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Isolation and characterization of virulent bacteriophages against Escherichia coli serogroups O1, O2, and O78 N. Jamalludeen,* Y.-M. She,† E. J. Lingohr,‡ and M. Griffiths*1 *Department of Food Science and the Canadian Research Institute for Food Safety, University of Guelph, Guelph, Ontario N1G 3W1, Canada; †Centre for Biologics Research, Biologics and Genetic Therapies Directorate (BGTD), Health Canada, 251 Sir Frederick Banting Driveway, Tunney’s Pasture A/L 2201E, Ottawa, Ontario K1A 0K9, Canada; and ‡Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario N1G 3W4, Canada ABSTRACT The goal of this study was to isolate and characterize a more complete set of phages that are active against Escherichia coli serogroups O1, O2, and O78, the main causative agents of avian colibacillosis. A mixture of E. coli (O1:K1), (O2:K1), and (O78:K80) was used as a host to isolate phages from wastewater and fecal samples from poultry processing plants. Seven phages were isolated; only 2 of them, EC-Nid1 and EC-Nid2, were selected for further characterization. It was found that EC-Nid1 and EC-Nid2 had icosahedral heads, necks, and contractile tails with tail fi-

bers and therefore belonged to Myoviridae. The phages had genome sizes of 67.06 to 68.04 kb and they lysed all tested strains of E. coli serotype O1, O2, and O78. The 2 phages were resistant to pH 5 to 9, and phage EC-Nid2 was slightly more resistant to acid and alkali environments. Two major protein bands were indicated in EC-Nid (A and D); band D at 45 kDa was a major coat protein and band A was identified as a homolog of endo-N-acetylneuraminidase. It was concluded that phage EC-Nid1 and EC-Nid2 are highly active against O1, O2, and O78 colibacillosis strains.

Key words: Escherichia coli, bacteriophage, serogroup, colibacillosis 2009 Poultry Science 88:1694–1702 doi:10.3382/ps.2009-00033

INTRODUCTION Avian colibacillosis, a disease caused by a group of bacteria called avian pathogenic Escherichia coli (APEC) in chickens, turkeys, and other avian species, is an infectious disease that often causes severe mortality and subsequently results in economic losses to the poultry industry (Dho-Moulin and Fairbrother, 1999; Gibbs et al., 2004). The disease is associated with a complete set of syndromes including septicemia, airsaculitis, pericarditis, and swollen head syndrome (Cheville and Arp, 1978; Rodriguez-Siek et al., 2005). Several E. coli isolates are commonly associated with colibacillosis in poultry, and the serogroups O1, O2, and O78 have been recognized as the predominant sources involved in this disease (Whittam and Wilson, 1988; McPeake et al., 2005). A high rate of antibiotic resistance was observed while testing these serogroups, which probably originates from the extensive use of antibiotics in the poul©2009 Poultry Science Association Inc. Received January 21, 2009. Accepted April 13, 2009. 1 Corresponding author: [email protected]

try industry (Allan et al., 1993), as well as by acquisition of R plasmids (Johnson et al., 2005b; Skyberg et al., 2006). Numerous concerns about the use of antibiotics in the poultry industry have been raised including the further selection of drug-resistant strains (Franklin, 1999; Angulo et al., 2004). There are also human health issues involved due to the potential transfer of E. coli from animals via the food chain (Angulo et al., 2004; Johnson et al., 2005a). This has attracted considerable attention from researchers who are seeking alternatives for control and treatment of colibacillosis in animals. One promising alternative to antibiotics is the use of virulent bacteriophage against E. coli serogroups O1, O2, and O78, a well-established approach that phages for these serogroups are able to be isolated and used in phage therapy against bacterial cells. Bacteriophages are a class of viruses that live and replicate in bacteria (Ackermann, 2000) and have the ability to attack a single species or subset of a species of bacterium, making them potential antibacterial agents. Previous studies on phage isolation and phage therapy have been reported in animals against E. coli infection (Smith and Huggins, 1982, 1983; Smith et al., 1987). Huff and colleagues have conducted several studies on the use of bacteriophage to prevent and treat

