5. c4. control of pathogenic vibrio spp. by bacillus subtilis

8/2/2019 5. C4. Control of Pathogenic Vibrio Spp. by Bacillus Subtilis

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Control of pathogenic Vibrio spp. by Bacillus subtilis BT23,a possible probiotic treatment for black tiger shrimpPenaeus monodon

B. Vaseeharan and P. RamasamyDepartment of Biotechnology, Life Sciences Building, University of Madras, Guindy Campus, Chennai, India

2002 170: received 6 June 2002, revised 22 October 2002 and accepted 1 November 2002

ABSTRACT

B . V A S E E H A R A N A N D P . R A M A S A M Y . 2 003.

Aims: The present study evaluated the in vitro and in vivo antagonistic effect of Bacillus against the pathogenic

vibrios.Methods and Results: Cell-free extracts of Bacillus subtilis BT23 showed greater inhibitory effects against the

growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon with black gill disease. The

probiotic effect ofBacillus was tested by exposing shrimp to B. subtilis BT23 at a density of 106)108 cfu ml)1 for 6 d

before a challenge with V. harveyi at 103)104 cfu ml)1 for 1 h infection. The combined results of long- and short-

term probiotic treatment of B. subtilis BT23 showed a 90% reduction in accumulated mortality.

Conclusions: This study reports that pathogenic vibrios were controlled by Bacillus under in vitro and in vivo

conditions.

Significance and Impact of the Study: Results indicated that probiotic treatment offers a promising alternative

to the use of antibiotics in shrimp aquaculture.

Keywords: Bacillus, P. monodon, probiotics, shrimp disease, Vibrio.

INTRODUCTION

Aquaculture is the worlds fastest growing food production

sector, with cultured shrimp and prawn growing at an annual

rate of 168% between 1984 and 1995 (Subasinghe et al.

1998). However, disease outbreaks have caused serious

economic losses in several countries. According to a recent

World Bank report, global losses due to shrimp diseases are

around US$ 3 billion (Lundin 1996). Vibrio species occur asthe dominant flora in various developmental stages ofPenaeus

monodon and have been described as the causal pathogens

(Lightner 1996; Sung et al. 2001). Potential negative conse-

quences of using antibiotics in aquaculture for the prophy-

lactic treatment of diseases are the development of drug-

resistant bacteria and reduced efficacy of antibiotic treatment

for human and animal diseases (Moriarty 1997). Increased

concern about antibiotic-resistant micro-organisms (Amabile

et al. 1995) has led to suggestions of alternative disease

prevention methods, including the use of non-pathogenic

bacteria as probiotic biocontrol agents. (Austin et al. 1995;

Moriarty 1997). Lactic acid bacteria have been tested as

probiotics in warm-blooded animals and attempts have been

made to use lactic acid bacteria as antagonists (probiotics) of

shrimp pathogens (Gatesoupe 1999; Skjermo and Vadstein

1999). Bacillus spores have been used as biocontrol agents toreduce vibrios in shrimp culture facilities (Skjermo and

Vadstein 1999; Rengipipat et al. 2000). Bacillus constitutes a

large part of the microflora of the gills, skin and intestinal

tracts of shrimps (Sharmila et al. 1996). Bacillus spp. are

often antagonistic against other micro-organisms, including

fish and shellfish pathogenic bacteria (Gatesoupe 1999;

Rengipipat et al. 2000). The present study investigated the

inhibitory activity of Bacillus subtilis BT23, isolated from

shrimp culture ponds, against pathogenic Vibrio harveyi

under in vitro and in vivo conditions.

Correspondence to: Dr B. Vaseeharan, Genetics and Biotechnology Division, Central

Institute of Brackish Water Aquaculture, 75 Santhome High Road, R.A. Puram,

Chennai 600 028, India (e-mail: [email protected]).

2003 The Society for Applied Microbiology

Letters in Applied Microbiology 2003, 36, 8387


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MATERIALS AND METHODS

Bacterial strains

A virulent strain of V. harveyi, obtained from P. monodon

with black gill disease (LD50 value 105 cfu ml)1 under

experimental infection of P. monodon juveniles), was used asa pathogenic strain and B. subtilis BT23, obtained from

shrimp culture ponds, was used as an antagonistic strain.

