Prevalence and virulence gene profiling of enteroaggregative Escherichia coli in malnourished and nourished Brazilian children

Abstract

The impact of enteroaggregative E. coli (EAEC) infection on childhood malnutrition and inflammation has been suggested, regardless of diarrhea. We investigated whether EAEC and its virulence-related genes (VRGs) are associated with malnutrition in a case-control study. Children aged 6-24 months from Brazil were enrolled as malnourished if weight-for-age Z-score (WAZ) ≤ -2 and nourished if WAZ > -1. Stools were cultured and examined for E. coli. DNA was extracted from fecal isolates and tested for EAEC by polymerase chain reaction (PCR). Positive samples were analyzed by 5 multiplex PCRs to identify 20 EAEC VRGs. Biomarkers of intestinal barrier function and inflammation were measured. The prevalence of EAEC was 39.94%. Samples that presented both aaiC and aatA genes were associated with malnutrition (P = 0.045). A high prevalence of VRGs was observed and the aafC gene was significantly associated with malnourished (P = 0.0101). Strains lacking aar and pic genes were associated with malnutrition (P = 0.018), while the concomitant presence of aar, pic, agg4A, and capU genes was associated with nourished (P = 0.031). These data reinforce the EAEC impact on malnutrition, the importance of aar as negative regulator and the great contribution of AAF/II fimbria for the pathobiology of EAEC.

Keywords: Enteroaggregative E. coli; Malnutrition; Virulence profile.

Fig. 1

Representative image of the classification tree topology (CART) analysis that shows combinations of EAEC virulence genes most associated to malnourished and nourished children. The tree is hierarchical in nature. Each tree branch ends in a terminal node defined by the presence or absence of the virulence genes in which statistical analysis was performed. Statistical significance (P < 0.05) was found only on the terminal nodes 1 and 2. The branches that ended in a non-statistical terminal node were not shown, but represented by dashed lines (− −).

Fig. 2

Host biomarkers associated with EAEC-related trait of virulence genes: Myeloperoxidase (MPO) (A), Serum amyloid A (SAA) (B), IgG anti-LPS (C) and lactulose/mannitol ratio (L/M) (D). Children had their specimens collected and frozen at −80 °C pending processing. Fecal MPO and serum SAA and IgG anti-LPS were measured by enzyme linked immunosorbent assays using specific kits, while urinary L/M ratio was measured by high-pressure-liquid chromatography after administration of a solution containing lactulose (250 mg/mL) and mannitol (50 mg/mL) and urine collection. Statistical analysis was performed by Mann–Whitney U test (n = 8 for malnourished and n = 5 for nourished). * Significantly different compared to Nourished, P = 0.0109 (MPO), P = 0.0054 (SAA), P = 0.0485 (IgG anti-LPS) and P = 0.0480 (L/M ratio).