Clostridium perfringens Contamination in Retail Meat and Meat-Based Products in Bursa,Turkey

Abstract

This study examined the incidence of Clostridium perfringens in raw, ready-to-cook (RTC), and ready-to-eat (RTE) meat and meat-based products (N = 306) collected from restaurants, supermarkets, and butcher shops in Bursa, Turkey. In addition, we investigated the presence of the C. perfringens enterotoxin (CPE), as well as cpe genes and their source (chromosomal or plasmid borne). In this study, tryptose sulfite cycloserine (TSC) agar for classic culture isolation and API and real-time polymerase chain reaction (RT-PCR) techniques were used to identify C. perfringens and detect cpa and cpe genes from these products, respectively. Seventeen C. perfringens isolates (5.6%) were isolated and identified with API 20A. In addition, 42 of 81 suspicious isolates (51.9%) were identified as C. perfringens using RT-PCR. Of the 81 suspicious isolates tested by RT-PCR, 22 (27.2%) carried the cpe gene either on the plasmid or chromosome. Twenty-one isolates were positive for chromosomal cpe (C-cpe), and one was positive for plasmid-borne cpe (P-cpe). CPE was detected in 31.8% (7/22) of the cpe positive isolates by the PET-RPLA test. In conclusion, C. perfringens and their CPEs were present in raw, RTC, and RTE meat and meat-based foods in this study. It is emphasized that the presence of C. perfringens and the cpe gene in these foods may be a potential risk for human health.

Introduction

C lostridium perfringens is a spore-forming, rod-shaped, anaerobic, Gram-positive pathogenic bacterium in the normal intestinal flora of humans and animals that also causes food poisoning (Aras and Hadimli, 2015). Food poisoning, characterized by watery diarrhea and abdominal pain, occurs by consuming food (usually meat and poultry products) contaminated by enterotoxin–producing C. perfringens (CPE) vegetative cells (Hailegebreal, 2017).

C. perfringens is classified into 5 types and produces >20 known toxins, of which 4 are used for toxinotyping classification. Alpha-toxin (CPA) is one of the most common toxins and is related to gas gangrene production in humans and some animal species. CPA (encoded by the cpa gene) is produced by most C. perfringens type (A-E) isolates. In addition, a few C. perfringens strains also harbor the cpe gene, which encodes CPE, causing food poisoning and antibiotic-associated diarrhea (Xiao et al., 2012).

This pathogen sporulates in the intestine and produces CPE, which is the virulence factor related to food poisoning. C. perfringens strains can carry the CPE-encoding gene (cpe) either on the chromosome (C-cpe) or the plasmid (P-cpe) (Freedman et al., 2016), and recent studies have found the same for C. perfringens isolated from food and clinical samples (Sarker et al., 2000; Sparks et al., 2001; Nakamura et al., 2004; Wen and Mcclane, 2004).

The first objective of this study was to determine the incidence of C. perfringens by classic culturing techniques in various retail food samples, including raw foods such as beef, minced meat, raw kokorech (a Turkish dish made of seasoned, skewered lamb intestines), beef offal, chicken meatballs, and chicken offal; ready-to-cook (RTC) foods (beef doner, meatballs, and chicken doner); and ready-to-eat (RTE) foods (beef, minced meat, meatballs, beef doner, grilled kokorech, sucuk, beef offal, chicken meat, and chicken doner) (N = 306) from restaurants, markets, and butcher shops in Bursa, Turkey, from June 2014 to October 2015. The second objective was to characterize the C. perfringens enterotoxigenic strains by the PET-RPLA test, and the third objective was to verify the isolates by API 20A and detect the cpe gene, which is responsible for producing the enterotoxin from C. perfringens isolates by real-time polymerase chain reaction (RT-PCR). The final objective was to detect the source of these genes (P-cpe or C-cpe).

Materials and Methods

In this study, raw (n = 109), RTC (n = 62), and RTE (n = 135) meat and meat-based product samples were purchased from markets, butchers, and restaurants in Bursa, Turkey, between June 2014 and October 2015. Samples (N = 306) were stored at 4°C for 1 day before processing.

