isolation and partial characterization of a virulent bacteriophage ihq1 specific for aeromonas...

HOST MICROBE INTERACTIONS

Isolation and Partial Characterization of a VirulentBacteriophage IHQ1 Specific for Aeromonas punctatafrom Stream Water

Irshad Ul Haq & Waqas Nasir Chaudhry &

Saadia Andleeb & Ishtiaq Qadri

Received: 4 July 2011 /Accepted: 10 September 2011 /Published online: 27 September 2011# Springer Science+Business Media, LLC 2011

Abstract Aeromonas punctata is the causative agent ofsepticemia, diarrhea, wound infections, meningitis, perito-nitis, and infections of the joints, bones and eyes.Bacteriophages are often considered alternative agents forcontrolling bacterial infection and contamination. In thisstudy, we described the isolation and preliminary charac-terization of bacteriophage IHQ1 (family Myoviridae)active against the Gram-negative bacterial strain A. punc-tata. This virulent bacteriophage was isolated from streamwater sample. Genome analysis indicated that phage IHQ1was a double-stranded DNA virus with an approximategenome size of 25–28 kb. The initial characterization ofthis newly isolated phage showed that it has a narrow hostrange and infects only A. punctata as it failed to infectseven other clinically isolated pathogenic strains, i.e.,methicillin-resistant Staphylococcus aureus 6403, MRSA17644, Acinetobacter 33408, Acinetobacter 1172, Pseudo-monas aeruginosa 22250, P. aeruginosa 11219, andEscherichia coli. Proteomic pattern of phage IHQ1,generated by SDS-PAGE using purified phage particles,showed three major and three minor protein bands withmolecular weights ranging from 25 to 70 kDa. Theadsorption rate of phage IHQ1 to the host bacterium wasalso determined, which was significantly enhanced by theaddition of 10 mM CaCl2. From the single-step growthexperiment, it was inferred that the latent time period ofphage IHQ1 was 24 min and a burst size of 626 phages percell. Moreover, the pH and thermal stability of phage IHQ1were also investigated. The maximum stability of the phage

was observed at optimal pH 7.0, and it was totally unstable atextreme acidic pH 3; however, it was comparatively stable atalkaline pH 11.0. At 37°C the phage showed maximumnumber of plaques, and the viability was almost 100%. Theexistence of Aeromonas bacteriophage is very promising forthe eradication of this opportunistic pathogen and also forfuture applications such as the design of new detection andphage typing (diagnosis) methods. The specificity of thebacteriophage for A. punctata makes it an attractivecandidate for phage therapy of A. punctata infections.

Introduction

Aeromonas punctata is a Gram-negative, rod-shapedbacterium that resides in water and soil [24]. The genusAeromonas comprises important human pathogens whichcause primary and secondary septicemia in immunocompro-mised individuals and wound infections in healthy persons aswell as in those patients who have undergone medicinal leechtherapy [24]. They also cause some less well-describedillnesses like meningitis, peritonitis, and infections of thejoints, bones, and eyes [24]. Aeromonas is considered to bean opportunistic or secondary pathogen. Most reports ofAeromonas infections describe patients who were eitherimmunologically compromised [2, 17] or having chronicdiseases [7, 16]. A. punctata harbors antibiotic resistanceplasmid pFBAOT6 which enables it to become immuneagainst all major classes of antimicrobial drugs [39].

Bacteriophages are the most abundant organisms onearth which infect bacteria. These bacterial viruses havegenetic material in the form of either DNA or RNA (single-or double-stranded), encapsidated by a protein coat [15].The lethal effect of bacteriophages on their bacterial hostshas been known since their discovery [28]. Phage biocon-

I. U. Haq (*) :W. N. Chaudhry : S. Andleeb : I. QadriNUST Center of Virology & Immunology (NCVI),National University of Sciences & Technology (NUST),Islamabad, Pakistane-mail: [email protected]

Microb Ecol (2012) 63:954–963DOI 10.1007/s00248-011-9944-2

trol of bacteria implicates the application of phages toreduce and inhibit pathogenic bacterial strains. The use ofbacterial viruses as therapeutic agents were interrupted bythe advent of antibiotics, whose wider spectrum of actionthan that of phages opened the possibility to applyempirical treatments. However, the selection of antibiotic-resistant bacterial strains and the frequent occurrence ofadverse effects associated with their use led to renewedinterest in phages as therapeutic agents [21].

