liquid based formulations of bacteriophages for the...

Indian Journal of Experimental Biology

Vol. 51, November 2013, pp. 1038-1045

Liquid based formulations of bacteriophages for the management of waterborne

bacterial pathogens in water microcosms

Sangeeta Ahiwale1, Sujata Tagunde

2, Sushama Khopkar

3, Mrudula Karni

3, Milind Gajbhiye

2 & Balasaheb Kapadnis

2 *

1Department of Environmental Sciences, University of Pune, Pune 411 007, India. 2Department of Microbiology, University of Pune, Pune 411 007, India

3Department of Microbiology, M. P. College, Pimpri, Pune 400 017

Received 9 December 2012; revised 2 March 2013

Water resources are contaminated by life-threatening multidrug resistant pathogenic bacteria. Unfortunately, these

pathogenic bacteria do not respond to the traditional water purification methods. Therefore, there is a need of

environmentally friendly strategies to overcome the problems associated with the antimicrobial resistant bacterial pathogens.

In the present study, highly potent lytic phages against multidrug-resistant Salmonella enterica serovar Paratyphi B,

Pseudomonas aeruginosa and Klebsiella pneumoniae were isolated from the Pavana river water. They belonged to the

Podoviridae and Siphoviridae families. These phages were purified and enriched in the laboratory. Monovalent formulations

of φSPB, BVPaP-3 and KPP phages were prepared in three different liquids viz., phage broth, saline and distilled water. The

phages were stable for almost 8-10 months in the phage broth at 4 °C. The stability of the phages in saline and distilled

water was 5-6 months at 4 °C. All of the phages were stable only for 4-6 months in the phage broth at 30 °C. The

monovalent phage formulation of φSPB was applied at MOI < 1, as disinfectant against an exponential and stationary phase

cells of Salmonella enterica serovar Paratyphi B in various water microcosms. The results indicated that there was almost 80

% reduction in the log phase cells of Salmonella serovar Paratyphi B in 24 h. In stationary phase cells, the reduction was

comparatively less within same period. At the same time, there was concomitant increase in the phage population by 80% in

all the microcosms indicating that φSPB phage is highly potent in killing pathogen in water. Results strongly support that

the formulation of φSPB in the phage broth in monovalent form could be used as an effective biological disinfectant for

preventing transmission of waterborne bacterial pathogens, including antimicrobial resistant ones.

Keywords: Bacteriophages, Drug resistant bacterial pathogens, Monovalent liquid formulations, 7-11 phages, T7-like

Waterborne bacterial diseases and sanitation related

infections are the major contributors to diseases and

mortality in human beings1,2

. Distribution of many

waterborne pathogens varies substantially from one

country to another3. Multidrug resistant P. aeruginosa,

Kl. pneumoniae and Salmonella group of organisms are

emerging worldwide. Salmonella group of organisms

are responsible for about 5.5 million deaths each year

due to the development of resistance to ampicillin,

chloramphenicol, streptomycin, spectinomycin,

sulphonamides and tetracycline4-6

. Besides, chlorine

resistant bacterial strains are also emerging worldwide7-9

.

Bacteriophages have attracted an interest as natural

antimicrobial agents to fight against bacterial diseases10

because of the emergence of antibiotic resistant

bacteria. Therefore, to overcome the problems

associated with such antimicrobial resistant bacterial

strains, bacteriophages could be an alternative weapon.

Bacteriophages are extensively studied with respect to

their applications in various fields. For the successful

application of phages as a biocontrol agent, phages are

needed to be isolated from the natural environment,

enriched and produced in sufficient number in the

laboratory for their application. Therefore, the main

objective of the present work is to isolate potent lytic

bacteriophages against S. enterica serovar Paratyphi B,

P. aeruginosa and Kl. pneumoniae, from the natural

environment, their laboratory scale production, and to

develop formulations in various liquids. Powder

formulations in skimmed milk, casein, pregelatinized

corn flour have already been reported earlier to

increase the longevity of phages11

.

