research article biological activities of tetrodotoxin-producing enterococcus … · 2019. 7....

Research ArticleBiological Activities of Tetrodotoxin-ProducingEnterococcus faecium AD1 Isolated from Puffer Fishes

Tu Hoang Khue Nguyen1 Huu Ngoc Nguyen1 Dat Van Nghe1 and Kim Hoang Nguyen2

1School of Biotechnology Hochiminh International University Vietnam National University Quarter 6Linh Trung Ward Thu Duc District Hochiminh City Vietnam2Retina Center 1715 N George Mason Drive Suite 101 Arlington VA 2220 USA

Correspondence should be addressed to Tu Hoang Khue Nguyen nhkhuetuyahoocom

Received 8 June 2015 Accepted 15 July 2015

Academic Editor Gang Liu

Copyright copy 2015 Tu Hoang Khue Nguyen et alThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Puffer fishes were collected from the central sea in Vietnam from spring to summer season The eggs were incubated in MRSbroth that was used to test the toxicity in mice and isolate the lactic acid bacteria community that could produce tetrodotoxin(TTX) Thin layer chromatography (TLC) and high performance lipid chromatography (HPLC) were used to detect and quantifyTTX As a result Enterococcus faecium AD1 which was identified by biochemical test and 16S rRNA analysis could produce TTX03mgmL when cultured in MRS broth The bacterium was optimized for TTX production and gave 018mgmL 007mgmLand 015mgmL in media prepared from the meat-washing water of freshwater fishes (Pangasius bocourtiOreochromis sp) and seafish (Auxis thazard) respectively that are also hopeful to answer some poisoning cases related to eating fishes Enterococcus faeciumalso showed the wide antimicrobial activities on yeast Gram-negative and -positive bacteria Extracted exopolysaccharide (EPS)that reacted with 22-diphenyl-1-picrylhydrazyl to give IC

50at 5mgmL equaled 11mgmL ascorbic acid which could show effects

on Hela-6 and Hep G2 using sulforhodamine B test Enterococcus faecium can be claimed as a promising source in tetrodotoxinand biological compounds

1 Introduction

Tetrodotoxin (TTX) is the most well-known marine toxinbut TTX has been proven to be useful compound in phar-maceutical science For example TTX might be used asneuroprotective agent for ischemic damage of brain followedby strokes [1] renoprotective agent [2] and antinocicep-tive agent [3] Up to now TTX has been isolated frommarine animals including xanthid crab Atergatis floridus[4] the blue-ringed octopus Octopus maculosus [5] starfishAstropecten polyacanthus [6] and several species from vari-ous countries In addition to animals TTX-producing bac-teria were reported to have been isolated from marine orfreshwater sediment [7ndash9] All the TTX-bearing animalswere infected by TTX-producing bacteria lying within theirbodies Bacterium that producedTTXwas reported inVibrioPseudomonas Alteromonas Bacillus Aeromonas Flavobac-terium Microbacterium arabinogalactanolyticum Serratia

marcescens andProvidentia rettgeri [10 11] It has been knownfor many years that toxicity of the ovaries of puffer fish is themost potent one although the toxicity is also detected in otherorgans

Because of TTX toxicity looking for the TTX-producingsource is always required so that the environmental safetyis ensured Most of researches found the TTX-producingmicroorganisms were not GRAS (generally recognized assafe) the study focused on lactic acid bacteria Thereforeselection of ovary for isolation of TTX-producing lactic acidbacteria will be interesting Lactic acid bacteria are importantorganisms recognized for their fermentative ability as wellas their health and nutritional benefits [12] They producevarious compounds such as organic acids diacetyl hydrogenperoxide and bacteriocin or bactericidal proteins duringlactic fermentations [13ndash15] The antimicrobial propertiesof lactobacilli are of special interest in developing stronglycompetitive starter cultures for food fermentation

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 973235 8 pageshttpdxdoiorg1011552015973235

2 BioMed Research International

Lactobacilli exert strong antagonistic activity againstmany microorganisms including food spoilage organismsand pathogens Bacteriocins are antimicrobial proteinaceouscompounds that are inhibitory towards sensitive strains andare produced by bothGram-positive andGram-negative bac-teria [16ndash20] Bacteriocins have widespread potential appli-cations in preservation for improving the safety and qualityof foods At present there are many antibiotic-resistantpathogens therefore it is necessary to identify develop orredesign antibiotics to fight themultidrug resistance bacteriaUp to now bacteriocins hold great promise as the nextgeneration of antimicrobials Beside facing drug resistancethe diseases which are due to the ldquooxidative stressrdquo are thechallenges that should be prevented

Oxidant stress is initiated by free radicals which pro-duced aerobic metabolism in the body and can cause oxida-tive damage of biological macromolecules such as proteinslipids and DNA in healthy human cells [21ndash23] Thesechanges contribute to oxidative stress that is among themajorcausative factors in the induction ofmany chronic and degen-erative diseases including atherosclerosis ischemic heartdiseases and diabetes mellitus cancer immunosuppressionneurodegenerative disease ageing [24ndash28] coronary heartdisease and Alzheimerrsquos disease [29ndash32] All human cellsprotect themselves against free radical damage by enzymessuch as superoxide dismutase and catalase or compoundssuch as ascorbic acid tocopherol and glutathione [33]However these protective mechanisms occurred by variouspathological processes and antioxidant supplements are nec-essary to combat oxidative stressTherefore the interest in thenatural compounds with strong antioxidant and anticancerproperties has steadily been increasing

From the above stated reasons the TTX-producing lacticacid bacteria were isolated in puffer fish and then optimizedthe ability of TTX production of bacteria in media preparedfrom freshwater fishes (Pangasius bocourti Oreochromis sp)and sea fish (Auxis thazard) Then this strain was usedto study antimicrobial radical scavenging and anticanceractivities to find out the potential lactic acid bacteria forpharmaceutical science

