Foodborne enterotoxigenic Escherichia coli: from gut ...

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Foodborne enterotoxigenic Escherichia coli: from gut pathogenesis to new preventive strategies involving probiotics Charlène Roussel1,2, Adeline Sivignon3, Tom Van de Wiele2 & Stéphanie Blanquet-Diot*,1 Enterotoxigenic Escherichia coli (ETEC) are a major cause of traveler’s diarrhea and infant mortality in developing countries. Given the rise of antibiotic resistance worldwide, there is an urgent need for the development of new preventive strategies. Among them, a promising approach is the use of probiotics. Although many studies, mostly performed under piglet digestive conditions, have shown the beneficial effects of probiotics on ETEC by interfering with their survival, virulence or adhesion to mucosa, underlying mechanisms remain unclear. This review describes ETEC pathogenesis, its modulation by human gastrointestinal cues as well as novel preventive strategies with a particular emphasis on probiotics. The potential of in vitro models simulating human digestion in elucidating probiotic mode of action will be discussed. First draft submitted: 27 May 2016; Accepted for publication: 30 September 2016; Published online: 16 December 2016 Background While Escherichia coli isolates form part of the endogenous microbiota of the human gut, some strains have evolved pathogenic mechanisms to cause significant diarrheal and extraintestinal diseases in humans. Pathogenic E. coli associated with gastrointestinal illness have been divided into eight pathotypes based on their virulence profiles: enteropathogenic E. coli; enterohaemorrhagic E. coli; enterotoxigenic E. coli (ETEC); enteroinvasive E. coli; enteroaggregative E. coli; diffusely adherent E. coli; adherent invasive E. coli; and shiga toxin-producing enteroaggregative E. coli [1] . Among them, ETEC infect both humans and several species of farm animals such as pigs. In pigs, enteric diseases due to strains of ETEC are the most commonly occurring form of colibacillosis. In humans, ETEC are the main bacterial cause of diarrhea in adults and children in developing countries and are also a leading cause (30–60%) of traveler’s diarrhea in people visiting or returning from endemic areas, including military personnel [2,3] . Overall, this pathogen is responsible of 210 million of diarrheal episodes worldwide per year [4] . The annual incidence of ETEC-associated diarrhea is highest in young children, with up to 500,000 infant deaths under the age of 5 years per year, mostly in low-income countries [5] . ETEC are transmitted primarily via contaminated water and weaning food [2] , increasing the burden of developing countries that lack the infrastructure to reliably provide treated drinking water. The clinical symptoms of ETEC infections can range from mild diarrhea to a severe cholera-like syndrome [2] . Major aspects of ETEC virulence are

Clermont Université, Université d’Auvergne, Centre de Recherche en Nutrition Humaine Auvergne, EA 4678 CIDAM, Conception Ingénierie et Développement de l’Aliment et du Médicament, 63000 Clermont-Ferrand, France 2 Cmet, Center for Microbial Ecology & Technology, Ghent University, 9000 Ghent, Belgium 3 Clermont Université, UMR 1071 INSERM/Université d’Auvergne, Clermont-Ferrand, France INRA, Unité Sous Contrat 2018, ClermontFerrand, France *Author for correspondence: Tel.: +33 473 178 390; Fax: +33 473 178 392; [email protected]

Keywords

• ETEC • foodborne pathogen • gastrointestinal cues • in vitro digestion models • nutritional strategies • pig • probiotics • ST/LT toxins • vaccine • virulence factors

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ISSN 1746-0913

Review Roussel, Sivignon, Van de Wiele & Blanquet-Diot colonization of the small intestine and secretion of enterotoxins which elicit diarrhea. To date, the treatment of ETEC-associated diarrhea is the same as that for any acute secretory diarrheal disease with oral rehydration and use of antimicrobials [6] . Nevertheless, the use of antibiotics is increasingly controversial due to side effects and ongoing surge of antibiotic-resistant bacteria. There is therefore a crucial need to develop nonantimicrobial prophylactic strategies aiming to tackle ETEC infections. A better understanding at a molecular level of the mechanisms mediating pathogenesis, especially in the human digestive environment, would help to speed up the development of such approaches. In this context, the current review will first provide a state of the art of ETEC physiopathology, focusing on the regulation of bacterial virulence traits in the human digestive environment. Then, it will give an overview of current treatments and novel preventive strategies against ETEC infections, with an emphasis on probiotics as an alternative to antibiotic therapy. The last part of the review on probiotics was extended to studies in pigs as a model of humans. ETEC physiopathology in humans ●●Reservoir & route of transmission

Humans are the major reservoir for human ETEC strains [7] . Transmission of ETEC occurs through the fecal–oral route, primarily via contaminated food or water. Contaminated waters (surface waters, bathing and water used for weaning food) are the most common source of infection, mainly in developing countries that lack appropriate sanitation and drinking water treatment facilities [2,8] . Fruits and vegetables that are washed with contaminated water and not cooked can also serve as vehicles of transmission. Direct person-to-person contact, through observed, is not a major route of transmission. Infectious doses are high and fluctuate between 10 6 to 1010 cells but can be lower in at-risk populations such as infants [9] . ETEC follow seasonality with infections being most frequent in warm periods, but can also occurred during natural disasters, such as floods, when acute deterioration in the quality of drinking water and sanitation happens [10] . ●●Clinical manifestations

The clinical symptoms of ETEC infections can range from minor discomfort to a severe choleralike syndrome. Following an incubation period

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of 10–72 h, ETEC may induce acute watery diarrhea leading to rapid dehydration, usually associated with nausea, vomiting, abdominal cramps and prostration [2] . ETEC cannot be distinguished from cholera on clinical grounds. Most patients suffering from travelers’ diarrhea recover within 4 days with supportive measures alone and do not require hospitalization. Nevertheless, some patients may have prolonged diarrheal illness lasting a week and rarely more than 3 weeks [11] . Symptoms are much more severe in children from developing countries where diarrhea and malnutrition combine to form a vicious cycle leading to declining health status and death. ●●Main virulence factors

Even if the virulence profiles of strains isolated from travelers seem to considerably differ from those observed in children [12] , ETEC pathogenesis (Figure 1) is mainly mediated by two plasmidencoded virulence factors. First, ETEC must penetrate the mucus barrier and adhere to the epithelium of the small intestine by means of several colonization factors (CFs). At the present time, there is no consensus on the site of bacterial colonization, from the upper jejunum [13] to the ileum [14] . Then, watery diarrhea is produced due to the effects of the heat-labile (LT) and/or heat-stable (ST) enterotoxins. CFs are antigenically and structurally diverse. Within the 25 CFs identified, seven are generally more prevalent than others: CFA/I (CF antigen) and CS1 to CS6 (coli surface antigen) [15] . Most CFs receptors have not been yet identified, but CFs are thought to bind to glycoprotein conjugates in mucus fraction from the small intestine and on the surface of host cells. Recently, mucin-degrading enzymes which allow temporary access to cell membrane receptors have been identified in ETEC: YghJ, a mucin-binding metalloprotease [16] and EatA, a member of serine protease autotransporters of the Enterobacteriaceae family [17] . Nonfimbrial adhesins such as TibA, a glycosylated autotransporter, Tia, an outer membrane protein, and EtpA, which acts as a molecular bridge binding host cell receptors to tips of ETEC flagella, have also been implicated in the pathogenesis [15] . Once a close contact established with the host cell, ETEC strains can produce ST and/or LT enterotoxins which induce fluid secretion and/or inhibition of electrolytes absorption by activation of the cystic fibrosis