Isolation and identification of C. perfringens

C. perfringens was isolated as per a previously described method. Briefly, a 25 g portion of each sample was aseptically placed in a sterile plastic bag containing 225 mL of Perfringens Enrichment Medium (PEM; Fluid Thioglycollate Medium, supplemented with Perfringens [TSC] supplement, Oxoid^®), homogenized by a Stomacher for ∼2 min, and incubated anaerobically at 45°C for 24 h. After the samples were enriched in PEM, one loopful was streaked onto tryptose sulfite cycloserine (TSC) agar (Oxoid), and the plates were further incubated anaerobically at 45°C for 48 h in a gas pack system (Oxoid).

Black colonies on the TSC agar were selected for morphological and biochemical identification (Erol et al., 2008). These colonies were identified as C. perfringens using the commercially available identification system, API 20A (BioMerieux, Paris, France). API 20 A test system was performed as per the manufacturer's instructions. Both API-positive and negative isolates were suspended in microbanks and stored at −80°C in Cooked Meat Medium (Oxoid) containing 40% glycerol (Sigma, St. Louis, MO) until RT-PCR and toxin detection analysis.

RT-PCR detection

All suspicious isolates (well-isolated presumptive colonies from the TSC agar plates) were examined for C. perfringens toxin genes (cpa, cpe, and dcm genes) using real-time PCR assay. Three reference strains of C. perfringens, type A strain ATCC 13124 cpa ⁺, NCTC 8237 cpa ⁺, and NCTC 8239 cpe ⁺, were used as controls.

Determination of cpa, chromosomal and plasmid-borne cpe genes of C. perfringens by RT-PCR analysis

CPA protein contains 370 amino acids, weighs 42.528 kDa, is thermostable, and has an LD₅₀ (mice) of 3 μg. The biological activity can be summarized as a lethal, necrotizing hemolytic smooth muscle contraction (Ferreira et al., 2016). cpa is a chromosomal gene located near the origin of replication, which is one of the most stable locations in the bacterial chromosome. Cultured isolates were confirmed as C. perfringens by amplifying the cpa gene.

The cpe gene is located between the purine permease (uapC) and quinolinate phosphoribosyltransferase (nadC) genes of the C. perfringens chromosome, and the cpe gene is adjacent to the dcm gene of the C. perfringens plasmid (Miyamoto et al., 2002). To determine whether the cpe gene was on the chromosome or plasmid, three different primers were designed. The first primer set targeted the cpe-nadC gene intersection, the second primer set targeted the uapC-cpe gene intersection, and the third primer set targeted the dcm-cpe gene intersection. To design these primers, their related gene sequences (cpa, uapC, nadC, and dcm) were retrieved from the DNA databank (National Center for Biotechnology Information, 2016).

After the related gene sequences were retrieved from the databank, primers were designed using Primer3 software (bioinfo.ut.ee/primer3-0.4.0/primer3). The specificity of the designed primers was confirmed by virtual PCR using the Primer-BLAST algorithm (NCBI/Primer-BLAST, 2016). The 155-, 204-, and 3679-bp fragments were amplified with the primers, cpa155F: 5′-TGACACAGGGGAATCACAAA-3′ and cpa155R: 5′-CGGCAGTAACATTAGCAGGA-3′; cpeF: 5′-ACAACTGCTGGTCCAAATGA-3′ and cpeR: 5′-AACACATCTTTCACCAGTTTCAA-3′; and dcmF: 5′-TAGGAGCTAGCCCAACCCTT-3′ and dcmR: 5′-AACACATCTTTCACCAGTTTCAA-3′, respectively.

These three primer sets determined whether C. perfringens carried the cpe gene on the plasmid, chromosome, or both. The cpa primer pair determined whether C. perfringens carried the toxicity gene. The cpe primer pair determined whether the cpe gene was on the chromosome, and the dcm primer pairs determined whether the cpe gene was on the plasmid.

