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J Clin Microbiol. Dec 2012; 50(12): 4035–4040.
PMCID: PMC3503007

Use of Clustered Regularly Interspaced Short Palindromic Repeat Sequence Polymorphisms for Specific Detection of Enterohemorrhagic Escherichia coli Strains of Serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by Real-Time PCR

Abstract

We explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of enterohemorrhagic Escherichia coli (EHEC) to design simplex real-time PCR assays for each of the seven most important EHEC serotypes worldwide. A panel of 958 E. coli strains investigated for their CRISPR loci by high-throughput real-time PCR showed that CRISPR polymorphisms in E. coli strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx and eae genes). The CRISPR sequences chosen for simplex real-time PCR amplification of EHEC strains belonging to the top 7 EHEC serogroups differentiated clearly between EHEC and non-EHEC strains. Specificity estimates for the CRISPR PCR assays varied from 97.5% to 100%. Sensitivity estimates for the assays ranged from 95.7% to 100%. The assays targeting EHEC O145:H28, O103:H2, and O45:H2 displayed 100% sensitivity. The combined usage of two simplex PCR assays targeting different sequences of the O26 CRISPR locus allowed detection of EHEC O26:H11 with 100% sensitivity. By combining two simplex PCR assays targeting different sequences of the EHEC O157 CRISPR locus, EHEC O157:H7 was detected with 99.56% sensitivity. EHEC O111:H8 and EHEC O121:H19 were detected with 95.9% and 95.7% sensitivity, respectively. This study demonstrates that the identification of EHEC serotype-specific CRISPR sequences is more specific than the mere identification of O-antigen gene sequences, as is used in current PCR protocols for detection of EHEC strains.

INTRODUCTION

Shiga toxin-producing Escherichia coli (STEC) strains are a diverse group of E. coli strains belonging to over 400 E. coli O:H serotypes, some of which cause outbreaks and sporadic cases of food-borne illnesses ranging from diarrhea to hemorrhagic colitis (HC) and the hemolytic-uremic syndrome (HUS) (11, 15, 16). According to their pathogenicity for humans, the latter strains were also designated enterohemorrhagic E. coli (EHEC) (17, 18). Numerous cases of HC and HUS have been attributed to EHEC O157:H7 strains (25), but it has now been recognized that other STEC serotypes belong to the EHEC group (8, 24). Cumulative evidence from numerous countries indicates that up to 30 to 60% of human EHEC infections are caused by non-O157 EHEC strains (7). There are seven “priority” EHEC serotypes most frequently implicated in outbreaks and sporadic cases of HC and HUS (8, 24). These comprise serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 and their nonmotile derivatives.

Although regulations are disparate throughout the world, many food inspection programs aim at detecting STEC strains that pose a significant threat to human health in foods that are the most likely to disseminate EHEC and to be consumed raw or undercooked. Some beef products are thus of particular interest in that aspect. The U.S. regulations have precisely been revised to add 6 additional serogroups (O26, O103, O45, O111, O121, and O145) to the existing O157:H7 regulation (22, 23). This regulation imposes testing of specified meat products (i.e., ground beef, beef scraps, and machine-tenderized steaks) for these 7 EHEC serogroups. However, detection of non-O157 EHEC strains is particularly challenging because they have no phenotypical characteristics that distinguish them from the large number of non-STEC strains that share the same habitats.

The current approach for detecting EHEC in food is to screen first for the presence of the stx1/stx2 genes and the eae gene (the latter is involved in the attaching and effacing phenotype) in DNA made from bacterial enrichment cultures. The CEN/ISO TS 13136 (8) and MLG 5B.01 (23) standard methods require the presence of both the stx1/stx2 and eae genes for further investigation of a suspected EHEC contamination. For this, specific sequences derived from the O-antigen genes associated with the seven priority serogroups are searched. However, the sequences derived from the O-antigen genes of the top 7 EHEC serogroups not only are present in EHEC strains but can be detected in a large number of Stx-negative E. coli strains as well (1). This sequential approach requires additional time and does not prove that EHEC strains are present in a sample, as multiple E. coli strains reacting with each of the PCRs will generate erroneous results. This is of particular interest in complex samples such as food, fecal, and environmental specimens (5, 9, 19). As a consequence, the CEN/ISO TS 13136 and MLG 5B.01 standard methods demand EHEC isolation from the sample to confirm that all the detected genes are associated with the particular EHEC strain (8, 22, 23). However, isolation of EHEC even from positive samples is not always successful and takes up to 4 days for confirmation (10, 20).

