Chapter 3

Characterization of bacteriophages of selected

pathogens

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59

Abstract

Multidrug resistant strains of Salmonella enterica Paratyphi B, Pseudomonas aeruginosa and

Klebsiella pneumoniae are ever emerging and their prevalence in the natural water resources is

also increasing. Literature survey indicated that, in the last few decades no effective drugs have

been discovered against these pathogens, Therefore, to overcome problems associated with

these pathogens, potent lytic bacteiophages could be a feasible alternative.To ascertain

feasibility of using these phages against bacterial pathogens, their characterization and

identification is desirable.Bacteriophages against such nuisance bacterial pathogens have been

characterized on the basis of plaque and phage morphology, their host range, adsorption and

growth kinetics, protein profile and the restriction digestion pattern of phage DNA. The

stability and infectivity of phages at different pH and temperatures were also studied.

Salmonella enterica serovar Paratyphi B phage (φSPB) was identified as a member of the

Podoviridae family, which is morphologically similar to the 7-11 phages of the C3 morphotype

of tailed phages, characterized by a very long, rounded -shaped head. The head measured about

162 × 58 nm and tail size was 12 × 7 nm. The phage was stable over a wide range of pH (4 to 9)

and temperature (4 to 40 °C). The optimal temperature for infection was 37

°C and the

adsorption rate constant was 4.7×10-10

. Latent and eclipse periods were 15 min and 10 min,

respectively, and the burst size was 100 pfu/infected cell after 25 min at 37 °C. The phage DNA

was 59 kb in size. On SDS-PAGE, 10 protein bands were observed. Pseudomonas aeruginosa

phage (BVPaP-3) was identified as a member of the family Podoviridae family, which is

morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58 to 59

nm in diameter and a short tail of 10 x 8 nm. It was stable within a wide range of pH (6 to 10)

and temperature (4 to 40 oC). Its optimal growth temperature for infectivity was 37

oC and the

adsorption rate constant was 1.19 x 10-9

. Latent and eclipse periods were 20 and 15 min,

respectively, and the burst size is 44 phage particles per cell after 35 min at 37 o

C. The phage

had a DNA size of 41.31 Kb and a proteome of 11 proteins. The major protein on the SDS-

PAGE was 33 kDa in size. Klebsiella pneumoniae phage (KPP) was identified as a member of

the family Myoviridae, which is morphologically similar to the FC3 phage group. The phage

has a head of 85.5 nm with the extended tail of 95 × 17 nm and a contracted sheath of 50 × 20

nm. It was stable within a wide range of pH (5 to 10) and temperatures (4 to 50 oC). Optimal

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60

temperature for infection was 37 o

C and the adsorption rate constant was 4.7 × 10-10

. Latent and

eclipse periods were 20 and 15 min, respectively, and the burst size was 120 phage particles per

infected cell after 45 min at 37 o

C. The phage DNA size was 54 kb and a proteome of 9 bands

in the gradient gel. The prevailing proteins had sizes of 90 kDa and 60 kDa.

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61

3.1 Introduction

Viruses are the obligate intracellular parasites that are present in the natural environment

abundantly. These viruses are of three types‟ viz., plant viruses, animal viruses and bacterial

viruses (bacteriophages). There are very few fundamental differences among the viruses

infecting plants, animals and bacteria (Primrose and Dimmock, 1980). The detailed study of

bacterial viruses offers as easy introduction to basic virology.

Bacteriophages are the viruses of prokaryotes, which can either instantly kill a bacterial cell or

integrate its DNA into the host genome (Medigan et al, 1997). Bacterial viruses are the most

numerous organisms on the earth surface therefore they can be easily isolated from a variety of

the natural sources, including water, sewage, intestinal contents, vegetables, and some insects

such as cockroaches and flies.

Bacteriophages were not recognized for almost 40 years after the beginning of

bacteriology in the 1880s. However, there are several reports in the literature that suggest the

presence of bacteriophages. Hankin reported that the waters of the Jumuna and Ganges rivers in

India could kill many kinds of bacteria (Hankin, 1896). In 1901, it was reported that a substance

in autolyzed cultures that caused the lysis of diverse bacterial cultures could also cure

experimental infections and provided prophylactic immunity to subsequent infections

(Emmerich et al, 1901). The first clear experiments on bacteriophages used bacterial cultures

spread on solid medium and were based on the observation of localized bacteriolysis i.e.

plaques. In 1915, Twort reported the phenomenon of “glassy transformation”. His interpretation

about the glassy transformation was that “…. It [the agent of glassy transformation] might

almost be considered as an acute infectious disease of Micrococcus” (Twort 1915).

In 1917, it was reported that a “microbe” lysed bacteria in liquid cultures and killed

bacteria in discrete patches, which he termed plaques, on the surface of solid medium

(d‟Herelle 1917). He for the first time called the “ultra viruses” that invaded bacteria and

multiplied at their cost as bacteriophages.

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62

3.1.1 Systematics of bacteriophages

Felix d‟Herelle believed that there was only one bacteriophage with many races, the

“Bacteriophagum intestinale” (d‟Herelle 1918). According to Burnet (1933), the enterobacterial

phages could be subdivided by serology, particle size as determined by filtration, host range and

stability tests due to their heterogenic properties. Ruska (1933) used electron microscopy first

time for the classification of viruses. In 1962, a classification scheme was proposed based on

the nature of virus nucleic acid (DNA or RNA), capsid shape, presence or absence of an

envelope and number of capsomers (Lwoff et al 1962), which was known as the LHT system

and was adopted by the provisional committee on Nomenclature of viruses (PCNV) then it also

included ssRNA and filamentous phages. In 1966, the PCNV became International Committee

on Taxonomy of Viruses (ICTV) in1973 (Mathews 1983). The ICTV is essentially concerned

with classifying viruses into higher taxa (i.e. orders, families and genera). The names of the

orders, families and genera reflect characteristic properties, are typically derived from Latin or

Greek roots and end in – virales, - viridae and – virus respectively.

a) Current phage classification system

Bacteriophages infect more than 140 bacterial genera (Ackermann 2001). More than 5500

bacteriophages have been examined by electron microscopy (Ackermann 2007).

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63

BACTERIOPHAGES

Tailed phages

(Order-Caudovirals

•Myoviridae family

(Contractile tails consisting of a

sheath and a contractile tube)

•Siphoviridae family

(Long non- contractile tails)

•Podoviridae family

(Short non-contractile tails)

Polyhedral phages

Polyhedral

DNA phages

Polyhedral

RNA phages

SS DNA phages

•Microviridae

DS DNA phages

•Corticiviridae

•Tectiviridae

SS RNA phages

•Leviviridae

DS RNA phages

•Cystoviridae

Filamentous phages

SS DNA phages

•Inoviridae

DS DNA phages

•Lipthrixviridae

•Rudiviridae

Pleomorphic phages

•Plasmoviridae (DS DNA)

•Fuselloviridae (DS DNA)

T7 – like viruses

P22 – like viruses

φ 29 – like viruses

Λ-like viruses

T1- like viruses

T5-like viruses

L5 –Like viruses

C2 –like viruses

ΨM1 –like viruses

T4 –like viruses

P1 – like viruses

P2 –like viruses

Mu – like viruses

SPO1- like viruses

φH – like viruses

Genera

PodoviridaeSiphoviridaeMyoviridae

Family

Tailed Phage Genera

b) Podoviridae phage family-Research status

i. International status

Literature survey indicated several research articles published recently on bacteriophages

belonging to the Podoviridae family. It is reported that since the introduction of negative

staining in 1959, almost 5568 phages have been examined in the electron micrograph, of which,

