Foodborne illnesses continue to be an important public health concern in developing, as well as in developed countries, thus prevention of foodborne illness outbreaks through effective and novel interventions should be given priority. The U.S. Public Health Service has identified ten important foodborne pathogens causing human illnesses, including pathogenic strains of Escherichia coli, Salmonella, Listeria, Clostridium botulinum, Shigella, and Campylobacter, which are associated with more than 250 known foodborne diseases (http://www3.niaid.nih.gov/topics/foodborne/default.htm).
In addition, according to the World Health Organization (WHO), antibiotic overuse in food animal production is a major contributor to the emergence of antibiotic resistant foodborne pathogens . The use of antibiotics in food animals for growth promotion and treatment disrupts the normal beneficial commensal bacterial microflora in the animal intestinal tract [2–6]. Recently, the human and chicken gut microbiome projects [7–12] have shed new light on the existence of a bacterial ‘phylogenetic core’  consisting of a wide diversity of gastrointestinal bacteria by using new technologies such as next generation sequencing, 16S rRNA screens, metagenomics, and metaproteomics. Healthful, commensal bacteria found in the GI tract might be key members of known or potential probiotic strains revealed in this ‘phylogenetic core’, which may include Bacillus clausii, Bacillus pumilus, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus rhamnosus GG (ATCC 53103), Bifidobacterium infantis, Saccharomyces boulardii, Lactobacillus ruminis, Lactobacillus johnsonii str. NCC 533, and many others. These known probiotic strains have been used as dietary supplements, as treatments for illnesses caused by foodborne pathogens, and for disease prevention. Use of probiotic strains not only reduce invasion by bacterial pathogens, but also restore and maintain an optimal balance of healthy commensal bacteria in the human gut via production of antimicrobials [14–23].
One of the major mechanisms recognized as being responsible for apoptosis, or programmed cell death, and production of toxic metabolites in bacteria is through the regulation of a wide variety of bacterial toxin-antitoxin modules [24–26], such as MazE/MazF, a chromosomal toxin-antitoxin module [27–30], plasmid-encoded par D [31–33], chpBIK[26, 34], relBE[35, 36], and the PhoQ-PhoP system [37–40]. MazEF is one of the most well-studied toxin–antitoxin (TA) systems and has been found on the chromosomes of many bacteria. The MazF protein has been recently categorized as an endoribonuclease [41, 42] or as a type of RNA interference enzyme . The link between this TA system and quorum sensing has also been explored recently through a small pentapeptide (NNWNN) called the Extracellular Death Factor (EDF) . This small peptide motif (such as NNWNN) is known to be an extra-cellular death factor in E. coli and other bacterial species. The necessity of an “extracellular death factor” (EDF) or “cell death factor” (CDF) via MazEF-mediated cell death is a population phenomenon requiring the activation of quorum-sensing (cell-to-cell signaling) peptides in bacteria. High cell density was found to be associated with elevated concentrations of EDF, and the presence of EDF resulted in MazF-induced cell death . From a food safety and public health perspective, use of EDF or a similar strategy may be used in place of antibiotics, resulting in less usage of antibiotics. We also noticed in one very interesting study that the induction of toxin MazF and the use of antibiotic share a similar mechanism by inhibiting the transcription and/or translation of the MazE antitoxin .
It has been theorized that there is “one toxin for one antitoxin” and interestingly MazF, in some bacteria, exhibits a selective inhibition of ribosomes and mRNAs [43, 46]. Numerous strains of probiotic bacteria, such as Lactobacillus spp., have been reported to produce antimicrobial agents , such as bacteriocins, that inhibit or kill closely-related species, or even different strains of the same species through the inhibition of transcription and translation by receptor binding. The antimicrobial activities of bacteriocins are due to a heterologous subgroup of ribosomally synthesized cationic peptides . Nisin, a polycyclic antibacterial peptide 34 amino acid residues long, is one of the most studied bacteriocins and is produced by many strains of Lactococcus lactis. It has been approved by the FDA for use as a food preservative, and certain probiotic Lactobacillus strains have been thoroughly studied and evaluated in vitro and in vivo. For example, L. reuteri controls diarrhea in children and suppresses the growth and pathogenicity of harmful foodborne pathogens such as Salmonella, E. coli, Staphylococcus, and Listeria[49, 50]. L. casei GG has been used to treat Clostridium difficile infections and to reduce intestinal permeability [51–54]. L. reuteri is known to produce a broad-spectrum antimicrobial agent, reuterin, composed of the natural metabolic compound 3-hydroxypropionaldehyde, which has been used on the surface of sausages to inhibit growth of harmful bacteria and fungi [15, 16]. However, the molecular mechanisms underlying the effectiveness of individual probiotic strains have not been systematically studied and characterized. Several potential mechanisms of action, including their ability to generate diverse natural toxic metabolites, lactic acid, and other organic acids, enzymes, vitamins, and hydrogen peroxide, as well as antimicrobial peptides such as nisin, have been well-described .
The work reported herein explores the experimental antimicrobial possibilities and/or procedures for (1) expression of an engineered, stress-induced recombinant secreted fusion gene encoded by MazF and a small extracellular cell death factor (CDF) on the surface or extracellular space of recombinant probiotic bacteria or (2) for potential application of a cocktail of natural probiotic strains via experimental in vitro selection to control foodborne pathogens for improving the safety and quality of foods, as well as improving human health. The use of engineered probiotic strains or the natural probiotic cocktail consisting of mixed probiotic strain populations for targeting foodborne pathogens will potentially be able to selectively inactivate these pathogens, even in complex food matrices. Through gene/motif reshuffling [55, 56] and computational molecular modeling, this engineered and secreted bacterial fusion protein, MazF-CDF, which contains an enterokinase (EK) cleavage site [57–60] for releasing active CDF and MazF after protein secretion, should have a narrow range of applicability, limited to inactivating only specific foodborne pathogens such as E. coli O157, Salmonella, Listeria, Campylobacter, and potentially other human pathogens. This pathogen specificity is due to the fact that the genetically-engineered MazF-CDF fusion protein could be modified by the inclusion of specific genomic DNA sequences from various commensal, as well as pathogenic bacterial strains, identified through DNA/protein structure and functional comparisons. Additionally, this engineered antimicrobial polypeptide and MazF will be non-toxic to beneficial (healthful/probiotic) bacteria, as well as to its host that expresses the protein/peptide. Moreover, these engineered and secreted CDFs and MazF proteins/peptides can easily pass through infectious foodborne pathogen cell barriers mediating cell death, and thus could potentially reduce the use of deleterious compounds such as antibiotics or other harmful chemicals in the feed and food industry.