For saving time and money, many laboratories use, therefore, only PCR-based strategies to diagnose the presence of putative EHEC in a test sample. The simple detection of the stx1/stx2, eae, and O-antigen genes in a food product may often result in its rejection even though it does not contain EHEC strains of the top 7 serotypes. In order to focus microbiological food inspection on the human-pathogenic STEC strains, the choice of selective genetic markers that allow more specifically the identification of the top 7 EHEC serotypes is desirable. In this work, we have introduced the clustered regularly interspaced short palindromic repeat (CRISPR) sequences as suitable genetic markers for selection of EHEC from sample material by PCR. We showed that CRISPR-directed real-time PCR procedures have a higher sensitivity and specificity to the top 7 EHEC serotype strains than any of the O-antigen gene-based real-time PCR protocols.

MATERIALS AND METHODS

Bacterial strains.

Strains of E. coli (n = 958) that were investigated for their CRISPR loci by high-throughput real-time PCR are reported in Table 1. The strains were isolated from human patients, animals, and food. Another panel of the E. coli strains investigated in this study includes 33 E. coli O157 strains expressing H types other than H7, namely, O157:HNT/H (n = 6), H12 (n = 2), H15 (n = 1), H16 (n = 10), H26 (n = 3), H39 (n = 4), H40 (n = 1), and H45 (n = 6). These were tested only with the O157 CRISPR PCR tests together with a set of 154 additional O157:H7 strains.

TABLE 1
E. coli strains

E. coli strains were divided into Shiga toxin-producing E. coli (STEC; n = 162), enteropathogenic E. coli (EPEC; n = 307), enterohemorrhagic E. coli (EHEC; n = 362), and nonpathogenic E. coli (n = 127). According to previous nomenclature, EHEC strains were defined as harboring both stx and eae genes, whereas STEC strains carried only one or more stx genes but no eae genes (18). Nonpathogenic E. coli strains were defined as stx- and eae-negative strains.

All strains investigated in this work were identified for the E. coli O (somatic) and H (flagellar) antigens and have been characterized for the stx and eae genes as previously reported (2). In addition to the E. coli strains, 35 strains of enterobacteria were investigated for their CRISPR loci by high-throughput real-time PCR. They belonged to the following bacterial species: Cronobacter sakazakii subsp. sakazakii, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter turinensis, Enterobacter amnigenus, Enterobacter cloacae, Enterobacter faecalis, Enterobacter gergoviae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter pyrinus, Buttiauxella agrestis, Citrobacter freundii, Citrobacter koseri, Escherichia hermannii, Escherichia vulneris, Ewingella americana, Hafnia alvei, Klebsiella pneumoniae, Klebsiella oxytoca, Kluyvera intermedius, Pantoea spp., Raoultella planticola, Raoultella terrigena, Salmonella enterica, Salmonella enterica serovar Agona, Salmonella enterica serovar Dublin, Salmonella enterica serovar Typhimurium, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Shigella flexneri, Shigella sonnei, and Yersinia enterocolitica.

For examination, bacteria were cultured to single colonies on Luria broth plates and grown overnight at 37°C. A single colony was taken for DNA extraction using the InstaGene matrix (Bio-Rad Laboratories, Marnes-La-Coquette, France). Extracted DNA was stored at −20°C until used in high-throughput real-time PCR testing.

High-throughput real-time PCR.

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) were 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), 300 nM (each) primer, and 300 nM (each) probe (corresponding to a 200 nM final concentration of each). Amplifications were performed using 6-carboxyfluorescein (FAM)- or hexachlorofluorescein (HEX)-labeled TaqMan probes. Primers and probes used for PCR amplifications are listed in Table 2. 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.

TABLE 2
Primers and probes

CRISPR locus sequencing.

The double-stranded DNA sequencing of the CRISPR loci was performed by Eurofins MWG Operon (Courtaboeuf, France).

Nucleotide sequence accession numbers.

The DNA sequences of the CRISPR loci of the following EHEC strains were identified and submitted to GenBank: EHEC O157:H7 strains CB4095 (accession number JX219753), CLC87 (accession number JX219754), and MF1380NM (accession number JX219755); EHEC O121:H19 strain CB8124 (accession number JX219756); EHEC O45:H2 strain A2619-C2 (accession number JX219757); EHEC O145:H28 strain VTH34 (accession number JX219758); EHEC O111:H8 strains ED313 (accession number JX219759) and EH240 (accession number JX402083); and EHEC O26:H11 strain H19 (accession number JX219760).

