Bacillus pumilus-Specific Bacteriophage Isolated from Ciapus River in Bogor, West Java

Bacillus pumilus-SPECIFIC BACTERIOPHAGE ISOLATED
FROM CIAPUS RIVER IN BOGOR, WEST JAVA

ANIK KUSMIATUN

POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

DECLARATION
I hereby declare that the thesis entitled Bacillus pumilus-Specific
Bacteriophage Isolated from Ciapus River in Bogor, West Java is true of my work
with the guidence of my academic supervisors and has not been submitted in any
form to any university. Sources information from published and unpublished
works of the other authors has been mentioned in the text and listed in the
references of this thesis.
I hereby assign the copyright of my papers to the Bogor Agricultural
University.
Bogor, September 2014

Anik Kusmiatun
G351120021

SUMMARY

ANIK KUSMIATUN. Bacillus pumilus-Specific Bacteriophage Isolated from
Ciapus River in Bogor, West Java. Supervised by SRI BUDIARTI and IMAN
RUSMANA.
Diarrhea disease is part of the public health problems in developed countries.
It is the main cause of mortality of diarrheal patients in Indonesia with Case
Fatality Rate (CFR) 1.79 %. In 2012, UPT Public Health Center Cigudeg, Bogor
reported that incident of diarrhea in Cigudeg was high (3180 patients). The main
factors that predispose children to diarrhea in the area is contamination of
pathogenic bacteria due to their poor sanitation. Salmonella sp, Shigella sp
Citrobacter, Vagococcus, Haemopillus, Micrococcus, and Streptococcus were
isolated from faeces samples of children sufferiety with diarrhea disease in
Cigudeg. Other bacteria i.e Enterococcus sp and Bacillus sp were isolated from
water of their drainase system in the area. Interestingly, the Bacillus sp isolate was
identified as Bacillus pumilus. It was a new report that B. pumilus found in an area
with high number of diarrhea disease. This bacterium is a spore-forming

bacterium commonly associated with food poisoning case. Characterization of its
patogenicity was shown that B. pumilus produced haemolisin toxin that lyses red
blood cells in culture media. Antibiotics were usually used to treat the disease
caused by bacteria. Antibiotic typing test of B. pumilus showed that this isolate
was resistant to ampicillin and clindamycin. It was a new report that B. pumilus
was resistant to these antibiotics.
An alternative way was by application bacteriophages (phages) as biocontrol
agents to reduce B. pumilus in environment. In this study, the phages was isolated
from Ciapus River using the double agar overlay method. It was found that the
phage isolate could infect and kill B. pumilus through lysis mechanism. Phage
FBa1 performed a 2 mm diameter translucent plaque with halo, FBa2 a 1mm
diameter translucent plaque with halo, and FBa3 a 0.5 mm diameter translucent
plaque without halo. The concentration of FBa1 phage was 10.2x108 PFU mL-1,
FBa2 phage was 5.9x108 PFU mL-1, and FBa3 phage was 8.5x108 PFU mL-1. High
concentration of B. pumilus-phage indicated that naturally phage was found in this
river.
Host range determination showed that phage FBa1, FBa2, and FBa3 had
specific activity to Bacillus pumilus. It was not infect other bacterial cells.
Morphological observation of phage FBa1 using TEM negative staining showed
an icosahedral head without tail (166.67 nm in diameter) of phage-like particles.

Characterization of phage FBa1 by SDS-PAGE showed five proteins band.
Molecular weight of phage FBa1 proteins was 70.9 kDa; 54.9 kDa; 33.8 kDa; 28.3
kDa; and 21.4 kDa respectively. The results of this research showed application
prospect of phage FBa1 as a biocontrol agent of B. pumilus in the environment.
Key words : Bacillus pumilus, bacteriophage, diarrhea

RINGKASAN

ANIK KUSMIATUN. Bakteriofag Spesifik Bacillus pumilus Diisolasi dari Sungai
Ciapus di Bogor, Jawa Barat. Dibimbing oleh SRI BUDIARTI dan IMAN
RUSMANA.
Penyakit diare merupakan masalah kesehatan masyarakat di negara-negara
berkembang. Penyakit ini menyebabkan kematian pasien diare di Indonesia
dengan Case Fatality Rate (CFR) sebesar 1.79%. Pada tahun 2012, UPT Pusat
Kesehatan Masyarakat Kecamatan Cigudeg, Bogor melaporkan bahwa jumlah
penderita diare di Cigudeg cukup tinggi sebanyak 3180 pasien. Faktor utama
penyebab diare pada anak-anak di daerah tersebut adalah kontaminasi bakteri
patogen karena sanitasi lingkungan yang buruk. Salmonella sp, Shigella sp
Citrobacter, Vagococcus, Haemopillus, Micrococcus, dan Streptococcus diisolasi
dari sampel tinja anak-anak penderita diare di Cigudeg. Bakteri lain yaitu

