Phylogenetic analysis to compare populations

of acid tolerant bacteria isolated from the

gastrointestinal tract of two different prawn
species Macrobrachium rosenbergii and
Penaeus monodon
1 1 1 1
1
FelixFeliatra, Iesje
1
Lukistyowati, 2Dessy Yoswaty, Haqqy
3
Rerian,
Deasy4 Melina, Wahid Hasyim, Titania T. Nugroho, Andi R.
Fauzi, Rofiza Yolanda

1
Laboratory of Marine Microbiology, Faculty of Fisheries and Marine Sciences, University
2
of Riau, Pekanbaru 28286 Riau Province, Indonesia; Laboratory of Enzyme,
Fermentation and Biomolecular, Department of Chemistry, Faculty of Mathematics and
3
Natural Sciences, University of Riau, Riau Province, Indonesia; English Education
Study Programme, Faculty of Teaching Training and Education, University of Pasir
4
Pengaraian, Rokan Hulu Regency 28557, Riau Province, Indonesia; Biology Education
Study Programme, Faculty of Teaching Training and Education, University of Pasir
Pengaraian, Rokan Hulu Regency 28557, Riau Province, Indonesia.
Corresponding author: F. Feliatra, feliatra@yahoo.com

Abstract. Probiotic is every preparation of microbe cell components which are beneficial to the health
and life of a host. The aim of this study was to evaluate the genetic differences of candidate probiotic
bacteria in the digestive tract of Macrobrachium rosenbergii and Penaeus monodon using phylogenetic
analysis of 16S rDNA sequences. The method used morphological, biochemical test and molecular
identification using 16s rDNA. 16S rDNA sequences was compared using a BLAST search to the data base
in Gen Bank. Eleven (11) candidate probiotic bacteria was isolated from M. rosenbergii. Isolate Iso A had
99% homology to Bacillus sp. BDU4. Ten (10) candidate probiotic bacteria was isolated from P. monodon.
There were none digestive track bacteria reported in Gen Bank which had more than 96% homology to
the isolates from P. monodon. The P. monodon isolates we conclude as original probiotic bacteria isolates
from Riau province.
Key Words: Probiotic bacteria, Macrobrachium rosenbergii, Penaeus monodon, Bacillus.

Introduction. The use of drugs or antibiotics in fisheries production as a prevention of

diseases is being reduced due to regulations prohibiting any residues of these chemicals
found in the fish. The use of antibiotics or antimicrobials as a additives in animal feed
lately decreased, it has been banned in some countries due to two main factors. First, the
possibility of antibiotics contaminating residues will be toxic for the consumers. Secondly,
antibiotics can increase the number of resistant microorganisms in the human or animal
body (especially pathogenic bacteria such as Salmonella sp., Escherichia coli and
Clostridium perfringens) (Feliatra et al 2011).
Probiotics are products composed by microbes or microscopic natural foods that
are beneficial and gives effect to increase the balance of intestinal tract microbial
communities in the host animal. Probiotics in aquaculture have a role in increasing the
growth rate, and improving the immune system by changing the bacterial community of
the intestines (Irianto & Austin 2003).
Probiotics can produce bacteriocins which work selectively against some
pathogenic strains. They also produce lactic acid, acetic acid, hydrogen peroxide,