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colibacillosis in broiler chickens (Huff et al., 2002, 2003, 2004, 2005); however, the research work was restricted only to the isolation of phages against serotype O2. The ultimate goals of this study were therefore to isolate a more complete set of phages that are active against serotypes O1, O2, and O78 and to characterize these species with respect to morphology, genome size and restriction endonuclease digestion, bacterial host range, activity assay, and proteome analysis using SDS-PAGE and matrix-assisted laser desorption ionization quadrupole time-of-flight (MALDI QqTOF) mass spectrometry (MS). Here we attempt to characterize the bacteriophages using molecular analyses at both genome and proteome levels.

MATERIALS AND METHODS Bacteria, Culture Media, and Chemicals Avian pathogenic E. coli strains EC101 (O1:K1) and EC99 (O78:K80) were isolated from colisepticemic chickens (Department of Pathobiology, University of Guelph). Strain EC317 (O2:K1) was isolated from a colisepticemic turkey. All of the APEC strains were kindly supplied by Carlton Gyles, University of Guelph. Luria-Bertani (LB) broth (Fisher Scientific, Nepean, Ontario, Canada), LB agar, and LB top agar (soft agar) were prepared as described by Sambrook et al. (1989). Bacteriophage broth was used to isolate the phage (Cappuccino and Sherman, 1992). The ingredients were dissolved in 1 L of distilled water containing peptone (100 g, Difco Laboratories, Detroit, MI), beef extract (30 g, Difco Laboratories), yeast extract (50 g, Fisher Scientific), NaCl (25 g, Fisher Scientific), and potassium dihydrogen phosphate (80 g, BDH Laboratory, Toronto, Ontario, Canada). The other reagents used included MgSO4, agarose (Invitrogen Canada, Burlington, Ontario, Canada), RNase I, DNase I, proteinase K (Qiagen, Mississauga, Ontario, Canada), ethanol (Commercial Alcohols Inc., Brampton, Ontario, Canada), and ethidium bromide (Sigma-Aldrich Canada, Oakville, Ontario, Canada).

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Bacteriophage Isolation and Purification Bacteriophages were isolated from wastewater and fecal samples collected from poultry processing plants in Ontario during the period of August and September 2007 according to the procedure described by Jamalludeen et al. (2007). Luria-Bertani broth was inoculated with a mixture of O1, O2, and O78 E. coli strains and incubated for 24 h at 37°C. A volume of 200 mL of wastewater or 200 g of fecal samples was homogenized and was aseptically poured into a sterile 1-L flask. Twenty milliliters of bacteriophage broth, 20 mL of LB with MgSO4, and 20 mL of a suspension of E. coli strains in broth culture [optical density at 600 nm (OD600) = 1.4] were aseptically added to the flask, and the mixture was subsequently incubated at 37°C for 24 h with shaking. After incubation, the total mixture was centrifuged at 4,000 × g for 15 min and the supernatant was collected into a clean flask, then filtered through a sterile 0.45-μm membrane filter (Fisher Scientific). The filtrate was sequentially diluted (10–1 to 10–9) in SM buffer (5.8 g of NaCl, 2 g of MgSO4·7H2O, 50 mL/L of 1 M Tris pH 7.5, 5 mL/L of gelatin in distilled water), which was used for storage and dilution of bacteriophage stocks. One hundred microliters of diluted filtrate was added to 100 μL of the E. coli strains (OD600 = 1.4) in a test tube and incubated at 37°C for 20 min. Then, 3 mL of top agar (7.0 g/L) prepared according to Sambrook et al. (1989) was added and the tube contents were mixed and poured onto the surface of an LB agar plate and allowed to solidify. The plates were incubated overnight at 37°C and examined for the presence of plaques. A previously described protocol (Sambrook et al., 1989; Jamalludeen et al., 2007) was used to isolate a single plaque. Only 2 phages, named EC-Nid1 and EC-Nid2, out of a total of 7 phages isolated were considered for further characterization. Phages Nid1 and Nid2 were propagated on EC101 and the titer of each phage was determined by plaquing 10-fold dilutions by the soft agar overlay method; this procedure was repeated 3 times to obtain purified phages. The phage preparations were stored at 4°C. The phage sus-

Figure 1. Electron microscope appearance of phages EC-Nid1 and EC-Nid2. The phages have a neck and a contractile tail and icosahedral head. Bar = 50 nm.