Enterococcus sp dan Bacillus sp diisolasi dari air lingkungan daerah Cigudeg.
Menariknya, isolat Bacillus sp diidentifikasi sebagai Bacillus pumilus. Hal ini
adalah laporan baru adanya B. pumilus yang ditemukan di daerah dengan jumlah
penyakit diare tinggi. Bakteri ini merupakan bakteri pembentuk spora yang
berkaitan dengan kasus keracunan makanan. Karakterisasi patogenisitas isolat B.
pumilus dapat menghasilkan toksin haemolisin yang melisiskan sel darah merah
dalam media kultur. Antibiotik biasa digunakan untuk mengobati penyakit yang
disebabkan oleh bakteri. Uji antibiotik terhadap B. pumilus menunjukkan bahwa
isolat ini resisten terhadap ampisilin dan klindamisin.
Cara alternatif untuk mengurangi B. pumilus yaitu dengan aplikasi
bakteriofag (fag) sebagai agen biokontrol B. pumilus di lingkungan. Fag pada
penelitian ini diisolasi dari Sungai Ciapus dengan menggunakan metode agar dua
lapis (double agar overlay). Isolat fag tersebut dapat menginfeksi dan membunuh
B. pumilus melalui mekanisme lisis. Ukuran diameter plak FBa1 sebesar 2 mm,
plak transparan dengan halo (cincin luar); plak FBa2 berukuran 1mm, plak
transparan dengan halo; dan plak FBa3 berukuran 0,5 mm, plak transparan tanpa
halo. Konsentrasi isolat fag FBa1 adalah 10.2x108 PFU mL-1, fag FBa2 adalah
5.9x108 PFU mL-1, dan fag FBa3 adalah 8.5x108 PFU mL-1. Konsentrasi fag B.
pumilus yang tinggi menunjukkan bahwa secara alami fag ditemukan di sungai ini.
Uji kisaran inang menunjukkan bahwa fag FBa1, FBa2, dan FBa3 memiliki

aktivitas spesifik untuk Bacillus pumilus. Fag tersebut tidak menginfeksi sel-sel
bakteri lainnya. Pengamatan morfologi fag FBa1 menggunakan Transmission
Electron Microscope (TEM) dengan pewarnaan negatif menunjukkan kepala fag
berbentuk ikosahedral tanpa ekor, diameter fag sebesar 166,67 nm, bentuk fag
menyerupai partikel fag. Karakterisasi fag FBa1 dengan SDS-PAGE menunjukkan
lima pita protein. Berat molekul masing-masing protein fag FBa1 adalah 70.9 kDa,
54.9 kDa, 33.8 kDa, 28.3 kDa, dan 21,4 kDa. Hasil penelitian ini menunjukkan
prospek fag FBa1 untuk diaplikasikan sebagai agen biokontrol B. pumilus di
lingkungan.
Kata kunci: Bacillus pumilus, bakteriofag, diare

© Copyright by Bogor Agricultural University 2014
All rights reserved
No part or all of this thesis may be excerpted without inclution or mentioning the
sources. Excerption only for research and education use, writing for scientific
papers, reporting, critical writing or reviewing of a problem.
Excerption does not inflict a financial loss in the proper interest of Bogor
Agricultural University.

Bacillus pumilus-SPECIFIC BACTERIOPHAGE ISOLATED

FROM CIAPUS RIVER IN BOGOR, WEST JAVA

ANIK KUSMIATUN

A thesis submitted as partial fulfillment of the requirement for the degree of
Master of Science in Microbiology

POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014

Thesis External Examiner: Dr Drh Sri Murtini, MSi

ACKNOWLEDGEMENT
In the name of Allah The Most Gracious and The Most Merciful. The theme
of this research was about bacteriophage, with the title Bacillus pumilus-Specific
Bacteriophage Isolated from Ciapus River in Bogor, West Java.
I would like to gratitude to my supervisors, Dr dr Sri Budiarti and Dr Ir Iman
Rusmana, MSi for their guidance, patience, and support during my study. I would

like to thank to Prof Dr Anja Meryandini, MS; Prof Dr Okky Setyawati
Dharmaputra; and Dr Drh Sri Murtini, MSi for their presence, patience and
guidance in my thesis examination. I would like to thank to all lecturer for their
guidance during my study in Microbiology Study Program.
I would also like to thank my parents, Mr. Saidi and Mrs. Siti Aminah; my
sisters, Tutik Supriyati and Muslimah; my brothers, Juprianto and Jalaludin Sayuti,
for their support and love that allowed me to continue my study.
I would also like to thank to Prof Dr Ir Suharsono, DEA and all staff of
Research Center for Bioresources and Biotechnology (PPSHB), Bogor
Agricultural University for their permission during my research in Biotechnology
of Animal and Biomedical Laboratory. I would also like to thank to Mrs. Dewi,
Mrs. Heni, Mr. Jaka, Mr. Aldi, and Mr. Pras for their guidance during my research
in laboratory. I would also like to thank to Mrs. Ita and Labibah for their help to
observed phage in TEM Laboratory, Eijkman Institute, Jakarta.
I would also like to thank to Bob Edwin Normande for help to isolated the
host bacterium from Cigudeg, Bogor. My appreciation to all lab members, Tini,
Novi, Rachmi, Ira, Fatin, Debby, Yeni, Leni, Ika, Lia, teteh Fitri for their
knowledge sharing to me and friendship.
Finally I would like to thank Microbiology students 2012 for their support
and friendship during my study in Bogor Agricultural University.

