This application is being filed as a PCT International Patent application on January 27, 2021, in the name of General Probiotics, Inc., a U.S. national corporation, applicant for the designation of all countries, and Yiannis John Kaznessis, a U.S. Citizen, and Kathryn Gayle Kruziki, a U.S. Citizen, and Dimitrios Nikolaos Sidiropoulos, a U.S. Citizen, inventor(s) for the designation of all countries, and claims priority to U.S. Provisional Patent Application No. 62/966,354 filed January 27, 2020, the contents of which are herein incorporated by reference in its entirety.

Reference to Sequence Listing

This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing titled "269-0002WOUl-SEQS_ST25.txt" created on January 27, 2021, and having a size of 38 KB. The sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.

Field

This disclosure relates to antimicrobial probiotics which may be used, for example, to control Clostridia perfringens ( C . perf) and necrotic enteritis in livestock. Embodiments herein relate to the use of engineered antimicrobial probiotics to improve animal health and well-being. Embodiments herein relate to the improvement of the safety and quality of end products derived from animals. More specifically, some embodiments relate to new probiotic bacteria isolated from natural environments and to the genetic engineering and/or screening of the isolated probiotic bacteria for the treatment of C. perf. infections in pre-harvest broilers. Background

Pressures on global meat production

Global food production is predicted to need to double in the next few decades, to meet the demand by earth’s population, which is projected to reach nearly 10 billion by 2050. This demand along with a confluence of environmental factors, including water scarcity and climate instabilities, are exerting pressure on global food security (FAO {Food and Agriculture Organization of the United Nations). 2017. The future of food and agriculture: Trends and challenges. Available at http://www.fao.Org/3/a-i6583e.pdf).

Due to the growing human population and historically high standards of living, there is a substantial increase in demand for animal protein diets. Global demand for beef, pork and chicken has nearly tripled in the past three decades and although there is pressure to reduce beef production for environmental reasons, pork and poultry production is projected to nearly double again in the following three decades.

The U.S. National Academy of Sciences concludes in a recent report “Science Breakthroughs to Advance Food and Agricultural Research by 2030” that “Today, the U.S. food and agricultural enterprise faces formidable challenges that will test its long-term sustainability, competitiveness, and resilience” {National Academies of Sciences, Engineering, and Medicine. 2018. Science Breakthroughs to Advance Food and Agricultural Research by 2030. Washington, DC: The National Academies Press doi: https://doi. org/10.17226/25059) .

Science-based improvements have historically enabled agriculture to produce food for a growing population. For decades, antibiotics have helped producers raise healthy livestock. Antibiotics have also often been used to promote growth and improve feed efficiency, even in the absence of infection ( White W. Medical Consequences of Antibiotic Use in Agriculture, Science, 1998, 279, 996). Arguably, antibiotics have facilitated and sustained, along with major advances in animal breeding and in production, major livestock productivity increases.

Use of antibiotics in livestock production

Of growing concern is the continuing emergence of microbial resistance to first line antibiotics {US CDC, Antibiotic Resistance Threats in the United States, 2013. Available at https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.p df). It is estimated that over 1 million people are sickened in the US by multidrug-resistant infections and over 20,000 die every year. The trends of increasingly frequent multidrug-resistant pathogens are disconcerting. In January 2017, the U.S. Center for Disease Control announced the death of a woman in Nevada by a bacterial strain that is pan-resistant, i.e. resistant to all antibiotics available in the U.S.

One possible source of drug-resistance emergence is the widespread use of antibiotics in farm animal production {US FDA, 2016 Summary Report on Antimicrobials Sold or Distributed for Use in Food-Producing Animals. Available at https://www.fda.gov/downloads/forindustry/userfees/animaldru guserfeeactadufa/ucm 588085.pdf). An estimated 14,000 tons of antibiotics, or approximately 70% of all antibiotics produced in the U.S., were administered to cattle, pigs and poultry in 2015.

Alarmingly, there has been substantial overlap between classes of antibiotics listed as critically important for human health by the World Health Organization and those antibiotics listed as critically important in agriculture by the World Organization for Animal Health {Joint FAO/WHO/OIE Expert Meeting on Critically Important Antimicrobials, Rome, Italy, Nov 2007, http://www.who. int foodborne disease resources Report (ΊA Meeling.pdf) . F or example, three classes of antibiotics; quinolones, 3rd and 4th generation cephalosporins, and macrolides, were reportedly used in agriculture, even though they are among the few viable therapeutic solutions against certain serious infections in humans.

The precise contribution of antibiotics used in livestock to human infections by antibiotic-resistant microbes is under debate {Editorial, Safety from farm to fork, Nature Reviews Microbiology, 2009, 7, 478). In complex systems such as food production, it is indeed difficult to establish causal relationships between the use of antibiotics in animal feed and gastrointestinal infections where antibiotic-resistant microbes affect human populations. Nevertheless, there is evidence that transmission of resistant strains to humans does occur through food {McEwen SA, Fedorka-Cray PJ. Antimicrobial use and resistance in animals. Clin Infect Dis 2002;34:S93 S106).

Antibiotics in livestock production

Because of these concerns, the European Union banned the use of antibiotics in food animal production in 2006. Attempts were made to pass similar measures in the U.S. with legislation introduced in the House and the Senate, entitled the “Preservation of Antibiotics for Medical Treatment Act” (H.R. 965/S. 1211). The law would result in the “Phased Elimination of Nontherapeutic Use in Animals of Critical Antimicrobial Animal Drugs Important for Human Health”.

The attempts to pass this law were met with resistance. There are, indeed, many important and demonstrated benefits of using antibiotics in livestock production. A widespread ban of antibiotics may then jeopardize the global supply of abundant, high-quality, nutritious, safe and relatively inexpensive food. In the U.S., a ban on the use of antibiotics, and in the absence of alternative antibiotic technologies, could result in substantially increased food prices (Karavolias, J. Raised without antibiotics: impact on animal welfare and implications for food policy, Translational Animal Science, 2(4), 2018, 337 348). This could diminish the enormous positive impact of the animal agriculture on the economy, estimated to $125 billion annually.

These negative effects notwithstanding, the FDA moved to curtail the use of medically important antibiotics for livestock production purposes. Drug companies have voluntarily adopted FDA Guidance #209 and Guidance #213, revising the FDA- approved labeled use conditions to remove the use of antimicrobial drugs for production purposes. The intent is to change the marketing status from over the counter to Veterinary Feed Directive (VFD) for antibiotics administered to animals.

With the VFD, beginning in 2017, over-the-counter antibiotics ceased being used in animal production. Antibiotics are now only prescribed for sick animals by licensed veterinarians. This step may help ensure judicious use of antibiotics. On the other hand, farmers now face the challenge of raising healthy animals without growth- promoting antibiotics.

Clostridia yerfrinsens and necrotic enteritis in poultry

Focusing on the poultry industry, phasing out of antibiotics is already resulting in higher frequency of necrotic enteritis (NE). NE is a result of broiler intestinal damage usually occurring from E. maxima in the presence of a toxicogenic C. perf strain. It is believed that coccidia weaken the mucus layer in the GI tract, enabling C. perf to reach and destroy epithelial cells {Prescott, J.F., The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review, Avian Pathology, 45(3), 2016, 288-294 ).

NE causes a significant negative economic impact in broiler production. The acute form of NE leads to increased mortality in the broiler flocks, which can reach 1% losses per day for consecutive days. The subclinical form of NE manifests as damage to the intestinal mucosa, decreased nutrient uptake, and reduced weight gain. Estimates of $6 billion annually have been reported as the economic worldwide impact of NE on the global poultry industry.

C. perf. in poultry is also a significant risk for foodborne transmission to humans. Type A and type C C. perf. strains cause type A diarrhea and type C necrotic enteritis, respectively, in humans. Approximately 1 million cases of C. perf. infections are reported annually in the U.S.

Sustainable production and welfare of livestock is at risk

Sustainability adds another important dimension to consider as a result of phasing out antibiotics in livestock production. Numerous pathogens impact the health of animals and result in losses in animal production. Arguably, limiting the use of antibiotics may result in considerable losses of animals because of illness, substantial amounts of food lost, and, as a consequence, a significantly negative impact on the environment.

On a global scale, food worth $750 billion is lost or wasted each year throughout the entire supply chain, with approximately 25% of food being lost during production. According to a study by the UN Food and Agriculture Organization (FAO), food loss and waste accounts for about 3.3 giga-tones of greenhouse gas emissions.

It has been estimated that without antibiotics, 700 million additional birds will need to be raised to meet poultry demand in the U.S. annually, requiring approximately 2 billion additional gallons of water and 5.4 million additional tons of feed per year.

Another important dimension relates to the well-being of animals. Animals raised without antibiotics are much more likely to suffer from painful medical conditions. For example, poultry raised without antibiotics are more than three times as likely to experience ammonia bums in their eyes.

Summary

Embodiments herein relate to the isolation, characterization, screening, selection, and/or engineering of antimicrobial probiotics for the treatment of C. perf. and necrotic enteritis in animals. In an embodiment, a composition for treatment of chickens is included. In an embodiment, the composition is administered to chickens in water. In an embodiment, the composition can include a bacterium originally isolated from the small intestinal tract of healthy chickens. In an embodiment, the bacterium can be tested for survival, growth, colonization, and metabolic activity in gastrointestinal tract environments. In an embodiment, the bacterium may be genetically engineered with two exogenous polynucleotides. A first exogenous polynucleotide can include a heterologous promoter and a polynucleotide that encodes an antimicrobial protein. A second exogenous polynucleotide can include a heterologous promoter and a polynucleotide that encodes proteins that function to secrete the antimicrobial protein to the extracellular environment. In an embodiment, the antimicrobial peptide can be effective in killing C. perf. inside the gastrointestinal tract of chickens.

This summary is an overview of some of the teachings of the present application and is not intended to be an exclusive or exhaustive treatment of the present subject matter. Further details are found in the detailed description and appended claims. Other aspects will be apparent to persons skilled in the art upon reading and understanding the following detailed description and viewing the drawings that form a part thereof, each of which is not to be taken in a limiting sense. The scope herein is defined by the appended claims and their legal equivalents.

Brief Description of the Figures

Aspects may be more completely understood in connection with the following figures, in which:

FIGURE l is a schematic diagram of the engineered Enterocin transcriptional unit including an assembled DNA sequence of a promoter, a ribosome binding site, enterocin A, and a transcription terminator site.

