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Mathipa and Thantsha Gut Pathog (2017) 9:28
DOI 10.1186/s13099-017-0178-9
REVIEW
Probiotic engineering:
towardsdevelopment ofrobust probiotic
strains withenhanced functional properties
andfor targeted control ofenteric pathogens
Moloko Gloria Mathipa and Mapitsi Silvester Thantsha*
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 develop-
ment 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 inhibi-
tion 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.
Keywords: Foodborne pathogens, Antibiotic resistance, Probiotics, Bioengineering
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
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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), lead-
ing 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., respec-
tively. 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), respec-
tively [3]. ese illnesses are not confined to developing
countries. In the United States, foodborne pathogens
cause an estimated 9.4 million illnesses, 55,961 hospitali-
zations and 1351 deaths each year [4]. Enteric pathogens
account for high morbidity and mortality and are con-
sidered to be the fifth leading cause of death at all ages
worldwide [5].
Open Access
Gut Pathogens
*Correspondence: mapitsi.thantsha@up.ac.za
Department of Microbiology and Plant Pathology, University of Pretoria,
New Agricultural Sciences Building, Pretoria 0002, South Africa
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Mathipa and Thantsha Gut Pathog (2017) 9:28
Probiotics have been used to restore the balance of the
gut microbial ecosystem and control pathogenic infec-
tions. ey are defined as “live microorganisms that when
administered in adequate amounts confer a health ben-
efit on the host” [6]. eir 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, modu-
lation 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. ey 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 behav-
iour of the pathogens and the mechanisms by which they
cause disease [11] provides possibilities to design new
probiotic strains with desired characteristics and func-
tionalities. rough genetic modification, novel bioen-
gineered probiotic strains can be produced. Functioning
of conventional probiotics in these novel strains can be
strengthened to influence critical steps in pathogenesis.
e strains can also be used to deliver drugs or vaccines,
target a specific pathogen or toxin, mimic surface recep-
tors and enhance an immune response within the host
[12].
Probiotics
Probiotics include mainly bacteria from the genera Strep-
tococcus, Enterococcus, Pediococci, Weissella and Lacto-
coccus [13] but the most common ones used belong to
Lactobacillus and Bifidobacteria spp. ese bacteria have
met the criteria necessary to consider them as probiot-
ics and they also have nutritional and therapeutic effects
[14]. One of the criteria that bacteria must meet in order
for them to be regarded as probiotics is that they have to
be able to survive and thrive throughout the GIT condi-
tions and confer their beneficial effects. It is therefore
important to understand their mechanisms of action in
order for them to be used both prophylactic and as treat-
ment options for the different foodborne diseases. e
presence of foodborne pathogens in the human GIT
affects the balance of the “good to bad” microorganisms.
Apart from the presence of the pathogens in the GIT,
there are other factors that can affect the balance of the
microorganisms in the host GIT. ese factors include
stress, illness or antibiotic treatment, which changes the
balance in the GIT in favour of harmful bacteria [15,
16]. One of the characteristics of probiotics is that they
are able to protect the host from microbial imbalance.
Table 1 gives a summarized overview of the different
mechanisms by which probiotics exclude pathogens
from the human GIT, which are discussed in more detail
below.
Probiotics’ mechanisms ofaction againstenteric
pathogens
Competitive exclusion
Probiotics can exclude or reduce the growth of other
microorganisms in the GIT either through competition
for nutrients or adherence space [17–19]. Microorgan-
isms in any environment require nutrients to multiply
and either cause or alleviate infections. e GIT is well
known for its abundance in nutrients, making it a suita-
ble environment for microbial colonization. e potential
of probiotics to outcompete pathogens for these nutri-
ents favours their growth over that of the pathogens [20].
During competition for nutrients, probiotics produce
metabolites such as volatile fatty acids reducing the pH of
the GIT. e reduction in the pH of the GIT makes it an
unfriendly environment for pathogens and will thus lead
to their inhibition because most of them cannot grow at
low pH [21, 22].
Competition for adherence space refers to the situation
when the presence of probiotics blocks pathogenic bac-
teria from colonizing favourite sites such as the intestinal
villi, goblet cells and the colonic crypts [22]. Attachment
to the surfaces of intestinal epithelial cells is a key patho-
genic factor of enteric pathogens [23]. At the same time,
colonization resistance, through which attachment and
multiplication of the pathogens on the intestinal mucosal
membrane is prohibited, is a critical function of the
microbiota [24, 25]. Probiotics bind to intestinal cells via
electrostatic interactions, steric forces or specific surface
proteins. is affords them the ability to bind to these
cells in high quantities [17, 26], thereby physically block-
ing the sites, leaving no space for the pathogens to adhere
and subsequently cause infection [27].
Probiotic LAB have a greater ability to adhere to the
epithelial cells than pathogens [28]. Lactobacilli and bifi-
dobacteria share carbohydrate-binding specificities with
some enteropathogens [29]. Bifidobacterium bifidum and
Lactobacillus reuteri bind to glycolipids on the surface of
the host cells to prevent attachment of certain pathogens
that also bind to specific surface glycolipids [8]. irabu-
nyanon and ongwittaya [30] reported in their study
that they observed a reduction in attachment of Salmo-
nella enteritidis to the surfaces of intestinal epithelial
cells in the presence of probiotic Bacillus subtilis NC11.
