Letters in Applied Microbiology 1997, 25, 325–326

Improved detection of enteroaggregative Escherichia coliusing formalin-fixed HEp-2 cells

J. Spencer, H. Chart, H.R. Smith and B. RoweLaboratory of Enteric Pathogens, Central Public Health Laboratory, London, UK

1471/97: received 13 March 1997 and accepted 15 April 1997

J. SPENCER, H. CHART, H.R. SMITH AND B. ROWE. 1997. Certain strains of enteroaggregativeEscherichia coli cause detachment of HEp-2 cells during adhesion tests, preventingobservation of the aggregative adherent phenotype. The use of formalin-fixed HEp-2cells prevents cell detachment and facilitates the detection of enteroaggregativeE. coli.

INTRODUCTION and grown on glass coverslips based on the method of Cra-vioto et al. (1979). Glass coverslips were placed into 12-well

Enteroaggregative strains of Escherichia coli (EAggEC) are antissue culture plates (Costar, Cambridge, MA) and seeded

important cause of persistent infantile diarrhoea in countrieswith HEp-2 cells (37 °C, 48 h, 5% CO2). Conventionally, cell

such as Chile (Nataro et al. 1987), Bangladesh (Baqui et al.monolayers were washed (×3) with Earle’s balanced salt

1992) and Mexico (Cravioto et al. 1991; Giron et al. 1991).solution (EBSS; Flow Laboratories, McLean, VA) and over-

EAggEC adhere to HEp-2 cell monolayers in an aggregativelayered with fresh BME medium, prior to inoculation with a

or ‘stacked brick’ formation, a characteristic that forms thetest organism. The modified method involved washing cell

basis for the principal method of detecting these strains of E.monolayers (×3) with EBSS followed by fixation of cells

coli. EAggEC can also be detected with gene probes (Baudrywith phosphate-buffered saline (PBS) containing formalin

et al. 1990; Smith et al. 1994). Certain strains of EAggEC(1% v/v, 30min, room temperature). Cell monolayers were

cause HEp-2 cell monolayers to detach during adhesionthen washed (×3) with sterile PBS and overlaid with fresh

assays, preventing the reliable observation of the aggregativeBME medium.

phenotype (Gunzburg et al. 1990; Elliott and Nataro 1995).Bacteria were grown statically in peptone water (37 °C,

This study describes an improved procedure for detecting16 h), and 50ml of bacterial suspension used for each cell

EAggEC, using formalin-fixed HEp-2 cells.test. Following incubation (37 °C, 3 h), cell monolayers werewashed (×3) with PBS, and fresh BME added for a second

MATERIAL AND METHODS 3 h incubation (37 °C). Cells were washed (×3) with PBS,fixed with absolute methanol (10min) and stained (30min)

Bacteria with Giemsa stain (Merck Ltd, UK). Glass slides werewashed (×3) and dehydrated using an acetone/xylene gradi-Four strains of E. coli belonging to serotypes O77:H18ent, and mounted onto glass microscope slides with DePeX(E58583A), O86:H19 (47697A), O111:H21 (57144A) and(Merck Ltd). Bacterial adhesion patterns were observedO126:H27 (E40104) were designated as enteroaggregativeunder oil immersion.based on their ability to adhere to HEp-2 cells with a ‘stacked

Adhesion tests were also performed with formalin-fixedbrick’ pattern and to hybridize with a DNA probe for EAg-HEp-2 cells using the initial 3 h incubation period only, andgEC (Smith et al. 1994). Bacteria were stored on Dorset’s eggwith fixed HEp-2 stored at 4 °C for up to 1 month.agar slopes in the culture collection held by the Laboratory

of Enteric Pathogens.

RESULTSPreparation of HEp-2 cells and bacteria

All four strains of EAggEC caused HEp-2 cells to detachHEp-2 cells were maintained in basal medium Eagle (BME)during the conventional adhesion assay; an example is showedtissue culture medium (Sigma Chemical Co. Ltd, Poole, UK)in Fig. 1a. However, these strains adhered to the fixed cellsCorrespondence to: Dr H. Chart, Laboratory of Enteric Pathogens, Central

Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK. with an aggregative phenotype without causing HEp-2 cell

© 1997 The Society for Applied Bacteriology

326 J. SPENCER ET AL.

4 out of 5 working d, allowing for overnight culture of teststrains.

