Environment Health Techniques Isolation and characterization of lytic vibriophage
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Research Paper Isolation and characterization of lytic vibriophage against Vibrio cholerae O1 from environmental water samples in Kelantan, Malaysia Ali Al-Fendi1, Rafidah Hanim Shueb1, Manickam Ravichandran2 and Chan Yean Yean1 1
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Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia Faculty of Applied Sciences, Department of Biotechnology, AIMST University, Semeling Bedong, Kedah, Malaysia
Water samples from a variety of sources in Kelantan, Malaysia (lakes, ponds, rivers, ditches, fish farms, and sewage) were screened for the presence of bacteriophages infecting Vibrio cholerae. Ten strains of V. cholerae that appeared to be free of inducible prophages were used as the host strains. Eleven bacteriophage isolates were obtained by plaque assay, three of which were lytic and further characterized. The morphologies of the three lytic phages were similar with each having an icosahedral head (ca. 50–60 nm in diameter), a neck, and a sheathed tail (ca. 90–100 nm in length) characteristic of the family Myoviridae. The genomes of the lytic phages were indistinguishable in length (ca. 33.5 kb), nuclease sensitivity (digestible with DNase I, but not RNase A or S1 nuclease), and restriction enzyme sensitivity (identical banding patterns with HindIII, no digestion with seven other enzymes). Testing for infection against 46 strains of V. cholerae and 16 other species of enteric bacteria revealed that all three isolates had a narrow host range and were only capable of infecting V. cholerae O1 El Tor Inaba. The similar morphologies, indistinguishable genome characteristics, and identical host ranges of these lytic isolates suggests that they represent one phage, or several very closely related phages, present in different water sources. These isolates are good candidates for further bio-phage-control studies. Abbreviations: VPUSM – Vibrio Phage Universiti Sains Malaysia; TEM – transmission electron microscopy; PFGE – pulsed field gel electrophoresis; MOI – multiplicity of infection; SMG – sodium/magnesium with Gelatin; PEG – polyethylene glycol Keywords: Lytic vibriophage / V. cholerae / Myoviridae / Environmental water Received: June 11, 2013; accepted: October 11, 2013 DOI 10.1002/jobm.201300458
Introduction Cholera is an acute food and waterborne communicable disease caused by Vibrio cholerae [1]. Cholera is a common cause of infant and adult deaths that has occurred frequently as epidemics in many developing countries since the early 19th century [2]. V. cholerae is divided into more than 206 serogroups based on their lipopolyCorrespondence: Dr. Chan Yean Yean, Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia E-mail: [email protected] Phone: þ609 7676258 Fax: þ609 7641615 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
saccharide composition, of which only the serogroups O1 and O139 have been associated with epidemic and pandemic cholera in humans [2]. Toxigenic V. cholerae O1 consists of two biotypes: classical and El Tor. Each biotype again has two major serotypes, namely Ogawa and Inaba. Out of the seven pandemics that have taken place, the classical biotype was responsible for the first six pandemics, while the seventh was due to the El Tor biotype and was exceedingly more extensive in geographic spread and duration [3]. In late 1992, V. cholerae O139 came into the limelight by causing a severe cholera epidemic in the Indian subcontinent [4]. The occurrence of epidemics is known to coincide with increased prevalence of the causative V. cholerae strain in the
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aquatic environment. In contrast, bacteriophage in the environment has been found to inversely correlate with the abundance rate of cholera [5]. Bacteriophages are viral cellular parasites that depend on bacterial cell processes to produce viral proteins and viral particles [6–8]. The ability of bacteriophage to replicate exponentially and lyse pathogenic strains of bacteria suggests that they could play a vital role in controlling bacteria populations in natural systems [9–11]. Thus, using bacteriophage as a therapeutic agent is one possible option to control pathogenic bacteria [12]. Previous trials in aquaculture systems have successfully eradicated pathogens using bacteriophages [10, 11, 13]. Vinod et al. [13] and Crothers-Stomps et al. [11] isolated phage against Vibrio harveyi and these phages have shown potential for use as biocontrol agents to combat vibriosis in the aquaculture systems. Therefore, this study was undertaken to isolate vibriophage with lytic activity against V. cholerae.
Materials and methods Sample collection Environmental water samples (250–300 ml) were obtained by dipping sterile sampling bag (Nasco, Salida, California, USA) from various sources including rivers, lake, sewages, fish farm, ditch, and ponds in Kelantan, Malaysia as described in Table 1. Following collection, samples were transported to the laboratory on ice. Upon arrival, the samples were immediately filtered using 0.22 mm pore cellulose acetate filters (Minisart, Goettingen, Germany) and processed for bacteriophage isolation. Bacterial strains Bacterial strains used in this study are listed in Table 2. Forty-six V. cholerae strains were obtained from the Department of Medical Microbiology and Parasitology, Universiti Sains Malaysia. The bacteria studied comprised 16 strains of V. cholerae O1 El Tor Ogawa, 14 strains V. cholerae O1 El Tor Inaba, 7 strains of V. cholerae O1 classical, 5 strains of V. cholerae O139, and 4 strains of V. cholerae Non-O1/O139. In addition, 16 other enteric bacterial species were also employed to determine the phage host range (Table 2). The V. cholerae strains used as host to detect bacteriophages were examined for the presence of endogenous phage using a minor modification of the method described by Oakey et al. [14]. All V. cholerae were cultured in 10 ml LB broth and then grown at 37 °C for 2–3 h until the optical density (OD) at 600 nm (OD600) was 0.4. The cultures were divided into two equal aliquots ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Table 1. Isolation of vibriophages from different water sources and sampling periods in Kelantan, Malaysia, between April 2011 and March 2012. Isolated vibriophages
Source of environmental water
NI Lake VPUSM 1 Lake VPUSM 2 Pond VPUSM 3 Lake NI Sewage Pond VPUSM 4a VPUSM 5 Ditch NI River NI Ditch NI Pond VPUSM 6 Sewage NI River NI Fish Farm Fish Farm VPUSM 7a Ditch VPUSM 8a NI River VPUSM 9 Lake NI Ditch VPUSM 10 Fish Farm VPUSM 11 Sewage Total: 11 vibriophages
Sampling period April 2011 May 2011 May 2011 June 2011 June 2011 July 2011 July 2011 August 2011 August 2011 September 2011 September 2011 October 2011 November2011 December 2011 December 2011 January 2012 February 2012 February 2012 March 2012 March 2012
No. of water samples 3 5 2 5 1 6 2 4 1 4 4 7 6 7 2 4 3 3 4 2 75
NI, no isolation. a These isolated vibriophages were lytic and further characterized in this study.
