硝酸镓对耐碳青霉烯肺炎克雷伯菌生长及其生物膜的抑制作用
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摘要
目的检测新型抗菌剂硝酸镓(gallium nitrate, GaN)对耐碳青霉烯肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumonia, CRKP)生长及其生物膜(biofilm, BF)的抑制作用。方法收集2015—2016年安徽省地区34家医院临床分离的CRKP共82株,采用VITEK-2Compact全自动微生物分析系统进行细菌鉴定,琼脂倍比稀释法进行药敏试验;微量肉汤稀释法测定GaN对CRKP的最低抑菌浓度(minimal inhibitory concentration, MIC);96孔板结晶紫染色法检测82株实验菌株BF的A_(570)值,筛选出BF强阳性菌株;检测1/2MIC的GaN对BF强阳性菌株的BF形成的抑制作用。比较GaN与庆大霉素(gentamicin, GEN)分别对CRKP的BF形成的抑制作用。激光共聚焦扫描显微镜观察GaN作用下的菌株生物膜三维立体结构。结果临床分离的CRKP呈多重耐药。GaN对82株CRKP的MIC范围为0.0625～2μmol/mL,其中MIC_(50)、MIC_(90)分别为0.25和0.5μmol/mL。BF强阳性菌株所占比例为37.8%(31/82)。三维立体图片显示CRKP在不同GaN浓度作用下BF形成的量不同。1/2MIC的GaN可显著抑制CRKP的BF形成。GaN对CRKP的BF形成的抑制作用优于GEN。结论 GaN对CRKP生长及其BF有明显抑制作用。
Objective To study the inhibitory activity of gallium nitrate(GaN) on the bacterial growth and biofilm formation of carbapenem-resistant Klebsiella pneumoniae(CRKP). Methods The 82 clinical CRKP isolates were collected from the 34 hospitals of Anhui province from 2015 to 2016. The VITEK 2 Compact automated microbial analyzer was employed to conduct the bacterial identification. The minimal inhibitory concentrations(MIC) of 17 antibacterial drugs for these isolates were determined by the agar double dilution assay. The MIC of GaN was determined by the Micro-dilution method. The crystal violet staining assay was used to measure the A_(570) of CRKP in order to screen out strong biofilm-positive strains, and to test inhibitory effects of GaN(1/2 MIC) on BF formation in 96-well plates. To compare the effects of Gallium nitrate with those of gentamicin on the biofilm formation of CRKP. The BF three-dimensional structure was visualized using the Confocal Laser Scanning Microscope. Results The clinical CRKP isolates demonstrated multidrug-resistance. In this study, the MIC of GaN for the 82 isolates was found to be in the range of 0.0625 to 2μmol/mL. The MIC_(50) was 0.25μmol/mL. The MIC90 was 0.5μmol/mL. The strong biofilm-positive strains accounted for 37.8%(31/82). Scanning electron microscopy imaging results revealed changes in amount of BF formation when the bacterial treated with GaN at different concentrations. The images demonstrated that GaN(1/2 MIC) could significantly inhibit the biofilm formation of CRKP. The effects of gallium nitrate on the biofilm formation was superior to those of gentamicin. Conclusion GaN could significantly inhibit the growth and BF formation of CRKP.
Objective To study the inhibitory activity of gallium nitrate(GaN) on the bacterial growth and biofilm formation of carbapenem-resistant Klebsiella pneumoniae(CRKP). Methods The 82 clinical CRKP isolates were collected from the 34 hospitals of Anhui province from 2015 to 2016. The VITEK 2 Compact automated microbial analyzer was employed to conduct the bacterial identification. The minimal inhibitory concentrations(MIC) of 17 antibacterial drugs for these isolates were determined by the agar double dilution assay. The MIC of GaN was determined by the Micro-dilution method. The crystal violet staining assay was used to measure the A_(570) of CRKP in order to screen out strong biofilm-positive strains, and to test inhibitory effects of GaN(1/2 MIC) on BF formation in 96-well plates. To compare the effects of Gallium nitrate with those of gentamicin on the biofilm formation of CRKP. The BF three-dimensional structure was visualized using the Confocal Laser Scanning Microscope. Results The clinical CRKP isolates demonstrated multidrug-resistance. In this study, the MIC of GaN for the 82 isolates was found to be in the range of 0.0625 to 2μmol/mL. The MIC_(50) was 0.25μmol/mL. The MIC90 was 0.5μmol/mL. The strong biofilm-positive strains accounted for 37.8%(31/82). Scanning electron microscopy imaging results revealed changes in amount of BF formation when the bacterial treated with GaN at different concentrations. The images demonstrated that GaN(1/2 MIC) could significantly inhibit the biofilm formation of CRKP. The effects of gallium nitrate on the biofilm formation was superior to those of gentamicin. Conclusion GaN could significantly inhibit the growth and BF formation of CRKP.
