产碳青霉烯酶肺炎克雷伯菌膜孔蛋白OmpK35与OmpK36基因转录水平
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摘要
目的通过对产碳青霉烯酶肺炎克雷伯菌携带耐药基因和分子流行特点及膜孔蛋白OmpK35、OmpK36基因转录水平、膜孔蛋白表达情况分析,了解膜孔蛋白OmpK35、OmpK36在耐药机制中的作用。方法对32株产碳青霉烯酶肺炎克雷伯菌,进行MLST基因分型;利用PCR技术对产碳青霉烯酶,头孢菌素酶、超广谱β内酰胺酶相关耐药基因和膜孔蛋白OmpK35、OmpK36基因进行检测,并对OmpK35、OmpK36基因进行序列分析;实时荧光定量RT-PCR检测膜孔蛋白OmpK35、OmpK36基因转录水平,用SDS-PAGE检测膜孔蛋白的表达情况。结果 32株耐碳青霉烯类肺炎克雷伯菌MLST分型,29株为ST11、2株ST15、1株为ST2193;PCR检测出31株KPC-2阳性,1株NDM-1阳性,32株都检出SHV,其中一株同时检出OXA-48基因。OmpK35、OmpK36基因序列存在点突变、单个碱基和小片段缺失;OmpK35、OmpK36基因转录水平与肺炎克雷伯菌ATCC 13883相比,转录水平不一,大部分明显上调;SDS-PAGE未检测有膜孔蛋白OmpK36缺失株。结论产KPC-2、NDM-1型碳青霉烯酶是导致肺炎克雷伯菌对碳青霉烯类药耐药并表现多药耐药的主要原因,而膜孔蛋白OmpK35、OmpK36在其耐药中作用未体现。
OBJECTIVE To explore the drug resistance genes in carbapenemase-producing Klebsiella pneumoniae, molecular epidemiological characteristics, transcription of porin genes OmpK35 and OmpK36 as well as expression of porin so as to understand the action mechanisms of the porin genes OmpK35 and OmpK36 in the drug resistance. METHODS Totally 32 strains of carbapenemase-producing K.pneumoniae were genotyped by means of MLST, the carbapenemase, cephalosporinase and extended spectrum β-lactamase related drug resistance genes and the porin OmpK35, OmpK36 genes were detected by using PCR, the OmpK35 and OmpK36 genes were sequenced, the levels of transcription of porin OmpK35, OmpK36 genes were detected by means of real-time fluorescence quantitative RT-PCR, and the expression of porin was detected with the use of SDS-PAGE. RESULTS The result of MLST showed that of the 32 strains of carbapenems-resistant K.pneumoniae, 29 were ST11, 2 were ST15, and 1 was ST2193. PCR showed that 31 were tested positive for KPC-2, 1 was positive for NDM-1, all of the 32 strains were detected with SHV, and one of them was detected with OXA-48 gene. Point mutation, single base and small fragment deletion were found in OmpK35 and OmpK36 gene sequences. As compared with K.pneumoniae ATCC 13883, the transcription levels of OmpK35 and OmpK36 genes were different, and most of them were up-regulated. The strain with deleted membrane porin OmpK36 was not detected by SDS-PAGE. CONCLUSION KPC-2 and NDM-1 carbapenemases are the main causes of carbapenems resistance in K.pneumoniae, but the roles of porin OmpK35 and OmpK36 are not reflected in carbapenems resistance.
OBJECTIVE To explore the drug resistance genes in carbapenemase-producing Klebsiella pneumoniae, molecular epidemiological characteristics, transcription of porin genes OmpK35 and OmpK36 as well as expression of porin so as to understand the action mechanisms of the porin genes OmpK35 and OmpK36 in the drug resistance. METHODS Totally 32 strains of carbapenemase-producing K.pneumoniae were genotyped by means of MLST, the carbapenemase, cephalosporinase and extended spectrum β-lactamase related drug resistance genes and the porin OmpK35, OmpK36 genes were detected by using PCR, the OmpK35 and OmpK36 genes were sequenced, the levels of transcription of porin OmpK35, OmpK36 genes were detected by means of real-time fluorescence quantitative RT-PCR, and the expression of porin was detected with the use of SDS-PAGE. RESULTS The result of MLST showed that of the 32 strains of carbapenems-resistant K.pneumoniae, 29 were ST11, 2 were ST15, and 1 was ST2193. PCR showed that 31 were tested positive for KPC-2, 1 was positive for NDM-1, all of the 32 strains were detected with SHV, and one of them was detected with OXA-48 gene. Point mutation, single base and small fragment deletion were found in OmpK35 and OmpK36 gene sequences. As compared with K.pneumoniae ATCC 13883, the transcription levels of OmpK35 and OmpK36 genes were different, and most of them were up-regulated. The strain with deleted membrane porin OmpK36 was not detected by SDS-PAGE. CONCLUSION KPC-2 and NDM-1 carbapenemases are the main causes of carbapenems resistance in K.pneumoniae, but the roles of porin OmpK35 and OmpK36 are not reflected in carbapenems resistance.