future science group

Foodborne enterotoxigenic Escherichia coli transmembrane regulator [15,18] . Different strains of ETEC can secrete either one or two toxins, those expressing both ST and LT leading to more severe diarrhea [19] . The LT toxins (eltAB gene) which share 80% homology to Cholera toxins, have an AB5 configuration consisting in a A enzymatic subunit linked to a pentameric ring of B subunits necessary for binding and internalization [2] . LT are mainly secreted associated with outer membrane vesicles and bind irreversibly to GM1 ganglioside on the host cell, the same identical ganglioside receptors that are recognized by the cholera toxin. A recent in vitro study has shown that EatA could enhance toxins access to their receptors thanks to mucin degradation [17] . In addition to causing diarrhea, LT plays multiple roles in modulating host cell function and providing a competitive advantage to ETEC adherence to cultured intestinal epithelial cells [20] . ST toxins are small cysteine-rich peptides (estAB gene). They can be divided into two structural and antigenically distinct groups: STa (methanol soluble, protease resistant) and STb (methanol insoluble, protease sensitive) which reversibly bind to guanylyl cyclase C (GC-C) and sulphatide, respectively [18] .

Review

ETEC-inflammation process, even if a recent study suggests that ETEC strain H10407 subverts NF-κβ signaling through secretion of a proteinaceous factor [24] . Lastly, links were established between ETEC toxins and inflammation. Wang et al. [25] have recently shown that LT activates both MAPK and NF-κβ pathways and that p38 MAPK activation is involved in LT-induced ETEC adherence. The GC-C signaling system triggered by ST may be also important to human intestinal inflammation. Modulation of ETEC survival & virulence in the human gut Both the survival of ETEC strains and the regulation of virulence genes in the human digestive environment are key factors in bacterial pathogenesis. Nevertheless, the influence of the main biotic and abiotic factors of the human gut on these factors remains largely unclear [26] . The available data are restricted to in vitro studies that have investigated the effect of specific parameters of the human digestive environment on ETEC survival and virulence, as described below and recapitulated in Figure 2. ●●pH gradient in the GI tract

●●Intestinal inflammation

ETEC are classically considered to cause noninflammatory watery diarrhea. Nevertheless, leucocytes, lactoferrin, IL-1β and IL-8 have been detected in fecal samples of ETECinfected patients [21] . This suggests that ETEC, like other noninvasive pathogens, could rather elicit a mild inflammatory response as a result of their interaction with the host’s enteric cells. Likewise, challenging human Caco-2 cells with ETEC causes strong upregulation of proinflammatory mediators that lead to membrane damage [22] . The means by which enteric pathogens elicit mucosal inflammation is complex and not completely understood. Intestinal epithelium cells first detect bacteria through their pathogen-associated recognition receptors, such as Toll-like receptors (TLR). Studies on TLR expression in porcine intestinal epithelial cells demonstrated that TLR4 is most strongly constitutively expressed [23] . Activation of epithelial and resident subepithelial immune cells results in a rapid burst of proinflammatory mediators (e.g. IL-1, IL-6 and IL-8) leading to the recruitment of effector cells such as neutrophils to the site of infection. MAPK and NF-κβ signaling pathways have been involved in the


Following ingestion, ETEC are exposed to the host digestive tract characterized by acidic conditions in the stomach followed by basic environment in the small intestine, where pH gradually increases from pH 6 in the duodenum to 7.4 in the terminal ileum. Few in vitro studies have investigated the effect of pH on human ETEC survival. Masters et al. [27] have shown that after exposure to pH 2, ETEC became undetectable by plate counting after 2 h but could be detected by PCR for up to 24 h (due to detection of both live and dead bacteria). A recent study using flow cytometry analysis indicates that there were no significant differences in the percentage of live bacteria when ETEC was subjected to pH 5, 7 or 9 [28] . There is no specific data in ETEC on the expression of genes conferring protection against acid conditions. Nevertheless, five acid resistance pathways have been described in E. coli, among them the glutamate decarboxylase system (AR2) is thought to offer the best protection below pH 3. Johnson et al. [29] suggested that the release of ST was not pH-dependent while it is acknowledged that extracellular pH has an influence on the release of LT toxin which increases with alkalinity [28] . ETEC seems to

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Review Roussel, Sivignon, Van de Wiele & Blanquet-Diot

CFs EtpA

Acute watery

EatA / YghJ

CFA/I

LTb Tb b

diarrhea

Cl-

TibA Tia STa

OMV

H2O +++

STb

GC-C Sulphatide

GM1

Mucus layer

CS6

P

LTa

CFTR cGMP

Ca2+ cAMP

Adenylate cyclase

AD

tion

syla

bo P ri

Figure 1. Physiopathology of enterotoxigenic Escherichia coli infections. The figure resumes the main features of enterotoxigenic E. coli physiopathology. (A) Contaminated waters mostly resulting from a lack of sanitation (drinking water and weaning foods) are the most common sources of ETEC infections. (B) After ingestion, ETEC have to face acidic conditions of the stomach before colonizing the small intestine. (C) Thanks to CFs (such a CS6 or CFA/I), ETEC will be able to adhere both to the mucus layer and enterocytes. Mucin-degrading enzymes EatA and YghJ will favor the adhesion of bacteria to enterocytes and later on the toxin access to their receptors. Non fimbrial adhesins (TibA, Tia and EtpA) also mediate the initial host/pathogen interactions. (D) ETEC adhesion promotes the secretion of LT and/or ST toxins. LTb subunits in OMV bind GM1 monoganglioside at the surface of the enterocyte. (E) The LTa subunit is endocytosed and joins the cytosol via retrograde transport. (F) LTa ADP-rybosylates the GSα subunit of adenylate cyclase leading to an increase in cAMP. (G) STa binds to the GC-C receptor and STb to sulphatide resulting in an increase in cGMP and Ca2+, respectively.

