I-Fa CRISPR-Cas System Of Acinetobacter Baumannii Upregulates The Virulence And Study On Antibacterial Effect Of Metformin Combined With Minocycline

The CRISPR-Cas system has a certain correlation with the drug resistance and virulence of bacteria.The study investigating the effect of the I-Fa CRISPR-Cas system on the drug resistance and virulence of A.baumannii Firstly,the cas3 gene of the I-Fa CRISPR-Cas in A.baumannii ATCC19606 was knocked out.The phenotypic changes such as drug resistance,virulence and biofilm formation ability were observed.It was found that the lose of cas3 gene had no effect on its drug resistance,but reduced virulence and biofilm formation.It is different from the previous research results that I-Fb type CRISPR-Cas system reduces the level of drug resistance and virulence of A.baumannii.It shows that different subtypes of CRISPR-Cas systems have different effects on the drug resistance and virulence of A.baumannii,and the specific mechanism needs to be further studied.Secondly,the related experimental research on the antibacterial effect of metformin and minocycline showed that metformin combined with minocycline could enhance the antibacterial effect of minocycline on A.baumannii by inhibiting the formation of biofilm,breaking the outer membrane and membrane potential.1.Correlation of different CRISPR-Cas subtypes with drug resistance and virulence in Acinetobacter baumanniiIn the previous study of our group,it was found that after the knockout of A.baumannii carrying the I-Fb CRISPR-Cas system,the bacterial drug resistance phenotype changed from sensitive to drug resistance,indicating that the complete CRISPR-Cas system can be maintained the susceptibility of A.baumannii to antibiotics.A.baumanniiAB43 carrying with deletion of I-Fb CRISPR-Cas system and bacterial resistance have obvious correlation.In this topic,firstly,in order to study the changes of gene expression levels of proteins related to the complete CRISPR-Cas system under the treatment of antibiotics,we treated A.baumannii AB43 and ATCC19606 strains with antibiotics,and qRT-PCR detected the gene expression of the CRISPR-Cas system.The AB43 strain and ATCC19606 carried complete type Ⅰ-Fa CRISPR-Cas and type Ⅰ-Fb CRISPR-Cas,respectively.The CRISPRCas-related cas/csy genes of the two subtypes showed different responsiveness to antibiotics.We speculate that the relationship between type Ⅰ-Fa and type Ⅰ-Fa CRISPR-Cas systems and drug resistance may be different.Therefore,in order to study the effect of the I-Fa CRISPR-Cas system on the drug resistance of A.baumannii,we knocked out the cas3 gene in the ATCC 19606 strain,and then conducted drug susceptibility experiments,biofilm formation,and virulence experiments.The results showed that the cas3 gene mainly affects the biofilm formation ability and virulence of A.baumannii,and the biofilm formation ability of the cas3 gene knockout strains Significantly and the virulence decreased,while the drug resistance did not change significantly.Our results suggest that the cas3 gene plays an important role in the biological activity of ATCC 19606 strain.It shows that different subtypes of CRISPR-Cas systems have different effects on the drug resistance and virulence of A.baumannii.Our findings extend the role of the CRISPR-Cas system in A.baumannii and link the CRISPR-Cas system to the biogenesis of bacterial resistance and virulence.2.Study on the synergistic antibacterial mechanism of metformin combined with minocyclineA.baumannii is an opportunistic bacterial pathogen considered to be the leading cause of nosocomial infections worldwide.In recent years,the drug resistance of A.baumannii has been increasing,and it has developed resistance to various antibiotics.New antimicrobial approaches need to be developed and researched to treat and reduce the occurrence of drug resistance.Minocycline maintains low resistance in A.baumannii,and attracts attention as a new "old drug",however,a few A.baumannii have developed resistance to minocycline medicinal properties.In this study,the combined application of metformin and minocycline found that it can not only inhibit the growth of bacteria,but also delay the occurrence of bacterial resistance.Three strains with different resistance to minocycline were selected for checkerboard test and time sterilization curve.The result preliminarily showed the bacteriostatic effect of metformin combined with minocycline.Then,taking minocycline-resistant A.baumannii as the research object,the effects of metformin combined with minocycline on its biofilm formation ability,outer membrane integrity,membrane permeability,membrane potential and ROS level were explored.It’s potential mechanism of action.The results show that the combination of the minocycline and metformin can reduce the formation ability of bacterial biofilm,break the outer membrane,and increase the membrane potential of bacteria to enhance the antibacterial effect of antibiotics.This study demonstrated that the combination of metformin and minocycline has the potential to restore minocycline activity against multidrug-resistant A.baumannii.