Published online September 3, 2007
PEDIATRICS Vol. 120 No. 4 October 2007, pp. e795-e803 (doi:10.1542/10.1542/peds.2006-2930)
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My File Cabinet
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liévin-Le Moal, V.
Right arrow Articles by Servin, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liévin-Le Moal, V.
Right arrow Articles by Servin, A. L.
Related Collections
Right arrow Infectious Disease & Immunity
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

ARTICLE

An Experimental Study and a Randomized, Double-Blind, Placebo-Controlled Clinical Trial to Evaluate the Antisecretory Activity of Lactobacillus acidophilus Strain LB Against Nonrotavirus Diarrhea

Vanessa Liévin-Le Moal, PhDa,b, Luis E. Sarrazin-Davila, MDc and Alain L. Servin, PhDa,b

a Faculté de Pharmacie, Institut National de la Santé et de la Recherche Médicale, Université Paris XI, UMR-S 756 Signalisation et Physiopathologie des Cellules Epithéliales, Châtenay-Malabry, France
b Faculté de Pharmacie, Université Paris-Sud 11, Châtenay-Malabry, France
C Department of Pediatrics, Hospital Clinica Kennedy, Guayaquil, Ecuador


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVE. Previous studies have shown that selected strains of Lactobacillus have the capacity to antagonize rotavirus-induced diarrhea. However, only a few reports have documented their efficacy against nonrotavirus diarrhea. This study involved an experimental investigation and a clinical trial of the antisecretory activity of Lactobacillus acidophilus strain LB in the context of nonrotavirus diarrhea.

METHODS. The activity of a culture of L acidophilus LB or of the lyophilized, heat-killed L acidophilus LB bacteria plus their spent culture medium was tested in inhibiting the formation of fluid-formed domes in cultured human intestinal Caco-2/TC7 cell monolayers infected with diarrheagenic, diffusely adhering Afa/Dr Escherichia coli C1845 bacteria. A randomized, double-blind, placebo-controlled clinical trial of male or female children who were 10 months of age and presented with nonrotavirus, well-established diarrhea was conducted to evaluate the therapeutic efficacy of a pharmaceutical preparation that contains 10 billion heat-killed L acidophilus LB plus 160 mg of spent culture medium.

RESULTS. Infection of the cells with C1845 bacteria that were treated with L acidophilus LB culture or the lyophilized, heat-killed L acidophilus LB bacteria plus their culture medium produced a dosage-dependent decrease in the number of fluid-formed domes as compared with cells that were infected with untreated C1845 bacteria. The clinical results show that in selected and controlled homogeneous groups of children with well-established, nonrotavirus diarrhea, adding lyophilized, heat-killed L acidophilus LB bacteria plus their culture medium to a solution of oral rehydration solution shortened by 1 day the recovery time (ie, the time until the first normal stool was passed) as compared with children who received placebo oral rehydration solution.

CONCLUSIONS. Heat-killed L acidophilus LB plus its culture medium antagonizes the C1845-induced increase in paracellular permeability in intestinal Caco-2/TC7 cells and produces a clinically significant benefit in the management of children with nonrotavirus, well-established diarrhea.

Key Words: children • nonrotavirus diarrhea • Lactobacillus • anti-secretory activity

Abbreviations: ORS—oral rehydration solution • DAEC—diffusely adhering Escherichia coli • PBS—phosphate-buffered saline • Sat—secreted autotransporter toxin • MRS—De Man, Rogosa, Sharpe • CFCS—cell-free culture supernatant • CFU—colony-forming units • LDH—lactate dehydrogenase

In developing countries, acute diarrhea is a major cause of morbidity and mortality, as a result of severe socioeconomic difficulties and the high prevalence of childhood malnutrition. Probiotic agents have been recently redefined as live microorganisms that, when consumed in adequate amounts as part of the diet, confer a health benefit to the host.1 Clinical reports have documented the therapeutic efficacy of live, selected Lactobacillus strains used as probiotic agents against infectious diarrhea.2,3 In particular, Lactobacillus rhamnosus strain GG,4–10 Lactobacillus casei strain DN-114 001,11,12 and Lactobacillus reuteri strain 211213,14 have demonstrated their therapeutic efficacy against diarrhea mainly of rotavirus origin. Van Niel et al15 and Szajewska et al16 conducted systematic reviews of published, randomized, double-blind, placebo-controlled trials on probiotic agents and concluded that the duration of rotavirus-induced diarrhea was significantly shorter in children who received live Lactobacillus strains than in those who received the placebo. Administering the nonprobiotic, lyophilized, heat-killed Lactobacillus acidophilus LB bacteria plus their spent culture medium in addition to oral rehydration solution (ORS) reduced the duration of rotavirus-induced, acute, watery diarrhea in children.17,18 However, it should be noted that probiotic agents have not shown any beneficial effect in children with severe disease.19,20

