Research Paper
Characterization of a virulent bacteriophage LK1 speci fi c for Citrobacter freundii isolated from sewage water
Waqas Nasir Chaudhry1, Irshad Ul Haq2, Saadia Andleeb1and Ishtiaq Qadri1
1Atta ur Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technology (NUST) H-12 Sector, Islamabad, 44000, Pakistan
2Department of Microbial Ecology, Center for Ecology and Evolutionary Studies (CEES), University of Groningen, Nikenborg 7, Groningen, 9747, Netherlands
Citrobacter freundii is a worldwide emerging nosocomial pathogen with escalating incidence of multidrug resistance.Citrobacter freundiiexists in natural environment, especially in health care settings and is difficult to eradicate. Phage therapy is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a virulent bacteriophage LK1 capable of specifically infecting Citrobacter freundii. A virulent bacteriophage LK1, specific forCitrobacter freundiiwas isolated from sewage water sample. TEM showed that phage Lk1 has an icosahedral head 70 nm in diameter and short tail of 17 nm, and can be classified as a member of the Podoviridae family. Restriction analysis indicated that phage LK1 was a dsDNA virus with an approximate genome size of 20–23 kb.
Proteomic pattern generated by SDS PAGE using purified LK1 phage particles, revealed three major and six minor protein bands with molecular weight ranging from 25 to 80 kDa. Adsorption rate of LK1 relative to the host bacterium was also determined which showed significant improvement in adsorption with the addition of CaCl2. In a single step growth experiment, LK1 exhibited a latent period of 24 min and burst size of 801 particle/cell. Moreover, pH and thermal stability of phage LK1 demonstrated a pH range of 5.0–6.0 and phage viability decreased to 0% at 65 °C. When LK1 was used to infect six other clinically isolated pathogenic strains, it showed relatively narrow host range. LK1 was capable of eliciting efficient lysis ofCitrobacter freundii, revealing its potential as a non-toxic sanitizer for controllingCitrobacter freundiiinfection and contamination in both hospital and other public environments.
Keywords:Bacteriophage / Phage therapy /Citrobacter freundii/ LK1 phage Received: November 27, 2012; accepted: January 5, 2013
DOI 10.1002/jobm.201200710
Introduction
Citrobacter freundii is a fermentative, motile, gram negative bacterium found in soil, water, food, sewage, and many health care units [1].Citrobacter freundiiis also a commensal microbe present in human intestine, which is the causative agent of pneumonia, meningitis, septicemia, urinary tract infection, and opportunistic infections especially in immune compromised individu-
als. Since its discovery Citrobacter freundii has gained resistance to many common antibiotics [2] due to intrinsic mechanisms and also its capability of acquiring drug resistance determinants [3]. The increasing preva- lence of multi drug resistant Citrobacter freundii strains found in clinics has included it among important nosocomial pathogens right next toPseudomonas aerugi- nosa[4].Citrobacter freundiiis resistant to dehydration, UV radiation, common chemical sanitizers, and detergents, thus making it extremely difficult to eradicate from hospital settings, especially from catheter related devices used in intensive care units (ICU). In fact, routinely used antimicrobial agents only inhibit its growth, while currently no such treatments are available to remove Citrobacter freundii in hospital environments. This
Correspondence: Waqas Nasir Chaudhry, Atta ur Rahman School of Applied Biosciences (ASAB), National University of Sciences & Technol- ogy (NUST) H-12 Sector, Islamabad 44000, Pakistan
E-mail:[email protected] Phone:0092 342 5278387 Fax:0092 51 90856102
Characterization of a virulent bacteriophage LK1 1
drawback of unavailability of potential bactericidals greatly increases the risk of infection for hospitalized patients [3].
Biological control agents such as bacteriophages have the potential to provide an alternative mechanism to control bacterial diseases both in human and animals.