引文
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[3] Hamzaoui Z,Ocampo-Sosa A,Fernandez Martinez M,et al.Role of association of OmpK35 and OmpK36 alteration and blaESBL and/or blaAmpC genes in conferring carbapenem resistance among non-carbapenemase-producing Klebsiellapneumoniae[J].Int J Antimicrob Agents,2018,52(6):898-905.
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[9] Clinical and Laboratory Standards Institute.M100S.Performance standards for antimicrobial susceptibility testing :twenty -sixth edition[S].Wayne,PA:clsi,2016.
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[11] Ansari M,Munir T,Saad N.Phenotypic identification,frequency distribution and antibiogram of carbapenemase-producing Enterobacteriaceae in clinical isolates[J].J Coll Physicians Surg Pak,2018,28(4):274-278.
[12] Ye Y,Xu L,Han Y,et al.Mechanism for carbapenem resistance of clinical Enterobacteriaceae isolates[J].Exp Ther Med,2018,15(1):1143-1149.
[13] da Silva KE,Maciel WG,Sacchi FPC,et al.Risk factors for KPC-producing Klebsiella pneumoniae:watch out for surgery[J].J Med Microbiol,2016,65(6):547-553.
[14] Calia C,Pazzani C,Oliva M,et al.Carbapenemases-producing Klebsiella pneumoniae in hospitals of two regions of Southern Italy[J].APMIS,2017,125(5):491-498.
[15] Hamzaoui Z,Ocampo-Sosa A,Maamar E,et al.An outbreak of NDM-1-producing Klebsiella pneumoniae,associated with OmpK35 and OmpK36 porin loss in Tunisia[J].Microb Drug Resist,2018,24(8):1137-1147.
[16] Zhang Y,Jiang X,Wang Y,et al.Contribution of β-lactamases and porin proteins OmpK35 and OmpK36 to carbapenem resistance in clinical isolates of KPC-2-producing Klebsiella pneumoniae[J].Antimicrob Agents Chemother,2014,58(2):1214-1217.
[17] Pages JM,Peslier S,Keating TA,et al.Role of the outer membrane and porins in susceptibility of β-lactamase-producing Enterobacteriaceae to ceftazidime-avibactam[J].Antimicrob Agents Chemother,2015,60(3):1349-1359.
[18] 吴水燕,丁云芳,陶云珍,等.多药耐药肺炎克雷伯菌β-内酰胺酶基因及膜孔蛋白突变检测[J].中华医院感染学杂志,2014,24(20):4948-4951.
[19] Hong JH,Clancy CJ,Cheng S,et al.Characterization of porin expression in Klebsiella pneumoniae carbapenemase (KPC)-producing K.pneumoniae identifies isolates most susceptible to the combination of colistin and carbapenems[J].Antimicrob Agents Chemother,2013,57(5):2147-2153.
[20] Ferenci T,Phan K.How porin heterogeneity and trade-offs affect the antibiotic susceptibility of gram-negative bacteria[J].Genes (Basel),2015,6(4):1113-1124.
[21] Simner PJ,Antar AAR,Hao S,et al.Antibiotic pressure on the acquisition and loss of antibiotic resistance genes in Klebsiella pneumoniae[J].J Antimicrob Chemother,2018,73(7):1796-1803.
[2] Turner KL,Cahill BK,Dilello SK,et al.Porin loss impacts the host inflammatory response to outer membrane vesicles of Klebsiella pneumoniae[J].Antimicrob Agents Chemother,2015,60(3):1360-1369.
[3] Hamzaoui Z,Ocampo-Sosa A,Fernandez Martinez M,et al.Role of association of OmpK35 and OmpK36 alteration and blaESBL and/or blaAmpC genes in conferring carbapenem resistance among non-carbapenemase-producing Klebsiellapneumoniae[J].Int J Antimicrob Agents,2018,52(6):898-905.
