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The bacterium Listeria monocytogenes is a gram-positive rod-shaped organism that causes the disease listeriosis in both humans and animals. In humans, mainly immunocompromised individuals are affected, and the symptoms include septicemia and/or meningitis. Pregnant women may have abortions, or the newborn child may be seriously compromised (9, 16). It has been suggested that ingestion of contaminated food is an important cause of listeriosis (7).

In investigations of food-borne listeriosis it is essential to type the isolated bacteria. A connection in time and space between patients and implicated food, combined with reliable typing results, is important for establishing the route of infection. Strain characterization is also important when contamination in food production plants is investigated.

L. monocytogenes is an intracellular pathogen that has the ability to induce its own uptake in different types of normally nonphagocytic cells (4, 8). The surface protein internalin, encoded by the inlA gene, mediates invasion of epithelial cells (8). Located downstream of inlA is inlB, which codes for a protein that is involved in uptake and replication of L. monocytogenes in hepatocytes (4, 10). Both inlA and inlB belong to the same gene family (8).

Serotyping and phage typing are established phenotypic methods for typing of L. monocytogenes. However, only a few laboratories have facilities to perform phage typing and complete serotyping. There are also sometimes problems with phage typing reproducibility, and some strains are even nontypeable (11). Recently, genetic methods for typing L. monocytogenes have been introduced, and pulsed-field gel electrophoresis is one commonly used method. We have previously shown that the sequence of inlB can be used for typing L. monocytogenes (6). In that study we observed that two nucleotides in the inlB gene, at positions 1575 and 1578, might be sufficient to distinguish four groups of L. monocytogenes strains (6). Strains of serovars 1/2a and 1/2c were grouped together, as were strains of serovars 1/2b and 3b, while strains of serovar 4b were divided into two groups. In the present study, we tested this hypothesis with a novel sequencing-by-synthesis method based on real-time pyrophosphate detection (14). The method is called pyrosequencing and is optimal for sequencing short sequences, including, for example, detection of single-nucleotide polymorphisms (1). The main application of pyrosequencing so far has been analysis of polymorphisms in the human genome in which single-nucleotide polymorphisms are the most common kind of sequence variation (3).

Bacterial strains. A total of 106 strains of L. monocytogenes were used in this study. The criteria used to confirm that a strain was a L. monocytogenes strain were those described by Unnerstad et al. (17). The strains were selected from the L. monocytogenes collection of the Department of Food Hygiene, Swedish University of Agricultural Sciences, Uppsala, Sweden, and were isolated in different years (1958 to 1998, except for NCTC 7973, which was isolated in 1926) and from different sources (48 strains from food, 43 strains from humans, 12 strains from animals, and 3 strains from the environment). The following serovars were represented: 1/2a (19 strains), 1/2b (16 strains), 1/2c (17 strains), 3a (1 strain), 3b (16 strains), 4b-I (20 strains), and 4b-II (17 strains). In a previous study, the 37 serovar 4b strains were divided into two groups, 4b-I (20 strains) and 4b-II (17 strains), by PCR-restriction enzyme analysis (PCR-REA) of most of the inlB gene and part of the inlA gene (5).

Production of PCR products for pyrosequencing. Primers LPP1 (5'-ATCGGTCTACCAAGGTAAAA-3'; positions 1518 to 1537) and LPP2 (biotin-5'-CGACCCAACCAATTACTTT-3'; positions 1603 to 1621) were used in a PCR. Sequence data used for primer construction were obtained from Ericsson et al. (6). Each 50-µl PCR mixture contained 1 µl of bacterial cells prepared and denatured as described by Ericsson et al. (5), 5 µl of GeneAmp 10× PCR Gold buffer (PE Biosystems), 2.0 mM MgCl₂, 200 µM (each) dATP, dTTP, dCTP, and dGTP, each primer at a concentration of 0.2 µM, and 1.0 U of AmpliTaq Gold DNA polymerase (PE Biosystems). The PCR was performed with a Perkin-Elmer GeneAmp PCR System 2400. The program was started by incubation at 95°C for 10 min, which was followed by 50 cycles consisting of 95°C for 30 s, 47°C for 45 s, and 72°C for 1 min. Each PCR product (5 µl) was visualized by electrophoresis in a 2% agarose gel and staining with ethidium bromide (1.5 µg/ml) for 15 min.

