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J Clin Microbiol. Nov 2012; 50(11): 3485–3492.
PMCID: PMC3486251

Specific Detection of Enteroaggregative Hemorrhagic Escherichia coli O104:H4 Strains by Use of the CRISPR Locus as a Target for a Diagnostic Real-Time PCR

Abstract

In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPRO104:H4) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPRO104:H4 PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwCO104, wzxO104, and wzyO104). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPRO104:H4 target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPRO104:H4 locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPRO104:H4 PCR (99.06% specificity).

INTRODUCTION

More than 400 serotypes of Shiga toxin (Stx)-producing Escherichia coli (STEC) strains have been described as agents of disease in humans, and some of these have been shown to be associated with severe diseases, such as hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). These strains were called enterohemorrhagic E. coli (EHEC) and were found to carry additional virulence markers besides Stx, such as effectors encoded by the locus of enterocyte effacement (LEE) and various non-LEE-encoded effectors. A concept of molecular risk assessment (MRA) was developed by Karmali et al. (13) and Coombes et al. (9) that employs PCR for identification of human-pathogenic EHEC. Using the MRA approach for screening STEC collections (6, 8), an increasing number of emerging EHEC types was detected.

During spring 2011, Europe faced its largest STEC outbreak involving an emerging enterohemorrhagic Escherichia coli O104:H4 strain (1). This EHEC strain presents an unusual virulence pattern that combines the production of Stx2a with enteroaggregative adherence which is encoded by genes of the pAA plasmid and chromosomally carried genes of enteroaggregative E. coli (EAEC) strains (1, 10). This new type of EHEC was designated enteroaggregative hemorrhagic E. coli since it shares virulence markers of both EHEC and EAEC strains. On the genome level (5, 17), the strain was found to be most closely related to an EAEC O104:H4 strain, strain 55989, that was isolated in Central Africa in 1995 (11). This hybrid EAEC STEC O104:H4 strain was found to be negative for the LEE-encoded effector and non-LEE-encoded effector (nle), both of which are presently being used by the current MRA approach to define human virulent EHEC types. Therefore, new diagnostic approaches needed to be developed for detection of EAEC STEC O104:H4 strains. The lack of unique biochemical traits of the hybrid EAEC STEC O104:H4 strains makes their detection with cultural and phenotypical tests difficult and time-consuming. Therefore, rapid molecular testing methods allowing for timely detection of these strains are deemed highly desirable.

During the course of the O104:H4 outbreak investigation, multitarget PCR assays have been used for rapid screening of samples; however, all of these assays require cultural isolation of the bacteria to confirm that all gene targets are present in the same strain. The used PCR assays (4, 12, 21, 26) combine multiple pairs of primers targeting, for example, genes encoding Shiga toxin 2 (stx2), O104 (rfbO104) and H4 (fliCH4) antigens, tellurite resistance (terD), and AggR (aggR), which is the master regulator of EAEC plasmid, as well as chromosomally inherited virulence genes (18). However, none of these gene targets was unique to the O104:H4 outbreak strain. Therefore, samples containing a mixed flora of bacteria, such as those collected from environmental and food sources, did not allow prediction that all targets were present in the same bacterial strain. Hence, these assays were suitable only for bacterial isolates and have limited use with clinical, food, or environmental samples.

Based on nucleotide sequence analysis of the genome of EAEC STEC O104:H4, we identified in the clustered regularly interspaced short palindromic repeats (CRISPR) locus of the epidemic strain two primers and one probe highly specific for the O104:H4 outbreak strain. This target was used as a single PCR target for a rapid identification and detection of EAEC STEC O104:H4 strains.

MATERIALS AND METHODS

Bacterial strains.