is led to a complete exclusion of this pathogen in the
GIT, which is the site where the infection process is ini-
tiated. is was corroborated with poor survival of the
pathogen attributed to limited nutrients for their growth
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Mathipa and Thantsha Gut Pathog (2017) 9:28
Table 1 Mechanisms ofaction ofprobiotics
Mechanism ofaction Probiotic bacteria Pathogen Functionality References
Competitive exclusion L. acidophilus; L. johnsonii S. enterica serovar Typhimurium, Enteropatho-
genic E. coli (EPEC), Yersinia pseudotuberculosis,
L. monocytogenes
Inhibited adherence of pathogens to Caco-2
cells [33]
L. casei subsp. rhamnosus EPEC, Enterotoxigenic E. coli (ETEC), Klebsiella
pneumoniae
Inhibited adherence of pathogens to Caco-2
cells [33]
L. rhamnosus; L. acidophilus E. coli O157:H7 Inhibited adherence of pathogens to T-84 epi-
thelial cell; inhibited colonization to Caco-2
cells
[33, 51]
L. acidophilus H. pylori Production of lacticins A164 and BH5 [37]
Production of inhibitory substances L. lactis E. coli, Salmonella Production of alyteserin-1a and A3APO [31]
B. longum Clostridium difficile, E. coli Production of bacteriocin [35]
L. salivarius L. monocytogenes Production of bacteriocin Abp118 [35]
L. sake L. monocytogenes Production of bacteriocin sakacin A [39]
L. plantarum L. monocytogenes Production of bacteriocins (plantaricins) [40]
L. lactis C. difficile Production of lacticin 3147 [45]
L. lactis, L. casei, L. acidophilus E. coli O157:H7 Reduced the growth of pathogen by lactic acid
production and pH reductive effect [33]
Immune system modulation L. reuteri Salmonella Increased the production anti-Salmonella IgM [35]
L. rhamnosus E. coli O157:H7 Increased intestinal anti-E. coli IgA responses
and blood leukocyte phagocytic activity [33]
L. acidophilus, L. rhamnosus GG, L. johnsonii La1 S. Typhimurium Increased the production of anti-Salmonella IgA [42, 43]
Improved barrier function L. plantarum, L. rhamnosus GG E. coli O157:H7 Subvert the adherence of pathogen by increas-
ing MUC2 and MUC3 in HT-29 epithelial cell
line
[45, 47]
L. acidophilus E. coli Protected against F-actin rearrangement, which
was induced in an epithelial cell line on expo-
sure to a pathogenic E. coli
[45]
S. thermophilus; L. acidophilus Enteroinvasive E. coli (EIEC) Increased transepithelial resistance, mainte-
nance and enhancement of cytoskeletal and
tight junctional protein phosphorylation in
HT29 and Caco-2 cell lines
[60]
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Mathipa and Thantsha Gut Pathog (2017) 9:28
and proliferation, and unavailability of adherent space in
the GIT.
Production ofinhibitory substances
In order to gain a competitive advantage when compet-
ing for space and nutrients, microorganisms release anti-
microbial compounds. Antimicrobial compounds have
a direct inhibition on several target pathogens [31]. e
mechanisms used by probiotics to inhibit pathogenic
bacteria are interconnected. As already mentioned,
exclusion of pathogens occurs due to the ability of pro-
biotics to secrete organic acids such as acetic and lactic
acids [32]. e production of these organic acids leads
to a decrease in the pH of the environment, making the
microenvironment acidic thereby excluding pathogens
that cannot survive acidic conditions [8]. e organic
acids also have an effect on the pathogen metabolism and
production of toxins, ultimately preventing disease.
e anti-pathogenic activity of probiotics is multifacto-
rial [33]. In addition to the acids mentioned above, pro-
biotics can produce other metabolites with antibacterial
properties, such as H2O2 and bacteriocins, also referred
to as non-lactic acid molecules [33–36, 41]. Bacteriocins
are small antimicrobial peptides produced for bacterial
competition in a natural ecosystem [31]. ey may act
as colonizing peptides by facilitating the introduction of
probiotics into an already occupied niche on the intesti-
nal epithelium. is competitive advantage allows for an
increase in the density of probiotic bacteria on the sur-
face of the host intestines [36]. ey can also act as kill-
ing peptides, by directly affecting pathogens. A study by
Kim etal. [37] evaluated the antimicrobial activity of the
bacteriocins: lacticin, pediocin and leucocin, produced
by lactic acid bacteria against Helicobacter pylori. ese
bacteriocins were able to significantly inhibit the growth
of H. pylori, with lacticin having the most inhibitory
effect against this gut pathogen.
Lactobacillus acidophilus has been reported to produce
metabolites such as acidophilin, lactocidin and acidolin
[35], whereas bifidobacteria produces bacteriocin-like
substances [38], all inhibiting bacteria such as Bacil-
lus, Salmonella, Staphylococcus and E. coli, Clostridium
perfringens, Listeria species, among others [35, 39, 40].
Fayol-Messaoudi etal. [41] reported that the antibacte-
rial effects of the probiotic Lactobacillus that inhibited
the growth and resulted in pathogen death were due
to the synergistic action of lactic acid and the secreted
non-lactic acid molecules. Certain probiotic strains can
also stimulate the increase in the expression of host cell
antimicrobial peptides. e intestinal cells of the host
are able to produce defensins which can inhibit the func-
tioning of pathogens thus aiding in the protection of the
intestinal barrier [36].
Immune system modulation
Probiotics can displace pathogens through stimulation
of host immunity [42]. ere is considerable evidence to
support the notion that probiotics displace pathogens
in the GIT through stimulation of specific and non-
specific immunity to inhibit bacteria causing intestinal
diseases [43, 44]. ey modulate the host’s immune
system against the pathogens harmful antigens by the
activation of lymphocytes and production of antibod-
ies [45]. ey can also stimulate the effects of different
cells involved in innate and adaptive immunity, such as
dendritic cells, macrophages, T cells and B cells, which
enhances phagocytosis of gut pathogens [46]. Probiotic
strains such as Lactobacillus rhamnosus and Lactoba-
cillus plantarum adhere to gut-associated lymphoid
tissue enhancing both systemic and mucosal immunity
[9]. ese probiotics strains enhance immunity by up-
regulating production of intestinal mucins (MUC2 and
MUC3), which disrupts the adherence of pathogens
to the intestinal epithelium, consequently prevent-
ing pathogen translocation. Furthermore, they induce
expression of TGFβ and interleukins (IL-10 and IL-6) by
epithelial cells, which enhances production and secre-
tion of IgA [47].
Probiotics can be recognized by the immune system
through pattern recognition molecules such as Toll-like
receptors. is recognition can lead to various intracel-
lular signal transduction cascades and enhancement or
reduction of pro and anti-inflammatory cytokines. ere
are different studies supporting this evidence. In 2000,
Fang etal. [43] divided 30 healthy volunteers into three
different treatment groups, with each group consum-
ing Lactobacillus GG, Lactococcus lactis and placebo
(ethyl cellulose), respectively, for 7days. All the treat-
ment groups were given an attenuated Salmonella typhi
Ty21a oral vaccine. e results showed that there was an
increase in the humoral immune response in the treat-
ment group receiving the probiotics as compared to the
control group. Probiotics are able to stimulate the pro-
duction of antibodies in the intestinal lumen, specifically
immunoglobulin A. Immunoglobulin A (IgA) represents
the first-line defense against infection and can inhibit the
adhesion of pathogenic bacteria to the intestinal epithe-
lia. It can interfere with adhesive cell receptors on the
pathogen’s cell surface and cause bacterial agglutination.
One study indicated that oral administration of Lactoba-
cillus casei enhanced the concentration of IgA in infants
suffering from diarrhoea, thereby shortening the dura-
tion of this symptom [46, 48]. Ng etal. [45] reported that
administration of L. rhamnosus resulted in enhanced
non-specific humoral responses reflected by an increase
in the levels of circulating IgG, IgA and IgM in children
with acute gastroenteritis.
