| Acinetobacter baumannii(A.baumannii)is a gram-negative,non-fermenting,conditionally pathogenic bacterium that is highly susceptible to infecting immunocompromised ICU patients.In recent years,the problem of multidrug resistance in A.baumannii has increased dramatically,and the therapeutic options for treating infections caused by this group of pathogens is limited.The resistance mechanisms in A.baumannii are related to various factors,including mainly horizontal gene transfer and other mechanisms such as altered targets,decreased permeability,increased enzyme production,overexpression of efflux pumps,metabolic changes,and biofilm formation.Horizontal gene transfer includes the spread of high-risk global multi-resistant genetic lineages and acquiring resistance genes on resistance plasmids and transposons.The CRISPR-Cas system is an adaptive immune defense system present in the genomes of bacteria and archaea that can effectively defend against the invasion of exogenous genetic elements,such as plasmids and phages.This system can further recognize and degrade exogenous DNA by integrating it into its chromosome,thus avoiding damaging itself by exogenous DNA.The process of horizontal transfer of drug-resistant genes is also a process of integrating exogenous DNA into the bacteria,and through the horizontal transfer of drug-resistant genes,the bacteria change from sensitive to resistant.The mechanism of the action of CRISPR-Cas is supposed to be the same as that which exists in drug-resistant genes.However,the relationship and mechanism between these two have not been clarified.Acinetobacter baumannii is an important pathogen causing hospital-acquired infections,and it shows multi-drug resistance and pan-drug resistance in the process of confronting antibiotics.Its resistance mechanism is very complex and variable,making the prevention and treatment of this type of pathogen very difficult.Therefore,it is imperative to explore emerging therapies.Traditional Chinese medicine attaches importance to treating the disease before it occurs,and it aims to regulate the patient’s health as a whole,improve immunity,restore the balance of flora in the body,and use herbs that can effectively resist pathogens through dialectic,to support the righteousness and drive away from the evil.Among them,the combined use of herbs and antibiotics is a significant feature of the clinical treatment of Chinese and Western medicine,which has an essential role in the clinical treatment of drug-resistant bacterial infectious diseases.Scutellaria baicalensis is a perennial herb with a bitter and cold taste,belonging to the family Labiatae,genus Scutellaria.In Scutellaria baicalensis,baicalein is one of the active ingredients of flavonoids,which has become an object of interest for researchers because of its low drug resistance rate and toxicity.The current studies on baicalin inhibition mainly focus on methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa.However,the inhibition of multidrug-resistant Acinetobacter baumannii is less reported,and the specific mechanism of baicalin inhibition alone or in combination has not been fully clarified.Therefore,this study focused on the relationship between CRISPR-Cas system and drug resistance and explored the mechanism of the inhibition of drug resistance and the mechanism of baicalin combined with doxycycline on the multidrug-resistant Gram-negative pathogens.Part Ⅰ.CRISPR-Cas inhibits antibiotic resistance in Acinetobacter baumanniiTo investigate the relationship between the CRISPR-Cas system and drug resistance in Acinetobacter baumannii,this project conducted screening and drug sensitivity assays on 245 clinical isolates of Acinetobacter baumannii and performed multi-locus sequence typing(MLST)on the screened CRISPR-Cas system-positive strains.The results showed that 26.12%(64/245)isolates containing the I-Fa CRISPR-Cas system,20%(49/245)isolates have the IFb CRISPR-Cas system.One strain with a complete I-Fa CRISPR-Cas system was AB105,another with a complete I-Fb CRISPR-Cas system was AB43,which were both sensitive strains.The other 111 clinical isolates of A.baumannii with incomplete CRISPR-Cas systems were mainly multidrug-resistant strains.The 113 CRISPR-Cas-positive A.baumannii clinical isolates covered 40 STs,among which ST1145,ST1696,and ST 195 were more commonly distributed,while the other STs were relatively few.In addition,we correlated the multidrug resistance phenotypes with the Cas genes of the I-Fa and I-Fb CRISPR-Cas systems.We found that the multidrug resistance rates of the Cas gene-negative strains were significantly higher than those of the positive strains.This part of the study initially revealed that the CRISPR-Cas system was negatively associated with drug resistance in A.baumannii.Part Ⅱ.Construction and biological characterization of AB43ΔCRISPR-CasBased on the Part I results,we further determined the relationship between the CRISPR-Cas system of Acinetobacter baumannii and drug resistance,biological traits and virulence.Using the RecAb system,the entire I-Fb CRISPR-Cas system of AB43 and each component(Cas1,Cas3,Csy1,Csy2,Csy3,Csy4,CRISPR)were individually knocked out and complemented to detect the drug resistance and biological traits of mutants to further explore the impact of the CRISPR-Cas system on the drug resistance,biological traits and virulence in A.baumannii.The drug susceptibility results showed that regardless of