Abstract：
Pseudomonas aeruginosa is one of the most important nosocomial pathogens that possess the ability to produce multiple antibiotic resistance and virulence factors. Elastase B (LasB) is the major factor implicated in tissue invasion and damage during P. aeruginosa infections, whose synthesis is regulated by the quorum sensing (QS) system. Anti-virulence approach is now considered as potential therapeutic alternative and/or adjuvant to current antibiotics\' failure. The aim of this study is primarily to find out the impact of the efflux pump inhibitor (EPI) phenylalanine arginyl β-naphthylamide (PAβN) on the production of elastase B and the gene expression of lasI quorum sensing and lasB virulence factor in clinical isolates of P. aeruginosa. Five P. aeruginosa isolates recovered from patients with respiratory tract infections were examined in this study. Antimicrobial susceptibility of isolates was performed by the disk agar diffusion method. Effect of the PAβN on imipenem susceptibility, bacterial viability, and elastase production was evaluated. The expression of lasB and lasI genes was measured by quantitative real-time PCR in the presence of PAβN. All isolates were identified as multidrug-resistant (MDR) and showed resistance to carbapenem (MIC = 64-256 µg/mL). Susceptibility of isolates to imipenem was highly increased in the presence of efflux inhibitor. PAβN significantly reduced elastase activity in three isolates tested without affecting bacterial growth. In addition, the relative expression of both lasB and lasI genes was diminished in all isolates in the presence of inhibitor. Efflux inhibition by using the EPI PAβN could be a potential target for controlling the P. aeruginosa virulence and pathogenesis. Furthermore, impairment of drug efflux by PAβN indicates its capability to be used as antimicrobial adjuvant that can decrease the resistance and lower the effective doses of current drugs.
摘要：
铜绿假单胞菌是重要的医院病原菌之一,具有产生多种耐药性和毒力因子的能力。弹性蛋白酶B(LasB)是铜绿假单胞菌感染过程中组织侵袭和损伤的主要因素,其合成由群体感应(QS)系统调节。抗毒力方法现在被认为是当前抗生素失败的潜在治疗替代和/或辅助方法。本研究的目的主要是找出外排泵抑制剂（EPI）苯丙氨酸精氨酰β-萘酰胺（PAβN）对弹性蛋白酶B的产生以及lasI群体感应和lasB毒力因子的基因表达的影响。临床分离株铜绿假单胞菌。在这项研究中检查了从呼吸道感染患者中回收的五种铜绿假单胞菌分离物。分离株的抗菌敏感性通过圆盘琼脂扩散法进行。PAβN对亚胺培南敏感性的影响,细菌活力，并对弹性蛋白酶产量进行了评价。在PAβN存在下,通过定量实时PCR测量lasB和lasI基因的表达。所有分离株均被鉴定为多药耐药（MDR），并对碳青霉烯具有耐药性（MIC=64-256µg/mL）。在存在外排抑制剂的情况下，分离株对亚胺培南的敏感性大大增加。PAβN显着降低了测试的三种分离物中的弹性蛋白酶活性，而不影响细菌生长。此外,在存在抑制剂的情况下,所有分离株的lasB和lasI基因的相对表达均降低.通过使用EPIPPAβN的外排抑制可能是控制铜绿假单胞菌毒力和发病机理的潜在靶标。此外，PAβN对药物外排的损害表明其能够用作抗微生物佐剂，可以降低耐药性并降低当前药物的有效剂量。