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Mathipa and Thantsha Gut Pathog (2017) 9:28
In addition to the above, probiotics can stimulate an
anti-inflammatory response, which can be used as an
approach to reduce inflammation caused by gastroenteri-
tis, enterocolitis and irritable bowel syndrome [9]. An anti-
inflammatory response is triggered when strains stimulate
the activation of dendritic cells which secrete interleukin
10 (IL-10), a cytokine that plays a role in reducing inflam-
mation. ey also cause a decrease in the levels of proin-
flammatory cytokines during inflammation [46].
Improved barrier function
e integrity of the intestinal barrier needs to be main-
tained in order to prevent pathogens from reaching the
intestinal cells, thereby leading to local and systemic infec-
tions. Gut pathogens have the ability to disrupt the barrier
when there is an imbalance in the microbial gut ecosystem
[49]. It has previously been reported that consumption of
probiotics can maintain the barrier function and mucosal
integrity, prevent chronic inflammation, thereby protect-
ing the host against infections [50]. Probiotics decrease
paracellular permeability, providing innate defense against
pathogens and enhancing the physical impediment of the
mucous layer [51]. ey are also able to repair this barrier
after damage that may have been caused by gut pathogens.
As an approach to repair the intestinal barrier, probiotics
can stimulate mucous secretion, chloride and water secre-
tion and the binding together of submucosa cells by tight
junctional proteins [8].
Goblet cells express rod-shaped mucins (MUCs), which
are either localized to the cell membrane or secreted into
the lumen to form the mucous layer [52, 53]. ere are 18
mucin-type glycoproteins that are expressed by humans
[49]. In the human intestinal cell lines, Lactobacillus spe-
cies increased mucin expression (MUC2 by Caco-2 cells;
MUC2 and MUC3 by HT29), thus blocking cellular adhe-
sion and invasion by pathogenic E. coli [54, 55]. Madsen
etal. [56] showed that the treatment of IL-10 gene-defi-
cient mice with a combination probiotic VSL#3 (L. casei,
L. plantarum, L. acidophilus, L. delbrueckii subsp. bulga-
ricus, Bifidobacterium longum, B. breve, B. infantis, and
Streptococcus salivarius subsp. thermophilus), resulted in
normalization of colonic physiologic function and barrier
integrity leading to significant improvement in histologic
disease [57].
Tight junctions (TJ) form the continuous intercellu-
lar barrier between epithelial cells, which is required to
separate tissue spaces and regulate selective movement
of solutes across the epithelium [58]. ere are different
proteins expressed on the TJ and the disruption of their
expression leads to a dysfunctional epithelial barrier [57].
A study by Qin et al. [59] reported that L. acidophilus
increases the expression of occludin, a major component
of TJ, in the gut mucosa of animals with cecal ligation
and perforation, leading to a reduced bacterial transloca-
tion. A different study by Resta-Lenert and Barrett [60]
reported that probiotic bacteria, specifically S. thermophi-
lus and L. acidophilus, prevented reduction in the entero-
invasive E. coli-induced phosphorylation of the proteins
occludin and zonula occludens 1 (ZO-1), thereby pre-
serving the TJ structure. Furthermore, Parassol etal. [61]
showed that L. casei prevents the redistribution of the TJ
protein ZO-1 away from the cell–cell contacts caused by
infection with enteropathogenic E. coli.
The use ofconventional probiotics forcontrol
ofselected food pathogens
Due to the widespread use of antibiotics as therapeu-
tic 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]. is 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. pro-
biotics. Prevention and treatment of infections caused
by the different pathogens is one of the reasons why pro-
biotics extensively studied [62]. When studying the pre-
vention and treatment of pathogens, it is important to
consider the complexity of the intestinal environment
where a network of interactions among the microorgan-
isms of the resident microbiota, epithelial and immune
cells associated with the GIT, and nutrients exist [63,
64]. e epithelial and the immune cells play a role in
the modulation of the immune functions and they pro-
vide the first line of defense against the pathogenic bacte-
ria. e resident microbiota have the ability to influence
the composition and activity of the gut microbiota [62].
ey also play a beneficial role in the treatment of dis-
ease 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 pre-
vent different intestinal disorders [62]. Valdez etal. [68]
reported that certain LAB probiotics inhibit apoptosis of
macrophage infected with Salmonella preventing salmo-
nellosis. Cano and Perdigón [69] studied the preventative
measure of L. casei CRL 431 against S. serovar Typhimu-
rium, reporting that administrating probiotics prevented
S. serovar Typhimurium infection (100% protection) after
14days of the re-nutrition diet in mouse models. Find-
ings of their study were confirmed by a different study
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Mathipa and Thantsha Gut Pathog (2017) 9:28
[62], where the preventative and continuous adminis-
tration of probiotic L. casei CRL 431 against S. sero-
var Typhimurium in a mouse model was studied. ey
reported that the study group fed the probiotic for 7days
before the introduction of the pathogen and post infec-
tion experienced less severe infection compared to the
control group which did not consume probiotics. ey
furthermore reported that 7-day administration of probi-
otics post infection resulted in better protection against
Salmonella infection. ey concluded that the continu-
ous administration of the probiotic diminished counts of
the pathogens in the intestine as well as their spread out-
side this organ.
More studies have been conducted on different patho-
gens to show the efficacy of probiotic strains. H. pylori
is a bacterium that plays a crucial role in the pathogen-
esis 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 impor-
tant 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]. ere are different therapeutic approaches that are
used to treat H. pylori, including but not limited to the
commonly used triple therapy with proton pump inhibi-
tor (PPI), clarithromycin and either amoxicillin or met-
ronidazole 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 treat-
ment and/or reduce side-effects is of particular interest
[72]. ere is growing evidence from different studies
emphasizing the efficacy of probiotics in the manage-
ment of H. pylori infection targeting different aspects
of this infectious disease [74, 75]. Cats etal. [74] inves-
tigated whether readily available commercial prepara-
tion containing L. casei inhibits the growth of H. pylori
invitro. ey reported that invitro L. casei inhibits the
growth of H. pylori; however, the probiotic cells have to
be viable. In a different study, Bernet-Camard etal. [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 etal. [77] studied if the regular ingestion
of a dietary product containing L. johnsonii La1 or L. par-
acasei ST11 would interfere with H. pylori colonization
in children. ey concluded that regular ingestion of the
dietary product containing L. johnsonii La1 may repre-
sent an interesting alternative to modulate H. pylori colo-
nization in children infected by this pathogen. Tursi etal.