96 % are tailed phages and only 3.7 % are polyhedral, filamentous and pleomorphic

(Ackermann, 2007). Gadaleta and Zorpopulos (1997) introduced a phage KvP1; a new member

of Podoviridae family that is phylogenitically related to the coliphage T7.Gill et al (2003)

reported bacteriophages belonging to the Podoviridae family specific for Erwinia amylovora. In

another study, the structural proteome of bacteriophage φKMV Pseudomonas aeruginosa that

was belonging to the Podoviridae family. Recently, the complete genomic sequence of the

bacteriophage φEcoM-GJ1, a novel phage was carried out. A gene for the RNA polymerase,

was similar to the T7 group of the Podoviridae family (Jamalludeen et al, 2008).In another

study, genomic and proteomic analysis of phiEco32, a novel Escherichia coli phage was carried

Chapter 3

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64

out which belongs to the Podoviridae group (Savalia et al,2008).The biological characteristics

of eight Podoviridae stx-2 converting phages from E.coli were also studied (Xia et al,2008).In a

study carried out by Son et al(2010), an antibacterial and biofilm removal activities of

Podoviridae Staphylococcus aureus phage SAP-2 was studied. Genome sequence of the lytic

phage φIBB-PF7A of Pseudomonas putida was carried out and found to be closely related to

P. putida gh-1 phage and coliphage T7 ( Sillankorva et al, 2011).KP34, a novel phage and a

member of φKMV-like bacteriophage belonging to the Podoviridae family is sequenced

completely (Kawa et al,2011). Lau et al (2010) reported a lytic phage, φEC1 was found to be

effective against the causative agent of Colibacillosis in Chickens belongs to the Podoviridae

family.

ii. National status

There are few reports from India regarding phages belonging to the Podoviridae family.

Five bacteriophages (Kpn 5, Kpn12 Kpn13 Kpn17 Kpn22) each having specificity against

Klebsiella pneumoniae B5055 were isolated and characterized (Kumari et al, 2010).BVPaP-3, a

T7-like lytic phage specific for Pseudomonas aeruginosa belonging to the Podoviridae family

was isolated from the Indian river and was Characterized based on its morphology, proteome

and genome studies (Ahiwale et al,2011). In one report, phages specific against Pseudomonas

aeruginosa PAO were isolated from sewage samples and all were assigned to the Podoviridae

family (Kumari et al, 2009). Morphological and general features of a lytic phage (SR3) of

Bradyrhizobium japonicum that belongs to the Podoviridae family was studied in detail

(Appunu et al, 2008). In another study, lytic phages of luminescent Vibrio harveyi isolated from

shrimp( Penaeus monodon ) hatcheries were studied in detail (Thiyagarajan et al, 2011). In one

more study, phage N4, a newly isolated phage belonged to the Podoviridae family was used in

the typing scheme for V. cholerae o1 biovar EIT strain (Gosh et al, 1995).

3.2 Characterization of bacteriophages

Bacteriophage characterization is the mandatory step prior to the practical application in

the various fields. Phages can be characterized based on the criteria that are recommended by

Ackermann (2009). The parameters used for the characterization are as follows:

Chapter 3

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65

Parameters that are normally used are

Nucleic acid

Nature – DNA, RNA, ss, ds

Anatomy – linear, circular, super coiled

Physical – molecular weight, length

Chemical – percent of particles, G+C

Genome – hybridization

Virion

Morphology – shape, capsid symmetry, number of capsomers

Physical – weight, buoyant density

Chemical – protein %, lipid %, number of proteins, protein molecular weight, amino acid

composition, lipid composition

Serology

Neutralization

Physiology

Adsorption site, adsorption velocity, burst size, latent period, helper function, virulent or

temperate

3.2.1 Selection of phages and bacterial pathogens for further studies

The following bacterial pathogens identified as described under (section 2.3.2) were

selected viz., Salmonella enterica Paratyphi B, Pseudomonas aeruginosa and Klebsiella

pneumoniae as they showed resistance to antibiotics tested (section 2.3.3 ) and lytic phages

specific to them that have potent lytic activity (section 2.3.4). Although many multidrug

resistant strains of these pathogens are ever emerging worldwide and are creating serious

problems, the frequency of occurrence of Salmonella enterica Paratyphi B, Pseudomonas

aeruginosa and Klebsiella pneumoniae in the Pavana river water was found to be very high (

84 %, 100 % and 100 % respectively samples).Therefore Salmonella enterica Paratyphi B,

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66

Pseudomonas aeruginosa and Klebsiella pneumoniae and their potent lytic phage (single phage

per bacterium) were selected for further studies. Besides, in recent years, problems associated

with Salmonella bacteria, mainly cases of human salmonellosis have increased in frequency and

severity. Medical investigators belive that antibiotics prolong the carrier state. Salmonella

enterica serovars Paratyphi A, B or C are estimated to cause 5.5 million cases of enteric fever

each year. Human cases of Salmonella enterica Paratyphi B resistant to ampicillin,

chloramphenicol, streptomycin, spectinomycin, sulphonamides and tetracycline have been

found in Canada, the United Kingdom, France and Australia (Mulvey et al, 2004; Threlfall et

al, 2005; Weill et al, 2005; Levings et al, 2006). Salmonella bacteria mainly contaminate

natural water resources including drinking water. Although, some vaccines are available against

salmonella for poultry and animals; human vaccines are available only for typhoid fever in an

oral and (Ty21) and injectable (VICPS) forms. Pseudomonas aeruginosa, an ubiquitous

opportunistic pathogen, has acquired resistance to almost all antibiotics (Harper et al, 2011

Strateva et al, 2009). In addition, P. aeruginosa is highly active in the formation of biofilms and

thus reduces the efficacy of multiple antibiotics. Therefore, P. aeruginosa infections are

declining the efficacy of conventional chemical antibiotic therapies.

Klebsiella pneumoniae is a gram negative opportunistic bacterial pathogen. It causes a

significant proportion of hospital- acquired urinary tract infections, viz., pneumonia, septicemia

and soft tissue infections (Podschun and Ullmann1998 ).In the literature ,it is reported that the

Extended- Spectrum Beta- Lactamase (ESBL) is a plasmid coded property and the resistance to

clavulanic acid is increasing among the strains of K. pneumoniae (Kamatchi et al,2009). About

80 % of nosocomial infections in immuno-compromised patients are caused by Multidrug

resistant strains of K. pneumoniae (Chhibber et al, 2008). Therefore, there is growing strong

interest towards the antimicrobial potential of lytic phages against such Multidrug resistance

pathogens. Therefore, P. aeruginosa and its potent lytic phage were selected for further studies

Chapter 3

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67

3.3 Materials and Methods

3.3.1 Propagation of the phages, φSPB, BVPaP-3, and KPP

Respective phages were selected after subsequent phage purification step (purification

step was carried out for 8-10 times). Then, a well isolated, single plaque of Salmonella enterica

Paratyphi B phage (6 mm diameter), Pseudomonas aeruginosa phage (3 mm diameter) and

Klebsiella pneumoniae phage (4 mm diameter) was transferred separately into three different

flasks containing sterile phage broth (250 ml) with 0.5 ml mid-log phase culture of the

respective host bacteria. The flasks were incubated at 37 °C for 24 h under shaking condition.

The contents were centrifuged and the supernatants were filtered through a membrane filter of

0.2 μm separately. These stock filtrates were stored at 4 °C. Hereafter, Salmonella enterica

Paratyphi B phage is designated as φSPB, Pseudomonas aeruginosa phage is designated as

BVPaP-3 and Klebsiella pneumoniae phage is designated as KPP. The phage titers of respective

lysate (φSPB, BVPaP-3, KPP) were determined by the double agar overlay technique.

