Immediate Inflammatory and long term Bowel effects Diseases of oral (IBD) application Crohn s disease of nonpathogenic (CD), Ulcerative E. colicolitis (UC) into newborns H. Tlaskalová-Hogenová, Lodinová-Žádníková R., Cukrowska B., Hudcovic T., Kozáková H., Štěpánková R., L. Schulze* Dept. of Immunology and Gnotobiology, Institute of Microbiology AS CR, Vídeňská 183, Prague, Czech Republic; * Ardey Pharma, Germany ISAPP Meeting, London, June 28th, 27
Raja Lodinova-Žádníková, MD, e-mail: annajan@volny.cz Vol. 26. 197
Characteristics of probiotic E. coli strain used for repeated oral application to children (Lodinová- Žádníková et al., 1967, 197, 1991, 23, 27) Serotype: 83:K2:H31 A human intestinal E.coli strain with usual biochemical properties. Sensitive to all commonly used antibiotics. It does not belong to any group of enteropathogenic E. coli strains and does not produce thermolabile or thermostabile enterotoxin. It is t1 fimbriated. It does not carry any plasmid and is a poor recipient for foreign plasmid DNA. After 3 years the strain has not changed in any of his properties, including genetical characteristics.. Monocolonization of GF pigs with E. coli O83 resulted in an increased expression of aminopeptidase N on the tips of villi, equivalent to that seen in CV piglets Histochemical staining of aminopeptidase N in cryosections of piglet duodenum (Kozáková et al., Microbes and Infection, 26)
11 % of ref. 1 6 1 1 IgA COLONIZED breast fed bottle fed 1 2 3 Řada1 Řada2 Řada3 Řada IgM CONTROLS breast fed bottle fed IgA and IgM antibodies to E. coli O83 in stools of infants after weeks lasting repeated oral application of live E. coli O83:K2:H31 started during the first 6 days of life (x1 8 three times a week) 6 1 1 2 3 before 2-3 -7 8-11 12-1 weeks Řada1 Řada2 Řada3 Řada (Lodinová-Žádníková et al., Ped. Res., 1991) 16 1 11 6 1 11 1 % of ref. IgA COLONIZED breast fed bottle fed 1 2 3 Řada1 Řada2 Řada3 Řada IgM CONTROLS breast fed bottle fed IgA and IgM antibodies to E. coli O83 in saliva of infants after weeks lasting repeated oral application of live E. coli O83:K2:H31 started during the first 6 days of life (x1 8 three times a week) 6 1 1 before 2 2-3 -7 3 8-11 12-1 weeks Řada1 Řada2 Řada3 Řada (Lodinová-Žádníková et al., Ped. Res., 1991)
2 2 1 1 SIgA Levels (mg/l) of total secretory immunoglobulins Řada1 Řada2 Řada3 mg/l 9 8 7 6 3 1 2 3 6 7 8 9 1 11 SIgM SIgA and SIgM in saliva of infants after application of E. coli O83 and controls Řada1 Řada2 Řada3 2 1 1 1 2 2 week 1 2 3 6 7 8 9 1 (Vančíková et al., Folia Microbiol., 23) log 2 of titer 7 6 3 2 1 E. coli O83 Milk antibodies 1 1 2 3 3 6 7 8 96 1 weeks 7 8 mg/ml 2 Milk secretory IgA E. coli O83 Level of secretory antibodies andimmunoglobulins in Řada1 maternal milk of infants Řada2 colonized Řada3 with E. coli O83 Řada Řada Řada6 Influence of oral application of E. coli O83 to the infants on the specific antibody level and secretory IgA in the maternal milk 1. 1. Řada1 Řada2 Řada3 mothers of colonized infants mothers of control infants 1 1 2 2 3 3 6 7 8 9 6 1 7weeks (Lodinová-Žádníková et al., Adv. Exp. Med. Biol. 371, 131-138,199)