2 Materials and Methods

21 Sample Preparation and Bacteria Cultures Puffer fisheswere collected at the sea in the center of Vietnam Nospecific permit was required for the described field studiesThe female fish weight was around 600 g kept in ice andtransported to the lab The female fishes were washed with70 and dissected to isolate the ovaries from abdomen bysterilized knife and scissor The ovaries (3 g) were homog-enized carefully and incubated directly in modified MRSbroth Samples were incubated for 48 hours in 5 CO

2

at room temperature Then the cultures were checked formicroorganims on MRS agar

22 Isolation and Identification of Enterococcus faeciumOne mL of culture was aseptically spreaded on MRS agar

plates and was incubated at 25∘C for 3ndash5 days Eventu-ally the forming colonies were transferred onto anothernew MRS agar plates until discrete colonies were obtainedEach discrete colony was further subcultured for severaltimes to ensure that a pure culture was obtained The purecultures were also characterized using bioassay for toxicitytest The toxin was extracted and purified for thin layerchromatography and high performance liquid chromatog-raphy Then the toxic strain was identified by biochemicalcharacterization based on the ability of utilization of differentcarbon sources by API 50CHL (bioMerieux Lyon France)Thenmolecular characterization using 16S rRNA sequencinganalysis was performed The forward primer was faec1 (51015840-ttggagagtttgatcctggctcaggac-31015840) and the reverse primer wasfaec2 (51015840-gctgctggcacgtagttagccgtggct-31015840) The PCR reactionwas performed as follows 95∘C for 5min 30 cycles of 95∘C for20 s 50∘C for 20 s and 72∘C for 3min and a final extensionat 72∘C for 10min The PCR product was stored at 4∘C ThePCR product was purified and sequenced The homologycomparison of the almost 16S rRNA gene sequence wasperformed using the NCBI BLAST database

23 Extraction and Purification of Tetrodotoxin from Bac-terial Cultures The isolation and purification proceduresof TTX from bacterial cultures were basically the same asthose used for puffer fish tissues Each broth culture wascentrifuged at 4000 rpm for 15min to remove the bacterialdebris The supernatant was mixed with an equal volume ofwater-washed activated charcoal under agitation and filteredthrough a Buchner funnel The charcoal on the funnel wasthoroughly washed with distilled water and TTX was elutedfor 3 times with 3 volumes of 1 acetic acid in 20 aqueousethanol [10 17 34 35] The eluate was heated at 100∘C for10min and then cooled and centrifuged to separate debrisThe eluatewas evaporated freeze-dried and then dissolved in10mL of 003M acetic acid for gel filtration Eluted TTX wasused for further toxicity bioassay thin layer chromatographyand high performance liquid chromatography

24 High Performance Liquid Chromatography (HPLC) forTetrodotoxin Detection The extracted samples were appliedto HPLC in order to detect and quantify the TTX existenceby UV detector The Shimadzu HPLC system with RP 18(250 times 46mm 5120583m) column was used as stationary phaseThe mobile phase was prepared by mixing of 001M sodiumheptanesulfonic and methanol at ratio 90 10 The flow ratewas 1mLmin at 30∘C wavelength was 205 nmThe authenticTTX (Tocris UK) at concentration 02mgmL was applied

25 Bioassay for Toxicity Detection Mice were injected intra-peritoneally with extraction [36] The toxicity was deter-mined by the death time of each injected mouse The deathtime was recorded at the grasping breath of mouse It wasrecommended not to use weight more than 23 g and reusemice

26 Thin Layer Chromatography (TLC) for TetrodotoxinDetection TLC analysis method was carried out [34] with

BioMed Research International 3

a little modification The eluted samples were applied onTLC silica gel 60 F256mdashMerckrsquos product The mobile systemwas n-butanol-acetic acid-distilled water (5 1 2) prepared inTLC chamber [10] The spots were observed under UV light(256 nm) and compared to TTX standard

27 Optimization of TTX-Producing Condition in BacteriaAs the concerning on the effects of media to optimize theTTX production abilities of bacteria the study had usedthe media prepared from fresh fishes (Pangasius bocourtiOreochromis) and sea fish (Auxis thazard) Each test wasperformed in triplicate After preparing all media they wereautoclaved at 121∘C in 15 minutes The starting inoculumwould be recorded in colony forming unit (CFU) permilliliter and the finishing inoculum as well [37] The TTXproduction was detected by bioassay TLC and HPLC

28 Antimicrobial Activity Test The indicator pathogens suchas Candida albicans ATCC 10231 Salmonella typhi ATCC19430 Pseudomonas aeruginosa ATCC 27853 Staphylococcussciuri ATCC 29061 Micrococcus luteus ATCC 10240 andStaphylococcus aureus ATCC 25923 were used in the studyAll pathogens (106 cfumL) were cultured in 200mL of LB for18 h

29 Preparation of Cell-Free Supernatant (CFS) of IsolatedStrain for Antimicrobial Test The isolated strain (106 cfumL)was cultured in 500mL of MRS broth for 24 h Cell-freesupernatant (CFS) of isolated strain was prepared followingthe method constructed by Ogunbanwo et al [38] Theculture was centrifuged at 10000 rpm for 20 minutes at 4∘CThen the CFSs were collected and stored in 4∘C for furtheruse

210 Test of Antimicrobial Activities of Isolated Strain Thestrain was tested for their antimicrobial activities to theindicator pathogens by well-agar diffusion assay of Tagg JR[39] Indicator pathogens were spread onto LB agar platesThe wells with 5mm diameter were carved on the platesThen they were filled up with 100120583L of CFS The plates wereincubated at 37∘C for 24 h and examined the antimicrobialactivities by observing the zones of inhibition appearingaround each well The experiment was triplicated

211 Effect of pH on the Growth of Isolated Strain In orderto study the effect of incubation temperature on growthand antimicrobial agent production the amount of 105ndash106 cfumL was inoculated into buffered MRS broth and wasadjusted at different pH values (1 15 2 25 3 4 5 65 758 and 85) before cultivation [39] The bacterial growth waschecked by counting the survival on MRS agar Then thegrowth percentage of bacteria in different pH was based onthe number of survival bacteria and the number of startingbacteria Each assay was performed in triplicate