Phages have several advantages over antibiotics and otherantimicrobial agents, such as host specificity, no side effects,and multiplication in the presence of their hosts [11, 23]. Theuse of phages might possibly lessen the burden of antibioticsin the control of pathogenic bacteria. Phages are highlyspecific, and their use as antibacterial agents is not likely toselect for phage resistance in nonspecific bacterial strains [38].Phage preparations can readily be changed in reaction tochanges in bacterial pathogen populations and susceptibility,while antibiotics have an extensive and affluent developmentcycle [38]. The specificity of phages sometimes may beconsidered as a possible disadvantage because there are muchmore pathogenic bacteria than expected to be targeted. Toaddress this problem, a cocktail of phages should be prepared.

The Food and Drug Administration of the United States ofAmerica recently approved some phages as “generallyregarded as safe” for food products to control Listeriainfections, which will definitely kindle the presentation ofphage biocontrol [6]. It has been postulated that about 1030

bacteriophages are present in the environment [4]. Despitethis rich reservoir of bacteriophages present in the environ-ment, only about 300 phages have been characterized [12].One of the major reasons for the failure of phage therapy inthe pre-antibiotic era was the application of uncharacterizedbacteriophage preparations that led to inconsistent outcomesof the therapy. Hence, it is pretty much important to isolateand characterize new phages especially in light of theobservation that most of the disease-causing organisms livein matrix-enclosed environments called biofilms [42] thatinherently show increased resistance toward all antibiotics[20]. In the present study, we have reported the isolation andpartial characterization of a virulent phage IHQ1 specific forA. punctata from stream water in Pakistan and to assess theirpotential for the abatement of A. punctata infections. To thebest of our knowledge, this is the first study to report on theexistence of bacteriophage against A. punctata.

Materials and Methods

Identification of Bacteria Strain

Bacteria strain A. punctata, isolated from wastewater, wasidentified on the basis of morphology and molecular

characterization. After an overnight incubation of bacterialstrain, established microbiological methods, such as colonymorphology and Gram’s staining, were used for theidentification of subject strain [19]. Molecular identificationof the bacterial isolate was confirmed by sequencing 16SrRNA gene. Bacterial genomic DNA was isolated using aFermentas kit. The 16S rRNA genes were amplified bypolymerase chain reaction (PCR) using primers RS-1 (5′-AAACTC-AAATGAATTGACGG-3′) and RS-3 (5′-ACGGGCGGTGTGTAC-3′) [43]. Denaturation of DNAat 110°C for 10 min was followed by 30 cycles ofamplification (95°C for 2 min, 52°C for 1 min, and 72°Cfor 2 min). The PCR product was electrophoresed on 1%agarose gel and was eluted from the gel using an Invitrogengel extraction kit. The PCR product of 0.5 kb was elutedfrom the gel using an Invitrogen gel extraction kit.Sequencing was carried out using CEQ 8000 GeneticAnalysis System (Beckman Coulter). Basic Local AlignmentSearch Tool (BLAST) at the NCBI data bank was used for the16S rRNA sequences to identify the sequence by alignment.

Bacteriophage Isolation and Enrichment

The water sample obtained from the stream (Badri stream,Shah Mansoor Swabi, Pakistan) was pre-incubated with thehost bacterial strain (A. punctata) for the enrichment of thephages present in the water sample. The water sample wascentrifuged (Centrifuge 5810R, Eppendorf Germany) at1,300 rpm for 10 min to remove any other cell debris andimpurities. The sample was filtered through 0.20-μm(Minisart, Salotrius Stedim Biotech) filters for purification(1% of an overnight culture of each strain).

