Abstract
Conserved single-nucleotide polymorphisms (SNPs) which characterize the allelic profile of the major epidemiological lineage ST-21 were identified from the alleles within the current Campylobacter jejuni multilocus sequence typing (MLST) database. Allelic discrimination assays were designed for the detection of SNPs, enabling rapid strain profiling for clonal complex ST-21. This method is suitable for epidemiological investigations and is complementary to full MLST.
Campylobacteriosis is the most commonly reported bacterial gastrointestinal infection throughout the United Kingdom; 41,406 laboratory-confirmed cases were reported in 2003 (www.hpa.org.uk/infections). The organisms are carried in the intestinal tracts of many animals and birds; epidemiological evidence has suggested this locale as a potential reservoir for human infections and as an entry point into the food chain (4, 6, 11). The majority of human infections are caused by Campylobacter jejuni, a highly diverse species that has been subdivided through current phenotyping and genotyping techniques (1, 2, 3, 6, 7, 8, 10, 12). Although these techniques have defined epidemiological groupings, they are unable to provide timely fingerprinting of isolates for robust early detection and case cluster recognition. Any effective intervention strategies for campylobacters would require the rapid identification of common sources of infection for clusters of cases, currently considered sporadic infections.
Multilocus sequence typing (MLST) (5), as described for C. jejuni by Dingle et al. (3), has the advantages of a method which provides a discriminatory molecular profile, is reproducible and easy to interpret (13), and provides data which are directly comparable between laboratories by use of the website http://pubmlst.org/campylobacter. Use of this method has resulted in the recognition of major genetic lineages or clonal complexes in C. jejuni populations from human infections and animal and environmental sources (2). Clonal complex ST-21 is one of the largest clonal complexes found to date, constituting 26% of all of the submitted isolates, with a total of 152 different sequence types (http://pubmlst.org/campylobacter). This clonal complex is frequently associated with cases of human disease and has been found in a wide range of food chain sources (3).
Conserved single-nucleotide polymorphisms (SNPs), which identify the allelic profile of major epidemiological lineages, such as ST-21, have been identified from all of the alleles within the current MLST database. Based upon this intelligence, the aim of this study was to identify informative SNPs within MLST alleles, develop rapid allelic discrimination assays to detect the SNPs, and verify the usefulness of SNPs in strain profiling for the ST-21 clonal complex.
Strain selection and preparation.
A total of 236 isolates received by the Campylobacter Reference Unit, Health Protection Agency, London, United Kingdom, and independent from isolates reported in any other C. jejuni MLST publication (excluding reference isolates) were used. They included isolates from a United Kingdom-wide retail poultry survey (n = 88) (www.foodstandard.gov.uk/multimedia/pdfs/campsalmsurvey.pdf); human enteritis, referred as part of the Campylobacter Sentinel Surveillance Scheme (n = 90) (4); animals (dogs, birds, and cattle) (n = 22); and animal products (n = 21). Also included were the 15 reference isolates for C. jejuni MLST described by Wareing et al. (15).
C. jejuni isolates were used to inoculate Columbia blood agar (CM331; Oxoid, Basingstoke, United Kingdom) containing 5% defibrinated horse blood. The samples were incubated for 24 h at 37°C in anaerobic jars (Don Whitley Scientific, Shipley, United Kingdom) under microaerobic conditions (5% CO2, 5% O2, 3% H2, 87% N2). DNA was isolated by using MagNApure with a bacterial DNA isolation kit according to the manufacturer's instructions (Roche, Lewes, United Kingdom).
MLST.
MLST was carried out as described by Dingle et al. (3), and sequenced products were separated and detected by using an ABI Prism 3700 or a Beckman CEQ 8000 sequencer. Contigs were assembled and edited by use of a sequence typing analysis and retrieval system (Man-Suen Chan and Nicki Ventress, University of Oxford), and allele numbers, sequence types, and clonal complexes were assigned by interrogation of the Campylobacter MLST website (http://pubmlst.org/campylobacter).
Identification of alleles for SNP assay design.