Bogor, September 2014

Anik Kusmiatun

TABLE OF CONTENT
LIST OF TABLES
LIST OF FIGURES
INTRODUCTION
Background
Objectives
LITERATURE REVIEW
Bacillus pumilus
Lytic Bacteriophages
Phage Therapy
MATERIALS AND METHOD
Place and Duration of the Study
Identification of Host Bacterium
Water Samples Collection
Isolation and Purification of Phage

Phage Quantification
Host Range Determination
Morphological Observation of Phage using Transmission Electron
Microscope (TEM)
Analysis of Phage Proteins
RESULTS
Identification of Host Bacterium
Isolation and Purification of Phage
Phage Quantification
Host Range Determination
Morphological Observation of Phage using Transmission Electron
Microscope (TEM)
Analysis of Phage Proteins
DISCUSSIONS
CONCLUSIONS
REFERENCES

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LIST OF TABLES
1 Identification of host bacterium isolated from water in Cigudeg, Bogor
2 Antibiotic susceptibility test results of Bacillus pumilus
3 Detection of phage in water samples
4 Phage isolates from Ciapus river water, Bogor
5 Phage quantification of FBa1, FBa2, and FBa3
6 Host range of phage FBa1, FBa2, FBa3

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LIST OF FIGURES
1 Photomicrograph of Bacillus pumilus
2 Bacillus pumilus isolate on blood agar
3 Morphology of plaque
4 Morphology of purified plaques
5 TEM images of phage negatively stained with 2% uranyl acetate
6 Molecular weight of phage FBa1 protein in SDS-PAGE gel

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INTRODUCTION
Background
Diarrhea disease is part of the public health problems in developed countries.
It is the main cause of mortality of diarrheal patients in Indonesia with Case
Fatality Rate (CFR) 1.79 % (Kemenkes RI 2012). In 2012, UPT Public Health
Center Cigudeg, Bogor reported that incident of diarrhea in Cigudeg was high
(3180 patients). The main factors that predispose children to diarrhea in the area is
contamination of pathogenic bacteria due to their poor sanitation. Salmonella sp,
Shigella sp, Citrobacter, Vagococcus, Haemopillus, Micrococcus, and
Streptococcus were isolated from faeces samples of children sufferiety diarrhea
disease in Cigudeg, Bogor (Normande 2013). Other bacteria include Enterococcus
sp and Bacillus sp were isolated from water of their drainase system in the area.
Interestingly, the Bacillus sp isolate was identified as Bacillus pumilus. It was a
new report that B. pumilus found in an area with high number of diarrhea disease.
Transmission of pathogenic bacteria that cause diarrhea are usually by faecal
contaminated water or food to person contact (Gosh et al. 1991).
Bacillus pumilus is a spore-forming bacterium commonly associated with
foodborne illness caused by intoxication. Suominen et al. (2001) reported that
incidents of food poisoning with diarrhea symptoms was occurred after ingestion
of foods containing large numbers of B. pumilus (105-107 cfu g-1). In 2007, From
et al. reported a food poisoning outbreak caused by B. pumilus in Norway.
Bacillus pumilus was found as contaminant from reheated rice in a Chinese
restaurant. The acute symptoms developed after meal, the people experienced
stomach cramps and diarrhea for several days. Large numbers of B. pumilus
strains produced a lipopeptides complex known as pumilacidins, a toxic
compounds. This toxin can destroy cell membranes through production of
selective cationic channels (Sheppard et al. 1991). The membrane destroying
capacity of this toxin explains that food poisoning often associated with stomach
cramps and diarrhea.
Antibiotics were used to treat bacterial infections. Antibiotic resistance in
bacteria is a global health problem. Antibiotic resistance in bacteria occurs via
either chromosomal mutations or foreign gene transfer by plasmids (Zhang et al.
2006). Transfer of resistant antibiotic gene between bacterial cells may cause a
new antibiotic resistant bacterial strain. Irrational application of antibiotics can
increase the antibiotics resistant, not only on pathogenic bacteria but also normal
flora in human gastrointestinal. Budiarti (2011) reported that E. coli as intestinal
normal flora isolated from faecal of new born children was resistant to antibiotic
bacitracin and erytromycin. It was showed that normal flora E. coli in new born
children has material genetic changes caused by transfer of the resistant E. coli
from mother or environment. Previously, antibiotic resistant of B. pumilus in
Indonesia have not been reported yet. In India, B. pumilus isolates were identified
resistant to penicillin (Parvathi et al. 2009). In Turkey, Ozkocaman et al. (2006)
reported that B. pumilus was resistant to amikacin, tobramycin, gentamycin,
penicillin, cefepim, cefotaxime, ampicillin-sulbactam, ceftazidime, piperacillin,
imipenem, meropenem, aztreonam, cefoperazone-sulbactam, piperacillin-

