Tingpej Pholawat, Smith Lucas, Rose Barbara, Zhu Hua, Conibear Tim, Al Nassafi Khaled, Manos Jim, Elkins Mark, Bye Peter, Willcox Mark, Bell Scott, Wainwright Claire, Harbour Colin
Department of Infectious Diseases and Immunology, Blackburn Building DO6, University of Sydney, 2006 NSW, Australia.
J Clin Microbiol. 2007 Jun;45(6):1697-704. doi: 10.1128/JCM.02364-06. Epub 2007 Mar 28.
The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P = 0.02), to show elastase and alkaline protease activity by zymography (P = 0.04 and P = 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P = 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P = 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.
强毒力铜绿假单胞菌克隆株的出现对全球囊性纤维化（CF）患者构成威胁。对铜绿假单胞菌克隆菌株进行特征分析对于了解其临床影响以及制定应对这一问题的策略至关重要。两种克隆菌株（AES-1和AES-2）在澳大利亚东部的CF中心内传播。在本研究中，比较了43株铜绿假单胞菌分离株（14株AES-1、5株AES-2和24株非克隆株）的表型特征，以深入了解克隆菌株的特性。所有43株分离株在vfr、rhlI、rhlR、lasA、lasB、aprA、rhlAB和exoS基因的PCR中均产生了预测大小的条带；42株lasI和lasR呈阳性，无一株有exoU。37株（86%）分离株在总蛋白酶检测中呈阳性；在酶谱分析中，24株（56%）产生弹性蛋白酶/葡萄球菌溶血素，22株（51%）产生碱性蛋白酶。克隆分离株比非克隆分离株更有可能在总蛋白酶检测中呈阳性（P = 0.02），在酶谱分析中表现出弹性蛋白酶和碱性蛋白酶活性（分别为P = 0.04和P = 0.01），并在弹性蛋白-刚果红试验中表现出弹性蛋白酶活性（P = 0.04）。与基因型无其他关联。总体而言，患者年龄增加与弹性蛋白酶活性降低相关（P = 0.03）。32株（74%）分离株通过薄层色谱法检测到至少一种N-酰基高丝氨酸内酯（AHL）。与rhl相关的AHL检测与总蛋白酶和弹性蛋白酶活性的产生及水平相关（所有P < 0.01）。33株（77%）分离株通过蛋白质印迹分析ExoS呈阳性，35株（81%）产生鼠李糖脂，34株（79%）表现出几丁质酶活性。研究结果表明，慢性感染期间的蛋白酶活性可能有助于这些克隆菌株的传播性或毒力。