and one of each pair was treated with 30 ng ml1 mitomycin C (Sigma–Aldrich, Castle Hill, NSW, Australia). The cultures were incubated at 37 °C and the OD600 was recorded periodically up to 8 h. Cell lysis, as indicated by a relative decrease in OD600 compared with the untreated control, was taken to indicate the presence of endogenous phage. A panel of V. cholerae strains, two of each V. cholerae El Tor Inaba, O1 El Tor Ogawa, O1 classical Inaba, O1 classical Ogawa, and O139, identified as not containing endogenous phage were challenged with environmental water samples to screen for vibriophages. Vibriophage isolation Two methods of vibriophage isolation were used with some modification. The first method was a spot plaque assay [5]. To prepare V. cholerae lawn on the plate, 0.5 ml (log phase) of each V. cholerae panel strain in LB broth was mixed in 3 ml of soft agar (LB broth containing 0.8% Agar, Merck) and the mixtures were overlaid on LB agar plates. Fifty milliliters of water samples were filtered through 0.22 mm pore filters to remove bacteria. One milliliter of the filtered water samples were added to young (0.4 OD600) V. cholerae panel cultures grown in 10 ml LB
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Table 2. Host range of isolated vibriophages. Plaque formation Species/Strain Vibrio cholerae, O1 El Tor Ogawa (n ¼ 16) Vibrio cholerae, O1 El Tor Inaba (n ¼ 14) Vibrio cholerae, O1 classical (n ¼ 7) Vibrio cholerae, O139 (n ¼ 5) Vibrio cholerae, nonO1/O139 (n ¼ 4) Vibrio vulnificus (n ¼ 2) Vibrio parahaemolyticus (n ¼ 2) Escherichia coli (n ¼ 2) Escherichia coli (EPEC) (n ¼ 2) Shigella dysenteriae (n ¼ 1) Shigella flexneri (n ¼ 2) Shigella sonnei (n ¼ 1) Proteus vulgaris (n ¼ 2) Proteus mirabillis (n ¼ 2) Klebsiella pneumonia (n ¼ 3) Klebsiella spp. (n ¼ 2) Salmonella typhimurium (n ¼ 4) Salmonella enteritidis (n ¼ 2) Morganella morganai (n ¼ 2) Serratia marcescens (n ¼ 2) Yerssnia enterocolitica (n ¼ 2) Enterococcus faecalis (n ¼ 1)
VPUSM VPUSM VPUSM 4 7 8 – þ – – – – – – – – – – – – – – – – – – – –
– þ – – – – – – – – – – – – – – – – – – – –
– þ – – – – – – – – – – – – – – – – – – – –
broth and further incubated at 37 °C with shaking for 6 h. The enriched cultures mixtures were centrifuged at 10,000g for 10 min at 4 °C. Aliquots of supernatant (25 ml) pre-filtered through 0.22 mm pore filters were inoculated on lawns of V. cholerae lawn. Plates were left at room temperature for 15 min and then incubated at 37 °C for 16 h, and inspected for clearing zone. In the second method to isolate lytic phage, water samples were enriched and filtrate was obtained as described in the first method and examined using the soft agar overlay method described by Phumkhachorn and Rattanachaikunsopon [15]. For each water sample, a 10fold dilution series (to 1010) was prepared and, for each dilution, an aliquot of 100 ml of each serial dilution was mixed with 100 ml of young (OD600 0.4) V. cholerae panel cultures and incubated at 37 °C for 15 min. The mixture was added to 5 ml of LB soft agar (0.8% agar), mixed gently by rolling between the palms to enhance good mixing and then overlaid on empty plates. After solidifying, the plates were incubated at 37 °C for 16 h and the presence of lytic phage in the form of plaques was examined. Purification of vibriophage All isolated vibriophages were purified by threefold successive single-plaque isolation method as mentioned in the soft agar overlay method. A single plaque was picked with a sterile pipette tip (Axygen Scientific, Mexico, Mexico) and inoculated into a log-phase ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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V. cholerae culture. After incubation at 37 °C with shaking (200 rpm) for 6 h and static incubation for 12 h, the vibriophage host mixture was centrifuged at 10,000g for 20 min at 4 °C and filtered through a 0.22 mm filter. The isolated vibriophage filtrates were designated as the vibriophage suspensions. Propagation and determination of vibriophage titers Vibriophage suspensions were propagated further to obtain higher titer using the aforementioned soft agar overlay method. A broth culture of the respective V. cholerae was infected with vibriophage at a multiplicity of infection (MOI) of 0.01 and adsorption was allowed to occur at 37 °C for 15 min. The mixture was overlaid and incubated at 37 °C until complete lysis occurred within 6–8 h. The soft agar layer was harvested with 3 ml of sodium/magnesium with gelatin (SMG) buffer (NaCl, 5.8 g; MgSO4 · 7H2O, 2 g; tris pH 7.5, 50 ml; gelatin, 0.1 g; distilled H2O, 950 ml) in 15 ml centrifuge tube (Cellstar tubes; Greiner Bio-One, Frickenhausen, Germany) and incubated at room temperature for 1 h with shaking, then centrifuged at 10,000g for 20 min at 4 °C. The propagated vibriophages were serially diluted in SMG buffer. Each dilution was subjected to plague assay using the aforementioned soft agar overlay method. Plaques were counted and expressed as plaque forming units/milliliter (pfu ml1). High-titer vibriophage stocks were used to test for their host range and morphology was determined by electron microscopy. Determination of host ranges of vibriophages The host ranges of isolated vibriophages were determined by both methods described above (Table 2). The plates were incubated at 37 °C for 16 h before the presence of a clear zone in the plates, indicating the ability of vibriophage to infect the tested bacterial strains, was examined. Concentration of vibriophages Two methods were used for concentration of vibriophage suspensions. The first method was a standard polyethylene glycol (PEG) precipitation described by Yamamoto et al. [16] where sodium chloride was added to vibriophage suspension to a final concentration of 1 M, and the suspension was stored with continuous mixing for 1 h on ice. Next, the vibriophage particles were precipitated by adding 10% w/v PEG 8000 (Sigma– Aldrich, Saint Louis, MO, USA) to the suspension and incubated at 4 °C for 6 h. The precipitated particles were pelleted by centrifuging at 12,000g for 20 min at 4 °C. The pellet was then washed and resuspended in 0.5 ml SMG buffer.