[78] demonstrated that a 10-day quadruple anti-helico-
bacter 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. is study concluded that the sup-
plementation of the therapy with the administration of
probiotics showed a slight improvement in the eradica-
tion of H. pylori. Probiotics can therefore be used as first
course of anti-H. pylori treatment or can be used in con-
jugation 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 5years in
developing countries [81, 82]. e 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 nec-
essary. Zhang etal. [84] studied the antimicrobial activ-
ity 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. ey reported that the
tested lactobacilii strains showed strong antimicrobial
activity against S. sonnei. In a study to screen for the anti-
microbial activity of probiotics against S. sonnei, Zhang
etal. [85] reported that L. johnsonii F0421 exhibited sig-
nificant inhibitory activity and excluded, competed and
displaced S. sonnei adhered to HT-29 cells. In a different
study, Mirnejad etal. [83] evaluated the nature of anti-
microbial substances and properties of L. casei against
multi-drug-resistant clinical isolates of S. flexneri and
S. sonnei. eir results indicated that L. casei showed
strong antimicrobial activity against S. flexneri and S. son-
nei, and they attributed pathogen inhibition to produc-
tion of organic acids by the test Lactobacillus. In another
study, Zou etal. [86] studied the antimicrobial activity of
nisin, a bacteriocin produced by L. lactis strains, against
L. monocytogenes, Staphylococcus aureus, Salmonella
typhimurium and Shigella boydii. ey reported that
there was a decline in pathogen populations, which was
ascribed to the changes in the fatty acid profiles, cell via-
bility, 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 sep-
ticemia in adults [88, 89]. ere 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
etal. [90] isolated six infant bifidobacterial strains from
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Mathipa and Thantsha Gut Pathog (2017) 9:28
breast-fed infant faeces, with a potential antimicrobial
activity against L. monocytogenes. ese isolates actively
inhibited L. monocytogenes by producing a heat-stable
proteinaceous substance. eir study indicated that the
use of bifidobacterial strains capable of competing with
pathogenic organisms following the probiotic approach
would advantageously improve intestinal bacterial ecol-
ogy and provides a useful alternative strategy for inhibit-
ing intestinal pathogens. In 2007, Corr etal. [91] studied
the pretreatment of C2Bbe1 cells, a clone of the Caco-2
human adenocarcinoma cell line with strains of Bifido-
bacterium and Lactobacillus to demonstrate that this
can significantly interfere with subsequent invasion by
L. monocytogenes. ey 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 Lac-
tobacillus 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 ofconventional 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 patho-
gens. 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. ere-
fore, a cocktail of various probiotic strains would need to
be produced in order to enhance the effects against dif-
ferent pathogens within the gut [93].
Contrary to earlier reports that probiotics exhib-
ited inhibitory effect against L. monocytogenes [90, 91],
according to Koo etal. [94], probiotics have a limited suc-
cess 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 per-
centages 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 con-
centration of the probiotic strain also failed to displace
the attached L. monocytogenes. e 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 lev-
els 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 lev-
els 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. e
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 reproduc-
ible 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 prep-
aration 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].
e 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 success-
fully, 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. us, 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.
The concept ofprobiotic bioengineering or
recombinant probiotics
e performance of the existing probiotic strains can
be improved through the use of bioengineering. Bioen-
gineering refers to the manipulation of a gene of a pro-
biotic strain in order to improve the tolerance to the
technological stress, including but not limited to tem-
perature extremes, oxygen and acidification, during food
production, and/or survival of the probiotic in the GIT,
to confer beneficial effects to the host [99]. is strategy
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Mathipa and Thantsha Gut Pathog (2017) 9:28
can be used in the design and construction of new pro-
biotic strains harbouring genes of interest derived from
the pathogens. It allows for the production of proteins
that were initially not present within the microorgan-
ism. Virulence factors of the pathogens can be cloned
and expressed into the probiotic strain and subsequent
administration of the resultant recombinant probiotic
strain will inhibit the development of infection and yield
no clinical presentation of the symptoms. Furthermore,
recombinant probiotics can be used to deliver drugs or
vaccines, target specific pathogens or toxins, enhance an
immune response and mimic cell surface receptors [100].
Most human receptors recognized by enteric pathogens
or their toxins are well characterized. Also, by targeting
a specific pathogen, this strategy deems the development
of resistance to the vaccine or treatment unlikely. Bioen-
gineering of probiotics is not entirely a new field, vari-
ous researchers have reported on the successes attained
with the use of such probiotics (Table2). Culligan etal.
[49] reported on the main advantages of using recombi-
nant probiotics in the treatment of enteric infection. e
next section of this review focuses on studies that were
conducted on bioengineered probiotics aimed at improv-
ing different functional properties of the conventional
strains.
Applications ofprobiotic bioengineering
Improvement ofstress tolerance
ere has been an increase in the use of probiotics due
to their known effects to confer beneficial health to the
host. However, there are still problems frequently asso-
ciated with the incorporation of probiotic strains into
food products. ese problems include but are not lim-
ited to poor temperature, salt and oxygen tolerance of
some species or strains. Different approaches including
pre-adaptation to stress, the use of oxygen-impermeable
containers, microencapsulation [101], incorporation of
nutrients, and selection of stress-resistant strains have
been used in an attempt to address these problems [102].
e use of bioengineering has been used in the field of
stress adaptation, and there have been promising results.
e ability to confer additional stress tolerance in
stress-sensitive cultures can lead to the development and
delivery of novel probiotics with maximal therapeutic
efficacy [103]. It has been reported that the two major
heat shock proteins, GroES and GroEL, are essential for
the survival of bacteria at all temperatures [104]. In a
study by Desmond etal. [101], the effect of overexpres-
sion of these heat shock protein chaperones (GroES and
GroEL) in the probiotic L. paracasei NFBC338 was inves-
tigated. Expression of these genes resulted in improved
thermotolerance (heat tolerance) as well as increased
solvent resistance by the probiotic strain. Furthermore,
they compared the survival of the non-adapted par-
ent strain, stress adapted and the recombinant probiotic
during exposure to heat stress. ey reported that the
recombinant probiotic survived 10- and 54-fold better
than the stress-adapted and non-adapted parent strains,
respectively.
e survival of pathogens is usually dependent on the
different systems that can help them overcome the differ-
ent stress conditions present in the GIT. ree transport
systems have to date been identified in L. monocytogenes
that have been linked to betaine and carnitine uptake
[105, 106]. e first of these is a gene encoding the sec-
ondary glycine betaine transporter, listerial betaine
uptake system (BetL), which is linked to salt tolerance of
Listeria [107, 108]. It has been reported that disrupting
BetL results in reduced growth at 37°C in complex media
of elevated osmolarity [107]. e reduction in the initial
betaine uptake in the absence of BetL leads to dimin-
ished intracellular solute pools [106], causing changes
in the cell volume, intracellular solute concentration and
the turgor pressure [109]. Sheehan et al. [110] studied
the heterologous expression of the BetL into the probi-
otic strain L. salivarius UCC118 using a nisin-controlled
expression system. ey reported that expression of BetL
led to an increase in the resistance of the probiotic to
several stresses (osmo, cryo, baro and chill), spray- and
freeze-drying. Later in another study these researchers
demonstrated that B. breve UCC2003 harbouring the
betaine uptake (BetL) gene displayed an improved toler-
ance to gastric juice and elevated osmolarity [111].
