J. Life Sci. Biomed. 6(1): 10-14, Jan 30, 2016  
JLSB  
© 2016, Scienceline Publication  
Journal of  
ISSN 2251-9939  
Life Science and Biomedicine  
Antimicrobial Activity of Bacillus cereus and Bacillus thuringiensis on  
Pathogenic Vibrio harveyi in Litopenaeus vannamei  
  
Marissa Maya Masitoh1 , A. M. Hariati2 and M. Fadjar2  
1Master Program on Faculty of Fishery and Marine Science, University of Brawijaya, Malang 65145, Indonesia  
2Department of Aquaculture, University of Brawijaya, Malang 65145, Indonesia  
ABSTRACT: The bacterial strain of Bacillus cereus and Bacillus thuringiensis has been known to produce  
antimicrobial activity against pathogenic Vibrio harveyi. The effect of B. cereus and B. thuringiensis were tested  
by in vitro and in vivo. In vitro test was used to analyze antagonism characteristic of bacteria using the paper  
disc diffusion method. In vivo test was applied to evaluate antimicrobial activity of B. cereus and B.  
thuringiensis (105 CFU ml-1) on survival rate and histopathology of Litopenaeus vannamei challenged with V.  
harveyi. The results showed that B. thuringiensis had a greater inhibitory activity of 18.60–35.97 mm. Both  
Bacillus bacteria treatment resulted in survival rate of 100%, compared with 75% in the treatment without  
Bacillus. It can be concluded that B. cereus and B. thuringiensis have potential applications for controlling  
pathogenic V. harveyi in shrimp aquaculture.  
Key words: Bacillus cereus, Bacillus thuringiensis, Inhibitory Activity, V. harveyi.  
INTRODUCTION  
Aquaculture is the world’s fastest growing food production sector. However, disease outbreaks have caused  
serious economic losses in several countries. Vibrio species are among the most important bacterial pathogens of  
cultured shrimp. They are responsible for several diseases and mortalities up to 100% due to vibriosis have been  
reported [1]. Using antibiotics in potential negative consequences of using antibiotics in aquaculture for the  
prophylactic treatment of diseases are the development of drug resistant bacteria and reduced efficacy of  
antibiotic treatment for human and animal diseases [2].  
In the search for more effective and environmentally friendly treatments, using bacteria like Bacillus  
provides a solution to these problems. Bacillus have antimicrobial (bacteriocin) which usually occurs in all  
growth phases and finishes at the end of logarithmic phase [3]. Luis-Villaseñor et al. [4] investigated that the  
effect of Bacillus showed trait inhibitory to Vibrio and ability to adhere and grow on intestinal mucus.  
The purpose of this study was to investigate antimicrobial activity of B. cereus and B. thuringiensis against  
Vibrio harveyi under in vitro and in vivo conditions.  
MATERIAL AND METHODS  
Bacterial strains  
A virulent strain of V. harveyi, was used as a pathogenic strain. Strain were taken from the stock culture  
collection of our laboratory. B. cereus and B. thuringiensis, obtained from isolated from the gastrointestinal of L.  
vannamei and cultured in duplicate in the general media (nutrient agar with 1.5% w/v NaCl) for 18-24 hours at a  
temperature of 30oC. Pure isolates were taken after subculture on Tryptic Soya Agar (TSA) was used as an  
antagonistic strain.  
Antagonism assay  
The methods of paper disc diffusion assays were used in this study. Both groups of the bacterial strains (the  
tested strains including the compared strain and the pathogenic strain) were briefly grown in tryptic soya broth  
(TSB), incubated at 32oC for 24 h. After 24 h, each sterilised paper disc was immersion with B. cereus and B.  
thuringiensis with different concentration (104, 105 and 106 CFU ml-1). And then placed on the surface of an agar  
plate (TSA) which was previously inoculated with the indicator pathogen at a concentration of about 107 CFU  
ml-1. The plate was then incubated at 32o C for 24 h and the inhibition zone around paper disc was recorded.  
To cite this paper: Masitoh M. M., Hariati A. M. and Fadjar M. 2016. Antimicrobial Activity of Bacillus cereus and Bacillus thuringiensis on Pathogenic Vibrio harveyi  
in Litopenaeus vannamei. J. Life Sci. Biomed. 6(1): 10-14.  
Journal homepage: www.jlsb.science-line.com  
10  
SDS-PAGE electrophoresis test for molecular weight  
For detection of bacteriocin activity use polyacrylamide gels and stained with silver nitrate to determine the  
molecular weight of the separated protein. Low molecular mass standards ranging from 10 to 225 kDa were  
purchased.  
