碳青霉烯耐药非产碳青霉烯酶肺炎克雷伯菌的药物敏感性及分子特征分析
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摘要
目的分析碳青霉烯耐药非产碳青霉烯酶肺炎克雷伯菌(carbapenem resistant Klebsiella pneumoniae without producing carbapenemase,CRKPPC)的抗菌药物敏感性、β-内酰胺酶基因和外排泵基因的分布及外排泵抑制剂的抑制作用。方法收集2012—2014年我院碳青霉烯不敏感肺炎克雷伯菌108株,用Mastdiscs combi Carba plus纸片系统检测碳青霉烯酶表型。用微量肉汤稀释法测定CRKPPC菌株对抗菌药物的敏感性;用PCR和DNA测序技术检测β-内酰胺酶耐药基因,包括超广谱β-内酰胺酶(extended-spectrum beta-lactamase,ESBL)和质粒介导的头孢菌素酶(Amp C)编码基因及外排泵基因acr AB-tolC、kex D、kde A、kpn EF和kpn GH;外排泵抑制剂羰基氰化物间氯苯腙(CCCP)和利血平抑制试验分析外排泵在碳青霉烯耐药中的作用;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析细菌外膜孔蛋白Omp K35和Omp K36的表达。结果碳青霉烯不敏感肺炎克雷伯菌菌株中有26株细菌为CRKPPC;这26株细菌对β-内酰胺类药物高度耐药,对阿米卡星、庆大霉素、左氧氟沙星和环丙沙星的耐药率均较高,但对替加环素、多黏菌素、头孢他啶/阿维巴坦和氨曲南/阿维巴坦等有很好的敏感性; blaCTX-M和blaSHV检出率依次为76.9%和57.7%;外膜孔蛋白Omp K35和Omp K36均有不同程度的缺失,缺失率分别为57.7%和19.2%;80%以上的细菌中存在acr AB-tolC、kex D、kde A、kpn EF和kpn GH基因; CCCP对碳青霉烯类药物具有显著的抑制作用,而利血平没有明显的抑制作用。结论替加环素、多黏菌素可以作为CRKPPC联合用药的基础药物。CRKPPC菌株中存在Amp C、ESBLs及不同程度的外膜孔蛋白Omp K35和Omp K36缺失,但外排泵对碳青霉烯类药物的外排作用是CRKPPC对碳青霉烯耐药的主要机制,外排泵抑制剂可能在这种细菌引起的感染治疗中有重要价值。
Objective To analyze the susceptibility and distribution of β-lactamase encoding and efflux pump genes as well as the inhibitory effect of efflux pump inhibitors on the carbapenem resistance of the carbapenem resistant Klebsiella pneumoniae without producing carbapenemase( CRKPPC). Methods One hundred and eight strains of carbapenem non-susceptible Klebsiella pneumoniae were collected from our hospital during 2012-2014. The strains producing carbapenemase were screened by Mastdiscs combi Carba plus disc system. For CRKPPC strains,the susceptibilities to antimicrobial agents were determined by micro-dilution broth methods. PCR and DNA sequencing technology were used to analyze the prevalence of β-lactamase encoding extended-spectrum β-lactamase( ESBL) and plasmid mediated Amp C genes as well as efflux genes including acr AB-tolC,kex D,kde A,kpn EF and kpn GH. The inhibitory experiments were implemented by using carbonylcyanide-m-chlorophenylhydrazone( CCCP) and Reserpine to observe the role of efflux pumps on the carbapenem resistance. Sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE) was performed to analyze the expression of outer membrane proteins Omp K35 and Omp K36. Results Twenty-six strains out of the 108 carbapenem non-susceptible Klebsiella pneumoniae were identified to be CRKPPC strains which displayed quite high resistance to β-lactam and high resistance to amikacin,gentamicin,levofloxacin and ciprofloxacin. Very good sensitivities were observed to tigecycline,polymyxin,ceftazidime/avibatan and aztreonam/avibatan. The high prevalence of blaCTX-Mand blaSHVwere also displayed with the prevalent rates being 76.9% and57.7%,respectively. The loss of outer membrane proteins Omp K35 and Omp K36 with varying degrees was observed,among which57.7% strains lost omp K35 and 19. 2% strains lost Omp K36. More than 80% strains contained efflux genes including acr AB-tolC,kex D,kde A,kpn EF and kpn GH. The results of inhibitory experiments showed that CCCP displayed quite obviously inhibitory effects on the carbepenem resistance whereas no inhibitory effect was observed for Reserpine. Conclusion Tigecycline and polymyxin could be used as the basis of combined drug for CRKPPC. The prevalence of Amp C,ESBLs and loss of Omp K35 and Omp K36 with varying degrees among these strains were observed,but the over-expression of efflux pumps should be the main mechanism of the carbapenem resistance in the Klebsiella pneumoniae strains,and efflux inhibitors may have potential value in treating the infections caused by these bacteria.
