Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Locascio, Mónica; González, Silvia; Apella, María C.; Bru de Labanda, Elena; Oliver, Guillermo bacteriotherapy in chronic infantile diarrhea Interciencia, vol. 27, núm. 7, julio, 2002, pp. 365-368 Asociación Interciencia Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative PROBIOTIC BACTERIOTHERAPY IN CHRONIC INFANTILE DIARRHEA

Mónica Locascio, Silvia González, María C. Apella, Elena Bru de Labanda and Guillermo Oliver

SUMMARY

A probiotic therapy based on the administration of a dairy clinical symptoms had been eliminated. Until the end of the product containing casei and Lactobacillus acido- study none of the patients suffered a relapse. Bacterial counts in philus was used against chronic diarrhea of 170 infants aging the feces suggested that the probiotic lactobacilli had restored from 4 months - 4 years. The mean period during which the the normal intestinal lactobacillus flora. The fermented product children suffered from diarrhea before the start of the probiotic tested in this work can be considered a beneficial food. treatment was of about 50 days. After 4 days of therapy the

Introduction pared with from com- the nineteenth century arise from intestinal mucosa mon human flora as a thera- (Metchnickoff, 1907) lactoba- (Nader de Macías et al., Good health implies an peutic measure in chronic and cilli, e.g. Lactobacillus casei 1992, 1993). Only after the equilibrium between bacterial recurrent infections. The main and Lactobacillus acidophilus, overcomes the non- populations in the gastrointes- indications for this kind of are known to inhibit growth specific host defense mecha- tinal tract. Disturbance of this therapy were catarrhal infec- of pathogenic intestinal bacte- nisms, specific host defense equilibrium will lead to intesti- tions of the respiratory sys- ria and therefore they are mechanisms are activated and nal disorders (Gebbers and tem, intestinal disorders, aller- considered to be potential will produce antibodies,

Laissue, 1982) e.g. diarrhea. gies and skin diseases as well biotherapeutic agents against mainly SIgA’s (Perdigón et al., For three decades, members of as several less well-defined (González et al., 1992). L. casei is able to the German Medical Associa- syndromes (Rusch et al., 1993; Apella et al., 1992, stimulate phagocytosis both tion for Microbiological Ther- 1980). However, for a long Romero et al., 1990). Protec- by the cell wall and the pepti- apy have used vaccines pre- time, starting at the end of tive immunity is suggested to doglycan, whereas it does not

KEYWORDS / Bacteriotherapy / Fermented Milk / Infantile Chronic Diarrhea / / Received: 09/20/2001. Modified: 05/06/2002. Accepted: 05/17/2002 Mónica Locascio. Ph.D. National Silvia González. Ph.D., UNT. Re- María C. Apella. Ph.D. National Guillermo Oliver. Ph.D. National University of Tucumán (UNT). searcher, CONICET-CERELA. University of La Plata. Re- University of Litoral. Superior Technical Researcher, National Associate Professor, Public searcher, CONICET. Associate Researcher, CONICET. Emeri- Research Council (CONICET), Health, UNT. Address: Centro Professor, UNT. tus Professor, UNT. Argentina. Centro de Referencia de Referencias para Lactobaci- Elena Bru De Labanda. Mathema- para Lactobacilos (CERELA), los (CERELA-CONICET), Cha- tician, UNT. Technical Re- Tucumán Argentina. cabuco 145, 4000, S.M. de Tu- searcher, CONICET-CERELA. cumán, Argentina. e-mail: [email protected]

JUL 2002, VOL. 27 Nº 7 0378-1844/02/07/365-04 $ 3.00/0 365 RESUMEN

La administración de un producto lácteo conteniendo Lacto- eliminaron al cabo de 4 días de terapia probiótica y ninguno de bacillus casei y Lactobacillus acidophilus fue usada como terapia los pacientes incurrió nuevamente en diarrea hasta el final del probiótica para diarrea crónica en 170 niños cuyas edades estudio. Los recuentos bacterianos en heces sugirieron que los estaban comprendidas entre 4 meses y 4 años. El tiempo lactobacilos probióticos habían normalizado la flora láctica in- promedio de duración de diarrea en estos niños, antes del testinal. El producto lácteo fermentado ensayado en este trabajo tratamiento probiótico, era de 50 días. Los síntomas clínicos se puede ser considerado un alimento beneficioso.

