Food and Nutrition Sciences, 2013, 4, 56-66
Published Online November 2013 (http://www.scirp.org/journal/fns)
http://dx.doi.org/10.4236/fns.2013.411A008
Open Access FNS
Isolation and Characterization of Bacteriophages from
Laban Jameed*
Murad Mohammad Ishnaiwer, Fawzi Al-Razem#
Biotechnology Research Center, Palestine Polytechnic University, Hebron, Palestine.
Received July 25th, 2013; revised August 25th, 2013; accepted September 2nd, 2013
Copyright © 2013 Murad Mohammad Ishnaiwer, Fawzi Al-Razem. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
ABSTRACT
Laban jameed is a dried salty dairy product obtained by fermentation of milk using a complex population of lactic acid
bacteria. Jameed is considered a traditional food product in most eastern Mediterranean countries and is usually made
from sheep or cow milk. The aim of this study was to assess phage contamination of jameed dairy product. Two phages
were isolated: one from sheep milk jameed (PPUDV) and the other from cow milk jameed (PPURV). Each of the two
bacteriophages was partially characterized. The PPUDV phage was identified as a single stranded DNA virus with an
approximately 20 kb genome that was resistant to RNase, whereas PPURV phage possessed a double stranded RNA
genome of approximately 20 kb and was resistant to DNase. The phage bacterial strain hosts were identified as Lacto-
bacillus h elveticus and Bacillus amyloliqu efaciens for PPUDV and PPURV, respectively. One step growth curve using
a double layer plaque assay test was carried out to monitor the phage life cycle after host infection. PPUDV showed a
latent period of about 36 h, burst period of 70 h and a burst size of about 600 Plaque Forming Units (PFU) per infected
cell. PPURV phage showed a latent period of about 24 h, burst period of 47 h and a burst size of about 700 PFU per
infected cell. SDS-PAGE analysis of total viral proteins showed at least three major bands (27, 40, and 45 kDa) for
PPUDV. This is the first study to report the isolation of both DNA and RNA bacteriophages from laban jameed. This
study adds new insights into the complexity of dairy contamination and fermentation microbiology of the jameed re-
vealing the existence of two viral genomes in this highly dried and salty dairy product.
Keywords: Lactic Acid Bacteria; Bacteriophages; Laban Jameed; DNA Viruses; RNA Viruses
1. Introduction
Laban jameed is a dried and salty dairy product that
forms part of an ancient traditional diet that is common
in the Middle East, particularly in Syria, Lebanon, Jordan,
Saudi Arabia and Palestine [1-3]. It is favored by
Bedouin communities because of the ease of storing this
dairy product for a long period of time and due to its high
stability and resistance to pathogens. Jameed is tradi-
tionally made from sheep and/or cow fermented milk and
is used in many Arab food dishes, such as Mansaf [1,2].
Milk fermentation occurs mostly through the action of a
highly complex microflora of lactic acid bacteria (LAB)
through continuous shaking, leading to a highly acidic
product, formation of butter (curd) and expulsion of the
milk serum liquid (whey). The curd is then slightly heat-
ed to accelerate the onset of the fermentation process and
to further ensure the full separation of whey. The whey is
decanted and the curd is added into a cheesecloth
container to remove the excess water. When it becomes a
thick paste, called labaneh in Palestine, it is kneaded or
sprinkled with sodium chloride salt (approximately 10%
salt contents) and placed to dry for a few days in the sun
to ensure that no dampness occurs, which could spoil the
product [1-3].
In most processed dairy products, the fermentation of
milk is facilitated by using a mixture of LAB, such as
Lactobacillus acidophilus, L. brevis, L. casei, L. fermen-
tum, L. kefir, L. parakefir, L. plantarum, and L. helviticus
[4,5]. Lactic acid bacteria are generally gram positive
and acid tolerant and producing lactic acid as a final
product of carbohydrate fermentation [5,6]. Similar to
*Authors’ contribution: M.M—I carried out the experimental work and
p
rovided the first draft of the manuscript. F. Al-R designed and super-
vised the work. Both authors have read and approved the final manu-
script.
#Corresponding author.

Isolation and Characterization of Bacteriophages from Laban Jameed 57
many other bacterial species, the LAB are susceptible to
several types of bacteriophages and some of these phage
particles have been isolated and characterized [5-8].
Most LAB known phages are tailed and are members of
the Caudovirales order [8].
With the expanding dairy product industries, phage
contamination is a growing problem. This contamination
is believed to affect the fermentation process and the
quality of dairy product [6,9]. Contamination could ori-
ginate from different sources, such as water, soil, air,
cattle feces, cattle udder and milk equipment [1,9]. The
phage contamination in dairy products becomes proble-
matic to dairy industries even with a minute amount of
phage particles due to phage ability to rapidly increase its
numbers once a bacterial host is available. To add to this
problem, phages usually tolerate high acidity and high
temperature values during the pasteurization process.
Phages can cause a collapse of the lactose-lactic acid
pathway and decrease the overall efficiency of the
fermentation process. These problems are detrimental to
dairy product quality and often make the food products
susceptible to spoilage by additional bacterial invasion,
which can further hurt the fermentation process [1,6].