DNA preparation

For the DNA extraction several optimization steps, including physical (bead homogenization and sonication), chemical (SDS, sodium dodecyl sulfate, Sigma-Aldrich^®; CTAB, hexadecyltrimethylammonium bromide, Sigma-Aldrich; GITC, Guanidine thiocyanate, Sigma-Aldrich), and biochemical (proteinase K), were applied as per previous studies (Sambrook and Russell, 2001). The most effective method was chosen by spectrophotometric parameters.

The DNA extraction protocol for C. perfringens was as follows. The culture was removed from the plate and placed at the bottom of the microcentrifuge tube. Two hundred fifty microliters of guanidine isothiocyanate and 250 μL of molecular grade water were added. The tube was vortexed for 1 min and then incubated for 10 min at 95°C. Next, the tube was centrifuged at 14,000 × g for 1 min, 250 μL isopropanol was added to the tube, and the sample was mixed. Next, 700 μL of the sample was transferred into the DNA column and centrifuged for 1 min at 14,000 × g. After the liquid portion was discarded, 400 μL of wash solution was added to the column and centrifuged at 14,000 × g for 1 min. The wash step was repeated once, and a new collection tube was placed under the DNA column for elution. Using 100 μL of molecular grade water, the DNA was extracted from the sample and stored at −20°C.

RT-PCR analysis

Total reaction volumes were 10 μL. The reaction mixture (5 μL) contained 6 mg/mL BSA, 11 mg/mL betaine, 20 mg/mL PEG 400, 0.2% Tween 20, 20 mM Tris-HCl at pH 8.0, 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM dNTP mix, 1U Hot Start Taq DNA Polymerase, 0.5 μL of forward primer, 0.5 μL of reverse primer, 3 μL MG water, and 1 μL of the template. The reaction solution was placed into a 96-well plate and inserted into a CFX connect device (185-5200; Bio-Rad).

To amplify the target region of the genes with the cpa, cpe, and dcm primers, suitable programs were written based on oligo properties and product length. These programs were optimized for the primer pairs and DNA templates. Quantitative PCR was performed with an initial denaturation step at 98°C for 60 s, followed by 40 denaturation cycles at 95°C for 20 s, annealing at 55°C for 60 s, a melt curve step at 65°C to 95°C (at 0.5°C increments) for 5 s/step, and a final cooling step at 37°C.

Toxin detection

The isolates' enterotoxigenicity was examined by the Reversed Passive Latex Agglutination (PET-RPLA) C. perfringens Enterotoxin Test Kit (Oxoid). RPLA is reported to be a reliable method for detecting CPE (Harmon and Kautter, 1986). The assay was performed as per the manufacturer's instructions. Briefly, the isolates were cultured in Cooked Meat Medium (Oxoid) at 37°C for 20 h and then heated at 75°C for 20 min in a water bath. A 0.8 mL aliquot (taken from the tube base) was subcultured into 18 mL of Modified Duncan Strong (DS) Medium (HiMedia) to promote enterotoxin production (Duncan and Strong, 1968). The tubes were incubated at 37°C for 24 h and then centrifuged at 900 × g for 20 min at 4°C, and the supernatant was used for the assay. If a lattice structure formed on the base of the microtiter plate V-well due to agglutination, the sample was evaluated as positive for CPE.

Results

The prevalence data with respect to the isolation and identification methods are shown in Table 1.

Table 1.

