新生儿早发型无乳链球菌感染的风险因素及耐药分析

Risk factors and drug resistance in early-onset neonatal group B streptococcal disease # # 新生儿早发型无乳链球菌感染的风险因素及耐药分析 1 first-author 2 3 4 4 2 2 5 5 3 6 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:chendong_wz@126.comwangzhen@hmc.edu.cn †E-mail:chendong_wz@126.comwangzhen@hmc.edu.cn chendong_wz@126.com 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:chendong_wz@126.comwangzhen@hmc.edu.cn †E-mail:chendong_wz@126.comwangzhen@hmc.edu.cn chendong_wz@126.com Department of Laboratory Medicine, Tiantai People’s Hospital, Taizhou 317200, China Department of Laboratory Medicine, Tiantai People’s Hospital, Taizhou 317200, China Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou 310014, China Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou 310014, China Department of Infectious Diseases, Eye Hospital of Wenzhou Medical University, Wenzhou 325035, China Department of Infectious Diseases, Eye Hospital of Wenzhou Medical University, Wenzhou 325035, China Department of Laboratory Medicine, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China Department of Laboratory Medicine, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China Department of Laboratory Medicine, Wenzhou People’s Hospital, Wenzhou 325000, China Department of Laboratory Medicine, Wenzhou People’s Hospital, Wenzhou 325000, China Department of Laboratory Medicine, The Sixth People Hospital of Wenzhou, Wenzhou 325015, China Department of Laboratory Medicine, The Sixth People Hospital of Wenzhou, Wenzhou 325015, China In recent years, group B streptococcus (GBS) has become an important pathogen that causes infections in many neonatal organs, including the brain, lung, and eye (Ballard et al., 2016 2016 ). A series of studies performed on GBS infections in western countries have revealed that GBS is one of the primary pathogens implicated in perinatal infection, and GBS infections are a major cause of neonatal morbidity and mortality in the United States (Decheva et al., 2013 2013 ). In China, GBS is mainly found by screens for adult urogenital tract and perinatal infections, and neonatal GBS infections have been rarely reported. The incidence rate of early-onset neonatal GBS disease is thought to be lower in China than in western countries; however, this data is controversial since it also reflects the clinical interest in GBS (Dabrowska-Szponar and Galinski, 2001 2001 ). 目的 2 新生儿早发型无乳链球菌(GBS)是一种可致婴儿脑、肺和眼部发生感染甚至死亡的细菌.筛查其感染风险因素并建立概率预测模型有助于疾病的预防和控制,进一步分析GBS菌株的耐药类型和耐药基因,为疾病的临床诊断和治疗提供指导意见. 创新点 2 首次归类了新生儿感染GBS的众多因素,按照孕妇、分娩过程、胎儿因素分为三类,统计筛选出重要风险因素并建立Logistic回归的概率预测模型,并通过聚合酶链式反应(PCR)筛查到了相关耐药基因. 方法 2 收集2007年1月至2015年12月,浙江大学医学院附属妇产科医院、温州医科大学附属眼视光医院和温州市人民医院的实验组135例早发型GBS感染和对照组234例无感染的新生儿及其母亲的临床资料和标本,进行数据统计分析并构建概率预测模型,采用传统的药敏实验对所有GBS阳性标本进行耐药分析,同时PCR筛查耐药基因并测定序列. 结论 2 这9年间新生儿早发型GBS感染率呈上升趋势.孕妇泌尿系统感染、胎膜早破、胎龄这三项因素与感染GBS的结局存在显著关联,由此得出的Logistic回归模型可用于预测感染GBS的概率.135株GBS对克林霉素、红霉素、四环素的耐药菌株比例分别为51.11%、80.00%、91.11%,同时未发现对万古霉素、青霉素、呋喃妥因、力奈唑胺的耐药性.PCR筛查发现 aac6 、 ant6-I 、 aph3-III 和 TEM 四个耐药基因,其中 aph3-III 仅存在于耐药株中,而测序发现 aac6 基因在非耐药株中相比耐药株存在一个缺失突变.临床上尽早对患者分离的GBS药敏试验,有助于合理用药和有效治疗. Neonate Group B streptococcus Risk factor Drug-resistance 新生儿 无乳链球菌 风险因素 耐药性 In recent years, group B streptococcus (GBS) has become an important pathogen that causes infections in many neonatal organs, including the brain, lung, and eye (Ballard et al., 2016 2016 ). A series of studies performed on GBS infections in western countries have revealed that GBS is one of the primary pathogens implicated in perinatal infection, and GBS infections are a major cause of neonatal morbidity and mortality in the United States (Decheva et al., 2013 2013 ). In China, GBS is mainly found by screens for adult urogenital tract and perinatal infections, and neonatal