The most common alleles at each locus within every clonal complex were identified by searching the MLST database. Alleles which were most specific for the ST-21 clonal complex were selected.
Downloading of MLST alleles and identification of SNPs.
All alleles were downloaded from the Campylobacter MLST website (http://pubmlst.org/campylobacter) into Bioedit Sequence Alignment Editor, version 4.0.9 (Tom Hall, North Carolina State University). SNPs unique for each chosen allele and in a location suitable to meet the primer and probe design parameters (Lightcycler probe design software; Roche) were identified from the alignments.
Design and application of allelic discrimination assays for the Lightcycler.
Two reactions using Lightcycler probe design software were designed to detect the ST-21 clonal complex: one duplex reaction to detect the two SNPs (A→G at bp 108 and C→T/A at bp 267) within the glnA1 allele and a separate, uniplex reaction to detect the one SNP (T→C at bp 330) within the tkt-1 allele. The Roche Lightcycler 1.2 instrument with version 3.5 Lightcycler software was used for all reactions. MLST amplification PCRs were performed by using MLST amplification primers for glnA1 and tkt-1 (3); 1 μl of first-round PCR product was added to a 10-μl Lightcycler Mastermix reaction. The latter included 0.3 μM concentrations of forward and reverse primers (MWG Biotech, Milton Keynes, United Kingdom), 0.1 μM concentrations of sensor probes (Metabion, Planegg-Martinsried, Germany), 0.1 μM concentrations of anchor probes (Metabion), 2.5 μl of Lightcycler Faststart hybridization probe Mastermix (Roche), and 2 to 4 mM MgCl2.
The primers (F, forward; R, reverse; S, sensor; A, anchor; phos, phosphate; fluo, fluorescein; LCred-640 and LCred-705, marker dyes) were as follows: glnF, GGATCAGGCGTAAAAGG; glnR, AACCCTTGAAAAAGTAGGTC; gln108S, LCred-640-GCCACTATTTTTAAGGTGTTCTATTGCT-phos; gln108A, TCTGGTCCAAAGTAAGCAGTATCAGCT-fluo; gln267S, LCRed-705-ATCGGTAAATTCTCTATCATCATTCCACT-phos; gln267A, TGTTTCTTGGCCTGTGTCCAGTATTGTAG-fluo; tktF, CCATCTCCGCAAAGACA; tktR, AGCACAAGGATTTGAAGT; tkt330S, LCRed-640-ATAGAGATATTGTTGCTATCATAAATAAGTATGAAGTTATCA-phos; and tkt330A, CGTTAAAGGCTAAACCTACATCGCCCTT-fluo.
Cycling consisted of an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 63°C for 10 s, and 72°C for 10 s. The melting steps consisted of denaturation at 95°C for 1 s, followed by 53°C for 1 min, and slow extension at a ramp rate of 0.1°C/s to 85°C, with continuous data acquisition. The highest melting temperature for each assay confirmed a perfect match between the probe and target sequences and hence was indicative of the SNP; melting temperatures that were 2 to 4°C lower indicated the absence of the SNP. Therefore, highest melting temperatures of 68°C for glnA1 (bp 108), 68°C for glnA1 (bp 267), and 66°C for tkt-1 (bp 330) would confirm the ST-21 clonal complex.
The glnA1 and tkt-1 alleles were selected for the ST-21 clonal complex due to their predicted specificities for ST-21 (0.88) and for this complex from the entire database (0.98) (Table 1). Additionally, both alleles had conserved informative SNPs (glnA, A→G at bp 108 and C→T/A at bp 267; tkt, T→C at bp 330), which enabled successful identification of the alleles.
TABLE 1.
