Study On Genotype Characterization And Susceptibility Of Plasmid-Mediated AmpC Beta-lactamase In Gram-negative Bacteria Isolated From Clinical In Taiyuan

Gram-negative bacteria are the present most prevalent pathogens causing nosocomial infection. Selected 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.AmpC beta-lactamases produced by gram-negative bacteria are the important cause resulting in resistance to many new extended-spectrum beta-lactam antibiotics.AmpC beta-lactamases are mainly mediated by chromosome,but dozens of plasmid encoded AmpC beta-lactamases were found in recent years,which can transmit horizontally widely among the same or different species,and could lead to outbreaks of nosocomial infection which cause the difficulty of therapy.In order to investigate the gram-negative bacteria resistance and the consumption of antibiotic,the occurrence of AmpC beta-lactamases and the distribution,epidemic state of plasmidmediated AmpC beta-lactamases from part hospitals of Taiyuan in China.We carry out series of this research,and the susceptibility test of the AmpC beta-lactamases produced bacteria are studied as well.Otherwise we also study the inoculum effect of Cefepime with bloodstream infections of Escherichia coli producing plasmid-mediated AmpC-type beta-lactamase.Materials and Methods1,Analysis of the resistance of gram-negative bacteria and the consumption of antibiotics from the year of 1998 to 2006 in one hospitalData origin:consumption of antibiotic drugs comes from the drugstore of one hospital from the year of 1998 to 2006.Consumption of an antibiotic drug was expressed as the number of defined daily dosages(DDD)per calendar year according to the WHO—recommended DDD method to probe into medication frequency(DDDs).Detected strains:The Detected strains were collected from clinical samples,including sputum,urine, pleural fluid,abdominal fluid,swab,and blood,between January 1998 and December 2006.They were identified by VITEK2.Antibiotic susceptive test was done by Kirby—Bauer method and VITEK2.The result was judged by the CLSI standard of 2004' S edition and the data were analyzed by WHONET5.3 software.The partial correlation between the resistance of gram-negative bacteria and the consumption of antibiotics is analyzed by SPSS statistic software. 2,Detection of AmpC beta-lactamase producing gram-negative bacteriaDetected strains:Clinical isolates of no repetitive gram-negative bacteria resistance to penicillin's, first-,second- and at least one third-generation cephalosporins were celected between September 2004 and August 2006 from four hospitals in Taiyaun of China.Species identification was performed by VITEK2Screening and confirmatory tests for clinical isolates producing AmpC beta-lactamases:Strains were detected with cefoxitin disks by Kirby-Bauer disk diffusion method.Those with cefoxitin zone diameter≤17 mm were suspicious of producing AmpC beta-lactamases.Cefoxitin three-dimensional extract tests were performed for suspicious strains to confirm whether produce AmpC beta-lactamases or not.Detection of clinical isolates producing AmpC beta-lactamases and ESBLs:ESBLs were determined further by ceftriaxone three-dimensional extract tests for those strains producing AmpC beta-lactamases.Phenotype screening test for AmpC beta-lactamases:Phenotype screening test is performed by five disks namely IPM.FEP,CD02,CD03,CTX.3,Study on susceptibility and genotype of plasmid-mediated AmpC beta-lactamases in gram-negative bacteriaDetected strains:Clinical isolates of no repetitive gram-negative bacteria of nosocomial infection were collected from four hospitals in Taiyaun of China between September 2004 and August 2006.Plasmid conjugation tests:Using E.coli J53Azi~R as the recipient strains.Using clinical isolates producing AmpC beta-lactamases as supply strains.Primer designation:The information ofplasmid-encoded ampC gene was collected from GenBank and reference s.Universal primers for ampC genes were designed by aligning their conserved encoding sequences.Five groups of universal primers were designed to amplify ampC genes:DHA group,ACT group,CMY-G1 group,CMY-G2 group,FOX groups.Likewise,nine groups of universal primers were designed to amplify ESBL genes.They are TEM group,SHV group, CTX-M1 group,CTX-M9 group,TeTA group,TeTB group,gyrA group,gyrB group and PArC group.Preparation of plasmids DNA:Plasmids DNA from clinical isolates of gram-negative