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Foodborne enterotoxigenic Escherichia coli

Review

Figure 1. Physiopathology of Enterotoxigenic Escherichia coli infections (cont. from facing page). (H) All these pathways induce CFTR phosphorylation, leading to a net efflux of electrolytes and water into the lumen of the small intestine and acute watery diarrhea. CF: Colonization factor; CFTR: Cystic fibrosis transmembrane regulator; GC-C: guanylate cyclase C; OMV: Outer membrane vesicle.

use the pH gradient in the GI tract to modulate LT toxin production and secretion: when bacteria reach the small intestine, alkaline pH induces both transcription and maximal release of LT [28] . ●●Bile & digestive enzymes

One major challenge that bacteria encounter in the small intestine is the high concentration of bile salts which act as an emulsifier of dietary fats but also as effective physiological antimicrobials. Surprisingly, no study has investigated the impact of bile on ETEC survival, but it has been shown that bile can serve as an environmental cue for the pathogen by modulating the expression of specific virulence factors. Chatterjee and Chowdhury [30] have shown that crude bile can prevent the binding of LT toxin to GM1. Among the bile components, this inhibitory effect was assigned to unsaturated fatty acids such as linoleic acid. Another study by Nicklasson et al. [31] has demonstrated that crude bile, sodium deoxycholate and bile salts sodium glycocholate hydrate induced expression of csfD gene encoding for CS5 (but not that of CS6), in a dose-dependent manner for the last one. These results suggest that expression of ETEC CFs may be differentially induced along the human intestine, as the concentrations of bile salts sequentially decrease from duodenum to ileum due to reabsorption. Finally, a global transcriptional analysis of two human ETEC strains has shown that the presence of bile salts in LB medium upregulated estA, eltA or etpA (encoding for STa, LTa enterotoxins and EtpA, respectively) while csoA and cstA (encoding for CS1 and CS3 CFs) were downregulated [32] . In this study, the transcriptional response to bile salts was straindependent, suggesting that the results should not be extrapolated to the entire pathovar without further investigation. Data on ETEC virulence gene regulation by digestive enzymes are scare. Trypsin, an endopeptidase secreted in the duodenum, is able to increase LT release [33] and its secretory activity [34] . These findings suggest that bile and intestinal enzymes may act to favor ETEC colonization and virulence in the small intestine.


●●Gut nutrients & gut microbiota generated

metabolites

Among carbon sources found in the intestinal lumen, available studies have mainly focused on the influence of glucose on ETEC virulence. A recent study by Wijemanne and Moxley [35] has shown that, at a concentration optimal for LT expression (0.25%), glucose enhances bacterial adherence to porcine intestinal epithelial cells, through the promotion of LT production. In contrast, glucose prevents transcription of estB (STb) and estA (STa) through catabolite repression [36] . Therefore, the concentration of glucose in the lumen of the small intestine may determine which enterotoxin is maximally expressed. A global transcriptomic analysis by Sahl and Rasko [32] also indicated that the presence of glucose in the growth medium upregulates the expression of fliC and eatA in the E24377A human strain while Haines et al. [37] found that it induces CFA/I surface expression. Mucin is also an important source of carbon for bacteria in the human gut. Two proteases have recently been described for their role in mucin degradation by ETEC [16,17] , indicating that this pathogen may sense and respond to the presence of mucus. The effect of mucin on ETEC virulence has been very poorly investigated. Only one study by Haines et al. [37] has shown that mucin positively influences CFAI, CS1 and CS3 expression. During passage through the human gut, enteric pathogenic bacteria such as ETEC also face a high number of commensal bacteria that compete with them for nutrients and space. A recent study in a cohort of infected patients from Bangladesh has shown that there is a rapid and reversible change in gut microbial community structure and gene abundance after ETEC infection in infants and adults [38] . However, we are not aware of how gut microbiota could influence ETEC virulence. Only one study by Takashi et al. [39] has investigated how short chain fatty acids (SCFAs), major end products of gut microbial fermentation, modulate the production of human ETEC enterotoxins. Addition of SCFAs from C-2 to C-7 (acetic, propionic, butyric, valeric, caproic and heptylic acids) at a concentration of 2 mg/ml in the culture medium significantly


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Review Roussel, Sivignon, Van de Wiele & Blanquet-Diot reduced or even abolished LT production. In the human gut, SCFAs concentrations varied from the small intestine to the colon. It would be of high interest to investigate how the various concentrations of SCFAs found in the different sections of the human intestine may influence the expression of ETEC virulence genes.

also been investigated as an environmental cue for ETEC [40] . Lyte et al. [41] demonstrated that norepinephrine (used in physiological concentrations) increased the in vitro growth of an ETEC strain isolated from calf, as well as the expression of the F5 fimbrial adhesin. On the contrary, Sturbelle et al. [42] did not observe any effect of norepinephrine or epinephrine on the in vitro growth of a piglet ETEC strain and Haines et al. [37] even found a significant inhibition of porcine ETEC by norepinephrine. However, a significant increase in motility and expression of F4 fimbriae and LT toxin encoding genes was shown in the ETEC culture supplemented with conditioned medium (containing autoinducer) and epinephrine [42] . Lastly, Haines et al. [37] found that norepinephrine

●●Cathecholamine hormones

Microbial endocrinology is a newly recognized microbiology research area investigating the interactions of bacteria with host stressassociated hormones. Among them, catecholamines such as epinephrine (adrenaline) and norepinephrine (noradrenaline) play roles as hormones and neurotransmitters. Both have

ETEC survival and virulence

ND

= ST LT (but not excreted)

[28,29]

=

eltA

[33,34]

ND

ND

[30,31,32]

ND

estA, eltA,etpA, astA, csfD ↓ csoA, cstA

[32,35,36,37]

ND

↓

[37]

ND

CFA/I, CS1, CS3

[39]

↓

[37,41,42]

↓ or no effect

Trypsin

Bile acids

Glucose

Mucin

↓

OH-

astA, fliC, eatA ↓ estB, estA

= ST LT

LT

↓ LT (the binding to GM1)

↓

OH-

↓

OH-

↓

↓

High pH

[27,28,29]

Ref.

↓

H+ H+ H+

Toxin production

↓

Low pH

Virulence genes

↓

Gastrointestinal parameters

Survival

LT

ND

SCFAs

eltA, ↓ CFA/I F4, F5 fimbriae

↓ ↓

↓

Norepi/Epinephrine

ND

↓ LT

ND

Figure 2. Modulation of enterotoxigenic Escherichia coli survival and virulence by gastrointestinal cues. The figure recapitulates how physicochemical parameters of the human gut may influence ETEC survival and expression of virulence genes, including toxin production. ETEC: Enterotoxigenic E. coli; ND: Not determined; LT: Heat-labile; SCFA: Short chain fatty acid; ST: Heat-stable.

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Foodborne enterotoxigenic Escherichia coli inhibited CFA/I expression in an ETEC strain isolated from human. Taken together, these results indicate that ETEC may use a variety of environmental cues, mainly present in the small intestine, to modulate toxin and CF expression. Nevertheless, for a more comprehensive understanding of ETEC pathogenesis, the effect of additional variables of the human digestive environment, such as gut microbiota, antimicrobial peptides, oxygen levels, fluid shear or neuroendocrine peptides remain to be explored. Current treatments & future prospects in the fight against ETEC infections in humans ●●Symptomatic treatments during ETEC

diarrhea

The treatment of diarrheal disease due to ETEC is the same as that for cholera or any other acute secretory diarrheal diseases. The Center for Control Diseases gives some recommendation to treat ETEC diarrhea in travelers and in children living in ETEC-endemic areas [6] . Clear liquids such as oral rehydration salts are of utmost importance to prevent dehydration and loss of electrolytes. Bismuth subsalicylate compounds can help reduce the number of bowel movements. Antimotility agents can effectively relieve ETEC-associated diarrhea and cramps but they may be not recommended as they prolong the time it takes the body to rid itself of the toxins [6] . The use of antimicrobials in the treatment of ETEC diarrhea is problematic since an etiologic diagnosis cannot be made rapidly, mainly in childhood diarrhea [2] . The antimicrobial treatment of traveler’s diarrhea has changed over the years because of the increasing resistance of ETEC to common antibiotics, including trimethoprim-sulfamethoxazole and ampicillin. Currently, fluoroquinolones are shown to be effective therapy in ETEC traveler’s diarrhea [6] . However, because resistance to antibiotics is increasing worldwide, the decision to use antibiotics should be carefully weighed against the severity of illness and the risk of adverse reactions, such as rash, antibiotic-associated colitis and vaginal yeast infections. ●●Vaccination strategies

