There is a growing concern about the increase in human morbidity and mortality caused by foodborne pathogens. Antibiotics were and still are used as the first line of defense against these pathogens, but an increase in the development of bacterial antibiotic resistance has led to a need for alternative effective interventions. Probiotics are used as dietary supplements to promote gut health and for prevention or alleviation of enteric infections. They are currently used as generics, thus making them non-specific for different pathogens. A good understanding of the infection cycle of the foodborne pathogens as well as the virulence factors involved in causing an infection can offer an alternative treatment with specificity. This specificity is attained through the bioengineering of probiotics, a process by which the specific gene of a pathogen is incorporated into the probiotic. Such a process will subsequently result in the inhibition of the pathogen and hence its infection. Recombinant probiotics offer an alternative novel therapeutic approach in the treatment of foodborne infections. This review article focuses on various strategies of bioengineered probiotics, their successes, failures and potential future prospects for their applications.

Probiotics, with their associated beneficial effects, have gained popularity for the control of foodborne pathogens. Various sources are explored with the intent to isolate novel robust probiotic strains with a broad range of health benefits due to, among other mechanisms, the production of an array of antimicrobial compounds. One of the shortcomings of these wild-type probiotics is their non-specificity. A pursuit to circumvent this limitation led to the advent of the field of pathobiotechnology. In this discipline, specific pathogen gene(s) are cloned and expressed into a given probiotic to yield a novel pathogen-specific strain. The resultant recombinant probiotic strain will exhibit enhanced species-specific inhibition of the pathogen and its associated infection. Such probiotics are also used as vehicles to deliver therapeutic agents. As fascinating as this approach is, coupled with the availability of numerous probiotics, it brings a challenge with regard to deciding which of the probiotics to use. Nonetheless, it is indisputable that an ideal candidate must fulfil the probiotic selection criteria. This review aims to show how Lacticaseibacillus rhamnosus, a clinically best-studied probiotic, presents as such a candidate. The objective is to spark researchers’ interest to conduct further probiotic-engineering studies using L. rhamnosus, with prospects for the successful development of novel probiotic strains with enhanced beneficial attributes.

Summary: Listeria monocytogenes has been implicated in a number of outbreaks including the recent largest outbreak in South Africa. Current methods for prevention of foodborne L. monocytogenes infection are inadequate, thus raising a need for an alternative strategy. Probiotic bioengineering is considered a prevailing approach to enhance the efficacy of probiotics for targeted control of pathogens. Here, the ability of Lactobacillus casei expressing the L. monocytogenes invasion proteins Internalins A and B ( inl AB ) to prevent infection was investigated. The inl AB operon was cloned and surface‐expressed on L. casei resulting in a recombinant strain, Lbc ^{Inl AB} , and subsequently, its ability to inhibit adhesion, invasion and translocation of L. monocytogenes through enterocyte‐like Caco‐2 cells was examined. Cell surface expression of Inl AB on the Lbc ^{Inl AB} was confirmed by Western blotting and immunofluorescence staining. The Lbc ^{Inl AB} strain showed significantly higher ( P  <   0.0001) adherence, invasion and translocation of Caco‐2 cells than the wild‐type L . casei strain (Lbc ^WT ), as well as reduced L. monocytogenes adhesion, invasion and transcellular passage through the cell monolayer than Lbc ^WT . Furthermore, pre‐exposure of Caco‐2 cells to Lbc ^{Inl AB} significantly reduced L. monocytogenes ‐induced cell cytotoxicity and epithelial barrier dysfunction. These results suggest that Inl AB ‐expressing L . casei could be a potential practical approach for prevention of listeriosis.

The success of the probiotics in delivery of health benefits depends on their ability to withstand the technological and gastrointestinal conditions; hence development of robust cultures is critical to the probiotic industry. Combinations of probiotic cultures have proven to be more effective than the use of single cultures for treatment and prevention of heterogeneous diseases. We investigated the effect of pre- adaptation of probiotics to multiple stresses on their stability under simulated gastrointestinal conditions and the effect of their singular as well as their synergistic antagonistic effect against selected enteric pathogens. Probiotic cultures were inoculated into MRS broth adjusted to pH 2 and incubated for 2 h at 37°C. Survivors of pH 2 were subcultured into 2% bile acid for 1 h at 37°C. Cells that showed growth after exposure to 2% bile acid for 1 h were finally inoculated in fresh MRS broth and incubated at 55°C for 2 h. The cells surviving were then used as stress adapted cultures. The adapted cultures were exposed to simulated gastrointestinal conditions and their non- adapted counterparts were used to compare the effects of stress adaptation. The combination cultures were tested for their antipathogenic effects on Escherichia coli and Staphylococcus aureus. Acid and bile tolerances of most of the stress-adapted cells were higher than of the non-adapted cells. Viable counts of all the stress-adapted lactobacilli and Bifidobacterium longum LMG 13197 were higher after sequential exposure to simulated gastric and intestinal fluids. However, for B. longum Bb46 and B. bifidum LMG 13197, viability of non-adapted cells was higher than for adapted cells after exposure to these fluids. A cocktail containing L. plantarum + B. longum Bb46 + B. longum LMG 13197 best inhibited S. aureus while E. coli was best inhibited by a combination containing L. acidophilus La14 150B + B. longum Bb46 + B. bifidum LMG 11041. A cocktail containing the six non- adapted cultures was the least effective in inhibiting the pathogens. Multi-stress pre-adaptation enhances viability of probiotics under simulated gastrointestinal conditions; and formulations containing a mixture of multi stress-adapted cells exhibits enhanced synergistic effects against foodborne pathogens.

The preadaptation of probiotics to sub-lethal levels of multiple stress factors boosts their survival and stability. However, little is known about how long-term cold storage affects the properties of such preadapted probiotics. This study examined the impact of long-term freezing on structural and functional properties of multi-stress (acid, bile and heat) adapted Lactiplantibacillus plantarum B411. Cell morphology was investigated using scanning electron microscopy, and then their selected functional (bile salt hydrolase (BSH) activity, surface hydrophobicity, auto-aggregative and antimicrobial) properties were evaluated. Furthermore, the survival of L. plantarum B411 cells in yoghurt and juices during storage and under simulated gastrointestinal (GIT) conditions was evaluated. Long-term freezing negatively affected the morphology, auto-aggregation ability, BSH and antimicrobial activities of L. plantarum B411. The viability of freshly adapted and old adapted L. plantarum B411 cells in foods was similar. Under simulated GIT conditions, the viability of the stress adapted cells from the freezer diminished more than that of freshly adapted cells. Prolonged freezing compromised some functional properties of stress adapted cells and their stability under simulated GIT conditions. Care should thus be taken to ensure that a method used to preserve stress adapted cells does not cause them to lose beneficial properties, nor revert to their pre-adaptation status.