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The second concentration method was carried out as described by Oakey et al. [14]. High-titer vibriophages (1010–1012) were subjected to ultracentrifugation at 200,000g for 2 h at 4 °C using a CP 80 Mx ultracentrifuge with a swing bucket rotor (Hitachi, Hitachinaka, Japan). After centrifugation, phage was eluted from the pellets by soaking overnight in 0.01 part of the original volume of sterile SMG buffer and then gently pipetting into 1.5 ml sterile centrifuge tubes. The concentrated vibriophages in both methods were titrated and stored at 4 °C for further work. Transmission electron microscopy (TEM) Vibriophage particles recovered by the ultracentrifuge method with a titer 108 pfu were negatively stained with 2% methylamine-tungstate stain (pH 6.5). A drop of vibriophage particles was deposited on a 400 mesh electron microscopic grid containing a copper–carbon gold film. A drop of methylamine-tungstate stain was then added and gently mixed with the vibriophage. After 1 min, excess fluid was withdrawn with a filter paper and the grid was allowed to dry. Stained particles were observed in a TEM-Philips CM12 apparatus (Philips Electron Optic, Eindhoven, The Netherlands). The size of the phages’ head and tail were measured in triplicates using SIS Docu Version 3.2 image software (Soft Imaging GmbH, Munster, Germany) and the mean and standard deviation was calculated using SPSS (IBM SPSS Statistics v20, New York, USA).
with some modifications. Briefly, 400 ml of vibriophage concentrated by PEG was mixed with 400 ml of 1.2% low melt agarose (Bio-Rad, Hercules, CA, USA) and the mixture was immediately transferred to PFGE plug casting mold. After solidified, plugs were treated with lysis buffer (50 mM tris, pH8.0, 50 mM EDTA, and 0.1% SDS) and 25 ml of proteinase K solution (20 mg ml1) for 2 h at 54 °C, followed by two washes in TE buffer (10 mM tris, pH 8.0, 1 mM EDTA) at 54 °C for 30 min. Plugs were loaded on 1% pulsed field certified agarose (Bio-Rad) in 0.5 TBE buffer (45 mM tris borate and 1 mM EDTA). The gels were run on CHEF-DRII electrophoresis system (Bio-Rad) in 0.5X TBE buffer at 14 °C, 6.0 V cm1, 120° angle, 1 s initial switch time, 6 s final switch time, and total run time of 18 h. “CHEF DNA size standards” for pulsed-field electrophoresis (Bio-Rad) was used to determine the DNA size. Results were visualized by staining with ethidium bromide. Nucleic acid type and restriction analysis The type of nucleic acid was analyzed by treatment with DNase I, RNase A, and S1 nuclease (Thermo Scientific). Eight restriction enzymes were used (BglII, EcoRI, EcoRV, Hin1ll, HindIII, ScaI, SmaI, and XbaI) and added into 1 mg of vibriophage nucleic acid using methods as recommended by supplier (Thermo Scientific). Uncut and digested nucleic acid was analyzed using 0.8% agarose gel at 60 V for 150 min.
Results Vibriophage nucleic acid extraction The nucleic acids of vibriophage particles were extracted from vibriophage suspension according to the method of Pickard [17] with modifications. RNase A (Sigma–Aldrich) to final concentration of 80 ml ml1 was added into 1.8 ml of vibriophage suspension (107 pfu) for 30 min at 37 °C. Then, sodium dodecyl sulfate (SDS) and proteinase K to final concentration of 1% and 170 ml ml1, respectively were added and the mixture was incubated at 37 °C for an additional 30 min. Proteins were removed by two phenol/ chloroform/isoamyl alcohol extractions and nucleic acid was precipitated with 100% isopropanol and 3 mol L1 of sodium acetate. After washing with 70% ethanol, the pellet was resuspended in TE buffer (10 mM tris, pH8.0, 1 mM EDTA). Nucleic acid concentration was determined by OD260/280 nm reading and the quality of the nucleic acid was observed by agarose gel electrophoresis (0.8% gel, 60 V, 90 min).
Isolation of vibriophages Seventy-five environmental water samples were collected from different areas in Kelantan, Malaysia (Table 1). Of these, 11 vibriophages were successfully isolated by both methods. However, the modified soft agar overlay method showed a better result for the isolation of the lytic vibriophages from environmental samples (data not shown). The lytic vibriophages produced clear and/or transparent plaques, compared with the spotting method, which sometimes produced cloudy or fuzzy plaques on host lawns. The isolated vibriophages were designated Vibriophage Universiti Sains Malaysia (VPUSM) 1 to VPUSM 11 based on the period and source of isolation. Most of the bacteriophages showed temperate activity (data not shown), but three of them (VPUSM 4, VPUSM 7, and VPUSM 8) showed lytic activity and produced small, round, and clear plaques with a diameter of 3–5 mm (Fig. 1).
Determination of bacteriophage genome size by PFGE Determination of vibriophage genomic DNA size using PFGE was performed as described by Talledo et al. [18]
Host range determination The host range of these three lytic vibriophages was determined by the soft agar overlay method. Only
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Figure 1. Plaque morphology of isolated vibriophages on V. cholerae O1 El Tor Inaba lawns culture. (a) VPUSM 4; (b) VPUSM 7; and (c) VPUSM 8.
V. cholerae O1 El Tor Inaba strains were sensitive and resulted in lysis. Strains of common enteric pathogens including Salmonella, Shigella, and Escherichia coli were not sensitive to these vibriophages including other V. cholerae biotypes and serogroups. These results suggest that VPUSM 4, VPUSM 7, and VPUSM 8 are specific vibriophages and have narrow host ranges with specificity to V. cholerae O1 El Tor Inaba (Table 2). Morphology of vibriophages Electron micrographs of the three isolated lytic vibriophages are shown in Fig. 2. The diameter of heads (distance between opposite apices) of VPUSM 4, VPUSM 7, and VPUSM 8 particles were 51.38 0.2, 55.32 1.1, and 58.12 1.4 nm, respectively, while the length of the tails were 90.7 6.4, 95.10 2.4, and 99.07 1.6 nm, respectively. TEM analysis showed that the predominant ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Figure 2. Electron micrograph of vibriophages isolated from environmental waters in Malaysia. (a) VPUSM 4; (b) VPUSM 7; and (c) VPUSM 8. Bars, 100 nm.
morphology of vibriophages against V. cholerae O1 El Tor Inaba was an icosahedral head with contractile, rigid, and partially sheathed tail and a neck was evident between the head and the tail. Based on morphology, the VPUSM 4, VPUSM 7, and VPUSM 8 particles most likely belong to the family Myoviridae [19, 20]. Genomic characterization of vibriophages Vibriophages concentrated by PEG method were assayed for genome size using PFGE (Fig. 3). The bands
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or closely related phages, and belong to Myoviridae family.
Discussion
Figure 3. Vibriophages isolated genomic DNA size determinated at 33.5 kb on PFGE. Lanes: M, ladder with standard DNA size 8– 48 kb (marker); lane 1, VPUSM 4; lane 2, VPUSM 7; and lane 3, VPUSM 8.