The use of phages to treat bacterial infections has a long history in the former Soviet Union and recently the Western scientific community has subjected phage therapy to clinical studies [5, 6]. Phage therapy typically involves isolation of diverse bacteriophages that are specific to a bacterial pathogen which can also be used in combination as a bacteriophage“cocktail [7]. The benefit of using phage therapy is the possibility that the infected bacterial cells will carry the phage deeper into infected tissue which upon lysis can infect other bacterial cells too.
United States Food and Drug Administration (FDA) approved the safe use of a bacteriophage on ready to eat meat and poultry products as an additive againstListeria monocytogenes, thereby increasing opportunities for phage application in the food industry [8]. Phage has been tried as bio-control of plant pathogens against Xanthomonas pruniassociated bacterial spot of peaches to control infections of peaches, cabbage and peppers.
Phages have also been used to control Ralstonia solana- cearumof tobacco. They have been successfully employed against Xanthomonas campestris which cause spots on tomatoes [9]. There have been a number of successful studies in animal models as well. Use of phages to treat experimental infections ofE. coliin mice, preventing and treating experimental disease of antibiotic resistant Pseudomonas aeruginosa [10] and Acinetobacter specie studies in mice and guinea pigs have suggested that phages might be efficacious in preventing infections of skin grafts used to treat burn patients. The goal of this research was to isolate phage that was active against Citrobacter freundiiand to characterize it with respect to morphology, structural proteins, growth patterns, and genome characteristics.
Materials and methods
Identification of bacterial isolate
After overnight incubation of bacterial strain, established microbiological methods (i.e., colony morphology, Gram staining and biochemical test) were used for identifica- tion of bacterial strain [1]. Analytical profile index (API) test kit [11], a standardized identification system for Enterobacteriaceaefamily, was used for biochemical tests.
For 16S rRNA (Ribotyping) DNA was isolated with Fermentas Genomic DNA isolation kit. For identification
of genomic origin, the 16S rRNA gene was amplified by using polymerase chain reaction (PCR) using 16S rRNA primers (RS-1; 50-AAACTC-AAATGAATTGACGG-30, RS-3;
50-ACGGGCGGTGTGTAC-30) [12]. PCR was carried out by denaturing DNA at 110 °C for 10 min followed by 30 cycles of amplification (95 °C for 2 min, 52 °C for 1 min, and 72 °C for 2 min). The PCR amplified product was electrophoresed on 1% agarose gel. The PCR product of 0.5 kb was eluted from gel using Invitrogen gel extrac- tion kit. Sequencing was carried out by CEQ 8000 Genetic Analysis System (Beckman Coulter). To identify the sequence by alignment, 16S rRNA sequences were analyzed with BLAST (www.ncbi.nlm.nih.gov/BLAST).
Phage enrichment and isolation
Bacteriophages specific to indicator bacterial strain were enriched with methods described by Stenholm with some modifications [13, 24]. To enrich the phage population, sewage water sample was pre-incubated with isolated host bacterial strain ofCitrobacter freundii. Sewage water was centrifuged at 1300 rpm for 10 min to remove algal cell and sewage debris.
Above prepared sample concentrates (5 ml) were added to a 30 ml log phaseCitrobacter freundii. Enriched cultures were incubated overnight at 37 °C with shaking at 150 rpm. Chloroform (1%) was added to 1.5 ml of sample to disrupt bacterial cell and release phages and then centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was filtered using 0.45 and 0.20
m
m(Minisart, Salotrius Stedim Biotech) syringe filters and transferred to a new tube. Phage isolation and detection was carried out by plaque assay on LB agar plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L) with soft agar (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 0.7%) [14]. For that overnight bacterial culture and phage sample were mixed in 0.7% LB soft agar (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 0.7%; at a temperature of 45 °C) and poured over LB plates. On solidification plates were incubated overnight at 37 °C and examined for plaques the following day. For a negative control, phage alone was added to the molten agar. Well isolated plaques were serially propagated until a single phage type was obtained. The purified phages were then stored in SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM, Tris–HCl [pH 7.5]), and 0.002% w/v gelatin at 4 °C with the addition of 7% dimethyl suloxide (DMSO) at–80 °C.