Up to now, there is no commercially available vaccine directed against ETEC bacteria. Nevertheless, given that ETEC share some similarities with Vibrio cholerae (same site of


Review

colonization, and structural, functional and immunological closely related toxins), Dukoral® (Chiron Healthcare SAS, Suresnes, France) can be prescribed to prevent traveler’s diarrheas due to ETEC, but the prescription is limited solely to Cholera in the EU and Australia. Dukoral® may provide protection (reaching 67%) against LT toxin of ETEC in traveler’s diarrhea [43] but protection of populations of children in endemic areas has been more difficult to demonstrate [44] . Economically, it would be more interesting to provide a vaccine that is effective on both travelers’ and children ETEC diarrheas, which is definitely not feasible with Dukoral. Among several vaccine candidates widely reported in the literature [45,46] , the most convincing and still on course could be classified into four groups: cellular candidates (ETVAX, ACE527); subunit candidates (antiadhesinbased subunit vaccine), antitoxin candidates (dmLT) and novel antigen candidates (Flagellin, EtpA, EatA, YghJ). These vaccines are actually under development from preclinical to Phase II assays, but the path seems to be long to counteract lack of protection and adverse effects in order to achieve a protective and long-term efficacy. Indeed, even if the development of an effective ETEC vaccine is a top priority for WHO and several public health institutions around the world, it is still limited by numerous challenges [45] : the serological heterogeneity of ETEC and the wide variety of structurally and functionally distinct CFs; the limited knowledge of ETEC bacterial structure and disease mechanisms; the high costs of each vaccine program. ●●Micronutrient strategies

There is a growing interest in the role of micronutrients as a ‘natural’ nonantibiotic alternative to combat ETEC infections. Zinc supplementation may be used in infant in low-income countries either in prophylaxis to reduce diarrhea morbidity or as a treatment to shorten the duration of symptoms [47] . Even if the mechanisms of this protective effect have not yet been elucidated, several in vitro and in vivo studies have provided some evidence. Zinc administration enhances innate immunity against ETEC infections in children [48] . Besides, zinc oxide protects cultured human enterocytes from the damage induced by ETEC by inhibiting the adhesion of bacteria, preventing the increase of tight junction permeability and modulating cytokine gene expression [49] . The effect


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Direct antagonism

Immunomodulation

Anti-inflammatory response

Lactobacillus spp. Bifidobacterium spp. Escherichia coli UM-2, UM-7 Saccharomyces cerevisiae

Down-regulation of virulence genes

Reduction of ETEC shedding Lactobacillus spp. Escherichia coli UM-2,UM-7 Saccharomyces cerevisiae

Exclusion

Lactobacillus reuteri

estA, estB

Villus

TLR4

Reduction of ST toxin production

Shaping the composition of the gut microbiota Lactobacillus rhamnosus

Toxins CD8

Reuteran, levan from Lactobacillus reuteri

+

P65

CD8+

CDs T cells IGA IL-1, IL-6, IL-8, TNF-α IL-10

Reduction of ETEC growth

Lactobacilli bifidobacteria Coliforms

Lactobacillus sobrius

Improving intestinal barrier integrity

Enterocyte

Lactobacillus plantarum Lactobacillus sobrius Saccharomyces cerevisiae

Decreasing bacterial adhesion

Digestive lumen

MAPK P38 NF-κβ

Saccharomyces cerevisiae Lactobacillus sobrius Pediococcus acidilactici

Paneth cells Piglet gut

Submucosa

Mucosa

Goblet cell

Tight junction proteins ZO-1, occludin and dephosphorylation of occludin TEER Blood vessels

Figure 3. Overview of probiotic activity against enterotoxigenic Escherichia coli under piglet digestive conditions. The figure recapitulates all the in vivo studies in piglets and in vitro studies in porcine intestinal epithelial cells reporting a beneficial effect of probiotics during ETEC infections. Probiotics may act following three modes of action: immunomodulation (in blue), direct antagonism (in green) or exclusion (in orange). ETEC: Enterotoxigenic E. coli; TEER: Transepithelial electrical resistance; TLR: Toll-like receptor; ZO: Zonula occludens.

on ETEC survival or virulence of other metals such as iron and silver has been also suggested. Addition of iron to growth media repressed the expression of CFA/I fimbriae by ETEC [50] while under iron starvation, production of the CFA/I fimbriae was increased in the H10407 strain [37] . Oral administration of silver nanoparticles to infant mice colonized with ETEC bacteria significantly reduced the colonization rate of the pathogen [51] . Other dietary components like phenolic compounds and vitamins might also have potential as new agents against ETEC infections. In vitro, polyphenol extracts inhibited the LT toxin binding to its intestinal receptor GM1 through aggregation [52] . In ETEC-infected piglets, dietary polyphenol and black tea extract reduced diarrhea prevalence [53,54] . A placebo-controlled double-blind study has shown that vitamin A supplementation led to shorter duration of

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ETEC infections among children in Mexico City [55] . The available data highlight some beneficial effects of micronutrients in ETEC infections but the level of evidence (from in vitro studies to clinical trial) widely depends on the tested compound. Besides, this strategy only focused on infantile diarrhea in malnourished children and was not yet applied to traveler’s diarrhea. Lastly, the therapeutic benefits attributable to such micronutrients might be modulated by differential levels of nutrient deficiency in individuals or populations within developing countries. Probiotic-based strategies in the prevention of ETEC infections Given growing concerns about effects of ETEC illness in human [3,5] , prevention remains an important area to address. Besides, in the face of antibiotic resistance increasing worldwide,


Foodborne enterotoxigenic Escherichia coli the development of nonantimicrobial strategies against pathogens is widely encouraged by the EU and WHO. In this respect, one of the most promising preventive strategies in the fight against ETEC infections is the use of probiotic micro-organisms. The internationally endorsed definition of probiotics is ‘Live micro-organisms that, when administered in adequate amounts, confer a health benefit on the host’ [56] . Most micro-organisms recognized to date as probiotics are Gram-positive lactic acid bacteria, with Lactobacillus and Bifidobacterium being the main species used, and yeasts from Saccharomyces genus. Up to date, most probiotics commercialized are single strains. Nevertheless, the efficacy and functionality of multistrain and multispecies probiotics is not called into question since they could be more effective and consistent than monostrain probiotics [57,58] . Even if the underlying mechanisms associated with probiotic protection against enteric pathogens are still largely unclear, three general classes of antipathogenic mechanisms have been proposed: direct antagonism, immunomodulation and competitive

Review

exclusion [59] . Here, we reviewed the in vitro and in vivo studies that have been carried out both in pigs (Figure 3) and in humans (Figure 4) and that show beneficial effects of probiotic bacteria and yeast against ETEC pathogens for each of the three mechanisms listed previously. This part of the review was extended to studies on pigs due to scare data in humans and to similarities between pigs and humans regarding digestive physiology and ETEC infectious process. All these studies are summarized in Table 1 (immunomodulation), Table 2 (direct antagonism) and Table 3 (competitive exclusion). ●●Immunomodulation

