wksl3, a New Biocontrol Agent for Salmonella enterica SerovarsEnteritidis and Typhimurium in Foods: Characterization, Application,Sequence Analysis, and Oral Acute Toxicity Study

Hyun-Wol Kang,a Jae-Won Kim,b Tae-Sung Jung,c Gun-Jo Wooa

Food Safety and Evaluation Laboratory, Department of Food Bioscience and Technology, Korea University, Anam-dong 5-ga, Seongbuk-gu, Seoul, Republic ofKoreaa; CJ Research Institute of Biotechnology, CJ CheilJedang, Seoul, Republic of Koreab; Department of Veterinary Medicine, Gyeongsang National University,JinJu, Republic of Koreac

Of the Salmonella enterica serovars, S. Enteritidis and S. Typhimurium are responsible for most of the Salmonella outbreaksimplicated in the consumption of contaminated foods in the Republic of Korea. Because of the widespread occurrence of antimi-crobial-resistant Salmonella in foods and food processing environments, bacteriophages have recently surfaced as an alternativebiocontrol tool. In this study, we isolated a virulent bacteriophage (wksl3) that could specifically infect S. Enteritidis, S. Typhi-murium, and several additional serovars. Transmission electron microscopy revealed that phage wksl3 belongs to the family Si-phoviridae. Complete genome sequence analysis and bioinformatic analysis revealed that the DNA of phage wksl3 is composedof 42,766 bp with 64 open reading frames. Since it does not encode any phage lysogeny factors, toxins, pathogen-related genes, orfood-borne allergens, phage wksl3 may be considered a virulent phage with no side effects. Analysis of genetic similarities be-tween phage wksl3 and four of its relatives (SS3e, vB_SenS-Ent1, SE2, and SETP3) allowed wksl3 to be categorized as a SETP3-like phage. A single-dose test of oral toxicity with BALB/c mice resulted in no abnormal clinical observations. Moreover, phageapplication to chicken skin at 8°C resulted in an about 2.5-log reduction in the number of Salmonella bacteria during the testperiod. The strong, stable lytic activity, the significant reduction of the number of S. Enteritidis bacteria after application tofood, and the lack of clinical symptoms of this phage suggest that wksl3 may be a useful agent for the protection of foods againstS. Enteritidis and S. Typhimurium contamination.

Each year, nontyphoid Salmonella is involved in approximately1.0 million estimated cases of salmonellosis, with 19,336 hos-

pitalizations and 378 deaths, in the United States (1). A recentreport by the U.S. Centers for Disease Control and Prevention(CDC) chronicled a continuous increase in Salmonella outbreaksfrom May to September 2010 throughout the United States; inthese outbreaks, 1,608 illnesses were found to be associated withthe consumption of shell eggs contaminated with Salmonella en-terica serovar Enteritidis (2). In the Republic of Korea, S. Enterit-idis and S. Typhimurium have been the main Salmonella serovarsresponsible for diarrhea and food-borne diseases due to salmonel-losis in humans (3). In addition, S. Enteritidis is the second-most-reported serovar that causes Salmonella-related human disease inthe United States, while S. Typhimurium is the most prevalentSalmonella serovar that causes salmonellosis (4). The industry hasbeen investigating an effective process for the production of S.Enteritidis-free eggs and chickens worldwide (5, 6). In addition tothe increasing incidence of salmonellosis, antimicrobial resistancein isolates associated with clinical outbreaks or food samples fromfood-borne outbreaks has been recognized as a new risk factor forSalmonella infection (7–10). The use of antibiotics in food-pro-ducing animals raised the prevalence of antimicrobial-resistantbacteria, and they have had adverse effects on the health of con-sumers via the food chain. The relationship between food-bornepathogens of human and animal origins has been well studied(11).

Widespread antibiotic resistance in isolates from varioussources has encouraged many researchers to investigate and re-search phages as alternative biocontrol agents (12, 13). The use ofphages as biological agents to control pathogens in foods has re-

cently been suggested (14, 15). The use of a six-listeriaphage mix-ture to surface treat ready-to-eat meat and poultry products wasapproved by the U.S. Food and Drug Administration (FDA) in2006, and in 2007, the U.S. FDA gave a generally recognized as safe(GRAS) designation to Listeria phage P100 (GRAS notice GRN000218) for all products; P100 had already been approved for usein ready-to-eat foods as a food additive (16). Recently, P100 waslisted by the Organic Materials Review Institute as an organic ma-terial classified as a processing nonagricultural ingredient andprocessing aid (http://www.omri.org/manufacturers/66440/ebi-food-safety-bv). The European Food Safety Authority also con-firmed the safety of phage P100 as an antibacterial agent againstListeria monocytogenes on the surface of raw fish (17).

The phage application field is now expanding to target variousfood-borne pathogens and food products. In addition to thephage application test against L. monocytogenes (18, 19), studiesinvestigating various food-borne pathogens, such as Salmonellaspp. (20, 21) and Escherichia coli O157:H7 (22), have shown thatphages are useful tools for the control of pathogens in foods with-out the risk of side effects. Since the regulatory clearance of the E.coli O157:H7-specific phage in the form of a food contact notifi-

Received 10 September 2012 Accepted 7 January 2013

Published ahead of print 18 January 2013

Address correspondence to Gun-Jo Woo, visionkorea@korea.ac.kr.

H.-W.K. and J.-W.K. contributed equally to this study.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02793-12

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cation (FCN), the product can now be applied to red meat (FCNno. 1018). Moreover, another product based on a Salmonellaphage is currently under review for FCN approval (Intralytix, Bal-timore, MD).

In this report, we describe the detailed characterization andgenetic information of Salmonella-specific virulent phage wksl3.Additional analyses, including phage host spectrum testingagainst S. Enteritidis, S. Typhimurium, and other serotypes; anti-microbial resistance profiles; oral toxicity tests; bioinformaticanalysis; and efficacy in the control of S. Enteritidis on chickenskin, were also carried out to evaluate the potential of wksl3 for useas a food additive.

MATERIALS AND METHODSBacterial strains and culture conditions. A total of 111 Salmonella strainswere used in this study (Table 1). Isolates from various food and clinicalsamples, such as ready-to-eat foods, livestock, fruits, vegetables, and clin-ical fecal samples, were collected from 2002 to 2010. The first S. Enteritidisisolate (SAL111-CF-KF10) producing the CTX-M-15 extended-spectrumbeta-lactamase, acquired from a pediatric patient suffering from gastro-enteritis in 2008 (23), was kindly provided by W. K. Song, Hallym Uni-versity College of Medicine. Antimicrobial resistance phenotypes are alsoindicated in Table 1. Salmonella strains were grown at 37°C in tryptic soybroth (Bacto TSB; BD, Sparks, MD) or Bacto TSB supplemented with1.5% agar. All strains were stored at �80°C in skim milk.