A collection of 1,321 E. coli strains was analyzed for their CRISPR loci by real-time PCR. The strains were isolated from human patients, animals, and food sources. Serotyping of O and H antigens was performed as previously described (15). The tested E. coli strains included 48 O104:H4 strains (including 1 serologically O-rough strain [Table 1]), 17 strains of serotype O104:[H21] which are also known to be associated with diarrhea and HUS (including 2 serologically O-rough strains), and 18 O104 strains expressing other H types (H2, H7, H11, and H12). Another panel of the E. coli strains analyzed in this study included the reference strains for E. coli O serogroups O1 to O186 and H1 to H56 and in particular 12 O174:H2, 8 O141:H2, and 4 O43:H2 strains that were available from the strain collection of the Federal Institute for Risk Assessment (BfR), Berlin, Germany. A subset of 117 E. coli strains was further examined for aggR, wzxO104, and flicH4 (Table 2). This subset comprised 48 O104:H4 strains, 28 O104 strains with flagellar types other than H4, and 41 E. coli strains belonging to other serogroups (including 4 E. coli K9 strains). For analysis, bacteria were cultured to single colonies on Luria broth plates and grown overnight at 37°C. Single colonies were picked for DNA extraction using the InstaGene matrix (Bio-Rad Laboratories, Marnes-La-Coquette, France). Extracted DNA was stored at −20°C until real-time PCR testing was performed.

Table 1
E. coli O104:H4 strains (including one serologically O-rough strain) tested by PCRa
Table 2
PCR examination of a subset of 117 E. coli strains for aggR, wzxO104, flicH4, and CRISPRO104:H4a

Detection of E. coli K9 strains.

E. coli K9 antiserum (Statens Serum Institut, Copenhagen, Denmark) was used for slide agglutination of live bacteria expressing the K9 capsule by following the recommendations of the manufacturer. Strain Bi 316-42 (O9:K9:H12) was used as a positive control.

Primers and probes used for real-time PCR.

Primers and probes used for detecting stx1, stx2, and eae as well as wzxO104 were described and evaluated previously (2, 7). Primers and probes for the detection of wxyO104, wbwCO104, flicH4, aggR, and CRISPRO104:H4 loci were developed in this work. All oligonucleotides were purchased from Eurofins MWG Biotech (Courtaboeuf, France), except for minor groove binder (MGB)-labeled oligonucleotides, which were from Applied Biosystems (Villebon sur Yvette, France) (Table 3).

Table 3
Primers and probes used in this study

A LightCycler 1536 (Roche, Meylan, France) was used to perform high-throughput real-time PCR amplifications. For PCR setup of the LightCycler 1536 multiwell plates, a Bravo liquid dispenser automat (Agilent Technologies, Massy, France) equipped with a chiller and a PlateLoc thermal microplate sealer (Agilent Technologies) was used. The PCR mixtures contained 0.5 μl DNA sample and 1 μl master mix containing 1× RealTime ready DNA Probes Master (Roche) (corresponding to 0.7× final concentration), 300 nM each primer, and 300 nM each probe (corresponding to a 200 nM final concentration for each). The following thermal profile was used for PCR: 95°C for 1 min, followed by 35 cycles of 95°C for 0 s and 60°C for 30 s and a final cooling step at 40°C for 30 s.

CRISPR locus sequencing.

The CRISPR loci of E. coli O104:H4 strains CB13344 and CB13410 (isolated in 2011 in Germany) and CB8983 (isolated in 2001 in Germany) were determined by DNA sequencing (GenBank accession numbers JX195533, JX195532, and JX195534). The CRISPR loci of E. coli O104 strains with H types other than H4 (O104:H2, O104:H7, O104:H11, O104:H12, and O104:H21) were also sequenced together with those of E. coli strains O43:H2, Ont:H2, O174:H2, and O141:H2 cross-reacting with the CRISPRO104:H4 assay (GenBank accession numbers JX195535 to JX195539 and JX195541 to JX195552). The CRISPR loci of E. coli O9:K9:H1 and O174:H2, which do not cross-react with the CRISPRO104:H4 assay, were also determined by sequencing (GenBank accession numbers JX195540, JX195553, and JX195554). The nucleotide sequences of the CRISPR loci were edited and aligned using BioEdit, version 7.1.3.0, and ClustalW2, respectively. Split decomposition analysis of the aligned sequences was performed using the SplitTree program, version 4.11.3, using neighbor net with uncorrected p distance. Evolution of the CRISPR locus by acquisition of foreign DNA (i.e., phages) is supported by incompatibilities in the phylogeny evidenced by the parallel edges of the resulting reticulated network. Split network derived from split decomposition analysis of the CRISPR locus of the sequenced strains is shown in Fig. 1.

