Multilocus Sequence Typing of Clostridium difficile

A total of 215 human stool specimens submitted to the Clinical Microbiology Laboratory, John Radcliffe Hospital, Oxford, United Kingdom, between 12 July and 17 October 2008 were included in this study. Stools were from both hospital and community patients. Stools were chosen so that half were sequential, enzyme-linked immunosorbent assay (ELISA) positive (n = 107) with sufficient stool remaining, and half were ELISA negative (n = 108) submitted during the same time period (Premier Toxins A&B Enzyme Immunoassay; Meridian Bioscience Europe, Villa Cortese, Italy). All stool samples underwent culture for C. difficile. Industrial methylated spirits ([IMS] 0.5 ml) was added to a 0.5-ml fecal sample (pea-sized portion if the stool was formed), and the sample was vortex mixed and incubated at room temperature for 1 h. A loopful was then cultured onto modified Brazier's cycloserine-cefoxitin-egg yolk (CCEY) agar (CCEY agar base containing cycloserine-cefoxitin supplement and 5% defibrinated horse blood), and the plates were incubated anaerobically at 37°C for up to 7 days. A single colony was subcultured onto a Columbia blood agar (CBA) plate and incubated for 48 h, after which colonies giving the characteristic odor and fluorescence under UV illumination were obtained. For long-term storage, isolates were emulsified in nutrient broth containing 10% glycerol and stored at −80°C.

An additional 50 isolates were obtained from a collection held at Leeds General Infirmary (reference laboratory for the C. difficile Ribotyping Network for England and Northern Ireland). They represented 45 different PCR ribotypes (plus five duplicates) chosen to represent the overall genetic diversity of C. difficile (determined by PCR ribotyping) and were used to validate the MLST scheme.

Total DNA was extracted from stool samples using a FastPrep homogenizer (MP Biomedicals Europe, Illkirch, France) to lyse cells and spores, followed by DNA purification using a FastDNA Spin kit for soil (MP Biomedicals). The manufacturer's protocol was followed with the following refinements. Stool samples (100 μl or equivalent volume if the stool was formed) were added to 978 μl of sodium phosphate buffer in impact-resistant 2.0-ml tubes containing matrix E, which comprises 1.4-mm ceramic spheres, 0.1-mm silica spheres, and one 4-mm glass bead in buffer. MT buffer (MP Biomedicals) (122 μl) was added, and stools were homogenized for 40 s at a speed of 6.0 m per s. The lysate was clarified by centrifugation at 13,000 rpm for 15 min. Proteins were precipitated using 250 μl of protein precipitation solution (PPS) and removed by centrifugation for 10 min. The supernatant was mixed with 1 ml of silica binding matrix for 2 min to take up DNA, and then the matrix was allowed to settle for 5 min. The binding matrix was transferred to a SPIN filter (MP Biomedicals) and washed using 500 μl of SEWS-M (salt-ethanol wash solution). After the sample was air dried at room temperature, DNA was eluted from the matrix in 100 μl of DNA elution solution ([DES] DNase and pyrogen-free water).

Isolates were cultured onto CBA and incubated anaerobically for 48 h. A few colonies were emulsified in TE (Tris-EDTA) buffer (Sigma-Aldrich Co., Ltd., Gillingham, United Kingdom) and heated at 100°C for 10 min. Debris was removed by centrifugation at 13,500 rpm for 2 min, and the supernatant was removed for use in MLST. DNA was stored at −20°C.

Ten publicly available C. difficile genome sequences, including six of PCR ribotype 027, were aligned using Mauve alignment software (5). The annotated C. difficile 630 genome (36) was included as a reference. This alignment contained several large gaps and was refined using BLAST to position fragments of each genome that were left unaligned by Mauve. Candidate regions for MLST were determined from the refined alignment as follows. Using windows of 500 bp (10 to 60 variable sites), the numbers of variable sites and the numbers of gaps were calculated. MLST loci were chosen such that there was a significant degree of divergence across the 500 bp, and no gaps were present. Fragments were annotated according to their orthologues in C. difficile 630 to ensure that they spanned housekeeping genes. Candidate fragments were tested in silico for suitability for primer design. The standard BLASTn search for “short nearly exact matches” was used, and this search is equivalent to BLASTn with the following parameters: word size, 7; low-complexity filter (DUST) off; expect value, 1,000. The database was the entire GenBank nonredundant database (nr). Details are found at: http://www.ncbi.nlm.nih.gov/blast/producttable.shtml#shortn . PCR was carried out on a subset of C. difficile isolates to verify amplification efficiency of the new MLST fragment. The seven loci and primers chosen for MLST are shown in Table 1 .