3.3.2 Determination of Multiplicity of infection values for φSPB, BVPaP-3 and KPP

The success of one step growth curve depends upon the standard host cell density that is

able to yield maximum plaques on plate. The actual number of phages that will enter any given

cell is a statistical process and that requires a mid-log growth phase cells. Therefore, the

information about the mid-log growth phase of the host bacteria is necessary. The multiplicity

of infection (MOI) is the ratio of infectious agent and the host cells.

a) Growth curve of Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa

and Klebsiella pneumoniae

Growth curves of Salmonella Paratyphi B, Pseudomonas aeruginosa and Klebsiella

pneumoniae were performed. Sterile flasks (3) containing phage broth (150 ml) were inoculated

separately with the overnight grown culture (0.05 ml) of respective host bacteria. Uninoculated

flask with phage broth was used as a negative control. Flasks were incubated at 37 ˚C for 24 h

under shaking conditions. Small aliquots were removed from the inoculated flasks after every

15 min interval for first 1-2 h and then after every 30 min. interval. The optical density of each

Chapter 3

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68

aliquot was determined at 650nm using uninoculated phage broth as blank using

spectrophotometer (Shimadzu, Asia Pacific PTE Ltd., Singapore ).The growth curves of the

above mentioned bacterial pathogens were (Time against O.D.) plotted and the lag, the log and

the stationary phases were determined.

b) Standardization of the host cell density for MOI determination

The flasks containing the sterile phage broths (200 ml) were inoculated with the overnight

grown culture of Salmonella enterica Paratyphi B, Pseudomonas aeruginosa and Klebsiella

pneumoniae separately into three flasks. Then the optical density of 5 h old (the mid-log phase)

bacterial suspension was determined as described above using un-inoculated broth as blank.

Then 5 h old culture of each bacterium was analyzed for the density in terms of viable count/ml

(TVC).Then the varied aliquots of the mid–log phase culture of Salmonella enterica Paratyphi

B, Pseudomonas aeruginosa and Klebsiella pneumoniae were mixed with the fixed volume

(0.1ml) of the appropriate dilution of respective phage lysate. Each mixture was then

transferred into soft agar (3 ml) and then poured separately onto the sterile nutrient agar

medium. Plates were incubated at 37 ˚C for 24 h. After incubation, plaques were recorded and

the average numbers of plaque forming units (pfu/ml) were compared with the aliquots of the

respective host bacterium. The host aliquot with the maximum plaque forming units was

considered the standard host density and was used to determine MOI value.

3.3.3 Characterization of φSPB, BVPaP-3 and KPP

The phages (φSPB, BVPaP-3 and KPP) were characterized by plaque and phage

morphology, host range, adsorption and growth kinetics, stability and infectivity of phage at

different pH values and temperatures, its protein profile and the restriction digestion pattern of

phage DNA.

a) Plaque morphology

The nature of plaque due to φSPB was studied on various media viz., Nutrient agar (NA)

A9, MacConkey' agar (MA)

A7, Salmonella–Shigella agar (SSA)

A14, Bismuth Sulphite agar

(BSA)A21

and Hektoen enteric agar (HEA)A4

. Plaque characteristics due to BVPaP-3 were

Chapter 3

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69

studied on various solid media viz., nutrient agar, MacConkey agar and Cetrimide agar and

plaque characteristics due to KPP were studied on nutrient agar and MacConkey agar. An

aliquot (1 ml) of respective phage lysate (φSPB; 2 x108, BVPaP-3; 6.6×10

9 and KPP; 2.5×10

10

(pfu/ml) was mixed with 1 ml of the mid-log phase culture of respective host viz., Salmonella

enterica Paratyphi B (OD650 0.8), Pseudomonas aeruginosa (OD650 0.05) and Klebsiella

pneumoniae (OD650 0.57). An aliquot of 0.1 ml of each mixture was spread onto the surface of

the respective medium. Plates were incubated at 37 °C and observed every 3 h over a period of

24 h for the development of plaques.

b) Phage morphology

Bacteriophage particles (φSPB, BVPaP-3 and KPP) were sedimented at 25,000 × g for 60

min using a Beckman J2-21 centrifuge (Beckman Instruments, Palo Alto, CA). Phages were

washed twice in 0.1 Molar ammonium acetate buffer (pH 7.0), stained with 2 %

phosphotungstate (pH 7.2) solution, deposited on carbon-coated Formvar films and examined

under a Philips EM 300 electron microscope.

c) Host range

The phages (φSPB, BVPaP-3 and KPP) were tested for their ability to infect 11 bacterial

strains belonging to different enterobacterial species viz., Citrobacter koseri (MTCC 1657),

Enterobacter aerogenes (MTCC 111), Escherichia .coli (MTCC1678), Klebsiella pneumoniae

(MTCC 39), Pseudomonas aeruginosa (MTCC 424), Proteus vulgaris (MTCC744), Salmonella

enterica Paratyphi A (MTCC 735), Salmonella typhimurium (MTCC 98), Shigella sonnei

(MTCC 2957), and Vibrio cholerae (MTCC 3906) were obtained from the Institute of

Microbial Type Culture Collection (IMTECH), Chandigarh, India. Salmonella enterica

Paratyphi B (ATCC 8759) was obtained from the American Type Culture Collection,

Manassas, VA, USA. An aliquot of 50 l of each mid- log phase culture was mixed with 4 ml

of soft agar (0.6 % w/v) and then poured onto a sterile nutrient agar plate. Once the overlay was

solid and dry, a volume of 100 μl of phage lysate (2.4 x 108

pfu/ml) was deposited at the centre

of each plate. Plates were incubated at 37 ˚C and examined for plaques after 6-10 h

(Manchester, 1997).A clear zone in the bacterial lawn was recorded as complete lysis.

Chapter 3

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70

d) Adsorption kinetics

A mid-log phase culture of Salmonella enterica Paratyphi B (OD650 0.8), Pseudomonas

aeruginosa (OD 650 0.05) and Klebsiella pneumoniae (OD650 0.57) (9 ml) grown in phage broth

separately was infected with 2 ml of φSPB [4x107 pfu/ml], 3 ml of BVPaP-3 (6.6×10

9 pfu/

ml)

and 2 ml of KPP(2.5 × 1010

pfu/ ml) respectively and introduced into sterile 100 ml flasks

separately equilibrated at 37 °C at time zero(t=0) with MOI of 0.035, 0.07 and 0.19 for

φSPB,BVPaP-3 and KPP respectively. The flasks were incubated at 37 °C in a shaker water

bath at 160 rpm for one hour. At one minute intervals, 50 µl aliquot was withdrawn from each

flask and transferred into the two separate tubes containing 950 μl phage broth, supplemented

with 5-6 drops of chloroform under cold conditions, for a period of 10 min. The contents of the

tubes were mixed thoroughly on a cyclomixer, serially diluted in phage broth and then plated on

sterile nutrient agar plates. Plaques were counted after overnight incubation at 37 °C.

e) One step growth curve

One-step growth curve was constructed as described by Hyman and Abedon (2009) with

few modifications. Briefly, 9 ml of the mid -log phase cultures of Salmonella enterica serovar

Paratyphi B (OD650 0.8), Pseudomonas aeruginosa (OD 650 0.05) and Klebsiella pneumoniae

(OD650 0.57) estimated on an spectrophotometer UV 1800 (Shimadzu, Asia Pacific PTE Ltd.,

Singapore) was mixed with respective phage lysates [2 ml of φSPB (4x107 pfu/ ml), 3 ml of

BVPaP-3 (6.6×109

pfu/ ml) and 2 ml of KPP (2.5 × 10

10 pfu/

ml)] in a 100 ml flask (with MOI

of 0.035, 0.07 and 0.19 respectively ). Phages were allowed to adsorb for 10 min at 37 °C. The

mixture was then centrifuged (10,000× g, 20 min, 4°C), the pellet formed was resuspended in a

10 ml fresh phage broth medium. Two aliquots of the suspension (0.1ml each) were withdrawn

at 5 min intervals over a period of 1 h, one aliquot was transferred to a tube containing 0.9 ml

of sterile phage broth and the second aliquot was transferred into a tube containing 0.9 ml of

phage broth with chloroform (1% v/v) kept on ice in order to determine the eclipse period. The

plaque forming units (pfu) in each tube were determined.