Distribution of Food Samples Clostridium perfringens Isolates and Presence of cpa Genes

Samples	Number of samples (%) n = 306	Number of TSC positive isolates (%)	Number of API positive isolates (%)	Number of positive for cpa gene (%); C. perfringens
Raw
Beef meat	32	5	1 (3.1)	0 (0)
Beef minced meat	32	14	1 (3.1)	4 (12.5)
Beef meatball	20	6	3 (15)	5 (25)
Raw kokorech	4	3	2 (50)	3 (75)
Beef offal	2	1	0 (0)	1 (50)
Chicken meat	16	2	0 (0)	0 (0)
Chicken meatball	1	1	1 (100)	1 (100)
Chicken offal	2	2	0 (0)	1 (50)
Total	109 (35.6)	34 (31.2)	8 (7.3)	15 (13.8)
Ready to cook
Beef doner	22	9	2 (9.1)	3 (13.6)
Beef meatball	29	5	0 (0)	1 (3.4)
Chicken doner	11	1	0 (0)	1 (9.1)
Total	62 (20.3)	15 (24.2)	2 (3.2)	5 (8.1)
Ready to eat
Beef meat	17	3	0 (0)	1 (5.9)
Beef minced meat	11	2	0 (0)	0 (0)
Beef doner	12	2	0 (0)	2 (8.3)
Meatball	23	5	1 (4.3)	5 (21.7)
Sucuk	16	3	0 (0)	3 (18.8)
Grilled kokorech	25	11	6 (24)	8 (28)
Beef offal	2	1	0 (0)	1 (50)
Chicken meat	22	4	0 (0)	1 (4.5)
Chicken doner	7	1	0 (0)	1 (14.3)
Total	135 (44.1)	32 (23.7)	7 (5.2)	22 (15.6)
Total	306 (100)	81 (26.5)	17 (5.6)	42 (13.7)

TSC, tryptose sulfite cycloserine.

Black colonies on the TSC agar were observed from 81 of the 306 (26.5%) food samples (Table 1). Thirty-four suspicious C. perfringens isolates were obtained from 109 raw food samples, 15 were isolated from 62 RTC food samples, and 32 were isolated from RTE food samples. Seventeen samples (5.6%) tested positive for C. perfringens, confirmed by both bacteriological and API identification methods. A total of 17 (5.6%) of the 306 food samples, including 8 raw foods (meat, minced meat, meatballs, raw kokorech, and chicken meatballs), 2 RTC foods (beef doner), and 7 RTE foods (grilled kokorech and meatballs), were contaminated with C. perfringens.

A comparison of the C. perfringens prevalence in the raw, RTC, and RTE foods showed that the contamination rates of the eight raw foods were higher (7.3%) than those of the seven RTE foods (5.2%) and the two RTC foods (3.2%).

Among the raw food samples, chicken meatballs had the highest C. perfringens detection rate (100%). Raw kokorech was contaminated with C. perfringens at the second-highest level (50%). C. perfringens was identified in 1 of 32 (3.1%) raw beef samples. In the raw food samples, beef offal, chicken meat, and chicken offal were free of C. perfringens.

Among the RTC food samples, beef doner (3.2%; 2/62) was the only food that was contaminated by C. perfringens. While no contamination was detected in the RTE food samples, including meat, minced meat, beef doner, beef offal, chicken meat, and chicken doner samples, C. perfringens was dominant in the grilled kokorech (24%) and meatball (4.3%) samples.

In the present investigation, the results obtained by the standard conventional bacteriological method and API identification conflicted with those obtained by the RT-PCR technique. All suspected isolates (n = 81) were examined for the cpa, UapC-cpe (C-cpe), and dcm-cpe (P-cpe) genes by RT-PCR.

From the RT-PCR identification results, overall positivity increased to 13.7% (42/306). Forty-two of the 81 suspected isolates (black colonies on TSC) (51.9%) harbored the alpha toxin gene (cpa), confirming them as C. perfringens. In short, although 81 (26.5%) of 306 diverse food samples investigated were culture positive, only 17 (21%) of these were API positive, of which 42 (51.9%) were PCR positive.

PCR assay revealed that cpa, UapC-cpe, and dcm-cpe genes were detected in 42 (51.9%), 21 (25.9%), and 1 (1.2%) isolates, respectively (Table 2). The dcm-cpe gene was only identified in one isolate (raw chicken meatball), for a total prevalence of 1.2% (1/81). This P-cpe isolate was positive by API identification and possessed the cpa gene, whereas CPE was negative. Among the raw food sample isolates, 15 were cpa positive (44.1%) and 11 were cpe positive (32.4%). Of the 15 C. perfringens isolates from the RTC food samples, 5 were found to carry cpa (33.3%), 1 carried C-cpe, and none carried dcm-cpe. Among the RTE food sample isolates, 22 were cpa positive (68.8%), 10 were C-cpe (31.3%), and none carried dcm-cpe.