2
tazobactam, and tetracycline. These report give information that antibiotic
resistant level in pathogenic and flora normal bacteria is very high. The phage
therapy was used to overcome the problem of antibiotic resistance.
Bacteriophages (phages) are viruses that infect bacteria. Phages have a
protein coat that encloses a nucleid acid (DNA or RNA). There are two life cycle
types of phages, lytic and lysogenic phages (Snyder and Champness 2007). Lytic
phage kills bacteria through lysis and releasing many particles of phages. Lytic
phages provide opportunity as biocontrol agent to decrease pathogenic bacteria.
Recently, the potential lytic phages could be used as a biocontrol agent of
foodborne pathogens. For example, phage FB4 could lyse EPEC K1.1 and this
phage expected as biocontrol to prevent foodborne disease in Indonesia (Budiarti
et al. 2011). Oral application in rat diet, phage FR38 to reduce Salmonella P38
had no effect on body weight, blood chemistry, kidney and liver functions in rat
(Sartika et al. 2012). Phage FGCSSa1 isolated from sewage had potential use for
biocontrol of Salmonella spp. in foods (Carey-Smith et al. 2006). Bacteriophage
cocktail as a biocontrol agent of Salmonella enterica serovar Typhimurium and S.
enterica serovar Enteritidis used in food process (Spricigo et al. 2013). Many
phages infecting Bacillus spp. were isolated; i.e. phages FWLBc1 and FWLBc2
were isolated from B. cereus (Lee et al. 2011). Tsamsa phage isolated from B.
antrhracis (Ganz et al. 2014). SPP1 phage was isolated from B. subtilis (Jakutytė
et al. 2011). PhiAGATE phage was isolated from B. pumilus (Barylski et al.
2014). However B. pumilus phages have not been reported in Indonesia yet. It was
very important to found a biocontrol agent to reduce B. pumilus in the
environment.
Objectives
The objectives of this research were to isolate and characterize B. pumilus
bacteriophage isolated from water sample of Ciapus River in Bogor, West Java.
The prospect of this research can be applied as a biocontrol agents of B.pumilus in
the environment.

LITERATURE REVIEW
Bacillus pumilus
Bacillus pumilus is a Gram-positive, rod-shaped, aerobic, spore-forming
bacteria (Figure 1). Bacillus pumilus can growth at pH 6-10 and temperature 1050 oC (Vos et al. 2009). Bacillus pumilus has peptidoglycan layer that play a role
in adhesion to host cells and other surfaces in the environment. Peptidoglycan is
composed of teichoic and lipoteichoic acids. These acids are composed
polyglycosyl phosphates i.e glycerol phosphate or ribitol phosphate (Kubler-Kielb
et al. 2004). Bacillus pumilus life cycle is similar to other Bacilli. It has three life
cycle stages: sporangium, vegetative cell, or free spore (Sonenshein 2000).
Bacillus pumilus will form a spore when the condition is not favourable for their

3
growth. The spore will germinate to be a vegetative cell when the condition is
favourable.

Figure 1 Photomicrograph of Bacillus pumilus viewed by phase-contrast
microscopy. Bar = 2 µm. (Logan 2011)
Human infection by B. pumilus is rarely reported, however an outbreak of
B. pumilus was found in reheated rice. It was responsible for 3 cases of food
poisoning outbreak. Bacillus pumilus produces a complex of lipopeptides called
pumilacidins. This complex is toxic to epithelial cells. The symptoms of this
infection were dizziness, headache, stomach cramps, and diarrhea (From et al.
2007). Parvathi et al. (2009) reported that B. pumilus produced protease and lipase.
This bacterium had cesA and cesB genes encoded cereulide synthetase. Cereulide
is a stable cyclic dodecadepsipeptide produced by some strains of B. cereus. This
toxin has high toxicity to humans (Agata et al. 1995; Yokohama et al. 1999).
Matarante et al. (2004) reported that hemolytic and lecithinase activities were
virulence factors in B. pumilus.
Lytic Bacteriophages
Bacteriophages were discovered in 1915 by Towart and in 1917 by Felix
d’Herelle (Duckworth 1976). They reported a filterable entity that capable to
destroyed bacterial culture. The destruction of the bacterial cell was noticeable at
the appearance of plaques. Plaques are clear spots formed in bacterial colonies
during the rupturing of the cells. D’Herelle categorized these filterable entities as
bacteriophages, meaning bacterial eaters.
Bacteriophages (phages) play important roles in maintaining the natural
abundance of microorganisms on earth. Phages have a life cycle within the host
bacteria which can be lytic or lysogenic. The lytic cycle occurs in five steps, i.e.
adsorption, penetration, replication, maturation, and lysis. During adsorption, the
phage attaches to the cell in order to inject its genetic material into the bacterial
cell (Inamdar et al. 2006). Infection into the bacterial cell is initiated by binding to
specific surface molecules or capsules. Binding sites differ depending on the type
of targeted bacteria, Gram positive or Gram negative. Penetration is a process of
injection of the genetic material. Typical viral genome is approximately 10 µm
long and its transfer from outside to inside the cell takes a variable amount of time,
from seconds to minutes (Inamdar et al. 2006). Replication was occurred after
penetration. The viral genetic material takes over the host and uses the bacterial

