Patent Application
20250160335
The instant application contains a Sequence Listing XML required by 37 C.F.R. § 1.831(a) which has been submitted in XML file format via the USPTO patent electronic filing system and is hereby incorporated by reference in its entirety. The XML file was created on 11/08/2024 is named Sequence_Listing-002023.xml and has 434 KB bytes.
The present disclosure provides for a combination of bacteriophages from various environmental sources combined to produce a phage mixture targeting Shiga-toxin-producing Escherichia coli strains. Techniques and methods of production and use of the phage combinations are also provided.
Shiga toxin-producing E. coli (STEC) strains—commensal to the gastrointestinal tract of ruminant animals, particularly cattle—have been linked to numerous foodborne outbreaks, causing approximately 265,000 illnesses, 3,600 hospitalizations, and 30 deaths every year in the United States (National Enteric Disease Surveillance: Shiga toxin-producing E. coli (STEC) Annual Report, 2017. Atlanta, Georgia: US Department of Health and Human Services, CDC). More than 30% of these outbreaks are associated with various produce—alfalfa sprouts, iceberg lettuce, and melon—contributing to over 350 identified cases in the United States between 2018 and 2021 (Glaize et al., Int'l J. Food Microbiol., (2021); 347:109196). Besides meat products, global epidemiologic data showed an estimated 147,000 cases of produce-related foodborne outbreaks reported from 1988 to 2020 contributed to 214 hospitalization and 58 deaths, primarily from the infection of STEC 0157 serogroup (Miyahira & Antunes, Int'l J. Food Microbiol., (2021): 352:109266). In 2018, a massive foodborne outbreak involving 22 states in the United States was linked to consuming E. coli O157:H7-contaminated romaine lettuce, causing 46 hospitalizations and 10 cases of the hemolytic uremic syndrome (CDC, 2018. Multistate Outbreaks of E. coli O157:H7 Infections Linked to Romaine Lettuce (Final Update)).
The use of antimicrobial intervention is a common practice regulated by regulatory agencies to ensure food safety during production and upon consumers′ consumption. Chemical-based antimicrobial treatment, such as chlorination, is commonly used in the produce industry because of its easy access, easy installation, and low cost. However, the major drawback is that frequent application results in the development of bacterial resistance, thus compromising the efficacy of these chemical antimicrobials. Additionally, chemical residuals may also negatively influence the produce quality (Fong et al., Front. Microbiol., (2017); 8:2193) and result in an environmental burden (Kraemer et al., Microorganisms, (2019); 7:180). Therefore, alternative interventional methods are critically needed to ensure microbiological safety in the produce industry with the minimum adverse impact (Mir & Kudva, Zonoses Pub. Health, (2019); 66:1-13).
Phages are bacteria-infecting viruses and are highly diverse and ubiquitous in the ecosystem where their bacterial hosts exist (O'Sullivan et al., Ann. Rev. Food Sci. Tech., (2019); 10:151-72). Phages bind specifically to the receptor proteins on the membrane of the target bacterial cells to initiate the infection (Milho et al., Scientific Rep., (2019); 9:18183; Pinto et al., Crit. Rev. Biotech., (2020); 40:1081-97). Due to the nature of the lytic cycle, phage infection always leads to the lysis of bacterial cells and the release of phage progenies for subsequent infection (Clokie et al., Bacteriophage, (2011); 1:31-45). Moreover, lytic phages can evolve with the constant changing of their bacterial hosts to combat the potential development of phage resistance. Consequently, we sought to examine a phage-based intervention technology to specifically address the challenge of STEC contamination using biological, genomic characterization, and in vitro antimicrobial tests to formulate a phage cocktail that can effectively control E. coli O157:H7 using environmentally isolated phages.
Provided herein are bacteriophage cocktails, wherein the cocktail comprises at least two isolated bacteriophages that target Escherichia coli selected from the group consisting of: (1) a bacteriophage having a genome sequence at least 95% identical to G1571w (PTA-127634)(SEQ ID NO:1); (2) a bacteriophage having a genome sequence at least 95% identical to Sa1571w (PTA-127635))(SEQ ID NO:2); and (3) a bacteriophage having a genome sequence at least 95% identical to Elw (PTA-127636)(SEQ ID NO:3). In a particular embodiment, the bacteriophage cocktail comprises a bacteriophage having a genome sequence at least 95% identical to G1571w (PTA-127634) and a bacteriophage having a genome sequence at least 95% identical to Elw (PTA-127636). In another particular embodiment, the bacteriophage cocktail comprises a bacteriophage having a genome sequence at least 95% identical to G1571w (PTA-127634)(SEQ ID NO:1), a bacteriophage having a genome sequence at least 95% identical to Sa1571w (PTA-127635)(SEQ ID NO:2), and a bacteriophage having a genome sequence at least 95% identical to Elw (PTA-127636)(SEQ ID NO:3). In a specific embodiment, the bacteriophage cocktail contains bacteriophage G1571w (PTA-127634), bacteriophage Sa1571w (PTA-127635); and bacteriophage Elw (PTA-127636).