Lactic acid bacteria adapted and modified a variety of
anti-phage defense mechanisms to escape phage infec-
tions. Such mechanisms were mostly observed against
dsDNA phages [8,10,11]. Phages, however, utilize
several tricky circumventions that were reported to sup-
press those mechanisms [8,9,11]. The most common
bacterial anti-phage defense mechanisms are developed
to suppress phage adsorption, DNA injection and recrui-
tment of restriction modification systems. Accordingly,
bacteria use specific proteins that mask the phage reco-
gnition site receptor located at bacterial cell surface or
even invoke a conformational change, which causes pha-
ges to mistake their attachment point. In addition,
bacteria can alternatively mask their receptors through
secreting specific sugar residues called exopolysaccha-
rides, which help in inhibiting phage adsorption. Phages,
however, can predominantly secrete specific polysaccha-
ride degrading enzymes called lyases that degrade those
bacterial residues [8,10,11].
Moreover, some LAB generate bacteriophage-insen-
sitive mutants (BIMs) in which a cascade of phage
mutations results in the alteration of recognition sites that
inhibit bacteriophage adsorption. Such point mutations
have been reported in chromosomal genes coding for
Lactococcus cell receptors [8,10,11]. Mutant phages
could, however, overcome this modification and infect
those resistant bacteria. In some cases, if bacteria
couldn’t inhibit phage adsorption, they instead secret
other specific proteins that affect genome translocations
to the cytoplasm through changing the injection site and
blocking the cell wall degradation [8,10,11]. Otherwise,
if phage genomes are able to adapt to these challenges
and successfully pass through the cytoplasm, bacteria
recruit other further defense mechanisms termed as res-
triction-modification systems to degrade unmethylated
genomes at specific sites.
Due to multiple antibiotic-resistant bacteria, the use of
bacterial viruses, i.e., bacteriophage therapy as alterna-
tive to conventional antibiotics is rapidly increasing.
Bacteriophages can be more specific than antibiotics.
One advantage of phage therapy is the specificity of
targeting only the host bacterial cells, while antibiotics
could also kill a wide range of bacteria in addition to the
targeted harmful one [9]. In addition, there are no repor-
ted cases of side effects following the use of LAB phages,
unlike most antibiotics that may enhance the side effects
[9].
The aim of this study was to isolate and characterize
jameed milk bacteriophages and identify their host cells.
To our knowledge this study represents the first isolation
of ssDNA and dsRNA bacteriophages from dairy sour-
ces.
2. Materials and Methods
2.1. Laban Jameed Bacterial Culture
Samples of laban jameed used in this study were
obtained from the cities of Tulkaram (north) and Dahriya
(south) of Palestine. The Tulkaram jameed was made of
cow fermented milk, whereas the Dahriya jameed was
made of sheep fermented milk. The isolated bacterio-
phages were named after the Palestine Polytechnic Uni-
versity (PPU) and its nucleic acid composition: PPUDV
(PPU DNA Virus) for phage isolated from Dahriya
jameed and PPURV (PPU RNA Virus) for phage isolated
from jameed obtained from the city of Tulkaram. Jameed
samples were stored at −80˚C until they were processed.
From each jameed sample, a small piece of approxi-
mately 1.0 gram was incubated for 2 days at 37˚C with
continuous shaking at 200 rpm in 100 ml skim milk
broth (5 g skim milk (SM) powder in 100 ml ddH2O)
until the O.D at 600 nm reached 0.2. This was followed
by plating 200 μl from each culture on SM agar plates
(100 ml SM broth added to it 1 g agar). Individual colo-
nies were then picked and re-cultured separately in new
flasks containing 100 ml SM broth to examine the lysis
of the bacteriophages. All bacterial stock cultures were
stored at −80˚C in SM containing 16% (v/v) glycerol.
When needed, frozen culture stock was allowed to thaw
and used to prepare an overnight culture in SM broth
before plating 200 μl from the overnight culture on SM
agar plates.
2.2. Phage Isolation
Fifty ml of two cultures of SM broth inoculated with a
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Isolation and Characterization of Bacteriophages from Laban Jameed 58
piece of jameed milk were tested for the presence of
phages. To ensure purified phage filtration from bacteria,
5 drops of chloroform were added to each sample, stored
for 15 min at room temperature before centrifugation at
13,000 rpm for 5 min. This step was repeated twice to
ensure that sufficient phage particles were purified. The
supernatant was then transferred to a new microfuge tube
and re-centrifuged again. It was finally filtered through
0.45 μm sterile filters and filtrates were named according
to the sample origin.
Phage filtrates were added upon overnight bacterial
culture plated on SM agar plates to monitor phage
sensitivity (lysis) or resistance (no lysis). Phage filtrates
from Tulkaram region were tested on both Tulkaram and
Dahriya bacterial colonies and the same was done to the
Dahriya filtrate. As a control, each time a tube containing
only bacteria without phage filtrates was used in each
manipulation, plates were kept for 15 min in a laminar
flow hood to dry, before incubation at 37˚C up to 3 days
until phage lysis was detected. Each bacteriophage lysis
was carried out for at least three subsequent times until a
pure phage was obtained. A small piece of each lysis was
stored in a small 1.5 ml Eppendorf tube in −80˚C freezer
as a stab culture.