Isolation of Salmonella phage. To isolate a Salmonella phage, we col-lected 25 chicken by-product samples from 16 traditional markets inGyeongGi-do, Republic of Korea. Three-gram samples were soaked in 30ml sodium chloride-magnesium sulfate (SM) buffer with gelatin (100mM NaCl, 10 mM MgSO4 [heptahydrate], 50 mM Tris-HCl [pH 7.5],0.01% gelatin). The tubes were vigorously vortexed for at least 5 min atroom temperature. After centrifugation of the suspension at 4,500 � g for30 min, the supernatant was filtered through a 0.20-�m membrane filter(Advantec Co., Ltd., Saijo City, Ehime, Japan). One hundred microlitersof filtrate from each sample was then added to 4 ml Luria-Bertani (LB)broth supplemented with 10 mM CaCl2 and 40 �l of an overnight brothculture of S. Enteritidis ATCC 13076 as a propagating host. After over-night incubation of the phage-Salmonella mixture at 37°C, each culturewas filtered (0.20-�m filter) and standard plaque assays were performedwith an indicator host (ATCC 13076) for each filtrate. Phage purificationwas carried out by picking single plaques with sterilized pipette tips, fol-lowed by serial purifications with amplifications from the same host(ATCC 13076), as described previously (24).

Determination of the phage host spectrum. To confirm the host lysisrange of Salmonella phage wksl3, spotting assays performed by the mod-ified bilayer standard plaque assay method (25) were used with 111 Sal-monella strains. After overnight cultivation of the Salmonella host, 40 �l ofeach solution was added to 4 ml of 50°C LB soft agar (0.75%) containingCaCl2 (final concentration, 10 mM) and then the mixture was pouredonto 1.5% LB agar plates. After a 30-min drying procedure in a laminar-flow closet, 5 �l of the diluted phage solution (1.1 � 108 PFU/ml) wasspotted onto the plate and incubated overnight at 37°C. All tests wereconducted at least three times with all of the strains used in this study.

Transmission electron microscopy (TEM). A purified wksl3 solution(1.2 � 1011 PFU/ml) was dropped onto a Formvar carbon-coated coppergrid (200 mesh). After 30 s of immobilization, water was removed withfilter paper and then 2% (wt/vol) uranyl acetate was dropped onto the gridfor negative staining of the phage. Electron micrographs were taken witha Carl Zeiss LEO 912AB transmission electron microscope operating at an80-kV accelerating voltage. Images were taken on the transmission elec-tron microscope at the National Academy of Agricultural Science, Suwon,Republic of Korea.

One-step growth curve. The standard one-step growth curve of phagewksl3 was determined at 37°C as described by Ellis and Delbruck (26). S.

Enteritidis ATCC 13076 was used as the host, and wksl3 was inoculated ata multiplicity of infection (MOI) of 0.01. Growth curves were plotted withSigma Plot 10.0 (SPSS Inc.) based on PFU data collected every 5 min. Thelatency period and burst size were then calculated.

Nucleotide extraction, sequencing, and genomic analysis. A high-titer (1.1 � 1011 PFU/ml) phage solution was prepared for wksl3 DNAextraction. Three milliliters of wksl3 suspension was incubated with 30 �lDNase I (10 �g/ml; Sigma-Aldrich, UK) and RNase A (10 �g/ml; Sigma-Aldrich, UK) at 37°C for 50 min. Following the addition of 0.5% sodiumdodecyl sulfate (SDS) and 50 �g/ml proteinase K, the phage suspensionwas incubated at 56°C for 1 h. After DNA extraction by the alkaline lysismethod, 3 M sodium acetate (pH 5.4, 0.1 volume) and cold ethanol (2.5volumes) were added for DNA precipitation as previously described (27).The phage genome was sequenced by the shotgun full-sequencing strategyon a 454 Genome Sequencer FLX titanium sequencer (Roche, Mannheim,Germany) at Macrogen Inc., Seoul, Republic of Korea. The whole genomesequence was also assembled by Macrogen Inc. with SeqMan II sequenceanalysis software (DNASTAR).

Possible open reading frames (ORFs) were predicted with the genomeannotation software GeneMarkS (28) and confirmed with FgenesV (Soft-Berry) and Glimmer 3.02 (29) by submitting the whole genome of wksl3.ATG, GTG, and TTG were considered gene start codons. Putative func-tions of conserved protein domains were identified with alignment searchtools (BLASTP, BLASTX, and BLASTN search) found at the NationalCenter for Biotechnology Information (NCBI) database. The PFAM 26.0online program was used to search specific protein domains with knownfunctions (30). Potential promoter regions for the wksl3 genome se-quence with cutoff scores of 0.90 and 0.95 were examined with the NeuralNetwork Promoter Prediction program (31) of the Berkeley DrosophilaGenome Project (http://www.fruitfly.org/seq_tools/promoter.html).FindTerm programs (Softberry, Inc., Mount Kisco, NY) were used toidentify nonoverlapping rho-independent terminators (at an energythreshold value of �11). Computed molecular weights and isoelectricpoints (pIs) of wksl3 putative protein products were predicted with pro-teomic tools (32) from ExPASy (http://www.expasy.org/proteomics). ThetRNAscan-SE 1.21 program (http://lowelab.ucsc.edu/tRNAscan-SE/) wasused to search for putative tRNAs (33). Sequence homologies of SETP3-like phages (wksl3, SETP3, SS3e, SE2, and vB_SenS-Ent1) were measuredwith the BLAST2 sequence tool (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). Comparisons of equivalent ORFs of wksl3, SETP3, andSS3e were also performed with the same software. TMHMM 2.0 was usedto predict transmembrane helices in proteins (34). To examine the simi-larity between wksl3 protein products and putative protein food allergens,the Allergenic Protein Sequence Searches program (35) of the Food Al-lergy Research and Resource Program database was used.

Oral toxicity studies with mice. Oral toxicity studies were conductedwith eight male BALB/c mice (Koatech, GyeongGi-Do, Republic of Ko-rea) 8 weeks old and weighing between 21 and 23 g each. An acute oraltoxicity test was performed according to the Good Laboratory PracticeStandards manual and Organization for Economic Cooperation and De-velopment (OECD) Guidelines for Acute Toxicity of Chemicals no. 420(36). Mice were housed in a temperature-controlled animal room on a12-h light-dark cycle. Fresh water and food were provided ad libitumthroughout the experimental period.

After an aliquot of wksl3 (1.1 � 1010 PFU/ml) suspended in SM bufferat pH 7.5 was prepared, test groups containing five animals were orallyadministered stock solutions according to body weight (1 ml/100 g bodyweight) as suggested by the OECD guidelines. The negative-control groupreceived SM buffer only in the same ratio as the test group. Feeding waspermitted 4 h after dosing.

Animals were weighed before the test started and 1 week after the testperiod. The development of abnormal behavior, changes in physical ap-pearance, and any other toxicological effects was observed within the first6 h after the test solution was administered.

