The systematic understanding of how various antimicrobial agents are involved in controlling biofilms is essential in order to establish an effective strategy for biofilm control, since many antimicrobial agents are effective against planktonic cells but are ineffective when they are used against the same bacteria growing in a biofilm state. Three different antimicrobial agents (chlorine, silver, and tobramycin) and three different methods for the measurement of membrane integrity (plate counts, the measurement of respiratory activity with 5-cyano-2,3-ditolyl tetrazolium chloride [CTC] staining, and BacLight Live/Dead staining) were used along with confocal laser scanning microscopy (CLSM) and epifluorescence microscopy to examine the activities of the antimicrobials on biofilms in a comparative way. The three methods of determining the activities of the antimicrobials gave very different results for each antimicrobial agent. Among the three antimicrobials, tobramycin appeared to be the most effective in reducing the respiratory activity of biofilm cells, based upon CTC staining. In contrast, tobramycin-treated biofilm cells maintained their membrane integrity better than chlorine-or silver-treated ones, as evidenced by imaging by both CLSM and epifluorescence microscopy. Combined and sequential treatments with silver and tobramycin showed an enhanced antimicrobial efficiency of more than 200%, while the antimicrobial activity of either chlorine or tobramycin was antagonized when the agents were used in combination. This observation makes sense when the different oxidative reactivities of chlorine, silver, and tobramycin are considered.
The biofilm formation of Pseudomonas aeruginosa, an opportunistic human pathogen, is developed by cell-to-cell signaling, so-called quorum sensing (QS). To control the biofilm formation, we designed and synthesized new QS inhibitors of P. aeruginosa based on the structure of the previously known QS inhibitor, furanone. Newly synthesized compounds were a series of analogs of (5-oxo-2,5-dihydrofuran-3-yl)methyl alkanoate, and the structures of all six synthesized compounds was confirmed by NMR and GC/MS analyses. These new QS inhibitor candidates could remarkably inhibit both Pseudomonas QS signaling and biofilm formation, which were assayed by using the recombinant reporter system and flow cell confocal microscopy. The degree of QS inhibition by these new inhibitors varied from 20% to 90%. For the profound understanding about inhibition mechanism, we tried to estimate the binding energy between QS receptor, LasR, and our inhibitors from the in silico modeling system. The predicted binding pattern from the modeling system and our experimental data about QS inhibition were in good agreement. From these results, we suggest a new approach to develop the QS inhibitors and biofilm control agents based on structural modeling.
Chlorine was more effective in inactivation of metabolically inactive dormant cells and also more effective under anaerobic conditions. The high oxidative reactivity and rapid decay of chlorine might influence the different antimicrobial actions of chlorine compared with antibiotics. This study contributes to understanding the effects of dormancy and the presence of oxygen on the susceptibility of P. aeruginosa biofilm to a wide range of antimicrobial agents.
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