2.3. Characterization of the Host Bacteria and
Marker Analysis
Genomic DNA for each bacterial host that showed
sensitivity to phage lysis was extracted using EZ-DNA
Kit (Biological Industries, Cat# 20-600-50). The primer
sequences for the two markers used to identify the host
strains isolated from the Jameed are shown in (Table 1).
The two markers were the recA gene, which is considered
specific for LAB and the gene encoding the 16S rRNA.
For both recA and 16S rRNA genes, the PCR reaction
contained 1 μl of template DNA for each host colony, 2.5
μl of 10 x PCR reaction buffer, 0.5 μM of each primer (10
pmol concentration), 2.5 μl of 20 mM MgSO4, 0.5 μl of 20
mM dNTPs and 1.25 U of thermostable Taq polymerase.
The mixture volume was completed with ultrapure water
Table 1. Primer sequences used to amplify the marker
genes, recA and 16S rDNA partial sequences. Information in
relative to their sequences and size of amplified fragment
are included.
Name Sequence Length
recA reverse 5’-T TY ATHGAY GCN GAR CAY GC-3’
recA forward 5’-CCW CCW GKWGTHGTYTCNGG-3’340 bp
16S rDNA
reverse 5’-GGACTACCAGGGTATCTAAT-3’
16S rDNA
forward 5’-AGTTTGATCCTGGCTC AG-3
780 bp
Y for (C or T), H for (A or C or T), R for (A or G), W for (A or T), N for (A
or C or G or T), M for (A or C), K for (G or T).
to a final volume of 25 μl. Amplification of the recA gene
was conducted using the following conditions: initial
denaturation was performed at 94˚C for 3 min and for 30
sec for the subsequent 30 cycles, followed by 30 sec for
primer annealing at 54˚C, elongation of the target gene
with taq polymerase at 72˚C for 30 sec. A final extension
of 5 min at 72˚C was followed by cooling down to 4˚C.
The 16S rRNA PCR reaction conditions were as follows:
95˚C for 5 min for the initial denaturation, 1 min dena-
turation for the subsequent 30 cycles, primer annealing at
51˚C for 1 min, target elongation at 72˚C for 1.30 min. A
final extension of 10 min at 72˚C was followed by cooling
down to 4˚C. For gene sequencing, PCR product puri-
fication was achieved by following instructions provided
in the AccuPrep PCR Purification Kit (Bioneer K-3035).
DNA sequences of the isolated DNA markers were
aligned manually, then similarity trees were constructed
using UPGMA method with MEGA V.5 software.
2.4. Bacteriophage Genome Isolation
Bacteriophage genomes were extracted according to a
protocol developed by Manasra and Barghouthi [12] with
minor modifications as follows. Two volumes (1000 μl)
of saturated ammonium sulfate containing 0.1% of 2-
mercaptoethanol were mixed with the phage filtrate (500
μl) for 5 min. Supernatant was removed after centri-
fuging at 13,000 rpm for 5 min at 4˚C. The pellet was
then dissolved in 0.2 ml 1% SDS and 0.2 ml 0.5 N NaOH
and centrifuged at 13,000 rpm for 5 min. To the clear
supernatant, 0.4 ml of 3 N sodium acetate was added in
addition to 0.6 volume of isopropanol to precipitate the
genome, and then held for 15 min at room temp. The
mixture was then centrifuged at 13,000 rpm for 10 min
and the resulting pellet was incubated with 100 μg/μl of
proteinase K at 37˚C for 30 min. Finally, the phage ge-
nome was precipitated using 70% ethanol and the pellet
was collected in 0.2 ml TE buffer after 2 min centri-
fugation at 8000 rpm.
2.5. Bacteriophage Genome Characterization
The entity of nucleic acids was determined via the treat-
ment of phage genome with DNase and RNase. Ten μl of
each genome sample was incubated with 3 μl of DNase
and the same with RNase for 35 min at 37˚C. The mix-
ture was then loaded on 0.7% agarose gel using undi-
gested genome as a control, and lambda phage ge nome-
treated with HindIII restriction enzyme was used as a
high molecular weight ladder.
The RNase A treatment at low and high concentrations
was used to determine ss/ds RNA genomes according to
the following references [13-17]. Briefly, 10 μl of
PPURV genome were incubated with 3 μl RNase A ei-
ther with low (0.1 M) or high (0.4 M) NaCl concen-
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Isolation and Characterization of Bacteriophages from Laban Jameed 59
tration for 1 h at 37˚C. Following treatment, samples
were mixed with 6× loading dye and loaded on 0.7% aga-
rose gel as above. To determine whether phage genomes
belong to dsDNA or ssDNA, 10 μl of genome was boiled
for 6 min, placed on ice, before rapidly being loaded on a
0.7% gel electrophoresis with un-boiled genome as a
control and lambda phage genome was used as a ladder.