Bacteriophages specific to the isolated bacterial strain ofA. punctata were enriched with the methods described byStenholm [10, 35], with some modifications. The preparedsample concentrates (5 mL) were added to a 30-mL logphase A. punctata strain grown overnight at 37°C. Theenriched cultures were incubated overnight at 37°C withshaking at 150 rpm (refrigerated shaking incubator TSS-40-250, Technico Scientific Supply, Lahore, Pakistan). Onemilliliter of the overnight enrichment culture was trans-ferred to the Eppendorf tube and one drop of 1%chloroform (Fisher Scientific, Fair Lawn, NJ, USA) wasadded to it for the disruption of bacterial cells and therelease of phages. It was then centrifuged at 14,000 rpm for10 min to pellet down bacterial cell debris.

The supernatant was filtered using 0.45- and 0.20-μmsyringe filters and transferred to a new tube; the phage wasdetected and isolated by a plaque assay [36]. The purifiedphage was then stored in an 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% dimethylsulfur oxide at −80°C.

Bacteriophage IHQ1 Specific for Aeromonas punctata 955

Isolation of Phage Nucleic Acids

The phage filtrate having 8% polyethylene glycol (PEG)and 4% NaCl was centrifuged at 26,000 rpm (Z 36 HKHERMLE, Germany) for 4 h. The supernatant wasdiscarded and the pellet was taken for further processing.PEG was removed by dissolving the pellet in 100 μLautoclaved distilled water and sonicating it for 3 min in awater bath sonicator (Elma E 30H Elmasonic, Germany).After sonication, it was centrifuged again for 5 min at14,000 rpm. The supernatant was collected having PEG-free viruses in suspension. One microliter of DNaseI wasadded and incubated at 37°C for 30 min. After incubation,4 μL of 2.5 X SDS-EDTA dye (0.4% SDS, 30 mM EDTA,0.25% bromophenol blue, 20% sucrose) was added to thetubes and kept at 80°C in Heat Block for 10 min. After heattreatment, the whole lysate was loaded onto 0.7% TAEagarose gel (Tris base, boric acid, EDTA, pH 8.3) containing0.5 μg/mL of ethidium bromide. The gel was run at avoltage of 90 V and the bands were visualized using a UVtransilluminater (Wealtec Corp., USA).

Morphology Study by Transmission Electron Microscopy

Morphology of phage IHQ1 was examined by transmissionelectron microscopy of purified phage particles. Phageswere negatively stained with 5% uranyl acetate afterwashing it three times with 0.1 M ammonium acetatesolution (pH 7.0). Examination of phage IHQ1 wasperformed with a JEOL JEM 1010 transmission electronmicroscope operating at 100 kV.

Analysis of Phage Proteins

Purified phage solution was washed three times with 0.1 Mammonium acetate solution (pH 7.0) to remove anyexisting remaining bacterial proteins. PEG-precipitatedpurified phage particles were subjected directly to SDS-PAGE as described previously [25], and the gel was stainedwith Commassie Blue G-250.

One-Step Growth, Latent Period, and Phage Burst

The one-step growth experiment was carried out accordingto the method previously described for determining thelatent period and burst size [3, 13, 37]. A. punctata culture(50 mL) was incubated to the mid-exponential phase O.D600 (0.4–0.6) and the cells were harvested by centrifuga-tion. The pellet obtained was resuspended in 0.5 mL Luria–Bertani (LB) broth media and mixed with 0.5 mL of phage(3.25×108 plaque-forming units (PFU)/mL). The phagewas allowed to adsorb to the bacteria for 1 min and themixture centrifuged at 13, 000 rpm for 30 s to remove free

phages. The pellet was then resuspended in 100 mL freshmedia and the culture incubated at 37°C continuously.Samples from the incubated flask were taken at 3-minintervals and the phage titer was determined by a double-layer agar technique.

Bacterial Reduction Assay

For the bacterial reduction assay, 1 mL of overnight-grownA. punctata bacterial culture was inoculated into twolabeled 100-mL LB broth flasks. One flask containing thebacterial culture was inoculated with 1 mL of phage filtrate.The other flask was taken as the control having no phages.The flasks were then incubated at 37°C in a shakingincubator at 120 rpm. The optical density (OD600) of thesamples taken at an interval of 2 h for 24 h was measuredby using a SP300 spectrophotometer from Optima (Japan).

