Journal of Medical Microbiology Papers in Press. Published February 13, 2015 as doi:10.1099/jmm.0.000040
Journal of Medical Microbiology Characterization of Siphoviridae phage Z and studying its efficacy against multi-drug resistant (MDR) Klebsiella pneumoniae planktonic cells and biofilm --Manuscript Draft-Manuscript Number:
JMM-D-14-00020R1
Full Title:
Characterization of Siphoviridae phage Z and studying its efficacy against multi-drug resistant (MDR) Klebsiella pneumoniae planktonic cells and biofilm
Short Title:
Phage Z against multi-drug resistant (MDR) Klebsiella pneumoniae
Article Type:
Standard
Section/Category:
Clinical microbiology and virology
Corresponding Author:
Saadia Andleeb ASAb-NUST Islamabad, other/autre PAKISTAN
First Author:
Muhsin Jamal
Order of Authors:
Muhsin Jamal Tahir Hussain Chythanya Rajanna Das Saadia Andleeb
Abstract:
Biofilm is involved in many serious consequences for public health and is a major virulence factor contributing to the chronicity of infections. The aim of the current study was to isolate and characterize a bacteriophage that inhibit multi-drug resistant Klebsiella pneumonia (M) in palnktonic form as well as biofilm. This phage designated as bacteriophage Z was isolated from waste water. Its adsorption rate to its host bacterium was significantly enhanced by MgCl2 and CaCl2. It has a wide range of pH and heat stability. From its one step growth, latent time and burst size was determined that were 24 min and about 320 virions per cell, respectively. As analyzed by transmission electron microscopy, phage Z had a head of width (76±10 nm) and length of (92±14 nm) with an icosahedrons' sides of 38 nm with a non- contractile (200±15 nm) long and (14-29 nm) wide tail belonging to family Siphoviridae of order Caudovirales. Six structural proteins ranging from 18 to 65 kDa were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its genome was found to be comprised of double stranded DNA with approximate size of 36 kb. Bacteria were grown in suspension and as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Phage Z was effective in reducing biofilm biomass after 24 h and 48 h and showed more than 2-fold and 3-fold reductions, respectively. Biofilm cells and stationary phase planktonic bacteria were killed at a lower rate than the log-phase planktonic bacteria.
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Characterization of Siphoviridae phage Z and studying its efficacy against multi-drug
2
resistant (MDR) Klebsiella pneumoniae planktonic cells and biofilm
3 4 5
Phage Z against multi-drug resistant (MDR) Klebsiella pneumoniae
6 7
Muhsin Jamal,1, 2 Tahir Hussain,1 Chythanya Rajanna Das2 and Saadia Andleeb1*
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1
Atta -ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences
10
and Technology (NUST), Islamabad, Pakistan.
11
2
Emerging Pathogens Institute (EPI), University of Florida (UF), Florida, USA.
12 13 14
*Corresponding Author:
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Saadia Andleeb
16
Atta-ur-Rahman School of Applied Biosciences (ASAB),
17
National University of Sciences and Technology (NUST),
18
Kashmir Highway, Sector H-12
19
Islamabad, Post code 44000, Pakistan
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Email ID: [email protected]
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Cell No: +92-3335188343
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Office:
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Fax No: +92-51-90856102
+92-51-90856133
1
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Summary
25
Biofilm is involved in many serious consequences for public health and is a major virulence
26
factor contributing to the chronicity of infections. The aim of the current study was to isolate and
27
characterize a bacteriophage that inhibit multi-drug resistant Klebsiella pneumonia (M) in
28
palnktonic form as well as biofilm. This phage designated as bacteriophage Z was isolated from
29
waste water. Its adsorption rate to its host bacterium was significantly enhanced by MgCl2 and
30
CaCl2. It has a wide range of pH and heat stability. From its one step growth, latent time and
31
burst size was determined that were 24 min and about 320 virions per cell, respectively. As
32
analyzed by transmission electron microscopy, phage Z had a head of width (76 ± 10 nm) and
33
length of (92 ± 14 nm) with an icosahedrons’ sides of 38 nm with a non- contractile (200 ±15
34
nm) long and (14-29 nm) wide tail belonging to family Siphoviridae of order Caudovirales. Six
35
structural proteins ranging from 18 to 65 kDa were revealed by sodium dodecyl sulfate
36
polyacrylamide gel electrophoresis (SDS-PAGE). Its genome was found to be comprised of
37
double stranded DNA with approximate size of 36 kb. Bacteria were grown in suspension and as
38
biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Phage Z was
39
effective in reducing biofilm biomass after 24 h and 48 h and showed more than 2-fold and 3-
40
fold reductions, respectively. Biofilm cells and stationary phase planktonic bacteria were killed
41
at a lower rate than the log-phase planktonic bacteria.
42
Key words: Bacteriophage, Waste water, Klebsiella pneumoniae, Multi drug-resistant (MDR),
43
Caudovirales.
44 45 46
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INTRODUCTION
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Klebsiella pneumoniae accounts for a significant proportion of hospital-acquired infections. The
49
most important reservoirs for transmission of Klebsiella are the gastrointestinal tract and the
50
hands of hospital personnel, hence they are crucially involved in causing outbreaks of
51
nosocomial infections (Podschun and Ullmann, 1998).
52
Among the nosocomial pathogens, K. pneumoniae is one of the most important biofilm forming
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bacteria which can cause a large number of infections (Podschun and Ullmann, 1998). Biofilms
54
have been involved in a large number of nosocomial infections associated with medical devices,
55
equipments used in hospital and other hard surfaces which can act as reservoirs for biofilm
56
acquired infections (Rao et al., 2005). It has been estimated that biofilm is involved in more than
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60 % nosocomial infections (Donlan and Costerton, 2002; Vinh and Embil, 2005). The National
58
Institutes of Health (NIH) claims that about 80 % of all chronic infections are caused by
59
biofilms (Monroe, 2007) and in all about 65 % microbial infections are related to biofilms
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(Potera, 1999) . These infections are very difficult to treat due to the resistance of bacteria
61
resistant to numerous antimicrobial agents (Costerton et al., 1999). As the threat of emergence of
62
antibiotic resistance and the inability to eradicate the biofilm structures has increased, the
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likelihood that novel strategies for preventing biofilm growth mode are urgently needed (Mah
64
and O'Toole, 2001).
65
Being obligate parasites of bacteria, the bacteriophages bind to microbial surfaces, injecting their
66
genetic material and replicating within the bacterial host causing lysis of the host cell
67
(Sulakvelidze et al., 2001). Phage therapy has gained an increasing attention because it has many
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advantages over antibiotic therapy. Phages are effective against multidrug resistant pathogenic
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bacteria because the mechanisms by which they induce bacteriolysis differ from those of the
3
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antibiotics (Nakai and Park, 2002). Phages have also been reported to produce depolymerases
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that are able to degrade biofilm exopolysaccharide matrix which acts as a barrier for
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antimicrobials and can cause extensive biofilm disruption (Hughes et al., 1998).
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It has been postulated that approximately 1030 bacteriophages are present in the biosphere
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(Ashelford et al., 2000). Despite this rich reservoir of phages present in the environment, very
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few phages (about 300) have been characterized (Casjens 2008). Hence, it is very important to
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isolate and characterize new phages especially in light of the observation that most of the
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disease-causing organisms live in matrix-enclosed environments called biofilms (Watnick and
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Kolter, 2000) that inherently show increased resistance toward all antibiotics (Gilbert et al.,
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1997). The objective of this study was to isolate and characterize a new lytic phage from waste
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water that infects K. pneumoniae (M) and also to investigate the phage lytic activity against the
81
bacterial planktonic cells as well as biofilms under controlled conditions in the laboratory.
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METHODS
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Bacterial identification
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A clinical strain of Klebsiella pneumoniae obtained from Railway General Hospital (RGH),
85
Pakistan, was identified by ribotyping. Sequencing of the 16S rRNA gene was performed. For
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molecular identification of Klebsiella pneumoniae, bacterial genomic DNA was isolated by using
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ZR Fungal/Bacterial DNA kit as instructed by the manufacturer. The 16S rRNA gene sequence
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was amplified by Polymerase Chain Reaction (PCR) using universal 16S rRNA primers (RS-
89
1:5ˊ -AAACTCAAATGAATTGACGG-3ˊ
90
described by Haq et al. (2012) . The PCR amplified product was electrophoresed on 1% agarose
91
gel. The PCR product was eluted from the gel using invitrogen gel extraction kit (Invitrogen™,
92
Carlsbad, USA). Purified PCR amplified product was sequenced at Cancer Genetics Department,
and RS-3: 5ˊ -ACGGGCGGTGTGTAC-3ˊ ) as
4
93
University of Florida, USA. The 16S rRNA sequence was identified by alignment using NCBI
94
BLAST.
95
Bacteriophage isolation and purification
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Bacteriophage Z was isolated from waste water sample collected from Rawalpindi, Pakistan. The
97
Phage Z was isolated by methods previously described by Jamalludeen et al. (2007) with some
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modifications. For determination of phage titer, plaque assay was performed by adding 100 µl of
99
diluted suspension and 100 μl of the K. pneumoniae (M) strain (OD600 = 1.0) into a tube
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containing 3 ml of soft LB agar (50 °C). The mixture was poured onto the surface of LB agar
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plates and allowed to solidify for 20 min. The plates were incubated overnight at 37 °C and were
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examined for the presence of plaques and a single clear plaque was isolated for purification of
103
the phage.
104
Host range determination
105
The host range of the phage Z was assessed on a range of Gram-positive and Gram-negative
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clinically isolated bacteria that were obtained from Microbiology Lab, Railway General
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Hospital, Pakistan (Table 1). The clinical bacterial strains used for host range determination
108
included strains of Klebsiella spp, E. coli spp, Pseudomonas spp, Staphylococcus spp, and
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Achromobacter xylosoxidans shown in Table 1. To test the susceptibility of bacterial isolates, a
110
spot test was used (Zimmer et al., 2002). After overnight incubation at 37 °C, plates were
111
checked for any plaque formation against an uninfected negative control.
112
Thermal stability of phage
113
Thermal stability tests for the phage were conducted according to the methodology described by
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Capra et al. (2006) with some modifications. Phage suspensions (9.0 × 105 c.f.u ml-1) were
115
poured in Eppendorf tubes and treated at 37 °C (control), 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70
5
116
°C and 80 °C for 1 h. After incubation at the respective temperatures, we used soft agar overlay
117
method to determine the rate of survival of each treated phage as described above.
118
pH stability
119
Experiments for testing pH stability were carried out as described by Capra et al. (2006) with
120
some modifications. We established a pH gradient ranging from 1 to 11 (pH 1, 3, 5, 7, 9, 11). We
121
added one ml of phage suspension to nine (9) ml trypic soy broth (TSB) media having specific
122
pH and incubated overnight at 37 °C. Each sample was tested after incubation against the host
123
bacteria by soft agar overlay method.