corresponding to the DNA of all three vibriophages were identical with size of approximately 33.5 kb. Nucleic acid of vibriophages VPUSM 4, VPUSM 7, and VPUSM 8 were then extracted and subjected to enzymatic digestion analysis. The genomic nucleic acid of these vibriophages were found to be a double stranded DNA following successful digestion with DNase 1 but not with RNase A and S1 nuclease. However, of eight restriction enzymes tested, only HindIII resulted in digestion of the nucleic acid of these three vibriophages and showed identical restriction digest profiles with fragments of approximately 9000, 6000, 5000, 4000, 3500, 2200, 2000, and 1750 bp (Fig. 4). Based on morphology and genomic characterization, the isolated lytic bacteriophages are probably identical
Figure 4. Vibriophage DNA digested with HindIII restriction enzyme. Lanes: M, 1 kb DNA ladder; lane C, vibriophage DNA undigested; lane 1, DNA digested for 1 h; lane 2, DNA digested for 2 h. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Phage therapy is one of the promising approaches to control human and environmental bacterial infections. Vibriophages can be isolated from the environment in which V. cholerae strains generally survive and can be found in sewage, feces, soil, and water [21, 22]. The characteristic of narrow host range of the vibriophage is a significant benefit for phage therapy; it is useful either individually or in combination with other V. cholerae lytic vibriophages as vibriophage cocktails to be employed as biological control agents in cholera endemic areas [5, 11, 23]. In this study, three lytic vibriophages were isolated from 75 environmental water samples after initial enrichment in broths seeded with V. cholerae. As a tailed virus of bacteria, the VPUSM 4, VPUSM 7, and VPUSM 8 vibriophages fell into the order Caudovirales that contains three families of tailed viruses that infect bacteria and archaea [24]. Possession of small icosahedral heads and defined collar/neck regions with contractile tail sheaths would tentatively place the bacteriophages in the family Myoviridae. The genomic size of myophages is typically >20 kb in size [25]. For example, K139, 33.1 kb [26]; HP2, 31.5 kb [27]; FP15, 29 kb [19]; VHML, 43.2 kb [14]; and FCTX, 35.5 kb [28]. Based on HindIII digestion analysis and PFGE, the isolated lytic vibriophages genomic DNA size was estimated at 33.5 kb, similar to the sizes of many bacteriophages in the family Myoviridae. Furthermore, the genomes of isolated lytic vibriophages are linear double-stranded DNA as the genomic nucleic acid could be digested with DNase 1 but not with RNase A and S1 nuclease. From previous studies, most of the vibriophages were found to be myophages with doublestranded DNA [20]. Morphologically, these vibriophages resembled the bacteriophages (k139 and FP15) previously reported to be associated with V. cholerae [18, 26]. By electron microscopy, all had icosahedral heads of approximately 60 nm diameter and defined collar/neck regions, contractile tail sheaths of approximately 100 nm. However, the K139 vibriophage and FP15 vibriophage have different host range and temperate activities to V. cholerae O139 and O1 El Tor, respectively [18, 26, 29]. In the current study, the host range of isolated vibriophages was relatively narrow and could only infect V. cholerae O1 El Tor Inaba. Some bacteriophages could infect more than one related species or even genus, while some may have a restricted host range,
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often only a single species or subspecies [30, 31]. Faruque et al. [5] suggested that vibriophages could play an important role in ending cholera epidemics. The absence of vibriophages in the aquatic environmental might promote cholera epidemic outbreak, particularly when V. cholerae is introduced for the first time. In late 2009, a cholera outbreak caused by V. cholerae O1 El Tor Ogawa was reported in Kelantan, Malaysia [3]. Hence, our data suggest that the presence of lytic vibriophage to V. cholerae O1 El Tor Inaba in Kelantan environmental water samples helps control the potential emergence of V. cholerae O1 El Tor Inaba outbreak in the state. In conclusion, we have isolated three lytic bacteriophages from different waters sources that infect V. cholerae O1 El Tor Inaba. The isolates were indistinguishable by the tests performed in the present study. Further detailed study of these phages is needed to determine if they are in fact identical and to explore their potential for use as biocontrol agents for phage therapy to prevent and treat cholera. Physiochemical studies like thermal and pH determinations, growth characteristic, structural proteins, and extensive study of their genomes will prove helpful in development of safe phage preparations having optimal efficacy against their specific bacteria hosts and on designing strategies for integrating this isolated vibriophage as biocontrol agent therapy for preventing and treating V. cholerae.
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and its relatedness to T7 viral supergroup. Intervirology, 55, 185–193. [3] Ang, G.Y., Yu, C.Y., Balqis, K., Elina, H.T. et al., 2010. Molecular evidence of cholera outbreak caused by a toxigenic Vibrio cholerae O1 El tor variant strain in Kelantan, Malaysia. J. Clin. Microbiol., 48, 3963–3969. [4] Sen, A., Ghosh, A.N., 2005. New Vibrio cholerae O1 biotype ElTor bacteriophages. Virol. J., 2, 28. [5] Faruque, S.M., Islam, M.J., Ahmad, Q.S., Faruque, A.S. et al., 2005. Self-limiting nature of seasonal cholera epidemics: role of host-mediated amplification of phage. Proc. Natl. Acad. Sci. USA, 102, 6119–6124. [6] Ackermann, H.W., 2001. Frequency of morphological phage descriptions in the year 2000. Brief review. Arch. Virol., 146, 843–857. [7] Boyd, E.F., Davis, B.M., Hochhut, B., 2001. Bacteriophage– bacteriophage interactions in the evolution of pathogenic bacteria. Trends Microbiol., 9, 137–144. [8] Chrisolite, B., Thiyagarajan, S., Alavandi, S.V., Abhilash, E.C. et al., 2008. Distribution of luminescent Vibrio harveyi, and their bacteriophages in a commercial shrimp hatchery in South India. Aquaculture, 275, 13–19. [9] Faruque, S.M., Naser, I.B., Islam, M.J., Faruque, A.S. et al., 2005. Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages. Proc. Natl. Acad. Sci. USA, 102, 1702–1707. [10] Karunasagar, I., Shivu, M.M., Girisha, S.K., Krohne, G. et al., 2007. Biocontrol of pathogens in shrimp hatcheries using bacteriophages. Aquaculture, 268, 288–292. [11] Crothers-Stomps, C., Hoj, L., Bourne, D.G., Hall, M.R. et al., 2010. Isolation of lytic bacteriophage against Vibrio harveyi. J. Appl. Microbiol., 108, 1744–1750. [12] Tanji, Y., Shimada, T., Fukudomi, H., Miyanaga, K. et al., 2005. Therapeutic use of phage cocktail for controlling Escherichia coli O157:H7 in gastrointestinal tract of mice. J. Biosci. Bioeng., 100, 280–287.
Acknowledgments This publication was financially supported by RUI grant (1001/PPSP/813045) from Universiti Sains Malaysia. The authors show their deep appreciation to staff of Public Health in Kota Bharu, Tumpat and Pasir Mas District of Ministry of Health Malaysia for their technical support in this work.
[14] Oakey, H.J., Cullen, B.R., Owens, L., 2002. The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML. J. Appl. Microbiol., 93, 1089–1098.
Conflict of interest statement
[15] Phumkhachorn, P., Ratianachaikunsopon, P., 2010. Isolation and partial characterization of a bacteriophage infecting the shrimp pathogen Vibrio harveyi. Afr. J. Microbiol. Res., 4, 1794–1800. [16] Yamamoto, K.R., Alberta, B.M., Benzinger, R., Lawhorne, L. et al., 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology, 40, 734–744.
No competing financial interests exist.
