Stool samples were collected from 83 dogs, 49 DD and 34 ND, between 2011 and 2013. All samples were obtained from hospitalized dogs at two animal hospitals: A (university animal hospital) and B (local animal hospital). Fecal samples were collected directly from the rectum by using a swab method. A sterilized cotton swab was inserted into the rectum and rotated gently to collect a sample of fecal matter. All feces were stored at 4℃ and were used for the isolation of C. perfringens within 24 h after collection.

A small portion of feces on a cotton swab was directly inoculated onto tryptose sulfite cycloserine (TSC; Oxoid, UK) agar with 5% egg yolk (Oxoid). The remaining sample was enriched in 10 mL of cooked meat media (Oxoid) followed by incubation at 37℃ for 24 h. After enrichment, a loopful of culture was inoculated onto TSC agar. All plates were incubated under anaerobic conditions at 37℃ for 24 to 48 h. Suspicious black colonies with lecithinase production from each plate were subcultured under aerobic and anaerobic conditions on blood agar. Colonies showing double hemolysis and growing only under anaerobic conditions were regarded as presumptive C. perfringens strains, and were selected and stored at −70℃.

A colony subcultured on blood agar was removed and genomic DNA was extracted by applying the boiling method [4]. Table 1 shows primer sequences and cycling conditions for PCR amplification of toxin genes cpa, cpb, cpe, cpb2, etx, and iap, encoding α, β, entero, β2, ε, and ι toxin, respectively [1321]. The amplification was conducted by using a PCR reaction mixture (20 µL) consisting of Frenche PCR PreMix (iNtRON Biotechnology, Korea), 50 ng of template DNA, and 500 nM of each primer. Each PCR product was analyzed on 1.5% agarose gel containing ethidium bromide (EtBr; Promega, USA). Gel images were visualized by using a GelDoc XR gel analyzer (Bio-Rad, USA) under ultraviolet light. Strains ATCC 3624, NCTR 3626, KNIH_CL_D, KNIH_CL_E, KNIH_Kim_1, and PM_S44 were used as the control strains for cpa, cpb, ext, iap, cpe, and cpb2 genes, respectively.

MIC values of antibiotics against C. perfringens were determined by using E-test kits (bioMérieux, France). Each isolate was suspended in Mueller-Hinton broth (Sigma-Aldrich, USA) to achieve a McFarland turbidity of 0.5 and then inoculated onto Brucella agar with 5% sheep blood, hemin, and vitamin K1 (BD Difco, USA). An E-test strip of each antibiotic (ampicillin, chloramphenicol, metronidazole, moxifloxacin, tetracycline, clindamycin, and imipenem) was placed on the plate and followed by incubation at 37℃ for 48 h under anaerobic conditions. The MIC value was noted according to the manufacturer's instructions, and susceptibility and resistance were determined according to the Interpretive Standards for Anaerobes in Clinical and Laboratory Standards Institute [6]. C. perfringens ATCC 13124 was used as the quality control strain.