引文
[1]吕继芳,郑焙文,张静.耐碳青霉烯类肺炎克雷伯菌分子流行病学和耐药基因分析[J].中国抗生素杂志,2016,41(5):356-360.
[2]Aparstek L,Carmeli Y,Navon-Venezia S,et al.Biofilm formation and susceptibility to gentamicin and colistin of extremely drugresistant KPC-producing Klebsiella pneumoniae[J].J Antimicrob Chemother,2014,69(4):1027-1034.
[3]Liu Y Y,Wang Y,Walsh T R,et al.Emergence of plasmidmediated colistin resistance mechanism MCR-1 in animals and human beings in China:A microbiological and molecular biological study[J].Lancet Infect Dis,2016,16(2):161-168.
[4]Singh S K,Gupta M.blaOXA-48 carrying clonal colistin resistant-carbapenem resistant Klebsiella pneumoniae in neonate intensive care unit,India[J].Microb Pathog,2016,100:75-77.
[5]Berglund B,Hoang N,T?rnberg M,et al.Insertion sequence transpositions and point mutations in mgrB causing colistin resistance in a clinical strain of carbapenem-resistant Klebsiella pneumoniae from vietnam[J].Int J Antimicrob Agents,2017,51(5):789-793.
[6]Xu J,Gao M,Hong Y,et al.Draft genome sequence of a tigecycline-resistant KPC-2-producing Klebsiella pneumoniae ST15 clinical isolate from China[J].J Glob Antimicrob Resist,2017,11:156-158.
[7]Reiter K C,Villa B,Paim T G,et al.Inhibition of biofilm maturation by linezolid in meticillin-resistant Staphylococcus epidermidis clinical isolates:Comparison with other drugs[J].J Med Microbiol,2013,62(3):394-399.
[8]Hess D J,Henry-Stanley M J,Lusczek E R,et al.Anoxia inhibits biofilm development and modulates antibiotic activity[J].J Surg Res,2013,184(1):488-494.
[9]Shiow-Fern N,Hon-Lunn L.Development of biofilmtargeted antimicrobial wound dressing for the treatment of chronic wound infections[J].Drug Develop Indust Pharm,2015,41(11):1902-1929.
[10]Mottola C,Matias C S,Mendes J J,et al.Susceptibility patterns of Staphylococcus aureus,biofilms in diabetic foot infections[J].Bmc Microbiol,2016,16(1):1-9.
[11]Sadowski S M,Neychev V,Cottledelisle C,et al.Detection of insulinoma using 68 Gallium-DOTATATE PET/CT:Acase report[J].Gland Surg,2014,4(3):E1-5.
[12]Gourni E,Mansi R,Jamous M,et al.N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists:examples of 68Ga-and 64Culabeled peptides for PET imaging[J].J Nucl Med Off Pub Soc Nucl Med,2014,55(10):1719-1725.
[13]Runci F,Bonchi C,Frangipani E,et al.Acinetobacter baumannii biofilm formation in human serum and disruption by gallium[J].Antimicrob Agents Chemother,2017,61(1):01563-16.
[14]葛新,王辉,董小青.硝酸镓对尿路致病性大肠埃希菌及其生物膜的抑制作用[J].中国抗生素杂志,2013,38(2):136-139.
[15]Zhang D,Xia J,Xu Y,et al.Biological features of biofilmforming ability of Acinetobacter baumannii,strains derived from 121 elderly patients with hospital-acquired pneumonia[J].Clin Exper Med,2016,16(1):73-80.
[16]Clinical and Laboratory Standards Institute.Performance standards for antimicrobial susceptibility testing[S].Twentysixth informational supplement,2016,M100S,26th Edition.
[17]Dzul S P,Thornton M M,Hohne D N,et al.Contribution of the Klebsiella pneumoniae capsule to bacterial aggregate and biofilm microstructures[J].Appl Environ Microbiol,2011,77(5):1777-17782.
[18]王玎,李兴禄.肺炎克雷伯菌生物被膜的体外模型建立[J].中国微生态学杂志,2009,21(2):135-136.
[19]Hallstoodley L,Stoodley P.Evolving concepts in biofilm infections[J].Cellular Microbiol,2009,11(7):1034.
[20]Kaneko Y,Thoendel M,Olakanmi O,et al.The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity[J].J Clin Invest,2007,117(4):877-888.
[21]Olakanmi O,Kesavalu B,Pasula R,et al.Gallium nitrate is efficacious in murine models of tuberculosis and inhibits key bacterial Fe-dependent enzymes[J].Antimicrob Agents Chemother,2013,57(12):6074-6080.
[22]Baldoni D,Steinhuber A,Zimmerli W,et al.In vitro activity of gallium maltolate against Staphylococci in logarithmic,stationary,and biofilm growth phases:comparison of conventional and calorimetric susceptibility testing methods[J].Antimicrob Agents Chemother,2010,54(1):157-163.