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[19] Ackermann, H.W., Dubow, M.S., Gershman, M., KarskaWysocki, B. et al., 1997. Taxonomic changes in tailed phages of enterobacteria. Arch. Virol., 142, 1381–1390. [20] Ackermann, H.W., 2003. Bacteriophage observations and evolution. Res. Microbiol., 154, 245–251. [21] Chakrabarti, A.K., Ghosh, A.N., Nair, G.B., Niyogi, S.K. et al., 2000. Development and evaluation of a phage typing scheme for Vibrio cholerae O139. J. Clin. Microbiol., 38, 44–49. [22] Imbeault, S., Parent, S., Lagace, M., Uhland, C.F. et al., 2006. Using bacteriophages to prevent furunculosis caused by Aeromonas salmonicida in farmed brook trout. J. Aquat. Anim. Health, 18, 203–214. [23] Nakai, T., Park, S.C., 2002. Bacteriophage therapy of infectious diseases in aquaculture. Res. Microbiol., 153, 13–18. [24] Pasharawips, T., Thaikua, S., Sriurairatana, S., Ruangpan, L. et al., 2005. Partial characterization of a novel bacteriophage of Vibrio harveyi isolated from shrimp culture ponds in Thailand. Virus Res., 114, 63–69. [25] Bernhardt, T.G., Wang, I.N., Struck, D.K., Young, R., 2002. Breaking free: “protein antibiotics” and phage lysis. Res. Microbiol., 153, 493–501.
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[26] Kapfhammer, D., Blass, J., Evers, S., Reidl, J., 2002. Vibrio cholerae phage K139: complete genome sequence and comparative genomics of related phages. J. Bacteriol., 184, 6592–6601. [27] Williams, B.J., Golomb, M., Phillips, T., Brownlee, J. et al., 2002. Bacteriophage HP2 of Haemophilus influenzae. J. Bacteriol., 184, 6893–6905. [28] Hayashi, T., Matsumoto, H., Ohnishi, M., Terawaki, Y., 1993. Molecular analysis of a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa: structure of the attPcos-ctx region and integration into the serine tRNA gene. Mol. Microbiol., 7, 657–667. [29] Reidl, J., Mekalanos, J.J., 1995. Characterization of Vibrio cholerae bacteriophage K139 and use of a novel minitransposon to identify a phage-encoded virulence factor. Mol. Microbiol., 18, 685–701. [30] Carlton, R.M., 1999. Phage therapy: past history and future prospects. Arch. Immunol. Ther. Exp. (Warsz), 47, 267– 274. [31] Lu, T.K., Koeris, M.S., 2011. The next generation of bacteriophage therapy. Curr. Opin. Microbiol., 14, 524–531.
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Research Paper Isolation and characterization of lytic vibriophage against Vibrio cholerae O1 from environmental water samples in Kelantan, Malaysia Ali Al-Fendi1, Rafidah Hanim Shueb1, Manickam Ravichandran2 and Chan Yean Yean1 1
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Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia Faculty of Applied Sciences, Department of Biotechnology, AIMST University, Semeling Bedong, Kedah, Malaysia
Water samples from a variety of sources in Kelantan, Malaysia (lakes, ponds, rivers, ditches, fish farms, and sewage) were screened for the presence of bacteriophages infecting Vibrio cholerae. Ten strains of V. cholerae that appeared to be free of inducible prophages were used as the host strains. Eleven bacteriophage isolates were obtained by plaque assay, three of which were lytic and further characterized. The morphologies of the three lytic phages were similar with each having an icosahedral head (ca. 50–60 nm in diameter), a neck, and a sheathed tail (ca. 90–100 nm in length) characteristic of the family Myoviridae. The genomes of the lytic phages were indistinguishable in length (ca. 33.5 kb), nuclease sensitivity (digestible with DNase I, but not RNase A or S1 nuclease), and restriction enzyme sensitivity (identical banding patterns with HindIII, no digestion with seven other enzymes). Testing for infection against 46 strains of V. cholerae and 16 other species of enteric bacteria revealed that all three isolates had a narrow host range and were only capable of infecting V. cholerae O1 El Tor Inaba. The similar morphologies, indistinguishable genome characteristics, and identical host ranges of these lytic isolates suggests that they represent one phage, or several very closely related phages, present in different water sources. These isolates are good candidates for further bio-phage-control studies. Abbreviations: VPUSM – Vibrio Phage Universiti Sains Malaysia; TEM – transmission electron microscopy; PFGE – pulsed field gel electrophoresis; MOI – multiplicity of infection; SMG – sodium/magnesium with Gelatin; PEG – polyethylene glycol Keywords: Lytic vibriophage / V. cholerae / Myoviridae / Environmental water Received: June 11, 2013; accepted: October 11, 2013 DOI 10.1002/jobm.201300458
Introduction Cholera is an acute food and waterborne communicable disease caused by Vibrio cholerae [1]. Cholera is a common cause of infant and adult deaths that has occurred frequently as epidemics in many developing countries since the early 19th century [2]. V. cholerae is divided into more than 206 serogroups based on their lipopolyCorrespondence: Dr. Chan Yean Yean, Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia E-mail: [email protected] Phone: þ609 7676258 Fax: þ609 7641615 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
saccharide composition, of which only the serogroups O1 and O139 have been associated with epidemic and pandemic cholera in humans [2]. Toxigenic V. cholerae O1 consists of two biotypes: classical and El Tor. Each biotype again has two major serotypes, namely Ogawa and Inaba. Out of the seven pandemics that have taken place, the classical biotype was responsible for the first six pandemics, while the seventh was due to the El Tor biotype and was exceedingly more extensive in geographic spread and duration [3]. In late 1992, V. cholerae O139 came into the limelight by causing a severe cholera epidemic in the Indian subcontinent [4]. The occurrence of epidemics is known to coincide with increased prevalence of the causative V. cholerae strain in the
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aquatic environment. In contrast, bacteriophage in the environment has been found to inversely correlate with the abundance rate of cholera [5]. Bacteriophages are viral cellular parasites that depend on bacterial cell processes to produce viral proteins and viral particles [6–8]. The ability of bacteriophage to replicate exponentially and lyse pathogenic strains of bacteria suggests that they could play a vital role in controlling bacteria populations in natural systems [9–11]. Thus, using bacteriophage as a therapeutic agent is one possible option to control pathogenic bacteria [12]. Previous trials in aquaculture systems have successfully eradicated pathogens using bacteriophages [10, 11, 13]. Vinod et al. [13] and Crothers-Stomps et al. [11] isolated phage against Vibrio harveyi and these phages have shown potential for use as biocontrol agents to combat vibriosis in the aquaculture systems. Therefore, this study was undertaken to isolate vibriophage with lytic activity against V. cholerae.