4
host machinery for phage replication. The RNA polymerase of the host recognises
strong phage promoters which results in the transcription of immediate early
genes. The early genes encode enzymes involved in DNA synthesis such as DNA
polymerase, primase, DNA ligase, and helicase (Snyder and Champness 2007).
These enzymes help the phage DNA to replicate. The late genes encode proteins
involved assembly of head and tail. After the phage particle is completed, the
DNA is packaged into procapsids which are icosahedral protein shells. The
mature phages then were released in a process known as lysis. Lytic phage
involve an enzyme, known as an endolysin which degradates the bacterial
peptidoglycan. Endolysins need a second lysis factor, a phage-encoded membrane
protein called holin. The holin-endolysin is essential for host lysis (Young 2002).
Phage Therapy
Multidrug resistant bacteria are a serious health problem these days.
Antibiotics are not bacteria specific and thus can result in distruption in the
balance of normal flora, which are bacteria that are naturally found in our bodies
(Giuliano et al. 1987). Phage therapy seems to be a good option for this problem.
Phage therapy is use of a specific bacteriophage to a bacterial cell. The
introduction of phage therapy was seen in the early 1930s (Levin and Bull 2004).
Phage therapy today will be used in possible applications to control foodborne
pathogens.
There are many research in the use of phage to control pathogenic bacteria.
Phage therapy against bacterial pathogens, for example, to control Salmonella
contamination on fresh cut fruit (Leverentz et al. 2001). Lytic bacteriophage was
used to decrease Salmonella and Campylobacter contamination on chicken
carcase (Goode et al. 2003). A cocktail of phages isolated from cattle feces
reduced E coli O157:H7 populations in the gut of sheep (Callaway et al. 2008).
Application of phage P100 on meat could reduced Listeria monocytogenes (Soni
and Nannapaneni 2010). Cocktail of phages application in milk decreased
contamination of S. aureus (Martínez et al. 2008). The advantages use phage as a
biocontrol agents are high specificity to target host bacterium, self-replication
because phage will multiply as long as there is still a host present, low toxicity
because phage consist mostly of nucleic acids and proteins, phage are relatively
cheap and easy to isolate, phage can add on food processing (Sillankorva et al.
2012).

MATERIALS AND METHODS
Place and Duration of this Research
This research was carried out from October 2013 up to May 2014 at
Biotechnology of Animal and Biomedical Laboratory, Research Center for
Bioresources and Biotechnology (PPSHB), Bogor Agricultural University and
TEM Laboratory, Eijkman Institute, Jakarta.

5
Identification of Host Bacterium
Host bacterium was isolated from water located in Cigudeg, Bogor,
Indonesia. Pathogenicity of Bacillus was determining using blood agar plates.
Identification was performed using API 50CHB (bioMérieux, Marcy I`Etoile,
France). The isolate was maintained aerobically on Tryptic Soy Agar (Difco), at
37 oC for 24 h. Antibiotics test with disk diffusion method was used to identify the
susceptibility of the bacterium to ampicillin, amoxicillin, ciprofloxacin, and
clindamycin (CLSI 2012). Antibiotics susceptibility was assessed by measuring
the diameter (in millimeters) of the zone inhibition surrounding disks.
Water Samples Collection
Water samples were collected from Ciapus River in Bogor. Each sample
(50 mL) from four station was stored in a sterile bottle at 4 oC overnigt. The crude
sample was centrifugated at 5,000 rpm for 20 min to remove bacterial cells and
debris. The supernatant was filtered through a 0.22 µm sterile filter and then
stored at 4 oC. This supernatant was used as a sample to isolate phage.
Isolation and Purification of Phage
Specific phages were sometimes difficult to isolate directly so enrichment
culture was used to multiply phages. Water sample of 4.5 mL was incubated
overnight with 0.5 mL of Nutrient broth (Difco) and 0.5 mL of host bacteria
culture (previously grown 24 h in Nutrient broth medium). The culture was
centrifugated at 5000 rpm for 25 min and then filtered through a 0.22 µm sterile
filter millipore. Stock solution of phage was stored in sterile tube at 4 oC.
The presence of a lytic phage in the filtrate was examined by using the
double layer method with some modification (Carey-Smith et al. 2006). Stock
solution of phage as much as 100 µL (dilutions of 10-5 to 10-8) was mixed with
Bacillus pumilus culture 100 µL (approximately 108 CFU mL-1), and incubated at
37 oC for 30 minutes. The culture bacterium with phage after incubation was
mixed with 7 mL of 0.8 % soft-agar (42 oC). It was poured as an overlay onto
Nutrient Agar (Difco) base plates, and incubated at 37 oC overnight. The presence
of a lytic phage in the form of plaques was detected after incubation of the plate.
Phage were purified by plaque isolation using Pasteur pipette. A plaque
plugs were diluted in 5 mL saline-magnesium (SM) buffer or phages can also be
diluted in the broth medium used to grow the host bacterium (without agar).
Saline-magnesium (SM) buffer is a buffer used for storage of phage stocks (5 M
NaCl, 1 M MgSO4, 1 M Tris-HCl pH 7.5, 1% gelatin in distilled water). The
suspension was mixed using vortex for 30s and then incubated at room
temperature for 1-2 h. The suspension was centrifugated at 5000 rpm for 20
minutes. After centrifugation, the supernatant was filtered through a 0.22 µm
sterile filter and transferred to sterile tubes. The stock solution of purified phage
was stored at 4 oC.

