Cocktails of probiotics pre-adapted to multiple stress factors are more robust under simulated gastrointestinal conditions than their parental counterparts and exhibit enhanced antagonistic capabilities against Escherichia coli and Staphylococcus aureus

BACKGROUND : 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. METHODS : 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. RESULTS : 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. CONCLUSION : 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.We would like to acknowledge National Research Foundation (NRF) and the University of Pretoria for funding this work.http://www.gutpathogens.comam201

The morphological and functional properties of lactiplantibacillus plantarum B411 subjected to acid, bile and heat multi-stress adaptation process and subsequent long-term freezing

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. Longterm 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.National Research Foundation (NRF) of South Africa, Competitive Programme for Rated Researchers and the University of Pretoria.https://www.mdpi.com/journal/microbiolresam2023BiochemistryGeneticsMicrobiology and Plant Patholog

Pre-adaptation of selected probiotic strains to multiple stress factors : consequent effect on their stability and probiotic properties

The interest in the use of probiotics has escalated in the last decade, this is consequent to an increase in the number of studies showing that these microorganisms have beneficial effects on the host’s health. Probiotics can be administered in different ways, as capsules (pharmaceuticals) and also incorporated in different food products. In the definition of probiotics, it is highlighted that for them to be beneficial, they have to be administered alive and in adequate numbers. Nevertheless, there have been a number of problems associated with the number of viable cells of probiotics, with reports that viable numbers decline drastically on exposure to various stresses including those that prevail in the GIT. This raised an interest in the probiotics research, focusing on the techniques that can yield stress resistant or tolerant probiotics. These techniques are aimed to increase the number of the surviving cells after the exposure to technological and gastrointestinal stress factors. There has, therefore, been an increase in the studies focusing specifically on how to adapt the probiotic cells to different stress factors. The mechanism of pre- adaptation or cross- protection has been one of the most studied areas. With these mechanisms, researchers pre- expose probiotics to different stress factors so that they can survive better when they are later exposed to the same stress factor. Pre- adaptation of probiotics to multiple stress factors will therefore offer tolerance to more stress factors. Taking that into consideration, the present study aimed at determining whether probiotic cells that have been pre- exposed to multiple stress factors (acid, bile and temperature) will have better tolerance to different gastric and intestinal conditions when compared to non- adapted cells. The first part of the research followed a stepwise stress adaptation mechanism for six probiotics (Bifidobacterium bifidum LMG 11041, B. longum LMG 13197, B. longum Bb46, Lactobacillus acidophilus La14 150B, L. fermentum and L. plantarum). The results obtained show that the stability of the probiotic cells improves when the cells are further adapted to more stress factors. After the probiotics were exposed to stress factors, the tolerance of these probiotics towards acid and bile was investigated. These are the stress factors the probiotics encounter through their GIT following their consumption. The acid and bile tolerances of the stress exposed cells were higher than those of the cells that were not exposed to stress factors. After sequential exposure of the cells to the simulated gastric and intestinal conditions, viability of the three Lactobacilli cells and B. bifidum LMG 11041 were higher than their non- adapted counterparts. The bile salt hydrolase (BSH) activity and the antibiotic profiles of the probiotics remained unchanged. From these results it was evident that multi- stress pre- adaptation of probiotics increases the chances of survival for these probiotics in the gastrointestinal tract, without negatively affecting their antibiotic sensitivity profile or their ability to produce the enzyme BSH, which is one criterion used for selection of probiotics, specifically those with cholesterol lowering properties. The observed better survival of multi- stress pre- adapted cells when exposed to simulated gastrointestinal conditions raised an interest in another study that was used in the treatment of diseases using probiotics, the use of multiple probiotics. This part of the study aimed to determine first the survival of multiple cells when exposed to acid and bile, and then investigating their ability to inhibit growth of enteric pathogens, specifically Staphylococcus aureus and Escherichia coli, when used individually as single- or multiple- stress adapted cells, combinations of multi-stress adapted cells and comparing them to a combination of the non- adapted cells. A cocktail containing L. plantarum, L. fermentum and B. longum Bb46 and the one containing all the six adapted cells survived better in 2% bile and pH 2, respectively. Interestingly, for both the acid and bile tolerance studies, a cocktail containing all the six non- adapted cells was the least resistant. In the antipathogenic tests, a combination containing L. plantarum, B. longum Bb46 and B. longum 13197 inhibited S. aureus better and combination containing all the six stress adapted cells inhibited E. coli better. It was evident that although the stress adapted single cells inhibited both pathogens, there was an increase in the inhibition when the stress- adapted combinations were used. In all cases the combination containing all the six non- adapted cells was the least effective of all cocktails in inhibition of E. coli and S. aureus. The results of this study revealed that multi- stress pre- adapted probiotics survive better than the single stress adapted cells and above all, the use of non- adapted cells. This was even demonstrated in the use of combinations, where the stress adapted combinations had better results than the non- adapted combinations. This study is of importance to consumers, food industries, pharmaceutical and the probiotic industry as a whole. This study shows the increase in surviving cells after the exposure to stress factors. This information can be used in the production of different products. For an increase in the number of surviving cells, they can use the pre- adaptation technique before the production of any products. The pharmaceutical industry can also apply the mechanisms of using multi- stress pre- adapted cells for their treatment of different diseases. The pre- adaptation of probiotic cells to multiple stress factors will be beneficial to the consumer because they will be getting the adequate number of live cells when they ingest probiotic products.Dissertation (MSc)--University of Pretoria, 2015.tm2015Microbiology and Plant PathologyMScUnrestricte

