Isolation and Characterization of some Bacteriophages and their Associated Bacteria in Sea Food : Phage-Host Interaction

Background: Economic loss due to diseases is a major problem in aquaculture. A number of bacterial illnesses may arise from the consumption of sea food that has been contaminated at source or during the processing. This study aims to isolate and characterize some bacteriophages specific for the most common sea food pathogens in addition to their associated bacteria. The work also extended to study the host-phage interaction and the potential use of phages as antibacterial agents to control sea food infecting pathogens. Materials and methods: Estimation of Vibrio spp and Aeromonas spp. in addition to their associated phages in different sources of sea food such as fish (Tilapia sp.), clam (Tapes decussatns) and crab (Callinectes sapidus) was carried out. Results: Crab exhibited the highest counts (200 CFU/g) of Vibrio spp., while the highest counts of Vibriophages (1500 PFU/g) were detected in clam. Fish harbored the highest counts (30 CFU/g) and (3000 PFU/g) of Aeromonas spp. and Aeromonas phages, respectively. Three different phages (VPS1, APS2 and APS3) in addition to the isolated bacteria were characterized. Results showed that VPS1, APS2 and APS3 were different, which was confirmed by molecular characterization using RAPD-PCR and the protein profile. APS3 was entrapped into calcium alginate beads and tested as antibacterial agent against Aeromonas hydrophila. Efficiency of APS3 was superior to the free particles (p<0.05) and realized 2.7 fold decrease in bacterial growth rate. Phage seeded beads were recycled for 7 successive cycles. Their activity was reduced up on reuse. Recommendations: This study revealed that phages remain an excellent potential tool for control of bacterial pathogens. Further research and manipulation of the isolated phages could produce novel effective biocontrol agents of sea food infectious diseases in marine aquaculture systems


INTRODUCTION
Sea food is a popular part of the diet in many parts of the world and in some countries constitutes the main supply of animal protein.Great economic losses in aquaculture are attributed to infection by different pathogens where microbial sea food-borne diseases represents 10 to 20% of the total food-borne outbreaks; most of them are from bacterial infections.(1,2)   The microbiological flora in the sea food Bull High Inst Public Health Vol.41 No. 4 [2011]   is believed to be a reflection of general contamination in the aquatic environment where accumulation and concentration of bacteria and viruses from the environment is generally taking place.
(3) Different pathogenic bacteria were previously isolated from sea food. (4)Vibrio spp.are an important cause of illnesses in molluscan bivalves that concentrate different particles during their filter feeding. (5)For a long time vibriosis caused by Vibrio anguillarum was the most serious disease in cultured populations.It causes a bacteraemia in salmonoid fish that leads to internal haemorrhage which leads to loss of yield and quality of fish after infection.(6)   Aeromonas hydrophila, the most common bacterial pathogen in fresh water fish, has been recognized to be the etiological agent of many pathological conditions, including tail rot, Aeromonas septicemia and epizootic ulcerative syndrome.(7)   Control of diseases in aquaculture was dependant on the use of chemical compounds, which resulted in the development of antibiotic resistance and its transfer among cells through plasmids or bacteriophages.Therefore there is an urgent need to have alternative tools for pathogen control in aquaculture.(8)   Bacteriophages are bacterial viruses extremely abundant in nature and believed to be important in controlling bacterial populations in natural systems. (9)Phage therapy may represent a viable alternative to antibiotics to inactivate pathogenic bacteria. (10)To find the ecological impact of marine phages on bacterial populations, it is necessary to carryout a detailed study of their diversity and host specificity.Also, virus-host interaction provides model systems to determine the effect and activity of the phage infection on the structure of the natural bacterial communities.

Although bacteriophages were
proposed for several applications in food safety to control the major pathogenic bacteria, (11)   only few applications in Sahar Wefky Mostafa Hassan 405 seafood were reported. (12)The treatment of microbial diseases is still difficult and might involve environmental hazards.A possible method to confront this problem might be the oral administration of antimicrobial materials to the larvae through the food chain, using the immobilization technique, (13)   in which the active components are covered by a layer of another material such as alginate, carrageenan and agarose.
Immobilization, either by covalent linkage to an insoluble matrix or by entrapment into gel of film support, could provide stability to phages as reported for enzymes and bacteriocins. (15)Immobilized phage could be particularly useful to create antimicrobial surfaces against pathogenic bacteria and was used in different applications.(16-19)   The aim of the present study was to estimate the counts of different groups of pathogenic bacteria such as Vibrio spp.
and Aeromonas spp., in addition to their specific phages in different sources of sea food.The work also extended to include isolation and characterization of some bacteriophages and their associated bacteria.Host-phage interaction was carried out to study the potential use of phages as ecofriendly alternatives to the chemotherapeutic agents.

