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A total of 535 samples were collected. Of all samples, 265 cases were collected from China Mei-tan General Hospital, 112 cases were collected from Civil Aviation General Hospital, 88 cases were collected from Peking University Third Hospital, and 70 cases were collected from Beijing Chao-yang Hospital. Of all samples, 523 cases (523/535, 97.76%) were throat swab specimens, and 12 cases (12/535, 2.24%) were bronchoalveolar lavage fluid specimens. All bronchoalveolar lavage fluid specimens were collected from Peking University Third Hospital in December 2016 (Table 1).
Table 1. Sample collection
Category Outpatient samples Inpatient samples China Mei-tan
General HospitalCivil Aviation General Hospital Beijing Chao-yang Hospital, Capital Medical University Peking University Third Hospital Throat swabs 265 112 70 76 Bronchoalveolar lavage fluid 0 0 0 12* Total 265 112 70 88 Note. *All bronchoalveolar lavage fluid specimens were collected from Peking University Third Hospital in December 2016. -
Table 1 shows that of 535 samples, 214 were detected positive by real-time PCR, and the detection rate was 40%. Furthermore, 68 samples (68/214, 31.78%) had no mutant in domain V of the 23S rRNA gene and were sensitive bacteria. Also, 142 samples (142/214, 66.36%) harbored a mutation in domain V of the 23S rRNA gene. Among these, 134 cases harbored an A2063G mutation. However, seven cases harbored an A2064G mutation, and in one case, both A2063 and A2064G mutations were detected (Figure 1). Four samples (4/214, 2%) were mixed with sensitive M. pneumoniae and macrolide-resistant M. pneumoniae. Among these, three cases (Figure 2) were mixed with sensitive and A2063G mutant M. pneumoniae, and one (Figure 2) was mixed with sensitive and A2064G mutant M. pneumoniae.
A total of 377 samples were collected from outpatients, whereas 131 samples (131/377, 34.75%) were detected positive by real-time PCR. Moreover, 50 samples were nonmutated M. pneumoniae, 78 samples harbored a mutation in domain V of the 23S rRNA gene (of which 71 harbored an A2063G mutation and seven harbored an A2064G mutation), and in three cases, macrolide-susceptible as well as A2063G macrolide-resistant strains were detected (Table 2). Of the 158 samples collected from inpatients, 83 (83/158, 52.53%) were detected positive by real-time PCR. Also, 18 samples were nonmutated M. pneumoniae, 64 harbored mutation in domain V of the 23S rRNA gene (63 harbored an A2063G mutation, and one had both A2063G and A2064G mutations), whereas no mutated and A2064G mutated M. pneumoniae were detected in one sample. The detection rate of outpatient samples (131/377, 34.75%) was lower than that of inpatient samples (83/158, 52.53%), and the difference was statistically significant (P < 0.0001). The 23S rRNA gene mutation rate of outpatient samples (78/131, 59.54%) was significantly lower than that of inpatient samples (64/83, 77.11%), and the difference was statistically significant (P = 0.001)
Table 2. Detection of M. pneumoniae and 23S rRNA gene mutation
Category Outpatient Inpatient Total P Positive No mutated 50 18 68 0.001 Mutateda 78 64 142 0.001 Mixedb 3 1 4 Negative 246 75 321 < 0.0001 Total 377 158 535 Note. aIncluding A2063G and A2064G mutations; A2063G and A2064G both mutated.
bIncluding the mixture of no mutant and A2063G mutant M. pneumoniae and the mixture of no mutant and A2064G mutant M. pneumoniae. -
A total of 81 M. pneumoniae isolates were obtained from 535 patients; the detection rate was 15.14% (81/535). Among these, 12 isolates (12/81, 14.81%) were no mutant strains, and 69 isolates (69/81, 85.19%) were A2063G mutant strains (Table 3). All A2064G mutant samples, mixed samples, and double mutant samples could not be cultured. The growth rate of 23S rRNA gene mutant M. pneumoniae collected from inpatients was significantly higher than that of the 23S rRNA gene mutant M. pneumoniae collected from outpatients, no mutant samples from outpatients or inpatients; the differences were statistically significant (P < 0.0001). However, the growth rate of no mutant M. pneumoniae showed no difference between outpatients (9/50, 18%) and inpatients (3/18, 16.67%). Finally, the growth rate of no mutant samples (9/50, 18%) and mutant samples (19/78, 24.35%) collected from outpatients showed no statistically significant difference (P = 0.016).
