观察一对紫绀型先天性心脏病双胞胎新生儿在接受手术及抗生素治疗前后的肠道菌群的动态变化,探究患儿肠道内阴沟肠杆菌在治疗期间耐药性如何发生改变。
通过采集患儿在不同治疗阶段的粪便样本,进行培养组学和16S rRNA基因测序分析。同时,从双胞胎哥哥不同治疗阶段的粪便样本中分离得到10株阴沟肠杆菌进行体外药敏试验。
培养组学结果显示使用抗生素导致术前粪便样本丰富度降低,仅可分离获得肠球菌属、不动杆菌属和肠杆菌属;长期医院环境暴露后,能分离出多种条件致病菌属,包括肠杆菌属、克雷伯菌属、不动杆菌属和假单胞菌属;母乳喂养4个月后,粪便菌群丰富度增加、构成发生改变,可分离获得乳杆菌属和双歧杆菌属等有益细菌。16S rRNA基因测序结果显示,在多种因素影响下不同治疗时间点α多样性指数和相对丰度最高的菌属不同。细菌耐药实验结果显示,治疗期间阴沟肠杆菌对哌拉西林逐渐产生耐药性。
接受抗生素治疗、院内环境暴露和母乳喂养共同影响患儿治疗和康复期间的肠道菌群组成;在不同治疗阶段仅使用一种抗生素,也会导致阴沟肠杆菌对不同药物的耐药性发生改变。
To observe the dynamic changes of gut microbiota in a pair of newborn twins with cyanotic congenital heart disease after surgery and antibiotic treatment and explore the variation of drug resistance of
The fecal samples of the twins were collected at different stages of treatment and analyzed by culturomics and 16S rRNA gene sequencing. The susceptibility of ten
Culturomics results suggested that antibiotics decreased the flora richness of fecal samples before surgery and only
Antibiotic treatment, exposure to hospital environment, and breast feeding all changed the gut microbiota structure of the twins during treatment and recovery. Even if only one antibiotic was used at different stages of treatment, the resistance of
新生儿主要从母体、分娩过程中和出生时所在的环境接受并获得肠道微生物[
接受抗生素治疗会明显降低人体肠道菌群多样性、干扰肠道微生物丰度,甚至在短时间内发生物种的快速更替,最终导致病原体的大量繁殖[
研究对象是在解放军总医院第七医学中心住院治疗的双胞胎新生儿,诊断为紫绀型先天性心脏病(CCHD)。此实验通过解放军总医院第七医学中心伦理委员会的伦理审批(审批号:2021034)。入院时,双胞胎哥哥体重2.1 kg,身长44 cm,双胞胎弟弟体重1.8 kg,身长44 cm,后续两患儿均接受完全型肺静脉异位引流术并痊愈出院,治疗期间部分实验室检查和临床监测数据见
双胞胎患儿治疗期间临床和实验室检查结果
Clinical and laboratory values of the twins during treatment
Inspection items | C1A | C1B | C1C | C1D | C2A | C2B | C2C | C2D |
Heart rate: beats/min; Breath: breaths/min; SBP: systolic blood pressure, mmHg; DBP: diastolic blood press, mmHg; PaO2: mmHg; PaCO2: mmHg; SaO2: %. | ||||||||
Heart rate | 154.0 | 134.0 | 155.0 | 135.0 | 153.0 | 171.0 | 168.0 | 163.0 |
Breath | 42.0 | 40.0 | 40.0 | 35.0 | 30.0. | 61.0 | 40.0 | 46.0 |
SBP | 80.0 | 70.0 | 65.0 | 79.0 | 63.0 | 73.0 | 79.0 | 82.0 |
DBP | 38.0 | 45.0 | 48.0 | 40.0 | 37.0 | 40.0 | 58.0 | 46.0 |
PaO2 | 46.4 | 34.7 | 92.9 | 103.8 | 48.9 | 60.4 | 64.9 | 97.0 |
PaCO2 | 47.2 | 44.7 | 35.1 | 50.9 | 45.6 | 50.4 | 31.0 | 51.5 |
SaO2 | 88.8 | 85.8 | 97.9 | 97.3 | 87.3 | 86.2 | 94.6 | 97.7 |
双胞胎粪便样本采集日期
Collection date of the fecal samples of the twins.