bacteria producing AmpC enzyme were extracted using plasmid mini—preps kit as templates for polymerase chain reaction(PCR).Amplification ofampC and ESBLs genes in producing AmpC beta-lactamases strains and sequencing:ampC and ESBLs genes on plasmids were amplified by PCR,the products were separated by electrophoresis on an 1.2%agarose gel with 0.5 u g/ml ethidium bromide and purified by gel extraction kit after photos.Nucleic acid sequence of the purified products were analyzed by Sanger dideoxy chain of termination method,and the results were compared with nucleic acid sequence of ampC genes provided by GenBank in order to identify their sub-genotype.4,Study on susceptibility and molecular epidemiology of gram-negative bacteria producing plasmid-mediated AmpC beta-lactamasesDetected strains:Plasmid-mediated AmpC beta-lactamases producing isolates were obtained from four hospitals in Taiyuan of China between September 2004 and August 2006.Preparation of chromosome DNA of bacteria:Chromosome DNA was extracted by boiling. Homology analysis of DNA:Enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR)was used for typing of cloning strains.Analysis of PCR products:The products were separated by electrophoresis on 1.5%agarose gel. Criterion of homology:The identical amplification bands of PCR was thought to be the same genotype;one band different was close correlate;2 or 3 bands different was possible correlate; exceeding 3 bands different was the different genotypes.Reproducibility tests:Reproducibility was examined by comparing patterns of E.coli 25922 and 2 strains randomly selected from 95 plasmid-mediated AmpC beta-lactamases producing isolates obtained on 3 different days.5,the inoculum effect of Cefepime in tests with Escherichia coli producing plasmid-mediated AmpC-type beta-lactamaseDetected strains:Plasmid-mediated AmpC beta-lactamases producing isolates were obtained from two teaching hospitals between September 2004 and August 2006.Detected antibiotics:Cefotaxime,ceftazidime,cefepime,sulperazon and imipenem were as detected drugs and their concentrations were adjusted to 512,256,128,64,32,16,8,4,2,1 and 0.5μg/ml.Antimicrobial susceptibility tests:The MIC of 10~5 and 10~7 cfu/mL inoculum concentration of five antibiotics was determined using a broth microdilution MIC method.The definition of a positive inoculum effect was an eight-fold or greater increase in MIC on testing with the higher inoculum.Results1,Analysis results to the resistances of gram-negative bacteria and the consumption of antibiotics from the year of 1998 to 2006 in one hospital.(1)There is an increasing tide of gram-negative bacteria isolates from the year of 1998 to of 2006 in one hospital,and an obviously increasing in A.baumannii strains.(2)The resistances to antibiotics were generally higher in No fermenting strains than in Enterrobact-eriaecae.strains the resistance rates of A.baumannii strains to almost antibiotics except imipenem exceeded 50%from the year of 2004.The resistance rates of P.Aeruginosa to ceftazidime kept about 20%,almost similar to imipenem.(3)There were very low resistance rates to imipenem in Enterrobact-eriaecae strains,almost to zero.The serious resistance is E.cloacae among Enterrobact-eriaecaes.Strains which resistance rates to most antibiotics exceeded 50%except imipenem.(4)During the 9 years of 1998-2006,the DDDs reached a peak in 2003 with a decrease afterwards.The total distributed sum of antibiotic has been rising during 1998-2006,but the highest component ratio of antibiotic to total medicine every year was in 2003.The total medication frequencies of Cephalosporins maintained a few change,but the total medication frequencies of the compound preparations ofβ-lactamase inhibitors sharply increased after 2003.There were small medication frequencies of Imipenem in the 9 years.The results of partial correlation by SPSS statistics software:There were positive correlations between the following resistances of gram-negative bacteria and the DDDs of antibiotics,and the following resistances of gram-negative bacteria were significantly related to the following consumption of antibiotics(p＜0.05).(1)The resistance rates of P.aeruginosa to Imipenem related to the DDDs of Imipenem and sulperazon.