Probiotics can interact with the host immune system to enhance the functionality of innate and/or adaptive immunity or to limit the ability of pathogen to induce an immune response. A large number of studies have investigated the immunomodulatory properties of probiotics in ETEC infections. Most of them have been carried out in vitro using porcine intestinal epithelial cells and the K88 ETEC strain

Immunomodulation Anti-inflammatory response

Enterococcus faecium Lactobacillus fructosus Lactobacillus amylovorus

Exclusion

HSP TLR 4

P65

Decreasing bacterial adhesion

Villus

Pediococcus pentasaceus Lactobacillus casei rhamnosus Bidobacterium adolescentis

IL-8

Enterocyte

Enterococcus faecium Lactobacillus fructosus

Bifidobacterium longum

BIF Mucus layer

Human gut

Tight junction proteins TEER Blood vessels

Submucosa

Mucosa

Goblet cell

Improving intestinal barrier integrity

Digestive lumen

NF - κβ

Figure 4. Overview of probiotic activity against enterotoxigenic Escherichia coli under human digestive conditions. The figure recapitulates all the in vitro studies in human intestinal epithelial cells showing a beneficial effect of probiotics during ETEC infections. Only two modes of action of probiotics have been investigated: immunomodulation (in blue) and exclusion (in orange). BIF: Binding inhibitory factor; ETEC: Enterotoxigenic E. coli; TEER: Transepithelial electrical resistance; TLR: Toll-like receptor.



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Zhou et al. (2014)



Finamore et al. (2014)

Therapy

Lactobacillus amylovorus strain DSM 16698

Lactobacillus fructosus C2

F4+

Lactobacillus sobrius strain DSM 16698 Enterococcus faecium NCIMB 10415

F4+

ETEC IMT 4818 and O149:K91/ K88 (F4+) F4+

F4+ad; O8:K87:H19 (56190); GN1034 F4+ F4+ad and O8:K87:H19 (56190)

Saccharomyces cerevisiae var. boulardii and β-galactomannan oligosaccharide Saccharomyces cerevisiae CNCM-I3856

Bifidobacteria longum BB536; Bifidobacteria breve M-16V

Lactobacillus delbrueckii TUA4408L and its extracellular polysaccharides

5.107 CFU/ml (PRO); 5.106 CFU/ml (ETEC)

Unknown

3 yeasts/cell (PRO); 10 μg/ml of βGM; 1×107 CFU/well (ETEC) 7.5 × 105 yeasts/ml (PRO); 1×107 CFU/well (ETEC) 109 CFU/ml (PRO); 108 CFU/ml (ETEC) 108 CFU/ml (PRO); 106 CFU/ml (ETEC)

Curative

Curative

Preventive

Preventive

Curative

Curative

F4+ and 108 CFU/ml (PRO Preventive O149:K88 (JG280) and ETEC) 987P and F4+ 5 × 107 CFU/ml Preventive (PRO); 5 × 107 CFU/ ml (ETEC); 100 μg/ml (NPS) 987P 5 × 107 CFU/ml (PRO Preventive and ETEC)

Lactobacillus reuteri CL9

5 × 107 CFU/ml (PRO Curative and ETEC)

Doses PRO/ETEC

987P

ETEC strain

Bifidobacterium breve MCC-17

Probiotic strain

AI: Anti-inflammatory; ETEC: Enterotoxigenic Escherichia coli; PI: Pro-inflammatory; PRO: Probiotic.

Yu et al. (2015)

Klingspor et al. (2015)

Co-culture in human intestinal cells

Roselli et al. (2007)

Zanello et al. (2011)

Badia et al. (2012)

Tomosada et al. (2013)

Wachi et al. (2014)

Murata et al. (2014)

Study (year)

Co-culture in piglet intestinal cells

In vitro

Model

Table 1. Immunomodulation.

Down-regulation of IL-8 and activation of IL-10 expression Decrease of IL-8 mRNA /protein production and reduction of HSP70 stress response Reduction of ERK and JNK activation. Decrease of IL-8 production Suppression of TLR4 activation, inhibition of HSP and P65 translocation; suppression of PI cytokine production

Decrease of PI cytokines (TNF, IL-1,6,8) production

AI effect by negative regulation of TLRs, NF-κβ, p38 MAPK and PI3K Decrease of IL-8 (PI) and increase of IL-10 (AI) production Reduction of NF-κβ, ERK, MAPK and TLRs activation; down regulation of PI cytokine expression (IL-6, IL-8) Reduction of TLR4, NF-κβ and MAPK activation; decrease of PI IL-6 and IL-8 Down-regulation of PI cytokines (TNF, IL-6) and chemokines (CCL2, CCL20, CXCL8) expression

Mechanisms

[68]

[22]

[67]

[66]

[65]

[64]

[63]

[62]

[61]

[60]

Ref.



109 CFU/ml (PRO); 105 CFU/ml (ETEC)

Lactobacillus plantarum Biocenol LP96


Lactobacillus rhamnosus GG ATCC 53103 F4+ ac, O149:K91


Lactobacillus rhamnosus ATCC F4+ ac, O149:K91 7469

AI: Anti-inflammatory; ETEC: Enterotoxigenic Escherichia coli; PI: Pro-inflammatory; PRO: Probiotic.

Zhang et al. (2010)

Li et al. (2012)

Chytilova et al. (2013)

O8:K88ab:H9


Zhu et al. (2014)

Lactobacillus rhamnosus ATCC F4+ ac, O149:K91 7469

109 to 109 CFU/ml (PRO and ETEC)

Doses PRO/ETEC

F4+

ETEC strain

Probiotic strain

E. coli UM-2, UM-7

Study (year)

Oral administration to Khafipour et al. (2014) piglets

In vivo

Model

Table 1. Immunomodulation (cont.). Mechanisms

Preventive Downregulation of PI cytokine and curative expression (IL-6, TNFα) in the serum Preventive Attenuation of CD3+ CD4+ CD8+ T cells increase in the small intestine Preventive Down-regulation of PI cytokine expression (IL-1α, IL-8) and upregulation of AI IL-10 in the small intestine Preventive Decrease of TNFα and attenuation of IL-8 and TLR4 increase in the small intestine Preventive Attenuation of IL-6 increase in the serum. Enhancement of intestinal antibody defense

Therapy

[73]

[72]

[71]

[70]

[69]

Ref.



