Study On Genotype Characteristics And Resistance Mechanism In AmpC Beta Lactamase From Clinical Isolates Of Escherichia Coli And Klebsiella Pneumoniae

PrefaceSelected for by pressure from the spectrum antibiotics, drug - resistance pathogens are more and more increasing by means of mutation and selection as well as the spread of resistance gene. According to the statistics materials in our country in 2001, clinical strains producing AmpC β - lactamase accounted for 17.3% of pathogens caused nosocomial infection and approximate 1/3 of Gram - negative bacilli ( GNB ) , which have gradually been the most prevelant pathogens in nosocomial infection.AmpC enzyme belong to Class C or group I β - lactamase and it is a serine cephalosporinase that can not be inhibited by clavelanic acid, most of them presumed to be chromosomally mediated, have been described in Enterobacter, Crtrobacter, Serration marcesans, Morganella morganii, Yersinia enterocolitica and Pseudomonas aeruginosa, which can be induced by β- lactam. The expression of inducible AmpC β - lactamase is controlled by multiplex amp operon, and it is made of five unlinkage genes, containing ampC,ampR,ampD,ampE and ampG. The production of the AmpC β - lactamase in Escherichia coli is not inducible and normally expressed at a low level, as are most class C cephalospo-rinases, because of the absence of ampR regulator gene. As a consequence, AmpC production depends mostly on the strength of the ampC promoter, although other factors, such as gene amplification or insertion of an IS2 sequence can cause AmpC hyperproduction, which cause resistant to almost all β - lactam exception with cefeime and imipenem. Different ampC promoters of Escherichiacoli clinical strains were cloned upstream of the chloramphenicol acetyltrans-ferase( CAT) gene in the pCAT3 reporter plasmids, to study the relationship between Escherichia coli hyperproducing AmpC p - lactamases and their mutations on promoter and attenuator, expound regulator mechanism of AmpC |3 - lacta-mase, supply a new target for designing a new antibiotic and the (3 - lactamase inhibitors.In recent years, an increasing number of ampC genes have been found on plasmids. These have mostly been acquired by ampC" deficient pathogenic bacteria, such as Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Salmonella spp, Enterobacter aerogens and Proteus mirabilis. Plasmids encoding AmpC (3 - lactamases have the typical biochemical features and resistance patterns of a class I P - lactamase , but they hydrolysis substrate more numerous and often carry multiple other resistances, including resistance to aminoglyco-sides, chloramphenicol, sulfonamide, tetracycline, trimethoprim and fluoro-quinolone, whose brought us great menace in antibiotic therapy. As already observed for AmpC - producing nosocomial isolates can be responsible for outbreaks , so clinical doctors should take the infection caused by AmpC (3 - lactamases seriously.It is demonstrated that ampC gene on plasmid transferred between plasmids as well as plasmids and chromosome by carriage on transpon or integron. Inte-grons are novel DNA elements capable of integrating or mobilizing individual gene cassettes encoding antibiotic resistance. The six classes of integron so far i-dentified are distinguished by their respective integrase gene, most integrons from clinical isolates belong to class I , which play an important role in dissemination of antibiotic resistance gene. A 5'- conserved segment contains a specific recombination site(attl) that captures external gene cassettes and leads to the rapid emergence of antibiotic resistance among clinical isolates of bacteria. As a natural expression vectors of resistance gene, integron mechanism has responsible for horizontal transfer of resistance gene. The purpose of our research is to investigate the susceptibility and genotype characteristics of Escherichia coli and Klebsiella pneumoniae producing plasmid - mediated AmpC p - lactamase, to detect inserted gene cassettes of class I integron - mediated multi - drug resist-ance located on plasmid , then to provide a useful suggestion for antibiotic use and controlling the spread of drug - resistance pathogens .MethodsExperiment MaterialsOrganisms sourceWe collected 43 strains of Escherichia coli and 67 strains of Klebsiella pneumoniae in our respiratory ward during January 2002 - May 2004.ReagentDrug sensitivity disc, E test strip, foxitin, cloxacillin, clavulanic acid, ni-trocefin, MH medium, LB medium, tryptic