(2)The resistance rates of A.Baumannii to Piperacillin/Tazobactam related to it's DDDs and the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.The resistance rates of A.Baumannii to ceftazidime and cefepime related to their DDDs severally.The resistance rates of A.Baumannii to sulperazon related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.(3)The resistance rates of E.cloacae to sulperazon related to it's DDDs and the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.(4)The resistance rates of K.pneumonia to Piperacillin related to it's DDDs.(5)The resistance rates of E.coli to ceftazidime related to it's DDDs,and resistance rates of E.coli to ceftazidime,Cefotaxime and Piperacillin fully related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.(6)At the analysis of total resistance of gram-negative bacteria to antibiotics,we found that the total resistance rates of gram-negative to ceftazidime and ceftazidime related to their DDDs,and the total resistance rates of gram-negative to Piperacillin and sulperazon related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.2,Results of detection of AmpC beta-lactamases producing gram-negative bacteria237 isolates were positive in three-dimensional tests in a total 867 clinical isolates,which the positive rate was 27.3%(237/867).Among 237 positive isolates,123 isolates were AmpC beta-lactamase producers only,114 isolates produce both AmpC beta-lactamase and ESBLs,which the positive rate was 14.2%and 13.1%,respectively.Comparison of phenotype screening test and three-dimensional test:Analysis by phenotype screening test,277 out of 867 isolates were detected to be high level AmpC beta-lactamases producing strains,which compared with three-dimensional test,the coincidence rate was 85.5%.3,Results of susceptibility and genotypes of plasmid-mediated AmpC beta-laetamase producing bacteriaThe positive rate of FOX three-dimensional test was 7.5%(237/3159).Amplified the above 237 clinical isolates by PCR with specific primers and then sequenced,95 strains were identified to produce plasmid-mediated AmpC beta-lactamases.The specific distribution among species was:for DHA-1:E.coli 0.9%(7/789),P.aeruginosa 0.8%(5/628),E.cloacae 1.5%(6/390),A.baumannii 2.4%(10/423);for AC-1:E.coli 1.6%(13/789),Klebsiella 2.5%(11/439),P.aeruginosa strains 1.0%(6/628),E.cloacae strains2.3%(9/390),A.baumannii 2.1%(9/423),Serratia 2.8%(7/246), freundii 1.6%(2/124);for CMY-G2:P.aeruginosa 0.3%(2/628).In addition,among 95 producing plasmid-mediated AmpC positive strains,45strains also produced ESBLs including E.coli 11 strains, P.aeruginosa 8 strains,beta-lactamases modified cefoxitin three-dimensional test suggested that 3 strains of E.coli,2 strains,E.cloacae 3 strains,Klebsiella10 strains,A.baumannii 7 strains,Serratia 5 strains and freundii 1 strains.There were posotive PCR results in TEM,CTX-M1,gyrA,gyrB and ParC.Nucleic acid sequences of ampC genes were compared with provided by GenBank.There were 99%(719/722)between DHA-1 gene and EF406115(blaDHA-1),98%(760/769)between ACT-1 gene and EF125014(blaACT-1),and 99%(711/714)consistency of nucleic acid sequences between CMY-G2 gene and AF373218(blaCMY-G2)in GenBank.Nucleic acid sequences of ESBL genes were compared with provided by GenBank.There were 99%(776/777)consistency between TEM gene and X56095,but only 20%consistency compared to blaTEM in GenBank.There were 99%((849/850))between CTX-M1 gene and DQ915953(bla CTX-M1),93%(452/528)between gyrA gene and X57174(blagyrA),99%(404/405)between gyrB gene and AB083954(blagyrB) and 99%(381/382)consistency between PArC gene and AB083954(blaPArC)in GenBank.Of 95 clinical ampC genes positive isolates tested,80(84.2%)strains succeeded to be transconjugants.Kirby-Bauer disk diffusion test was used for identifying the susceptibility 80 transconjugants,and compared their susceptibilities to susceptibilities before to be transconjugants. The transconjugants remained resistance to cefoxitin and most susceptible to extended-spectrum third-generation cephalosporins.And,transconjugants and most of clinical isolates were susceptible to imipenem.By analyzing the differences between transconjugants and their clinical isolates by SPSS software,the differences of Cefepime,Cefotaxime,Cefoperazon,sulperazon and Piperacillin/Tazobactam were significant(p＜0.05).4,Results of susceptibility and molecular epidemiology of gramnegative bacteria producing plasmid-mediated AmpC beta-lactamasesStains with plasmid-mediated AmpC were consistently resistant to cefoxitin of cephamycin and above 80%resistant to the third generation