A wide variety of Clostridium perfringens type A food-borne isolates that carry a chromosomal cpe gene belong to one multilocus sequence typing cluster

Abstract

Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)(5) fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods.

Fig 1

Clustering of (GTG)₅-rep patterns of identified pure Clostridium spp. food-borne isolates in this study with reference strains (types A to E). The resulting fingerprints were analyzed using the BioNumerics version 5.10 software package. The left dendrogram shows the similarity among (GTG)₅-rep patterns of test strains/isolates (curve-based cosine coefficient). The cpe-positive food isolates and reference strains are boxed. *, sporulation ability in mDS sporulating culture was estimated using phase-contrast microscopy (magnification, ×1,000), and four levels of spore production were defined: no phase-bright spores/view (−), 1 to 3 spores/view (+), less than 50% spores (++), more than 50% spores formed (+++). Spore counts (sporea/ml) of cpe-positive C. perfringens strains are noted in brackets. The details are described in Materials and Methods. **, cpe types were determined using a multiplex PCR genotyping assay. Strains VWA012 and NCTC11144 could carry the IS1151-cpe gene on the plasmid. ***, a PET-RPLA kit was used to detect in vitro enterotoxin release in mDS sporulating cultures of cpe-positive strains. The results were interpreted following the instructions supplied by the manufacturer.

Fig 2

PCR results from the evaluation of the proximity of the cpe gene to the IS1470 elements for the studied enterotoxic strains, using one primer specific for the cpe gene (forward primer cpe4F) and the other specific for the IS1470 element (reverse primer IS1470R1.3). For all IS1470-cpe-type strains, a 1.3-kb product was found, and no product was amplified for two IS1151-type strains (VWA012 and NCTC11144), indicating that the fragment size between the cpe gene and the IS1470 element is 1.3 kb.

Fig 3

Minimal spanning tree from MLST analysis of 17 C. perfringens strains in this study, combined with results of a worldwide survey, revealing three clusters correlating to environmental niches. The seven chromosomal cpe food isolates are clustered with all recorded food poisoning strains (group I, light green); P-cpe, gas gangrene, and healthy human isolates are clustered in group II (red); group III (yellow) mainly contains non-cpe food isolates. The tree was constructed by using BioNumerics version 5.10 (Applied Maths, Sint-Martens-Latem, Belgium) with allowed hypothetical types and a categorical coefficient. The sizes of the nodes indicate the numbers of strains sharing the same MLST, and the node colors show C-cpe type A in red, P-cpe type A in dark green, non-cpe type A in blue, and other types in white. The clusters were created for at least three types within 5 changes of neighbor distances.