124
Calcium and magnesium ion effect on the adsorption rate of the phage
125
A 50 ml of K. pneumoniae (M) culture was divided into two flasks of 25 ml each. One flask and
126
was inoculated with 250 μl (2.8× 108 p.f.u) phage, while the other flask with 250 μl phage and
127
250 μl CaCl2 or MgCl2 (each at a conc. of 10 mM). Samples were taken from both flasks at time
128
intervals of 0, 10, 20, and 30 minutes to measure the number of free phages in control and
129
calcium or magnesium added suspension. Calcium or magnesium ion effect was evaluated by
130
adsorption on the basis of the percentage of free phages by using the formula: Percentage of free
131
phages = N/N0 x 100, where N0 is the p.f.u ml-1 at T = 0 min while N is p.f.u ml-1 at T = 10, 20,
132
30 min (Capra, et al., 2006).
133
One-step growth
134
The one-step growth experiment for determination of latent time and burst size were carried out
135
according to the methodology previously described by Adams (1959). K. pneumoniae culture (50
136
ml) was incubated to a late log phase OD600 (0.4–0.6) and the bacterial cells were harvested by
137
centrifugation. The pellet obtained was resuspended in 0.5 ml LB broth media and mixed with
138
0.5 ml of phage (2.5 × 108 p.f.u ml-1). The phage was allowed to adsorb to the bacteria for 1
6
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minute and the mixture was centrifuged at 15, 000 rpm for 30 sec to remove unadsorbed free
140
phages. We then resuspended the pellet in 100 ml fresh media and the bacterial culture was
141
incubated at 37 °C continuously. Samples from the incubated culture flask were taken at 3-min
142
intervals and soft agar overlay method was used to determined phage titer.
143
Phage morphology by transmission electron microscopy
144
Phage suspension was pelleted down by ultracentrifugation at 32,000 revolutions per minutes
145
(RPM) for four hours. Phage Z morphology was examined by transmission electron microscopy.
146
A high titer (approximately 1010 p.f.u ml-1) of phage tenfold diluted in a 1X Phosphate Buffer
147
Saline (PBS) was applied to the surface of a formvar carbon film (200 mesh copper grids). Then
148
the samples were negatively stained with 2% uranyl acetate, blotted off immediately with a filter
149
paper, and the grid were air dried. The grids were then loaded into a transmission electron
150
microscope (Hitachi, H-7000, Tokyo, Japan) operated at 100 kV at Interdisciplinary Center for
151
Biotechnology Research (ICBR), University of Florida (UF) USA. The phage Z was classified
152
according to the guidelines of the International Committee on Taxonomy of Viruses (ICTV)
153
based on their morphological features (van Regenmortel et al., 2000).
154
Analysis of phage proteins
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Phage particles were pelleted down through ultracentrifugation at 32,000 rpm for 4 hours and the
156
supernatant was discarded. Then phage particles were resuspended in 1 X PBS (pH 7.0) solution
157
and were pelleted down again to remove the bacterial residual proteins. The pallet was washed
158
further three times with 0.1 M ammonium acetate solution (pH 7.0) to remove any existing
159
remaining bacterial proteins and finally suspended in PBS (1X) solution. The phage suspension
160
was added into an Eppendorf tube and boiled in water bath at 100 °C for 10 min and about 10-
161
15µl of phage suspension were mixed with loading dye and suspensions were separated by SDS-
7
162
PAGE on a 12 % acrylamide gel as described previously (Laemmli, 1970). The gel was stained
163
with Commassie Blue G-250.
164
Extraction of phage DNA and restriction with EcoR1 enzyme
165
Phage genomic DNA was isolated by using QIAGEN® Lambda kit (Cat no 12523). Chloroform
166
(2 % v/v) was added to a 50 ml of phage filtrate and incubated at 37 ºC for 30 min to enhance
167
lysis efficiency. Phage suspension was centrifuged at 15,000 rpm for 20 min to remove bacterial
168
debris, and supernatant was retained. This was followed by several steps of additions of different
169
buffers and centrifugations as recommended by the manufacturer which resulted in the isolation
170
of DNA. Phage DNA was treated with the restriction enzyme EcoRI, (New England Biolab,
171
Ontario, Canada) following standard procedures (Sambrook et al., 1989). Briefly
172
deoxyribonucleicacids (40 μl) were digested for 16 h at 37 ºC and then cleaved nucleic acids
173
were subjected to electrophoresis in a 0.7 % (w/v) agarose gel and observed with the help of UV
174
transluminator.
175
Susceptibility of planktonic cultures to phage Z
176
Bacterial susceptibility was determined as previously described by Cerca et al. (2007). Bacterial
177
strain susceptible to the phage at a cell suspension adjusted to ≤ 2 × 108 cells ml-1 in 0.9 % NaCl
178
was added to 20 ml of tryptic soy broth (TSB) and incubated at 37 °C with shaking at 130 rpm,
179
until a cell density of ≥ 2 × 108 cells ml-1 was reached. Then, phage was added at different
180
multiplicities of infection (MOI) of 0, 0.1, 0.5, 1 and 5 and growth was allowed to occur during 5
181
h. Samples were collected at different time points and the OD600 was determined. Each sample
182
was also diluted 10-fold and plated in triplicate in tryptic soy agar (TSA). The plates were then
183
incubated overnight at 37 °C. This experiment was repeated three times. To determine the
8
184
concentration of phage in suspension during bacterial growth, a sample was collected every hour
185
and phages were quantified by serial dilution method as described earlier.
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Susceptibility of biofilm to phage Z
187
For clinical bacterial strain K. pneumoniae (M), susceptible to their respective phage, biofilm
188
was formed as described previously with some modifications (Cerca et al., 2007). Briefly, 10 µl
189
of a bacterial culture (2.5 × 109 cells ) grown over night was diluted 100 times in TSB and 100µl
190
of cell suspensions at 2.0 × 109 cells ml-1, prepared in a 0.9 % NaCl solution were added to 96
191
wells microtiter plates containing tryptic soy broth with glucose (TSBG) (TSB + 1 % glucose).
192
Biofilm formation was allowed to occur during 24 h and 48 h at 37 °C while rotating at 130 rpm.
193
Each biofilm was washed twice in 0.9 % NaCl to remove planktonic cells. Then the phage (titre
194
5.2 ×109 p.f.u ml-1) was diluted in 0.9 % NaCl and added to half of the wells, while normal saline
195
was added to other half of the well as a negative control. These 96-well plates were incubated for
196
24 h or 48 h at 37 °C with constant shaking at 130 rpm .The biofilms were washed twice in 0.9
197
% NaCl, and the total biomass of the biofilm was determined by crystal violet staining as
198
described previously with some modifications. Briefly, biofilms were washed two times with 0.9
199
% NaCl solution, dried in inverted position, and stained with 1% crystal violet for 20 min. The
200
plates were washed with distilled water, and air-dried. An aliquot of 200 µl 0.9 % NaCl solution
201
was added to each well, and the OD570 was measured in an ELISA plate reader (BioTek, USA).
202
For each condition studied, three separate experiments were performed.
203
Biofilm-grown cells versus planktonic cultures susceptibility to phage
204
Susceptibility of biofilm and planktonic cells was performed by the methods previously
205
described by Cerca et al. (2007). K. pneumoniae (M) biofilm was formed for 24 h in TSBG as
206
described above. The biofilm was then scraped from the surface and resuspended in 0.9 % NaCl.
9
207
Resuspended biofilms were then vortexed for 20 sec and sonicated for 5 sec at 10 W, to
208
disaggregate the bacteria in an appropriate way to minimize to cell disruption. Planktonic
209
bacteria were grown for 24 h in TSB, in order to obtain cells in the stationary growth phase. The
210
suspension was centrifuged at 10,000 xg for 5 min and resuspended in 0.9 % NaCl by vortexing
211
for 20 sec and sonication for 5 sec at 10 W. Then both suspensions were diluted in a nutrient-
212
poor medium (10 % TSB diluted in 0.9 % NaCl) to an OD600 of about 0.4. Bacterial cell count
213
was determined by serial dilution method. Phage at an appropriate MOI was added to each
214
suspension and OD600 reduction was monitored by spectrophotometer (Biomate3 Thermoscientic
215
Corporation) during 5 h, when compared with a control having no phage. This experiment was
216
repeated three times in triplicates.
217
Statistical analysis
218
Data are expressed as means and standard deviation (SD) of mean and statistical analysis was
219
performed with Excel 2007, using student’s t test for biofilm experiments. Difference at p ≤ 0.05
220
was considered statistically significant.
221
RESULTS
222
Bacterial identification by ribotyping
223
Bacterial strain of K. pneumoniae (M) was identified by ribotyping and the sequence
224
information derived from its 16S rRNA gene. A 470-bp amplicon was amplified and subjected to
225
DNA sequencing from both orientations. The resulted sequence was deposited to a database
226
(GenBank Accession ID: KJ438818) and aligned to search for the most similar sequences. In the
227
BLAST analysis, it showed a high nucleotide sequence identity of 99 % to K. pneumoniae.
228 229
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230
Isolation of phage
231
A phage was isolated from a waste water sample against multi-drug resistant (MDR) K.
232
pneumoniae (M). Antibiotic resistance profile of K. pneumoniae (M) is shown in Table S1. The
233
phage produced clear plaques on the lawn of the host, indicating that it was a virulent phage. The
234
phage has a plaque size ranging from 1.0 to 3.0 mm in diameter and well-defined boundaries.
235
The isolated phage was designated Z (Fig. 1).
236
Host range determination
237
All 34 strains of bacteria were used to determine the host range of phage Z by using the spot test
238
method. It infected K. pneumoniae (M), K. pneumoniae-3206, K. pneumonia-3, Achromobacter
239
xylosoxidans and P. aeruginosa-2995 as shown in Table 1. The other bacterial strains used in
240
this study were insensitive to the phage. These results suggested that the phage had a narrow host
241
range among different bacterial strains.
242
Thermal stability
243
A thermal stability test was carried out to analyze the heat resistant capability of phage at pH 7.0.
244
The phage retained almost 100 % infection activity (8.4 × 105 c.f.u ml-1) after incubation at 37
245
°C. The results suggested that the phage was stable at temperatures ranging between 37 °C and
246
70 °C. At 80 °C, there were no plaques (Fig. 2).
247
pH Stability
248
Optimal pH for phage was determined by testing the stability of phage at different pH values
249
incubating at 37 °C for 16 hours. Phage showed maximum stability at pH 7 while also showing
250
good stability at pH 5, 9 and 11 while at pH 1.0 and 3.0 there were no active infectious phages to
251
be detected. The results showed that low pH might pose hindrance to phage stability. At pH 1.0
252
and 3.0, no plaques were observed, while the number of plaques was found to be increased with
11
253
increasing pH, reaching the highest number at pH 7.0. A low decrease was observed in the
254
number of plaques when the phage was incubated at pH above 7.0 (Fig. 3).