212 Bacteriocin Precipitation The strain was grown inMRS broth at 37∘C in 48 h Supernatant was collected after

centrifugation at 10000 rpm for 15min at 4∘C Ammoniumsulfate was added slowly to the cell-free culture super-natant with constant stirring to achieve the concentrationof ammonium sulfate (40) The mixture was centrifugedat 10000 rpm for 30min at 4∘C The collected pellet wasdissolved in phosphate buffered saline (PBS) pH 74 and wasready for the antimicrobial test The remaining supernatantfraction was subsequently adjusted with ammonium sulfateto 60 and 80 The precipitant in each ammonium sulfatefraction was dissolved in PBS (pH 74) and then was readyfor antimicrobial test Bacteriocin was checked on sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE 15 discontinuous gel) The molecular weight of thefractionated proteins was compared with standard markers(Bio-Rad)

213 Preparation of Polysaccharides The isolate was culturedin (MRS) and incubated at 37∘C under aerobic condi-tions Cultures were centrifuged at 10000 rpm for 30minto remove debris The culture supernatant was added withthree times volume of absolute cold ethanol (EtOH) and leftovernight The precipitant was collected after centrifugationat 10000 rpm for 30min The obtained pellet was resus-pended in distilled water and further precipitated by addingthree times volume of cold ethanol The overnight solutionwas centrifuged to collect the water-soluble exopolysaccha-rides (EPS) The crude EPS was dried at 60∘C to a constantweight EPS stocks were dissolved in distilled water andthen filtered through the 022120583m pore-size filters (MilliporeBedford MA) before use

214 Antioxidant Activity Using DPPH Radical ScavengingAssay In order to perform the 22-diphenylpicrylhydrazyl(DPPH assay) amounts of 43mg of DPPH were dissolved in33mL methanol in a test tube [40] Solution was protectedfrom light by covering the test tubes with aluminum foil150 120583L of the above solution was added to 3mL methanolThis solution was measured at 517 nm on UV spectropho-tometer Methanol was used as blank For standard (ascorbicacid) curve the aliquots of different concentration rangewereprepared After 50 120583L of tested EPS (10mgmL) and standardascorbic acid in the various concentration were diluted withmethanol up to 3mL 150120583L DPPH was added All thesesamples were taken after 12 h and measured at 517 nm onUV-visible spectrometer (Shimadzu UV-1601 Japan) TheDPPH free radical scavenging activity was calculated usingthe following formula

(1 minus 119860119904)

119860119888

times 100 (1)

where 119860119904and 119860

119888are the absorbance of control and sample

respectively

215 Anticancer Activity Test In order to perform antitumoractivity test the crude EPS sample was prepared accordingto the above prescribed procedure The sulforhodamine B(SRB) assay was used in the study according to the method of


Longo-Sorbello with a slight modification [41] Each cancercell line was seeded in a 96-well plate (10 times 104 cells per well)After 24 h of incubation the test samples (1153 times 106 cfumLof cell-free supernatant and 20mgmL of crude EPS) wereadded to the cancer cells 5 CO

2for 48 h at 37∘C For this

incubation time no significant differences were observed inthe pH of medium Thereafter 50120583L of 50 TCA (4∘C)was added to each well containing 200 120583L of medium toreach a final concentration of 10 TCA in each well andlet the 96-well plate for 1 h at 4∘C to allow cell fixationAfter 1 h of incubation the culture medium in each wellwas removed and the plate was gently washed with water(200120583Lwell) five times and dried at room temperature for12ndash24 h 02 SRB (wv) solution was added after the timefor incubation to each well and left at room temperaturefor 5ndash20min Then washing the plate with 1 acetic acidwas performed five times in order to remove unbound SRBDrying the plate for 12ndash24 h before adding 200120583L Trizma-base 10mM in order to solubilize bound SRB is necessaryThe last step is plating the 96-well plate on a plate shakerfor at least 10min Absorbance was measured at 492 nm and620 nm using an enzyme-linked immunosorbent assay platereader (Molecular Devices Sunnyvale CA USA) DMSOwas used as a negative control The percentage of viablecells was calculated based on the difference of the opticaldensity of experimental group and control group with theoptical density of control groupThe SPSS 160 software (SPSSInc Chicago IL USA) was used to calculate the means andstandard deviations in any experiments involving triplicateanalyses of any samples The statistical significance of anyobserved difference was evaluated by one-way analysis ofvariance (one-way ANOVA) using the Bonferroni multiplecomparisons test

3 Results and Discussion

31 Identification of Enterococcus faecium After detecting thetoxicity of the puffer fish ovaries in MRS the isolation ofTTX-producing bacteria was done There were 5 dominantbacterial strains isolated from ovaries of the puffer fish Onlyone of them was determined to be toxic in mouse bioassayThe bacterium could produce 15ndash3 120583g ranging within 30ndash80MU (mouse per unit) in 100mL of broth medium Thedeath time occurred in a short time at average time of 6mins(Figure 1) The culture showed that the mouse toxicity waspurified and each purified fraction was used to test toxicity(Figure 1)

In the toxicity detection using mouse bioassay therelationship between dose and time to death is used toquantify each sample This relationship is approximated bylogMU = 23 log(1 + 119879minus1) where MU is the number ofmouse units of tetrodotoxin injected and 119879 is time to deathin hours The mouse assay has been traditionally used but itis unsuitable as a market test There are other disadvantagessuch as the variation in mouse weight that must be limitedinvolving a large breeding colony of the mice and the deathtime relationship to dose is nonlinear Therefore the studycombined TLC and HPLC analyses for qualification and

Purifi

ed-T

Oreochrom

is-T

Auxis-

T

Pangasius-

T

MRS

-T

Media

05

1015202530354045

Dea

th ti

me (

min

)