The most important task after isolating phages is to

maintain their stability during storage. Therefore,

there is need of carriers that will sustain structural

integrity of phages required for the infection. The

high titers of phages can be prepared in the laboratory

______________

*Correspondent author

Telephone: +91-20-25690643

Fax: +91-20-25690087

E-mail: [email protected]; [email protected]

AHIWALE et al.: BACTERIOPHAGES & MANAGEMENT OF BACTERIAL PATHOGENS

1039

using liquids or powders as carriers. Phage survival

(stability and infectivity) is an important criterion in

order to use them as biocontrol agents. To date, little

work has been focused on their formulation with

respect to the controlled release and passive targeting.

Phages are stabilized in a suspension form, viz., SM

buffer, sugar solutions, salt solutions, protein

solutions etc. Various powder formulations of

bacteriophages were prepared to increase their shelf-

life. There are reports regarding the encapsulation of

bacteriophages in the biodegradable polyester

microcapsules12

. Phages were also stabilized in

freeze-dried cakes of polyethylene glycol plus

sucrose13

. Various methods have also been employed

for storing phages that included freezing at low

temperatures and lyophilization in the liquid media.

All these methods have shown varying degree of

success at maintaining the high titer of active phages.

Bacteriophage formulations were tested for the

stability by subjecting them to various temperatures.

Jepson and March18

disclosed that a liquid suspension

of phages (either in SM buffer or 1: 200 dilution of

SM buffer in the water) was stable for 6 months at

4 °C and at -70 °C. Warren et al14

reported a

significant decrease in the titer and viability of phages

in suspension (without the stabilizers) at 4 °C while

storage at -20 and 20 °C resulted in the greater

survival of phages. In the present study, an attempt

has been made to prepare low-cost and stabilized

liquid formulations in saline, distilled water and

phage broth of phages specific for Salmonella

Paratyphi B, Kl. pneumoniae and P. aeruginosa,

isolated from the Pavana river, Pimpri, India.

Materials and Methods

The Pavana river water was collected in 250 mL

sterile screw capped bottle. The phages and the

pathogens were isolated from the water samples using

standard microbiological methods.

Isolation of pathogensWater sample was serially

diluted and an aliquot (0.1 mL) of specific dilution

was plated on media, viz., nutrient agar, Mac

Conkey’s agar, Salmonella-Shigella agar, Hektoen

Enteric agar to isolate Salmonella Paratyphi B. The

Pseudomonas aeruginosa and Klebsiella pneumoniae

were isolated from the hospital environment viz.,

toilets, table tops, floor etc. using a sterile cotton

swab, on sterile nutrient agar, Cetrimide agar, Mac

Conkey’s agar and Eosine Methylene blue agar

media. Plates were incubated at 37 °C for 24 h. The

suspected colonies on the plates were identified

according to the identification scheme recommended

in Bergey’s Manual of Determinative Bacteriology15

.

The suspected colonies on nutrient agar, Mac

Conkey’s agar, Salmonella-Shigella agar, Hektoen

Enteric agar, Cetrimide agar and on Eosin Methylene

blue agar were presumptive forms, identified using

Gram stain and motility test. The bacterial isolates

were confirmed using a set of biochemical tests that

included IMViC (Indole, Methyl red, Voges

Proskauer and Citrate), triple sugar iron agar (TSI),

sugars (glucose, xylose, mannitol), Gelatinase and

Oxidase tests. Antibiotic resistance pattern was

studied by Kirby-Bauer disc diffusion technique.

Phage isolationWater sample from the Pavana

river (100 mL) was treated with 2 mL chloroform

(1:50), shaked vigorously and then kept in the

refrigerator for 12-18 h. The aqueous layer was

filtered through membrane filter of pore size 0.2 µm

(Machery-Nagel, Dueren, Germany). The filtrate was

used as a source of phages (lysate). Phages from the

filtrate were isolated by Double Agar Layer plaque

assay technique16

.

Plaque morphologyThe mid-log phase culture

(1 mL) of the respective bacterial pathogen and an

aliquot of (1 mL) filtrate (source of phages) were

mixed and 0.1 mL of this mixture was spread onto the

surface of the nutrient agar medium. The plates were

incubated at 37 °C and observed at an interval of 3 h

until the development of plaques.

