Identification of unconventional intestinal pathogenic Escherichia coli isolates expressing intermediate virulence factor profiles by using a novel single-step multiplex PCR

Abstract

Intestinal pathogenic Escherichia coli represents a global health problem for mammals, including humans. At present, diarrheagenic E. coli bacteria are grouped into seven major pathotypes that differ in their virulence factor profiles, severity of clinical manifestations, and prognosis. In this study, we developed and evaluated a one-step multiplex PCR (MPCR) for the straightforward differential identification of intestinal pathotypes of E. coli. The specificity of this novel MPCR was validated by using a subset of reference strains and further confirmed by PCR-independent pheno- and genotypic characterization. Moreover, we tested 246 clinical E. coli isolates derived from diarrhea patients from several distinct geographic regions. Interestingly, besides strains belonging to the defined and well-described pathotypes, we identified five unconventional strains expressing intermediate virulence factor profiles. These strains have been further characterized and appear to represent intermediate strains carrying genes and expressing factors associated with enteropathogenic E. coli, Shiga toxin-producing E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli alike. These strains represent further examples of the extraordinary plasticity of the E. coli genome. Moreover, this implies that the important identification of specific pathotypes has to be based on a broad matrix of indicator genes. In addition, the presence of intermediate strains needs to be accounted for.

FIG. 1.

MPCR analysis of reference strains. Twelve primer pairs were designed for the specific detection of the seven major categories of intestinal pathogenic E. coli. All of the primer pairs yield specific gene products indicating the appropriate pathotypes and generate no unspecific products. The amplicons can be differentiated by agarose gel electrophoresis (2%). Lanes M contained molecular size markers. The other lanes contained EPEC strain E2348/69 (uidA positive, LEE positive, bfp positive), ATEC strain 9812 (uidA positive, LEE positive), STEC strain EDL933 (uidA positive, LEE positive, stx₁ positive, stx₂ positive), STEC strain 04-3175 (uidA positive, stx₁ positive, stx₂ positive), ETEC strain 164/82 (uidA positive, elt positive, estIa positive), ETEC strain 117/86 (uidA positive, estIb positive), EIEC strain 99-10282 (uidA positive, invE positive), EAEC strain 02-1850 (uidA positive, astA positive, aggR positive, pic positive), E. coli C600 (uidA positive), and a negative control lacking template DNA.

FIG. 2.

Comparative PCR analysis of STEC strains. Strains were analyzed by MPCR (top) and single-PCR approaches (bottom; EP1/EP2, bfp; SK1/SK2, eae; LP43/LP44, stx₂; KS7/KS8, stx₁). All strains exhibited the same gene pattern in the MPCR and single PCR assays and were positive for uidA; LEE (escV, eae); and stx₁, stx₂, or both. Lanes: M, marker; 1, 11062; 2, Y113; 3, 493/88; 4, 5720/96; 5, EDL933; 6, E. coli 04-2936; 7, E. coli 04-2938; 8, E. coli 04-3313; 9, E. coli 04-3453; 10, E. coli 04-4080.

FIG. 3.

MPCR analysis of intermediate strains. Single colonies of intermediate strains were directly incubated in the PCR mixture, lysed by the initial denaturation step, and analyzed by MPCR as described in Materials and Methods. All intermediate strains, i.e., 2771/97 (uidA positive, stx₂ positive, estIa positive), 04-3908 (uidA positive, stx₂ positive, estIa positive, astA positive), 265-1 (uidA positive, bfpB positive), and 4932-53 (uidA positive, bfpB positive), exhibit unusual genotypes. The negative control was tested with the MPCR set of primer pairs but lacked template DNA. Lanes M contained molecular size markers.

FIG. 4.

Adherence patterns of intermediate intestinal pathogenic E. coli strains. Adherence behavior of unconventional E. coli strains to HeLa cells and induction of actin polymerization were monitored by FAS assay. Strain 2771/97 (A; stx₂ positive, estIa positive) adheres in a diffuse pattern to target cells, whereas strains 04-3908 (B; stx₂ positive, estIa positive, astA positive) and 03-7355 (C; estIa positive, astA positive) exhibit aggregative adherence. Strains 4932-53 (D; LEE negative, bfp positive) and 265-1 (E; LEE negative, bfp positive) show localized adherence patterns and develop microcolonies. All strains are not able to induce actin polymerization in HeLa cells (A₂, B₂, C₂, D₂, E₂). Actin filaments were labeled with phalloidin-Texas Red (red), and DNA was visualized with DAPI (blue). Bars = 10 μm.

FIG. 5.

Verification of BFP pheno- and genotypes of intermediate pathogens. BFP expression was confirmed by detection of bfpB cDNA by RT-PCR (A) and by electron microscopy analysis (B). Whole RNAs of strains 265-1 (LEE negative, EAF positive), 4932-53 (LEE negative, EAF positive), E2348/69 (LEE positive, EAF positive), and 3431-4/86 (LEE positive, EAF negative) were isolated and transcribed to cDNAs. Transcription of bfpB could be detected for strain 265-1 (A, lane 1), 4932-53 (A, lane 2), and E2348/69 (A, lane 3). Strain 3431-4/86 cDNA (A, lane 4) did not yield a PCR product, like the negative controls with mRNAs of strains 265-1 (A, lane 5), 4932-53 (A, lane 6), and E2348/69 (A, lane 7) as the template and the sample without a template (A, lane 9). The positive control with genomic DNA of strain E2348/69 (A, lane 8) as the template produced the expected 910-bp PCR product. Lane M contained molecular size markers. (B) Production of BFP was visualized by electron microscopy analysis of strain 265-1 (magnification, ×21,000).