Fig 1
Split network derived from split decomposition analysis of the CRISPR loci of the sequenced strains. E. coli strains that reacted with the CRISPRO104:H4 assay are noted in bold.

Sensitivity of detection of O104:H4 strains with the CRISPRO104:H4 real-time PCR assay.

Single colonies of E. coli O104:H4 were grown at 37°C in tryptone soy broth (TSB) medium until a logarithmic growth phase was reached. The titer of the cultures was determined by plating 100-μl aliquots of 10-fold serial dilutions of the culture on tryptone soy agar (TSA). Aliquots of the stock culture as well as each dilution (1 ml) were pelleted by centrifugation, and DNA from bacterial pellets was extracted using InstaGene matrix (Bio-Rad). For real-time PCR, 1-μl aliquots of appropriate dilutions were used as the template for PCR. To determine the dynamic range of the assay, the obtained threshold cycle (CT) values were correlated to the total number of bacteria as gene equivalents for each PCR.

RESULTS

Serotyping assays using O104 and K9 antisera.

All E. coli O104 strains with various H types reacted with the O104 antiserum, whereas E. coli strains from other O serogroups tested negative. Exceptions were the K9-positive E. coli strains which cross-reacted with the O104 antiserum (data not shown). Three E. coli O8 and five O9 strains from our collection were found to react with the K9 antiserum in slide agglutination tests and were serotyped as O8:K9:H10 (n = 2), O8:K9:H45, O9:K9:H1, O9:K9:H51, and O9:K9:H12 (n = 3). Interestingly, the K9 antiserum agglutinated also live cultures of E. coli O104 strains investigated in this study, indicating cross-reactivity between the K9 and the O104 antigens. This cross-reaction was also observed when boiled cultures of K9-positive and O104-positive strains were used for agglutination assays with K9 and with O104 antiserum (data not shown). E. coli strains belonging to other O serogroups were not found to be agglutinated by the K9 antiserum, indicating its specificity toward the K9 and O104 antigens.

Detection of the E. coli O104 antigen-specific wbwCO104, wzxO104, and wzyO104 genes.

Evaluation of the PCR assays based on detection of the genes wbwCO104, wzxO104, and wzyO104, which are associated with the determination of the somatic O104 antigen, has been performed on eight strains of serotypes O8:K9 and O9:K9 and on nine E. coli O104 strains of serotypes O104:H4, O104:H2, O104:H7, O104:H11, O104:H12, and O104:H21. The PCR assays reacted with all tested O104 strains, including one Or:H4 strain, and reacted also with the eight K9-positive E. coli strains (data not shown). Accordingly, PCR assays directed to the O104 antigen genes were not found to be specific for E. coli strains expressing only the O104 lipopolysaccharide.

The PCR assay based on the detection of the wzxO104 gene was further evaluated by employing a subset of 117 strains as reported in Table 2. Of these, 80 E. coli strains tested positive for the wzxO104 gene, namely, 48 epidemic and nonepidemic EAEC STEC O104:H4 strains (including the Or:H4 strain), 28 E. coli O104-positive and H4-negative strains (O104:H2, O104:H7, O104:H11, O104:H12, and O104:H21), and 4 E. coli strains of serotypes O8:K9:H10, O9:K9:H1, O9:K9:H12, and O9:K9:H51. All the other E. coli strains investigated tested negative for the gene wzxO104.

Detection of the E. coli flagellar type H4 (fliCH4) gene.

We designed a real-time PCR assay for targeting the fliCH4 gene. The assay was first tested on non-O104 E. coli strains of serotypes O113:H4, O141:H4, O126:H4, O105:H4, and O139:H4. The fliCH4 PCR assay gave a positive result for these strains and was further evaluated on a panel of 117 E. coli strains as shown in Table 2. All E. coli O104:H4 and Or:H4 strains that tested positive for wzxO104 (see above) were also shown to be positive for the fliCH4 gene encoding flagella of serotype H4. E. coli strains expressing flagellar types other than H4 all tested negative for the fliCH4 gene, indicating the specificity of the E. coli fliCH4 PCR assay (Table 2). All motile strains carrying a fliCH4 gene agglutinated with H4-specific antiserum. By selection of strains that had shown positive results for both the wzxO104 and the fliCH4 genes, all EAEC STEC O104:H4 strains and the Or:H4 strain were clearly identified, while no other strain gave positive results for both of these genes.

