Bacteria strain

XN9, an AHPND-causing isolate, was kindly provided by Nha Trang University 14. It was streaked from glycerol stock on Thiosulfate-citrate-bile salts-sucrose agar (TCBS, Himedia, India). One colony was picked up for overnight culture in BHI medium at optimal culture condition (pH 8.5, 2.5% NaCl, 30C, and static condition) described previously (14). For assessing the effect of culture condition on the activity of extracellular enzymes, pH, temperature, and oxygen level were adjusted around the optimal culture condition (pH 8.5, 2.5% NaCl, 30C, and static condition). So, the testing conditions were 7.5, 8.0, 8.5 and 9.0 for pH; 25C, 30C and 35C for temperature, and 120 rpm shaking or static condition with the presence of Oxygen absorber package (O-Buster, Hsiao Sung, Non-Oxygen Chemical Co. Ltd., China) adhered to the bottom side of the falcon cap for oxygen level.

Extracellular enzymatic testing

Egg-yolk agar containing 1mL of Egg Yolk Emulsion (Himedia), Tributyrin Agar (Himedia) with added Tributyrin (Himedia) (10mL/L), BHI agar plates containing 1.5% (w/v) skim milk, 8% gelatin and 2% (w/v) colloidal chitin was used for lecithinase, lipase, caseinase, gelatinase, and chitinase, respectively. The colloidal chitin was prepared, as previously described 15. The overnight culture of V. parahaemolyticus was adjusted to OD600nm of 0.08- 0.1, then 10 µL of this bacterial suspension was dropped onto agar plates corresponding to the tested enzymes mentioned above. In the case of assessing the effect of oxygen, the overnight culture was dropped on the plate either without or with AnaeroPack® (Mitsubishi Gas Chemical, Japan) and plastic wrap. Positive controls used in these tests included ATCC29213 for caseinase and lipase, for lecithinase and gelatinase and for chitinase. After inoculation, the plates were incubated 24 hours for caseinase, gelatinase, and lipase and 48 hours for lecithinase and chitinase. For gelatinase, before reading the result, the agar plate was flooded with saturated ammonium sulfate ((NH)SO) to precipitate the undegraded gelatin. Clear halos surrounding the bacterial drop indicated the activity of the tested enzymes 16, 17, 18. All the tests were triplicated.

Data analysis

Enzyme activity (EA) was calculated using the formula: where D is the diameter of the bacterial drop plus the clear halo zone (mm), and dis the diameter of the bacterial drop itself (mm). It is graded (-) if there was no visible hydrolytic area; (+) if the EA value is less than 2 mm and (++) if equal or higher than 2 mm (15). Each test was triplicated, and the obtained data were analyzed using two-way ANOVA (Excel software, Microsoft 7) 14.

Effects of pH on extracellular enzymatic activities of

Following the increase of pH from 7.5 to 9.0, caseinase activity decreased significantly by nearly 88% from 3.55 ± 0.25 (++) to 0.41 ± 0.08 mm (+) and lipase activity decreased by roughly 44%, from 1.83 ± 0.29 (+) to 1.21 ± 0.25 mm (+). In contrast, there was a significant increase of gelatinase activity from an undetectable level at pH 7.5 to 1.38 ± 0.17 mm (+) at pH 9.0. On the other hand, lecithinase activity of was recorded as strong (++), in all tested pHs with the highest value obtained at pH 8.5 (2.96 ± 0.13 mm). Chitinase activity was not observed in any tested pHs (Figure 1, Table 1). In the increasing pH from 7.5 to 9.0, exhibited significant differences in enzymatic activities between four pH levels (p-value < 0.05, Supplementary Table 2 A).

Effects of temperature on extracellular enzymatic activities of

The rise of temperature from 25C to 35C led to strong decomposition of gelatin in the BHI medium with EA value increased by 56% from 0.86 ± 0.14 (+) to 1.37 ± 0.25 mm (+). It also resulted in slight increase of caseinase from 0.67 ± 0.10 (+) to 0.85 ± 0.13 mm (+). At 30C, the obtained EA value was highest for lecithinase (2.96 ± 0.13 mm (++)) but lowest for lipase (1.4 ± 0.1 mm (+)). Chitinase activity was again not observed in any tested temperatures (Figure 2, Table 1). Temperature significantly affected the extracellular enzymatic activities of (p-value< 0.05, Supplementary Table 2 B).

Investigating the effects of oxygen on extracellular enzymatic activities of

Under the limited oxygen presence, most of the tested enzyme activities were low or even not observed. Caseinase activity declined considerably from 1.10 ± 0.31 mm (+) in the case of both fully oxygenated culture and testing to 0 mm (-) in case of both limited oxygen culture and testing. Similar trend was seen in case of lipase and gelatinase, with enzyme activity decreased markedly about 49% from 2.33± 0.23 mm (++) to 1.17± 0.20 mm (+) and 25% from 1.59 ± 0.11 (+) to 1.18 ± 0.13 mm (+) respectively. In case of lecithinase, this enzyme activity expressed in most tested conditions with lowest activity (1.62 ± 0.29 mm (+)) in case of both limited oxygen culture and testing and highest activity (2.92 ± 0.08 mm (++)) in case of oxygenated overnight culture followed by limited oxygen testing condition. No activity of chitinase was observed in any case (Figure 3, Table 1). Under four tested oxygen conditions, the activity of tested extracellular enzymes was different significantly (p-value< 0.05, Table 2 C).