Bioengineered Lactobacillus casei expressing internalin AB genes for control of Listeria monocytogenes infection

Listeria monocytogenes is one of the common food pathogens implicated in different outbreaks. It has recently (2017-2018) been implicated in the South African listeriosis outbreak, ever reported, where 1060 people were infected resulting in 214 deaths. The ability of listeriosis to cause high case fatality rate (20 to 30%) when compared to most foodborne pathogens makes it an important pathogen and a substantial public health concern. Listeria is an intracellular pathogen that employs different virulence factors to cross the three significant barriers, namely, the intestinal epithelial, the blood-brain endothelial, and the feto-placental endothelial cell barrier, thereby causing listeriosis. As it is the case for most pathogenic infections, antibiotics have been the first line of defence against listeriosis, however, these undesirable effects in the gastrointestinal (GI) infections keep increasing and thus posing major clinical problems. Coupled with that is the increase in the number of bacteria referred to as “superbugs”, those bacteria which have developed resistance against most of the commonly used antibiotics. The rise in these clinical problems, the increase in foodborne infections and the development of antibiotic resistance have led to a need for an alternative solution for these infections. There has been a growing interest in exploring probiotics as an alternative to antibiotics. Probiotics offer beneficial effects to the host and are able to inhibit pathogens through the use of different mechanisms including among others, competing for food and space with foodborne pathogens. They grow rapidly and colonize the gastrointestinal tract (GIT) either permanently or temporarily, consequently alleviate and prevent foodborne infections through mechanisms such as competitive exclusion. However, these probiotics are generic in their action, that is, they are non discriminatory in their action. Furthermore, they are not equally effective in all hosts nor against all pathogens. These limitations inspired the development or design of probiotics strains that will be targeted against specific pathogens. This can be achieved through a systematic understanding of the infection cycle of the pathogens, their virulence factors and disease mechanisms. Virulence genes from food-borne pathogens are cloned and expressed into probiotics through bioengineering in an effort to offer them direct competition for the same receptor sites to which pathogens attach, or for enhanced production of antimicrobial peptides and ultimately inhibition of the specific pathogen. Listeria monocytogenes in its disease progression uses virulence factors such as Listeria adhesion protein (LAP), autolysin amidase (AmiA) for adhesion, while the bacterial surface proteins internalin A (InlA) and internalin B (InlB) are responsible for invasion through the host cells. Cloning and expression of these virulence factors into probiotics will potentially offer the recombinant probiotics an enhanced ability to compete and ultimately inhibit L. monocytogenes. Taking this into consideration, the current study intended to determine whether cloning and expressing the invasion proteins internalins A and B of L. monocytogenes into Lactobacillus casei using the expression vector pLP401-T would alleviate or prevent the Listeria associated damages in vitro. The current study and its findings are organized into the five chapters of this thesis as follows. The first chapter of this thesis (Chapter 1- Literature Review) gives an overview of L. monocytogenes characteristics and pathogenesis, highlighting the virulence genes important for its infection. The various control measures used in clinical environment and food industry, their advantages and disadvantages are discussed. The limitations of these control measures and the need for an alternative measure are justified. Then probiotics as an alternative control for L. monocytogenes are described, taking into consideration their different modes of action. This gives a comprehensive explanation of the limitations of the wild type probiotics, including that they at times fail to inhibit pathogens, which emphasizes the demand for a robust strategy for their improvement. This is followed by discussion of the concept of probiotic engineering as an alternative strategy for improving the efficiency of probiotics for enhanced and targeted control of specific pathogens, explaining some applications where such recombinant strains have been explored. Recombinant probiotics are genetically modified organisms, therefore, due to the ethical reasons surrounding genetically modified organisms, safety concerns regarding recombinant probiotics were briefly addressed. This chapter ends by giving future perspectives regarding the use of recombinant probiotics. In the first experimental chapter (Chapter 2- Construction of recombinant Lactobacillus casei strain expressing the invasion proteins internalins A and B of Listeria monocytogenes), the research followed a stepwise procedure to clone and express the proteins. Firstly, the genomic DNA from L. monocytogenes F4244 (serotype 4b) was extracted and using the specific InlAB primers, the genes was amplified using PCR. The amplification of the InlAB genes was successful, and the genes was subsequently purified for cloning. Using the specific restriction digestion enzymes, the genes and expression vector pLP401- T were digested and ligated using T4 DNA Ligase. Ligation of the two was successful and this was visualized by a band larger than that of the vector alone. The ligated pLP401- InlAB was transformed into L. casei through electroporation. A total of twenty-five transformants were obtained, which were subsequently tested for the presence of InlA, InlB and InlAB genes with their specific primers using PCR. The full length InlA, InlB and the genes InlAB were all amplified confirming their presence in the transformants (recombinant L. casei). The SDS-PAGE and Western blot were used to determine whether the internalins were expressed in the recombinants. The results showed that both InlA and InlB were expressed by the recombinant L. casei but not in its wild- type counterpart. The growth patterns of the wild-type L. casei strains (L. casei WT(LbcWT)), L. casei with the vector without InlAB (LbcV) and L. casei with InlAB (LbcInlAB)) were compared. Interestingly, there was no difference in the growth patterns of all the L. casei strains. The results from this chapter demonstrates that the cloning and expression of the proteins InlAB into the probiotic was successful and that expression of the foreign genes did not have observable negative effects on L. casei growth characteristics as growth curves of all the L. casei strains were comparable. The successful cloning and expression of the invasion proteins InlAB allowed an opportunity to test if there were any differences in the effects that the recombinant L. casei would have on the inhibition of L. monocytogenes in vitro. In the second experimental chapter (Chapter 3- Prevention of Listeria monocytogenes adhesion, invasion and translocation in vitro by the recombinant Lactobacillus casei expressing the internalin AB), the study investigated the ability of L. casei expressing the invasion genes internalin AB (InlAB) (LbcInlAB) to affect L. monocytogenes progression in vitro using the Caco-2 cells grown and maintained in the cell culture medium, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS). This construct was compared with a previously developed L. casei expressing Listeria adhesion protein (LAP) (LbcLAP). To achieve this aim, the ability of the L. casei strains to adhere to, invade and translocate through the Caco-2 cells were first investigated. The results showed a difference in all stages, with the recombinant L. casei showing enhanced activity then the wild- type counterpart. For microorganisms to be deemed a probiotic, they have to be able to competitively exclude pathogens. Taking that into consideration, the ability of all L. casei strains to inhibit L. monocytogenes adhesion using three different mechanisms, namely, CompeThesis (PhD)--University of Pretoria, 2019.Microbiology and Plant PathologyPhDUnrestricte