255
Calcium and magnesium ions effect on the adsorption rate of phage
256
The effect of calcium and magnesium ions on the adsorption of phage was analyzed by adding
257
(10 mM) calcium chloride and magnesium chloride to the phage and the K. pneumoniae (M)
258
mixture. The number of free phages left in the solution (which were not bound to the bacteria)
259
was detected at different time intervals of 0, 10, 20 and 30 min using the plaque assay. Data
260
analysis showed a significant difference between the control and the Ca2+/Mg2 ion-treated phage
261
Z. The results showed that calcium and magnesium ions stabilize the process of adsorption. The
262
numbers of free phages are decreased, as shown by the lower line in the figure as compared with
263
the upper line representing the control (Fig. 4).
264
Latent time period and burst size
265
The single-step growth experiment was performed for determining the latent time period and
266
burst size of the phage. A triphasic curve was obtained that has the latent phase, log or rise
267
phase, and stationary or plateau phase. From the data, the latent time period was calculated to be
268
24 min. The burst size of the phage was 320 phages per cell. Determination of burst size was
269
based on the ratio of the mean yield of phage that infected the bacterial cells to the mean phage
270
particles liberated (Fig. 5).
271
Morphology of phage
272
As analyzed by transmission electron microscopy, Z phage had a head of width (76 ± 10 nm)
273
and length of (92 ± 14 nm) with an icosahedrons’ sides of 38 nm with a non contractile (200 ± 15
274
nm) long and (14-29 nm) wide tail, therefore falling into the family Siphoviridae of order
275
Caudovirales. All values were determined as means ± SD from 3 measurements (Fig. 6).
12
276
Phage structural proteins
277
Ultracentrifuge purified phage particles were subjected to 12 % SDS-PAGE, and protein bands
278
were obtained after Commassie Blue G-250 staining and de-staining. A total of six proteins
279
composing phage Z were detected by SDS-PAGE. Their molecular weight was ranging
280
approximately from 18 to 65 kDa (Fig. 7).
281
Genome isolation of bacteriophage
282
Phage genome was detected by agarose gel. The genome was found to be approximately 36 kb
283
(Fig. 8a) and upon EcoR1 restriction produced two bands of different sizes (Fig. 8b). The
284
genome was found to be double-stranded DNA because phage nucleic acid was digested by the
285
EcoR1.
286
Susceptibility of planktonic cultures to phage Z
287
The highly susceptible K. pneumoniae (M) strain was treated with different MOI of phage Z. The
288
lytic activity of phage Z against planktonic cultures of K. pneumoniae (M) in the exponential
289
phase of growth is illustrated in Fig. 9. The Bacteria showed high susceptibility of the phage Z at
290
all MOIs. An abrupt c.f.u reduction was observed in the very first hour with MOI 1.0 and 5.0
291
from 6.0 × 108 to 7.0 × 104 and 1.0 × 105 c.f.u ml-1, respectively. While the MOI 0.1 and 0.5
292
showed a little gradual reduction but after 2 h all MOI’s showed approximately similar efficacy
293
i.e. 4.0 × 105 to 1.0 × 105 c.f.u ml-1.
294
Susceptibility of biofilms to phage Z
295
To determine the action of phage Z in K. pneumoniae (M) biofilms, the biofilm-forming
296
susceptible strain was grown in TSBG in microtiter plates (coaster) for 24 h and 48 h, after
297
which biofilms were challenged with phage Z. The biomass reduction of the biofilms compared
298
with the controls was evident (Fig. 10). Phage Z showed interesting results on both 24 h as well
13
299
as 48 h biofilms. The biofilm formed in 24 hour showed 2.5-fold reduction while 48 hour biofilm
300
also showed
301
(statistically) determined by paired samples t-test (p < 0.05) when compared with the control.
302
Biofilm-grown cells versus planktonic cultures susceptibility to the phage
303
To compare planktonic and biofilm-grown cell susceptibilities to phage Z, the lytic assay was
304
performed using strain K. pneumoniae (M) planktonic cells at the stationary growth phase (after
305
24 h of growth) and 24 h biofilm-grown cells, in a low-nutrient medium to slow down the growth
306
rate of cells and to preserve the biofilm cells for the longest possible time. Fig. 11 presents the
307
results of the effect of phage Z at MOI of one, in biofilm grown cells and also in planktonic
308
stationary phase cells. Biofilm grown cells and stationary phase planktonic cells demonstrated
309
little susceptibility to phage Z. The biofilm cells were little more resistant as compare to
310
stationary planktonic cells. In the case of biofilm cells drop in OD600 occurred relatively slow as
311
compared to planktonic stationary cells, while in control case the stationary planktonic cell
312
showed a slightly higher growth as compared to biofilm cells (Fig.11).
313
DISCUSSION
314
Biofilms can be found everywhere and have been implicated in a variety of nosocomial
315
infections associated with medical devices, hospital equipment and other hard and moist surfaces
316
(Rao et al., 2005). Microscopic observations have proved that most bacteria (99.9 %) are capable
317
of producing biofilms on a wide variety of biotic and abiotic surfaces (Costerton et al., 1978).
318
There is a renewed interest in phage therapy due to a very high emergence of antimicrobial
319
resistance in healthcare institutions worldwide due to frequent usage of antimicrobials
320
(Archibald et al., 1997; Tenover, 2001). Bacteriophages are often considered alternative agents
321
for controlling bacterial infection and contamination and increased antibiotic resistance in
about 3-fold reduction but no total removal was observed in both cases
14
322
bacteria. In the last few years, researchers are looking forward to controlling the emergence of
323
antibiotic resistance bacteria through phage therapy (Nakai and Park, 2002)
324
Most of the known phages interact only with a specific set of bacteria that expresses specific
325
binding sites. This narrow host range is also a challenge for phage therapy. Consequently, there
326
is no known phage that is lytic for all strains of K. pneumonia. This high specificity of phage-
327
host relationship leads to a need for phages to inhibit newly isolated K. pneumoniaa.
328
Phages are generally isolated from environments that are habitats for the respective host bacteria
329
(Nakai and Park, 2002). Phage Z was isolated from sewage water. It is known that sewage
330
generally contains a large diversity of micro-organisms due to contamination from fecal material
331
and hospital drainage water (Piracha et al., 2014). Phage Z was highly lytic and capable of
332
producing clear plaques ranging from 1.0 to 3.0 mm. It has a narrow host range infecting only K.
333
pneumonia (M), K. pneumoniae-3206, K. pneumoniae-3, Achromobacter xylosoxidans and
334
Pseudononas aeruginosa-2995. Many phages have been reported that are found to be greatly
335
specific for their receptors present on the host cell surface. They only show interface with their
336
specific receptors but do not interact with receptors having different structures (Piracha et al.,
337
2014).
338
Several studies have documented that phages varied in thermal and pH stability depending upon
339
strains of phages. The phage was tolerant to relatively high temperature ranging from 37 to 65 °C
340
was killed at 70 °C. Also showing good pH stability over broad range of pH values ranging from
341
5-11 and a maximum stability at pH 7.0. The results are consistent with the previous
342
observations by Ackermann and Dubow (1987) and Jamalludeen et al. (2007) that most phages
343
are able to survive well over a wide range of pH (5 to 9) at physiological conditions that maintain
344
the native virion structure and stability. The inactivity of the phage at lower pH values of 1 and 3
15
345
in our study can be attributed to protein denaturation in acidic environment (Hazem, 2002).
346
These characteristics may be useful for the application of the phage in different environments.
347
The infectivity of phage Z was shown to be increased at 10 mM calcium chloride or magnesium
348
chloride solution concentrations. According to Guttman et al.(2005) cofactors such as Ca2+,
349
Mg2+, divalent cations or sugars may be required for successful binding to occur. Ca2+/Mg2+ ions
350
stabilize the weak interaction of virion with receptors during the adsorption. Diverse quantities of
351
calcium ions give maximum infectivity for various phages (Donlan, 2005; Reese et al., 1974). It
352
is also postulated that Ca2+ ions may increase the concentration of phage particles at the host
353
surface or alter the structure of a cell surface receptor thereby increasing accessibility to the
354
receptor molecules or transfer of phage nucleic acids (Russel et al., 1988; Watanabe and
355
Takesue, 1972). Yang et al. (2010) have described the burst size and the duration of the latent
356
phase which nearly correlates with our results (latent period of 24 min and burst size of 320
357
virions per cell) while Sillankorva et al. (2004) have described small burst size and less duration
358
of the latent phase. Thus a lot of variations have been reported in literature regarding latent time
359
and burst size of bacteriophages.
360
For Phage classification, ICTV recognizes one order, 13 families and 31 genera of
361
bacteriophages. As our phage was a non-contractile tailed virus, phage Z fell into the to family
362
Siphoviridae of order Caudovirales that contains three families of tailed viruses that infect
363
bacteria and archaea (van Regenmortel et al., 2000). Possession of an icosahedral head and a
364
long non-contractile tail would place it in the family Siphoviridae (van Regenmortel et al.,
365
2000).
16
366
Phage specific for a bacteria can infect biofilm cells by first degrading the EPS and then
367
ultimately lysing the bacterial cells. There is evidence that phage-induced depolymerases could
368
affect biofilms and have potential for biofilm control (Hughes et al., 1998; Roy et al., 1993).
369
Carson et al. (2010) have reported the potential utility of bacteriophages to reduce bacterial
370
biofilms on medical device surfaces and in prevention of biofilm via direct incorporation of
371
phages and reported approximately 90 % reduction in E. coli biofilm formation on
372
bacteriophage-treated catheters when compared with untreated controls.
373
Out of the ordinary was the fact that planktonic cells of K. pneumoniae (M) in exponential
374
growth phase were much more sensitive to phage Z lysis than in stationary growth phase. This
375
effect was previously demonstrated on P. fluorescens planktonic cultures (Sillankorva et al.,
376
2004) and susceptibility of S. epidermidis planktonic cells (Cerca et al., 2007). It seems that
377
biofilms are slowly killed by phage Z, not due to a specific biofilm cells, but probably due to the
378
low metabolic activity of biofilm cells (Briandet et al., 2008; Corbin et al., 2001; Costerton,
379
1995). Corbin et al. (2001) studied phage T4 impact on E. coli biofilm and observed one and a
380
half log reduction while Moons et al. (2006) studied the effect of phage T7 on E. coli biofilms.
381
They gave phage T7 treatment for one hour at a concentration of 1 × 1010 c.f.u ml-1 and observed
382
about 2-fold reductions. Similarly we observed slightly more then 2-fold reduction on both 24 h
383
and 48 h biofilms when treated with a phage titer of 4.5 × 109 p.f.u ml-1.