Figure 1 Death time of mice injected with different bacterialcultures prepared from MRS purified tetrodotoxin from MRSculture Oreochromis sp Auxis thazard and Pangasius bocourti

1 2 3

Figure 2 Thin layer chromatography analysis on tetrodotoxindetection 1 standard tetrodotoxin 2 MRS medium and 3 extractfrom bacterium The arrow showed tetrodotoxin spot

quantification The TTX production of the bacterium wasdetected on TLC (Figure 2)

The spots of the standard TTX and purified toxin ofthe isolated strain had the same 119877

119891value (065) under UV

illumination In comparison with the negative control asthe media the bacterium could produce the spots with thesame 119877

119891of the TTX standard this bacterium could produce

suspected TTX By HPLC analysis the sample prepared frombacteria in this study also showed the peak at retention time(13494min) that was similar to the retention time of the peakof standard TTX (13376min) (Figure 3) Based on mousebioassay test and TLC and HPLC analysis this bacteriumcould produce TTX

After determining the bacteria that could produce toxinby bioassay TLC and HPLC the bacterium was identified


13376

15 2010

(min)

(a)

13494

15339

15 2010

(min)

(b)

Figure 3 High performance liquid chromatography analysis oftetrodotoxin (a) Standard tetrodotoxin (b) Purified tetrodotoxinfromMRS culture

by biochemical tests and 16S rRNA sequence analysis Thebacterium was found to be a facultative anaerobe which canutilize and produce acid by fermenting d-glucose mannitolrhamnose d-sorbitol mannose d-galactose d-fructose and10 lactose The partial 16S rRNA sequence was 540 bpdeposited in DDBJ (Accession number AB828395) Thepartial 16S rRNA gene sequence was analyzed by using NCBIBLAST showing 99 of the homology to Enterococcus fae-cium Aus004 and 97 of the homology to Enterococcus fae-ciumDSM 20477 There was no information of tetrodotoxin-producing Enterococcus faecium announced before There-fore the isolated strain was identified Enterococcus faeciumAD1

This study is the first report of TTX-producing Entero-coccus faecium isolated from puffer fishes in Vietnam andthe world The discovery of TTX-producing Enterococcusfaecium supports the theory that TTX is originated from themicroorganisms found inside the TTX-bearing organismsNormally Enterococcus faecium itself could not produce TTXexcept this bacterium surviving in TTX in puffer fishes Thisindicated that there was an interaction of puffer fish andbacteria Interestingly the bacterium which could produceTTX in puffer fishes could produce TTX in some kinds offish like TTX-producing Providentia rettgeri [10] TTX isconventionally obtained by the extraction and purificationfrom toxic organs of the puffer fishes that gave an extremelylow TTX yield about 1 ton of toxic puffer ovaries requiredto yield 1 g of pure TTX [35] Study on the biosynthesisof TTX directly from TTX-producing Enterococcus faecium

Table 1 TTX concentration (mgmL) produced by E faecium inmedia prepared from different fishes

Samples Concentration (mgmL)Standard 02Auxis thazard 015Pangasius bocourti 007Oreochromis sp 018

AD1 in vitro is much more economical and efficient thanthe traditional method Besides when Enterococcus faeciumAD1 produces TTX TTX formulation as probiotic will behopeful application in using TTX without further TTXpurification that affects the living environmentThus in orderto obtain mass production of TTX from TTX-producingbacteria in vitro it is necessary to develop the optimummedium composition such as the temperature pH salinityrate of shaking and light intensity as well as the effectivemethods of isolating TTX from the culture media (batchor continuous methods) In this study we focused on themedium composition originated in freshwater fishes and seafishes

32 Optimization of TTX Production in Enterococcus fae-cium AD1 All the extracts from Pangasius bocourti Ore-ochromis sp andAuxis thazardwere used formouse bioassay(Figure 1) As seen in Figure 1 TTX produced in mediumprepared from Pangasius bocourti was the lowest The TLCanalysis for the TTX production in the fish media was alsoperformed The spots of the standard TTX and those of theextracts had the same 119877

119891value (065) under UV illumination

in TLC analysis By HPLC analysis the sample preparedfrom Pangasius bocourti Oreochromis sp and Auxis thazardin this study also showed the peak at retention time of13330 12802 and 13324min respectively The retentiontime in different samples was evaluated using standarddeviations and maximum response Using the United StatesPharmacopoeia (USP) method the tailing factors should notexceed 2 and the peak response is acceptable The standarddeviation was less than 2 The other separate peaks are notof concern to this study Those retention time values wereclose to the retention time of the peak of standard TTX(13376min) which meant that these fish cultures could bethe good substrates for Enterococcus faecium AD1 to produceTTXThe yield produced by this bacterium in fishmedia wassummarized in Table 1

From Table 1 the trends of TTX production have partlyshown the suitable condition in natural life The mediumprepared from Oreochromis sp (018mgmL calculated onthe final product in 003M acetic acid) was higher thanin Auxis thazard (015mgmL) The hypothesis is pointingthat this TTX-producing bacterium can take place in anykinds of fishes fed in lake or in sea For media preparedfrom freshwater fishes with the inoculum of 1014 cells in10mLmedia TTX amount of cultured sample inOreochromissp equaled 018mgmL which was larger than in Panga-sius bocourti (007mgmL) It makes sense when making


Cand

ida

albicans

Salm

onella

typhim

urium

Pseudomonas

aeruginosa

Staphylococcus

sciuri

Staphylococcus

aureus

Micrococcus

luteus

Pathogen

24h32h

48h72h

0

5

10

15

20

25

30

Dia

met

er o

f inh

ibiti

on zo

ne (m

m)