MATERIALS AND METHODS
Cross section and experimental designs were followed.Fresh samples of fish ( The prepared samples were diluted in sterile sea water and 0. Phages were isolated directly from the supernatant of fish kidney, clam and crab homogenates as previously described.Serial dilutions of each supernatant were prepared in sterile seawater.Aliquots of each dilution were adsorbed to 0.2 ml of log-phase host cultures for 15 min, and virulent phages were detected by using the soft-agar overlay technique. (22)The plating medium and soft-agar overlay were prepared with CPM medium supplemented with 1.5 and 0.7% Bacto Agar (Difco), respectively.The plates were incubated at 30°C, and plaques were detected at 24 -shape and size were isolated, purified and propagated to represent different phage isolates, namely VPS1, APS2 and APS3.
The titre of each isolate was determined.
Recovery of purified intact phage was achieved according to Sambrook et al. (23) To suspension system was removed by pipetting and optical density (O.D.) was measured at 550 nm. (26)The optical density of the free phage samples was also measured as mentioned above.The active entrapped phage particles were recycled for seven successive cycles.

Statistical analysis
Results were analyzed by numerical techniques using the simple matching coefficient (SsM) (27) and clustering was achieved by unweighted pair group average linkage (UPGMA).(28,29)   The computations were performed by using SYSTAT-PC program V7.On the other hand, clam harbored the highest counts of vibriophages (15000 PFU/g) followed by fish (5000 PFU/ g), while only fish exhibited high counts of Aeromonas phages (3000 PFU/g).Phenon C: This phenon was the minor group (2 strains) with 84.2% similarity.They were isolated from clam and fish.
One strain of each phenon was selected and identified by the aid of Bergy's Manual of Systematic Bacteriology.Member of phenon A was identified as V. parahaemolyticus and member of phenon B was identified as V.
alginolyticus while member of phenon C was identified as Aeromonas hydrophila .

Characterization of the isolated phages 1-Electron microscopy examination and identification of the phage isolates
The study was extended to isolation of different phages depending on morphology and size as shown in figure 3.
Electron micrographs showed that VPS1 (figure 3a) has icosahedral head and long tail and thus belongs to family Siphoviridae morphotype 1 as it has collar like structure between the head and the tail with no additional appendages on its head or tail.On the other hand, APS2 (figure 3b) belongs to family Myoviridae morphotype 2 as it has icosahedral head and long tail with no special appendages.APS3 (figure 3c) has icosaherdal head and short tail and thus belongs to family Podoviridae.4 shows that all the used primers were able to differentiate between the three phages.
RAPD-PCR results obtained using primer 2 showed a specific band of molecular size 1188 bp for APS2, which was absent completely in case of VPS1 and APS3.
RAPD results with primer 3 were also able to differentiate between the three phages.

Phage-host Interaction: Antibacterial activity of APS3 against A. hydrophila
The aim of this part was to study the  provide important information for control of water quality. (31)In the present study, Vibrio spp.and their associated phages detected in the tested sea food samples outnumbered the counts of Aeromonas spp.and their associated phages.Also, there was complete absence of Aeromonas spp. in crab which was consistent with that reported by Boutaib et al. (4) This may be attributed to high tolerance of Vibrio spp.and their phages to a wide range of salinity and their tend to be more common in warm water, when temperature exceeds 17°C.In the present study, ten of the isolated bacterial spp.were chosen and characterized.The general characteristics of temperature and Na Cl requirements were indicative of their marine origin as was described by Wichels et al. (34) The identified strains were previously isolated from sea food samples. (35)These included