Review

10.2217/fmb-2016-0101

Review Roussel, Sivignon, Van de Wiele & Blanquet-Diot (F4+) which is the most prevalent in pig postweaning diarrhea. Probiotic strains from the Lactobacillus [61,62,66] and Saccharomyces [64,65] genus significantly reduced the expression of proinflammatory cytokines, such as IL-1, IL-6 and IL-8 and induced an upregulation of the anti-inflammatory IL-10. These effects have been observed both in prophylaxis and curative treatments. The mechanisms associated with the anti-inflammatory effect of probiotic strains were further explored for Bifidobacterium [60,63] and Lactobacillus strains [62] . These probiotics inhibited ETEC-mediated MAPK and NF-κB

activation by upregulating TLR negative regulators [60,62,63] . Only two studies [67,68] have investigated the effects of bacterial probiotics on ETEC-induced inflammatory response in human Caco-2 intestinal cells, but still using the porcine K88 strain. Both Enterococcus faecium [67] and Lactobacillus amylovorus [68] inhibited the overproduction of proinflammatory cytokines induced by ETEC and blocked the upregulation of heat shock protein (Hsp), especially that of Hsp72 and Hsp90 which are critical for TLR4 function. In the study by Finamore et al. [68] , both the Lactobacillus

Table 2. Direct antagonism. Model

Study (year)

Probiotic strain

ETEC strain

Doses PRO/ETEC

Lactobacillus acidophilus RY2; Lactobacillus salivarius MM1; Lactobacillus paracasei En4 Lactobacillus spp.

109CFU/ml (PRO); BCRC 15372 108CFU/ml (ETEC) & BCRC 41443 and UM4247 and EK06 F4+ ac 107CFU/ml (PRO); 108CFU/ml (ETEC)

Zhou et al. (2014)

Lactobacillus reuteri CL9

F4+, O149:K88 (JG280)

108CFU/ml (PRO and ETEC)

Oral administration to piglets

Trevisi et al. (2015)

F4+ ac; O149

Yang et al. (2015)

Saccharomyces cerevisiae CNCM I-4407 Lactobacillus reuteri TMW1.656 Lactobacillus reuteri LTH5794

5.1010CFU/ml (PRO); 108CFU/ml (ETEC) 107 CFU/ml (PRO and ETEC)

Lee et al. (2015)

Lactobacillus plantarum CJLP243

F4+ ac

Khafipour et al. (2014)

E. coli UM-2, UM-7

F4+

Zhang et al. (2010) Lactobacillus F4+ ac; rhamnosus GG ATCC O149:K91 53103 Konstantinov et al. Lactobacillus sobrius F4+ (2008) DSM 16698

Therapy

Mechanisms

Ref.

In vitro Co-culture in culture Tsai et al. (2008) medium


Hillman et al. (1995)

Preventive Inhibition of ETEC growth due to antimicrobial activity of lactic acid

[74]

Preventive Inhibition of ETEC growth (in continuous culture of porcine intestinal bacteria) Preventive Reduction of ETEC virulence genes expression (estA, estB)

[75]

Preventive Reduction of ETEC fecal shedding

[76]

Curative

Reduction of ETEC colonization and number of ST gene copies in feces and in colonic digesta by feed fermentation with L. reuteri (reuteran and levan) Reduction of ETEC fecal shedding

[77]

[69]


Preventive Reduction of ETEC fecal and shedding curative Preventive Reduction of ETEC fecal shedding

1010 CFU/ml (PRO and ETEC)

Preventive Reduction of ETEC levels in the ileum

[79]

[61]

In vivo

ECL13795

1010 CFU/kg (PRO); 5 × 109 CFU/ml (ETEC) 107 to 109 CFU/ml (PRO and ETEC)

Curative

ETEC: Enterotoxigenic Escherichia coli.

10.2217/fmb-2016-0101



[78]

[73]

Foodborne enterotoxigenic Escherichia coli strain and its secreted products showed anti-inflammatory properties. In vivo studies in ETEC-infected piglets confirmed the anti-inflammatory potential of probiotic strains, mainly that of Lactobacillus spp strains. Challenged pigs fed with Lactobacillus probiotic strains had lower levels of proinflammatory cytokines in intestinal tissues [70–72] . Zhu et al. [70] have also investigated the modulation of systemic and intestinal lymphocyte T cells subpopulations by Lactobacillus rhamnosus and showed that the probiotic strain can attenuate the ETEC-induced increase in CD3+ CD4 + CD8+ T cells in the small intestine. Li et al. [72] found that the same probiotic strain reduced the increase of TLR4 expression observed both at the mRNA and protein levels in the jejunum of infected piglets. Of note, the results of these two studies suggest that pretreatment with a low dose of Lactobacillus rhamnosus might be more effective than with a high one. ●●Direct antagonism

In direct antagonism, probiotics kill or inhibit the growth of pathogen to limit the spread of the infection or they downregulate the expression of virulence factors, such as toxins or adhesins, required for pathogenesis. Only two in vitro studies showed the inhibition of the growth of ETEC strains isolated from human or pig, following co-incubation of the pathogen with the culture supernatant of Lactobacillus species. In the study by Tsai et al. [74] , the inhibitory activity was partially affected by lactate deshydrogenase treatment showing that it may result from lactic acid production. Strains of Lactobacillus spp also increased the rate of decline of ETEC K88 in a continuous culture of porcine intestinal bacteria, which provides a more realistic in vitro situation for the examination of probiotic potential than co-culture does [75] . In piglets orally challenged with ETEC strain K88 or ECL13795, ingestion of diets containing Escherichia coli [69] , Lactobacillus plantarum [78] , Lactobacillus reuteri [77] or Saccharomyces cerevisiae [76] , probiotic strains led to a lower fecal shedding of the pathogen. Similarly, Lactobacillus sobrius significantly reduced the prevalence of ETEC K88 in the ileum of piglets supplemented with the probiotic [79] . This antagonistic effect was not associated with any change in luminal pH. The reduced levels of ETEC in the ileum and feces may result from colonization of gut mucosa by the probiotic thereby reducing the attachment of the pathogen to the intestinal surface or from


Review

host microbiota modulation (see the section on ‘Exclusion’). Only two studies have assessed the effect of a probiotic strain on the expression of ETEC virulence genes. Zhou et al. [61] have shown that Lactobacillus reuteri reduced the expression of ST encoding genes (estA and estB) but not that of LT (elt), in ETEC K88 strain JG280 at the early stage of its infection to porcine intestinal epithelial cells. The underlying mechanisms have not been explored by these authors but Yang et al. [77] have shown in weaning piglets that reuteran-containing diets (an exopolysaccharide produced by Lactobacillus reuteri) reduces the copy number of ST gene and the toxin level in samples from the ileum, cecum and colon. ●●Exclusion

Exclusion is used to describe all mechanisms that make the gastrointestinal environment less hospitable for pathogens. These mechanisms include decreasing luminal pH, modulating gut microbiota, improving epithelium barrier function, interfering with pathogen binding and translocation and stimulating production of defense-associated factors, such as mucins and defensins. Even if most of the available studies have investigated the probiotic activity of lactic acid bacteria, none of them has associated their beneficial effect with a decrease in luminal pH. Only two studies in piglets have investigated how probiotics may modulate gut microbiota during ETEC infections. In both studies, oral administration of Lactobacillus rhamnosus counteracted the rise in the fecal shedding of coliforms in ETEC-infected animals and increased the number of Lactobacilli and Bifidobacteria [72,73] . These findings suggest that treatment with specific Lactobacillus strains may be used in piglets to restore the homeostasis of an impaired microbial ecosystem associated with weaning [72] and/or ETEC challenge [73] . In vitro studies using both pig and human enterocytes in culture and in vivo studies in piglets also showed that probiotic strains from Lactobacillus genus and Enterococcus faecium spp may protect the integrity of intestinal epithelial barrier damaged by ETEC. In vitro, treatment of intestinal cells with probiotics prevented the ETEC-induced decrease of transepithelial resistance [67,80,81] , reduced the permeation of tracers such as dextran [22] and regulated tight


10.2217/fmb-2016-0101

10.2217/fmb-2016-0101



Li et al. (2012)

Lactobacillus rhamnosus ATCC 7469

F4+ ac; O149:K91

ETEC: Enterotoxigenic Escherichia coli; TEER: Trans-epithelial electrical resistance; ZO: Zonula occludens.

