CS21 positive multidrug-resistant ETEC clinical isolates from children with diarrhea are associated with self-aggregation, and adherence

Abstract

Background: Enterotoxigenic Escherichia coli (ETEC) colonize the human intestinal mucosa using pili and non-pili colonization factors (CFs). CS21 (also designated Longus) is one of the most prevalent CFs encoded by a 14 kb lng DNA cluster located in a virulence plasmid of ETEC; yet limited information is available on the prevalence of CS21 positive ETEC isolates in different countries. The aim of this study was to evaluate the prevalence of CS21 among ETEC clinical isolates from Mexican and Bangladeshi children under 5 years old with diarrhea and to determine the phenotypic and genotypic features of these isolates.

Methods: ETEC clinical isolates positive to lngA gene were characterized by genotype, multidrug-resistance, self-aggregation, biofilm formation, and adherence to HT-29 cell line.

Results: A collection of 303 E. coli clinical isolates were analyzed, the 81.51% (247/303) were identified as ETEC, 30.76% (76/247) were st (+)/lt (+), and 25.10% (62/247) were positive for the lngA gene. Among the lngA (+) ETECs identified, 50% of isolates (31/62) were positive for LngA protein. The most frequent serotype was O128ac:H12 found in 19.35% (12/62) of lngA (+) ETEC studied. Multidrug-resistance (MDR) lngA (+) ETEC isolates was identified in 65% (39/60), self-aggregation in 48.38% (30/62), and biofilm formation in 83.87% (52/62). ETEC lngA (+) isolates were able to adhere to HT-29 cells at different levels. Two lngA isogenic mutants were constructed in the ETEC E9034A and ETEC73332 clinical isolate, showing a 77% and 98% reduction in adherence, respectively with respect to the wild type.

Conclusion: ETEC isolates that have the lngA gene showed features associated with self-aggregation, and adherence to HT-29 cells, important characteristics in the human gut colonization process and pathogenesis.

Keywords: CS21; ETEC; biofilm; multidrug-resistance; self-aggregation and adherence.

Figure 1

Resistance profiles for ETEC positive isolates for the lngA gene. Sixty-two ETEC isolates were tested for resistance against different antibiotic categories. ^*One ETEC isolate was resistant to a third generation cephalosporin, therefore an Extended Spectrum Beta Lactamases (ESLB) assay was performed, and was positive for this test. n = number of positive ETEC lngA isolates.

Figure 2

Genotypes found in MDR ETEC isolates positive for the lngA gene. Sixty-five percent (39/60) of ETEC isolates were resistant to at least three antibiotic families. The association of multidrug-resistance to the genotypes was determined. n = number of positive ETEC lngA isolates.

Figure 3

Self-aggregation in ETEC isolates positive for the lngA gene. Forty-eight point eighty-three percent of ETEC isolates analyzed showed weakly (E9034A), moderately (115340), and strongly (45162) self-aggregation phenotypes. n = number of positive ETEC lngA isolates.

Figure 4

Biofilm in ETEC isolates positive for the lngA gene. From the 62 lngA⁺ ETEC isolates analyzed, 83.87% were able to form biofilm. Biofilm formation is critical in the pathogenesis of many human bacterial pathogens. ETEC lngA⁻ was used as negative control and its absorbance to classify as weakly biofilm formers (0.129–0.258); moderately biofilm formers (0.259–0.516); and strongly biofilm formers (≥0.517). Enteroaggregative E. coli 042 shows as rhomb on gray (positive control).

Figure 5

Adherence in ETEC isolates positive for the lngA gene. Quantitative adherence assay determined in CFU when HT-29 cells were infected. The classification of adherence was done according to the positive control (Enteroaggregative E. coli 042) mean (11.97 × 10⁶ CFU/ml), grouped in three phenotypes: weakly (0.3–2.99 × 10⁶ CFU/ml), moderately (3.0–5.98 × 10⁶ CFU/ml), and strongly adherent (=5.99 × 10⁶ CFU/ml). Enteroaggregative E. coli 042 shows as rhomb on gray (positive control).

Figure 6

Role of Longus in ETEC isolates E9034A and 73332. (A) Quantitative adherence assay in CFU after 6 h of infection to HT-29 cells. The percentage of each isolate is shown in a bar graph. (B) Growth curve in PPLO medium to corroborate growth rate among wild type isolates and isogenic mutants in the lngA gene. h, hours.