RESULTS

Identification of specific DNA sequences targeting the CRISPR loci of EHEC O157:H7.

The whole-genome sequences of the 5 strains of EHEC O157:H7 available in GenBank showed a high degree of nucleotide sequence conservation of their CRISPR loci. We identified from our strain collection three E. coli O157:H7 strains which differed for their O157 CRISPR loci. The CRISPR sequences of these strains (CB4095, CLC87, and MF1380NM) have been determined, and based on these sequences and the published CRISPR sequence of the strains EDL933, EC4115, TW14359, Sakai, and Xuzhou21, three real-time PCR assays were designed (SP_O157_A, SP_O157_B, and SP_O157_C) for detecting EHEC O157:H7. The specificity and sensitivity of each assay were first tested against a panel of 958 E. coli strains, including 75 strains of EHEC O157:H7 (Table 1). The PCR tests proved to be highly sensitive and specific for EHEC O157:H7. The sensitivity of the assays ranged from 92.0% to 97.3%, with 69/75 strains being detected with SP_O157_A, 73/75 strains being detected with SP_O157_B, and 71/75 strains being detected with SP_O157_C. The specificity of the PCR tests was high, ranging from 99.7 to 100%. The PCR assay SP_O157_B was the only test reacting with non-O157:H7 strains. It gave positive results with the unique EHEC O55:H7 strain tested and with 2 out of the 26 stx-negative and eae-positive O55:H7 strains tested. By combining the PCR assays SP_O157_B and SP_O157_C, all the 75 EHEC O157:H7 strains were correctly detected (100% sensitivity) and only 3 isolates of serogroup O55 were found positive (99.7% specificity). For further testing the specificity of the O157 CRISPR PCRs, we additionally investigated 33 E. coli O157 strains expressing H types other than H7, namely, HNT, H12, H15, H16, H26, H39, H40, and H45. None of these 33 strains reacted with any of the CRISPR O157 PCR assays. In order to investigate further the sensitivity of the CRISPR O157 PCR assays, we examined 154 additional O157:H7 strains. Only one isolate (strain C799-96) was found negative with each of the CRISPR O157 PCR assays. We showed by PCR flanking the CRISPR locus that this strain has lost its CRISPR locus (data not shown). Altogether, 229 EHEC O157:H7 strains were investigated by the CRISPR O157 PCR assays, resulting in only 1 false negative (99.6% sensitivity) and 3 false positives (99.7% specificity) when 916 non-O157:H7 E. coli strains were tested by the combined set of PCR assays SP_O157_B and SP_O157_C (Table 3). None of the other enterobacteria tested in this study were positive by the CRISPR O157 PCR assays.

TABLE 3
Sensitivity and specificity estimates of the CRISPR PCR assays

Identification of specific DNA sequences targeting the CRISPR locus of EHEC O145:H28.

The CRISPR locus of EHEC O145:H28 strain VTH34 has been characterized by sequencing (accession number JX219758). A PCR assay (SP_O145) has been designed from this CRISPR sequence to target EHEC O145:H28. Among the 958 E. coli strains that were investigated with this PCR test, only the 29 EHEC O145:H28 and 4 EPEC O28:H28 strains tested positive (Table 3). Strains of E. coli O145 having an H type other than H28, such as EHEC O145:H25 (n = 5), EPEC O145:H34 (n = 5), and nonpathogenic E. coli O145:H2 (n = 2) strains, tested negative. Sensitivity and specificity estimates of the PCR assay SP_O145 were 100% and 99.6%, respectively. None of the other enterobacterial species tested was found positive by the CRISPR O145 PCR assay.

Identification of specific DNA sequences targeting the CRISPR locus of EHEC O111:H8.

Based on the sequence of the CRISPR locus of EHEC O111:H8 strain ED313 (accession number JX219759) and the CRISPR locus of strain 11128 (accession number AP010960), a real-time PCR assay combining one forward primer with two reverse primers and one probe has been designed (SP_O111) to detect EHEC O111:H8 (Table 2). Investigation of 958 E. coli strains by the PCR assay SP_O111 gave positive results for 47 EHEC O111:H8 strains out of the 49 O111:H8 strains tested. Only two EPEC strains of serotype O45:H11 and one nonpathogenic E. coli O45:H11 strain also tested positive. It is noteworthy that E. coli O111 strains with H types other than H8 (O111:HND, O111:H2, O111:H9, O111:H10, O111:H11, O111:H12, O111:H19, O111:H21, O111:H25, and O111:H45) were found negative regardless of whether they belong to STEC, EPEC, or nonpathogenic E. coli pathogroups. Sensitivity and specificity estimates of this PCR assay were high, 95.9% and 99.7%, respectively (Table 3). The CRISPR locus of the two EHEC O111:H8 strains that tested negative has been sequenced. Strain EH240 (accession number JX402083), which is positive for stx1 and eae, displayed some spacer deletions in its CRISPR (data not shown) which explain the negative result. The other strain (ED608), which is positive for stx1, stx2, and eae genes, did not carry a CRISPR locus in this region of the genome and accordingly tested negative by the O111 CRISPR PCR assay. The PCR assay SP_O111 also showed negative results for all the other enterobacteria examined in this study.

