Screening Of Marine Microorganisms With Quorum Sensing Inhibitory Activity And Activity Evaluation On Inhibitors

Bacterial resistance refers to the tolerance of pathogens to antimicrobial agents, or bacteria are not sensitive to antibiotics. The prevalence of antibiotic resistant bacteria addresses an important public health concern. Many diseases are caused by microbial pathogens, which can produce virulence factors. Through the underlying mechanisms of bacterial pathogenecity, expression of many virulence factors is regulated by quorum sensing. It has been reported that mutations in quorum sensing have a significant decrease in virulence production. Compared with traditional antibiotics, the potential antipathogenic drugs that specifically target quorum sensing systems do not affect the bacterial growth, but inhibit pathogenic behaviors; it is unlikely to pose a selective pressure for the development of resistant mutants. This strategy can greatly reduce the development of resistant bacteria. The main purpose of this paper is to screen quorum sensing inhibitors from the secondary metabolites in marine microorganisms.All of the cultural extracts from marine microorganisms were screened by PAO MWI (pW-4) and CV026. PAOMWI (pW-4) is based on P. aeruginosa QS systems. The lasB promoter is fused to levansucrase-encoding gene sacB, which lead to cell death in the presence of 3-oxo-C12-HSL and sucrose, but the cells can be rescued in the presence of QSI compounds. CV026 has the ability to respond C6-HSL. A positive QSI result was indicated by the lack of pigmentation of the indicator organism.One actinomycete and two fungal extracts were found to exhibit quorum sensing inhibitory activity. The fragment of 18S rDNA of QY013 was compared with those of reference strains deposited in GenBank (NCBI) databases by a BLAST search. Results showed that the sequences of QY013 exhibited high identity with Penicillium decumbens. It has not been reported that such species of Penicillium have quorum sensing inhibitory activity. From QY013 extract, one active compound with quorum sensing inhibitory activity was isolated. By means of spectroscopic method (1H NMR, 13C NMR), structure of this compound was elucidated as 5, 6-dihydro-4-methyl-2H-pyran-2-ketone. It was the first report that 5, 6-dihydro-4-methyl-2-H-pyran-2-ketone could interfere with the P. aeruginosa and C. violaceum quorum sensing system. This compound could attenuate pathogen virulence factors of P. aeruginosa such as activity of elastase and pyocyanin production, without affecting bacterial growth in the effective range of concentration. It can effectively inhibit biofilm formation of P. aeruginosa. Based on RT-PCR, the compound had a negative effect on the expression of the key QS regulatory genes. It could also reduce violacein production and inhibit QS-controlled genes of C. violaceum.The chemical structure of quorum sensing signal molecules in Gram-negative bacteria has some similarity. They have a similar lactone ring structure; the main difference is the length of fatty acid chain (C) on the lactone ring and its saturation. According to this similarity and difference, 18 compounds were compared with N-acyl homoserine lactones (AHLs) based on the marine natural compounds laboratory (School of Medicine and Pharmacy, Ocean University of China), which were tested according to the screening system mentioned above. Four compounds were found to exhibit quorum sensing inhibitory activity. In 2004, the compound HD-ZWM-242 (penicillic acid) has been identified as being biologically active QSI compounds. The compound HD-ZWM-169 can interfere with P. aeruginosa and C. violaceum quorum sensing system. It can inhibit P. aeruginosa virulence factors production, bacterial swarming motility and downregulate gene expression of quorum sensing.In our study, the quorum sensing inhibitors have different mechanism of action compared with traditional antibiotics. They can only inhibit bacterial quorum sensing regulated pathogenic behavior, but they do not impose harsh selective pressure on bacterial growth, which could significantly reduce the probability of bacterial resistance. Our study has an important theoretical significance for basic research. Meanwhile, it is believed that this work can provide new ideas for combating bacterial resistance and introduce new methods for developing marine active products.