knocking out any component of the CRISPR-Cas system,its drug sensitivity to other antibiotics except polymyxin B and minocycline changed from sensitive to resistant.In addition,this study also tested the auto-aggregation ability,resistant to ultraviolet light,adhesion capacity and invasion ability in these mutants.These results showed that the mutants with incomplete CRISPR-Cas system had significantly stronger auto-aggregation ability,UV resistance,adhesion and invasion ability to 16HBE than the strains with complete CRISPR-Cas system.Furthermore,the virulence of strains with incomplete CRISPR-Cas system also enhanced in Galleria mellonella and mice.In conclusion,this part determined that the drug resistance and virulence of the strain with incomplete CRISPR-Cas systems were significantly higher than those of strains with complete CRISPR-Cas systems.Part Ⅲ.Mechanisms of drug resistance inhibition by the CRISPR-Cas system of Acinetobacter baumanniiBased on Part Ⅰ and Part Ⅱ results,it determined that the entire CRISPR-Cas system of A.baumannii could inhibit drug resistance.To further investigate the specific mechanism of drug resistance inhibition by the CRISPR-Cas system,transcriptome sequencing analysis found that the differentially expressed genes were not completely consistent after each component of the CRISPR-Cas system was knocked out.But the two-component regulatory system,ABC transporter protein,TCA cycle,and oxidative phosphorylation pathways were consistently changed in each component deletion strain.The efflux pump gene and function of the deletion strain were detected,and it was found that the efflux gene and efflux ability of A.baumannii with incomplete CRISPR-Cas system were significantly higher than those of the wild strain.The major energy source of proton pump power(PMF)and ATP required for drug efflux were also significantly higher than those of the wild strain.The ability of the deletion strain to form biofilms was significantly stronger than that of the wild or complemented mutants.In contrast,the cell membrane permeability of the deletion strains were significantly reduced.The cell membrane of the wild strain was more likely depolarization than the deletion strain.Subsequently,it was also found that the total ROS production of the deletion strain was reduced,but the SOD activity was enhanced.Furthermore,the CRISPR-Cas system significantly facilitated the production of H2O2 and the TCA cycle.In the transcriptome KEGG clustering analysis,we observed significant changes in quorum sensing-related genes,and also found that the quorum sensing synthetase gene abaI contained a region matching the CRISPR spacer,and the AHLs activity of AB43ΔCRISPRCas was stronger than that of the wild strain.Further research found that the key gene that acts on abaI in the CRISPR-Cas system is Cas3.Next,abaI deletion strain and complementation strain were successfully constructed.Compared with AB43,the results of drug sensitivity of AB43ΔabaI were almost unchanged,and the results of real-time quantitative PCR showed that the drug-resistant genes in AB43ΔCRISPR-Cas and AB43ΔCas3 strains were higher than those in the wild strain,but the multiple drug-resistant genes in the AB43ΔabaI strain were lower than those in the wild strain.In general,this part of the study systematically expounded that the mechanism of the CRISPR-Cas system of A.baumannii to suppress drug resistance is multifaceted.It can not only regulate a variety of drug resistance genes by targeting abaI mRNA endogenously,but also can activate the intracellular TCA cycle to increase the level of ROS and inhibit the intracellular antioxidant response.At the same time,it can not only directly inhibit drug efflux,but also inhibit the energy required for drug efflux.In addition,the CRISPR-Cas system also inhibited lipid A expression and biofilm formation.Part Ⅳ.The mechanism of antibacterial effect of baicalein combined with doxycyclineWe used the multidrug-resistant strain AB43ΔCRISPR-Cas,which was successfully constructed in Part II,as a model strain to screen out doxycycline that has a good synergistic effect with baicalein.In order to further study the antibacterial spectrum of the combined drug,we applied the combination of baicalein and doxycycline to other A.baumannii clinical strains(including drug-resistant,sensitive and doxycycline-resistant strains)and common clinical doxycycline-resistant Gram-negative bacteria through a checkerboard synergy testing.The results showed that the drug combination was effective against the above bacterial strains.And the results of hemolysis assay of sheep erythrocytes and cell safety test showed that this drug combination has good biological safety.In addition,a 31day drug resistance development experiment was carried out on the strains treated with the combination of drugs,and it was found that the combination did not induce drug resistance in the strains.Then we evaluated the effect of baicalin on a cell membrane using propidium iodide(PI),β-galactosidase,N-phenyl-l-naphthylamine(NPN),and extracellular ATP content.The fluorescence values of propidium iodide,β-galactosidase,extracellular ATP content,and NPN were significantly increased with the gradual increase of baicalein concentration.In addition,we observed the morphological changes of bacteria after baicalin treatment that only part of the surface of the bacterial cells had depressions,while the surface of the bacterial