Escherichia coli are important and often overlooked causes of severe diarrhea in children . Several molecules have been postulated to be critical pathogenicity factors, including Shiga toxins (Stx), produced by (1-3) E. coli O157:H7 and other serotypes of E. coli (designated Stx-producing E. coli (STEC) : intimin, encoded by (1) eae , which has also been associated with (4) diarrhea, both in epidemiologic and volunteer studies (5) and enterohemorrhagic (6) E. coli (EHEC)-hemolysin, which is encoded on the large plasmid of E. coli O157:H7 , which can also be found in (7,8) E. coli that do not produce Stx.
E. coli can also cause serious extraintestinal complications in children, such as hemolytic uremic syndrome (HUS) which is caused by STEC. Currently available data do not support the use of specific antimicrobial therapy for the treatment of childhood infections caused by STEC or other diarrheagenic E. coli . The clinical and economic impact of infection with these bacteria, including hospital admission for dehydration or for HUS, has only recently been elucidated (9) . (10)
Colostrum is an important defense against a variety of microbial pathogens. In many mammalian species, these protective factors are transferred from the mother to immunologically naive offspring. In humans, breast feeding during the first months of life decreases infant morbidity and mortality secondary to diarrheal and systemic infections
. Whole (11) bovine colostrum and immunoglobulin-enriched colostrum fractions have been used in infants and immunocompromised adults to treat or prevent enteric infections . (12-14)
We performed an exploratory study to test the hypotheses that
bovine colostrum containing antibodies against E. coli virulence factors, in particular Stx, intimin, and EHEC-hemolysin, hasten the elimination of a variety of diarrheagenic E. coli and reduce the frequency of stools in children infected with these organisms. PATIENTS, MATERIALS, AND METHODS
diarrhea whose stool cultures yielded E. coli containing eae which encodes intimin, in addition to Stx1, Stx2, or both or EHEC-hemolysin were considered eligible for enrollment. To exclude the presence of typical enteropathogenic E. coli, cultured strains had to be negative for the enteropathogenic E. coli adherence factor . (15)
Screening for the pathogenic
E. coli strains was performed by polymerase chain reaction (PCR) analysis of overnight stool cultures from sorbitol MacConkey agar to detect the presence of sequences homologous to the genes encoding Stx1, Stx2, intimin, and EHEC-hemolysin . For PCR, colonies grown overnight (approximately 1500 colonies) were harvested in 1 ml of saline solution (0.85% NaCl). The PCR reactions were performed with a commercial system (GeneAmp 9600; Perkin Elmer-Applied Biosystems, Weiterstadt, Germany). Amplifications were carried out in a total volume of 50 µl containing 15 µl bacterial suspension (10 (7,16-18) 6 cells), each deoxynucleoside triphosphate at 200 µM, 30 pM of each primer, 5 µl of 10-fold concentrated polymerase synthesis buffer, 1.5 mM MgCl 2, and 2.0 U of DNA polymerase (Ampli Taq; Perkin Elmer-Applied Biosystems). The primer sequences and PCR conditions are shown in Table 1. After 30 cycles had been completed, a 5-µl-aliquot of each PCR sample was analyzed by submarine gel electrophoresis on 1.5% (wt/vol) agarose gel and visualized by staining with ethidium bromide. To distinguish between Stx2 and Stx2c, restriction endonuclease analysis of PCR products obtained with Stx2-specific primers was performed with HaeIII and FokI, as described . To identify colonies of (17) E. coli containing these virulence genes in PCR-positive samples, colony-blot hybridization with 100 to 200 individual colonies was performed by using digoxigenin-labeled probes specific for the sequences of Stx1, Stx2, and intimin, respectively, as described . Production of Stx by (19) E. coli strains was tested by using the Vero cell cytotoxicity assay . Enterohemolytic phenotype was verified on enterohemolysin agar (20) . All stools were also screened for the presence of other enteropathogenic bacteria by standard culture techniques and for rotavirus antigen by enzyme immunoassay. (7) TABLE 1: Primers used in polymerase chain reaction assays for the detection of virulence factors in Escherichia coli strains grown from stools of children with diarrhea
Patients (age 1 month to 18 years) admitted to the hospital in either of Würzburg's two children's hospitals because of
diarrhea caused by E. coli were entered into the trial from July 1993 through June 1996. The frequency of isolation of Stx-producing E. coli in this population is about 2.8% of all patients with diarrhea . Two patients treated for established HUS in the Children's Hospital of the University of Erlangen, Germany, were also entered into the trial. The parents of the children and the adolescent patients were informed about the trial both orally (duration of interview, >60 minutes) and in writing. All parents and adolescents consented in writing to participate. (1)
A complete history, including the time of the onset of
diarrhea, a thorough physical examination, and laboratory values including complete blood count, urinalysis, blood gases, serum electrolytes, and other tests were recorded when appropriate. Exclusion criteria were unknown time of onset of diarrhea, a history of bovine milk intolerance, treatment of diarrhea with drugs, and breast-feeding. In addition, patients were excluded from the final evaluation if vomiting interfered with administration of the study medication.