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71

f) Optimal pH and temperature for phage stability

The stability of φSPB, BVPaP-3 and KPP was studied at varied pH (0.05 mol l-1

citrate

bufferB7

: pH 4, 5 and 6; 0.05 mol l-1

phosphate bufferB8

: pH 7, 8 and 9 and 0.05 mol l- of glycine

- NaOH bufferB1

of pH 10, 11, and 12). An aliquot of 1ml of the lysate [φSPB (4x107 pfu/ ml),

BVPaP-3 (6.6×109

pfu/ ml) and KPP (2.5 × 10

10 pfu/

ml)] was suspended in 9 ml of the

respective buffers. Tubes were incubated at room temperature for 1 h. Contents of the tubes

were serially diluted in SM buffer (pH 7.5) A15

and a suitable dilution was used to determine the

number of pfu (plaque forming units) in the lysate by the soft agar overlay technique (Adams,

1959).The stability of φSPB, BVPaP-3 and KPP was evaluated at different temperatures (4, 10,

20, 30, 37, 40 and 50 °C). Aliquots (5 ml) of phage lysate [φSPB (4x10

7 pfu/ ml), BVPaP-3

(6.6×109

pfu/ ml) and KPP (2.5 × 10

10 pfu/

ml)] in sterile screw capped tubes were incubated at

the selected temperatures for 1 h. Phages were titrated by the double agar overlay technique.

g) Protein profile

The purified respective phage lysates (50 ml each) (φSPB, BVPaP-3 and KPP) were used

for extraction of proteins. The filtrate was concentrated by precipitation using 10 % PEG 8000

and centrifuged at 12,000 × g for 25 min after overnight incubation at 4 °C. The supernatant

was decanted and the glazy pellet was resuspended in SM buffer (pH 7.5). The concentrated

phage filtrate was further concentrated by means of an Amicon kit (Millipore India Pvt. Ltd,

Bangalore, India) using a 3 kDa cut-off membrane. Phage particles were disrupted by Triple

Freeze Thawing Cycle in 6M urea, 50 mM Tris/HCl, pH 8 and 5 mM dithiothreitol. The

disrupted phage particles were heat denatured for 5 minute at 95 ºC and subsequently alkylated

by addition of an equal volume of 100 mM iodoacetamide in 50 Mm ammonium hydrogen

carbonate plus three additional volumes of 50 Mm ammonium hydrogen carbonate for

incubation at room temperature in dark for 45 min to denature and reduce proteins (Lavigne et

al, 2006). The phage protein (100 µg ml-1

) was loaded on a 12 % SDS-PAGE gel along with a

standard molecular weight marker (Laemmili, 1970). Protein bands were visualized after

staining with Coomassie dye G-250 (Sigma-Aldrich, Bangalore, India).

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72

h) Restriction digestion pattern of phage DNA

The purified phage lysates (φSPB, 5.0 × 108 pfu/ml; BVPaP-3, 2.8 × 10

9 pfu/ml; and

KPP, 2× 1010

pfu/ml) ( 50 ml each ) were used for the extraction of DNA as described by

Ausubel et al (1992) and purified by cesium chloride density gradient centrifugation as

described by Davis et al, (1980) using a Hitachi 55P Ultracentrifuge (Hitachi Koki Co. Ltd.,

Tokyo, Japan) at 64,000 × g for 24 h. Phage DNA (φSPB-0.5 μg/μl ; BVPaP-3 0.3 μg/μl and

KPP- 0.5 μg/μl ) was digested with EcoRI and Hind III restriction enzymes according to the

manufacturer's instructions (Fermentas International Inc., Glen Burnie, MD, USA). The

reaction mixture comprised: Phage DNA 20 µl, 10X assay buffer 2.5 µl, nuclease free water 1.5

µl and 1 µl. The reaction mixture was incubated in a water bath at 37 °C for 1 h. Molecular

weights of fragments were determined by using an electrophoresis unit (BioEra Life Sciences

Pvt. Ltd., Pune, India) at 100 V for 9 h on a 0.5 % agarose gel. A broad range DNA molecular

weight marker (BioEra Life Sciences Pvt. Ltd., Pune, India) was used for control. The

molecular weight of the products was determined using Alpha Imager Software (BioEra Life

Sciences Pvt. Ltd., Pune, India).

i) Statistical analysis

The One-way ANOVA test (Khan and Khanum, 2008) was used to evaluate the effect of

nutrient media on the nature of plaques.

3.4 Results

3.4.1 MOI values of phages

MOI is the ratio defined by the number of infectious virus particles divided by the number

of target host cells. The MOI value or the ratio of number of phage to number of host bacteria

should not exceed 1 in order to determine one step growth curve. If the multiplicity of infection

is 1 or > 1, that means the number of phages is equivalent to the number of host bacteria. To

determine MOI value, specific host cell density is required.

Chapter 3

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73

i. Bacterial growth curves

Bacterial growth curves were constructed to find out the mid-log phase of the bacterial

hosts. The lag phase lasted for approximately for 2 h and the mid-log phase of Salmonella

Paratyphi B was achieved after 5h.The stationary phase was achieved after 18 h. (Fig.3.1a).

Pseudomonas aeruginosa requires approximately 2.5 h to complete its lag phase The mid-log

phase of Pseudomonas aeruginosa was achieved after 6 h and the stationary phase was

achieved after 20 h (Fig.3.1b). In case of Klebsiella pneumoniae, the lag phase lasted for

approximately 2 h mid-log phase was achieved after 5 h and the stationary phase was achieved

after 16 h. (Fig.3.1 c).

Fig. 3.1 Growth curves of (a); Salmonella enterica serovar Paratyphi B, (b); Pseudomonas aeruginosa and

(c); Klebsiella pneumoniae carried out in phage broth medium at 37 ˚C.

Chapter 3

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74

ii. Host cell density

The purpose of the standardization of the host cell density was to determine the exact

proportion of phages to cells ratio i.e., the multiplicity of infection (MOI).The concentration of

the mid-log phase culture of the respective host that gives the maximum number of phages was

considered for the further studies (Table 3.1,3.2 and 3.3).