Detection of the EAEC virulence gene master regulator (aggR) gene.

The specificity of aggR gene detection by PCR was examined using a subset of 117 strains of E. coli (Table 2). Only EAEC strains gave positive results for aggR, including the EAEC reference strain 17-2 (serotype O3:H2) and six EAEC strains of serotypes O111:HND, O111:H10 (n = 3), O55:H21, and O86:H2. The aggR gene was also detected in all 48 epidemic and nonepidemic O104:H4 strains (including 1 Or:H4 strain which was positive for O104 antigen genes by PCR; see above). All the other strains were negative for aggR, including 28 E. coli O104 strains with H types other than H4.

Detection of the E. coli O104:H4 CRISPR locus.

The PCR assay targeting the CRISPR locus of E. coli O104:H4 was first evaluated on 1,297 strains of E. coli, including representatives of the 186 known O serogroups and 56 H types. This PCR assay gave positive results with the 48 EAEC STEC O104:H4 isolates (including 1 Or:H4 isolate) from this study (Table 1). All 35 strains of E. coli O104 having H types other than H4 as well as E. coli O8 and O9 strains carrying the K9 capsular antigen (see above) tested negative. Five (0.40%) of the 1,249 non-O104:H4 E. coli strains reacted positive in the CRISPRO104:H4 PCR. These strains belonged to serotypes Ont:H2, O43:H2, O141:H2, and O174:H2 (n = 2). In order to explore whether the CRISPRO104:H4 locus is associated with particular other serotypes of E. coli, we have additionally tested 12 O174:H2, 8 O141:H2, and four O43:H2 strains that were available from the BfR strain collection. The CRISPRO104:H4 PCR assay was positive in 3/12 of the O174:H2, 3/4 of the O43:H2, and 1/8 of the tested O141:H2 strains. These results indicate that the CRISPRO104:H4 sequence is highly specific for the O104:H4 serotype and rarely found in other E. coli serotype strains. In conclusion, a total of 1,321 E. coli strains were investigated using the CRISPRO104:H4 PCR assay. They were divided into 48 EAEC STEC O104:H4 and 1,273 non-O104:H4 strains. Of the 60 CRISPRO104:H4 PCR-positive isolates, 12 (20%) were not EAEC STEC O104:H4. All together, the specificity estimate of the CRISPRO104:H4 PCR assay was 99.06%.

CRISPR locus sequencing.

The CRISPR loci of EAEC STEC O104:H4 strains CB13344 and CB13410, which were isolated during the 2011 outbreak in Germany, were identical to the CRISPR locus of strain CB8983, which was isolated in 2001 in Germany from a child suffering from hemorrhagic colitis. The nucleotide sequences of the CRISPR loci of these strains matched that of the CRISPR locus of EAEC O104:H4 strain 55989, which was isolated in Central Africa in 1995 from a patient suffering from persistent diarrhea (11). In contrast, the nucleotide sequences of the CRISPR loci of E. coli O104 strains expressing H types other than H4 were found to be significantly different from that of EAEC STEC O104:H4, with nucleotide sequence identities of 18% (O104:H2), 29% (O104:H12), 53% (O104:H7), 58% (O104:H11), and 59% (O104:H21). Interestingly, none of the spacers of the O104:H4 CRISPR locus was found in the CRISPR loci of E. coli O104 strains expressing H types other than H4. This finding demonstrates very high polymorphism of CRISPR loci among unrelated serotypes (Fig. 1).

The genetic relatedness of the few isolates that cross-reacted with the CRISPRO104:H4 PCR assay was confirmed by nucleotide sequence analysis of their CRISPR loci. All of the strains were found to share a relative identity with the E. coli O104:H4 CRISPR locus (Fig. 1). The CRISPR of the Ont:H2 isolate exhibited 72% sequence identity with the O104:H4 CRISPR locus. Strains of E. coli O43:H2 have a nucleotide sequence identity ranging from 59% to 66% with the O104:H4 CRISPR locus. Strains of E. coli O141:H2 were all identical regarding their CRISPR locus and exhibited 75% identities with the CRISPR locus of O104:H4. The nucleotide sequences of the CRISPR loci of E. coli O174:H2 strains were found to be more diverse. The CRISPR loci of these strains displayed 59% to 73% identity with that of EAEC STEC O104:H4.