Calcium Ion Effect on the Adsorption Rate of Phage

In order to measure divalent metal ions effects on theadsorption rate of phage, 1 M CaCl2 was added to theinfected culture. A culture of 50 mL was divided into twoautoclaved flasks, 25 mL each. One flask was inoculatedwith 500 μL (3.25×108 PFU/mL) phage only, while thesecond flask was inoculated both with 500 μL (3.25×108 PFU/mL) phage and 250 μL CaCl2 (1 M) andincubated at 37°C with constant shaking at 90 rpm.Samples were taken at different time intervals of 0, 10,20, and 30 min to measure the number of free phages in thecontrol and the calcium chloride-added mixture [8]. Thenumber of free phages was determined using the double-layer agar assay. The evaluation of calcium ion effect wasmade on the basis of the percentage of free phages using thefollowing formula:

Percentage of free phages ¼ N=N0ð Þ � 100

where N0 is the PFU per milliliter of phages at T=0 min,while N is PFU per milliliter at T=10, 20, and 30 min.

Effect of Temperature on Phage and Thermostability

Thermal stability tests for the phage were performed accord-ing to the method described in [8, 9]. Phage filtrates weretaken in Eppendorf tubes and treated at 37°C (control), 45°C,50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and 80°C for 1 h.After incubation, the rate of survival of each treated phagewas determined by the double-layer agar assay.

Determination of pH Stability

The experiment for testing pH stability was carried outas previously described in [8, 9]. Briefly, 500 μL

956 I. U. Haq et al.

(3.25×108 PFU/mL) of phages was treated under specific pHconditions and incubated at 37°C for 1 h. After incubation,each treated sample was tested against the host in a double-layer agar assay to check the viability of phage.

Bacteriophage Host Range

The host range of phage IHQ1 was evaluated on anumber of pathogenic bacterial strains including Pseu-domonas aeruginosa 22250, P. aeruginosa 11219,methicillin-resistant Staphylococcus aureus (MRSA)6403, MRSA 17644, Acinetobacter 33408, Acinetobacter1172, and Escherichia coli DH5 alpha, which wereobtained from the microbiology laboratory, PakistanInstitute of Medical Sciences Islamabad. The drop-on-lawn technique [44] was used to test the susceptibility ofbacterial isolates.

Results

Identification of A. punctata Using Ribotyping

A. punctata was identified both by a biochemical test(Gram staining) and the sequence information derived fromtheir 16S rRNA gene (ribotyping). Gram staining resultsrevealed that it is a Gram-negative bacterial strain. A 470-bp amplicon was amplified and subjected to DNAsequencing from both orientations (Fig. 1a). The resultedsequence was deposited to a database (accession no.HQ124005) and aligned to search for the most similarsequences. In the BLAST analysis, it showed a high

nucleotide sequence identity of 99% to A. punctata. Aphylogenetic tree based on the 16S rRNA gene sequencewas also constructed (Fig. 1b).

Isolation of Bacteriophage

Phage was isolated from the water sample collectedfrom a stream (Badri) in Shah Mansoor (Swabi) usingthe double-layer agar assay technique after enrichmentof the phage. Plaques were obtained after incubation ofplates at 37°C overnight. The phage has a plaque sizeranging from 0.1 to 0.5 mm in diameter and uniqueclear pin point morphology with well-defined boundaries(Fig. 2).

Morphology of Phage IHQ1

Purified and concentrated solutions of phage IHQ1 wereexamined by electron microscopy (Fig. 3). A number offeatures were observed: an isometric head with contractiletail as well as base plate with short tail fibers. Phage IHQ1had an average head diameter of 128 nm, while the tail hasa diameter of 108 nm. Based on these morphologicalcharacteristics, phage IHQ1 belongs to the Myoviridaefamily.