In the past 10 years, it has been demonstrated using cellular and animal models that L acidophilus strain LB exhibits antibacterial activity.21–26 This study involved clinical and experimental investigations of the antisecretory activity of L acidophilus strain LB in the context of nonrotavirus diarrhea. An experimental study was conducted to demonstrate the antisecretory activity of the heat-killed, lyophilized L acidophilus LB bacteria plus their spent culture medium, in a context of increased paracellular permeability leading the inducing of fluid accumulation in cultured human intestinal Caco-2/TC7 cell monolayers by the diarrheagenic, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain C1845.27 In addition, to assess the therapeutic efficacy of lyophilized, heat-killed L acidophilus LB bacteria plus their spent culture medium as an adjunct to ORS, we conducted a clinical trial in children who had nonrotavirus, well-established, acute diarrhea and were hospitalized at the Department of Pediatrics, Hospital Clínica Kennedy (Guayaquil, Ecuador).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Bacterial Strains
The L acidophilus strain LB was provided by the Laboratoire du Lactéol (Houdan, France). The wild-type Afa/Dr DAEC strain C184527 was a gift of S. Moseley (Washington University, Seattle, WA). The recombinant Escherichia coli strain AAEC185 expressing the secreted autotransporter toxin (Sat; AAEC185 psat) was used.28 The stock culture was maintained on 10% glycerol at –80°C. Before the experiments, the bacterial strain was transferred onto fresh Luria-Bertani agar (Difco Laboratories, Detroit, MI) and incubated at 37°C for 24 hours. For each experiment, bacteria were subcultured in Luria-Bertani broth (Difco Laboratories) at 37°C for 18 hours. For the experiment, the bacteria were washed 3 times with sterile phosphate-buffered saline (PBS) and recovered in an appropriate medium as described. Bacterial cells were counted in a Salumbini chamber and then adjusted to the desired concentration. L acidophilus strain LB was grown in De Man, Rogosa, Sharpe (MRS) broth (Difco Laboratories) for 18 hours at 37°C. Lactobacillus cell-free culture supernatant (LB-CFCS) was obtained by centrifuging at 10000g, for 30 minutes at 4°C. After being centrifuged, CFCS was passed through a sterile 0.22-µm Millex GS filter unit (Millipore, Molsheim, France). Batches of heated and lyophilized L acidophilus LB culture plus their spent culture medium were provided by Laboratoire du Lactéol. The lyophilized, heat-killed LB culture was reconstituted before use by dissolving 1000 mg of a batch preparation in 5 mL of sterile PBS.

Cell Culture
The cells used were human intestinal Caco-2/TC7 cells.29 These cells were routinely grown using Dulbecco's modified Eagle's minimal essential medium (Invitrogen, Cergy-Pontoise, France; 25 mM glucose), supplemented with 15% heat-inactivated (30 minutes, 56°C) fetal calf serum (Invitrogen) and 1% nonessential amino acids. For maintenance purposes, cells were passaged weekly using 0.02% trypsin in Ca2+Mg2+-free PBS that contained 3 mM EDTA. The experiments and cell maintenance were conducted at 37°C in an atmosphere of 10% CO2/90% air. The culture medium was changed daily.

Cell Infection
For assays of infection, fully differentiated Caco-2/TC7 cell monolayers were used at postconfluence after 15 days in culture.24 Briefly, the cell monolayers were washed twice with sterile PBS. Infecting bacteria were suspended in cell culture medium, and a total of 108 colony-forming units (CFU) per well of this suspension was added to each well of the tissue culture plate. The infection assay was conducted in the presence of 1% mannose to prevent type 1 fimbriae-mediated binding. The plates were incubated at 37°C in 10% CO2/90% air for 3 hours. The monolayers were then washed 3 times with sterile PBS. Each assay was conducted in triplicate, with 3 successive passages of Caco-2/TC7 cells.

The inhibition of C1845-induced dome formation was determined by pretreating the bacteria (108 CFU/mL) for 1 hour with L acidophilus LB culture, or lyophilized, heat-killed LB culture, or not. The bacterial co-culture was centrifuged at 5500g for 5 minutes at 4°C, the culture medium was discarded, and the bacteria were washed once with sterile PBS before being resuspended in cell culture medium. Caco-2/TC7 monolayers were infected with untreated C1845 or L acidophilus LB culture–treated or lyophilized, heat-killed LB culture–treated C1845 bacteria for 3 hours at 37°C in 10% CO2/90% air.

Measurement of Cell Integrity
Cell integrity was determined by measuring the lactate dehydrogenase (LDH) activity in the culture medium (Enzyline LDH kit, Biomérieux, Dardilly, France) according to the manufacturer's instructions. Proteins were assayed by the bicinchonic acid protein assay. Results were expressed as milliunits of LDH activity per milligram of protein.

Bacterial Adhesion
For determination of the levels of cell-associated C1845 bacteria, infected Caco-2/TC7 cell monolayers were lysed with H2O2 for 10 minutes. Bacteria were suspended by vigorous pipetting, and bacterial CFU/mL of the lysates were determined by plating serially diluted aliquots on tryptic soil agar.

Determination of Fluid-Formed Domes in Caco-2/TC7 Cell Monolayers
For visualization of the domes that formed in control or infected Caco-2/TC7 cell monolayers, the cells were fixed, embedded in Epon, and then reembedded to cut sections perpendicular to the bottom of the flask. Semithin sections were examined by light microscopy. In addition, the number of domes was counted in 20 random microscopic fields per monolayer under phase-contrast microscopy. Domes were counted by 2 different technicians to eliminate bias.

Clinical Study
Children who were hospitalized at the Department of Pediatrics, Hospital Clínica Kennedy, were selected on the basis of their having acute diarrhea (at least 4 liquid stools passed during the last 24 hours) of suspected infectious origin (viral or bacterial) that had developed recently (<72 hours before inclusion). The severity of the diarrhea was assessed according to the following parameters: number of stools and their consistency, the presence or absence of mucus, bloating, pain, vomiting, dehydration, fever, and blood in stools. Rotavirus was detected by a latex agglutination test.

