Acta Paediatr 81: 266-7. 1992

SHORT COMMUNICATION

Colostrum and human milk inhibit localized adherence of enteropathogenic Escherichia coli to HeLa cells MLM Silva and CMS Giampaglia Escola Paulisla de Medicina, Deparimeni of Microbiology, Immunology and Parasiiology. Rua Boiucaiu, Siio Paulo, Brazil

In many developing countries (1) enteropathogenic Escherichia cofi (EPEC) are the most common cause of diarrhoea in children aged less than one year. E P E t include several specific serotypes, 01 1 l:H-, 01 1lab:HZ and 0119:H6 being the most frequent agents of diarrhoea found in Brazil (2). EPEC strains d o not produce enterotoxins or invade the intestinal mucosa (1, 3) but adherence to enterocytes with subsequent destruction of microvilli is believed to represent their main mechanism of virulence (4). Adherence to host cells can be studied in vitro using HeLa or HEp-2 cells (5) to which EPEC serotypes show a localized adherence (6) pattern. Breast feeding has been related to protection from intestinal or respiratory infections (7). Secretory IgA (8) and cell receptor analogues (9, 10) may be responsible for this protection, inhibiting the attachment of certain bacteria to host cells. It is shown in this study that colostrum and human milk inhibit localized adherence of different EPEC serotypes to HeLa cells. Adherence tests of eight EPEC strains belonging to serotypes 055:H-, 055:H6,055:H7,086:H34,011l:H-, 01 1 lab:H2, 01 19:H6 and 0142:H6 were performed with HeLa cells either in the presence of milk collected from three women, four, nine and 14 months after delivery (milk I , 2 and 3, respectively) or a pool of five colostrum samples. Adhesion tests were carried out as described previously (6) with some modifications. HeLa cells ( los cells ml-', 300 pl) were grown for 48 h in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with lo'% foetal bovine serum in Lab-Tek chamber slides for tissue culture (Nunc Inc., Naperville, Illinois), with eight chambers per slide. The medium was replaced with I50 pl of colostrum or milk in addition to 150 pl of DMEM with 2% foetal calf serum and 1 ' X Dmannose, followed by 100 p1 of an exponential-phase bacterial culture (2 x lo8cells ml-') grown in Trypticase Soy Broth (Difco). Unattached bacteria were removed by washing six times with phosphate-buffered saline after 30 min. Subsequently, 300 pl of the same medium without colostrum or milk was added to each chamber and the slides were incubated for 3 h a t 37°C (multiplication period). The cells were then washed three times with phosphate-buffered saline, fixed with methanol and stained with May-Grunwald and Giemsa as described previously (6). The effect of colostrum or milk upon

Table 1. Inhibition of localized EPEC adherence to HeLa cells by colostrum and three samples of human milk.

Inhibition of adherence (%) in the presence of Serotypes of EPEC strains 055:H055:H6 055:H7 086:H34 01 I I:H01 1lab:HZ 01 19:H6 0142:H6

Colostrum

Milk I

Milk 2

Milk 3

92 96 94 55 97 96 44 87

88 66 24 64 92 73 89 NT

91 24 71 28 96 86 75 61

91 69 94 72 88 79 81 79

All results were significant (chi-square test). NT= Not tested.

EPEC adherence to HeLa cells was determined by calculating the percentage of cells with six or more attached bacteria in relation to the control, carried out under the same conditions without colostrum or milk. No bactericidal or bacteriostatic effect was observed in growth curves carried out with and without a pool of the three samples of milk, under the same conditions as the test. Colostrum and the three samples of milk inhibited the adherence of all EPEC strains (Table 1). High percentages of inhibition were observed with strains of serotypes 055:H-, 01 1 I:H- and 01 1 lab:H2, the last two being the most frequently found in Brazil (46% of EPEC isolates) (2). The lowest percentages of inhibition were observed with strains of serotypes 055:H6 and 086:H34. Localized adherence inhibition was not dependent on lactation time under the test conditions described and the differences observed may be related to the particular characteristics of each mother. The fact that common (01 1 l:H-, H2 and 01 19:H6) and rare (055:H-, H6 and H7, 086:H34 and 0142:H6) EPEC serotypes were equally inhibited by colostrum or milk suggests that receptor analogues may be involved in inhibition. Another possibility is that Brazilian mothers exposed to the adhesins of 01 1 I and 01 19 strains may have antibodies to these antigens in their colostrum or milk, which would then be responsible for cross-reactive inhibition of antigenically similar adhesins of less common serotypes.

Inhibition of E. coli by colosirum and human milk

ACTA PEDIATR (81) 1992

References I . Levine MM. Escherichiucoli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis 1987;155:377-89 2. Toledo MRF, Alvariza MCB, Murahovschi J, Ramos SRTS, Trabulsi LR. Enteropathogenic Escherichiu coli serotypes and endemic diarrhea in infants. Infect Immun 1983;39:586-9 3. Law D. Virulence factors of enteropathogenic Escherichiu coli. J Med Microbiol 1988;26:1-10 4. Knutton S, Lloyd DR, McNeish AS. Adhesion of enteropathogenic Escherichiu coli to human intestinal enterocytes and cultured intestinal mucosa. Infect Immun 1987;55:69-77 5. Nataro JP, Scaletsky ICA, Kaper JB, Levine MM, Trabulsi LR. Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichiu coli. Infect Immun 1985;48:378-83 6. Scaletsky ICA, Silva MLM, Trabulsi LR. Distinctive patterns of

7. 8.

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adherence of enteropathogenic Escherichiu coli to HeLa cells. Infect Immun 1984;45:534-6 Hanson LA, Adlerberth I, Carlsson B, et al. Breastfeeding protects against infections and allergy. Breast Rev 1988;13:19-22 Boedeker EC, Cheney CP, Cantey JR. Inhibition of enteropathogenic Escherichiu coli (strain RDEC-I) adherence to rabbit intestinal brush borders by milk immune secretory immunoglobulin A. Adv Exp Med Biol 1987;216:919-29 Andersson B, Porras 0, Hanson LA, LagerdArd T, SvanborgEdtn C. Inhibition of attachment of Streptococcus pneumoniue and Huemophilus inzuenzue by human milk and receptor oligosaccharides. J Infect Dis 1986;153:232-7 Ashkenazi S, Mirelman D. Nonimmunoglobulin fraction of human milk inhibits the adherence of certain enterotoxigenic Escherichiu coli strains to guinea pig intestinal tract. Pediatr Res 1987;22:130-3

Received Jan. 14, 1991. Accepted June 11, 1991

Colostrum and human milk inhibit localized adherence of enteropathogenic Escherichia coli to HeLa cells.

Acta Paediatr 81: 266-7. 1992 SHORT COMMUNICATION Colostrum and human milk inhibit localized adherence of enteropathogenic Escherichia coli to HeLa...
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