Complete gross pathological examinations of the skin, lymph nodes,

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TABLE 1 Antimicrobial resistance profiles and phage susceptibilities of the Salmonella strains used in this study

Host strain Sample origin Serotype

Antibiotic resistancea

Phage wksl3susceptibilitybResistance Intermediate

S. EnteritidisSAL1-FF-KW02 Processed foods Enteritidis NA ����SAL2-FF-KW02 Processed foods Enteritidis NA CF ���SAL5-FF-KW02 Processed foods Enteritidis S ���SAL6-FF-KW02 Processed foods Enteritidis ��SAL7-FF-KW02 Processed foods Enteritidis NA ����SAL10-FF-KW02 Processed foods Enteritidis NA ����SAL11-FF-KW02 Processed foods Enteritidis NA ����SAL13-FF-KW02 Processed foods Enteritidis NA ��SAL16-FF-KW02 Processed foods Enteritidis NA ��SAL17-FF-KW02 Processed foods Enteritidis S, NA ���SAL18-FF-KW02 Processed foods Enteritidis ��SAL19-FF-KW02 Processed foods Enteritidis ��SAL23-FF-KW02 Processed foods Enteritidis ��SAL24-FF-KW02 Processed foods Enteritidis ���SAL28-FF-KW02 Processed foods Enteritidis AM CF, S ���SAL29-FF-KW02 Processed foods Enteritidis AM ���SAL30-FC-KW02 Livestock Enteritidis NA ���SAL32-FC-KW02 Livestock Enteritidis NA ���SAL33-FC-KW02 Livestock Enteritidis NA ���SAL57-FC-KF04 Livestock Enteritidis NA ����SAL58-FC-KF04 Livestock Enteritidis NA ���SAL61-FC-KF04 Livestock Enteritidis AM, S, C, TE ����SAL62-FF-KF04 Processed foods Enteritidis AM, S, C ����SAL63-FF-KF04 Processed foods Enteritidis AM, S, C S ����SAL65-FC-KF05 Livestock Enteritidis S, NA, TE ����SAL66-FC-KF05 Livestock Enteritidis NA �SAL67-FC-KF05 Livestock Enteritidis AM, S, C ����SAL74-UI-KK Unidentified Enteritidis S ����SAL97-FF-KK09 Livestock Enteritidis AM, CF, S, NA, C SXT ����SAL99-FC-KK09 Livestock Enteritidis NA S ����SAL105-FC-KK09 Livestock Enteritidis AM, CF, CTX, S, GM, NA, TE ���SAL111-CF-HU10 Clinical feces Enteritidis AM, CF, CTX, NA, TE FOX, SXT ���

S. TyphimuriumSAL3-FF-KW02 Processed foods Typhimurium NA ���SAL4-FF-KW02 Processed foods Typhimurium S, NA ���SAL8-FF-KW02 Processed foods Typhimurium NA ���SAL9-FF-KW02 Processed foods Typhimurium C ���SAL12-FF-KW02 Processed foods Typhimurium NA ���SAL14-FF-KW02 Processed foods Typhimurium ���SAL15-FF-KW02 Processed foods Typhimurium ���SAL20-FF-KW02 Processed foods Typhimurium S, NA ���SAL21-FF-KW02 Processed foods Typhimurium ���SAL22-FF-KW02 Processed foods Typhimurium S ���SAL25-FF-KW02 Processed foods Typhimurium S ���SAL26-FF-KW02 Processed foods Typhimurium S ���SAL27-FF-KW02 Processed foods Typhimurium S, NA ���SAL34-FV-KW02 Fruit-vegetables Typhimurium S ���SAL35-FV-KW02 Fruit-vegetables Typhimurium ���SAL36-FV-KW02 Fruit-vegetables Typhimurium ���SAL37-FV-KW02 Fruit-vegetables Typhimurium ���SAL38-FV-KW02 Fruit-vegetables Typhimurium ���SAL39-FV-KW02 Fruit-vegetables Typhimurium ���SAL40-FV-KW02 Fruit-vegetables Typhimurium ���SAL41-FV-KW02 Fruit-vegetables Typhimurium ���SAL42-FV-KW02 Fruit-vegetables Typhimurium ���SAL43-FE-KW02 Other foods Typhimurium ���SAL44-FV-KW02 Fruit-vegetables Typhimurium ���SAL45-FV-KW02 Fruit-vegetables Typhimurium ���

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TABLE 1 (Continued)

Host strain Sample origin Serotype

Antibiotic resistancea

Phage wksl3susceptibilitybResistance Intermediate

SAL46-FV-KW02 Fruit-vegetables Typhimurium ���SAL47-FV-KW02 Fruit-vegetables Typhimurium ���SAL48-FV-KW02 Fruit-vegetables Typhimurium ���SAL49-FV-KW02 Fruit-vegetables Typhimurium ���SAL50-FV-KW02 Fruit-vegetables Typhimurium ���SAL51-FV-KW02 Fruit-vegetables Typhimurium ���SAL52-FV-KW02 Fruit-vegetables Typhimurium ���SAL53-FV-KW02 Fruit-vegetables Typhimurium ���SAL54-FC-KW02 Livestock Typhimurium S, NA ���SAL56-FF-KW02 Processed foods Typhimurium ���SAL72-UI-KK Unidentified Typhimurium AM, S, C, TE AmC ��

Other S. enterica serotypesSAL101-FC-KK09 Livestock Agona S ���SAL70-EW-KK Environments Agona S ���SAL71-FF-KK Processed foods Anatum S �SAL78-EW-KU09 Environments Arizonae S TE �SAL79-VS-KU09 Vet feces Arizonae AM, S, C, TE �SAL83-VS-KU10 Carcasses Arizonae AM, CF, S, TE AmC �SAL84-VS-KU10 Carcasses Arizonae AM, CF, S, SXT, TE AmC, GM, NA, CIP �SAL85-VS-KU10 Carcasses Arizonae AM, CF, C, TE SXT �SAL86-VS-KU10 Carcasses Arizonae AM, CF, S, SXT, TE AmC, GM, NA, CIP �SAL87-FP-KU10 Livestock Arizonae AM, CF, S, GM, C, SXT, TE �SAL94-EW-KU10 Environments Arizonae S, TE �SAL108-VS-KU10 Carcasses Arizonae AM, CF, S, NA, TE AmC, CTX �SAL109-VS-KU10 Carcasses Arizonae AM, CF, S, NA, TE AmC �SAL110-VS-KU10 Carcasses Arizonae AM, S, C, TE AmC, CF, SXT �SAL103-FC-KK09 Livestock Dessau NA S �SAL106-FC-KK09 Livestock Dessau GM, NA S �SAL31-FC-KW02 Livestock Haardt AM, S, NA, TE CF �SAL55-FC-KW02 Livestock Haardt AM, S, NA, TE CF �SAL59-FC-KF04 Livestock Haardt AM, S, NA, TE �SAL60-FC-KF04 Livestock Haardt AM, S, GM, NA, TE �SAL73-UI-KK Unidentified Heidelberg S, GM ��SAL68-FF-KK Processed foods Infantis S �SAL69-FE-KK Other foods Javiana S ����SAL77-FM-KK Marine foods Kentucky S �SAL95-FF-KK09 Livestock London S �SAL98-FF-KK09 Livestock London S �SAL76-FF-KL Processed foods Montevideo S �SAL100-FC-KK09 Livestock Montevideo NA �SAL102-FC-KK09 Livestock Montevideo NA S �SAL75-FM-KK Marine foods Poona AmC S �SAL64-FB-KF05 Livestock Rissen TE S �SAL96-FF-KK09 Livestock Rissen S, C, TE �SAL104-FC-KK09 Livestock Weltevreden �SAL80-CF-KU09 Clinical feces Nontyped AM, S, TE GM ��SAL81-CF-KU09 Clinical feces Nontyped AM, S, NA, C, TE �SAL82-CF-KU09 Clinical feces Nontyped AM, S, TE ���SAL88-EM-KU10 Environments Nontyped S �SAL89-EM-KU10 Environments Nontyped S �SAL90-EM-KU10 Environments Nontyped S �SAL91-EM-KU10 Environments Nontyped S �SAL92-EM-KU10 Environments Nontyped S �SAL93-EM-KU10 Environments Nontyped S �SAL107-FC-KK09 Livestock Nontyped GM, NA S �

a Ampicillin, AM; amoxicillin-clavulanic acid, AmC; cephalothin, CF; cefoxitin, FOX; cefotaxime, CTX; streptomycin, S; gentamicin, GM; nalidixic acid, NA; ciprofloxacin, CIP;chloramphenicol, C; trimethoprim-sulfamethoxazole, SXT; tetracycline, TE.b ����, complete lysis with secondary infection; ���, complete lysis; ��, lysis; �, turbid lysis; �, growth inhibition zone; �, no plaques.

