Detection Of AmpC Beta-lactamase In Isolates Of Klebsiella Pneumoniae From Several Hospitals Of Anhui Province And Analysis Of The Resistance Of AmpC-producing Isolates

ObjectiveTo detect of AmpC beta-lactamase in 247 clinical isolates of Klebsiella pneumoniae in several hospitals in Anhui province in 2005 and study the resistance of AmpC-producing isolates against 12 antibiotics.To investigate distribution of plasmid-mediated beta-lactamase of the AmpC-producing isolates.To investigate distribution of other drug resistance genes of the AmpC-producing isolates.To study the isoelectric point and the enzymatic hydrolyzing kinetics of the novel enzyme.Materials and MethodsThe 247 clinical isolates of Klebsiella pneumoniae were collected from 25 hospitals of Anhui province in 2005.A total of 247 clinical isolates of Klebsiella pneumoniae were consecutively collected from different clinical significance specimens in 25 hospitals of Anhui province in September 2005(from 1 September,2005 to 30 September,2005),and were identified by the standard methods in each cooperative hospitals in the entire month(from 1 September,2005 to 30 September,2005).All isolates were identified with the API identification System,and excluded the repeated isolates from the same patients.The AmpC-produeing isolates were choosed by cefoxitin firstly,and identified by the three-dimensional test,extracting plasmid and multiple PCR,the susceptibility to Antibiotics of AmpC-produeing isolates of Klebsiella pneumoniae were studied by agar dilution method.The conjugation experiment was performed in AmpC-producing isolates,according to multiple PCR results,extracting plasmid of the conjugant,three pairs of entire coding gene primers were designed,and performed in the consequence experiments and the sequence was determined by direct sequencing of PCR products carried out by the dideoxy chain termination procedure of Sanger on an ABI377 automatic sequencer (Invotrigen Biocompany,Shanghai,China).Adopting PCR methods,universal primers of bla_CTX-M,bla_sHV,bla_TEM and bla_OXA were used for ESBLs⁺ isolates screening,and analyse the other genotypes of the AmpC-producing isolates.Moreover,we test the novel AmpC beta-lactamase' isoelectric point and its hydrolysis rate to 6 antimicrobial agents.We desired southern blot hydridization to demonstrate the novel gene was in plasmid.Results 49 isolates were intermediate-susceptible resistant to cefoxitin were choosed by means of Kirby-Bauer(K-B),and 17 AmpC-producing isolates were confirmed by the three-dimensional test and multiple PCR,and accounted for 40.8%(20/49).From the size of PCR products(DHA-405bp,EBC-302bp,CIT-462bp),the genotypes of AmpC beta-lactamase can be concluded.Of 20 AmpC-producing isolates,resistance to eeftazidim,ceftriaxone,ciprofloxacin,piperacillin,cefotaxim,cefoxitin and levofloxacin was above 55%,resistance to cefepime,piperacillin-tazobactam, imipenem and meropenem was 40%,0%and 0%,respectively.A total of DNA from 17 transeonjugants was used in PCR by three pairs of entire coding gene primers.Then BLAST program was used to ascertain the genotypes of the sequencing of PCR products at GenBank.The amino acid sequence showed the gene of 11 isolates had 100%identity with that of DHA-1,the gene of 1 isolate(K. pneumoniae 462) had 99%identity with that of ACT-2,the nucleotide sequence data was presented to GeneBank,and the accession number was acquired(EF125013).The results of PCR with the universal primers of bla_CTX-M,bla_SHV,bla_TEM and bla_OXA showed 1 isolates carried bla_CTX-M-1,8 bla_CTX-M-9,2 bla_SHV,9 bla_TEM.1 bla_OXA-1.And there existed above 3 drug resistance genes in 1 strain.The novel AmpC beta-laetamase(K.pneumoniae 462) is mediated by the plasmid and its pI is 8.6,and it hydrolyzes cefoxitin with high measurable hydrolysis rate.Southern blot hydridization have demonstrated the novel AmpC beta-lactamase(K.pneumoniae 462) is mediated by the plasmid.Conclusions AmpC-producing isolates in several hospitals of Anhui province have powerful resistace to antibiotics,including the third-generation cephalosporins,but suspective to the eephalosporins withβ-lactamase inhibitions,the forth-generation cephalosporins and carbapenems.20 isolates of plasmid-mediated AmpCβ-lactamase had been found in 247 clinical isolates,and 1 novel genotype had been found.A novel plasmid-encoded class Cβ-Lactamase with an isoelectric point of 8.6 hydrolyzes cefoxitin with high measurable hydrolysis rate.We designate it ACT-3,GenBank accession number EF 125013.There existed other genotypes in AmpC-producing isolates.