6
Phage Quantification
Phage quantification (plaque assay) was examined using double layer
method as mentioned earlier. Stock solution of purified phage was serially diluted
(10-1 to 10-8) in 0.85 % NaCl. Each dilution was subjected to plaque assay.
Plaques were counted on the plate that contain 30-300 plaques and expressed as
plaque forming unit per milliliter (PFU mL-1).
Phage titer (PFU mL-1) = number of plaques x 10 x reciprocal of counted dilution
Host Range Determination
Exponential phase cultures of the host bacteria (Bacillus pumilus, Proteus
mirabilis, Photobacterium damselae, Salmonella sp., EPEC K1.1) were prepared,
and agar overlays inoculated with a host (100 µL) and phage stocks (100 µL). The
plates were incubated at 37 oC for 24 h and the plaques were observed for each
host.
Morphological Observation of Phage by Using Transmission Electron
Microscope (TEM)
The morphology of phage FBa1 was examined by transmission electron
microscope. Stock solution phage (5 µL) was dropped in to grid for 30 seconds,
and then dried up with filter paper. Uranyl acetate 2% solution (5 µL) was also
dropped in to grid for 1 minute, and then dried up with filter paper for 60 minutes.
The grid placed in holder and left for perfect dry. Specimen was observed using
transmission electron microscope (TEM JEOL JEM-1010 model) at 80 kV, and
phages were examined at 20000x – 80000x magnification.
Analysis of Phage Proteins
The protein composition of phage FBa1 was examined by Sodium Dodecyl
Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Stock phage were
mixed with buffer sample (4:1), and then boiled for 5 – 10 minutes. As much as
60 µL phage protein samples were loaded onto a SDS-PAGE gel (12%
acrilamide) at 20 mA, 50 volt, for 3.5 h. Silver staining was used for visualization
of the result.

RESULTS
Identification of Host Bacterium
The study of B. pumilus contaminated in water have not been reported in
Indonesia previously. The bacterium was isolated from water in Cigudeg, Bogor,
West Java, Indonesia. Gram staining of host bacteria revealed that the isolate was
Gram-positive Bacillus. The colonies on blood agar plates showed a clear zone

7
around the colonies, it was indicated that the isolate exhibited -haemolytic
(Figure 2). Several biochemical tests on API 50CHB were used to identify species
of the bacterial isolate (Table 1). The isolate was identified as B. pumilus with
99.9 % match in identity by API 50CHB. The antibiotic test of B. pumilus showed
that the isolate was resistant to ampicillin and clindamycin (Table 2).
a
b

Figure 2 Bacillus pumilus isolate on blood agar (a); -haemolytic zone (b).

Table 1 Identification of host bacterium isolated from water in Cigudeg, Bogor
Identification
Results
test
API 50 CHB Positive : glycerol, L-arabinose, D-ribose, D-glucose, Dfructose, D-mannose, D-mannitol, amygdalin, arbutin, esculin
ferric citrate, salicin, D-cellobiose, D-saccharose (sucrose),
D-trehalose, D-tagatose
Negative : erythritol, D-arabinose, D-xylose, L-xylose, Dadonitol, D-galactose, L-sorbose, L-rhamnose, dulcitol,
inositol, D-sorbitol, methyl-αD-mannopyranoside, methylαD-glucopyranoside, N-acetylglucosamine, D-maltose, Dlactose, D-melibiose, inulin, D-melezitose, D-raffinose,
amidon, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose,
D-fucose, L-fucose, D-arabitol, L-arabitol, potassium
gluconate, potassium 2-ketogluconate, potassium 5ketogluconate

Table 2 Antibiotic susceptibility test results of Bacillus pumilus

Antibiotic

Disk
content*
(μg/mL)

Ampicillin
Amoxicillin
Ciprofloxacin
Clindamycin

10
20
5
2

CLSI standard of
antibiotic susceptibility
(mm)
S
R
> 29
< 28
> 20
< 19
> 21
< 15
> 21
< 14

*Standards by CLSI (2012), S: sensitive, R: resistant

Zone
diameter of
inhibition
(mm)
23
22
27
14

Results
R
S
S
R

8
Isolation and Purification of Phage
The phage isolate of B. pumilus was found in a water sample collected from
Ciapus River in Bogor. Samples were collected from river water because of phage
is naturally part of environmental ecosystem. Lytic activity were detected by
plaques using double agar method. Only one sample that positive contained
paghes specific to B. pumilus (Table 3). Treatment phages inoculate to B. pumilus
culture resulted in complete lysis of the bacterial cells. Plaques were formed after
3 h of incubation of agar plates. Differences in plaque morphology and sizes were
observed. The lytic activity formed two types of plaque, haloing plaque and
plaque without halo (Figure 3). Plaque diameter average was 0.5 to 2 mm after 18
h incubation at 37 oC.
Three phages were successfully isolated and they had different on plaque
morphology (Table 4). The phages were named FBa1, FBa2, and FBa3. Plaques
were purified by single-plaque isolations. Three isolates of phage were purified
based on plaque morphology, FBa1, phage from large plaque with halo; FBa2,
phage from medium plaque with halo; and FBa3, phage from small plaque
without halo. Purified phage was diluted in SM buffer and stored at 4 oC. Purified
phages were test on B. pumilus for three times. Plaques morphology were the
same as plaques morphology of isolation step, however different in plaque size or
diameter (Figure 4).
Table 3 Detection of phage in water samples
Station Water sample (mL)
1
50
2
50
3
50
4

Lytic phage
+

50

-

+ : plaques; - : no plaque

Figure 3 Morphology of plaques : FBa1, large plaque with halo (a); FBa2,
medium plaque with halo (b); FBa3, small plaque without halo (c). Bar
= 2 mm
Table 4 Phage isolates from Ciapus River, Bogor
Phage
isolate
FBa1
FBa2
FBa3

Morphology of plaque
Translucent plaque, presence of halo
Translucent plaque, presence of halo
Translucent plaque, absence of halo

Plaque size (mm)
2
1
0.5

9

Figure 4 Morphology of purified plaques: FBa1 (a); FBa2 (b); FBa3 (c).

