the plugs were placed in TE buffer (10 mM Tris-HCl [pH 8.0], and 1 mM EDTA[pH 8.0] containing 1 mM phenylmethylsulfonyl fluoride) at room temperature for 3 h.
The plugs were washed three times in TE buffer for 15 min at room temperature and placed in TE buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA [pH 8.0]). For restriction endonuclease digestion, the plugs were incubated in restriction enzyme buffer for 30 min at room temperature to remove EDTA and then incubated in restriction enzyme buffer with 20 U of ApaI at 37°C for 16 h. Electrophoresis was performed using a CHEF Mapper (Bio-Rad Laboratories, Hercules, Calif.). Separation of fragments was carried out at 5.7 V/cm at 14°C for 18 h.
RESULTS
Clinical isolates of M. pneumoniae.
PCR detection of M. pneumoniae was routinely conducted on all clinical specimens, such as nasopharyngeal and pharyngeal secretions and sputum collected from pediatric outpatients with RTI. Cultivation of M. pneumoniae was only carried out with PCR-positive samples.
As shown in Table 2, 195 M. pneumoniae strains were isolated from 2,462 subjects. Almost all isolates were from cases of pneumonia (169 isolates; 86.7%) and then acute bronchitis (19 isolates; 9.7%). The remaining were isolates from acute otitis media, upper respiratory tract infection, and acute tonsillitis cases.
TABLE 2.
Diseases and M. pneumoniae-positive cases in pediatric outpatients
| Disease | Cases (n) |
M. pneumoniae
|
|
|---|---|---|---|
| No. (%) PCR positive | No. (%) culture positive | ||
| Pneumonia | 1,184 | 218 (18.4) | 169 (14.3) |
| Acute bronchitis | 576 | 29 (5.0) | 19 (3.3) |
| Acute upper respiratory infection | 329 | 2 (0.6) | 2 (0.6) |
| Acute tonsillitis | 173 | 6 (3.5) | 1 (0.6) |
| Acute otitis media | 63 | 1 (1.6) | 1 (1.6) |
| Other | 137 | 6 (4.4) | 3 (2.2) |
| Total | 2,462 | 262 (10.6) | 195 (7.9) |
Susceptibility to 10 agents for M. pneumoniae.
MICs in this study were determined for erythromycin and clarithromycin of the 14-ML; josamycin, midecamycin, and rokitamycin of 16-membered ring ML (16-ML); azithromycin of azalides; telithromycin of ketolides; minocycline; levofloxacin; and sitafloxacin.
Table 3 shows the results of 183 M. pneumoniae strains that were susceptible to ML, together with the results of the control M129 strain. The MIC90s of each agent for these strains were excellent, in the following order: 0.00195 μg/ml for azithromycin and telithromycin, 0.0078 μg/ml for clarithromycin, 0.0156 μg/ml for erythromycin and rokitamycin, 0.0625 μg/ml for sitafloxacin and josamycin, 0.125 μg/ml for midecamycin, 0.5 μg/ml for minocycline, and 1 μg/ml for levofloxacin.
TABLE 3.
In vitro antimicrobial activity of 10 oral agents against macrolide-susceptible Mycoplasma pneumoniae strains (n = 183)
| Antibiotic | MIC (μg/ml)
|
|||
|---|---|---|---|---|
| Range | 50% | 90% | M129a | |
| Erythromycin | 0.00195-0.0313 | 0.0078 | 0.0156 | 0.0156 |
| Clarithromycin | 0.00049-0.0313 | 0.0039 | 0.0078 | 0.0078 |
| Azithromycin | 0.00024-0.00195 | 0.00098 | 0.00195 | 0.00195 |
| Josamycin | 0.0156-0.0625 | 0.0313 | 0.0625 | 0.0313 |
| Midecamycin | 0.0625-0.25 | 0.0625 | 0.125 | 0.0078 |
| Rokitamycin | 0.0039-0.0313 | 0.0156 | 0.0156 | 0.0156 |
| Telithromycin | 0.00024-0.0039 | 0.00098 | 0.00195 | 0.00195 |
| Minocycline | 0.0313-2 | 0.25 | 0.5 | 0.5 |
| Levofloxacin | 0.25-1 | 0.5 | 1 | 1 |
| Sitafloxacin | 0.0313-0.0625 | 0.0313 | 0.0625 | 0.0625 |
Standard strain.