384
In conclusion, this study suggests that phage Z is a tailed, DNA lytic phage, having a good heat
385
tolerance and wide range of pH stability. It also had an activity against multi-drug resistant K.
386
pneumoniae (M) in both plankonic cells and biofilms but does not result in total eradication of K.
387
pneumonia (M) biofilms. Thus for efficient and complete eradication of biofilms a combination
388
of phages (phage cocktail) may be used.
17
389
ACKNOWLEDGMENTS
390
We would like to acknowledge Kalina Rosenova Atanasova from Emerging Pathogens Institute
391
(EPI), Department of Periodontology, University of Florida, USA for helping us during lab
392
experiments and editing this manuscript. We also are very thankful to Karen Kelley, electron
393
microscopy manager from Interdisciplinary Center for Biotechnology Research (ICBR)
394
University of Florida, USA for transmission electron microscopy. We are also thankful to Dr
395
Farida Nighat, incharge Microbiology Lab, Railway General Hospital, Pakistan for providing
396
bacterial strains to carry out phage host range experiments. We are thankful to the Higher
397
Education Commission (HEC) of Pakistan for providing funding to support the current study.
398
REFERENCES
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Ackermann, H. and Dubow, M. (1987). Viruses of prokaryotes: general properties of bacteriophages. Boca Raton, Fla. CRC Press, Inc. Adams, M. H. (1959). Bacteriophages. New York, Interscience Publishers, Inc. Archibald, L., Phillips, L., Monnet, D., Mcgowan, J. E., Tenover, F. and Gaynes, R. (1997). Antimicrobial resistance in isolates from inpatients and outpatients in the United States: increasing importance of the intensive care unit. Clin Infect Dis 24, 211-215. Ashelford, K. E., Norris, S. J., Fry, J. C., Bailey, M. J. and Day, M. J. (2000). Seasonal population dynamics and interactions of competing bacteriophages and their host in the rhizosphere. Appl Environ Microbiol 66, 4193-4199. Briandet, R., Lacroix-Gueu, P., Renault, M., Lecart, S., Meylheuc, T., Bidnenko, E., Steenkeste, K., Bellon-Fontaine, M.-N. and Fontaine-Aupart, M.-P. (2008). Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms. Appl Environ Microbiol 74, 2135-2143. Capra, M., Quiberoni, A. and Reinheimer, J. (2006). Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains. J Appl Microbiol 100, 334-342. Carson, L., Gorman, S. P. and Gilmore, B. F. (2010). The use of lytic bacteriophages in the prevention and eradication of biofilms of Proteus mirabilis and Escherichia coli. FEMS Immunol Med Microbiol 59, 447-455. Cerca, N., Oliveira, R. and Azeredo, J. (2007). Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K. Lett Appl Microbiol 45, 313-317. Corbin, B. D., Mclean, R. J. and Aron, G. M. (2001). Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm. Canadian J Microbiol 47, 680-684. Costerton, J. (1995). Overview of microbial biofilms. J Indust Microbiol 15, 137-140. Costerton, J., Stewart, P. S. and Greenberg, E. (1999). Bacterial biofilms: a common cause of persistent infections. Sci 284, 1318-1322. Costerton, J. W., Geesey, G. and Cheng, K. (1978). How bacteria stick. Scientific American, 238 (1): 86.
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Donlan, R. M. (2005). New approaches for the characterization of prosthetic joint biofilms. Clin Orthopaed Related Res 437, 12-19. Donlan, R. M. and Costerton, J. W. (2002). Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15, 167-193. Gilbert, P., Das, J. and Foley, I. (1997). Biofilm susceptibility to antimicrobials. Advances in Dental Res 11, 160-167. Guttman, B., Raya, R. and Kutter, E. (2005). Basic phage biology. Bacteriophages: Biol Applications 4. Haq, I. U., Chaudhry, W. N., Andleeb, S. and Qadri, I. (2012). Isolation and Partial Characterization of a Virulent Bacteriophage IHQ1 Specific for Aeromonas punctata from Stream Water. Microbial Ecol 63, 954-963. Hazem, A. (2002). Effects of temperatures, pH-values, ultra-violet light, ethanol and chloroform on the growth of isolated thermophilic Bacillus phages. The New Microbiologica 25, 469-476. Hughes, K. A., Sutherland, I. W. and Jones, M. V. (1998). Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. Microbiol 144, 3039-3047. Jamalludeen, N., Johnson, R. P., Friendship, R., Kropinski, A. M., Lingohr, E. J. and Gyles, C. L. (2007). Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli. Vet Microbiol 124, 47-57. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Mah, T.-F. C. and O'toole, G. A. (2001). Mechanisms of biofilm resistance to antimicrobial agents. Trends in Microbiol 9, 34-39. Monroe, D. (2007). Looking for chinks in the armor of bacterial biofilms. PLoS Biol 5, e307. Moons, P., Werckx, W., Van Houdt, R., Aertsen, A. and Michiels, C. W. (2006). Resistance development of bacterial biofilms against bacteriophage attack. Commun Agric Appl Biol Sci 71, 297-300. Nakai, T. and Park, S. C. (2002). Bacteriophage therapy of infectious diseases in aquaculture. Res Microbiol 153, 13-18. Piracha, Z., Saeed, U., Khurshid, A. and Chaudhary, W. N. (2014). Isolation and Partial Characterization of Virulent Phage Specific against Pseudomonas Aeruginosa. Global J Med Res 14. Podschun, R. and Ullmann, U. (1998). Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11, 589-603. Potera, C. (1999). Forging a link between biofilms and disease. Sci 283, 1837-1839. Rao, V., Ghei, R. and Chambers, Y. (2005). Biofilms research-implications to biosafety and public health. Appl Biosafety 10, 83. Reese, J. F., Dimitracopoulos, G. and Bartell, P. F. (1974). Factors influencing the adsorption of bacteriophage 2 to cells of Pseudomonas aeruginosa. J Virol 13, 22-27. Roy, B., Ackermann, H., Pandian, S., Picard, G. and Goulet, J. (1993). Biological inactivation of adhering Listeria monocytogenes by listeriaphages and a quaternary ammonium compound. Appl Environ Microbiol 59, 2914-2917. Russel, M., Whirlow, H., Sun, T. and Webster, R. (1988). Low-frequency infection of F-bacteria by transducing particles of filamentous bacteriophages. J Bacteriol 170, 5312-5316. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning, Cold spring harbor laboratory press New York. Sillankorva, S., Oliveira, R., Vieira, M. J., Sutherland, I. and Azeredo, J. (2004). Bacteriophage Φ S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms. Biofouling 20, 133-138. Sulakvelidze, A., Alavidze, Z. and Morris, J. G. (2001). Bacteriophage therapy. Antimicrobial Agents and Chemother 45, 649-659. Tenover, F. C. (2001). Development and spread of bacterial resistance to antimicrobial agents: an overview. Clin Infect Dis 33, S108-S115. 19
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Van Regenmortel, M. H., Fauquet, C. M., Bishop, D. H., Carstens, E., Estes, M., Lemon, S., Maniloff, J., Mayo, M., Mcgeoch, D. and Pringle, C. (2000). Virus taxonomy: classification and nomenclature of viruses. Seventh report of the International Committee on Taxonomy of Viruses, Academic Press. Vinh, D. C. and Embil, J. M. (2005). Device-related infections: a review. J Long-Term Effects of Med Implants 15. Watanabe, K. and Takesue, S. (1972). The requirement for calcium in infection with Lactobacillus phage. J Gen Viro 17, 19-30. Watnick, P. and Kolter, R. (2000). Biofilm, city of microbes. J Bacteriol 182, 2675-2679. Yang, H., Liang, L., Lin, S. and Jia, S. (2010). Isolation and characterization of a virulent bacteriophage AB1 of Acinetobacter baumannii. BMC Microbiol 10, 131. Zimmer, M., Scherer, S. and Loessner, M. J. (2002). Genomic analysis of Clostridium perfringens bacteriophage φ3626, which integrates into guaA and possibly affects sporulation. J Bacteriol 184, 43594368.
488 489 490 491 492 493 494 495 496 497 498 499 20
500
Figure legends
501
Fig. 1. Soft Agar overlay plates, (a) showing spot test assay and (b) a higher dilution (10-7) of
502
phage titer showing clear plaques of 1.0 – 3.0 mm in diameter.
503
Fig. 2. Stability of phage Z treated with different temperature for 60 min. All values represent
504
means of 3 determinations with ± standard deviations.
505
Fig. 3. Stability of phage Z treated with different pH overnight at 37 °C. All values represent
506
means of 3 determinations with ± standard deviations.
507
Fig. 4. Test for phage adsorption rate. Effect of divalent metal ions on phage adsorption rate by
508
adding divalent metal ions by adding 10 mM CaCl2 or MgCl2 solution to the mixture of phage Z
509
and K. pneumonia (M). All values represent means of 3 determinations with ± standard
510
deviations.
511
Fig. 5. One-step growth experiment. Latent time and burst size of phage Z were inferred from
512
the curve with a triphasic pattern. All values represent means of 3 determinations with ± standard
513
deviations.
514
Fig. 6. Transmission electron micrographs of the purified phage Z using scale bars of 200 nm.
515
Three representative images (a), (b) and (c) are shown.
516
Fig. 7. SDS-PAGE analysis of phage Z structural proteins. Lane 1, broad range protein
517
molecular weight markers (Precision Plus Protein™, Bio-Red); Lane 2, phage Z proteins.
518
Fig. 8. (a) Genome of phage: 0.6% (w/v) agarose gel. Lane 1 shows DNA Ladder (GeneRuler
519
High Range) and Lane 2, shows band of phage DNA having a size of approximately 36 kb while
520
(b) Lane 1, shows 1kb DNA Ladder (New England Biolabs) and Lane 2 shows restriction
521
analysis of phage Z DNA with EcoR1.
21
522
Fig. 9. Kill curves of exponential growth phase planktonic K. pneumoniae (M) by phage Z at
523
different multiplicity of infections (MOIs): 0, 0.1, 0.5, 1 and 5. All values represent means of 3
524
determinations.
525
Fig. 10. Reduction of biofilm biomass after 24 h and 48 h of challenge with 5.2 × 109 p.f.u ml-1of
526
phage Z. White bars represent control biofilms without phage and dark bands represent biofilm
527
infected with phage.*Significant reduction in biomass compared with control (light bands; paired
528
samples t-test, p < 0.05). All values represent means of 3 determinations with ± standard
529
deviations.
530
Fig. 11. Kill curves of K. pneumoniae (M) in different growth stages: stationary phase bacteria
531
(control and with phage), or biofilm grown bacteria (control and with phage). All values
532
represent means of 3 determinations.