Association of alleles within clonal complex ST-21
| Allele [SNP site(s)] | Specificity of allele for clonal complex | Specificity of allele for each clonal complex within MLST database
|
|
|---|---|---|---|
| Assigned to clonal complex ST-21 | Not assigned to clonal complex ST-21 | ||
| glnA1 (bp 108 and 267) | 0.93 | 0.9 | 0.04 (ST-49), 0.003 (ST-206, ST-22, ST-257, ST-353, ST-52), 0.002 (ST-177, ST-48) |
| tkt-1 (bp 330) | 0.94 | 0.67 | 0.15 (ST-48), 0.12 (ST-206), 0.01 (ST-45), 0.003 (ST-257, ST-61, ST-42, ST-443, ST-573, ST-658) |
| Combined glnA1 and tkt-1 | 0.88 | 0.98 | 0.004 (ST-206), 0.002 (ST-48) |
Evaluation of the SNP assay for ST-21 with the MLST reference isolate panel (15) demonstrated specificity for ST-21 (isolate 13254) with the highest melting temperature for each SNP reaction; therefore, this isolate possessed the three SNPs (Table 2). Other reference isolates showed combinations of lower melting temperatures. The study isolates (n = 221) were found to be 100% concordant with all 51 clonal complex ST-21 isolates identified by full MLST and confirmed to be ST-21 by the SNP assay (Table 3).
TABLE 2.
Results of SNP assay for ST-21 for MLST reference isolates (n = 15) and identification of isolates assigned to ST-21 by MLST and SNP analysis
| Isolate | Source | Allele profile
|
ST (clonal complex) | Melting temp (°C) in SNP assay fora:
|
Clonal complex assigned by SNP analysisb | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| aspA | glnA | gltA | glyA | pgm | tkt | uncA | glnA1 (bp 108) | glnA1 (bp 267) | tkt-1 (bp 330) | ||||
| 13254 | Beef offal | 2 | 1 | 1 | 3 | 2 | 1 | 5 | 21 | 68 | 68 | 66 | 21 |
| 13257 | Human stool | 4 | 7 | 10 | 4 | 1 | 7 | 1 | 45 | 66 | 68 | 64 | NA |
| 13263 | Human stool | 2 | 21 | 5 | 37 | 2 | 1 | 5 | 206 | 66 | 68 | 66 | NA |
| 13261 | Beef offal | 1 | 4 | 2 | 2 | 6 | 3 | 17 | 61 | 62 | 64 | 64 | NA |
| 13258 | Lamb offal | 2 | 4 | 1 | 2 | 7 | 1 | 5 | 48 | 62 | 64 | 66 | NA |
| 13264 | Human stool | 9 | 2 | 4 | 62 | 4 | 5 | 6 | 257 | 66 | 68 | 60 | NA |
| 13256 | Human stool | 1 | 2 | 3 | 4 | 5 | 9 | 3 | 42 | 66 | 68 | 64 | NA |
| 13262 | Sand | 17 | 2 | 8 | 5 | 8 | 2 | 4 | 177 | 66 | 68 | 62 | NA |
| 13260 | Lamb offal | 9 | 25 | 2 | 10 | 22 | 3 | 6 | 52 | 62 | 68 | 64 | NA |
| 13265 | Human stool | 8 | 10 | 2 | 2 | 11 | 12 | 6 | 354 | 66 | 68 | 64 | NA |
| 13255 | Human stool | 1 | 3 | 6 | 4 | 3 | 3 | 3 | 22 | 62 | 64 | 64 | NA |
| 13266 | Human stool | 1 | 2 | 49 | 4 | 11 | 66 | 8 | 362 | 66 | 68 | 64 | NA |
| 13259 | Human stool | 3 | 1 | 5 | 17 | 11 | 11 | 6 | 49 | 68 | 68 | 64 | NA |
| 13268 | Unknown | 10 | 27 | 16 | 19 | 10 | 7 | 5 | 403 | 66 | 64 | 64 | NA |
| 13267 | Unknown | 7 | 17 | 5 | 2 | 10 | 6 | 3 | 353 | 62 | 68 | 62 | NA |
Bold type indicates the highest melting temperatures. The highest melting temperature profile for all three SNPs was detected only for the isolate assigned to ST-21.
NA, not applicable.
TABLE 3.
