Abstract
Quorum sensing (QS) has been recognized as a general phenomenon in microorganisms and plays an important role in many pathogenic bacteria. In this report, we used the Agrobacterium tumefaciens biosensor strain NT1 to rapidly screen for autoinducer-quenching inhibitors from bacteria. After initial screening 5389 isolates obtained from land and beach soil, 53 putative positive strains were identified. A confirmatory bioassay was carried out after concentrating the putative positive culture supernatant, and 22 strains were confirmed to have anti-LasR activity. Finally, we determined the strain JM2, which could completely inhibit biofilm formation of Pseudomonas aeruginosa PAO1, belonged to the genus Pseudomonas by analysis of 16S rDNA. Partially purified inhibitor factor(s) F5 derived from culture supernatants specifically inhibited LasR-controlled elastase and protease in wild type P. aeruginosa PAO1 by 68% and 73%, respectively, without significantly affecting growth; the rhl-controlled pyocyanin and rhamnolipids were inhibited by 54% and 52% in the presence of 100 μg/mL of F5. The swarming motility and biofilm of PAO1 were also inhibited by F5. Real time RT-PCR on samples from 100 μg/mL F5-treated P. aeruginosa showed downregulation of autoinducer synthase (LasRI and rhlI) and cognate receptor (lasR and rhlR) genes by 50%, 28%, 48%, and 29%, respectively. These results provide compelling evidence that the F5 inhibitor(s) interferes with the las system and significantly inhibits biofilm formation.
Highlights
Pseudomonas aeruginosa is one of the most difficult pathogens to treat clinically, and infects vulnerable patients including those with postoperative immune suppression
In searching for novel quorum-quenching bacteria from soil samples, we focused on screening the las Quorum sensing (QS) system, and obtained an isolate that strongly inactivated autoinducing activity and reduced the PAO1 biofilm formation
In the initial plate screening after incubation overnight, a blue color in the indicator bacteria NT1 occurred without inhibitors
Summary
Pseudomonas aeruginosa is one of the most difficult pathogens to treat clinically, and infects vulnerable patients including those with postoperative immune suppression. Molecule, N-decanoyl-L-homoserine benzyl ester, was found to down-regulate total protease and elastase activities, as well as the production of rhamnolipid, without affecting bacterial growth. It had synergistic interactions with several antibiotics, including tobramycin, gentamycin, cefepime, and meropenem [14]. A more highly purified preparation (4 μg/mL) from concentrated culture supernatants of this isolate inhibited rhl-controlled pyocyanin and rhamnolipid production in wild type P. aeruginosa PAO1 by 49%, without significantly affecting growth. A description of a novel autoinducer-quenching strain is presented here, including the anti-LasR fragment from culture supernatant extract, and its inhibition of biofilm formation and QS dependent virulence factors. This study emphasizes the potential of JM2 to produce bioactive agents with anti-LasR and anti-biofilm properties that are novel drug candidates
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