A method of treating a surface, comprising administering any of the bacteriophage cocktails recited above to the surface. In some embodiments, the surface is a plant or plant part, such as a seed, pre-harvest crop or leafy vegetable (e.g., lettuce or spinach). In other embodiments, the surface is a food contact surface, such as food packaging or a food processing surface.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the claims. Features and advantages of the present invention are referred to in the following detailed description, and the accompanying drawings of which:
Strains representative of the inventions disclosed herein were deposited on Sep. 13, 2023 under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC). A representative bacteriophage G1571w (Tequatrovirus genus under the Straboviridae family) was deposited under ATCC Reference No. PTA-127634. A representative bacteriophage Sa1571w (Kuttervirus genus under the Ackermannviridae family) was deposited under ATCC Reference No. PTA-127635. A representative bacteriophage Elw (Tequintavirus genus under the Demerecviridae family) was deposited under ATCC Reference No. PTA-127636. The microorganism deposits were made under the provisions of the “Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure”. All restrictions on the availability to the public of these deposited microorganisms will be irrevocably removed upon issuance of a United States patent based on this application. For the purposes of this invention, any bacteriophage strains having the identifying characteristics of PTA-127634, PTA-127635, or PTA-127636, including subcultures and variants thereof which have the identifying characteristics and activity as described herein are included.
Lytic phages are innovative antimicrobials that can target bacterial pathogens without killing background flora (Mohammad et al., Int'l J. Food Microbiol., (2019), 289:57-63). Most importantly, they do not contribute to the development of antibiotic resistance in the target bacteria, commonly seen from current chemical interventions (Moye et al., J. Food Safety, (2020), 40:e12763). These phages can combat antibiotic-resistant strains in the agricultural field (Kahn et al., Ann. New York Acad. Sci., (2019), 1441:31-9) and minimize any environmental burden on subsequent terrestrial surroundings (O'Sullivan, et al., supra). Furthermore, FDA has authorized several commercial phage products as Generally Recognized As Safe (GRAS) since 2006 to ensure safe application directly on ready-to-eat food or during food production (Vikram et al., J. Food Protection, (2020), 83:668-76). Therefore, phage-based technology sheds new light on developing a green and novel antimicrobial intervention measurement. It also helps provide the resolution of antibiotic resistance in the food industry.
The instant disclosure focuses on the formulation of phage cocktails, including the three-phage cocktail of G1571w+Sa1571w+Elw, from five different lytic phages isolated from agricultural-associated environments. During the formulation process, we used whole-genome sequencing technology to ensure the diversity of phages and the novelty compared to the reference phages published in the National Center for Biotechnology Information (NCBI). Additionally, sequencing technology is used to thoroughly screen the unwanted genes, such as toxin, lysogenic, and antibiotic-resistant genes, in the phage genomes that could be potential threats to human health and jeopardize the phage application.
As detailed herein, phage G1571w is included in each version of the cocktails, as it showed the most significant antimicrobial activity against E. coli O157:H7 as a single phage and the largest burst size among all the phages in the cocktail. Phage cocktails of the present disclosure show a wide host range capable of infecting other top 6 non-O157 STEC and provide additional mitigating effects against bacterial pathogens other than E. coli O157:H7. G1571w was isolated from the compost sample of food scraps and yard trimming, and phages Sa1571w and Elw were isolated from produce pre-harvest surface water and municipal sewage water samples, respectively. Geographical differences in the source of isolation ensure the natural diversity of the phages besides the genomic variety. Many studies showed that lytic phages were highly associated with the presence of their bacterial host (Liao et al., PLoS One, (2018), 13:e0190534; Wang et al., Can. J. Microbiol., (2015), 61:467-75). Therefore, the samples with high animal contaminations, such as animal feces and sewages, are usually favorable for isolating phages specific to foodborne pathogens, like E. coli O157:H7. However, two (G1571w and Sa1571w) of three phages in this phage cocktail were from the environmental samples with low animal contamination, making our phages, particularly G1571w, unique for developing a phage cocktail.
The instant disclosure provides in vitro antimicrobial activities of various phage combinations to determine whether combining different phages had facilitating, neutral or antagonistic effects compared to that of fewer phages combined. As a result, a cocktail comprising phages G1571w and Elw was an effective combination among all possible two-phage combinations in reducing single E. coli O157:H7, contributing to non-detectable bacterial level, with 7 log reduction (99.99999% reduction) at a 6-h time point. Furthermore, a three-phage cocktail, G1571w+Sa1571w+Elw, demonstrated stronger in vitro antimicrobial effects compared to the two-phage cocktail (G1571w+Elw) at 24-h treatment, with 1.1 log more reduction (92.3% reduction). Surprisingly, the three-phage cocktail was able to mitigate a single E. coli O157:H7 strain and a four-strain E. coli O157:H7 cocktail more than a commercial phage cocktail in vitro after 24-h treatment by 6 log (99.9999% reduction) and 3.4 log reduction (99.96% reduction), respectively.