YCFA培养基,MPYG培养基,TSB培养基,哥伦比亚血平板,LB培养基均购自赛默飞公司。
上机血培养瓶(BC120,郑州安图),澄清瘤胃液(A386-01,上海ELITE-MEDIA),无菌脱纤维羊血(H0011,北京宝特医疗有限公司),肠杆菌药敏检测试剂盒(30061303MB01,郑州安图),麦氏比浊仪(Densi CHEK Plus,梅里埃),厌氧培养箱(Concept 400,Ruskinn),恒温振荡培养箱(HZQ-F160,宿州培英实验设备有限公司),MALDI-TOF质谱仪及检测试剂(Autof MS1000,郑州安图)。
新鲜收集的粪便样本,用10 mL无菌PBS混悬,取上清1 mL分别加入需氧和厌氧预增菌培养瓶(40 mL上机血瓶+5 mL澄清瘤胃液+5 mL无菌脱纤维羊血)中。最后根据对气体不同的需求,将预增菌血培养瓶放入37 ℃恒温振荡培养箱或厌氧培养箱中培养共计35 d (
培养组学流程图
Protocol of culturomics.
从第0天(取样当天)开始,在0、3、6、9、12、15、18、21、24、27、30和35 d共12个时间点从预增菌体系中均匀取样,进行梯度稀释,涂布在固体YCFA、MPYG和哥伦比亚血平板上,随后将平板置于需氧和厌氧条件下进行分离培养。其中需氧条件下培养24 h,厌氧条件下培养48–72 h。待培养到相应时间后,选取单克隆数目在50–500个的固体平板进行单克隆挑取和液体培养基扩增。
蘸取1 μL菌液在对应固体平板上进行三区划线,继续放入37 ℃恒温培养箱或厌氧培养箱中培养。待相应培养时间后对长出的纯菌落进行细菌MALDI-TOF质谱鉴定或16S rRNA基因测序鉴定。取1 mL液体扩增菌液按照0.3 mL甘油+1 mL菌液的比例进行甘油保菌,每株细菌保存1支,充分混匀后冻存于–80 ℃。纯菌落按照拓展法进行MALDI-TOF质谱鉴定,2 h内上机检测。检测结果评分大于9则为种水平可信。
由深圳华大基因科技服务有限公司对所有粪便样本进行16S rRNA基因测序并进行分析。使用MagPure Stool DNA KF kit B (Magen)试剂盒提取粪便基因组,使用Qubit Fluorometer:QubitⓇ dsDNA BR Assay kit (Invitrogen)进行总DNA质量检查。使用V3–V4区域的通用引物进行PCR扩增完成文库构建。检测合格的文库选择Hiseq 2500平台进行测序。使用软件FLASH (Fast Length Adjustment of short reads,v1.2.11)进行序列拼接,利用重叠关系将双末端测序得到的成对reads组装成一条序列,得到高变区的Tags。利用软件USEARCH (v7.0.1090)将拼接好的Tags在97%相似度下聚类为OTU。利用UCHIME (v4.2.40)将PCR扩增产生的嵌合体和已有的嵌合体数据库进行比对,将其中的嵌合体序列从OTU代表序列中去除。同时选取OTUs的代表性序列。将该代表性序列与RDP数据库进行比对,最小置信阈值为0.6,根据比对结果对每个样本的OTU进行物种分类,所得到物种丰度进行后续分析。使用Qiime软件(v1.9.1)计算Shannon指数。16S rRNA基因测序数据已上传至NCBI中SRA数据库,Submission ID:SUB10209395,BioProject ID:PRJNA755453。
在液体LB培养基中复苏冻存于–80 ℃的C1患儿入院、术前、术后和转出4个治疗时间点分离到的共10株阴沟肠杆菌(
对该对患儿在5个治疗时间点采集的10个粪便样本在属水平上的组成同时进行了培养组学(
10个粪便样本的培养组学属水平组成图
Bacteria composition of the 10 fecal samples identified by culturomics at genus level. The thickness of the column represents the number of species of each genus.