The study protocol was designed in accordance with the regulations that govern this type of study at the Hospital Clinica Kennedy and was approved by an internal review board. Parents or legal guardians of eligible children were informed, and their consent was obtained. The study was performed in accordance with the Declaration of Helsinki. Ninety-three patients were screened, and 80 of them were enrolled. In accordance with World Health Organization guidelines,30 all children enrolled received ORS (at least 100 mL/kg). The randomized, placebo-controlled, double-blind trial involved the use of the pharmaceutical preparation Lactéol Fort sachet (Laboratoire du Lactéol), which contains 10 billion heat-killed L acidophilus LB plus 160 mg of spent culture medium. Placebo sachets, identical in appearance to the Lactéol Fort sachets, were specially prepared for this study and contained sucrose, ferrous oxides, silicic acid, and banana-orange flavoring but no LB bacteria or spent culture medium. The Lactéol Fort sachets and placebo sachets were provided free of charge for the study by Laboratoire du Lactéol. The patients were divided into 2 groups (L acidophilus LB-ORS and placebo-ORS). The sachets were numerically coded to ensure sequential distribution. Both groups of patients received a 72-hour treatment that consisted of an initial dose of 2 sachets (L acidophilus LB-ORS or placebo-ORS) and subsequently 1 sachet every 12 hours, with a total of 8 sachets ingested between meals. The final assessment was made after 96 hours and was based on the clinical signs displayed by the patients. The influence of the treatment on the duration of the disease was assessed in terms of recovery time, which was defined as the time from inclusion until the first normal stool was passed. For determination of this end point, the time and the consistency of every stool were recorded.

Statistical Analyses
For the experimental studies, Student's t test was used. For the clinical study, the statistical analyses performed depended on the treatment administered (L acidophilus LB-ORS or placebo-ORS). All mean values are expressed as means ± SD. The {chi}2 test and a nonparametric test (Mann-Whitney) were used to compare the results obtained. We also performed a survival analysis on the main criterion, with a log rank test. The level of significance was 5%.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
L acidophilus LB Inhibits the C1845-Induced Increase in the Fluid Domes That Formed in Cultured Human Intestinal Caco-2/TC7 Monolayers
Cultured human intestinal Caco-2/TC7 cell monolayers form fluid-formed domes (henceforth referred to as fluid domes).31 This cell model can be used to evaluate antisecretory activity. The wild-type Afa/Dr DAEC strain C1845, isolated from an infant who had diarrhea in Mexico,27 was chosen for use as the test diarrheagenic bacterium inducing an increase in fluid accumulation, because Peiffer et al32 previously demonstrated that this enterovirulent E coli strain increased the paracellular passage of fluid and electrolytes in Caco-2 cells. Interferential phase-contrast light microscopic examination of a Caco-2/TC7 cell monolayer reveals a fluid dome in C1845-infected cells (Fig 1A). Figure 1B shows that strain C1845 increased the number of fluid domes formed in Caco-2/TC7 cell monolayers. We wanted to find out whether the L acidophilus LB culture and/or the lyophilized, heat-killed LB culture would inhibit the C1845-induced increase in the number of fluid domes formed. Because L acidophilus LB cultures and heat-killed LB cultures are known to display killing activity against diarrheagenic Gram-negative bacteria,23–26 we used concentrations of the L acidophilus LB culture or the lyophilized, heat-killed LB culture with no killing activity (bacterial counts after 1 hour of contact C1845 + MRS: 8.20 ± 0.2 log; C1845 + L acidophilus LB culture: 8.02 ± 0.4; C1845 + lyophilized heat-killed LB culture: 8.11 ± 0.4 log CFU/mL). When the wild-type C1845 bacteria had been pretreated before being infected with the L acidophilus LB culture, a very significantly fewer fluid domes were observed in the infected cells than in cells that were infected with untreated, wild-type C1845 bacteria (Fig 2A). It was noted that no change in the number of fluid domes develops in noninfected cell monolayers that are treated with the L acidophilus strain LB. Treating the wild-type C1845 bacteria with MRS that contained lactic acid at the concentration found in the LB cultures (MRS-LA, 60 mM, pH 4.5) did not affect the number of C1845-induced domes (Fig 2A). A highly significant reduction in the number of fluid domes was observed in the cells that were infected with C1845 that had been pretreated before infection with the LB-CFCS (Fig 2A). The reduced number of fluid domes observed in the cells that were infected with LB-CFCS–treated C1845 was the same as that observed in the cells that were infected with the LB culture–treated C1845 bacteria (Fig 2A). When the wild-type C1845 bacteria had been pretreated for 1 hour with increasing concentrations of lyophilized, heat-killed LB culture, a dosage-dependent decrease in the number of fluid domes formed was observed (Fig 2B). Moreover, observation under light microscopy of semithin sections of Caco-2/TC7 cell monolayers that were infected with lyophilized, heat-killed LB culture–treated, wild-type C1845 showed that the fluid domes that were formed (Fig 3B) were much smaller than those that were formed in Caco-2/TC7 monolayers that were infected with untreated, wild-type C1845 bacteria (Fig 3A). To demonstrate that the blocking effect observed did not result from a reduction of the adhesiveness of the C1845 bacteria as a result of exposure to the L acidophilus LB bacteria, we checked the levels of C1845 bacteria that adhered to the cultured cells. We found that the levels of bacterial adhesion were the same for the untreated wild-type, C1845 bacteria; the C1845 bacteria that were treated with the L acidophilus LB culture; and the C1845 bacteria that were treated with lyophilized, heat-killed LB culture (untreated C1845: 7.45 ± 0.4 log CFU/mL; L acidophilus LB culture–treated C1845: 7.02 ± 0.7; lyophilized, heat-killed LB culture–treated C1845: 7.11 ± 0.6 log CFU/mL).


