Background
Poor hygiene and sanitation during food preparation can lead to the presence of different foodborne pathogens in food. Some of these pathogens or their toxins produced either before or after ingestion of such foods can either act locally within the gastrointestinal tract (GIT), leading to development of illnesses, or disseminate to other parts of the body and damage cells/tissues and ultimately the immune system [
1]. Incidences of foodborne illnesses are high in most developing countries as food control is a low priority issue due to limited funds. As a result of this, food pathogens are the leading cause of illnesses and death in these countries [
2]. Most foodborne illnesses cause diarrhoea, which is the primary symptom. Most societies consider diarrhoea a normal, natural condition; therefore, it goes unnoticed and/or untreated. Recently, the World Health Organization reported that of the 600 million global total cases of foodborne illness recorded in 2010, 550 million were due to infectious agents causing diarrhoea, of which 120 million and 96 million cases were caused by norovirus and
Campylobacter spp., respectively. Diarrhoeal disease agents were responsible for approximately 55% (230,000 out of 420,000) deaths, with 59,000, 37,000, 35,000 and 26,000 deaths attributed to non-typhoidal
Salmonella enterica, enteropathogenic
E. coli (EPEC) and enterotoxigenic
E. coli (ETEC), respectively [
3]. These illnesses are not confined to developing countries. In the United States, foodborne pathogens cause an estimated 9.4 million illnesses, 55,961 hospitalizations and 1351 deaths each year [
4]. Enteric pathogens account for high morbidity and mortality and are considered to be the fifth leading cause of death at all ages worldwide [
5].
Probiotics have been used to restore the balance of the gut microbial ecosystem and control pathogenic infections. They are defined as “
live microorganisms that when administered in adequate amounts confer a health benefit on the host” [
6]. Their administration assists in the prevention and control of foodborne illnesses, through a number of mechanisms including but not limited to, competitive exclusion of pathogens in the GIT, modulation of the host immune system and strengthening of the intestinal barrier [
7‐
9]. Although probiotics have proven successful in the control of enteric pathogens, they do have limitations. They are generic in nature and often fail to inhibit the attachment of certain pathogens at specific sites of infection and induce low levels of an immune response [
10]. A thorough understanding of the limitations of conventional probiotics, the behaviour of the pathogens and the mechanisms by which they cause disease [
11] provides possibilities to design new probiotic strains with desired characteristics and functionalities. Through genetic modification, novel bioengineered probiotic strains can be produced. Functioning of conventional probiotics in these novel strains can be strengthened to influence critical steps in pathogenesis. The strains can also be used to deliver drugs or vaccines, target a specific pathogen or toxin, mimic surface receptors and enhance an immune response within the host [
12].
The use of conventional probiotics for control of selected food pathogens
Due to the widespread use of antibiotics as therapeutic agents and the misuse of these antibiotics, there has been an increase in the antibiotic resistance of bacteria, an imbalance of normal microflora and the presence of drug residues in food products [
41]. This brought about a requirement for new intervention in the treatment of bacterial pathogens, leading to an escalation in the research field of the beneficial microorganisms, i.e. probiotics. Prevention and treatment of infections caused by the different pathogens is one of the reasons why probiotics extensively studied [
62]. When studying the prevention and treatment of pathogens, it is important to consider the complexity of the intestinal environment where a network of interactions among the microorganisms of the resident microbiota, epithelial and immune cells associated with the GIT, and nutrients exist [
63,
64]. The epithelial and the immune cells play a role in the modulation of the immune functions and they provide the first line of defense against the pathogenic bacteria. The resident microbiota have the ability to influence the composition and activity of the gut microbiota [
62]. They also play a beneficial role in the treatment of disease caused by foodborne pathogens [
65,
66]. Different microorganisms infect different parts of the host GIT, for example,
H. pylori, infects the gastric and duodenal mucosa,
Salmonella spp. and
Clostridium difficile cause inflammation in ileum and colon, while
Shigella sp. clearly prefers the colonic mucosa [
67].
Previous studies have shown the effects of probiotics, that when consumed as part of the daily diet, they can maintain the immune system in an active state and prevent different intestinal disorders [
62]. Valdez et al. [
68] reported that certain LAB probiotics inhibit apoptosis of macrophage infected with
Salmonella preventing salmonellosis. Cano and Perdigón [
69] studied the preventative measure of
L. casei CRL 431 against
S. serovar Typhimurium, reporting that administrating probiotics prevented
S. serovar Typhimurium infection (100% protection) after 14 days of the re-nutrition diet in mouse models. Findings of their study were confirmed by a different study [
62], where the preventative and continuous administration of probiotic
L. casei CRL 431 against
S. serovar Typhimurium in a mouse model was studied. They reported that the study group fed the probiotic for 7 days before the introduction of the pathogen and post infection experienced less severe infection compared to the control group which did not consume probiotics. They furthermore reported that 7-day administration of probiotics post infection resulted in better protection against
Salmonella infection. They concluded that the continuous administration of the probiotic diminished counts of the pathogens in the intestine as well as their spread outside this organ.