GBS infections have been rarely reported. The incidence rate of early-onset neonatal GBS disease is thought to be lower in China than in western countries; however, this data is controversial since it also reflects the clinical interest in GBS (Dabrowska-Szponar and Galinski, 2001 2001 ). The clinical manifestations of neonatal GBS infections can be divided into two subtypes based on the time of onset of postnatal symptoms: early and late onset. Early-onset GBS infections occur by vertical (maternal-infant) transmission under normal conditions (Barbadoro et al., 2011 2011 ). Crago et al. ( 2012 2012 ) have identified the risk factors associated with different fetus prognoses in mothers with GBS infections. However, few researchers have examined the diverse risk factors associated with neonatal GBS disease or the effect of interactions. In this study, factors associated with neonatal GBS disease were divided into three categories: factors of gravidas, delivery, and neonates. We collected samples that included 135 cases (mother-neonate pairs) of early-onset neonatal GBS disease and 234 controls in accordance with clinical diagnostic criteria between January 2007 and December 2015 in Women’s Hospital of Zhejiang University School of Medicine, the Eye Hospital of Wenzhou Medical University, and Wenzhou People’s Hospital, China. There was an increasing trend in the overall incidence of early-onset neonatal GBS disease (Fig. 1a 1a ). Twenty-two parameters associated with disease were selected for univariate analysis (Table 1 1 ). Five factors (gravida urinary tract infection, premature rupture of membranes, gestational age, bilirubin of neonates, and phototherapy of neonates) were statistically significant ( P < 0.05). Values for case and control are expressed as mean±standard deviation or number/number. Apgar5: 5-min Apgar score. P < 0.05 Factor Case ( n =135) Control ( n =234) χ 2 or t P -value Factors of gravidas  Puerpera age (year) 30.4±6.1 29.5±4.0 −1.242 0.215  Parity (each time) 1.6±0.9 1.9±1.2 1.186 0.237  Delivery time (each time) 1.2±0.5 1.2±0.4 −1.043 0.298  Pregnancy hypertension syndrome (no/yes) 132/3 221/13 1.560 0.212  Gestational diabetes mellitus (no/yes) 123/12 210/24 0.182 0.670  Endocrine disease (no/yes) 132/3 227/7 0.011 0.916  Urinary tract infection (no/yes) 129/6 207/27 5.291 0.021 * * Factors of delivery  Embryo number (1/(2 or 3)) 132/3 222/12 1.184 0.277  Premature rupture of membrane (<18 h/≥18 h) 78/57 222/12 77.484 1.34×10 −18 * *  Character of amniotic fluid (normal/abnormal) 114/21 195/39 0.078 0.781  Volume of amniotic fluid (normal/abnormal) 132/3 221/13 1.560 0.212  Fetal distress (no/yes) 93/42 178/56 2.262 0.133  Mother’s fever (no/yes) 126/9 223/11 0.645 0.422  Way of delivery (normal/abnormal) 87/48 128/106 3.342 0.068 Factors of neonates  Gestational age (mature/premature) 134/1 186/48 27.371 1.68×10 −7 * *  Gender (male/female) 75/60 132/102 0.025 0.873  Weight (g) 3101±602 3078±729 −0.204 0.838  Neonatal Apgar5 score (point) 9.7±1.4 9.8±1.2 0.312 0.755  Neonatal respiratory disease (normal/abnormal) 120/15 201/33 0.677 0.411  Neonatal survival (survival/death) 134/1 232/2 0.014 0.907  High bilirubin (no/yes) 63/72 165/69 20.619 5.61×10 −6 * *  Phototherapy (no/yes) 81/54 193/41 22.628 1.97×10 −6 * * A binary multivariate logistic regression model (IBM SPSS 21.0) was performed, revealing that the gravida urinary tract infection, premature rupture of membranes, and gestational age were statistically significant ( P < 0.05, Table 2 2 ). Thus, the probability prediction model of early-onset neonatal GBS disease was as follows: $$P = {{\text{e}}^y}/\left( {{\text{1}} + {{\text{e}}^y}} \right)$$ , $$y = - {\text{3}}.{\text{8}}A + {\text{4}}.{\text{1}}B - {\text{3}}.