F4+ ac; O149:K91

Lactobacillus plantarum CGMCC 1258

Yang et al. (2014)

F4+ ac; O149

Saccharomyces cerevisiae CNCM I-4407

Pb 176 (human)

LMG 3083 (human) Unknown

O149:K91:K88 (F4+) ETEC IMT 4818 O149:K91/K88 (F4+) F4+

Oral administration to Trevisi et al. (2016) piglets

In vivo

Lactobacillus fructosus C2

Enterococcus faecium NCIMB 10415 Enterococcus faecium NCIMB 10415

F4+ ad; O8:K87:H19 (56190); GN1034 F4+

Saccharomyces cerevisiae var. boulardii and β-galactomannan oligosaccharide (Salmosan®, prebiotic) Lactobacillus sobrius strain DSM 16698 F4+

F4+ ac, O149:K91

ETEC strain

Lactobacillus plantarum CCGMCC1258

Probiotic strain

Osmanagaoglu et al. Pediococcus pentosaceus (2010) OZF Zhong et al. (2004) Bifidobacterium adolescentis 1027 Fujiwara et al. (2001) Bifidobacterium longum SBT2928 (BL2928)

Yu et al. (2015)

Roselli et al. (2007)

Lodemann et al. (2015) Klingspor et al. (2015)

Co-culture in human intestinal cells

Badia et al. (2012)

Wu et al. (2016)

Study (year)


In vitro

Model

Table 3. Competitive exclusion.

Curative

Preventive

Preventive

Preventive

Curative

Preventive

Preventive

Preventive

Curative

Preventive

Therapy

1010 CFU/ml (PRO) 109 CFU/ml (ETEC)

Preventive

5.1010 CFU/kg of diet Preventive (PRO); 108 CFU/ml (ETEC)

5.1010 CFU/kg (PRO); 108 CFU/ml (ETEC)

108 CFU/ml (PRO); 108 CFU/ml (ETEC) 108 CFU/ml (PRO and ETEC) 0.5g/ml (BIF protein); 109 CFU/ml (ETEC)

Unknown

108 CFU/ml (PRO); 108 CFU/ml (ETEC) 108 CFU/ml (PRO); 106 CFU/ml (ETEC)


3 yeasts/cell (PRO); 10 μg/ml of βGM; 1 × 107 CFU/well (ETEC)

108 CFU/well (PRO) 6.5 × 107 CFU/ml (ETEC)

Doses PRO/ETEC

Limitation of early activation of genes related to the impairment of the jejunal mucosa associated with ETEC infection Protection from ETEC-induced membrane damage, decrease of ZO-1 and occludin (tight junction proteins) expression in the jejunum Modulation of pig fecal microbiota composition: increase in Lactobacilli and Bifidobacteria

Reduction of intestinal permeability and maintain of barrier integrity Decrease in the number of adhering ETEC Competitive inhibition of ETEC adherence by adhesin production Inhibition of interactions with ETEC receptor GA1 by BIF issued from the extracellular protein fraction of probiotic culture

Maintain of barrier integrity by inhibiting bacterial adhesion and ETECinduced occludin dephosphorylation Reduction of ETEC-induced decrease of TEER Reduction of ETEC-induced decrease of TEER

Inhibition of the reduction of tight junctions proteins and improvement of epithelial barrier integrity by maintaining TEER Inhibition of bacterial adhesion

Mechanisms

[72]

[86]

[85]

[84]

[83]

[82]

[22]

[67]

[81]

[66]

[64]

[80]

Ref.


[73]

Modulation of pig fecal microbiota composition: increase in Lactobacilli and Bifidobacteria

[87]


ETEC: Enterotoxigenic Escherichia coli; TEER: Trans-epithelial electrical resistance; ZO: Zonula occludens.

O149:F4+ (ECL8559)

Oral administration to Daudelin et al. (2011) piglets (cont.) Zhang et al. (2010)

Pediococcus acidilactici; Saccharomyces cerevisiae boulardii Lactobacillus rhamnosus GG; ATCC 53103

F4+ ac, O149:K91

9

Preventive

Preventive

10 CFU/pig/day (PRO) 109 CFU/5ml (ETEC) 1010 CFU/ml (PRO) 109 CFU/ml (ETEC)

Reduction of ETEC F4 attachment to the ileal mucosa

Therapy Doses PRO/ETEC ETEC strain Probiotic strain Study (year) Model

Table 3. Competitive exclusion (cont.).

Mechanisms

Ref.


Review

junction and cytoskeleton proteins by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin and dephosphorylation of occludin caused by ETEC [66,80] . Such in vitro beneficial effects of probiotic Lactobacillus strains have been strengthened by an in vivo study in piglets. Yang et al. [86] showed that Lactobacillus plantarum prevented the damage to intestinal morphology and greater intestinal permeability (measured by a functional lactulose-mannitol absorption test) induced by ETEC K88 challenge and lowered plasma endotoxin concentrations. In addition, the reduction in ZO-1 and occludin mRNA abundance observed in the jejunum after ETEC infections was inhibited in piglets fed Lactobacillus plantarum. Lastly, a recent study by Trevisi et al. [85] have also shown that feeding pigs with Saccharomyces cerevisiae was effective in counteracting the detrimental effect of ETEC infection by limiting the early activation of genes related to the impairment of the jejunal mucosa. In addition, several in vitro studies on pig [64] or human [82] intestinal cells in culture have shown that probiotic bacteria or yeast may decrease the number of adhering ETEC. In these studies, ETEC strains from both porcine and human origins have been tested. Only an in vivo study in piglets has demonstrated that Pediococcus acidilactici and Saccharomyces boulardii significantly reduced attachment of ETEC K88 to the ileal mucosa [87] . Little is known about the mechanism of inhibition of ETEC adhesion by probiotics. It has been assumed that probiotics may (i) impede the access of pathogens to tissue receptors by nonspecific steric hindrance, (ii) interact with the levels of mucins produced and thus impair the adhesion of pathogens or (iii) block the binding site of ETEC to intestinal epithelial cells by receptor competition. This last hypothesis was the most studied with regards to ETEC pathogens. Purified adhesin of Bifidobacterium adolescentis effectively inhibits ETEC adherence to intestinal epithelial cells in vitro [83] . Fujiwara et al. [84] reported that Bifidobacterium longum produces a proteinaceous inhibitory factor termed binding inhibitory factor (BIF), which prevents the binding of ETEC to GA1 in a dose-dependent manner and also inhibits their adherence to HCT-8 human epithelial cells. Other authors rather suggest the role of carbohydrate structures in the inhibition of adhesion [64,82] .