cells had multiple depressions,shrinkage,and even the collapse and disintegration of the cytoskeleton in the combined treatment group.The effect of baicalein on the antibacterial activity of gram-negative bacteria was detected by adding different concentrations of pre-extracted lipopolysaccharide(LPS)from A.baumannii to the bacterial culture medium.The results showed that the addition of exogenous purified LPS dose-dependently reduced the antibacterial activity of baicalein and the synergistic effect with doxycycline against A.baumannii.The effects of eight different cations(K+,Ca2+,Mg2+,Cu2+,Fe3+,Zn2+,Mn2+,Na+)on the antibacterial activity of baicalein were then tested.The results showed that K+,Ca2+,Mg2+,Cu2+ and Zn2+ all inhibited the antibacterial activity of baicalein,especially the addition of Mg2+ could greatly inhibit the antibacterial activity of baicalein,and high levels of Mg2+(480 μg/ml)reduced the synergistic effect of doxycycline and baicalein.Moreover,it was also found that with the increase of baicalein concentration,the key gene tetA of doxycycline resistance also showed a dose-dependent decrease.We attempted to compare the antibacterial activity of baicalein with three phospholipids on the cell membrane of gram-negative bacteria,including phosphatidylethanolamine(PE),phosphatidylglycerol(PG)and cardiolipin(CL,also known as diphosphate Glycerides),it was found that after exogenously adding phospholipids from gram-negative bacteria,the MIC value of baicalein showed a dose-dependent increase.Transcriptome analyses showed that,compared to the doxycycline alone group,the up-regulated genes in the combination group were mainly in the biosynthesis of ironcontaining non-ribosomal peptides,two-component system,ROS,fatty acid synthesis,and tryptophan metabolism pathway.The down-regulated genes were mainly in ribosome synthesis,ATP synthesis,NADH genes required for the TCA cycle,antioxidant response,multidrug efflux,ABC transporter protein genes,and outer membrane protein-related genes.Consistent with the transcriptome results,the NAD+/NADH value in the combined drug group was lower than that in the doxycycline alone,and it was dose-dependent with the concentration of baicalein.Additionally,the intracellular ROS and hydrogen peroxide production of gram-negative bacteria showed a dose-dependent increase,and correspondingly,the antioxidant response SOD showed a dose-dependent decrease.The results of the Laurdan probe detection of cell membrane fluidity showed that as the concentration of baicalein gradually increased,the cell membrane fluidity of gram-negative bacteria decreased significantly,and the cell membrane fluidity of the combined group was higher than that of the doxycycline alone.Using the fluorescent probe BCECF-AM to assess the proton pump of gram-negative bacteria,the ΔpH value showed a dose-dependent decrease related to the concentration of baicalein,and the ΔpH value of the combination group was significantly lower than that of the doxycycline alone.In addition,by detectingΔΨ with the fluorescent dye DiSC3(5),it was found that baicalein promoted the increase of the membrane potential fluorescence value of gram-negative bacteria in a dose-dependent manner,and the combined drug group had higher membrane potential fluorescence value when doxycycline was administered alone.Ethidium bromide(EtBr)was used to detect the baicalein,doxycycline,or their combination-induced effect on efflux activity.The results showed that baicalin could inhibit the efflux of EtBr,and the efflux inhibition effect was better than that of medication alone.The intracellular ATP content and biofilm formation ability were consistent with the efflux results.We then evaluated the effect of the combination in both in vivo and in vitro models of gram-negative bacterial infections.The results showed that the combined drug group could effectively inhibit gram-negative bacteria from invading 16HBE cells in vitro.ELISA test found that baicalein can stimulate MH-S cells to produce pro-inflammatory cytokines(such as TNF-α,IL-1β,IFN-γ and IL-8),among which the secretion of IL-8 increased nearly 2 times compared with the control group.The findings in vivo were consistent with those in vitro.The combined treatment group not only increased the survival rate of Galleria mellonella and mice,but also reduced the amounts of bacteria in each organ of the mice,and at the same time,the secretion of inflammatory cytokines in the serum were also reduced.The above results suggest that baicalin can disrupt bacterial cell membranes and help antibiotics enter bacterial cells more rapidly through multiple effects,and also can kill bacteria by promoting TCA circulation,promoting reactive oxygen species production,inhibiting multidrug efflux,and inhibiting biofilm,which can lock antibiotics firmly in bacterial cells.In addition,baicalein can also prevent bacteria from entering the host cells and enhancing the host immune response,ultimately leading to bacterial death.In summary,this study illustrates the mechanism of the CRISPR-Cas system of A.baumannii to inhibit drug resistance and deepens the understanding of the complete CRISPR-Cas system to inhibit drug resistance.The antibacterial effect and mechanism of baicalein combined with doxycycline were clarified,which provided new ideas for the clinical treatment of multidrug-resistant A.baumannii infection. |
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