Regulation On Biofilm Formation And Drug Resistance Of Acinetobacter Baumannii AB43 Strain By CRISPR-Cas System

Acinetobacter baumannii(AB)is a non-fermenting Gram-negative coccobacillus which distributes widely in water,soil,and especially in hospital.The infections caused by A.baumannii include hospital acquired pneumonia,bacteremia,meningitis,large area wound infections,urinary tract infections and other diseases.In recent years,with the broad use of antibiotics,most of the clinical isolates of A.baumannii are multi-drug resistant,making the bacteria become one of the important opportunistic pathogens in hospitals.1.Analysis of the correlation between drug resistance and biofilm formation ability of A.baumanniiAcquired resistance may lead to variations in virulence,and the ability of resistant A.baumannii strains to form biofilms is stronger,but there are also reports of conflicting conclusions.In order to explore the relationship between the ability of biofilm formation and drug resistance of A.baumannii,the genome of ATCC19606 strain was used as the template for primers design of 16S rRNA gene.ATCC19606 strain and 68 clinical isolates collected by our lab were re-identified by PCR.The drug resistance of re-identified A.baumannii to 9 types of 21 antibiotics was detected by disk diffusion method or micro-liquid dilution method,and the biofilm formation level of each strain was determined by 0.5%crystal violet quantitative staining method.The results showed that 67 of the 68 clinical isolates were A.baumannii,which maintained 100%sensitivity only to minocycline.To the left 20 antibiotics there were resistant strains.The resistance rate to 14 antibiotics was more than 50%.The proportion of multi-drug resistance strains and extensively resistant strains was as high as 92.65%.The resistance rate to the preferred drug nowadays,meropenem,was 65.71%,and to the last line of defense drug polymyxin B reached 74.29%.94.12%of the isolates and ATCC 19606 formed biofilms.Statistical analysis of t-test revealed that the biofilm forming level of the strains resistant to P-lactam/p-lactamase inhibitor complex(ampicillin/sulbactam)and tetracycline(tetracycline and doxycycline)was stronger than that of non-resistant strains with statistically significant.Biofilm formation levels of resistant strains to penicillins,cephalosporins,carbapenems,aminoglycosides.quinolones,lipopeptides,and folate metabolic pathway inhibitors were slightly higher than non-resistant strains.These data suggested that the acquisition of resistance promoted the biofilm formation ability of A.baumannii,thereby enhancing the adaptability and survival probability of the bacteria in new environments.2.Screening of the A.baumannii CRISPR-Cas system and its regulation of target genesThe acquisition of A.baumannii resistance and virulence is associated with horizontal gene transfer,while bacterial CRISPR-Cas systems are resistant to horizontal gene transfer.Casl in A.baumannii 1-Fb CRISPR-Cas system may be associated with drug resistance and biofilm formation.To study the structures and functions of the CRISPR-Cas systems of A.baumannii clinical isolates,we first analyzed the published A.baumannii CRISPR-Cas systems through the CRISPR database.BLAST homology analysis tools of the NCBI database were used to design primers of the CRISPR clusters.And then ATCC 19606 strain and the 67 clinical isolates were used to screen CRISPR clusters by PCR and sequencing.The target genes of the CRISPR clusters were forecasted by bioinformatics analysis of the homology of those genes and spacers in the CRISPR clusters.The results showed that 10 A.baumannii strains carrying CRISPR-Cas systems have been published at present.There are 12 CRISPR clusters,2 gene sequences for casl,cas3 or csy4,and 1 gene sequence for csyl,csy2 or csy3.Analysis of the homology of 100 bp DNA upstream of the CRISPR cluster obtained 2 sets of sequences,therefore 2 universal forward primers were designed accordingly.The 100 bp DNA downstream of the CRISPR cluster has low homology,and 6 different reverse primers were designed after merging the same sequence.PCR analysis of CRISPR clusters was performed on ATCC 19606 strain and 67 clinical isolates by forward and reverse primer combinations.Two CRISPR clusters were cloned from 2 strains(AB43 and ATCC19606).The CRISPR cluster of AB43 strain was 6339 bp long and contained 103 spacers.The CRISPR cluster of ATCC 19606 strain was 1702 bp long and contains 26 spacers.Primers were designed for each cas gene sequence,and 5 cas genes,including casl,cas3,csy2,csy3,and csy4,were amplificated by PCR method in AB43 strain;however 3 Cas genes,including casl,cas3,csy4,were screened in ATCC 19606 strain.Considering that PCR can only screen out DNA fragments of known sequences,AB43 strain was subjected to genome sequencing to avoid omission of CRISPR-Cas information,and the csyl gene was found to be carried.The CRISPR-Cas systems of the two A.baumannii strains targeted many self genes,including type IV secretion protein Virb5,banded adhesion toxin protein,phage protein,DNA polymerase,etc.The type IV secretion protein Virb5 is a secreted protein necessary for the formation of pili,and the band-like adhesion toxin protein regulates the genes involved in pili formation.It is indicated that the A.baumannii CRISPR systems might inhibit the expression of Virb5 and band-like adhesion toxin protein,which had a certain regulatory effect on biofilm formation.3.Construction of A.baumannii AB43?cas1::Kanr mutant and detection of its biological propertiesIn order to explore the regulation of biofilm formation and drug resistance of A.baumannii by CRISPR-Cas system,one-step knockout method of RecAb system was used to construct a casl deletion mutant AB43?cas1::Kanr of A.baumannii.Variations of the mutant in drug resistance,serum resistance,biofilm formation,growth,and survival rate of infected mice were examined.Detection of resistance to 15 antibiotics by AB43?cas1::Kanr mutant was performed with Kirby-Bauer disc diffusion method.The survival of AB43?cas1::Kanr mutant in normal human serum and heat inactivated serum was investigated.The biofilm formation level of AB43?cas1::Kanr mutant was analyzed by 0.5%crystal violet staining.The growth curve of the mutant strain was drawn by using ultraviolet spectrophotometer analysis.Mouse model of pneumonia was developed by tracheal intubation method,with which the virulence of AB43?cas1::Kanr mutant was detected.The results showed that the AB43 wild-type strain was sensitive to 15 antibiotics,while the mutant strain was only sensitive to gatifloxacin and resistant to the left 14 antibiotics.There was no statistical difference in serum resistance between wild and mutant strains.The biofilm formation level of the mutant strain was higher than that of the wild strain,and the growth rate was slightly faster.The mortality of mice infected with mutant strains was lower than that of wild strains.These results indicated that the CRISPR-Cas system of A.baumannii AB43 strain inhibited drug resistance,biofilm formation level and growth rate in vitro,while promoted in vivo pathogenicity of the bacteria.