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lactoperoxide, lipopolysaccharide, and several antimicrobials. In addition, important
nutrients in the host immune system and metabolism, such as B vitamine (pantothenic
acid), pyridoxine, niacin, folic acid, cobalamin, biotin and antioxidants such as K vitamine
can be produced (Adam 2009).
According to Munoz-Quezada et al (2013), probiotics must be able to control the
digestive tract and increase the health of its host through metabolic activity. In particular,
probiotics must survive with the acid conditions and high levels of salt with 2.0 and 7.0
pH as a control.
Feliatra et al (2004) found nine species of bacteria that have the potential as
probiotics in the gastrointestinal tract of Ephinephelus fuscogutatus. They are
Lactococcus sp., Carnobacterium sp., Staphylococcus sp., Bacillus sp., Eubacterium sp.,
Pseudomonas sp., Lactobacillus sp., Micrococcus sp. and Bifidobacterium sp. Those
bacteria have a potential as probiotic because of having resistance at pH 2 as a main
indicator of probiotic bacteria. According to Yulvizar (2013) there are five isolates which
have a potential as probiotics from the stomach and intestines of Rastrelliger sp. All the
isolates consist of three genera of the bacteria such as Micrococcus, Staphylococcus,
Bacillus and one species of bacteria that is Hafnia alvei. Yulvizar et al (2015) found three
isolates have the ability to inhibit and eliminate the pathogenic bacteria in aquaculture
systems.
Modabberi et al (2014) examined the effect of different doses of probiotic
Bactocell in the diet of rainbow (Oncorhynchus mykiss) trout on growth parameters and
bacterial flora. These authors found that the probiotic Bactocell is promising candidate to
affect the weight gain and capable of improving the efficacy of bacterial flora of rainbow
trout and Sharibi et al (2015) found the use of Bactocell as dietary supplement notably
increases growth and nutritional parameters in the benni fish fingerlings. The optimal
level could be the 0.2 grams of probiotic Bactocell per kg of diet. In this direction,
incorporating 0.2 grams of Bactocell per kg diet stands the chance to improve the growth
and nutritional performance of benni fish fingerlings.
Based on these findings, this study was conducted to find out the genetic diversity
of probiotics in the digestive tract of Macrobrachium rosenbergii and Penaeus monodon.
Isolated probiotic candidate bacteria were characterized by phenotype characterization
(morphological observation, physiological and biochemical test) and genotype
characterization based on phylogenetic analysis of 16S rDNA sequences and nucleotide
sequence analysis with the Basic Local Alignment program Search Tool (BLAST) against
sequences published in GenBank.

Material and Method. This study was conducted on October 2013 at the Microbiology
and Biotechnology Laboratory of Fisheries and Marine Sciences Faculty, University of
Riau, Pekanbaru to isolate the candidate probiotic bacteria. Furthermore, molecular
examination, purification gel electrophoresis and sequencing DNA were done at the
Institute of Biotechnology Serpong, Tangerang, Banten.
The samples of the study were Macrobrachium rosenbergii (15-30 cm in length),
obtained from fish farmers derived from Teratak Buluh, Pekanbaru, Riau and Penaeus
monodon (7-9 cm in length) samples derived from shrimp farms in Bengkalis, Riau. They
o
were included in the ice box with 5 C then they were brought to Marine Microbiology
laboratory. Nutrient Agar (NA) and physiological solution pH 2 were used as the media to
isolate and purify the bacteria. Test Gram staining test was done with crystal violet
solution, distilled water, iodine solution, 95% ethyl alcohol and safranin solution. The
solution of 3% hydrogen peroxide (H 2O2) was used in the catalase test. MR-VP media and
methyl red indicator were used in the methyl red test. Oxidative/fermentative (O/F)
medium was used in O/F test. SIM medium (Sulfide-Indol-Motility) was used in the
Indole, H2S and motility test. Simmon's citrate media was used in the citrate agar test.
Triple Sugar Iron Agar (TSIA) media was used in the use of sugar test. In additions, NA
medium was used to test the growth of bacteria at different temperatures.
For DNA isolation cultures of probiotic candidate bacteria were grown in 4 mL of
o
liquid Luria-Bertani medium (LB) at 37 C for 24 hours. The cultures were then moved to
1.5 mL microtubes and centrifuged at a speed of 13,000 rpm (revolutions per minute) at
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 o
4 C for 2 minutes. The supernatant was discarded, and the pellet dried. Dissolved in 40
L of of Tris-EDTA (TE) buffer solution with 8 pH and 20 L of lysozyme added to the
o
solution. The solution was inverted 3-5 times, and incubated for 10 minutes at 37 C.
After incubator 50 L of 10% Sodium dodecyl sulfate (SDS) was added and mixture was
gently rotated (20 rpm) for 5-10 minutes on a rotamix rotator. Added 550 V of Phenol:
Chloroform: Isoamyl alcohol (25:24:1) was added to the mixture and the mixture further
rotated gently (20 rpm) for 10 minutes. The mixture was then centrifuged at 7.000 rpm
for 10 minutes. The upper aqueos phase was transferred to a new microtube, and an
equal volume of Chloroform:Isoamyl alcohol (24:1) was added to the tube. The mixture
was gently rotated (20 rpm) on a rotamix rotator for 10 minutes. The mixture was then
centrifuged at 7.000 rpm for 10 minutes. The upper aqueos phase was transferred to a
new microtube and 3 M sodium acetate was added (40% volume) to the aqueos phase.
Two times volume of 99% cold alcohol was then added to this solution to precipitate the
o
DNA, and the solution chilled at -20 C for 30 minutes. The precipitated DNA was
o
centrifuged at 13.000 rpm, 4 C for 10 minutes and the supernatant discarded. The DNA
pellet was washed in 1 mL 70% cold ethanol centrifuged for another 10 minutes at
o
13.000, 4 C. The supernatant discarded, and resulting DNA pellet was then air dried. The
pellet was dissolved in 40 LTE-buffer (pH 8) and 2.5 L of RNAse solution were added
o
and homogenized using vortex mixer and incubated at 37 C for 1 hour. Finally, the DNA
o
of probiotic candidate bacteria was ready to be used, or stored -20 C.