soy broth, plasmid extraction kit, PCR ampliation kit, primer, lambda DNA, pGEM -T vector, agarose, ethidi-um dromide, DNA fragment purification kit, E. coli Electro - cells DH5a,am-pholine -polyacrylamide(PH3.5 -10) , pCAT3 basic vector, agarose gel DNA purification kit, restriction endonuclease Sac H , pst I , Hind M and Klenow fragment.Experiment procedureOrganisms collection and identificationA total of 110 strains were isolated from the patients hospitalized in our respiratory ward during January 2002 - May 2004, 43 strains of them were Escherichia coli, 67 strains of them were Klebsiella pneumoniae. These isolates were identified by using the API 20E system. Antibiotics susceptibility was detected by E - test method or disk diffusion method . The result were read based on NC-CLS in 2002.Detection of ESBLSConfirmatory test : Etest method. Isolates were considered ESBLS producers when a rate 5=8 in the MIC of ceftazidime or cefotaxime tested in combination with clavulanic acid, compared to the MIC recorded when ceftazidime or cefotaxime was tested alone.Detection of AmpC enzymeScreening test were measured by cefoxitin disk diffusion method. Isolateswith zone dameters of cefoxitin sS 17mm, ceftazidimes* 18 mm, or/and cefotaxime ^22mm were first screened as strains -producing AmpC enzyme.Confirmatory test : a cefoxitin three - dimensional test.Plasmids extraction and electroporation experimentPlasmids DNA from clinical isolates of Escherichia coli and Klebsiella pneumoniae producing AmpC enzyme were extracted using plasmid mini - preps kit. Transformation of large Plasmids DNA was performed using standard elec-troporations techniques with DH5a electeocompetent E. coli. The conditions for electroporation were as follows-, voltage 1.5kv,resistance 200ft,electric capacity 25 uf. Transformants were selected on LB agar plates supplemented with 64ug of cefoxitin per ml.Isoelectric focusing analysis (IEF) and enzyme inhibition assayEEF was performed with an LKB multiphor apparatus on prepared ampholine - polyacrylamide plates ( PH3.5 - 10). Enzyme activities of p - lactamase were detected by overlaying the gel with 0.5mM nitrocefin in 0.1M phosphate buffer ( PH =7.0). Inhibition assay was carried out by overlaying the gel with 0. 5mM nitrocefin with or without 0. 3 mM cloxacillin or 0. 3 mM clavulanic acid in 0. 1M phosphate buffer(PH =7.0).PCR amlificationDNA templatesPlasmids DNA from transformants were used as templates in PCR reactions.PrimersThe entire blaDHA gene was amplified with the oligonucleotide primers DHA Fl(5' - ATGGCGGTTGCCGTCTC - 3') and DHAR1 (5' - TGACTCTTTCGG-TATTCGGG -3') , the amplifying length was 967bp fragment.The Int I gene was amplified with primers Int I - F (5' - TGATG-GCGACGCACGAC - 3') and Int I - B (5' - TTGGGCAGCAGCGAAGT - 3'), the amplifying length was 587 bp fragment.Inserted gene cassettes of class I integron were amplified with primers 5' CS - F(5' - GGCATCCAAGCAGCAAGC - 3') and 3'CS - B (5' - AAG-C AGACTTGACCTGAT - 3').Reaction conditionPCR reactions were performed in a final volume of 50ul containing 1 x PCR buffer, 1.5mmol/L MgCl2,0. 2mmol/L of each nucleotide,0. lrnmol/L of each primer, 1.25 units of Tag DNA polymerase and lOOng target DNA.Cycle conditionPCR amplification of ampC gene: After 5min denaturation at 94X,35 PCR cycles were performed, each consisting of 30s denaturation at 94X, 60s annealing at 56 X ,and lmin extension at 72^1, a final extension step of 7min at 72 X was performed.PCR amplification of Int I gene: After 5min denaturation at 941 ,35 PCR cycles were performed, each consisting of 40s denaturation at 94X., 40s annealing at 5&X ,and 45min extension at 12X ,a final extension step of 5min at 721 was performed.PCR amplification of Inserted gene cassettes of class I integron : After 5min denaturation at 94T! ,35 PCR cycles were performed, each consisting of 60s denaturation at 94X , 60s annealing at 55t ,and 3min extension at 12X ,a final extension step of 5min at 72X was performed.DNA sequencingThe amplicons purified with DNA fragment purification kit were ligated to pGEM - T vector. The ligation mixture was used to transform highly competent JM109 cell. Transformants were selected on LB agar plates supplemented with 50ug of ampicillin per ml. Extract plasmids DNA from transformants using plas-mid mini - preps kit, then sequenced by Shanghai Sangon Biological Energying Technology Corporation.Clone the Escherichia coli ampC promoterPrimer Eel (5' - GATCGTTCTGCCGCTGTG - 3' ) and Ec2 (5' -GGGCAGCAAATGTGAGCAA - 3' ) were used to amplify a 271 bp fragment containing the 35 box, the 10 box and the attenuator from the Escherichia coli ampC promoter. The 271 bp PCR fragment was cloned in pGEM -T vector. Extract recombinant plasmids DNA pTp , then digested with restriction endonucle-ase Sac II and pst I , the fragment containing the ampC promoter was recovered with agarose gel DNA purification kit, pCAT3 basic vector was digested with Hind HI, their blunt ended with Klenow fragment of Escherichia coli DNA poly-merase I and then ligated with T4 ligase. After 1 hour incubation at hothuuse, these products were used to transform highly competent JM109 cell. Transforma-nts were selected on LB agar plates supplemented with 50ug chloramphenicol of per ml. Extract plasmids DNA pCATp from transformants, then sequenced by Shanghai Sangon Biological Energying Technology Corporation.CAT assaysThe chloramphenicol MIC was determined by broth microdilution method.ResultsIncidence of ESBLS and AmpC enzymeAmong 110 clinical isolates of Escherichia coli and Klebsiella pneumoniae, AmpC enzyme, AmpC enzyme combined with ESBLS, and ESBLS produing strains were found in 6(5. 45% ), 1(0. 90% ) , 20 (18. 2% ) strains, respectively. AmpC enzyme was detected in 6.98% of Escherichia coli and 5.97 % of Klebsiella pneumoniae; ESBLS was detected 16. 3% of Escherichia coli and 20. 9% of Klebsiella pneumoniae.Resistance phenotype of AmpC enzyme producersDrug - sensitivity test of 8 isolates producing AmpC enzyme to 11 antibiotics showed all were resistant to cefoxitin, they had high - level resistant to the third - generation cephalosporins, whose resistant rate from high to low in turn was ceftriaxone, cefotaxime, ceftazidime , and relative low - level resistant to cefeime as well as p - lactams combined with the 3 - lactamase inhibitors ( cef-operazone/sulbactam) , part of these strains were resistant to aztreonam, amika-cin, trimethoprim/sulfamethoxazole and ciprofloxacin in some degree, none of them was resistant to imipenem.Transfer of resistanceIn all of 8 isolates producing AmpC enzyme, 4 strains Klebsiella pneumoniae and 1 strains Escherichia coli transferred cefoxitin - resistance to recipient by electroporation. A cefoxitin three - dimensional test of transformants was positive , biological features of them were similar to recipient and different from their donors, transformants kept drug - resistance of AmpC enzyme and were distin-guished between recipient. A single large plasmid was detected in each transfor-mant, the molecular size of them estimated by electrophoresis on 0. 8% agarose gels was 20 kb or so.Identification of 3 - lactamaseIEF demonstrated all 5 isolates producing plasmid - mediated AmpC enzyme dislayed a brand of fj - lactamase activity with pis of 7. 8, which was inhibited by 0.3 mM cloxacillin but not by 0. 3 mM clavulanic acid, so was their transformants.PCR and sequence analysisA 967 bp fragment was amplified with the blaDHA - specific primers for all 5 isolates producing plasmid - mediated AmpC enzyme. The amino acid sequence of the PCR products deduced from the sequence analysis were identical to the plasmid - mediated cephalosporinase DHA - 1 from S. enterica serovar Enteriti-dis.Four of the five AmpC (3 - lactamase producing plasmids were found to comprise class I integron sequence by PCR assay with integron - specific primers , three kinds integrons were detected. DNA sequencing demonstrated the plasmid termed 5T had only the integron conserved segments without inserted DNA, the other harbored 2 drug - resistance gene cassettes encoding resistance to aminoglycosides ( aacA4 and aadA5 ) , trimethoprin ( dfrA17 ) , chlorampheni-col (cmlA4) and an openreading frame (ORF5) of unkown function.Sequence analysis of the cloned fragmentsA 271 bp fragment containing the promoter of the gene for four different ampC p - lactamase from four Escherichia coli clinical strains was cloned upstream of the CAT gene of the reporter plasmids pCAT3, the sequences of the cloned fragments were as follow: the promoter named 5 with seven different mutations mainly at positions - 32 and + 22^+26^+32 ; the promoter name 113 with five different mutations at position - 88N - 82^ - 18^ -1 and + 58; the promoter name E6 with six different mutations mainly at position -1 and +58.Strength of the different ampC promotersWith untransformed JM109 cells, MIC of chloramphenicol was low. In the same cells transformed with pCAT3 basic vector , MIC of chloramphenicol did...