Lysate preparation and its titer

determinationLysate was prepared by transferring a

single plaque of the respective phage in a flask

containing 500 mL phage broth with the mid-log phase

culture of respective host viz., Salmonella serovar

Paratyphi B, P. aeruginosa and Kl. pneumoniae. After

incubation, the content of the flask was treated with

chloroform (1:50) with vigorous shaking. Then the

supernatant was centrifuged (10,000 rpm, 20 min,

4 °C) and the supernatant was passed through the

Millipore membrane filter of pore size 0.20 µm

(diameter 47 mm). The phage titer of the filtrate was

determined by double agar layer plaque assay

technique. Hereafter the phages of Salmonella serovar

Paratyphi B, P. aeruginosa and Kl. pneumoniae are

designated as φSPB, BVPaP-3 and KPP respectively.

Phage morphologyPhage particles were

sedimented using a Beckman J2-21 centrifuge

(Beckman Instruments, Inc., Palo Alto, USA) at

25,000 rpm for 60 min. Phages were washed with 0.1 M

INDIAN J EXP BIOL, NOVEMBER 2013

1040

ammonium acetate buffer (pH 7.0) deposited on

carbon-coated Formvar films, stained with 2%

phosphotungstate and examined under a Philips EM

300 electron microscope (TEM) (H-W Ackermann,

Laval University, Quebec, Canada) at magnification

297,000 times.

Liquid formulation of bacteriophagesThe lysate

(phage broth) obtained was subjected to plaque assay

technique onto 60 nutrient agar plates. Plates were

incubated till the confluent lysis (the term confluent

lysis refers to agar plates where borders of the plaques

in it are touching each other) was obtained. Out of the

60 nutrient agar plates, a set of 20 plates was used per

suspending medium. The respective set of assay

plates was separately flooded with the suspending

media viz., sterile saline, sterile distilled water and

sterile phage broth. All the plates were kept for 12-16 h

in the refrigerator (4 -6 °C). With the help of a sterile

glass spreader, soft agar layer was scraped and

dispensed into three respective sterile bumper tubes.

The content of each tube was treated with chloroform

(1:50) and kept in the refrigerator for 12-16 h., The

supernatant was centrifuged (10000 rpm, 20 min.,

4 °C) and filtered through the membrane filter (0.20 µm).

The lysate of each suspending medium was stored in

the refrigerator until use.

Survival of phagesThe phage titer in each

formulation was determined by the plaque assay

technique. Each formulation (saline, distilled water

and phage broth based) was equally dispensed into

two sterile screw capped bottles and stored at 4 and

30 °C. The density of phages (residual phage titer) in

each formulation was monitored for one year, at an

interval of one month. The observations were

recorded, analyzed and presented graphically.

Water microcosm studiesThe microcosms were

prepared using water from swimming pool, river, bore

well and aquaculture. Each type of water (90 mL) was

autoclaved separately in screw capped containers

(500 mL) at 15 lb pressure (121 °C) for 30 min in

triplicate. In all, 16 water microcosms were prepared.

The water microcosms were spiked with the saline

suspension of the mid-log (5 h old, 1 × 106

colony

forming units mL-1

) and stationary phase

(24 h old, 1 × 106

colony forming units mL-1

) cells of

S. enterica serovar Paratyphi B separately. The

monovalent phage broth preparation of S. enterica

serovar Paratyphi B phage (φ SPB, 1 × 102

plaqu

forming units mL-1

) was applied (MOI < 1) to each of

the microcosm except the host control flask

(respective water sample + host bacterium). The

microcosm flasks were incubated at 35 ± 2 °C for 24

h under shaking conditions (60 rpm)., The aliquots

(0.1 mL) were withdrawn from each microcosm and

serially diluted in sterile saline and then spread on

sterile MacConkey agar medium in triplicates. After

incubation at 37 °C for 24 h the number of colonies

were recorded. The plaque forming units were also

determined in each aliquot by Double Agar Layer

(DAL) plaque assay technique. Percentage decrease

or increase respectively in the colony forming units

and plaque forming units in each aliquot was

determined.

Statistical analysisThe analysis of data for

significant difference was done by Post Hoc Test

(Tukey’s HSD procedure) using SPSS 18. A 95%

confidence level was used for the analysis so that P ≤

0.05 were considered to be statistically significant.

Values given are mean±SD from 3 replicates each.