RESUMO

A administração de um produto lácteo contendo Lactobacil- eliminados em 4 dias de terapia probiótica e nenhum dos lus casei and Lactobacillus acidophilus foi usada como terapia pacientes incorreu novamente em diarréia até o final do estudo. probiótica para diarréia crônica em 170 crianças cujas idades As contagens bacterianas em fezes constataram que os estavam compreendidas entre 4 meses e 4 anos. O tempo médio lactobacilos probióticos haviam normalizado a flora láctica in- de duração de diarréia nestas crianças, antes do tratamento testinal. O produto lácteo fermentado testado neste trabalho probiótico, era de 50 dias. Os sintomas clínicos foram pode ser considerado um alimento com muitos benefícios.

produce changes in IgA. L. (vi) bacterial origin of the Collection (CRL) stock, iso- the first period of probiotic acidophilus, on the other diarrhea and (vii) no evi- lated initially from feces of supplementation, and at the hand, produces an increase in dence of systemic infection. healthy children, were used. 8th day after the end of the the levels of IgA without As controls, the clinical his- Just before preparation of the probiotic therapy. Each swab modifying phagocytosis (Mo- tories of the same patients probiotic product the bacilli was placed into a Lapt-soft rata de Ambrosini et al., selected for this study were had been cultured in MRS- agar medium (Raibaud et al., 1998). An extracellular lectin- used, as they all showed re- medium at least twice in suc- 1961) containing agar (0.8% like substance is characterized sistance to conventional ther- cession (De Man et al., 1960), w/v) to maintain viability and in L. casei, when it is ex- apy. Due to the results the concentrated and washed by each duplicate was carried to tracted, it does not show any infants selected were their centrifugation (4000g for 10 the laboratory under anaerobic stimulation of the immune own controls. Initially, 123 min in 0.1M phosphate buffer, conditions. Previously, the system (Morata de Ambrosini infants aging between 4 and pH 7), resuspended separately tare of each sterile swab was et al., 1997). In order to 24 months (mean age: 10 in 10-fold diluted reconstituted determined. On the other eliminate diarrhea in children months) constituted group A milk, and incubated anaerobi- hand, each tube containing a simple probiotic dairy prod- and 47 children aging be- cally at 37°C during 4-6 hr. A Lapt-soft agar medium was uct prepared by addition of tween 24 and 48 months mixture (the final probiotic weighed. Finally the wet both L. casei and L. acidophi- (mean age: 30 months) con- dairy product) of equal weight of each fecal sample lus to reconstituted milk, fol- stituted group B. Since the amounts of each suspension was calculated and the results lowed by a short fermentation collected results of both (final concentration of each were expressed as Colony process (metabolic activation groups were not essentially lactobacillus strain: 107-108 liv- Forming Units per gram of of lactobacilli). In this study different, all results were ing cells/ml) was stored at 4ºC feces (CFU/g). All samples 170 infants suffering from combined. All infants were until used. The final probiotic were analyzed the day of col- chronic diarrhea were treated ambulatory patients and re- dairy product was used before lection. Dilutions of the with this product and the mi- ceived normal food at home. a maximum storage of 7 days. samples were plated out (in crobiological and clinical re- They belonged to a low so- duplicate) onto a selective sults are presented. cioeconomic stratum and all Probiotic treatment lactobacillus medium (LSB; parents authorized the pro- Rogosa et al., 1951). After Materials and Methods biotic therapy. The CERELA The total bacteriotherapeu- incubation for 48 hr at 37ºC