V. parahaemolyticus, V. alginolyticus, and
A. hydrophila and also were documented as hosts for phages.(21)   Electron microscopy was used to classify the isolates into their appropriate taxonomic position. (36)In the present study, VPS1 and APS2 assigned to order caudovirales as they have long tail and double stranded DNA.Our results were in accordance with that reported by Fattouh et al., (16) and Pereira et al., (10) where the tailed phages predominated other morphotypes.VPS1, APS2 and APS3 were found to belong to family Siphoviridae, Myoviridae and Podoviridae, respectively as was proposed by Murphy et al. (37) APS3 belonged to family Podoviridae.Frank and Moebus (38) isolated a number of phages belonging to Podoviridae.DePaola et al. (21) also reported the existence of phages belonging to Podoviridae in bivalves.
Random amplification of polymorphic DNA (RAPD) technique is used extensively for the epidemiological investigation and differentiation of many microorganisms.
( 42) .In the present study, RAPD technique detected some polymorphic regions and differences between the three isolates and confirmed that they were not identical.
In the present study, SDS PAGE analysis of APS3 showed three major bands with molecular masses of 46, 30 and 17 KDa.This range nearly agreed with that of Elshayeb et al., (43) who found that SDSPAGE analysis of purified phages showed three major bands with apparent molecular masses of 47, 34 and 16 kDa.It was also consistent with the results reported by Barbian and Minnick. (44)There were common bands among the three Tilapia sp.), clam (Tapes decussatns) and crab (Callinectes sapidus) were purchased from the market in January, 2010.Samples were held at 5 to 10°C during shipment for 24 to 30 hours prior to analysis.Ten of each sample type were scrubbed, shucked, mixed with an equal (1:1) weight of Butterfield's phosphate-buffered saline and blended.

( 21 )
All media and diluents were prepared in seawater and diluted with deionized seawater.Casamino acids peptone marine (CPM) broth (5.0 g of Casamino Acids[Difco], 5.0 g of Bacto Peptone [Difco], and 1.0 liter of seawater, autoclaved for 15 min at 121°C) was used as a growth medium.
phage lysate tube, RNase A and DNase I (Sigma Chemical Co.) were added to a final concentration of 1 μg/ml and incubated at 37°C for 30 min.Polyethelene glycol (PEG, molecular biology grade, MW= 8,000) and sodium chloride were added to the tubes, each at 9.3 g and 5.8 g per 100 ml of lysate, respectively.The tubes were inverted several times to dissolve PEG and sodium chloride completely.The tubes were kept on ice for two hours and the precipitate was then recovered by centrifugation at 10,000 rpm for 20 min at 4°C.The supernatant was allowed to drain, and the precipitate containing the purified intact phage isolates was each resuspended by gentle vortexing in 2 ml of phage buffer.The morphology of the three phage isolates VPS1, APS2 and APS3 were investigated with electron microscopy.Phages were negatively stained with 2% sodium tungestate in bi-distilled water at pH 6-7.5.Five μl of each phage suspension were dropped onto a carboncoated grid.The excess liquid was removed with filter paper after 1 min.Five μl of dye solution were added and after 1 min, the grid was dried.The grids were then examined and electron micrographs were taken with a T. E. M. (JEOL 100 CX) operating at 80 kv.Phage DNA was isolated and purified as follows: each supernatant containing the purified phage was transferred to another tube and 100μl of 10 % sodium dodecyl sulphate (SDS) and 100 μl of 0.5 M EDTA pH 8.0 per 100 ml of phage lysate were added.The tubes were incubated at 68°C for 20 min, after which phenol/chloroform extraction was carried out at 12,000 rpm for 5 min at 4°C.The upper aqueous phase was then transferred to a clean tube, an equal volume of isopropanol was added and the tubes were kept at -20°C Bull High Inst Public Health Vol.41 No.4 [2011] for 1 hour, after which the DNA was collected by centrifugation at 12,000 rpm for 20 min at 4°C.Each supernatant was drained carefully and the pellets were washed with 1 ml of 70% ethanol, dried at room temperature for 15 min and finally resuspended in 200 μl of sterile distilled water.(23) The purified phage DNA isolated from each phage isolate was analyzed using Random Amplification of Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) Amplification reactions were performed in a total volume of 50 μl containing 100 μM each of dATP, dTTP, dCTP, and dGTP, 0.2M of each RAPD primer, 25 ng of template DNA and 1.25 units of Taq polymerase in 1x PCR buffer containing 2.5 mM MgCl 2 .The reaction mixtures were subjected to amplification as follows: 45 cycles of 1 min at 94°C, 1 min at 37°C and 1 min at 72°C.After the last cycle, samples were maintained at 72°C for 10 min.Amplification products were analyzed by agarose gel (1%) electrophoresis, stained with ethidium bromide and DNA profiles were documented and analyzed using Alfa Imager 1200 Tm.Faint, <1 % of total intensity, and inconsistent bands most likely the result of poor primer-template matching, were excluded from the analysis.Amplification reactions were done using one of the following primers: primer 1: 6-d: 5`-(AAGAGCCCGT)-3`; primer 2: 6-d :5`(AACGCGCAAC)-3` and primer 3: 6-d: 5`(CCCGTCAGCA)-3`.The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli, (24) using SDS-PAGE (10%).Protein samples were prepared by mixing 50 µg proteins with 5X sample application buffer [0.6 M Tris-HCl, pH 6.8, 1% (w/v) SDS, 10 % β-mercaptoethanol, 10 % sucrose and 0.05 % bromophenol blue] to give a final concentration 1X.Samples (50 ug each) were then boiled in water bath for 3 minutes at 95C.Samples were applied to the slab gel along with a molecular Sahar Wefky Mostafa Hassan 409 weight marker.Electrophoresis was carried out at a constant voltage 150 volts for about 2 hours.The gel was then stained with Coomassie Brilliant Blue R-250 [0.1 % Coomassie Brilliant Blue R-250 in 50 % Methanol, 10 % acetic acid] for 2 hours with gentle agitation at room temperature.The gel was destained overnight using a destain solution (100 ml methanol, 70 ml acetic acid and 830 ml distilled water).The isolated bacteria, in addition to three reference strains of V. anguillarum, A. hydrophila and P. fluorescens, were grown to log phase and plated on CPM medium by the soft-agar overlay technique.After 1 hour, 4 µl from a phage stock was spotted onto the plates, and the plates were incubated overnight at 30°C.Bacterial strains were considered susceptible to phages that produced either clear or turbid plaques.APS3 particles were entrapped using sodium alginate (2% w/v).Sodium alginate was purchased from Sisco Research Laboratories Pvt. Ltd., India.Beads were obtained by mixing the active phage with sodium alginate solution, then homogenized and dropped from a hydrodermic syringe to 100 ml of calcium chloride solution (2%) with constant stirring at room temperature.The formed beads were maintained in the gelling bath to harden for 1 hour.Then, they were filtered through a Whatman No. 1 paper and washed with sterile distilled water.To test the antibacterial activity of the free and entrapped phage, the test bacteria were grown at 30 o C for 24 hours on nutrient broth.A cell suspension of each microorganism was used for the antimicrobial test.The antibacterial activity was evaluated by using the shake-flask method. (25)In this test, 50 ml of each cell suspension and predetermined amounts of beads were placed in a sterilized flask and continuously shaken at 150 rpm on a rotary shaker.At prescribed time intervals, 1.0 ml of sample solution from the bead/microbial Bull High Inst Public Health Vol.41 No.4 [2011]

