Based On Metagenomic Technology To Explore The Origin Of Pathogenic Bacteria And Carbapenems Resistance Genes In Medical-related Environments

Objective:The monitoring and tracing of pathogenic bacteria in the medical-related environments are important elements of forensic microbiology research.It can not only assist in determining the storage and dispersal of pathogenic bacteria in the medical-related environments but also explore the origin and distribution based on strain identification and genotyping.Thus,it can provide the necessary support for prevention and control of bioterrorism and disposal of biocrime.The microbial community diversity analysis was used to infer the transmission and storage patterns of pathogenic bacteria in the environment.Among the superpathogens that pose a risk of global spread,the Environment Carbapenem-Resistant Enterobacteriaceae（e CRE）has caused serious medical hazards and greatly increased the risk of pathogenic infections.However,there is a lack of technical methods to precisely quantify its transmission and to meet the forensic traceability requirements of pathogenic microorganisms.This study analysed the diversity of microbial communities using metagenome sequencing techniques,and to analyse the retention,dispersal and distribution of e CRE and their resistance genes in the medical-related environments in combination with pathogenic bacteria culture identification methods.The study also explored the traceability of pathogenic bacteria and the diversity of microbial communities for forensic microbiology practice.Methods:First,followed by experimental investigation of multiple sampling methods and different pre-sequencing sample treatments,the sampling sites were designed and characterized,and a standardised experimental procedure was established finally.Second,samples from specific environmental sites were periodically obtained and sequenced for the V3-V4 region of 16S rDNA,and bioinformatics tools were used to complete alpha diversity analysis such as sample community abundance,species composition analysis,beta diversity analysis,similarity of bacterial communities across spatial distribution and robustness to disturbance across time.The magnitude of the association of bacterial communities between environmental sampling sites was analysed comprehensively.In parallel with the environmental microbial community analysis,samples from the same sampling sites were isolated and cultured for e CRE,and strains were identified and DNA extracted using commercial colour development techniques.The common carbapenem resistance genes were amplified by PCR and typed by polyacrylamide gel electrophoresis（PAGE）,and the Sanger sequencing results were compared with the resistance gene database to identify the types of resistance genes carried by e CRE.Finally,a comprehensive analysis of the retention and dissemination characteristics of e CRE in the medical-related environments was performed.The feasibility of traceability was analysed and its efficacy was explained by combining multiple methods.Results:The first phase of the experiment validated the different sampling and sample pre-processing methods based on the sequencing analysis of seven representative samples.It was confirmed that the ESwab Collection&Transport System sampling method and direct sequencing of samples resulted in the highest species diversity and a high number of valid sequences.In the second phase of the experiment,34 samples（from 10sampling sites）were sequenced in the V3-V4 region of 16S rDNA with depths ranging from 11949–57456 and OTU clusters ranging from 156-1560.The alpha diversity analysis of the spatially distributed bacterial diversity showed the Shannon index and Simpson index of the microorganisms differed significantly between the sampling sites simultaneously,with the Ce site showing a higher community diversity.The community diversity of the samples was robust across the sampling periods（except for the 6th）.The species composition of the microbial communities in the medical-related environments showed various and high variation in abundance between groups.Comparative analysis of beta diversity between sampling sites showed the bacterial community composition in the medical-related indoor environment was less variable,and did not change significantly over time.A large number of carbapenem-resistant bacteria were cultured from samples taken in the medical-related environments.The system was based on the analysis of 11 common resistance genes(bla_IMP,bla_NDM,bla_KPC,etc.)in five species of bacteria,including Klebsiella pneumoniae and Acinetobacter baumannii,and the culture-active carbapenem-resistant bacteria were confirmed to carry multiple resistance genes types,including SPM,OXA,VIM,AIM and NDM.The highest abundance of VIM and other resistance genes was found in the hospital waste treatment center,and the SPM and SIM types included were verified by sequencing to match the database.Conclusion:The medical-related indoor microbial communities show higher similarity and robustness to disturbance than outdoor,and such environmental spaces and their microbial distribution communities can be correlated and comparatively analysed by period.Multidrug-resistant active e CRE was confirmed to be present in both indoor and outdoor areas of the medical-related environments,and having potential to spread.The transmission model of e CRE still needs to be accurately described in the transmission pathway and forensic microbial traceability.Combined with high-throughput sequencing-based microbial community analysis,pathogenic bacteria culture identification and carbapenem resistance genotyping techniques allowed us to detect for forensic traceability of biothreatening microbes preliminary.