Materials and methods Sample collection Environmental water samples (250–300 ml) were obtained by dipping sterile sampling bag (Nasco, Salida, California, USA) from various sources including rivers, lake, sewages, fish farm, ditch, and ponds in Kelantan, Malaysia as described in Table 1. Following collection, samples were transported to the laboratory on ice. Upon arrival, the samples were immediately filtered using 0.22 mm pore cellulose acetate filters (Minisart, Goettingen, Germany) and processed for bacteriophage isolation. Bacterial strains Bacterial strains used in this study are listed in Table 2. Forty-six V. cholerae strains were obtained from the Department of Medical Microbiology and Parasitology, Universiti Sains Malaysia. The bacteria studied comprised 16 strains of V. cholerae O1 El Tor Ogawa, 14 strains V. cholerae O1 El Tor Inaba, 7 strains of V. cholerae O1 classical, 5 strains of V. cholerae O139, and 4 strains of V. cholerae Non-O1/O139. In addition, 16 other enteric bacterial species were also employed to determine the phage host range (Table 2). The V. cholerae strains used as host to detect bacteriophages were examined for the presence of endogenous phage using a minor modification of the method described by Oakey et al. [14]. All V. cholerae were cultured in 10 ml LB broth and then grown at 37 °C for 2–3 h until the optical density (OD) at 600 nm (OD600) was 0.4. The cultures were divided into two equal aliquots ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Table 1. Isolation of vibriophages from different water sources and sampling periods in Kelantan, Malaysia, between April 2011 and March 2012. Isolated vibriophages
Source of environmental water
NI Lake VPUSM 1 Lake VPUSM 2 Pond VPUSM 3 Lake NI Sewage Pond VPUSM 4a VPUSM 5 Ditch NI River NI Ditch NI Pond VPUSM 6 Sewage NI River NI Fish Farm Fish Farm VPUSM 7a Ditch VPUSM 8a NI River VPUSM 9 Lake NI Ditch VPUSM 10 Fish Farm VPUSM 11 Sewage Total: 11 vibriophages
Sampling period April 2011 May 2011 May 2011 June 2011 June 2011 July 2011 July 2011 August 2011 August 2011 September 2011 September 2011 October 2011 November2011 December 2011 December 2011 January 2012 February 2012 February 2012 March 2012 March 2012
No. of water samples 3 5 2 5 1 6 2 4 1 4 4 7 6 7 2 4 3 3 4 2 75
NI, no isolation. a These isolated vibriophages were lytic and further characterized in this study.
and one of each pair was treated with 30 ng ml1 mitomycin C (Sigma–Aldrich, Castle Hill, NSW, Australia). The cultures were incubated at 37 °C and the OD600 was recorded periodically up to 8 h. Cell lysis, as indicated by a relative decrease in OD600 compared with the untreated control, was taken to indicate the presence of endogenous phage. A panel of V. cholerae strains, two of each V. cholerae El Tor Inaba, O1 El Tor Ogawa, O1 classical Inaba, O1 classical Ogawa, and O139, identified as not containing endogenous phage were challenged with environmental water samples to screen for vibriophages. Vibriophage isolation Two methods of vibriophage isolation were used with some modification. The first method was a spot plaque assay [5]. To prepare V. cholerae lawn on the plate, 0.5 ml (log phase) of each V. cholerae panel strain in LB broth was mixed in 3 ml of soft agar (LB broth containing 0.8% Agar, Merck) and the mixtures were overlaid on LB agar plates. Fifty milliliters of water samples were filtered through 0.22 mm pore filters to remove bacteria. One milliliter of the filtered water samples were added to young (0.4 OD600) V. cholerae panel cultures grown in 10 ml LB
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Table 2. Host range of isolated vibriophages. Plaque formation Species/Strain Vibrio cholerae, O1 El Tor Ogawa (n ¼ 16) Vibrio cholerae, O1 El Tor Inaba (n ¼ 14) Vibrio cholerae, O1 classical (n ¼ 7) Vibrio cholerae, O139 (n ¼ 5) Vibrio cholerae, nonO1/O139 (n ¼ 4) Vibrio vulnificus (n ¼ 2) Vibrio parahaemolyticus (n ¼ 2) Escherichia coli (n ¼ 2) Escherichia coli (EPEC) (n ¼ 2) Shigella dysenteriae (n ¼ 1) Shigella flexneri (n ¼ 2) Shigella sonnei (n ¼ 1) Proteus vulgaris (n ¼ 2) Proteus mirabillis (n ¼ 2) Klebsiella pneumonia (n ¼ 3) Klebsiella spp. (n ¼ 2) Salmonella typhimurium (n ¼ 4) Salmonella enteritidis (n ¼ 2) Morganella morganai (n ¼ 2) Serratia marcescens (n ¼ 2) Yerssnia enterocolitica (n ¼ 2) Enterococcus faecalis (n ¼ 1)
VPUSM VPUSM VPUSM 4 7 8 – þ – – – – – – – – – – – – – – – – – – – –
– þ – – – – – – – – – – – – – – – – – – – –
– þ – – – – – – – – – – – – – – – – – – – –
broth and further incubated at 37 °C with shaking for 6 h. The enriched cultures mixtures were centrifuged at 10,000g for 10 min at 4 °C. Aliquots of supernatant (25 ml) pre-filtered through 0.22 mm pore filters were inoculated on lawns of V. cholerae lawn. Plates were left at room temperature for 15 min and then incubated at 37 °C for 16 h, and inspected for clearing zone. In the second method to isolate lytic phage, water samples were enriched and filtrate was obtained as described in the first method and examined using the soft agar overlay method described by Phumkhachorn and Rattanachaikunsopon [15]. For each water sample, a 10fold dilution series (to 1010) was prepared and, for each dilution, an aliquot of 100 ml of each serial dilution was mixed with 100 ml of young (OD600 0.4) V. cholerae panel cultures and incubated at 37 °C for 15 min. The mixture was added to 5 ml of LB soft agar (0.8% agar), mixed gently by rolling between the palms to enhance good mixing and then overlaid on empty plates. After solidifying, the plates were incubated at 37 °C for 16 h and the presence of lytic phage in the form of plaques was examined. Purification of vibriophage All isolated vibriophages were purified by threefold successive single-plaque isolation method as mentioned in the soft agar overlay method. A single plaque was picked with a sterile pipette tip (Axygen Scientific, Mexico, Mexico) and inoculated into a log-phase ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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V. cholerae culture. After incubation at 37 °C with shaking (200 rpm) for 6 h and static incubation for 12 h, the vibriophage host mixture was centrifuged at 10,000g for 20 min at 4 °C and filtered through a 0.22 mm filter. The isolated vibriophage filtrates were designated as the vibriophage suspensions. Propagation and determination of vibriophage titers Vibriophage suspensions were propagated further to obtain higher titer using the aforementioned soft agar overlay method. A broth culture of the respective V. cholerae was infected with vibriophage at a multiplicity of infection (MOI) of 0.01 and adsorption was allowed to occur at 37 °C for 15 min. The mixture was overlaid and incubated at 37 °C until complete lysis occurred within 6–8 h. The soft agar layer was harvested with 3 ml of sodium/magnesium with gelatin (SMG) buffer (NaCl, 5.8 g; MgSO4 · 7H2O, 2 g; tris pH 7.5, 50 ml; gelatin, 0.1 g; distilled H2O, 950 ml) in 15 ml centrifuge tube (Cellstar tubes; Greiner Bio-One, Frickenhausen, Germany) and incubated at room temperature for 1 h with shaking, then centrifuged at 10,000g for 20 min at 4 °C. The propagated vibriophages were serially diluted in SMG buffer. Each dilution was subjected to plague assay using the aforementioned soft agar overlay method. Plaques were counted and expressed as plaque forming units/milliliter (pfu ml1). High-titer vibriophage stocks were used to test for their host range and morphology was determined by electron microscopy. Determination of host ranges of vibriophages The host ranges of isolated vibriophages were determined by both methods described above (Table 2). The plates were incubated at 37 °C for 16 h before the presence of a clear zone in the plates, indicating the ability of vibriophage to infect the tested bacterial strains, was examined. Concentration of vibriophages Two methods were used for concentration of vibriophage suspensions. The first method was a standard polyethylene glycol (PEG) precipitation described by Yamamoto et al. [16] where sodium chloride was added to vibriophage suspension to a final concentration of 1 M, and the suspension was stored with continuous mixing for 1 h on ice. Next, the vibriophage particles were precipitated by adding 10% w/v PEG 8000 (Sigma– Aldrich, Saint Louis, MO, USA) to the suspension and incubated at 4 °C for 6 h. The precipitated particles were pelleted by centrifuging at 12,000g for 20 min at 4 °C. The pellet was then washed and resuspended in 0.5 ml SMG buffer.