The observation that EAggEC adhered to formalin-fixedHEp-2 cells demonstrated that EAggEC attach withoutinvolving the morphological changes made by HEp-2 cells.This demonstrates that the EAggEC adhesion process con-trasts with that, for example, of enteropathogenic E. coli(EPEC), where adhesion to HEp-2 cells involves changes inthe arrangement of actin filaments within HEp-2 cells (Bald-win et al. 1991). The HEp-2 adhesion method described herenot only facilitates the detection of enteroaggregative E. coli,but may also provide a better understanding of the mech-anisms involved in bacterial attachment to eukaryotic cells.

REFERENCES

Baldwin, T.J., Ward, W., Aitken, A., Knutton, S. and Williams,P.H. (1991) Elevation of intracellular free calcium levels in HEp-2 cells infected with enteropathogenic Escherichia coli. Infectionand Immunity 59, 1599–1604.

Baqui, A.H., Sack, R.B., Black, R.E. et al. (1992) Enteropathogensassociated with acute and persistent diarrhea in Bangladeshi chil-dren ³5 years of age. Journal of Infectious Diseases 166, 792–796.

Baudry, B., Savarino, J.S., Vial, P., Kaper, J.B. and Levine, M.M.(1990) A sensitive and specific DNA probe to identify entero-aggregative Escherichia coli, a recently discovered diarrhoealpathogen. Journal of Infectious Diseases 161, 1249–1251.

Cravioto, A., Gross, R.J., Scotland, S.M. and Rowe, B. (1979) Anadhesive factor found in strains of Escherichia coli belonging to

Fig. 1 (a) Dislodged HEp-2 cells of enteroaggregative strains of the traditional enteropathogenic serogroups. Current MicrobiologyEscherichia coli during adhesion assays. (b) With formalin- 3, 95–99.fixed HEp-2 cells, the adhesion phenotype was readily observed Cravioto, A., Tello, A., Navarro, A. et al. (1991) Association of(bar = 100 mm) Escherichia coli HEp-2 adherence patterns with type and duration

of diarrhoea. Lancet 337, 262–264.Elliott, S.J. and Nataro, J.P. (1995) Enteroaggregative and diffusely

adherent Escherichia coli. Reviews in Medical Microbiology 6, 196–damage (Fig. 1b). Formalin-fixed HEp-2 cell monolayers,206.stored at 4 °C in PBS, could be used for adhesion assays for

Giron, J.A., Jones, T., Millan-Velasco, F. et al. (1991) Diffuse-up to 3 weeks after preparation. Tests done after this time adhering Escherichia coli (DAEC) as a putative cause of diarrheashowed that the HEp-2 cells were slipping off the glass in Mayan Children in Mexico. Journal of Infectious Diseases 163,coverslip and did not stain as well as freshly formalin-fixed 507–513.cells, and so gave poorer results. Gunzburg, S.T., Burke, V. and Bettelheim, K.A. (1990) HEp-2 cell

adherence and Vero cell cytotoxin production by EPEC strainsisloted from children with diarrhoea in New Zealand. FEMS

DISCUSSION Microbiology Letters 69, 181–186.Nataro, J.P., Kaper, J.B., Robins-Browne, R., Prado, V. and Levine,Strains of EAggEC may cause HEp-2 cell detachment (Nat-

M.M. (1987) Patterns of adherence of diarrheagenic Escherichiaaro et al. 1995). However, fixing HEp-2 cell monolayerscoli to HEp-2 cells. Paediatric Infectious Diseases Journal 6, 829–with formalin prevented cell detachment and improved the831.observation and detection of E. coli expressing the EAggEC

Nataro, J.P., Yikang, D., Cookson, S. et al. (1995) Heterogeneityphenotype. The conventional HEp-2 test, using viable cells, of enteroaggregative Escherichia coli virulence demonstrated ininvolves harvesting HEp-2 cells from stock tissue culture volunteers. Journal of Infectious Diseases 171, 465–468.flasks and the preparation of cell tests 2 d prior to use. This Smith, H.R., Scotland, S.M., Willshaw, G.A., Rowe, B., Cravioto,permits the HEp-2 adhesion assay to be performed on only a A. and Eslava, C. (1994) Isolates of Escherichia coli, O44:H18 oflimited basis during a standard working week. In contrast, diverse origin, are enteroaggregative. Journal of Infectious Diseases

170, 1610–1613.formalin-fixed monolayers can be made in bulk and used on

© 1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 25, 325–326