Effects of pH, temperature and oxygen-limited condition on the virulence of Vibrio parahaemolyticus

Science & Technology Development Journal, 23(3):569-575 Open Access Full Text Article Research Article School of Biotechnology, International University, Vietnam National University of HCMC Correspondence Thi Thu Hoai Nguyen, School of Biotechnology, International University, Vietnam National University of HCMC Email: ntthoai@hcmiu.edu.vn History  Received: 2020-04-06  Accepted: 2020-07-12  Published: 2020-07-27 DOI : 10.32508/stdj.v23i3.2057 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Effects of pH, temperature and oxygen-limited condition on the virulence of Vibrio parahaemolyticus Van Nhi Tran, Phuong Nhat Vi Nguyen, Thi Thu Hoai Nguyen* Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Vibrio parahaemolyticus is a popular Gram-negative bacterium in themarine and es- tuarine 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 enzy- matic activity of V. parahaemolyticus. Methods: V. parahaemolyticusXN9, an AHPND-causing strain, was cultured in Brain Heart Infusion (BHI) medium at different pHs (7.5, 8.0, 8.5 and 9.0), tempera- tures (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 li- pase 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 condi- tion 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. para- haemolyticus is significantly influenced by environmental conditions.No particular testing condi- tion 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. Key words: Vibrio parahaemolyticus, Acute Hepatopancreatic Necrosis Disease (AHPND), Early Mortality Syndrome (EMS), extracellular enzymatic activity, oxygen-limited, pH, temperature, virulence INTRODUCTION Vibrio parahaemolyticus is a halophilic Gram- negative bacterium. It lives ubiquitously as a free-living organism in the marine environment or a colonizer of many different kinds of marine organisms1. This motile, curved shaped bacterium, is a well-known causative agent of food-borne acute gastroenteritis in humans due to the consumption of raw or undercooked seafood2–4. V. parahaemolyticus is also known to cause Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPND), which affects penaeid shrimp, causing massive death in larvae and young adults5,6. In recent years, AHPND has brought devastating effects to the shrimp industry of various countries such as China, Vietnam, Malaysia, Philippines, Thailand, and Mexico7–9. AHPND pathogenesis is mainly caused by a binary toxin PirA/B encoded on a plasmid in V. parahaemolyticus 10. However, whether other toxins may also take part in causing this disease is still under investigation. The potential of a pathogen to cause so-called disease virulence reflects its ability to colonize, invade, escape the immune system, and obtain nutrition from the host. An important part of bacterial virulence is the ability to produce and secret extracellular enzymes to break down and digest nutrients from the environment11,12. However, the production of these enzymes is highly influenced by environmental factors such as nutrient supplement, dissolved oxygen, pH, temperature...13. In this study, 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. Effects of pH, temperature andoxygen-limited condition on the virulence of Vibrio parahaemolyticus. Sci. Tech. Dev. J.; 23(3):569-575. 