Infected bacterial cells (SEM), phage morphology (TEM)
Purified phages (107PFU/ml) were dialyzed to remove
<10 kDa bacterial proteins. Phages added in dialysis bag
(10 kDa cut off dialysis tube), dialyzed in 250 ml pre-chilled phage buffer (10 ml 1 M Tris (pH 7.5), 10 ml 1 M MgSO4, 4 g NaCl, 980 ml d H2O) plus calcium for several hours, changing the buffer at least once. Insert the magnetic bar and allow the tube to spin slowly over night. Infected (5
m
l)and uninfected bacteria (control) were applied to aluminum tape and coated with Gold (250 A° thickness) with Auto Quick Quoter containing ion sputtering device and the grid was observed under analytical scanning electron micro- scope (JEOL JSM 6490A Japan).
The phage LK1 morphology was examined by trans- mission electron microscopy. After washing the samples three times with 0.1 M ammonium acetate solution (pH 7.0), phages were negatively stained with 5% uranyl acetate and then examined in a JOEL JEM 1010 transmission electron microscope operated at 100 kV.
The phage was classified according to the guidelines of the International Committee on Taxonomy of Viruses (ICTV, 1995) based on their morphological features.
Bacteriophage genome isolation
The phage filtrate having 8% PEG and 4% NaCl was centrifuged at 26,000 rpm (Z 36 HK HERMLE, Germany) for 4 h. Supernatant was discarded. Polyethylene glycol (PEG 8000) was removed by dissolving pellet in 100
m
lautoclaved distilled water and by later on sonicating the tube for 3 min in water bath sonicator (Elma E 30H Elmasonic, Germany), and then centrifuged again for 5 min at 14,000 rpm. Supernatant was collected having PEG free viruses in suspension. DNaseI (1
m
l) was added and incubated at 37 °C for 30 min. After incubation 4m
lof 2.5 SDS–EDTA dye (SDS 0.4%, EDTA 30 mM Bromophenol blue 0.25% sucrose 20%) was added to the tubes and kept in Heating Block at 80 °C for 10 min.
The whole lysate was loaded on the 0.7% TAE agarose gel (Tris Base, Boric Acid, EDTA pH: 8.3) containing 0.5
m
g/ml of ethidium bromide. The gel was run at a voltage of 90 V and the bands were visualized using a UV transilluminator. DNA bands were extracted from gel by using Silica bead DNA gel extraction kit Fermentas.
Restriction analysis
For molecular manipulation, phage purified genomic DNA was digested with several restriction endonucleases and their combinations, includingHindIII,XbaI,MboI, and SmaI (Takara Bio, Inc., Japan) and subsequently subjected to electrophoresis on 1% Agarose gel. Lambda DNA/
HindIII, 2 (Fermentas) was used as molecular marker.
Proteomic analysis of phage structural proteins To remove any residual bacterial proteins and phage SDS PAGE analysis, purified phage stock was washed three
times with 0.1 M ammonium acetate solution (pH 7.0).
Polyethylene glycol 8000 (PEG 8000) precipitated purified phage particles were subjected to 12% SDS PAGE directly and the gel was stained with Coomassie Blue G-250.
Analysis of calcium ion effect on phage adsorption Bacterial culture (50 ml) of optical density 0.5 OD600was divided into two autoclaved flasks of 25 ml each. One flask was inoculated with phage 500
m
l (2.56109PFU/ml) only (control), while the secondflask was inoculated both with 500
m
l phage (2.56109PFU/ml) and 250m
l1 M CaCl2 and incubated with constant shaking at 90 rpm at 37 °C. Samples were taken from bothflasks at different time intervals of 0, 10, 20, and 30 min to measure the number of free phages in control and calcium chloride added mixture [15]. The evaluation of calcium ion effect was made on the basis of the percentage of free phages by the following formula.
