Enterotoxigenic Bacteroides fragilis: epidemiologic studies of its role as a human diarrhoeal pathogen.

Strains of Bacteroides fragilis which produce enterotoxin(s) (ETBF) have been associated with diarrhoeal diseases in young domestic animals and have also been isolated from humans with diarrhoea. We have determined epidemiologically that ETBF are significantly associated with diarrhoea in humans. We studied Apaches, primarily children, with diarrhoea attending an outpatient facility in Whiteriver, Arizona, from July 1986 through July, 1988. Stool cultures for isolation of ETBF and other diarrhoeal pathogens were taken from these persons as well as from age and time-matched control persons who did not have diarrhoea. ETBF were isolated significantly more often from persons with diarrhoea (12%) than from controls (6%), p = 0.03. Isolation was highest (20-24% of stool cultures positive) during the second and third years of life. The diarrhoeal syndrome associated with ETBF was non-specific, and most characteristic of a secretory, rather than inflammatory, type of diarrhoea. ETBF are significantly associated with acute diarrhoea in Apache children, and may be an important newly described cause of diarrhoea in humans.

Enterovirulence of enterotoxigenic Bacteroides fragilis in gnotobiotic pigs.

A porcine isolate of enterotoxigenic Bacteroides fragilis colonized the intestinal tract and caused watery, nonhemorrhagic diarrhea when given orally to 12, 1- to 2-day-old gnotobiotic pigs. Diarrhea occurred 2 to 3 days post-inoculation and continued throughout the 4 to 6 day post-inoculation period. Diarrheic pigs became mildly anorexic and dehydrated. They developed intestinal lesions characterized by swelling, vacuolation, and exfoliation of enterocytes, and crypt hyperplasia throughout the large intestine and, to a lesser extent, in the distal small intestine. Bacterial adherence to, or invasion of, the intestinal mucosa was not detected. A porcine isolate of nonenterotoxigenic B. fragilis was administered orally to six control pigs. The isolate colonized the intestinal tract, but the pigs did not develop clinical disease or intestinal lesions. The pathogenetic mechanism of the disease may involve mediation by a soluble enterotoxin (or toxins) elaborated by B. fragilis.

Rabbit model to evaluate enterovirulence of Bacteroides fragilis.

Enterotoxigenic Bacteroides fragilis elicited fluid accumulation in the intestinal tract and exfoliation of epithelial cells in the proximal colon of the 2-week-old rabbit. The rabbit model was employed to screen isolates of B. fragilis for enterovirulence and to study the pathogenesis of disease caused by enterotoxigenic B. fragilis.

Enumeration of enterotoxigenic Bacteroides fragilis in municipal sewage.

Of 237 isolates of Bacteroides fragilis from sewage influent at the Bozeman, Mont., wastewater treatment plant, 22 (9.3%) were enterotoxigenic, as indicated by the ability to elicit fluid accumulation in the lamb ileal loop test. It appears that enterotoxigenic B. fragilis is endemic in the human population at a moderately high level.

Diarrheal disease caused by enterotoxigenic Bacteroides fragilis in infant rabbits.

Enterotoxigenic Bacteroides fragilis caused severe, nonhemorrhagic, watery diarrhea when 10(9) CFU of a porcine or human isolate was administered orogastrically to 3-day-old rabbits. The bacterium colonized the intestinal tract with a predilection for the large intestine (10(9) CFU/g of cecal contents). Diarrhea occurred at an average of 4.6 days postinoculation, and 84% of rabbits were dead or moribund at an average of 8.8 days postinoculation. The disease was characterized by watery diarrhea and dehydration. Severe histologic lesions including inflammation, exfoliation of epithelial cells, and crypt hyperplasia were observed throughout the colon. There was no indication of bacteremia or of bacterial adherence to or invasion of intestinal epithelial cells. Rabbits inoculated with nonenterotoxigenic B. fragilis were colonized with B. fragilis but did not develop clinical disease or intestinal lesions. While the pathogenesis of this disease is undefined, clinical signs of disease and histologic changes were consistent with a mechanism of net secretion of fluid into the small intestine and decreased absorption of fluid from the large intestine. Enteric disease caused by enterotoxigenic B. fragilis in infant rabbits was similar to naturally occurring enteric disease associated with the bacterium in humans and livestock. This study established that enterotoxigenic B. fragilis is enteropathogenic in intact infant rabbits.

