| Objectives1.To understand the distribution,structure and evolution characteristics of CRISPR/Cas system in Laribacter hongkongensis(LH)from different source hosts,we detect CRISPR and cas genes in LH,and analyze the structure characteristics of repeat sequence and spacer.2.The drug resistance analysis and related resistance genes detection of LH were conducted to investigate the drug-resistance characteristics and the carrying of drug-resistant genes of LH.3.To provide evidence for the regulation mechanism of CRISPR/Cas system on drug resistance.we analyze the relationship between CRISPR/Cas system and drug resistance of LH.Method1.There were 118 LH in this study,including 90 live LH stored in the lab,DNA of 22 LH,and 4 sequences from the whole genome sequencing of the laboratory,and 2 NCBI database sequences.2.CRISPRfinder online software was used to detect the CRISPR/Cas system of the 6 LH sequences that were stored in NCBI database and obtained by the whole gene sequencing of this experiment.And the CRISPR/Cas system of the remaining 112 LH was amplified by PCR,and then we detected CRISPR/Cas after sequencing.3.Using ClustalX 2.1 to analyze repeat sequences,the spacers were analyzed with CRISPRTarget and BLASTN.4.The drug-resistant phenotype of 13 antibiotics in the 90 LH was determined by disk diffusion method.5.PCR was used to detect 9 kinds of transferable drug-resistant genes and analyzed the detection rate of genes.6.SPSS20.0 statistical software was used to analyze the relationship between CRISPR and drug resistance.The comparation between two samples mean was conducted by independent samples T test,and the comparison between more than three samples was used by analysis of variance,and the comparison difference of the rate of compared with chi-square test or Fisher’s exact probability test,both bilateral inspection,inspection level α=0.05.Results1.Distribution of 118 LH CRISPR:CRISPR4.1 and CRISPR4.2 were detected in 91.53%(108/118)and 72.03%(85/118)of LH respectively,and the upstream of the CRISPR4.1 array was closely connected to the cas genes cluster:cas1-cas3-csy1-csy3-csy3-csy4,which belongs to Ypest subtype I-F,and CRISPR4.2 was a orphan locus without cas genes around.2.The CRISPR spacer characteristics of 118 LH:Among 118 LH,2562 spacers were found,980 were unique spacers,including 1613 spacers,579 unique spacers in CRISPR4.1,949 spacers,401 unique spacers in CRISPR4.2.Only 18 spacers were matched to homologous spacers mainly on bacteriophage(31)and plasmid(19),being compared with the CRISPRDB.Furthermore,it was found that(ⅰ)in CRISPR4.1 and CRISPR4.2,the average spacer number of human-origin and frog-origin LH was more than that fish-origin LH(p<0.05).Unique spacers accounted for the largest proportion(97.46%,98.63%)in human-origin LH,followed by frog-origin LH(56.63%,70.86%)(p<0.05).The number of alleles of human-origin and frog-origin was more than fish-origin LH(p<0.05).(ⅱ)CRISPR4.1 spacers were more abundant than CRISPR4.2(p<0.05).3.The detection of drug-resistance phenotype and genes:90 LH showed different levels of drug-resistant to 13 antibiotics,and mainly resisted to rifampin,cefalotin,ampicillin,sulfamethoxazole,erythromycin,tetracycline,streptomycin,ciprofloxacin,aztreonam,cefepime,chloramphenicol,amikacin,imipenem,and the drug-resistant rate was 82.2%,68.9%,45.6%,26.7%,21.1%,16.7%,14.4%,8.9%,7.8%,7.8%,6.7%,2.2%,6.7%,respectively.Our study revealed that fish-origin LH had higher drug-resistant proportion than frog-origin LH in ampicillin,cefalotin,erythromycin,rifampicin,while frog-origin LH showed higher resistance rates to tetracycline,ciprofloxacin,sulfamethoxazole and streptomycin than frog-origin LH.The detection rates of 6 drug resistance genes sull,sul2,tetA,tetB,ereA and aac(6)-ib were 20.0%,7.8%,35.6%,26.7%,5.9%and 4.5%,respectively.The three resistance genes of tetM,ermB and qnrA were not detected.4.The relationship between CRISPR,CRISPR spacer and LH drug-resistant:CRISPR4.1 was not significantly associated with 13 antibiotics,the number of CRISPR4.1 spacer was associated with cefalotin.The multi-drug resistance rate and the drug resistance rate of cefalotin,ampicillin,streptomycin(45.16%,61.29%,37.10%,8.06%,respectively)in LH with CRISPR4.2 were lower than that LH without CRISPR4.2(78.57%,85.71%,64.29%,28.57%,respectively)(P<0.05).The average spacer of antibiotic-resistant LH was smaller than that of LH with non-drug-resistant(P<0.05).Among different source-origin LH:in fish-origin LH,CRISPR4.1 and the number of spacer was not significantly associated with 13 antibiotics.CRISPR4.2 and spacer were mainly correlated with ampicillin.The antibiotic resistant rate in LH with CRISPR is lower than the CRISPR-negative LH resistant rate;The average spacer of antibiotic-resistant LH was smaller than that of LH with non-resistant(p<0.05).In frog-origin LH,there was no statistically significant difference between the number of CRISPR4.1 and spacer and the distribution of multidrug resistance and 13 single antibiotic resistance in(p<0.05).CRISPR4.2 had mainly a negative correlation with multiple drug resistance(p<0.05).5.The relationship between CRISPR and CRISPR4.1 spacer and drug-resistant genes of LH:the sul1 carrying rate of LH with CRISPR4.2 was high(p=0.012),and the number of CRISPR spacer in frog-origin LH with sul1 was more than that of LH without sul1.Conclusion1.The CRISPR structure is widespread in LH,and the CRISPR4.1 locus and the cas genes cluster constitute the CRISPR4.1/Cas system,while the CRISPR4.2 locus is orphan locus.2.The number of spacers in CRISPR4.1 and CRISPR4.2 are abundant,and the genetic polymorphism of LH from human and frog is significantly higher than that of LH from fish.In comparison,LH from frog are closer to LH from human.CRISPR4.1 is more active than CRISPR4.2.3.The drug resistance is serious for LH from Guangdong’s freshwater products,and LH from fish and frog have different resistance models.The detection of sul1,sul2,tetA,tetB,ereA,aac(6’)-ib resistance genes can explain the resistance mechanism of the corresponding antibiotics to a certain extent.4.CRISPR4.1 has no significant relationship with multiple drug resistance distribution,and the number of spacers of CRISPR4.1 is related to the distribution of cefalotin.CRISPR4.2 is negatively correlated with multidrug resistance,cefalotin ampicillin,streptomycin resistance.The number of CRISPR4.2 spacer is negatively correlated with multidrug resistance and cefalotin resistance,the average spacer number of in LH with drug-resistant is smaller than that of LH with non-drug-resistant.The sul1 carrying rate is high in CRISPR4.2 positive LH.5.In the analysis of relation between CRISPR,CRISPR spacer and drug-resistant,the analysis results of the fish-origin and the frog-origin LH is not consistent,and the relationship is mainly reflected in the frog-origin LH. |
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