Lactobacillus Prophylaxis for Diarrhea Due to Enterotoxigenic Escherichia coli

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUly 1981, p /81/ $02.00 Vol. 20, No. 1 Lactobacillus Prophylaxis for Diarrhea Due to Enterotoxigenic Escherichia coli MARY L. CLEMENTS,* MYRON M. LEVINE, ROBERT E. BLACK, ROY M. ROBINS-BROWNE,t LUIS A. CISNEROS, GEORGE L. DRUSANO, CLAUDIO F. LANATA, AND ALFRED J. SAAH Center for Vaccine Development, Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland Received 24 December 1980/Accepted 24 April 1981 In vitro and animal experiments indicated that lactobacilli might prevent Escherichia coli from colonizing the intestine and may produce substances counteracting enterotoxin. Lactinex, a commercial preparation of dried Lactobacillus acidophilus and L. bulgaricus, is marketed for uncomplicated diarrhea. Preliminary experiments in nonfasting volunteers indicated that lactobacilli in this preparation colonized the small intestine for up to 6 h. To evaluate the protective efficacy of Lactinex, a double-blind randomized study was carried out in which 48 volunteers (23 receiving Lactinex and 25 receiving placebos) were challenged with E. coli strains that produced heat-stable or heat-labile enterotoxins or both. No significant differences between the two groups were noted with respect to attack rate, incubation period, duration of diarrhea, volume and number of liquid stools, and coproculture yields. These data suggest that this lactobacillus preparation does not prevent or alter the course of enterotoxigenic E. coli diarrhea in adults. Lack of efficacy occurred despite efforts to maximize small bowel colonization, including administration of Lactinex in milk and in a 6-hour-interval regimen during 36 h before and 96 h after challenge. The rationale for treating intestinal disorders with yogurt, buttermilk, or other agents containing lactobacilli stems from a long-standing belief that nonpathogenic aciduric flora alter the intestinal milieu, thereby inhibiting or eliminating enteric pathogens. As early as 1908, Metchnikov advocated the use of oral lactobacilli to arrest intestinal putrifaction and to disinfect the intestine (18). Since that time, numerous publications, based on uncontrolled clinical observations, have suggested a role for lactobacilli in the prevention or management of diarrheas (1, 3, 4, 9, 27). In other studies, however, antidiarrheal effects with lactobacillus therapy were not observed (20, 21). More recently, animal and in vitro experiments have indicated that lactobacilli inhibit Escherichia coli-induced enterotoxin reactions perhaps by preventing E. coli from colonizing the jejunum and also by producing substances directed against the enterotoxins (6, 10, 19). These findings have led to renewed interest in the possible value of lactobacillus therapy against traveler's diarrhea, an illness usually caused by enterotoxigenic strains of E. coli (ETEC) (6, 17). t Present addres: Department of Microbiology, University of Natal, Congella 4013, South Africa. Lactinex is a dried preparation of Lactobacillus acidophilus and Lactobacillus bulgaricus that is available without prescription for uncomplicated diarrhea. Preliminary studies in nonfasting volunteers have demonstrated that most of the lactobacilli in Lactinex granules, when given in milk, can survive passage through the stomach and remain in the upper small bowel for up to 6 h (22a). At the Center for Vaccine Development, a randomized double-blind study with healthy adult volunteers was undertaken to determine the efficacy of Lactinex to protect against diarrhea due to ETEC. MATERIALS AND METHODS Volunteers. The 48 volunteers participating in these studies were college students and other healthy adults (mean age, 23.1 years; range, 18 to 35 years; 39 males). Challenge studies were carried out in the 22- bed Isolation Ward of the Center for Vaccine Development and were a part of a long-term program studying the pathogenesis, dose reponse, immune response to, and control of ETEC diarrhea. Protocols were approved by the University of Maryland Human Volunteer Research Committee and the Clinical Research Sub-Panel of the National Institute of Allergy and Infectious Diseases. Informed consent was obtained; the informed nature of consent was confirmed before challenge by requiring all volunteers to pass a written examination on all aspects of the study, including its purpose, hazards, procedures, bacteriology, and im- 104