Objective To analyze the susceptibility and distribution of β-lactamase encoding and efflux pump genes as well as the inhibitory effect of efflux pump inhibitors on the carbapenem resistance of the carbapenem resistant Klebsiella pneumoniae without producing carbapenemase( CRKPPC). Methods One hundred and eight strains of carbapenem non-susceptible Klebsiella pneumoniae were collected from our hospital during 2012-2014. The strains producing carbapenemase were screened by Mastdiscs combi Carba plus disc system. For CRKPPC strains,the susceptibilities to antimicrobial agents were determined by micro-dilution broth methods. PCR and DNA sequencing technology were used to analyze the prevalence of β-lactamase encoding extended-spectrum β-lactamase( ESBL) and plasmid mediated Amp C genes as well as efflux genes including acr AB-tolC,kex D,kde A,kpn EF and kpn GH. The inhibitory experiments were implemented by using carbonylcyanide-m-chlorophenylhydrazone( CCCP) and Reserpine to observe the role of efflux pumps on the carbapenem resistance. Sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE) was performed to analyze the expression of outer membrane proteins Omp K35 and Omp K36. Results Twenty-six strains out of the 108 carbapenem non-susceptible Klebsiella pneumoniae were identified to be CRKPPC strains which displayed quite high resistance to β-lactam and high resistance to amikacin,gentamicin,levofloxacin and ciprofloxacin. Very good sensitivities were observed to tigecycline,polymyxin,ceftazidime/avibatan and aztreonam/avibatan. The high prevalence of blaCTX-Mand blaSHVwere also displayed with the prevalent rates being 76.9% and57.7%,respectively. The loss of outer membrane proteins Omp K35 and Omp K36 with varying degrees was observed,among which57.7% strains lost omp K35 and 19. 2% strains lost Omp K36. More than 80% strains contained efflux genes including acr AB-tolC,kex D,kde A,kpn EF and kpn GH. The results of inhibitory experiments showed that CCCP displayed quite obviously inhibitory effects on the carbepenem resistance whereas no inhibitory effect was observed for Reserpine. Conclusion Tigecycline and polymyxin could be used as the basis of combined drug for CRKPPC. The prevalence of Amp C,ESBLs and loss of Omp K35 and Omp K36 with varying degrees among these strains were observed,but the over-expression of efflux pumps should be the main mechanism of the carbapenem resistance in the Klebsiella pneumoniae strains,and efflux inhibitors may have potential value in treating the infections caused by these bacteria.
引文
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[5]Ohsaki Y,Kubo R,Hobson J,et al.MASTDISCS combi Carba plus,a simple method for discriminating carbapenemase-producing Enterobacteriaceae,including OXA-48-type producers[J].Microbiol Immunol,2018,62(1):60-65.
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[8]Dallenne C,Da Costa A,Decre D,et al. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae[J]. J Antimicrob Chemother,2010,65(3):490-495.