Results of SNP assay for the ST-21 clonal complex for 221 isolates from different clonal complexes
| MLST clonal complexa (no. of isolates) | Melting temp (°C) in SNP assay forb:
|
Clonal complex assigned by SNP analysisc | ||
|---|---|---|---|---|
| glnA1 (bp 108) | glnA1 (bp 267) | tkt-1 (bp 330) | ||
| ST-21 (51) | 68 | 68 | 66 | ST-21 |
| ST-257 (29) | 66 | 68 | 60 | NA |
| ST-61 (3) | 62 | 64 | 66, 64 | NA |
| ST-48 (21) | 62, 61 | 64, 68 | 66, 62, 60 | NA |
| ST-45 (28) | 66 | 68 | 66, 64, 60 | NA |
| ST-49 (4) | 68 | 68 | 64, 62 | NA |
| ST-206 (5) | 66, 61 | 68, 64 | 66 | NA |
| ST-22 (7) | 62, 61 | 64 | 66, 64 | NA |
| ST-177 (2) | 66 | 68 | 64, 62 | NA |
| ST-353 (4) | 66, 62 | 68 | 65, 62 | NA |
| ST-42 (10) | 66 | 68 | 64, 62, 60 | NA |
| ST-52 (7) | 62, 61 | 68 | 66, 65, 64 | NA |
| ST-354 (10) | 66, 62 | 68 | 66, 64, 62 | NA |
| ST-403 (4) | 66 | 64 | 64, 62 | NA |
| ST-362 (1) | 66 | 68 | 64, 61 | NA |
| Other clonal complexes and unassigned isolates (35) | ND | ND | ND | NA |
Determined by full MLST (3).
Bold type indicates the highest melting temperatures.
NA, not applicable.
Using melting curve analyses with the Lightcycler, it was possible to design an assay to recognize the presence of informative SNPs that can identify alleles specific to clonal complex ST-21. The melting peak analyses are sufficiently robust, since each SNP reaction showed an obvious shift in melting temperatures between different alleles on the basis of sequences. The assignment of a melting temperature profile of 68, 68, and 66°C with the SNP assay identifies isolates belonging to clonal complex ST-21 in less than 4 h at a cost of US$2.40 per isolate.
The drawbacks of the SNP approach are that the data in the current MLST database are representative only of isolates that have been both typed by MLST and submitted; therefore, as the database expands, there is the potential for new alleles to be missed. The process of assigning clonal complexes from SNP analysis might not suit all areas of research where MLST might be considered. For population biology, the full MLST data set would be required; alternative strategies for MLST, such as the use of high-density DNA arrays like those described for MLST of Staphylococcus aureus, might be more applicable (14). Also, MLST or the SNP approach might not be suitable in certain situations. A supplementary technique, such as sequencing of the short variable region of the flaA gene, was described as being necessary for adequate discrimination in C. jejuni outbreak investigations (9).
Nevertheless, we have established a timely and discriminatory method for the identification of isolates belonging to the ST-21 clonal complex, the greatest benefit being its applicability for rapid screening. This work is currently being extended to include the other major important clonal complexes of C. jejuni and an additional real-time platform to achieve high-volume throughput. Furthermore, this method has the potential for rapid allelic discrimination of C. jejuni directly from clinical specimens and food and environmental samples. This approach is complementary to full MLST characterization of C. jejuni.
Acknowledgments
We thank Martin Maiden, Frances Colles, and Roisin Ure (University of Oxford) for help in setting up MLST and the use of sequencing facilities. We thank the staff of the Molecular Epidemiology Department, Health Protection Agency North West Laboratory, Manchester, and the Campylobacter Reference Unit, Laboratory of Enteric Pathogens, Health Protection Agency, London, United Kingdom, and Ankur Agrawal for help in setting up the sequence typing analysis and retrieval system software.
We acknowledge the Health Protection Agency (HPA) for funding through an HPA studentship. During the course of this study we used the Campylobacter MLST website (http://pubmlst.org/campylobacter) developed by Man-Suen Chan and Keith Jolley and located at the University of Oxford. Initial development of this website was funded by the Wellcome Trust; maintenance is funded by DEFRA.
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