The study medication was either
bovine colostrum or placebo. Bovine colostrum concentrate was prepared following the guidelines for the preparation of infant's milk and contained 80% protein with >65% immunoglobulin, mainly IgG (Lactobin, Biotest Pharma, Dreieich, Germany) . (21) Bovine colostrum used was from a single batch that originated from more than 100 carefully supervised cows not immunized against E. coli strains and contained high titers of neutralizing antibodies against Stx1, Stx2, and EHEC-hemolysin . Gelatin (92% protein, Töpfer, Dietmannsried, Germany), an innocuous preparation devoid of antibodies but similar in chemical composition and identical in appearance with (22) bovine colostrum, served as placebo.
Patients meeting the entry criteria and still in the hospital at the time of the bacteriologic diagnosis were randomly allocated to receive either
bovine colostrum or placebo administered double-blind as three daily doses of 7 g before meals for 14 days. Patients were examined every other day during their hospital stays, at least once weekly thereafter for the duration of treatment, and on days 15 (first day after treatment cessation) and 21. While in the hospital, the patients or parents of the patients were instructed in how to follow the study guidelines by specially trained nurses. Stool frequency was noted daily and compared with the stool frequency recorded on diary cards by the parents. Discrepancies between parental report and hospital records were reconciled by interview with the parents. Parents also recorded the consumption of the study medication and other events. Adherence to the study protocol after discharge from hospital was ascertained by telephone calls to the patients' homes.
It was assumed that 15 patients per group in a parallel group setting were sufficient to identify relevant treatment effects and to enable appropriate sample sizes to be determined in subsequent confirmatory studies. Data were analyzed using the Mann-Whitney test or Fisher's exact test.
P at the 5% level was regarded as significant. The study was approved by the ethics committee of the Medical Faculty of the University of Würzburg. RESULTS
Thirty children with
diarrhea caused by infection with E. coli expressing Stx1, Stx2, or both; intimin; or EHEC-hemolysin were entered into the study. No patient met the exclusion criteria at enrollment. In 1 patient each, Salmonella enterica and rotavirus antigen were also found. In three children with Stx-producing E. coli infection, study medication was discontinued because of preexisting continuous vomiting: Two of these patients had HUS before entry into the study, whereas the third patient was an infant with severe developmental retardation and wasting secondary to preexisting severe feeding problems. Vomiting in these three children was not considered a side effect of the study medication (two in the bovine colostrum group, one in the placebo group) but to be related to the preexisting illnesses. In the third patient who had HUS before entry into the study, treatment was administered. No patient experienced development of HUS after initiation of study treatment. Table 2 shows the characterization of the isolated strains of E. coli including bacteria of a variety of sero-types. The demographic and clinical data of the patients treated with bovine colostrum ( n = 13) and those treated with placebo ( n = 14) showed no obvious or significant differences ( Table 3). TABLE 2: Microbiologic characterization of Escherichia coli expressing Shiga toxin 1 or 2, Intimin (eae) or EHEC-hemolysin (Hly) isolated from patients with diarrhea and treated with bovine colostrum or placebo TABLE 3: Clinical and demographic data of 27 children with Escherichia coli diarrhea caused by infection with expressing Shiga toxin 1 or 2, intimin, or EHEC-hemolysin who were treated with bovine colostrum or placebo
The study medication was well tolerated. Six children treated with
bovine colostrum and seven administered placebo reported minor symptoms (poor appetite, abdominal colic, and occasional vomiting).