Trehalose is a non-reducing disaccharide ubiquitously
distributed in nature and is well known for its role in pro-
tecting cells against a variety of stresses [112]. In E. coli,
it is synthesized in response to high osmolarity [113].
Termont etal. [114] cloned the trehalose synthesis gene
(ostAB) from E. coli into L. lactis and reported that there
was an enhanced probiotic’s survival during freeze-dry-
ing, in high bile concentrations and its resistance to gas-
tric acid. In a different study, Carvalho etal. [115] studied
the expression of the trehalose synthesis in the same pro-
biotic L. lactis and reported that trehalose plays a definite
role in the protection of this bacterium against damage
caused by acid, cold or heat shock. ese studies provide
evidence that expression of genes from pathogenic spe-
cies to improve stress tolerance of probiotics has been
explored with promising results. However, further sci-
entific assessment is still required to analyse the benefit
of using these genes and interpretation by risk–benefit
analysis [103].
Production ofantimicrobial peptides
e rise in development of antibiotic resistance of patho-
gens has led to a dire need for alternative methods to treat
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Mathipa and Thantsha Gut Pathog (2017) 9:28
infections. Antimicrobial peptides (AMPs) have been
explored as an alternative method for effective control of
multi-drug resistant (MDR) pathogens [116]. As already
mentioned, some probiotics produce several antimicro-
bial compounds and peptides as a defense mechanism
against pathogens [12] but they are not specific. Probiot-
ics can therefore be used as candidates for the production
and delivery of therapeutic antimicrobial peptides within
the host GIT targeting a specific action or pathogen.
e current methods for production of AMPs have been
reported to have several limitations. Synthesis of pep-
tides is not only expensive, but also time-consuming too;
in some cases, the peptides eventually kill the producing
cells or are secreted as inclusion bodies. Oral administra-
tion of the peptides subjects them to degradation before
they can reach the target site. ey are also difficult to
administer systemically as they are rapidly identified and
directed for restoration of the immune system before
they can reach the site of infection. erefore, an alterna-
tive strategy will be to use probiotic strains to express the
different AMPs resulting in a combination strategy where
hosts will get the probiotic effects with the production of
the different AMPs [116].
Volzing etal. [31] chose L. lactis as an ideal vehicle
for production and delivery of AMPs to the site of GI
infection due to its ability to survive within the human
Table 2 Applications ofbioengineering
Applications Probiotics Genes/receptors expressed Action References
Improvement of stress
tolerance L. paracasei Heat shock protein chaperones
(GroES and GroEL) Improved thermotolerance (heat tolerance) of
probiotic; increased solvent resistance by the
probiotic strain
[104]
L. salivarius Listerial betaine uptake system
(BetL) Increase in the resistance of the probiotic to
several stresses [110]
L. lactis Trehalose synthesis gene (ostAB) Enhanced probiotic’s resistance to gastric acid
protection of the probiotic against damage
caused by acid, cold, or heat shock
[114, 115]
Production of antimicro-
bial peptides L. lactis A3APO and alyteserin Successfully inhibited E. coli and Salmonella [31]
Probiotic E. coli Cell receptor (ganglioside) for
cholera toxin or ETEC heat-
labile toxin
Enterotoxins are sequestered by the probiotic
E. coli thus protecting host against diarrheal
infection
[12]
L. reuteri Heat-stable (ST) and heat-labile
(LT) enterotoxins Successfully bound to the enterotoxins and
prevented enterotoxicity in a mouse model [12]
Enhancement of anti-
inflammatory response L. lactis Elafin Significant reduction in inflammation [118]
L. lactis TGF-β Overall reduction of inflammation and colitis [120]
L. lactis IL-10 Successfully prevented colitis in murine models [121]
L. lactis Anti-TNF-α nanobodies Reduced the colonic inflammation [123]
L. lactis Internalin A Enhanced efficient internalization of L. lactis in
the human intestinal cell line Caco-2 [124]
Enhancement of coloniza-
tion exclusion L. paracasei Listeria adhesion protein (LAP) Inhibited the adhesion of Listeria to host cells [94]
L. lactis Surface-associated flagellin Inhibited the binding and adhesion of patho-
genic E. coli and S. enterica
[126]
L. acidophilus K99 fimbriae Reduced the attachment of ETEC to porcine
intestinal brush border [129]
Receptor mimicry system
and toxin neutralization E. coli Nissle 1917; L.
lactis
Galactosyl-transferase genes;
Tetanus toxin fragment C
(TTFC)
Recombinant bacteria neutralized shiga toxins,
Stx1 or Stx2 [132]
Increased IgA levels led to protection of the
host against the infections of the mucous
membrane
[135, 136]
E. coli Nissle 1917 Receptor GM1 Protected infant mice from challenge with
virulent V. cholerae
[139]
E. coli Nissle 1917; L.
casei
AI-2 co-expressed CAI-1 80% reduction in Ctx binding to the intestines of
mice which reduced numbers of V. cholerae in
treated mouse intestines
[140]
Adhesins K99 Protected 80% of the vaccinated mice after
challenge with a lethal dose of strains of ETEC
K99 and K88
[142]
Vaccination L. lactis Virus spike protein VP8 Provided 100% protection against rotavirus
infection [145]
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Mathipa and Thantsha Gut Pathog (2017) 9:28
gastrointestinal tract and its amenability to heterologous
gene overexpression. In their study, they engineered a L.
lactis strain to inducibly express and secrete AMPs with
high activity against Gram-negative pathogens, specifi-
cally E. coli and Salmonella strains. e AMPs of inter-
est, A3APO and alyteserin were selected and then cloned
into L. lactis for the expression of the heterologous pep-
tides. An expression cassette containing a codon-opti-
mized sequence for alyteserin was fused with an Usp45
secretion signal sequence. is expression cassette was
cloned under the control of a nisin inducible promoter
and transformed into L. lactis. When the resultant L.
lactis recombinant strain was induced to express and
secrete these peptides, and the effect of their expression
on growth and viability of E. coli and Salmonella was
tested, the results indicated successful inhibition of both
these pathogens while viability of the host (i.e. the L. lac-
tis expressing the peptides) was maintained. Inhibition of
these pathogens by alyteserin was observed from concen-
trations ranging from 0.125–1mg/ml, while the L. lactis
strains remained viable when exposed to the alyteserin
supernatant at 1mg/ml. is system showed potential as
a therapeutic alternative to antibiotics in order to target
and inhibit Gram-negative bacteria.