Figure 4 Antimicrobial activities of E faecium in pathogens indifferent incubation times

the relation to many poisoned cases in consuming Ore-ochromis sp in this situation Oreochromis sp tissues are theideal places for bacteria to take place especially for Entero-coccus as we analyzed above TTX production in different fishmedia was interesting that might be the significant differencein the contents of calcium sodium and potassium in thesefishes These ions might be affected on the TTX-producingpathway of bacterium More studies should be performed toclarify the mechanism In practice these kinds of fishes didnot contain TTX It was thought that there were immuneresponses to TTX in these alive fishesThe study will performmany experiments to study the poisoning ability in pufferfishes and other kinds of fishes infected with TTX-producingbacterium to bring up the detoxifyingmechanisms For TTX-producing strategies to apply on many fields such as medicalsciences and pharmaceuticals we have to produce on largescale so the economic point of view ismore importantMeat-washingmediumprepared from freshwater is thewaste of fishindustry we can make use of it for targeted purposes

33 Antibiotic Activities The antibiotic activities of E fae-cium AD1 were shown in Figure 4 E faecium AD1 couldinhibit Candida albicans Salmonella typhi Pseudomonasaeruginosa Staphylococcus sciuri Staphylococcus aureus andMicrococcus luteus As seen in Figure 4 the activity showedstrong effect at 48 h culture When bacterium was culturedless than 48 h the exponential phase was reached andthere were small amounts of antimicrobial agents producedOtherwise for the 72 h culture the antimicrobial activitybecame lower than the 48 h culture which meant that therewere some degraded antimicrobial agents for example theproteins could inhibit the pathogens but they were lysed byprotease produced by E faecium AD1 itself

The activities on pathogens in different stages of culturewere not similar that might be the antagonist activities ofdifferent antimicrobial agents probably happening in theculture stages More study should be done in the future

1 2 3 4 5 24

Time (h)

0

20

40

60

80

100

120

Viab

ility

()

15

2

25

3

4

5

65

75

8

85

Figure 5 Effects of pH on growth of Enterococcus faecium

to clarify these actions By bacteriocin precipitation twobacteriocins that had the estimatedmolecularweight of 4 kDaand 45 kDa could be collected and showed antimicrobialactivities

34 Effect of Medium pH on Bacteria Growth Besides theculture period that was studied in antimicrobial activitymedium pHwas also important factor that affects cell growthas well as bacteriocin productionThe bacteriocin was treatedwith a wide range of pH values from 15 to 85This bacteriumcould survive at pH 2 in 5 h However it could survive atpH from 25 to 85 (Figure 5) With the advantage of theadaptability to a wide pH range this bacterium could be usedin different formulations Moreover this bacterium couldsurvive in gastrointestinal tract that was of benefit to orallypharmaceutical formulations for tetrodotoxin formulationfrom Enterococcus faecium without further purification toprevent the TTX contamination to environment

35 DPPH Radical Scavenging Assay In this present studythe antioxidant activity of the crude EPS was done by usingtheDPPH scavenging assayThe IC

50of crude EPS (5mgmL)

equaled 11mgmL ascorbic acid to give the antioxidantactivities according to the standard curve (119910 = 17204119909 +43475 1198772 = 09993) From the results of antioxidant activityit was recognized clearly that the bacterium could producethe highest amount of antioxidant activity

36 Anticancer Activities As illustrated in Table 2 CFSand EPS were effective to Hela and Hep G2 cancer cellsDepending on the differentmechanisms like receptor bindingability to cancer cell lines the cytotoxicity percentage ofeach sample was also different For Hela cancer cell thecytotoxicity percentage of EPS was 9042 plusmn 2965 In caseof Hep G2 cancer cell the cytotoxicity percentage of crudeEPS was 7028 plusmn 3125 These results indicated that the EPS


Table 2 Inhibitory effects of cell-free supernatant and exopolysac-charide on the growth of two human cancer cell lines

Cancer cell line cytotoxicityCell free supernatant Crude EPS

Hela 4015 plusmn 5469 9042 plusmn 2965Hep G2 6051 plusmn 6568 7028 plusmn 3125

isolated from E faecium constituted the major fraction thatinhibits the proliferation of cancer cells The CFS gave thelower activities in Hela (4015 plusmn 5469) and Hep G2 (6051 plusmn6568) as compared to EPS because EPS in the study wasconcentrated from 100mL of cell-free supernatant that wasdirectly used to test cancer cells More studies should be doneso far Remarkably Enterococcus faecium AD1 isolated couldbe the potent source for bioactivity studies

4 Conclusions

The study has identified a TTX-producing Enterococcus fae-ciumAD1 andoptimized theTTXproduction in cheap sourceand also suggested that the fishes could be infected withTTX However because of the immune response they willor will not prevent TTX in their life The study will uncoverthe mechanism to prevent TTX infection so far MoreoverTTX-producing Enterococcus faecium AD1 could survive indifferent pH and produced antimicrobial antioxidant andanticancer activities that might supply a potential probioticused to yield TTX in reducing pain in cancer without furtherTTX purification as well as producing the activities for cancerand pathogen treatment

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

All authors read and approved the final paper

Acknowledgment

The research is financed by Vietnam National UniversityHochiminh City (VNU-HCM) under Grant no C2013-28-02

References

[1] W J Koroshetz and M A Moskowitz ldquoEmerging treatmentsfor stroke in humansrdquo Trends in Pharmacological Sciences vol17 no 6 pp 227ndash233 1996

[2] P J Garvin M L Niehoff and S M Robinson ldquoEffectsof tetrodotoxin and OKY-046 in renal ischemia reperfusionrdquoJournal of Surgical Research vol 85 no 2 pp 273ndash278 1999

[3] P Beaulieu and S Lu ldquoAntinociceptive effects of tetrodotoxin(TTX) in rodentsrdquo British Journal of Anaesthesia vol 96 no 6pp 761ndash768 2006

[4] T Noguchi O Arakawa K Daigo and K Hashimoto ldquoLocaldifferences in toxin composition of a xanthid crab Atergatisfloridus inhabiting Ishigaki Island Okinawardquo Toxicon vol 24no 7 pp 705ndash711 1986

[5] D D Sheumack M E H Howden I Spence and R J QuinnldquoMaculotoxin a neurotoxin from the venom glands of theoctopus Hapalochlaena maculosa identified as tetrodotoxinrdquoScience vol 199 no 4325 pp 188ndash189 1978