533 534 535 536 537 538 539 540 541 542 543 544 545 22
546
Table 1. Spot test of phage Z on different bacterial species S:No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
547
Bacterial Strain Klebsiella pneumoniae (M) Klebsiella pneumoniae-3206 Klebsiella pneumoniae-3 Klebsiella pneumoniae-M 37 Klebsiella pneumoniae-3025 Klebsiella pneumoniae-3011 Klebsiella pneumoniae -3033 Klebsiella pneumoniae-3019 Klebsiella pneumoniae-3114 Klebsiella pneumoniae-2825 Klebsiella pneumoniae-3015 Klebsiella pneumoniae-3018 Klebsiella pneumoniae-2870 Klebsiella pneumoniae-2908 Klebsiella pneumoniae-3202 Klebsiella pneumoniae-3311 Escherichia coli-3 Escherichia coli -F Escherichia coli LF-1969 Escherichia coli LF-1990 Escherichia coli-3051 Escherichia coli LF-1968 Pseudomonas aeruginosa-3098 Pseudomonas aeruginosa-2995 Pseudomonas aeruginosa-2949 Pseudomonas aeruginosa-3048 Pseudomonas aeruginosa-3068 Pseudomonas aeruginosa-2830 Pseudomonas aeruginosa-3178 Staphaylococcus aureus-2895 Staphaylococcus aureus-2975 Staphaylococcus aureus-2938 Staphaylococcus aureus-2895 Achromobacter xylosoxidans + = lysis,
Activity (+/₋) + + + + ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ +
- = no lysis
548 549
23
Supplementary Material Files Click here to download Supplementary Material Files: Table S1, Sensitivity of K. pneumonia to different group of antibiotics.pdf
Figure 2 Click here to download high resolution image
Figure 1 Click here to download high resolution image
Figure 3 Click here to download high resolution image
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Journal of Medical Microbiology Characterization of Siphoviridae phage Z and studying its efficacy against multi-drug resistant (MDR) Klebsiella pneumoniae planktonic cells and biofilm --Manuscript Draft-Manuscript Number:
JMM-D-14-00020R1
Full Title:
Characterization of Siphoviridae phage Z and studying its efficacy against multi-drug resistant (MDR) Klebsiella pneumoniae planktonic cells and biofilm
Short Title:
Phage Z against multi-drug resistant (MDR) Klebsiella pneumoniae
Article Type:
Standard
Section/Category:
Clinical microbiology and virology
Corresponding Author:
Saadia Andleeb ASAb-NUST Islamabad, other/autre PAKISTAN
First Author:
Muhsin Jamal
Order of Authors:
Muhsin Jamal Tahir Hussain Chythanya Rajanna Das Saadia Andleeb
Abstract:
Biofilm is involved in many serious consequences for public health and is a major virulence factor contributing to the chronicity of infections. The aim of the current study was to isolate and characterize a bacteriophage that inhibit multi-drug resistant Klebsiella pneumonia (M) in palnktonic form as well as biofilm. This phage designated as bacteriophage Z was isolated from waste water. Its adsorption rate to its host bacterium was significantly enhanced by MgCl2 and CaCl2. It has a wide range of pH and heat stability. From its one step growth, latent time and burst size was determined that were 24 min and about 320 virions per cell, respectively. As analyzed by transmission electron microscopy, phage Z had a head of width (76±10 nm) and length of (92±14 nm) with an icosahedrons' sides of 38 nm with a non- contractile (200±15 nm) long and (14-29 nm) wide tail belonging to family Siphoviridae of order Caudovirales. Six structural proteins ranging from 18 to 65 kDa were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its genome was found to be comprised of double stranded DNA with approximate size of 36 kb. Bacteria were grown in suspension and as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Phage Z was effective in reducing biofilm biomass after 24 h and 48 h and showed more than 2-fold and 3-fold reductions, respectively. Biofilm cells and stationary phase planktonic bacteria were killed at a lower rate than the log-phase planktonic bacteria.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Paper K.P (M) Final Revision 2.docx
1
Characterization of Siphoviridae phage Z and studying its efficacy against multi-drug
2
resistant (MDR) Klebsiella pneumoniae planktonic cells and biofilm
3 4 5
Phage Z against multi-drug resistant (MDR) Klebsiella pneumoniae
6 7
Muhsin Jamal,1, 2 Tahir Hussain,1 Chythanya Rajanna Das2 and Saadia Andleeb1*
8 9
1
Atta -ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences
10
and Technology (NUST), Islamabad, Pakistan.
11
2
Emerging Pathogens Institute (EPI), University of Florida (UF), Florida, USA.
12 13 14
*Corresponding Author:
15
Saadia Andleeb
16
Atta-ur-Rahman School of Applied Biosciences (ASAB),
17
National University of Sciences and Technology (NUST),
18
Kashmir Highway, Sector H-12
19
Islamabad, Post code 44000, Pakistan
20
Email ID: [email protected]
21
Cell No: +92-3335188343
22
Office:
23
Fax No: +92-51-90856102
+92-51-90856133
1
24
Summary
25
Biofilm is involved in many serious consequences for public health and is a major virulence
26
factor contributing to the chronicity of infections. The aim of the current study was to isolate and
27
characterize a bacteriophage that inhibit multi-drug resistant Klebsiella pneumonia (M) in
28
palnktonic form as well as biofilm. This phage designated as bacteriophage Z was isolated from
29
waste water. Its adsorption rate to its host bacterium was significantly enhanced by MgCl2 and
30
CaCl2. It has a wide range of pH and heat stability. From its one step growth, latent time and
31
burst size was determined that were 24 min and about 320 virions per cell, respectively. As
32
analyzed by transmission electron microscopy, phage Z had a head of width (76 ± 10 nm) and
33
length of (92 ± 14 nm) with an icosahedrons’ sides of 38 nm with a non- contractile (200 ±15
34
nm) long and (14-29 nm) wide tail belonging to family Siphoviridae of order Caudovirales. Six
35
structural proteins ranging from 18 to 65 kDa were revealed by sodium dodecyl sulfate
36
polyacrylamide gel electrophoresis (SDS-PAGE). Its genome was found to be comprised of
37
double stranded DNA with approximate size of 36 kb. Bacteria were grown in suspension and as
38
biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Phage Z was
39
effective in reducing biofilm biomass after 24 h and 48 h and showed more than 2-fold and 3-
40
fold reductions, respectively. Biofilm cells and stationary phase planktonic bacteria were killed
41
at a lower rate than the log-phase planktonic bacteria.
42
Key words: Bacteriophage, Waste water, Klebsiella pneumoniae, Multi drug-resistant (MDR),
43
Caudovirales.
44 45 46
2
47
INTRODUCTION
48
Klebsiella pneumoniae accounts for a significant proportion of hospital-acquired infections. The
49
most important reservoirs for transmission of Klebsiella are the gastrointestinal tract and the
50
hands of hospital personnel, hence they are crucially involved in causing outbreaks of
51
nosocomial infections (Podschun and Ullmann, 1998).
52
Among the nosocomial pathogens, K. pneumoniae is one of the most important biofilm forming
53
bacteria which can cause a large number of infections (Podschun and Ullmann, 1998). Biofilms
54
have been involved in a large number of nosocomial infections associated with medical devices,
55
equipments used in hospital and other hard surfaces which can act as reservoirs for biofilm
56
acquired infections (Rao et al., 2005). It has been estimated that biofilm is involved in more than
57
60 % nosocomial infections (Donlan and Costerton, 2002; Vinh and Embil, 2005). The National
58
Institutes of Health (NIH) claims that about 80 % of all chronic infections are caused by
59
biofilms (Monroe, 2007) and in all about 65 % microbial infections are related to biofilms
60
(Potera, 1999) . These infections are very difficult to treat due to the resistance of bacteria
61
resistant to numerous antimicrobial agents (Costerton et al., 1999). As the threat of emergence of
62
antibiotic resistance and the inability to eradicate the biofilm structures has increased, the
63
likelihood that novel strategies for preventing biofilm growth mode are urgently needed (Mah
64
and O'Toole, 2001).
65
Being obligate parasites of bacteria, the bacteriophages bind to microbial surfaces, injecting their
66
genetic material and replicating within the bacterial host causing lysis of the host cell
67
(Sulakvelidze et al., 2001). Phage therapy has gained an increasing attention because it has many
68
advantages over antibiotic therapy. Phages are effective against multidrug resistant pathogenic
69
bacteria because the mechanisms by which they induce bacteriolysis differ from those of the
3
70
antibiotics (Nakai and Park, 2002). Phages have also been reported to produce depolymerases
71
that are able to degrade biofilm exopolysaccharide matrix which acts as a barrier for
72
antimicrobials and can cause extensive biofilm disruption (Hughes et al., 1998).
73
It has been postulated that approximately 1030 bacteriophages are present in the biosphere
74
(Ashelford et al., 2000). Despite this rich reservoir of phages present in the environment, very
75
few phages (about 300) have been characterized (Casjens 2008). Hence, it is very important to
76
isolate and characterize new phages especially in light of the observation that most of the
77
disease-causing organisms live in matrix-enclosed environments called biofilms (Watnick and
78
Kolter, 2000) that inherently show increased resistance toward all antibiotics (Gilbert et al.,
79
1997). The objective of this study was to isolate and characterize a new lytic phage from waste
80
water that infects K. pneumoniae (M) and also to investigate the phage lytic activity against the
81
bacterial planktonic cells as well as biofilms under controlled conditions in the laboratory.
82
METHODS
83
Bacterial identification
84
A clinical strain of Klebsiella pneumoniae obtained from Railway General Hospital (RGH),
85
Pakistan, was identified by ribotyping. Sequencing of the 16S rRNA gene was performed. For
86
molecular identification of Klebsiella pneumoniae, bacterial genomic DNA was isolated by using
87
ZR Fungal/Bacterial DNA kit as instructed by the manufacturer. The 16S rRNA gene sequence
88
was amplified by Polymerase Chain Reaction (PCR) using universal 16S rRNA primers (RS-
89
1:5ˊ -AAACTCAAATGAATTGACGG-3ˊ
90
described by Haq et al. (2012) . The PCR amplified product was electrophoresed on 1% agarose
91
gel. The PCR product was eluted from the gel using invitrogen gel extraction kit (Invitrogen™,
92
Carlsbad, USA). Purified PCR amplified product was sequenced at Cancer Genetics Department,
and RS-3: 5ˊ -ACGGGCGGTGTGTAC-3ˊ ) as
4
93
University of Florida, USA. The 16S rRNA sequence was identified by alignment using NCBI
94
BLAST.
95
Bacteriophage isolation and purification
96
Bacteriophage Z was isolated from waste water sample collected from Rawalpindi, Pakistan. The
97
Phage Z was isolated by methods previously described by Jamalludeen et al. (2007) with some
98
modifications. For determination of phage titer, plaque assay was performed by adding 100 µl of
99
diluted suspension and 100 μl of the K. pneumoniae (M) strain (OD600 = 1.0) into a tube
100
containing 3 ml of soft LB agar (50 °C). The mixture was poured onto the surface of LB agar
101
plates and allowed to solidify for 20 min. The plates were incubated overnight at 37 °C and were
102
examined for the presence of plaques and a single clear plaque was isolated for purification of
103
the phage.