During treatment with
bovine colostrum, median stool frequency decreased from three stools per day to one, whereas during treatment with placebo, the median stool frequency did not change during the observation period ( P < 0.05; Table 4). The treatment period required for a reduction in stool frequency of at least 50% was shorter in patients treated with bovine colostrum ( P < 0.05). TABLE 4: Effect of treatment with Escherichia coli bovine colostrum or placebo in children with diarrhea caused by infection with expressing Shiga toxin 1 or 2, intimin, or EHEC-hemolysin
The excretion of
E. coli expressing intimin and EHEC-hemolysin by patients treated with bovine colostrum was not significantly different from that in patients treated with placebo ( Table 4). DISCUSSION
Bovine colostrum was well tolerated in children infected with diarrheagenic E. coli, specifically Shiga toxin-producing E. coli and E. coli expressing intimin and EHEC-hemolysin. However, two of the three patients in whom vomiting precluded continued administration had HUS. Thus, although it may be reasonable to treat children with HUS with bovine colostrum in an attempt to minimize the absorption of toxins from the bowel, children with HUS sometimes may not tolerate this treatment or other orally administered treatments early in the course because of vomiting. However, therapy with colostrum late in the course of HUS, after the appetite has returned and vomiting has abated, may play a role in reducing the number of bowel movements and thereby reduce potential secondary spread at that point.
Our study was not intended to demonstrate that
bovine colostrum prevents HUS in children infected with STEC. However, our demonstration that bovine colostrum reduced the stool frequency in children infected with diarrheagenic E. coli suggests that such an intervention may prevent secondary cases of STEC infection. Stx-producing E. coli may be excreted for several weeks after acute infection . It has been shown that exposure to a family member with (23) diarrhea or other direct contact is a risk factor for infection with STEC, leading to diarrhea and HUS . Reducing the frequency of (24,25) diarrhea may curtail transmission of such pathogens in homes and in day care centers if administered earlier in the course of diarrhea than was achieved in this study. However, a larger number of patients and their contacts would be necessary to test these hypotheses.
The interval between hospital admission secondary to
diarrhea and initiation of treatment with the study medication was 1 to 2 days. This was because the causative E. coli strain had to be grown from stools, identified by PCR, and informed consent obtained for participation in the trial. We do not know whether this delay adversely affected the efficacy of the bovine colostrum treatment. Therefore, the possibility exists that if colostrum therapy had commenced at the time of presentation, and not 1 or 2 days later, the difference in effects between the two groups may have been even greater.
Antibiotics are not recommended in infections with STEC for several reasons
: Antibiotic treatment may release Stx, which may be systemically absorbed (26) , may increase Stx production (27) , may be ineffective in children with STEC-associated enteritis (28) , and may increase the risk of development of HUS (29) . In this regard, it is somewhat reassuring to note that the colostrum did not accelerate clearance of the STEC from the stool of infected patients, as might have been expected had the antibodies elicited an intraintestinal bactericidal effect, with potentially increased toxin release. (30-32)
bovine colostrum reduces stool frequency in children infected with diarrheagenic E. coli and is well tolerated. These findings warrant extension of this treatment method to larger populations infected with diarrheagenic E. coli, ideally, targeting infected children early in the course of their illnesses to determine whether early administration prevents HUS and has an even greater effect on diarrhea than was demonstrated in this study. Acknowledgment: The authors thank Dr. Fricke, Kinderklinik am Mönchberg, Würzburg, and PD Dr. Ruder, Universitätskinderklinik, Erlangen, for allowing us to study their patients; Dr. Phillip Tarr, Seattle, Washington, for critical review of the manuscript; the nurses of the infectious diseases wards of the two Würzburg children's hospitals caring for our patients; and Barbara Plaschke for excellent technical assistance. REFERENCES
1. Huppertz HI, Busch D, Schmidt H, Aleksic S, Karch H.
in young children associated with
non-O157 organisms that produce Shiga-like toxins.
2. Huppertz HI, Rutkowski S, Aleksi S, Karch H. Acute and chronic
and abdominal colic associated with enteroaggregative
in young children living in western Europe.
3. Nataro JP, Kaper JB. Diarrheagenic
Escherichia coli. Clin Microbiol Rev
4. Jerse AE, Yu J, Tall BD, Kaper JB. A genetic locus of enteropathogenic
necessary for the production of attaching and effacing lesions on tissue culture cells.
Proc Natl Acad Sci USA
5. Bokete TN, Whittam TS, Wilson RA, et al. Genetic and phenotypic analysis of
with enteropathogenic characteristics isolated from Seattle children.
J Infect Dis
6. Donnenberg MS, Tacket CO, James SP, et al. Role of the
gene in experimental enteropathogenic
J Clin Invest
7. Schmidt H, Beutin L, Karch H. Molecular analysis of the plasmid-encoded hemolysin of
O157:H7 strain EDL 933.
