Escherichia coli and community-acquired gastroenteritis, Melbourne, Australia

Emerging Infectious Diseases, Oct, 2004 by Roy M. Robins-Browne, Anne-Marie Bordun, Marija Tauschek, Vicki R. Bennett-Wood, Jacinta Russell, Frances Oppedisano, Nicole A. Lister, Karl A. Bettelheim, Christopher K. Fairley, Martha I. Sinclair, Margaret E. Hellard

Twenty-two EPEC isolates (11 from healthy persons and 11 from persons with diarrhea) were isolated in pure culture. Representative colonies of each were then serotyped with hyperimmune rabbit antisera (16). These strains were also subjected to PCR with the primers and conditions listed in Table 3 to determine intimin subtype and to investigate the presence of selected virulence-associated genes. The same 22 strains were also examined for their ability to adhere to and invade HEp-2 epithelial cells.

Bacterial Adhesion and Invasion of HEp-2 Cells

The Center for Vaccine Development (University of Maryland, Baltimore, MD) method was used to determine the pattern of bacterial adherence to HEp-2 epithelial cells (12). Bacterial strains were designated nonadherent if <10 of 200 HEp-2 cells had five or more bacteria attached. The fluorescent actin staining (FAS) assay, which correlates with the ability of E. coli to produce attaching-effacing lesions in the intestine, was performed by using a 6-h incubation period (22). At the completion of the assay, cells were examined by fluorescence and phase-contrast microscopy to confirm that fluorescent areas corresponded to attached bacteria. Bacterial strains that gave equivocal or negative results in the FAS assay were investigated for DNA corresponding to regions of the LEE, other than eae, by colony blotting using the LEE-A, LEE-B, and LEE-D DNA probes and hybridization conditions described previously (23). Quantitative assessment of the ability of E. coli to invade HEp-2 cells was performed with the gentamicin-protection assay (24). Results were expressed as the number of bacteria recovered from HEp-2 cells after treatment with gentamicin as a percentage of the total number of cell-associated bacteria (24).

Statistical analysis was performed with InStat version 3.0 (GraphPad Software Inc., San Diego, CA). A p value < 0.05 was considered significant.

Results

Association of E. coil Pathotypes with Gastroenteritis

After excluding samples for which patient data were incomplete (12 samples), which were negative by PCR for lacZ (45 samples), or which gave equivocal results in the PCR or DNA hybridization assays (8 samples), 1,185 (94.8%) samples of the original 1,250 were available for analysis: 696 from patients with gastroenteritis and 489 from healthy persons. The results of the assays are summarized in Table 4. Enterotoxigenic (ETEC) and enteroinvasive (EIEC) strains of E. coli and Bfp-positive EPEC were identified in <0.5% of healthy persons or patients with gastroenteritis. EHEC were present in 4 (0.6%) of 696 of samples from persons with gastroenteritis and in no healthy persons, but the difference between the two groups was not significant (p = 0.15, Fisher exact test, 2-tailed). In contrast, both EAEC and atypical (Bfp-negative) EPEC were identified in >5% of patients, and the difference between the symptomatic and baseline groups was significant for each of these pathotypes (Table 4). Analysis of the data pertaining to patients in whom EAEC and atypical EPEC were identified showed that atypical EPEC were more frequent in younger persons; patients with atypical EPEC were a median age of 3.4 years, compared with 7.4 years for the symptomatic group overall (p < 0.0001; Mann-Whitney test, 2-tailed). Of all atypical EPEC in study participants with gastroenteritis, 75 (84%) were identified in children <10 years old, compared with 14 (16%) in patients [greater than or equal to] 10 years (relative rate [RR] 3.4, 95% confidence interval [CI] 2.0-5.9). In contrast, the median age of patients infected with EAEC was 7.3 years.