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中华实验和临床感染病杂志(电子版) ›› 2018, Vol. 12 ›› Issue (01) : 20 -27. doi: 10.3877/cma.j.issn.1674-1358.2018.01.005

所属专题： 文献；

临床论著

新生儿侵袭性感染B族链球菌的耐药表型及耐药机制
李娟1, 高坎坎2, 容莉莉2, 关小珊2, 邓秋连2, 钟华敏2, 龙燕2, 高秀蓉2, 张丽华3, 穆小平4, 袁春雷5, 计文婧6, 刘海英2,†()   
  1. 1. 510070　广州市，广州医科大学附属肿瘤医院检验科；510623　广州市，广州市妇女儿童医疗中心检验科
    2. 510623　广州市，广州市妇女儿童医疗中心检验科
    3. 523110　东莞市，东莞东华医院检验科
    4. 511400　广州市，广东省妇幼保健院
    5. 528403　中山市，南方医科大学附属中山博爱医院
    6. 710061　西安市，西安交通大学药物院药事管理与临床药学系
  • 收稿日期:2017-04-28 出版日期:2018-02-15
  • 通信作者: 刘海英
  • 基金资助:
    广东省科技计划项目(No. 2015A030401007); 广州市卫生局西医类一般项目(No. 20151A010040，No. 20161A010026，No. 20151A010034)

Resistance patterns and mechanism of group B Streptococcus isolated from infants with invasive disease

Juan Li1, Kankan Gao2, Lili Rong2, Xiaoshan Guan2, Qiulian Deng2, Huamin Zhong2, Yan Long2, Xiurong Gao2, Lihua Zhang3, Xiaoping Mu4, Chunlei Yuan5, Wenjing Ji6, Haiying Liu2,†()   

  1. 1. Department of Clinical Laboratory, Guangzhou Medical University Affiliated Tumor Hospital, Guangzhou 510070, China
    2. Department of Clinical Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou 510623, China
    3. Department of Clinical Laboratory, Donghua Hospital of Dongguan, 523110 Dongguan, China
    4. Maternal and Child Health Hospital of Guangdong Provincial, 511400 Guangzhou, China
    5. Boai Hospital of Zhongshan, Affiliated to Southern Medical University, 528403 Zhongshan, China
    6. Management and Clinical Pharmacy, Xi’an Jiaotong University, 710061 Xi’an, China
  • Received:2017-04-28 Published:2018-02-15
  • Corresponding author: Haiying Liu
  • About author:
    Corresponding author: Liu Haiying, Email:
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李娟, 高坎坎, 容莉莉, 关小珊, 邓秋连, 钟华敏, 龙燕, 高秀蓉, 张丽华, 穆小平, 袁春雷, 计文婧, 刘海英. 新生儿侵袭性感染B族链球菌的耐药表型及耐药机制[J/OL]. 中华实验和临床感染病杂志(电子版), 2018, 12(01): 20-27.

Juan Li, Kankan Gao, Lili Rong, Xiaoshan Guan, Qiulian Deng, Huamin Zhong, Yan Long, Xiurong Gao, Lihua Zhang, Xiaoping Mu, Chunlei Yuan, Wenjing Ji, Haiying Liu. Resistance patterns and mechanism of group B Streptococcus isolated from infants with invasive disease[J/OL]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2018, 12(01): 20-27.

目的

通过分析侵袭性感染新生儿的B族链球菌（GBS）临床分离株的耐药性和耐药基因型，为新生儿GBS感染的防治和抗菌药物的合理应用提供理论依据。

方法

收集2013年1月至2016年6月就诊于广东省4家医院GBS侵袭性感染< 90 d新生儿病例。使用VITEK-2 Compact全自动细菌鉴定及药敏分析系统对GBS分离株进行鉴定和抗菌药物最小抑菌浓度（MIC）测定；应用PCR方法进行红霉素和四环素耐药基因检测。

结果

确诊新生儿GBS侵袭性感染93例，其中34例（36.6%）早发型，59例（63.4%）晚发型。93株GBS分离株药物敏感试验结果显示，GBS对青霉素类、头孢菌素类、利奈唑胺和万古霉素均100%敏感；对左氧氟沙星、氧氟沙星和阿奇霉素的耐药率较低，分别为8.6%、2.2%和1.1%。对红霉素、克林霉素和四环素的耐药率高，分别为60.2%、78.5%和93.5%；耐药表型以固有表型（cMLSB）为主，占53.9%，其次为L表型（26.3%）、诱导表型（iMLSB）（15.8%）和M表型（3.9%）。56株红霉素耐药菌株ermB基因的携带率为85.7%，2株（3.6%）耐药菌株同时携带ermB和mefA基因，均未检出ermA。87株四环素耐药菌株中，四环素耐药基因tetO和tetM携带率分别为74.7%和46%，其中25株同时携带tetO和tetM，均未检出tetL和tetK基因。

结论

青霉素和氨苄西林仍是治疗新生儿侵袭性GBS感染及高危新生儿预防GBS感染的首选药物。广东地区GBS红霉素耐药机制以ermB基因介导的核糖体靶位改变为主，四环素耐药机制以tetO和tetM基因为主。

关键词: 新生儿, B族链球菌, 微生物敏感性试验, 耐药基因
Objective

To investigate the antibiotics resistance rates and the resistance mechanisms of erythromycin and tetracycline among group B Streptococcus (GBS)-positive isolates cultured from infants with invasive infection, and to provide a reliable evidence for the prevention and treatment of infants with invasive GBS infection.