Percentage of infants with positive rectal cultures for E. coli O83 and for E. coli of other serotypes after application of E. coli O83 Breast-fed Bottle-fed 1 3 1 3 (day) (day) E. coli O83 % 67% 9% % % 6% Other E. coli 2% 17% 1% 2% % % (Slavíková et al., Adv. Exp. Med. Biol. 371, 21-23, 199) Detection of the E. coli strain O83 after oral application in infants (3 times/week, 1 9 /ml) O83 and others O83 only % 7 6 % 3 2 1 3 days 3 months 6 months 1 year 2 years 3 years (Lodinová-Žádníková et al., J.Clin.Exp. Allergy, in press)
Mannose-sensitive adherence of E. coli O83 isolates recovered from breast-fed and bottle-fed infants Adherence to intestinal epithelial cell line HT29 is expressed as the mean number of adhering bacteria per cell (Slavíková et al., Adv. Exp. Med. Biol. 371, 21-23, 199) The influence of repeated oral application of E. coli O83 on the presence of bacterial pathogens in infants 3 Application of E. coli O83 Number of infants 2 2 1 Infants with pathogens Řada2 Řada1 1 Infants without pathogens 1I II 2 III 3 IV V VI 6 VII 7 VIII 8 IX 9 1 X XI 11 XII 12 13 I Months (Lodinová-Žádníková et al., Quad.Ped., 23)
Incidence of infections, number of isolated pathogenic bacterial strains in stools and elsewhere (nose, throat, blood culture, stomach, other) and need for antibiotics in colonized and control premature infants Group Colonized Controls N = 23 % N = 2 % Number of sick infants 19 37 Total number of infections after colonization 28 Need for antibiotics 27 6 Pathogens detected during hospitalization in stool 62 83 Number and % of disappeared pathogens 86 2 (Lodinová-Žádníková et al., 23) Influence of oral colonization of the intestine after birth with a probiotic E. coli strain on the frequency of allergic diseases and infections after 1 and 2 years 1 8 % 6 % 2 colonized infants 1 years after E. coli colonization * p <.1 23 % 8 % * p <.1 * p <.1 12 % 33 % control infants 2 years after E. coli colonization 16 % 32 % 9 % 6 % Allergy n = Infections n = 77 Allergy n = 1 Infections n = 1 (Lodinová-Žádníková et al., Int.Arch. Allerg.Immunol., 23)
Allergy incidence rate during the follow-up period in allergic families: clinical symptoms Specific IgE antibodies to common allergens are low, no significant differences were found % 3 3 non allergic controls allergic non-colonized allergic colonized * ** 2 2 1 1 * ** 3 6 12 2 36 months * p<. * * p<.1 (Lodinová-Žádníková et al., in press) Production of IL-1 by PBMC from healthy adults cultivated in vitro with various probiotic bacteria stimulatory index 3 2 1 E.coli E.c.83 E.coli E.c.Nis Nissle Lactobacillus Lac.imm. imm. 2hodin hrs 72hodin hrs (Kokešová, Bártová et al., 2)
Expression of cellular activation markers on PBMC cultivated with probiotic strains Activated cells CD2-% CD2 aktivovaných -% activated cells buněk 6 6 33 2 1 E.c.O83 E.c.Niss. imm. Controls E.c.83 E.c.Nis Lac.im m. kontrola CD19 CD3 CD19 CD8 CD3 CD8 Conclusion I. More than 3 infants were repeatedly treated by oral application of E. coli O83:K2:H31 to prevent or treat nosocomial infections. The strain exert short term clinical effects (oubreaks of infection) as well as delayed effects (development of allergy). Activation of the mucosal immune system and systemic immunity after E. coli application was demonstrated. No adverse effects after oral application were found. This E. coli strain is commercially available from 1997 in Czech Republic ( Colinfant, Dyntec) and is widely used by pediatrists.