569 Science & Technology Development Journal, 23(3):569-575 temperatures and oxygen levels. MATERIALS- METHODS Bacteria strain Vibrio parahaemolyticus XN9, an AHPND-causing isolate, was kindly provided by Nha Trang Uni- versity14. It was streaked from glycerol stock on Thiosulfate-citrate-bile salts-sucrose agar (TCBS, Hi- media, India). One colony was picked up for overnight culture in BHI medium at optimal culture condition (pH 8.5, 2.5%NaCl, 30oC, and static condi- tion) described previously (14). For assessing the ef- fect of culture condition on the activity of extracellular enzymes, pH, temperature, and oxygen level were ad- justed around the optimal culture condition (pH 8.5, 2.5% NaCl, 30oC, and static condition). So, the test- ing conditions were 7.5, 8.0, 8.5 and 9.0 for pH; 25oC, 30oC and 35oC for temperature, and 120 rpm shaking or static condition with the presence of Oxygen ab- sorber 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 con- taining 1.5% (w/v) skim milk, 8% gelatin and 2% (w/v) colloidal chitin was used for lecithinase, li- pase, caseinase, gelatinase, and chitinase, respectively. The colloidal chitin was prepared, as previously de- scribed15. The overnight culture of V. parahaemolyti- cus was adjusted to OD600nm of 0.08- 0.1, then 10 mL of this bacterial suspension was dropped onto agar plates corresponding to the tested enzymesmen- tioned above. In the case of assessing the effect of oxygen, the overnight culture was dropped on the plate either without or with AnaeroPack® (Mit- subishi Gas Chemical, Japan) and plastic wrap. Pos- itive controls used in these tests included Staphylo- coccus aureus ATCC29213 for caseinase and lipase, Vibrio cholerae for lecithinase and gelatinase and Vib- rio alginolyticus for chitinase. After inoculation, the plates were incubated 24 hours for caseinase, gelati- nase, and lipase and 48 hours for lecithinase and chiti- nase. For gelatinase, before reading the result, the agar plate was flooded with saturated ammonium sulfate ((NH4)2SO4) to precipitate the undegraded gelatin. Clear halos surrounding the bacterial drop indicated the activity of the tested enzymes16–18. All the tests were triplicated. Data analysis Enzyme activity (EA) was calculated using the for- mula: where D is the diameter of the bacterial drop plus the clear halo zone (mm), and d is the diame- ter 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. RESULTS Effects of pH on extracellular enzymatic ac- tivities of V. parahaemolyticus Following the increase of pH from7.5 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 un- detectable level at pH 7.5 to 1.38 0.17mm (+) at pH 9.0. On the other hand, lecithinase activity ofV. para- heamolyticus 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, V. parahaemolyticus exhibited signif- icant differences in enzymatic activities between four pH levels (p-value < 0.05, Supplementary Table 2 A). Effects of temperature on extracellular enzymatic activities of V. parahaemolyticus The rise of temperature from 25oC to 35oC 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 in- crease of caseinase from 0.67 0.10 (+) to 0.85 0.13 mm (+). At 30oC, the obtained EA value was high- est 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 (Fig- ure 2, Table 1). Temperature significantly affected the extracellular enzymatic activities ofV. parahaemolyti- cus (p-value< 0.05, Supplementary Table 2 B). Investigating the effects of oxygen on ex- tracellular enzymatic activities of V. para- haemolyticus Under the limited oxygen presence, most of the tested enzyme activities were low or even not observed. Ca- seinase activity declined considerably from 1.10  570 Science & Technology Development Journal, 23(3):569-575 Ta b le 1: Eff ec to fp H ,t em p er at u re an d ox yg en le ve lo n th e ex tr ac el lu la re n zy m at ic ac ti vi ty of Vi br io pa ra ha em ol yt icu sX N 9. Te st ed p H s in cl u d ed 7. 5, 8, 8. 