We also sequenced the CRISPR loci of E. coli O9:K9:H1 and O174:H2, which tested negative with the CRISPRO104H4 PCR assay. We confirmed that the CRISPR loci of these strains were significantly different from that of EAEC STEC O104:H4.

Sensitivity of the CRISPRO104:H4 real-time PCR detection assay for EAEC STEC O104:H4 in pure cultures.

The sensitivity of the real-time PCR assay targeting the CRISPR locus of E. coli O104:H4 was determined from 10-fold serial dilutions of pure culture of EAEC STEC O104:H4. Positive amplification was achieved in all replicates when 6 or more E. coli genome equivalents were present in the reaction (Table 4). Fifty-six percent (5/9) of the reaction mixtures containing 0.6 CFU per PCR gave positive results, indicating that as few as 1 E. coli genome equivalent per reaction could be detected. The CT values of 9 replicates indicated that the assay was highly reproducible.

Table 4
Limit of detection of EAEC STEC O104:H4 in pure cultures by using CRISPRO104:H4 as the target sequence

DISCUSSION

Enteroaggregative hemorrhagic E. coli O104:H4 strains have been reported since 2001 as agents of diarrhea and HUS in Europe and Asia (3). EAEC STEC O104:H4 caused a large outbreak in May and June 2011 in Germany, with 3,842 patients and 855 HUS cases (1, 10), and a smaller outbreak in June 2011 in France (14). The hybrid EAEC STEC O104:H4 strains isolated between 2001 and 2011 differ from other STEC and EHEC strains by their virulence properties (35).

Multiplex PCRs targeting the major virulence genes (stx2, aggR, and aggA) of these strains were used in association with those associated with the somatic (rfbO104 and wzxO104) and H4 flagellar (fliCH4) antigens (4, 12, 21, 26). In this study, the PCR assays targeting the O104 antigen sequences tested positive for all E. coli O104 strains having H types other than H4 and showed cross-reactivity with E. coli strains carrying the K9 capsular antigen and thus were not specific to the O104:H4 strains. The results are consistent with those reported by Wang et al., indicating that the E. coli K9 capsular antigen has the same structure as the O104 O antigen (25). Parolis et al. have also reported that O104 has the same structure as the E. coli K9 antigen, which is usually associated with the O8 or O9 antigens (19).

The gene aggR was detected in all EAEC STEC O104:H4 strains but is also present in EAEC strains belonging to serogroups other than O104. The PCR assay targeting the fliCH4 gene reacted with all E. coli H4 strains independent of their O group and was not specific for detection of EAEC STEC O104:H4 strains. As many E. coli strains express the H4 antigen, the fliCH4 PCR can be used only for confirmation of the presence of the fliCH4 gene and not for specific detection of EAEC STEC O104:H4.

PCR assays targeting the O104 antigen-encoding genes, aggR and the fliCH4 gene, may cause ambiguous results when clinical, environmental, and food samples, which frequently contain multiple types of bacteria, are investigated (3). For confirmation of EAEC STEC O104:H4, the causative agent has to be isolated from the sample and the presence of the virulence genes has to be associated with the particular isolate. In outbreak situations and for routine screening of samples, there is a need for rapid and unambiguous detection of E. coli O104:H4 in any kind of sample and independent of the isolation of the bacterium (3). Therefore, a simple and single PCR indicating the presence of this pathogen has to be developed.

In an attempt to identify discriminatory diagnostic primers for EAEC STEC O104:H4, Pritchard et al. used a novel alignment-free strategy against whole genome assemblies of EAEC STEC O104:H4 and E. coli sequences from public databases (20). They selected a number of individual primer sets which exhibited 100% sensitivity against 21 EAEC STEC O104:H4 strains and 82% to 94% specificity for EAEC STEC O104:H4, with false-positive rates between 9% and 22% for non-EAEC STEC O104:H4 strains. With the aim to design a unique set of diagnostic primers able to target with higher specificity and sensitivity the EAEC STEC O104:H4 strains and to discriminate them from closely related strains, we explored the genetic diversity of the CRISPR regions of E. coli. The CRISPR loci have been identified within the genomes of many bacterial species, including E. coli (24). These loci encode tandem sequences containing direct repeats of 21 to 47 bp and separated by unique spacers of similar size. Spacers are postulated to be derived from foreign nucleic acids, such as phages or plasmids, and might protect bacteria from subsequent infection by homologous phages and plasmids.