Phage Quantification
Natural phage number in water samples was too low to produce a
quantifiable titer. Enrichment method was used to isolate phages and to produce
quantifiable titer. Phage FBa1 had the highest titer, approximately 10.2x108 PFU
mL-1 (Table 5).
Table 5 Phage quantification of FBa1, FBa2, and FBa3
Phage isolate
FBa1
FBa2
FBa3

Plaque count
102
59
85

Titer (PFU mL-1)
10.2x108
5.9x108
8.5x108

Host Range Determination
The host range of phages is defined by what bacterial cellscan lyse. The
phages of FBa1, FBa2, and FBa3 were tested on exponential-phase cultures of
other pathogenic bacteria i.e. Photobacterium damselae, Proteus mirabilis,
Salmonella sp., and EPEC K1.1. FBa1, FBa2, and FBa3 phage isolates were lytic
on Bacillus pumilus but they did not infect the other tested bacterial isolates
(Table 6).
Table 6 Host range of phage FBa1, FBa2, FBa3
Host bacteria
Bacillus Photobacterium Proteus
Salmonella sp.
pumilus
damselae
mirabilis
FBa1
+
FBa2
+
FBa3
+
+ : clear plaques; - : no plaque
Phage
Isolate

EPEC
K1.1
-

10
Morphological Observation of Phage Using Transmission Electron
Microscope (TEM)
Phage FBa1 was selected for further characterization according to the
morphological feature. Phage FBa1 was assigned to phage-like particles with
166.67 nm in diameter (Figure 5). It was a phage with icosahedral capsids without
tail.

Figure 5 TEM images of phages with negative staining with 2% uranyl acetate.
Phage FBa1 morphology was icosahedral. Diameter of phage was
166.67 nm. Magnification x25,000. Bar = 200 nm.

Analysis of Phage Proteins
Analysis of phage FBa1 proteins by SDS-PAGE showed five protein bands
(Figure 6). Molecular weight of phage FBa1 proteins was 70.9 kDa; 54.9 kDa;
33.8 kDa; 28.3 kDa; and 21.4 kDa respectively.

Figure 6 Molecular weight of phage FBa1 proteins on SDS-PAGE gel. Lane 1,
molecular size marker (PageRuler TM, Prestined Protein Ladder);
lanes 2, 3, and 4, phage FBa1 proteins.

11

DISCUSSIONS
Bacillus pumilus was isolated from water in Cigudeg, Bogor, West Java. It
indicated that water environment in this location was contaminated with B.
pumilus. There was no report about contamination of B. pumilus from water
samples in Cigudeg, Bogor, previously. The B. pumilus isolate produced a toxin
that lysed red blood cells in culture media. Hoult and Tuxford (1991) reported that
B. pumilus had haemolytic activity. Haemolysin toxin produced by B. pumilus
caused diarrhea in human. Haemolysin is a toxin having dermonecrotic and
vascular permeability activities and causing fluid accumulation. It implicated in
diarrheal illness.
Most studies on bacterial antibiotic resistance focused on pathogenic
microorganisms. Very limited information on antimicrobial susceptibility profiles
of B. pumilus was available. Ozkocaman (2006) reported that B. pumilus was
resistant to penicillin and sensitive to ciprofloxacin. This study showed that B.
pumilus isolate was resistant to ampicillin and clindamycin, but it was still
sensitive to amoxicillin and ciprofloxacin. Resistant of antibiotic would be a
problem. It could overcome by alternative solution of bacteriophage therapy.
Phage was isolated from water. Water sample was collected from river
because phages can be found in the environments where their bacterial host
inhabit. This study found phage isolate that can infect and kill B. pumilus through
cell lysis. Phage progeny was released after host bacterial cell lysed. The progeny
could diffuse in soft agar and then infect the surrounding cells. Lysis of the cells
results a circular clear zone called a plaque. Interestingly, halos were formed
around some of plaques. A secondary lysis zone around the centre of plaques was
due to endolysins activity. Some bacterial cells were not yet fully lysed in
secondary lysis zone. Plaque morphology (plaque haloing) in this results was
similar to plaque of B. pumilus phage reported by Grilione and Carr (1960).
Plaque morphology of phage FBa1 was a 2 mm diameter translucent plaque
with halo, plaque of FBa2 was a 1 mm diameter translucent plaque with halo, and
plaque of FBa3 was a 0.5 mm diameter translucent plaque without halo. The
different of plaque size in this results may be caused by delays in adsorbtion.
Delays in adsorbtion makes a lower adsorbtion rate and results a smaller plaque
size (Abedon et al. 2001). Plaque size was also influenced by numerous factors,
such as reducing the agar concentration, condition of incubation, and log phase
cells of host bacteria (Clokie and Kropinski 2009). Reducing the agar
concentration in the medium makes the phage diffuses easily. Condition of
incubation must suitable for host bacteria to produces many phage progeny.
The differences of plaques morphology indicated the presence of different
phages in the samples from where they were isolated. Phages were purified by
single-plaque isolation. Three phage isolates were purified based on their plaque
morphology. Plaques morphology were the same as plaques morphology of
isolation step, hoever different in plaque size or diameter. Concentration of FBa1
phage was 10.2 x 108 PFU mL-1, FBa2 phage was 5.9x108 PFU mL-1, and FBa3
phage was 8.5x108 PFU mL-1.
The titer of FBa1 phage was higher than that of FBa2 phage and FBa3
phage. It means that FBa1 phage population in environment was higher than the
other phages. Titer values could indicated phage generation time. The phage