Polymerase Chain Reaction (PCR). The PCR reaction was done by using 8 L DNA of
each probiotic candidate bacteria as template, and adding 23 L of H 2O, 4 L of 10x Taq
polyrmerase, 0.5 L of 12mM dNTPmix, 2 L for each of universal primers consisting of
forward primer 24F (5'AGAGTTTGATCCTGGCT-3') and reverse primer 1541R
(5'AAGGAGGTGATCCAGCCGCA-3') and 0.5 L Taq polymerase. PCR conditions were: hot
o o
start for 2 minutes at 94 C, followed by 30 cycles of denaturation for 1 minute at 94 C,
o o
annealing for 40 seconds at 50 C, and strand extention for 1 minute at 72 C.

Purification of DNA PCR fragments. After PCR, the DNA obtained was separated on a
1% agarose gel by electrophoresis. PCR DNA bands were purified from the agarose gels
using the Qiagen High Pure PCR Purification Kit (Japan), following instructions by the
manufacturer.

Sequencing, BLAST and phylogenetic analysis. Purified PCR products were

sequenced with ABI 3010 Genetic Analyzer XL Applied Biosystems. Sequencing was done
at the Institute for Biotechnology Serpong, Tangerang, Banten. BLAST analysis was done
using the tools and data of GenBank accessed at http://www.nebi.nih.gov/blast. DNA
sequences were aligned using CLUSTALX 2.1. (Larkin et al 2007). Phylogenetic trees were
generated using the tools provided by CLUSTAL X 2.1., and trees were visualized using
TreeView X version 0.5.0.

Results. From the digestive tract of M. rosenbergii and P. monodon 21 probiotic

candidate bacteria could be isolated, consisting of 11 isolates derived from M. rosenbergii
and 10 isolates from P. monodon. From the 21 probiotic candidates bacteria isolates, 12
isolates were gram positive, 6 isolates were gram negative and 3 isolates were gram
positive leads to negative (Table 1).
Based on the observation of morphological and biochemical potential bacterial
strains found in the digestive tract of M. rosenbergii and P. monodon, each strain showed
different characteristics from each other. Colonies morphology was dominated by round
and irregular shapes. The dominant colors of the colony were creamy with various kinds
from creamy beige thick, yellowish and dull beige. The shape of the cells were dominated
by round and followed by rods. For gram staining of M. rosenbergii, equal numbers were
found between gram positive and negative bacteria, where as the strains of P. monodon
were dominated by gram positive bacteria (Table 1).
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 Table 1
The results of morphological observations and biochemical potential of bacterial strains found in the
digestive tract of Macrobrachium rosenbergii and Penaeus monodon

Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Macrobrachium rosenbergii
Iso A Irregular FC rod Carbol-fuchsin + + - + O + - + - -
and spread stain
Iso B round CC round Gram stain - + - + O + + - + + +
Iso C round FC round Gram stain - - - + O - + + + -
Iso D round FC round Gram stain - - - + O - + + - -
Iso E round FC round Methylene + - - + O + + + + - -
blue stain
Iso F Irregular FC rod Pappenheims + - - + O - - + + -
stain
Iso G Irregular CC rod Gram stain + - - + O + - - + - +
Iso H round YC rod Hiss capsole - - - + O - - + - -
stain
Iso I Irregular CC rod Gram stain + - - + O - - + - -
Iso J Fibrous CC rod Carbol-fuchsin - - - + O + - - + + -
stain
Iso K round YC rod Gentian violet + - - + O + - + + -
stain
Penaeus monodon
HQS1 round FC rod Gram stain + + - + F + + - -
HQS2 round CC rod Pappenheims - + - + O- + - - +
stain
HQS3 Irregular FC rod Vn Vn + - + F + - - +
HQS4 round FC rod Vn Vn + - + F + - - +
HQS5 round FC rod Gram stain + + - + F + - - -
HQS6 round FC rod Gram stain + - - + F + - + -
HQS7 Irregular CC rod Gram stain + + - + F + - - -
HQS8 Irregular YC rod Gram stain + - - + F Yello - + -
wish
green
HQS9 Irregular CC rod Vn Vn + - + F + - + -
HQS10 Irregular CC rod Gram stain + + - + F Green + + +
1 - isolate; 2 - colonies forms; 3 - colonies color (FC - faded cream, CC - concentrated cream, YC - yellowish
cream); 4 - cell shape; 5 - type of coupling; 6 - Gram; 7 - catalase; 8 - motilitas; 9 - different temperatures (28-
0
37 C); 10 - O/F; 11 - Indole; 12 - Metil Red; 13 - Citrat; 14 - Glucose; 15 - Sucrose; 16 - H2S.

PCR products using primers for 16S rDNA formed DNA bands on agarose gel
electrophoresis with molecular weights between 1500 to 2000 base pairs/bp (Figures 1
and 2). The size was suitable with the expectation of the 16S rDNA genes of bacteria
(Sabdono & Rajasa 2006). The results of BLAST of potential probiotic bacteria derived
from M. rosenbergii indicated that only isolates A had a degree of homology of 99% with
the bacterial strain Bacillus sp. BDU4, while 10 others bacterial isolates only had a level
of homology between 91-96% with the Bacillus genus. Likewise, the result of BLAST
candidates from P. monodon was also leading to the Bacillus genus. For digestive isolates
from P. monodon, there were none which had homologous isolates approaching 97%, so
it was concluded that all isolates were putative new isolates not reported yet in Gen
Bank. All isolates from P. monodon showed homology to Bacillus sp. with the closest
homology of 96% to Bacillus cereus NC7401 reported by Takeno et al (2012).
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 Figure 1. The results of gel agarose electrophoresis of 16S rRNA PCR products of bacteria isolated
from the digestive tract of Macrobrachium rosenbergii (isoA - Iso K).

Figure 2. The results of gel agarose electrophoresis of 16S rRNA PCR products of bacteria isolated
from the digestive tract of Penaeus monodon (HQS1- HQ S10).

