Isolation and characterization of bacteriophages against enterotoxigenic Escherichia coli strains causing swine diseases

Six lytic bacteriophages (JH1-JH6) lytic against O8 ETEC were isolated from 40 individual fecal samples and 30 sewage samples from pig farms in Nanjing, China. Two of the bacteriophages (JH1 and JH2) with icosahedral heads, necks, contractile tails belonged to the Myoviridae family. The other four bacteriophages (JH3, JH4, JH5 and JH6) had relatively thin, long, non-contractile and belonged to the Siphoviridae family. Unlike typical Siphoviridae family members’ morphologyes had flexible tails, whereas JH5 had the straight tails and fibers. All bacteriophages produced clear plaques in double agar, with JH6 producing the largest plaques. Phages JH1 to JH6 lysed 50 to 100% of 12 O8: F18 ETEC strains, 50 to 75% of 20 O8:F4 ETEC, 5 to 42.5% of 40 non-O8 ETEC and 4.76 to 9.52% of 21 chickens E. coli strains. No lysis of E. coli strain from cows, as well as Escherichia coli in other O serogroups (O2, O5, O9, O11, O138, O139, O141 and O149), was observed. All bacteriophages titers were unaffected by exposure to pH 5 to 9 for 16 h. Sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) obtained from proteins was extracted from the various bacteriophages. The proteomes of JH1 and JH2 appear to be identical while that of the other four phages are all different indicating they are probably different. These six bacteriophages are potential candidates for prophylaxis and therapeutic strategies against diarrhea in piglets caused by O8 ETEC.


INTRODUCTION
Bacteriophages are diverse organisms in our world and exert a major influence over the microbial world (RW et al., 1999).While phages have been proposed for the treatment of bacterial diseases (Abhilash et al., 2009), the nature of phage-host interactions, poor understanding of mechanisms of bacterial pathogenesis and introduction of antibiotics have hampered the investigations into their role in therapy (Soothill, 1992).Antibiotics have been commonly used in the treatment of infectious diseases, but their wide spread and improper use has led to antibiotic resistance in porcine colibacillosis (Lu and Koeris, 2011).Recently, scientists have reported on the isolation and application of phage in the treatment of animals with resistant Escherichia coli infections (Jamalludeen et al., 2009a).
The goal of this present study was to isolate phages with lytic activity against enterotoxigenic E. coli (ETEC) serotype O8, to characterize their morphology and biologi-cal file.

Media and chemicals
Luria Bertani (LB) broth, LB agarose, and LB top agarose (soft agarose) were prepared as described by Sambrook et al. (1989).Each litre of bacteriophage broth contained trypton 10 g, yeast extract 5 g and NaCl 10 g, pH 7.5 and TS buffer (8.5 g of NaCl and 1 g tryptone per litre).The following reagents were also used: RNase I, DNase I (Roche, Basel, Switzerland, cat no: 10104159001), proteinase K, and ethidium bromide (Invitrogen, Carlsbad, CA, USA).

Bacteriophage isolation and purification
Bacteriophages were isolated from 40 individual fecal samples and 30 sewage samples from four Nanjing pig farms (during the period of October 2011 to Jun 2012).LB broth was inoculated with a mixture of equal proportions of the six E. coli VN1-O8: F18 ETEC, VN2-O8: F4 ETEC, VN3-O8:F4 STEC, VN4-O8:F18 ETEC, VN5-O8 ETEC and VN6-O8 ETEC strains and incubated for 5 h at 37°C.The samples (fecal sample in TS buffer, or sewage samples) were centrifuge the samples before filtering through a membrane filtered (0.45-µm membrane) to remove impurities and bacteria before being added to the host suspension.Twenty milliliters of LB broth and 20 ml of a suspension of E. coli strains in broth culture (OD600 = 1.4) and sample were then added to the flask incubated at 37°C for 24 h shaking to enrich E. coli bacteriophages.After incubation, the culture was centrifuged twice at 4,000 × g for 15 min at 4°C, the supernatant was collected into a sterile flask and filtered through a sterile 0.45-µm membrane filter (Fisher Scientific).To detect the presence of phage in the filtrate, spot testing was perfored as described previously (Kropinski et al., 2009).Phage preparation were obtained as described (Jamalludeen et al., 2009b) elsewhere and stored at 4°C.

Electron microscopy
Phage pareparations were applied to a carbon film and fixed to a copper grid being negatively stained with phosphotungstic acid (PTA, 2% w/v).Electron micrographs were taken with an H_7650 (HITACHI, Japan) transmission electron microscope (TEM) operating at 80 kV.Both phage morphology and dimension were capsid in diameter and having a lengthy tail (Bai et al., 2013).