Sequencing the CRISPR loci of EAEC STEC O104:H4 strains allowed identification of different spacers that can be used for specific PCR identification of EAEC STEC O104:H4 strains issued from different geographical locations and associated with human infections. Hence, all 48 EAEC STEC O104:H4 isolates (including 1 Or:H4 isolate) related to the outbreak occurring in May 2011 and to one O104:H4 clinical isolate reported in 2001 were detected, together with isolates issued from other geographical regions and time periods. It is also noteworthy that the sequenced CRISPR loci of these isolates were identical to the CRISPR locus of enteroaggregative E. coli O104:H4 strain 55989 (GenBank accession number NC_011748), which was isolated in 1995 from a patient suffering from chronic diarrhea (11). This would suggest that the CRISPRO104:H4 is highly stable across time and space.

The specificity of the PCR assay targeting the CRISPR locus of E. coli O104:H4 has been evaluated using 1,321 E. coli strains, including all known O serogroups and H types. The specificity of the CRISPRO104:H4 PCR was found to be 99.06% for detection of E. coli O104:H4 strains. Interestingly, only 12 strains (0.94%) belonging to other E. coli serotypes (Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive with the CRISPRO104:H4 PCR. STEC O43:H2 strains represented only 0.17% of the 597 STEC isolates from different food categories (16) and have not been described for human patients (22). All STEC O43:H2 isolates from this study were collected from deer meat. STEC O141:H2 and STEC O174:H2 were from cattle or cattle food products and were rarely found (<0.16% and 0.84%, respectively) among food-borne STEC isolates (16). The last two STEC serotypes have already been recorded as human pathogens (22). As not all tested O43:H2, O141:H2, and O174:H2 strains were found to be positive in the CRISPRO104:H4 PCR used in this study, it seems likely that this locus is polymorphous in strains belonging to these E. coli serotypes. The nucleotide sequence identities of the CRISPR loci of these strains with that of EAEC STEC O104:H4 ranged from 59% to 75%, showing the relative relatedness of the loci. Given the very low number of E. coli strains showing a false-positive reaction with the CRISPRO104:H4 PCR assay, this test has the potential to be used as a rapid-screening single PCR assay, and any CRISPRO104:H4-positive result may then be confirmed by further testing for the presence of the stx, aggR, wzxO104, and fliCH4 genes.

The sensitivity of the CRISPRO104:H4 PCR allowed the detection of one genome equivalent of E. coli O104:H4 by PCR. The specificity and sensitivity of the CRISPRO104:H4 PCR make it suitable for detection of samples containing low numbers of E. coli O104:H4 organisms. We demonstrated here that the CRISPRO104:H4 spacer content is strongly correlated with serotype O104:H4 and has the potential to be used to develop a robust, highly discriminatory and practical PCR assay for detecting EAEC STEC O104:H4 isolates. The CRISPRO104:H4 locus proved to be the most specific target that could be used as a single PCR target for identification and detection of EAEC STEC O104:H4 compared to the PCR assays targeting the O104 antigen (wbwCO104, wzxO104, and wzyO104) and the H4 flagellar antigen (fliCH4). Given the rapidity, specificity, and sensitivity of the CRISPRO104:H4 PCR assay, it should have a major impact on EAEC STEC O104:H4 surveillance and outbreak investigation and is likely to be of benefit to public health.

ACKNOWLEDGMENTS

We are grateful to N. A. Strockbine (CDC, Atlanta, GA), B. A. Lindstedt (NIH, Oslo, Norway), and A. Gill (Health Canada, Ottawa, Canada) for providing some O104:H4 isolates. We are grateful to Sabine Haby, Karin Pries, and Katja Steege (NRL E. coli) for technical assistance. We acknowledge the collaboration with the food inspection laboratories in Germany. We thank Pia D. Vogel (SMU, Dallas, TX) for her support in reviewing the manuscript.

Footnotes

Published ahead of print 15 August 2012

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