Further disclosed herein are methods of treating various known contamination sources, such as agricultural irrigation water, commonly susceptible to external contamination and associated with various foodborne outbreaks (Lacombe et al., Front. Food Sci. Tech., (2022), 2:1068690), was used to determine the efficacy of our phage cocktail. The phage cocktail, G1571w+Sa1571w+Elw, formulated in this study continued to mitigate a four-strain E. coli O157:H7 spiked in agricultural well water throughout 24-h treatment, with a maximum 1.3 log reduction (95% reduction) at a 6-h time point. Moreover, the efficacy of the phage cocktail, G1571w+Sa1571w+Elw, was superior to a commercial phage cocktail product in controlling the pathogenic bacteria. Furthermore, a previous study showed that the single phage Sa1571w reduced E. coli O157:H7 on mung bean seeds by 1.1 log (92.3% reduction). Based on the current in vitro results, the phage cocktails of the instant disclosure provide surprisingly better antimicrobial efficacy than Sa1571w alone. Phage cocktails of the instant disclosure may have significant versatile application values with the potential GRAS approval for the application in animal feeds, directly on food, food-processing environments, and food packaging, in addition to the pre-harvest agricultural environment, such as in-farm application and irrigation treatment.
Preferred embodiments of the present invention are shown and described herein. It will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the included claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are covered thereby.
Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the instant invention pertains, unless otherwise defined. Reference is made herein to various materials and methodologies known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1989; Kaufman et al., eds., “Handbook of Molecular and Cellular Methods in Biology and Medicine”, CRC Press, Boca Raton, 1995; and McPherson, ed., “Directed Mutagenesis: A Practical Approach”, IRL Press, Oxford, 1991. Standard reference literature teaching general methodologies and principles of genetics useful for selected aspects of the invention include: Sherman et al. “Laboratory Course Manual Methods in Yeast Genetics”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986 and Guthrie et al., “Guide to Yeast Genetics and Molecular Biology”, Academic, New York, 1991.
Any suitable materials and/or methods known to those of skill can be utilized in carrying out the instant invention. Materials and/or methods for practicing the instant invention are described. Materials, reagents and the like to which reference is made in the following description and examples are obtainable from commercial sources, unless otherwise noted. This invention teaches methods and describes tools for producing bacteriophage cocktails to target STEC.
As used in the specification and claims, use of the singular “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
The terms isolated, purified, or biologically pure as used herein, refer to material that is substantially or essentially free from components that normally accompany the referenced material in its native state.
The term “about” is defined as plus or minus ten percent of a recited value. For example, about 1.0 g means 0.9 g to 1.1 g and all values within that range, whether specifically stated or not.
The term “consisting essentially of” excludes additional method (or process) steps or composition components that substantially interfere with the intended activity of the method (or process) or composition and can be readily determined by those skilled in the art (for example, from a consideration of this specification or practice of the invention disclosed herein).
The term “antimicrobial activity”, and grammatical variations thereof, refers to the ability of a composition of the present invention to impede growth of a microorganism, or kill a microorganism, when present in an effective amount. “Antibacterial” refers specifically to the capability of a composition to impede growth of, or kill, bacteria when present in an effective amount.
The term “controlling microbial growth”, as used herein, denotes any activity for completely inhibiting or at least reducing the growth of bacteria (such as E. coli) in a given environment. The term “inhibiting”, as used herein, is to be understood as not only to include the prevention of further growth of but also to killing of any given bacteria. The term “reducing”, as used herein, denotes any decrease in a bacteria's growth (or growth rate), for example, a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% as compared to control conditions (i.e. in the absence of antimicrobial agents according to the present invention). Levels of reduction or inhibition of bacterial growth can be indicated by log reduction, for example 1 log reduction=90% reduction (e.g. bacterial counts dropped from 100 CFU to 10 CFU); 1.1 log reduction=92.3% reduction (e.g. bacterial Bacterial counts dropped from 13 CFU to 1 CFU); 1.3 log reduction=95% reduction (e.g. bacterial counts dropped from 20 CFU to 1 CFU); 2 log reduction=99% reduction (e.g. bacterial counts dropped from 100 CFU to 1 CFU); 3.4 log reduction=99.96% reduction (e.g. bacterial counts dropped from 2,500 CFU to 1 CFU); 6 log reduction=99.9999% reduction (e.g. bacterial counts dropped from 1,000,000 CFU to 1 CFU); 7 log reduction=99.99999% reduction (e.g. bacterial counts dropped from 10,000,000 CFU to 1 CFU).
The term “effective amount” of a bacteriophage mixture composition provided herein refers to the amount of the composition capable of performing the specified function for which an effective amount is expressed. The exact amount required can vary from composition to composition and from function to function, depending on recognized variables such as the compositions and processes involved. An effective amount can be delivered in one or more applications. Thus, it is not possible to specify an exact amount, however, an appropriate “effective amount” can be determined by the skilled artisan via routine experimentation.
The term “fresh produce” as used herein refers generally to farm-produced fruits and vegetable crops including, but not limited to fruit and vegetable crops such as e.g., lettuce, spinach, cilantro, cabbage, almonds, cucumbers, cantaloupes, etc.
A “phage cocktail” as used herein refers to a. composition comprising two or more bacteriophages combined according to the methods of the instant invention which can provide a control for at least one strain of E.coli when administered to an organic or inorganic surface.
The term “plant” includes whole plants, plant organs, progeny of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm-like bodies (PLBs), and suspensions of plant cells. Plant organs comprise, e.g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seeds (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like).