5 an d 9. A ct iv it y w as ex p re ss ed vi a EA va lu e in m ill im et er (m m ). Te st ed te m p er at u re s in cl u d ed 25 ,3 0 an d 35 o C .O xy g en co n d it io n s in cl u d ed sh ak in g ov er n ig h tc u lt u re fo llo w ed b y p la te te st in g w it h ou tA n ae ro Pa ck ® (N or -N or ), sh ak in g ov er n ig h tc u lt u re fo llo w ed b y p la te te st in g w it h A n ae ro Pa ck (N or -L i) ,s ta ti c ov er n ig h tc u lt u re w it h O xy g en ab so rb er p ac ka g e fo llo w ed b y p la te te st in g w it h ou t A n ae ro Pa ck ® (L i- N or ), an d st at ic ov er n ig h tc u lt u re w it h O xy g en ab so rb er p ac ka g e fo llo w ed b y p la te te st in g w it h ou tA n ae ro Pa ck ® (L i- Li ). A ct iv it y w as ex p re ss ed vi a EA va lu e in m ill im et er (m m ). St an da rd : pH Te m pe ra tu re O xy ge n co nd iti on pH 8.5 /3 0o C 7.5 8.0 9.0 25 oC 35 oC No r-N or No r-L i Li -N or Li -L i C a se in as e 0.8 2 0.1 7 (+ ) 3.5 5 0.2 5 (+ +) 2.8 5 0.1 4 (+ +) 0.4 1  0.0 8 (+ ) 0.6 7  0.1 0 (+ ) 0.8 5  0.1 3 (+ ) 1.1 0 0.3 1 (+ ) 0.4 6 0.0 6 (+ ) 0.3 3 0.1 3 (+ ) _ Le cit hi na se 2.9 6 0.1 3 (+ +) 2.7 2 0.1 2 (+ +) 2.1  0.3 9 (+ +) 2.5 7 0.3 9 (+ +) 1.1 0 0.1 5 (+ ) 1.8 7 0.1 5 (+ ) 2.5 6 0.1 9 (+ +) 2.9 2 0.0 8 (+ +) 1.9 4 0.1 8 (+ ) 1.6 2 0.2 9 (+ ) Ge lat in as e 0.8 9 0.1 5 (+ ) _ 0.2 8 0.1 4 (+ ) 1.3 8 0.1 7 (+ ) 0.8 6 0.1 4 (+ ) 1.3 7 0.2 5 (+ ) 1.5 9 0.1 1 (+ ) 1.5 0 0.2 1 (+ ) 1.2 7 0.2 0 (+ ) 1.1 8 0.1 3 (+ ) L i pa se 1.4  0.1 (+ ) 1.8 3 0.2 9 (+ ) 1.7 7 0.2 5 (+ ) 1.2 1 0.2 5 (+ ) 2.6 7 0.2 2 (+ +) 2.2 5 0.2 3 (+ +) 2.3 3 0.2 3 (+ +) 2.2 0 0.2 2 (+ +) 1.6 9 0.1 4 (+ ) 1.1 7 0.2 0 (+ ) Ch iti na se _ _ _ _ _ _ _ _ _ _ 571 Science & Technology Development Journal, 23(3):569-575 Table 2: Two-way ANOVA in analyzing the effect of A) four pH levels (7.5, 8.0, 8.5 and 9.0); B) three temperatures (25, 30 and 35oC); C) four tested oxygen conditions (Nor-Nor; Nor-Li; Li-Nor and Nor-Li) on the enzymatic activities of V. parahaemolyticus A) Source of Variation SS df MS F p-value F crit Sample 50.3623567 4 12.5905892 335.719203 3.4326E-30 2.60597495 Columns 2.31145833 3 0.77048611 20.5444701 3.2315E-08 2.8387454 Interaction 23.97035 12 1.99752917 53.26271 1.0043E-20 2.0034594 Within 1.50013333 40 0.03750333 Total 78.1442983 59 B) Source of Variation SS df MS F p-value F crit Sample 27.6018133 4 6.90045333 324.982104 3.1206E-24 2.68962757 Columns 0.35015111 2 0.17507556 8.24531659 0.00140067 3.3158295 Interaction 7.94242667 8 0.99280333 46.7568289 7.5498E-15 2.26616327 Within 0.637 30 0.02123333 Total 36.5313911 44 C) Source of Variation SS df MS F p-value F crit Sample 42.5853233 4 10.6463308 253.032224 8.0542E-28 2.60597495 Columns 5.012045 3 1.67068167 39.7072292 4.5515E-12 2.8387454 Interaction 2.85693 12 0.2380775 5.65840761 1.4569E-05 2.0034594 Within 1.683 40 0.042075 Total 52.1372983 59 0.31 mm (+) in the case of both fully oxygenated cul- ture 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 activ- ity 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 conditionswith lowest activity (1.62 0.29mm (+)) 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 extra- cellular enzymes was different significantly (p-value< 0.05, Table 2 C). DISCUSSION Our data indicated that the production of extracel- lular enzymes in V. parahaemolyticus was highly af- fected by environmental factors. In inappropriate conditions, the production of some enzymes can be minimized to undetected levels such as gelatinase in case of pH 7.5 or caseinase in case of limited oxygen condition. On the other hand, some enzymes, such as 572 Science & Technology Development Journal, 23(3):569-575 Figure 1: Extracellular enzymatic activity of Vibrio parahaemolyticus under different culturing pHs. No activity of chitinase was observed in any tested culturing pH. Figure 2: Extracellular enzymatic activity of Vibrio parahaemolyticus under different culturing temperatures. No activity of chitinase was observed in any tested culturing temperature. lecithinase seemed to be constantly and strongly pro- duced in most conditions tested in our study. The strong production of lecithinase was observed not only in V. parahaemolyticus but also in other Vibrio species17,19–21. We did not detect chitinase activity in any tested conditions. Chitinase is a typical virulence factor of marine bacteria that can breakdown glyco- sidic bonds in the chitin of shrimp and other marine organisms22. The absence of chitinase activity indi- cated that this AHPND strain may not utilize chiti- nase attack aquatic crustaceans or chitinase might not be induced in vitro. Regarding pH, our data showed that while most of the tested enzymes showed the highest activity at pH 7.0, gelatinase only expressed its activity at alkaline conditions. This is in agreement with previous stud- ies showing that V. parahaemolyticus had a high rate of hydrolysis of gelatin in alkaline environments23,24. Gelatinase, together with lecithinase and protease, are constantly expressed in most disease-causing Vibrio species, particularly V. parahaemolyticus strains21,25. 573 Science & Technology Development Journal, 23(3):569-575 Figure 3: Extracellular enzymatic activity of Vibrio parahaemolyticus under different oxygen conditions: shaking overnight culture followed by plate testingwithout AnaeroPack® (Nor-Nor), shaking overnight culture followed by plate testing with AnaeroPack® (Nor-Li), static overnight culture with Oxygen absorber package followed by plate testing without AnaeroPack® (Li-Nor), static overnight culture with Oxygen absorber package followed by plate testing without AnaeroPack® (Li-Li). No activity of chitinase was observed in any tested culturing temperature. Temperature is a well-known factor that affects the growth of V. parahaemolyticus. It was shown that the minimal growth temperature of V. parahaemolyticus was 13oC, and its optimal growth temperature was 30oC14,26. However, for extracellular enzyme pro- duction, the optimal temperature was varied for dif- ferent types of enzymes. Gelatinase, for examples was found to express the highest activity in Vibrio species at 24C23. In our study, the optimal temperature was 35C for caseinase, gelatinase and lipase, and 30C for lecithinase. V. parahaemolyticus, like otherVibrio species, are fac- ultative anaerobe. Its growth is only hindered by strict anaerobic conditions but not limited oxygen condi- tion. Some in vivo environmental study even showed that the number ofV. parahaemolyticus in low oxygen marine water was higher than in high oxygen sam- ples27. Our data showed a decrease in the activity of extracellular enzymes for all tested enzymes, of which caseinase was the most affected one. No activity of caseinase was found when V. parahaemolyticus was cultured and tested in limited oxygen conditions. It was in agreement with a previous study showing that the production of proteolytic enzymes was negatively affected by a low dissolved oxygen level28. CONCLUSION pH, temperature, and oxygen condition are essen- tial factors affecting not only the growth of V. para- haemolyticus as previously shown (14) but also its ex- tracellular enzyme activity. No culturing condition resulted in the highest activity for all extracellular en- zymes was found. However, warm temperature (30/ 35oC), mildly alkaline pH (pH 8.0), and fully oxy- genated condition could increase the overall extracel- lular enzymatic activity of V. parahaemolyticus, thus increase its potential virulence. 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