中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2014年
5期
294-299
,共6页
杨晓军%王晓红%梁志娟%张小亚%王妍柏%王振海
楊曉軍%王曉紅%樑誌娟%張小亞%王妍柏%王振海
양효군%왕효홍%량지연%장소아%왕연백%왕진해
呼吸机相关性肺炎%痰液%细菌培养%16S rDNA%测序
呼吸機相關性肺炎%痰液%細菌培養%16S rDNA%測序
호흡궤상관성폐염%담액%세균배양%16S rDNA%측서
Ventilator-associated pneumonia%Sputum%Bacterial culture%16S rDNA%Sequencing
目的 用16S rDNA测序研究呼吸机相关性肺炎(VAP)患者痰液中致病菌的种类和丰度,探讨VAP病原学诊断的新方法.方法 对31例VAP患者进行支气管肺泡灌洗,提取痰液标本细菌DNA,进行聚合酶链反应(PCR)鉴定,同时进行痰液细菌培养.利用16S rDNA测序技术进行宏基因测序及生物信息学分析,并将测序结果与细菌培养结果比较.结果 ①31例VAP患者中27例患者的痰液标本550 bp处扩增出特异的DNA产物用于测序分析.16S rDNA测序共得到103 856条序列,39 Mb的原始数据.Tag物种注释27份样本均可注释到属的水平.②样本复杂度分析:Alpha多样性分析显示27份样本Shannon指数约为1.20,Simpson指数约为0.48,表明VAP痰液样本中细菌物种丰富且较为复杂.稀释曲线趋势分析提示部分VAP痰液样本还存在较多未被测序检测到的物种.③样本多样品分析:VAP痰液样本16S rDNA测序分析共出现放线菌门、拟杆菌门、硬壁菌门和变形菌门4个细菌门.以属为分类标准,优势菌属为链球菌属88.9％(24/27),变性菌属77.8％(21/27),不动杆菌属70.4％(19/27),鞘氨醇单胞菌属63.0％(17/27),普氏菌属63.0％(17/27),克雷伯菌属55.6％(15/27),假单胞菌属55.6％(15/27),水杆菌属55.6％(15/27),棒状杆菌属55.6％(15/27).④16S rDNA测序法与细菌培养结果比较:测序法检测可发现普通细菌培养未检测到的普氏菌属、变性菌属、水杆菌属、鞘氨醇单胞菌属;两种方法共同检测到的7种菌属中,测序法检测到的链球菌属[88.9％(24/27)]、克雷伯菌属[55.6％(15/27)]、不动杆菌属[70.4％(19/27)]、棒状杆菌属[55.6％(15/27)]的阳性率均高于普通细菌培养[分别为18.5％(5/27)、18.5％(5/27)、37.0％(10/27)、7.4％(2/27),P＜0.05或P＜0.01],假单胞菌属阳性率较普通细菌培养法有增高趋势[55.6％(15/27)比25.9％(7/27),P=0.050];而测序法与细菌培养检测到葡萄球菌属[7.4％(2/27)比11.1％(3/27)]、奈瑟菌属[18.5％(5/27)比3.7％(1/27)]的阳性率则均无差异(均P＞0.05).结论 16S rDNA测序分析证实VAP患者的痰液致病菌菌种复杂多样,物种丰富,多种多重耐药菌株定植.与普通细菌培养比较,16S rDNA宏基因组测序发现病原体阳性率更高.16S rDNA基因测序技术可能成为VAP病原学诊断的新方法.