[6] H Narita S Matsubara and N Miwa ldquoVibrio alginolyti-cus a TTX-producing bacterium isolated from the starfishAstropecten polyacanthusrdquoNippon Suisan Gakkaishi vol 53 pp617ndash621 1987

[7] H K Do K Hamasaki K Ohwada et al ldquoPresence oftetrodotoxin and tetrodotoxin-producing bacteria in freshwatersedimentsrdquo Applied and Environmental Microbiology vol 59no 11 pp 3934ndash3937 1993

[8] H K Do K Kogure C Imada T Noguchi K Ohwada andU Simidu ldquoTetrodotoxin production of actinomycetes isolatedfrommarine sedimentrdquo Journal of Applied Bacteriology vol 70no 6 pp 464ndash468 1991

[9] H K Do K Kogure andU Simidu ldquoIdentification of deep-sea-sediment bacteria which produce tetrodotoxinrdquo Applied andEnvironmental Microbiology vol 56 no 4 pp 1162ndash1163 1990

[10] N Tu Q Tu H Tung D Hieu and S Romero-Jovel ldquoDetec-tion of tetrodotoxin-producing Providencia rettgeri T892 inLagocephalus pufferfishrdquo World Journal of Microbiology andBiotechnology vol 30 no 6 pp 1829ndash1835 2014

[11] N Tu and Q Tu ldquoTetrodotoxin production in Providenciarettgeri T892 isolated in Vietnamese puffer fishrdquo WulfeniaJournal vol 21 no 4 pp 268ndash285 2014

[12] S E Gilliland ldquoHealth and nutritional benefits from lactic acidbacteriardquo FEMS Microbiology Reviews vol 87 no 1-2 pp 175ndash188 1990

[13] M A Daeschel H P Fleming and R F McFeeters ldquoMixedculture fermentation of cucumber juice with Lactobacillusplantarum and yeastsrdquo Journal of Food Science vol 53 no 3 pp862ndash864 1988

[14] S E Lindgren and W J Dobrogosz ldquoAntagonistic activitiesof lactic acid bacteria in food and feed fermentationsrdquo FEMSMicrobiology Letters vol 87 no 1-2 pp 149ndash163 1991

[15] T R Klaenhammer ldquoBacteriocins of lactic acid bacteriardquoBiochimie vol 70 no 3 pp 337ndash349 1988

[16] J R Tagg A S Dajani and L W Wannamaker ldquoBacteriocinsof gram-positive bacteriardquo Bacteriological Reviews vol 40 no3 pp 722ndash756 1976

[17] S Karaoglu M Halici and A Baktir ldquoAn unidentified pitfall ofEndobutton use case reportrdquo Knee Surgery Sports Traumatol-ogy Arthroscopy vol 10 no 4 pp 247ndash249 2002

[18] N L Tatsadjieu N Y Njintang S T Kemgang B Daoudouand C M F Mbofung ldquoCharacterization of lactic acid bacteriaproducing bacteriocins against chicken Salmonella enterica andEscherichia colirdquo African Journal of Microbiology Research vol3 no 5 pp 220ndash227 2009

[19] D Nghe and T Nguyen ldquoCharacterization of antimicrobialactivities of Pediococcus pentosaceus Vtcc-B-601rdquo Journal ofApplied Pharmaceutical Science vol 4 no 5 pp 61ndash64 2014

[20] D T Nguyen and T H Nguyen ldquoDetection on antioxidant andcytotoxicity activities of exopolysaccharides isolated in plant-originated Lactococcus lactisrdquo Biomedical amp Pharmacology Jour-nal vol 7 no 1 pp 33ndash38 2014


[21] G-C Yen and H-Y Chen ldquoAntioxidant activity of varioustea extracts in relation to their antimutagenicityrdquo Journal ofAgricultural and Food Chemistry vol 43 no 1 pp 27ndash32 1995

[22] JM CGutteridge andBHalliwell ldquoInvited review free radicalsin disease processes a compilation of cause and consequencerdquoFree Radical Research Communications vol 19 no 3 pp 141ndash158 1993

[23] B Halliwell ldquoHow to characterize an antioxidant an updaterdquoBiochemical Society Symposium vol 61 pp 73ndash101 1995

[24] M E Buyukokuroglu I GulcIn M Oktay and O IKufrevioglu ldquoIn vitro antioxidant properties of dantrolenesodiumrdquo Pharmacological Research vol 44 no 6 pp 491ndash4942001

[25] T P A Devasagayam J C Tilak K K Boloor K S Sane SS Ghaskadbi and R D Lele ldquoFree radicals and antioxidants inhuman health current status and future prospectsrdquo Journal ofAssociation of Physicians of India vol 52 pp 794ndash804 2004

[26] I Gulcin M Oktay O I Kufrevioglu and A Aslan ldquoDeter-mination of antioxidant activity of lichen Cetraria islandica (L)Achrdquo Journal of Ethnopharmacology vol 79 no 3 pp 325ndash3292002

[27] F Shahidi P K Janitha and P D Wanasundara ldquoPhenolicantioxidantsrdquo Critical Reviews in Food Science and Nutritionvol 32 no 1 pp 67ndash103 1992

[28] G L Squadrito and W A Pryor ldquoOxidative chemistry ofnitric oxide the roles of superoxide peroxynitrite and carbondioxiderdquo Free Radical Biology and Medicine vol 25 no 4-5 pp392ndash403 1998

[29] B N Ames ldquoDietary carcinogens and anticarcinogens oxygenradicals and degenerative diseasesrdquo Science vol 221 no 4617pp 1256ndash1264 1983

[30] K F Gey ldquoThe antioxidant hypothesis of cardiovascular diseaseepidemiology and mechanismsrdquo Biochemical Society Transac-tions vol 18 no 6 pp 1041ndash1045 1990