More studies have been conducted on different pathogens to show the efficacy of probiotic strains.
H. pylori is a bacterium that plays a crucial role in the pathogenesis of chronic active gastritis and peptic ulcer disease in both adults and children [
70] with increasing amount of evidence supporting the hypothesis that it is an important co-factor in the development of gastric cancer [
71].
H. pylori has been linked to cancer; however, there is no vaccine licensed to prevent infection with this organism [
72]. There are different therapeutic approaches that are used to treat
H. pylori, including but not limited to the commonly used triple therapy with proton pump inhibitor (PPI), clarithromycin and either amoxicillin or metronidazole or dual-therapy high-dosage amoxicillin and PPI; however, there have been reports that suggest that some patients still remain infected after administration of these treatment [
73]. Administration of alternative compounds that may increase the efficacy of the treatment and/or reduce side-effects is of particular interest [
72]. There is growing evidence from different studies emphasizing the efficacy of probiotics in the management of
H. pylori infection targeting different aspects of this infectious disease [
74,
75]. Cats et al. [
74] investigated whether readily available commercial preparation containing
L. casei inhibits the growth of
H. pylori in vitro. They reported that in vitro
L. casei inhibits the growth of
H. pylori; however, the probiotic cells have to be viable. In a different study, Bernet-Camard et al. [
76] reported that probiotics such as
L. johnsonii La1 (La1) or
L. rhamnosus GG exert bacteriostatic or bactericidal activities against a wide range of pathogens, including
H. pylori. Cruchet et al. [
77] studied if the regular ingestion of a dietary product containing
L. johnsonii La1 or
L. paracasei ST11 would interfere with
H. pylori colonization in children. They concluded that regular ingestion of the dietary product containing
L. johnsonii La1 may represent an interesting alternative to modulate
H. pylori colonization in children infected by this pathogen. Tursi et al. [
78] demonstrated that a 10-day quadruple anti-helicobacter therapy with ranitidine bismuth citrate (RBC) plus proton pump inhibitors (PPI), amoxicillin and tinidazole obtains a high eradication rate, whereas supplementation with
L. casei significantly increased the eradication rate of
H. pylori infection. This study concluded that the supplementation of the therapy with the administration of probiotics showed a slight improvement in the eradication of
H. pylori. Probiotics can therefore be used as first course of anti-
H. pylori treatment or can be used in conjugation with the first-line therapeutic approaches.
Shigella is an antibiotic-resistant bacterium [
79,
80] that has been reported to cause gastroenteritis-induced deaths in 3–5 million children aged less than 5 years in developing countries [
81,
82]. The emergence of multiple drug resistance to cost-effective antimicrobials against
Shigella is a matter of concern in developing countries, and resistance pattern of this bacterium is the cause of numerous clinical problems worldwide [
83]. Due to increased prevalence of its antibiotic resistance, the need for alternative treatment has therefore been deemed necessary. Zhang et al. [
84] studied the antimicrobial activity of the probiotics
L. paracasei subsp.
paracasei M5-L,
L. rhamnosus J10-L,
L. casei Q8-L and
L. rhamnosus GG (LGG) against
Shigella sonnei. They reported that the tested lactobacilii strains showed strong antimicrobial activity against
S. sonnei. In a study to screen for the antimicrobial activity of probiotics against
S. sonnei, Zhang et al. [
85] reported that
L. johnsonii F0421 exhibited significant inhibitory activity and excluded, competed and displaced
S. sonnei adhered to HT-29 cells. In a different study, Mirnejad et al. [
83] evaluated the nature of antimicrobial substances and properties of
L. casei against multi-drug-resistant clinical isolates of
S. flexneri and
S. sonnei. Their results indicated that
L. casei showed strong antimicrobial activity against
S. flexneri and
S. sonnei, and they attributed pathogen inhibition to production of organic acids by the test
Lactobacillus. In another study, Zou et al. [
86] studied the antimicrobial activity of nisin, a bacteriocin produced by
L. lactis strains, against
L. monocytogenes,
Staphylococcus aureus,
Salmonella typhimurium and
Shigella boydii. They reported that there was a decline in pathogen populations, which was ascribed to the changes in the fatty acid profiles, cell viability, membrane permeability and depolarization activity in response to nisin.