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The second concentration method was carried out as described by Oakey et al. [14]. High-titer vibriophages (1010–1012) were subjected to ultracentrifugation at 200,000g for 2 h at 4 °C using a CP 80 Mx ultracentrifuge with a swing bucket rotor (Hitachi, Hitachinaka, Japan). After centrifugation, phage was eluted from the pellets by soaking overnight in 0.01 part of the original volume of sterile SMG buffer and then gently pipetting into 1.5 ml sterile centrifuge tubes. The concentrated vibriophages in both methods were titrated and stored at 4 °C for further work. Transmission electron microscopy (TEM) Vibriophage particles recovered by the ultracentrifuge method with a titer 108 pfu were negatively stained with 2% methylamine-tungstate stain (pH 6.5). A drop of vibriophage particles was deposited on a 400 mesh electron microscopic grid containing a copper–carbon gold film. A drop of methylamine-tungstate stain was then added and gently mixed with the vibriophage. After 1 min, excess fluid was withdrawn with a filter paper and the grid was allowed to dry. Stained particles were observed in a TEM-Philips CM12 apparatus (Philips Electron Optic, Eindhoven, The Netherlands). The size of the phages’ head and tail were measured in triplicates using SIS Docu Version 3.2 image software (Soft Imaging GmbH, Munster, Germany) and the mean and standard deviation was calculated using SPSS (IBM SPSS Statistics v20, New York, USA).
with some modifications. Briefly, 400 ml of vibriophage concentrated by PEG was mixed with 400 ml of 1.2% low melt agarose (Bio-Rad, Hercules, CA, USA) and the mixture was immediately transferred to PFGE plug casting mold. After solidified, plugs were treated with lysis buffer (50 mM tris, pH8.0, 50 mM EDTA, and 0.1% SDS) and 25 ml of proteinase K solution (20 mg ml1) for 2 h at 54 °C, followed by two washes in TE buffer (10 mM tris, pH 8.0, 1 mM EDTA) at 54 °C for 30 min. Plugs were loaded on 1% pulsed field certified agarose (Bio-Rad) in 0.5 TBE buffer (45 mM tris borate and 1 mM EDTA). The gels were run on CHEF-DRII electrophoresis system (Bio-Rad) in 0.5X TBE buffer at 14 °C, 6.0 V cm1, 120° angle, 1 s initial switch time, 6 s final switch time, and total run time of 18 h. “CHEF DNA size standards” for pulsed-field electrophoresis (Bio-Rad) was used to determine the DNA size. Results were visualized by staining with ethidium bromide. Nucleic acid type and restriction analysis The type of nucleic acid was analyzed by treatment with DNase I, RNase A, and S1 nuclease (Thermo Scientific). Eight restriction enzymes were used (BglII, EcoRI, EcoRV, Hin1ll, HindIII, ScaI, SmaI, and XbaI) and added into 1 mg of vibriophage nucleic acid using methods as recommended by supplier (Thermo Scientific). Uncut and digested nucleic acid was analyzed using 0.8% agarose gel at 60 V for 150 min.
Results Vibriophage nucleic acid extraction The nucleic acids of vibriophage particles were extracted from vibriophage suspension according to the method of Pickard [17] with modifications. RNase A (Sigma–Aldrich) to final concentration of 80 ml ml1 was added into 1.8 ml of vibriophage suspension (107 pfu) for 30 min at 37 °C. Then, sodium dodecyl sulfate (SDS) and proteinase K to final concentration of 1% and 170 ml ml1, respectively were added and the mixture was incubated at 37 °C for an additional 30 min. Proteins were removed by two phenol/ chloroform/isoamyl alcohol extractions and nucleic acid was precipitated with 100% isopropanol and 3 mol L1 of sodium acetate. After washing with 70% ethanol, the pellet was resuspended in TE buffer (10 mM tris, pH8.0, 1 mM EDTA). Nucleic acid concentration was determined by OD260/280 nm reading and the quality of the nucleic acid was observed by agarose gel electrophoresis (0.8% gel, 60 V, 90 min).
Isolation of vibriophages Seventy-five environmental water samples were collected from different areas in Kelantan, Malaysia (Table 1). Of these, 11 vibriophages were successfully isolated by both methods. However, the modified soft agar overlay method showed a better result for the isolation of the lytic vibriophages from environmental samples (data not shown). The lytic vibriophages produced clear and/or transparent plaques, compared with the spotting method, which sometimes produced cloudy or fuzzy plaques on host lawns. The isolated vibriophages were designated Vibriophage Universiti Sains Malaysia (VPUSM) 1 to VPUSM 11 based on the period and source of isolation. Most of the bacteriophages showed temperate activity (data not shown), but three of them (VPUSM 4, VPUSM 7, and VPUSM 8) showed lytic activity and produced small, round, and clear plaques with a diameter of 3–5 mm (Fig. 1).
Determination of bacteriophage genome size by PFGE Determination of vibriophage genomic DNA size using PFGE was performed as described by Talledo et al. [18]
Host range determination The host range of these three lytic vibriophages was determined by the soft agar overlay method. Only
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Figure 1. Plaque morphology of isolated vibriophages on V. cholerae O1 El Tor Inaba lawns culture. (a) VPUSM 4; (b) VPUSM 7; and (c) VPUSM 8.