Figure 1
View larger version (54K):
[in this window]
[in a new window]

  		FIGURE 1 Increase in the number of fluid domes in C1845-infected Caco-2/TC7 monolayers. Caco-2/TC7 cell monolayers were infected with 108 CFU per well of C1845 bacteria for 3 hours at 37°C in 10% CO2/90% air. The fluid domes were counted in 20 randomly selected microscopy fields by examining monolayers by phase-contrast light microscopy. A, Fluid dome observed by phase-contrast microscopy in a C1845-infected Caco-2/TC7 monolayer (magnification: x100). B, Number of fluid domes formed in C1845-infected Caco-2/TC7 monolayers. Each value shown is the mean ± SD from 3 experiments, each conducted in triplicate. a Values were significantly different than the noninfected value (P < .01).

 

Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 2 L acidophilus LB culture and lyophilized, heat-killed LB culture reduced the number of fluid domes in C1845-infected Caco-2/TC7 cell monolayers. Before cell infection, the C1845 bacteria (108 CFU per well) were exposed for 1 hour at 37°C to L acidophilus LB culture or to increasing concentrations of lyophilized, heat-killed LB culture. The cell monolayers were infected and processed for phase-contrast light microscopy as described for Fig 1. A, Number of fluid domes formed in Caco-2/TC7 monolayers that were infected with LB culture–treated C1845 bacteria. B, Number of fluid domes formed in Caco-2/TC7 monolayers that were infected with C1845 treated with increasing concentrations of lyophilized, heat-killed LB culture. Each value shown is the mean ± SD from 3 experiments, each conducted in triplicate. a Values were significantly different from those for the C1845-infected cells (P < .01).

 

Figure 3
View larger version (65K):
[in this window]
[in a new window]

  		FIGURE 3 Observation of fluid domes in Caco-2/TC7 cell monolayers that were infected with C1845 bacteria or infected with lyophilized, heat-killed LB culture–treated C1845 bacteria. Cell monolayers were processed for light-microscopic examination of semithin sections perpendicular to the bottom of the flask (magnification: x100). A, Fluid dome in the Caco-2/TC7 cell monolayer infected with C1845 bacteria. B, Fluid dome in the Caco-2/TC7 cell monolayer infected with lyophilized, heat-killed LB culture–treated C1845 bacteria. The hatched line indicates the bottom of the culture cell. The micrographs are representative of 2 experiments.

 
L acidophilus LB Inhibits the Sat-Induced Increase in Fluid Domes in Cultured Human Intestinal Caco-2/TC7 Monolayers
Bacterial pathogens promote changes in fluid and ion transport, leading to diarrhea, mainly as a result of their toxins.33,34 Our group recently reported that the Sat expressed by Afa/Dr DAEC, including strain C1845, increases the paracellular passage of fluids and nonionic macromolecules in monolayers of Caco-2/TC7 cells.28 An experiment was conducted to find out whether the blockade of the wild-type C1845 induced an increase in paracellular permeability by L acidophilus LB culture or by the lyophilized, heat-killed LB culture was attributable to inhibition of the Sat-induced effect. To do this, Caco-2/TC7 cell monolayers were infected with the recombinant E coli AAEC185 psat. This recombinant E coli lacks adhesive factor35 and so did not adhere to the Caco-2/TC7 cells and is known to secrete the Sat.28 As for the C845, the E coli AAEC1845 psat was pretreated before cell infection with or without the L acidophilus LB culture or the lyophilized, heat-killed LB culture. Counting the fluid domes formed showed that there were significantly fewer domes in the E coli AAEC185 psat–infected Caco-2/TC7 monolayers in the presence of L acidophilus LB culture or lyophilized, heat-killed LB than in cells that were infected with E coli AAEC185 psat alone (Fig 4).


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 4 L acidophilus LB culture and lyophilized, heat-killed LB culture reduced the number of fluid domes formed in Caco-2/TC7 cell monolayers that were infected with the recombinant E coli AAEC185 expressing the sat gene encoding for the Sat. Caco-2/TC7 cell monolayers were infected with untreated, L acidophilus LB culture–treated or lyophilized, heat-killed LB culture–treated E coli AAEC185 psat (108 CFU per well) for 3 hours at 37°C in 10% CO2/90% air. The cell monolayers were processed for counting the fluid domes as described for Fig 1. Each value shown is the mean ± SD from 3 experiments, each conducted in duplicate. a Values were significantly different from those for the C1845-infected cells (P < .01).