Identification of specific DNA sequences targeting the CRISPR locus of EHEC O121:H19.

The CRISPR locus of EHEC O121:H19 strain CB8124 has been sequenced in this study (accession number JX219756). A PCR assay (SP_O121) has been designed from this sequence to target EHEC O121:H19. Among the 958 E. coli strains tested by the PCR assay SP_O121, only one STEC O104:H7 strain and 22 out of the 23 EHEC O121:H19 strains tested positive (Table 3). This assay also showed positive results for one stx-negative, eae-positive O121:H19 strain and one stx- and eae-negative O121:H19 strain. Interestingly, E. coli O121 strains with H types other than H19 (O121:H7, O121:H10, O121:H11, O121:H14, and O121:H45) were found negative regardless of their E. coli pathogroup. In addition, all the other enterobacteria examined in this study by the PCR assay SP_O121 were found negative. The specificity and sensitivity estimates of the assay were high, 99.7% and 95.7%, respectively.

Identification of specific DNA sequences targeting the CRISPR loci of EHEC O103:H2 and O45:H2.

Based on the sequence determination of the CRISPR locus of EHEC O45:H2 strain A2619-C2 (accession number JX219757) and the sequence of the CRISPR locus of EHEC O103:H2, issued from strain 12009 (accession number AP010958), a PCR assay (SP_O45) has been designed and has shown positive results for the 17 strains of EHEC O45:H2, one stx-negative O45:H2 strain, and all the 37 EHEC O103:H2 strains investigated in this study. Thus, the PCR assay SP_O45 has shown high sensitivity (100%) for EHEC O103:H2 and O45:H2. This test has 98.7% specificity when tested on a large panel of E. coli strains, reacting additionally with only 11 strains of the following serotypes: O118:H8, O128:H2, O128:H8, O46:H38, O8:H8, O142, and O145:H2 and one O103 strain that tested negative for the flagellar H2 (Table 3). All other O103 strains (O103:HND, O103:H8, O103:H21, and O103:H25) and O45 strains (O45:HND, O45:H4, O45:H9, O45:H11, and O45:H16) did not react with the O103/O45-specific PCR assay. The test found negative all the other enterobacteria examined in this study as well.

Identification of specific DNA sequences targeting the CRISPR locus of EHEC O26:H11.

Based on the sequences of the CRISPR loci of EHEC O26:H11 strains 11368 (accession number AP010953) and H19 (accession number JX219760), two real-time PCR assays were designed (SP_O26_C and SP_O26_D) for specific detection of EHEC O26:H11 strains. The specificity and sensitivity of the assays were tested against a panel of 958 E. coli strains. These two PCR tests proved to be sensitive and specific for detecting EHEC O26:H11 (Table 3). Each PCR assay detected 41 strains out of the 43 EHEC O26:H11 strains tested, but the combined usage of the two simplex PCR assays allowed the detection of all EHEC O26:H11 strains (100% sensitivity). The EHEC O26-specific PCR tests detected in addition 29 strains of E. coli O26:H11 which were stx negative and eae positive. It is likely that these strains are remnants of EHEC strains which have lost their stx genes on subculture. It was previously described that such strains are frequent among E. coli O26:H11 isolates and still carry sequences of truncated stx phages in their genomes (3).

The specificity of the PCR test SP_O26_C was 98.9% (10 non-EHEC O26:H11 strains detected), whereas the specificity of the PCR test SP_O26_D was 98.5% (14 non-EHEC O26:H11 strains detected). By combining the PCR assays SP_O26_C and SP_O26_D, only 23 non-EHEC O26:H11 strains were found positive (97.5% specificity). Interestingly, O26 strains with H types other than H11 (O26:HND, O26:H31, O26:H34, and O26:H32) were all negative when tested by the PCR assays SP_O26_C and SP_O26_D. The two PCR assays did not detect any of the other enterobacteria investigated in this study.

