Degradation of AHLs by isolated strains.

<p>Residual C4-HSL (A) and C6-HSL (B) were detected by <i>C. violaceum</i> CV026. Decreased or loss of violacein indicated AHL inactivation after incubation for 24 h. <i>B. cereus</i> known for carrying a lactonase was used as positive control. <i>E. coli</i> DH5α and PBS served as negative controls.</p

Mass spectrometry identification of different quorum sensing signaling molecules from bacterial isolates spent supernatants.

<p>Mass spectrometry identification of different quorum sensing signaling molecules from bacterial isolates spent supernatants.</p

Summary of phenotypic properties of isolated bacterial strains from infected tilapia.

<p>N.D^a Not Detectable.</p><p>+ Positive result.</p><p>− Negative result.</p

Phylogenetic analysis of the 16S rDNA sequences of isolated bacterial strains.

<p>The tree is based on maximum-likelihood (Tamura-Nei model) analysis. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points. Solid triangles indicate the positions of isolated bacteria in phylogenetic analysis. Bar represents evolutionary distances as 0.05 changes per nucleotide position. W4.2: <i>Klebsiella</i> sp.; W2.3: <i>Serratia</i> sp.; W3 and W3.1: <i>Pseudomonas</i> sp.; and W2.2: <i>Bacillus</i> sp.</p

Extracellular enzyme activity of isolated strains.

<p>The overnight cultures of various strains were spotted on (A) 5% v/v sheep blood agar for haemolytic activity and (B) 1.5% v/v skim milk agar for proteolytic activity. A transparent halo zone around the colony was considered to indicate enzyme production. W2.6 showed high similarity with W2.3 in their 16S rDNA sequences (data not shown), hence only isolate W2.3 is selected for further analysis.</p

Screening of AHL production using <i>Chromobacterium violaceum</i> CV026 as biosensor.

<p>The test strains were cross-streaked against CV026. The positive control (+ve) strain is <i>E. carotovora</i> GS101 and the negative control (−ve) strain is <i>E. carotovora</i> PNP22 (AHL synthase deficient mutant). Only W3.1 isolate showed positive response.</p

Thin layer chromatography analysis of AHLs.

<p>AHL standards from Sigma (C12-HSL (**) and 3-oxo-C16-HSL (*)) and 10 µl of W3.1 and W2.3 extract were spotted on normal phase TLC plate. The TLC plate was chromatographed with dichloromethane and methanol as mobile phases. The locations of AHLs were revealed by overlay of <i>Agrobacterium tumefaciens</i> NTL4 pZLR4 as evident by blue pigmentation. Based on the R<i>_f</i> values, W2.3 produced C12-HSL.</p