Discussion. Based on molecular characteristics between species or strains of the same

species, phylogenetic tree was useful to show the genetic relationship of each species. All
analyzed samples make a group. Isolates Iso consisted of Iso I and Iso F have a level of
genetic relationship with the bacteria B. cereus strains (Figure 3), where as isolates HQ
S7 has genetic relationship with Bacillus subtilis strain SRS-35GU056813.1. According to
Blackwood & Buyer (2004) there are at least two kinds of subspecies of B. subtilis, B.
subtilis and subspecies subtilis and B. subtilis subspecies spizizenii. There have previously
done some researches related to the benefits of these bacteria which could associate to
the inside and outside of the body of another living creature (Silva et al 2013).
Hagstrom et al (2000) stated that the isolates which have 16S rDNA sequence
similarity greater than 97% may represent the same species. For those who have
similarities between 93-97% sequence could represent the identity at the level of genus
but they are probably different at the species level. Possibly other isolates with homology
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under 97% are putative probiotic bacteria that have not been reported yet in Gen Bank.
Additional analysis, using other marker genes still needs to be done, to verify this.
Isolates bacterial: Iso D, Iso C, Iso A, Iso G, Iso K and Iso E, formed separate clusters.
Based on the genetic relationship, the closest bacteria was B. cereus strains
JDA1GU056812.1, but closer to the HQ S9, HQ S3, HQ S6, HQ S1, HQ S8 which clustered
together with Bacillus cereus JDA-1GU056812.1 (Figure 3).

Figure 3. Phylogenetic tree (cladogram) potential isolate probiotic bacteria from the digestive of
Macrobrachium rosenbergii (Iso A - Iso K) and Penaeus monodon (HQ S1-S10).

Iso J bacteria had a cluster with B. cereus strains JSYM2816S, and Iso I had the same
cluster with the Bacillus anthracis, while Iso F had the same cluster with the B. cereus
(Figure 3). Bacillus sp. was proteolytic bacterium that could break down proteins into
amino acids. Amino acid was used to multiply, so that it could increase the feed protein
and decrease the crude fiber. Moreover, this bacterium was also able to decipher
disaccharides and polysaccharides into simple sugars which had pectinolytic nature with a
capability in producing a complex carbohydrate pectin (Anggraini et al 2012).
Bacteria S5 and S4 were closer to the cluster of Bacillus thuringiensis (Figure 3).
This bacterium could produce two types of toxin: crystals toxin (crystal, Cry) and
cytolytic toxin (cytolytic, Cyt). Cyt toxin could streng then Cry toxin widely used to
improve the effectiveness in controlling insects. Several studies have been able to isolate
and purify the bacteriocin of Bacillus sp.: cerein produced by B. cereus (Oscariz &
Pisabarro 2000), and toxin produced by B. thuringiensis (Paik et al 1997).
According to Doi & Mcgloughlin (1992), two main properties which distinguish
Bacillus from other bacteria forming endospores are the ability of Bacillus to live aerobe
(although some of them are facultative anaerobes) and the majority of its kind to
produce catalase (positive catalase). Endospores produced by Bacillus had a high
resistance to chemical and physical factors, such as extreme temperatures, alcohol, and
so on. These kinds entirely contained dipicolinic acid (DPA) and they had a degree of
unparallel dormancy on the other life forms. The spores brought the development cycle
where vegetative cells could form spore then it could germinate into vegetative cells.
While the S9 isolates candidate had genetic relationship with B. cereus.
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B. cereus bacterium was one of the pathogen agents having a great potential to be used
as biological control. This bacterium has a specific host which is not harmful for the
natural enemies of pests and other non-target organisms, easily biodegradable by the
environment and its pathogenicity can be increased by genetic engineering techniques
(Khetan 2001). B. cereus could grow well on NA medium incubated in the laboratory
which had an average temperature of 28.8C. According to Gordon et al (1973) B. cereus
can grow well at a maximum temperature of 35 to 45C and a minimum temperature of
10 to 20C. The B. cereus surface colonies that grow on NA media looks shiny and
reflective. Eight-day bacteria can make a foul odor (Binoto 2001). B. cereus had the
3 -1
ability to do lysis toward the Cyanobacteria (BGA) at a concentration of 10 cells mL in
waterpool. It could generate Cyanobacteriocyde so that it was suitable to be used as a
biological control toward blooming blue green algae.
The kinds of Bacillus (B. cereus, B. clausii and B. pumilus) are included in the five
commercial probiotic products for colonization, immunostimulation, and antimicrobial
activity (Duc et al 2004). Twenty-one (21) probiotic candidate bacteria of Bacillus types
were expected to be used to improve the productivity of fish and shrimp farming either
for growing or improving the carrying capacity of the pool or pond.
Conclusions. Isolation of 21 strains of bacteria have potential as probiotics. They were
found in the digestive tract of Macrobrachium rosenbergii and Penaeus monodon. Only
one isolate had 99% homology to Bacillus sp. It could be ascertained as the same species
because the lowest limit of the same species was 97%, while there was not found
homologous bacterial approaching 97% from the other 20 bacteria isolates so that it can
be said that they were bacteria whos 16S rDNA sequences have not been reported yet in
Gen Bank. Because 20 of these bacteria had a survival at pH 2 and were able to develop
well as the main indicators as probiotic bacteria, it could be concluded that they had the
potential as probiotics.