Table 2.

Prevalence of Enterotoxin Gene–Carrying Clostridium perfringens and A Comparison of Identification by API, cpa, UapC-cpe, and dcm-cpe Genes by PCR and Detection of Enterotoxin by PET-RPLA in Isolates

Food product	Number (%) of isolates^a tested/food product	Number (%) of isolates
		cpa+	cpe+		API+ cpa+ UapC+ dcm-CPE+	API+ cpa+ UapC+ dcm-CPE−	API+ cpa+ UapC− dcm+ CPE−	API+ cpa+ UapC− dcm-CPE−	API+ cpa− UapC− dcm-CPE−	API− cpa+ UapC+ dcm-CPE−	API− cpa+ UapC+ dcm-CPE−	API− cpa+ UapC− dcm-CPE−
		cpa+	C-cpe	P-cpe	API+ cpa+ UapC+ dcm-CPE+	API+ cpa+ UapC+ dcm-CPE−	API+ cpa+ UapC− dcm+ CPE−	API+ cpa+ UapC− dcm-CPE−	API+ cpa− UapC− dcm-CPE−	API− cpa+ UapC+ dcm-CPE−	API− cpa+ UapC+ dcm-CPE−	API− cpa+ UapC− dcm-CPE−
Raw	34/109	15	10	1	1	2	1	2	2	5	2	2
Ready to cook	15/62	5	1	0	0	1	0	0	1	0	0	4
Ready to eat	32/135	22	10	0	1	3	0	3	0	3	3	9
Total	81/306 (26.5)	42 (51.9)	21 (25.9)	1 (1.2)	2 (2.5)	6 (7.4)	1 (1.2)	5 (6.2)	3 (3.7)	8 (9.9)	5 (6.2)	15 (18.5)

These isolates isolated by classical cultural method and evaluated as C. perfringens suspicious colonies.

CPE, C. perfringens enterotoxin.

Forty-two of 81 suspicious C. perfringens isolates carried the cpa gene, while 22 carried both the cpa and cpe genes. Of the 45 C. perfringens isolates identified by API and RT-PCR (42 carried the cpa gene and 3 were API positive), 7 (15.6%) produced C. perfringens enterotoxin (CPE) (Table 3). Although all 22 isolates were shown to express the cpe gene as determined by RT-PCR, only 7 of them (31.8%) were found to be positive for CPE. In detail, this toxin was found in two raw beef and minced meat samples and in one each of the raw beef meatballs, RTE chicken meat, RTE beef doner, RTE beef meatball, and RTE grilled kokorech. No C. perfringens isolates cultured from the RTC food samples were positive for this toxin. The highest CPE toxin level (+++) was detected in the RTE grilled kokorech isolate. This isolate was positive for API detection and cpa and cpe genes.

Table 3.