MLST was performed as described below by setting up PCR and sequencing reactions in 24-, 48-, or 96-well plates (Fig. 3 shows the procedure for 24 samples, but it can easily be scaled up to 48 samples). Seven PCR amplicons were obtained for each isolate using the primers shown in Table 1. Each 50-μl PCR mixture contained 39.75 μl of molecular biology-grade water (Sigma-Aldrich Co., Ltd.), 5 μl of 10× PCR buffer (Qiagen Ltd., Crawley, United Kingdom), 1 μl of a 10 μM concentration of each forward and reverse primer, 1 μl of 10 mM deoxynucleoside triphosphate (dNTP) mix (Invitrogen Corp., Paisley, United Kingdom), 0.25 μl of HotStart Taq DNA polymerase (Qiagen Ltd.), and 2 μl of C. difficile chromosomal DNA (approximately 10 ng) or extracted total stool DNA. The amplification conditions were 95°C for 15 min, followed by 35 cycles of 94°C for 30 s, 50°C for 40 s, and 72°C for 70 s, with a final extension at 72°C for 5 min and storage at 15°C. The amplification products were purified by precipitation with 20% polyethylene glycol (molecular weight, 8,000) and 2.5 M NaCl, and their nucleotide sequences were determined on each DNA strand using the amplification primers and BigDye Ready Reaction Mix (Applied Biosystems, Warrington, United Kingdom) as follows. Each 10-μl sequencing reaction mixture comprised 2 μl of PCR amplicon, 4 μl of a 1:15 dilution of either forward or reverse PCR primer (0.66 μM), 0.25 μl of BigDye Ready Reaction Mix, 1.875 μl of 5× sequencing buffer (20 ml of stock solution comprised 200 μl of 1 M MgCl₂, 8 ml of 1 M Tris-HCl, pH 9, and 11.8 ml of molecular biology-grade water [all from Sigma-Aldrich Co., Ltd.]), and 1.875 μl of molecular biology-grade water. Dilution of the BigDye Ready Reaction Mix using 5× sequencing buffer reduces the cost of high-throughput sequencing without any compromise in sequence quality. The reaction conditions were 30 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, and extension at 60°C for 2 min. Unincorporated dye terminators were removed by precipitation of the termination products with 2 volumes of ethanol and 0.1 volume of 3 M sodium acetate (pH 5.2), followed by centrifugation, and the resulting pellet was then washed with 70% ethanol. The reaction products were separated and detected using a 3730 XL DNA analyzer (Applied Biosystems). For each sample, the program STARS (Sequence Typing Analysis Retrieval System [http://pubmlst.org/software/assembly/ ]) was used to rapidly collate paired reads, determine sequences, and identify alleles. The data for C. difficile alleles and STs were deposited in a newly developed C. difficile MLST database, which is accessible at http://pubmlst.org/cdifficile . Phylogenetic analysis was performed using the program MEGA, version 4 (Molecular Evolutionary Genetics Analysis [http://www.megasoftware.net/ ]).