 
L acidophilus LB Ameliorates the Outcome of Nonrotavirus Diarrhea in Children From Ecuador
The antisecretory effect of L acidophilus LB reported could be just an in vitro effect. Because of the high human specificity of the virulence factors expressed by the Afa/Dr DAEC strains,36 it was impossible to use an animal model to examine the antisecretory effect of the L acidophilus LB culture or lyophilized, heat-killed LB. So to evaluate the in vivo efficacy of L acidophilus strain LB plus its spent culture medium, we conducted a double-blind, placebo-controlled, and randomized study in a population of children who had nonrotavirus, acute, watery diarrhea and were hospitalized at the Department of Pediatrics, Hospital Clínica Kennedy. This clinical study investigated the effect of the lyophilized, heat-killed L acidophilus LB bacteria plus its spent culture medium as an adjunct to ORS. In total, 93 children who had well-established diarrhea (<24 hours) were screened for enrollment in the study (Table 1). The infection status showed that only 12 of the children screened were positive for rotavirus infection, agreeing with previous epidemiologic studies in Ecuador.37–39 The 12 children who were positive for rotavirus were not subsequently enrolled, and 1 other child was not enrolled because the parents did not give their consent. The 80 children who fulfilled the study criteria were assigned to treatment groups (Table 2). Our findings show that when administered in addition to ORS, the lyophilized, heat-killed L acidophilus LB bacteria plus its spent culture medium led to a significant reduction in the duration of diarrhea (Table 3). Indeed, the diarrhea lasted 1 day less in the group of infants who received the L acidophilus LB-ORS than in the placebo-ORS group. The diarrhea was resolved in a significantly higher proportion of children in the L acidophilus-ORS group than in the placebo-ORS group. Moreover, examination of the consistency of the stools showed that for the children in the L acidophilus LB-ORS group, the stools were formed, whereas those of the children of the placebo-ORS group tended to remain fluid or semifluid.


View this table:
[in this window]
[in a new window]

  		TABLE 1 Clinical Characteristics of the Children Screened

 

View this table:
[in this window]
[in a new window]

  		TABLE 2 Clinical Characteristics of the Children Enrolled

 

View this table:
[in this window]
[in a new window]

  		TABLE 3 Comparison of Outcome Measures of All Children Treated With L acidophilus LB-ORS or Placebo-ORS

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Very little information is available about the potential role of probiotic agents in nonrotavirus diarrheal episodes. We chose to investigate here the antisecretory activity of the L acidophilus strain LB against the Afa/Dr DAEC strain C1845, which belongs to class 6 of human enterovirulent E coli.40 Epidemiologic studies have demonstrated an age-related incidence for Afa/Dr DAEC in diarrhea in children.41–45 At the brush border of enterocytes, Afa/Dr DAEC recognizes the human decay accelerating factor (CD55) and the human carcinoembryonic antigen (CD66e) as receptors. It has been previously reported that wild-type Afa/Dr DAEC strain C1845 promotes structural and functional lesions in the brush border, opens the junctional domain of polarized cells, and induces a proinflammatory response.36 In particular, it has been demonstrated that Afa/Dr DAEC increases the paracellular permeability in human intestinal Caco-2/TC7 cells that form monolayers in culture and that this in turn promotes an increase in the formation of fluid domes as a result of an accumulation of fluids.28,32 Here, using the Caco-2/TC7 cells, we demonstrate that the live L acidophilus strain LB, LB-CFCS, and the lyophilized, heat-killed L acidophilus LB bacteria plus their culture medium exhibited antisecretory activity. Indeed, after treatment of the pathogen with L acidophilus LB culture, LB-CFCS, or the lyophilized, heat-killed L acidophilus LB bacteria plus their culture medium, we observed a decrease in the C1845-induced formation of fluid domes. Similar activity was previously reported for a few number of probiotic Lactobacillus strains. For example, L casei strain DN-114 001 is able to reduce the increase in paracellular permeability induced by the prototype EPEC strain E2348/69 in monolayer-forming, cultured, human, intestinal T84 colonic cells.46Lactobacillus plantarum strain 299v abolished the E coli–induced increase in intestinal permeability in rats.47 However, the mechanism(s) of action supporting the inhibitory activity of these probiotic strains remain(s) to be determined.

Diarrheagenic bacteria are known to act by various mechanisms to induce a common intestinal injury characterized by a dramatic increase in the secretion of fluid and electrolytes.33,34 We conducted experiments to identify the mechanism by which L acidophilus strain LB blocks the C1845-induced increase in fluid domes. The wild-type strain C1845 expresses a toxin, known as Sat,48 that displays enterotoxic effects that are characterized by a pronounced accumulation of intestinal fluid49 and an increase in the paracellular permeability of Caco-2/TC7 cells.28 We show that both the live L acidophilus strain LB and the lyophilized, heat-killed L acidophilus LB bacteria plus their culture medium inhibit the increase in the number of fluid domes in Caco-2 cell monolayers that are infected with a recombinant strain of E coli AAEC185, that secretes Sat.28 It is tempting to hypothesize that the molecule(s) present in the CFCS of the L acidophilus strain LB could block the secretion of the Sat by the bacteria. Another hypothesis is that the molecule(s) present in the CFCS of the L acidophilus strain LB block the Sat-induced signaling necessary for the changes in the organization of the junctional domain that controls paracellular passage in monolayers of polarized cells.28 We are attempting to demonstrate the mechanism that uses the molecule(s) present in the CFCS of the L acidophilus strain LB.