FIGURE 2 is a schematic diagram of the secretion system transcriptional unit, including the genes cvaA and cvaB of the Microcin V secretion systems, the diagram specifically illustrating an assembled DNA sequence of a promoter, a ribosome binding site for cvaA, cvaA, a ribosome binding site for cvaB, and a transcription terminator site.

FIGURE 3 is a schematic diagram of the plasmid map used to engineer cellbots for the expression of an antimicrobial peptide, the diagram specifically illustrating plasmid pKG00255 (SEQ ID No: 13) for the expression of an antimicrobial peptide (AMP) and the secretion machinery. The selection marker confers resistance to chloramphenicol.

FIGURE 4 is a snapshot of stab-on-agar tests demonstrating antimicrobial activity of cellbot GP00837 (General Probiotics Inc. Accession Number 00837) against four different C. perf. strains. FIGURE 4 specifically illustrates the activity of E. coli GP00837 constitutively expressing Enterocin A against four different C. perf. strains in an agar diffusion assay. The white dot at the center is the modified GP00837. The light background indicates C. perf. growth white the dark region is a zone of C. perf. growth-inhibition resulting from EntA secreted by GP00837.

FIGURE 5 is a graph of growth of C. perf. in rich media versus time showing how the supernatant containing antimicrobial peptides Enterocin B, Hiracin JM79 and Enterocin A inhibits the growth of the pathogen. Growth is measured at specific time points (0 hours, 3 hours, 6 hours, 24 hours) by plating and enumerating colony forming units (CFU) per ml. Growth is measured in the presence of supernatant from a culture of a cellbot producing Enterocin B, Hiracin JM79 and Enterocin A (BHA).

FIGURES 6A and 6B are graphs of growth of four cellbots versus time, demonstrating that cellbots grow variably in different environments. Growth is measured in terms of optical density at 600nm. FIGURE 6A shows growth in rich media and FIGURE 6B shows growth in small intestinal mucus contents.

FIGURES 7A and 7B are graphs of growth of C. perf. versus time, showing how antimicrobial peptides inhibit the pathogen in various environments. Growth is measured at specific time points (0 hours, 3 hours, 6 hours, 24 hours) by plating and enumerating colony forming units (CFU) per ml. Growth is measured either with supernatant from a culture of a cellbot producing Enterocin B, Hiracin JM79 annd Enterocin A (BHA), or from a culture of cellbot without an antimicrobial peptide. FIGURE 7A shows growth in small intestinal contents. FIGURE 7B shows growth in cecal mucus contents.

FIGURE 8 is a graph demonstrating the lower mortality rate in birds challenged with C. perf. and necrotic enteritis treated with cellbot GP00837 (named GPEC2019005 in the animal study) compared to the mortality rate of untreated birds.

Detailed Description

Probiotics or direct-fed microbials (DFMs) are considered a potential alternative to antibiotic feed additives to lower carriage of pathogens in livestock. The administration of DFMs is also purported to induce better nutrient digestion and enhance meat production in animals such as cattle and poultry. In addition, these products are thought to modulate the immune response of animals.

The FAO defines probiotics as “live micro-organisms that, when administered in adequate amounts, confer a health benefit on the host.” A plethora of microbes are considered probiotics, including lactobacilli, bifidobacteria, bacilli and enterococci {FAO, Probiotics in Animal Nutrition, http://www.fao.Org/3/a-i5933e.pdj).

An official list of microbes that can be marketed as generally regarded as safe (GRAS) DFMs is compiled by the Association of American Feed Control Officials (AAFCO). These DFMs are considered either as fermentation products or yeast products, and are accepted by the FDA as safe.

However, the performance and efficacy of probiotics have not been consistent. There are gaps in the understanding of probiotic organisms, especially in the context of the complex environment of the gastrointestinal tract. The modes of action of specific probiotics are generally not well understood. Competitive exclusion has been long believed to be an important mechanism of action, with probiotic organisms colonizing the gut and inhibiting pathogens from taking hold. Inhibition may occur simply as a result of limited resources, or more actively by the expression and secretion of inhibiting substances. More specifically probiotics are known to produce bacteriocins.

Bacteriocins are antimicrobial peptides (AMPs) produced by a wide range of bacteria. Unlike antibiotic peptides such as the gramicidins, polymyxins, or glycopeptides which are formed by multienzyme complexes, bacteriocins are ribosomally synthesized, i.e., their sequence is gene encoded. The exact biological role of many bacteriocins is still unknown but it is believed that bacteriocins have a vital role in ecology as they influence the composition of the microbial flora in certain growth habitats, e.g., the gastrointestinal tract of humans and animals (Nissen-Meyer, Ribosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action Arch Microbiol. 1997, 167(2/3), 67-77 and references therein).

Numerous bacteriocins exert their antimicrobial effect by interfering with the cell membrane integrity of target bacteria, and they share several physicochemical features. They are generally heat-stable, small in size, often cationic and have amphiphilic or hydrophobic structure. However, they differ greatly from eukaryotic AMPs which often serve as the first line of defense against invading pathogens in mammals: bacteriocins can be very potent and may be able to act at pico- to nanomolar concentrations, whereas micromolar concentrations are often required for the activity of eukaryotic AMPs. Most bacteriocins also have a somewhat narrow target spectrum; individual bacteriocins are usually active against just few species or genera. In contrast, eukaryotic AMPs as well as traditional antibiotics are generally less specific, targeting a large diversity of different bacteria. Consequently, in terms of potency and specificity, bacteriocins may be superior to traditional antibiotics and eukaryotic AMPs.

Bacteriocins can thus be very useful in therapeutic treatments where a particular pathogen is to be removed from a complex multi-species environment (such as in the gut) without causing adverse secondary effects as normally occur with common antibiotics.

For all the promise of bacteriocins, in particular, and antimicrobial peptides, in general, a critical barrier in using these compounds as therapeutics exists. It is difficult to administer AMPs orally or intravenously for therapeutic purposes. As proteins they are quickly degraded, and in high initial dosages they may become toxic to host cells. A technology that could safely deliver bacteriocins at the site of infection, such as inside the GI tract of animals, would be advantageous.

Described herein is the development of engineered antimicrobial probiotics, “cellbots” for short, which affect the viability of C. perf. in the GI tract of livestock. The desired outcome is a cellbot that can safely and effectively reduce the amount of C. perf. in the gut. This may lower the risk of necrotic enteritis, morbidity and mortality, and lower the risk of production losses to livestock producers.

While not wishing to be bound by theory, it is believed that only certain specific natural isolates can be engineered to colonize the jejunum of chickens, be metabolically active inside the jejunum, express and secrete antimicrobial peptides that target C. perf, and/or reduce necrotic enteritis-induced morbidity and mortality in chickens.

“Effective” herein means measurably and consistently lowering C. perf carriage inside the length of the GI tract of animals, and consequently lowering the incidence of necrotic enteritis and NE-induced animal morbidity and mortality.

“Safe” herein means the following risks are minimal: 1) the cellbot infects birds; 2) the cellbot negatively impacts the gastro-physiology of chicken; 3) the cellbot disrupts the normal gut microflora of chicken; 4) the cellbot has toxic effects; 5) the cellbot has immunogenic effects; 7) the cellbot cannot be contained and spreads to the environment.

Methods are presented for the following primary performance criteria to be met for a safe, therapeutic effect against C. perf. :

1. The cellbot survives the path to the GI tract through the stomach

2. The cellbot colonizes the gut in the vicinity of the pathogen

3. The cellbot is metabolically active inside the gut

4. The cellbot expresses and secretes antimicrobial proteins

5. The antimicrobial proteins are stable inside the gut

6. The antimicrobials proteins preferentially and effectively kill the pathogen

7. There are no adverse effects on the host

Herein we describe an iterative method for all the primary performance criteria to be met. To our knowledge it is challenging to make a linear, reductionist approach work in engineering of cellbots. We can choose each component diligently, starting with the probiotic organism, and adding the synthetic DNA construct component by component, but the overall performance may not be sufficient inside the intestinal tract of an animal for an antimicrobial effect. We can measure the performance outcome, but when we change a different component, the outcomes observed with the first component may shift in unpredictable ways, hampering design efforts. For example, in rich media, overexpressing the secretion gene by using a strong constitutive promoter, predictably results in higher titer of secreted peptide. When this strong promoter is used in low pH or low nutrient environments the growth of the bacteria may be hampered, severely limiting the amount of peptide expressed and secreted.

In accordance with embodiments herein, linear development processes were replaced with iterative ones in the following four cycles:

Cycle 1. Build a library of organisms that can be engineered into cellbots.

The first cycle of the method consists of the following steps:

1. In various embodiments, a library of bacteria is isolated from a specific area of the intestinal tract of healthy animals, as described in Example 1 below. In various embodiments the intestinal location is the jejunum area. In other embodiments the intestinal location is the ileum or the cecum. In some embodiments the animals are infected with a pathogen. In some embodiments, the pathogen belongs to Clostridia spp., Salmonella spp., Campylobacter spp., Lawsonia spp., Escherichia spp., or Streptococcus spp.

In various embodiments, the isolated strains of bacteria are E. coli. In other embodiments, the isolated strains belong to Bacillus spp. Lactobacillus spp., and Enterococcus spp. In various embodiments isolates will re-colonize the area they have been isolated from upon administration to an animal. In various embodiments, the isolates will be metabolically active inside the intestinal tract of animals. For example, cellbots selected and engineered to target C. perf. must colonize the jejunum and be metabolically active in the jejunum because the jejunum is a main location of C. perf. colonization.

2. In various embodiments, the absence of virulence factors will be established with the experimental technique described in Example 2 below. In one embodiment, the absence of virulence factors associated with Avian Pathogenic A. coli (APEC) virulence will be established. In one embodiment, the absence of the following set of virulence factors will be established: cvaC, iroN, ompTp, hlyF, etsB, iss, aerJ/iutA, ire A, papC.

In various embodiments, antibiotic resistance markers will be absent. In some embodiments, the selected isolates will be susceptible to the following antibiotics, using the technique in Example 3 below: rifampicin, nalidixic acid, chloramphenicol, ampicillin, kanamycin, and spectinomycin.

In various embodiments, all isolated bacteria are characterized with the fingerprinting technique described in Example 4 below.

In various embodiments the serotype of the isolated bacteria will be determined with the method described in Example 5 below. In various embodiments, the serotype of selected isolated E. coli strains will not be of the following types: 026, 055, 0103, 0111, 0121, 0129abc, 0145, 0157, and 045.

In various embodiments, a library will be built with distinct isolates with no known virulence factors, toxins, and undesired resistance markers.

Cycle 2. Design DNA sequences that encode DNA promoters, antimicrobial proteins and secretion senes.