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pension was purified using CsCl gradient according to the protocol of Sambrook et al. (1989).

Electron Microscopy The phages EC-Nid1 and EC-Nid2 were examined by electron microscopy of negatively stained preparations. A drop of pure phage preparations suspended in 0.5 mL of sterile 0.1 M HEPES buffer (Boehringer Mannheim, Montreal, Quebec Canada) was applied to the surface of a formvar-coated grid (200 mesh copper grids). The samples were negatively stained with 2% uranyl acetate and then examined in a LEO 912AB energy-filtered transmission electron microscope operated at 100 kV (Guelph Reginal STEM Facility, University of Guelph). The phages (EC-Nid1 and EC-Nid2) were classified according to the guidelines of the International Committee on Taxonomy of Viruses (ICTV, 1995) based on their morphological features.

Extraction of Phage DNA Phage DNA was extracted using the Lambda Maxi Kit (Qiagen) according to the instructions of the manufacturer. A supernatant from phage lysate was precipitated using the kit buffers. After centrifugation at 10,000 × g for 10 min, the pellet was resuspended, washed, eluted, and a pure phage DNA was prepared in respect of the protocol of the manufacturer.

Host Range Determination Isolates EC101, EC99, EC317, and the 72 strains that comprise the E. coli reference (ECOR) collection (Ochman and Selander, 1984) were used to test the spectrum of virulence of phage EC-Nid1 and EC-Nid2 according to the spot test procedure described by Sambrook et al. (1989). A log phase culture of EC101 bacteria [OD600 = 1.4] (5 μL) was spread over each square on an LB agar plate, which was divided into 4 squares by marking the bottom surface of the plate. The plates were allowed to dry and phage suspension (109 pfu/ mL; 10 μL) was dropped in the center of each square. After incubation at 37°C overnight, these plates were examined for lysis. A clear zone in the bacterial lawn was recorded as complete lysis.

Acidity and Alkalinity Resistance The method of Jamalludeen et al. (2007) was used to evaluate the activity of phages to survive at different pH levels. Briefly, phage suspension was exposed to a certain pH value adjusted from 1 to 11 using 0.1 M HCl or NaOH over 16 h of incubation at 37°C and then checked for survival. Figure 2. Pulsed-field gel electrophoretogram of phages ECNid1 and EC-Nid2 genome. M = Marker: Xba1 digested Salmonella Braenderup, 1% Seakem Gold agarose (Mandel Scientific, Guelph. Ontario, Canada); Nid1 = DNA from phage EC-Nid1; Nid2 = DNA from page EC-Nid2. EC-Nid1 = 67.06 kb and EC-Nid2 = 68.04 kb.

Analysis of Phage Proteins Proteins of EC-Nid1 and EC-Nid2 were further purified by a CsCl gradients procedure (Sambrook et al.,

AVIAN COLIBACILLOSIS, PHAGES, ESCHERICHIA COLI O1, O2, AND O78 Table 1. Survival of phages EC-Nid1 and EC-Nid2 after exposure to pH 1 to 11 Titer of surviving, viable phages (pfu/mL) pH

EC-Nid1

1 and 2 3 4 5 to 9 10 11 Control

1

ND 7 × 106 1.3 × 107 ≥108 5 × 106 4 × 106 ≥108

EC-Nid2 ND 2.3 × 108 5.5 × 108 ≥108 2.2 × 108 2 × 107 ≥108

1

Not detected.