Toxin Producing Isolates

Sample No.	Sample name	API+	cpa ⁺	cpe ⁺	CPE
Samples
Raw
63	Meatball	+	−	−	−
68	Minced meat	+	+	+	++
70	Meatball	−	+	+	−
75	Meat	+	−	−	−
79	Minced meat	−	+	+	−
84	Minced meat	−	+	+	−
90	Meatball	−	+	+	+
91	Meatball	−	+	+	−
200	Grilled kokorech	−	+	−	−
205	Minced meat	−	+	+	++
206	Chicken offal	−	+	+	−
214	Beef offal	−	+	−	−
291	Meatball	+	+	+	−
296	Chicken meatball	+	+	+^a	−
302	Grilled kokorech	+	+	+	−
303	Grilled kokorech	+	+	−	−
304	Meatball	+	+	−	−
Total		8	15	11	3
Ready to cook
16	Meatball	−	+	−	−
17	Chicken doner	−	+	−	−
61	Beef doner	−	+	−	−
93	Beef doner	−	+	−	−
294	Beef doner	+	+	+	−
306	Beef doner	+	−	−	−
Total		2	5	1	0
Ready to eat
14	Sucuk	−	+	−	−
21	Meat	−	+	−	−
56	Sucuk	−	+	−	−
78	Chicken meat	−	+	+	+−
85	Meatball	−	+	+	−
87	Beef doner	−	+	+	+−
88	Beef doner	−	+	+	−
92	Meatball	−	+	+	+
97	Grilled kokorech	+	+	+	−
98	Meatball	+	+	−	−
102	Meatball	−	+	+	−
152	Beef offal	−	+	−	−
198	Grilled kokorech	−	+	−	−
209	Meatball	−	+	−	−
222	Sucuk	−	+	−	−
246	Chicken doner	−	+	−	−
277	Grilled kokorech	+	+	+	−
278	Grilled kokorech	+	+	+	+++
281	Grilled kokorech	−	+	−	−
282	Grilled kokorech	+	+	−	−
284	Grilled kokorech	+	+	+	−
287	Grilled kokorech	+	+	−	−
Total		7	22	10	4

P-cpe (dcm sequences).

CPE, Clostridium perfringens enterotoxin.

Discussion

Interestingly, the C. perfringens incidence obtained in the study (5.6%; 17/306) appeared to be lower than several reports: Lin and Labbe (2003) reported that 31% of retail food samples were positive for C. perfringens. In another study, Miwa et al. (1998) reported that 37% of meat and poultry samples were positive for this pathogen. In our study, this pathogen was detected in 42 of the 81 suspicious isolates from 306 samples (13.7%) using RT-PCR. However, with API identification, C. perfringens was undetected in 25 of these RT-PCR positive isolates, suggesting that RT-PCR test is a more sensitive method in detecting C. perfringens contamination than API test.

The C. perfringens prevalence in raw beef and raw beef minced meat was 3.1% in the present study. These findings are lower than those of Gökmen and Alişarlı (2003), who reported an overall incidence of 15% for C. perfringens in raw beef minced meat samples. In another study, Stagnitta et al. (2002) reported that 38.1% of the raw meat and meat product samples were positive for C. perfringens. In contrast to our results, Aras and Hadimli (2015) and Wen and Mcclane (2004) isolated C. perfringens from 40% and 21% of raw beef samples, respectively.

In this study, no C. perfringens was isolated from raw chicken meat samples. These findings contradict the results of Nowell et al. (2010), Doosti et al. (2017), and Aras and Hadimli (2015) who reported overall incidences of 66%, 42%, and 31% for C. perfringens in raw chicken meat samples, respectively.

Kayisoglu et al. (2003) reported that 40% of cooked beef and 60% of cooked chicken doners contained C. perfringens. In addition, Jöckel and Stengel (1984) and Stolle et al. (1993) reported that 10% and 18% of RTE doner samples contained C. perfringens, respectively. In this study, none of the RTE beef, chicken meat, and doner meat samples was positive for this pathogen. The percentage of C. perfringens positive foods is much lower than in other studies. These differences may be due to differences in the hygienic conditions of the process and products (Miwa et al., 1998). In addition, the use of only black colonies, as opposed to white colonies from TSC agar plates, may have contributed to this outcome.

In this study, 13.8% of the 109 raw sample isolates were positive for the cpa gene. Aras and Hadimli (2015) reported that 95 strains, isolated from 305 raw meat samples (31.1%), were positive for the cpa gene. Hanifehnezhad et al. (2015) found that 46.7% of broiler raw heart-liver was positive for C. perfringens. All isolates carried the cpa gene, but none were positive for the cpe gene. The prevalence of cpe-positive isolates was 27.2% (22/81) in the present study. Our results indicated that 7.2% (22/306) of the animal-derived foods sampled in this survey were contaminated with cpe-positive C. perfringens isolates. This is surprisingly high. These findings are higher than those of Lin and Labbe (2003), who reported that no isolates carried the cpe gene in retail foods.