Extraction of bacteriophage DNA
Bacteriophage DNA was extracted as described previously (Pickard, 2009;Sambrook et al., 1989).All bacteriophages were allowed to completely lyse host E. coli strains in a soft agar overlay.The overlay was added to SM buffer, and bacteriophages were allowed to diffuse into the buffer at 4°C for 3 to 4 h with gentle shaking as previously described.After the suspension was centrifuged at 4000 × g for 15 min, the supernatant was collected.Solid NaCl was added to a final concentration of 1 M and dissolved by swirling.Following incubation on ice for 1 h, the suspension was centrifuged at 11,000 × g for 10 min at 4°C (Beckman Coulter, J2-MC Centrifuge).The supernatant was collected, solid polyethylene glycol (PEG 8000) was added to a final concentration of 10% (w/v), and the mixture was stirred slowly at room temperature.After cooling in ice water and left to stand for 1 h on ice, the mixture was centrifuged at 14,000 × g for 10 min at 4°C.The bacteriophage pellet was then resuspended in 1 mL of SM buffer.An equal volume of chloroform was added to the phage suspension and mixed by vortexing for 30 s.The phages were separated by centrifugation at 3000 × g for 15 min at 4°C and the aqueous phages was recovered.Pancreatic DNase I and RNase I were added to a final concentration of 5 and 1 µg/mL, respectively, and allowed to digest substrates for 30 min at 37°C.EDTA, pH 8.0 was added to a final concentration of 20 mM.Proteinase K was added to a final concentration of 50 μg/mL, then sodium dodecyl sulfate (SDS, 10%) was added to a final concentration of 0.5% and the mixture was inverted several times prior to incubation at 56°C for 2 h.An equal volume of phenol: chloroform:isoamyl alcohol (25:24:1, v/v/v) was mixed into the sample.The aqueous phase was collected after centrifugation at 10,000 × g for 10 min and extracted with an equal volume of chloroform:isoamyl alcohol (24:1, v/v).Centrifugation was repeated and the aqueous phase was collected.Two volumes of ice-cold 95% ethanol were added and the mixture was kept at room temperature for 20 mins.The precipitate was collected by centrifugation at 10,000 × g for 10 min at 4°C and the pellet was washed with cold 70% ethanol.Following centrifugation at 10,000 × g at 4°C for 30 min the pellet was air dried and dissolved in 20 to 35 µL Tris-EDTA (TE) buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

Assessment of bacteriophage resistance to acidity and alkalinity
Resistance to acidity and alkalinity in suspensions of each of the six bacteriophages were evaluated by exposure to various pH conditions ranging from 1 to 11 and checked for survival over a 16-h period as described by Jamalludeen et al. (2007).In brief, a 100-µl bacteriophage suspension 10 7 (pfu/ml) and 900 µl of normal saline, pH 7.2, was also incubated at 37°C for 16 h.After incubation, a 100-µl volume of the bacteriophage suspension was serially diluted 10-fold, mixed with 100 µl of host bacterium (10 8 cfu/ml) and incubated for 15 min at 37°C before being added to 4 ml of soft agar and spread over an LB plate.Titers of the surviving bacteriophage were determined by plaque assays with 10-fold dilutions using the soft agar overlay method.

Agarose gel electrophoresis analysis of bacteriophage DNA
Genome sizes of undigested or digested bacteriophage were determined by electrophoresis (Sambrook and Russell, 2001).The bands were stained with ethidium bromide (EB) and compared against a λ phage DNA digested with Hind III marker; where indicated, purified bacteriophage DNA samples were digested with restriction enzyme Dra I before comparison by gel electrophoresis (Chakrabarti et al., 2000).

Protein of purified particles of phage
Bacteriophages particles by centrifugation through a glycerol step gradient were determined by Sambrook and Russell. (2001) after purified particles were subjected to SDS-PAGE on precast 4 to 15% gradient TRIS acrylamidegels (BioRad) along with protein molecular weight markers (Kropinski et al., 2012).The phage suspensions (approximately 10 10 pfu/ml) were boiled for 5 min and separated by SDS-PAGE loading buffer (50 mM Tris-HCl, 3% SDS, 1% β-mercaptoethanol, 20% glycerol, 0.7% bromophenol blue pH 6.8) on 12.5% acrylamide gel.Electrophoresis was initiated at 80 V until samples had run through the stacking gel (approximately 30 min).The voltage was subsequently increased to 120 V and electrophoresis was continued until the tracking dye had reached the bottom of the gel (approximately 2 h).Proteins were stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich).

Plaque characteristics and morphology of bacteriophage
All bacteriophages produced similar plaques that were clear and medium-sized, ranging from 1.5 to 3.5 mm in diameter.The approximate diameter of plaques formed by the bacteriophages was: JH1 and JH2, 1.5 mm; JH3 and JH4, 2.5 mm; JH5, 2 mm; JH6, 3.5 mm (Figure 1).Electron microscopy confirmed that two of the six bacteriophages (Figure 2); JH1 and JH2 showed icosahedral heads, necks and contractile tails belonged to the Myoviridae family.The other four bacteriophages (JH3, JH4, JH5 and JH6) had relatively thin, long, non-contractile and belonged to the Siphoviridae family.Unlike typical Siphoviridae family members' morphologyes had flexible tails, whereas JH5 had the straight tails and fibers (Ackermann, 2009).All six lytic bacteriophages (JH1 to JH6) belonged to the order Caudovirales (Ackermann, 2011).Table 1 shows the measurements of the heads and tails of the bacterio-phage obtained from electron microscopic images.The diameter of its capsid was estimated at ranging from 50±1.58 to 75 ±0.84;while all of the bacteriophages had long tails, ranging from 90 to 160 nm.