1989), concentrated by precipitation with 10% polyethelene glycol and 1 M NaCl, and then resuspended in Tris-EDTA buffer. The phage suspensions were boiled for 5 min and separated by SDS-PAGE on 12.5% acrylamide gel. Proteins were stained with Simply Blue safe stain (Invitrogen Canada). Four bands of each phage were excised and identified by MS at Queen’s University, Ontario, Canada. The excised protein bands were crushed into small pieces and destained with a mixture of 25 mM NH4HCO3 and acetonitrile. The protein in the gels was reduced with 10 mM dithiothreitol solution (in 100 mM NH4HCO3) at 56°C for 1 h, then incubated with 55 mM iodoacetamide at room temperature for another 45 min. After drying of the gel particles in a SpeedVac centrifuge (Savant; Fisher Scientific), the protein was digested overnight with 10 ng of sequencing grade trypsin (Calbiochem, San Diego, CA) in 25 mM NH4HCO3. The proteolytic peptides were sequentially extracted using 0.1% formic acid, 60% acetonitrile/0.1% formic acid, and pure acetonitrile with the aid of sonication. Finally, the collected fraction was dried by SpeedVac centrifugation and cleaned by C18 Ziptip (Millipore, Billerica, MA) for MALDI QqTOF MS and tandem MS (MS/ MS) analyses. Data from the matrix-assisted laser desorption ionization MS analysis were acquired using an Applied Biosystems/MDS Sciex QStar XL QqTOF mass spectrometer equipped with an oMALDI II source and a nitrogen laser operating at 337 nm (Applied Biosystems, Foster City, CA). The sample was prepared at the ratio of 1:1 (vol/vol) of the peptide digest to matrix (i.e., 2,5-dihydroxybenzoic) and subsequently dried on a stainless steel matrix-assisted laser desorption ionization plate. After matrix-assisted laser desorption ionization MS mapping, the peptide sequences were then identified by MS/MS measurements using argon as the collision gas. The peptide fingerprinting masses were initially searched by the MS-Fit program against the National Center for Biotechnology Information (NCBI) database using ProteinProspector at the University of California, San Francisco Web site (http://prospector.ucsf. edu), whereas the MS/MS ions search on each tandem mass spectrum was performed using the Mascot search

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engine (MatrixScience, http://www.matrixscience. com). These searches took account of up to 2 missed trypsin cleavage sites, and possible modifications of methionine oxidation, carbamidomethylation, deamidation of asparagine and glutamine to asparate acid and glutamic acid, and N-terminal pyroglutamation. The mass tolerance between calculated and observed masses used for the database search was considered at the range of ±0.1 Da for MS peaks and ±0.2 Da for MS/MS fragment ions. When no confident protein identification was found in the NCBI databases listed above, then de novo peptide sequencing was performed by MS/MS analyses on the tryptic peptides and the deduced partial sequence tags were used for protein homology database search with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). When a sufficient amount of the material was available from samples Nid2A and Nid2D, the tryptic digests were subsequently derivatized by N-terminal sulfonation as described by Wang et al. (2004). It has been found that MS/MS analysis of the sulfonated peptides with low-energy collision dissociation yields a set of Cterminal fragments ions (yn), which enable a reliable and rapid sequence identification of the unknown peptides.

Pulsed-Field Gel Electrophoresis Genome sizes of EC-Nid1 and EC-Nid2 were determined by using pulsed-field gel electrophoresis (PFGE). Phages embedded in 1.0% Seakem Gold agarose (Mandel Scientific, Guelph, Ontario, Canada) were electrophoresed in 0.5 × Tris-borate-EDTA buffer at 14°C for 18 h, using a Chef DR-III Mapper electrophoresis system (Bio-Rad, Mississauga, Ontario, Canada), with pulse times of 2.2- to 54.2-s pulses, at 6 V/cm. The bands were visualized under UV transillumination after staining with ethidium bromide. The PFGE results were analyzed using BioNumerics software (Applied Maths Inc., Austin, TX).