Percentage of free phages¼ ðN=NoÞ 100
whereNois the PFU/ml of phages atT¼0 min whileNis PFU/ml atT¼10, 20, 30 min.
One step growth experiment, latent period, and burst size
One step growth experiment was carried out according to previously described method for determining the latent period and burst size [17, 17]. Bacterial culture (50 ml) was incubated to mid exponential phase (0.4–0.6 OD600) and the cells were harvested by centrifugation. Pellet was re-suspended in 0.5 ml LB broth media and mixed with 0.5 ml phage (2.56109). Phage was allowed to adsorb on bacteria for 1 min and the mixture was centrifuged at 13,000 rpm for 30 s to remove free phage particles. The pellet was suspended in 100 ml fresh media and the culture was incubated at 37 °C. Samples from the incubated flask were taken after every 3 min time interval for 45 min and the phage titer was determined by double layer agar assay.
Determination of pH and thermal stability
Three parallel experiments were carried out for testing pH stability as described by Capra et al. [16].
Phages (500
m
l) having 2.56109PFU/ml were incubat- ed at 37 °C for 1 h under specific pH conditions. Each treated sample was tested against the host in double layer agar assay for phage viability. Thermal stability tests were performed according to the method described by Capra et al. [15, 18]. Phage filtrates were taken in microfuge tubes and treated under different temperature condi- tions (37 °C [control], 45, 50, 55, 60, 65, 70, 75, and 80 °C)for 1 h. After incubation, plaque assay was performed for each temperature treated sample. Results of three parallel experiments were compared with control.
Host range determinations
The host range of the isolated phage was assessed on a range of different bacteria, i.e., methicillin resistant Staphylococcus aureus(MRSA) 6403, MRSA 17644,Acineto- bacterspecie 33408,Acinetobacterspecie 1172,Pseudomonas aeroginosa 22250, Pseudomonas aeroginosa 11219 and Escherichia coli. All these tested bacterial strains were clinical pathogen, obtained from Microbiology lab, Pakistan Institute of Medical Sciences Islamabad, Pakistan. All phage host combinations were evaluated by spot testing 107PFU/ml on air dried bacterial lawn (100
m
l overnight culture plated directly on LB agar plates) in three independent experiments. After over- night incubation at 37 °C, plates were checked for the presence of a lysis zone against a negative, uninfected control. Susceptibility of various bacterial isolates was tested using the drop on lawn technique [19].Bacterial reduction assay
Four sets of experiments were carried out for bacterial reduction analysis by tube lysis assay in broth culture.
In each case, 1 ml of overnight grown Citrobacter freundii bacterial culture was inoculated in 100 ml LB brothflasks. OD600of eachflask was measured. OD of phage inoculated cell was compared to that of mock infected control cells. Flask 1 was inoculated with 2 ml of LK1 phage,flask 2 with 4 ml andflask 3 with 6 ml of phage having 6.7 105PFU/ml while flask 4 was control having no phages and incubated at 37 °C in shaking incubator at 120 rpm. Samples were taken after every 2 h from 0 to 30 h. Results were compared with control.
Results
Identification of bacterial strain
In this study, Citrobacter freundiiwas identified both by biochemical tests and sequence information derived from their 16S rRNA gene (ribotyping). Gram staining results showed that it is a Gram negative bacillus.
Biochemical test API 20E showed susceptible isolate to be Citrobacter freundii which belongs to Enterobacteriaceae family. An expected size amplicon (470 bp) was ampli- fied, using ribotyping method (Fig. 1) which was subjected to DNA sequencing from both orientations.
The sequence is available in the databases under accession number [GenBank: HM640009]. In Basic Local
Alignment Search Tool (BLAST) analysis, 100% nucleotide sequence identity toCitrobacter freundiiwas scored.