The terms “G1571w” and “PTA-127634” refer to bacteriophage of the Tequatrovirus genus under the Straboviridae family having the biological and genetic characteristics of the virus deposited under ATCC Reference No. PTA-127634. The terms include genetically modified versions of this virus. A representative genome is available as GenBank accession #OK331996 and this sequence is incorporated in its entirety (SEQ ID NO: 1).
The terms “Sa1571w” and “PTA-127635” refer to bacteriophage of the Kuttervirus genus under the Ackermannviridae family having the biological and genetic characteristics of the virus deposited under ATCC Reference No. PTA-127635. The terms also include genetically modified versions of this virus. A representative genome is available as GenBank accession #OK322699 and this sequence is incorporated in its entirety (SEQ ID NO:2).
The terms “Elw” and “PTA-127636” refer to bacteriophage of the Tequintavirus genus under the Demerecviridae family having the biological and genetic characteristics of the virus deposited under ATCC Reference No. PTA-127636. The terms also include genetically modified versions of this virus. A representative genome is available as GenBank accession #OR621298 and this sequence is incorporated in its entirety (SEQ ID NO:3).
As disclosed herein, a plurality of individually isolated phage was obtained from diverse environmental sources. Members of the same bacteriophage species detailed herein (e g., PTA-127634, PTA-127635, and PTA-127636) have not been shown to reside together in nature, but can be collected from diverse environmental locations, including soil, water treatment plants, raw sewage, sea water, lakes, rivers, streams, standing cesspools, animal fecal matter, organic substrates, biofilms, or medical/hospital sources. The present disclosure includes phage mixtures produced by combining phage of the same species as reported herein, or phages with genomes having 95.0%, 95.1%, 95.2%, 95.3, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6, 96.7%, 96.8%, 96.9, 97.0%, 97.1%, 97.2% 97.3%, 97.4%, 97.5%, 97.6%, 97.7, 97.8% 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.1%, 99.2%, 99.3% 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% genomic identity to SEQ ID NO:1, SEQ ID NO:2, and/or SEQ ID NO:3.
Individual phage utilized in the instant disclosure can be purified by any purification technique known in the art, including, but not limited to cesium chloride gradient ultracentrifugation, removing cesium chloride, differentiation filtration, column chromatography and phase partition chromatography.
Any of these compositions described herein can be used to treat bacterial contamination in an environment, or on specific surfaces. Exemplary environments and surfaces that can be treated include, but are not limited to, meat processing facilities, fresh produce, unharvested crops (e.g., lettuce), sprayed or dipped leafy greens (such as mung bean sprouts and lettuce), irrigation water, ready-to-eat foods, seeds (such as mung bean seeds), controlled environment agriculture systems, biofilms, food-contact surfaces, and water troughs.
The embodiments illustratively disclosed herein suitably may be practiced in the absence of any element [e.g., method (or process) steps or composition components)] which is not specifically disclosed herein. Thus, the specification includes disclosure by silence. Written support for a negative limitation may also be found through the absence of the excluded element in the specification, known as disclosure by silence.
Having generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
Isolation and characterization of candidate bacteriophages
Escherichia phage “Ro1571w”, “G1571w”, “UDF1571w”, “Sa1571w”, and “Elw” were previously isolated with E. coli O157:H7 (RM18959, ATCC 43888, or ATCC 35150) from various environmental samples, such as non-fecal compost (Ro1571w and G1571w), bovine feces (UDF1571w), surface water in a produce-growing area (Sa1571w), and sewage water (Elw). Phage propagation was performed by mixing 50 μL phage lysate (˜109 PFU/mL) with 45 mL of the log-phase bacterial host culture in tryptic soy broth (TSB; Becton Dickinson, Sparks, MD) and CaCl2 at 10 mM for incubation at 37° C. for 20 hours. The propagated phages were filtered through a 0.22-μm filter membrane, following centrifugation at 8,000×g for 10 minutes before downstream analysis. A proprietary commercial phage, ECOSHIELD, was used to compare with our developed phage cocktails.
A collection of the top 6 non-O157 STEC—the serogroups O26, O45, O103, O111, O121, O145-E. coli O157:H7 and generic E. coli strains were obtained from the Produce Safety and Microbiology (PSM) Research Unit at the U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), Western Regional Research Center, Albany, CA, USA for this study. E. coli O157:H7 (ATCC 43888) was used for propagation and quantification of Elw, Sa1571w, and G1571w, E. coli O157:H7 (RM 18959) was used for Ro1571w, and E. coli O157:H7 (ATCC 35150) was used for UDF1571w. Fresh bacterial culture was prepared by inoculating 10 mL TSB with 1 μL loopful of each strain for overnight incubation at 37° C. before use.
The phage cocktail formulation started with the determination of phage varieties based on morphological and genomic characterizations. Additionally, the whole-genome sequencing data of individual phages was used to ensure no toxin genes or lysogenic genes were encoded in each phage genome for the safety of the application. Later, the bacterial host range and productive infection of individual phages against the top six non-O157 and 0157 serogroups of STEC were obtained as a preliminary antimicrobial criterium. The phages with a wide host range were promising for cocktail development; therefore, phage G1571w was selected as the core phage for phage cocktail development.
