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Bacteriophage Preparation Lytic for Shigella Significantly Reduces Shigella Sonnei Contamination in Various Foods

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Bacteriophage Preparation Lytic for Shigella Significantly Reduces Shigella Sonnei Contamination in Various Foods RESEARCH ARTICLE Bacteriophage preparation lytic for Shigella significantly reduces Shigella sonnei contamination in various foods Nitzan Soffer*, Joelle Woolston, Manrong Li, Chythanya Das, Alexander Sulakvelidze Intralytix, Inc., Baltimore, Maryland, United States of America * [email protected] a1111111111 a1111111111 a1111111111 Abstract a1111111111 a1111111111 ShigaShield™ is a phage preparation composed of five lytic bacteriophages that specifically target pathogenic Shigella species found in contaminated waters and foods. In this study, we examined the efficacy of various doses (9x105-9x107 PFU/g) of ShigaShield™ in remov- ing experimentally added Shigella on deli meat, smoked salmon, pre-cooked chicken, let- 7 7 OPEN ACCESS tuce, melon and yogurt. The highest dose (2x10 or 9x10 PFU/g) of ShigaShield™ applied to each food type resulted in at least 1 log (90%) reduction of Shigella in all the food types. Citation: Soffer N, Woolston J, Li M, Das C, Sulakvelidze A (2017) Bacteriophage preparation There was significant (P<0.01) reduction in the Shigella levels in all phage treated foods lytic for Shigella significantly reduces Shigella compared to controls, except for the lowest phage dose (9x105 PFU/g) on melon where sonnei contamination in various foods. PLoS ONE reduction was only ca. 45% (0.25 log). The genomes of each component phage in the cock- 12(3): e0175256. https://doi.org/10.1371/journal. tail were fully sequenced and analyzed, and they were found not to contain any ªundesirable pone.0175256 genesº including those listed in the US Code for Federal Regulations (40 CFR Ch1). Our Editor: Mikael Skurnik, University of Helsinki, data suggest that ShigaShield™ (and similar phage preparations with potent lytic activity FINLAND against Shigella spp.) may offer a safe and effective approach for reducing the levels of Shi- Received: January 16, 2017 gella in various foods that may be contaminated with the bacterium. Accepted: March 22, 2017 Published: March 31, 2017 Copyright: © 2017 Soffer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which Introduction permits unrestricted use, distribution, and reproduction in any medium, provided the original Shigella is an important cause of morbidity and mortality throughout the world, causing author and source are credited. approximately 125 million Shigella infections / year and an estimated 14,000 deaths, mostly Data Availability Statement: All phage genomes among children <5 years of age [1,2]. In the United States, Shigella is the third most common are available from the NCBI database (accession causes of gastroenteritis, with at least 500,000 cases of shigellosis linked diarrheal events in the numbers KX130865.1; KX130864.1; KX130863.1; USA annually [3,4]. General symptoms of shigellosis include bloody watery diarrhea, fever, KX130862.1; and KX130861.1). nausea, and tenesmus (pain in the bowel), while complications include post-infection arthritis, Funding: Our studies were supported, in part, by a sepsis, seizures (in young children) and hemolytic-uremic syndrome [4]. The two main routes Small Business Innovation Research (SBIR) award of transmission include (i) through contaminated foods, and (ii) water contaminated with W911NF-11-C-0074 and Contract No. W911QY- human waste; Shigella are easily transmitted through human contact due to its low infectious 09-C-0079 from the United States Army (to A.S.). The funders had no role in study design, data dose of 10±200 cells [5]. Shigella have been isolated from almost all food types, including salads collection and analysis, decision to publish, or (potato, tuna, shrimp, macaroni, or chicken), fresh fruits and vegetables, poultry, milk and preparation of the manuscript. dairy products, deli meats, and seafood [6]. In the USA, the main Shigella species isolated from PLOS ONE | https://doi.org/10.1371/journal.pone.0175256 March 31, 2017 1 / 11 Bacteriophages reduce Shigella contamination in various foods Competing interests: Disclosure of possible contaminated foods is Shigella sonnei (found in ca. 80% of tested foods during 1998±2008). conflicts of interest: NS, JW, CD, ML and AS hold Internationally, Shigella flexineri (66%) and then Shigella sonnei (18%) are the most common an equity stake in Intralytix, Inc., a Maryland food contaminant Shigella species [6,7]. The ªready-to-eatº foods (i.e., foods that are ready to corporation developing bacteriophage preparations (including ShigaShield™) for various applications. be eaten, without any additional processing) are of particular concern because of the lack of This does not alter our adherence