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bladder, testes, stomach, intestines, cecum, colon, spleen, pancreas, liver,kidneys, heart, thymus, and oral cavity of all test animals were conducted.All animals were euthanized by carbon dioxide asphyxiation. The nec-ropsy results of all test animals were compared with those of negative-control animals.

Application of phages to control Salmonella on chicken skin.Chicken skin samples were collected from broiler carcasses to verify theeffectiveness of wksl3 for the control of experimentally contaminated S.Enteritidis on the surface of chicken skin. Sample preparation was per-formed as previously described, with modifications (37), and a total of 50skin pieces (4 cm2) were prepared.

SAL111-CF-KF10 (5 � 104 CFU/ml; selective marker is nalidixic acidand cefotaxime resistance) (23) was sprayed onto chicken skin with adisposable hand sprayer that transferred 0.2 ml per operation to give aninitial concentration of approximately 103 to 104 CFU/cm2. Each skinpiece was inoculated individually. Sprayed samples were dried underblowing air for 1 h at 8°C, which is the average temperature of a domesticrefrigerator (38). Phage solutions (2.2 � 108 PFU/ml) were diluted withphosphate-buffered saline (PBS) and inoculated at an MOI of approxi-mately 5 � 103 by the same spraying method as previously described.Phage solutions were applied to the chicken skin one at a time shortly afterSalmonella contamination. Twenty-five skin pieces were treated withphage solutions, and the remaining skin pieces were treated with the samevolume of PBS as a control. Each skin piece was homogenized and dilutedin 4 ml PBS. Viable cells were counted immediately after phage adminis-tration (day 0) and on test days 1, 2, 3, 5, and 7 by using four pieces eachday. To determine the number of viable strains per skin piece, LB agarplates containing 128 �g/ml nalidixic acid and 8 �g/ml cefotaxime wereused as selection media.

Statistical analysis. Differences in weight changes and the numbers ofviable bacteria between phage-treated and untreated groups were statisti-cally analyzed with paired t tests and Duncan’s multiple-range tests. Pvalues of less than 0.05 were considered statistically significant. All testswere conducted with IBM SPSS Statistics 20 software.

Nucleotide sequence accession number. The complete genome se-quence of virulent Salmonella phage wksl3 was deposited in GenBankunder accession number JX202565.

RESULTSBacteriophage wksl3 isolation and host spectrum determina-tion. Ten bacteriophage types were recovered from chicken by-product samples with S. Enteritidis ATCC 13076 as an indicatorstrain. Spotting assays were performed with various Salmonellastrains, and one broad host spectrum phage, designated wksl3,was selected for further analysis. Phage wksl3 showed 100% lysisactivity against all of the S. Enteritidis (n � 32) and S. Typhimu-rium (n � 36) strains tested and also infected S. enterica serovarsAgona, Heidelberg, and Javiana, as well as 68% of the Salmonellastrains tested (111 isolates). Positive lytic reactions were observedregardless of the host’s antimicrobial resistance, showing thatboth drug-sensitive and -resistant S. Enteritidis and S. Typhimu-rium were phage susceptible (Table 1).

Phage wksl3 microscopy. Morphological analysis revealedthat phage wksl3 belongs to the order Caudovirales and familySiphoviridae morphotype B1 (the isometric head was 63 nm, andthe long. noncontractile tail was 121 by 7.9 nm, with a 20-nm-wide baseplate with tailspikes), similar to the morphology ofphage SETP3 (62.5 nm, icosahedral). Tails were rigid and noncon-tractile, measured 120 by 7 nm, and exhibited a 20-nm-wide base-plate with spikes (39).

One-step growth curve. More than 90% of the wksl3 particlesattached to the host cell within the first 10 min. Phage wksl3 dis-

played a 19-min latency period with a calculated average burst sizeof 51 PFU/cell (data not shown).

DNA sequence analysis. According to sequence analysis, thewksl3 genome is composed of 42,766 bp, including 133-bp directrepeats at the end of the genome, with a total G�C content of49.81%, which is similar to that of another Siphoviridae phage,SETP3 (42,572 bp in length with a G�C content of 49.85%). Atotal of 54 putative promoters, 20 transcriptional termination re-gions, and 64 ORFs, representing 91.6% of the phage sequence,were predicted in its genome. Genes were located in a region ofhigh density (1.501 genes/kb), and the average length of each genewas 620 bp. Many short overlapping regions between contiguousgenes were commonly detected. No predicted tRNA genes werediscovered by tRNAscan-SE software. On the basis of transcrip-tional direction, the genome was predicted to be clustered intofour groups. Twenty-three gene products showed significant ho-mology to reported functional genes. Two ORFs were found to bemembers of the helix-turn-helix (HTH) superfamily, and oneORF was found to be a member of the immunity to superinfectionmembrane superfamily. While 38 ORFs were shown to encodehypothetical proteins, one ORF in wksl3 (gp33) showed no obvi-ous homology to any other bacterium-, phage-, or prophage-re-lated genes in the current GenBank database.

Bioinformatic studies of all 64 gene products of phage wksl3showed no similarities to any other known virulent, toxin, orpathogen-associated protein family or gene product of Salmonellaor any other pathogenic bacterium. With a 0.01 E-value cutoff,none of the protein products from the 64 predicted wksl3 geneswere matched with polypeptides or protein sequences containedin the food allergenic protein sequence database.

According to the homology search-based annotation of func-tional genes, the wksl3 genes were categorized into three func-tional groups (Fig. 1): cell wall lysis genes (gp1, putative amidase;gp14, lysozyme; gp15, putative holin), phage structural genes(gp2, structural protein; gp3, terminase; gp4, terminase small sub-unit; gp38, tailspike protein; gp39, tail protein; gp43, tape measureprotein; gp51, 53, 54, 58, and 63, tail proteins; gp59, head protein;gp60, coat protein; gp64, head morphogenesis protein), and me-tabolism-related genes (gp25, helicase-primase; gp32, DNA poly-merase; gp34, restriction endonuclease; gp36, helicase; gp48,HNH endonuclease, gp50, DNA-binding protein). ORF informa-tion, such as the positions of genes, amino acid lengths, directionsof transcription, sizes, functions, and homologies between wksl3genes and other phage-related genes, is shown in Table 2.