10个粪便样本的16S rRNA基因测序属水平组成图
Bacteria composition of the 10 fecal samples detected by 16S rRNA gene sequencing at genus level.
10个粪便样本菌群Shannon指数
Shannon index of bacterial community in the 10 fecal samples.
该对患儿入院后接受抗生素治疗(哌拉西林)是改变术前时间点粪便样本细菌组成和多样性的主要因素。培养组学结果显示,入院粪便样本中可分离到的葡萄球菌属和乳杆菌属,因为手术前使用抗生素治疗,在术前粪便样本中无法分离获得,导致每个术前粪便样本仅可分离到2个菌属,表明肠道细菌丰富度降低(
随着住院时间延长,培养组学方法可从术后(C1C、C2C)和转出(C1D、C2D)样本分离出医院常见的几种条件致病菌属,包括肠杆菌属、克雷伯菌属、不动杆菌属和假单胞菌属(
在离开医院环境和母乳喂养的共同影响下,培养组学结果显示复诊的C1E和C2E两个样本可以分离培养到的细菌增加到15个属以上。与转出的C1D和C2D两个样本不同的是,在离开医院环境且母乳喂养4个月后,在C1E中无法分离到肠杆菌属和假单胞菌属,在C2E中无法分离到肠杆菌属和丙酸杆菌属(
上述结果表明,培养组学方法可分离到细菌种类较多的2个治疗时间点是转出(D)和复诊(E)。我们比较了这2个时间点从双胞胎中分离出的细菌,如
双胞胎患儿转出和复诊样本培养组学分离到的共同和差异菌株
Common and different species isolated by culturomics at the D and E timepoints from the twins. A: the bacteria isolated under different conditions at the D timepoint from the twins; B: the bacteria isolated under different conditions at the E timepoint from the twins. The red square indicated the bacteria existed both in C1 and C2. The bacteria isolated under different culture conditions at each timepoint were represented by horizontal lines of different colors.
我们比较了该对患儿5个治疗时间点共计10个样本培养组学和测序结果在属水平之间的差异(
属水平培养组学和16S rRNA基因测序结果对比
Comparison of 16S rRNA gene sequencing and culturomics at genus level. A–E represent the comparison between the genera identified by 16S rRNA gene sequencing and the genera isolated by culturomics from the fecal samples at A–E timepoints.
在入院、术前、术后和转出4个时间点中,C1患儿均可分离获得阴沟肠杆菌,为了研究不同治疗时间点的阴沟肠杆菌菌株(EC1–EC10)的药物敏感性,我们将这些菌株进行了复苏、培养和质谱鉴定。体外耐药性检测显示,4个治疗阶段的EC1–EC10菌株对亚胺培南(术前1 d开始连续服用8 d)的敏感性相似(
10株阴沟肠杆菌体外药敏试验检测结果
Detection of antibiotic resistance of the 10
微生物培养对于表型、生态学和抗菌药物敏感性的实验验证是非常宝贵的资源[
抗生素、喂养方式和外界环境等许多因素都会影响婴儿的肠道微生物群。本研究通过观察该对患儿整个治疗过程的肠道菌群动态变化,可以很好地反映这3个因素的共同作用,为新生儿住院期间肠道菌群的扰动和恢复提供参考。医院环境是影响肠道微生物群的重要因素。通常,低出生体重和/或患有严重疾病的新生儿将在新生儿重症监护病房的培养箱中接受护理[
研究显示,不同的喂养模式似乎不会在出生后的第一周对新生儿的肠道菌群产生显著影响[
我们还检测了阴沟肠杆菌的耐药性是否在抗生素使用过程中发生变化,这对于改善住院患者抗生素治疗期间逐渐表现出对所用抗生素耐药的临床现状至关重要。据报道,细菌所携带的抗生素耐药基因在抗生素治疗后大量增加,并可持续保留至少1年[
综上所述,该对患儿住院期间肠道菌群构成和多样性发生明显改变,在接受母乳喂养后肠道菌群内益生菌开始定殖,肠道菌群多样性水平恢复。另外治疗期间患儿体内阴沟肠杆菌逐渐对哌拉西林耐药,为以后关注新生儿抗生素的临床应用和肠道菌群动态变化提供参考。
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