3. Build/ a library of DNA sequences, as described in Example 6 below, including DNA encoding for: a. antimicrobial peptides. In various embodiments DNA sequences will be assembled that encode for antimicrobial peptides that belong to the following classes: microcins, protegrins, ovispirins, and enterocins. In various embodiments DNA sequences will be assembled that encode for antimicrobial peptides that belong to the class of endolysins.

In various embodiments, the DNA sequence encodes for one of the following set of antimicrobial peptides: Enterocin A, Enterocin B, Enterocin P, Camobacteriocin, Hiracin JM79, and Plantaricin EF, described in Table 3. In various embodiments, the DNA sequence encodes for two distinct antimicrobial peptides resulting in a polycistronic mRNA molecule. In various embodiments, the DNA sequence encodes for three distinct antimicrobial peptides, resulting in a polycistronic mRNA molecule. In various embodiments, the polycistronic DNA sequence encodes for two or three of the antimicrobial peptides in the following set: Enterocin A, Enterocin B, Enterocin P, Camobacteriocin, Hiracin JM79, and Plantaricin EF. b. DNA promoters. In various embodiments, DNA promoters are constitutive, stationary state, anaerobic state, and chloride-inducible DNA promoters. In various embodiments, DNA promoters are selected from the set of promoters described in Table 5. c. secretion genes. In various embodiments, secretion genes are genes of the Microcin V secretion system. In various embodiments, secretions genes are genes of the Microcin N secretion system. In various embodiments, secretion genes are genes of the Microcin C7 secretion system. d. Ribosome binding sites and termination sites. In one embodiment, the ribosome binding sites is the synthetic bicistronic design (SEQ ID No 11) as described by Mutalik ( Mutalik VK, et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013,

10(4): 354-360). In one embodiment, the termination site is the synthetic transcriptional terminator (SEQ ID No 12).

4. Assemble DNA constructs combining promoters, peptides and the secretion machinery. In one embodiment, a constitutive DNA promoter is operably linked to RBS sequence (SEQ ID No. 11) and to the DNA sequence encoding for Enterocin A which is linked to terminator site (SEQ ID No 12), followed by a constitutive promoter operably linked to RBS sequence and to the DNA encoding the secretion genes of the Microcin V system followed by termination site sequence (all contained in SEQ ID No 2).

The number of possible embodiments of distinct DNA sequences resulting by combining single peptide, promoters and secretion machinery is N= (M available peptides (times) P available promoters for the peptides (times) R available promoters for the secretion machinery (times) S distinct secretion gene sets).

The number of possible embodiments of distinct DNA sequences resulting by combining two distinct peptides, promoters and secretion machinery is N= (( available peptides choose 2) (times) P available promoters for the peptides (times) R available promoters for the secretion machinery (times) S distinct secretion gene sets).

The number of possible embodiments of distinct DNA sequences resulting by combining three distinct peptides, promoters and secretion machinery is N= (( available peptides choose 3) (times) P available promoters for the peptides (times) R available promoters for the secretion machinery (times) S distinct secretion gene sets).

5. Transform DNA constructs in library of /bacterial isolates selected in step 2. Select cellbots that contain the DNA plasmid. A maximum number of distinct embodiments of cellbots is the number of isolates times the number of distinct DNA sequences (C = I (times) N). In various embodiments, the transformation of DNA constructs in a library of isolates will be conducted as described in Example 7 below.

Cycle 3. Conduct performance tests and select best performing candidates.

6. Screen and rank-order systems against C. perf.

In one embodiment, the activity of a cellbot can be tested against C. perf with a stab-on-agar test as described in Example 8 below.

In one embodiment, the activity of a cellbot can be tested against C. perf. with a supernatant test as described in Example 9 below. In one embodiment, the activity of a cellbot can be tested against an indicator strain with a liquid culture test as described in Example 9. In one embodiment, the indicator strain will be Enterococcus spp., as in Example 9.

7. Screen and rank-order systems for survival and growth in the following biomatrix assays: a. stomach contents and low pH environments. In various embodiments, the experimental technique followed will be the one described in Example 10 below. b. bile acid contents from the duodenum area. In various embodiments, the experimental technique followed will be the one described in Example 11 below. c. mucus layer scraped from the jejunum, ileum and colon of animals. In various embodiments, the experimental technique followed will be the one described in Example 12.

8. Screen and rank-order systems for metabolic and antimicrobial activity in biomatrix assays. In various embodiments, experiments will be conducted to grow cellbots and C. perf. in these environments and measure the activity of peptides against C. perf. as described in Example 13 below. In various embodiments, experiments will be conducted to grow cellbots and an indicator strain in gut- mimicking environments and measure the activity of peptides against the indicator strain. In one embodiment, the indicator strain can be Enterococcus faecium. In one embodiment, the indicator strain can be Enterococcus faecalis.

9. Rank-order systems and select top cellbots to move into animal experiments. In various embodiments, the ranking and selecting of cellbots can be conducted as described in Example 14 below.

Cycle 4. Conduct proof of concept animal experiments.

In various embodiments, the animal experiments will be conducted with the experimental protocol described in Example 15 below.

Terms

As used herein, the term “protein” refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term “protein” also includes molecules which contain more than one protein joined by a disulfide bond, or complexes of proteins that are joined together, covalently or noncovalentiy, as mui timers (e g., dimers, trimers, tetramers). Thus, the terms peptide, oligopeptide, enzyme, subunit, and protein are all included within the definition of protein and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the protein is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded RNA and DNA. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide may be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non coding regions such as regulatory regions.

As used herein, the terms “coding region,” “coding sequence,” and “open reading frame” are used interchangeably and refer to a nucleotide sequence that encodes a protein and, when placed under the control of appropriate regulator sequences expresses the encoded protein. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A “regulatory sequence” is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term “operably linked” refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

As used herein, a “polycistronic mRNA” refers to a transcription product that includes two or more coding regions. Expression of the two or more coding regions is controlled by a single promoter, and the series of the two or more coding regions that are transcribed to produce a polycistronic mRNA is referred to as an operon.

As used herein, “genetically modified bacterium” refers to a bacterium which has been altered “by the hand of man.” A genetically modified bacterium includes a bacterium into which has been introduced an exogenous polynucleotide, e.g., an expression vector.

As used herein, a "vector" is a nucleic acid (e.g , DNA) used as a vehicle to artificially carry genetic material (e.g., an engineered nucleic acid) into a cell where, for example, the nucleic acid can be replicated and/or expressed. A non-limiting example of a vector is a plasmid. Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmids typically contain an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert. Plasmids may have more features, including, for example, a "multiple cloning site," which includes nucleotide overhangs for insertion of a nucleic add insert, and multiple restriction enzyme consensus sites to either side of the insert.

As used herein, an "‘exogenous protein” and ‘"exogenous polynucleotide” refers to a protein and polynucleotide, respectively, which is not normally or naturally found in a microbe, and/or has been introduced into a microbe. An exogenous polynucleotide may be separate from the genomic DNA of a cell (e.g., it may be a vector, such as a plasmid), or an exogenous polynucleotide may be integrated into the genomic DNA of a cell.

As used herein, a “heterologous” polynucleotide, such as a heterologous promoter, refers to a polynucleotide that is not normally or naturally found in nature operably linked to another polynucleotide, such as a coding region. As used herein, a ‘"heterologous” protein or "‘heterologous” amino acids refers to amino acids that are not normally or naturally found in nature flanking an amino acid sequence.

As used herein, the term “variant” refers to a polypeptide that comprises one or more differences in the amino acid sequence of the variant relative to a reference sequence. For example, a “variant” polypeptide may include one or more deletions, additions or substitutions relative to a reference sequence. The term “variant” is not intended to limit the variant polypeptide to only those polypeptides made by the modification of an existing polypeptide or nucleic acid molecule encoding the reference sequence, but may include variant polypeptides that are made de novo or starting from a polypeptide other than the reference sequence.

As used herein, the term “conservative variant” shall refer to sequences which reflect the incorporation of conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W. H. Freeman and Company (Eds) and Table 1 below).

TABLE 1 : Examples of Conservative Amino Acid Substitut ons

As used herein, a protein may be ‘ ^' structurally similar” to a reference protein if the amino acid sequence of the protein possesses a specified amount of sequence similarity and/or sequence identity compared to the reference protein. Thus, a protein may be “structurally similar” to a reference protein if, compared to the reference protein, it possesses a sufficient level of amino acid sequence identity, amino acid sequence similarity, or a combination thereof. Structural similarity of two proteins can be determined by aligning the residues of the two proteins (for example, a candidate protein and any appropriate reference protein described herein) to optimize the number of identical amino acids along the lengths of their sequences: gaps In either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. A reference protein may be a protein described herein. A candidate protein is the protein being compared to the reference protein. A candidate protein may be isolated, for example, from a microbe, or can be produced using recombinant techniques, or chemically or enzymatically synthesized. Unless modified as otherwise described herein, a pair-wise comparison analysis of amino acid sequences can be carried out using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix=BLOSUM62; open gap penalty^! 1, extension gap penalty=l, gap x dropoff=50, expect=10, wordsize=3, and filter on. Alternatively, polypeptides may be compared using the BESTFIT algorithm in the GCG package (version 10.2. Madison Wis.).

The term "percent identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and " similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins et al., CABIOS. 5:151 (1989)) with the default parameters (GAP PENALTY = 10, GAP LENGTH PENALTY = 10). Default parameters for pairwise alignments using the Clustal method may be selected: KTUPLE 1, GAP PENALTY = 3, WINDOW = 5 and DIAGONALS SAVED = 5.

Thus, as used herein, a candidate protein useful in the methods and compositions described herein includes those with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88'%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to a reference amino acid sequence. Alternatively, as used herein, a candidate protein useful in the methods and compositions described herein includes those with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino add sequence Identity to the reference amino acid sequence.

Nucleic acids useful in the methods and compositions herein includes those with at least at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at leas! 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid sequence identity to the reference nucleic acid sequence.

Conditions that are “suitable” for an event to occur, such as expression of an exogenous polynucleotide in a cell to produce a protein, or production of a product, or "‘suitable’' conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.

As used herein, an “animal” includes members of the class Mammalia and members of the class Ayes, such as human, avian, bovine, caprine, ovine, porcine, equine, canine, and feline.

As used herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

As used herein, “probiotics” are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”, as defined by The Food and Agriculture Organization of the United Nations (FAO). A plethora of microbes are considered probiotics and can be used in an engineered state in accordance with embodiments herein, including E. coli. lactobacilli, bifidobacteria, bacilli and enterococci.