Bacteriophage isolation
Citrobacter freundiiwas used as indicator strain for phage isolation. After enrichment, phage containing samples were placed on semisolid LB medium with Citrobacter freundiiforming lawn. Three phages LK1, LK2, and LK3 were isolated that ranged from 4.5 to 5 mm in diameter on lawn of indicator strain by plaque analysis (Figs. 2 and 3). These phages were isolated from sewage water, the same reservoir as their host. Later experiments showed that the observed plaques of all three phages were similar in all aspects. Purified LK1 phage produced clear plaques on lawn of host bacterium Citrobacter freundii(Fig. 3).
Figure 1. PCR amplification of 16S ribosomal RNA gene of Citrobacter freundii, 100 bp DNA ladder (M), PCR product of Citrobacter freundii(lanes 1–4).
Figure 2. Master plate of plaque assay for phage isolation from sewage water againstCitrobacter freundii, marked points show the presence of bacterial growth clearance in the form of round zones (plaque).
Electron microscopy
Citrobacter freundii cells (0.4 OD600) were analyzed for Scanning Electron Microscopy (SEM) analysis. After washing with PBS cells a drop of the cell suspension was applied to aluminum taped grid as described in materials and methods. Infected and uninfected Citro- bacter freundii cells observed at 16,000. Infected cells were shown as burst cells because of phage release while uninfected (control) cells were intact in shape showing bacterial cell viability (Fig. 4). The resolution of the transmission electron micrographs was good enough to reveal details in thefine structure of Phage. LK1 particle had icosahederal head and very short stumpy non- contractile tails, assigning it to the orderCaudoviralesand
family Podoviridae (Fig. 5). Measurements taken from these images showed diameter of 70 nm and tail of 17 nm.
LK1 phage genome isolation and restriction analysis of DNA
Extracted phage genomic DNA when analyzed on 1%
agarose gel showed it to be of approximately 20–23 kb in size (Fig. 6). Phage genome was eluted from agarose gel for further molecular analysis (Fig. 7). Phage DNA was not digested with endonucleaseI indicating that it’s a double stranded DNA molecule. Purified genomic DNA was digested with several restriction endonucleases and their combinations, includingHindIII,XbaI,MboI, andSmaI and
Figure 3. Double layer agar plates showing plaque purified after repeating plaque assay three times andfiltering phage lysate through 0.2mm
filter. Calculated PFU/ml was 3.81011and observed plaque size was 4.5–5 mm. Plate (A) shows lower dilution of viral titer and plate (B) shows higher dilution of viral titer.
Figure 4. Scanning electron micrograph of Citrobacter freundii. Sample (A) uninfectedCitrobacter freundii; arrow shows bacterial cells in intact form; sample (B)Citrobacter freundiiinfected with LK1 phage; arrow indicates visible burst cells showing phage release from bacteria. The bar represents a length of 1mm.
subsequently subjected to analysis by gel electrophoresis.
Based on the digestion profiles, the genome size was observed to be approximately in the range of 20–23 kb (Fig. 8).
Proteomic analysis LK1 phage protein
Polyethylene glycol (PEG 8000) precipitated phage particles were subjected to SDS PAGE analysis after
washing with 0.1 M ammonium acetate three times in order to remove any residual bacterial protein. Proteomic patterns were obtained after Coomassie Blue G 250 staining and destaining steps. A total of three major and six minor protein bands were observed on the gel, with molecular weight ranging from 25 to 80 kDa with reference to Spectra™ Multicolor Protein Ladders (10– 260; Fig. 9).
Figure 5. Transmission electron micrograph of newly isolatedCitrobacter freundiiLK1 phage, scale bars correspond to 100 nm.
Figure 6. Image of 0.7% Agarose gel taken by Gel Documentation System, showing genome of phage LK1. Lanes 1–3; Phage genome DNA and M; Lambda DNA/HindIII, 2 marker.
