A Cause-effect Relationship Between GyrA Mutation And Quinolone Resistance In Escherichia Coli Isolated From Animals

Quinolone,a broad-spectrum antibiotic has been used for a long history in treating bacterial infections in humans and animals.With the increasing frequency of its use,the bacterial resistance to quinolone increased gradually theses years.The therapeutic effect of quinolone on clinic was influenced heavily by the increasing resistant bacteria strains.At present,the quinolone resistance situation of Escherichia coli and other Gram-negative bacteria in China are significantly worse than those in other countries.There are many mechanisms about E.coli resistance to quinolone drugs,including the changes of target bar,mutations of coding gene,achieving of plasmid with resistance genes,the ability of cell membrane permeability,the active drug efflux pump.Mutations in the target genes are the point in the establishment of antibacterial resistance for instance mutations in gene coding gyrase and topoisomerase IV result in increasing resistance to quinolone in E.coli.The studies reported recently suggested the causation of quinolone resistance but not the cause-effect relationship between gyrA gene and quinolones resistance.In this study,580 clinical isolated of E.coli were detected for resistance to nalidixic acid,enrofloxacin and ciprofloxacin by bacteria susceptibility testing,and muctions on gyrA gene by FRET-qPCR.The relationship between gene mutation and antibiotic resistance was analyzed with these data.The causal relationship between mutations of the gyrA gene and the quinolone resistance of E.coli was studied by site-directed mutagenesis of the standard and clinical E.coli strains Based on CRISPR/Cas9.1.Correlation of mutation in gyrA gene and quinolone resistanceThe Kirby-Bauer disc diffusion method recommended by the World Health Organization(WHO)was used to determine the sensitivity of E.coli to three quinolone antibiotics according to the criteria of CLSI(2012).The results showed that the resistance rate of E.coli to quinolone antibiotics was very high,and the resistance rates of 580 clinical E.coli to nalidixic acid,ciprofloxacin and enrofloxacin were 75.34%(437/580),47.07%(273/580)and 48.62%(282/580),respectively.The mutation situation of gyrA gene mutation was performed on 580 strains of E.coli with FRET-qPCR and gene sequencing.The results indicated that the genomic mutations were occurred on 248th,255th,259th,260th,261th,273th and 300th base mutation in the gyrA gene.According to the genetic codon table,nucleotide mutation of gyrA gene on 83 and 87 lead to mutations of amino acid but not other sites,mainly 255th,273th,300th.Here,we found that the amino acid at position 83 mutated from serine to leucine,and the amino acid at 87th mutated from aspartate to asparagine,glutamic,glycine and tyrosine.The simultaneous mutations of 83Ser→Leu and 87Asp→Asn were the main type(49.3%)in this study.The same amino acid mutations type was also advantaged in the E.coli strains with strong quinolone resistance which indicated that the correlation of this type amino acid mutation and quinolone resistance.2.Study on the cause-effect relationship between GyrA gene mutation and quinolone resistance in E.coli based on CRISPR/Cas9The genomic mutation of gyrA gene with E.coli ATCC25922 and reverse mutation with clinical strain were performed with CRISPR/Cas9 system used in this study.Since the mutated amino acid sites of the gyrA gene which were related to quinolone resistance were sites 83 and 87,we mutated the 248 and 259 nucleotide sites in the gyrA gene of E.coli standard strains,and the 248th and 259th sites of E.coli clinical strain by the CRISPR/Cas9 system.Four mutated strains of E.coli were obtained in this study,mainly E.coli ATCC25922 D87Y(amino acid 87 from aspartic acid to tyrosine),E.coli ATCC25922 S83L&D87Y(amino acid 83 from serine to leucine),E.coli ATCC25922 D87Y(amino acid 87 from serine to leucine and aspartate to asparagine),and E.coli S462231 L83S(amino acid 83 from leucine to serine).The results of growth curve,minimum inhibitory concentration test and susceptibility test demonstrated that the gyrA gene mutations in 248 and 259 had a causal relationship with quinolone resistance.In summary,we studied 580 strains of E.coli from clinical isolates resistance to quinolone,and the correlation between the gyrA gene mutation-and quinolone resistance.We also conducted mutations in E.coli ATCC25922 and clinical strains by CRISPR/Cas9 technology to study E.coli resistance to quinolone causal relationship.In this investigation,we researched on the resistance of E.coli to quinolone and provided the extra experimental basis for the study of the resistance mechanism of E.coli to quinolone.