Genome Extraction of Bacteriophage

Phage was amplified and its genomic DNA extractedusing the PEG/sodium chloride (NaCl) precipitationmethod. The genome was found to be double-strandedDNA because phage nucleic acids were not digested by

Figure 1 a Ribotyping PCR-based amplification of A. punctata 16SrRNA gene. Lanes 2 and 3 show bands of amplified DNA atapproximately 470 bp. Lane 1 shows a 1-kb ladder. b Neighbor-

joining tree based on the 16S rRNA sequences showing theintraspecies relationships of the genus Aeromonas


the endonucleaseI enzyme, indicative of the fact thatphages are double-stranded DNA phages. The genomesize was determined to be approximately 25–28 kbrunning it with standard lHindIII ladder on 0.8% agarosegel (Fig. 4).

Analysis of Phage Structural Proteins

PEG-precipitated phage particles were subjected to 12%SDS-PAGE, and protein bands were obtained after Com-massie Blue G-250 staining and destaining. A total of three

Figure 2 Double-layer agarplates, arrows showing pinpoint plaques of 0.1–0.5 mmin diameter. a Lower dilution(10−3) of phage titer. b Higherdilution (10−4) of phage titer

A

FED

CB

Figure 3 Transmission electron micrographs of the purified A. punctata phages. Negatively stained phage preparations were viewed. Scale bars,100 nm. Six representative images are shown


major protein bands and three minor protein bands wereobserved on the gel, with different molecular weightsranging from 25 to 70 kDa (Fig. 5).

Calcium Ion Effect on the Adsorption Rate of Phage

The effect of calcium ions on the adsorption of phage wasanalyzed by adding (10 mM) calcium chloride to the phageand the A. punctata mixture. The number of free phages leftin the solution (which was not bound to the bacteria) was

detected at different time intervals of 0, 10, 20, and 30 minusing the plaque assay. Data analysis showed a significantdifference between the control and the calcium ion-treatedphage IHQ1. The results showed that calcium ions stabilizethe process of adsorption. The numbers of free phages aredecreased, as shown by the lower line in the figure ascompared with the upper line representing the control(Fig. 6).

Latent Time Period and Burst Size

The single-step growth experiment was performed fordetermining the latent time period and burst size of phage.A triphasic curve was obtained that has the latent phase, logor rise phase, and stationary or plateau phase. From thedata, the latent time period was calculated to be 24 min.The burst size of the phage was 626 phages per cell.Determination of burst size was based on the ratio of themean yield of phage that infected the bacterial cells to themean phage particles liberated (Fig. 7).

Determination of pH Stability

Optimal pH for phage was determined by testing thestability of phage at different pH values. No reduction wasobserved in the infectious phage almost after 1-h incubation atpH 7.0. The number of phages was found to be reduced atpH 5.0, 9.0, and 11.0, while at pH 3.0 there were no activeinfectious phages to be detected. The results showed thatextreme pH might pose hindrance to phage stability. AtpH 3.0, no plaques were observed, while the number ofplaques was found to be increased with increasing pH,reaching the highest number at pH 7.0. A gradual decreasewas observed in the number of plaques when the pH wasincreased above 7.0 (Fig. 8).

Figure 5 SDS-PAGE analysis of PEG-precipitated IHQ1 phage.Arrows show protein bands in lane 2, while lane 1 contains theprotein ladder

Figure 4 Genome of phage: 0.8% agarose gel. Lane 1 showslHindIII marker. Lanes 2, 3, 4, and 5 show bands of phage DNAhaving a size of approximately 25–28 kb

Figure 6 Adsorption rate test. At different time intervals, sampleswere taken from the supernatant to measure the number of free phageparticles. The effect of divalent ions on the adsorption rate wasexamined by adding 10 mM CaCl2 to the mixture of IHQ1 phage andA. punctata cells


Effect of Temperature on the Stability of Phage

A thermal stability test was carried out to analyze the heat-resistant capability of phage at pH 7.0. The phage retainedalmost 100% infection activity after incubation at 37°C.The results suggested that phage was amazingly stable attemperatures ranging between 37°C and 65°C. But at 70°C,a single plaque was lacking (Fig. 9).