Analysis of calcium ion on LK1 phage adsorption Adsorption is often affected by the presence of divalent metal ions in the solution. Calcium ions were added in phage bacterium mixture. Phage LK1 and Citrobacter freundii cells were mixed in test tube in presence of 10 mM CaCl2. Free phages were detected at different time intervals for 30 min. Result showed significant differ- ence between the two groups (Fig. 10). Calcium ions might stabilize phage adsorption process.
Latent time and burst dize
One step growth experiment was performed to deter- mine the latent time and burst size of phage LK1. A tri- phasic curve including the latent, rise and plateau phage was obtained (Fig. 11). The latent time was determined to be about 24 min and the burst size of phage LK1 was 801 phages per bacterial cell. Determination of burst size was based on the ratio of mean yield of phage particles liberated to the mean number of phage particles that infected bacterial cells. There was no difference in burst size between a 1-month-old phage stock and a freshly prepared phage stock.
pH and thermal stability test
Optimal pH was determined by testing the stability of phage LK1 under different pH conditions. Phage stability decreased as pH was increased from 5 to 9. No plaques were observed at pH 3 and 11. Phage became inactive at both extremes of pH. Incubation at different temper- atures showed maximum phage stability at 37 °C and gradually decreased as the temperature was increased up to 65 °C. At 70 °C phage became completely inactive and no PFU was observed (Fig. 12).
Host range
The infectivity of phage LK1 was investigated with six other clinically isolated pathogenic strains of methicillin resistant Staphylococcus aureus (MRSA) 6403, MRSA 17644, Acineto- bacter specie 33408, Acinetobacter 1172, Pseudomonas aero- ginosa22250,Pseudomonas aeroginosa11219, andEscheria coli.
All phage host combinations were evaluated by spot testing (107 PFU) on air dried bacterial lawn (100
m
l overnight culture plated directly on LB agar plates) in three independent experiments. Phage LK1 showed growth inhibition in Acinetobacter species only by spot test. The results indicated that phage LK1 had a narrow host range.Phages specifically targetingCitrobacter freundiihave narrow host range, usually one host one phage.
Bacterial reduction assay
In order to investigate lytic potential of LKI onCitrobacter freundii, tube lysis assay was applied in broth culture. This
Figure 7. Image of 0.7% Agarose gel of eluted LK1 phage genome.
Lane 1; LK1 phage genome (20–23 kb) and M; Lambda DNA/HindIII, 2 marker.
Figure 8. Image of 0.7% Agarose gel showing restriction digestion analysis of LK1 Phage DNA. M1; 1 kb DNA marker, lane 1; LK1 genome digested withMboI, lane 2;HindIII, lane 3;XbaI, lane 4;SmaI and M2; Lambda DNA/HindIII, 2 marker.
test is more labor intensive than the spot test but offers a more rigorous assessment of bacterial lysis activity.
Infection of Citrobacter freundii with different concen- trations (PFU/ml) of phage LK1 was monitored for 30 h.
Phage infection drastically decreased growth of Citro-
bacter freundiias compared to control (Fig. 13). The OD of phage infected LKI cultures lagged from the beginning behind the OD development of the uninfected culture.
However, a renascent of the OD600 was observed after 16 h culture incubation. The renascent of growth activity
Figure 9. 12% SDS–PAGE of PEG precipitated LK1 phage. Black arrows indicate major proteins bands, hollow arrows show minor proteins bands and M; protein marker.
Figure 10. Adsorption rate of LK1 at different time intervals. Samples were taken from the supernatants to measure free phage particles.
Divalent metal ions effect on adsorption rate was analyzed by adding 10 mM CaCl2to the mixture of phage LK1 andCitrobacter freundii cells.
Figure 11. One step growth curve experiment showing the latent period (24 min) and the average burst size (801 viral particles per host cell). Latent time and burst size of phage LK1 were inferred from the curve with a tri phasic pattern. L, latent phase; R, rise phase; P, plateau phase.
was most probably due to adaptation of phage resistant cells.