All selected phages were purified through a CsCl gradient and subsequently subjected to DNA extraction and DNA library preparation prior to sequencing as previously described (Liao et al., Fronts. Microbiol., (2022) 13:1053583). Later, the samples were loaded to a MiSeq Reagent Kit v2 (500-cycle) and sequenced on the MiSeq platform (Illumina, San Deigo, CA, USA). The raw 2×250-bp paired-end reads were subjected to quality control via FASTQC and Trimmomatic with the setting of Q30. De novo assembly was conducted on the resulting quality reads using Unicycler v0.5.0, followed by annotation via the Prokka pipeline Galaxy 1.13 (Seeman, T., Bioinformatics, (2014); 30:2068-69) with default settings. Subsequently, the annotation was manually curated with Universal Protein Resource (UniProt) database (Bairoch et al., Nucl. Acids Res., (2005); 33: D154-D159) using Geneious (v11.0.3, Biomatters, New Zealand). tRNAscan-SE (v2.0) server was used to confirm the predicted tRNAs in the phage genome (Lowe & Chan, Nucl. Acids Res., (2016); 44:W54-W57). The final annotated sequences were deposited in National Center for Biotechnology Information (NCBI) database. The presence of antibiotic-resistance genes and virulence genes in the phage genome was identified using the ResFinder 4.1 webserver (Florensa et al., Microbial Genomics, (2022); 8:000748) and VirulenceFinder (Malberg Tetzchner et al., J. Clin. Microbiol., (2020); 58:e01269-01220).
Purified phages were purified using a CsCl gradient and subsequently subjected to negative staining as previously described (Liao et al., 2022 I, supra). The specimens were then examined in a transmitted electron microscope (FEI Tecnai G2).
The host range tests for the phages included in this study were conducted against non-pathogenic E. coli, E. coli O157:H7, top six non-0157 STEC, and various Salmonella strains using a spot test assay as previously described (Liao et al., Antibiotics, (2019), 8:74). Briefly, each bacterial culture with 100 μL was mixed with 50% molten TSA and then poured into a Petri dish containing 10-mL bottom TSA. After solidifying, 5 μL of each phage at approximately 7 log PFU/mL was spotted on the plate, followed by incubation at 37° C. The phage-sensitive strains were subsequently tested with productive infection of each phage using the efficiency of plating (EOP) assay described in a previous study (Liao et al., J. Virology, (2011), 85:6567-78). Briefly, fresh cultures were prepared in TSB at 37° C. overnight and used for quantification of Sa1571w using double-layer plaque assay with diluted phage lysate with four successive dilutions (10-3 to 10-7). The plates were incubated at 37° C. for 18 h. The experiment was conducted in 3 replications. Generally, a high phage-producing efficiency had EOP of 0.5 or more; a medium-producing efficiency had EOP above 0.1 but below 0.5; a low-producing efficiency had EOP between 0.001 and 0.1; inefficient phage production was any value lower than 0.001.
Growth factors of the phages included in the phage cocktail were determined using E. coli O157:H7 (ATCC 43888) based on the previous method with subtle modification (Liao et al., Microbiol. Spectrum, (2022 II), 10:e02220-02221). Briefly, 0.2 mL of fresh overnight culture in TSB was sub-cultured in sterile 19.8 mL of TSB and incubated for 2 h at 37° C. to reach the log phase of bacterial growth. Later, a single phage was added to the log-phase bacterial solution (MOI of 0.01) with CaCl2 at 10 mM and incubated at 37° C. for 10 min, allowing phage adsorption onto the bacterial membranes. The phage-bacterial mixture was centrifuged at 10,000 xg for 5 min at 4° C. to discard the supernatant. After washing, the bacterial pellet was resuspended with 20 mL of fresh TSB before further 10-fold dilution (0.3 mL of resuspension in 29.7 mL of TSB). The sample was then incubated at 37° C. for a specific period (varied by different phages). Meanwhile, the phage-infected bacterial cells were quantified before incubation (time 0) by mixing 50 μL of the 30-mL phage-bacterial mixture (no filtration) with 100 μL of fresh overnight bacterial culture for a double-layer plaque assay. Subsequently, a 1 mL phage-bacterial mixture was obtained from the selected time points and filtered using a 0.22-μm filter membrane. The phage titers at each time point were determined using the double-layer plaque assay. The experiment was conducted in three replications to estimate the latent period and burst size of the selected phages (G1571w & Elw).
A quick antimicrobial activity screening on various two-phage combinations from the first included phages G1571w, Sa1571w, UDF1571w, and Ro1571w was conducted using a spectrophotometer (Elw phage was isolated later to increase the morphological variety). Detailed information regarding the experiment and assay used in the formulation step is described below.
