Introduction

In recent years, a challenge in clinical treatment has developed due to bacterial resistance to antibiotics. One of the new mechanisms against infections is virulence factor inhibition. Many virulence factors are controlled by quorum sensing pathways such as biofilm formation and pyocyanin production. The goal of the present study was to investigate the effect of an obligate halophilic bacterial strain on Pseudomonas aeruginosa and Staphylococcus aureus, due to its halo-tolerant substances and enzymes.

Methods

The effect of Halobacillus karajensis on bacterial growth and production of virulence factors was studied in this work. The obligate halophile cells and supernatant fractions were extracted by the methanol/chloroform method and characterized by Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Gas Chromatography–Mass Spectrometry (GC-MS), and zymography. The effects of these fractions were studied on biofilm formation in P. aeruginosa and S. aureus as well as on pyocyanin production in P. aeruginosa. The effective protein in the fraction was analyzed by the SDS-PAGE method, and all protein fragments were studied for pyocyanin inhibition.

Results

The crude supernatant extract, MMS fraction, from H. karajensis was effective for the biofilm reduction in S. aureus (74%) and P. aeruginosa (27%). Two proteases in this fraction, which were recognized by zymography on skim milk, were the probable causes for extracellular polymeric substances (EPS) hydrolysis in the biofilm matrix. Also, halide crystals and branched fatty acids, 12methyl-tetradecanoic acid, in the other fractions decreased the biofilm by 18% in S. aureus. The results showed that a new 25 kD protein, which was obtained from MMS fraction, inhibited pyocyanin production by 60% in P. aeruginosa. The zymogram and bioinformatics studies showed that this protein was a serine alkaline metalloprotease and had an interaction with AHL molecules.

Conclusion

The inhibitory effects of the non-toxic natural substances and proteases on biofilm formation and pyocyanin production, specifically the 25 kD protease, are novel in this study and make them a good candidate for infected wound healing and inhibiting the virulence factors.

中文翻译：

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天然物质和新型蛋白酶对群体感应控制的毒力因子的抑制作用，得自卡拉耶克氏菌

介绍

近年来，由于细菌对抗生素的耐药性，临床治疗中出现了挑战。抵抗感染的新机制之一是抑制毒力因子。许多毒力因子受群体感应途径控制，例如生物膜形成和绿脓素生成。本研究的目的是研究专性的嗜盐细菌菌株对铜绿假单胞菌和金黄色葡萄球菌的耐盐物质和酶的作用。

方法

在这项工作中，研究了哈洛杆菌（Halobacillus karajensis）对细菌生长和毒力因子产生的影响。通过甲醇/氯仿方法提取专性的嗜盐细胞和上清液部分，并通过傅立叶变换红外（FTIR）光谱，X射线衍射（XRD），气相色谱-质谱（GC-MS）和酶谱进行表征。研究了这些馏分对铜绿假单胞菌和金黄色葡萄球菌的生物膜形成以及铜绿假单胞菌的绿蛋白生成的影响。通过SDS-PAGE方法分析级分中的有效蛋白质，并研究了所有蛋白质片段的绿脓素抑制作用。

结果

来自karajensis的粗上清提取物MMS馏分可有效减少金黄色葡萄球菌（74％）和铜绿假单胞菌（27％）的生物膜。该部分中的两种蛋白酶已通过脱脂乳上的酶谱法识别，可能是生物膜基质中细胞外聚合物质（EPS）水解的原因。同样，在其他组分中，卤化物晶体和支链脂肪酸（十二甲基十四烷酸）使金黄色葡萄球菌的生物膜减少了18％。结果表明，从MMS馏分中获得的一种新的25 kD蛋白可抑制铜绿假单胞菌中的绿脓素生成60％。。酶谱图和生物信息学研究表明，该蛋白是一种丝氨酸碱性金属蛋白酶，与AHL分子有相互作用。

结论

无毒天然物质和蛋白酶对生物膜形成和绿脓素生成的抑制作用，特别是25 kD蛋白酶，在本研究中是新颖的，使其成为感染伤口愈合和抑制毒力因子的良好候选者。