In accordance with embodiments herein, probiotic bacteria include bacteria discovered inside the gastrointestinal tract of animals.

As used herein, “cellbots” are probiotics that are modified using synthetic biology techniques to express and deliver antimicrobial proteins/peptides (including, but not limited to, bacteriocins). The present disclosure is based, at least in part, on unexpected findings showing that not all bacteria isolated from the gastrointestinal tract of animals can be engineered to re-colonize the gut of animals and maintain the metabolic activity for protein production. Methods are disclosed for isolating bacteria and for testing these isolated bacteria for their colonization and metabolic activity inside the gut. We find that only a small number of probiotic bacteria isolates can re colonize the gut of animals and express and secrete antimicrobial peptides in suitable quantities to target pathogenic bacteria.

Although pathogens can be found throughout the GI tract, they also preferentially adhere to different parts of the GI tract. Clostridia spp. for example preferentially colonize the jejunum area of the small intestine of chickens. A probiotic organism genetically modified to target C. perf. will perform better if it can colonize the jejunum and if it can be metabolically active and grow in the area of the jejunum. As discussed in Examples, only a small number of probiotic A. coli isolated from the jejunum area of the small intestine of chickens can re-colonize the jejunum and upon genetic modification express and secrete antimicrobial peptides.

Antimicrobial Peptides

As used herein, “antimicrobial peptides ^’ ’ are small proteins, typically between about 10 and about 100 amino acids in length that inhibit, and often kill, certain bacteria. As such, an antimicrobial peptide has antimicrobial activity that inhibits or kills a target microbe.

The target microbe may be a Gram-positive bacterium that is member of the genus Clostridia. Examples of Clostridia include, for instance, Clostridia perfringens and Clostridia difficile.

The target microbe may be i n vitro or in vivo. For instance, in one embodiment, a target microbe may be one that is present in the gastrointestinal tract or urogenital system of a subject, and optionally may be pathogenic to the subject. Tor instance, in another embodiment, a target microbe may be one that is present in the ovaries of hens, contaminating the eggs inside the chicken before the shells are formed.

Whether an antimicrobial peptide has antimicrobial activity can be determined using different indicator strains. Examples of indicator strains include but are not limited to Enterococcus spp. and Escherichia spp. Examples of suitable indicator strains include, but are not limited to those listed in Table 2 below. In one embodiment, an indicator strain is a member of the genus Enterococcu , such as E. faecalis and E. faecium. Methods for testing the activity of an antimicrobial peptide include, but are not limited to, the stab-on-agar test as well as other methods useful for evaluating the activity of bacteriocins. Such methods are known in the art and are routine. TABLE 2. Example indicator strains

An antimicrobial peptide may be naturally occurring or may be engineered. Antimicrobial peptides are produced by ad classes of organisms, including mammals, bacteria, and phage. Examples of antimicrobial peptides are shown in Table 3.

TABLE 3. Exemplary antimicrobial peptides

Sources for Table 3 i. Aymerich et al, 1996, Appl Environ Microbiol. 62:1676-1682; 2. Cintas et al., 1997, Appl Environ Microbiol., 63:4321-4330; 3. Sanchez et al., 2007, FEMS Microbiol Lett. 270:227-236; 4. Proenpa et al, 2012, Microb Drug Resist., 18:322-332; 5. Fahmer et al., 1996, Chemistry & Biology 3:543-550: 5. Yoong et al., 2004. 1. Bacterid. 186:4808-4812; 6. Uchiyama et al ., 2011, Appl Environ Microbiol. 77:580-585; 7. Son et al, 2010, 1. Appl Microbiol. 108:1769-1779; 8. Proenga ei al., 2012, .Microb Dnxg Resist. 9. Hauge et al. 1999,1 Bacterid., 181(3):740-7; 10. Zhang et al. Biochi m Biophys Acta. 2016 Feb;1858(2):274~80. Corsini et al., 2010, FEMS Microbiol Lett. 312(2) : 119-25. 12. Casans et al., 1997. Microbiology. 143 (Pt 7):2287-94. Exmples of antimicrobial peptides also include those that are essentially identical to any one of the antimicrobial peptides in Table 3. As used herein in the context of a protein “essentially identical” refers to a protein that differs from one of the proteins disclosed herein by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, but shares activity. For example, a protein that is essentially identical to an antimicrobial peptide differs from one of the antimicrobial peptides in in Table 3 at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues and has antimicrobial activity.

In one embodiment, the difference is a conservative substitution. Conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class 1: Ala, Giy, Ser, Thr, and Pro (representing small aliphatic side chains and hydroxyl group side chains); Class 2:

Cys, Ser, Thr, and Tyr (representing side chains including an ^. OH or ^. SH group);

Class 3: Glu, Asp, Asn, and Gin (carboxyl group containing side chains): Class 4: His, Arg, and Lys (representing bas c side chains); Class 5: lie, Val, Leu, Phe, and Met (representing hydrophobic side chains); and Class 6: Phe, Trp, Tyr, and His (representing aromatic side chains).

Most bacteriocins also have a very narrow target spectrum; individual bacteriocins are active against a just few species or genera. On the contrary, eukaryotic AMPs as well as traditional antibiotics are generally much less specific, targeting a large diversity of different bacteria. Consequently, in terms of potency and specificity, bacteriocins may be superior to traditional antibiotics and eukaryotic AMPs.

Bacteriocins can thus be very useful in therapeutic treatments where a particular pathogen is to be removed from a complex multi-species environment (such as in the gut) without causing adverse secondary effects as normally occur with common antibiotics.

Bacteriocins include class I and class II bacteriocins. An example of class II bacteriocins includes members of the subclass lla bacteriocins. Class Ha bacteriocins are small (usually 37 to 48 amino acid), heat-stable, and non-post-translationally modified proteins that are typically positively charged and may contain an N-terroinal consensus sequence -Tyr-Giy-Asn-G!y-(Val/Lys)-Xaa-Cys- (SEQ ID NO: 50). Examples of class II a bacteriocins include, but are not limited to, those described in TABLE 5. Another example of class II bacteriocins includes members of the subclass lib bacteriocins. Class lib bacteriocins are heterodimeric bacteriocins that require two different molecules at approximately equal concentrations to exhibit optimal activity. Examples of class lib bacteriocins include, but are not limited, to those described in TABLE 5.

Another example of antimicrobial peptides includes endolysins. Endolysins are double-stranded DNA bacteriophage-encoded peptidoglycan hydrol ses produced in phage-infected bacterial cells, and cause rapid lysis when applied to Gram-positive bacteria (Fenton et at., 2010, Bioeng Bugs. 1 :9-16; Fischetti, 2008, Cun: Opin Microbiol. 11:393-400).

A nucleotide sequence of a coding sequence encoding an antimicrobial peptide may be easily predicted based on reference to the standard genetic code. When an antimicrobial peptide is to be expressed in a particular microbe, a nucleotide sequence encoding the antimicrobial peptide may be produced with reference to preferred codon usage for the particular microbe.

A coding sequence encoding an antimicrobial peptide may further include nucleotides encoding a secretion signaling protein, such that the antimicrobial peptide and the secretion signaling protein are fused and expressed as a single protein. A secretion signaling protein targets a protein for secretion out of the cell, and is usually present at the amino-terminal end of a protein. Secretion signaling proteins useful in prokaryotic microbes are known in the art and routinely used. Examples of secretion signaling proteins useful in lactic acid bacteria, including L. lactis, Lb. acidophilus,

Lb. acidophilus, Lb. bulgaricus, Lb. reuteri , and Lb. plantarum are known. One example of a useful secretion signaling protein is from the protein Usp45 (Van Asseldonk et ak, 1990, Gene, 95, 155-160). Several variations on Usp45 have been explored and may also be employed (Ng and Sarkar, 2012, Appl. Environ. Microbiol., 79:347-356). Additionally, lactobacillus secretion tags including but not limited to Lp_3050 and Lp_2145 may be used in L. lactis and Lactobacilli spp.

In addition to the signal peptides mentioned above which rely on the general Sec secretion machinery, many antimicrobial peptides also have their own dedicated secretion machinery with corresponding secretion tags. These tags are typically associated with the antimicrobial peptide natively secreted by these transport systems, however, these tags can also be used to secrete non-native antimicrobial peptides. An example of this mechanism of secretion is a double-glycine-type leader, which has been used to secrete colicin V from L. lactis. In the majority of microcin transport systems, secretion systems are associated with self-immunity or proteolytic cleavage of the microcin precursor. The Class II microcin gene clusters often encode for a dedicated ABC transporter and an accessory protein.

In embodiments herein, genetically engineered bacteria can express and secrete one or more AMPs. In various embodiments herein, genetically engineered bacteria can express and secrete combinations of AMPs, such as two or more distinct AMPs.

Promoters and Sigma Factors

Naturally occurring bacteria monitor environmental conditions and they respond by modifying the expression pattern of their genes. Transcription of genes is carried out by a single species of RNA polymerase (RNAP). The core enzyme of RNAP executes RNA polymerization reactions, but it cannot recognize a DNA promoter, bind to it and initiate transcription. The task of promoter recognition in bacteria is left to one of a few protein subunits called sigma factors. Each sigma factor binds to its cognate promoter and connects with the RNAP core enzyme, forming the fully functioning RNAP holoenzyme. In E. coli there are seven known sigma factors and each bind to DNA promoters under different conditions. For example, Sigma 70 binds to its cognate DNA promoters at all times. Sigma 38 binds to its DNA cognate promoters in stationary state. Thus, expression of a gene of interest can be controlled by employing promoters that interact with sigma factors that are dominant under the desired expression condition. For example, by employing a promoter capable of binding sigma 38 but not sigma 70, gene expression would be upregulated in stationary phase rather than in exponential phase.

A complete list of known sigma factors in E. coli is presented in Table 4. TABLE 4. Known Sigma Factors for E. coli Source for Table 4. Gruber TM, Gross CA (2003). "Multiple sigma subunits and the partitioning of bacterial transcription space". Annual Review of Microbiology. 57: 441-66. The sigma factors of E. coli are exemplified above. However, it will be appreciated that each bacterium may have different sigma factors.

Promoters used herein include but are not limited to, high, medium, and low expression constitutive promoters, promoters that respond to stress, nutrient limitations, varying pH, varying osmotic pressure, and promoters that activate in stationary state.

A list of example promoters is presented in Table 5.