Inhibition of pathogenic V. Harveyi, in vivo  
L. vannamei were introduced into 5 treatment (each in tripled) filled with filtered seawater at salinity of 20  
ppt. The set A was inoculated with B. cereus at the concentrations of 105 CFU ml-1 in rearing medium to facilitate  
attachment or colonization on the larvae for 24 hours. After one day was then exposed to test pathogen (V.  
harveyi) at 107 CFU ml-1 for 2 hour. The set B was inoculated with B. thuringienis at the concentrations of 105 CFU  
ml-1. After one day was then exposed to test pathogen (V. harveyi) at 107 CFU ml-1 for 2 h. The set C was inoculated  
with B. cereus and B. thuringiensis (combined treatment) at the concentrations of 105 CFU ml-1. After one day was  
then exposed to test pathogen (V. harveyi) at 107 CFU ml-1 for 2 h. The set K(n) received no bacterial inoculums  
and served as control. The set K(-) was inoculated with pathogen alone at 107 CFU ml-1 in rearing medium to serve  
as negative control. The number of CFU ml-1 in overnight culture of baceria were standardized from OD  
measurements at 600 nm. Mortality in each set was recorded for twelve days and no water exchange was done  
during that period.  
Statistical Analysis  
Results were presented as mean ± mean standard deviation of three replicates. Statistical analysis was  
performed using SPSS 20.0. To determine the effect of treatment on the response of each parameter used ANOVA  
analysis followed by Duncan's multiple range test. Levels of significance are expressed as P < 0.05.  
RESULTS AND DISCUSSION  
Antagonism assay  
Antimicrobial activity of B. cereus and B. thuringiensis against V. harveyi were performed by paper disc with  
different concentrations. The diameters of the inhibitory zones of B. thuringiensis was 18.60–35.97 mm (Table 1).  
We suggested that B. thuringiensis showed a greater inhibitory activity than others. The antagonistic effect of  
these isolates on the growth of indicator pathogen could be determined by the appearance of clear inhibition  
zones around the paper disc. Bacillus species could produce a large number of antimicrobials [3].  
Table 1. Antagonistic activity of B. cereus and B. thuringiensis strains against V. harveyi  
Inhibition zones (mm)  
Bacteria  
104  
105  
106  
B. cereus  
16.47±0.67  
18.60±1.06  
13.79±0.81  
22.80±1.14  
25.13±1.07  
20.81±1.47  
31.70±1.96  
35.97±1.58  
25.46±0.64  
B. thuringiensis  
B. cereus + B. thuringiensis  
SDS-PAGE electrophoresis test for molecular weight  
Direct detection of the bacteriocin was performed by SDS-PAGE. Following electrophoresis, several  
contaminating proteins were detected in sample. The band had an apparent molecular mass of about 10-225 kDa.  
Many other antimicrobial polypeptides of intermediate size (10–30 kDa) and other large antimicrobial  
proteins produced by Abriouel et al. [5]. The results (Fig. 1) showed that B. cereus had molecular weights of 15.53  
kDa and 25.21 kDa while B. thuringiensis had molecular weights of 12.19 and 23.25 kDa which there are  
antimicrobial polypeptides of intermediate size.  
Inhibition of pathogenic V. harveyi in vivo  
Survival rate: The results revealed that B. cereus and B. thuringiensis lead to reduce shrimp mortality under  
in vivo conditions. Both Bacillus treatments resulted 100 % survival rate of shrimp, while survival rate of 75%  
was obtained in not treated shrimp (Figure 2). Bacillus significantly increased survival rate and some digestive  
enzyme activities of shrimp larvae [6].  
To cite this paper: Masitoh M. M., Hariati A. M. and Fadjar M. 2016. Antimicrobial Activity of Bacillus cereus and Bacillus thuringiensis on Pathogenic Vibrio harveyi  
in Litopenaeus vannamei. J. Life Sci. Biomed. 6(1): 10-14.  
Journal homepage: www.jlsb.science-line.com  
11  
Figure 1. SDS-PAGE analysis protein profile. Line (M): Silver nitrate-stained gel, low range protein standard; line  
(A1 and A2) protein profile B. cereus; line (B1 and B2) protein profile B. thuringiensis.  
Figure 2. Survival rate recorded during twelve days  
Total Haemocyte Counts (THC) : The total haemocyte counts showed significant differences among the  
treatments. The average of total haemocyte was 5.93 x 105 sel mm-3 – 89.17 x 105 sel mm-3 (Table 2). THC of  
crustaceans plays an important role in regulating the physiological functions including hardening of exoskeleton,  
wound repair, carbohydrate metabolism, transport and storage of protein and amino acid, haemolymph  
coagulation and the confinement of invasive organisms by clot formation, phagocytosis, and encapsulation [7].  