Results

Pathogen identification and their antibiotic

resistance patternThe pathogens were identified

based on cultural, morphological and biochemical

characteristics (Table 1). The antibiotic resistance

pattern of the isolates (Salmonella serovar Paratyphi

B, Pseudomonas aeruginosa and Klebsiella

pneumoniae) were determined and found to be

resistant to almost all antibiotics tested (Table 2).

Table 1—Biochemical characteristics of bacterial pathogens

Test Klebsiella

pneumoniae

Pseudomonas

aeruginosa

S. enterica serovar

Paratyphi B

Oxidase - + -

Urease + - -

TSI Acid butt

Acid slant with gas

- Acid butt

Alkaline slant with

excess H2S

MR + - +

VP - - -

Citrate + + +

Indole - - -

Nitrate + (48 h ) + +

Gelatin - + -

Glucose A+G + A+G

Xylose A+G - A+G

Mannitol A+G - A+G

Lactose A+G - -

Sucrose A+G - -

Maltose A+G - -

+: positive, - : negative, A+G: acid and gas


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Plaque morphologyThe average plaque diameter

(mm) of Salmonella Paratyphi B (φSPB); Pseudomonas

aeruginosa (BVPaP-3) and Klebsiella pneumoniae (KPP)

phages were 6, 2 and 5 mm respectively (Fig. 1 a-c).

Plaques of φSPB and BVPaP-3 were clear and circular.

Plaques of KPP were circular with small clear center

surrounded by hazy ring (halo). Such morphological

feature of plaque is observed in case of FC3 phage group17

.

Phage morphologyTransmission electron

microscopic studies revealed that φSPB and BVPaP-3

phages belonged to the Podoviridae family. The phage

φSPB was morphologically similar to the 7-11 phages of

C3 morphotype and BVPaP-3 is T7-like lytic phage

(Fig. 2 a and b). KPP phage belonged to the Siphoviridae

family and is a member of FC-3 group. (Fig. 2 c). All the

above mentioned phages are tailed phages.

Table 2—Antibiotic (µg) resistance pattern of bacterial isolates

Host bacterium CXM

(30)

OF

(5)

TET

(30)

Gm

(10)

AOCL

(30)

CTZ

(25)

DOH

(30)

CRO

(30 )

Cm

(30)

Ap

(10)

S. enterica serovar

Paratyphi B

I R R R I S I R R S

P. aeruginosa I R R R R I I S R R

K. pneumoniae S S R I R I I S R R

(Results- as per CLSI guidelines) CXM: Cefuroxime, OF: Ofloxacin, TET: Tetracyclin, Gm: Gentamycin, AOCL:Amoxyclave, CTZ:

trimoxazole, DOH: Doxycycline-HCl, CRO: Ceftrioxone, Cm: Chloramphenicol, Ap: Ampicillin,

Fig. 1—Plaque morphology of phage φSPB (a), phage BVPaP-3 (b) and phage KPP (c)

Fig. 2—Electron micrograph of phage φSPB (a), phage BVPaP-3 (b), and KPP (c) (Bar = 100 nm)


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Survival of φSPB Phage at 4 and 30 °CPhage

broth formulation of φSPB was stable for almost

seven months at 4 °C; i.e. the active residual phage

titer in the lysate was 100 % in comparison with the

initial phage titer which decreased gradually upto

60% at the end of 12 months. Distilled water phage

formulations were stable upto 4 months and with

further incubation the titer decreased upto 50% at the

end of 9 months, and finally reduced to zero in the

next 3 months. Saline suspension of φSPB was stable

upto first 4 months with 100% survival, and then

there was gradual decrease in the active phage titer.

Salmonella Paratyphi B phages suspended in phage

broth, saline and distilled water stored at 30 °C were

stable for 4 months, and 3 months respectively as

compared to suspensions stored at 4 °C.

Survival of BVPaP-3 phage at 4 and 30 °CThe

phages of Pseudomonas aeruginosa (BVPaP-3)

suspended in phage broth at 4 °C were almost 100%

stable upto 7 months and then reduced to almost 70%

at the end of eleventh month. Saline suspension of

BVPaP-3 was stable for the first 5 months with almost

100% stability. Distilled water suspension was stable

upto first 6 months. BVPaP-3 suspended in saline,

distilled water and in phage broth, stored at 30 °C was

less stable as compared to the same suspensions

stored at 4 °C. Phage broth suspension was showing

good titer for 2 months, the titer was reduced by 40%

in the fifth month. Distilled water suspension was

stable for 4 months and the saline suspension was

stable for 2 months only.

