Mucus-Activatable Shiga Toxin Genotype stx2d in Escherichia coli O157:H7.

We identified the mucus-activatable Shiga toxin genotype stx2d in the most common hemolytic uremic syndrome-associated Escherichia coli serotype, O157:H7. stx2d was detected in a strain isolated from a 2-year-old boy with bloody diarrhea in Spain, and whole-genome sequencing was used to confirm and fully characterize the strain.

The foodborne zoonotic pathogen Shiga toxin (Stx)–producing Escherichia coli (STEC) is responsible for human diseases ranging from uncomplicated diarrhea to the life-threatening hemolytic-uremic syndrome (HUS) (). Stx production is the most determining virulence factor implicated in HUS, and the intimin, encoded by eae, is the most common adherence factor in HUS-associated STEC (). Stx2d is a Stx2 variant in which cytotoxicity is increased (from 35- to 350-fold) by the action of elastase in intestinal mucus (). This mucus-enhanced toxicity is termed “activation,” and activatable Stx2d proteins are designated Stx2dact. Stx2dact production and Stx2dact genotype (stx2d) have been associated primarily with eae-negative STEC and considered a predictor for severe clinical outcome in such infections ().

All STEC strains received or isolated in the Reference and Research Laboratory of Food and Waterborne Bacterial Infections (Majadahonda, Spain) are routinely tested for stx1 and stx2 subtypes by a PCR subtyping method (). For serotyping, O antigen is identified with both commercial antiserum and PCR (), and H antigen is identified by PCR amplification of the fliC gene () and further sequencing of the PCR product. During 2012–2016, stx2d was identified in 7 (3%) of 236 STEC strains isolated from patients with HUS and/or diarrhea in Spain (193 eae-positive and 43 eae-negative strains). Six were eae-negative non-O157 STEC belonging to serotypes O73:H18 (2 strains), O91:H21, O148:H8, O181:H49, and ONT:H21. Strikingly, the other stx2d-positive strain identified (CNM-2140/12) belonged to serotype O157:H7 and contained stx2d in combination with stx2c, apart from eae. The strain had been isolated from a 2-year-old boy with bloody diarrhea in July 2012, and it fermented sorbitol after overnight incubation on sorbitol MacConkey agar (Becton Dickinson, Sparks, MD, USA). We confirmed Stx2 production using the enzyme immunoassay kit SHIGA TOXIN QUIK CHEK (TechLab, Blacksburg, VA, USA). The activatable property of the toxin was confirmed by partial sequencing of the stx2 gene (), analysis of the resulting nucleotide sequence by comparison with the published stx2 reference sequences, and comparison of the resulting amino acid sequences. The nucleotide sequence of stx2d (GenBank accession no. MF094370) was 100% identical to that of strain 06–5231 (O55:H7, GenBank accession no. EF584538). The sequence was translated to amino acids (Technical Appendix Figure), and the activatable property of Stx2d was confirmed with the combined presence of the “activatable tail,” the last 10 aa in the C-terminal end of the A subunit (KSQSLYTTGE), and the END motif at position 14–16 in the N-terminal end of the B subunit (). Stx-associated bacteriophage insertion sites genotyping was performed (). According to the Stx-associated bacteriophage insertion genotype nomenclature proposed by Shringi et al. (), the strain belonged to the SY2c genotype; both sbcB and yehV loci were occupied, the former with a Stx2c-associated bacteriophage, plus the additional insertion of the Stx2d-associated bacteriophage within the yecE locus, as recently reported for stx2d-positive STEC O26:H11 ().

To confirm the genotypic traits mentioned earlier, we sequenced the strain using an Illumina NextSeq 500 next-generation sequencer system (Illumina, San Diego, CA, USA). We extracted DNA using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) and generated a genomic DNA paired-end library using a Nextera XT DNA Sample Preparation Kit (Illumina). A total of 1,371,617,258 bp were obtained, providing ≈250-fold coverage and 9,083,558 reads. The sequencing reads have been deposited in the SRA-NCBI public sequence repository (accession no. SRP107062). From the raw whole-genome sequence data, we confirmed serotype O157:H7 using SerotypeFinder; investigated the virulence gene profile, including stx subtypes, using VirulenceFinder (Table); and identified acquired antimicrobial resistance genes using ResFinder (all publicly available on the Center for Genomic Epidemiology server, https://cge.cbs.dtu.dk//services/all.php). In addition, we detected mutations in the quinolone resistance–determining regions of the gyrA and parC genes in silico on the whole-genome sequence, and no acquired antimicrobial resistance gene or mutation conferring quinolone resistance were identified.

Table

Virulence gene profile of the stx2d-positive Shiga toxin–producing Escherichia coli O157:H7 strain CNM-2140/12*

Virulence factor	Identity	Protein function	GenBank accession no.
ehxA	100.00	Enterohemolysin	AB011549
etpD	100.00	Type II secretion protein	AB011549
nleA	100.00	Non-LEE encoded effector A	AE005174
espP	99.92	Extracellular serine protease plasmid-encoded	AB011549
nleB	99.90	Non-LEE encoded effector B	AE005174
gad	98.50	Glutamate decarboxylase	BA000007
iss	98.30	Increased serum survival	CP001509
eae	100.00	Intimin	AF071034
iha	100.00	Adherence protein	AE005174
tir	100.00	Translocated intimin receptor protein	EU871626
stx2B	100.00	Shiga toxin 2, subunit B, variant c	AB071845
iss	98.54	Increased serum survival	CU928160
astA	100.00	EAST-1 heat-stable toxin	HM099897
astA	91.96	EAST-1 heat-stable toxin	AB042005
nleC	100.00	Non-LEE encoded effector C	AP010960
espA	100.00	Type III secretions system	AE005174
gad	98.50	Glutamate decarboxylase	BA000007
nleB	99.90	Non-LEE encoded effector B	AE005174
espJ	99.85	Prophage-encoded type III secretion system effector	AE005174
katP	100.00	Plasmid-encoded catalase peroxidase	AB011549
espB	100.00	Secreted protein B	AE005174
nleC	100.00	Non-LEE encoded effector C	AE005174
stx2B	100.00	Shiga toxin 2, subunit B, variant d	EF584538

*Determined from raw whole-genome sequence data using VirulenceFinder (https://cge.cbs.dtu.dk//services/all.php). A percent identity threshold of 90% between the input and the best matching database gene was selected. Bold indicates the mucus-activatable Shiga toxin genotype. EAST, enteroaggregative E. coli heat-stable enterotoxin; LEE, locus of enterocyte effacement.

Despite its usual association with eae-negative STEC, we identified the mucus-activatable Stx genotype also in STEC O157:H7, the most common HUS-associated eae-positive STEC serotype. This rare virulence gene combination (stx2d/eae) was recently described in STEC O26:H11 isolated from several HUS patients () and in STEC O80:H2 causing HUS and bacteremia in France (). Although its clinical implications remain unknown—because the strain in our current study was isolated from a patient with bloody diarrhea but not HUS, whereas those isolated in France originated from patients with HUS—these examples show the potential for acquiring and transferring important STEC virulence factors, which can lead to unusual and potentially more virulent strains. Further studies are needed to better determine whether stx2d-positive O157:H7 and other eae-positive non-O157 STEC strains carrying this stx subtype are statistically significant in humans and to determine their clinical implications.

Technical Appendix

Alignment with reference strains 06–5231, 5905, C165–02, and B2F1 of the amino acid sequences of the C-terminal end of the A subunit and the N-terminal end of the B subunit of the stx2d-positive Shiga toxin–producing Escherichia coli O157:H7 strain CNM-2140/12.