12
generation time devided into three period, (1) diffusion of phage progeny to new
host cells; (2) the phage eclipse period; (3) a period of progeny maturation
(Abedon et al. 2001). The phage generation was controlled by the phage lysis time.
The phage lysis time was defined by the infective phage particles released from
the host bacteria. The longer lysis time results the larger burst size (Wang 2006).
The FBa1 phage with larger burst size associated with longer lysis time. In the
longer lysis time, FBa1 phage could continue to accumulate progeny virions
before lysis. The larger burst size was formed the larger plaque size. Host cell
growth in logarithmic phase was also effected the phage replication (Woody and
Cliver 1995). In this study, log phase of host cell was used for optimum phage
replication and produced large burst size.
This study was showed that phage FBa1, FBa2, and FBa3 had lytic activity
to B. pumilus. It was not infect Photobacterium damselae, Proteus mirabilis,
Salmonella sp, and EPEC K1.1. FBa1, FBa2, and FBa3 phage isolates had a
narrow host range. It was specific to B. pumilus. Specificity interaction of phage
with bacterial cell is determined by specificity of adsorption, dependent on the
structural of reseptors on bacterial cell surface (Braun and Hantke 1997). Phages
bind to all possible bacterial cell surface receptors including pili (Roncero et al.
1990), flagella (Shade et al. 1967; Lovett 1972), capsule (Suthereland et al. 2004),
teichoic acid (Raisanen et al. 2007), surface protein (Davison et al. 2005), and
lipopolysaccharide (Lindberg et al. 1978).
Morphological observation of phage FBa1 using TEM negative staining.
Uranyl acetate was used for negative staining in this study. Uranyl acetate is
acidic, acts as afixative, inactives phages. Morphology of phage FBa1 showed an
icosahedral capsid without tail of phage-like particles. The phage-like particles are
phage without tail and were more abundant than tailed phage in environment
(Ashelford et al. 2003). Phage FBa1 in this result were different from previously
discovered of phiAGATE. Phage phiAGATE infecting B. pumilus by TEM
analysis showed that virions phiAGATE had tail morphology and assumed that
phage was a member of Myoviridae family (Barylski et al. 2014).
Phage FBa1 did not have a tail to attach Bacillus pumilus. Phage FBa1 may
be have adsorption mechanism like phage infecting Pseudomonas syringae (Von
Seggern et al. 1999). The virion is fixed to the distal part of pilus. The virion
moves closer to cell surface and then binding to the baseplate of pilus. Pilus
retraction brings the virion to contact with the host’s outer membrane, and
crossing the outer membrane deposits the nucleocapsid in the periplasm. Phageencoded lytic enzyme on the nucleocapsid surface digests the peptidoglycan layer,
and then brings the nucleocapsid into contact with the host’s plasma membrane.
Electrochemical membrane potential, ATP molecules, enzymatic splitting of
peptidoglycan layer may be important for penetration of genetic material phage
inside the bacterial cell (Rakhuba et al. 2010).
Molecular weight of phage proteins was analyzed using SDS-PAGE. Five
bands were observed at SDS-PAGE gel, they may correlated to structural and
functional protein of phage FBa1. Three bands were observed at 70.9 kDa, 54.9
kDa, and 33.8 kDa, they may correlated to structural proteins of phage FBa1.
Capsid proteins of phage SPP1 infecting B. subtilis was reported between 47.5
kDa and 32.5 kDa (Vinga et al. 2006). While 28.3 kDa protein was similar to
purified protein of LysB4. LysB4 (28 kDa) is an endolysin from the Bacillus

13
cereus-infecting bacteriophage B4 (Son et al. 2012). And 21.4 kDa protein may
correlated to endolysin of phage. Endolysin molecular weights of phage BtCS33
infecting Bacillus thuringiensis was 24 kDa and 11 kDa respectively (Yuan et al.
2012).
Lytic phage was produced holin and endolysin to degradate bacterial cell
wall (Hanlon 2007). The combinated action of the holin-lysin proteins was
important in releasing of new phage particles from an infected cell (Daniel et al.
2007). Holin forms a hole in the cell membrane, and endolysin passes through the
hole and destroys the peptidoglycan structure. Two gene products were
determined to be the possible lysin, an N-acetylmuramoyl-L-alanine amidases
and/or L-alanoyl-D-glutamate peptidase. The endolysin have two domains
connected by a short linker: the N-terminal catalytic domain is responsible for cell
lytic activity and the C-terminal cell wall binding domain that recognizes and
binds a specific substrate in the cell wall of target bacteria (Fischetti 2008).

CONCLUSIONS
Phage FBa1, FBa2, FBa3 were isolated from Ciapus River in Bogor, West
Java. They were capable to lyse bacterial cells and specific for Bacilllus pumilus.
The phage FBa1 was typical icosahedral capsid phage-like particles with 166.67
nm in diameter. Molecular weight of phage FBa1 proteins was 70.9 kDa; 54.9
kDa; 33.8 kDa; 28.3 kDa; and 21.4 kDa respectively. Phage FBa1, FBa2, and
FBa3 had potency for biocontrol of Bacilllus pumilus in the environment and to
be applied in food production process.

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