Isolation of enterotoxigenic Bacteroides fragilis from humans with diarrhea.

Enterotoxigenic Bacteroides fragilis was isolated from stool specimens of 8 of 44 diarrheic individuals (ages, 4 months to 69 years). The individuals had watery diarrhea and intestinal cramping; and infants had hyperthermia, vomiting, and blood in the stools. No recognized enteric pathogens were detected in seven of the eight diarrheic individuals positive for enterotoxigenic B. fragilis. The bacterium produced an enterotoxin detectable in concentrated broth that supported bacterial growth. Fifteen adult rabbits with ligated ceca developed fatal enteric disease following intraileal injection with 5 x 10(9) CFU of enterotoxigenic B. fragilis. Conversely, eight control rabbits injected with nonenterotoxigenic B. fragilis remained clinically normal. As few as 5 x 10(3) CFU of enterotoxigenic B. fragilis caused fatal enteric disease in the rabbit model. Disease in rabbits was characterized by mucoid, often hemorrhagic, diarrhea. The bacterium colonized the caudal small intestine and the colon of the rabbits and caused moderate to severe necrotizing colitis. Enterotoxigenic B. fragilis is widespread in the intestinal tract of diarrheic humans and is enteropathogenic in adult rabbits with ligated ceca. Its possible role in the enteric disease complex merits further study.

Diarrhea associated with enterotoxigenic Bacteroides fragilis in foals.

Enterotoxigenic Bacteroides fragilis (ETBF) was isolated from the feces of 10 of 40 Thoroughbred foals with naturally acquired diarrhea. Of the 10 foals positive for ETBF, 6 were less than or equal to 7 days old. Fecal specimens from 4 of the 10 foals also were positive for rotavirus, and one fecal specimen was positive for Salmonella enteritidis. Clinical or hematologic differences were not evident between foals infected with ETBF only and those infected with ETBF and another recognized enteric pathogen. Only 1 of 10 foals infected with ETBF died. Of 25 adult rabbits with ligated ceca, 23 developed mucoid, often hemorrhagic, diarrhea after inoculation of 5 X 10(9) viable ETBF cells into the ileum. Nine of 13 (69%) rabbits inoculated with 1 of 3 isolates of ETBF died, but none of 12 inoculated with 1 of 6 other isolates of ETBF died. Enteric disease did not develop in 15 rabbits inoculated with nonenterotoxigenic B fragilis.

Association of enterotoxigenic Bacteroides fragilis with diarrheal disease in young pigs.

Enterotoxigenic Bacteroides fragilis (ETBF) was isolated from intestinal contents of 10 of 45 pigs with diarrhea. Of 10 ETBF-positive pigs, seven were 1 to 4 weeks of age, whereas three pigs had diarrhea after weaning (6 to 8 weeks of age). Of ten pigs infected with ETBF, nine were also infected with K88-positive Escherichia coli, rotavirus, coronavirus, coccidia, or Cryptosporidium. Of 11 conventional pigs exposed to ETBF at 1 to 2 days of age, two subsequently developed fatal postweaning diarrhea. An ETBF isolate, serologically similar to the challenge-exposure strain, was isolated from the feces of the two diarrheal pigs, but not from three healthy littermates. Fatal enteric disease occurred in 11 of 12 adult rabbits with ligated ceca after approximately 5 X 10(9) colony-forming units of ETBF were injected into the ileum. Disease in rabbits was characterized by anorexia and severe, mucoid, hemorrhagic diarrhea. Of 11 rabbits given non-ETBF or sterile brain-heart infusion broth, ten remained clinically normal during the 7-day observation period.

Antigenic characteristics of enterotoxigenic and nonenterotoxigenic isolates of Bacteroides fragilis.