Table 3.1 Standardization of Salmonella enterica Paratyphi B density to determine MOI

value

Tube No. S. Paratyphi B*

( ml)

(φSPB )

Lysate**

(ml)

Plaque forming

unit/ml of host

culture

1 0.1 0.1 820

2 0.2 0.1 850

3 0.3 0.1 1250

4 0.4 0.1 1300

5 0.5 0.1 1500

6 0.6 0.1 1200

7 0.7 0.1 1210

8 0.8 0.1 1000

9 0.9 0.1 1157

10 1.0 0.1 887

*Host density- 2.6 × 108 cfu/ml ** φSPB titer- 4x107 pfu/ml

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75

Table 3.2 Standardization of Pseudomonas aeruginosa density to determine MOI value

Tube No. P. aeruginosa*

( ml)

(BVPaP-3)

Lysate**

(ml)

Plaque forming

unit/ml of host

culture

1 0.1 0.1 600

2 0.2 0.1 610

3 0.3 0.1 720

4 0.4 0.1 900

5 0.5 0.1 1200

6 0.6 0.1 690

7 0.7 0.1 570

8 0.8 0.1 560

9 0.9 0.1 550

10 1.0 0.1 560

* Host density- 2.8 × 1010 cfu/ml **BVPaP-3 titer- 6.6 ×109pfu/ml

Table 3.3 Standardization of Klebsiella pneumoniae density to determine MOI value

Tube

No.

K. pneumoniae*

(ml)

(KPP)

Lysate*

(ml)

Plaque forming

unit/ml of host

culture

1 0.1 0.1 850

2 0.2 0.1 920

3 0.3 0.1 1120

4 0.4 0.1 1480

5 0.5 0.1 3000

6 0.6 0.1 2900

7 0.7 0.1 2000

8 0.8 0.1 1320

9 0.9 0.1 1000

10 1.0 0.1 9000

Host density- 2.9×1010 cfu/ml KPP titer- 2.5×1010 pfu/ml

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76

iii. The MOI values for respective phages

The MOI values for φSPB, BVPaP-3 and KPP were 0.035, 0.07 and 0.14 respectively.

The MOI value was found to be the highest for the KPP phage and for φSPB the MOI value

was the lowest one (Table 3.4).

Table 3.4 MOI values determined for phages

Host bacteria Phage MOI value

(pfu/cfu)

Salmonella Paratyphi B φSPB 0.035

Pseudomonas

aeruginosa

BVPaP-3 0.07

Klebsiella pneumoniae KPP 0.14

3.4.2 Titers of φSPB, BVPaP-3 and KPP phages

The titer of φSPB, BVPaP-3 and KPP in the lysates was 4x107pfu/ml, 6.6×10

9pfu/ml and

2.5×1010

pfu/ml respectively.

3.4.3 Characteristics of φSPB, BVPaP-3 and KPP

a) Plaque morphology: effect of medium

The φSPB plaque was clear and circular in nature (Fig.3.2 a) with an average plaque

diameter of 6 mm on all growth media except on nutrient agar (5 mm). There was no significant

difference in the average plaque diameter (p > 0.05) on the various growth media. The average

number of phage particles per plaque varied with different media. On nutrient agar, the average

number of phage particles per plaque was 3×1010

, whereas on Bismuth Sulphite and on Hektoen

Enteric agar, the average number of phage particles was 1.6×1010

per plaque. There was a

significant difference in the average value of phage particles per plaque (p ≤ 0.05) on different

growth media. In conclusion, φSPB multiplied differently in the host cells on different growth

media (Table 3.5).

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77

BVPaP-3 plaque was clear and circular in nature (Fig.3.2 b) with an average plaque

diameter of 2mm on MacConkey agar and Cetrimide agar media respectively, but was different

on nutrient agar medium (3 mm). There was no significant difference in the average plaque

diameter (p< 0.05) on all the growth media. The plaque was clear and circular (3.2 b).The

average number of phage particles per plaque varied with the medium. On nutrient agar

medium, the average number of phage particles per plaque was 5.6×107, whereas on

MacConkey agar and Cetrimide agar media, the average number of phage particles per plaque

was 5.4×107 and 5.2×10

7, respectively. There was no significant difference in the average

number of phage particles per plaque (p< 0.05) on the different growth media. Thus, from the

results, it can be concluded that BVPaP-3 multiplies at the same rate in the host cells on

different growth media. KPP plaque was also clear and but was surrounded by a turbid

hallo(Fig 3.2 c).The average plaque diameter of KPP was 5 mm and the average number of

phage particles per plaque was 5 × 105 pfu on nutrient agar as well as on MacConkeys agar

media. There was no significant difference in the average plaque diameter value (p≤0.05) as

well as average number of phage particles per plaque (p≤0.05) on nutrient agar as well as on

MacConkeys agar media.

(a) φSPB (b) BVPaP-3 (c) KPP

Plaque, Size-5 mm

Clear, Circular, indicating

lytic nature

Plaque, Size-2 mm

Clear circular , indicating

lytic nature

Plaque, Size-5 mm

Clear, circular surrounded

by a turbid hallo, indicating

lytic nature

Fig.3.2 Plaque characteristics of φSPB, BVPaP-3 and KPP on nutrient agar medium

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78

Table 3.5 Nature of Plaque of phage φSPB on different growth media

Growth

medium

Nature of Plaque

Plaque morphology Diameter

(mm )*

No. of phages/plaque

(pfu/plaque)**

NA Well defined, clear, round 5±0.20 3x1010

± 0.34

MA Well defined, clear, round 5±0.23 2x1010

± 0.55

SSA Well defined, clear circular

turbid halo

5±0.25 1.8x1010

± 0.22

BSA Well defined, clear, round 5±0.56 1.6x1010

± 0.10

HEA Well defined, clear, turbid

halo, round

5±0.30 1.6x1010

± 0.33

NA, nutrient agar; MA, MacConkeys agar: SSA, Salmonella-Shigella agar; BSA Bismuth Sulphite agar; HEA,

Hektoen Enteric agar. Each data point represents average ±standard deviation

b) Phage morphology

Transmission electron microscopy (TEM) showed that φSPB belonged to the C3

morphotype of the Podoviridae family. The phage had a rounded head of 162 × 58 nm and a

short tail 12 × 7 nm (Fig.3.3 a) with the cigar-shaped body.BVPaP-3 has a short non-contractile

tail with a hexagonal head of 58 to 59 nm in diameter and a tail size of about 10×8 nm,

belonging to the Podoviridae family, subfamily Autographivirinae and the genus T7-like

phages that includes phage gh-1. It has the same morphology as phage gh-1(Fig.3.3 b).

Transmission electron microscopy (TEM) revealed that KPP belongs to the Myoviridae family

and resembles Citrobacter FC3 phage. The phage has a head of 85.5 nm with the extended tail

of 95 × 17 nm and contracted sheath of 50×20 nm. The phage has no neck, base plate but the

tail fibers are folded along the tail (Fig.3.3 c). KPP is found to be a very stable phage.

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79

(a)φSPB (b)BVPaP-3 (c) KPP

Podoviridae family Podoviridae family Myoviridae family

Very long, cigar shaped

head with short tail

The hexagonal head with a

short tail

The hexagonal head with a

very long tail and contracted

sheath

Similar to the 7-11 phages

of the C3 morphotype

Similar to the T7- like

phage gh-1

Similar to the phages

belonging to the FC3 group

Fig.3.3 Transmission electron micrograph of a; φSPB. b; BVPaP-3, c; KPP phage X297, 000. The bar

indicates 100 nm.

c) Host range

The host range studies indicated that φSPB produced complete lysis (clear zones) on

Salmonella enterica Paratyphi B (ATCC8759), and lysogenic activity (turbid zones) on

Citrobacter koseri (MTCC1657) and Shigella sonnei (MTCC2957). The Phage did not lyse

Enterobacter aerogenes (MTCC 111), Escherichia.coli (MTCC1678), Klebsiella pneumoniae

(MTCC 39), Pseudomonas aeruginosa (MTCC 424), Proteus vulgaris (MTCC744), Salmonella

Paratyphi A (MTCC 735), Salmonella typhimurium (MTCC 98) and Vibrio cholerae (MTCC