Table 2. Total haemocyte counts among different treatments  
Treatment  
THC (x 105 sel mm-3)  
12.75 ± 0.75d  
15.73 ± 0.75a  
37.3 ± 1.53b  
K (n)  
K (-)  
A
B
89.17 ± 3.75a  
5.93 ± 0.12c  
C
Fig K(n) : not infected with V. harveyi bacteria; Fig K(-): Infected with 107 CFU/ml V. harveyi bacteria; Fig A: Adding B. cereus and infected V. harveyi;Fig B: Adding  
B. thuringiensis and infected V. harveyi; Fig C: Adding B. cereus and B. thuringiensis than infected V. harveyi.  
THC in crustaceans rapidly drops following the injection of foreign material. B. cereus and B. thuringiensis in  
L. vannamei can be increased significantly after the injection of V. harveyi. This study demonstrated promising  
To cite this paper: Masitoh M. M., Hariati A. M. and Fadjar M. 2016. Antimicrobial Activity of Bacillus cereus and Bacillus thuringiensis on Pathogenic Vibrio harveyi  
in Litopenaeus vannamei. J. Life Sci. Biomed. 6(1): 10-14.  
Journal homepage: www.jlsb.science-line.com  
12  
results for immune response stimulation in L. vannamei. Moreover, Rengpipat et al. [8] explained that Bacillus S11  
surface antigens, or their metabolites might act as immunogens for shrimp immune defense. Bacillus S11 cell wall  
peptidoglycan might elicit an immune function in shrimps.  
Histopathology of Hepatopancreas  
The hepatopancreas of control K(n) maintained a good shape and colour. The hepatopancreas of infected  
larvae were grey and showing vacuolization (Figure 3). The hepatopancreas or digestive gland is a sensitive  
indicator of diverse physiological states, such as metabolic level, ecdysis phase, nutritional status, disease, etc., so  
that its use as a general indicator of the physiological condition of the shrimp has been proposed [9].  
Figure 3 detected the sites of bacterial presence in the tissue, and the changes caused by the pathogens could  
be evaluated. Obviously, death will occur when a sufficiently large number of hepatopancreatic cells are damaged  
by the pathogen, rendering the organ non functional. Robertson et al. [10] explained that the hepatopancreas of  
chronically infected larvae were grey and aciated, with balls of necrotic tissue. Shrimp with infected by virus or  
bacteria, pathology of hepatopancreas showing severe necrosis, loss of structure, atrophy of tubule epithelial  
cells, vacuolation and rounding and sloughing of cells into the lumen [11].  
K(n)  
K(-)  
V
L
A
B
C
Figure 3. Histopathology of L. vannamei hepatopancreas (arrows indicated vacuolation)  
Fig K (n): Hepatopancreas of control shrimps group (not exposed to V. harveyi bacteria ); Fig K (-): Infected hepatopancreas of shrimps  
exposed to 107 CFU/ml V. harveyi bacteria. There are many vacuolation of cells (black arrow destruction of hepatopancreas tissue); Fig A:  
Adding B. cereus and infected V. harveyi. Some of tissues are healthy (stars/L) also vacuolation is observed; Fig B: Adding B. thuringiensis and  
infected V. harveyi. Some of tissues are healthy (stars/L) also vacuolation is observed but low than Fig A; Fig C: Adding B. cereus and B.  
thuringiensis and infected V. harveyi. There are many vacuolation of cells (black arrow), cells are finally destroyed. L (lumen) and V  
(vacuolation).  
To cite this paper: Masitoh M. M., Hariati A. M. and Fadjar M. 2016. Antimicrobial Activity of Bacillus cereus and Bacillus thuringiensis on Pathogenic Vibrio harveyi  
in Litopenaeus vannamei. J. Life Sci. Biomed. 6(1): 10-14.  
Journal homepage: www.jlsb.science-line.com  
13  
Acknowledgments  
The authors would like to thank Dr. Ating Yuniarti for providing the B. cereus, B. thuringiensis and V. harveyi  
strains.  
Competing interests  
The authors declare that they have no competing interests.  
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To cite this paper: Masitoh M. M., Hariati A. M. and Fadjar M. 2016. Antimicrobial Activity of Bacillus cereus and Bacillus thuringiensis on Pathogenic Vibrio harveyi  
in Litopenaeus vannamei. J. Life Sci. Biomed. 6(1): 10-14.  
Journal homepage: www.jlsb.science-line.com  
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