Sources for Table 5: 1. http://parts.igem.org/Promoters/Catalog/Ecoli/Constitutive; 2. Piraner DI, et al. Tunable thermal bioswitches for in vivo control of microbial therapeutics, Nat Chem Biol. 2017 Jan;13(l):75-80; 3. Suhyun K, et al. Quorum Sensing Can Be Repurposed To Promote Information Transfer between Bacteria in the Mammalian Gut, ACS Synth. Biol. 2018, 7, 9, 2270-2281; 4. Schlegel S, et al. Isolating E. coli strains for recombinant protein production, Cell Mol Life Sci. 2017 Mar;74(5):891-908; 5. Kammerer W, et al. Functional dissection of E. coli promoters: information in the transcribed region is involved in late steps of the overall process, EMBO J. 1986 Nov; 5(11): 2995-3000; 6. Courbet A, et al. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates, Sci Transl Med. 2015 May 27;7(289):289ra83; 7. Royo JL, et al. In vivo gene regulation in Salmonella spp. by a salicylate -dependent control circuit, Nat Methods. 2007 Nov;4(l l):937-42; 8. Tucker DL, et al. Gene Expression Profding of the pH Response in E. coli, J. Bacteriol. 183: 6551-6558; 9. Boulanger A, et al. Multistress Regulation in E. coli: Expression of osmB Involves Two Independent Promoters Responding either to oS or to the RcsCDB His-Asp Phosphorelay, J Bacteriol. 2005 May;187(9):3282-6; 10. Sanders JW, et al. A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis. Appl Environ Microbiol. 1997 Dec;63(12):4877-82.; 11. Gutierrez C, et al. Osmotic induction of gene osmC expression in E. coliK12, J Mol Biol. 1991 Aug 20;220(4):959-73.; 12. Boysen A, et al. Translational Regulation of Gene Expression by an Anaerobically Induced Small Non-coding RNA in E. coli, J Biol Chem. 2010 Apr 2;285(14): 10690-702; 13. Kang Y, et al. Genome-Wide Expression Analysis Indicates that FNR of E. coli K-12 Regulates a Large Number of Genes of Unknown Function, J Bacteriol. 2005 Feb; 187(3): 1135-1160; 14. Nasr R, et al. Construction of a Synthetically Engineered nirB Promoter for Expression of Recombinant Protein in E. coli, Jundishapur J Microbiol. 2014 Jul; 7(7): el5942.; 15. Chiuchiolo MJ, et al. Growth-Phase-Dependent Expression of the Cyclopeptide Antibiotic Microcin J25, J Bacteriol. 2001 Mar; 183(5): 1755-1764.; 16. Shimada T, et al. Classification and Strength Measurement of Stationary- Phase Promoters by Use of a Newly Developed Promoter Cloning Vector, J Bacteriol. 2004 Nov; 186(21): 7112-22; 17. Hurme R, et al. A proteinaceous gene regulatory thermometer in Salmonella. Cell. 1997 Jul 1 l;90(l):55-64.; 18. Valdez-Cruz NA, et al. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters, Microb Cell Fact. 2010 Mar 19;9: 18.; 19. Dobson R, et al. Characterization of a rationally engineered nitric oxide, nitrate and nitrite biosensor linked to a hybrid bacterial mammalian promoter, available online: https://doi.org/10.6084/m9.figshare.1103248.vl; 20. Kottula JW, et al. Programmable bacteria detect and record an environmental signal in the mammalian gut, PNAS April 1,

2014 111 (13) 4838-4843; 21. Prindle A, et al. A sensing array of radically coupled genetic ‘biopixels’. Nature. 2011 ;481 :39— 44.; 22. Jayaraman P, et al. Repurposing a Two-Component System-Based Biosensor for the Killing of Vibrio cholerae. ACS Synth Biol. 2017 Jul 21;6(7): 1403-1415

In various embodiments, constitutive promoters J23100-109 (SEQ ID NOS: 3- 5) perform best in nutrient-rich environments of the GI tract - their differences in strength of gene expression are also used as a way to produce antimicrobial peptides, maturation factors and secretion machinery at the most optimal ratios.

In various embodiments, the FNR promoter (SEQ ID NO: 6) acts as a constitutive control in the most anerobic environments of the GI tract, as it originates from a switch system in E. coli between aerobic and anaerobic metabolism, the FNR regulon.

In various embodiments, GadA/B promoters (SEQ ID NOS: 7-8) are pH sensitive, which makes them useful for the highly acidic components of the GI tract.

In various embodiments herein, rpoS promoters can be used. In various embodiments herein, anaerobically-inducible promoters can be used. In various embodiments herein, chloride-inducible promoters can be used. In various embodiments herein, stationary-phase promoters can be used.

In various embodiments, promoter osmB (SEQ ID NO: 9) is a stress- responsive rpoS promoter intended for nutrient-poor environments with a high salt/ion content (osmotic stress).

Compositions

In an embodiment, a composition for treatment of an animal is included. The composition can include a bacterium isolated from the intestinal tract of an animal and comprising an exogenous polynucleotide, the polynucleotide comprising a first heterologous promoter and a first polynucleotide that encodes an antimicrobial protein, wherein the first polynucleotide is operably linked to the first heterologous promoter. In various embodiments, the polynucleotide of the composition can further include a second heterologous promoter and a second polynucleotide that encodes suitable secretion genes, wherein the second polynucleotide is operably linked to the second heterologous promoter.

In some embodiments, the bacterium can include the E. coli strain GP00700 deposited under ATCC Accession No. PTA-126596. In some embodiments, the composition can further include a pharmaceutically acceptable carrier. In some embodiments, the bacterium can include the E. coli strain GP00695.

In some embodiments, the antimicrobial peptide has bacteriolytic or bacteriostatic activity against Clostridia perfringens. In some embodiments, the treated animal is a chicken. In some embodiments, the bacterium is isolated from the jejunum of healthy chickens. In some embodiments, the bacterium recolonizes the gastrointestinal tract of chicken infected with Clostridia perfringens. In some embodiments, the bacterium is metabolically active within the gastrointestinal tract of chicken.

In some embodiments, the first heterologous promoter and the second heterologous promoter are selected to respond to different sigma factors selected from the group consisting of o70(RpoD), s19 (Feci), s24 (RpoE), s28 (RpoF), s32 (RpoH), s38 (RpoS), and s54 (RpoN).

In some embodiments, the composition of any of claims 2-10, wherein the first heterologous promoter and the second heterologous promoter are selected to respond to different exogenous environmental conditions found in the gastrointestinal tract of animals, the exogenous environmental conditions defined by one or more of nutrient content, oxygen content, pH and bile concentration.

In some embodiments, at least one of the first heterologous promoter and the second heterologous promoter are selected from the group of constitutive promoters, exogenously-inducible promoters, pH-inducible promoters, osmotic pressure- inducible promoters, anaerobically-inducible promoters, starvation-inducible promoters, temperature-inducible promoters, inflammation-inducible promoters, and quorum-sensing promoters. In some embodiments, the genetically engineered bacterium is a probiotic bacterium. In some embodiments, the genetically engineered bacterium is selected from the group consisting of Bacillus, Bacteroides, Bifidobacterium, Escherichia, Lactobacillus, and Lactococcus. In some embodiments, the genetically engineered bacterium is an E. coli strain that does not belong to the serotypes 026, 055, 0103, 0111, 0121, 0129abc, 0145, 0157, and 045. In some embodiments, the genetically engineered bacterium is an E. coli strain does not encode for Shiga toxin. In some embodiments, the genetically engineered bacterium is an E. coli strain that does not encode for the following virulence genes cvaC, iroN, ompTp, hlyF, etsB, iss, aerJ/iutA, ireA, papC. In some ;embodiments, the genetically engineered bacterium is an E. coli strain that is susceptible to the following antibiotics: rifampicin, nalidixic acid, chloramphenicol, ampicillin, kanamycin, and spectinomycin.

In some embodiments, the antimicrobial protein is selected from the group containing Enterocin A, Enterocin B, Enterocin P, Carnobacteriocin, Plantaricin EF, and Hiracin JM79, or conservative variants thereof.

In some embodiments, the heterologous promoters and the polynucleotides that encode the antimicrobial proteins are located on the chromosome of the bacterium. In some embodiments, the heterologous promoters and the polynucleotides that encode the antimicrobial proteins are located on a plasmid in the bacterium.

In some embodiments, the bacterium is isolated from the intestinal tract of a healthy animal.

In some embodiments, the exogenous polynucleotide that encodes an antimicrobial protein having at least 70, 80, 85, 90, 95, 98, 99, or 100% sequence identity with SEQ ID NO: 1.

In some embodiments, a pharmaceutically acceptable composition is included herein. The composition can include any of the aforementioned compositions. In some embodiments, a composition is formulated for oral administration. In some embodiments, a composition is formulated for incorporation in the water supply of the animal. In some embodiments, a composition is formulated for incorporation in the feed supply of the animal.

In some embodiments, a composition for treatment of an animal is included. The composition can include a bacterium isolated from the intestinal tract of an animal and transfected with a plasmid, the plasmid comprising an exogenous polynucleotide, the polynucleotide comprising a first heterologous promoter and a first polynucleotide that encodes an antimicrobial protein, wherein the first polynucleotide is operably linked to the first heterologous promoter.

In an embodiment, the plasmid can include at least about 70, 80, 85, 90, 92,

95, 98, 99 or 100% sequence identity with SEQ ID NO: 13, or a degree of sequence identity falling within a range between any of the foregoing.

SEQ ID No: 13 includes promoter J23100 (SEQ ID No. 5) at 77-111; DNA sequence of construct to express Enterocin A (SEQ ID No. 1 - which itself includes SEQ ID No. 5 and SEQ ID No. 12) at positions 77-500; DNA sequence of synthetic transcriptional terminator (SEQ ID No. 12) at positions 372-500; and DNA sequence of construct to secrete Enterocin A (SEQ ID No. 2) at positions 513-4144.

Embodiments herein can specifically include a bacterium isolated from an intestinal tract of an animal comprising an exogenous polynucleotide including SEQ ID No. 1 (or a polynucleotide operative for expressing Enterocin A including at least about 70, 80, 85, 90, 92, 95, 98, 99 or 100% sequence identity with SEQ ID NO: 1, or a degree of sequence identity falling within a range between any of the foregoing) and including SEQ ID No. 2 (or a polynucleotide operative for expression of components for the secretion of Enterocin A including at least about 70, 80, 85, 90, 92, 95, 98, 99 or 100% sequence identity with SEQ ID NO: 2, or a degree of sequence identity falling within a range between any of the foregoing).