Enhancement ofanti‑inammatory response
A group of chronic inflammatory disorders known as
inflammatory bowel diseases (IBD) are responsible for
the inflammation of the digestive tract. e two forms of
the IBD are Crohn’s disease and ulcerative colitis, which
are both characterized by an uncontrolled inflamma-
tory response in the intestines [117]. e treatment of
IBDs poses a challenge as the current treatment options
are either costly or cause severe side-effects in patients.
ere have been a number of studies on the treatment
of IBDs and recent research has reported that probiotic
bacteria may counteract the chronic inflammatory pro-
cess [118]. Elafin, is a protease inhibitor expressed in the
intestinal epithelium, which contributes to the reduc-
tion of inflammation. During inflammation, there is an
increase in elastase and myeloperoxidase (MPO) activ-
ity, elafin can inhibit the function of proteases, thereby
reducing inflammation [119]. Bermúdez-Humarán etal.
[118] bioengineered L. lactis to express elafin in mice
suffering from colitis. e gene encoding for elafin was
fused in frame with a gene encoding for a ribosome-bind-
ing site and with an Usp45 secretion signal sequence and
inserted into an expression vector. e recombinant plas-
mid was thereafter transformed into L. lactis and expres-
sion was induced under the control of a nisin-induced
promoter. Colonic inflammation was then induced in
mice with dextran sodium sulphate and then the mice
were subsequently orally treated with either wild-type
or recombinant L. lactis. Analysis of mice colons for
inflammation parameters such as colonic thickness,
elastase activities and granulocyte infiltration after 7days
indicated that mice treated with recombinant L. lactis
secreting the elafin showed a significant reduction in all
inflammation parameters. However, mice treated with
wild-type probiotics did not show the same significant
decrease in inflammation parameters, their response
was similar to that of the control-untreated mice. Fur-
thermore, comparison of efficiency of recombinant L.
lactis secreting elafin to those expressing either the anti-
inflammatory cytokine IL-10 or TGF-β1 (to be discussed
next) showed that elafin-secreting strain was the most
efficient. ese results suggested that elafin was the most
efficient anti-inflammatory molecule to be delivered by a
probiotic strain at the mucosal surface in order to treat
inflammation [118].
Chronic inflammation of IBD patients can also be
reduced through the administration of anti-inflammatory
cytokines such as interleukin 10 (IL-10). IL-10 plays a
central role in down-regulation of inflammatory cascades
and in the establishment of tolerance in the mucosa [9].
Interferons (IFN), including IFN-α and IFN-β, are widely
expressed cytokines involved in innate responses and
additionally, these cytokines have an immunomodulatory
role in the anti-inflammatory host response. e use of
probiotic bioengineering to treat IBD has been studied
and it has been reported that this can indeed be used as
an alternative. Several studies have been carried out with
regard to probiotics expressing cytokines and other anti-
inflammatory molecules such as IL-10 and TGF-β instead
of elafin, using similar cloning procedures used for elafin.
After transformation, recombinant probiotic strains were
induced with nisin in order to either express IL-10 or
TGF-β and orally administered to mice suffering from
colitis. Recombinant L. lactis expressing TGF-β displayed
beneficial effects by reducing MPO levels, overall reduc-
ing inflammation and colitis in 40% of the mice. However,
the protective effects against colitis were higher in mice
treated with recombinant probiotics expressing elafin
than those treated with probiotics expressing IL-10 [120].
Another study reported that intra-gastric administration
of L. lactis expressing recombinant IL-10, a cytokine used
in clinical trials for treatment of IBD, could successfully
prevent colitis in murine models [121].
McFarland etal. [122] investigated the effects of local
administration of IFN-β on a murine model of colitis.
ey developed a transgenic L. acidophilus strain that
constitutively expresses IFN-β and reported that the
resultant recombinant strain secreting IFN-β resulted
in the exacerbation of colitis. Tumor necrosis factor α
(TNF-α) is a cytokine that mediates the clinical symp-
toms of IBD [9]. In a study by Vandenbroucke etal. [123],
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Mathipa and Thantsha Gut Pathog (2017) 9:28
they constructed a recombinant L. lactis to produce anti-
TNF-α nanobodies and reported that daily administra-
tion of this strain reduced the colonic inflammation.
Enhancement ofcolonization exclusion
Enhancement of probiotic adhesion to the intestinal
mucosal surface can be seen as a potential strategy in
order to prevent adhesion and colonization of pathogenic
bacteria. Strategies include using gene products of tar-
get pathogens such as adhesins or secretory systems in
probiotic bacteria to create a competitive environment
for colonization [94]. A number of researchers investi-
gated the efficiency of this approach in improvement of
competitive exclusion by enhancing binding or adhesion
efficacy of the probiotics to host cells. When internalin
A from L. monocytogenes was cloned and expressed into
the L. lactis strain, there was enhanced binding to human
epithelial cells and bacterial internalization [124]. A more
recent study, Koo etal. [94] developed a recombinant
probiotic L. paracasei harbouring the Listeria adhesion
protein (LAP) in order to control L. monocytogenes infec-
tion. LAP interacts with a heat shock protein 60 recep-
tor in host cells and promotes adhesion of Listeria to host
cells. Conventional and recombinant probiotic L. para-
casei were added to Caco-2 cell monolayers separately,
thereafter these monolayers were Giemsa-stained. Pre-
exposure of Caco-2 cell monolayers to recombinant L.
paracasei expressing LAP followed by the addition of L.
monocytogenes led to a reduction of adhesion and trans-
location of the pathogen. e wild-type probiotic strain
had no significant reduction in the adhesion of the L.
monocytogenes to the cell monolayer, while the recombi-
nant strain resulted in a 60% reduction of adhesion.