Table 3. Cultures of outpatient and inpatient samples
Category Outpatient Inpatient No mutant 23S rRNA mutationa Mixedb No mutant 23S rRNA mutationa Mixedb PCR 50 78 3 18 64 1 Cultures 9 19 0 3 50 0 Growth Rate 18% 24.36% 0 16.67% 78.13% 0 Note. aIncluding A2063G and A2064G mutations; A2063G and A2064G both mutated.
bIncluding the mixture of no mutant and A2063G mutant M. pneumoniae and the mixture of no mutant and A2064G mutant M. pneumoniae. -
A total of 67 strains were randomly selected from all 81 isolates to test the MICs of erythromycin, azithromycin, levofloxacin, and tetracycline. The MIC of erythromycin for no mutant M. pneumoniae strains was 0.002–0.003 µg/mL. In contrast, the MIC for A2063G mutant M. pneumoniae was 8–1,024 µg/mL; the MIC90 was 512 µg/mL, and the MIC50 was 256 µg/mL. All erythromycin-resistant isolates also showed resistance to azithromycin. The MIC of azithromycin for no mutant M. pneumoniae strains was 0.0005–0.004 µg/mL, whereas the MIC for the A2063G mutant M. pneumoniae was 8–512 µg/mL; the MIC90 was 256 µg/mL, and the MIC50 was 128 µg/mL. All 67 selected isolates in this study were susceptible to tetracycline and levofloxacin.
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MLVA genotyping was performed on 65 A2063G mutation samples and nine no mutant samples (Table 4). Except for one A2063G mutation sample, the remaining 73 samples were successfully typed, including ten cases with type 3-5-6-2 and 63 were type 4-5-7-2. Among the type 3-5-6-2 samples, three had an A2063G mutation, and seven had no mutant. In contrast, among the type 4-5-7-2 samples, 61 had the A2063G mutation, and two had no mutant.
Table 4. MLVA genotyping and the mutation
Category 3-5-6-2 4-5-7-2 Failed Total No mutant 7 2 1 10 A2063G mutation 3 61 0 64 Total 10 63 1 74
doi: 10.3967/bes2020.125
Mycoplasma pneumoniae Macrolide Resistance and MLVA Typing in Children in Beijing, China, in 2016: Is It Relevant?
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Abstract:
Objective The aim of this study is to investigate the macrolide resistance rate and molecular type with multiple-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae of Beijing in 2016 in pediatric patients. Methods Real-time quantitative polymerase chain reaction (PCR) was used to identify M. pneumoniae, and MLVA was performed. The domain V of the 23S rRNA was sequenced to detect macrolide-resistant point mutations. We also investigated the activities of antibiotics against M. pneumoniae isolates in vitro. Results The PCR detection rate of M. pneumoniae in children in Beijing was 40%, and the macrolide resistance rate was 66%. The A2063G mutation in the 23S rRNA V region is the dominant mutation (137/146, 93.84%), whereas the A2064G mutation is rare (9/146, 6.16%). Seventy-three samples were typed successfully by MLVA typing, including 86.3% (63/73) were MLVA type 4-5-7-2, and 13.7% (10/73) were MLVA type 3-5-6-2. No other types were found. No strains were resistant to levofloxacin or tetracycline. Conclusion In 2016, a specific decrease in the macrolide resistance rate occurred in Beijing. The detection rate and macrolide resistance rate of outpatients are lower than those of inpatients. The A2063G mutants M. pneumoniae have high levels of resistance to erythromycin and azithromycin. The primary MLVA type is 4-5-7-2, followed by 3-5-6-2. No other MLVA types were detected. No strains resistant to tetracycline or levofloxacin were found in vitro. -
Key words:
- Culture /
- Macrolide resistance /
- MLVA type /
- Mycoplasma pneumoniae
注释: -
Table 1. Sample collection
Category Outpatient samples Inpatient samples China Mei-tan
General HospitalCivil Aviation General Hospital Beijing Chao-yang Hospital, Capital Medical University Peking University Third Hospital Throat swabs 265 112 70 76 Bronchoalveolar lavage fluid 0 0 0 12* Total 265 112 70 88 Note. *All bronchoalveolar lavage fluid specimens were collected from Peking University Third Hospital in December 2016. Table 2. Detection of M. pneumoniae and 23S rRNA gene mutation
Category Outpatient Inpatient Total P Positive No mutated 50 18 68 0.001 Mutateda 78 64 142 0.001 Mixedb 3 1 4 Negative 246 75 321 < 0.0001 Total 377 158 535 Note. aIncluding A2063G and A2064G mutations; A2063G and A2064G both mutated.
bIncluding the mixture of no mutant and A2063G mutant M. pneumoniae and the mixture of no mutant and A2064G mutant M. pneumoniae.Table 3. Cultures of outpatient and inpatient samples
Category Outpatient Inpatient No mutant 23S rRNA mutationa Mixedb No mutant 23S rRNA mutationa Mixedb PCR 50 78 3 18 64 1 Cultures 9 19 0 3 50 0 Growth Rate 18% 24.36% 0 16.67% 78.13% 0 Note. aIncluding A2063G and A2064G mutations; A2063G and A2064G both mutated.
bIncluding the mixture of no mutant and A2063G mutant M. pneumoniae and the mixture of no mutant and A2064G mutant M. pneumoniae.Table 4. MLVA genotyping and the mutation
Category 3-5-6-2 4-5-7-2 Failed Total No mutant 7 2 1 10 A2063G mutation 3 61 0 64 Total 10 63 1 74 -
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