应用硼酸联合吡啶二羧酸检测铜绿假单胞菌ESBLs
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摘要
目的对美国临床实验室标准化研究所(CLSI)推荐超广谱β-内酰胺酶(ESBLs)表型确证实验进行修饰,对比推荐实验与修饰试验对铜绿假单胞菌ESBLs表型的检出率。方法挑选78株多重耐药铜绿假单胞菌纳入试验。首先采用VITEK-compactⅡ分析仪鉴定细菌,使用K-B法进行药敏试验,利用聚合酶链反应(PCR)和荧光定量PCR(RT-PCR)技术检测相关耐药基因,然后在CLSI确证实验基础上使用硼酸联合吡啶二羧酸作为抑制剂检测待测菌ESBLs表型,并比较CLSI实验与修饰实验表型检测结果。结果 78株铜绿假单胞菌菌中,32株携带ESBLs,其中12株携带MBLs,9株染色体Amp C过度表达。其余46株均未检出ESBLs,其中30株携带MBLs,12株染色体Amp C过度表达。修饰实验对32株基因型阳性的铜绿假单胞菌表型检出率为100%。CLSI确证实验仅在12个ESLBs/MBLs均阳性菌株中检出6株,20株仅产ESBLs菌株中检出17株。在46株ESBLs基因阴性待检菌株中,修饰实验对所有菌株均显示阴性而CLSI确证实验在除4个分离株之外的所有菌株均显示阴性结果。结论无论MBLs、Amp C存在与否,硼酸联合吡啶二羧酸修饰实验对铜绿假单胞菌ESBLs表型检测优于CLSI推荐表型确证实验。
Objective To compare detection rate of ESBLs phenotypic in Pseudomonas aeruginosa between the CLSI confirmation test and the modified test. Methods 78 strains of multidrug-resistant pseudomonas aeruginosa were selected in this study. First,identification and of clinically isolation of bacteria were performed by VITEK-compact Ⅱ automatic system,and drug sensitivity test was performed using the K-B method,PCR and RT-PCR technology were performed to detect drug resistance genes in Pseudomonas aeruginosa. Then,based on the CLSI confirmation test,combined boric acid with dipicolinic acid as the inhibitors was used to detect the ESBLs in Pseudomonas aeruginosa. The results of CLSI experiment and modified experiment were compared. Results Among the 78 strains of Pseudomonas aeruginosa,32 strains were ESBLs-positive,in which 12 strains were the MBLs-positive and 9 strains of chromosome AmpC were overexpressed. The remaining 46 strains were ESBLs-negative,in which 30 strains were MBLs-positive and 12 strains of Amp C were overexpressed. The detection rate of the 32 genotype-positive Pseudomonas aeruginosa with the modifited test was 100%,only 6 of the 12 ESBLs/MBLs positive strains and 17 of 20 ESBLs-positive strains were detected with the CLSI confirmation test. The modified test was negative for all 46 ESBLs genotype-negative strains,CLSI confirmation test showed negative results in all strains except 4 isolates. Conclusion Regardless of the coexisting of MBLs or overexpressed AmpC,the boric acid combined with the dipicolinic acid modification test is superior to the CLSI confirmation test for the detection of ESBLs in Pseudomonas aeruginosa.
Objective To compare detection rate of ESBLs phenotypic in Pseudomonas aeruginosa between the CLSI confirmation test and the modified test. Methods 78 strains of multidrug-resistant pseudomonas aeruginosa were selected in this study. First,identification and of clinically isolation of bacteria were performed by VITEK-compact Ⅱ automatic system,and drug sensitivity test was performed using the K-B method,PCR and RT-PCR technology were performed to detect drug resistance genes in Pseudomonas aeruginosa. Then,based on the CLSI confirmation test,combined boric acid with dipicolinic acid as the inhibitors was used to detect the ESBLs in Pseudomonas aeruginosa. The results of CLSI experiment and modified experiment were compared. Results Among the 78 strains of Pseudomonas aeruginosa,32 strains were ESBLs-positive,in which 12 strains were the MBLs-positive and 9 strains of chromosome AmpC were overexpressed. The remaining 46 strains were ESBLs-negative,in which 30 strains were MBLs-positive and 12 strains of Amp C were overexpressed. The detection rate of the 32 genotype-positive Pseudomonas aeruginosa with the modifited test was 100%,only 6 of the 12 ESBLs/MBLs positive strains and 17 of 20 ESBLs-positive strains were detected with the CLSI confirmation test. The modified test was negative for all 46 ESBLs genotype-negative strains,CLSI confirmation test showed negative results in all strains except 4 isolates. Conclusion Regardless of the coexisting of MBLs or overexpressed AmpC,the boric acid combined with the dipicolinic acid modification test is superior to the CLSI confirmation test for the detection of ESBLs in Pseudomonas aeruginosa.