Five lytic phages isolated from various environmental samples are in genetic variation (Table 1) and morphological differences (
Tequintavirus
Wifcevirus
Rogunavirus
Kuttervirus
Tequatrovirus
Among all five phages, G1571w has a broader host range targeting different STEC serogroups, including 045, 0103, and 0145, than the other phages in addition to infecting E. coli O157:H7 strain (Table 2; H=host strain used for the phage isolation and R=the bacterial strain is resistant to the phage). For Table 2, the EOP was calculated by the ratio of phage titer on a test bacterium versus the primary bacterial host. High production efficiency is EOP≥0.5, medium production efficiency is 0.5>EOP≥0.1, low production efficiency is 0.1>EOP>0.001, and the inefficiency of phage production is EOP 0.001. H means the host strain used for the phage isolation, and R means the bacterial strain is resistant to the phage infection. Additionally, these phages express a variety of productive infections against different E. coli O157:H7 strains. The phenomenon is favorable as the development of a phage cocktail. Phage Sa1571w was a polyvalent phage able to infect various Salmonella enterica serovars: Heidelberg, Saintpaul, Agona, Typhimurium, and Enteritidis (Liao et al., 2022 I, supra). However, these phages show a wide infection capability against generic E. coli strain. Growth factors—the latent period and burst size—were examined on the three phages included in the cocktail. The results show that phages G1571w and Sa1571w have similar latent periods of 30 min, whereas Elw has around 40 min (Table 3). Additionally, G1571w has the largest burst size of approximately 1,220 PFU/CFU among all the phages.
E. coli O45:H16
E. coli O103:H2
E. coli O145:H28
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7
E. coli O157:H7 (ATCC 43888).
A bacterial challenge assay was performed to measure the effects of phages Ro1571w, UDF1571w, and G1571w with different MOIs on the cocktail of two E. coli O157:H7 (RM 18959 and E. coli O157:H7 (RM 35150) based on bacterial optical density at a wavelength of 600 nm (OD600) as previously described with minor change (Zhang et al., Microorganisms, (2021), 9:1527). In brief, fresh bacterial cultures of E. coli O157:H7 (ATCC 35150) and E. coli O157:H7 (RM18959) were prepared in TSB at 37° C. overnight and further diluted in TSB to 1×106 CFU/mL. An aliquot of 200 μL diluted bacterial solution containing both bacterial cultures per well was added to a 96-well plate. Later, 10 μL of individual phage with different titers was added to the wells to reach MOIs of 1, 10, and 100; the control group contained only bacterial solution without phage. The reading of OD600 was measured using a spectrophotometer (Promega, Madison, WI, USA) at 25° C. every 30 min for 12 h. The experiment was conducted in 3 replications.
A culture of E. coli O157:H7 (RM9995) was used to evaluate the antimicrobial activity of an individual and different combinations of phages using the method as previously described with subtle modifications (Liao et al., 2022 II, supra). In brief, the bacterial culture was prepared in 10 ml TSB at 37° C. for 18 h, followed by dilution in LB broth (Invitrogen, Carlsbad, CA, USA) to reach the final concentration at approximately 4 log CFU/ml for the experiment. Single phages or multi-phage cocktails in SM buffer was added to 4-ml bacterial solution at an MOI of 1. For the control, SM buffer, with the same volume as the phage used in the treatment, was also added to 4-ml bacterial solution. Both control and treatment groups were incubated at 25° C. or 30° C., and the bacterial counts were quantified at various time points (0, 3, 4, 6, 8 or 24 hours) during the incubation. Bacterial counts were quantified on Sorbitol MacConkey agar (SMAC; BD, Franklin Lakes, NJ) overlayered thin TSA (Thin Agar Layer Method, TAL) (Wu, V., Food Microbiol, (2008), 25:735-744).
Agricultural water from a produce-growing area was collected and used to evaluate the efficacy of a phage cocktail formulated in this study versus a commercial phage cocktail in reducing a four-strain E. coli O157:H7 cocktail (RM9995, RM18419, RM6416, and ATCC35150). The water sample was filtered through a 0.22-mm membrane before use. Each bacterial culture was prepared in 10 ml TSB at 37° C. overnight. An aliquot of 0.1 ml per bacterial strain was added to a sterile tube to prepare the bacterial cocktail used to inoculate 60-ml water sample at the final concentration of 5 log CFU/ml. The E. coli O157:H7-inoculated water was evenly dispensed into three conical tubes (20 ml per tube) and treated separately with a three-phage cocktail and the commercial phages at an MOI=50. The SM butter, with the same volume used for the phage cocktails, was added to a 20-ml bacterial cocktail as the control group. The control and treatment tubes were incubated at 25° C. for 24 h with shaking. Bacterial counts were evaluated for control and treatment groups by plating on duplicated SMAC TAL at 0, 4, 6, and 24 h. The plates were incubated at 24° C. overnight for bacterial quantification.
A quick screening assay was used to determine antimicrobial suppression regarding two-phage combinations of G1571w, Sa1571w, and Ro1571w against a cocktail of E. coli O157:H7. The results showed that the order of antimicrobial activities for the combination groups from high to low is G1571w+Sa1571w or G1571w+UDF1571w and G1571w+Ro1571w (results not shown). As a result, the combination with Ro1571w had the worst antimicrobial effect among all and thus was removed. Therefore, phage Ro1571w was excluded from the subsequent antimicrobial activity test in LB broth.
