Virulence characteristics and molecular epidemiology of enteroaggregative Escherichia coli isolates from hospitalized diarrheal patients in Kolkata, India

Abstract

Enteroaggregative Escherichia coli (EAEC) is an important diarrheal enteropathogen defined by aggregative adherence to cultured epithelial cells. We have detected EAEC from 121 (6.6%) of 1,826 hospitalized patients admitted with diarrhea to the Infectious Diseases Hospital in Kolkata, India. Watery diarrhea was recorded significantly (P = 0.0142) more often in children. The majority of the EAEC isolates were not serotypeable (62%) and showed resistance to five or more antibiotics (76%). We studied different virulence genes and the molecular epidemiology of 121 EAEC isolates recovered from diarrheal patients. A PCR assay for detection of virulence genes, an assay for determination of clump formation in liquid culture, and a HeLa cell adherence assay were carried out to characterize the EAEC isolates. Investigations were also conducted to correlate the virulence gene profiles with diarrheal symptoms and molecular epidemiology by pulsed-field gel electrophoresis (PFGE). Two or more virulence genes were detected in 109 (90.1%) EAEC isolates. In the cluster analysis, some isolates with specific gene profiles and phenotypes formed a group or subcluster. This study highlights the comparative distributions of three fimbrial adhesins and other virulence genes among EAEC isolates. The diverse virulence gene and PFGE profiles, along with the existence of diverse serotypes and antibiograms, suggests that the EAEC isolates are genetically heterogeneous in Kolkata.

FIG. 1.

XbaI-restricted PFGE profiles of the 45 EAEC isolates from hospitalized patients with diarrhea. The identity of the isolate and its serogroup are indicated above each lane. The bacteriophage lambda DNA ladder standard for PFGE (New England Biolabs, Beverly, Mass.) was used in the lane labeled PFGE Marker as a DNA molecular size marker. ONT, O nontypeable.

FIG. 2.

The XbaI restriction patterns were digitized and analyzed by using the Diversity Database fingerprinting software (version 2.2.0; Bio-Rad) to calculate the similarity matrix by use of Dice coefficients of correlation, and clustering correlation coefficients were determined by the UPGAMA method. The scale indicated at the top collates the levels of pattern similarity. ONT, O nontypeable.

FIG. 3.

Virulence gene profile relationships of the EAEC isolates recovered as the sole pathogens from diarrheal patients. The five major phylogenetic clusters (clusters A1, A2, B1, B2, and C) are labeled. The dendrogram was obtained by cluster analysis (by the UPGAMA method) of the virulence gene profiles shared by 102 EAEC isolates. The average distance between clusters was determined by the cluster analysis, based on the similarity matrices of the EAEC isolates exhibiting diversified gene profiles. The virulence genes were coded 1 through 14 for aafA, aap, afaBC, aggA, agg3A, aggC, agg3C, aggR, astA, fliC, irp2, pet, pic, and shf, respectively. The number of EAEC isolates harboring the respective virulence profile is indicated in parentheses. The similarity scale is shown above the dendrogram.