Bacillus BT21, Bacillus BT22 and B. subtilis BT23 were

isolated from shrimp culture ponds. The bacteria used in

this study were identified using standard morphological,

physiological and biochemical plate and tube tests (Holt

et al. 1994). All strains were taken from the stock culture

collection of our laboratory and had been stored in Luria-

Bertani (LB) broth cultures with sterile glycerol (15% v v).

Antagonism assay

The initial screening of antagonism by Bacillus BT21, Bacillus

BT22 and B. subtilis BT23 was by the agar well diffusion plate

assay method (Bauer et al. 1966). Vibrio harveyi, V. anguil-

larum, V. vulnificus and V. damsela were precultured in LB

broth incubated at 28 C for 2 d and 50 ll of this culture were

spread over the agar plates. Bacillus spp. culture (3 d old) was

centrifuged at 9600 rev min)1 for 15 minand the supernatant

fluid filtered through a 022-lm membrane filter (Sartorius,

Bedford, MA, USA) to obtain cell-free extracts (culture

supernatant fluid). A volume (100 ll) of Bacillus cell-free

extracts was introduced into the wells of the agar medium and

incubated for a period of 2448 h at 28 C. Antibacterial

activity was defined as the diameter (mm) of the clear

inhibitory zone formed around the well.

Co-culture experiments

Bacillus subtilis BT23 and V. harveyi were precultured

separately in LB broth at 28 C for 3 d. Vibrio harveyi was

inoculated into LB broth at an initial cell density of approx.

103 cfu ml)1, whereas the initial levels of B. subtilis BT23

were 105, 107, 108 and 109 cfu ml)1. All combinations were

performed in triplicate. The co-culture plates were incuba-

ted at 28 C and samples withdrawn daily for the deter-

mination of V. harveyidensities. The numbers of V. harveyiwere estimated by preparing 10-fold serial dilutions and

01 ml from each dilution was inoculated into thiosulphate

citrate bile salts sucrose agar plates.

Effect of Bacillus subtilis BT23 cell-free extracts

Bacillus subtilis BT23 was precultured in LB broth and then

used to inoculate 50 ml of LB broth in the same four

combinations at an initial cell density of 104)108 cfu ml)1.

The flasks were incubated at 28 C with agitation (200

rev min)1) and samples withdrawn daily and the number of

cfu determined. Sterile filtered supernatant fluid (2 ml) was

tested by adding 1 ml of supernatant fluid to 1 ml of fresh

LB broth in test tubes and inoculating it with 100 ll of

V. harveyi in LB broth, yielding approx. 104 cfu ml)1.

Controls were made by inoculating V. harveyi (100 ll) in2 ml of LB broth without B. subtilis BT23 cell-free extracts.

Each combination was tested in triplicate and the growth of

the V. harveyi monitored by recording the optical density at

600 nm with a spectrophotometer.

Experimental infection of shrimp and Bacillus

subtilis BT23 treatment

Bacillus subtilis BT23 was grown for 3 d at 28 C

(150 rev min)1) in LB broth and V. harveyi was grown for

24 h in tryptone soy broth. Two hundred P. monodon, each

approx. 56 g, were divided equally into eight groups, eachhoused in a 300-l tank. Four of the tanks were treated with

B. subtilis BT23 for 5 d at a level of 106 cfu ml)1 at 28 C

(long-term treatment) by adding the bacteria to the water.

After 5 d, shrimp in all of the four tanks were infected with

V. harveyi (104)106 cfu ml)1) for 1 h and two of the tanks

were again treated with B. subtilis BT23 (106 cfu ml)1) for

1 h along with V. harveyi (combined treatment).Of the

remaining four tanks, two were treated with B. subtilis BT23

(106 cfu ml)1) for 1 h and the animals were then transferred

to a tank in which V. harveyi (104)106 cfu ml)1) was

maintained for 1 h (short-term treatment); the other two

tanks were infected with V. harveyi (104)106 cfu ml)1)

alone for positive control. The cumulative mortality of the

shrimp was recorded and analysed using analysis of variance.