3906). The cross infectivity of BVPaP-3 was evaluated to check its spectrum of activity against

other bacterial genera. BVPaP-3 could only lyse P. aeruginosa (MTCC424), but was unable to

lyse P. fluorescens (MTCC671) and strains of the other bacterial genera. KPP produced

complete lysis (clear zone) on E. coli (MTCC1678) and Klebsiella pneumoniae (MTCC 39) and

incomplete lysis (turbid zone) of Citrobacter koseri (MTCC 1657) and Shigella sonnei (MTCC

2957). The results are listed in Table 3.6.

a b c

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80

Table 3.6 Host range of phages

Host φSPB

activity

BVPaP-3

activity

KPP

activity

E.coli (MTCC1678) – – –

Proteus vulgaris (MTCC744) – – –

Pseudomonas aeruginosa (MTCC 424) – + –

Salmonella typhi (MTCC 733) – – –

Salmonella Paratyphi A (MTCC 735) – – –

Salmonella Paratyphi B (ATCC 8759) + – –

Klebsiella pneumoniae (MTCC 39) – – +

Salmonella Typhimurium (MTCC 98) – – –

Vibrio cholera (MTCC 906) – – –

Enterobacter aerogens (MTCC 111) – – –

Citrobacter koseri * (MTCC1657) Turbid zone – Turbid zone

Shigella sonnei * (MTCC2957) Turbid zone – Turbid zone

+ Complete lysis; - No lysis zone; * Lysogenic activity

d) Phage growth kinetics

Adsorption studies were performed to identify the adsorption rate constant of phage φSPB

on Salmonella enterica serovar Paratyphi B, phage BVPaP-3 on Pseudomonas aeruginosa and

KPP on Klebsiella pneumoniae. The affinity of the phage for its host was calculated as

described by Krueger (1931). Ecliped period may be determined in conjunction with the latent

period, since chloroform treatment allow us to to delay plating.Phage φSPB adsorbed to its host

rapidly. The number of free phages was 48 % after 6 min and declined to 48 % after 10 min.

The adsorption rate constant of φSPB was 4.7×10-10

phage particles/cell/ml/min (Fig.3.4 a).

Latent eclipse, and rise period and the burst size were determined from the change in the

numbers of free and total phages. Eclipse and latent periods were 10 and 15 min, respectively.

The burst size of the phage was 100 pfu per infected cell after 25 min at 37 °C (Fig.3.5). The

BVPaP-3 phage adsorbed to its host rapidly. The number of free phages was below 20 % in the

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81

first 7 min and after a period of 10 min, the number of free phages was below 1. The adsorption

rate constant of Pseudomonas phage (BVPaP-3) was found to be 1.19×10-9

phages/cell/ml/min

(Fig.3.4b). The phage growth parameters, latent period, eclipse period, rise period and burst

size, were determined from the dynamical change of the number of free and total phages. The

eclipse and latent periods of BVPaP-3 were 20 and 15 min, respectively. The burst size of the

phage was 44 pfu per infected cell after 35 min at 37 °C (Fig.3.6). KPP phage adsorbed to its

host rapidly. The number of free phages was 50 % in the first 6 min. and declined to 9 % after

10 min. The affinity of KPP for Klebsiella pneumoniae i.e. the adsorption rate constant was 4.7

× 10-10

phage particles/cell/min (Fig.3.4 c) Latent, eclipse, rise period and the burst size were

determined from the change in the number of free and total phages. Eclipse and latent periods

were 20 and 15 min. respectively. The burst size was 120 pfu per infected cell after 45 min at

37˚C (Fig.3.7).

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82

Fig.3.4 Adsorption rate kinetics of (a); φSPB to Salmonella Paratyphi B cells at MOI 0.035, (b); BVPaP-3 to

Pseudomonas aeruginosa at MOI 0.07 and (c); KPP to Klebsiella pneumoniae at MOI 0.14. Data are

averages of three determinations ± SD.

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83

Fig.3.5 One-step growth curve of φSPB on Salmonella enterica serovar Paratyphi B at 37˚C in phage

broth. log10 (pfu) per infected cell in chloroform treated (-■-) and untreated (-▲-) cells. Each data point

represents averages of three determinants ± SD.

Fig.3.6 One-step growth curve of BVPaP-3 on Pseudomonas aeruginosa at 37˚C in phage broth. log10 (pfu)

per infected cell in chloroform treated (-■-) and untreated (-▲-) cells. Each data point represents averages

of three determinants ± SD.

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84

Fig.3.7 One-step growth curve of KPP on Klebsiella pneumoniae at 37˚C in phage broth. log10 (pfu) per

infected cell in chloroform treated (-■-) and untreated (-▲-) cells. Each data point represents averages of

three determinants ± SD.

e) Stability of φSPB, BVPaP-3 and KPP phages at different pH and temperatures

The phage φSPB was stable within wide pH range viz., 4 to 12 and 100 % stable at pH

7.However, the stability was below 90 % in the acidic and alkaline pH range and it was zero

beyond pH 9 (Fig. 3.8 a).The stability of the phage φ SPB was 100 % at 4 °C and decreased

gradually with increasing temperature as shown in Fig. (3.8 b). BVPaP-3 was stable within pH

range 6-10 (90 % survivors), but inactivated to 50% at pH 11. At pH 7, the stability of the

phage was 100 %. Phage stability at pH 12 was zero (Fig. 3.9 a). BVPaP-3 was quite stable

within the temperature range 4 to 30 °C, with 100 % stability. The stability was 90 % and 20 %

at 40 °C and 50 °C, respectively (Fig. 3.9 b). KPP was stable within wide pH range, 5 to 10.The

stability was 100% at pH 7 to 9.However, the stability decreased to 90 % at pH 6 and 9

(Fig.3.10 c). The stability of KPP was 100 % within temperature range 4 to 30˚C. Stability was

decreased beyond 30˚C (Fig.3.10c).

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85

Fig.3.8 Stability of φSPB at varying pH (a) and temperature (b)

(a); % stability: (N/N0) x100, where N is the number of viable phages after 1 h of incubation, N0 is the

initial number of phages. % stability with reference pfu/ml at 7 pH. (b); % stability: (N/N0) x100, where N is

the number of viable phages after 1 h of incubation, N0 is the initial number of phages; % stability with

reference pfu/ml at 4˚C. Data are averages of three determinations± SD.

Fig.3.9 Stability of BVPaP-3 at varying pH (a) and temperature (b)

(a); % stability: (N/N0) x100, where N is the number of viable phages after 1 h of incubation, N0 is the initial

number of phages. % stability with reference pfu/ml at 7 pH. (b); % stability: (N/N0) x100, where N is the

number of viable phages after 1 h of incubation, N0 is the initial number of phages; % stability with

reference pfu/ml at 4˚C. Data are averages of three determinations± SD.

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86

Fig.3.10 Stability of KPP at varying pH (a) and temperature (b)

(a); % stability: (N/N0) x100, where N is the number of viable phages after 1 h of incubation, N0 is the

iinitial number of phages. % stability with reference pfu/ml at 7 pH. (b); % stability: (N/N0) x100, where N

is the number of viable phages after 1 h of incubation, N0 is the initial number of phages; % stability with

reference pfu/ml at 4˚C. Data are averages of three determinations± SD.

f) Protein profile of φSPB, BVPaP-3 and KPP

Protein profile of φSPB indicated at least 10 bands that could be clearly distinguished in

the SDS-PAGE gel. The prevailing protein had a size of 28.9 kDa and was tentatively identified

with the major capsid protein (Fig.3.11a). Protein profile of BVPaP-3 on SDS-PAGE indicated

the occurrence of eleven proteins that may comprise various proteins of the phage like the

capsid (head), tail, tail fibres and tail pins, among others (Fig. 3.11b).In case of KPP, a protein

band with molecular size 33 kDa was found to be the most prominent protein. The SDS-PAGE

revealed at least 9 bands in the gradient gel. The prevailing protein had size of 90 kDa

(Fig.3.11c).