Furthermore, isolated genomes were treated with a set
of specific dsDNA restriction enzymes including, MluI,
BamHI, EcoRI, HindIII and PvuI following protocols
provided by the manufacture. Briefly, the same reaction
mixture of 20 μl was prepared for all restriction enzymes
at which 16 μl nuclease-free water was mixed with 2 μl
10× buffer, 1 μl from each restriction enzyme and 1 μl of
DNA template. Samples were then incubated at 37˚C for
8 and 16 h periods before the reaction mixtures were
loaded on a 1% agarose gel.
The SDS-PAGE was carried out according to [18,19].
Total phage proteins were separated on a 10% SDS-
PAGE, stained with 0.1% (w/v) Coomassie Brilliant Blue
(Applichem/ A3480,0010) before documentation using a
digital camera.
2.6. One Step Growth Curve
The double layer Plaque assay method was used accord-
ing to published work [20,21] as follows: Previously
prepared high titer filtrates were used, a single plaque
was picked up from an agar plate, mixed with log phase
bacteria O.D600 nm of 0.2, and then incubated for 3 hours.
Samples were then purified as above and used for one
step growth curve. 10-fold serial dilutions were
performed with each dilution prepared by mixing 0.1 ml
of stock phage suspension in 0.9 ml water (tube labeled
as tube 1). The 0.1 ml from tube 1 sample was trans-
ferred into tube 2, filled with 0.9 ml water; the same was
done for the other remaining dilutions. From each titer,
0.1 ml bacteriophage suspension dilution was inoculated
to 0.5 ml of O.D600 nm = 0.2 bacterial culture, incubated
at 37˚C for 40 min, and then added to a tube containing 3
ml of 0.7% soft agar heated at 49˚C and gently mixed.
Finally it was poured onto a separated prepared mono-
layer SM plate. Plates were then incubated upside down
at 37˚C. Plaque formation was monitored and data were
recorded.
3. Results
3.1. Isolation of Bacterial Strains and Phage
Filtrates
Three bacterial colonies from each skimed milk agar
plate representing different jameed samples (see M&M
Section 2.1) were selected for further testing against
phage filtrates. Each colony was labeled in reference to
the bacteria (B) and colony number (1 - 6), (i.e., BC1 for
bacteria colony 1, BC2 for bacteria colony 2, etc.). Thus,
the bacterial colonies BC1, BC2, BC3 were selected
from Dahriya samples, whereas BC4, BC5, BC6 were
selected from Tulkaram samples. Phage filtrates were
prepared from jameed samples as described above.
Filtrates were tested against all bacterial colonies from
both cities, Dahriya virus was tested on Tulkaram and
Dahriya, the same was done for the other filtrate.
Screening of phage filtrate effects on the bacterial
lawns showed two clear round plaques, each with
approximately 1.5 cm diameter (Figure 1). The viruses
which could cause the plaques were named PPUDV for
phage isolated from Dahriya sheep milk jameed (Figure
1(a)) and PPURV for phage isolated from Tulkaram cow
milk jameed (Figure 1(b)). The PPUDV bacteriophage
propagated on bacterial colony 1 (BC1), while PPURV
bacteriophage lysis bacteria colony 4 (BC4) bacterial
hosts, the results were confirmed by triplicate experi-
ments. In each case, a control plate consisting of bacterial
strain devoid of phage was prepared. Separated plates
with two sides were used, for example bacterial colony 1
cultured on both sides of the plate, then drops of phage
filtrates spotted on one of the sides, while keeping the
other side as a control with just bacteria without virus.
3.2. Characterization of Phage Host Bacteria
To identify the bacteria which were susceptible to phage
lysis, two marker genes for the 16S rRNA and recA were
amplified from genomic DNA isolated from the BC4 and
BC1 host bacteria. The PCR amplification results show-
ed clear bands matching the expected sizes of the two
fragments (Figures 2(a) and (b)).
The partial sequences targeted, as commonly assessed
Figure 1. Bacterial cultures on skim milk plates showing
bacteriophage lysis. Phage filtrate (30 μl) was added to each
bacterial colony. (a) The PPUDV phage was able to lyse the
bacterial host (BC1) isolated from jameed made of sheep
fermented milk. Lysis appeared as a clear circle at the right
side. The left side is a control with the host bacteria, but
with no phage added. (b) the PPURV phage lysed the
bacterial host (BC4) isolated from jameed made of cow
fermented milk with clear circle at the left side. The right
side represents the control host bacteria showed no lysis.
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Isolation and Characterization of Bacteriophages from Laban Jameed 60
Figure 2. Amplification of two marker genes from the BC4
and BC1 host cells. Amplicons were separated on 1.5%
agarose gel and visualized by ethidium bromide staining
according to standard protocols [18] using 1.5 kb DNA
Ladder. (a) PCR results of the 16S rRNA partial sequence
shows the expected fragment size of 780 bp. Lane 1: DNA
ladder (Promega/G5711), lane 2 is a negative control (mas-
ter mix with no DNA template). Lanes 3 & 4 show the 16S
rRNA amplicons for BC4 and BC1, respectively. (b) PCR
results of the recA gene partial sequence. Lane 1 shows the
DNA ladder (Promega/G5711) Lane 2 is a negative control
(master mix with no DNA template). Lanes 3 & 4 show the
recA gene amplicon for BC4 and BC1, respectively.
by PCR amplification for the 16S rRNA and recA were
approximately (780 bp and 340 bp), respectively.