Bacterial Reduction Assay

Infections of A. punctata with different concentrations (PFUper milliliter) of phage was monitored for 24 h. Phageinfection drastically decreased the A. punctata cultureturbidity in comparison to control. However, an increase ofthe turbidity (OD600) was observed after 9 h of culture

incubation. This increase of turbidity was most probably dueto the growth of phage-resistant cells (Fig. 10).

Host Range of Phage IHQ1

The effect of phage IHQ1 on non-host bacteria was alsoinvestigated to determine the host range of the phage.Seven pathogenic bacterial strains, viz., MRSA 6403,MRSA 17644, Acinetobacter 33408, Acinetobacter 1172,P. aeruginosa 22250, P. aeruginosa 11219, and E. coli DH5alpha were tested against phage IHQ1. None of thesestrains was found susceptible to the phage. The resultsindicated that phage IHQ1 has a narrow host range.

Discussion

A. punctata and other members of the genus Aeromonadscause primary and secondary infections in immunocompro-

Figure 9 Thermal stability test of phage IHQ1. Samples were treatedat various temperatures mentioned to calculate titer of the survivinginfectious phages in PFU per milliliter

Figure 8 pH stability test of phage IHQ1. Phage was incubated for1 h under different pH conditions before determining the number ofactive infectious phage particles

Figure 10 Bacterial reduction assay. Effect of IHQ1 phage on logphase A. punctata culture compared with the control having no IHQ1phages

Figure 7 One-step growth experiment. Curve with triphasic patternshows latent period (24 min), rise phase, plateau phase, and theaverage burst size of 626 virions per cell


mised individuals, and they are also notorious for theirwound infections in healthy persons. A. punctata is alsoassociated with joint infections, meningitis, peritonitis, andinfection of the eyes, while their involvement in gastroen-teritis remains controversial to some degree [24]. Theinfection rate of A. punctata is higher in individuals whohave either chronic diseases [7, 16] or a compromisedimmune system [2, 17]. A. punctata harbors antibioticresistance plasmid pFBAOT6 which enables it to becomeimmune against all major classes of antimicrobial drugs[39]. The fact that A. punctata colonies are found in waterand soil also suggests that they cause infection in marineand freshwater animals, most importantly fishes. Aeromo-nas physiologically resemble certain members of theEnterobacteriaceae. Some of the Aeromonas spp. that havebeen isolated from human sources made it important formedical bacteriologists [18].

In the current study, a bacteriophage IHQ1 which canefficiently lyse A. punctata was isolated from stream water.The isolation of the phage from the stream water suggeststhat A. punctata-specific phage subsist in aquatic environ-ment. The verity that Aeromonas causes infection in marineand freshwater animals also proposes that phage IHQ1might be related to other phages specific to fish pathogensreported previously [22, 30, 34]. Phage IHQ1 isolatedagainst A. punctata was lytic and produced clear plaques of0.1–0.5 mm in diameter, which are in accordance withprevious observations for the Aeromonas hydrophila phageAeh1 [14].

Previous studies on bacteriophage infections have shownthat the progression of infection starts when virion interactsprecisely with receptor molecules on the surface of the hostcell [33]. Many phages have been found to be greatlyspecific for their receptors present on the host cell surface.They show no interaction with receptors having a differentstructure. The specificity turns out to be the basis of phagetyping methods used for the identification of bacterialstrains. The results obtained clearly showed that phageIHQ1 for A. punctata was highly specific.

One-step growth curve reveals all stages involved in themultiplication of bacteriophages. It has been found that forevery environmental setting, there is a progressive relation-ship between burst size and latent time period such that anoptimal latent time period leads to high phage fitness [40,41]. Furthermore, an upsurge in burst size may contribute toplaque size or larger plaques with higher burst size [1].Phage IHQ1 has a burst size of 626 virions per bacterialcell. The burst size of phage IHQ1 is much higher thanphages Aeh1 and Aeh2, with virions per cell number of 17and 92, respectively [14]. The latent time period of phageIHQ1 was found to be 24 min, which suggests that thephage inside A. punctata stays for a long time, and onemight get the feeling that the phage has a complex life cycle