[9]Yazgan B,Türkel I,Gü9kan R,et al. Comparison of biofilm formation and efflux pumps in ESBL and carbapenemase producing Klebsiella pneumoniae[J]. J Infect Dev Ctries,2018,12(3):156-163.
[10]Srinivasan VB,Singh BB,Priyadarshi N,et al. Role of novel multidrug efflux pump involved in drug resistance in Klebsiella pneumoniae[J]. PLoS One,2014,9(5):e96288.
[11]Grundmann H,Glasner C,Albiger B,et al. Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing Enterobacteriaceae(EuSCAPE):a prospective,multinational study[J]. Lancet Infect Dis,2017,17(2):153-163.
[12]Khoshnood S,Heidary M,Mirnejad R,et al. Drug resistant gramnegative uropathogens:A review[J]. Biomed Pharmacother,2017,94:982-994.
[13]Li XZ,Plésiat P,Nikaido H. The challenge of efflux-mediated antibiotic resistance in gram-negative bacteria[J]. Clin Microbiol Rev,2015,28(2):337-418.
[14]王于莉,姜昕汝,郑月娟.外排泵介导肺炎克雷伯菌耐药的研究进展[J].微生物学通报,2016,43(6):1351-1357.
[2]Zhang Y,Jiang X,Wang Y,et al. Contribution of beta-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.
[3]Hamzaoui Z,Ocampo-Sosa A,Fernandez Martinez M,et al. Role of association of OmpK35 and OmpK36 alteration and blaESBLand/or blaAmpCin conferring carbapenem resistance among non-producing carbapenemase-Klebsiella pneumoniae[J]. Int J Antimicrob Agents,2018,52(6):898-905.
[4]Zhang R,Liu L,Zhou H,et al. Nationwide surveillance of clinical carbapenem-resistant Enterobacteriaceae(CRE)strains in China[J]. EbioMedicine,2017,19:98-106.
[5]Ohsaki Y,Kubo R,Hobson J,et al.MASTDISCS combi Carba plus,a simple method for discriminating carbapenemase-producing Enterobacteriaceae,including OXA-48-type producers[J].Microbiol Immunol,2018,62(1):60-65.
[6]Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing,28th informational supplement.M100-S28[S]. Wayne PA:CLSI,2018.
[7]Poirel L,Walsh TR,Cuvillier V,et al. Multiplex PCR for detection of acquired carbapenemase genes[J]. Diagn Microbiol Infect Dis,2011,70(1):119-123.
[8]Dallenne C,Da Costa A,Decre D,et al. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae[J]. J Antimicrob Chemother,2010,65(3):490-495.
[9]Yazgan B,Türkel I,Gü9kan R,et al. Comparison of biofilm formation and efflux pumps in ESBL and carbapenemase producing Klebsiella pneumoniae[J]. J Infect Dev Ctries,2018,12(3):156-163.
[10]Srinivasan VB,Singh BB,Priyadarshi N,et al. Role of novel multidrug efflux pump involved in drug resistance in Klebsiella pneumoniae[J]. PLoS One,2014,9(5):e96288.
[11]Grundmann H,Glasner C,Albiger B,et al. Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing Enterobacteriaceae(EuSCAPE):a prospective,multinational study[J]. Lancet Infect Dis,2017,17(2):153-163.
[12]Khoshnood S,Heidary M,Mirnejad R,et al. Drug resistant gramnegative uropathogens:A review[J]. Biomed Pharmacother,2017,94:982-994.
[13]Li XZ,Plésiat P,Nikaido H. The challenge of efflux-mediated antibiotic resistance in gram-negative bacteria[J]. Clin Microbiol Rev,2015,28(2):337-418.
[14]王于莉,姜昕汝,郑月娟.外排泵介导肺炎克雷伯菌耐药的研究进展[J].微生物学通报,2016,43(6):1351-1357.