The purified gel products for the two genes, 16S rRNA
and recA were sequenced in the Hereditary Research
Laboratory/Life Science Department/Bethlehem Uni-
versity. The identity for each sequence was determined
and confirmed by Blast analysis and by aligning against
available sequences on the GenBank
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) using BLAST N
program. Both host bacteria were identified by matching
the sequence with the highest maximum identity score.
To determine the relationship of the host strains with
LAB that are mostly found in dairy products, multiple
sequence alignments for both recA and 16S rRNA se-
quences were constructed using the CLUSTAL W
software. Then phylogenetic trees were constructed using
UPGMA method with MEGA V.5 software comprising
all highly similar alignments, most common dairy bac-
teria and BC4, BC1 recA and 16S rRNA sequences, to
better illustrate the homology with available bacterial
strains (Figures 3 and 4). All closely related sequences
were obtained from the National Center for Biotech-
nology Information (NCBI) databases. In addition, the
16S rRNA and recA partial sequences of the Strepto-
coccus thermophilus (a gram positive bacteria that play a
role in milk fermentation) were used, because several
phylogenetic studies recommended its use as an outgroup,
since other LAB strains are too closely related to serve as
a suitable phylogenetic outgroup [22,23]. From the mar-
ker analyses of the two genes encoding the 16S rRNA
and recA, results showed that the BC4 host is a close
relative to the Bacillus amyloliquefaciens (Figures 3(a)
and (b)), whereas the BC1 is a close relative to the
Lactobacillus helveticus strain (Figures 4(a) and (b)).
Figure 3. The phylogenetic trees for BC4 using marker
genes of the 16S rRNA (a) and recA (b). Trees were con-
structed using UPGMA method. Trees were built using high
sequence similarity from the alignment with the most com-
mon LAB sequences form GenBank. Streptococcus thermo-
philus was used as an outgroup as shown. The recA of BC4
clustered with Bacillus amyloliquefaciens, whereas in 16S
rRNA tree, it was difficult to determine whether BC4 did
actually belong to Bacillus amyloliquefaciens.
Figure 4. The phylohenetic trees for BC1 using marker
genes of the 16S rRNA (a) and recA (b). Trees were con-
structed using UPGMA method. Streptococcus thermophilus
was used as an outgroup as shown. The recA and 16S rRNA
phylogenetic trees of the BC1 clustered with the Lactoba-
cillus helveticus, one of the common LAB.
3.3. Characterization of Isolated Bacteriophages
Two phages representing RNA and DNA genomes were
isolated. The PPURV bacteriophage that was able to
cause lysis to (Bacillus amyloliquefaciens) was found to
be an RNA bacteriophage as it was sensitive to RNase
and resistant to DNase digestion (Figure 5(a)). The PPUDV,
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Isolation and Characterization of Bacteriophages from Laban Jameed 61
on the other hand, was a DNA bacteriophage sensitive to
DNase, but resistant to RNase digestion (Figure 5(b)).
Genome sizes, however, seemed to be very similar, ap-
proximately 20 kb each (Figure 5).
Confirmation that the PPURV genome is dsRNA co-
mes from it being sensitive to RNase A digestion at low
NaCl treatment, but was resistant to RNase A treatment
at high NaCl concentration (Figure 6(a)). The PPUDV
bacteriophage genome, on the other hand, was a ssDNA
as confirmed by agarose gel. Boiled and un-boiled ge-
nomes migrated to the same distance on the agarose gel
(Figure 6(b)).
3.4. One Step Growth Curve of the Isolated
Bacteriophages
To reveal the nature of virus replication upon infection of
host bacteria, one step growth curve assays were design-
ed to allow estimating the titers of bacteriophage stocks
through using a “Plaque Forming Assay” (PFU). This re-
flects how much the original infected cells release viral
progeny. Consequently, the titer calculation in PFU/ml
were obtained through multiplying the number of plaques
with dilution factor then dividing on the inoculum volu-
me [24]. It relies first on constructing a table showing
each titration with the resulting plaques. This helps in
measuring the (PFU) as previously described as only the
first titration is capable of forming plaques.
The data from the plaque assays were analyzed. The
number of PFU per bacterial cell was plotted on the Y
axis against time required to form plaques on the X axis.
Consequently, for each bacteriophage, the latent, burst
period and burst size were as a result determined (Figure
7). For PPURV phage, the latent period was about 24 h,
burst period was 47 h and the burst size was about 700
Figure 5. Identity and sizes of PPUDV and PPURV bac-
teriophages. Both genomes were treated with DNase and
RNase before they were loaded on 0.7% agarose gel elec-
trophoresis. (a) the PPURV genome is an RNA phage.