Figure 1 .
Figure 1.Average total counts of Vibrio spp.and Aeromoas spp.(CFU/g) and their corresponding phages (PFU/g) in different sea food samples (Fish, clam and crab)

Figure 3 .
Figure 3. Electron micrographs showing a) VPS1, b) APS2 and c) APS3 Figure There were bands of molecular sizes 185 and 130 pb for APS2 and bands of molecular sizes 165 and 115 pb for APS3, which were completely absent from VPS1.There were relatively identical pattern of bands mobility in case of VPS1 and APS3, however they were different from the mobility pattern of bands obtained from APS2 using primer 1.

Figure 6 .
Figure 6.Effect of entrapment of APS3 on the interaction between it and A. hydrophilaRecycling of the entrapped phage particlesAPS3 seeded alginate beads were tested for their efficiency in reducing the growth rate of A. hydrophila for seven successive cycles.figure7showed that the entrapped phage particles were better than free

Figure 7 .
Figure 7. Effect of recycled APS3 seeded alginate beads on the growth rate of A. hydrophila aquatic environment and in foods of marine origin is a function of the geographic and hydrographic conditions in the area, and varies according to the time of year and location within the lagoon systems.e b a c t e r i a F r e e p h a g e c y c l e 1 c y c l e 2 c y c l e 3 c y c l e 4 c y c l e 5 c y c l e 6 c y c l e 7Bull High Inst Public Health Vol.41 No.4 [2011]