Restriction Enzyme Digestion Patterns Phage nucleic acids (2 μg) were treated with the restriction enzymes AccI, EcoRI, and XhoI (New England Biolab, Ontario, Canada) following standard procedures (Sambrook et al., 1989). Deoxyribonucleic acids (3-μL vol) were digested for 8 h at 37°C and the cleaved nucleic acids were subjected to electrophoresis in a 1% (wt/vol) agarose gel and stained with ethidium bromide.

RESULTS Isolation of Phages Seven phages were isolated using a mixture of O1, O2, and O78 avian E. coli strains as hosts. The phages were named (EC-Nid1 to EC-Nid 7). Phages EC-Nid1

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and EC-Nid2 were selected for further characterization according to their morphological features (Figure 1).

Morphology of the Phages EC-Nid1 and EC-Nid2 and Genome Sizes Phages were morphologically classified into the family Myoviridae, The electron microscopy preparations showed that the phages possessed icosahedral heads,

necks, and contractile tails, with tail fibers (Figure 1). The head dimensions for EC-Nid1 and EC-Nid2 were 66 × 52 nm and 68 × 55 nm, and tail dimensions were 61 × 15 nm and 57 × 15 nm, respectively. The data were acquired based on unbiased experiments; 6 images were measured and the mean values were recorded. Based on PFGE of the entire genome of these 2 phages (Figure 2), phage EC-Nid1 had a genome size of 67.06 kb and phage EC-Nid2 had a size of 68.04 kb.

Figure 3. Electrophoresis on 1% agarose of AccI and EcoRI restriction enzymes digest of phages EC-Nid1 and EC-Nid2 genome. M = marker; Nid1 = DNA from phage EC-Nid1; Nid2 = DNA from page EC-Nid2.

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Host Range Phage EC-Nid1 and EC-Nid2 lysed all the strains that were tested. These included 4 O1 (3 in ECOR), 5 O2 (4 in ECOR), and 3 O78 (2 in ECOR).

Resistance to Acidity and Alkalinity The 2 phages were resistant to pH 5 to 9. In contrast, phage EC-Nid2 was slightly more resistant to acidic and alkaline environments than phage EC-Nid1 (Table 1).

Restriction Enzyme Digestion Patterns Phages EC-Nid1 and EC-Nid2 appear to have similar profiles of the nucleic acid fragments generated by digestion of their DNA with AccI and EcoRI. The patterns for those enzyme-cleaved products are shown in Figure 3.

Protein Identification by MS Based on a side-by-side comparison, MALDI QqTOF MS analyses on the in-gel tryptic digests of 2 sets of proteins in EC-Nid1 and EC-Nid2, separated by SDS-PAGE gels (Figure 4), revealed similar profiles of the peaks between 2 horizontal parallel protein bands with closely related molecular masses (not shown). The typical MS spectra of EC-Nid2A to D are illustrated in Figure 5. Because the initial database searches by peptide mass fingerprinting were inconclusive, peptide sequencing by MS/MS was then employed to identify these unknown peptides/proteins. With the aid of chemical derivitization (i.e., sulfonation) of the tryptic peptides at the N-termini (Supplemental Figure 1; http://ps.fass.org/ content/vol88/issue8/), we were able to gain enough sequence information to identify 2 major protein bands EC-Nid A and D as indicated in Table 2. The most intense band D at 45 kDa appeared to be a major coat protein, and the top protein band A of 107 kDa was identified as a homolog of endo-N-acetylneuraminidase. In the cases of the weak bands of Nid B and C, the manual interpretation of protein sequences were always difficult because of the poor peptide fragmentation in the matrix-assisted laser desorption ionization MS/MS spectrum. Fortunately, 1 peptide sequence of the highabundance peptide at m/z 1,423.72 at 92-kDa band has been exactly matched to the 94-kDa protein (NCBI accession no. 119952228) and the other peptide at m/z 1,944.97 was confirmed to have a homologous sequence that localized at the same protein [Supplemental Figure 2, (F) to (G); http://ps.fass.org/content/vol88/issue8/]. Despite the identification of some partial peptide sequences by MS/MS measurements in the 74-kDa protein band [Table 2 and Supplemental Figure 2, (H) to (J)], many other peptides are still unknown and the protein identity remains to be solved.

