Study On β-lactamases, Qnr And Target Enzyme Alteration Mediate Resistance Mechanism In Clinical Isolates Of Citrobacter

Objective To study the drug-resistant pattern of multiresistant Citrobacter strains isolated from the First Affiliated Hospital of Anhui Medical University and the genotype disttribution ofβ-lactamases produced by Citrobacter strains,and to investigate the mechanisms ofβ-lactam resistance in Citrobacter strains and to instruct clinical application of antibiotics reasonably.Methods A total of 52 Citrobacter strains were isolated in the First Affiliated Hospital of Anhui Medical University from January 2004 to September 2006.Standard agar dilution method was used to determine the minimal inhibitory concentration(MIC) of 52 clinically isolated Citrobacter strains, modified three-dimensional tests were performed to detect the strains of ESBLs-producing,AmpC-producing and MBL-producing in 52 Citrobacter strains, thenβ-lactamases-encoding genes were amplified by polymerase chain reaction(PCR). Furthermore,PCR products ofβ-lactamase gene in Citrobacter strains were sequenced and their homology were compared to the knownβ-lactamase gene counterparts reported in Genbank.Results 31β-lactamases-producing Citrobacter strains have been detected by modified three-dimensional tests.Among the 31 Citrobacter strains,24 isolates produced ESBLs,3 isolates produced high-level AmpCβ-lactamase,and 2 isolates produced both high-level AmpCβ-lactamase and ESBLs.No isolates produced metalβ-lactamase.31β-lactamases-producing Citrobacter strains were resistant to many antibiotics.The resistant rates ofβ-lactamases-producing strains to other 14 antibiotics were much higher than that of non-β-lactamases-producing.All tested strains however were susceptible to imipenem and meropenem and resistant to ampicillin.20 strains carried bla_TEM gene,1 strain carried bla_SHV gene,8 strains carried bla_CTX-M-1 gene, 15 strains carried bla_CTX-M-13 gene,and 5 strains carried bla_CIT gene by PCR.5 of TEM gene was sequenced,they carried broad-spectrum beta-lactamases TEM-1 gene;2 of CTX-M-1 gene was sequenced,they were CTX-M-3 subtype;3 of CTX-M-13 gene was sequenced,two of them were CTX-M-14 subtype(GenBank accession No. EF416289) and one were CTX-M-65 subtype(GenBank accession No.EF394372),they are both the first report that the CTX-M-14 subtype and CTX-M-65 subtype were found in C.freundii;4 of CIT gene was sequenced,1 was CMY-2 subtype,DNA sequence of 1 strain revealed 98%identities of the CMY-2 amino acid sequence,thirteen points mutation lead to three amino acid change,this novel CMY subtype ofβ-lactamases was designated CMY-35(GenBank accession No.EF394371) and DNA sequence of 2 strains revealed 99%identities of the CMY-2 amino acid sequence,three points mutation lead to two amino acid change,this novel CMY subtype ofβ-lactamases was designated CMY-34(GenBank accession No.EF394370).Conclusions Modified three-dimensional test can detect isolates producing simultaneous ESBLs and high-level AmpCβ-lactamase.The positive rates ofβ-lactamases in Citrobacter strains were high, so we should pay much attention to detectingβ-lactamase-producing strains.The clinical isolates of Citrobacter strains in the hospital showed high resistance toβ-lactam antibiotics,fluoroquinolones,and aminoglycosides but susceptibility to carbopenems. Therapy forβ-lactamase-producing Citrobacter strains was limited to imipenem and meropenem.The resistant rates ofβ-lactamases-producing strains to antibiotics were higher than that of non-β-lactamases-producing.The main types ofβ-lactamases produced by Citrobacter strains were ESBLs and AmpC B-lactamases.It was one of the main mechanisms of resistantance toβ-lactam antibiotics that producing ESBLs and AmpC B-lactamase in clinical isolates of Citrobacter strains.CTX-M-type is the most common genotype in ESBLs-producing Citrobacter strains in our hospital,secondly,is TEM-type.A great diversity ofβ-lactamases production was found in Citrobacter strains and a single strain could contain severalβ-Iactamases simultaneously. Objective To investigate the drug resistance of Citrobacter strains isolated from clinical specimens and mechanisms of its resistance to fluoroquinolones,and analysis the qnr genes and the correlation of alterations in GyrA,GyrB,ParC and ParE with