{\text{2}}C - 0.{\text{7}}$$ , where P is probability, A represents gravida urinary tract infection (no, A =0; yes, A =1), B represents premature rupture of membrane ( < 18 h, B =0; ≥18 h, B =1), and C represents gestational age (mature, C =0; premature, C =1). B : regression coefficient; SE: standard error; OR: odds ratio Factor B SE OR P -value Gravida urinary tract infection −3.8 0.86 19.62 9.4×10 −6 Premature rupture of membranes 4.1 0.73 30.81 2.8×10 −8 Gestational age −3.2 1.02 9.58 2.0×10 −3 Constant value −0.7 0.14 27.04 2.0×10 −7 A study of drug susceptibility in 135 cases was performed by the disk diffusion (K–B) method. We found that the strains were not resistant to vancomycin, penicillin, nitrofurantoin, or linezolid. However, 51.11%, 80.00%, and 91.11% of the strains were resistant to clindamycin, erythromycin, and tetracycline, respectively (Fig. 1b 1b ). Twelve strains of GBS were screened for subsequent experiments, revealing that two were sensitive to clindamycin, erythromycin, tetracycline, and levofloxacin, whereas the other ten were drug-resistant. We then amplified 25 candidate drug-resistance genes (polymerase chain reaction (PCR) primers are shown in Table S1 S1 ), revealing the aac6 , ant6-I , aph3-III , and TEM genes in the GBS samples (Fig. 2 2 ). We obtained aac6 amplicons of about 250 bp, and this gene showed low expression in the two drug-sensitive strains. The ant6-I amplicons of about 600-bp products in all strains, as well as that of aph3-III , were not expressed in drug-sensitive strains, but a 300-bp product in some (6/10) drug-resistant strains was found. Finally, a TEM amplicon of about 500 bp in some drug-sensitive strains (1/2) and drug-resistant strains (9/10) was found. Therefore, GBS drug resistance may be closely related to the presence of aph3-III (Fig. 2c 2c ). (a) aac6 (220 bp); (b) ant6-I (597 bp); (c) aph3-III (292 bp); (d) TEM (535 bp); (e) Drug-sensitive strains in acc6 ; (f) Drug-resistant strains in acc6 . M: marker PCR products of drug-resistant genes were sequenced by Sanger sequencing (Invitrogen, Shanghai, China), and sequences were aligned and visualized using the BioEdit 7.2.2. We found that aac6 , ant6-I , and TEM were expressed in both drug-sensitive and drug-resistant strains; hence, we sequenced the PCR products of these three genes in the 12 GBS strains. The aac6 sequences had a deletion mutation (T) in the drug-sensitive strains but not in the drug-resistant strains (Figs. 2e and 2f 2e and 2f ), suggesting that the mutation attenuated the GBS drug resistance. As we obtained samples over the years, the rate of early-onset neonatal GBS disease detected in blood cultures has increased, suggesting that more attention should be paid to GBS prevention. Notably, our logistic regression analysis revealed three factors that were associated with variations in GBS: premature rupture of membranes was identified as a risk factor, and the other two factors were found to be protective factors. Perhaps the bacteria involved in gravida urinary tract infections have a competitive relationship with GBS and other bacteria (Yu et al., 2016 2016 ). To our knowledge, the low GBS infection rate of premature neonates has not been previously reported. The US Center for Disease Control and Prevention (CDC) recommended penicillin as the first-choice drug for early-onset neonatal GBS disease treatment in China (Li et al., 2009 2009 ). Erythromycin and clindamycin were recommended for penicillin-resistant strains or for patients who were allergic to penicillin (Juncosa-Morros et al., 2014 2014 ). The high level of resistance to clindamycin, erythromycin, and tetracycline may reflect overuse of this drugs in the general population. The Clinical and Laboratory Standards