Table 4 shows the MICs of all agents for 12 M. pneumoniae strains classified as ML resistant. The strains showed high resistance for erythromycin, clarithromycin, and azithromycin with MICs of ≥32 μg/ml. The MICs of telithromycin, josamycin, and midecamycin ranged from 2 to ≥64 μg/ml, except for the ARD-185 strain. Only rokitamycin of the 16-ML had good MICs for these resistant strains, ranging from 0.0156 μg/ml to 16 μg/ml. The MICs of minocycline, levofloxacin, and sitafloxacin were the same as for ML-susceptible strains. Of the 12 strains, 10 originated from pneumonia cases and 2 originated from bronchitis cases.
TABLE 4.
In vitro antimicrobial activity of 10 agents against macrolide-resistant Mycoplasma pneumoniae strains (n = 12)
| Strain | Patient age (yr) | MIC (μg/ml)a
|
Nucleotide changes, 23S rRNAb | PFGE patternc | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ERY | CLR | AZM | TEL | JOS | MDM | RKI | MIN | LVX | SFX | ||||
| ARD-176 | 12 | 32 | 32 | 32 | 32 | 4 | 8 | 0.25 | 1 | 1 | 0.0625 | A2063G | IIb |
| ARD-185 | 10 | 64 | 64 | 32 | 64 | 0.0625 | 0.0313 | 0.0156 | 0.5 | 0.5 | 0.0625 | None | IIb |
| ARD-1909 | 6 | 64 | 64 | 32 | 32 | 4 | 4 | 0.25 | 0.0625 | 0.5 | 0.0313 | A2063G | IIb |
| ARD-1940 | 6 | 64 | 64 | 64 | 64 | 2 | 2 | 0.0313 | 0.0625 | 0.5 | 0.0313 | A2063G | I |
| ARD-1986 | 10 | 64 | 64 | 64 | 64 | 8 | 8 | 0.125 | 0.125 | 1 | 0.0313 | A2063G | I |
| ARD-2000 | 9 | 64 | 64 | 64 | 64 | 8 | 64 | 0.125 | 0.0625 | 0.5 | 0.0313 | A2064G | I |
| ARD-2081 | 9 | >64 | 16 | 16 | 2 | >64 | >64 | 8 | 0.5 | 0.5 | 0.0313 | A20634 | I |
| ARD-2159 | 11 | >64 | >64 | >64 | >64 | 8 | 8 | 0.125 | 0.125 | 0.5 | 0.0313 | A2063G | IIb |
| ARD-2313 | 11 | 64 | 64 | 32 | 64 | 64 | >64 | 16 | 0.25 | 1 | 0.0313 | A2063G | I |
| ARD-2316 | 13 | >64 | >64 | >64 | >64 | 8 | 8 | 0.125 | 0.125 | 1 | 0.0313 | A2063G | I |
| ARD-2323 | 3 | >64 | >64 | 64 | 4 | >64 | >64 | 16 | 0.0313 | 0.5 | 0.0156 | A2064G | IIb |
| ARD-2381 | 12 | >64 | >64 | >64 | >64 | 8 | 8 | 0.125 | 0.25 | 1 | 0.0313 | A2063G | I |
ERY, erythromycin; CLR, clarithromycin; AZM, azithromycin; TEL, telithromycin; JOS, josamycin; MDM, midecamycin; RKI, rokitamycin; MIN, minocycline; LVX, levofloxacin; SFX, sitafloxacin (DU-6859a).
M. pneumoniae numbering.
PFGE typing was classified into group I and subgroup II b as described by Cousin-Allery et al. (2).
Mutation on the 23S rRNA gene in ML-resistant M. pneumoniae.
The full-length 23S rRNA gene (8) in ML-resistant M. pneumoniae strains was sequenced, and the results of the transition mutation on the gene are shown in Table 4 along with MICs. Nine strains had an A2063G transition in domain V, and two strains had an A2064G transition. All these strains had no C785 transition in domain II. MICs of telithromycin for the latter strains were 2 μg/ml and 4 μg/ml, obviously different from strains with the A2063G transition. The strain numbered ARD-185 had no transition in the 23S rRNA gene.
No mutation that effects to amino acid substitution was identified on each gene encoding ribosomal proteins L4 and L22 (data not shown).
PFGE typing.
PFGE typing was performed on ML-susceptible strains selected randomly in addition to all ML-resistant strains. Figure 1 shows the results of those and M129 strain as a control digested by the ApaI restriction enzyme. All strains were classified into two groups, group I and subgroup IIb, as described by Cousin-Allery et al. (2).