104
Host range determination
105
The host range of the phage Z was assessed on a range of Gram-positive and Gram-negative
106
clinically isolated bacteria that were obtained from Microbiology Lab, Railway General
107
Hospital, Pakistan (Table 1). The clinical bacterial strains used for host range determination
108
included strains of Klebsiella spp, E. coli spp, Pseudomonas spp, Staphylococcus spp, and
109
Achromobacter xylosoxidans shown in Table 1. To test the susceptibility of bacterial isolates, a
110
spot test was used (Zimmer et al., 2002). After overnight incubation at 37 °C, plates were
111
checked for any plaque formation against an uninfected negative control.
112
Thermal stability of phage
113
Thermal stability tests for the phage were conducted according to the methodology described by
114
Capra et al. (2006) with some modifications. Phage suspensions (9.0 × 105 c.f.u ml-1) were
115
poured in Eppendorf tubes and treated at 37 °C (control), 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70
5
116
°C and 80 °C for 1 h. After incubation at the respective temperatures, we used soft agar overlay
117
method to determine the rate of survival of each treated phage as described above.
118
pH stability
119
Experiments for testing pH stability were carried out as described by Capra et al. (2006) with
120
some modifications. We established a pH gradient ranging from 1 to 11 (pH 1, 3, 5, 7, 9, 11). We
121
added one ml of phage suspension to nine (9) ml trypic soy broth (TSB) media having specific
122
pH and incubated overnight at 37 °C. Each sample was tested after incubation against the host
123
bacteria by soft agar overlay method.
124
Calcium and magnesium ion effect on the adsorption rate of the phage
125
A 50 ml of K. pneumoniae (M) culture was divided into two flasks of 25 ml each. One flask and
126
was inoculated with 250 μl (2.8× 108 p.f.u) phage, while the other flask with 250 μl phage and
127
250 μl CaCl2 or MgCl2 (each at a conc. of 10 mM). Samples were taken from both flasks at time
128
intervals of 0, 10, 20, and 30 minutes to measure the number of free phages in control and
129
calcium or magnesium added suspension. Calcium or magnesium ion effect was evaluated by
130
adsorption on the basis of the percentage of free phages by using the formula: Percentage of free
131
phages = N/N0 x 100, where N0 is the p.f.u ml-1 at T = 0 min while N is p.f.u ml-1 at T = 10, 20,
132
30 min (Capra, et al., 2006).
133
One-step growth
134
The one-step growth experiment for determination of latent time and burst size were carried out
135
according to the methodology previously described by Adams (1959). K. pneumoniae culture (50
136
ml) was incubated to a late log phase OD600 (0.4–0.6) and the bacterial cells were harvested by
137
centrifugation. The pellet obtained was resuspended in 0.5 ml LB broth media and mixed with
138
0.5 ml of phage (2.5 × 108 p.f.u ml-1). The phage was allowed to adsorb to the bacteria for 1
6
139
minute and the mixture was centrifuged at 15, 000 rpm for 30 sec to remove unadsorbed free
140
phages. We then resuspended the pellet in 100 ml fresh media and the bacterial culture was
141
incubated at 37 °C continuously. Samples from the incubated culture flask were taken at 3-min
142
intervals and soft agar overlay method was used to determined phage titer.
143
Phage morphology by transmission electron microscopy
144
Phage suspension was pelleted down by ultracentrifugation at 32,000 revolutions per minutes
145
(RPM) for four hours. Phage Z morphology was examined by transmission electron microscopy.
146
A high titer (approximately 1010 p.f.u ml-1) of phage tenfold diluted in a 1X Phosphate Buffer
147
Saline (PBS) was applied to the surface of a formvar carbon film (200 mesh copper grids). Then
148
the samples were negatively stained with 2% uranyl acetate, blotted off immediately with a filter
149
paper, and the grid were air dried. The grids were then loaded into a transmission electron
150
microscope (Hitachi, H-7000, Tokyo, Japan) operated at 100 kV at Interdisciplinary Center for
151
Biotechnology Research (ICBR), University of Florida (UF) USA. The phage Z was classified
152
according to the guidelines of the International Committee on Taxonomy of Viruses (ICTV)
153
based on their morphological features (van Regenmortel et al., 2000).
154
Analysis of phage proteins
155
Phage particles were pelleted down through ultracentrifugation at 32,000 rpm for 4 hours and the
156
supernatant was discarded. Then phage particles were resuspended in 1 X PBS (pH 7.0) solution
157
and were pelleted down again to remove the bacterial residual proteins. The pallet was washed
158
further three times with 0.1 M ammonium acetate solution (pH 7.0) to remove any existing
159
remaining bacterial proteins and finally suspended in PBS (1X) solution. The phage suspension
160
was added into an Eppendorf tube and boiled in water bath at 100 °C for 10 min and about 10-
161
15µl of phage suspension were mixed with loading dye and suspensions were separated by SDS-
7
162
PAGE on a 12 % acrylamide gel as described previously (Laemmli, 1970). The gel was stained
163
with Commassie Blue G-250.
164
Extraction of phage DNA and restriction with EcoR1 enzyme
165
Phage genomic DNA was isolated by using QIAGEN® Lambda kit (Cat no 12523). Chloroform
166
(2 % v/v) was added to a 50 ml of phage filtrate and incubated at 37 ºC for 30 min to enhance
167
lysis efficiency. Phage suspension was centrifuged at 15,000 rpm for 20 min to remove bacterial
168
debris, and supernatant was retained. This was followed by several steps of additions of different
169
buffers and centrifugations as recommended by the manufacturer which resulted in the isolation
170
of DNA. Phage DNA was treated with the restriction enzyme EcoRI, (New England Biolab,
171
Ontario, Canada) following standard procedures (Sambrook et al., 1989). Briefly
172
deoxyribonucleicacids (40 μl) were digested for 16 h at 37 ºC and then cleaved nucleic acids
173
were subjected to electrophoresis in a 0.7 % (w/v) agarose gel and observed with the help of UV
174
transluminator.
175
Susceptibility of planktonic cultures to phage Z
176
Bacterial susceptibility was determined as previously described by Cerca et al. (2007). Bacterial
177
strain susceptible to the phage at a cell suspension adjusted to ≤ 2 × 108 cells ml-1 in 0.9 % NaCl
178
was added to 20 ml of tryptic soy broth (TSB) and incubated at 37 °C with shaking at 130 rpm,
179
until a cell density of ≥ 2 × 108 cells ml-1 was reached. Then, phage was added at different
180
multiplicities of infection (MOI) of 0, 0.1, 0.5, 1 and 5 and growth was allowed to occur during 5
181
h. Samples were collected at different time points and the OD600 was determined. Each sample
182
was also diluted 10-fold and plated in triplicate in tryptic soy agar (TSA). The plates were then
183
incubated overnight at 37 °C. This experiment was repeated three times. To determine the
8
184
concentration of phage in suspension during bacterial growth, a sample was collected every hour
185
and phages were quantified by serial dilution method as described earlier.
186
Susceptibility of biofilm to phage Z
187
For clinical bacterial strain K. pneumoniae (M), susceptible to their respective phage, biofilm
188
was formed as described previously with some modifications (Cerca et al., 2007). Briefly, 10 µl
189
of a bacterial culture (2.5 × 109 cells ) grown over night was diluted 100 times in TSB and 100µl
190
of cell suspensions at 2.0 × 109 cells ml-1, prepared in a 0.9 % NaCl solution were added to 96
191
wells microtiter plates containing tryptic soy broth with glucose (TSBG) (TSB + 1 % glucose).
192
Biofilm formation was allowed to occur during 24 h and 48 h at 37 °C while rotating at 130 rpm.
193
Each biofilm was washed twice in 0.9 % NaCl to remove planktonic cells. Then the phage (titre
194
5.2 ×109 p.f.u ml-1) was diluted in 0.9 % NaCl and added to half of the wells, while normal saline
195
was added to other half of the well as a negative control. These 96-well plates were incubated for
196
24 h or 48 h at 37 °C with constant shaking at 130 rpm .The biofilms were washed twice in 0.9
197
% NaCl, and the total biomass of the biofilm was determined by crystal violet staining as
198
described previously with some modifications. Briefly, biofilms were washed two times with 0.9
199
% NaCl solution, dried in inverted position, and stained with 1% crystal violet for 20 min. The
200
plates were washed with distilled water, and air-dried. An aliquot of 200 µl 0.9 % NaCl solution
201
was added to each well, and the OD570 was measured in an ELISA plate reader (BioTek, USA).
202
For each condition studied, three separate experiments were performed.
203
Biofilm-grown cells versus planktonic cultures susceptibility to phage
204
Susceptibility of biofilm and planktonic cells was performed by the methods previously
205
described by Cerca et al. (2007). K. pneumoniae (M) biofilm was formed for 24 h in TSBG as
206
described above. The biofilm was then scraped from the surface and resuspended in 0.9 % NaCl.
9
207
Resuspended biofilms were then vortexed for 20 sec and sonicated for 5 sec at 10 W, to
208
disaggregate the bacteria in an appropriate way to minimize to cell disruption. Planktonic
209
bacteria were grown for 24 h in TSB, in order to obtain cells in the stationary growth phase. The
210
suspension was centrifuged at 10,000 xg for 5 min and resuspended in 0.9 % NaCl by vortexing
211
for 20 sec and sonication for 5 sec at 10 W. Then both suspensions were diluted in a nutrient-
212
poor medium (10 % TSB diluted in 0.9 % NaCl) to an OD600 of about 0.4. Bacterial cell count
213
was determined by serial dilution method. Phage at an appropriate MOI was added to each
214
suspension and OD600 reduction was monitored by spectrophotometer (Biomate3 Thermoscientic
215
Corporation) during 5 h, when compared with a control having no phage. This experiment was
216
repeated three times in triplicates.
217
Statistical analysis
218
Data are expressed as means and standard deviation (SD) of mean and statistical analysis was
219
performed with Excel 2007, using student’s t test for biofilm experiments. Difference at p ≤ 0.05
220
was considered statistically significant.
221
RESULTS
222
Bacterial identification by ribotyping
223
Bacterial strain of K. pneumoniae (M) was identified by ribotyping and the sequence
224
information derived from its 16S rRNA gene. A 470-bp amplicon was amplified and subjected to
225
DNA sequencing from both orientations. The resulted sequence was deposited to a database
226
(GenBank Accession ID: KJ438818) and aligned to search for the most similar sequences. In the
227
BLAST analysis, it showed a high nucleotide sequence identity of 99 % to K. pneumoniae.
