铜绿假单胞菌中T3SS毒力基因分布、表达及与抗菌药物耐药相关性研究
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摘要
目的探究临床分离铜绿假单胞菌中T3SS毒力基因分布、表达及与耐药的相关性。方法收集2015年温州医科大学附属第一医院分离的铜绿假单胞菌共68株。琼脂稀释法检测抗菌药物的敏感性; PCR法检测不同来源菌株exo U、exo S、exo T、exo Y 4种毒力基因的分布,并对结果进行统计学分析;实时荧光定量PCR检测部分菌株中转录调控相关基因ptrA、exs A以及效应蛋白基因exo T、exo S的表达情况,并对4种基因的相对表达量进行Pearson相关性分析。结果 68株铜绿假单胞菌中,exo T和exo Y检出率较高,分别为79.4%和75.0%,其中exo T在创面来源菌株中的检出率高于痰液(97.0%vs 61.8%,P<0.01),且以exo U-/exo S+基因型最常见,占51.5%(35/68)。痰液来源的菌株对亚胺培南、美罗培南的耐药率显著高于创面来源菌株(47.1%vs 8.8%,47.1%vs 14.7%,P<0.01);携带exo U基因的菌株对碳青霉烯类、氨基糖苷类和氟喹诺酮类药物耐药率均高于其他3种基因型菌株。Pearson相关性分析显示ptrA基因表达量与exo T、exo S、exs A基因表达水平均呈负相关(P<0.05)。结论本院临床分离的铜绿假单胞菌T3SS毒力基因exo T和exo Y的携带率较高,毒力基因与铜绿假单胞菌耐药性相关,ptrA可能是T3SS毒力基因表达的潜在负调控基因。
Objective To investigate the distribution and expression of T3 SS virulence genes in clinically isolated Pseudomonas aeruginosa( P. aeruginosa) strains and its correlation with drug resistance. Methods A total of 68 P. aeruginosa isolates were collected from the First Affiliated Hospital of Wenzhou Medical University in 2015. The antimicrobial susceptibility was detected by the agar dilution method. The distribution of virulence genes such as exo U,exo S,exo T and exo Y from different isolates was detected by PCR. The expression levels of transcriptional regulator genes( ptrA and exs A) and effector-related genes( exo T and exo S) in some isolates were determined by real-time fluorescence quantitative PCR,and the Pearson correlation analysis was used to analyze the results. Results The detection rates of exo T and exo Y in 68 P. aeruginosa isolates were higher,accounting for 79.4% and 75.0%,respectively. The detection rate of exo T in wound-sourced isolates was significantly higher than that in sputum( 97.0% vs 61.8%,P<0.01). In addition,the genotype of exo U-/exo S+was the most common,accounting for 51.5%( 35/68). The resistance rates of sputum-sourced isolates to imipenem and meropenem were significantly higher than that of wound-sourced isolates( 47.1% vs 8.8%,47.1% vs 14.7%,P<0.01).The resistance rates of isolates carrying exo U gene to carbapenems,aminoglycosides and fluoroquinolones were higher than those of isolates carrying exo S,exo T or exo Y genes. Pearson correlation analysis showed that the expression level of ptrA gene was negatively correlated with those of exo T,exo S and exs A genes( P<0.05). Conclusion The P. aeruginosa isolates from our hospital carrying T3 SS virulence genes exo T and exo Y are common,and the virulence genes are related to the drug resistance of P. aeruginosa. In addition,ptrA may be a potential negative regulatory gene for the expression of T3 SS virulence genes.
Objective To investigate the distribution and expression of T3 SS virulence genes in clinically isolated Pseudomonas aeruginosa( P. aeruginosa) strains and its correlation with drug resistance. Methods A total of 68 P. aeruginosa isolates were collected from the First Affiliated Hospital of Wenzhou Medical University in 2015. The antimicrobial susceptibility was detected by the agar dilution method. The distribution of virulence genes such as exo U,exo S,exo T and exo Y from different isolates was detected by PCR. The expression levels of transcriptional regulator genes( ptrA and exs A) and effector-related genes( exo T and exo S) in some isolates were determined by real-time fluorescence quantitative PCR,and the Pearson correlation analysis was used to analyze the results. Results The detection rates of exo T and exo Y in 68 P. aeruginosa isolates were higher,accounting for 79.4% and 75.0%,respectively. The detection rate of exo T in wound-sourced isolates was significantly higher than that in sputum( 97.0% vs 61.8%,P<0.01). In addition,the genotype of exo U-/exo S+was the most common,accounting for 51.5%( 35/68). The resistance rates of sputum-sourced isolates to imipenem and meropenem were significantly higher than that of wound-sourced isolates( 47.1% vs 8.8%,47.1% vs 14.7%,P<0.01).The resistance rates of isolates carrying exo U gene to carbapenems,aminoglycosides and fluoroquinolones were higher than those of isolates carrying exo S,exo T or exo Y genes. Pearson correlation analysis showed that the expression level of ptrA gene was negatively correlated with those of exo T,exo S and exs A genes( P<0.05). Conclusion The P. aeruginosa isolates from our hospital carrying T3 SS virulence genes exo T and exo Y are common,and the virulence genes are related to the drug resistance of P. aeruginosa. In addition,ptrA may be a potential negative regulatory gene for the expression of T3 SS virulence genes.