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87

Fig.3.11 Protein profile of phages (a); φ SPB, (b); BVPaP-3 and (c); KPP

Brand- BioEra/Cat. No. Cust/BE/O61) Lane 1: High molecular weight protein marker (range 3 -205 kDa

Marker Lane (KDa): 200- Myosin; 150 -Alcohol Dehydrogenase; 116 β-galactosidase; 97-hosphorylase-B;

66- Albumin, Bovine Serum; 55- Glutamic Dehydrase; 45- Ovalbumin, Chicken Egg; 36–Glyceraldehyde 3-

phosphate dehydrogenase; 29 - Carbonic Anhydrase; 14- α-lactalbumin; 8.8- Prokineticin-2.Lane 2: Protein

profile of the respective phage

g) Restriction digestion pattern of DNA

When the genomic DNA of φSPB was digested with EcoRI and HindIII, four and ten

bands, respectively, were visible and indicated an appreciative genome size of 59 kb (Fig.3.12

a). The size of the genomic DNA of BVPaP-3 was 41.13 Kb when compared to the standard

molecular weight marker. When the genomic DNA was digested with EcoRI and Hind III, four

and ten bands were visible, respectively, indicating the occurrence of these restriction sites on

the phage genomic DNA (Fig.3.12 b). When the genomic DNA of KPP was digested with

EcoRI and HindIII, nine and one band respectively were visible and indicated an approximate

Chapter 3

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88

size of 56.8 Kb. Restriction digestion pattern also revealed only one band after digestion with

HindIII indicating no restriction site for HindIII on KPP genome (Fig.3.12 c).

Fig. 3.12 Restriction digestion pattern of phage DNA, (a); φ SPB, (b); BVPaP-3 and (c); KPP

Lane M: Broad range DNA marker designed by BioEra Life Sciences Pvt. Ltd., Pune, India (300-62,500 bp

where molecular weight of 62500 bp was achieved with TP114 plasmid and molecular weight of 48502 bp

was achieved with Lambda DNA and other molecular weights were part of BioEra's High Range DNA

Marker),

Lane 1: Restriction digestion pattern of φSPB DNA with EcoRI, Lane 2: Restriction digestion pattern of

φSPB DNA with HindIII.

3.5 Identification of phage isolates

From this detailed study, the phage isolates viz., φSPB, BVPaP-3 and KPP were

identified as a member of C3 morphotype of 7-11 group belonging to the Podoviridae family

Chapter 3

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89

(φSPB), a T7-like lytic phage that belongs to Podoviridae family (BVPaP-3) and FC3-9 phage

of Citrobacter intermedius C3 (KPP) respectively.

3.6 Discussion

Characterization of bacteriophages is a mandatory step in order to explore them in various

fields (Ackermann, 2009). In this study, bacteriophages specific against the antimicrobial

resistant Salmonella enterica serovar Paratyphi B, Pseudomonas aeruginosa and Klebsiella

pneumoniae are characterized in detail in order to use them as „biological disinfectant.‟ It has

been observed that, amongst the phages studied so far, 96 % phages are tailed phages and

remaining 3.7 % are polyhedral, filamentous and pleomorphic (Ackermann, 2007). Here also,

phages under study are tailed phages. Phage specific for Salmonella enterica serovar Paratyphi

B (φSPB) belongs to Podoviridae family of tailed phages that are characterized by short, non-

contractile tails. This phage has a rounded-shaped elongated body and is a member of C3

morphotype of 7-11 phage group. C3 morphotype phages are characterized by long heads and

are very rare members of the family of the Podoviridae family and constitute only 0.5 % of the

over 5,500 phages that have been examined (Kropinski et al, 2011). There are scanty reports

about 7-11 phages in the literature. (Ackermann et al,1974; Moazamie et al, 1976). This is the

first report of 7-11-like phages from India. The rounded - shaped head structure reflects

deviation in size from that of the already reported 7-11 phages viz., Esc-7-11 and phiEco32

(Kropinski et al, 2011). This observation is contradictory to the previous observations in the

literature. Apart from S.enterica serovar Paratyphi B, Shigella and Citrobacter spp. were also

lysed by φSPB. This is not surprising because these two pathogens belong to

Enterobacteriaceae family and phages consider enteric bacteria as a single host “genus‟‟ (Bielke

et al, 2007). This indicates that φSPB, has a narrow host range as that of coliphage phiEco32

(Savalia et al, 2008). The genome size of φSPB is around 59 kb which differs from that of other

members of the 7-11 group, that display sizes of 90 and 77 kb for phages 7-11 and phieEco32,

respectively (Kropinski et al, 2011, Savalia et al, 2008). Restriction digestion of the DNA

yielded four and five bands which add up to the total genome size of around 59 kb. This is

probably much too low and demands confirmation by sequencing. Proteomic studies revealed

Chapter 3

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90

the presence of 10 different bands of proteins. These proteins may be the structural components

of the capsid (head), tail, and tail fibers (Savalia et al, 2008). A protein band with a molecular

mass of 28.9 kDa was the most prominent protein that can be compared with a protein of 26

kDa of Phage phiEco32 has which is known to function as a RNAP (RNA polymerase) sigma

factor (Savalia et al, 2008). Further studies of genes and related proteins are required for the

identification of these proteins. Bacteriophage activity is generally evident when the

environmental and nutritional conditions favor the growth of host bacteria. φSPB was stable

and survived at pH values between 4 and 9. These results are consistent with the previous

observations (Ackermann and DuBow, 1987; Jamalludeen et al, 2007) that most phages are

able to survive easily over the range of pH 5 and 9. Phage φSPB is very stable over a wide

temperature range, i.e., from 4 °C to 40

°C, since bacteriophages can enter the lytic cycle once

their habitats are warmed (Greer and Dilts, 1990). Experimental evidence indicates survival of

Salmonella bacteriophages on chicken skin for 48 h at 4 °C and survival on cheeses at 14

°C for

several days (Schellekens et al, 2007). Previous publications have described the inactivation of

experimental Salmonella on food with the lytic phages that were belonging to Podoviridae;

Siphoviridae and Myoviridae families (Leverentz et al, 2001; Goode et al, 2003; McLaughlin et

al, 2008). φSPB phage has the potential to infect its host at a wide temperature range.

Therefore, φSPB should be explored in the food industry as a preservative to control

Salmonella enterica serovar Paratyphi B infections. φSPB may also be used as a biological

disinfectant in water systems including ornamental aquariums, that are an emerging source for

multiple drug resistant Salmonella enterica serovar Paratyphi B strains (Levings et al, 2006).

