Abstract
Introduction: Vibrio parahaemolyticus is a popular Gram-negative bacterium in the marine and estuarine regions. It can cause Early Mortality Syndrome (EMS), now named Acute Hepatopancreatic Necrosis Disease (AHPND), which resulted in severe losses to the shrimp culture. This study aimed to investigate the effect of pH, temperature, and oxygen-limited condition on the extracellular enzymatic activity of V. parahaemolyticus.
 Methods: V. parahaemolyticus XN9, an AHPND-causing strain, was cultured in Brain Heart Infusion (BHI) medium at different pHs (7.5, 8.0, 8.5 and 9.0), temperatures (25oC, 30oC, and 35oC) and different oxygen conditions (either 120rpm shaking or static with the presence of oxygen absorber packages). The activity of five extracellular enzymes, including caseinase, lecithinase, chitinase, gelatinase, and lipase, was assessed using the agar-based method with the corresponding media.
 Results: When pH was increased from 7.5 to 9.0, caseinase and lipase activity was decreased significantly by 88% and 44%. In contrast, gelatinase activity increased markedly from 0 to 1.38 ± 0.17 (+) mm, and lecithinase reached the highest activity, which was 2.96 ± 0.13 mm (++) at pH 8.5. Regarding effect of temperature, highest activity of caseinase (0.85 ± 0.13 mm (+)) and gelatinase (1.37 ± 0.25 mm (+)) was obtained at 350C, lecithinase at 30oC and lipase at 25oC. Regarding the effect of oxygen level, the activity of most tested enzymes decreased significantly following the decrease of oxygen level. The highest activity of caseinase, gelatinase, and lipase was observed when the bacteria were cultured and tested in a fully oxygenated condition while lecithinase showed the highest activity when the bacteria were cultured in oxygenated condition but tested in oxygen-limited condition. No chitinase activity was observed in any of the tested conditions.
 Conclusion: Our data suggested that extracellular enzymatic activity of V. parahaemolyticus is significantly influenced by environmental conditions. No particular testing condition resulted in the highest activity for all tested enzymes. However, warm temperature (30/ 35oC), mildly alkaline pH (pH 8.0), and fully oxygenated condition could increase the overall extracellular enzymatic activity of V. parahaemolyticus, thus increase its potential virulence.
Highlights
Introduction: Vibrio parahaemolyticus is a popular Gram-negative bacterium in the marine and estuarine regions. It can cause Early Mortality Syndrome (EMS), named Acute Hepatopancreatic Necrosis Disease (AHPND), which resulted in severe losses to the shrimp culture
This study aimed to investigate the effect of pH, temperature, and oxygen-limited condition on the extracellular enzymatic activity of V. parahaemolyticus
AHPND pathogenesis is mainly caused by a binary toxin PirA/B encoded on a plasmid in V. parahaemolyticus 10
Summary
Vibrio parahaemolyticus is a popular Gram-negative bacterium in the marine and estuarine regions It can cause Early Mortality Syndrome (EMS), named Acute Hepatopancreatic Necrosis Disease (AHPND), which resulted in severe losses to the shrimp culture. This study aimed to investigate the effect of pH, temperature, and oxygen-limited condition on the extracellular enzymatic activity of V. parahaemolyticus. Vibrio parahaemolyticus is a halophilic Gramnegative bacterium It lives ubiquitously as a free-living organism in the marine environment or a colonizer of many different kinds of marine organisms 1. The activity of five extracellular enzymes, including caseinase, lecithinase, gelatinase, lipase, and chitinase, was examined under the different pHs, Cite this article : Tran V N, Nguyen P N V, Nguyen T T H.
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