Dot plot analysis of wksl3 and other Salmonella phages(SETP3, SS3e, vB_SenS-Ent1, and SE2) revealed that they showsignificant homology at the nucleotide sequence level (Fig. 2B),suggesting that these phages can be clustered into a tentative rela-tional group. BLAST2 sequence comparisons with other SETP3-like phages revealed that the full wksl3 genome sequence shows84% homology to that of SETP3 (GenBank accession numberEF177456), 89% with that of vB_SenS-Ent1 (GenBank accessionnumber HE775250), 90% with that of SE2 (GenBank accessionnumber JQ007353), and 93% with that of SS3e (GenBank acces-sion number AY730274). CoreGenes 3.0 analysis, with theBLASTP threshold score set at 75, revealed that these five phageshave a total of 40 homologous genes in common, while wksl3 has50, 53, 56, and 54 homologous genes in common with SETP3,SS3e, SE2, and vB_SenS-Ent1, respectively (40). To compare thehomologies of the ORFs in wksl3, SS3e, and SETP3, we used

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BLASTN and BLASTP analyses and confirmed the colinear andreverse complemented arrangement of functionally homologousgenes or orthologous proteins (Fig. 2A), suggesting that wksl3 hasthe same ancestry as other SETP3-like phages, including SETP3,vB_SenS-Ent1, SE2, and SS3e.

Like other lytic SETP3-like phages, no proteins encoded bywksl3 genes showed homology to other phage lysogenic-cycle-related genes, such as integrases, in GenBank. In addition,PHACTS (41) results predicted that phage wksl3 is a lytic phagewith a PHACTS output value of 0.532, corresponding to the frac-tion of trees in the algorithm supporting this conclusion. In fact,SETP3 and SE2 have also been reported as lytic (42, 43). This valueindicates that wksl3 also has strong lytic activity; in comparison,the well-known, strongly virulent Listeria phage P100 (GenBankaccession number DQ004855) was predicted to be lytic with a0.525 value.

As demonstrated in Fig. 1, wksl3 genes were arranged into fourtranscriptional clusters representing the regulator operon (gp46to gp50), the replication operon (gp23 to gp37), and two late oper-ons. Structural genes and cell wall lysis-related genes were foundin the late operons. A relatively large gene-free region (857 bp) wasobserved between gp22 and gp23, and this region was also ob-served in vB_SenS-Ent1 (957 bp) and SE2 (994 bp), located be-tween genes orthologous to wksl3 gp22 and gp23.

The two subunits of gp1 of wksl3 were located at the right andleft ends of the linear genome structure. Both subunits appearedto be transcribed in the same direction. Since circularly permuted

genomes are commonly observed in head-full DNA packagingschemes (44), it is possible that the two gp1 subunits, which wereseparated by the terminal redundant region, may be translated asone single gene. The gp1 orthologs SE2 gp33 and SETP3 gp32 alsoshowed single-gene translation. Moreover, gp1 exhibited signifi-cantly better homology to SE2 gp33 and SETP3 gp32 in terms ofamino acid length and sequence identity than the combined orseparate gene products of the two subunits.

Metabolism-related operons. The phage wksl3 regulatoroperon is composed of five genes (gp46 to gp50) and is locatedbetween two structural gene clusters. gp50 shows high homologyto phage vB_SenS-Ent1 gp20 (88%) and SETP3 gp47 (87%), eachof which encodes a DNA-binding protein. These three ortholo-gous genes also contained both the phage regulatory protein Rha(pRha) domain and the phage antirepressor protein KilAC do-main (ANT). Phage SE2 gp49 showed significantly high sequencehomology (99%) with gp20 but differed in that it lacked an N-ter-minal pRha domain. The P22 antirepressor ant encodes an anti-repressor protein that binds to and inhibits the CII repressor pro-tein of phage P22 (45). In addition, P22 ant activity against lambdarepressor CI has also been observed (46). We expect that the an-tirepressor domain of wksl3 will function in lytic-activity devel-opment, similar to the P22 antirepressor protein (47). An HNHendonuclease was predicted to be encoded by gp48, which showed99% homology to vB_SenS-Ent1 gp22 and 62% homology tophage T1 endonuclease, whereas no homologous gene was iden-tified in the SETP3 genome. gp46 was predicted to possess immu-

FIG 1 Genomic structure of wksl3, including the terminal redundant region. Arrows represent predicted genes and indicate their directions of transcription.Different colors denote different functional groups of bacteriophage genes.

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TABLE 2 Full genome sequence of wksl3

ORF

wksl3 coordinates(amino acidlength) Strand

Molecularmass(kDa) pI Best match (%) Function Best E value

gp1 42539–559 (216) � 24.66 6.45 SETP3 orf32 (73) Putative amidase 3E-107gp2 590–2065 (491) � 53.92 4.71 SETP3 orf31 (94) Putative structural protein 0gp3 2078–3349 (423) � 47.52 5.86 SETP3 orf30 (96) Terminase 0gp4 3339–3845 (168) � 18.78 5.99 vB_SenS-Ent1 orf1 (98) Putative terminase small subunit 1E-116gp5 3874–4005 (43) � 4.71 4.22 SE2 orf29 (86) 6E-18gp6 4141–4248 (35) � 3.93 4.56 SS3e orf18 (100) 3E-16gp7 4245–4547 (100) � 11.60 9.52 SS3e orf19 (100) 3E-64gp8 4547–4771 (74) � 8.58 9.34 SS3e orf20 (100) 6E-47gp9 4768–4917 (49) � 5.93 5.4 SS3e orf21 (98) 1E-23gp10 4914–5144 (76) � 8.87 5.62 SS3e orf22 (99) 4E-46gp11 5141–5311 (56) � 6.20 10.29 SS3e orf23 (100) 2E-32gp12 5293–5451 (52) � 6.00 4.3 vB_SenS-Ent1 orf54 (79) 4E-08gp13 5448–5633 (61) � 6.89 7.89 SETP3 orf24 (93) 3E-32gp14 5820–6308 (162) � 17.50 9.71 SETP3 orf23 (93) Lysozyme 3E-97gp15 6286–6576 (96) � 10.61 9.8 vB_SenS-Ent1 orf51 (83) Putative holin 9E-52gp16 6578–6859 (93) � 10.29 7.87 vB_SenS-Ent1 orf50 (98) 6E-60gp17 6939–7373 (144) � 15.38 4.63 SS3e orf27 (100) 7E-101gp18 7379–7744 (121) � 13.82 10.52 SS3e orf28 (100) 1E-82gp19 7741–7944 (67) � 7.32 7.79 SS3e orf29 (100) 6E-41gp20 7941–8546 (201) � 23.54 5.73 SS3e orf30 (100) 1E-123gp21 8539–8652 (37) � 4.10 3.4 SS3e orf31 (100) 3E-16gp22 8715–8879 (54) � 6.41 11.57 SETP3 orf18 (89) 1E-26gp23 9736–9921 (61) � 7.28 8.84 SS3e orf32 (100) DNA-binding helix-turn-helix superfamily protein 9E-37gp24 9918–10151 (77) � 8.60 9.61 SS3e orf33 (99) 2E-46gp25 10208–12394 (728) � 80.57 4.97 vB_SenS-Ent1 orf43 (98) Putative replicative helicase-primase 0gp26 12386–12628 (80) � 8.84 8.8 SETP3 orf14 (100) Helix-turn-helix family protein 2E-44gp27 12762–13274 (170) � 19.16 4.65 SE2 orf11 (99) 2E-112gp28 13318–13521 (67) � 7.66 7.87 SE2 orf10 (100) 1E-41gp29 13518–13790 (90) � 10.78 9.2 SE2 orf9 (100) 8E-62gp30 13787–15061 (424) � 47.16 6.35 SE2 orf8 (99) 0gp31 15143–15772 (209) � 23.63 4.85 SE2 orf7 (98) 1E-134gp32 15830–18031 (733) � 82.51 6.93 SETP3 orf10 (98) DNA polymerase 0gp33 18119–18271 (50) � 5.42 7.81gp34 18268–18552 (94) � 10.82 9.56 vB_SenS-Ent1 orf36 (96) Putative restriction-endonuclease 9E-58gp35 18584–18775 (63) � 7.03 9.45 vB_SenS-Ent1 orf35 (100) 1E-36gp36 18772–21237 (821) � 92.29 8.87 SETP3 orf6 (98) Putative helicase 0gp37 21234–21455 (73) � 8.72 4.85 SS3e orf48 (100) 1E-41gp38 21574–23604 (676) � 72.11 5.22 SETP3 orf4 (95) Tailspike protein 0gp39 23641–26127 (828) � 90.96 5.18 vB_SenS-Ent1 orf31 (98) Putative tail protein 0gp40 26190–26453 (87) � 9.94 5.41 vB_SenS-Ent1 orf30 (95) 1E-54gp41 26552–27067 (171) � 19.15 4.46 SE2 orf58 (99) 3E-120gp42 27064–27564 (166) � 16.49 4.59 SS3e orf54 (98) 2E-118gp43 27566–29899 (777) � 83.14 4.75 vB_SenS-Ent1 orf27 (98) Putative tape measure protein 0gp44 29892–30251 (119) � 13.61 4.69 SE2 orf55 (97) 2E-81gp45 30257–30673 (138) � 15.87 5.28 vB_SenS-Ent1 orf25 (99) 6E-96gp46 30843–31022 (59) � 6.60 9.7 SETP3 orf50 (100) Immunity to superinfection membrane superfamily protein 3E-32gp47 31085–32203 (372) � 42.22 8.66 SETP3 orf49 (99) 0gp48 32256–32756 (166) � 19.28 9.92 vB_SenS-Ent1 orf22 (99) Putative HNH endonuclease 5E-117gp49 32766–32951 (61) � 6.89 6.01 SETP3 orf48 (82) 2E-21gp50 33112–33783 (223) � 25.63 6.24 vB_SenS-Ent1 orf20 (88) Putative DNA-binding protein 3E-138gp51 33812–34981 (389) � 41.20 4.64 vB_SenS-Ent1 orf19 (99) Putative tail protein 0gp52 34981–35400 (139) � 15.06 4.56 SETP3 orf45 (100) 9E-97gp53 35400–35795 (131) � 14.47 9.75 SETP3 orf44 (97) Putative tail protein 2E-88gp54 35792–36151 (119) � 12.98 9.4 vB_SenS-Ent1 orf16 (96) Putative tail protein 71E-76gp55 36151–36756 (201) � 20.69 6.59 SE2 orf44 (94) 9E-131gp56 36759–37268 (169) � 17.81 4.84 SE2 orf43 (95) 4E-113gp57 37272–37460 (62) � 7.15 4.92 vB_SenS-Ent1 orf13 (98) 3E-35gp58 37497–37847 (116) � 12.23 4.38 vB_SenS-Ent1 orf12 (95) Putative Hoc protein 3E-72gp59 37859–38143 (94) � 9.37 9.77 SETP3 orf38 (96) Putative head protein 1E-46