引文
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[15]Odumosu BT,Adeniyi BA,Chandra R.Occurrence of aminoglycoside-modifying enzymes genes(aac(6')-I and ant(2″)-I)in clinical isolates of Pseudomonas aeruginosa from Southwest Nigeria[J].Afr Health Sci,2015,15(4):1277-1281.
[16]Dreier J,Ruggerone P.Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa[J].Front Microbiol,2015,6:660.
[17]郝巧歆,苏建荣.基层医院与三级医院耐亚胺培南铜绿假单胞菌耐药表型及耐药机制研究[J].临床和实验医学杂志,2017,16(1):91-93.
[18]高春波,苏丽菊,柴淼,等.哈尔滨地区铜绿假单胞菌β-内酰胺酶耐药基因的研究[J].中国卫生检验杂志,2017,27(13):1965-1976.
[2]葛称,顾金花,邹洁,等.ICU抗菌药物使用量与铜绿假单胞菌耐药相关性分析[J].临床和实验医学杂志,2018,17(1):102-105.
[3]Gallah S,Benzerara Y,Tankovic J,et al.βLACTA test performance for detection of extended-spectrumβ-lactamase-producing Gramnegative bacilli directly on bronchial aspirates samples:a validation study[J].Clin Microbiol Infect,2018,24(4):402-408.
[4]Peymani A,Naserpour-Farivar T,Zare E,et al.Distribution of blaTEM,blaSHV,and blaCTX-M genes among ESBL-producing P.aeruginosa isolated from Qazvin and Tehran hospitals,Iran[J].J Prev Med Hyg,2017,58(2):E115-E160.
[5]Hammoudi D,Moubareck CA,Sarkis DK,et al.How to detect carbapenemase producers?A literature review of phenotypic and molecular methods[J].J Microbiol Methods,2014,107:106-118.
[6]Kimura S,Ishii Y,Yamaguchi K.Evaluation of dipicolinic acid for detection of IMP-or VIM-type metallo-β-lactamase-producing Pseudomonas aeruginosa clinical isolates[J].Diagn Microbiol Infect Dis,2005,53(3):241-244.
[7]李小燕,吴爱武.产AmpC酶铜绿假单胞菌对亚胺培南耐药机制的研究[J].中国感染与化疗杂志,2008,8(6):423-428.
[8]王桂东,王晓燕,马璨粲,等.某院348株铜绿假单胞菌耐药性分析及多重耐药铜绿假单胞菌耐药基因检测[J].中国现代医学杂志,2017,27(25):119-122.
[9]Clinical and Laboratory Standards Institute.Tests for Extended-Spectrumβ-Lactamases in Klebsiella pneumoniae,Klebsiella oxytoca,Escherichia coli,and Proteus mirabilis[S].M100-91,CLSI,Wayne,PA(2017).
[10]陈贤君,李克诚,韩立中,等.产超广谱β-内酰胺酶肠杆菌科细菌的基因型研究[J].检验医学,2007,22(3):283-286.
[11]茅利明,许德顺.铜绿假单胞菌中OXA基因的分布与耐药性研究[J].中华医院感染学杂志,2011,21(16):3329-3331.
[12]倪朝辉,黄红兰,李凡.多药耐药铜绿假单胞菌产超广谱β-内胺酶检测及同源性分析[J].中华医院感染学杂志,2013,23(3):484-487.
[13]向波,陈定强,徐霞.铜绿假单胞菌耐药性分析及其超广谱β-内酰胺酶的流行分布[J].热带医学杂志,2016,16(6):737-740.
[14]Murugan N,Malathi J,Therese KL,et al.Application of six multiplex PCR's among 200 clinical isolates of Pseudomonas aeruginosa for the detection of 20 drug resistance encoding genes[J].Kaohsiung J Med Sci,2018,34(2):79-88.
[15]Odumosu BT,Adeniyi BA,Chandra R.Occurrence of aminoglycoside-modifying enzymes genes(aac(6')-I and ant(2″)-I)in clinical isolates of Pseudomonas aeruginosa from Southwest Nigeria[J].Afr Health Sci,2015,15(4):1277-1281.
[16]Dreier J,Ruggerone P.Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa[J].Front Microbiol,2015,6:660.
[17]郝巧歆,苏建荣.基层医院与三级医院耐亚胺培南铜绿假单胞菌耐药表型及耐药机制研究[J].临床和实验医学杂志,2017,16(1):91-93.
[18]高春波,苏丽菊,柴淼,等.哈尔滨地区铜绿假单胞菌β-内酰胺酶耐药基因的研究[J].中国卫生检验杂志,2017,27(13):1965-1976.