A Study On Plasmid-mediated AmpCβ-Lactamase Genotypes And Mechanism Of Integron Mediated Multi-resistance From Clinical Isolates Of Escherichia Coli

[Objective](1)To investigate plasmid-mediated AmpCβ-Lactamase genotypes and epidemiology fromclinical isolates of Escherichia coli in Anhui province.(2)To detect whether a new type ofβ-lactamase, extended-spectrum AmpCβ-lactamases(ESACs), emerges in clinical isolates of Escherichia coli.(3) To study resistance of the plasmid-mediated AmpCβ-Lactamase-producingEscherichia coli against 12 antibiotics, and offer resistance statistics to clinical therapy.(4)To investigate the molecular mechanism of classⅠand classⅡintegrons participatedmulti-resistance in plasmid-mediated AmpCβ-Lactamase-producing Escherichia coli.Purpose to provide science reasons for prophylaxis and control of resistant organism andR-plasmid disseminations.[Methods](1)Cefoxitin susceptibility disk test (Kirby-Bauer, K-B method) was used for ScreeningAmpC-producing Escherichia coli..(2)The isolates which resist to cefoxitin were investigated by indirect cefoxitinthree-dimensional extract test for the confirming of hyperproducting AmpC strains.(3)Multiplex PCR method was used to screen plasmid-mediated AmpC gene and theirgenotypes after extracted plasmid from cefoxitin-resistant clinical strains. Using theX~2-test and the symmetric measures for categorical variables of cefoxitinthree-dimensional extract test and multiplex PCR test.(4)Plasmid DNA was extracted by alkaline lysis. According to the universal primers PCRresults, six pairs of entire coding gene primers were designed, and the sequences were carried out by the dideoxy chain termination procedure of Sanger on an ABI 377automatic sequencer. Then blastn program was used to ascertain the genotypes at GenBank.(5)PCR and DNA sequencing were used to determine the genotypes of ESBLs forplasmid-mediated AmpCβ-Lactamase-producing Escherichia coli.(6)Conjugation experiment was used to study the transfer of cefoxitin resistance. Agardilution method was used to determine MIC of 12 antibiotics against wild-type isolates andthe transconjugants.(7)Agar dilution method was used to determine MIC of 12 antibiotics amongplasmid-mediated AmpC -producing wild-type Escherichia coli isolates.(8)ClassⅠand classⅡintegrons were detected by integrase gene PCR. The variableregion were amplified by variable gene PCR, and the PCR products were sequenced.[Results](1)Of 407 strains,302 isolates were suspected to hyperproducting AmpC enzyme bycefoxitin K-B method.(2)Of the 302 positive isolates, 37 strains were positive by indirect cefoxitinthree-dimensional confirming test, with a positive rate of 12.2%.(3)Among 302 strains of Escherichia coli, 30 strains were proved to hyperproductplasmid-mediated AmpC by multiplex PCR test, with a positive rate of 10.6%.(4)The X~2-test showed significant difference in outcome between the cefoxitinthree-dimensional extract test and multiplex PCR test. The symmetricmeasures(K=0.756, P＜0.01) illustrated better coincidence in two methods.(5)Six pairs of entire coding gene primers were designed and amplicated. Results ofPCR and DNA sequencing confirmed them to be blaCMY-2 gene (7 strains), blaDHA-1gene (4 strains)and blaACT-2 gene (3 strains). A novel CMY genotype was detected, itsnucleotide fragment length was 1165bp. The deduced amino acid sequence has SXXK.YXN and KTG .BLASTN revealed that thisβ-lactamase gene is most similar to CMY-2(97% homology),differing from CMY-2 by 27 ponits mutation of nucleotide fragment and 10 ponits amino acid substitution. It has been designated EF054895 by GenBank.(6)Of the 15 isolates that produced plasmid-mediated AmpCβ-Lactamase, 2 isolateswere successfully conjugated to the recipient. The transconjugants have reservatedresistant characteristics of the donors and distinguished from the recipient. MultiplexPCR test was positive for the transconjugants . Compared with parent isolates, thetransconjugants have retained resistance for most ofβ-lactam antibiotics but increasedsusceptibility to ciprofloxacin and levofloxacin.(7)ESBLs positive amplification results were observed for 14 E.coli isolates whichproduced plasmid-mediated AmpCβ-Lactamase. The most of genotypes of ESBLs wasCTX-M group and 9 strains have generated two types of ESBLs simultaneously. A strainwas confirmed positive for CTX-M-1 group, TEM-1 group and SHV group respectively.(8)All wild-type isolates which produced plasmid-mediated AmpCβ-Lactamase exhibitedthe highest resistant rate(93.3%)to piperacillin, and the higher resistant rate(＞60%)to allthird generation cephalosporin. The sensitivity rates of 15 strains to imipenam, cefepimeand amikacin were 100%, 80% and 80% respectively. The sensitivity rates of 15 strains toother antimicrobial agents were lower However, 10 strains plasmid-mediatedAmpC-producing accompanied with CTX-M group expressed the least susceptibility rate tocefotaxime and less susceptibility rate(50%) to cefepime.(9)70% (21/30) of the strains were shown to be ClassⅠintegron positive. No classⅡintegron was detected.(10)90.5% (19/21)of the positive strains of ClassⅠintegron contained a gene cassette, whichsized from 500bp to＞2000bp , and no classⅡintegron variable region was detected.DNA sequencing was used to identify the genetic content of the integron variable regions.5'CS or 3'CS backbone structure, intI1, dfrV, qacE△1 ,Sull and ORF5 were common genein integron.[Conclusions](1)Multiplex PCR technique and indirect three-dimensional test can detect hyperproducting AmpC accurately. Multiplex PCR technique is a sensitive, specificand quick method for detecting plasmid-mediated AmpCβ-lactamase gene and can beused in clinical routine detection.(2)CMY-2 , DHA-1 and ACT-2 plasmid-mediated AmpCβ-Lactamase were detectedfrom clinical isolates of Escherichia coli in Anhui province. CMY-2 was the mostcommon genotype in plasmid-mediated AmpC gene. It was the first reported strain of E.coli producing ACT-2 plasmid-mediated AmpCβ-lactamase in China. A novel CMYgenotype was detected and has been designated EF054895 by GenBank.(3) Severe drug resistance and multi-resistance was extensively existed in theplasmid-mediated AmpCβ-lactarnase-producing Escherichia coli.(4)Some of clinical strains producing plasmid-mediated AmpCβ-lactamase wereaccompanied by extensively producing ESBLs. The expression of ESBLs in the highAmpCβ-lactamase producing strains resulted in a significant rise in resistance level,which is regarded as a great challenge for clinical antibiotic therapy.(5)The antibiotics for infections caused by AmpC-producing Escherichia coli should beselected according to drug sensitive test results so as to choose rational and effectivedrugs. The fourth generation cephalosporins and carbapenems could be better choicesfor the treatment of infection caused by AmpCβ-lactamase producers. To severeinfections caused by strains producing plasmid-mediated AmpCβ-lactamase,carbapenems is the best choice.(6)ClassⅠintegron is widespread in plasmid-mediated AmpC-producing Escherichiacoli. Integron takes part in multiple resistance in AmpC-producing Escherichia coli .Weshould pay more attention to integron-mediated horizontal transference of multidrugresistance.