Fitness Cost Study Of Model Microorganism And Clinical Study On Microorganism Co-infected With COVID-19

First part Study on the fitness cost of model microorganism Objective: Fitness cost means that individuals with specific functional genes have lower adaptability than those without this functional gene under no corresponding selection pressure.In this study,two model microorganisms,Escherichia coli and Saccharomyces cerevisiae,were used as the research objects,and the evolutionary method was used to verify whether the resistance genes in microorganisms produced adaptive cost.The target genes included tetracycline resistance gene(TCR)and ampicillin resistance gene(APR)in Escherichia coli,histidine synthase gene(HIS),leucine synthase gene(Leu)and tryptophan synthase gene(Trp)in Saccharomyces cerevisiae.Methods:First part-study on the fitness cost of model microorganism: Firstly,the transformation plasmid marker gene of the wild strain was modified.The parent genotype of Escherichia coli DH5αwas TCR+APR+,and the mutant genotype of TCR-APR + and TCR + APR-were obtained after modification.The parent genotype of Saccharomyces cereviscera strain F169 was His + Leu + Trp+,and the mutant genotypes His-,Leu-,Trp-,His-Trp-,His-Leu-Trp-were obtained after modification.Secondly,vitro growth rate measurement experiment and competitive growth experiment were carried out on wild bacteria and mutant bacteria in the same system to analyze whether the fitness cost of the target gene exists.Finally,the mutation sites were sequenced and analyzed after continuous subgeneration.Results: First part-study on the fitness cost of model microorganism: the Tet mutant(TCR-APR +)strain in Escherichia coli showed stronger adaptability than the wild-type strain(TCR-APR +)in the growth rate measurement experiment and competitive growth experiment,indicating that the tetracycline resistant TCR-gene in Escherichia coli does have a fitness cost.The Amp mutant strain of Escherichia coli(APR-TCR +)showed a higher growth rate in the growth rate determination experiment,but no significant change in the competitive growth experiment,compared with the wild strain,and even showed a lower adaptability and was eliminated by the wild type strain.These results indicated that no detectable adaptive changes were found in the APR gene of ampicillin resistance in E.coli.The results showed that the growth rate of the three trophic deficiency marker gene mutation(His-Leu-Trp-)was significantly higher than that of the single mutation(His-Leu-,Trp-)and the two mutation(His-Trp-),but the repeatability was poor in the competitive growth experiment,and no adaptive change was observed.Conclusion: The tetracycline resistant TCR gene in Escherichia coli had fitness cost,but no adaptive cost was found in APR gene,and no obvious fitness change was observed in Saccharomyces cerevisiae.Second part Clinical research of COVID-19 co-infected with microbes Objective: Through the retrospective analysis of wuhan vulcan hill hospital COVID-19 patients with clinical data,and related data of the test results of bacteria and fungi infection,to make detailed statistics and analysis of COVID-19 with microbes co-infection,which is of certain guiding significance about protection measures of epidemic diseases and antimicrobial drug use.Methods: The clinical data of patients from Wuhan Huoshenshan Hospital were retrospectively studied,combined with the related detection experiments of bacteria and fungi,including bacterial culture and gene chip for bacteria detection,and fungus culture,serological detection,gene chip for fungi detection,and comprehensively analyse the COVID-19 with microbial co-infection.Results: The suspected positive rate of co-infection was 15%,and the top three strains were Klebsiella pneumoniae,methicillin-resistant Staphylococcus,and Haemophilus influenzae.The suspected positive rate of combined fungal infection was 34%,mainly Candida and Aspergillus.A total of 5 deaths in the positive group were all related to Aspergillus infection,and Aspergillus was the most common among the dead and Candida was the most common among the survivors.Conclusion: More attention should be paid to COVID-19 with Aspergillus co-infection.