As has recently been pointed out,2,3 it is difficult to extrapolate from in vitro effects to the in vivo gastrointestinal situation. The resident microbiota and mucus and the peristaltic flow that continuously washes over the gastrointestinal epithelium could considerably modify or even abolish the effects of exogenous probiotic lactic acid bacteria and/or their active metabolites. We therefore conducted a clinical trial to evaluate the clinical efficacy of the heat-killed L acidophilus LB bacteria plus their culture medium against nonrotavirus diarrhea. Our results show that when administered concomitantly with ORS, the lyophilized, heat-killed L acidophilus LB bacteria plus their culture medium were able to halve the duration of diarrhea in children who had nonrotavirus diarrhea. The shortening by 1 day of the recovery time from diarrhea was statistically significant, despite the relatively small number of infants in our study. A similar finding was recently reported, showing that in a subgroup in a randomized, double-blind, placebo-controlled clinical trial, conducted in infants and young children who were 4 to 24 months of age and presented with diarrhea, the live Lactobacillus paracasei strain ST11 significantly shortened the duration of non–rotavirus-induced diarrhea but had no effect against the rotavirus-induced diarrhea.20


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
We have shown experimentally that L acidophilus LB culture or heat-killed L acidophilus LB bacteria plus spent culture medium were able to induce dosage-dependent blockade of the C1845-induced increase in the number of fluid domes in cultured, human intestinal Caco-2/TC7 cells. We have also shown that the lyophilized, heat-killed L acidophilus LB bacteria plus spent culture medium reduced the duration of nonrotavirus diarrhea in children who had bacteria-induced diarrhea. It is interesting that it was noted that the antisecretory effect observed was attributable to heat-resistant molecules that were present in the CFCS of the L acidophilus strain LB, because the lyophilized, heat-killed L acidophilus LB bacteria plus spent culture medium had the same effect as the L acidophilus LB culture. This is an important finding, because the variable storage quality of live probiotic cultures results in unpredictable losses of dose and activity in the preparations administered to patients with acute watery diarrhea, and this is a technologic problem that is difficult to solve, particularly in developing countries. Lyophilized, heat-killed L acidophilus LB bacteria plus spent culture medium has the advantage of providing a potentially stable pharmaceutical preparation with reproducible activity.


   ACKNOWLEDGMENTS
 
We are grateful to R. Amsellem for assistance with the cell cultures. We are also indebted to J. Guignot for the generous gift of recombinant E coli AAEC185 strain expressing the sat gene.


   FOOTNOTES
 
Accepted Feb 23, 2007.

Address correspondence to Alain L. Servin, PhD, INSERM UMR-S 756, Faculté de Pharmacie, 92296 Châtenay-Malabry cedex, France. E-mail: alain.servin{at}cep.u-psud.fr