Probiotic engineering: towards development of robust probiotic strains with enhanced functional properties and for targeted control of enteric pathogens

Abstract 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

Lacticaseibacillus rhamnosus : a suitable candidate for the construction of novel bioengineered probiotic strains for targeted pathogen control

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.The National Research Foundation of South Africa Competitive Programme for Rated Researchers.http://www.mdpi.com/journal/foodsdm2022BiochemistryGeneticsMicrobiology and Plant Patholog

Lactobacillus casei expressing Internalins A and B reduces Listeria monocytogenes interaction with Caco-2 cells in vitro

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 (inlAB) to prevent infection was investigated. The inlAB operon was cloned and surface-expressed on L. casei resulting in a recombinant strain, LbcInl 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 InlAB on the LbcInl AB was confirmed by Western blotting and immunofluorescence staining. The LbcInl AB strain showed significantly higher (P < 0.0001) adherence, invasion and translocation of Caco-2 cells than the wild-type L. casei strain (LbcWT ), as well as reduced L. monocytogenes adhesion, invasion and transcellular passage through the cell monolayer than LbcWT . Furthermore, pre-exposure of Caco-2 cells to LbcInl AB significantly reduced L. monocytogenes-induced cell cytotoxicity and epithelial barrier dysfunction. These results suggest that InlAB-expressing L. casei could be a potential practical approach for prevention of listeriosis.The National Research Foundation, the Department of Agriculture National Institute of Food and Agriculture and the USDA Agricultural Research Service.https://sfamjournals.onlinelibrary.wiley.com/journal/17517915pm2020BiochemistryGeneticsMicrobiology and Plant Patholog