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nity to superinfections via three predicted transmembrane do-mains (TMHMM), and this gene showed not sequence butfunctional homology to the vB_SenS-Ent1 immunity protein(gp24).

The replication operon (gp23 to gp37) of wksl3 encodes a rep-licative helicase-primase, a helicase, a DNA polymerase, a restric-tion endonuclease, two putative HTH DNA-binding regions, andnine hypothetical proteins. gp25 encodes a replicative helicase-primase containing an AAA_25 (pfam13481) domain and a hexa-meric replicative helicase RepA region. gp36 encodes a helicaseconsisting of a homing endonuclease intein domain of 346 aminoacids (pfam05203) and an SNF2 family N-terminal domain. A

BLASTP search of the wksl3 helicase revealed 98% sequence ho-mology to the SETP3 helicase and the vB_SenS-Ent1 intein-con-taining helicase precursor. Without an endonuclease domain,gp36 showed significant identity with SE2 gp1 (99%) and SS3egp47 (97%). The DNA polymerase encoded by gp32 showed ho-mology to the SETP3 DNA polymerase (98%) and the vB_SenS-Ent1 intein-containing DNA polymerase precursor (98%), whilethe wksl3 DNA polymerase lacked a 299-amino-acid intein do-main. gp34, which encodes a restrictive endonuclease, showedhigh similarity to the putative restriction endonuclease (96%) ofvB_SenS-Ent1, gp44 (92%) of SS3e, and gp8 (89%) of SETP3.gp26 contains a Cro/C1-type HTH DNA-binding domain with a

TABLE 2 (Continued)

ORF

wksl3 coordinates(amino acidlength) Strand

Molecularmass(kDa) pI Best match (%) Function Best E value

gp60 38204–39253 (349) � 37.89 4.84 SETP3 orf37 (97) Putative coat protein 0gp61 39257–39958 (233) � 25.72 5.63 SS3e orf10 (99) 2E-162gp62 40149–40538 (129) � 14.39 9.27 SS3e orf11 (100) 4E-89gp63 40856–41314 (152) � 16.40 4.59 SETP3 orf34 (95) Tail protein 1E-95gp64 41317–42360 (347) � 38.58 6.39 SETP3 orf33 (96) Head morphogenesis protein 0

FIG 2 (A) Three homologous SETP3-like Salmonella phages. Rectangles represent the genes of each phage, and the numbers above the rectangles are ORFnumbers. Numbers in parentheses under the rectangles indicate the corresponding wksl3 gene numbers that show high sequence homology. The color of eachrectangle represents the functional group of each gene, predicted with BLAST programs. The scale is in kilobase pairs. (B) Dot plot image displaying sequencehomology comparisons of wksl3 and four SETP3-like phages (SETP3, SS3e, vB_SenS-Ent1, and SE2).

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transcriptional direction that is the opposite of that of the replica-tion operon, suggesting that gp26 may act as a regulator protein.gp23 also contains an HTH transcription regulator MerR super-family region.

Structure- and cell wall lysis-related operons. Two late oper-ons (gp38 to gp45 and gp51 to gp64/gp1 to gp22) contained genesinvolved in phage structure and host cell lysis. The arrangement ofgenes encoding wksl3 structure assembly proteins followed theconserved synteny and gene orders of Siphovirus (48). Entire geneproducts encoded by the wksl3 late operon had orthologous geneproducts in other SETP3-like phages. PSI-BLAST and Pfam anal-ysis results against a putative phage structural protein (gp2) sug-gested that the gene product immediately downstream of the twoterminase subunits (gp3 and gp4) was predicted to be a portalprotein. The major tail protein of wksl3 (gp51) showed 99% ho-mology to the vB_SenS-Ent1 major tail protein and exhibited 99%and 93% similarity to the SE2 and SETP3 tail proteins, respec-tively. Comparisons of the tape measure protein lengths andTEM-based tail lengths of wksl3 and SETP3 confirmed that the taillength corresponded to the length of the tape measure protein,similar to other Siphoviridae phages (49, 50). BLASTP analysis ofgp39 showed significant homology to the tail fiber proteins ofvB_SenS-Ent1 (98%) and SE2 gp60 (98%). Interestingly, wksl3gp39 also showed sequence similarity to putative tail proteins ofEscherichia phages K1ind3 (62%), K1ind1 (61%), K1dep1 (61%),and K1dep4 (62%). gp38 encodes a tailspike protein that containsa phage P22 tailspike domain in the C-terminal region. The C-ter-minal sequence of the P22 tailspike protein, which possesses oli-gosaccharide-binding and endorhamnosidase activities, is crucialfor binding to the O-antigen region of the bacterial lipopolysac-charide (51). These results indicate that wksl3 may be able to rec-ognize lipopolysaccharide (LPS) O-antigenic repeating units toinfect host cells. O-antigen affinity was also observed in phageSETP3 (39). BLASTP results showed significantly high homologyto the tailspike proteins of Siphoviridae phages SE2 (98%),SETP13 (97%), SS3e (96%), vB_SenS-Ent1 (96%), SETP3 (95%),SETP7 (94%), SETP5 (95%), and SETP12 (95%). High local sim-ilarity to the P22 tailspike region in wksl3 gp38 was also found inSalmonella Podoviridae phages ST104 (80%), P22 (80%), SETP14(80%), SPN9CC (79%), SE1 (79%), ST64T (79%), SETP1 (79%),and 15 (79%).