Embodiments herein can specifically include the intestinal tract bacterium E. coli strain GP00700 deposited under ATCC Accession No. PTA-126596, the bacterium further comprising an exogenous polynucleotide including SEQ ID No. 1 (or a polynucleotide operative for expressing Enterocin A including at least about 70, 80, 85, 90, 92, 95, 98, 99 or 100% sequence identity with SEQ ID NO: 1, or a degree of sequence identity falling within a range between any of the foregoing) and SEQ ID No. 2 (or a polynucleotide operative for expression of components for the secretion of Enterocin A including at least about 70, 80, 85, 90, 92, 95, 98, 99 or 100% sequence identity with SEQ ID NO: 2, or a degree of sequence identity falling within a range between any of the foregoing).

Embodiments herein can include a polynucleotide including SEQ ID No. 1 (or a polynucleotide operative for expressing Enterocin A including at least about 70, 80, 85, 90, 92, 95, 98, 99 or 100% sequence identity with SEQ ID NO: 1, or a degree of sequence identity falling within a range between any of the foregoing) and including SEQ ID No. 2 (or a polynucleotide operative for expression of components for the secretion of Enterocin A including at least about 70, 80, 85, 90, 92, 95, 98, 99 or 100% sequence identity with SEQ ID NO: 2, or a degree of sequence identity falling within a range between any of the foregoing).

In some embodiments, a method for preventing, alleviating or treating necrotic enteritis in an animal caused by Clostridia perfringens is included herein. The method can include a step of identifying an animal in need thereof. The method can include a step of administering an effective amount of a composition (such as any of the aforementioned compositions) to an animal in need thereof. In some embodiments, the animal is a mammal. In some embodiments, the mammal is a human, a dog, a cat, or a pig. In some embodiments, the animal is a bird. In some embodiments, the bird is chicken, a turkey or a duck. In some embodiments, the animal is a fish.

In some embodiments, a method for restoring rate of weight gain in an animal that has necrotic enteritis caused by C. perf. is included herein. In some embodiments, the method can include a step of administering to an animal in need thereof an effective amount of a composition such as any of the aforementioned compositions. In some embodiments, the animal is a mammal. In some embodiments, the mammal is a pig. In some embodiments, the animal is a bird. In some embodiments, the bird is a chicken, a turkey or a duck.

It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a bacterium" includes a composition with more than one bacterium. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.

It should also be noted that, as used in this specification and the appended claims, the word “engineered” describes a system, apparatus, or other structure that is constructed or configured to perform a particular task or adopt a particular configuration. The phrase "engineered" can be used interchangeably with other similar phrases such as arranged and configured, constructed and arranged, constructed, manufactured and arranged, and the like.

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. Embodiments described herein are not to be taken in isolation from the rest of the disclosure and, as such, can be combined with other features or embodiments as may be appropriate based on the general knowledge and understanding of a skilled reader.

All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

Aspects may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments but are not intended as limiting the overall scope of embodiments herein.

EXAMPLES

Herein, we describe the application of this invention for the delivery of the antimicrobial peptide Enterocin A to the intestines of animals.

Enterocin A is a bacteriostatic peptide with activity against Clostridia spp. Application of this invention results in low counts of Clostridia perfringens in the jejunum of chickens.

In embodiments herein, new A. coli strains are isolated from the intestinal tracts of healthy chickens. Embodiments herein include natural E. coli isolates GP00492-551, GP00666-811 (General Probiotics Inc. accession numbers 00492- 00551 and 00666-00811).

In embodiments herein, isolated A. coli strains are characterized for virulence factors, antibiotic susceptibility and DNA fingerprints.

In embodiments herein, selected A coli isolates are engineered for the expression and secretion of Enterocin A.

Enterocin A expression vectors are included for various A coli probiotic species with the expression of all parts relying on recombinant expression systems.

Embodiments herein include genetically engineered A coli isolates GP00492- 551, and GP00666-811.

In embodiments herein, the DNA promoter used in a recombinant construct for the expression of Enterocin A is chosen from promoters presented in Table 5, including but not limited to the following group: constitutive (SEQ ID NOS: 3-5), FNRs (SEQ ID NO: 6), stationary phase (SEQ ID NO: 10), GadA/B (SEQ ID NOS: 7-8), and OsmB (SEQ ID NO: 9).

In embodiments herein, engineered E. coli are tested in assays that mimic the gastrointestinal tract environment. In embodiments herein, engineered E. coli are rank-ordered, screened and selected for animal experiments.

In embodiments herein, selected E. coli are administered to animals demonstrating the decrease of C. perf- induced necrotic enteritis.

Example 1. E. coli Isolation from Intestinal Tracts of Chickens

In this example, we describe isolating a library of E. coli from the jejunum of healthy chickens and using these isolates as cellbot candidates.

Intestinal tracts of healthy chickens were excised from the birds and frozen at - 20°C prior to isolation. On the day of isolation, intestinal tracts were thawed at ~37°C. The small intestines were then spread out and separated into five approximately 12” long sections beginning at the proximal end of the duodenum. A wash bottle containing sterile water was then inserted in the proximal end of each segment and used to rinse the lumen contents out of each segment. Segments were then opened lengthwise using sterile surgical scissors and a sterile razor blade was used to separate the mucus layer from the intestinal wall. The mucus was then stored in a sterile 1.5 mL microcentrifuge tube.

To isolate E. coli from the mucus, 100 uL of mucus was directly plated on a 150 mm diameter MacConkey agar plate (Remel). Additional samples were also generated by adding 2 mL of lysogeny broth (Miller) to the remaining mucus (-500 uL to 1 mL of mucus) and allowed to incubate for 2 hours at 37°C to assist in E. coli recovery.

The sample was then centrifuged at 3000 ref for 2 minutes and 50 uL of the pellet was plated on another MacConkey agar plate. When necessary, 10 and lOOx dilutions were performed prior to plating to obtain single colonies. The plates were incubated overnight (-20 hours) at 37°C. Colonies exhibiting lactose-fermenting A. coli morphology on MacConkey agar were saved for further evaluation.

To further verify that colonies on MacConkey plates were indeed E. coli , two independent colony Polymerase Chain Reaction (PCRs) were performed using primers that bind to the gadA and gadB promoter regions in the E. coli chromosome. PCRs were performed using GoTaq Green Mastermix with the parameters shown below. The primers, annealing temperatures, and expected band sizes are listed in Table 6.

E. coli Nissle 1917 as well as another verified E. coli isolate were used as positive controls. Detection of one or both PCRs with the expected band size signified positive E. coli identity. Five positive isolates from each segment from each bird were grown overnight in lysogeny broth and saved as freezer stocks at -80°C.

PCR Conditions: Initial Denaturation: 95°C 2 minutes. Repeat 30x: 95°C 1 minute, 45°C 1 minute. 72°C 30 seconds. Final extension: 5 minutes

Example 2. Virulence Marker Identification

In this example, we describe ensuring the isolated cellbot candidates do not contain certain virulence factors.

The following genes have been identified as being associated with Avian Pathogenic E. coli (APEC) virulence; cvaC, iroN, ompTp, hlyF, etsB, iss, aerJ/iutA, ireA, papC ( Logue CM, et al. Comparative analysis of phylogenetic assignment of human and avian ExPEC and fecal commensal Escherichia coli using the (previous and revised) clermont phylogenetic typing methods and its impact on avian pathogenic Escherichia coli (APEC) classification. Front. Microbiol. 8, 2017). Isolates were screened for the presence of these genes using colony PCR. PCRs were run for each gene using the primers listed in Table 7. Primer sequences were obtained from Logue et al. 2017. PCRs were performed using GoTaq Green Mastermix with the parameters shown below. Control strains known to contain or 5 lack each gene of interest were included in all PCRs. Strains exhibiting PCR products at the expected DNA length were considered positive for a virulence gene. In general, strains exhibiting none of the above virulence factors were preferentially selected. Note that the presence of a virulence gene does not necessarily indicate pathogenicity of a strain. However, the genes are enriched in APEC strains compared to non- 10 pathogenic E. coli (, Johnson TJ, et al. Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J. Clin. Microbiol. 2008, 46, 3987 3996 ).

PCR Conditions: Initial Denaturation: 95°C 2 minutes. Repeat 30x: 95°C 1 minute, See Annealing Temperatures in Table 7, 1 minute, 72°C 1 minute. Final 15 extension: 5 minutes.

TABLE 7. Primers used for virulence factor screening

Example 3. Antibiotic Resistance Screening

In this example, we describe characterizing the susceptibility of cellbot 5 candidates to various classes of antibiotics.

Antibiotic resistance was tested for the following antibiotics: rifampicin (100 ug/mL), nalidixic acid (20 ug/mL), chloramphenicol (20 ug/mL), ampicillin (100 ug/mL), kanamycin (50 ug/mL), and spectinomycin (100 ug/mL). E. coli isolates were struck out on lysogeny broth (LB) agar and grown overnight at 37°C. The 10 following day, E. coli from the LB plates was struck on LB agar growth plates containing the above concentrations of antibiotic. The plates were incubated overnight at 37°C. Growth was recorded as positive, negative, or minor depending on the density of the resulting E. coli patch. Isolates exhibiting no antibiotic resistance were preferentially used for further study.

15

Example 4. E. coli Isolate Fingerprinting

In this example, we describe fingerprinting the library of E. coli isolates.

DNA fingerprinting was used to characterize isolates and to ensure that isolates were in fact unique from one another. This is particularly important when 20 isolating different strains within the same bird. DNA fingerprinting uses a single primer that binds randomly but reproducibly throughout the genome of a species. When a PCR is performed on a strain using these primers, it generates a unique set of product bands which can be used to differentiate between strains (Dombek PE, et al. Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli 25 isolates from human and animal sources. Appl. Environ. Microbiol. 2000, 66, 2572 2577). The BOX AIR primer (5’-CTACGGCAAGGCGACGCTGACG-3’) was used differentiate among E. coli isolates. ³ PCRs were performed using GoTaq Green Master Mix (Promega) with the following PCR conditions: Initial Denaturation: 95°C 2 minutes. Repeat 30x: 95°C 1 minute, 50°C 1 minute, 72°C 8 minutes. Final extension: 8 minutes.

Example 5. Serotyping of E. coli Isolates

All isolated E. coli were tested for the following serotypes: 026, 055, 0103, 0111, 0121, 0128abc, 0145, 0157, 045. These serotypes include “the Big Six” A. coli serotypes that are monitored in the US meat industry. These serotypes, along with 0157, are the most common E. coli serotypes found in food. They produce Shiga toxins that can result in severe intestinal infections in humans.

The 045 single antiseria (product number 85042) and the O Pool 1 EPEC & VTEC/STEC (product number 48229) tests from Statens Serum Institut (SSI) were used to test for the aforementioned serotypes.