(Centro de Referencia para tic period consisted of 5 suc- in CO2 atmosphere colonies Children Lactobacilos) Committee of cessive periods of 7 days: 3 were counted and results ex- Ethics approved the protocol periods of probiotic supple- pressed as colony forming Criteria for inclusion in used for these studies. mentation were alternated units per gram of wet feces this study were: (i) age from Diarrhea was defined as 3 with periods without this (CFU/g). To identify the Lac- 4 months to 4 years, (ii) di- or more unformed stools with supplementation. During the tobacilli in feces, colonies arrhea for more than 30 a total volume of ≥200ml periods of supplementation from LBS agar plates were days, (iii) failure of previous within 48 hr or a single volu- the infants got daily 240ml subcultured in MRS broth for conventional therapies (sub- minous (≥300ml) liquid stool. skim milk supplemented with 24-48 hr at 37ºC. Microorgan- stitution of milk by soy milk 15ml of the probiotic product. isms were observed under the or lactose-free milk, and Probiotic product microscope to determine mor- sometimes thera- Microbiological assays phological characteristics and py), (iv) absence of antibiotic Lactobacillus casei subsp Gram reaction. Tests for cata- therapy during the last 7 casei CRL 431 and Lactoba- Fecal samples were col- lase activity, carbohydrate uti- days before probiotic therapy, cillus acidophilus CRL 730 lected by rectal swabs before lization and growth at 37 and (v) absence of malnutrition, were from the Cerela Culture treatment, at the 7th day of 45°C were carried out.

366 JUL 2002, VOL. 27 Nº 7 Figure 1. Frequencies of lactobacillus concentrations in fecal samples from infants (N= 170) before (1a), during (1b) and after (1c) probiotic bacteriotherapy. Counts of lactobacilli are given as the log of CFU/g wet feces.