DISCUSSION

The emergence of O157 and non-O157 EHEC strains in severe and epidemic human disease is of great global concern (4, 7, 8). Non-O157 EHEC strains have been shown to be important pathogens, despite being severely underreported, because in contrast to O157:H7, there are no phenotypical traits that can be used for their specific selection on cultivation media. Current methods for detection of non-O157 EHEC in food samples are based on real-time PCR detection of stx and eae genes followed by detection of EHEC-associated O-antigen genes (O26, O45, O103, O111, O121, O145, and O157) (8, 22, 23). This approach bears the disadvantage that a large number of non-EHEC strains are found among members of the above-mentioned serogroups (1).

In order to increase the specificity of the above-mentioned real-time PCR-based detection systems, tests are needed that not only identify the targeted EHEC serogroups but also differentiate between strains of these serogroups that carry EHEC virulence factors and those that do not. In an attempt to identify discriminatory diagnostic primers for identifying with high specificity and sensitivity strains of the top 7 EHEC serotypes, we explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of E. coli. CRISPRs have been identified within the genomes of many bacterial species, including E. coli (14, 21). CRISPRs encompass tandem sequences containing direct repeats of 21 to 47 bp that are separated by spacers of similar size. Spacers appear to be derived from foreign nucleic acids, such as phages or plasmids (12, 13), and may protect bacteria from subsequent infection by homologous phages and plasmids (13). A recent study by Touchon et al. (21) has examined a collection of diverse E. coli strains and demonstrated that approximately 90% of strains cluster into appropriate phylogenetic groups by use of CRISPR analysis. In another very recent study, a single PCR for the CRISPRO104:H4 target proved to be sufficient for a highly specific identification of the recently emerged enteroaggregative E. coli (EAEC) type that has acquired an Stx2 phage (EAEC STEC O104:H4) (6). These findings suggest that the CRISPR locus of E. coli might be closely related to clonal types of E. coli, which are characterized by expression of certain O:H antigens and virulence factors. The present study focuses on the CRISPR loci of EHEC strains associated with the world's most frequent clinical cases with the aim to identify conserved CRISPR motifs associated with EHEC strains of the top 7 serotypes.

Sequencing the CRISPR loci of various EHEC strains has shown the genetic diversity of the CRISPR sequences derived from EHEC O157:H7, O26:H11, O145:H28, O103:H2, O111:H8, O121:H19, and O45:H2 strains. Analysis of the spacer sequences located between the short palindromic repeats within the CRISPR loci allowed identification of useful genetic markers able to detect with high sensitivity (95.65% to 100%) and specificity (97.5% to 100%) EHEC strains of the top 7 serotypes. Interestingly, the CRISPR PCR assays developed in this study strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx and eae genes). Strains belonging to the top 7 serogroups but with H types different than the above-mentioned H types tested negative regardless of their E. coli pathogroup. These findings support clearly the application and utility of the CRISPR loci in identification and molecular serotyping of EHEC O157:H7, O26:H11, O145:H28, O103:H2, O111:H8, O121:H19, and O45:H2. The CRISPR PCR assays showed negative results for all the other enterobacteria investigated in this study. In conclusion, the CRISPR sequences chosen for simplex real-time PCR amplification of EHEC strains belonging to the top 7 EHEC serotypes differentiated clearly between EHEC and non-EHEC strains. The number of strains and the diversity of serotypes and pathogroups that were investigated in this study provide a solid base for a future utilization of the CRISPR real-time PCR tests not only on pure bacterial isolates but also on food and stool samples. These assays would work potentially with DNA extracted from enriched food (19) or stool samples (26) suspected to be positive. This needs to be confirmed with further evaluation of the CRISPR PCR tests on spiked and naturally contaminated samples. A complete evaluation of these tests in real samples will be crucial to definitively know how they can be applied in EHEC surveillance and outbreak investigations.

ACKNOWLEDGMENTS

We are grateful to F. Scheutz (WHO Collaborating Centre for Reference and Research on Escherichia and Klebsiella, Copenhagen, Denmark), A. Gill (Health Canada, Ottawa, Canada), L. Chui (University of Alberta Hospital, Alberta, Canada), M. Rivas (INEI-ANLIS, Buenos Aires, Argentina), G. H. Loneragan (Texas Tech University, Lubbock, TX), Peter Feng (U.S. FDA, College Park, MD), and C. DebRoy (The Pennsylvania State University, State College, PA) for providing some E. coli isolates or DNA extracts from E. coli.

Footnotes

Published ahead of print 3 October 2012

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