Listeria monocytogenes is a foodborne pathogen that causes devastating effects in the human host, causing disease conditions ranging from premature delivery and stillbirth in perinatal cases [
87] to meningitis and septicemia in adults [
88,
89]. There have been many studies using different probiotics to combat this food pathogen. In a study to demonstrate the activity of the antibacterial substances produced by bifidobacterial isolates, Touré et al. [
90] isolated six infant bifidobacterial strains from breast-fed infant faeces, with a potential antimicrobial activity against
L. monocytogenes. These isolates actively inhibited
L. monocytogenes by producing a heat-stable proteinaceous substance. Their study indicated that the use of bifidobacterial strains capable of competing with pathogenic organisms following the probiotic approach would advantageously improve intestinal bacterial ecology and provides a useful alternative strategy for inhibiting intestinal pathogens. In 2007, Corr et al. [
91] studied the pretreatment of C2Bbe1 cells, a clone of the Caco-2 human adenocarcinoma cell line with strains of
Bifidobacterium and
Lactobacillus to demonstrate that this can significantly interfere with subsequent invasion by
L. monocytogenes. They reported that the pretreatment of intestinal epithelial cells with probiotic bacteria prior to infection with
L. monocytogenes EGDe resulted in a significant decrease in listerial invasion (60–90%). In yet another study testing for the antagonistic effect of
Lactobacillus strains against
E. coli and
L. monocytogenes, it was reported that
L. plantarum WS4174 exhibited a stronger inhibitory effect against the Gram-positive
L. monocytogenes LMO26, possibly due to the accumulation of lactic acid and higher sensitivity of
L. monocytogenes to low pH [
92].
Limitations of conventional probiotics
Although probiotics provide numerous benefits to the host, they do have certain limitations. Certain studies have provided evidence that probiotic strains may be inefficient or ineffective in response to specific gut pathogens. Probiotics may release antimicrobial compounds that have a broad antimicrobial spectrum; however, reports have suggested that there are limitations in the success of probiotics targeting specific pathogens. Therefore, a cocktail of various probiotic strains would need to be produced in order to enhance the effects against different pathogens within the gut [
93].
Contrary to earlier reports that probiotics exhibited inhibitory effect against
L.
monocytogenes [
90,
91], according to Koo et al. [
94], probiotics have a limited success in preventing the attachment of
L. monocytogenes to intestinal monolayers. In their study, which used three experimental approaches of competitive exclusion, inhibition of adhesion or displacement, to determine whether selected lactobacilli would reduce adhesion of
L.
monocytogenes to Caco-2 cells, they showed that the percentages of
L.
monocytogenes adhesion in the presence and absence of probiotics were fairly similar. None of the lactobacilli and other LAB were able to significantly reduce adhesion or colonization on epithelial cells, even at higher numbers. Furthermore, an increase in the concentration of the probiotic strain also failed to displace the attached
L. monocytogenes. The data from the study indicated the conventional LAB strains could not prevent adhesion of this pathogen.
Another report indicated that probiotics may also stimulate low levels of an immune response and low levels of an anti-inflammatory response [
10].
L. salivarius and
B. infantis were orally administered to mice suffering from colitis. Results indicated that TGF-β levels in mice treated and untreated with probiotics remained the same. TGF-β is an anti-inflammatory cytokine, and the levels of this cytokine were not significantly increased but still maintained by
L. salivarius; however, these were not maintained in the presence of
B. infantis.
Most probiotics are administered as food or capsules; therefore, they have to be able to withstand both the technological and gastrointestinal stress factors. The broad mode of action of probiotics and the differences from one probiotic to another is also an obstacle in their efficacy. It has been reported that the beneficial attributes of one strain or a cocktail of strains may not be reproducible and may vary from person to person [
95]. In addition to that, the strain of the probiotic, the dosage, the route of administration, and the formulation of probiotic preparation can also affect their efficacy [
94]. Taking these studies into consideration, it is evident that probiotics are still non-specific and non-discriminatory in their mode of action or ineffective in certain hosts [
96].
The limitations discussed above introduce the need for more novel and innovative approaches in the use of probiotics for the prevention and treatment of foodborne pathogens. Previous literature has reported that the use of probiotics has been extended to deliver therapeutic and prophylactic molecules to the mucosal barrier of the host [
94,
97,
98]. However, for that to be done successfully, a thorough understanding of the behaviour of the pathogens and their disease mechanisms is needed [
11]. Such knowledge can then be used to increase the efficacy of the probiotics and later use of a specific probiotic for a specific pathogen or toxin. Thus, novel probiotic strains with enhanced or even targeted probiotic functioning can be produced. Bioengineering techniques offers an opportunity for the design of such recombinant probiotic strains.