V. cholerae O1 El Tor Inaba strains were sensitive and resulted in lysis. Strains of common enteric pathogens including Salmonella, Shigella, and Escherichia coli were not sensitive to these vibriophages including other V. cholerae biotypes and serogroups. These results suggest that VPUSM 4, VPUSM 7, and VPUSM 8 are specific vibriophages and have narrow host ranges with specificity to V. cholerae O1 El Tor Inaba (Table 2). Morphology of vibriophages Electron micrographs of the three isolated lytic vibriophages are shown in Fig. 2. The diameter of heads (distance between opposite apices) of VPUSM 4, VPUSM 7, and VPUSM 8 particles were 51.38 0.2, 55.32 1.1, and 58.12 1.4 nm, respectively, while the length of the tails were 90.7 6.4, 95.10 2.4, and 99.07 1.6 nm, respectively. TEM analysis showed that the predominant ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Figure 2. Electron micrograph of vibriophages isolated from environmental waters in Malaysia. (a) VPUSM 4; (b) VPUSM 7; and (c) VPUSM 8. Bars, 100 nm.
morphology of vibriophages against V. cholerae O1 El Tor Inaba was an icosahedral head with contractile, rigid, and partially sheathed tail and a neck was evident between the head and the tail. Based on morphology, the VPUSM 4, VPUSM 7, and VPUSM 8 particles most likely belong to the family Myoviridae [19, 20]. Genomic characterization of vibriophages Vibriophages concentrated by PEG method were assayed for genome size using PFGE (Fig. 3). The bands
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or closely related phages, and belong to Myoviridae family.
Discussion
Figure 3. Vibriophages isolated genomic DNA size determinated at 33.5 kb on PFGE. Lanes: M, ladder with standard DNA size 8– 48 kb (marker); lane 1, VPUSM 4; lane 2, VPUSM 7; and lane 3, VPUSM 8.
corresponding to the DNA of all three vibriophages were identical with size of approximately 33.5 kb. Nucleic acid of vibriophages VPUSM 4, VPUSM 7, and VPUSM 8 were then extracted and subjected to enzymatic digestion analysis. The genomic nucleic acid of these vibriophages were found to be a double stranded DNA following successful digestion with DNase 1 but not with RNase A and S1 nuclease. However, of eight restriction enzymes tested, only HindIII resulted in digestion of the nucleic acid of these three vibriophages and showed identical restriction digest profiles with fragments of approximately 9000, 6000, 5000, 4000, 3500, 2200, 2000, and 1750 bp (Fig. 4). Based on morphology and genomic characterization, the isolated lytic bacteriophages are probably identical
Figure 4. Vibriophage DNA digested with HindIII restriction enzyme. Lanes: M, 1 kb DNA ladder; lane C, vibriophage DNA undigested; lane 1, DNA digested for 1 h; lane 2, DNA digested for 2 h. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Phage therapy is one of the promising approaches to control human and environmental bacterial infections. Vibriophages can be isolated from the environment in which V. cholerae strains generally survive and can be found in sewage, feces, soil, and water [21, 22]. The characteristic of narrow host range of the vibriophage is a significant benefit for phage therapy; it is useful either individually or in combination with other V. cholerae lytic vibriophages as vibriophage cocktails to be employed as biological control agents in cholera endemic areas [5, 11, 23]. In this study, three lytic vibriophages were isolated from 75 environmental water samples after initial enrichment in broths seeded with V. cholerae. As a tailed virus of bacteria, the VPUSM 4, VPUSM 7, and VPUSM 8 vibriophages fell into the order Caudovirales that contains three families of tailed viruses that infect bacteria and archaea [24]. Possession of small icosahedral heads and defined collar/neck regions with contractile tail sheaths would tentatively place the bacteriophages in the family Myoviridae. The genomic size of myophages is typically >20 kb in size [25]. For example, K139, 33.1 kb [26]; HP2, 31.5 kb [27]; FP15, 29 kb [19]; VHML, 43.2 kb [14]; and FCTX, 35.5 kb [28]. Based on HindIII digestion analysis and PFGE, the isolated lytic vibriophages genomic DNA size was estimated at 33.5 kb, similar to the sizes of many bacteriophages in the family Myoviridae. Furthermore, the genomes of isolated lytic vibriophages are linear double-stranded DNA as the genomic nucleic acid could be digested with DNase 1 but not with RNase A and S1 nuclease. From previous studies, most of the vibriophages were found to be myophages with doublestranded DNA [20]. Morphologically, these vibriophages resembled the bacteriophages (k139 and FP15) previously reported to be associated with V. cholerae [18, 26]. By electron microscopy, all had icosahedral heads of approximately 60 nm diameter and defined collar/neck regions, contractile tail sheaths of approximately 100 nm. However, the K139 vibriophage and FP15 vibriophage have different host range and temperate activities to V. cholerae O139 and O1 El Tor, respectively [18, 26, 29]. In the current study, the host range of isolated vibriophages was relatively narrow and could only infect V. cholerae O1 El Tor Inaba. Some bacteriophages could infect more than one related species or even genus, while some may have a restricted host range,
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often only a single species or subspecies [30, 31]. Faruque et al. [5] suggested that vibriophages could play an important role in ending cholera epidemics. The absence of vibriophages in the aquatic environmental might promote cholera epidemic outbreak, particularly when V. cholerae is introduced for the first time. In late 2009, a cholera outbreak caused by V. cholerae O1 El Tor Ogawa was reported in Kelantan, Malaysia [3]. Hence, our data suggest that the presence of lytic vibriophage to V. cholerae O1 El Tor Inaba in Kelantan environmental water samples helps control the potential emergence of V. cholerae O1 El Tor Inaba outbreak in the state. In conclusion, we have isolated three lytic bacteriophages from different waters sources that infect V. cholerae O1 El Tor Inaba. The isolates were indistinguishable by the tests performed in the present study. Further detailed study of these phages is needed to determine if they are in fact identical and to explore their potential for use as biocontrol agents for phage therapy to prevent and treat cholera. Physiochemical studies like thermal and pH determinations, growth characteristic, structural proteins, and extensive study of their genomes will prove helpful in development of safe phage preparations having optimal efficacy against their specific bacteria hosts and on designing strategies for integrating this isolated vibriophage as biocontrol agent therapy for preventing and treating V. cholerae.
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Acknowledgments This publication was financially supported by RUI grant (1001/PPSP/813045) from Universiti Sains Malaysia. The authors show their deep appreciation to staff of Public Health in Kota Bharu, Tumpat and Pasir Mas District of Ministry of Health Malaysia for their technical support in this work.
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Conflict of interest statement
[15] Phumkhachorn, P., Ratianachaikunsopon, P., 2010. Isolation and partial characterization of a bacteriophage infecting the shrimp pathogen Vibrio harveyi. Afr. J. Microbiol. Res., 4, 1794–1800. [16] Yamamoto, K.R., Alberta, B.M., Benzinger, R., Lawhorne, L. et al., 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology, 40, 734–744.
No competing financial interests exist.
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