susceptibilities to fluoroquinolones in clinical isolates of Citrobacter.Methods A total of 52 Citrobacter strains were isolated in the First Affiliated Hospital of Anhui Medical University from January 2004 to September 2006.Corresponding group of the qnr genes and gyrA,gyrB,parC,and parE were amplified by polymerase chain reaction (PCR),the sequence and homology of PCR products were analyzed,determining the genotypes of qnr and mutations of the quinolone resistance-determining regions(QRDR) of DNA gyrase(gyrA and gyrB) and topoisomeraseⅣ(parC and parE).Conjugation experiments were done with azide-resistant E.coli J53 as the recipient and qnr-positive strains as the donors,transconjugants were selection with azide and ampicillin.Standard agar dilution method was used to determine the minimal inhibitory concentration(MIC) for 52 Citrobacter strains,E.coli J53,and transconjugants of seventeen antibacterials. Results The qnr genes were identified in 22 of 52 Citrobacter strains,5 strains had qnrA gene and 11 strains had qnrB2 gene,1 strain had qnrB4 gene.2 of qnrA gene was sequenced,DNA sequence of they revealed 100%identities of the qnrA1 amino acid sequence of Enterobacter cloacae(GenBank No.DQ989302),3 of qnrB2 gene was sequenced,DNA sequence of they revealed 97%identities of qnrB2 amino acid sequence of Citrobacter freundii(GenBank No.AB281054),seventeen points mutation leads to two amino acid change,this is a novel QnrB variant(GenBank accession No. EF526508);1 qnrB4 gene(GenBank accession No.EF543140) was sequenced,DNA sequence revealed 100%identities of the qnrB4 amino acid sequence of Escherichia coli(GenBank No.DQ303921).This is the first report that strain containing qnrB2 can also produce the AmpC-typeβ-lactamase CMY-2 and qnrB4-positive isolate produceβ-lactamases TEM,SHV and CTX-M13.The results of seven strians showed that the sequenced regions of gyrA in three strains had 99%homology with these of Citrobacter freundii ATCC 8090(GenBank No.AF052253) and the others had 98%homology with these of Escherichia coil K12(GenBank No.U00096),and the sequenced regions of gyrB,parC and parE had 98%,95%and 88%homology with these of Escherichia coil K12 respectively.Among five fluroquinolone-resistance isolates,the principal mutations were those resulting in the substitutions Ile for Thr-83 or Leu for Ser-83 and Asn for Asp-87 of gyrA,and lle for Ser-80 and Gly for Glu-84 of parC.The results did show some novel mutations at codon 417 of gyrB(Leu→His) and codon 127 of parC (Lys→Arg),which were not related to fluroquinolone resistance.There was no amino acid change in parE.Only one Citrobacter strain plasmid transfer of qnrA gene was achieved and the other transconjugants were detected carrying beta-lactamases gene. The rates of 52 Citrobacter strains resistance to levofloxacin,ciprofloxacin and gatifloxacin were 84.6%,86.5%and 76.9%respectively.The MICs of transconjugants to fifteen antibacterials were decreased differently.Conclusions qnrB-type is the most common genotype in qnr positive Citrobacter strains in our hospital,secondly,is qnrA-type.Compared toβ-lactamases-negative isolates,the presence ofβ-lactamases enhances the level of resistance to fluoroquinolones inβ-lactamases-producing isolates. The expressing of qnr not only confers resistance to quinolones,but also enhances the action of other mechanisms of antibiotic resistance.The Qnr determinant alone confers low-level quinolone resistance,but not to provide resistance to fluoroquinolone.The presence of qnr facilitates the selection of chromosomal mutants in the presence of a quinolone,resulting in higher levels of quinolone resistance,so qnr can supplement resistance via altered quinolone target enzymes,efflux pump activation,or deficiencies in outer membrane porin channels.For Gram-negative rod,DNA gyrase is the primary target enzyme for fluoroquinolones and topoisomeraseⅣis the secondary target,a single mutation in codon 83 of gyrA was associated with decreased susceptibility or low levels of resistance to fluoroquinolones,and that the accumulation of amino acid changes in GyrA with the simultaneous presence of alterations in ParC contributes to increase this resistance to a high level.This study indicate the positive rate of the qnr genes in Citrobacter strains isolated is higher,which should be highly concerned.