inside the bacterial cell. Earlier, Chow and Rouf [14]showed latent time periods of 39 and 52 min for phagesAeh1 and Aeh2, respectively. Calcium ions have a positiveeffect on phage adsorption rate. The infectivity of phageIHQ1 was shown to be increased on 10 mM calciumchloride concentration. It is suggested that ions have anelectrostatic bonding effect in the interactions of phage withthe surface receptors of bacteria [29, 32]. Calcium ionsstabilize the weak interaction of virion with receptorsduring the adsorption. Diverse quantities of calcium ionsgive maximum infectivity for various phages [3, 32].

It was found that highly acidic pH 3.0 was lethal tophage IHQ1, while a pH around 5–9 was favorable for itsmaximum activity and viability. Previously isolated A.hydrophila phages Aeh1 and Aeh2 were also shown to beinactive at extreme acidic pH 3.0 [14]. Phage IHQ1 showedmaximum activity at pH 7.0. Most surprisingly, phageactivity was also observed at basic pH 11.0. Popoff [31]observed that Aeromonas salmonicida phages are fairlyresistant to alkaline conditions (pH 12 for 30 min at 26°C).This behavior of IHQ1 phage might have some connectionwith its natural habitat from where it was isolated. PhageIHQ1 has shown less thermal stability. Maximum infectiv-ity was observed at 37°C, which gradually decreased aftertemperature reached 65°C. It remained active at 4°C forquite a long time, and its viability was also observed at65°C. Beyond 65°C, phage IHQ1 became inactivecompletely.

A drastic decrease in culture turbidity of A. punctatawas observed for 10 h when treated with phage IHQ1.There was again a slow and gradual rise in the opticaldensity (l=600 nm) after 10 h, which can be attributed tothe immunity shown by the host bacterium to phageinfection [5]. Surviving resistant cells may have startedmultiplying again after 10 h and increased A. punctataculture turbidity in the solution. This behavior of phage isconsidered to be a hindrance in phage therapy. Somestudies have shown that bacteria resistant to phage losetheir capsule and flagella, which are receptor sites forphage adsorption. Such loss of bacterial virulence in mutantbacterial strains has been demonstrated previously in fishpathogens resistant to phages [30]. Resistance to phageinfections reduces the fitness of bacteria, making it competehostilely with its phage-sensitive ancestors [26, 27].

Conclusions

This study provides the first evidence for the existence ofbacteriophages able to infect A. punctata. The discovery oflytic phage IHQ1 which specifically infects A. punctata isan important step toward the control of A. punctatainfections. Characterization of phage IHQ1 showed that it


was very efficient in lysing A. punctata, combined with itsoutstanding thermal and pH stability; it could be a goodcandidate to be used as an alternative to antibiotics.However, host range tests showed that phage IHQ1 didnot infect other pathogenic bacterial strains included in thisstudy. Such narrow host range suggests that more virulentbacteriophages specific to different bacteria need to bescreened and isolated in the future. A pool of lytic phagesmight be more useful against other bacterial strains forpossible phage therapy. Hence, future directions will delveon: (1) isolating additional phages that infect some of thenotorious super bugs; (2) evaluating the protective effectsof lytic A. punctata phage on infected animal models; (3)evaluating various mechanisms of phage delivery inexperimental infections; and (4) determining and annotatingthe whole genome sequence of phage.

Acknowledgments We would like to acknowledge Dr. SohailHamid and Mr. Javed Iqbal from the National Institute of Biotech-nology and Genetic Engineering (NIBGE), Faisalabad, for transmis-sion electron microscopy. We are thankful to Mr. MuhammadShafique, Microbiology lab, Pakistan Institute of Medical Sciences(PIMS), Pakistan, for providing bacterial strains to carry out phagehost range experiments. We are thankful to the Higher EducationCommission (HEC) and the Ministry of Science and Technology(MoST), Pakistan, for providing funding to Dr. Ishtiaq Qadri.

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isolation and partial characterization of a virulent bacteriophage ihq1 specific for aeromonas...

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