Probiotics were grown overnight in Brain Heart Infusion (BHI) medium. The following day, the cultures were autoclaved (along with a negative BHI control lacking cells) at 121°C for two hours to remove possible K capsules and cross reactivity. Cultures were then allowed to settle for 1 hour at room temperature. 80 uL of the antiserum was then combined with 80 uL of autoclaved cultures and controls in a sterile round-bottom 96 well plate. The plate was then sealed in a Ziplock bag and incubated overnight at 50°C.

Cultures exhibiting agglutination were deemed positive for the serotypes. Positive controls were included (ex. E. coli 0157 H7 EDL #933 was used for the O Pool 1) and were found to agglutinate. Additionally, auto agglutinating was tested by using phosphate buffered saline (PBS) rather than O antiserum. Cultures that exhibited agglutination in PBS were not able to be tested for serotype and were eliminated as candidates.

Example 6. Assembly of a set of DNA sequences for the expression and secretion of Enterocin A.

To generate the modified GP strains, E. coli was transformed with plasmid pKG00255. The engineered strain expresses Enterocin A and employs the Microcin V secretion genes (cvaA and cvaB ) to secrete the peptide. This genetic construct contains promoters from the Anderson promoter collection (J23100-J23119) as well as promoters derived from then native E. coli microcin V genetic cluster. Specifically, J23100 was used as promoter controlling the expression of Enterocin A, and native secretion system promoter as promoter controlling the expression of Microcin V secretion genes. These components are incorporated in a high copy number plasmid containing the pMBl origin of replication for if coli.

FIGURE l is a schematic diagram of the engineered Enterocin transcriptional unit including an assembled DNA sequence of a promoter, a ribosome binding site, enterocin A, and a transcription terminator site. FIGURE 2 is a schematic diagram of the secretion system transcriptional unit, including the genes cvaA and cvaB of the Microcin V secretion systems, the diagram specifically illustrating an assembled DNA sequence of a promoter, a ribosome binding site for cvaA, cvaA, a ribosome binding site for cvaB, and a transcription terminator site. FIGURE 3 is a schematic diagram of the plasmid map used to engineer cellbots for the expression of an antimicrobial peptide, the diagram specifically illustrating plasmid pKG00255 (SEQ ID No: 13) for the expression of an antimicrobial peptide (AMP) and the secretion machinery. The selection marker confers resistance to chloramphenicol.

Example 7. Transformation of DNA in Cellbot Variants

DNA constructs were first generated in standard cloning strains (ex. E. coli JM109 and E. coli MC1061 F’). Constructs were then isolated, sequence-verified, and transformed into the desired delivery host. E. coli isolated from poultry intestines were made electrocompetent using standard methods, including natural E. coli isolates GP00492-551 and GP00666-811.

Briefly, 100 mL of lysogeny broth (LB) was inoculated with 400 uL of an overnight culture of E. coli then incubated at 37C under agitation until the culture reaches an OD600 of -0.4. The cells were pelleted down by centrifugation at 4000xg for 10 minutes and washed three times in ice cold deionized water. The final pellet was resuspended in 400 uL ice cold 10% glycerol in deionized water.

For transformation, 50 uL of electrocompetent cells were thawed on ice then mixed with 50-200 ng of vector. E. coli were transformed at 2400 V, 25 uF, 200 Ohms in a 0.2 um gap cuvette. The transformation was then incubated in 400 uL SOC medium for 1 hr at 37C and plated on LB agar containing the appropriate selective antibiotic.

Example 8. Stab-on-agar tests of antimicrobial activity of cellbots against C nerf.

To test the activity of the modified cellbots, 50-150 uL C. perf. overnight culture was spread on Brain Heart Infusion (BHI) with agar plates to generate a lawn. A colony of the modified GP00837 was swabbed then stabbed into the agar and incubated anaerobically overnight at 37°C. Figure 4 shows the result of this assay. The white dot at the center is the modified GP00837. The light background indicates C. perf. growth white the dark region is a zone of C. perf. growth-inhibition resulting from EntA secreted by GP00837. This example shows that engineered E. coli with pKG00255 shows high activity against strains of C. perf. in an agar diffusion assay in high nutrient agar.

Example 9. Supernatant assays and liquid culture assays of antimicrobial activity of cellbots against indicator strain

To conduct these assays, colonies of the cellbots to be compared were inoculated in growth medium. Cultures are grown for 24 hours in an aerobic environment (shaking) at 37°C. After 24 hours, the cultures were centrifuged for one minute at 13,000 x g to pellet the cells. The supernatant was then transferred to a new tube and sterilized by boiling at 100°C for 10 minutes.

Peptide concentration of the supernatants was then compared by serially diluting each supernatant and testing the dilutions abilities to inhibit the indicator strain, or a strain known to be susceptible to the peptides. This essentially estimates a minimal inhibitory concentration (MIC) of each supernatant. The supernatant with the lowest MIC is the most potent.

In the experiment shown here, Enterococcus faecium 8E9 (EF) was used as the indicator strain rather than C. perf. because of its robust growth. This enabled more reliable quantitative comparisons across producer strains. These results were shown to be translatable to C. perf. by demonstrating a linear correlation between EF and C. perf. susceptibility to the cellbots in question.

To determine the MIC, the indicator strain was grown overnight in rich medium. The following day, the indicator strain was diluted in rich medium to give -10E3-10E4 CFU/mL. 30 uL of the probiotic supernatants were loaded into the first two rows of a sterile 96 well plate. 30 uL of phosphate buffered saline (PBS) was loaded into the remaining rows. 2x serial dilutions are performed from row 2 to row 8. 270 uL of the diluted indicator strain culture was then added to each well. This gives a series of 82x dilutions of supernatant giving concentrations from 10 % v/v to 0.08 % v/v. The indicator plate was covered and incubated statically for 24 hours at 37°C. The following day, the last dilution exhibiting no growth was recorded for each supernatant tested. These data were then used to compare potency of each supernatant.

TABLE 8 shows the activities of twelve different probiotic organisms containing three different constructs (total of 36 probiotic strains) grown in rich growth medium (lysogeny broth). Cellbot activities are shown as the reciprocal of the lowest volumetric fraction of supernatant capable of inhibiting the indicator strain. For example, 1 % v/v supernatant from probiotic GP00835 with the high constitutive promoter was sufficient to inhibit pathogen growth. The reciprocal is taken only to make the data more intuitive so that a higher value indicates a higher activity level.

GP Inhibitory

Strain Species Strain Promoter Fraction Activity

00632 E. coli Chicken Ceca Isolate anaerobic 0.04 28

00639 E. coli Human Isolate anaerobic 0.04 28

00816 E. coli Chicken SI 1 anaerobic 0.10 10

00817 E. coli Chicken SI 2 anaerobic 0.17 6

00818 E. coli Chicken SI 3 anaerobic 0.20 5

00819 E. coli Chicken SI 4 anaerobic 0.17 6

00820 E. coli Chicken SI 5 anaerobic 0.05 20

00821 E. coli Chicken SI 6 anaerobic 0.04 24

00822 E. coli Chicken SI 7 anaerobic 0.20 5

00823 E. coli Chicken SI 8 anaerobic 0.05 20

00824 E. coli Chicken SI 9 anaerobic 0.03 34

00825 E. coli Chicken SI 10 anaerobic 0.10 10

00635 E. coli Chicken Ceca Isolate high constitutive 0.05 20

00638 E. coli Human Isolate high constitutive 0.05 20

00833 E. coli Chicken SI 1 high constitutive 0.02 48

00834 E. coli Chicken SI 2 high constitutive 0.05 20

00835 E. coli Chicken SI 3 high constitutive 0.01 95

00836 E. coli Chicken SI 4 high constitutive 0.20 5

00837 E. coli Chicken SI 5 high constitutive 0.03 34

00838 E. coli Chicken SI 6 high constitutive 0.03 40

00839 E. coli Chicken SI 7 high constitutive 0.08 12

00840 E. coli Chicken SI 8 high constitutive 0.03 34

00841 E. coli Chicken SI 9 high constitutive 0.04 24

00842 E. coli Chicken SI 10 high constitutive 0.05 20

00633 E. coli Chicken Ceca Isolate medium constitutive 0.10 10

00636 E. coli Human Isolate medium constitutive 0.04 28

00843 E. coli Chicken SI 1 medium constitutive 0.05 20

00844 E. coli Chicken SI 2 medium constitutive 0.20 5

00845 E. coli Chicken SI 3 medium constitutive 0.08 12

00846 E. coli Chicken SI 4 medium constitutive 0.20 5 00847 E. coli Chicken SI 5 medium constitutive 0.20 5 00848 E. coli Chicken SI 6 medium constitutive 0.10 10 00849 E. coli Chicken SI 7 medium constitutive 0.20 5 00850 E. coli Chicken SI 8 medium constitutive 0.20 5 00851 E. coli Chicken SI 9 medium constitutive 0.10 10 00852 E. coli Chicken SI 10 medium constitutive 0.05 20

TABLE 8. Supernatant minimum inhibitory activities to compare Enterocin production

Figure 5 shows a graph of C. perf. growth in nutrient rich media (thioglycolate 5 with 2% beef extract) over time comparing the effects of supernatant from a probiotic producing Enterocin A, Enterocin B, and Hiracin JM79 under high-nutrient conditions versus no treatment. This example shows that the peptides secreted by the probiotic engineered with a high nutrient promoter suppresses C. perf. growth in nutrient rich media.

10 A supernatant colony-counting assay was used to obtain these data. For this assay 10 uL of an overnight culture of C. perf. was inoculated into 900 uL Thioglycollate+2% beef extract. 100 uL of supernatant from either a probiotic producing no peptides (Control) or the same strain producing the three peptides Enterocin A, Enterocin B, and Hiracin JM79 (BHA) was then added to the culture.

15 The culture was then incubated at 37°C under anaerobic conditions.

10 uL samples of each culture was taken at 0 hours, 3 hours, 6 hours, and 24 hours and serially diluted in a series of 6 lOx dilutions. Dilutions were plated on selective agar (Brain heart infusion with 100 ug/mL rifampicin and 30 ug/mL nalidixic acid). Plates were incubated overnight under anaerobic conditions at 37°C

20 and colonies of C. perf. were counted. Based on the number of colonies, the colony forming units (CFU) of C. perf. per mL of culture were determined for each time point.