Acknowledgements. We would like to express our gratitude to the Head of Research

Institute of the University of Riau and the staff. In cooperation with the Head of
Biotechnology Research Center Serpong, Tangerang, Banten and staff who have helped in
this study. Thanks also to all those who have given criticism and advice in the preparation
of research reports. This research is part of Grant Competence of Directorate General of
Higher Education in 2013/2014.

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AACL Bioflux, 2016, Volume 9, Issue 2.
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Received: 13 February 2016. Accepted: 27 March 2016. Published online: 16 April
2016. Authors:
Felix Feliatra, Laboratory of Marine Microbiology, Faculty of Fisheries and Marine Sciences, University of Riau,
Pekanbaru 28286 Riau Province, Indonesia, e-mail: feliatra@yahoo.com
Iesje Lukistyowati, Laboratory of Marine Microbiology, Faculty of Fisheries and Marine Sciences, University of
Riau, Pekanbaru 28286 Riau Province, Indonesia, e-mail: iesjel@yahoo.com
Dessy Yoswaty, Laboratory of Marine Microbiology, Faculty of Fisheries and Marine Sciences, University of Riau,
Pekanbaru 28286 Riau Province, Indonesia, e-mail: Dyoswaty@yahoo.com
Haqqy Rerian, Laboratory of Marine Microbiology, Faculty of Fisheries and Marine Sciences, University of Riau,
Pekanbaru 28286 Riau Province, Indonesia, e-mail: qrerian@gmail.com
Deasy Melina, Laboratory of Marine Microbiology, Faculty of Fisheries and Marine Sciences, University of Riau,
Pekanbaru 28286 Riau Province, Indonesia, e-mail: dkilway_91@yahoo.com
Wahid Hasyim, Laboratory of Marine Microbiology, Faculty of Fisheries and Marine Sciences, University of Riau,
Pekanbaru 28286 Riau Province, Indonesia, e-mail: wahid_hasyimi@ymail.com
Titania T. Nugroho, Laboratory of Enzyme, Fermentation and Biomolecular, Department of Chemistry, Faculty of
Mathematics and Natural Sciences, University of Riau, Riau Province, Indonesia, e-mail:
titanianugroho@gmail.com
Andi R. Fauzi, English Education Study Programme, Faculty of Teaching Training and Education, University of
Pasir Pengaraian, Rokan Hulu Regency 28557, Riau Province, Indonesia, e-mail: andi_gundoel@yahoo.com
Rofiza Yolanda, Biology Education Study Programme, Faculty of Teaching Training and Education, University of
Pasir Pengaraian, Rokan Hulu Regency 28557, Riau Province, Indonesia, e-mail: padangers@gmail.com
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source are credited.
How to cite this article:
Feliatra F., Lukistyowati I., Yoswaty D., Rerian H., Melina D., Hasyim W., Nugroho T. T., Fauzi A. R., Yolanda R.,
2016 Phylogenetic analysis to compare populations of acid tolerant bacteria isolated from the gastrointestinal
tract of two different prawn species Macrobrachium rosenbergii and Penaeus monodon. AACL Bioflux 9(2):360-
368.

AACL Bioflux, 2016, Volume 9, Issue 2.

http://www.bioflux.com.ro/aacl 36