Phage wksl3 was expected to encode a holin (gp15), a lysozyme(gp14), and an amidase (gp1), which would affect the phage’smechanism of host cell wall lysis. In wksl3, lysis-related genes werelocated in the late operon. gp1 showed 76% and 73% C-terminalhomology to the amidase of phages SE2 and SETP3. As in manyother double-stranded DNA phages, gp15, a predicted holin com-posed of 96 amino acids with three helical transmembrane regions(34), and gp14, which encodes a lysozyme with 93% homology tothe SETP3 lysozyme, seemed to be involved in the holin-endolysinsystem. Regarding the lysis cassette of the dual-protein system(52), up to five proteins (gp9 to gp13) encoded by the cassette werenot assigned to any other auxiliary lysis functions. This systemdiffers from that of Salmonella phage SE2 (42), which was pre-dicted to use a holin-independent lytic system. Considering thepredicted promoter and terminator regions, as well as the direc-tion of gene transcription, the amidase and dual-lysin systemsseemed to act independently on host cell lysis.

Acute oral toxicity study with mice. No mice in either the testor the control group died during the 8-day study period. No clin-

ical signs of wksl3-mediated toxicity were observed in the testgroup to which a single dose of 1011 PFU/kg body weight wasadministered. No development of abnormal behavior, changes inphysical appearance (such as hair loss or wound formation), orany other toxicological effects, including inflammation, allergy, ordiarrheal symptoms, were observed in any mouse during the ex-perimental period (data not shown). The mean body weights ofthe phage-treated and control groups before treatment did notdiffer significantly. However, average increases of 3.62 g (phage-treated group) and 2.77 g (phage-untreated group) were observedby day 8 and these changes were significantly different from thebaseline measurements (P � 0.05). Moreover, the absoluteweights of the mice in the two groups differed significantly as aresult of these changes in weight (P � 0.05), with phage-treatedmice showing a greater increase in body weight.

Macroscopic examination of the organs of all of the phage-treated mice revealed normal color compared to those of un-treated animals (data not shown). Moreover, no obvious differ-ences or gross lesions were found in the skin, lymph nodes,bladder, testes, stomach, intestines, cecum, colon, spleen, pan-creas, liver, kidneys, heart, thymus, or oral cavity in either treatedor untreated mice at postmortem examination. Thin sectionswere not observed in the gastrointestinal tracts of phage-treatedmice. Autopsy results of stomach tissues from wksl3-treated miceshowed that no adverse inflammatory effects occurred at eitherthe outer or the inner surface of the stomach (data not shown). Allexaminations were performed and confirmed by a doctor of vet-erinary medicine (DVM). According to the DVM’s opinion oftheir gross pathology, there were no noticeable abnormalities inphage-treated mice, supporting the conclusion that phage wksl3 isnontoxic.

Effectiveness of wksl3 in the control of Salmonella onchicken skin. Figure 3 demonstrates the effects of phage wksl3 onSalmonella strains used to inoculate chicken skin. Skin pieces wereexperimentally sprayed with approximately log10 3.25 CFU of Sal-monella Enteritidis/cm2 of skin. A single-dose application ofphage wksl3 resulted in a 3.04-log decrease (P � 0.001) in thenumbers of viable Salmonella bacteria after 24 h of storage at 8°C.

FIG 3 Effect of phage wksl3 on growth of S. Enteritidis on chicken skin incu-bated at 8°C. Diluted Salmonella cells (103 CFU/cm2 skin) were applied at day0. Phage wksl3 (1 � 107 PFU/ml, open circles) and buffered peptone water(negative control, closed circles) were applied after 2 h of stabilization at 4°C.

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Viable Salmonella cell concentrations were reduced by log10 0.3CFU/cm2 (mean, 2 CFU/cm2) at day 1, but growth resumed afterday 2. According to our statistical analysis, no significant growthwas observed after days 2 to 7 (P � 0.13). Phage wksl3-treatedsamples maintained a decrease in the average viable Salmonellalevel of log10 2.43 CFU/cm2 (P � 0.01) from day 2 to day 7. Forty-eight randomly selected viable cells recovered from wksl3-treatedchicken skins after day 7 retained their susceptibility to phagewksl3.

DISCUSSION

Bacteriophages (or, more simply, phages) are viruses that infectand kill bacterial cells. Generally, phages are found near the hostbacteria (53) and recognize specific bacterial hosts (54). While theemergence of pathogenic bacteria with increasing resistance tocurrently used antimicrobial agents is growing, phages have nowresurfaced as potential biocontrol and therapeutic agents (55–60).The majority of human-infecting Salmonella strains originatefrom poultry and poultry-derived food products (61). On thisbasis, we screened and isolated Salmonella-specific phages fromchicken carcasses and by-products purchased from local marketsin the Republic of Korea. A wide-host-spectrum virulent phage,wksl3, was selected and characterized to investigate its host spec-trum in relation to antibiotic resistance patterns, genomic charac-terization, oral toxicity, and efficacy of model phage application toprocessed chicken carcasses from chicken processing plants forassessment of its adequacy as a potential biocontrol agent.

Phage wksl3 was obtained with S. Enteritidis ATCC 13076 as anindicator strain; the phage had strong lytic activity against S. Ty-phimurium and S. Enteritidis and also inhibited the growth of S.Agona, S. Heidelberg, and S. Javiana. Phage wksl3 formed differ-ent plaque turbidities with variable efficiencies of plating, regard-less of the antimicrobial resistance patterns of the strains (Table1). While S. Typhimurium and S. Enteritidis have been proven tobe the major serotypes responsible for the majority of Salmonella-related outbreaks, this result implied that application of this phagecould be an effective tool to reduce S. Enteritidis- and S. Typhi-murium-related food-borne salmonellosis by inactivating thegrowth of S. Enteritidis and S. Typhimurium, including a fewadditional serotypes from the field. We were able to classify Sal-monella phage wksl3 in the order Caudovirales and the family Si-phoviridae (Fig. 4) on the basis of its morphology (an icosahedralhead with a noncontractile tail) (62).

