Detection and Toxin Typing ofClostridium perfringensin Formalin-Fixed, Paraffin-Embedded Tissue Samples by PCR

Abstract

Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases.Clostridium perfringens causes several forms of enteric diseases, including food poisoning and fatal enterotoxemia (15,17). Based on the presence of four major lethal toxins (alpha-, beta-, epsilon-, and iota-toxins), C. perfringens is classified into five toxigenic types (A through E), and each type can cause different diseases. The most commonly encountered type A (alpha-toxin) strain causes gas gangrene (myonecrosis), diarrhea, and food-borne illness in humans (4). Type B (alpha-, beta-, and epsilon-toxins) and type D (alpha-and epsilontoxins) strains are the causative agents of fatal enterotoxemia in domestic animals and occasionally humans (16).

Degradation of the extracellular matrix components by bacterial-derived metalloproteases

“…Five lL of the overnight cultures (grown in TSB with cysteine) were spotted onto TSA plates containing elastin-Congo red and L-cysteine. The plates were incubated for 16 hours at 37 C under anaerobic conditions and subsequently stored at 4 C for 3 days. Elastinolytic activity was detected by the formation of a clear halo were elastin was degraded.…”

“…Gelatin strips (Key Scientific Products, Stamford, TX) were immersed in 100 lL culture supernatant of C. perfringens and 100 lL TSB supplemented with 0.5 g/L Lcysteine and incubated for 72 hours at 37 C under anaerobic conditions. Gelatinase activity was evidenced by the visibility of the blue color of the base material when the gelatin was degraded.…”

“…Individual colonies displaying zonal changes (opaque zones) around them were considered to be gelatinase-positive and were purified by restreaking onto TSA (0.5 g/L L-cysteine) plates. A single, well-isolated colony was then inoculated into TSB containing 0.5 g/L L-cysteine, grown overnight at 37 C under anaerobic conditions, and served as a stock culture. The purity of these cultures was confirmed by Gram staining.…”

“…C. perfringens total bacterial DNA, extracted as described above, was used as template for PCR amplification with the following primers: colA-F 5 0 -TGTAGTTCCTGGAATGTG GG-3 0 and colA-R 5 0 -CCATCAGTTCTAGTTGAACCTG-3 0 for colA gene fragment (108 bp); cpa-F 5 0 -GCTAATGT TACTGCCGTTG-3 0 and cpa-R 5 0 -TCTGATACATCGTG TAAG-3 0 for cpa gene fragment (323 bp). The primers used for the detection of cpb and etx genes encoding betaand epsilon-toxin, respectively, were previously described by Wu et al 37 The sepA gene encoding an extracellular metalloprotease in Staphylococcus epidermidis was confirmed by PCR using total bacterial DNA and the primers: sepA-F 5 0 -GAA CAACGTGCAGGTGTAGATG-3 0 and sepA-R 5 0 -CTTAA GGCATCTCCGCCTACAC-3 0 .…”

Prevalence and characterization of Clostridium perfringens from the faecal microbiota of elderly Irish subjects

“…Primers corresponding to the a toxin (cpa), b toxin (cpb) or e toxin (etx) gene were used as described by Wu et al (2009) to assess the C. perfringens isolates for toxin production capability. In addition, production of the b2 toxin, encoded by cpb2 gene, was assessed as outlined by Greco et al (2005).…”