Financial Disclosure: Laboratoire du Lactéol (Houdan, France) provided strain LB and batches of lyophilized, heat-killed LB bacteria plus their culture medium to Dr Servin and Lactéol Fort sachets and placebo sachets to Dr Sarrazin-Davila. Dr Liévin-Le Moal indicated she has no financial relationships relevant to this article to disclose.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Food and Agriculture Organization/World Health Organization. Joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Cordoba, Argentina 1 to 4 October 2001. Available at: www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf. Accessed July 26, 2007
  2. Servin AL. Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev. 2004;28 :405 –440[Web of Science][Medline]
  3. Reid G, Burton J. Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect. 2002;4 :319 –324[CrossRef][Web of Science][Medline]
  4. Guandalini S, Pensabene L, Zikri MA, et al. Lactobacillus GG administered in oral rehydration solution to children with acute diarrhea: a multicenter European trial. J Pediatr Gastroenterol Nutr. 2000;30 :54 –60[CrossRef][Web of Science][Medline]
  5. Majamaa H, Isolauri E, Saxelin M, Vesikari T. Lactic acid bacteria in the treatment of acute rotavirus gastroenteritis. J Pediatr Gastroenterol Nutr. 1995;20 :333 –338[Web of Science][Medline]
  6. Mastretta E, Longo P, Laccisaglia A, et al. Effect of Lactobacillus GG and breast-feeding in the prevention of rotavirus nosocomial infection. J Pediatr Gastroenterol Nutr. 2002;35 :527 –531[CrossRef][Web of Science][Medline]
  7. Oberhelman RA, Gilman RH, Sheen P, et al. A placebo-controlled trial of Lactobacillus GG to prevent diarrhea in undernourished Peruvian children. J Pediatr. 1999;134 :15 –20[CrossRef][Web of Science][Medline]
  8. Shornikova AV, Isolauri E, Burkanova L, Lukovnikova S, Vesikari T. A trial in the Karelian Republic of oral rehydration and Lactobacillus GG for treatment of acute diarrhoea. Acta Paediatr. 1997;86 :460 –465[Web of Science][Medline]
  9. Isolauri E, Juntunen M, Rautanen T, Sillanaukee P, Koivula T. A human Lactobacillus strain (Lactobacillus casei sp strain GG) promotes recovery from acute diarrhea in children. Pediatrics. 1991;88 :90 –97[Abstract/Free Full Text]
  10. Szajewska H, Kotowska M, Mrukowicz JZ, Armanska M, Mikolajczyk W. Efficacy of Lactobacillus GG in prevention of nosocomial diarrhea in infants. J Pediatr. 2001;138 :361 –365[CrossRef][Web of Science][Medline]
  11. Pedone CA, Bernabeu AO, Postaire ER, Bouley CF, Reinert P. The effect of supplementation with milk fermented by Lactobacillus casei (strain DN-114 001) on acute diarrhoea in children attending day care centres. Int J Clin Pract. 1999;53 :179 –184[Web of Science][Medline]
  12. Pedone CA, Arnaud CC, Postaire ER, Bouley CF, Reinert P. Multicentric study of the effect of milk fermented by Lactobacillus casei on the incidence of diarrhoea. Int J Clin Pract. 2000;54 :568 –571[Web of Science][Medline]
  13. Shornikova AV, Casas IA, Isolauri E, Mykkanen H, Vesikari T. Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children. J Pediatr Gastroenterol Nutr. 1997;24 :399 –404[CrossRef][Web of Science][Medline]
  14. Shornikova AV, Casas IA, Mykkanen H, Salo E, Vesikari T. Bacteriotherapy with Lactobacillus reuteri in rotavirus gastroenteritis. Pediatr Infect Dis J. 1997;16 :1103 –1107[CrossRef][Web of Science][Medline]
  15. Van Niel CW, Feudtner C, Garrison MM, Christakis DA. Lactobacillus therapy for acute infectious diarrhea in children: a meta-analysis. Pediatrics. 2002;109 :678 –684[Abstract/Free Full Text]
  16. Szajewska H, Mrukowicz JZ. Probiotics in the treatment and prevention of acute infectious diarrhea in infants and children: a systematic review of published randomized, double-blind, placebo-controlled trials. J Pediatr Gastroenterol Nutr. 2001;33(suppl 2) :S17 –S25[CrossRef]
  17. Xiao SD, Zhang de Z, Lu H, et al. Multicenter, randomized, controlled trial of heat-killed Lactobacillus acidophilus LB in patients with chronic diarrhea. Adv Ther. 2003;20 :253 –260[Web of Science][Medline]
  18. Simakachorn N, Pichaipat V, Rithipornpaisarn P, Kongkaew C, Tongpradit P, Varavithya W. Clinical evaluation of the addition of lyophilized, heat-killed Lactobacillus acidophilus LB to oral rehydration therapy in the treatment of acute diarrhea in children. J Pediatr Gastroenterol Nutr. 2000;30 :68 –72[CrossRef][Web of Science][Medline]
  19. Costa-Ribeiro H, Ribeiro TC, Mattos AP, et al. Limitations of probiotic therapy in acute, severe dehydrating diarrhea. J Pediatr Gastroenterol Nutr. 2003;36 :112 –115[CrossRef][Web of Science][Medline]
  20. Sarker SA, Sultana S, Fuchs GJ, et al. Lactobacillus paracasei strain ST11 has no effect on rotavirus but ameliorates the outcome of nonrotavirus diarrhea in children from Bangladesh. Pediatrics. 2005;116(2) . Available at: www.pediatrics.org/cgi/content/full/116/2/e221
  21. Chauviere G, Coconnier MH, Kerneis S, Darfeuille-Michaud A, Joly B, Servin AL. Competitive exclusion of diarrheagenic Escherichia coli (ETEC) from human enterocyte-like Caco-2 cells by heat-killed Lactobacillus. FEMS Microbiol Lett. 1992;70 :213 –217[Medline]
  22. Coconnier MH, Bernet MF, Chauviere G, Servin AL. Adhering heat-killed human Lactobacillus acidophilus, strain LB, inhibits the process of pathogenicity of diarrhoeagenic bacteria in cultured human intestinal cells. J Diarrhoeal Dis Res. 1993;11 :235 –242[Web of Science][Medline]
  23. Coconnier MH, Lievin V, Bernet-Camard MF, Hudault S, Servin AL. Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB. Antimicrob Agents Chemother. 1997;41 :1046 –1052[Abstract]
  24. Coconnier MH, Lievin V, Lorrot M, Servin AL. Antagonistic activity of Lactobacillus acidophilus LB against intracellular Salmonella enterica serovar Typhimurium infecting human enterocyte-like Caco-2/TC-7 Cells. Appl Environ Microbiol. 2000;66 :1152 –1157[Abstract/Free Full Text]
  25. Coconnier-Polter MH, Lievin-Le Moal V, Servin AL. A Lactobacillus acidophilus strain of human gastrointestinal microbiota origin elicits killing of enterovirulent Salmonella enterica serovar Typhimurium by triggering lethal bacterial membrane damage. Appl Environ Microbiol. 2005;71 :6115 –6120[Abstract/Free Full Text]