In silico full-genome sequence analysis revealed that none of thewksl3 gene products showed any similarity to known pathogenic-bacterium-related toxin, pathogenic, or virulence-encoding genes.Bioinformatics also failed to indicate homology between any of thewksl3 gene products and potential immunoreactive food allergens.High homology in terms of genome size and sequence identity sug-gested that wksl3 is related to SETP3, SE2, vB_SenS-Ent1, and SS3e(39, 42, 63). In particular, we identified wksl3 as a novel member ofthe group of SETP3-like phages that is classified in the family Sipho-viridae in an International Committee on Taxonomy of Viruses re-port released in 2011 (63) and in the NCBI taxonomy.

Bacteriophages are known to recognize various outer cell wallcomponents, including flagella (64–66), LPS (67–70), and severalouter membrane proteins (71–74). While phage cocktails are gen-erally composed of phages with a spectrum of two or more hostsusing different receptors, receptor identification is the first step inthe measurement and evaluation of their potential applications in

the biocontrol of target bacteria (75) and is useful for producingmore efficient phage cocktails. According to our current genomeanalysis, the tailspike protein of wksl3 exhibits a high level of ho-mology to various Salmonella phages, including SETP3-likephages. Tailspike proteins encoded by SETP3-like phages containa phage P22 tail-spike domain in the C-terminal region. Bothphages P22 and SETP3 are known to recognize the O antigen as areceptor (39, 51). Phage wksl3 encodes two endonucleases thatplay important roles in phage replication. Nuclear disruption ofhost DNA occurs because of the action of endonucleases, anddisrupted nucleotide particles are recombined into progeny phageDNA (76). Methyltransferases or methylases in phages protectDNA from self or host cell restriction enzymes (77). The DNAmethylase N-4/N-6 domain protein was encoded in SE2 (42),while other SETP3-like phages did not have homologous genes orputative methylase functional genes in their genomes. However,wksl3 gp35 showed 30 and 50% homology to Anaeromyxobacterdehalogenans 2CP-C lysine N-methyltransferase and Planctomycesbrasiliensis DSM5305 guanine-N(2)-methyltransferase, respec-tively. gp47 also contained a domain in the N-terminal regionsimilar to the Saccharomonospora cyanea NA-134 N-6 DNA meth-ylase domain, with 28% similarity. gp35 and gp37 of phage wksl3can be predicted to function as potential methylases and/or meth-yltransferases.

In our oral acute-toxicology study with mice to observe phagewksl3-related toxicity effects, a high-titer (1.1 � 1011 PFU/kg bodyweight) single dose of phage wksl3 produced no test substance-related clinical signs, gross lesions, or deaths. The titer used in thisstudy corresponds to 6.82 � 1012 phage particles per average hu-man body weight (62 kg) (78). According to a previous study (79),the total surface area of a 1.5-kg chicken carcass corresponds to1,940 cm2 and an uncooked chicken carcass will contain approx-imately 3.1 � 1010 phage particles. More than 200 chickens wouldhave to be consumed by an individual to equal the phage titertested. Considering global chicken and poultry consumption peryear (80), this value is quite high. Previous studies have also re-ported that no deaths, abnormalities, or adverse effects were ob-served after the administration of phages to animals (18) and that

FIG 4 Transmission electron micrograph of lytic bacteriophage wksl3. Bar,200 nm.

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oral administration of phages to humans is also safe, with no ad-verse effects (81). Advanced toxicity testing, including long-termtoxicity tests and histopathological analysis, is required to confirmthe safety of phage wksl3.

Phage wksl3 was first applied to LB broth at different MOIs andtemperatures (22 and 8°C; data no shown). Phage application(1.1 � 1011 PFU/ml) with overnight culture of the host strain atlow temperatures resulted in complete elimination of Salmonellacells from the broth. Tests performed at 22°C also showed a sig-nificantly large (4- to 5-log) reduction but not complete eradica-tion of viable Salmonella cells on day 1. However, phage-treatedsamples incubated at 8°C showed complete elimination of hostcells over the 1-week test period. Hence, the present study dem-onstrated the optimal application conditions for phage particleson artificially contaminated chicken skin (low temperature andappropriate MOI). The Salmonella count was successfully reducedbelow the detection limit (30 CFU/ml). Regarding the phage sus-ceptibility of the remaining strains in Fig. 3, escape from contactwith phage particles because of their immobilization on foodseems to cause incomplete Salmonella reduction. A standardizedphage amount per unit of meat surface area and a method ofhomogeneous distribution on food are necessary to achieve highlyefficacious pathogen reduction (19). For effective application ofwksl3 to foods, the time points, frequency, and dosage of thephage on different types (forms, textures, etc.) of foods shouldalso be optimized.

In a previous investigation at a chicken slaughtering plant,phage application in chiller water showed more efficient reduc-tion (J. W. Kim et al., unpublished data), so it would be useful totest wksl3 in a processing plant in a future study.

The emergence of multiple-antibiotic-resistant pathogens hassparked interest in alternative antibiotics. Phages are predators ofspecific pathogens, and many Western countries have begun toinvestigate whether this natural bacterial enemy may be able tocontrol pathogens that cannot be controlled by available drugs(82). Phages have merits in various contexts; (i) phage-resistantstrains are still susceptible to other phages targeting different re-ceptors and (ii) isolation of novel phages requires relatively lesstime than the development of new antibiotics (83). The most in-teresting finding in this study is that Salmonella phage wksl3showed a bactericidal effect against all of the S. Enteritidis and S.Typhimurium strains tested, regardless of their antimicrobial re-sistance. This may suggest that this phage could be useful in thecontrol of S. Enteritidis and S. Typhimurium, including antimi-crobial-resistant strains, and in the protection of foods from bac-terial contamination as a food preservative. In addition, thesecharacteristics of phages suggest that further investigation of thisnatural predator may give us many advantages in the war againstpathogens. Additional bioinformatic analyses with genome se-quences of applicable phages should be conducted to provide uswith information to evaluate the potential usefulness of phages orrisk factors associated with phage consumption. Combined use ofphages and other agents, such as bacteriocins or essential oils, mayalso be advantageous.

In conclusion, this study was carried out to characterize andanalyze the novel SETP3-like phage wksl3. The strong lytic activityand broad host spectrum of phage wksl3 indicate that it has po-tential as a biocontrol agent against S. Enteritidis and S. Typhimu-rium. Our data indicate that this phage may be effective in S.Enteritidis reduction strategies, regardless of drug resistance, to

ensure food safety and public health protection. In addition, noclear harmful effects were noted after oral administration to mice.This encouraging result implies that wksl3 may be a potentialtherapeutic agent not only for the prevention of contamination offoods but also for the treatment of humans or animals in clinicalcontexts. Further model application studies are needed to com-mercialize and expand the use of phage functions to other types offood products. Moreover, further genomic, proteomic, and hostmutational studies of phage wksl3 may provide a greater under-standing of the potential ecological role of wksl3 homologues inphage-host interactions during farm-to-table development, ex-tending the range of phage applications.

ACKNOWLEDGMENTS

This work was supported by grants from the National Antimicrobial Re-sistance Management Program (11092NARMP158) of the Korean Foodand Drug Administration.

We thank In-Seok Cha (Gyeongsang National University, Jinju, Re-public of Korea) for helpful confirmation of gross pathological examina-tions. We also thank the Korea University Food Safety Hall and Instituteof Food and Biomedicine Safety for allowing the use of their equipmentand facilities.

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