[23]Chitambar C R.Medical applications and toxicities of gallium compounds[J].Int J Environ Res Public Health,2010,7(5):2337-2361.
[2]Aparstek L,Carmeli Y,Navon-Venezia S,et al.Biofilm formation and susceptibility to gentamicin and colistin of extremely drugresistant KPC-producing Klebsiella pneumoniae[J].J Antimicrob Chemother,2014,69(4):1027-1034.
[3]Liu Y Y,Wang Y,Walsh T R,et al.Emergence of plasmidmediated colistin resistance mechanism MCR-1 in animals and human beings in China:A microbiological and molecular biological study[J].Lancet Infect Dis,2016,16(2):161-168.
[4]Singh S K,Gupta M.blaOXA-48 carrying clonal colistin resistant-carbapenem resistant Klebsiella pneumoniae in neonate intensive care unit,India[J].Microb Pathog,2016,100:75-77.
[5]Berglund B,Hoang N,T?rnberg M,et al.Insertion sequence transpositions and point mutations in mgrB causing colistin resistance in a clinical strain of carbapenem-resistant Klebsiella pneumoniae from vietnam[J].Int J Antimicrob Agents,2017,51(5):789-793.
[6]Xu J,Gao M,Hong Y,et al.Draft genome sequence of a tigecycline-resistant KPC-2-producing Klebsiella pneumoniae ST15 clinical isolate from China[J].J Glob Antimicrob Resist,2017,11:156-158.
[7]Reiter K C,Villa B,Paim T G,et al.Inhibition of biofilm maturation by linezolid in meticillin-resistant Staphylococcus epidermidis clinical isolates:Comparison with other drugs[J].J Med Microbiol,2013,62(3):394-399.
[8]Hess D J,Henry-Stanley M J,Lusczek E R,et al.Anoxia inhibits biofilm development and modulates antibiotic activity[J].J Surg Res,2013,184(1):488-494.
[9]Shiow-Fern N,Hon-Lunn L.Development of biofilmtargeted antimicrobial wound dressing for the treatment of chronic wound infections[J].Drug Develop Indust Pharm,2015,41(11):1902-1929.
[10]Mottola C,Matias C S,Mendes J J,et al.Susceptibility patterns of Staphylococcus aureus,biofilms in diabetic foot infections[J].Bmc Microbiol,2016,16(1):1-9.
[11]Sadowski S M,Neychev V,Cottledelisle C,et al.Detection of insulinoma using 68 Gallium-DOTATATE PET/CT:Acase report[J].Gland Surg,2014,4(3):E1-5.
[12]Gourni E,Mansi R,Jamous M,et al.N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists:examples of 68Ga-and 64Culabeled peptides for PET imaging[J].J Nucl Med Off Pub Soc Nucl Med,2014,55(10):1719-1725.
[13]Runci F,Bonchi C,Frangipani E,et al.Acinetobacter baumannii biofilm formation in human serum and disruption by gallium[J].Antimicrob Agents Chemother,2017,61(1):01563-16.
[14]葛新,王辉,董小青.硝酸镓对尿路致病性大肠埃希菌及其生物膜的抑制作用[J].中国抗生素杂志,2013,38(2):136-139.
[15]Zhang D,Xia J,Xu Y,et al.Biological features of biofilmforming ability of Acinetobacter baumannii,strains derived from 121 elderly patients with hospital-acquired pneumonia[J].Clin Exper Med,2016,16(1):73-80.
[16]Clinical and Laboratory Standards Institute.Performance standards for antimicrobial susceptibility testing[S].Twentysixth informational supplement,2016,M100S,26th Edition.
[17]Dzul S P,Thornton M M,Hohne D N,et al.Contribution of the Klebsiella pneumoniae capsule to bacterial aggregate and biofilm microstructures[J].Appl Environ Microbiol,2011,77(5):1777-17782.
[18]王玎,李兴禄.肺炎克雷伯菌生物被膜的体外模型建立[J].中国微生态学杂志,2009,21(2):135-136.
[19]Hallstoodley L,Stoodley P.Evolving concepts in biofilm infections[J].Cellular Microbiol,2009,11(7):1034.
[20]Kaneko Y,Thoendel M,Olakanmi O,et al.The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity[J].J Clin Invest,2007,117(4):877-888.
[21]Olakanmi O,Kesavalu B,Pasula R,et al.Gallium nitrate is efficacious in murine models of tuberculosis and inhibits key bacterial Fe-dependent enzymes[J].Antimicrob Agents Chemother,2013,57(12):6074-6080.
[22]Baldoni D,Steinhuber A,Zimmerli W,et al.In vitro activity of gallium maltolate against Staphylococci in logarithmic,stationary,and biofilm growth phases:comparison of conventional and calorimetric susceptibility testing methods[J].Antimicrob Agents Chemother,2010,54(1):157-163.
[23]Chitambar C R.Medical applications and toxicities of gallium compounds[J].Int J Environ Res Public Health,2010,7(5):2337-2361.