PFGE was performed for subtyping of the 52 C. perfringens isolates from 49 DD and 34 ND according to the procedure described by Matushek et al. [18] with a few modifications. A single colony was grown in 10 mL of brain heart infusion broth (Sigma-Aldrich) at 37℃ for 48 h under anaerobic conditions and centrifuged. Cultures were suspended in 1 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to achieve an absorbance level of 1.5 to 2.0 at 600 nm. The cells (100 µL) were mixed with 80 µL of 2× lysis buffer (10 mM Tris [pH 8.0], 0.2 M EDTA [pH 8.0], 1 M NaCl, 0.5% sodium lauryl sarcosine, 0.2% sodium deoxycholate, and 0.5% polyethylene glycol hexadecyl ether) and 200 µL of 1.5% low melting point agarose (Life Technologies, USA), and the mixture was then placed in a rectangular mold and solidified. The solid plugs were incubated overnight in 5 mL of lysis buffer with lysozyme (1 mg/mL; Sigma-Aldrich) and mutanolysin (20 U/mL; Sigma-Aldrich) at 50℃ with agitation at 95 rpm. The lysis buffer was removed and each plug was incubated at 37℃ for 4 h in 5 mL of buffer (0.5 M EDTA, pH 8.0, 1% sodium lauryl sarcosine) with proteinase K (1 mg/mL; Research Products International, USA). Plugs were rinsed twice with TE buffer and then incubated at 50℃ for 30 min in TE buffer containing 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Each plug was rinsed twice in TE buffer and sliced into small pieces. The plugs were digested overnight in restriction buffer with 40 units of SmaI (Fermentas, USA). Electrophoresis was carried out in 1% of SeaKem Gold agarose gels (Lonza, Switzerland) prepared in electrophoresis buffer (0.5% Tris-borate-EDTA buffer; Sigma-Aldrich). The digested DNA was separated by using a CHEF Mapper (Bio-Rad) with the following conditions: low molecular weight (MW) = 30 kb, high MW = 600 kb, initial switch time = 0.5 sec, final switch time = 40 sec, included angle = 120°, and running time = 21 h. The gels were stained with EtBr for 30 min and then washed in distilled water for 20 min. The gels were visualized on the Gel Doc (Bio-Rad) and the images were saved as TIFF files. Banding patterns and dendrograms were created by determining the distance matrices and using an unweighted pair group method with arithmetic mean clustering method. Subtyping patterns were analyzed by using BioNumerics software (ver. 7; Applied Maths, USA). For the construction of dendrograms, a 1% position tolerance shift between similar bands was used.

The prevalences of C. perfringens in samples from 49 DD and 34 ND and their toxin gene profiles are presented in Table 2. The prevalence of C. perfringens was not statistically different (p > 0.05) between the DD (31 of 49, 63.3%) and ND (21 of 34, 61.8%) groups. All presumptive C. perfringens strains were positive for cpa, and none was positive for cpb, iap, or etx. Thus, all isolated strains were identified as type A C. perfringens. All isolates screened by PCR were cpe-negative. However, cpb2 was identified in 83.9% (26 of 31) and 61.9% (13 of 21) of isolates from DD and ND, respectively, and the difference between the groups was statistically significant (p < 0.05).

The MIC values of antibiotics against C. perfringens are summarized in Table 3. The antibiotic susceptibility profile of each strain is presented in Fig. 1. Most isolates were susceptible to the tested antibiotics, but 25.0% and 21.2% of isolates were resistant to tetracycline and clindamycin, respectively (Table 3). Among the 52 isolates, four strains from hospital A (KDD_3, 12, 19, and KND_19; Fig. 1) were resistant to three different classes of antibiotics, indicating multidrug resistance. No notable differences were observed in terms of antibiotic resistance between strains from DD and ND.

The dendrogram generated from the obtained PFGE patterns, virtual gel images, and profiles of each strain is summarized in Fig. 1. It has been reported that PFGE analysis can strongly discriminate phenotypically similar C. perfringens isolated from human clinical samples, food products, and veterinary samples [2324]. Based on the PFGE banding patterns with ≥ 90% similarity, the strains were classified into 48 pulsotypes (Fig. 1). The PFGE banding patterns of the isolates were not associated with the source hospital, distribution of toxin genes, or antibiotic resistance patterns (Fig. 1). Moreover, there was no remarkable difference in PFGE banding patterns between the isolates from DD and ND. Although some strain pairs, such as KND_5 and 6 and KDD_12 and 13, showed high similarity (> 95%) in banding patterns, most strains were genetically unrelated, indicating genetic diversity among the C. perfringens isolates from fecal samples of the DD and ND groups (Fig. 1). Each strain pair showing high similarity, KND_5 and 6 (96.3%) and KDD_12 and 13 (96.5%), could be distinguished because they had different antibiotic resistance and toxin profiles.