百日咳疑似患儿及家属百日咳鲍特菌阳性情况及菌株耐药分析

Analysis of antibiotic resistance and infection of Bordetella pertussis in children with suspected pertussis and close family members

目的了解浙江省儿童疑似百日咳病例及其密接家庭成员中百日咳鲍特菌的检出情况及菌株分离率,探究百日咳鲍特菌的耐药特征及其耐药机制。方法采集2022年5—10月杭州市儿童医院收治的百日咳疑似患儿及其密接家庭成员的鼻咽拭子共273份作为研究样本。使用木炭选择性培养基进行菌株的分离培养;荧光定量PCR法对采集样本进行百日咳靶标基因的检测;E-test法检测百日咳鲍特菌株对不同抗菌药物的敏感性;全基因组测序分析百日咳鲍特菌株对大环内酯类抗菌药物的耐药机制,并对分离的菌株进行基于核心基因组多位点序列分型(cgMLST)的进化分析。结果273株百日咳疑似患儿及其密接家庭成员样本中,荧光定量PCR阳性的样本为168份,占比61.54%,成功分离培养30株百日咳鲍特菌,阳性率为10.98%。另外,143份密接家庭成员样本中,54.55%(78/143)的样本PCR检测为阳性,9.79%(14/143)培养阳性,提示家庭成员在百日咳的家庭传播中具有重要意义,且母亲占比较高。药敏结果显示,96.67%(29/30)的菌株对大环内酯类抗菌药物阿奇霉素的MIC值>256 mg/L,显示出高度耐药性,其耐药机制均为23S rRNA发生A2047G突变。基于cgMLST的进化分析显示2022年分离的百日咳鲍特菌株已经形成两个单独的分支。结论疑似百日咳患儿的密接家庭成员中百日咳鲍特菌阳性率较高且以母亲为主,对疑似患儿家庭成员的监测及实验室检测具有重要意义;浙江省百日咳鲍特菌对大环内酯类抗菌药物呈现出高度耐药性,应进一步加强耐药性监测,为临床治疗及用药提供理论依据。

Objective To investigate the positive rate and isolate Bordetella pertussis in children with suspected whooping cough and their close family members in Zhejiang Province,and further explore the susceptibility and resistant mechanism of Bordetella pertussis to antibiotics.Methods A total of 273 nasopharynx swabs specimens from children with suspected whooping cough in Hangzhou Children′s Hospital from May 2022 to October 2022 were collected.The strains were isolated and cultured using charcoal select agar plate.Pertussis target genes were detected by RT-PCR.E-test method was used to detect the sensitivity of Bordetella pertussis strains to different antibiotics.The mechanism of resistance of Bordetella pertussis to macrolides was analyzed by whole genome sequencing.The phylogenetic analysis of isolated strains was based on core genome multilocus sequence typing(cgMLST).Results Among 273 clinical samples of children with suspected pertussis and their close family members,168 samples were positive by fluorescence quantitative PCR,accounting for 61.54%,and 30 pertussis strains were successfully isolated with a positive rate of 10.98%.In addition,among the 143 samples of close family members,54.55%(78/143)samples were positive by RT-PCR and 9.79%(14/143)samples were positive by culture,suggesting that the close family member are important in family transmission of pertussis.Besides,most of the positive samples were from mothers.The results of E-test showed that 96.67%(29/30)strains showed high resistance to azithromycin with MIC value>256 mg/L,and the resistant mechanism of azithromycin was A2047G mutation in 23S rRNA.The phylogenetic analysis based on the cgMSLT showed that the isolated strains were clustered into two new different clades.Conclusions The positive rate of Bordetella pertussis in close family members is at a high level and the mother may be the main source of infection,which is of great significance for monitoring and laboratory detection of suspected children′s family members.Bordetella pertussis shows high resistance to macrolides in Zhejiang Province,so monitoring of the antimicrobial resistance should be further strengthened to provide theoretical basis for clinical treatment and drug guidance.