228 229
10
230
Isolation of phage
231
A phage was isolated from a waste water sample against multi-drug resistant (MDR) K.
232
pneumoniae (M). Antibiotic resistance profile of K. pneumoniae (M) is shown in Table S1. The
233
phage produced clear plaques on the lawn of the host, indicating that it was a virulent phage. The
234
phage has a plaque size ranging from 1.0 to 3.0 mm in diameter and well-defined boundaries.
235
The isolated phage was designated Z (Fig. 1).
236
Host range determination
237
All 34 strains of bacteria were used to determine the host range of phage Z by using the spot test
238
method. It infected K. pneumoniae (M), K. pneumoniae-3206, K. pneumonia-3, Achromobacter
239
xylosoxidans and P. aeruginosa-2995 as shown in Table 1. The other bacterial strains used in
240
this study were insensitive to the phage. These results suggested that the phage had a narrow host
241
range among different bacterial strains.
242
Thermal stability
243
A thermal stability test was carried out to analyze the heat resistant capability of phage at pH 7.0.
244
The phage retained almost 100 % infection activity (8.4 × 105 c.f.u ml-1) after incubation at 37
245
°C. The results suggested that the phage was stable at temperatures ranging between 37 °C and
246
70 °C. At 80 °C, there were no plaques (Fig. 2).
247
pH Stability
248
Optimal pH for phage was determined by testing the stability of phage at different pH values
249
incubating at 37 °C for 16 hours. Phage showed maximum stability at pH 7 while also showing
250
good stability at pH 5, 9 and 11 while at pH 1.0 and 3.0 there were no active infectious phages to
251
be detected. The results showed that low pH might pose hindrance to phage stability. At pH 1.0
252
and 3.0, no plaques were observed, while the number of plaques was found to be increased with
11
253
increasing pH, reaching the highest number at pH 7.0. A low decrease was observed in the
254
number of plaques when the phage was incubated at pH above 7.0 (Fig. 3).
255
Calcium and magnesium ions effect on the adsorption rate of phage
256
The effect of calcium and magnesium ions on the adsorption of phage was analyzed by adding
257
(10 mM) calcium chloride and magnesium chloride to the phage and the K. pneumoniae (M)
258
mixture. The number of free phages left in the solution (which were not bound to the bacteria)
259
was detected at different time intervals of 0, 10, 20 and 30 min using the plaque assay. Data
260
analysis showed a significant difference between the control and the Ca2+/Mg2 ion-treated phage
261
Z. The results showed that calcium and magnesium ions stabilize the process of adsorption. The
262
numbers of free phages are decreased, as shown by the lower line in the figure as compared with
263
the upper line representing the control (Fig. 4).
264
Latent time period and burst size
265
The single-step growth experiment was performed for determining the latent time period and
266
burst size of the phage. A triphasic curve was obtained that has the latent phase, log or rise
267
phase, and stationary or plateau phase. From the data, the latent time period was calculated to be
268
24 min. The burst size of the phage was 320 phages per cell. Determination of burst size was
269
based on the ratio of the mean yield of phage that infected the bacterial cells to the mean phage
270
particles liberated (Fig. 5).
271
Morphology of phage
272
As analyzed by transmission electron microscopy, Z phage had a head of width (76 ± 10 nm)
273
and length of (92 ± 14 nm) with an icosahedrons’ sides of 38 nm with a non contractile (200 ± 15
274
nm) long and (14-29 nm) wide tail, therefore falling into the family Siphoviridae of order
275
Caudovirales. All values were determined as means ± SD from 3 measurements (Fig. 6).
12
276
Phage structural proteins
277
Ultracentrifuge purified phage particles were subjected to 12 % SDS-PAGE, and protein bands
278
were obtained after Commassie Blue G-250 staining and de-staining. A total of six proteins
279
composing phage Z were detected by SDS-PAGE. Their molecular weight was ranging
280
approximately from 18 to 65 kDa (Fig. 7).
281
Genome isolation of bacteriophage
282
Phage genome was detected by agarose gel. The genome was found to be approximately 36 kb
283
(Fig. 8a) and upon EcoR1 restriction produced two bands of different sizes (Fig. 8b). The
284
genome was found to be double-stranded DNA because phage nucleic acid was digested by the
285
EcoR1.
286
Susceptibility of planktonic cultures to phage Z
287
The highly susceptible K. pneumoniae (M) strain was treated with different MOI of phage Z. The
288
lytic activity of phage Z against planktonic cultures of K. pneumoniae (M) in the exponential
289
phase of growth is illustrated in Fig. 9. The Bacteria showed high susceptibility of the phage Z at
290
all MOIs. An abrupt c.f.u reduction was observed in the very first hour with MOI 1.0 and 5.0
291
from 6.0 × 108 to 7.0 × 104 and 1.0 × 105 c.f.u ml-1, respectively. While the MOI 0.1 and 0.5
292
showed a little gradual reduction but after 2 h all MOI’s showed approximately similar efficacy
293
i.e. 4.0 × 105 to 1.0 × 105 c.f.u ml-1.
294
Susceptibility of biofilms to phage Z
295
To determine the action of phage Z in K. pneumoniae (M) biofilms, the biofilm-forming
296
susceptible strain was grown in TSBG in microtiter plates (coaster) for 24 h and 48 h, after
297
which biofilms were challenged with phage Z. The biomass reduction of the biofilms compared
298
with the controls was evident (Fig. 10). Phage Z showed interesting results on both 24 h as well
13
299
as 48 h biofilms. The biofilm formed in 24 hour showed 2.5-fold reduction while 48 hour biofilm
300
also showed
301
(statistically) determined by paired samples t-test (p < 0.05) when compared with the control.
302
Biofilm-grown cells versus planktonic cultures susceptibility to the phage
303
To compare planktonic and biofilm-grown cell susceptibilities to phage Z, the lytic assay was
304
performed using strain K. pneumoniae (M) planktonic cells at the stationary growth phase (after
305
24 h of growth) and 24 h biofilm-grown cells, in a low-nutrient medium to slow down the growth
306
rate of cells and to preserve the biofilm cells for the longest possible time. Fig. 11 presents the
307
results of the effect of phage Z at MOI of one, in biofilm grown cells and also in planktonic
308
stationary phase cells. Biofilm grown cells and stationary phase planktonic cells demonstrated
309
little susceptibility to phage Z. The biofilm cells were little more resistant as compare to
310
stationary planktonic cells. In the case of biofilm cells drop in OD600 occurred relatively slow as
311
compared to planktonic stationary cells, while in control case the stationary planktonic cell
312
showed a slightly higher growth as compared to biofilm cells (Fig.11).
313
DISCUSSION
314
Biofilms can be found everywhere and have been implicated in a variety of nosocomial
315
infections associated with medical devices, hospital equipment and other hard and moist surfaces
316
(Rao et al., 2005). Microscopic observations have proved that most bacteria (99.9 %) are capable
317
of producing biofilms on a wide variety of biotic and abiotic surfaces (Costerton et al., 1978).
318
There is a renewed interest in phage therapy due to a very high emergence of antimicrobial
319
resistance in healthcare institutions worldwide due to frequent usage of antimicrobials
320
(Archibald et al., 1997; Tenover, 2001). Bacteriophages are often considered alternative agents
321
for controlling bacterial infection and contamination and increased antibiotic resistance in
about 3-fold reduction but no total removal was observed in both cases
14
322
bacteria. In the last few years, researchers are looking forward to controlling the emergence of
323
antibiotic resistance bacteria through phage therapy (Nakai and Park, 2002)
324
Most of the known phages interact only with a specific set of bacteria that expresses specific
325
binding sites. This narrow host range is also a challenge for phage therapy. Consequently, there
326
is no known phage that is lytic for all strains of K. pneumonia. This high specificity of phage-
327
host relationship leads to a need for phages to inhibit newly isolated K. pneumoniaa.
328
Phages are generally isolated from environments that are habitats for the respective host bacteria
329
(Nakai and Park, 2002). Phage Z was isolated from sewage water. It is known that sewage
330
generally contains a large diversity of micro-organisms due to contamination from fecal material
331
and hospital drainage water (Piracha et al., 2014). Phage Z was highly lytic and capable of
332
producing clear plaques ranging from 1.0 to 3.0 mm. It has a narrow host range infecting only K.
333
pneumonia (M), K. pneumoniae-3206, K. pneumoniae-3, Achromobacter xylosoxidans and
334
Pseudononas aeruginosa-2995. Many phages have been reported that are found to be greatly
335
specific for their receptors present on the host cell surface. They only show interface with their
336
specific receptors but do not interact with receptors having different structures (Piracha et al.,
337
2014).
338
Several studies have documented that phages varied in thermal and pH stability depending upon
339
strains of phages. The phage was tolerant to relatively high temperature ranging from 37 to 65 °C
340
was killed at 70 °C. Also showing good pH stability over broad range of pH values ranging from
341
5-11 and a maximum stability at pH 7.0. The results are consistent with the previous
342
observations by Ackermann and Dubow (1987) and Jamalludeen et al. (2007) that most phages
343
are able to survive well over a wide range of pH (5 to 9) at physiological conditions that maintain
344
the native virion structure and stability. The inactivity of the phage at lower pH values of 1 and 3
15
345
in our study can be attributed to protein denaturation in acidic environment (Hazem, 2002).
346
These characteristics may be useful for the application of the phage in different environments.
347
The infectivity of phage Z was shown to be increased at 10 mM calcium chloride or magnesium
348
chloride solution concentrations. According to Guttman et al.(2005) cofactors such as Ca2+,
349
Mg2+, divalent cations or sugars may be required for successful binding to occur. Ca2+/Mg2+ ions
350
stabilize the weak interaction of virion with receptors during the adsorption. Diverse quantities of
351
calcium ions give maximum infectivity for various phages (Donlan, 2005; Reese et al., 1974). It
352
is also postulated that Ca2+ ions may increase the concentration of phage particles at the host
353
surface or alter the structure of a cell surface receptor thereby increasing accessibility to the
354
receptor molecules or transfer of phage nucleic acids (Russel et al., 1988; Watanabe and
355
Takesue, 1972). Yang et al. (2010) have described the burst size and the duration of the latent
356
phase which nearly correlates with our results (latent period of 24 min and burst size of 320
357
virions per cell) while Sillankorva et al. (2004) have described small burst size and less duration
358
of the latent phase. Thus a lot of variations have been reported in literature regarding latent time
359
and burst size of bacteriophages.
360
For Phage classification, ICTV recognizes one order, 13 families and 31 genera of
361
bacteriophages. As our phage was a non-contractile tailed virus, phage Z fell into the to family
362
Siphoviridae of order Caudovirales that contains three families of tailed viruses that infect
363
bacteria and archaea (van Regenmortel et al., 2000). Possession of an icosahedral head and a
364
long non-contractile tail would place it in the family Siphoviridae (van Regenmortel et al.,
365
2000).