FIG. 1.
Pulsed-field gel electrophoresis patterns generated by ApaI digestion of Mycoplasma pneumoniae strains. M129, reference strain; strain numbers ARD-1940 to ARD-2323, ML resistance (MLr); strain numbers ARD-11 to ARD-1987, ML susceptible (MLs); M, marker of lambda ladder.
Of the 12 ML-resistant strains, 7 belonged in group I, and 5 belonged in subgroup IIb (Fig. 1 and Table 4). The ML-resistant strains were isolated from three different areas (Tokyo, Osaka, and Chiba, Japan). The difference in PFGE patterns was not recognized between samples from different areas.
DISCUSSION
M. pneumoniae is a common etiologic agent of RTI in communities, particularly in school-aged children and young adults. This agent accounts for 10 to 30% of CAPs (4, 6, 13).
Generally, 14-ML and azithromycin have been prescribed as first-choice agents for CAP and RTI due to M. pneumoniae when pathogen-specific therapy is used. Minocycline and fluoroquinolones may be used as alternatives.
Recently, telithromycin of the ketolide compounds, a new class of antimicrobial agent, has been developed based on resistance to ML in S. pneumoniae (1, 19). The chemical structure of telithromycin is characterized by a ketone group at the 3-position, a methyoxy group at the 6-position, and an alkyl/aryl extension from carbamate group at positions 11 and 12 (5, 18), which prevents methylation of 23S rRNA by methylase in ML-resistant strains. Telithromycin had excellent MICs of 0.063 to 0.25 μg/ml for ML-resistant S. pneumoniae possessing resistant determinants (23) and has been introduced clinically.
Meanwhile, ML-resistant M. pneumoniae was first isolated in 2000 from a 9-year-old girl with pneumonia (12, 16). These results suggest emergence of ML-resistant M. pneumoniae in Japan, not yet reported worldwide.
As described in the results, 93.8% of M. pneumoniae strains showed excellent susceptibilities to 14-ML, azithromycin, and telithromycin with MIC90s ranging from 0.0156 to 0.00195 μg/ml. However, 12 (6.2%) ML high-resistance M. pneumoniae strains were detected in this study. These cause clinical problems, since concentrations that overcome high resistance are not attainable by oral administration.
ML-resistant M. pneumoniae has been known for more than 20 years, although the mechanisms of resistance have been uncertain (15, 20). Transition mutations identified on the 23S rRNA gene in resistant strains were reported previously by Lucier et al. (11) using the M129 standard strain for in vitro mutation experiments; mutations at A2063G and A2064G in domain V correspond to A2058 and A2059 in Escherichia coli (18), respectively. All but one strain tested showed resistance, not only for 14-ML and azithromycin but also for all 16-MLs except rokitamycin, which has been developed in Japan and used in a few countries in Asia. From the current understanding of the mechanism of ML resistance, we speculate that telithromycin resistance is as follows: A784 detected in domain II of S. pneumoniae (9), which is supposed to be necessary for the expression of activity, was originally C785 in M. pneumoniae. This position in domain II in M. pneumoniae also corresponds to A752 in E. coli (18). The resistance mechanism in one strain, ARD185, is unknown, so further studies will be necessary.
Molecular typing by PFGE for M. pneumoniae has been carried out with isolates from two countries (2). These isolates were classified into two groups, group I and group II, with subgroups IIa and IIb. As shown in Fig. 1, all strains tested by PFGE typing belonged to either group I or group IIb, and ML-resistant strains were confirmed in both groups. These findings suggest that ML-resistant M. pneumoniae isolates may gradually be increasing in parallel with the increase in number of ML prescriptions to outpatients. The expansion of 14-ML, azithromycin, and telithromycin usage for RTI worldwide may increase ML-resistant M. pneumoniae. Further evaluation for new quinolones, such as sparfloxacin (10) and sitafloxacin, will be necessary in vivo and in vitro.
Although clinical symptoms appeared to be prolonged in patients with ML-resistant M. pneumoniae infections, their clinical course will be described in another paper.
In conclusion, studies are needed to clarify the prevalence of ML-resistant M. pneumoniae in adults and monitoring of global trends.
Acknowledgments
This work was supported in part by two grants, one from the Meiji Seika Kaisha and one from the Japanese Ministry of Education, Culture, Sport, Science and Technology (the 21st Century COE program).
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