It has been shown that flagellins from Bacillus cereus
are responsible for the adhesion of the bacteria to
mucosal cells [125]. Gut pathogens may also use fimbriae
or flagella which are extended appendages on the surface
of the cell wall, to adhere to host cell receptors. ere-
fore, expression of these specific appendages in probiotic
strains would allow them to bind to the intestinal epi-
thelium, excluding pathogenic binding. Taking that into
consideration, Sánchez et al. [126] cloned the surface-
associated flagellin of B. cereus CH and expressed it in
the probiotic L. lactis. e recombinant strain adhered
strongly to the mucin-coated polystyrene plates in an
invitro experiment and competitively inhibited the bind-
ing and adhesion of pathogenic E. coli and S. enterica.
Enterotoxigenic Escherichia coli (ETEC) K99 fim-
briae have been reported to enhance the production of
mucosal IgA and serum IgG1 fimbria-specific responses
[127], thereby increasing the immune responses at
mucosal surfaces such as the gastrointestinal (GI) tract,
the respiratory tract, and the vaginal tract [128]. Chu
etal. [129] cloned and expressed the K99 fimbriae from
ETEC into the probiotic L. acidophilus and reported that
the recombinant L. acidophilus was able to reduce the
attachment of ETEC to porcine intestinal brush border
in a dose-dependent manner. e reduction of the adher-
ence of the pathogen by the recombinant probiotic pre-
vents the binding of the pathogen, therefore inhibiting
the infection.
Receptor mimicry system andtoxin neutralization
One mechanism that pathogens use to invade the host
cells and cause infection is through the production of
toxins. ese pathogens secrete toxins, and sometimes
express adhesins that bind to host cells via oligosaccha-
ride receptors displayed on surface glycolipids or gly-
coproteins. e interaction between the released toxin
and the specific oligosaccharide receptors on the surface
of the human intestinal cells is an essential step during
pathogenesis [130]. erefore, toxins or secretory sys-
tems of pathogens may also serve as potential targets in
development of therapeutics [131]. Taking this into con-
sideration, it thus becomes apparent that interfering with
the toxin receptor binding and adhesion can be used as
a strategy to exclude the pathogen and subsequently
minimize or control its infection [130]. A therapeutic
strategy would be to express toxin receptors on the cell
surface of probiotic strains in order to mimic the recep-
tor [132]. is expression produces a lipopolysaccharide
that mimics a host cell receptor, which, e.g. cholera toxin
or ETEC heat-labile toxin could recognize and bind to.
erefore, upon infection, enterotoxins would bind to
probiotics and become sequestered, protecting the host
from a pathogenic infection [130]. at is, the toxin is
sequestered when, instead of binding to the receptor on
the surface of the host cell, it binds with high avidity to
the receptor mimic expressed on the surface of the pro-
biotic cell. is hinders the interaction between the toxin
and the host cells, which is a crucial step in the disease
process [130, 134]. Studies of probiotics expressing toxin
receptor mimics were mostly biased towards impact on
the disease progression without monitoring of the probi-
otic-toxin complex. However, Paton etal. [130] reported
that the receptor mimic probiotic was spontaneously
eliminated from the GIT of mice a day or two after the
end of its administration. erefore, more studies track-
ing the probiotic-toxin complex are required to establish
their fate.
ere are a number of pathogens that secrete these tox-
ins and among them are, Vibrio cholerae, Shiga toxigenic
Escherichia coli (STEC), ETEC and Clostridium difficile,
just to name a few. Shiga toxigenic E. coli and ETEC both
cause enteric infections, they cause gastrointestinal dis-
ease and diarrhoeal disease in humans, respectively. If
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Mathipa and Thantsha Gut Pathog (2017) 9:28
left untreated, these pathogens can cause severe bloody
diarrhoea associated with haemorrhagic colitis [133]. In
an earlier study by Paton etal. [134], the galactosyl-trans-
ferase genes from Neisseria gonorrhoeae were cloned
and expressed into a non-pathogenic E. coli. e results
showed that the recombinant E. coli was 100% effective
in treating mice infected with the normally fatal shiga
toxigenic E. coli. en later on in another study, these
researchers cloned the glycosyltransferase gene, Neisseria
meningitidis toxin-specific receptor, into the probiotic E.
coli, creating a competitive environment for toxin binding
to the host cells. Expression of these genes created a cell
surface mimic of a shiga toxin receptor. is led to com-
petitive exclusion of the pathogen by the probiotic and
subsequently inhibiting its infection. is recombinant
strain had a high binding capacity and efficacy in mouse
models and was effective in neutralizing shiga toxin vari-
ants (stx1 and stx2) [132]. Norton etal. [134] cloned and
expressed a tetanus toxin fragment C (TTFC) in L. lactis.
ey then reported that there were increased IgA levels
in the host after oral administration of the recombinant
probiotic, which led to protection of the host against the
infections of the mucous membrane. ese results were
supported by other studies, where mice immunized with
this recombinant probiotic showed more resistance to
the lethal challenge with tetanus toxin than those that
were not immunized [136, 137].
Pathogens are able to control the expression of their
virulence genes by sensing signals from their own spe-
cies, other bacteria or their environment, a phenomenon
termed quorum sensing [12]. Interruption of quorum
sensing of the pathogen can be used as an alternative
strategy to control the pathogen. Cholera is a life-threat-
ening gastrointestinal infection [138] that is caused by
ingestion of water or food (usually undercooked shellfish)
contaminated with V. cholerae [130]. Following inges-
tion, V. cholerae passes through the stomach, colonizes
the small intestine and then release cholera toxin (Ctx),
which is responsible for its virulence. It has been hypoth-
esized that neutralization of Ctx in the gut should pre-
vent the disease from developing or at least speed up
recovery from an established V. cholerae infection [130].
e cloning and expression of Ctx receptor into probiot-
ics can therefore be used as an alternative strategy for the
treatment of cholera. Focareta et al. [139] constructed
a probiotic E. coli encoding receptor GM1 (to express
the GM1 ganglioside) on its surface, which is capable of
binding large amounts of Ctx and protecting infant mice
from challenge with virulent V. cholerae. e resultant
recombinant E. coli was capable of binding purified Ctx
with high avidity and adsorbing >5% of its own weight of
toxin invitro. V. cholerae releases cholera autoinducer-1
(CAI-1) and autoinducer-2 (AI-2), which depending of
population density, can down- or up-regulate expression
of virulence genes [12, 140]. Virulence genes involved
are Ctx, which causes diarrhoea, and toxin-coregulated
pilus (TCP), which facilitates attachment of vibrios to the
intestinal wall. When cell densities are high, expression of
genes encoding these virulence factors is reduced, while
proteases expressed degrade the attachment matrix with
consequent flushing out of the bacterial cells with diar-
rhoeal fluids. In order to determine the possibility for use
of bioengineered probiotic for control of cholera, Duan
and March [140] constructed an AI-2 producing E. coli
Nissle that co-expressed CAI-1. ey reported an 80%
reduction in Ctx binding to the intestines of mice pre-
treated with recombinant probiotic, which reduced the
chances of infection. ese results showed the poten-
tial for use of bioengineered E. coli Nissle co-express-
ing CAI-1 and AI-2 for the prevention or treatment of
cholera.
