难治性肺炎支原体肺炎患儿支气管肺泡灌洗液中肺炎支原体耐药基因检测分析

Detection of drug-resistance genes of Mycoplasma pneumoniae in bronchoalveolar lavage fluid of children with refractory Mycoplasma pneumoniae pneumonia

摘要:

目的 分析难治性肺炎支原体肺炎（refractory Mycoplasma pneumoniae pneumonia，RMPP）患儿支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中肺炎支原体（Mycoplasma pneumoniae，MP）耐药性及耐药基因分布，并阐明23SrRNA Ⅴ区中心环2063位等位基因与抗菌药物耐药性的关系。方法 收集我院2016年3月至2020年12月收治的RMPP患儿BALF标本245例，采用快速培养基培养药敏法检测MP分离株对9种常用抗菌药物的耐药性，并采用Real-Time PCR法检测MP DNA，PCR产物采用直接测序方法检测MP 23SrRNA Ⅴ区中心环基因突变情况。结果 245例RMPP患儿BALF标本中，MP DNA检测阳性207例，阳性率为84.5%；药敏试验结果显示，RMPP患儿对大环内酯类抗菌药物耐药率均较高（> 70%），耐药率由高到低依次为克拉霉素、罗红霉素、克林霉素、乙酰螺旋霉素、红霉素及阿奇霉素，对喹诺酮类抗菌药物耐药率均较低（< 5%）；207例MP DNA阳性标本中，41例（19.8%）无耐药基因突变，166例（80.2%）存在耐药基因突变，其中154例（74.4%）突变位点为23SrRNA Ⅴ区中心环2063位A→G点突变，7例（3.4%）突变位点为2064位A→G点突变，5例（2.4%）为2063和2064双位点突变，166例MP 23SrRNA基因点突变中2063位点突变率达95.8%（159/166）；23SrRNA Ⅴ区中心环2063位A→G点突变对大环内酯类抗菌药物耐药影响较大，克拉霉素、罗红霉素、克林霉素、乙酰螺旋霉素、红霉素及阿奇霉素耐药病例的2063位等位基因分布比较，差异均有统计学意义（P < 0.05）。结论 RMPP患儿BALF中MP对大环内酯类抗菌药物耐药率较高，大环内酯类抗菌药物耐药与23SrRNA位A→G点突变密切相关，23SrRNA Ⅴ区中心环2063位点突变可能影响MP耐药机制。

Abstract:

Objective To study the drug resistance of Mycoplasma pneumoniae (MP) in the bronchoalveolar lavage fluid (BALF) of children with refractory Mycoplasma pneumoniae pneumonia (RMPP) and the distribution of drug-resistance genes, as well as the association of alleles at 2063 locus of 23SrRNA V region central ring with resistance to antimicrobial drugs. Methods BALF specimens were collected from 245 children with RMPP who were admitted to the Children's Hospital Affiliated to Zhengzhou University from March 2016 to December 2020. A rapid cultured drug sensitivity assay was used to detect the resistance of MP isolates to nine commonly used antimicrobial drugs. The real-time PCR was used to measure MP DNA. The direct sequencing was used to detect gene mutations in MP 23SrRNA V region central ring. Results Among the 245 BALF specimens, 207 tested positive for MP DNA, with a positive rate of 84.5%. The results of drug susceptibility test showed that the children with RMPP had a resistance rate of > 70% to macrolide antimicrobial drugs, with the highest resistance rate to clarithromycin, followed by roxithromycin, clindamycin, acetylspiramycin, erythromycin, and azithromycin, and these children had a resistance rate of < 5% to quinolone antimicrobial drugs. Among the 207 MP DNA-positive specimens, 41 (19.8%) had no drug-resistance gene mutations and 166 (80.2%) had drug-resistance gene mutations, among which 154 (74.4%) had an A→G mutation at 2063 locus of 23SrRNA V region central ring, 7 (3.4%) had an A→G mutation at 2064 locus, and 5 (2.4%) had mutations in both 2063 and 2064 loci. Among the 166 specimens with point mutations of the MP 23SrRNA gene, 159 (95.8%) had point mutations at 2063 locus. The A→G point mutation at 2063 locus of 23SrRNA V region central ring had a great impact on resistance to macrolide antimicrobial drugs. There was a significant difference in the distribution of alleles at 2063 locus between the children with resistance to clarithromycin, roxithromycin, clindamycin, acetylspiramycin, erythromycin, and azithromycin (P < 0.05). Conclusions MP in the BALF of children with RMPP has a relatively high resistance rate to macrolide antimicrobial drugs. Resistance to macrolide antimicrobial drugs is closely associated with the A→G point mutation in the 23SrRNA gene, and the point mutation at 2063 locus of 23SrRNA V region central ring may affect the drug-resistance mechanism of MP.