Lytic as well as lysogenic phages of P. aeruginosa are present in the environment such as

sewage, soil and water (Duran et al, 2002). Therefore, in the present study, the lytic phage

specific to P. aeruginosa was isolated from river water. BVPaP-3 was selected for further

characterization because it was found to be highly potent against a wide range of P. aeruginosa

clinical strains (Ahiwale et al, 2011). BVPaP-3 did not lyse other bacterial strains in the cross

infectivity studies, indicating that it is highly specific for P. aeruginosa. These results are

contrary to those in the literature (Bielke et al, 2007). BVPaP-3 phage belongs to the

Podoviridae family and is a relative of the Pseudomonas phage gh-1. It is isolated from river

Chapter 3

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91

water using P. aeruginosa as the host. However, gh-1 phage was originally isolated from

sewage using P. putida as the host (Lee and Boize, 1966), suggesting that gh-1 phages are able

to infect different species of Pseudomonas but BVPaP-3 phage is specific to P. aeruginosa

only. A literature survey indicated that previously isolated gh-1 phage is known to infect P.

putida and P. fluorescens under psychrotrophic conditions (Lee and Boize, 1966; Sillankorva,

2011), but infection of P. aeruginosa with BVPaP-3 was found to be optimum at 37°C.The

adsorption rate of BVPaP-3 was 1.19×10-9

phages/cell/ml/min, suggesting that BVPaP-3 has a

strong affinity for the host cell receptors with a very high amount of tail fibre proteins involved

in the recognition and binding to the specific cellular receptors. As reported earlier, the tail fibre

proteins are involved in the recognition and binding to the host surface (Schall, 2001). These

observations are contradictory to those of previous reports on other T7 phages, with the

adsorption rate varying between 4.5×10-10

and 8.9 ×10-10

phages/cell/ml/min (Sillankorva,

2008). BVPaP-3 was stable and survived at pH values between 6 and 9. These results are

consistent with the previous observations made by Ackermann and Du Bow (1987) and

Jamalluddin et al. (2007). At physiological conditions, BVPaP-3 has the potential to infect its

host within a wide temperature range, with 100% infectivity at 37°C. Therefore, BVPaP-3 has a

potential to be used as a biological disinfectant to control P. aeruginosa in the environment.

The protein profile indicated the presence of eleven major proteins that is close to the number

of proteins reported for the phage gh-1 (Kovalyova and Kropinski, 2003). This report described

the occurrence of proteins of similar molecular mass (63, 56 and 41 kDa), which form the

structural proteins in gh-1 phages. There are similar reports on the predictive size of various

proteins of T7 and T7-like phages (Lavigne, 2003). Mass spectroscopic studies are required to

further confirm the molecular mass of proteins of BVPaP-3. The genome size of BVPaP-3 is

41.3kb, which reflects variations in the results when compared with the P. putida gh-1 lytic

phage genome of 37.36 kb (Kovalyova and Kropinski, 2003). BVPaP-3 is found to be very

close to the T7 phage φ IBBPF7A, which has a genome size of 42 kb (Sillankorva, 2008), φ

KMV (42.5kb), a P. aeruginosa phage, and Escherichia coli T7-like phage, which has a 39.9 kb

genome. This is well within the range of genome size of other T7-like phages; the smallest T7-

like phage genome sequenced so far is that of E. coli phage T3, with a genome size of 38.2 kb,

while the largest is that of the T7-like lytic phage of Vibrio parahaemolyticus, which has a size

Chapter 3

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92

of 45.8 kb (Kwan et al, 2006; Lavigne, 2003). Genome sequencing of BVPaP-3 would help to

reveal how close it is related to the gh-1 phage of P. putida and the φ IBBPF7A phage.

Bacteriophages specific for Klebsiella pneumoniae are widely spread in the nature and can be

isolated from the fresh water environments. Plaque morphological features of KPP phage

revealed that the plaque has a diameter of 5 mm with small clear center surrounded by hazy

ring (halo). Such morphological feature of plaque is observed in case of FC3 phage group. The

plaque features of KPP are found to be similar to FC3-9 phage of Citrobacter intermedius C3

(Regue et al, 1986).The presence of the halo might suggest the production of the soluble phage

enzymes, for e.g. polysaccharide depolymerases, as suggested by Huges et al (1998). It has also

been reported that FC3-9 phage show capsular depolymerase activity (Regue et al,

1986).Electron microscopic studies revealed that KPP has an icosahedral head of about 85.5 nm

in diameter and contractile tail of about 95 nm long and belongs to the Myoviridae family.

Morphologically, KPP resembles FC3-9 phage of Citrobacter intermedius that has the capsid of

about 80 nm and tail about 110 nm long (Regue et al, 1986).FC3 phages are reported in

Klebsiella pneumoniae and Citrobacter spp. (Camprubi et al,1991; Regue et al,

1981).Bacteriophage FC3-9 is one of the several FC3 group phages of Klebsiella pneumoniae

C3 that is reported .Mutants resistant to this bacteriophages are also isolated and found to be

devoid of lipopolysaccharides O antigens (Benedi et al, 1989). FC3 phage members require

capsular receptor (lipopolysaccharides) for their binding (Tomas and Jofre, 1885).Therefore, it

can be concluded that the host (Klebsiella pneumoniae) isolated in this study is a wild type

strain. Apart from Klebsiella pneumoniae (MTCC 39), KPP lysed E. coli (MTCC 1678),

Shigella (MTCC 2957) and Citrobacter (MTCC 1657) This is not surprising because these

pathogens belong to Enterobacteriaceae family and phages infecting Enterobacteriaceae

consider enteric bacteria as a single host “genus‟‟ (Bielke et al, 2007; Hernandez and Alberti,

1995).Growth properties of KPP revealed that the latent period for FC3-9 phage is 15 min and

the burst size is 120 pfu/infected cell. These results are found to be contradictory to the

previous results of FC3 phages where the range of latent period for FC3-1 to FC3-9 was 30 -50

min. and the burst size was similar to FC3-4 phage (Regue et al, 1981).Storage stability is

important parameter in order to apply phages as biocontrol agent in the environment. KPP is

found to be stable over a wide range of pH (5 to 9) and temperature (4 to 50 C˚) indicating their

wide applications to control K. pneumoniae infections. The proteome analysis revealed total 9

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bands on gel, with three prominent protein bands viz., 143, 90.8 and 60 kDa and four less

prominent bands viz., 42, 32, 18 kDa were detected. Similar proteins were also detected (16, 30,

33, 90 kDa) in FC3-11phage of K. pneumoniae (Hernandez and Alberti, 1995). Similar proteins

(94, 62, 42, 30, 20 kDa) were also found in the φKMV-like virus particle of K. pneumoniae that

belongs to the Podoviridae family (Kawa et al, 2011).This indicates that these proteins may be

the common structural proteins in these phages. The restriction digestion pattern indicated that

KPP DNA is resistant to the digestion by EcoRI and sensitive to HindIII restriction enzyme.

The genome size of KPP is 57 kb that is found to be close to the FC3-11 phage of K.

pneumoniae genome size 48-50 kb (Hernandez and Alberti, 1995). This newly isolated K.

pneumoniae phage (KPP) shows properties similar to the FC3-9, FC3-11, FC3-4 phages that

belong to the Myoviridae family and to the φKMV-like virus particle of Podoviridae family

indicating the genetic evolution.

3.7 Conclusions

This newly isolated phage of Salmonella enterica serovar Paratyphi B (φSPB) has many

unique features such as a short generation time and a high stability over a wide range of pH and

temperature, making it a promising biocontrol agent for drug resistant strains of Salmonella

enterica serovar Paratyphi B. This is the first report on the isolation and characterization of rare

phage of the C3 morphotype from India with a therapeutic potential.

This is perhaps the first report of the isolation of the phage gh-1 from India using P.

aeruginosa as a host and has many features similar to that of the phage gh-1 of P. putida and P.

fluorescens. This newly isolated T7-like phage gh-1 has many features such as a very fast

adsorption ability and short replication time that makes the phage a promising agent for

sanitation and therapeutic applications.

Phage (KPP), a member of FC3 phage group can able to infect few genera of the

Enterobacteriaceae family and therefore can be used as a biocontrol agent against those

pathogens.

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