2 VOL. 20, 1981 LACTOBACILLUS PROPHYLAXIS FOR DIARRHEA 105 munology (12). The prechallenge health status of volunteers was verified by medical history (which included questions regarding lactose intolerance and milk allergies), physical and psychological examinations, chest X-ray, electrocardiogram, complete blood counts, urinalysis, blood chemistries, and serological tests for syphilis and hepatitis B surface antigen. Preparation and administration of test suspensions. Lactinex granules and placebo granules were obtained from Hynson, Westcott, and Dunning of Baltimore, Md. Each 1-ounce (ca. 30-ml) packet of Lactinex granules contained approximately 2 x 100 dried, viable L. acidophilus and L. bulgaricus in equal proportions, whey powder, evaporated milk, lactose, and sucrose. The 1-ounce packaged placebo consisted of lactose, dextrose, powdered acacia, evaporated milk, and caramel and yellow coloring. Quantitative cultures of Lactinex packets documented that each contained 1.4 x 10" to 6.8 x 10' viable lactobacilli per package. Volunteers were randomly assigned to receive either Lactinex granules or identical-appearing placebo granules, and the study was conducted in a double-blind manner. To optimize the chances for intestinal colonization, the volunteers ingested 240 ml of skim milk containing a packet of either Lactinex or placebo granules at 6-h intervals beginning 36 h before and continuing for 96 h after challenge with virulent ETEC. The milk served to neutralize gastric acidity, thereby enhancing the passage of viable bacteria to the intestine. Challenge strains. Volunteers ingested 108 to 1010 organims of E. coli strains which produce heat-labile (LT) or heat-stable (ST) enterotoxins or both. Strains (and types) of E. coli employed for challenge were: LT, TD 255C4 (075:H9); ST, (nontypable) (14, 25); and LT/ST, B7A (0148:H28) (2) or H10407 (078: Hil) (5). All of the above strains, except for 214-4, possess type 1 somatic pili (16). The colonization factor antigen I is found only on strain H10407 (16). Inocula and challenge. Inocula of virulent ETEC were prepared by harvesting 18-h Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) cultures of the challenge strains with saline and making dilutions with saline (2, 26). Inoculum size was determined by replicate pour-plate technique before and after challenge. At 36 h after starting Lactinex or placebo, 36 volunteers drank 120 ml of a solution containing 2 g of NaHCO3 dissolved in 150 ml of distilled water. After 1 min, they drank the remaining 30 ml, in which the E. coli were suspended. Volunteers took no other drink or food during the 90 min before and after challenge. A slightly different regimen (previously described) (15) was employed to administer NaHCO3 buffer and challenge inocula of 1010 B7A organims to 12 volunteers. Clinical observations. Volunteers were interviewed daily, and oral temperatures were taken every 6 h and repeated within 5 min if they were 37.80C or above. Volumes of all stools and vomitus were measured. The consistency of the stools was graded by the following scale: grades 1 (fully formed) and 2 (soft) were considered normal; grade 3 denoted a thick liquid (assuming the shape of the container) stool, grade 4 was watery, and grade 5 was a rice-water stool. To replace diarrheal fluid losses, volunteers were required to drink glucose electrolyte solution amounting to 150% of the volume of their liquid stool. Beginning 96 to 120 h after inoculation, volunteers received 500 to 750 mg of neomycin every 6 h for 5 days to eliminate carriage of the pathogen. Definition of diarrheal illness. Diarrhea was specifically defined as three or more unformed stools (grades 3 to 5), or two unformed stools totaling at least 200 ml within 