Identification of the Shiga Toxin-Producing Escherichia coli O104:H4 Strain Responsible for a Food Poisoning Outbreak in Germany by PCR

FAST-TRACK COMMUNICATION

An outbreak caused by Escherichia coli serotype O104:H4 strains has been affecting northern Germany since May 2011, with 3,222 patients infected and 39 of them dead by 18 June (4). Around 25% of the patients developed hemolytic-uremic syndrome (HUS) (1, 3), a rate which is much higher than was previously reported for patients infected with Shiga toxin-producing E. coli strains (6, 8). The outbreak spread quickly over northern Germany, with some cases in other European countries, and has become one of the biggest outbreaks of E. coli infections reported so far. As rapid pathogen detection from clinical and environmental samples is crucial for patient management and disease control, we developed a rapid PCR method targeting two virulence determinants of the outbreak strain, based on a whole-genome comparative analysis, and then validated the specificity and sensitivity of the method.

HUS is most commonly triggered by enterohemorrhagic E. coli (EHEC), a type of highly virulent E. coli that produces Shiga toxin (Stx) and usually colonizes the intestines of healthy domestic animals (5, 7). However, according to the whole-genome sequencing analysis (2), the outbreak strain (TY2482) is genetically closer to enteroaggregative E. coli (EAEC; represented by strain 55989), which is of lower virulence and is associated with cases of acute or persistent diarrhea in children and adults worldwide (5). Comparative genome analysis (against strain 55989) identified around 405 kb of TY2482-unique sequences. Among the regions of these sequences, we selected the aggregative adherence-associated gene cluster (including four genes, aggA to aggD), termed the aggregative adherence fimbria I (AAF/I) gene cluster, and the Shiga toxin-encoding genes stxA₂ and stxB₂ as targets for primer design. A comparison of the nucleotide sequences of the AAF/I genes and stx₂ against available sequence data from 4,547 strains representing 2,183 species using BLASTn confirmed that no single strain was positive for both targets simultaneously. Two pairs of primers were designed for each target and experimentally evaluated (see Table S1 in the supplemental material).

Amplification was performed successfully using annealing temperatures of 55°C to 62°C (see Fig. S1 in the supplemental material). Therefore, we used 58°C as the annealing temperature in the following experiments. In total, 392 strains were analyzed in the experiments (see Table S2 in the supplemental material), and representative results are shown in Table 1 and in Fig. S2 in the supplemental material. Of the 54 Shiga toxin-producing isolates from patients presenting with diarrhea at the emergency department of the University Medical Center Hamburg-Eppendorf, 52 tested positive for the AAF/I genes and stx₂, one isolate was negative for both targets, and one isolate was positive only for stx₂. The AAF/I gene-negative isolates were identified as an stx₁-positive strain and a sorbitol-nonfermenting, stx₂-positive E. coli O157 strain, respectively, with neither of them related to the current outbreak. The DNA samples from 338 isolates (71 E. coli strains and 267 strains other than E. coli, including 92 bacterial species) were used as a control, and no PCR product was amplified from these samples, with only two strains of E. coli positive for stx₂. All evaluated primer pairs showed excellent specificity. The limit of detection for primer pairs AAF/I-2, Stx2-1, and Stx2-2 was 10² target gene copies, while primer pair AAF/I-1 showed a lower analytical sensitivity (10⁵ target gene copies) (see Fig. S3 in the supplemental material). Based on our experimental results, primer pairs AAF/I-2 and Stx2-2 are recommended for routine use (see Table S1 in the supplemental material).

Table 1.

Characteristics and PCR results of Escherichia coli isolates used in this study

Pathotype (presence of virulence genes)a	Sourcee	No. of strains	PCR result for indicated target
			stx2	AAF/I genes
EHEC (SLT-1, +; SLT-2, +)	Laboratory collection	1	+	−
EHEC (SLT-1, −; SLT-2, +)	Laboratory collection	1	+	−
EHEC (SLT-1, +; SLT-2, −)	Laboratory collection	2	−	−
ETEC (ST, −; LT, +)	Laboratory collection	9	−	−
ETEC (ST, +; LT, +)	Laboratory collection	3	−	−
ETEC (ST, +; LT, −)	Laboratory collection	4	−	−
EPEC	Laboratory collection	5	−	−
EIEC	Laboratory collection	3	−	−
E. colib	Hospital 302, Beijing, Chinac	11	−	−
E. colib	UMC Hamburg-Eppendorfc	32	−	−
STEC	UMC Hamburg-Eppendorfd	1	−	−
STEC	UMC Hamburg-Eppendorfd	1	+	−
STEAEC	UMC Hamburg-Eppendorfd	52	+	+

^aSLT, Shiga-like toxin; ETEC, enterotoxigenic E. coli; ST, heat-stable enterotoxin; LT, heat-labile enterotoxin; EPEC, enteropathogenic E. coli; EIEC, enteroinvasive E. coli; STEC, Shiga toxin-producing E. coli; STEAEC, Shiga toxin-producing enteroaggregative E. coli.

^bUnknown pathotype.

^cFrom patients with non-EHEC-related diarrhea.

^dFrom patients with EHEC-related diarrhea.

^eUMC Hamburg-Eppendorf, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.

Based on the results described above, the proposed two-target PCR is a simple and specific tool to detect and identify the 2011 outbreak strain that spread in Germany and northern Europe. The development of a simple screening test following rapid sequencing and computational analysis of an outbreak strain genome, as employed in this study, could be helpful for control of future emerging infectious diseases.