目的 用16S rDNA測序研究呼吸機相關性肺炎(VAP)患者痰液中緻病菌的種類和豐度,探討VAP病原學診斷的新方法.方法 對31例VAP患者進行支氣管肺泡灌洗,提取痰液標本細菌DNA,進行聚閤酶鏈反應(PCR)鑒定,同時進行痰液細菌培養.利用16S rDNA測序技術進行宏基因測序及生物信息學分析,併將測序結果與細菌培養結果比較.結果 ①31例VAP患者中27例患者的痰液標本550 bp處擴增齣特異的DNA產物用于測序分析.16S rDNA測序共得到103 856條序列,39 Mb的原始數據.Tag物種註釋27份樣本均可註釋到屬的水平.②樣本複雜度分析:Alpha多樣性分析顯示27份樣本Shannon指數約為1.20,Simpson指數約為0.48,錶明VAP痰液樣本中細菌物種豐富且較為複雜.稀釋麯線趨勢分析提示部分VAP痰液樣本還存在較多未被測序檢測到的物種.③樣本多樣品分析:VAP痰液樣本16S rDNA測序分析共齣現放線菌門、擬桿菌門、硬壁菌門和變形菌門4箇細菌門.以屬為分類標準,優勢菌屬為鏈毬菌屬88.9％(24/27),變性菌屬77.8％(21/27),不動桿菌屬70.4％(19/27),鞘氨醇單胞菌屬63.0％(17/27),普氏菌屬63.0％(17/27),剋雷伯菌屬55.6％(15/27),假單胞菌屬55.6％(15/27),水桿菌屬55.6％(15/27),棒狀桿菌屬55.6％(15/27).④16S rDNA測序法與細菌培養結果比較:測序法檢測可髮現普通細菌培養未檢測到的普氏菌屬、變性菌屬、水桿菌屬、鞘氨醇單胞菌屬;兩種方法共同檢測到的7種菌屬中,測序法檢測到的鏈毬菌屬[88.9％(24/27)]、剋雷伯菌屬[55.6％(15/27)]、不動桿菌屬[70.4％(19/27)]、棒狀桿菌屬[55.6％(15/27)]的暘性率均高于普通細菌培養[分彆為18.5％(5/27)、18.5％(5/27)、37.0％(10/27)、7.4％(2/27),P＜0.05或P＜0.01],假單胞菌屬暘性率較普通細菌培養法有增高趨勢[55.6％(15/27)比25.9％(7/27),P=0.050];而測序法與細菌培養檢測到葡萄毬菌屬[7.4％(2/27)比11.1％(3/27)]、奈瑟菌屬[18.5％(5/27)比3.7％(1/27)]的暘性率則均無差異(均P＞0.05).結論 16S rDNA測序分析證實VAP患者的痰液緻病菌菌種複雜多樣,物種豐富,多種多重耐藥菌株定植.與普通細菌培養比較,16S rDNA宏基因組測序髮現病原體暘性率更高.16S rDNA基因測序技術可能成為VAP病原學診斷的新方法.
목적 용16S rDNA측서연구호흡궤상관성폐염(VAP)환자담액중치병균적충류화봉도,탐토VAP병원학진단적신방법.방법 대31례VAP환자진행지기관폐포관세,제취담액표본세균DNA,진행취합매련반응(PCR)감정,동시진행담액세균배양.이용16S rDNA측서기술진행굉기인측서급생물신식학분석,병장측서결과여세균배양결과비교.결과 ①31례VAP환자중27례환자적담액표본550 bp처확증출특이적DNA산물용우측서분석.16S rDNA측서공득도103 856조서렬,39 Mb적원시수거.Tag물충주석27빈양본균가주석도속적수평.②양본복잡도분석:Alpha다양성분석현시27빈양본Shannon지수약위1.20,Simpson지수약위0.48,표명VAP담액양본중세균물충봉부차교위복잡.희석곡선추세분석제시부분VAP담액양본환존재교다미피측서검측도적물충.③양본다양품분석:VAP담액양본16S rDNA측서분석공출현방선균문、의간균문、경벽균문화변형균문4개세균문.이속위분류표준,우세균속위련구균속88.9％(24/27),변성균속77.8％(21/27),불동간균속70.4％(19/27),초안순단포균속63.0％(17/27),보씨균속63.0％(17/27),극뢰백균속55.6％(15/27),가단포균속55.6％(15/27),수간균속55.6％(15/27),봉상간균속55.6％(15/27).④16S rDNA측서법여세균배양결과비교:측서법검측가발현보통세균배양미검측도적보씨균속、변성균속、수간균속、초안순단포균속;량충방법공동검측도적7충균속중,측서법검측도적련구균속[88.9％(24/27)]、극뢰백균속[55.6％(15/27)]、불동간균속[70.4％(19/27)]、봉상간균속[55.6％(15/27)]적양성솔균고우보통세균배양[분별위18.5％(5/27)、18.5％(5/27)、37.0％(10/27)、7.4％(2/27),P＜0.05혹P＜0.01],가단포균속양성솔교보통세균배양법유증고추세[55.6％(15/27)비25.9％(7/27),P=0.050];이측서법여세균배양검측도포도구균속[7.4％(2/27)비11.1％(3/27)]、내슬균속[18.5％(5/27)비3.7％(1/27)]적양성솔칙균무차이(균P＞0.05).결론 16S rDNA측서분석증실VAP환자적담액치병균균충복잡다양,물충봉부,다충다중내약균주정식.여보통세균배양비교,16S rDNA굉기인조측서발현병원체양성솔경고.16S rDNA기인측서기술가능성위VAP병원학진단적신방법.
