Relationship And Role Of CRISPR-Cas System In Antimicrobial Resistance Of Campylobacter Jejuni

Campylobacter jejuni(C.jejuni)is a gram negative bacterium,and considered as one of the leading cause of bacterial gastroenteritis all over the world.C.jejuni is responsible for approximately 90% cases of human campylobacteriosis.The infection or illness is considered to be self-limited where symptoms resolves within few days.However,antibiotics are used for the treatment of immune-compromised patients in particular that suffer from extra-intestinal infection or bacteremia.The rationale use of antimicrobials plays an important role in safeguarding the food producing animals and human health as well,while inappropriate usage may cause antimicrobial resistance.Antimicrobial resistance against different antibiotics,such as fluoroquinolones,macrolides,beta-lactams and aminoglycosides has been reported in Campylobacter species.Cross resistance for fluoroquinolones and macrolides,in particular,is associated with clinical isolates of C.jejuni.A clustered regularly interspaced short palindromic repeat(CRISPR)is an adaptive immune system protecting the prokaryotes against foreign genetic elements.C.jejuni has a CRISPR-cas immune system that not only identify but also interfere against invaders genomic DNA.This system is divided into three types such as CRISPR-system type I,II and III.CRISPR-system type II(Nmeni subtype)is present in C.jejuni comprised of cas1,cas2 and cas9/csn1 proteins.Apart from defending bacteria from invaders,a controversial role of CRISPR-cas system is yet under-debate in enhancing the antimicrobial resistance.Some suggests a significant role in development of antimicrobial resistance.While others couldn’t find even a single evidence of CRISPR-cas system in facilitating horizontal gene transfer.In fact,some reported a role of said system that limits horizontal gene transfer.Therefore,the purpose of present study was to find out the involvement of cas9 gene in antimicrobial resistance of C.jejuni.We explore the role of cas9 gene in antimicrobial resistance through the phenotypic and genotypic methods.By the combination of these methods,one can fully understand the relationship between cas9 and antimicrobial resistance,and provide more theoretical basis for solving the risk management and hazard control in C.jejuni.Poultry isolates of C.jejuni were subjected to antibiotic sensitivity testing and it was observed that these isolates have high antimicrobial resistance against different antibiotics.CRISPR system was identified from these isolates by using the PCR.Furthermore,CRISPR-spacers were found by using the CRISPR-finder program from the poultry isolates of C.jejuni after sequencing.Upon further analysis,it was noted that poultry isolates of C.jejuni spacers have homology to other strains of C.jejuni and C.coli.This indicates that poultry isolates may have exposure to different antibiotics and adopt or developed resistance against different drugs by integrating antibiotic resistant genes from the environment.Additionally,to determine relationship between CRISPR-cas system and antimicrobial resistance,C.jejuni NCTC11168 and C.coli ATCC33559 strains were exposed to various antibiotics.An increased expression of CRISPR related genes suggests a relationship between CRISPR-cas system and antimicrobial resistance.To inactivate Cj1523c(cas9)gene from C.jejuni NCTC11168,the gene along with its flanking sequences(upstream and downstream)were amplified by PCR.Kanamycin gene was also amplified and three fragments were then ligated by fusion PCR.After sequencing,ligated product was cloned with pGEM-Teasy vector to construct homologous recombination vector p3HKan5 H.This vector was then transformed into C.jejuni NCTC11168 by electro-transformation,and cas9 mutants were selected after verification.The role of cas9 gene in antimicrobial resistance was investigated by subjecting both Δcas9 mutant and C.jejuni NCTC11168 strains to antibiotic susceptibility testing,in vitro resistance development and exposure of both strains to different antibiotics to determined growth rate.It was observed that Δcas9 mutant strain have more antibiotic susceptibility than its parent strain as well as poultry isolates of C.jejuni.We also observed that resistance development in Δcas9 mutant strain is less as compared to wild strain.Additionally,significant difference in growth rate of Δcas9 mutant strain was observed than C.jejuni NCTC11168 upon the exposure of different antimicrobials.These phenotypic findings suggest that cas9 gene might be involved in promoting the antimicrobial resistance.For further investigation,comparative gene expression profile of Δcas9 mutant and C.jejuni NCTC11168 was done using DE-Seq2 transcriptome and results were verified by RT-qPCR analysis.In Δcas9 mutant strain,a total of 116 differently expressed genes were found.Out of 116,only 8 genes were down-regulated and 108 genes were up-regulated.It was observed that down-regulated genes such as Cj0309 c,Cj0037c,Cj1702 c,Cj1522c and Cj1521 c,along with up-regulated genes like Cj0411,Cj0412,and Cj1373 have role in antimicrobial resistance.Based on expression profile of genes,it was revealed that cas9 gene regulate several genes to promote antimicrobial resistance.Additionally,this gene expression profile also supported our results of in vitro experiments.On the basis of phenotypic determination of antibiotic resistance development and transcriptomic profiling it is concluded that CRSIPR-cas9 gene is involved in the regulation and enhancement of antibiotic resistance in C.jejuni.High antimicrobial resistance along with spacer homology of poultry isolates to other Campylobacter strains also indicates the involvement of this system in resistance.Present study elucidates the genetic picture of C.jejuni in term of their up or down-regulation.Further investigations could reveal the molecular characters of cas9 gene and their correlation with resistance.This comparative study provides new opportunities for further work on molecular mechanisms to prevent resistance development in bacteria and to ensure public health.