10.2217/fmb-2016-0101

Review Roussel, Sivignon, Van de Wiele & Blanquet-Diot ●●Human trials involving probiotics

Up to date, the vast majority of in vivo studies involving ETEC and probiotic strains have been carried out in piglets, most often in the specific context of the postweaning phase. Only two studies have investigated the effect of probiotics in human volunteers when orally challenged with live attenuated ETEC strains. Unfortunately, these two studies showed that supplementation with either a single strain of Lactobacillus acidophilus [88] or a blend of probiotic bacteria and yeast [89] was ineffective in reducing ETEC infection symptoms in healthy men. Besides, the study by Ouwehand et al. [88] provides useful data on changes in human intestinal microbiota associated with ETEC infections, the authors showing a reduction of the fecal levels of Bacteroides-Prevotella, Bifidobacterium and Clostridium clusters XIVa and b. The ineffective effects of probiotics may be linked to the challenge model or to the fact that in these studies the disease mechanisms was toxin independent (ETEC strains do not produce LT and ST toxins). Other studies have shown a significant reduction in the risk of traveler’s diarrhea when probiotics such as S. boulardii are given [90] , but ETEC strains have not been clearly involved in the etiology of diarrhea. Then, a significant number of in vitro or in vivo studies have shown the beneficial effects of probiotics against ETEC pathogens, by interfering with their survival, adhesion to mucosa or expression of virulence genes. Nevertheless, most of these studies involve strains such as K88 which are pathogenic for piglets but not for human and the two only in vivo studies in humans were performed with non toxigenic strains. As it is highly probable that the outcomes would be strain-dependent, additional studies involving strains pathogenic for humans are required. Future perspective Among the new preventive therapies in ETEC infections, probiotics emerge as a relevant strategy, a significant number of in vitro and in vivo studies showing their beneficial effects through immunomodulation, direct antagonism or exclusion. Probiotics may use a combination of these mechanisms, giving the opportunity to consider a multifaceted approach in the fight against ETEC pathogens, definitely more relevant than a targeted one. Nevertheless, up to date, most of

10.2217/fmb-2016-0101


these studies have been carried out under piglet digestive conditions that can markedly differ from those observed in the human GI tract. Notable divergences between pig and human digestive conditions are known to be the following ones: dietary intake and coprophagy; volume of stomach and length of intestinal tract; gastric lipase biochemical characterization and bile acid composition; gut microbiota composition; and privileged caecal fermentation in pigs [91–97] . In addition, ETEC physiopathology shows specific features in pigs: most of strains pathogenic in pigs are not pathogenic in humans; animal CFs are distinct from those in human isolates (the most common in pigs are F4 -K88-, F5 -K99and F6 -987P-); pigs produce STp (STa toxins undercategorized) and LT-Ip enterotoxins structurally and antigenically different from human toxins; and host genetic susceptibility can vary with the pig genotype [2,96,97] . Besides, the available studies remain merely descriptive and further efforts have to be made to better understand the underlying modes of action. In particular, even if probiotics may act through modulation of human intestinal microbiota, no study has yet investigated the interactions between ETEC, probiotics and gut microbiota. As studies on human volunteers are ethically inconceivable with nonattenuated ETEC strains, a relevant alternative to explore interactions between pathogen and probiotics would be to use human microbiota-associated pigs [98] or in vitro models that mimic the conditions found in the human digestive tract. To date, a large number of artificial digestive systems have been developed but most of them are simulating oversimplified conditions compared with the in vivo situation [99,100] . Only two well-validated dynamic multicompartmental systems are currently available, the TNO (Nederlandse Organisatie Voor Toegepast) GastroIntestinal Model (TIM) and the Simulator of Human Intestinal Microbial Ecosystem (SHIME). These models simulate the complex evolution of the main physicochemical (e.g. pH, digestive secretions, transit time) and biotic (gut microbiota) parameters of human digestion, which are key parameters in the survival, virulence and/or activity of pathogens and probiotics. In particular, TIM and SHIME are of great interest to: assess how the dose and mode of administration of probiotics (e.g. galenic forms or functional foods) may influence their viability in the human GI tract and their antagonistic


Foodborne enterotoxigenic Escherichia coli effects against ETEC bacteria [101] ; and investigate how probiotics and ETEC interact with luminal and mucosal gut microbiota under physiological fluid shear stresses and microaerobic conditions [102] . They could be therefore advantageously used as a complement to in vivo assays to better define the cellular and molecular mechanisms of probiotic strains and their appropriate conditions of use, which are questions that need to be addressed before commercialization and use of probiotics in ETEC infected patients. Here, the most likely scenario for probiotics is their marketing as food supplement with a specific health claim on ‘disease risk

Review

reduction’ (Article 14 from the EC Regulation No. 1924/2006 on nutrition and health claims made on foods). Financial & competing interests disclosure This work was supported by a grant from Lesaffre Company (Marcq-en-Baroeul, France) to Charlène Roussel. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary ETEC physiopathology ●●

Enterotoxigenic Escherichia coli (ETEC) are major food and water-borne pathogens responsible for cholera-like diarrhea.

●●

ain virulence traits of the pathogen are colonization factors mediating intestinal adhesion and production of M enterotoxins.

Modulation of ETEC survival & virulence in the human gut ●●

F ew in vitro studies have addressed the influence of biotic and abiotic factors of the human gut on ETEC survival and virulence.

●●

E TEC may use a variety of gastrointestinal cues, such as variations in pH, bile, short chain fatty acids concentrations or host hormones, to modulate toxin and colonization factor expression.

Current treatments & future prospects in the fight against ETEC ●●

T reatment of ETEC infections is essentially symptomatic and antibiotics should be used with caution due to the growing burden of antimicrobial resistance worldwide.

●●

Main alternative prophylactic treatments include vaccination and nutritional strategies.

Probiotic-based strategies in the prevention of ETEC infections ●●

Use of probiotics is among the most promising preventive strategies in ETEC infections.

●●

I n vitro and in vivo studies have shown the beneficial effects of probiotics, mainly lactic acid bacteria, against ETEC through immunodulation, direct antagonism or exclusion.

●●

Most of these studies were carried out under piglet digestive conditions and remain merely descriptive.

●●

ynamic human digestion models could be advantageously used to better describe the interactions between ETEC, D probiotics and human gut microbiota. prevention. Clin. Microbiol. Rev. 18(3), 465–483 (2005).

References Papers of special note have been highlighted as: • of interest; •• of considerable interest 1

2

Clements A, Young JC, Constantinou N, Frankel G. Infection strategies of enteric pathogenic Escherichia coli. Gut Microbes 3(2), 71–87 (2012). Qadri F, Svennerholm AM, Faruque ASG, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and


•• Gives a comprehensive review on enterotoxigenic Escherichia coli (ETEC) pathogen in developing countries from historical aspects to treatments. 3

Kotloff KL, Nataro JP, Blackwelder WC et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382(9888), 209–222 (2013a).

4

Steffen R, Castelli F, Dieter Nothdurft H, Rombo L, Jane Zuckerman N. Vaccination against enterotoxigenic Escherichia coli, a cause of traveler’s diarrhea. J. Travel. Med. 12(2), 102–107 (2005).

5

Lanata CF, Fischer-Walker CL, Olascoaga AC et al. Global causes of diarrheal disease mortality in children