16
366
Phage specific for a bacteria can infect biofilm cells by first degrading the EPS and then
367
ultimately lysing the bacterial cells. There is evidence that phage-induced depolymerases could
368
affect biofilms and have potential for biofilm control (Hughes et al., 1998; Roy et al., 1993).
369
Carson et al. (2010) have reported the potential utility of bacteriophages to reduce bacterial
370
biofilms on medical device surfaces and in prevention of biofilm via direct incorporation of
371
phages and reported approximately 90 % reduction in E. coli biofilm formation on
372
bacteriophage-treated catheters when compared with untreated controls.
373
Out of the ordinary was the fact that planktonic cells of K. pneumoniae (M) in exponential
374
growth phase were much more sensitive to phage Z lysis than in stationary growth phase. This
375
effect was previously demonstrated on P. fluorescens planktonic cultures (Sillankorva et al.,
376
2004) and susceptibility of S. epidermidis planktonic cells (Cerca et al., 2007). It seems that
377
biofilms are slowly killed by phage Z, not due to a specific biofilm cells, but probably due to the
378
low metabolic activity of biofilm cells (Briandet et al., 2008; Corbin et al., 2001; Costerton,
379
1995). Corbin et al. (2001) studied phage T4 impact on E. coli biofilm and observed one and a
380
half log reduction while Moons et al. (2006) studied the effect of phage T7 on E. coli biofilms.
381
They gave phage T7 treatment for one hour at a concentration of 1 × 1010 c.f.u ml-1 and observed
382
about 2-fold reductions. Similarly we observed slightly more then 2-fold reduction on both 24 h
383
and 48 h biofilms when treated with a phage titer of 4.5 × 109 p.f.u ml-1.
384
In conclusion, this study suggests that phage Z is a tailed, DNA lytic phage, having a good heat
385
tolerance and wide range of pH stability. It also had an activity against multi-drug resistant K.
386
pneumoniae (M) in both plankonic cells and biofilms but does not result in total eradication of K.
387
pneumonia (M) biofilms. Thus for efficient and complete eradication of biofilms a combination
388
of phages (phage cocktail) may be used.
17
389
ACKNOWLEDGMENTS
390
We would like to acknowledge Kalina Rosenova Atanasova from Emerging Pathogens Institute
391
(EPI), Department of Periodontology, University of Florida, USA for helping us during lab
392
experiments and editing this manuscript. We also are very thankful to Karen Kelley, electron
393
microscopy manager from Interdisciplinary Center for Biotechnology Research (ICBR)
394
University of Florida, USA for transmission electron microscopy. We are also thankful to Dr
395
Farida Nighat, incharge Microbiology Lab, Railway General Hospital, Pakistan for providing
396
bacterial strains to carry out phage host range experiments. We are thankful to the Higher
397
Education Commission (HEC) of Pakistan for providing funding to support the current study.
398
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Donlan, R. M. (2005). New approaches for the characterization of prosthetic joint biofilms. Clin Orthopaed Related Res 437, 12-19. Donlan, R. M. and Costerton, J. W. (2002). Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15, 167-193. Gilbert, P., Das, J. and Foley, I. (1997). Biofilm susceptibility to antimicrobials. Advances in Dental Res 11, 160-167. Guttman, B., Raya, R. and Kutter, E. (2005). Basic phage biology. Bacteriophages: Biol Applications 4. Haq, I. U., Chaudhry, W. N., Andleeb, S. and Qadri, I. (2012). Isolation and Partial Characterization of a Virulent Bacteriophage IHQ1 Specific for Aeromonas punctata from Stream Water. Microbial Ecol 63, 954-963. Hazem, A. (2002). Effects of temperatures, pH-values, ultra-violet light, ethanol and chloroform on the growth of isolated thermophilic Bacillus phages. The New Microbiologica 25, 469-476. Hughes, K. A., Sutherland, I. W. and Jones, M. V. (1998). Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. Microbiol 144, 3039-3047. Jamalludeen, N., Johnson, R. P., Friendship, R., Kropinski, A. M., Lingohr, E. J. and Gyles, C. L. (2007). Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli. Vet Microbiol 124, 47-57. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Mah, T.-F. C. and O'toole, G. A. (2001). Mechanisms of biofilm resistance to antimicrobial agents. Trends in Microbiol 9, 34-39. Monroe, D. (2007). Looking for chinks in the armor of bacterial biofilms. PLoS Biol 5, e307. Moons, P., Werckx, W., Van Houdt, R., Aertsen, A. and Michiels, C. W. (2006). Resistance development of bacterial biofilms against bacteriophage attack. Commun Agric Appl Biol Sci 71, 297-300. Nakai, T. and Park, S. C. (2002). Bacteriophage therapy of infectious diseases in aquaculture. Res Microbiol 153, 13-18. Piracha, Z., Saeed, U., Khurshid, A. and Chaudhary, W. N. (2014). Isolation and Partial Characterization of Virulent Phage Specific against Pseudomonas Aeruginosa. Global J Med Res 14. Podschun, R. and Ullmann, U. (1998). Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11, 589-603. Potera, C. (1999). Forging a link between biofilms and disease. Sci 283, 1837-1839. Rao, V., Ghei, R. and Chambers, Y. (2005). Biofilms research-implications to biosafety and public health. Appl Biosafety 10, 83. Reese, J. F., Dimitracopoulos, G. and Bartell, P. F. (1974). Factors influencing the adsorption of bacteriophage 2 to cells of Pseudomonas aeruginosa. J Virol 13, 22-27. Roy, B., Ackermann, H., Pandian, S., Picard, G. and Goulet, J. (1993). Biological inactivation of adhering Listeria monocytogenes by listeriaphages and a quaternary ammonium compound. Appl Environ Microbiol 59, 2914-2917. Russel, M., Whirlow, H., Sun, T. and Webster, R. (1988). Low-frequency infection of F-bacteria by transducing particles of filamentous bacteriophages. J Bacteriol 170, 5312-5316. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning, Cold spring harbor laboratory press New York. Sillankorva, S., Oliveira, R., Vieira, M. J., Sutherland, I. and Azeredo, J. (2004). Bacteriophage Φ S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms. Biofouling 20, 133-138. Sulakvelidze, A., Alavidze, Z. and Morris, J. G. (2001). Bacteriophage therapy. Antimicrobial Agents and Chemother 45, 649-659. Tenover, F. C. (2001). Development and spread of bacterial resistance to antimicrobial agents: an overview. Clin Infect Dis 33, S108-S115. 19
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488 489 490 491 492 493 494 495 496 497 498 499 20
500
Figure legends
501
Fig. 1. Soft Agar overlay plates, (a) showing spot test assay and (b) a higher dilution (10-7) of
502
phage titer showing clear plaques of 1.0 – 3.0 mm in diameter.
503
Fig. 2. Stability of phage Z treated with different temperature for 60 min. All values represent
504
means of 3 determinations with ± standard deviations.
505
Fig. 3. Stability of phage Z treated with different pH overnight at 37 °C. All values represent
506
means of 3 determinations with ± standard deviations.
507
Fig. 4. Test for phage adsorption rate. Effect of divalent metal ions on phage adsorption rate by
508
adding divalent metal ions by adding 10 mM CaCl2 or MgCl2 solution to the mixture of phage Z
509
and K. pneumonia (M). All values represent means of 3 determinations with ± standard
510
deviations.
511
Fig. 5. One-step growth experiment. Latent time and burst size of phage Z were inferred from
512
the curve with a triphasic pattern. All values represent means of 3 determinations with ± standard
513
deviations.
514
Fig. 6. Transmission electron micrographs of the purified phage Z using scale bars of 200 nm.
515
Three representative images (a), (b) and (c) are shown.
516
Fig. 7. SDS-PAGE analysis of phage Z structural proteins. Lane 1, broad range protein
517
molecular weight markers (Precision Plus Protein™, Bio-Red); Lane 2, phage Z proteins.
518
Fig. 8. (a) Genome of phage: 0.6% (w/v) agarose gel. Lane 1 shows DNA Ladder (GeneRuler
519
High Range) and Lane 2, shows band of phage DNA having a size of approximately 36 kb while
520
(b) Lane 1, shows 1kb DNA Ladder (New England Biolabs) and Lane 2 shows restriction
521
analysis of phage Z DNA with EcoR1.
21
522
Fig. 9. Kill curves of exponential growth phase planktonic K. pneumoniae (M) by phage Z at
523
different multiplicity of infections (MOIs): 0, 0.1, 0.5, 1 and 5. All values represent means of 3
524
determinations.
525
Fig. 10. Reduction of biofilm biomass after 24 h and 48 h of challenge with 5.2 × 109 p.f.u ml-1of
526
phage Z. White bars represent control biofilms without phage and dark bands represent biofilm
527
infected with phage.*Significant reduction in biomass compared with control (light bands; paired
528
samples t-test, p < 0.05). All values represent means of 3 determinations with ± standard
529
deviations.
530
Fig. 11. Kill curves of K. pneumoniae (M) in different growth stages: stationary phase bacteria
531
(control and with phage), or biofilm grown bacteria (control and with phage). All values
532
represent means of 3 determinations.
533 534 535 536 537 538 539 540 541 542 543 544 545 22
546
Table 1. Spot test of phage Z on different bacterial species S:No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
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Bacterial Strain Klebsiella pneumoniae (M) Klebsiella pneumoniae-3206 Klebsiella pneumoniae-3 Klebsiella pneumoniae-M 37 Klebsiella pneumoniae-3025 Klebsiella pneumoniae-3011 Klebsiella pneumoniae -3033 Klebsiella pneumoniae-3019 Klebsiella pneumoniae-3114 Klebsiella pneumoniae-2825 Klebsiella pneumoniae-3015 Klebsiella pneumoniae-3018 Klebsiella pneumoniae-2870 Klebsiella pneumoniae-2908 Klebsiella pneumoniae-3202 Klebsiella pneumoniae-3311 Escherichia coli-3 Escherichia coli -F Escherichia coli LF-1969 Escherichia coli LF-1990 Escherichia coli-3051 Escherichia coli LF-1968 Pseudomonas aeruginosa-3098 Pseudomonas aeruginosa-2995 Pseudomonas aeruginosa-2949 Pseudomonas aeruginosa-3048 Pseudomonas aeruginosa-3068 Pseudomonas aeruginosa-2830 Pseudomonas aeruginosa-3178 Staphaylococcus aureus-2895 Staphaylococcus aureus-2975 Staphaylococcus aureus-2938 Staphaylococcus aureus-2895 Achromobacter xylosoxidans + = lysis,
Activity (+/₋) + + + + ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ ₋ +
- = no lysis
548 549
23
Supplementary Material Files Click here to download Supplementary Material Files: Table S1, Sensitivity of K. pneumonia to different group of antibiotics.pdf
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