Example 10. Testing and screening of cellbots in stomach-like environments

25 In order to be delivered to the site of infection intact, it is beneficial that the cellbots survive passage through the acidic conditions of the stomach. Cellbots were thus tested for their sensitivity to stomach contents using survival assays in vitro. For these assays, contents were removed from the gizzards of healthy chickens and were mixed with water (~1 : 1 ratio). The mixture was then centrifuged for 1 minute at 3500 ref to remove large solids. The pH was measured for each experiment (pH ~2). Gizzard contents were then inoculated with ~10 ⁷ CFU/mL of each probiotic. The cultures were then incubated anaerobically at 37°C for 45 minutes. This time was selected as a conservative estimate of the residence time in the chicken gizzard.

The probiotic was enumerated at 0 hours and at 45 minutes by dilution plating on selective agar (BHI or LB+150 ug/mL rifampicin). Plates were incubated overnight at 37°C and the CFU/mL were determined for both time points. The survival ratio of each strain was then calculated as follows: Survival Ratio = (CFU/mL after incubation) / (CFU/mL before incubation)

Table 9 shows the survival ratios of 30 different probiotics tested. Probiotics exhibiting particularly low survival ratios (ex. <0.2) were retested.

TABLE 9. Survival of probiotics in chicken gizzard contents

In addition to testing survival in stomach contents, survival was also tested in M9 minimal medium that had been adjusted to a pH of 2. This was done to test the impact of pH on the cells in the absence of any other variable biological components. Table 10 shows the survival ratios of 11 different isolates in stomach contents and M9 with HC1 after 24 hours of incubation. Note that despite the same pH of ~2 in both media, isolates appear more sensitive in M9 adjusted with HC1.

TABLE 10. Survival ratios of probiotics after 24 hours in stomach contents or M9

(pH=2)

Example 11. Testing and screening of cellbots in bile acid contents. In order to be delivered to the site of infection intact, it is beneficial that the cellbots also survive passage through the bile of the duodenum. Cellbots were thus tested for their sensitivity to chicken bile using survival assays in vitro. For these assays, contents were removed from the duodenums of healthy chickens centrifuged for 1 minute at 3500 ref to remove large solids. Duodenum contents were then inoculated with ~10 ⁷ CFU/mL of each probiotic. The cultures were then incubated anaerobically at 37°C for 2 hours. This time was selected as a conservative estimate of the residence time in the chicken duodenum.

The probiotic was enumerated at 0 hours and at 2 hours by dilution plating on selective agar (BHI or LB+150 ug/mL rifampicin). Plates were incubated overnight at 37°C and the CFU/mL were determined for both time points. The survival ratio of each strain was then calculated as follows:

Survival Ratio = (CFU/mL after incubation) / (CFU/mL before incubation) Table 11 shows the survival ratios of 30 different probiotics tested. Probiotics exhibiting particularly low survival ratios (ex. <0.2, shown in grey) were retested.

TAB ,E 11. Survival of probiotics in chicken bile

Example 12. Growth and stability of cellbots in ieiunum-like environments

For the cellbots to be effective, they are preferably metabolically active inside the poultry intestinal tracts act the site of infection. In NE, the C. perf proliferation largely occurs in the mucosal layer of the small intestinal tract. Thus, it is desirable to select a delivery strain that can proliferate in the SI mucus layer, indicating metabolic activity. To test this parameter, SI mucus from the jejunum and ileum were isolated. The mucus was then diluted 1 Ox in M9 minimal medium and sterile filtered to generate the biomatrix growth medium. A similar assay can be done using mucus isolated from the ceca.

To perform the assay, the probiotic was first grown overnight in lysogeny broth (LB). 2.5 uL of the overnight culture was then added to 250 uL of the biomatrix growth medium in a sterile 96 well plate. The plate was then incubated at 37°C with agitation in a plate reader. Growth was monitored continuously for 20 hours based on optical density at 600 nm.

Figure 6 shows the growth curves of four different intestinal isolates in rich medium (LB) as well as lOx diluted SI mucus. Note that GP00105 exhibits poor growth compared to the other three strains in SI mucus but not in rich medium. This suggests that this particular strain may exhibit sub-optimal metabolic activity inside the gut of animals.

Example 13. Antimicrobial activity in biomatrix assays

C. perf. is largely believed to proliferate in the mucosal layer in the small intestines of chickens. It is thus desirable that the probiotics be active against C. perf. under the conditions found in this mucus layer. Supernatant activity assays, similar to those in Example 8, were used to verify that the peptides are active in the nutrients available at the site of infection.

For these assays, intestines were isolated from healthy chickens that were sacrificed at a poultry research facility. The intestines were then divided into their primary components- the duodenum, the ileum, the jejunum, and the ceca. Each section was then rinsed with sterile deionized water to remove the lumen contents. The sections were then cut lengthwise using a sterile razor blade and the mucus layer was gently scraped and saved in a sterile vial.

Figure 7 shows the supernatant inhibition assay performed in dilute small intestinal and ceca mucus. For this assay, mucus was harvested from the ileum and jejunum (for small intestinal) or the ceca of healthy chickens as described above. The mucus was diluted 1:10 with M9 minimal medium then filter-sterilized. 10 uL of an overnight culture of C. perf. was inoculated into 900 uL of the diluted mucus. 100 uL of supernatant from either a probiotic producing no peptides (Control) or the same strain producing the three peptides Enterocin A, Enterocin B, and Hiracin JM79 (BHA) was then added to the culture. The culture was then incubated at 37°C under anaerobic conditions.

10 uL samples of each culture was taken at 0 hours, 3 hours, 6 hours, and 24 hours and serially diluted in a series of 6 lOx dilutions. Dilutions were plated on selective agar (Brain heart infusion with 100 ug/mL rifampicin and 30 ug/mL nalidixic acid). Plates were incubated overnight under anaerobic conditions at 37°C and colonies of C. perf. were counted. Based on the number of colonies, the colony forming units (CFU) of C. perf. per mL of culture were determined for each time point. Based on the results from the figures, it appears the peptides are active in the nutrients available in both the small intestine and ceca mucosal layers but to different extents.

Example 14. Rank-ordering and screening of cellbots For each of the screening experiments described in previous Examples, a scheme was employed to rank-order the E. coli isolates. For example, in Example 12 a procedure is described for assessing the growth of isolates in environments mimicking the jejunum mucus of chickens. The OD600 measurement is recorded at various time points and the isolates are ranked accordingly. In Table 12, 10 strains are ranked based on the OD600 measurement at 40 hours.

TABLE 12. Example rank-ordering of isolates according to their growth in chicken jejunum mucus environments (Example 12). In Table 13, E. coli engineered isolates are ranked according to the diameter of the halo measured in a stab-on-agar plate experiment, as described in Example 8.

TABLE 13. Example rank-ordering of cellbots according to their antimicrobial activity measured with the stab-on-agar test (Example 8)

The rankings were compiled for 360 systems engineered and tested. A linear combination of ranking was calculated and a new global ranking order was determined. The top five leading candidates, GP00824, GP00834, GP00632, GP00839, and

GP00837 were renamed GPEC2019001 (or T5), GPEC2019002 (or T6),

GPEC2019003 (or T7), GPEC2019004 (or T8), and GPEC2019005 (or T9). These five cellbots were prepared for proof of concept animal experiments, described in Example 14.

Example 14. Proof of concept animal experiment.

In this example, we describe animal experiments conducted for assessing the efficacy and safety of engineered E. coli isolates that have been tested and screened as described in Examples 1-13. An animal experiment was conducted to test the efficacy and safety of five engineered probiotic E.coli on the control of gross lesion scores and mortality due to C. perf- induced necrotic enteritis, when medicated through water from 0-21 days, in broilers raised in battery cages for 22 days.

This study utilized 540 single-sex commercial broilers from a commercial hatchery. Each cage was considered an experimental unit. The study began on Study Day 0 (arrival of birds). There were nine (n=9) treatment groups (two negative controls, one positive control, five test article groups with challenge and one test article group without challenge) as shown in Table 14.

Each treatment group had 6 replicates. Each replicate contained 10 birds.

There were 60 birds per treatment group. Treatment groups were represented as Tl, T2, T3, T4, T5, T6, T7 T8 and T9.

On study day 0, eleven apparently healthy chicks were enrolled into each cage. On study day 7, each cage was corrected to have exactly 10 chicks per cage.

Except Tl and T3 treatment groups, all other treatment groups were medicated with their respective test article from very first (Day 0) drinking water until the last day of the study (Day 21).

Except Tl and T2 group, all other treatment groups were administered with mixture of E. maxima (10,000 sporulated oocyst/mL/bird) + E. acervulina (no limit) on day 14 through oral gavage.

Except Tl & T2 group, all other treatment groups were administered with C. perf. strain #16 at the rate of 2-9 x 108 cfu/mL/bird on day 17 & 18 through oral gavage.

During the period of day 17-22, all the mortalities were necropsied.

TABLE 14. Description of groups in animal experiment (Example 14). Probiotic E. coli strains #1, #2, #3, #4, and #5 are GP00824, GP00834, GP00632, GP00839, and GP00837, respectively. “Best construct system alone” refers to GP00824, which had the highest global ranking.

The mortality results of the study are the following:

• There were no mortalities observed in T1 & T2 groups.

• The untreated challenge control group T3 had mortality of 32.8%.

• Positive control T4 also had no mortality.

• T5 construct system (GPEC2019001) showed highest mortality of 42.6%.

• T6 (GPEC2019002) resulted in mortality of 14.8% (P<0.05).

• T7 & T8 did not show statistically significant improvements in terms of mortality.

• T9 (GPEC2019005) resulted in mortality of 11.3% (P<0.05).

T9 (GPEC2019005 or GP00837) is a cellbot with demonstrated performance in vivo , proving that a probiotic-based technology can have a profound effect on the mortality of broiler chickens caused by C. perf- induced necrotic enteritis. Cellbot GP00837 (GPEC2019005) was formed using A. coli strain GP00700 deposited under ATCC Accession No. PTA-126596. This shows that GP00837 (GPEC2019005) is a highly useful E. coli strain for the creation of cellbots for treatment of animals and, in specific, chickens.

What was unexpected was that only two out of the five cellbots that ranked at the top of 360 engineered cellbots decreased necrotic enteritis induced mortality in chickens.

We did not observe any adverse effects on birds consuming probiotics, establishing the safety of the cellbots administered to chickens at the prescribed dosing regimen.

FIGURE 8 is a graph demonstrating the lower mortality rate in birds challenged with C. perf. and necrotic enteritis treated with cellbot GP00837 (named GPEC2019005 in the animal study) compared to the mortality rate of untreated birds.

The embodiments described herein are not intended to be exhaustive or to limit the invention to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices. As such, aspects have been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope herein.