Pyrosequencing. Biotinylated PCR templates (25 µl) were immobilized on 150 µg of streptavidin-coated paramagnetic Dynabeads M-280 (Dynal AS) in BW buffer (5 mM Tris-HCl, 1 M NaCl, 0.5 mM EDTA, 0.05% Tween 20; pH 7.6). Samples were incubated for 15 min at 65°C with an Eppendorf Thermomixer Comfort set to constant agitation at 1,400 rpm. After immobilization, the bead-template complexes were washed in 100 µl of 10 mM Tris-acetate (pH 7.6) and transferred to 50 µl of 0.5 M NaOH in order to generate single-stranded DNA. After 5 min the samples were washed twice in 100 µl of 10 mM Tris-acetate (pH 7.6). The beads were then added to 45 µl of annealing buffer (20 mM Tris-acetate, 5 mM magnesium acetate; pH 7.6) containing 15 pmol of the sequencing primer 5'-GCCAAAACACCAATTAC-3', which was located next to position 1575 and 4 nucleotides from position 1578 in the inlB gene. Annealing took place at 95°C for 1 min, after which the samples were left at room temperature while the sequencing reaction mixture was prepared. All transfers of the bead-template complexes were performed with a PSQ 96 Sample Prep station (Pyrosequencing AB). The sequencing reaction was performed automatically with a PSQ 96 system (Pyrosequencing AB) by using a SNP reagent kit.

Southern blot analysis. A 104-bp inlB probe labeled with digoxigenin was produced with a PCR DIG probe synthesis kit (Roche Molecular Biochemicals). The primers used were LPP1 and LPP4 (5'-CGACCCAACCAATTACTTT-3'; positions 1603 to 1621). Cells of one L. monocytogenes serovar 1/2a strain, one serovar 1/2b strain, one serovar 1/2c strain, one serovar 3b strain, one serovar 4b-I strain, and one serovar 4b-II strain were harvested from 2.5-ml portions of overnight cultures. Total genomic DNA was extracted by the method of Pandiripally et al. (13). Ten micrograms of DNA from each strain was cleaved with EcoRI and, in a second experiment, with NcoI. These two restriction enzymes were chosen because it was assumed that they do not cut within the hybridization target. This assumption was based on sequence data from Gaillard et al. (8) and Ericsson et al. (6). DNA was separated on a 0.9% agarose gel (SeaKem GTG) in 1× TAE (40 mM Tris-acetate, 1 mM EDTA) and was then transferred to a nylon membrane (Hybond N+; Amersham Pharmacia Biotech) by using a VacuGene XL vacuum blotting system (Amersham Pharmacia Biotech) as recommended in the manufacturer's manual. The filter was prehybridized for 30 min at 40°C in Dig Easy Hyb solution (Roche Molecular Biochemicals). The prehybridization solution was replaced by fresh Dig Easy Hyb solution that included the probe, and hybridization was performed at 40°C overnight. The membrane was washed twice in 2× SSC (1× SSC is 0.15 M sodium chloride plus 15 mM sodium citrate) with 0.1% sodium dodecyl sulfate for 5 min at 40°C and twice in 0.5× SSC with 0.1% sodium dodecyl sulfate for 15 min at 40°C. The digoxigenin-labeled probe was visualized with a Dig luminescent detection kit (Roche Molecular Biochemicals).

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Pyrograms obtained by pyrosequencing of L. monocytogenes strains belonging to different serovars, based on positions 1575 and 1578 of the inlB gene. Incorporated nucleotides are shown below each pyrogram. Relative light units are indicated on the ordinate axes. Additions of enzyme (E) and substrate (S) mixtures and nucleotides are indicated on the horizontal axes. (A) Serovar 1/2a strain SLU 1906 representing the serovar 1/2a-serovar 1/2c group as determined by T and A at the two variable positions. (B) Serovar 3b strain SLU 1182 representing the serovar 1/2b-serovar 3b group as determined by T and T at the two variable positions. (C) Serovar 4b strain SLU 249 representing the serovar 4b-I group as determined by C/T and G/T at the two variable positions. (D) Serovar 4b strain SLU 28 representing the serovar 4b-II group as determined by T and G/T at the two variable positions.

View larger version (49K): [in this window] [in a new window]   FIG. 2.   Southern blot hybridization with a 104-bp inlB probe of EcoRI-digested (A) and NcoI-digested (B) genomic DNAs from L. monocytogenes strains belonging to different serovars. Lane 1, serovar 1/2a strain SLU 83; lane 2, serovar 4b-I strain SLU 520; lane 3, serovar 1/2c strain SLU 524; lane 4, serovar 4b-II strain SLU 559; lane 5, serovar 3b strain SLU 594; lane 6, serovar 1/2b strain SLU 2324; lane M,  DNA HindIII marker. Molecular sizes (in kilobases) are indicated on the left.