Discussion
Members ofCitrobacterspecie are rarely isolated from the hospitalized and non-hospitalized patients, however, it is an emerging nosocomial multidrug resistant pathogen [2] across the globe and particularly in Asia [3]. It causes meningitis in infants with a mortality rate of approximately 30.0% while, among survivors;
80.0% show some degree of mental retardation [20].
Citrobacter freundiihas beta lactamase gene in its plasmid and therefore is resistant to penicillins and cephalospor- ins. Presence of Plasmid Associated, and Extended Spectrum Beta Lactamase (PABL and ESBL) genes in Citrobacter freundiihas made it a highly resistant pathogen
to antibiotics. A sudden increase in prevalence of gentamicin resistantCitrobacter freundiiwas observed in Danish population in 2001 and resistant gene was found to be on Tn21 like transposon [21]. Thesefindings suggest the need of an alternative therapy for this bacterial disease. Clinical trial results are promising for bacterio- phage therapeutics development [22].
In this study, our results showed that a newly isolated bacteriophage LK1 can lyse the actively growing cells of Citrobacter freundiiefficiently. Phages can be isolated from a wide variety of sources such as sea water, sewage water/
sludge ponds etc. They mainly“feed”on the organisms that are present in their natural habitat. They are host specific and evolve along with their host. Phage LK1 was isolated from sewage water. Sewage in general, contains a large diversity of enterobacteriaceae family due to contamination from fecal and hospital drainage water.
Therefore sewage water is a reservoir for many enteric pathogens. This newly isolated phage LK1 was highly lytic and produced large plaque of 5 mm. Phage LK1 had an icosahedral head with a short non-contractile tail, and its genome was a molecule of double stranded DNA, so it was tentatively classified as a member of Podoviridaefamily.
Similar phages Kpn5, Kpn12, Kpn13, Kpn17, and Kpn22 of Podoviridae family having genome of 23–24 kb have been reported previously from India against K.
pneumonia[19].
It was found that phage LK1 showed growth clearance ofAcenitobacterspecies 33408 and 1172 on agar plate by spot test but no infection was observed in plaque assay (Table 1). Growth clearance on agar plate might be due to the presence of bacteriocins in LK1 phage lysate produced byCitrobacter freundiiwhich inhibit growth of Acenitobacterspecies. Studies on bacteriophage infections have revealed that the process of infection starts when virion interacts with host cell surface receptor
Figure 12. Effect of temperature on phage LK1. Phages were incubated for 1 h at respective temperature (°C) and titer of surviving phage particles was calculated in PFU/ml.
Figure 13. Lysis ofCitrobacter freundiiby the LK1 phage in broth culture. OD development of an uninfected control culture (flask 4) and parallel cultures infected with 2, 4, 6 ml of phage LK1 (6.7105PFU/
ml) inflask 1,flask 2, andflask 3, respectively.
Table 1. Host range analysis of phage LK1.
# Bacterial strains
Phage infectivity (spot test)
Phage infectivity (plaque test) 1 Methicillin resistant
Staphylococcus aureus (MRSA) 6403
ve ve
2 Methicillin resistant Staphylococcus aureus (MRSA) 17644
ve ve
3 Acinetobacter33408 þve ve
4 Acinetobacter1172 þve ve
5 Pseudomonas aeroginosa22250 ve ve 6 Pseudomonas aeroginosa11219 ve ve
7 Escherichia coli ve ve
ve, no infection;þve, phage LK1 infection.
molecules [23]. Many bacteriophages have been found to be highly specific for their receptors and have shown little or no interaction with receptors having even slight differences. This specificity becomes the basis of phage typing methods for the identification of bacterial species or sub-species. The results clearly indicated that the bacteriophage forCitrobacter freundiiwas highly specific against its respective host organism.