Survival of KPP phage at 4 and 30 °CKlebsiella

pneumoniae phages (KPP) suspended in saline,

distilled water and in phage broth stored at 4 °C was

stable with 100% phage titer at the end of seventh

month and then the titer was reduced to almost 80% at

the end of twelth month. Saline suspension of phages

was also quite stable with almost 80% survival of

phages till the end of ninth month and then the titer

was reduced gradually. The distilled water suspension

was stable in the first 3 months, with 100% survival

of active phages and then was reduced to 50 % at the

end of seventh month. KPP phages suspended in

saline, distilled water and phage broth stored at 30 °C

showed decrease in the titer when compared to the

initial phage titer in the lysates. Phages suspended in

the phage broth were stable for first 5 months only,

whereas saline and distilled water suspensions were

stable for first 2 months only.

Bioremediation of pathogen spiked water

microcosm using monovalent φSPB phage

formulationIn case of exponential phase cells of

Salmonella enterica serovar Paratyphi B, φSPB could

lyse the cells actively in all the types of microcosms.

Maximum reduction (80%) was observed in

aquaculture microcosm in 24 h. In case of swimming

pool and bore well water microcosms, almost 60%

reduction was seen. Less reduction was seen in the

river water microcosm. There was an increase in

φSPB phage population by 70-90%; with maximum

(90%) in case of aquaculture microcosm (Fig. 3a).

In case of stationary phase cells, reduction in

Salmonella population was comparatively less. Only

20-40% reduction was observed. Maximum reduction

was observed in aquaculture and river water

microcosms. The increase in the phage population

was only 20-40% as compared to the initial φSPB

titer (Fig. 3b).

The analysis of data for significant difference was

done by Post Hoc Test (Tukey’s HSD procedure)

Fig. 3—Effect of φSPB on (A) exponential phase, (B) stationary

phase cells of Salmonella enterica serovar Paratyphi B in various

water microcosms. Host control, % reduction in Salmonella

population , � initial φSPB titer -●-, and % increase in φSPB

phage population -▲- . Each data point is mean±SD of 3

determinations. a; b values denote statistically significant

difference.


1043

using SPSS 18. A 95% confidence level was used for

the analysis so that P ≤ 0.05 were considered to be

statistically significant.

Discussion

Bacteriophages have attracted researchers all over

the world as a new biocontrol tool against nuisance

bacteria. For the successful application of phages as

biocontrol agent in the environment, phages are

required to be isolated, purified, enriched from the

natural resources. Most importantly, preparation of an

eco-friendly formulation is an important step. In this

particular study, potent lytic phages specific for

Salmonella enterica serovar Paratyphi B,

Pseudomonas aeruginosa and Klebsiella pneumoniae

were isolated from the Pavana river water. Laboratory

scale production and development of monovalent

phage formulations in various liquid media. The

present study indicated that φSPB, BVPaP-3 and KPP

phages are tailed phages and are lytic in nature.

Amongst the phages studied so far, 96% phages are

tailed and remaining 3.7% are polyhedral, filamentous

and pleomorphic18.

. Phages in the present study

(φSPB, BVPaP-3 and KPP) are tailed phages and

survived better in phage broth, distilled water and

saline at 4 °C than at 30 °C. This result is consistent

with previous results that tailed phages were the most

resistant to storage conditions and showed longest

viability1. The ingredient of phage broth (Ca

2+ and

Mg2+

) supported viability of phages. As described in

the literature, metal ions help in the stabilization of

phages19

. Olsen et al 20

recommended that 4 °C is the

optimal temperature for phage storage but

lyophilization of phages is not recommended to

preserve phages because of its damaging action21

.

Jepson and March22

also observed good phage

stability at 4 °C for more than six months in SM

buffer but not at 37 °C. Phages under this study,

retained stability for 2-3 months at 30 °C in phage

broth, saline and distilled water. The present results of

distilled water formulation are similar to the previous

results where phages were found to be more stable in

distilled water at ambient temperature22

. From the

present results, it can be concluded that phage broth,

distilled water and saline played key roles in

maintaining active phages. Phages have been explored

in various fields as biocontrol agent. However, there

are scanty reports regarding their application in the

water systems23,24

as this is emerging as new field. It

has been observed that disinfection by-products

remained in the water bodies leave behind many

health risks. Therefore, use of bacteriophages in the

decontamination of water is now exploring as an

emerging field in the research area. Much of the work

done was related to the application of phages in the

activated sludge systems. Dias and Bhat25

, indicated

that E. coli phages were applied in laboratory scale

activated sludge systems were not functional enough.

