Preliminary Study On Intestinal Colonization Of CRE And Intestinal Microbiome Diversity Of Patients

【Objective】Carbapenem-resistant Enterobacteriaceae(CRE)are bacteria that are resistant to any of the carbapenem antibiotics(for those who are naturally resistant to imipenem,refer to the sensitivity results of other carbapenem antibiotics)or carbapenemase-producing Enterobacteriaceae.The World Health Organization(WHO)suggested that CRE intestinal colonization often precedes or coincides with CRE infection,and it has been reported that the intestinal microbiome may be the source of CRE infection in the body.In order to clarify the changes of intestinal microbiome in patients with intestinal colonization of CRE,this study will examine the intestinal microbiome of patients with intestinal colonization of CRE,clarify the characteristics of the changes of relative abundance of intestinal microbiome in patients with intestinal colonization of CRE,understand the clinical risk factors of patients with intestinal colonization of CRE,and observe the ratio of their peripheral blood lymphocyte subpopulation,so as to provide a theoretical basis for clinical prevention and control of CRE infection.【Method】1.Screening of inpatients(excluding the same patients)from January 1,2022 to December 31,2022 at the Affiliated Hospital of Yunnan University using self-made imipenem-containing Mac Conkey medium,and strain identification using the VITEK2fully automated system for strains grown on the medium,combined with m CIM and e CIM tests for carbapenemase phenotype confirmation.2.The department,age,and gender of the experimental and control groups were matched,and according to the 1:1 case-control study method,the patients with CRE growth detected were the experimental group,and the control group were the patients with no CRE growth detected and complete clinical data during the same period.The clinical data of patients in the experimental and control groups were also collected,including basic information(department,gender,age),past history(heart,liver,kidney and other related chronic diseases,history of hospitalization in the past 6 months),treatment(invasive operation,antibiotics,antifungal drug use,etc.),whether the current hospitalization was combined with CRE systemic infection and other drug-resistant bacteria.SPSS25.0 software was used to analyze the clinical data of the two groups for univariate and multifactorial analysis of clinical risk factors in patients with intestinal colonization of CRE.3.Peripheral whole blood samples were collected from patients in the experimental and control groups,and the proportion of lymphocyte subpopulations was measured using flow cytometry,and the results were statistically analyzed using SPSS25.0 software,and P<0.05 was statistically significant.4.Fecal samples of the experimental group and the control groups were collected,total fecal DNA was extracted,and V3-V4 regions were amplified by 16Sr RNA high-throughput sequencing method.The structure and composition of intestinal microbiome in the two groups were analyzed and statistically processed by Illumina platform sequencing.【Results】1.Sixty patients with intestinal colonization of CRE were selected from the inpatients(excluding the same patient)of the Affiliated Hospital of Yunnan University from January 1,2022 to December 31,2022,including 19 patients in the department of geriatrics,13 patients in the department of rehabilitation,13 patients in the department of general medicine,8 patients in the department of respiratory medicine,and 7 patients in other departments.Among the CRE colonized strains,57 were carbapenem-resistant Klebsiella pneumoniae,3 were carbapenem-resistant Escherichia coli,among which 56were m CIM(+)and e CIM(-),indicating the production of serine carbapenemase,and 4were m CIM(+)and e CIM(+),indicating the production of metal-β-lactamase.2.Clinical risk factor analysis of patients with CRE intestinal colonization versus those without colonization:tracheotomy,indwelling gastric tube,deep vein placement,indwelling urinary catheter,carbapenem antibiotics,cefoperazone sulbactam,fluoroquinolone antibiotics,and carriage of other multi-drug resistant bacteria in univariate analysis P<0.05,indicating that these were clinical risk factors for CRE intestinal colonization;logistic regression was used for Multi-factor analysis showed that indwelling gastric tube and tracheotomy OR>1,P<0.05,indicating that indwelling gastric tube and tracheotomy were independent risk factors for patients with CRE intestinal colonization.3.Characteristics of peripheral blood lymphocyte subsets:the proportions of CD3~+T cells and CD19~+B cells decreased in CRE intestinal colonization patients(P<0.05),while the proportions of CD16~+CD56~+NK cells increased(P<0.05).4.Intestinal microbiome diversity analysis:theα-diversity Chao1 index and Shannon index of CRE intestinal colonized patients were lower than those of uncolonized patients(P<0.05).In theβ-diversity analysis,the results of principal coordinates analysis(PCo A)showed that the intestinal microbiome of CRE intestinal colonized patients and non-colonized patients were significantly different and the composition of intestinal microbiome was significantly different.5.Analysis of the relative abundance of intestinal microbiome in CRE colonized patients:at the phylum level,the dominant microbiome in CRE colonized patients were Firmicutes 51.8%,Proteobacteria 28.8%,Actinobacteria 10.9%,Bacteroides 4.3%and Verrucomicrobia 4.1%.The relative abundance of Firmicutes and Bacteroides decreased in CRE intestinal colonized patients compared with uncolonized patients,while the relative abundance of Proteobacteria increased.The rank sum test showed that the relative abundance of Firmicutes,Bacteroidetes and Proteobacteria were significantly different between the two groups(P<0.05).【Conclusion】1.Clinical risk factors for intestinal colonization of CRE included deep vein catheterization,indwelling gastric tube,indwelling urinary tube,tracheotomy,carbapenems antibiotics,fluoroquinolones antibiotics,cefoperazone sulbactam,and other multi-drug resistant bacteria,among which tracheotomy and indwelling gastric tube were independent risk factors.2.The proportion of CD3~+T cells,CD19~+B cells and CD16~+CD56~+NK cells in peripheral blood of patients with colonization decreased and the proportion of CD16~+CD56~+NK cells increased,suggesting that patients may have immune disorders and other conditions.Monitoring the proportion of lymphocyte subsets in patients may prevent CRE infection.3.The diversity and composition of intestinal microbiome in patients with intestinal colonization of CRE are changed.Monitoring the intestinal flora of patients with risk factors for intestinal colonization of CRE may be able to identify the intestinal colonization of CRE in advance.