Safety concerns regarding bioengineered probiotics
Bioengineered probiotics are increasingly being studied as vehicles that can express and target delivery of specific genes directed towards a specific foodborne pathogen. One of the main drawbacks of working with bioengineered probiotics is that they are classified as genetically modified organisms (GMO) [
144]. The nature of such probiotics regarded as GMO presents a major limitation to their widely applications. It is well known that some consumers have ethical reasons for not consuming GMO for fear that such organisms may pose danger to one’s life [
145]. Other concerns about GMO relate to their release into the environment and their survival and propagation in this environment, dissemination of antibiotic selection markers or other genetic material to other organisms [
146]. Introduction of the GMO into the environment can impact there directly by competing with natural species, or indirectly by changing the balance between native species [
147]. However, these modified microorganisms have a great potential to address novel approaches for prevention and treatment of different human and animal pathological conditions. It is therefore, important to establish criteria that can be used for the assessment of the environmental safety and tracing the fate of recombinant DNA in vitro and in vivo, which are both of significant importance [
148]. Hence, safety of these strains needs to be guaranteed in order for them not to possess antibiotic selection markers or to transfer genetically modified DNA to other bacteria [
144]. Biological containment systems can be used to prevent dissemination of genetic material to other bacteria and to prevent a significant uncontrolled increase of probiotic cells into the natural environment [
145]. The organism is genetically programmed to only grow in the laboratory and to die in the natural environment [
149]. The use of the thymidine-deficient strains is one of the promising strategies for biological containment of bioengineered probiotics. In these strains, the gene of interest is cloned into the chromosomal thymidylate synthase gene (
thyA), which codes for production of thymine essential for growth of
L. lactis. This disruption of the
thyA gene makes the recombinant strain dependent on external supplementation of thymidine or thymine in the growth medium for growth and survival. Thymine is absent in the environment or its levels are limiting in vivo, and this ensures that the recombinant strain dies rapidly due to the absence of an essential growth component. In addition, chromosomal location of the introduced gene provides stability and reduces the risk of horizontal gene transfer [
146,
150].
When cloning and expressing the different virulent traits into probiotics, only traits that will not make the probiotics pathogenic should be used. It is also crucial that each bioengineered strain be carefully evaluated for virulence determinants and sensitivity to clinically relevant antibiotics before being deemed suitable as a probiotic [
151]. When cloning probiotics, therapeutic safety of recombinant probiotic carrier organisms is crucial, especially when the strain has to be used in individuals who are already infected with a pathogen. The risk exposure determination, risk assessment and safety assessment are essential to ensure protection for the population against any unintended consequences of the use of probiotics [
152].
Conclusions and future perspective
The rise in morbidity and mortality due to foodborne pathogens remains a serious concern worldwide and the need for an alternative strategy for the control and treatment of infections caused by pathogens is equally crucial. The application of probiotics in food for control of enteric pathogens has been explored and the probiotic market is growing worldwide. The ability of probiotics to inhibit human enteric pathogen has been well researched and documented and this has led to their use as a therapeutic approach for treatment of enteric infections. These studies showed both their successes and limitations, mainly highlighting the generic nature of their mode of action and their failure in controlling some specific pathogens. These limitations can be overcome and functions of conventional probiotics enhanced to create a greater beneficial effect through the use of bioengineering. The modification of conventional probiotics by use of bioengineering technology has a significant potential for design and development of novel therapeutic approaches for effective treatment of pathogens.
Thorough understanding the life cycle of pathogens post ingestion, and knowledge of the virulence factors they use to cause infections offers a strategy for development of bioengineered probiotics strains tailored to control-targeted pathogens. By targeting a specific pathogen, the efficacy of the probiotics inhibiting both the pathogens and infection will be increased. Although still in the early stages, researchers have made impressive strides towards design of such probiotics, producing strains geared towards enhancement of various functional and/or technological probiotic properties. Results from most of such studies showed positive effects although in some cases no benefits were reported. The bioengineered probiotics thus offer important potential to be used as novel therapeutic approach for the prevention and treatment of foodborne infections. More studies targeting different virulence genes and pathogens, including the less studied and emerging ones, are necessary in order to establish the future of this field of research and determine how it will impact on the food and health industries.
In addition to the above, most bioengineered probiotics are designed to be orally administered; therefore, they must still be able to survive through both technological and gastrointestinal stresses. It is also crucial that these strains have scientifically validated health properties, demonstrated safety and good technological properties to be produced on a large scale [
147]. They should remain viable in large numbers so as to confer the beneficial effects to the host and should not develop unpleasant flavours or textures upon their incorporation into foods [
148]. Furthermore, studies on bioengineered probiotics, specifically for targeted control of pathogens, have focused on the impact of the recombinant probiotic strain on the pathogen(s) of interest. The influence of administration of these probiotics on commensal bacteria or the whole microbiota has not been the subject of studies. These aspects should also be addressed in future studies on bioengineered probiotics.