引文
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[12]蒋月婷,欧阳浩新,吴爱武,等.多重耐药铜绿假单胞菌毒力基因与耐药的相关性[J].热带医学杂志,2016,16(5):604-607.
[13]鞠晓红,李瑶,王月华,等.铜绿假单胞菌毒力基因exo S、exo U临床分布及耐药性研究[J].中国医院药学杂志,2017,37(1):48-51.
[14]阳志勇.耐碳青霉烯类铜绿假单胞菌的药物敏感性与耐药机制研究[D].湖南:南华大学,2010:1-41.
[15]李昕,刘周,姚杰,等.合肥地区铜绿假单胞菌Ⅲ型分泌系统毒力基因检测分析[J].中华医院感染学杂志,2017,27(2):251-254.
[16]Agnello M,Wong-Beringer A.Differentiation in quinolone resistance by virulence genotype in Pseudomonas aeruginosa[J]. PLoS One,2012,7(8):e42973.
[17]Shen EP,Hsieh YT,Chu HS,et al. Correlation of Pseudomonas aeruginosa genotype with antibiotic susceptibility and clinical features of induced central keratitis[J]. Invest Ophthalmol Vis Sci,2014,56(1):365-371.
[18]董晨晓,宋诗铎,王悦,等. 43株临床铜绿假单胞菌exo S、exo U基因的携带及其耐药性[J].中国感染控制杂志,2010,9(2):93-96.
[19]类承斌,晁艳,孙相红,等.铜绿假单胞菌毒力基因检测及临床意义[J].临床检验杂志,2011,29(4):301-303.
[2]钱远宇.多重耐药铜绿假单胞菌感染的临床治疗[J].临床药物治疗杂志,2010,8(3):25-28.
[3]Xu X,Yu H,Zhang D,et al. Role of ppGpp in Pseudomonas aeruginosa acute pulmonary infection and virulence regulation[J].Microbiol Res,2016,192:84-95.
[4]Vareechon C,Zmina SE,Karmakar M,et al. Pseudomonas aeruginosa effector ExoS inhibits ROS production in human neutrophils[J].Cell Host Microbe,2017,21(5):611-618.
[5]Ha UH,Kim J,Badrane H,et al. An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the typeⅢsecretion system[J]. Mol Microbiol,2004,54(2):307-320.
[6]Anantharajah A,Mingeot-Leclercq MP,Van Bambeke F. Targeting the type three secretion system in Pseudomonas aeruginosa[J].Trends Pharmacol Sci,2016,37(9):734-749.
[7]Jabalameli F,Mirsalehian A,Khoramian B,et al. Evaluation of biofilm production and characterization of genes encoding typeⅢsecretion system among Pseudomonas aeruginosa isolated from burn patients[J]. Burns,2012,38(8):1192-1197.
[8]Clinical and Laboratory Standards Institute(CLSI).Performance standards for antimicrobial susceptibility testing; Twenty-sixth informational supplement.CLSI document M100-S26E[S].Wayne PA:CLSI,2016.
[9]Wu YT,Tam C,Zhu LS,et al. Human tear fluid reduces culturability of contact lens-associated Pseudomonas aeruginosa biofilms but induces expression of the virulence-associated typeⅢsecretion system[J]. Ocul Surf,2017,15(1):88-96.
[10]Finck-Barbancon V,Goranson J,Zhu L,et al. ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury[J]. Mol Microbiol,1997,25(3):547-557.
[11]Soong G,Parker D,Magargee M,et al. The typeⅢtoxins of Pseudomonas aeruginosa disrupt epithelial barrier function[J]. J Bacteriol,2008,190(8):2814-2821.
[12]蒋月婷,欧阳浩新,吴爱武,等.多重耐药铜绿假单胞菌毒力基因与耐药的相关性[J].热带医学杂志,2016,16(5):604-607.
[13]鞠晓红,李瑶,王月华,等.铜绿假单胞菌毒力基因exo S、exo U临床分布及耐药性研究[J].中国医院药学杂志,2017,37(1):48-51.
[14]阳志勇.耐碳青霉烯类铜绿假单胞菌的药物敏感性与耐药机制研究[D].湖南:南华大学,2010:1-41.
[15]李昕,刘周,姚杰,等.合肥地区铜绿假单胞菌Ⅲ型分泌系统毒力基因检测分析[J].中华医院感染学杂志,2017,27(2):251-254.
[16]Agnello M,Wong-Beringer A.Differentiation in quinolone resistance by virulence genotype in Pseudomonas aeruginosa[J]. PLoS One,2012,7(8):e42973.
[17]Shen EP,Hsieh YT,Chu HS,et al. Correlation of Pseudomonas aeruginosa genotype with antibiotic susceptibility and clinical features of induced central keratitis[J]. Invest Ophthalmol Vis Sci,2014,56(1):365-371.
[18]董晨晓,宋诗铎,王悦,等. 43株临床铜绿假单胞菌exo S、exo U基因的携带及其耐药性[J].中国感染控制杂志,2010,9(2):93-96.
[19]类承斌,晁艳,孙相红,等.铜绿假单胞菌毒力基因检测及临床意义[J].临床检验杂志,2011,29(4):301-303.