Detection of non-enterotoxigenic and enterotoxigenic Bacteroides fragilis in stool samples from children in São Paulo, Brazil

Abstract

SUMMARYNon-enterotoxigenic bacteria of the Bacteroides fragilis group and enterotoxigenic B. fragilis were identified from children with and without aqueous acute diarrhea. In this study, 170 stool samples from 96 children with and 74 without diarrhea were analyzed. Enterotoxin production and the toxin gene detection were detected by cytotoxicity assay on HT-29/C 1 cells and by PCR, respectively. B. fragilis species was prevalent in both groups and enterotoxigenic B. fragilis strains were isolated from two children with diarrhea. More studies are important to evaluate the role of each bacteria of the B. fragilis group, including enterotoxigenic strains play in the diarrheal processes in children. KEYWORDS:Bacteroides fragilis group; ETBF; Acute diarrhea; Children.Bacteroides fragilis is considered to be an important member of the human intestinal indigenous microbiota and in various experimental animal models B. fragilis has been reported to be more virulent than the other species from the B. fragilis group 3 .The involvement of B. fragilis in the etiology of human and animal diarrhea has been the subject of some investigations in the last years 6,11 . Childhood diarrhea is one of the most important causes of morbidity and mortality worldwide 5 . MYERS et al. 10 reported B. fragilis strains producing an enterotoxin (ETBF) that causes diarrhea in young farm animals and which is associated with diarrhea in man 2,7,14,15 .The enterotoxin or fragilisyn has been characterized as a zincmetalloprotease with a molecular weight of 20,000 Da, and with activity against tight junction proteins, disrupting the intestinal epithelial barrier 9,12,13 and inducing fluid losses 19. In this study, the isolation and identification of bacteria of the B. fragilis group and the detection of the ETBF in stool samples of children with and without diarrhea were performed.Fresh stool samples from ninety-six hospitalized children with aqueous acute diarrhea from two hospitals (Menino Jesus Hospital and Instituto da Criança, São Paulo, SP, Brazil) and from 74 healthy children from two different day care centers, aged one month to 12 years old, without sex distinction were analyzed. The Ethic Commission from both hospitals approved this study. Collected stools from children with or without diarrhea were immediately plated onto a selective B. fragilisbile-esculin agar 8 (BBE, Difco Laboratories) and incubated in atmosphere with 90% N 2 /10% CO 2, at 37 °C, for 72 hours. Characteristic dark colonies grown in BBE were subcultured on blood agar (BHI agar -Difco, enriched with 5% sheep desfibrined blood). Isolates were identified by using identification kits API 32-A (bioMérieux) and stored in 10% skim milk at -70 °C.Two of the enterotoxin-positive strains were used as control: B. fragilis ATCC 43858 and B. fragilis GAI 97124, kindly provided by Dr Annalisa Pantosti (Laboratory of Bacteriology and Medical Mycology, "Istituto Superiore di Sanità", Rome, Italy) and by Dr Naoki Kato (Institute of Anaerobic Bacteriology, Gifu University School of Med...

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

“…The evaluation of the enterotoxin activity by the HT-29/C 1 cells assay was successful. Studies have shown that ETBF were recovered in 3.1% from feces of immunodeficient children (Bressane et al 2001) and in 2.1% in children with diarrhea (Krzyzanowsky & Avila-Campos 2003). Moreover, Cáceres et al (2000) showed the presence of ETBF in 8.4% of Nicaraguan children.…”

EnterotoxigenicBacteroides fragilis: a Rogue among Symbiotes

“…Of 17 studies with controls (Table 2), 5 did not show an association between ETBF and diarrheal disease in differing patient populations (4,8,48,77,84). Of these studies, a follow-up study of the same patient population by the same authors subsequently did show an association of ETBF with diarrheal disease (59a, 84), one study had a B. fragilis isolation rate of only 6% (77), and three studies were from Brazil (4,8,48).…”

“…Of these studies, a follow-up study of the same patient population by the same authors subsequently did show an association of ETBF with diarrheal disease (59a, 84), one study had a B. fragilis isolation rate of only 6% (77), and three studies were from Brazil (4,8,48). Excluding the one study (128) focused on adults, all but one (10) of the remaining studies confirmed the original observations of Sack et al (95) suggesting that, similar to the case with Clostridium difficile, there is no association between ETBF isolation and diarrheal illnesses in children of Ͻ1 year old, whereas diarrheal disease due to ETBF emerges after age 1.…”

Enterotoxigenic and nontoxigenic Bacteroides fragilis strains isolated in Brazil

“…A fragment of expected size was amplified from one B. fragilis strain (0.9%) which was classified as ETBF. Other studies in Brazil already reported low numbers of ETBF strains, ranging from 1.5-3.0% (Bressane et al 2001, Antunes et al 2002, Krzyzanowsky & Avila-Campos 2003. Nevertheless, high numbers of ETBF strains have been isolated in several other regions of the world (Kato et al 1996, Pantosti et al 1997.…”