[31] M N Diaz B Frei J A Vita and J F Keaney Jr ldquoAntioxidantsand atherosclerotic heart diseaserdquo The New England Journal ofMedicine vol 337 no 6 pp 408ndash416 1997

[32] M A Smith G P P Richey L M Sayre V E Anderson M FBeal and N Kowal ldquoTest for oxidative damage in AlzheimerrsquosrdquoNature vol 382 pp 120ndash121 1996

[33] E Niki H Shimaski andMMinoAntioxidantism-Free Radicaland Biological Defense Gakkai Syuppn Center Tokyo Japan1994

[34] K D Simon A G Mazlan and U Gires ldquoToxicity of pufferfishes (Lagocephalus wheeleri Abe Tabeta and Kitahama 1984and Lagocephalus sceleratus Gmelin 1789) from the East Coastwater of Peninular Malaysiardquo Journal of Biological Sciences vol9 pp 482ndash487 2009

[35] C H Yu Detection and biosynthesis of Pufferfish toxin frombacterial culture for novelmedical application [MS thesis] HongKong Polytechnic University Hong Kong 2007

[36] T Kawabata ldquoAssay method for tetrodotoxinrdquo in Food HygieneExamination Manual vol 2 pp 232ndash240 1978

[37] S Gallacher and TH Birkbeck ldquoEffect of phosphate concentra-tion on production of tetrodotoxin byAlteromonas tetraodonisrdquoApplied and Environmental Microbiology vol 59 no 11 pp3981ndash3983 1993

[38] S T Ogunbanwo A I Sanni andA A Onilude ldquoCharacteriza-tion of bacteriocin produced by Lactobacillus plantarum F1 andLactobacillus brevis OG1rdquo African Journal of Biotechnology vol2 no 8 pp 223ndash235 2003

[39] J R Tagg and A R McGiven ldquoAssay system for bacteriocinsrdquoApplied Microbiology vol 21 no 5 article 943 1971

[40] R Bromberg I Moreno R R Delboni H C Cintra and PT V Oliveira ldquoCharacteristics of the bacteriocin produced byLactococcus lactis subsp cremoris CTC 204 and the effect of thiscompound on themesophilic bacteria associatedwith rawbeefrdquoWorld Journal of Microbiology and Biotechnology vol 21 no 3pp 351ndash358 2005

[41] G S Longo-Sorbello G Saydam D Banerjee and J R BertinoldquoCytotoxicity and cell growth assaysrdquo in Cell Biology J E CelisN Carter K Simons J V Small and T Hunter Eds pp 315ndash324 Elsevier Science 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Lactobacilli exert strong antagonistic activity againstmany microorganisms including food spoilage organismsand pathogens Bacteriocins are antimicrobial proteinaceouscompounds that are inhibitory towards sensitive strains andare produced by bothGram-positive andGram-negative bac-teria [16ndash20] Bacteriocins have widespread potential appli-cations in preservation for improving the safety and qualityof foods At present there are many antibiotic-resistantpathogens therefore it is necessary to identify develop orredesign antibiotics to fight themultidrug resistance bacteriaUp to now bacteriocins hold great promise as the nextgeneration of antimicrobials Beside facing drug resistancethe diseases which are due to the ldquooxidative stressrdquo are thechallenges that should be prevented

Oxidant stress is initiated by free radicals which pro-duced aerobic metabolism in the body and can cause oxida-tive damage of biological macromolecules such as proteinslipids and DNA in healthy human cells [21ndash23] Thesechanges contribute to oxidative stress that is among themajorcausative factors in the induction ofmany chronic and degen-erative diseases including atherosclerosis ischemic heartdiseases and diabetes mellitus cancer immunosuppressionneurodegenerative disease ageing [24ndash28] coronary heartdisease and Alzheimerrsquos disease [29ndash32] All human cellsprotect themselves against free radical damage by enzymessuch as superoxide dismutase and catalase or compoundssuch as ascorbic acid tocopherol and glutathione [33]However these protective mechanisms occurred by variouspathological processes and antioxidant supplements are nec-essary to combat oxidative stressTherefore the interest in thenatural compounds with strong antioxidant and anticancerproperties has steadily been increasing

From the above stated reasons the TTX-producing lacticacid bacteria were isolated in puffer fish and then optimizedthe ability of TTX production of bacteria in media preparedfrom freshwater fishes (Pangasius bocourti Oreochromis sp)and sea fish (Auxis thazard) Then this strain was usedto study antimicrobial radical scavenging and anticanceractivities to find out the potential lactic acid bacteria forpharmaceutical science

2 Materials and Methods

21 Sample Preparation and Bacteria Cultures Puffer fisheswere collected at the sea in the center of Vietnam Nospecific permit was required for the described field studiesThe female fish weight was around 600 g kept in ice andtransported to the lab The female fishes were washed with70 and dissected to isolate the ovaries from abdomen bysterilized knife and scissor The ovaries (3 g) were homog-enized carefully and incubated directly in modified MRSbroth Samples were incubated for 48 hours in 5 CO

2

at room temperature Then the cultures were checked formicroorganims on MRS agar

22 Isolation and Identification of Enterococcus faeciumOne mL of culture was aseptically spreaded on MRS agar

plates and was incubated at 25∘C for 3ndash5 days Eventu-ally the forming colonies were transferred onto anothernew MRS agar plates until discrete colonies were obtainedEach discrete colony was further subcultured for severaltimes to ensure that a pure culture was obtained The purecultures were also characterized using bioassay for toxicitytest The toxin was extracted and purified for thin layerchromatography and high performance liquid chromatog-raphy Then the toxic strain was identified by biochemicalcharacterization based on the ability of utilization of differentcarbon sources by API 50CHL (bioMerieux Lyon France)Thenmolecular characterization using 16S rRNA sequencinganalysis was performed The forward primer was faec1 (51015840-ttggagagtttgatcctggctcaggac-31015840) and the reverse primer wasfaec2 (51015840-gctgctggcacgtagttagccgtggct-31015840) The PCR reactionwas performed as follows 95∘C for 5min 30 cycles of 95∘C for20 s 50∘C for 20 s and 72∘C for 3min and a final extensionat 72∘C for 10min The PCR product was stored at 4∘C ThePCR product was purified and sequenced The homologycomparison of the almost 16S rRNA gene sequence wasperformed using the NCBI BLAST database