The remaining phages and phage Elw were subjected to in vitro antimicrobial activity tests on two-phage, three-phage, and four-phage combinations to determine “facilitating” (greater efficacy than the most effective constituent phages acting alone), “neutral” (no better or worse than the most effective constituent phages acting alone) or “antagonistic” (lower efficacy than the most effective constituent phages acting alone) effects among different phages used in the same cocktail. First, antimicrobial effects of individual phages of G1571w, Sa1571w, and UDF1571w were tested against E. coli O157:H7 (RM11781) at 30° C. for 3 h. The results showed that phage G1571w was the best among the three phages, resulting in more than 6 log bacterial reduction (99.9999% reduction) in 3 h (Table 4; *numbers represent the viable cell counts of bacteria (log10 CFU/ml)). Later, G1571w was used as a core phage in combination with Sa1571w or UDF1571w to test a two-phage cocktail against the same bacterial strain. The results showed that the phage combination of G1571w and Sa1571w had an enhanced antimicrobial effect in mitigating E. coli O157:H7 (RM11781) in comparison to phages G1571w and UDF1571w combined (Table 5; *numbers represent the viable cell counts of bacteria (log10 CFU/ml)). The other two-phage combination (G1571w and UDF1571w) had a similar bacterial reduction level compared to the single phage G1571w. Furthermore, phage Elw was tested in combination with G1571w to compare with the two-phage cocktail of G1571w and Sa1571w. The results showed that combining phage Elw greatly increased the antimicrobial activity of G1571w alone at 6 h and 24 h of the treatment at 25° C. (Table 6; *numbers represent the viable cell counts of bacteria (log10 CFU/ml); αO represents a non-detectable bacterial count). Additionally, the combination of G1571w and Elw had better antimicrobial activity than G1571w and Sa1571w combined in LB broth.
Next, phage Elw was added to the combination of G1571w and Sa1571w against E. coli O157:H7 (ATCC 35150) at 25° C. for 6 h. The results showed that the three-phage cocktail combination containing Elw reduced more bacterial cells of E. coli O157:H7 at 4-h and 6-h time points than the two-phage cocktail (G1571w and Sa1571w), with 1.3 and 1.1 log reduction, respectively (Table 7, *numbers represent the viable cell counts of bacteria (log10 CFU/ml); α0 represents a non-detectable bacterial count). Subsequently, the phage cocktail of G1571w, Sa1571w, and Elw was combined with UDF1571w to combat E. coli O157:H7 for 24 h; however, no synergistic effect was observed (results not shown). Subsequently, a three-phage cocktail, containing G1571w, Sa1571w, and Elw, was used to compare with ECOSHIELD, the sole commercial phage product tested herein, to mitigate single E. coli O157:H7 strain (ATCC 35150) or a four-strain E. coli O157:H7 cocktail (ATCC 35150, RM6416, RM9995, and RM18419) at 25° C. for 24 h. The results showed that the three-phage cocktail in this study reduced single E. coli O157:H7 to the non-detectable level after 6 h to 24 h of the phage treatment (Table 8, *numbers represent the viable cell counts of bacteria (log10 CFU/ml); α0 represents a non-detectable bacterial count). For the commercial phage product, the bacterial growth rebounded starting from 6 h. For the antimicrobial effect against a four-bacteria cocktail, our phage cocktail was able to reduce the bacterial levels from 6.5 log CFU/ml to below 1 log CFU/ml up to 8 h (Table 9, *numbers represent the viable cell counts of bacteria (log10 CFU/ml)). Although bacteria grew back after 24 h of treatment, the bacterial levels mitigated by our three-phage cocktail were significantly lower than that caused by the commercial phage product by more than 3 log.
The efficacy of phage application was determined in treating agricultural well water inoculated with a four-strain E. coli O157:H7 cocktail. The results showed that using the MOI of 50, the 3-phage cocktail continued to mitigate the bacterial levels examined at 4 and 6 h time points at 25° C., with a maximum reduction of 1.3 log compared to the control group. Although the E. coli O157:H7 bounced back at 24 h, the treatment with the 3-phage combination kept the bacterial level lower than the control group by 0.6 log and that treated with a commercial phage. Besides the antimicrobial activities, some nutrients might be present in the commercial phage product to facilitate bacterial growth in the water sample (Table 10, *numbers represent the viable cell counts of bacteria (log10 CFU/ml)).
Experiments subjected to statistical analysis were conducted in three individual replications. The quantification of bacteria and phages was calculated as CFU/g and PFU/mL, respectively, with logarithmical conversion for statistical analysis.
Antimicrobial Activity of Phage Cocktails Against Pathogenic E. coli Other than STEC
The susceptibility of the tested strains, such as EPEC, ETEC, and EIEC, to phage cocktails, was determined using a spot test assay. Later, the susceptible strains with strong lysis were used to determine EOP. If the tested strains showed medium to high EOP with the 3-phage cocktail, further experiments would be used for an in vitro antimicrobial activity test.
The results showed that the 3-phage cocktail (G1571w+Sa1571w+Elw) did not infect EIEC or EPEC and rendered weak antimicrobial activity against two ETEC strains of the serogroups 025 and 0117, with low efficiency and inefficiency, respectively (Table 11; the EOP was calculated by the ratio of phage titer on a test bacterium versus the primary bacterial host. E. coli O157:H7 ATCC43888 was used as host strain for EOP. High production efficiency is EOP≥0.5, medium production efficiency is 0.5>EOP≥0.1, low production efficiency is 0.1>EOP>0.001, and the inefficiency of phage production is EOP≤0.001. R means the phage cocktail did not infect and lyze the bacterial strains). The results suggested that the 3-phage cocktail is specific to STEC strains and has little mitigating effects on the pathogenic E. coli other than STEC O157:H7.