  26. Lievin-Le Moal V, Amsellem R, Servin AL, Coconnier MH. Lactobacillus acidophilus (strain LB) from the resident adult human gastrointestinal microflora exerts activity against brush border damage promoted by a diarrhoeagenic Escherichia coli in human enterocyte-like cells. Gut. 2002;50 :803 –811[Abstract/Free Full Text]
  27. Bilge SS, Clausen CR, Lau W, Moseley SL. Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells. J Bacteriol. 1989;171 :4281 –4289[Abstract/Free Full Text]
  28. Guignot J, Chaplais C, Coconnier-Polter MH, Servin AL. The secreted autotransporteur toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in junctional domain of polarized epithelial cells. Cell Microbiol. 2006;9 :204 –221[CrossRef][Web of Science][Medline]
  29. Chantret I, Rodolosse A, Barbat A, et al. Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation. J Cell Sci. 1994;107 :213 –225[Abstract]
  30. World Health Organization, Programme for the Control of Diarrhoeal Disease. A Manual for the Treatment of Diarrhoea for Use by Physicians and Other Senior Health Workers. Geneva. Switzerland: World Health Organization; 1990. Report WHO/CDD/SER/80.2ev.2
  31. Grasset E, Pinto M, Dussaulx E, Zweibaum A, Desjeux JF. Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters. Am J Physiol. 1984;247 :C260 –C267[Web of Science][Medline]
  32. Peiffer I, Blanc-Potard AB, Bernet-Camard MF, Guignot J, Barbat A, Servin AL. Afa/Dr diffusely adhering Escherichia coli C1845 infection promotes selective injuries in the junctional domain of polarized human intestinal Caco-2/TC7 cells. Infect Immun. 2000;68 :3431 –3442[Abstract/Free Full Text]
  33. Berkes J, Viswanathan VK, Savkovic SD, Hecht G. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation. Gut. 2003;52 :439 –451[Abstract/Free Full Text]
  34. Sears CL, Kaper JB. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev. 1996;60 :167 –215[Free Full Text]
  35. Blomfield IC, McClain MS, Princ JA, Calie PJ, Eisenstein BI. Type 1 fimbriation and fimE mutants of Escherichia coli K-12. J Bacteriol. 1991;173 :5298 –5307[Abstract/Free Full Text]
  36. Servin AL. Pathogenesis of Afa/Dr diffusely adhering Escherichia coli. Clin Microbiol Rev. 2005;18 :264 –292[Abstract/Free Full Text]
  37. Bourgeois AL, Gardiner CH, Thornton SA, et al. Etiology of acute diarrhea among United States military personnel deployed to South America and West Africa. Am J Trop Med Hyg. 1993;48 :243 –248[Abstract/Free Full Text]
  38. Brussow H, Sidoti J, Link H, et al. Age-specific prevalence of antibody to enterotoxigenic Escherichia coli in Ecuadorian and German children. J Infect Dis. 1990;162 :974 –977[Web of Science][Medline]
  39. Pan American Health Organization; World Health Organization. Ecuador. In: Health in the Americas. Washington, DC: Pan American Health Organization; 1998:240–257. Available at: www.paho.org/english/HIA1998/Ecuador.pdf. Accessed July 26, 2007
  40. Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2 :123 –140[CrossRef][Web of Science][Medline]
  41. Levine MM, Ferreccio C, Prado V, et al. Epidemiologic studies of Escherichia coli diarrheal infections in a low socioeconomic level peri-urban community in Santiago, Chile. Am J Epidemiol. 1993;138 :849 –869[Abstract/Free Full Text]
  42. Gunzburg ST, Chang BJ, Elliott SJ, Burke V, Gracey M. Diffuse and enteroaggregative patterns of adherence of enteric Escherichia coli isolated from aboriginal children from the Kimberley region of Western Australia. J Infect Dis. 1993;167 :755 –758[Web of Science][Medline]
  43. Scaletsky IC, Fabbricotti SH, Carvalho RL, et al. Diffusely adherent Escherichia coli as a cause of acute diarrhea in young children in Northeast Brazil: a case-control study. J Clin Microbiol. 2002;40 :645 –648[Abstract/Free Full Text]
  44. Baqui AH, Sack RB, Black RE, et al. Enteropathogens associated with acute and persistent diarrhea in Bangladeshi children less than 5 years of age. J Infect Dis. 1992;166 :792 –796[Web of Science][Medline]
  45. Quiroga M, Oviedo P, Chinen I, et al. Asymptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives. Rev Inst Med Trop Sao Paulo. 2000;42 :9 –15[Medline]
  46. Parassol N, Freitas M, Thoreux K, Dalmasso G, Bourdet-Sicard R, Rampal P. Lactobacillus casei DN-114 001 inhibits the increase in paracellular permeability of enteropathogenic Escherichia coli-infected T84 cells. Res Microbiol. 2005;156 :256 –262[Medline]
  47. Mangell P, Nejdfors P, Wang M, et al. Lactobacillus plantarum 299v inhibits Escherichia coli-induced intestinal permeability. Dig Dis Sci. 2002;47 :511 –516[CrossRef][Web of Science][Medline]
  48. Taddei CR, Moreno AC, Fernandes Filho A, Montemor LP, Martinez MB. Prevalence of secreted autotransporter toxin gene among diffusely adhering Escherichia coli isolated from stools of children. FEMS Microbiol Lett. 2003;227 :249 –253[CrossRef][Web of Science][Medline]
  49. Taddei CR, Fasano A, Ferreira AJ, Trabulsi LR, Martinez MB. Secreted autotransporter toxin produced by a diffusely adhering Escherichia coli strain causes intestinal damage in animal model assays. FEMS Microbiol Lett. 2005;250 :263 –269[CrossRef][Web of Science][Medline]
PEDIATRICS (ISSN 1098-4275). ©2007 by the American Academy of Pediatrics

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Facebook Facebook   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:


Home page
 CLIN PEDIATRHome page
S.-T. T. Li, E. J. Klein, P. I. Tarr, and D. M. Denno
Parental Management of Childhood Diarrhea
Clinical Pediatrics, April 1, 2009; 48(3): 295 - 303.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My File Cabinet
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liévin-Le Moal, V.
Right arrow Articles by Servin, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liévin-Le Moal, V.
Right arrow Articles by Servin, A. L.
Related Collections
Right arrow Infectious Disease & Immunity
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?