Vaccination
Probiotics may induce low levels of the immune response.
erefore, probiotics can be bioengineered to deliver
immunogenic molecules to the intestinal mucosal surface
to enhance the immune response. Recombinant probi-
otics can act as a vaccine arming the host immune sys-
tem to deal with gut pathogens [141]. In order to exploit
a safe and effective vaccine for the prevention against
K99 infections of ETEC, Wen et al. [142] cloned and
expressed ETEC adhesins K99 into the probiotic L. casei.
ey reported that there was an increase in the efficacy
of the recombinant probiotic and that more than 80% of
the vaccinated mice were protected after challenge with a
lethal dose of standard strains.
Non-bactericidal infections can be treated with bio-
engineered probiotics through an approach using vacci-
nation delivery systems. Rotavirus is the most common
cause of diarrhoea in children. It damages cells within the
small intestine (enterocytes) and thereafter causes gas-
troenteritis. e viral proteins can disrupt the reabsorp-
tion of water within the human intestine and can also
cause an inefficiency to digest lactose, resulting in milk
intolerance for infants. Symptoms include nausea, vom-
iting, diarrhoea, fever and dehydration [143]. Gardlik
etal. [145] bioengineered L. lactis to express virus spike
protein VP8, which induced anti-VP8 antibodies and IgA
antibodies in mice. is induction occurred systemically
and locally within the mouse intestine providing 100%
protection against rotavirus. With oral vaccination being
favoured above the other types of vaccination, using pro-
biotics with their ability to withstand the GIT conditions
can be used as an alternative mode of vaccination. ere
are several other advantages of delivery of vaccines using
recombinant probiotics such as easy administration by
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Mathipa and Thantsha Gut Pathog (2017) 9:28
consumers, a decreased risk in transmission of food-
borne diseases and the stimulation of both innate and
adaptive immunity [9].
Safety concerns regardingbioengineered
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 bioengi-
neered probiotics is that they are classified as genetically
modified organisms (GMO) [144]. e 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 selec-
tion markers or other genetic material to other organisms
[146]. Introduction of the GMO into the environment
can impact there directly by competing with natural spe-
cies, 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 ani-
mal 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 recom-
binant DNA invitro and invivo, which are both of sig-
nificant 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 con-
tainment systems can be used to prevent dissemination
of genetic material to other bacteria and to prevent a sig-
nificant uncontrolled increase of probiotic cells into the
natural environment [145]. e organism is genetically
programmed to only grow in the laboratory and to die in
the natural environment [149]. e use of the thymidine-
deficient strains is one of the promising strategies for bio-
logical containment of bioengineered probiotics. In these
strains, the gene of interest is cloned into the chromo-
somal thymidylate synthase gene (thyA), which codes for
production of thymine essential for growth of L. lactis.
is disruption of the thyA gene makes the recombinant
strain dependent on external supplementation of thymi-
dine or thymine in the growth medium for growth and
survival. ymine is absent in the environment or its lev-
els are limiting invivo, and this ensures that the recombi-
nant 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 rele-
vant antibiotics before being deemed suitable as a probi-
otic [151]. When cloning probiotics, therapeutic safety of
recombinant probiotic carrier organisms is crucial, espe-
cially when the strain has to be used in individuals who
are already infected with a pathogen. e 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 andfuture perspective
e 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. e application of probiotics in food for control
of enteric pathogens has been explored and the probi-
otic market is growing worldwide. e ability of probi-
otics 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 infec-
tions. ese studies showed both their successes and lim-
itations, mainly highlighting the generic nature of their
mode of action and their failure in controlling some spe-
cific pathogens. ese limitations can be overcome and
functions of conventional probiotics enhanced to create
a greater beneficial effect through the use of bioengineer-
ing. e 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.
orough understanding the life cycle of pathogens
post ingestion, and knowledge of the virulence factors
they use to cause infections offers a strategy for develop-
ment of bioengineered probiotics strains tailored to con-
trol-targeted pathogens. By targeting a specific pathogen,
the efficacy of the probiotics inhibiting both the patho-
gens 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. e bioengineered pro-
biotics thus offer important potential to be used as novel
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 14 of 17
Mathipa and Thantsha Gut Pathog (2017) 9:28
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 probiot-
ics 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 proper-
ties to be produced on a large scale [147]. ey should
remain viable in large numbers so as to confer the benefi-
cial effects to the host and should not develop unpleasant
flavours or textures upon their incorporation into foods
[148]. Furthermore, studies on bioengineered probiot-
ics, specifically for targeted control of pathogens, have
focused on the impact of the recombinant probiotic
strain on the pathogen(s) of interest. e influence of
administration of these probiotics on commensal bacte-
ria or the whole microbiota has not been the subject of
studies. ese aspects should also be addressed in future
studies on bioengineered probiotics.
Abbreviations
GIT: gastrointestinal tract; LAB: lactic acid bacteria; IgA: immunoglobulin A; IL-
10: interleukin 10; RBC: ranitidine bismuth citrate; BetL: listerial betaine uptake
system; AMPs: antimicrobial peptides; MDR: multi-drug-resistant; IBD: inflam-
matory bowel diseases; MPO: myeloperoxidase; LAP: Listeria adhesion protein;
ETEC: enterotoxigenic Escherichia coli; STEC: Shiga toxigenic Escherichia coli;
TTFC: tetanus toxin fragment C; GMO: genetically modified organisms; TCP:
toxin coregulated pilus; thyA: thymidylate synthase gene.
Authors’ contributions
MST conceived the original concept and outline for the review. MST and MGM
collected information, drafted and edited the manuscript. Both authors read
and approved the final manuscript.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
Data sharing not applicable to this article as no datasets were generated or
analysed during the current study.
Funding
We are grateful for funding received from the National Research Foundation of
South Africa and the University of Pretoria. Both funders were not involved in
the design of the study and collection, analysis and interpretation of data and
in writing the manuscript.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 2 February 2017 Accepted: 27 April 2017
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