Objective To study the species and amount of bacteria in sputum of patients with ventilator associated pneumonia (VAP) by using 16S rDNA sequencing analysis,and to explore the new method for etiologic diagnosis of VAP.Methods Bronchoalveolar lavage sputum samples were collected from 31 patients with VAP.Bacterial DNA of the samples were extracted and identified by polymerase chain reaction (PCR).At the same time,sputum specimens were processed for routine bacterial culture.The high flux sequencing experiment was conducted on PCR positive samples with 16S rDNA macro genome sequencing technology,and sequencing results were analyzed using bioinformatics,then the results between the sequencing and bacteria culture were compared.Results ① 550 bp of specific DNA sequences were amplified in sputum specimens from 27 cases of the 31 patients with VAP,and they were used for sequencing analysis.103 856 sequences were obtained from those sputum specimens using 16S rDNA sequencing,yielding approximately 39 Mb of raw data.Tag sequencing was able to inform genus level in all 27 samples.② Alpha-diversity analysis showed that sputum samples of patients with VAP had significantly higher variability and richness in bacterial species (Shannon index values 1.20,Simpson index values 0.48).Rarefaction curve analysis showed that there were more species that were not detected by sequencing from some VAP sputum samples.③ Analysis of 27 sputum samples with VAP by using 16S rDNA sequences yielded four phyla:namely Acitinobacteria,Bacteroidetes,Firmicutes,Proteobacteda.With genus as a classification,it was found that the dominant species included Streptococcus 88.9％ (24/27),Limnohabitans 77.8％ (21/27),Acinetobacter 70.4％ (19/27),Sphingomonas 63.0％ (17/27),Prevotella 63.0％ (17/27),Klebsiella 55.6％ (15/27),Pseudomonas 55.6％ (15/27),Aquabacterium 55.6％ (15/27),and Corynebacterium 55.6％ (15/27).④ Pyrophosphate sequencing discovered that Prevotella,Limnohabitans,Aquabacterium,Sphingomonas might not be detected by routine bacteria culture.Among seven species which were identified by both methods,pyrophosphate sequencing yielded higher positive rate than that of ordinary bacteria culture [Streptococcus:88.9％ (24/27) vs.18.5％ (5/27),KlebsieHa:55.6％ (15/27) vs.18.5％ (5/2 7),Acinetobacter:70.4％ (19/27) vs.37.0％ (10/27),Corynebacterium:55.6％ (15/27) vs.7.4％ (2/27),P＜0.05 or P＜0.01].Sequencing positive rate was found to increase positive rate for culture of Pseudomonas [55.6％ (15/27) vs.25.9％ (7/27),P=0.050].No significant differences were observed between sequencing and ordinary bacteria culture for detection Staphylococcus [7.4％ (2/27) vs.11.1％ (3/27)] and Neisseria bacteria genera [18.5％ (5/27) vs.3.7％(1/27),both P＞0.05].Conclusions 16S rDNA sequencing analysis confirmed that pathogenic bacteria in sputum of VAP were complicated with multiple drug resistant strains.Compared with routine bacterial culture,pyrophosphate sequencing had higher positive rate in detecting pathogens.16S rDNA gene sequencing technology may become a new method for etiological diagnosis of VAP.