Nucleic acid of bacteriophages
Nucleic acid of all the six bacteriophages had a DNA.All the bacteriophages DNA samples were digested with restriction enzyme Dra I (Figure 3).

Bacteriophage resistance to acidity and alkalinity
Survival of bacteriophages over the range of pH 1 to 11 is shown in Table 3.The six bacteriophages were highly susceptible to acidity at pH 1 to 2 and susceptible in varying degrees to overnight exposure to pH 3 to 4. All of the bacteriophages were resistant to the range of pH 5 to 9. Bacteriophages JH4 and JH6 appeared to be slightly more resistant to acidic conditions than the others, as they survived at pH 3.

Protein of purified particles of phage
Distinct profiles of the particles were observed among the six bacteriophages tested (Figure 4).Bacteriophage particles derived from two of the bacteriophages (JH1 and JH2) contained four abundant protein bands with approximate molecular sizes of 92, 55, 43 and 9 kDa; lanes 1 and 2).Two bacteriophages (JH3 and JH4) consisted of three abundant proteins with estimated molecular sizes of 40, 38 and 34 kDa (Figure 4, lanes 3 and 4).The remaining two bacteriophages consisted of three abundant proteins with slightly different estimated molecular sizes (JH5, 32, 26 and 15 kDa; JH6, 32, 28 and 20 kDa) (Figure 4, lanes 5 and 6).

DISCUSSION
The goal of this study was to isolate and characterize bacteriophages against O8 E. coli, with special emphasis on those infecting O8:F18 ETEC, which were reported to be the most common in feces.In this study, sewage was collected from waste drainage of entire pig farms, which was considered to be a good source from which to isolate bacteriophages against O8 ETEC, especially since these farms had a record of post-weaning E. coli-related diarrhea.The percentages of bacteriophages isolated from sewage samples and fecal were 13 and 5%, respectively.This difference may be likely due to the pooling of feces from large numbers of animals that occurs in sewage and the use of 100 ml sewage samples In this study, the rates of recovery of bacteriophages from sewage samples were 50% from March until June and 16.7% in November and December suggesting a substantial seasonal effect on the isolation of bacteriophages.Furthermore, higher isolation rates in the warmer months have been reported for other bacteriophages (Comeau and Krisch, 2005).Morphological characteristics seen under an electron microscope are considered important in bacteriophage taxonomy and used to identify 96% of all bacteriophages investigated in the last 45 years, especially members of the Siphoviridae, Myoviridae and Podoviridae families (Kumari et al., 2009).Of the six isolated bacteriophages (JH1, JH2, JH3, JH4, JH5 and JH6) which lysed O8 E. coli in this study, JH1 and JH2 were determined to be members of the Myoviridae family based on their morphological features and their contractile tails.This family consists of six genera, characterized by having icosahedral or elongated heads and contractile tails that were more or less rigid, long and relatively thick (80 to 455 nm × 16 to 20 nm).The four bacteriophages (JH3, JH4, JH5 and JH6) were found to belong to the Siphoviridae family of bacteriophages, each consisting of a head, a tail and no envelope.
The six bacteriophages were highly susceptible to acidity at pH 2 and susceptible to varying degrees to overnight exposure to pH 3 to 4 and ability to survive well over the pH range between 5 to 9 is a common feature in bacteriophages.JH4 and JH6 bacteriophage were susceptible to low pH while bacteriophage JH5 was susceptible than the others.Hazem (2002) had reported that bacteriophages are often quite sensitive to protein denaturation in an acidic environment, which may result in a loss of viability of the bacteriophages.He found that many bacteriophages are stable at pH 3 or 4. The pH in the stomach of weaned pigs may be as low as one to two before a meal and may rise quickly to four to five after the meal, depending on the diet and the feeding regime (Snoeck et al., 2004).The six bacteriophages will likely undergo a marked reduction in titer following oral administration to pigs unless steps are taken to reduce their exposure to low pH in the stomach and upper small intestine (Jamalludeen et al., 2007).

Conclusion
Among the six bacteriophages isolated and characterized in this study, JH1 was related to JH2, and JH3 was related to JH4, based on the spectrum of activity against O8:F18 ETEC, morphology and restriction enzyme digestion patterns.These bacteriophages all exhibited high activity against O8:F18 ETEC and genomic analyses will provide further indication of the extent of their relationships.The information gained from the present study provides a basis for selection of bacteriophages for their potential therapeutic and prophylactic use against O8:F18 ETEC infections in piglets.

Table 1 .
Estimated dimensions a and genome sizes b of six bacteriophages from pig sewage or feces.
a Determined from TEM micrographs of negatively stained phage particles from concentrated phage lysates.b Determined by restriction endonuclease digestion and electrophoresis.

Table 2 .
Summary of lytic activity of six bacteriophages against porcine O8 ETEC and other E. coli strains.Obtained Department of Microbiology and Immunology, Nanjing Agricultural University, China.
*Strain number.abObtained from the Central Vietnam Veterinary Institute.