The oligonucleotide primers used to detect the tcdA (encoding toxin A), tcdB (encoding toxin B), and tcdC (encoding a negative regulator of toxins A and B) sequences found within the pathogenicity locus operon (PaLoc) are summarized in Table 1. The tcdA assay was published by Lemee et al. (21) and amplifies a 369-bp amplicon for toxin A-positive B-positive (A⁺ B⁺) strains and a 110-bp amplicon for A-negative (A⁻) B⁺ strains, which contain a deletion in the tcdA gene. The reaction conditions were 95°C for 15 min, followed by 35 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 40 s, with a final extension at 72°C for 5 min and storage at 15°C. The tcdB primers amplify a 688-bp amplicon under the same reaction conditions used for tcdA, except an annealing temperature of 50°C for 40 s and extension of 72°C for 70 s were used. The tcdC primers (33) amplify the 5′ region of the tcdC gene, giving a 475-bp amplicon under the same conditions used for tcdA. The absence of the PaLoc was demonstrated using primers lok1 and lok3 (3), which amplify a 769-bp amplicon in strains without the PaLoc. The reaction conditions were the same as those for tcdB above.

All PCR ribotyping of reference isolates and cultured isolates described in the present study was performed at the reference laboratory for the C. difficile Ribotyping Network for England and Northern Ireland, Leeds General Infirmary. PCR ribotyping was performed as described previously, with modifications (30). Briefly, bacterial growth was harvested from cultures raised on modified Brazier's CCEY agar with the omission of egg yolk and addition of 5 mg/liter lysozyme (CCEYL) (BioConnections, Wetherby, United Kingdom) for 48 h at 37°C (44). Template DNA was prepared using a QIAxtractor automated nucleic acid extraction system (Qiagen Ltd). Amplification reactions were performed in 50-μl volumes containing 50 pmol of both forward and reverse primers, 25 μl of HotStart Taq Plus PCR Master Mix (Qiagen Ltd.), 19 μl of water, and 5 μl of DNA template. The reaction mixtures were activated by heating to 95°C for 5 min and then subjected to 30 cycles of 92°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min. A final cycle of 95°C for 1 min, 55°C for 45 s, and 72°C for 5 min was added. The resultant amplimer was concentrated to a final volume of approximately 20 μl by heating the opened reaction tubes at 75°C for 30 min. Amplification products were subjected to electrophoresis using 3% Metasieve agarose (Flowgen Bioscience, Nottingham, United Kingdom) at a field strength of 7.5 V/cm for approximately 2.5 h. Agarose gels were imaged using a GeneGenius camera system (Syngene, Cambridge, United Kingdom) after ethidium bromide staining. DNA profiles were analyzed and identified against a library of known PCR ribotypes using BioNumerics, version 4.6, software (Applied Maths, Belgium).

The index of discrimination (ID) for MLST and PCR ribotyping was calculated according to Hunter and Gaston (11). The ID expresses the average probability that two individuals in the collection will have the same MLST type.

The MLST scheme was designed to be technically robust, generating high amplicon yields for all C. difficile genotypes, under the same PCR conditions for all seven loci. Loci were chosen that were widely distributed around the chromosome (Fig. 1), had demonstrable discriminatory ability, and lacked insertions or deletions that caused changes in length. The longest possible sequence was used at each locus to maximize resolution, trim sites were located so that primer sequences were removed, and null alleles were avoided (as far as possible, given current information on the species) to ensure 100% typeability.

The ability of the MLST primers to amplify and sequence all C. difficile genotypes was assessed using DNA from 152 cultured isolates. These included 50 isolates of 45 different PCR ribotypes (from a collection held at Leeds General Infirmary, the reference laboratory for the C. difficile Ribotyping Network for England and Northern Ireland) representing the known diversity of the species (Fig. 2) and an additional 102 isolates from recent clinical samples (91 from 107 ELISA-positive stools and 11 from 108 ELISA-negative stools) (Tables 2 and 3). The clinical isolates were also PCR ribotyped, and consequently four additional PCR ribotypes were included, giving a total of 49 in the study as a whole (Fig. 2 and Tables 2 and 3). All isolates were successfully typed by MLST, and a total of 40 STs were identified (Fig. 2). These data have been made available at http://pubmlst.org/cdifficile/ .