8. Bauer ME, Welch RA. Characterization of an RTX toxin from enterohemorrhagic
9. Bell BP, Griffin PM, Lozano P, Christie DL, Kobayashi JM, Tarr PI. Predictors of hemolytic-uremic syndrome in children during a large outbreak of
10. Tarr PI.
O157:H7: Clinical, diagnostic and epidemiological aspects of human infection.
Clin Infect Dis
11. Cunningham AS. Morbidity in breast-fed and artificially fed infants.
12. Mietens C, Kleinhorst H, Hilpert H, Gerber H, Amster H, Pahud JJ. Treatment of infantile
gastroenteritis with specific bovine anti-
Eur J Pediatr
13. Brunser O, Espinoza J, Figueroa G, et al. Field trial of an infant formula containing anti-rotavirus and anti-
milk antibodies from hyperimmunized cows.
J Pediatr Gastroenterol Nutr
14. Davidson GP. Passive protection against diarrheal disease.
J Pediatr Gastroenterol Nutr
15. Jerse AE, Martin WC, Galen JE, et al. Oligonucleotide probe for detection of the enteropathogenic
(EPEC) adherence factor of localized adherent EPEC.
J Clin Microbiol
16. Rüssmann H, Kothe E, Schmidt H, Franke S, Harmsen D, Caprioli A, Karch H. Genotyping of Shiga-like toxin genes in non-O157
strains associated with
hemolytic uremic syndrome
J Med Microbiol
17. Rüssmann H, Schmidt H, Heesemann J, Caprioli A, Karch H. Variants of Shiga-like toxin II constitute a major toxin component in
O157 strains from patients with haemolytic uraemic syndrome.
J Med Microbiol
18. Schmidt H, Plaschke B, Franke S, et al. Differentiation in virulence patterns of
Med Microbiol Immunol
19. Schmidt H, Rüssmann H, Schwarzkopf A, Aleksic S, Heesemann J, Karch H. Prevalence of attaching and effacing
in stool samples from patients and controls.
Int J Med Microbiol Virol Parasitol Infect Dis
20. Schmidt H, Geitz C, Tarr PI, Frosch M, Karch H. Non-O157 pathogenic Shiga toxin-producing
Phenotypic and genetic profiling of virulence traits and evidence for clonality.
J Infect Dis
21. Stephan W, Dichtelmüller H, Lissner R. Antibodies from colostrum in oral immunotherapy.
J Clin Chem Clin Biochem
22. Lissner R, Schmidt H, Karch H. A standard immunoglobulin preparation produced from bovine colostra shows antibody reactivity and neutralization activity against Shiga-like toxins and EHEC-hemolysin of
23. Karch H, Rüssmann H, Schmidt H, Schwarzkopf A, Heesemann, J. Long-term shedding and clonal turnover of enterohemorrhagic
O157 in diarrheal diseases.
J Clin Microbiol
24. Rowe PC, Orrbine E, Ogborn M, et al. Epidemic
O157:H7 gastroenteritis and hemolytic-uremic syndrome in a Canadian Inuit community: Intestinal illness in family members as a risk factor.
25. Parry SM, Salmon RL, Willshaw GA, Cheasty T. Risk factors for and prevention of sporadic infections with vero cytotoxin (shiga toxin) producing
26. Tapper D, Tarr P, Avner E, Brandt J, Waldhausen J. Lessons learned in the management of
hemolytic uremic syndrome
J Pediatr Surg
27. Karch H, Strockbine NA, O'Brien AD. Growth of
in the presence of trimethoprim-sulfamethoxazole facilitates detection of Shiga-like toxin-producing strains by colony blot assays.
FEMS Microbiol Lett
28. Walterspiel JN, Ashkenazi S, Morrow AL, Cleary TG. Effect of subinhibitory concentrations of antibiotics on extracellular Shiga-like toxin 1.
29. Proulx F, Turgeon JP, Delage G, Lafleur L, Chicoine L. Randomized controlled trial of antibiotic therapy for
30. Ostroff SM, Kobayashi JM, Lewis JH. Infections with
O157:H7 in Washington State: The first year of statewide disease surveillance.
31. Pavia AT, Nichols CR, Green DP, et al. Hemolytic-uremic syndrome during an outbreak of
O157:H7 infections in institutions for mentally retarded persons: Clinical and epidemiologic observations.
32. Carter AO, Borczyk AA, Carlson JA, et al. A severe outbreak of
O157:H7-associated hemorrhagic colitis in a nursing home.
N Engl J Med