48 h, or a single voluminous liquid stool (2300 ml). Severe diarrhea was defined as an illness with a stool volume of 2 liters or more. The observational period for diarrheal illness began after challenge and ended when the first dose of neomycin was given. Data were analyzed by the Student t, chi-square, or Fisher's exact tests. Stool culture. Stool specimens or rectal swabs collected daily were plated onto MacConkey and Levine eosin-methylene-blue agar. Ten colonies with a typical E. coli metallic sheen were subcultured on slants of trypticase soy agar (BBL) in screw-top tubes. After 18 h of incubation at 370C, the E. coli were tested for agglutination by specific antiserum (the challenge E. coli serving as positive control and saline serving as a negative control) which was prepared in rabbits as described elsewhere (13, 14). RESULTS Overall, 16 (70%) of 23 recipients of Lactinex granules and 17 (68%) of 25 volunteers who received placebo granules developed diarrhea after experimental challenge with ETEC. Among the volunteers who developed diarrhea, a slightly greater percentage of severe illnesses occurred in the Lactinex group than in the placebo group (25 versus 18%, respectively). The proportion of volunteers ill in either treatment group did not vary by strain of E. coli ingested, by type of toxins elaborated, or by presence of colonization factor antigen I or type 1 somatic pili (Table 1). Likewise, incubation periods, stool output (number and volume), and durations of diarrheal illness on the average were similar for the Lactinex and placebo groups. In each treatment group, the percentage of individuals who experienced clinical symptoms (ranging from fever to vomiting and abdominal complaints) was approximately equal (Table 2). No significant differences between the two groups were noted with respect to any of these parameters. All volunteers, except for one in the Lactinex group who ingested 10i B7A E. coli, excreted the challenge pathogen in the stool. There was essentially no difference in the time of onset of ETEC fecal excretion between the two treatment groups overall or by E. coli challenge strain. DISCUSSION In humans, L. acidophilus and several lactobacillus species are normal inhabitants of the

3 106 CLEMENTS ET AL. TABLE 1. ANTIMICROB. AGENTS CHEMOTHER. Clinical response of Lactinex- and placebo-treated volunteers after challenge with ETEC Study group Incubation Total no. of Total vol of Duration of Challenge inocula and strain (attack rate) period (h) diarrheal diarrhea (ml) illness (h) stools" (ST)b Lactinex (5/7) 28.4 ± 99.c 5.2 ± ± ± 15.1 Placebo (4/5) 24.5 ± ± ± ± TD225-C4 (LT) Lactinex (3/4) 6.5 ± ± ± ± 9.9 Placebo (2/5) 9.6 ± ± ± ± 17.1 i05 H10407 (ST/LT) Lactinex (3/5) 36.4 ± ± 2.9 2,497 ± ± 10.3 Placebo (3/4) 57.5 ± ± 3.7 1,335 ± ± B7A (ST/LT) Lactinex (1/3) Placebo (3/3) 23.4 ± ± ± ± B7A (ST/LT) Lactinex (4/4) 24.1 ± ± 2.9 1,146 ± ± 21.7 Placebo (5/8) 14.4 ± ± 4.8 1,494 ± ± 16.4 Definitions of diarrhea are explained in the text. b Form of enterotoxin produced. c Mean ± standard error of the mean. Challenge orga- TABLE 2. Clinical symptoms of ETEC diarrhea in volunteers by treatment group No. (%) of volunteers with: Study group Fever' Vomiting Borbor- Abdominal Anorexia Malaise Lactinex (n =7) (86) 5 (71) 3 (43) 2 (29) Placebo (n =5) 1 (20) 0 4 (80) 3 (60) 4 (80) 4 (80) TD225-C4 Lactinex (n = 4) 0 1 (25) 2 (50) 3 (75) 2 (50) 2 (50) Placebo (n =5) 1 (20) 0 3 (60) 4 (80) 2 (40) 3 (60) H10407 Lactinex (n =5) 1 (20) 2 (40) 3 (60) 3 (60) 3 (60) 0 Placebo (n = 4) 1 (25) 1 (25) 3 (75) 4 (100) 1 (25) 0 B7A Lactinex (n = 7) (71) 5 (71) 2 (28) 1 (14) Placebo (n = 11) 0 1 (9) 8 (73) 9 (82) 7 (62) 5 (45) Total Lactinex (n = 23) 1 (4) 3 (13) 16 (70) 16 (70) 10 (43) 5 (22) Placebo (n = 25) 3 (12) 2 (8) 18 (72) 20 (80) 14 (56) 12 (48) aoral temperature of 37.8 C or higher. colon as well as of the oral cavity, rectum, and vagina (23, 24). Although they probably do not constitute true indigenous flora of the small bowel, L. acidophilus have been commonly recovered from the distal ileum and, in smaller numbers, from the upper snall intestine. It