The probiotic drug for life. MUTAFLOR Escherichia coli strain Nissle 1917 EcN contains four genomic islands (GEI I-IV): GEI I Nissle 1917 Determinants for 2 microcins (mch and mcm), F1C fimbriae (foc), Fe-uptake system (iro) GEI II Nissle 1917 Genes for capsule biosynthesis (kps, kfi), Fe-uptake system (iuc) GEI III Nissle 1917 Roughly homologous to the sequence known for E. coli CFT73 GEI IV Nissle 1917 Fe-uptake system (ybt) Grozdanov et al.: J. Bacteriol. 2,186: 32-1. Analysis of the genome structure of the nonpathogenic probiotic E. coli strain Nissle 1917. E. coli Nissle 1917 chromosome (Grozdanov et al., J.Bacteriol., 2)
Characteristics of the groups of premature infants Groups Caesarian Birth weight Birth lenght Gestat.age Mother s milk of infants section (g) (cm) (weeks) feeding Colonized 26 (76%) 176 2 33 26 (76%) n = 3 Noncolon. 22 (81%) 1829 2 33 21 (78%) n = 27 (Cukrowska et al., Scand.J.Immunol., 22) Specific antibodies in the blood of premature infants after oral application of probiotic E. coli Nissle 1917 6 Standard serum (%) 3 2 1 Control infants Colonized infants n=27 n=3 * ### *** ** IgM IgA day 1 days day 1 days Days after birth (Cukrowska et al., Scand.J.Immunol. 22)
SI Dynamics of the cellular response (H 3 incorporation) of cord and peripheral lymphocytes of premature infants after the oral application of probiotic E. coli Nissle 1917 12 1 heat inact. E. coli 8 6 Řada1 Řada2 Řada3 sonicate LPS 2 day 1 days 21 days 1 2 3 Days after birth (Cukrowska et al., Scand.J.Immunol., 22) Proliferative response of blood cells of premature infants after oral application of probiotic E. coli Nissle 1917(n=3) and of noncolonized (control) infants (n=27) 12 1 8 Control infants n=27 n=3 Colonized infants *** # *** # *** # SI 6 *** # 2 day 1 days day 1 days Days after birth (Cukrowska et al., Scand.J.Immunol.,22)
Colonization of E. coli Nissle 1917 (Mutaflor ) in premature infants 1 % 9 % 8 % 8,1 81,8 1, 9,9 81,8 7 % Proportion of Infants 6 % % % 3 % 2,3 2,3 29, 3,2 Mutaflor No Statement No statement includes no recultivation possible 2 % 1 % 2, not received in laboratory no information % Day 3 Day Day 7 Day 1 Day 21 Day 28 Month 12 Month 2 (unpublished, data on file MU 96 9) grading 2, 2 1, 1, E.coli 83 Protective (antiinflammatory) effect of probiotic bacteria in mouse model of human IBD E.coli Nissle Lactobac. imm. PBS Clinical symptoms (rectal prolaps, bleeding etc.) in BALB/c mice with DSS induced colitis treated repeatedly with various probiotics 6 grading Histopathological changes present in colon of BALB/c mice with DSS induced colitis treated repeatedly with various probiotic bacteria 3 3 E.coli 83 E.coli Nissle Lactobac. imm. PBS
Conclusion II. Mechanisms of Action of E. coli Nissle 1917 in the Gut Direct antagonistic activity Inhibition of growth/ killing of pathogenic bacteria Mucin and defensin production Immunomodulation Antiinflammatory effect (Inhibition of TNFα effect on IL-8) Inhibition of invasion (Salmonella, EIEC, AIEC, Shigella, Yersinia, Listeria) Darmepithel Gut epithelium ACKNOWLEDGEMENT R. Lodinová-Žádníková B. Cukrowska T. Hudcovic H. Kozáková R. Štěpánková L. Schulz Graphic Design: Veronika Patrovská e-mail addreses: Tlaskalová: tlaskalo@biomed.cas.cz Lodinová: annajan@volny.cz Dept.Immunology and Gnotobiology Inst. of Microbiology AS CR, Prague and Nový Hrádek The Children s Memorial Health Inst., Poland Ardey Pharma, Germany Supported by Grants NIH Fogarty Grant (USA) H13 936-3, EU Project LABDEL QLK3-2-3 and Project GA AS CR, Ministery of Health CR.