| Objective To investigate the detection rates of class 1,2 and 3 integrons in clinical gram-negative strains and their antimicrobial resistance gene cassettes' category and molecular structure.To know the horizontal transmission state of antimicrobial resistance mediated by integron in gram-negative bacteria.To analyse isogenetic dependability among integron positive strains in local area.Methods To design primers based on sequences published at genebank and PCR amplify class 1,2 and 3 integron integrase(intI1,intI2,intI3) genes in clinical gram-negative strains,and furthermore,muti-PCR was used to amplify intI1 and intI2 genes.Some intI1 gene positive strains were selected to PCR amplify resistance gene cassettes and 3' conserved regions of class 1 integrons with corresponding primers.After that,PCR products of antimicrobial resistance gene cassettes were sequenced and then analyzed by comparing with sequences published before.Designing primers of gene dfrA1,sat,aadA1,ereA, orfX,tnsE,tnsD,tnsC,tnsB,tnsA to amplify intI2 gene positive strains,and then PCR mapping was used to amplify different combination of antimicrobial resistance gene cassettes by touchdown PCR to find wheather any other gene was contained between common antimicrobial resistance gene cassettes found in class 2 integrons or not and to find arrangements of gene cassettes.Sequencing was performed after the DNA products were retrieved and purified,and then we analyzed the sequences by comparing with those had been published on NCBI blast before after splicing all fragments.In order to know wheather integrons located on plasmids or not,transconjugation experiment was performed to integron positive strains and followed by PCR amplications to transconjugants with intI1 and intI2 gene primers to identify integrons.To further locate integrons,we select some strains to extract their plasmids and then make Southern blotting experiment with probes of intI1 and intI2 which labeled by digoxin, the last,integron location was judged by coloration of Southern blotting.Plate dilution method was preformed to clinical isolates which have been successfully transconjugated and their conjugants to measure their MICs.Antimicrobial resistance differences were analyzed between clinical wild strains and their transconjugants,moreover,relations between integrons and antimicrobial resistance of bacteria were also analyzed Molecular epidemiology features of integron -containing bacteria on local area were analyzed by ERIC-PCR connecting with integron location analysises by conjugation and Southern blotting experiments.Results IntI1 and intI2 genes were found in 54.0%(399/739) and 3.0%(22/739) of gram-negative clinical isolates,no intI3 gene was detected,and firstly found class 2 integron in K.pneumoniae and K.oxytoca. Primers designed for detection of intI1 and intI2 genes were proved to be effective and differentiable to them.Variable gene cassettes and 3' conserved region were detected in 80 strains of randomly selected intI1 gene positive gram- negative bacteria by PCR amplication to find 9 different lengths and 17 different combination models of gene cassette products in 64 strains,77 containing of the 3'conserved region in 80 strains of this study.Gene cassettes in 2 bacteria containing class 1 integron were dfrA15, aadA2,and in another one were aadB,aadA1 and cmlA6.184 strains of the class 1 and 2 integrons positive bacteria were successfully transconjugated,among them,31 transconjugants containing integrase.Southern blotting proved that 60%(18/30) of class 1 integrons,but none of class 2 integrons located on plasmids.MICs of clinical isolates and their transconjugants showed that the MICs of most clinical isolates were higher than their transconjugants to commonly used antimicrobials and many factors participated in antimicrobial resistance.MICs of integrase positive transconjugants were totally higher than integrase negative ones.ERIC-PCR showed that there were some the same clones prevalent among class 1 integron-containing clinical gram-negative isolates in local area.Conclusions Class 1 integrons were widespread but low detection rate of class 2 integron among gram-negative bacteria in local area.There were many gene cassettes in class 1 integrons and the combination models variable in different bacteria.Most gene cassettes in class 2 integrons were immobile.Many factors including integrons participated in antimicrobial resistance. There were some the same clones prevalent among class 1 integron-containing clinical gram-negative isolates in local area.We should strengthen surveillance and control of integrons to avoid them spread among clinical bacterial in large scale. |
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