Fecal isolates of enterotoxigenic (44 isolates) and nonenterotoxigenic (25 isolates) Bacteroides fragilis were obtained from diarrheic calves (62 isolates), lambs (2 isolates), and pigs (5 isolates). Using a Microtiter whole-cell agglutination test and gel double-diffusion analysis, the isolates were reacted with nonabsorbed rabbit antisera prepared against 13 isolates of enterotoxigenic B fragilis (ETBF). Isolates of B fragilis were antigenically diverse. Thirty-seven (84%) of the 44 isolates of ETBF comprised 13 serogroups on the basis of reaction in the agglutination test. Fourteen (56%) of the 25 isolates of non-ETBF comprised 4 of the 13 groups. Compared with results of the gel-diffusion test, most isolates had a different agglutination test reaction pattern against the 13 antisera. Isolates of ETBF could not be distinguished from non-ETBF. Antigenic heterogeneity of B fragilis facilitated differentiation of individual isolates, a capability that may be useful in future epidemiologic and virulence studies.

Isolation of Bacteroides fragilis from the feces of diarrheic calves and lambs.

A selective medium was developed for the isolation of Bacteroides fragilis directly from ovine and bovine fecal samples. The medium (tryptose blood agar plus polymyxin B, triclosan (Irgasan), novobiocin, and nalidixic acid) permitted growth of B. fragilis and several other species of Bacteroides but inhibited growth of most other intestinal bacteria.

Serologic analysis of isolates of Pasteurella haemolytica and Staphylococcus aureus from mastitic ewes.

Milk samples (10 ml) were collected aseptically from infected and healthy mammary glands of 20 range ewes in the early stages of unilateral acute mastitis. The ewes were on 5 different ranches in the Northern Rocky Mountain region of the United States. The samples were plated on tryptose blood agar and examined for bacteria of possible etiologic significance. Twelve of the 20 ewes were infected with Pasteurella haemolytica, 4 ewes with Staphylococcus aureus, and 1 ewe with both bacteria. Twelve of the P haemolytica infections were in pure culture as were 4 S aureus infections. The 13 isolates of P haemolytica represented 6 different serotypes. Isolates of P haemolytica from ewes on the same ranch were as serologically diverse as were isolates from ewes in different herds. The 5 isolates of S aureus were similar antigenically. Bacterial isolates were not obtained from the milk of clinically healthy mammae.

Prevalence of enteric pathogens in the feces of healthy beef calves.

Fecal specimens from 136 healthy beef calves (1 day to 12 weeks of age) were examined for the presence of infectious agents known to cause enteric disease in calves. The calves were selected from 22 herds in which all calves were free of clinically apparent enteric disease. Salmonella sp, enterotoxigenic Escherichia coli, Cryptosporidium, and coronavirus were not detected in any of the calves. Three calves were infected with rotavirus and 1 calf was infected with Yersinia enterocolitica. Campylobacter-like bacteria were isolated from 50 of 130 calves, with 36 of the calves positive for C jejuni. Seemingly, clinically normal calves may be infected more often with enterotoxigenic E coli, Cryptosporidium, coronavirus, or rotavirus in herds in which some calves have enteric disease than in herds free of major enteric disease. Campylobacter jejuni was well adapted to the bovine host and was of similar prevalence in diarrheal and nondiarrheal calves. The K99 positive, nonenterotoxigenic E coli was isolated from the feces of 16 healthy calves. Information in addition to the presence of K99 antigen is useful when diagnosing enterotoxic colibacillosis in calves.

Bacteroides fragilis: a possible cause of acute diarrheal disease in newborn lambs.

Bacteroides fragilis with enterotoxin-like activity (BFEL) was isolated from the feces of 24- to 48-h-old lambs with acute diarrheal disease on three different sheep ranches in the Northern Rocky Mountain region of the United States. The lamb intestinal loop test was used to enrich for the bacterium before its initial isolation. Pure cultures of B. fragilis from the feces of diarrheic lambs caused fluid accumulation in five of five lamb intestinal loop tests and in three of three calf intestinal loop tests. Two different serogroups of BFEL were found in diarrheic lambs, and a third serogroups was found in the feces of a diarrheic ewe. Enteric disease characterized by diarrhea, depression, and inappetence occurred in three of eight newborn, colostrum-fed lambs after oral challenge inoculation with viable BFEL. One of the three lambs died 32 h after challenge. Clinical signs of disease were similar to those observed in the naturally occurring disease. B. fragilis is an obligately anaerobic bacterium found in the intestinal tract of most animals and humans. It is a serious cause of extraintestinal infection in humans. The bacterium has not been reported to cause fluid accumulation in the intestine or to cause diarrhea in any species of animal.