Statistical analysis cillus flora. The presence of Univariate repeated mea- justified by the results of lectin-like substances in bacte- sure (F-test) was 90.43 and well-controlled animal studies Significant differences were ria of the genus Lactobacillus its related significance’s (Romero et al., 1990; Per- tested using the Tukey HSD- has been rarely reported probability was p=0.9·10-15. digón et al., 1992). Perdigón test (Minitab Student R12; (Morata de Ambrosini et al., Since the F-test was strongly et al. (1998) demonstrated Rossman and Chance, 1998). 1997; Mukai et al., 1992). significant, multiple compari- that feeding mice with L. ca- These substances would play a sons (Tukey HSD-test) were sei, L. acidophilus or a mix- Results and Discussion critical role in the bacterial at- used to determine the statisti- ture of both strains, produce a tachment to the gastrointestinal cal significant differences be- remarkable immunostimula- The data obtained from se- epithelium, and under these tween measures performed tory effect on the host, both quential ingestion of fer- conditions the immune stimu- before, during and after treat- on the lymphocytic and reti- mented milk by children (0-4 lation would be enhanced. ment. Statistical significant culoendothelial systems. Pro- years old) suffering chronic The mean period during differences were found be- duction of secretory IgA is diarrhea are presented in Fig- which the infants suffered di- tween a) during and before important because it consti- ure 1. Lactobacillus counts in arrhea just before the begin- treatment (p=0.2·10-4) and b) tutes the first line of defense feces began before the first ning of the probiotic treat- after and before treatment against microbial pathogens administration of fermented ment was of 51 ±33 days (p=0.2·10-4). No significant (Tomasi and Bienenstock, milk and are shown in Figure (N= 170; range 30-200 days). differences were found be- 1968; Tomasi et al., 1980). 1a. Lactobacillus concentra- In all infants the probiotic tween after and during treat- The results presented, to- tions obtained during and af- therapy was very effective, ment (p=0.47). So, mean ini- gether with those obtained in ter probiotic treatment are since clinical symptoms were tial lactobacillus flora differs previous studies using the shown in Figures 1b and 1c eliminated within the first pe- significantly from the mean same probiotic product in or- respectively. Since long-term riod with probiotic supple- flora during or after probiotic der to prevent diarrhea (Gon- probiotic therapy with rather mentation. The therapeutic ef- bacteriotherapy. zález et al., 1990) or to treat high concentrations of lacto- fect was seen after 4.1 ±1.6 Some patients with high diarrhea due to post-gastroen- bacilli might change the in- days (N= 170; range 2-7 initial lactobacillus counts (up teritis syndrome (González et testinal microflora, lactoba- days). These results agree to 109-1010 UFC/g) also al., 1995), show that levels of cilli in feces were counted. largely with those of Rusch et showed chronic diarrhea. 106 lactobacilli/g feces Before therapy more intents al. (1982), who proved the Probably in these patients strongly contribute to paraim- showed counts of 104 and 106 safety and efficacy of probi- physiological deficiencies of munization (Mayr et al., lactobacilli/g (Figure 1a). By otic therapy in numerous the small bowel were in- 1979). Our probiotic therapy day 7 after the first supple- case-reports. After the probi- volved; nevertheless, the posi- confirms that daily consump- mentation period, the fre- otic therapy till the end of tive probiotic effect in these tion of high amounts of vi- quency peak was highest at this study none of the patients patients is not well under- able alters the fecal counts of 108 lactoba- suffered again from diarrhea. stood. The effectiveness of intestinal environment (Zoppi cilli/g (Figure 1b). These data This post-therapy period the therapy may be explained et al., 1982). A successful confirm that at least a part of ranged from 34 days (last pa- on the basis of adhesion and probiotic ought not only to the ingested lactobacilli pass tient) to 630 days (first pa- immunologic properties of our enhance the immune re- the stomach. At the 8th day tient) with a median of 250 lactobacilli. The binding of sponse, but also to be free after the completed therapy days. This observation could the lectin-like structures to from adverse side effects. Al- the frequency peak was high- be explained by the remaining specific carbohydrates would though further research is est at fecal counts of 107 lac- immune stimulating effect explain the adhesion phenom- needed to elucidate the mech- tobacilli/g (Figure 1c). These found, for both whole cells enon of lactobacilli to epithe- anism by which L. casei and results strongly suggest that and cell walls of L. casei, af- lial cells (Morata de Am- L. acidophilus eliminate in- in general the probiotic lac- ter oral administration (Per- brosini et al., 1997). The hy- fantile diarrhea, clinical appli- tobacillus flora has restored digón et al., 1992; Morata de pothesis that these bacteria cations of our oral probiotic the normal levels of lactoba- Ambrosini et al., 1998). stimulate immune activities is dairy product are possible.