The MLST primers amplified DNA from both toxigenic and nontoxigenic strains. The 91 isolates from ELISA-positive stools were presumed to be toxigenic, and all were typed by MLST. The 11 isolates from ELISA-negative stools were assessed by PCRs designed to amplify fragments of three genes located within the PaLoc (Table 3). Nine of 11 isolates were toxigenic. The other two were nontoxigenic and were assigned a unique genotype, ST-15. They both yielded a 769-bp amplicon with the lok1/lok3 primer pair (Tables 1 and 3), confirming the absence of the PaLoc. The MLST primers have been further validated using a collection of 37 isolates (including 26 nontoxigenic strains) from healthy infants aged under 2 years and a large collection of ∼1,600 clinical isolates collected at the John Radcliffe Hospital, Oxford, United Kingdom, between September 2006 and April 2009 (molecular epidemiology data are beyond the scope of this paper).

The level of discrimination achieved with MLST was compared to PCR ribotyping. The total number of isolates with both ST and PCR ribotype data available was 152, comprising 50 in the Leeds PCR ribotype collection, 91 from ELISA-positive stools (Table 2), and 11 from ELISA-negative stools (Table 3). Among the 152 isolates, 32/40 STs occurred with a single PCR ribotype, but 8 STs were associated with two or more PCR ribotypes (Fig. 2). Conversely, the PCR ribotype was not always predictive of the ST, with three PCR ribotypes being associated with more than one ST (Fig. 2). The multiple PCR ribotypes associated with single STs had a high degree of similarity in DNA profile. For example, when PCR ribotypes share an ST (n = 8), 6/8 STs were PCR ribotypes that differed by a single band. The other two were PCR ribotypes 023 and 063 (both ST-5) (Fig. 2), which differ by four bands, and PCR ribotype 009 (ST-3), which differs from the other PCR ribotypes associated with ST-3 by four bands.

The set of 102 clinical isolates was used to compare the discriminatory ability of the two methods. The IDs (11) for MLST and PCR ribotyping were 0.90 and 0.92, respectively, a difference that is unlikely to be statistically significant.

The robustness of the MLST scheme was validated by typing strains contained in total DNA extracted from stool samples. C. difficile DNA isolated from cultured cells is a straightforward template for PCR amplification. However, when the same target is mixed in human feces, successful amplification requires efficient lysis of C. difficile cells and spores, PCR inhibitor removal (19, 29), and specific amplification from a complex mixture of DNAs (8, 22). To test the approach, MLST data were generated from both cultured isolates and total stool DNA extracts for 95 clinical samples (89/107 ELISA-positive and 6/108 ELISA-negative samples) (Tables 2 and 3). The direct and culture MLST data were generated by two different people, each without prior knowledge of the other's results. Direct MLST data were examined carefully for evidence of primer cross-reaction with other clostridial species in stools and coinfections with multiple C. difficile genotypes. Among 93 of the 95 stools, there was 100% agreement of the direct and culture MLST data. The two exceptions yielded occasional mixed peaks in the sequence chromatograms (2.1%), indicating a coinfection of more than one C. difficile genotype. This compares to 3/23 (13%) stools reported by Wroblewski et al. (45) using PCR detection of binary toxin gene(s) to detect heterogeneous isolate populations, and 2/23 (9%) patients with a first episode of CDI detected by van den Berg et al. (43) using PCR ribotyping. One additional stool DNA extract (Table 2, sample 104+) yielded very low levels of PCR amplicons, insufficient for sequencing at all seven loci.

The relationships among the 40 STs were examined using the concatenated sequences of the seven MLST loci to construct a neighbor-joining tree (Fig. 2). The total number of variable nucleotide sites was 103/3,501 (2.9%), falling to 59/3,501 (1.7%) if the outlier ST-11 (078) was excluded. The comparable data for amino acid sites were 22/1,167 (1.9%) and 17/1,167 (1.5%). Despite the relatively low level of genetic diversity, the STs clustered into four groups with one outlier (ST-11) (Fig. 2). The majority of sequence types clustered in group 1 with very low internal bootstrap values. Group 2 contained ST-1 (027), the hypervirulent clone; group 3 contained two STs, both of which were associated with PCR ribotype 023; and group 4 contained the toxin A⁻ B⁺ ST-37 (PCR ribotype 017). The outlier ST-11 is associated with PCR ribotype 078, which has been reported as an emergent hypervirulent clone.