RESULTS

Antagonism assay

The cell-free extract of Bacillus BT21, Bacillus BT22 and

B. subtilis BT23 showed inhibitory activity against Vibrio

spp. Of these, B. subtilis BT23 showed a higher inhibitory

activity than the other two Bacillus spp. tested. BT23

showed inhibitory activity against 112 Vibrio spp., V. harveyi

(39 isolates), V. anguillarum (24 isolates), V. vulnificus(30 isolates) and V. damsela (19 isolates) obtained from

P. monodon culture hatcheries and ponds (Table 1). The

diameters of the inhibitory zones around the growth of

Vibrio spp. were about 36 mm (Fig. 1).

Co-culture experiments

The growth of pathogenic V. harveyi was inhibited by

B. subtilis BT23 culture inoculated at an initial level of

105)109 cfu ml)1 (Fig. 2). Lower concentrations of

84 B . V A S E E H A R A N A N D P . R A M A S A M Y

2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 8387


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B. subtilis BT23 (105 and 107 cfu ml)1) allowed initial

growth of V. harveyi, but cfu densities never reached the

level of the control. High concentrations (109

cfu ml)1

) ofB. subtilis BT23 allowed an initial increase of V. harveyi

followed by a decrease in the total viable counts (Fig. 2).

Co-culture experiment results showed that, when the

concentration of B. subtilis BT23 increased, the growth of

V. harveyi was controlled under in vitro conditions.

Effect of cell-free extracts of Bacillus subtilis BT23

Cell-free extracts of B. subtilis BT23 inhibited the growth of

V. harveyi in liquid culture under aerobic conditions. The

inhibitory efficiency was high in B. subtilis BT23 cell-free

extracts of 108 cfu ml)1 and low in 104 cfu ml)1. Bacillus

subtilis BT23 cell-free extracts did not restrict the growth of

V. harveyi for 2 d and after that the growth was remarkably

controlled (Fig. 3) when compared with the growth of

V. harveyi without B. subtilis BT23.

Experimental infection of shrimp and probiotictreatment

The studies on the probiotic treatment and infection of

shrimp revealed that the mortality of shrimp by V. harveyi

infection was reduced by B. subtilis BT23 strains under

in vivo conditions. The cumulative mortality of infected

P. monodon not treated with B. subtilis BT23 reached 50%

on the 9th day after infection with V. harveyi and 100% on

the 17th day. However, in the case of probiotic treatment

Table 1 Inhibitory activity of Bacillus subtilis BT23 against Vibrio harveyi, V. anguillarum, V. vulnificus and V. damsela obtained from Penaeus

monodon culture hatcheries and ponds

B. subtilis with inhibitory effects against

Source of Vibrio spp. V. harveyi isolates V. anguillarum isolates V. vulnificus isolates V. damsela isolates

Vibriosis-infected post-larval P. monodon 7 3 12 4

MBV-infected post-larval P. monodon 9 6 11 8

Black gill-diseased P. monodon juveniles 12 7 4 3

WSSV-infected P. monodon gills 10 8 3 4

Total 39 24 30 19

MBV, Monodon baculovirus; WSSV, white spot syndrome virus.

Fig. 1 Petri dishes containing cell-free extract of (a) Bacillus BT21,

(b) Bacillus BT22 and (d) Bacillus subtilis BT23 showed inhibitory

zones against the growth of (A) Vibrio harveyi, (B) V. anguillarum and

(C) V. damsela. No inhibitory zone was found in the control (c). Note

the Bacillus subtilis BT23 (d) showing the greatest inhibitory zones. The

strain was identified as B. subtilis BT23 and used for further in vivo and

in vitro studies

0

1

2

3

4

5

6

7

8

9

10

Time at 30 C (d)

Log(cfuml1)

1 2 3 4 5 6 7

Fig. 2 Growth pattern of Vibrio harveyi at 28 C with and withoutBacillus subtilis BT23 at different initial concentrations (colony-forming

units; cfu). r, Without B. subtilis; j, B. subtilis 105 cfu ml)1;

m, B. subtilis 107 cfu ml)1;s, B. subtilis 108 cfu ml)1 andd, B. subtilis

109 cfu ml)1

E F F E C T O F B A C I L L U S O N S H R I M P P A T H O G E N I C V I B R I O S 85



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groups, the mortality levelled off after 5 d when the

cumulative mortality was 10% in the combined treatment

(Fig. 4). Both the long- and short-term treatments with

B. subtilis BT23 caused a decrease in cumulative mortality,

to 32 and 60%, respectively. No mortality was found in

control tanks which were not exposed to V. harveyi. The

effect of probiotic treatment was most pronounced during

the first day of infection.