JUL 2002, VOL. 27 Nº 7 367 ACKNOWLEDGMENTS enteropathogens by lactobacilli Nader de Macías ME, Romero NC, Romero NC, Nader de Macías ME, strains used in fermented milk. Apella MC, González SN, Apella MC, Oliver G (1990) J. Food Prot. 56: 773-776. Oliver G (1993) Prevention of Inhibition of Listeria mono- This work was supported infections produced by E. coli cytogenes by strains of lactic by the National Research González SN, Apella MC, Cardozo R, Oliver G (1995) Biothera- and Listeria monocytogenes by acid bacteria used as ferments. Council of Argentina, grants peutic role of fermented milk. feeding milk fermented with Microbiologie-Aliments-Nutri- from CIUNT (D-220) pro- Biotherapy 8: 129-134. lactobacilli. J Food Prot. 56: tion 5: 175-179. 401-405. gram and from a SanCor- Mayr A, Raettig H, Stickl H, Rossman AJ, Chance BL (1998) Cerela-Conicet joint venture. Alexander M (1979) Paramuni- Perdigón G., Alvarez S, Médici M Workshop Statistics: discovery tat, Paramunisierung, Paramu- (1992) Systemic and local aug- with data and Minitab. Sprin- mentation of the immune re- ger-Verlag, New York. pp. 279- REFERENCES nitatsinducer. Fortschr. Med. 97: 1159-1165. sponse in milk by feeding with 325. milk fermented with Lactoba- Rusch V, Hyde RM, Luckey TD Apella MC, González SN, Nader Metchnickoff, E (1907) The prolon- cillus acidophilus and/or Lac- (1980) Documentation on the de Macías ME, Romero NC, gation of life. Heinemann. tobacillus casei. In Paubert- identity, manufacture, safety, Oliver G (1992) In vitro stud- London. Braquet M, Dupont Ch, Pao- efficacy and stability of SYM- ies on the inhibition of growth Morata de Ambrosini V, Salado C, letti R (Eds). Foods, Nutrition BIOFLOR-products. Microecol. of Shigella sonnei by Lactoba- González S, Oliver G (1997) and Immunity. Vol. 1. Dynamic Ther. 10: 173-203. cillus casei and Lact. acidophi- Lectin-like substance in Lacto- Nutrition Research. Karger. Rusch V, Hyde RM, Luckey TD lus. J. Appl. Bacteriol. 73: bacillus casei CRL 431. Mi- Paris. pp. 66-76. (1982) Microbiological thera- 480-483. crobiologie-Aliments-Nutrition Perdigón G, Nader de Macías ME, py: oral and parenteral applica- 15: 285-289. De Man JC, Rogosa M, Sharpe Alvarez S, Pesce de Ruiz tion of normal intestinal bacte- ME (1960) A medium for the Morata de Ambrosini V, González Holgado A (1998) Systemic rial vaccines in humans and cultivation of lactobacilli. J. S, Perdigón G, P. de Ruiz augmentation on the immune animals. Microecol. Ther. 12: Appl. Bacteriol. 23: 130-135. Holgado A, Oliver G (1998) response in mice by feeding 71-80. Immunostimulating activity of fermented milks with L. casei Tomasi TB, Bienenstock J (1968) Gebbers JO, Laissue JA (1982) Bar- cell walls from lactic acid bac- and L. acidophilus. Immunol- Secretory immunoglobulins. rier function of the colonic mu- teria and related species. Food ogy 63: 17-23. Adv. Immunol. 9: 1-5. cosa: morphological aspects. Agric. Immun. 10: 183-191. Microecol. Ther. 12: 31-45. Raibaud P, Caulet M, Galpin JV, Tomasi T, Lawson B, Challacombe Mukai T, Arihara K, Itoh H (1992) Mocquot G (1961) Studies on S, Mc Nabb P (1980) Mucosal González SN, Albarracín G, Lo- Lectin-like activity of Lactoba- the bacterial flora of the ali- immunity: the origin and mi- cascio de Ruiz Pesce M, Malé cillus acidophilus strain JCM mentary tract of pigs II. Strep- gration patterns of cells in the M, Apella MC, Pesce de Ruiz 1026. FEMS Microb. Lett. 98: tococci: selective enumeration secretory system. J. Allergy Holgado A, Oliver G (1990) 71-74. and differentiation of the Clin. Immunol. 65: 12-19. Prevention of infantile diar- dominant group. J. Appl. Zoppi G, Deganello A, Benoni G, rhoea by fermented milk. Nader de Macías ME, Apella MC, Romero NC, González SN, Bacteriol. 24: 285-291. Saccomani F (1982) Oral Microbiologie-Aliments-Nutri- Oliver G (1992) Inhibition of Rogosa M, Mitchel DA, Weiseman bacteriotherapy in clinical tion 8: 349-354. Shigella sonnei by Lactobacil- RF (1951) A selective medium practice. I. The use of different González SN, Apella MC, Romero lus casei and Lactobacillus for the isolation and enumera- preparations in infants treated NC, Nader de Macías ME, acidophilus. J Appl. Bacteriol. tion of oral and faecal lactoba- with . Eur. J. Pediat. Oliver G (1993) Inhibition of 73: 407-411. cilli. J. Bacteriol. 62: 132-133. 139: 18-21.

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