Figure 4. Sodium dodecyl sulfate-PAGE for phages EC-Nid1 and EC-Nid2. The phages were purified by CsCl gradient procedure. The bands (A, B, C, and D) were identified by mass spectrometry. M = molecular weight markers; Nid1 = DNA from phage EC-Nid1; Nid2 = DNA from page EC-Nid2. EC-Nid1 bands: A = 109.9 kD; B = 93.3 kD; C = 74.8 kD; and D = 45.8 kD; EC-Nid2 bands: A = 107.6 kD; B = 92.4 kD; C = 74.2 kD; and D = 45.2 kD.

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Figure 5. Matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry spectra of the tryptic digests of EC-Nid2 protein bands. amu = atomic mass unit.

DISCUSSION With increasing concerns on the emergence of antibiotic resistance on farms (Kuehn, 2007), we conducted this study to isolate and characterize phages that were active against avian colibacillosis strains (O1, O2, and O78), as an alternative to antibiotics. We isolated phages EC-Nid1 and EC-Nid2 active among the predominant strains that cause colibacillosis in poultry from wastewater and poultry processing plants, which are the main sources for these phages. Previous studies by Huff et al. (2002) also identified phages, designated SPRO2 and DAF6, which were active only against E. coli serotype O2 in broiler chickens. Phages isolated from our study target all of the dominant strains from

Ontario, Canada, and are broadly active against E. coli serotype O1, O2, and O78. Phages EC-Nid1 and EC-Nid2 are members of the family Myoviridae based on their morphological features and their contractile tails (Figure 1). Myoviridae are characterized by those having icosahedral or elongated heads and contractile tails that are more or less rigid, long, and relatively thick (ICTV, 1995). Deoxyribonucleic acid structure, protein composition, base sequence similarity, host range, and infection characteristics also define the tailed virus species (Ackermann et al., 1992; Maniloff and Ackermann, 1998). The phages were tested for their host ranges on the O1, O2, and O78, the predominant avian colibacillosis strains, as well as their host range among the 72 E.

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Table 2. Peptide identification by tandem mass spectrometry (MS/MS) de novo sequencing and National Center for Biotechnology Information (NCBI) database searching on the tryptic digests of Nid2A, 2B, 2C, and 2D Protein band identification (kDa)

m/z (Meas.)1

107

74

Peptide position

Peptide sequence3

1,088.579 2,219.089 2,302.200

Endo-N-acetylneuraminidase (gi|77118205) [Enterobacteria phage K1F] 896.510 355 to 361 LHVSWVR 980.523 399 to 406 LFAMIETR 1,250.667 397 to 406 NRLFAMIETR 1,266.662 397 to 406 NRLFAMIETR (Met-OX) 1,318.691 867 to 877 IVYNGAEHLFR 1,345.715 586 to 596 YFDGLLYVVTR 1,347.727 636 to 647 VGNELILFGTER 1,604.876 280 to 293 VTSLPDISRFVNTR 2,127.090 785 to 804 TVPAPMEFTGDLGLGHVTIR (Met-OX) 2,705.441 277 to 298 TYKVTSLPDISRFINTRFVYER 3,009.503 294 to 318 FVYERIPGQPLYYASEEFVQGELFK 94-kDa protein (gi|119952228) [Enterobacteria phage N4] 1,423.733 116 to 126 QNELVLNYQFR 1,944.966 132 to 141 VIDDYLHSVVDDGTGIAR Unknown protein4 1,088.574 — EPL(/I)NVDVFR — — …PL(/I)DCADL(/I)ASY…TAR — — …GTPTVDVTNTEN…R

