基于16S rRNA测序分析CRE定植对ICU患者肠道微生物的影响

Impact of CRE colonization on gut microbes in ICU patients based on 16S rRNA sequencing

摘要:

目的 采用16S rRNA测序方法分析耐碳青霉烯类肠杆菌(CRE)定植对重症监护病房(ICU)患者肠道微生物的影响。 方法 收集ICU患者新鲜粪便或肛拭子标本, 接种于含有2 μg/mL厄他培南的MacConkey平板上培养。采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对菌株进行鉴定, 纸片扩散法对菌株进行药敏分析, 采用聚合酶链式反应(PCR)对初步筛查出的CRE菌株进行16S rDNA片段扩增, 采用微量肉汤稀释法进一步确定CRE。最后, 分别提取CRE定植组与非定植组患者的粪便或肛拭子标本基因组DNA在HiSeq平台进行16S rRNA测序分析。 结果 共收集ICU患者粪便或肛拭子标本241份, 其中CRE阳性标本17份。16S扩增子V3~V4区测序共分离到726 OTUs, CRE未定植组有631 OTUs, CRE定植组有480 OTUs, 两组间共有385 OTUs。这些共有OTUs绝大多数属于厚壁菌门、变形菌门、拟杆菌门、疣微菌门及放线菌门。在纲水平上, CRE定植前后γ-变形菌纲(W=193.000, P＜0.001, FDR=0.004)和互营养菌(W=37.500, P=0.001, FDR=0.018)相对丰度差异有统计学意义。在种水平上, 两组间肺炎克雷伯菌(W=195.000, P＜0.001)、解糖肠球菌(W=153.000, P=0.038)及弯曲杆菌(W=63.500, P=0.050)比较差异有统计学意义。未发现肠道CRE定植对ICU患者肠道微生物KEGG功能通路有显著影响。 结论 CRE定植会降低ICU患者肠道微生物的多样性, CRE定植组在肺炎克雷伯菌、解糖肠球菌及弯曲杆菌的丰度上与非定植组存在显著差异。

Abstract:

Objective To analyze the impact of carbapenem-resistant Enterobacterales (CRE) colonization on gut microbes in patients in intensive care unit (ICU) based on 16S rRNA sequencing. Methods Fresh stool or anal swab specimens from ICU patients were collected and inoculated on MacConkey plates containing 2 μg/mL ertape-nem for incubation. Strains were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility analysis was performed by disk diffusion method. 16S rDNA fragments of preliminarily screened CRE strains were amplified by polymerase chain reaction (PCR), and CRE strains were further determined using micro-broth dilution method. Finally, genomic DNA of fecal or anal swab specimens from CRE colonized and non-colonized patients was extracted for 16S rRNA sequencing analysis on the HiSeq platform. Results A total of 241 stool or anal swab specimens were collected from ICU patients, including 17 CRE-positive specimens. 726 OTUs were isolated by sequencing the V3-V4 region of the 16S amplicon, 631 in the non-colonized group and 480 in the colonized group, with a total of 385 OTUs in two groups. Most of these shared OTUs belonged to the Firmicutes, Proteobacteria, Bacteroidetes, Verrucomicrobia and Actinobacteria. At the class level, there was a significant difference in the relative abundance of γ-proteobacteria (W=193.000, P < 0.001, FDR=0.004) and Synergistia (W=37.500, P=0.001, FDR=0.018) before and after CRE colonization. At the genus level, Klebsiella pneumoniae (W=195.000, P < 0.001), Enterococcus saccharolyticus (W=153.000, P=0.038) and Campylobacter hominis (W=63.500, P=0.050) between the two groups had significant diffe-rences. No significant impact of intestinal CRE colonization on the KEGG functional pathway of gut microbiota in ICU patients was found. Conclusion CRE colonization can decrease the diversity of gut microbes in ICU patients. CRE colonization group showed significant differences in the abundance of Klebsiella pneumoniae, Enterococcus saccharolyticus and Campylobacter hominis compared to the non-colonization group.