to PLOS ONE the heat processing step (e.g., cooking) that could kill Shigella and render those foods safer to policies on sharing data and materials. eat. Shigella also present a major concern to military personnel and travelers, especially when the deployment and/or travel is in the endemic areas where commercial food sanitation stan- dards are poor and enforcement of those standards is lenient [8]. For example, several studies have shown a considerable loss of person-hours because of traveler's diarrhea among U.S. mili- tary personnel deployed to the Persian Gulf during operations Desert Shield and Desert Storm, well as during peacetime operations [9±12]. The problem is further exacerbated by the increase of Shigella strains that are resistant to many commonly available antibiotics. For example, in 2013, the Centers for Disease Control and Prevention (CDC) declared antibiotic- resistant Shigella an urgent threat in the United States [13]. Also, an increasing prevalence of antibiotic resistance has been recently reported in the USA for the Shigella strains transmitted through sexual contact among men (nearly a quarter of Shigella isolates tested in New York City showed decreased susceptibility or resistance to recommended antibiotics) [14]. The prevalence of antibiotic-resistant Shigella strains also appears to be on the rise in foods; for example, in a recent study where more than 1,600 food samples (seafood, fresh vegetables, and meats) were examined, 89% of the Shigella strains were found to be multidrug resistant [6]. In these settings, novel non-antibiotic approaches are required to reduce the prevalence and lev- els of Shigella in various foods, which may help reduce the risk of shigellosis in the civilian (including young children) and military (including in US troops stationed in developing coun- tries) populace. Lytic bacteriophages may provide one such relatively novel, environmentally friendly ªgreenº approach. Bacteriophages are bacterial viruses that are the most abundant biological entities in the world, in one ml of seawater there is an estimated 107 phages, with approximately 1030±1031 in the world [15]. Lytic phages have a potent, highly specific bactericidal activity against their tar- geted bacterial cells±a feature that enables a targeted killing of specific problem-causing bacte- ria in various settings, without disturbing the normal±and often beneficial±microflora. Various bacteriophage-based food additives have been approved by the FDA for food safety applications, including (listed in chronological order of receipt of regulatory approval in the USA): ListShield™, Listex P-100™, EcoShield™, SalmoFresh™ and Salmonelex™ [16]. Here, we report the results of studies in which the ability of ShigaShield™ to reduce Shigella levels was evaluated in various foods experimentally contaminated with a S. sonnei strain. Various Shi- gella phage preparations have been extensively used therapeutically previously to prevent and/ or treat shigellosis in humans [17] reviewed by [18]; however, to the best of our knowledge, there is only one previous report of successful use of a Shigella phage preparation in food safety applications [19]. The phage preparation described in this communication (i.e., ShigaShield™) is currently undergoing FDA and USDA review for the GRAS (Generally Recognized As Safe) status (GRN 672). Materials and methods Bacteriophage preparation ShigaShield™ is a bacteriophage ªcocktailº essentially identical to the previously-described Shi- gActive™ preparation [20]. It is composed of 5 lytic phages (mixed in approximately equal con- centrations): SHSML-52-1 (ATCC PTA-121241), SHFML-11 (ATCC PTA-121234), SHSML- 45 (ATCC PTA-121238), SHFML-26 (ATCC PTA-121236), and SHBML-50-1 (ATCC PTA- PLOS ONE | https://doi.org/10.1371/journal.pone.0175256 March 31, 2017 2 / 11 Bacteriophages reduce Shigella contamination in various foods 121239). The same lot (# 1112I210158) was used in all studies. The phage preparation was sup- plied in normal saline solution (0.1 M NaCl, pH 6.5±7.5), and was stored refrigerated (2±8ÊC) until use. An additional 36 strains were examined for their susceptibility to ShigaShield™ employing the same method used on the other Shigella strains in our previous study [20]. Genomic sequencing of component bacteriophages Each phage included in ShigaShield™ was sequenced, annotated, and the sequences were deposited in GenBank at the National Center for Biotechnology Information (NCBI) (Acces- sion numbers: KX130865.1; KX130864.1; KX130863.1; KX130862.1; and KX130861.1). Briefly, each monophage was sequenced on a MiSeq (Illumina, San Diego, CA), assembled and anno- tated on the CLC Bio Genomic Workbench software, using default settings (version 7.0.2; CLC Bio, Cambridge, MA) at the BioAnalytical Laboratory of the Institute of Marine and Environmental Technology (IMET). Annotations were
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