Results of one step growth curve experiment demon- strated all the stages involved in multiplication of bacteriophage. It has been shown that for any ecological setting there is a positive relationship between latent period and burst size, also an optimal latent period leads to high phagefitness [24, 25]. Additionally an increase in burst size may contribute to larger plaque size [26]. Phage burst size and latent period both are individual character- istics of a specific phage. Phage LK1 was observed with a burst size of 801 per bacterial cell. This higher burst size and larger plaque size results are consistent with Abedon and Culler’sfindings [26].
A number of factors, i.e., ions (e.g., Mgþ2 and Caþ2), pH, temperature, and the growth medium can influence phage adsorption rate. Previously, it has been reported that calcium and magnesium ions have positive effect on phage bacteria attachment. These ions have an electrostatic bonding effect on the interactions of phage bacterium systems [27, 28].
Calcium ions stabilize the week interaction of virion with receptors. Different concentrations of calcium ions have shown maximum effects on infectivity of different phages [16, 27]. Phage LK1 showed significantly more infectivity with a 10 mM Calcium chloride concentra- tion. It was observed that acidic pH (5–6) gave more stability to LK1 phage while any pH level lower or higher than this optimum was lethal for the virus. This behavior of LK1 phage andCitrobacter freundiimight be due to its adaptation in its natural sewage water habitat which also has slightly acidic pH. Phage LK1 has shown a very broad range of thermal stability. It remained active for 6 months at 4 °C and viable at 65 °C for 1 h incubation time and became completely inactive at temperatures higher than this, while maximum infec- tivity was observed at 37 °C. This behavior of the phage can also be explained by the presence of Citrobacter freundiias part of the normalflora of human body with 37 °C temperature. Phage LK1 produced drastic de- crease in Citrobacter freundii growth for 16 h as compared to control. During this time period there was maximum bacterial cell destruction but some cells showed resistance to virus infection which might be due to the development of host bacterium immunity to phage infection [29]. Resistant cells, though their
number was far less initially, started multiplying and after 16 h there was again an increase in the growth of Citrobacter freundii. This behavior of phage is considered to be a great hindrance in phage therapy. Some studies have shown that phage resistant bacteria lose their virulence factor, i.e., capsule, flagella and these viru- lence factors are the receptor sites for phage infections.
Such loss of virulence factors in a phage resistant host mutant has also been reported infish pathogens [30].
Basically resistance reduces thefitness of the bacteria rendering it to compete unfavorably with its phage sensitive ancestors [31, 32].
Concluding remarks
Characterization of newly isolated phage LK1 showed that it is highly efficient in lysingCitrobacter freundii, as it has shown some outstanding aspects including rapid growth nature, high pH and thermal stability. All these characteristics make this phage very promising for possible application in eradication ofCitrobacter freundii contaminations and treatment of Citrobacter freundii infections. Phage LK1 has shown narrow host range, so for the broad-spectrum elimination of bacteria; a
“cocktail” with a pool of lytic phages might be more useful against present and other bacterial strains. Also a better understanding of phage biology, combined with knowledge of lytic enzyme biology and the previous experiences attained in several European countries should facilitate the development of these new therapies.
Acknowledgments
This study was supported by Higher Education Commis- sion (HEC) and Ministry of Science and Technology (MoST). Authors would like to acknowledge Dr. M.
Mujahid and Mr. Shamas Uddin, School of Chemical and Material Engineering (SCME) NUST, for analytical Scanning Electron Microscopy, Dr. Sohail Hameed and Mr. M. Javed Iqbal, National Institute of Biotechnology and Genetic Engineering (NIBGE) Faisalabad, for TEM analysis, Mr. Muhammad Shafique, Microbiology Lab Pakistan Institute of Medical Sciences (PIMS) Pakistan, for generously providing bacterial strains to carry out host analysis experiment. WNC carried out all research experimental work. IH helped in data analysis and experiment acquisition. S.A. helped in manuscript preparation and interpretation of results. I.Q. supervised TEM analysis and manuscript preparation.
Conflict of interests
The author(s) declare that they have no competing interests.
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