Institute (CLSI) recommends that clinical trials can be conducted without the need for antimicrobial susceptibility testing and experience with penicillin-based antimicrobial therapy (Schrag et al., 2000 2000 ). Persson et al. ( 2008 2008 ) reported that the prevalence of penicillin-resistant infections had increased year-over-year in Sweden. Moreover, the erythromycin resistance rate had reached 14%–20%, and the clindamycin resistance rate had reached 10%–16%. However, in our study, the results of early-onset GBS infection in in vitro susceptibility tests revealed that the sensitivity of penicillin was 100%, and the erythromycin and clindamycin resistance rates were 80.00% and 51.11%, respectively. Therefore, the applicability of the US CDC guidelines for treatment is questionable. When GBS-positive cases are identified to avoid the possibility of serious consequences for neonates, we suggest the application of early and adequate doses of penicillin or ampicillin. These coupled with other forms of neonatal prophylaxis and treatment can effectively reduce neonatal mortality (Dabrowska-Szponar and Galinski, 2001 2001 ). For penicillin-resistant or allergic cases, GBS should be isolated for susceptibility testing as soon as possible, and an effective drug should be identified from drug susceptibility test results. Strains carrying drug-resistant genes are common causes of drug resistance (Yang et al., 2015 2015 ). In this study, PCR analyses revealed that 4 of 25 well-known drug-resistance genes were present in different GBS samples. Among these, the aminoglycoside antibiotic resistance genes aac6 and ant6-I were expressed both in drug-sensitive and drug-resistant strains, while aph3-III was expressed only in drug-resistant strains (6/10). Sequencing results revealed that the PCR products of the aac6 gene contained a deletion mutation that may lead to decreased expression of this drug-resistance gene and thus decreased resistance to relevant antibiotics. The β-lactamase gene TEM , which confers penicillin resistance in many strains (Pimenta et al., 2014 2014 ), was also detectable in the GBS strains (10/12). However, no penicillin resistance was found in this study, suggesting that the TEM gene may not be the major cause of the GBS resistance to penicillin. Finally, the ermB and mefA genes (Harimaya et al., 2007 2007 ), which are closely related to the TetM gene for tetracycline resistance (Fischer et al., 2013 2013 ), are worthy of further studies and may be associated with tumors (Hao et al., 2017 2017 ; Zhang et al., 2017 2017 ). Our study systematically analyzed the influence of various factors on GBS infection. Using the chi-square test and t -tests, 5 out of 22 factors showed a significant relationship with infection. We examined three major factors, gravida urinary tract infection, premature rupture of membranes, and gestational age, further using logistic regression to build a model of the GBS infection incidence rate, in the hope that this would contribute to the development of methods to prevent neonatal GBS infection. Moreover, drug resistance analyses of GBS patients offer a useful reference dataset that can be used to treat GBS more effectively. 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All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. 10.1631/jzus.B1800165 Project supported by the Natural Science Foundation of Zhejiang Province (No. Y18H040003), the Medicine and Health Research Foundation of Zhejiang Province (Nos. 2019RC012, 2019KY017, 2016KYA197, 2015KYA028, and 2013RCA025), and the Outstanding Young Scientific Research Funds of Zhejiang Provincial People’s Hospital (No. zry2015A005), China 2018-04-29 2018-09-09 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 19