Lane1 contains 23 kb ladder and lane 2 contains undigested
PPURV genome. Lane 3 contains PPURV genome treated
with DNase and Lane 4 the PPURV genome treated with
RNase. (b) the PPUDV genome is a DNA phage. Lane 1
contains a 23 kb ladder and lane 2 contains the undigested
PPUDV genome. Lane 3 contains PPUDV genome treated
with DNase and Lane 4 PPUDV genome treated with
RNase.
Figure 6. The PPURV is a dsRNA whereas PPUDV is a
ssDNA phage. (a) Lane 1 contains a 23 kb ladder, lane 2
contains the PPURV genome treated with RNase A in the
presence of 0.4 M NaCl and lane 3 contains the PPURV
genome treated with RNase (a) at 0.1 M NaCl. (b) the
PPUDV is a ssDNA phage. Lane 1 contains a 23 kb ladder,
lane 2 contains the PPUDV genome incubated on ice and
Lane 3 contains PPUDV genome after 6 min incubation in
boiling water. All treatments and ladders were loaded on
0.7% agarose gels.
Figure 7. One step growth curves for PPUDV and PPURV
bacteriophages. The curves showed the latent, burst period
and burst size for each phage through its replication after
bacteria intrusion. It is based on counting the number of
formed plaques that were developed, then measuring PFU
and comparing it against time. Results are the measure of
three time trials. For PPURV phage, the latent period was
about 24 h, burst period was 47 h and the burst size of
approximately 700 PFU per infected cell. Whereas, the
latent period for PPUDV was about 36 h with burst period
of about 70 h and a burst size of about 600 PFU/infected
cell.
PFU per infected cell. Whereas, the latent period for
PPUDV was about 36 h with burst period of about 70 h
and a burst size of about 600 PFU/cell.
3.5. SDS-PAGE Analysis of the Isolated
PPUDV Bacteriophage
The phage total proteins were analyzed on SDS-PAGE.
Samples were electrophoresed on a 10% polyacrylamide
gel in the presence of SDS. Stained gels showed three
distinct protein bands for PPUDV phage particles (Fig-
ure 8). The band sizes were estimated to be approxi-
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Isolation and Characterization of Bacteriophages from Laban Jameed 62
Figure 8. SDS-PAGE gel image of PPUDV phage proteins.
Lane 1 shows three bands and lane 2, empty well with no
phage sample. Lane 3 contains high molecular weight pro-
ein ladder (Sigma/S8320). As shown, at least three major
bands (27, 40 and 45 kDa) were detected.
mately 27 KDa, 40 KDa and 45 KDa.
4. Discussion
4.1. Bacteriophages PPUDV and PPURV
Genome Characterizations
Dairy products are essential components in food indus-
tries and individual nutrition in Palestine. The quality of
dairy products is quite critical and any contamination is
considered detrimental and results in major economic
losses. Contamination that is caused by bacteriophages
generally decreases the quality of food products and is
considered undesirable by dairy industries.
We reported the isolation of two bacteriophages from a
traditional dairy product called laban jameed: a dsRNA
PPURV and ssDNA PPUDV hosting on Bacillus amylo-
liquefaciens and Lactobacillus helveticus, respectively.
The majority of identified Bacillus amyloliquefaciens
and Lactobacillus helveticus bacteriophage genomes be-
long to dsDNA [25-28]. However, little is known about
dsRNA and ssDNA bacteriophages that host on Bacillus
amyloliquefaciens and Lactobacillus helveticus strains. A
dsDNA Lactobacillus helveticus with genome size 36,566
bp from Grana Padano cheese product was isolated [28].
Also, Sechaud et al. [26] characterized 35 cheese whey
Lactobacillus helveticus dsDNA bacteriophages, but
without providing information on their genome size. Most
lactic acid bacteriophages are believed to be dsDNA
[5,27]. In addition, the isolation of 235 baceriophages
affecting L. helveticus, L. delbrueckii subsp bulgaricus
and L. delbrueckii subsp lactis strains, all with dsDNA
and belonged to Siphoviridae, Myoviridae and Podoviri-
dae were also described [27].
There are no reports of dsRNA bacteriophages that host
on Bacillus amyloliquefaciens, although a dsDNA
PBA12 Bacillus amyloliquefaciens bacteriophage was
isolated from soil [25]. While Qiao et al. (2010) [29]
described a dsRNA bacteriophage from radish leaves with
genome size of 12684 bp hosting on Pseudomonas sy-
ringae bacteria. But so far, this study represents the first
isolation of dsRNA phage that hosts on Bacillus amylo-
liquefaciens and ssDNA phage that hosts on Lactobacillus
helveticus from dairy jameed food product. The bacte-
riophage genomes were isolated under reducing condi-
tions to minimize degradation. The -mercaptoethanol
was added to inhibit the activity of DNase and RNase
degrading enzymes by weakening their disulfide bonds
[12].
To liberate phage genomes from DNA and RNA
binding proteins, phage proteins were denatured and pre-
cipitated through the addition of NaOH and SDS and the
phage genomes were precipitated from the supernatant
through the addition of sodium acetate in the presence of
-mercaptoethanol as reported by other studies [14,30].
