Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries

Abstract

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea among children, human immunodeficiency virus-infected patients, and travelers to developing regions of the world. The pathogenesis of EAEC strains involves the production of biofilm. In this study, we determined the association between presence of putative EAEC virulence genes and biofilm formation in 57 EAEC isolates (as defined by HEp-2 adherence) from travelers with diarrhea and in 18 EAEC isolates from travelers without diarrhea. Twelve nondiarrheagenic E. coli isolates from healthy travelers were used as controls. Biofilm formation was measured by using a microtiter plate assay with the crystal violet staining method, and the presence of the putative EAEC virulence genes aap, aatA, aggR, astA, irp2, pet, set1A, and shf was determined by PCR. EAEC isolates were more likely to produce biofilm than nondiarrheagenic E. coli isolates (P = 0.027), and the production of biofilm was associated with the virulence genes aggR, set1A, aatA, and irp2, which were found in 16 (40%), 17 (43%), 10 (25%), and 27 (68%) of the biofilm producers versus only 4 (11%), 6 (6%), 2 (6%), and 15 (43%) in non-biofilm producers (P = 0.008 for aggR, P = 0.0004 for set1A, P = 0.029 for aatA, and P = 0.04 for irp2). Although the proportion of EAEC isolates producing biofilm in patients with diarrhea (51%) was similar to that in patients without diarrhea (61%), biofilm production was related to the carriage of aggR (P = 0.015), set1A (P = 0.001), and aatA (P = 0.025). Since aggR is a master regulator of EAEC, the presence of aap (P = 0.004), astA (P = 0.001), irp2 (P = 0.0006), pet (P = 0.002), and set1A (P = 0.014) in an aggR versus an aggR-lacking background was investigated and was also found to be associated with biofilm production. This study suggests that biofilm formation is a common phenomenon among EAEC isolates derived from travelers with or without diarrhea and that multiple genes associated with biofilm formation are regulated by aggR.

FIG. 1.

Biofilm formation by EAEC isolates derived from travelers' with diarrhea or without diarrhea. Biofilm formation on polystyrene surface after 18 h was assessed by crystal violet staining. Each dot indicates the mean values with standard deviations. The biofilm assay was performed three times, with quadruplicates in each assay. Biofilm formation by nondiarrheagenic E. coli isolates was also tested. E coli isolates were deemed biofilm formers if the OD₅₇₀ readings exceeded the mean plus two standard deviations of the E. coli strains HS and DH5α negative control for non-biofilm producers (i.e., >0.46).

FIG. 2.

Biofilm formation by EAEC isolates based on the presence of various genes in aggR background. Biofilm formation on a polystyrene surface after 18 h was assessed by crystal violet staining. Values are based on pooled median biofilm OD₅₇₀ values shown by the box-and-whiskers plot which represents an upper quartile, median, and lower quartile.