is believed that lactobacilli may live transiently in the upper small intestine after being swallowed in fermented foods or in saliva, whereas their presence in the ileum is probably also due to backwash from the cecum (8, 22a). In contrast, L. bulgaricus, the original yogurt strain, is not normally found in humans, and thus far, numerous attempts to colonize the human gut with these bacteria have not been successful (11, 22). Lactobacillus therapy for diarrhea has been promoted for many years without verification of its efficacy in controlled clinical trials. Two controlled experiments (6, 10) in rabbit ileal loops provided evidence that Lactinex and some of its components (L. bulgaricus and L. acidophilus) can effect a significant reduction of intestinal fluid induced by enterotoxigenic (LT) E. coli 334A. Since lactobacilli (in milk) can traverse the stomach and remain viable in the jenunum for 4 to 6 h in humans (22a), it was conceivable that they might then inhibit the enterotoxin activity of E. coli in the small intestine. We therefore investigated whether this preparation might be an effective bioprophylactic agent for ETEC traveler's diarrhea. The results of our study, however, indicated that the commercially available preparation (Lactinex granules) containing

4 VOL. 20, 1981 high counts of L. acidophilus and L. bulgaricus did not prevent or alter the course of ETEC diarrhea in adults. The E. coli challenge strains used were those implicated as causal agents in traveler's diarrhea in different geographic areas. Lack of protection occurred despite efforts to maximize the small bowel implantation of lactobacilli, which included admninistration of Lactinex in milk on a six-hour-interval schedule. Our findings were consistent with those of a small-scale field trial in Mexico to evaluate the prophylactic effect of Lactinex tablets on traveler's diarrhea (21). Although the incidence of diarrhea during the field trial was very low overall, no differences in incidence or duration of the acute diarrhea were observed between the group receiving Lactinex and the control group. It is not possible to compare attack rates for E. coli diarrhea in the two groups since etiological studies were not performed. It is conceivable that the challenge dose of E. coli in the volunteer model might have overwhelmed any protection offered by lactobacilli. In earlier studies to investigate ETEC immunity (15), the same levels of challenge organisms were ingested by volunteers without overwhelming the natural protection against diarrhea conferred by previous ETEC illness. Since various parameters of illness appear to be dose related, it is noteworthy that the clinical symptoms, incubation periods, and duration of illnesses of control volunteers (and in Lactinex recipients) mimicked those observed in nature with traveler's diarrhea due to ETEC (7). The data from this study do not suggest that Lactinex therapy prevents or alters the course of ETEC diarrhea in adults. The apparent lack of efficacy occurred despite attempts to maximize small bowel colonization. However, this study does not refute the possibility that lactobacilli in the presence of smaller inocula of ETEC could prevent adhesion of E. coli or inhibit the enterotoxigenic effects in the human jejunum or both. To answer these questions, LACTOBACILLUS PROPHYLAXIS FOR DIARRHEA 107 additional experimental studies would be required. The effects of lactobacillus preparations on other types of diarrhea, such as antibioticassociated diarrhea in particular, may also warrant further study (1, 9, 20). ACKNOWLEDGMENTS This work was supported in part by Public Health Service research contract NO I-A with the National Institute of Allergy and Infectious Diseases, research contract DAMD17-78-C-8011 with the U.S. Army Research and Development Command, and a research grant from Hynson, W,escott, and Dunning, Baltimore, Md. LITERATURE CITED 1. Beck, C., and H. Necheles Beneficial effects of administration of LactobaciUus acidophilus in diarrheal in other intestinal disorders. Am. J. 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