[4] Pulzova L,Navratilova L,Comor L.Alterations in outer membrane permeability favor drug-resistant phenotype of Klebsiella pneumoniae[J].Microb Drug Resist,2017,23(4):413-420.
[5] Sugawara E,Kojima S,Nikaido H.Klebsiella pneumoniae major porins OmpK35 and OmpK36 allow more efficient diffusion of β-lactams than their Escherichia coli homologs OmpF and OmpC[J].J Bacteriol,2016,198(23):3200-3208.
[6] Protonotariou E,Poulou A,Politi L,et al.Hospital outbreak due to a Klebsiella pneumoniae ST147 clonal strain co-producing KPC-2 and VIM-1 carbapenemases in a tertiary teaching hospital in Northern Greece[J].Int J Antimicrob Agents,2018,52(3):331-337.
[7] Balabanian G,Rose M,Manning N,et al.Effect of porins and blaKPC expression on activity of imipenem with relebactam in Klebsiella pneumoniae:can antibiotic combinations overcome resistance[J].Microb Drug Resist,2018,24(7):877-881.
[8] Wassef M,Abdelhaleim M,AbdulRahman E,et al.The role of OmpK35,OmpK36 porins,and production of β-lactamases on imipenem susceptibility in Klebsiella pneumoniae clinical isolates,Cairo,Egypt[J].Microb Drug Resist,2015,21(6):577-580.
[9] Clinical and Laboratory Standards Institute.M100S.Performance standards for antimicrobial susceptibility testing :twenty -sixth edition[S].Wayne,PA:clsi,2016.
[10] Song W,Hong SG,Yong D,et al.Combined use of the modified Hodge test and carbapenemase inhibition test for detection of carbapenemase-producing Enterobacteriaceae and metallo-β-lactamase-producing Pseudomonas spp[J].Ann Lab Med,2015,35(2):212-219.
[11] Ansari M,Munir T,Saad N.Phenotypic identification,frequency distribution and antibiogram of carbapenemase-producing Enterobacteriaceae in clinical isolates[J].J Coll Physicians Surg Pak,2018,28(4):274-278.
[12] Ye Y,Xu L,Han Y,et al.Mechanism for carbapenem resistance of clinical Enterobacteriaceae isolates[J].Exp Ther Med,2018,15(1):1143-1149.
[13] da Silva KE,Maciel WG,Sacchi FPC,et al.Risk factors for KPC-producing Klebsiella pneumoniae:watch out for surgery[J].J Med Microbiol,2016,65(6):547-553.
[14] Calia C,Pazzani C,Oliva M,et al.Carbapenemases-producing Klebsiella pneumoniae in hospitals of two regions of Southern Italy[J].APMIS,2017,125(5):491-498.
[15] Hamzaoui Z,Ocampo-Sosa A,Maamar E,et al.An outbreak of NDM-1-producing Klebsiella pneumoniae,associated with OmpK35 and OmpK36 porin loss in Tunisia[J].Microb Drug Resist,2018,24(8):1137-1147.
[16] Zhang Y,Jiang X,Wang Y,et al.Contribution of β-lactamases and porin proteins OmpK35 and OmpK36 to carbapenem resistance in clinical isolates of KPC-2-producing Klebsiella pneumoniae[J].Antimicrob Agents Chemother,2014,58(2):1214-1217.
[17] Pages JM,Peslier S,Keating TA,et al.Role of the outer membrane and porins in susceptibility of β-lactamase-producing Enterobacteriaceae to ceftazidime-avibactam[J].Antimicrob Agents Chemother,2015,60(3):1349-1359.
[18] 吴水燕,丁云芳,陶云珍,等.多药耐药肺炎克雷伯菌β-内酰胺酶基因及膜孔蛋白突变检测[J].中华医院感染学杂志,2014,24(20):4948-4951.
[19] Hong JH,Clancy CJ,Cheng S,et al.Characterization of porin expression in Klebsiella pneumoniae carbapenemase (KPC)-producing K.pneumoniae identifies isolates most susceptible to the combination of colistin and carbapenems[J].Antimicrob Agents Chemother,2013,57(5):2147-2153.
[20] Ferenci T,Phan K.How porin heterogeneity and trade-offs affect the antibiotic susceptibility of gram-negative bacteria[J].Genes (Basel),2015,6(4):1113-1124.
[21] Simner PJ,Antar AAR,Hao S,et al.Antibiotic pressure on the acquisition and loss of antibiotic resistance genes in Klebsiella pneumoniae[J].J Antimicrob Chemother,2018,73(7):1796-1803.