Comparative Analysis Of The Gene Structure Of The CRISPR-Cas System In The Genome Of Klebsiella And Its Relationship With Antibiotic Resistance

Objective Klebsiella is a group of spore-free,flagella-free gram-negative bacillus,mainly including Klebsiella pneumoniae and Klebsiella oxytoca,which often parasitic on the respiratory or intestinal tract of humans or animals.It is an opportunistic pathogen,which is one of the pathogens that cause human pneumonia.In addition,it can cause hysteritis,mastitis and other purulent inflammation,and even sepsis.In recent years,the resistance of Klebsiella pneumoniae to common antibacterials has increased year by year,and even multi-drug resistant bacteria have appeared,posing severe challenges to the control and treatment of clinical infections.The CRISPR-Cas system is an adaptive immune defense system that exists in the genome of bacterial and can resist the invasion of foreign genetic material.The purpose of this study is to analyze the distribution and genetic structure of the CRISPR-Cas system in the genome of Klebsiella bacteria systematically and comprehensively.In addition,the common Klebsiella pneumoniae was used as the research object to explore the relationship between the CRISPR-Cas system and drug-resistance,and to provide new ideas for controlling the horizontal spread of resistance genes between bacteria.Methods This study is mainly divided into two parts.The CRISPR loci of the bacteria that collected form CRISPRdb can be collected directly on this website and the CRISPR loci of the bacteria that downloaded by NCBI can be searched through CRISPRFinder.CRISPR-Cas Finder was used to search for cas genes.MEGA7.0 was used to perform multiple sequence alignment and genetic evolution analysis on all cas genes and repeats,and construct a phylogenetic tree.The results of phylogenetic analysis of the repeates were submitted to the CRISPRmap online software for analysis.RNA secondary structure and prediction of minimum free energy(MFE)are performed through RNAfold.The search of the exogenous gene sequence with homology of the spaces was performed by Blastn,and the targeting criteria were set to be both coverage and matching rate greater than 85%.The leader sequence was intercepted by the EditSeq software of DNAStar.Promoter 2.0 was used to predict the presence of a promoter in the leader sequence.Conservation of the leader sequence is predicted by Weblogo.The MLST typing of Klebsiella pneumoniae was performed through the MLST database.The resistant genes were searched by annotating the genome of Klebsiella pneumoniae.χ~2 test and Fishers’exact probability method were used to study the relationship between the presence of the CRISPR-Cas system of Klebsiella pneumoniae and the rate of drug-resistant genes.The strains in the second part of the study were collected from the laboratory of the Affiliated Hospital of Qingdao University.Primer design was based on the data from the first part of the bioinformatics analysis.Based on the analysis of the cas1gene of the Klebsiella pneumoniae,two pairs of primers were designed in the I-Ea and I-Eb cas1 genes using Primer premier 5 software.The relationship between the presence of the CRISPR-Cas system and the bacterial resistance phenotype of clinical isolates was further analyzed by statistical analysis.Results 1.The positive rate of confirmed CRISPR array in the genome of 441 Klebsiella bacteria was 37.64%,and the proportion of cas gene cluster was 27.21%.The positive rate of confirmed CRISPR array in the genome of Klebsiella pneumoniae was 40.53%,and the proportion of cas gene cluster was 31.95%.Most of the CRISPR arrays of Klebsiella are located on the chromosome,but a few are on the plasmid.The positive rate of cas1gene of clinical isolates collected in this study was 34%.Most of the CRISPR-Cas systems of Klebsiella belong to the type I-E and a few belong to the type I-F.The CRISPR-Cas system of Klebsiella pneumoniae all belong to the type I-E and can be divided into I-Ea and I-Eb two subtypes.2.A total of 242 confirmed repeats were found in the genome of 441 Klebsiella isolates.After removing the same and reverse complementary sequences,a total of 28 repeats were found,ranging in length from 24 to 48 bp,most common at 30 bp and an average length of 29 bp.We performed multiple sequence alignments and phylogenetic analysis on28 confirmed repeats and found that they can be roughly divided into two categories.The repeats of subtype I-Ea are more conserved,while the repeats of I-Eb are more variable.A total of 4350 spacers were found in the confirmed CRISPR array of 441 Klebsiella isolates.A total of 1050 different spacers were screened after removing the same sequence by multiple sequence alignment.We performed a Blast search of 1050 different spacers in Genbank and found that 24.8%of the spacers were homologous to phages,26%were homologous to plasmids,and 41.2%were homologous to non-Klebsiella bacterial gene sequences.In addition,our study also found that 9%of the spacers were homologous to gene sequences of other species.3.Through multiple sequence alignment,we removed the 194 identical leader sequences and selected 33 different leader sequences.Phylogenetic analysis revealed that they can be roughly divided into two categories,and each category can be divided into multiple subclasses.This indicates that the leader sequences of Klebsiella are not conserved.No promoter was found in the leader sequence of Klebsiella pneumoniae.The leader sequence of subtype I-Ea is rich in AT content between 75-95bp,and there is a continuous"AAAA",while the leader sequence of subtype I-Eb has a lower AT content,but it also has"AATAT".4.This study analyzed the relationship between the MLST typing and the CRISPR-Cas system of Klebsiella pneumoniae,and found that bacteria containing I-Ea subtype CRISPR-Cas systems were concentrated in ST147,163,and 253,273,34,383,392,45,485,66,67,941 types,containing I-Eb subtype CRISPR systems are concentrated in ST111,13,14,23,15,1518,1660,1941,234,374,493,505,91 types,the ST type distribution of the two subtypes rarely cross.5.This study found that the carrying rate of KPC and aminoglycosidase resistance genes of ANT in the CRISPR-Cas positive group was lower than negative group.For the CRISPR array-positive group,the carrying rates of TEM,KPC,APH and ANT were significantly lower than those of the negative group.In addition,we also found that the carrying rates of TEM,KPC,and OXA were significantly lower in the group containing the subtype I-Eb CRISPR-Cas system,while the carrying rates of SHV were higher than in the subtype I-Ea group.For clinical isolates,the resistance rate of cas gene positive group to ciprofloxacin was lower than that in negative group.The resistance rate of subtype I-Eb to multiple antibiotics such as aztreonam,ceftazidime,ceftriaxone,and cefotetan were significantly lower than those of the I-Ea group.Conclusion The proportion of confirmed CRISPR arrays in the genome of Klebsiella is slightly lower than the average in most bacteria.The proportion of complete CRISPR-Cas systems in the genome of Klebsiella pneumoniae is higher than that of other Klebsiella species.Most of the CRISPR-Cas systems of Klebsiella belong to type I-E,and a few are type I-F.Type I-E can be further divided into two subtypes,I-Ea and I-Eb.The repeats in subtype I-Ea are more conserved,while the repeats in I-Eb are more variable.The repeats are highly conserved within the same locus,however,it does not mean that they are conserved among different species of Klebsiella and Klebsiella pneumoniae.There are some differences in the distribution of CRISPR-Cas systems between different ST types of Klebsiella pneumoniae.There is a certain correspondence between CRISPR typing and MLST typing.The existence of CRISPR-Cas system may only affect the carrying rate of some resistant genes and the resistance phenotype.