Thomas et al26

reported that phages may act as an

active components of the activated sludge systems.

An increase in total phage concentration during an

activated sludge process, suggesting active replication

of phages via, host infection and lysis as described by

Ewert and Paynter27

and Hantula28

demonstrated the

presence of active phage-host relationship. There are

scanty reports where phages are used in the

decontamination of water under controlled

conditions29

. In this particular study, phage broth

formulation was explored in various water

microcosms to kill infested Salmonella population.

The carrier that supports the stability of phages should

also support the host to remain in the log phase

because such cells are killed rapidly by phages.

Application of phage broth formulation of phages at

MOI<1 (multiplicity of infection) in laboratory scale

studies showed that different water microcosms

infested with exponential phase and stationary phase

cells of Salmonella enterica serovar Paratyphi B

reduced the population of the host bacteria. This

reduction in the Salmonella population was due to

inclusion of phage and not due to potential die off

factors associated with Salmonella. This can be

explained on the basis of comparison of the test

microcosms with that of the host control microcosms

where Salmonella organisms were infested in various

water microcosms showed no reduction in the host

population in 24 h. In this study various water

samples were tested that may differ in their physico-

chemical properties, like pH, temperature, dissolved

organic matter in the form of detergents, sugars,

pesticides etc. Many kinds of metal ions can also be

found in such water samples. pH of water do not have

a major effect on phage adsorption and replication30

.

Bacteriophages were found to be resistant to natural

inactivation by water treatment and they survive

better there31,32

. Phages have the potential to multiply

at ambient temperatures33

and they don’t have any

adverse effect on phage stability. Metal ions may be

present in water, but literature survey indicated that

they do not have an adverse effect on phage


1044

stability34

. Anionic and cationic detergents may have

an adverse effect on phage stability30

. Bacteriophages

in water were found resistant to UV light, this might

be due to the absorption of UV light by the water

matrix that results in the loss of activity of UV light35

.

This is the real fact that viruses survive better in water

and therefore are considered an indicator of faecal

pollution of water. The phage broth carrier contains

nutrients that helped the phage to remain in the active

state. Powder formulations of bacteriophages are

applied worldwide for disease treatment, prevention

and sanitation purposes but in powder formulations,

phages are adsorbed on the surface of the matrix, as a

result, they may get inactivated due to various

environmental conditions36

. Adhesion of phage to the

matrix is so strong that they cannot be released upon

appropriate re-suspension in the medium. Therefore,

phages in the liquid formulations would be the best

alternative as biocontrol agent to be explored in the

water systems to kill life threatening infectious

bacterial pathogens. In the present study, an attempt

has been made to find out the base that will retain the

viability of phages for the longer period of time

during the storage at a specific temperature. Phage

broth that was used as an enrichment medium for

phages retained very good active residual phage titers

at 4 °C for almost one year and at 30 °C only for 3-5

months.

Conclusion

Phage broth acts as the best suspending medium to

increase the longevity of phages at 4 and 30 °C.

Saline and distilled water also maintained the viability

of phages at 4 °C. Incidentally, these liquid

formulations are low-cost, feasible and effective. The

observation of this study indicated that application of

bacteriophage based technology in water system

could be an ideal and effective for the removal of

pathogens in water. Phage broth formulation in

monovalent form can be used as an effective

biological disinfectant against targeted antimicrobial

susceptible or resistant bacterial pathogens in areas

where people use natural water resources for drinking

purposes. Therefore, these liquid formulations of

phages would be applied directly as a biological

disinfectant against the pathogens in the environment.

Acknowledgement SA is grateful to the University Grants

Commission (UGC) New Delhi for awarding the

Teacher Fellowship and the financial assistance.

Thanks are also due to Dr. H W Ackermann for TEM

studies.

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liquid based formulations of bacteriophages for the...

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