948.538 1,069.601 1,101.591 1,463.773 1,615.852 1,883.954 2,464.165 2,581.275

Major coat protein (gi|119952248) 948.537 114 to 121 1,069.600 104 to 113 1,101.590 211 to 219 1,463.781 243 to 255 1,615.858 352 to 366 1,883.939 38 to 53 2,464.170 72 to 95 2,581.298 291 to 315

896.512 980.517 1,250.668 1,266.657 1,318.681 1,345.720 1,347.706 1,604.864 2,127.080 2,705.451 3,009.463

92

[MH+] (Calc.)2

1,423.715 1,944.972

45

[Enterobacteria phage N4] VNRVGFTR LPLLTENGGR LDQILTENR VMYVGSELVPELK NLMRLDQILTENR KEQYFMPLASVTNMPK NINDQGIDANGATIVDGNLYGSSR IIQVPEMLHWAGAGAQATTNPGYR

1

m/z (Meas.) = measured value of mass-to-charge ratio. [MH+] (Calc.) = calculated molecular mass of the protonated peptide ion. 3 The underlined residues represent the differences from the amino acid sequences of the proteins in the NCBI database. The supporting evidence is available in the supplementary materials online. 4 The amino acid sequence of the 74-kDa protein is not in the NCBI database, and only partial sequence information was obtained by MS/MS measurements. Leucine and Ile are unable to be distinguished because of the identical masses. 2

coli of the ECOR collection, which is a widely used set of reference strains isolated between 1973 and 1983 from different hosts and geographical locations that represents the range of genotypic variation in E. coli (Ochman and Selander, 1984). Both phages were highly susceptible to acidity at pH 1 and 2. It seems that EC-Nid1 is more susceptible to pH 3 than EC-Nid2 (Table 1). Phages are often sensitive to protein denaturation in an acidic environment (Hazem, 2002). On the other hand, both phages were stable and survive at close to neutral pH values between 5 and 9. The results are consistent with the previous observations by Ackermann and DuBow (1987) and Jamalludeen et al. (2007) that most phages are able to survive well over a wide range of pH 5 to 9 at physiological conditions that maintain the native virion structrue and stability. Both phages appear to be closely related based on similar patterns of the DNA fragments obtained from AccI and EcoRI restriction enzymes (Figure 3), which established a close genetic relationship to each other. Similar observations have been reported previously (Jamalludeen et al., 2007). In our next experiments, the mass spectrometric analyses on the proteome have

demonstrated the identical protein expression in ECNid1 and EC-Nid2 and unambiguously identified 3 major protein components in which the protein sequences are highly homologous to those of enterobacteria phage N4. The efforts to resolve all of the protein sequences are hampered by the difficulty in collecting sufficient materials, especially of the low-abundance proteins, for MS/MS sequencing. Much more information can be obtained by sequencing the complete genome of these 2 phages, whereupon exact differences may be highlighted. Endo-N-acetylneuraminidase associated with bacteriophage particles (Kwiatkowski et al., 1982; Hallenbeck et al., 1987) is a tail fiber spike enzyme that specifically recognizes and degrades polysialic acid (Jakobsson et al., 2007). These polysaccharide coats act as recognition sites for bacteriophages and are involved in both adsorption to the cell surface and penetration into the cell by enzyme degradation of polysaccharide (Gross et al., 1977; Scholl et al., 2001). We identified 110 kDa of endo-N-acetylneuraminidase in the phages of ECNid1 and EC-Nid2, which may catalyze the cleavage of a-2,8-linked poly-N-acetylneuraminic acid carbohydrate polymer of the K1 capsule.

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In conclusion, phage EC-Nid1 and EC-Nid2 are highly active against O1, O2, and O78 colibacillosis strains. However, more work on host range needs to be done before these phages can be recommended for phage therapy for colibacillosis. There are slight differences in morphology and in their tolerance of acidic environments, but other characteristics indicate that they are closely related.

ACKNOWLEDGMENTS This work was supported by the Poultry Industry Council (PIC). We are grateful to Bob Harris (Department of Microbiology, University of Guelph) for kind assistance in electron microscopy.

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