Involvement of Acylated Homoserine Lactones (AHLs) of Aeromonas sobria in Spoilage of Refrigerated Turbot (Scophthalmus maximus L.)

Abstract

One quorum sensing strain was isolated from spoiled turbot. The species was determined by 16S rRNA gene analysis and classical tests, named Aeromonas sobria AS7. Quorum-sensing (QS) signals (N-acyl homoserine lactones (AHLs)) were detected by report strains and their structures were further determined by GC-MS. The activity changes of AHLs on strain growth stage as well as the influence of different culture conditions on secretion activity of AHLs were studied by the punch method. The result indicated that strain AS7 could induce report strains to produce typical phenotypic response. N-butanoyl-dl-homoserine lactone (C₄-HSL), N-hexanoyl-dl-homoserine lactone (C₆-HSL), N-octanoyl-dl-homoserine lactone (C₈-HSL), N-decanoyl-dl-homoserine lactone (C10-HSL), N-dodecanoyl-dl-homoserine lactone (C12-HSL) could be detected. The activities of AHLs were density-dependent and the max secretion level was at pH 8, sucrose culture, 1% NaCl and 32 h, respectively. The production of siderophore in strain AS7 was regulated by exogenous C₈-HSL, rather than C₆-HSL. Exogenous C₄-HSL and C₈-HSL accelerated the growth rate and population density of AS7 in turbot samples under refrigerated storage. However, according to the total viable counts and total volatile basic nitrogen (TVB-N) values of the fish samples, exogenous C₆-HSL did not cause spoilage of the turbot fillets. In conclusion, our results suggested that QS was involved in the spoilage of refrigerated turbot.

Keywords: Aeromonas sobria; quorum sensing; siderophore; spoilage; turbot.

Figure 1

The phylogenetic relationships of strain AS7 and other Aeromona.

Figure 2

Streak assays for the production of short chain (N-acyl homoserine lactones (AHLs)) in test strain (screening for AHLs production using C. violaceum CV026 and A. tumefaciens A136 cross parallel streaking with C₄–HSL (C₄-homoserine lactones) and C₆–HSL as positive control, respectively. Negative controls were the monitor strains themselves.).

Figure 3

Relationship between growth kinetics and short chain AHL secretion under different culture time of strain AS7 and effect of different conditions on short chain AHLs production by AS7 (a): Agar well diffusion assay to detect AHLs secretion under different culture time; (b): the graph of relationship between kinetics and AHLs; (c,d): NaCl concentration 0: blank control, 1: NaCl 0.5%, 2: NaCl 0.7%, 3: NaCl 1%, 4: NaCl 2%, 5: NaCl 3%, 6: NaCl 4%; (e,f): carbon source 0: blank control, 1: glucose, 2: sucrose, 3: frutose, 4: xylose, 5: lactose, 6: maltose; (g,h): 0: blank control, 1: pH 5, 2: pH 6, 3: pH 7, 4: pH 8, 5: pH 9.

Figure 3

Relationship between growth kinetics and short chain AHL secretion under different culture time of strain AS7 and effect of different conditions on short chain AHLs production by AS7 (a): Agar well diffusion assay to detect AHLs secretion under different culture time; (b): the graph of relationship between kinetics and AHLs; (c,d): NaCl concentration 0: blank control, 1: NaCl 0.5%, 2: NaCl 0.7%, 3: NaCl 1%, 4: NaCl 2%, 5: NaCl 3%, 6: NaCl 4%; (e,f): carbon source 0: blank control, 1: glucose, 2: sucrose, 3: frutose, 4: xylose, 5: lactose, 6: maltose; (g,h): 0: blank control, 1: pH 5, 2: pH 6, 3: pH 7, 4: pH 8, 5: pH 9.

Figure 3

Relationship between growth kinetics and short chain AHL secretion under different culture time of strain AS7 and effect of different conditions on short chain AHLs production by AS7 (a): Agar well diffusion assay to detect AHLs secretion under different culture time; (b): the graph of relationship between kinetics and AHLs; (c,d): NaCl concentration 0: blank control, 1: NaCl 0.5%, 2: NaCl 0.7%, 3: NaCl 1%, 4: NaCl 2%, 5: NaCl 3%, 6: NaCl 4%; (e,f): carbon source 0: blank control, 1: glucose, 2: sucrose, 3: frutose, 4: xylose, 5: lactose, 6: maltose; (g,h): 0: blank control, 1: pH 5, 2: pH 6, 3: pH 7, 4: pH 8, 5: pH 9.

Figure 4

GC-MS chromatogram in SIM mode at m/z 143 of a standard mixture of AHLs (a) and an extract of cell-free supernatant of AS7 (b).

Figure 5

The effects of exogenous autoinducers on siderophore formation in AS7 (c)—(a): (2), 10 μM C₆–HSL; (3), 20 μM C₆–HSL; (4), 40 μM C₆–HSL and (b): (6), 10 μM C₈–HSL; (7), 20 μM C₈–HSL; (8), 40 μM C₈–HSL. (1) and (5) was as the control. Data were presented as the mean ± standard deviation (n = 3; * p < 0.05; ** p < 0.01).

Figure 6

The effects of C₄–HSL (20 μM), C₆–HSL (20 μM) and C₈–HSL (20 μM) on (a) microbial counts; and (b) TVB-N production in turbot blocks stored at 4 °C. Data were presented as the mean ± standard deviation (n = 6; * p < 0.05; ** p < 0.01).