The PPUDV phage was found to be a DNA genome as no
bands appeared on the 0.7% agarose gel when treated with
DNase. This is in contrast to PPURV phage which was
insensitive to DNase. Unlike PPUDV, it was responsive to
RNase digestion as observed on the agarose gel (Figures
5(a) and (b)). Both PPUDV and PPURV genomes ap-
peared to have the same sizes, approximately 20 kb ± 1 kb
as observed on agarose gel using a 23 kb ladder.
The nature of the bacteriophage genomes was further
studied. The RNA genome was determined to be a dsRNA
(Figure 6(a)), whereas the DNA genome was a ssDNA
genome (Figure 6(b)). There are other assays, such as the
Hyperchromicity test, ds/ssRNase specific enzyme deg-
radation and RNase III specific for breaking down dsRNA
that were reportedly used to differentiate between ds/ss
genomes, but these methods were reported to possess low
efficiency in working on small amounts of RNA [13,30].
In this study, the RNase A assay was preferred and used.
The RNase A digests both ssRNA and dsRNA under low
NaCl concentration (0.1 M), while it digests only ssRNA
under high NaCl concentration (0.3 M) [13-17,30,31]. A
previous study reported the resistance of dsRNA of vi-
ruses infected plant and fungi to RNase A treatment under
0.3 M NaCl even after 24 h incubation [13]. Following
treatment with high salt concentration, clear bands were
still visible, but disappeared under low (0.1 M) salt treat-
ment. In addition, ssRNA extracted from other viruses
was also examined. It completely disappeared under 0.1
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Isolation and Characterization of Bacteriophages from Laban Jameed 63
M and 0.3 M NaCl treatments [13]. The PPUDV bacte-
riophage genome, on the other hand, was a ssDNA. The
ssDNAs are usually smaller in size than dsDNAs, thus
having more fragile structures and are expected therefore
to run much faster on agarose gels. The heated PPUDV
genome migrated similarly to the un-boiled PPUDV ge-
nome on the gel, thus confirming its ssDNA identity. The
single strandedness entity was further confirmed by the
resistance of genomes to restriction enzyme treatment that
cut dsDNA, no bands were detected in both reactions
incubated at 8 h or 16 h (data not shown).
The ssDNA phage genomes are mostly related to either
the Microviridae, circular ssDNA genomes with nonen-
veloped and isometric shapes, or Inoviridae families, cir-
cular ssDNA genomes with nonenveloped and filamen-
tous shapes as appeared by morphological studies using
the electron microscope [32,33]. Phages that belong to the
Cystoviridae family are dsRNA and possess segmented
genomes with enveloped capsids and spherical shapes for
the phage genomes. However, up to 2009, 88% of com-
pletely sequenced genomes in GenBank phage databases
are related to dsDNA, mainly distributed among the
Caudoviridae orders. The PPUDV and PPURV bacte-
riophages are therefore possibly belong to the Microviri-
dae, or Inoviridae (ssDNA nucleic acid) or Cystoviridae
(dsRNA) family, respectively.
To identify the phases of bacteriophage infection, one
step growth curves were constructed. These curves were
performed using the double layer plaque assay, a com-
monly used and highly accurate, fast and easy to handle
method [21]. It comprises an agar layer at the plate surface,
overlaid with another soft layer (0.7%) of agar containing
a phage-bacterial suspension. The phage-bacterial sus-
pension is first incubated at 37˚C to ensure phage ad-
sorption to bacteria, then the soft agar is added in a steril-
ity conditions to avoid any other microorganism con-
taminations. The latent period, burst period and burst size
are then determined.
The latent period illustrates the time period from the
adsorption of phage to the host bacteria, until the onset of
cell lysis and burst. Host cells rapidly lyse and release
infective phage at burst period, occurring after the latent
period. The average number estimated for phage progeny
to be formed per infected bacterial host cell symbolizes
the burst size (Figure 7). Results are recorded as the mean
of three trials; measures of PFUs were approximately
conserved. This means the relationship between phage
titer and phage infection on the related host cells are the
same in the three trials. Both PPURV and PPUDV bacte-
riophages have long latent periods. This indicates that
further conditions should be manipulated as to detect
optimum bacteriophage activity after infecting their host
bacteria. Thus, such conditions could be the culture media
itself, as skim milk broth and agar that were used. Other
studies preferred the MRS broth media being used for the
detection of dairy bacteriophages [34,35].
Three major protein bands appeared for PPUDV bac-
teriophage with estimated molecular weights of ap-
proximately, 27, 40 and 45 kDa (Figure 8). These results
indicate that aggregations of several proteins accumulate
at each band and/or that some proteins are repetitive
copies encoded from the same gene. This is very common
in viruses, for example, the capsid surface proteins are
usually repetitive of small number of genes [36]. For
PPURV bacteriophage, it was difficult to obtain good
resolution of the total PPURV proteins (data not shown).
This is probably due to the fact that RNA binding proteins
are susceptible to degradation.
