1260. Analysis of diffusely adhering Escherichia coli (EAEC) and enteroaggregative E. coli (EAEC) adherence to HEp-2 cells by flow cytometry (FC) reveals distinct patterns of attachment and cell death caused by isolates from visitors to Mexico and India with travelersí diarrhea (TD)
Session: Poster Abstract Session: Travel/Tropical Medicine and Parasitology
Saturday, October 22, 2011
Room: Poster Hall B1
Background: EAEC and DAEC cause diarrhea in indigenous children and TD in developing countries. EAEC and DAEC show characteristic patterns of adherence to HEp-2 epithelial cells when observed by conventional light microscopy; however, interpretation is subjective and non-quantitative, time consuming, and provides little information on the pathogenicity of individual isolates.

Methods: Reference strains of E. coli (EAEC 042, DAEC DC1845, and the non-pathogenic HS strain) and 19 clinical isolates of EAEC from US and European travelers to Mexico and India with TD were studied for mannose resistant adherence to HEp-2 by conventional methods.  The presence of aat, a marker of EAEC was studied by colony PCR.  For FC studies, bacteria and HEp-2 cells were stained with the fluorescent dyes PKH 67 FITC (green) and PKH 26 PE (red) respectively.  After co-incubation for 1 and 3 hrs in suspension at 37°C in 5% CO2 with shaking, the percent and patterns of adherence were estimated by multicolored FC using a Beckman Coulter Gallios flow cytometer gated on stained HEp-2 cells.  Assays in which at least 40% of E. coli remained stained at 3 hrs were considered suitable for interpretation.  Adherence to >5% HEp-2 by FC was considered significant.

Results: Adhesion kinetics by FC demonstrated that the HS strain did not adhere to HEp-2 after 1 or 3 hrs, whereas DAEC DC1845 adhered rapidly (<1 hrs), persisted at 3 hrs and was not associated with cell death.  By contrast, adherence of EAEC 042 (aat+) was minimal at 1 hr but increased at 3 hrs and was associated with cell death as evidenced by a reduction in PHK 26 fluorescence.  Six of 9 (67%) aat+ EAEC and 3 of 10 (30%) aat- EAEC showed >5% adherence to HEp-2 by FC (p=0.08).  Isolates adherent by FC were more likely to cause cell death (6/9; 67%) than non adherent (0/10) isolates (p=.003).

Conclusion: Analysis of E. coli adherence by FC at 1 and 3 hrs distinguishes DAEC from EAEC based on adherence kinetics and fluorescence intensity. FC provides a quantitative method to determine cell adherence of EAEC and to identify cytopathic isolates.  We plan studies that correlate cytopathic EAEC with the presence of definable virulence properties, disease severity and response to antibiotic therapy.


Subject Category: T. Travel/tropical medicine and parasitology

Pablo Okhuysen, MD1,2,3, Dorothy Lewis, PhD1, Lily Carlin, BS1, Aaron Orozco, PhD1, Daniela Gomez1, Malik Raynor4, Zhi-Dong Jiang, MD, PhD3 and Herbert DuPont, MD2,3,5, (1)Division of Infectious Diseases, The University of Texas Medical School at Houston, Houston, TX, (2)Section of Infectious Diseases, Baylor College of Medicine, Houston, TX, (3)Center for Infectious Diseases, The University of Texas, School of Public Health, Houston, TX, (4)The University of Texas Graduate School of Biological Sciences at Houston, Houston, TX, (5)St. Luke's Episcopal Hospital and Kelsey Research Foundation and Kelsey-Seybold Clinic, Houston, TX

Disclosures:

P. Okhuysen, None

D. Lewis, None

L. Carlin, None

A. Orozco, None

D. Gomez, None

M. Raynor, None

Z. D. Jiang, None

H. DuPont, None

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