cephalosporins,but resistance to the fourth generation cephalosporins cefepime was much lower(44.2%).The enzymes also provided different resistant to aztreonam,ofloxacin,amikacinl,but all stains susceptible to imipenem except 3 strains of Pseudomonas Aeruginosa.Amplified bands of ERIC-PCR ranged from 1 to 8 bands,and the size of bands ranged from 100 to 3000 bp.Of the 57 isolates of ACT-1 producing strains,E.coli32,34,36,37,38,and A.Baumannii 41,42,43,44,46,47,48,49and 50 were the same clone.P.aeruginosa 1,6,8,9,and E. cloacae 36,37,38 and Serratia 51 and 52 were the same clone.K pneumoniae 21,22,24,26, 27and 28 were the same clone.Of the 28 isolates of DHA -1 producing strains,E.coli 73,77,79, 80 and E.cloacae 67 and 71 were the same clone.P.aeruginosa 63 and 70 was the same clone,and A.Baumannii 64,65,74 and 75 was the same clone.Of the 2 isolates of CMY-G2 were similar genes with one difference.Of the 8 isolates ACT-1 and DHA-1 were different clones.The above same clone strains were isolated from the same hospital and also from the different hospitals.Reproducibility tests demonstrated that only brightness of the electrophoresis bands showed a little different in 3 strains detected,the reproducibility result was good.5,Results of inoculum effect of Cefepime in tests with Escherichia eoli producing plasmid-mediated AmpC-type beta-lactamaseOf 19 strains of E.coli blood isolates producing plasmid-mediated AmpC beta-lactamases, antimicrobial susceptibility of Cefotaxime,ceftazidime,sulperazon,cefepime and imipenem were 0,0,5.3%,84.2%and 100%respectively at Standard inoculum.Cefepime had strong inoculum effect.Because its MIC of had above 100 times in the higher inoculum tests than in the standard inoculum tests.However,the inoculum effect was least frequently detected with imipenem.Conclusions1,There was a increasing tide of gram-negative bacteria isolates,a decreasing tide of DDDs of antibiotics,and a increasing tide of consumption money,and the ranks of antibiotics were more and more advanced during nine years. 2,Imipenem remained good susceptibility against gram-negative bacteria especially Enterobacter. Fluoroquinolones had badly resistance to gram-negative bacteria especially Enterobacter.3,Resistances of gram-negative bacteria to many antibiotics were related to the total DDDs of Antibiotic/beta-lactamase inhibitor preparations.4,Of nosocomial infection bacteria producing AmpC beta-lactamases,about half are producers only, and another half are AmpC and ESBL enzyme producers.5,Three-dimensional test can be used to identify clinical Gram-negative isolates whether they produce AmpC enzyme,ESBIs or Metallo-beta-lactamases.Phenotype screening test is easily operated and can be the use as the primary screening method to detect AmpC beta-lactamases.6,Three genotypes of plasmid-mediated AmpC beta-lactamases were identified that is DHA-1,ACT -1 and CMY-G2 in Taiyuan.There have been not reports which we investigated on P.aeruginosa of producing CMY-G2 in China.7,Three genotypes are transferable plasmids to recipient strains.8,Of AmpC and ESBL enzyme,genotypes of ESBL enzyme mostly are CTX-M1,and also are gyrA, gyrB and ParC.Positive TEM gene by PCR is not the sequences of TEM by nucleic acid sequencing.9,Transconjugants remain resistance to extended-spectrum third-generation cephalosporins,but show decreasing resistance to fourth-generation cephalosporins and Antibiotic/beta-lactamase inhibitor preparations.All transconjugants are susceptible to imipenem.10,Carbapenems could be better choice for treatment of infections caused by AmpC enzyme producers,and could also choose fourth-generation cephalosporins as secondary antibiotic to treat the infection of AmpC enzyme producers on the basis of susceptibility tests.11,Gram-negative bacteria producing ACT 1,DHA-1 and CMY-G2 demonstrate the multi-cloning patterns,however,there are high similar among them.Evidences show that there is more possibility of diffusion and prevalence with producing plasmid-mediated AmpC gram-negative bacteria in medium and small cities.12 In higher inoculum tests,cefepime was dramatically affected.However,the inoculum effect was least frequently detected with imipenem.Although the inoculum effect is an in vitro laboratory phenomenon,these results suggest that cefepime may be a less than reliable agent for therapy in cases of high inoculum infections caused by AmpC[beta]-lactamase-producing Escherichia coli.