Application of Phage Cocktails on Mung Bean Seeds Contaminated with E. coli O157:H7
Mung beans were disinfected with 2% of NaClO for 15 min and then rinsed with sterile water five times to remove NaClO residue. The sterilized seeds were air-dried in a biosafety hood. Before the experiment, the sterilized mung beans were homogenized with 20 mL sterile 0.1% peptone water for 2 min and plated on Sorbitol MacConkey (SMAC) with a thin TSA overlay to check the beans were free of background flora.
Mung beans (100 g) were submerged in 140 mL bacterial solution containing a four-strain E. coli O157:H7 cocktail (RM9995, RM6416, RM18419, and ATCC35150) at approximately 5.5 log CFU/mL for 1 h at room temperature (˜20° C.). After air drying under a biosafety hood, inoculated beans were stored in a sterile zip-loc bag at 4° C. overnight before the experiment. Later, three groups of ten grams of inoculated mung beans were measured and subjected to the control, a test 3-phage cocktail (G1571w+Sa1571w+Elw), and commercial phage treatments, respectively. For the phage cocktail treatment, mung beans were submerged in 50 mL phage solution with ˜8 log PFU/mL for 1 h at room temperature (˜20° C.). For the control, mung beans were mixed with 50 mL 0.1% peptone water under the same condition. After removing the phage solution and peptone water, the control and treated beans were dried on a separate sterile aluminum foil sheet under the hood for 2 h. Subsequently, the bacterial levels were quantified by plating on SMAC with thin TSA overlay plates.
The results showed that the 3-phage cocktail (G1571w+Sa1571w+Elw) reduced the four-strain E. coli O157:H7 cocktail on the contaminated mung beans by 1 log after phage treatment for 1 hour (
E. coli O157:H7 (RM9995) culture was prepared in 10 mL TSB and incubated at 37° C. for 20 h. Later, an aliquot of 0.1 ml of the overnight culture was placed on each sterile stainless coupon (n=9), put and sealed in a sterile 12-well plate to grow the biofilm at 22° C. for 48 h without shaking. After incubation, the planktonic cells were removed from the stainless surface by washing with 1 mL 0.1% peptone water twice. Later, three coupons per group were individually subjected to 0.1% peptone water, a 3-phage cocktail (G1571w+Sa1571w+Elw), and ECOSHIELD (commercial phage product) for 1 h at 22° C. (room temperature). In brief, each coupon was submerged in 3 mL peptone water or phage solutions at ˜8 log PFU/mL for 1 h. After treatment, each coupon was washed with peptone water, followed by biofilm cells being removed from the surface into 10 mL peptone water using ultrasonication before bacterial quantification on SMAC TAL plates.
The results showed that both phage cocktails with ˜8 log PFU/mL significantly mitigated 48-h E. coli O157:H7 biofilm on the stainless surface (
Additional phage cocktail experiments in bacterial culture and additional experiments in agricultural water were carried out substantially as described in Example 2 above, including bacterial culture preparation, single phage or multi-phage cocktails, agricultural water handling, incubation times and media utilized.
The results from these experiments showed that G157+Elw had the best antimicrobial activity within 6-h treatment at 25° C. (Table 12; *numbers represent the viable cell counts of bacteria (log10 CFU/ml)). However, Elw+Sa1571w had further reduced bacterial levels to 1.2 log CFU/ml, with the highest reduction among all treatment groups. We selected G157+Elw as the two-phage cocktail for the following experiment because this two-phage combination reached the maximum bacterial reduction faster than Elw+Sa1571w.
The results also showed that both two-phage and three-phage cocktails are better than the commercial phage product over a 24-h treatment period, even though the commercial product (ECOSHIELD) reduced the bacteria to the minimum levels (in 4 h) faster than both of our phage cocktails (in 6 h) (Table 13; *numbers represent the viable cell counts of bacteria (log10 CFU/ml)). Additionally, the three-phage cocktail (G1571w+Sa1571w+Elw) continued to reduce the bacterial population to the non-detectable levels at the 24-h timepoint; however, the bacteria grew back at 24 h from the treatment of the two-phage cocktail and the commercial phage cocktail.
The results showed that both two-phage and three-phage cocktails are better than the tested commercial phage product (ECOSHIELD) upon application in reducing a four-strain E. coli O157:H7 cocktail (ATCC 35150, RM18419, RM6416, and RM9995) in agricultural water (Table 14; *numbers represent the viable cell counts of bacteria (log10 CFU/ml)). Additionally, the three-phage cocktail (G1571w+Sa1571w+Elw) continued to reduce the bacterial population over a 24-h treatment period, contributing to a 1.6 log reduction compared to the control. However, the bacterial levels were suppressed by the commercial phage product until the 6-h timepoint but grew back at 24 h.
While the invention has been described with reference to details of the illustrated embodiments, these details are not intended to limit the scope of the invention as defined in the appended claims. The embodiment of the invention in which exclusive property or privilege is claimed is defined as follows:
The present application claims priority to U.S. Provisional Patent Application Ser. No. 63/599,751 filed Nov. 16, 2024, the content of which is expressly incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63599751 | Nov 2023 | US |