23 Extraction and Purification of Tetrodotoxin from Bac-terial Cultures The isolation and purification proceduresof TTX from bacterial cultures were basically the same asthose used for puffer fish tissues Each broth culture wascentrifuged at 4000 rpm for 15min to remove the bacterialdebris The supernatant was mixed with an equal volume ofwater-washed activated charcoal under agitation and filteredthrough a Buchner funnel The charcoal on the funnel wasthoroughly washed with distilled water and TTX was elutedfor 3 times with 3 volumes of 1 acetic acid in 20 aqueousethanol [10 17 34 35] The eluate was heated at 100∘C for10min and then cooled and centrifuged to separate debrisThe eluatewas evaporated freeze-dried and then dissolved in10mL of 003M acetic acid for gel filtration Eluted TTX wasused for further toxicity bioassay thin layer chromatographyand high performance liquid chromatography

24 High Performance Liquid Chromatography (HPLC) forTetrodotoxin Detection The extracted samples were appliedto HPLC in order to detect and quantify the TTX existenceby UV detector The Shimadzu HPLC system with RP 18(250 times 46mm 5120583m) column was used as stationary phaseThe mobile phase was prepared by mixing of 001M sodiumheptanesulfonic and methanol at ratio 90 10 The flow ratewas 1mLmin at 30∘C wavelength was 205 nmThe authenticTTX (Tocris UK) at concentration 02mgmL was applied

25 Bioassay for Toxicity Detection Mice were injected intra-peritoneally with extraction [36] The toxicity was deter-mined by the death time of each injected mouse The deathtime was recorded at the grasping breath of mouse It wasrecommended not to use weight more than 23 g and reusemice

26 Thin Layer Chromatography (TLC) for TetrodotoxinDetection TLC analysis method was carried out [34] with












(1 minus 119860119904)

119860119888

times 100 (1)

where 119860119904and 119860


respectively









Purifi

ed-T

Oreochrom

is-T

Auxis-

T

Pangasius-

T

MRS

-T

Media

05

1015202530354045

Dea

th ti

me (

min

)


1 2 3










13376

15 2010

(min)

(a)

13494

15339

15 2010

(min)

(b)












Cand

ida

albicans

Salm

onella

typhim

urium

Pseudomonas

aeruginosa

Staphylococcus

sciuri

Staphylococcus

aureus

Micrococcus

luteus

Pathogen

24h32h

48h72h

0

5

10

15

20

25

30

Dia

met

er o

f inh

ibiti

on zo

ne (m

m)





1 2 3 4 5 24

Time (h)

0

20

40

60

80

100

120

Viab

ility

()

15

2

25

3

4

5

65

75

8

85













4 Conclusions






Acknowledgment


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












(1 minus 119860119904)

119860119888

times 100 (1)

where 119860119904and 119860


respectively









Purifi

ed-T

Oreochrom

is-T

Auxis-

T

Pangasius-

T

MRS

-T

Media

05

1015202530354045

Dea

th ti

me (

min

)


1 2 3










13376

15 2010

(min)

(a)

13494

15339

15 2010

(min)

(b)












Cand

ida

albicans

Salm

onella

typhim

urium

Pseudomonas

aeruginosa

Staphylococcus

sciuri

Staphylococcus

aureus

Micrococcus

luteus

Pathogen

24h32h

48h72h

0

5

10

15

20

25

30

Dia

met

er o

f inh

ibiti

on zo

ne (m

m)





1 2 3 4 5 24

Time (h)

0

20

40

60

80

100

120

Viab

ility

()

15

2

25

3

4

5

65

75

8

85













4 Conclusions






Acknowledgment


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Purifi

ed-T

Oreochrom

is-T

Auxis-

T

Pangasius-

T

MRS

-T

Media

05

1015202530354045

Dea

th ti

me (

min

)


1 2 3










13376

15 2010

(min)

(a)

13494

15339

15 2010

(min)

(b)












Cand

ida

albicans

Salm

onella

typhim

urium

Pseudomonas

aeruginosa

Staphylococcus

sciuri

Staphylococcus

aureus

Micrococcus

luteus

Pathogen

24h32h

48h72h

0

5

10

15

20

25

30

Dia

met

er o

f inh

ibiti

on zo

ne (m

m)





1 2 3 4 5 24

Time (h)

0

20

40

60

80

100

120

Viab

ility

()

15

2

25

3

4

5

65

75

8

85













4 Conclusions






Acknowledgment


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


13376

15 2010

(min)

(a)

13494

15339

15 2010

(min)

(b)












Cand

ida

albicans

Salm

onella

typhim

urium

Pseudomonas

aeruginosa

Staphylococcus

sciuri

Staphylococcus

aureus

Micrococcus

luteus

Pathogen

24h32h

48h72h

0

5

10

15

20

25

30

Dia

met

er o

f inh

ibiti

on zo

ne (m

m)





1 2 3 4 5 24

Time (h)

0

20

40

60

80

100

120

Viab

ility

()

15

2

25

3

4

5

65

75

8

85













4 Conclusions






Acknowledgment


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cand

ida

albicans

Salm

onella

typhim

urium

Pseudomonas

aeruginosa

Staphylococcus

sciuri

Staphylococcus

aureus

Micrococcus

luteus

Pathogen

24h32h

48h72h

0

5

10

15

20

25

30

Dia

met

er o

f inh

ibiti

on zo

ne (m

m)





1 2 3 4 5 24

Time (h)

0

20

40

60

80

100

120

Viab

ility

()

15

2

25

3

4

5

65

75

8

85













4 Conclusions






Acknowledgment


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Conclusions






Acknowledgment


References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article biological activities of tetrodotoxin-producing enterococcus … · 2019. 7....

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