DISCUSSION

The present study showed that the growth of pathogenic V.

harveyi was controlled by non-pathogenic B. subtilis BT23

under in vivo and in vitro conditions. The control of fish and

shellfish pathogenic Vibrio, particularly using non-patho-

genic bacterial strains and disease prevention, has received

much attention during the last decade (Sugita et al. 1998;

Rengipipat et al. 2000). Fuller (1989) defined a probiotic as a

live microbial feed supplement which benefits the host

animal by improving its intestinal microbial balance.

Co-culture experiments showed that the inhibitory activity

of B. subtilis BT23 increased with increasing density of the

antagonist. A high concentration of B. subtilis BT23

(antagonist) was required to inhibit V. harveyi in the

co-culture experiments. The present study showed that the

antagonist must be present at significantly higher levels than

the pathogen and the degree of inhibition increased with the

level of antagonist. During the co-culture, 107)

109 cfu ml)

1

were required to inhibit the growth of the pathogen

V. harveyi. Therefore, a potential probiotic co-culture must

either be supplied on a regular basis or be able to colonize

and multiply on or in the host. The Bacillus spp. used as

probiotics for terrestrial livestock are of telluric origin and

are not autochthonous in the gastrointestinal tract but they

may be active during intestinal transit (Gouthier et al. 1994).

Kennedy et al. (1998) isolated a strain of B. subtilis from

common snook (Centropomus undecimalis). The inoculation

of this strain into the rearing water resulted in the apparent

elimination of Vibrio spp. from the snook larvae. Smith and

Davey (1993) reported that Pseudomonas fluorescens reduceddiseases caused by Aeromonas solmonicida in fish. Austin

et al. (1995) also observed a similar phenomenon, that

V. alginolyticus, used as a probiotic strain, reduced the

diseases caused by Aerom. solmonicida, V. anguillarum and

V. ordalli in P. monodon. Maedo and Liao (1992) reported

the use of a soil bacterial strain, PM-4, that promoted the

growth of P. monodon nauplius, probably acting as a food

source. This strain also showed an in vitro inhibitory effect

against V. anguillarum. Rengipipat et al. (1998) reported that

inoculation of Bacillus S11, a saprophytic strain, resulted in

greater survival of the post-larval P. monodon that were

challenged by pathogenic luminescent bacterial culture.

These works strongly suggest the effective control of

microflora in fish and shellfish in culture environments by

antibiotic-producing bacteria. Purification and characteriza-

tion of the antibacterial substance would help to understand

the mechanism of antibacterial activity of Bacillus strains.

Probiotic treatment offers a very promising alternative to

the use of antibiotics in fish and shrimp aquaculture. Further

study is needed to elucidate the exact mode of action of the

observed beneficial effects and to understand the possibilities

and limitations of microbial control in aquaculture.

0

01

02

03

04

05

06

07

08

09

1

1 2 3 4 5

Time at 30 C (d)

Opticaldensity(600nm)

Fig. 3 Growth of Vibrio harveyi at 28 C with cell-free extracts of

Bacillus subtilis BT23 extracted by different cell densities. With

B. subtilis cell-free extracts of: r, 104

cfu ml)1

; j, 105

cfu ml)1

;

m, 106 cfu ml

)1; s, 107 cfu ml

)1 and d, 108 cfu ml

)1

20

0

20

40

60

80

100

1 2 4 5 6 7 8 9 10 11 12 13 14 15

Time after infection (d)

Averageaccu

mulatedmortality(%)

3

Fig. 4 Cumulative mortality of Penaeus monodon juveniles infected

with Vibrio harveyi with and without probiotic treatment of Bacillus

subtilis BT23. s, Control; m, long-term treatment; j, short-term

treatment and r, combined treatment

86 B . V A S E E H A R A N A N D P . R A M A S A M Y



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ACKNOW LEDGEMENTS

The authors thank Dr Junda Lin (Florida Institute of

Technology, Melbourne, FL, USA) for his comments on an

earlier version of the manuscript.

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5. c4. control of pathogenic vibrio spp. by bacillus subtilis

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