Thirty-nine of the 132 (30%) retail food samples, including various meat products, were positive for the cpa gene of C. perfringens, but none possessed the cpe gene. In the present study, cpe-positive C. perfringens was isolated from 11 (13.6%) raw food samples. This rate contrasts with the prevalence rates from previous studies in Turkey (Guran and Oksuztepe, 2013; Aras and Hadimli, 2015). Aras and Hadimli (2015) reported that cpe-positive C. perfringens type A was isolated from 1 (1%) beef and 1 (1%) chicken meat sample. No cpe-positive isolates were found in raw chicken samples. Similar results were obtained in 2003 by Lin and Labbe and were also consistent with those of Nowell et al. (2010), Erol et al. (2008), and Sariguzel (2005). Sariguzel (2005) reported that 58% of minced turkey meat was positive for C. perfringens, whereas none of the isolates had the cpe gene.

Wen and Mcclane (2004) detected C. perfringens in a high percentage of chicken meat samples, and one of these was cpe positive. In addition, these authors detected a high percentage of C. perfringens in other foods, as well as chicken. Cooper et al. (2013) reported that 69.6% of raw chicken livers were positive for C. perfringens, while none of the isolates had the cpe gene. Johansson et al. (2006) reported that the cpe gene was found in all isolates originating from food poisoning outbreaks. In the present investigation, it is interesting to note that only a small number (1/15; 6.6%) of the C. perfringens isolates from RTC food samples were positive for the cpe gene.

The prevalence was 0.3% (1/306; in the raw chicken meatball) for plasmid-borne cpe (P-cpe), which causes antimicrobial drug-associated diarrhea and sporadic diarrhea, and 7.2% (21/306) for chromosomal cpe (C-cpe). Overwhelming presence of chromosomal cpe isolates (vs. plasmid-borne cpe isolates) agrees with Wen and Mcclane (2004)'s results and helps to explain why the chromosomal cpe strains are so strongly associated with food poisoning. As previously reported in the literature on C-cpe, the C. perfringens that causes food poisoning was present in food samples (Wen and Mcclane, 2004), in the feces of healthy food handlers (Heikinheimo et al., 2006) and in food poisoning outbreak isolates (Nakamura et al., 2004).

In this study, 15.6% (7/45) of the isolates were positive for CPE. The rates of CPE in food samples detected in our study were close or below those reported by other researchers (Saito, 1990; Stagnitta et al., 2002; Lin and Labbe, 2003). In contrast to our results, Augustynowicz et al. (2002) reported that 30% of food sample isolates were positive for CPE. In addition, 31.8% (7/22) of the isolates containing the cpe gene produced CPE detectable by RPLA. Likewise, Augustynowicz et al. (2002) showed that 30% (6/20) of C. perfringens isolates from food samples with the cpe gene produced CPE. The absence of CPE in the remaining 15 isolates may have been caused by a variety of factors, including inadequate growth and poor spore formation. In addition, the amount of CPE produced in these isolates could be lower than the detection limit (<2 ng/mL) of the PET-RPLA test.

Conclusion

Our findings suggest that retail meat and meat-based food samples serve as a rich reservoir for cpe ⁺ and CPE⁺ C. perfringens and may play a role in the etiology of gastrointestinal diseases caused by this pathogen. To our knowledge, this is the first study to determine both CPE and the source of the C. perfringens cpe gene in retail meat and meat-based food samples in Turkey.

Footnotes

Acknowledgments

This work was supported by the Project Unit of Scientific Research Projects, Uludag University (Project No: KUAP(V)-2014/06). This article was edited before submission by American Journal Experts (AJE).The authors thank Assoc. Prof. Dr. Sahan Guran (Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Dicle University) for kindly providing the positive control NCTC 8239.

Disclosure Statement

No competing financial interests exist.

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