4.2. Bacterial Hosts of PPUDV and PPURV
The molecular characterization was used to identify the
two host bacteria that were susceptible to bacteriophage
lysis. Two phylogenetic markers that are universally dis-
tributed in bacteria and commonly used in bacterial strain
identifications, the 16S rRNA and recA genes were tar-
geted [37-39]. The16S rRNA is used as a useful marker in
bacterial characterization as it has conserved sequence
and function among bacteria, though it shows several
limitations due to its inability to differentiate between
closely related species that share 99% or higher sequence
identity, like in some LAB strains [38]. Such inability was
clearly observed in the case of the L. plantarum and L.
pentosus [38]. Therefore, to further confirm the identity of
the host bacteria in this study, the recA gene was also used
as a marker commonly used in lactic acid bacterial iden-
tifications. In this regard, the recA gene has a fundamental
advantage over the gene encoding the 16S rRNA, its abil-
ity to differentiate closely related species.
Sequence analysis and GenBank blast of the two
marker genes used for the identification of BC4 and BC1
revealed their identities as Bacillus amyloliquefaciens for
BC4 and Lactobacillu s helveticu s for the BC1 (Figures 3
and 4). The 16S rRNA was less informative in determin-
ing the relatedness of the BC4 bacteria. Using the highest
maximum identity score, it was initially difficult to de-
termine whether BC4 was a close relative to Bacillus
vallismortis, or Bacillus subtilis, or Bacillus amylolique-
faciens. The use of the recA easily confirmed, however,
the identity of the BC4 host to be more closely related to
Bacillus amyloliquefaciens (Figure 3). The B. amyloliq-
uefaciensis is a gram positive bacteria, discovered and
normally found in soil. It is considered a useful industrial
microorganism, representing, for example, an ample sour-
ce for producing amylase enzyme commercially used in
starch hydrolysis [40,41]. It is also a source of protease
enzyme that catalyzes the breakdown of proteins and is
used in detergent industries [40]. There is no previous
study to report the natural presence of the B. amylolique-
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Isolation and Characterization of Bacteriophages from Laban Jameed 64
faciens in dairy products and that it plays a role in milk
fermentation similar to LAB. However, it was shown to be
a useful source for a milk-clotting enzyme in some in-
dustrial dairy processing fermentations [42] and in the
fermentation of other food products, such as soybean-
fermented food [43]. Otherwise, the explanation for its
existence is to be a result of jameed contamination with
soil. During the process of jameed drying, it is usually
kept uncovered on the ground in sunny areas for a few
days to reduce the moisture content and so it is possible
that this strain is just a contaminant from the nearby soil.
Milk microbial contaminants belong mostly to the genus
Bacillus, including the B. amyloliquefaciens and the B.
cereus, which produce a toxin that can cause diarrhea and
another that causes vomiting [44]. Bacillus cereus spores
are heat-resistant and may survive pasteurization. There
have even been very rare cases linked to dried milk and
dried infant formulas [45]. The source of contamination in
dairy products could be multifarious, like soil, air and
water [44,46]. The dairy industries implement several
strategies to reduce contaminations, such as pasteurization
under high temperature, but even so, contamination still
could occur due to some microbial heat resistant spores.
Dairy processing that relies on traditional methods is
subject to a higher possibility of contaminations. Tradi-
tional laban jameed production is dependent on personnel
in milk transport and processing under mostly unsterile
environment and storage conditions.
The BC1 host was clearly identified to be a close rela-
tive to the Lactobacillus helveticus strains (Figure 4).
This strain is one of the common lactic acid producing
gram positive bacteria [6,47]. It helps in maintaining good
food flavor and acidic conditions that inhibit the spoilage
of milk products. Furthermore, the relationships of
PPUDV and BC1 and PPURV and BC4 host bacteria with
other related species were determined by the phylogenetic
trees, which were constructed using the highest identities
as appeared by the two markers, recA and 16S rRNA se-
quence blasts. The trees included the most common Lac-
tobacillus bacteria that occupy the highest percentage of
the microbial population; L. pentosus, L. casei, L. fer-
mentum, L. plantarumand L. paraplantarum, while those
with lower identities were masked and excluded.
5. Conclusions
Several studies demonstrated the isolation of dairy prod-
uct bacteriophages, but so far no phages have been iso-
lated from laban jameed. Most of the isolated dairy bac-
teriophages were characterized as dsDNA. To our know-
ledge, this study reports the first isolation of ssDNA and
dsRNA bacteriophages from laban jameed dairy product.
Moreover, as there is a significant increase in antibi-
otic resistance among bacteria, there is a notion to use
phage therapy as an alternative for antibiotics. The iso-
lated bacteriophages from jameed milk may have the
potential for future uses as alternatives for antibiotics if
they prove to lyse pathogenic bacteria, particularly those
that exist in close proximity to milk, such as those caus-
ing mastitis disease. It is believed that this study may
provide further information on the complex interactions
between phages and their hosts, and promote studies on
phage therapy.
6. Acknowledgements
The authors would like to thank Dr. Robin Abu Ghazaleh,
Dr. Rami Arafeh, and Dr. Sameer Barghouthi for care-
fully reading the manuscript and Dr. Yaqoub Ashhab for
advice, Mrs. Amal Abu Rayan for carrying out the se-
quencing, and Mrs. Asma Tamimi and Mr. Hasan Alta-
rada for technical support.
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