Methods

From January 2013 to June 2016, the GBS strains isolated from infants with invasive diseases of < 90 days from four teaching hospitals were collected to investigate the current antimicrobial resistance status and resistance genes of these strains. The GBS isolates were identified and the minimum inhibitory concentration (MIC) of the antibiotics was determined by VITEK-2 Compact automatic bacterial identification system. Genes associated with resistance to erythromycin (ermA, ermB, mefA) and tetracycline (tetK, tetL, tetM, tetO) were investigated using PCR amplification and DNA sequencing.

Results

Total of 93 non-duplicate colonizing GBS isolates were collected from infants with invasive GBS infection, among whom, 34 cases with early-onset infection and 59 cases with late-onset infection were diagnosed. All the isolates were susceptible to penicillins, cephalosporins, linezolid and vancomycin. The resistance of the isolates to levofloxacin, ofloxacin and azithromycin were 8.6 %, 2.2% and 1.1%, respectively. The resistance of isolates to erythromycin, clindamycin and tetracycline were significantly high, which were 60.2%, 78.5% and 93.5%, respectively. The most predominant phenotype was cMLSB (53.9%), followed by L phenotype (26.3%), iMLSB (15.8%) and M phenotype (3.9%). Among the resistance genes investigated, tetM (46%) and tetO (74.7%) were detected among tetracycline-resistant isolates and ermB (85.7%) and mefA (3.6%) were found among macrolide-resistant isolates. But ermA was not detected.

Conclusions

For neonates and young infants, empirical treatment with penicillins and ampicillin at birth for all high-risk neonates were recommended. Ribosomal modification by ermB gene play major role in erythromycin resistance of GBS, and the tetracycline resistance mediated by tetO and tetM genes were dominated.

Key words: Infant, Group B Streptococcus, Antimicrobial sensitivity test, Resistance gene
表1 红霉素和克林霉素耐药基因引物序列及产物
引物 核酸序列（5'→3'） 产物长度（bp）
ermA-F TCT AAA AAG CAT GTA AAA GAA 645
ermA-R CTT CGA TAG TTT ATT AAT ATT AGT
ermB-F GAA AAG GTA CTC AAC CAA ATA 639
ermB-R AGT AAC GGT ACT TAA ATT GTT TAC
mefA-F CGTAGCATTGGAACAGC 316
mefA-R TGCCGTAGTACAGCCAT
tetL-F TGAACGTCTCATTACCTG 993
tetL-R ACGAAAGCCCACCTAAAA
tetO-F AAC TT AGGCAT TC TGGC TCAC 515
tetO-R TCCC AC TGT TCCA TAT CG TCA
tetK-F TCCTGGAACCATGAGTGT 189
tetK-R AGATAATCCGCCCATAAC
tetM-F TTATCAACGGTTTATCAGG 397
tetM-R CGTATATATGCAAGACG
表2 93例GBS感染的标本分布构成比 [例（%）]
疾病类型 血液 脑脊液 血液+脑脊液 关节腔积液
GBS-EOD 28（30.1） 1（1.1） 5（5.4） 0（0.0）
GBS-LOD 34（36.6） 16（17.2） 8（8.6） 1（1.1）
合计 62（66.7） 17（18.3） 13（14.0） 1（1.1）
表3 93株GBS对11种抗菌药物的耐药情况 [例（%）]
抗菌药物 耐药 中介 敏感
株数 耐药率 株数 中介率 株数 敏感率
青霉素G 0 0.0 0 0.0 93 100.0
氨苄西林 0 0.0 0 0.0 93 100.0
头孢曲松 0 0.0 0 0.0 93 100.0
万古霉素 0 0.0 0 0.0 93 100.0
利奈唑胺 0 0.0 0 0.0 93 100.0
四环素 87 93.5 0 0.0 6 6.5
克林霉素 73 78.5 0 0.0 20 22.6
红霉素 56 60.2 17 18.3 20 21.5
左氧氟沙星 8 8.6 1 1.1 84 90.3
氧氟沙星 2 2.2 0 0.0 91 97.8
阿奇霉素 1 1.1 0 0.0 92 98.9
图1 2013至2016年侵袭性B族链球菌对四环素、红霉素和克林霉素的耐药率变迁
表4 红霉素和克林霉素耐药表型与基因型的相关性
耐药表型 菌株 [株（%）] 耐药基因（株） 耐药特征
cMLSB 41（53.9） ermB（33） 红霉素和克林霉素同时耐药
ermB + mefA（2）
iMLSB 12（15.8） ermB（11） 红霉素耐药、克林霉素诱导耐药
M表型 3（3.9） ermB（2） 红霉素耐药、克林霉素敏感
L表型 20（26.3） ermB（18） 克林霉素耐药、红霉素敏感或中介
ermB + mefA（1）
合计 76 ermB（64）
ermB + mefA（3）
图2 红霉素和四环素耐药基因PCR产物电泳图
图3 红霉素和四环素耐药基因部分基因序列图
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