The Molecular Biology Property Of Plasmid-mediated Qui No Lone Resistance Determinants In Citrobacter Freundii And The Analysis Of QnrB24

ObjectivesThis study was conducted to detect and analyse the presence of plasmid-mediatedquinolone resistance (PMQR) determinants [qnr, aac(6′)-Ib-cr and qepA] among clinicalisolates of Citrobacter freundii strains isolated from patients in Anhui, China. Thegenotyping of them was investigated. The I,II,III Class integron and ESBL gene wereinvestigated.Materials and MethodsIsolatesIn2009,31Citrobacter strains were collected from the first affiliated hospital of Anhuimedical university.MethodPCR was used to detect PMQR genes. The class I, II and III integrons as well asExtended-spectrum β-lactamase were also detected by PCR, and their contribution toresistance of antimicrobial agents were analysed.Conjugation experiments were conducted to determine whether the qnr-carrying plasmidswere self-transferable. The relatedness of PMQR positive isolates was analysis by PFGE, ERIC-PCR.The susceptibility of the positive isolates and transconjugants were tested by agar dilutionmethod according to CLSI guidelines. The minimum inhibitory concentrations (MICs) ofciprofloxacin and levofloxacin were determined by Etest strips according to themanufacturer's instructions.The qnrB24PCR product was purified by double-enzyme cleavage method, cloned intoPHSG398, and expressed in E. coli JM109competent cells. The plasmid DNA oftransformant was extracted, then the recombinant plasmid was comfirmed by restrictionenzyme analysis and sequenced.Southern blotting was used to reveal that the novel gene located on a plasmid.The TAIL-PCR was used to understand the genetic environment of the qnrB24gene.ResultThe qnr genes were detected in eight isolates, whereas aac(6′)-Ib-cr and qepA were notidentified in these isolates. qnrA1, qnrB1, qnrB2, qnrB4, qnrB10and qnrB24werepresent in6.5%,3.2%,6.5%,3.2%,3.2%and3.2%of Citrobacter freundii isolatesrespectively. Sequence analysis identified one novel qnrB variant (qnrB24).The results of PFGE, ERIC-PCR showed that PMQR positive isolates were not clonerelated.87.5%qnr-positive clinical isolates were resistant to quinolone resistance. Most of themwere also resistant to various antibiotics, including aminoglycosides, β-lactams, et al. The qnr genes were transferred from four clinical isolates to their transconjugants. All MICs oftransconjugants showed reduced susceptibility to fluoroquinolones.The ESBL gene and I class integron were found in almost qnr positive isolates.Sequence analysis revealed that the qnrB24gene had a maximal identity of98.7%to theqnrB10gene. Three nucleotide changes were observed. The C→A at nucleotide position139resulted in Leu→Met, the C→T at position257produced a Ala→Val change, and theA→G at position670produced a Ile→Val change.Susceptibility results showed that the MIC of transformant carrying qnrB24gene againstciprofloxacin, levofloxacin was both0.25μg/ml. The transconjugant of carrying qnrB24gene against ciprofloxacin, levofloxacin was0.125μg/ml,0.19μg/ml respectively. Thesensitivity of the transformant and transconjugant was lower than the recipient starin, butthe drug resistance was lower than the clinical isolate.Southern hybridization of plasmid DNA from transconjugant of qnrB24revealed thatthe gene was located on about60kb plasmid.The quinolone resistance of qnrB24could be transferred by conjugation.There was putative transposase adjust to5'flank of qnrB24gene.ConclusionOur study shows that qnr gene has occurred in Citrobacter freundii isolates from AnhuiProvince, China. qnr gene was therefore present in both quinolone-resistant and-susceptible isolates andsome of them could be transferred by conjugation experiments.qnr positive isolates strains showed multi-resistance and no clone was spread found inthese isolates.Class I integrons and ESBL genes were prevalence among PMQR positive isolates.Meanwhile, The qnrB24gene was discovered firstly. qnrB24gene could increase the drugresistance to fluoroquinolones slightly. The qnrB24could be transferred by plasmid.PMQR provide the low-level quinolone resistance shown in vitro to facilitate theemergence of higher-level resistance in the presence of quinolones at therapeutic levels.Our study suggests that surveillance for PMQR determinants should be undertaken on aregular basis.