Genotyping and Antimicrobial Susceptibility of Clostridium perfringens and Clostridioides difficile in Camel Minced Meat

The present study aimed to determine the occurrence, genotypes, and antimicrobial resistance of Clostridium perfringens (C. perfringens) and Clostridioides difficile (C. difficile) in camel minced meat samples collected from small butcher shops and supermarkets in Al-Ahsa Governorate, Saudi Arabia. A total of 100 camel minced meat samples were randomly collected from small butcher’s shops (n = 50) and supermarkets (n = 50) in Al-Ahsa Governorate, Saudi Arabia. C. perfringens and C. difficile were isolated and identified using the VITEK-2 compact system and 16S rRNA gene amplification. Genotypes, toxin genes, and antimicrobial susceptibility of the isolates were determined. Moreover, ELISA was used to detect C. perfringens and C. difficile toxins. C. perfringens and C. difficile were isolated from 14% and 4% of the tested minced meat samples, respectively. Out of the 14 C. perfringens isolates, type A (64.3%), type B (7.1%), type C (21.5%), and type D (7.1%) were detected. However, out of the four C. difficile isolates, three (75%) were type A+B+ and one (25%) was type A−B+. None of the C. perfringens or C. difficile toxins were identified using ELISA. C. perfringens and C. difficile isolates exhibited a high rate of resistance to tetracycline (56% and 75%, respectively). However, all isolates were susceptible to amoxicillin-clavulanate. Multidrug resistance was observed in three (21.4%) C. perfringens and one (25%) C. difficile isolates. In conclusion, camel minced meat was contaminated with C. perfringens and C. difficile, which present a potential risk of food poisoning. The majority of the isolates were resistant to at least one antimicrobial, and some isolates were multidrug-resistant. Therefore, food safety standards and frequent inspections of abattoirs, small butcher shops, and supermarkets should be enforced.


Introduction
Food hygiene is described as all the conditions and measures necessary to ensure that food is safe and fit for human consumption during production, processing, storage, distri-Pathogens 2021, 10, 1640 2 of 14 bution, and preparation [1]. Foodborne diseases are caused by food contamination and can occur at any stage of the food production, delivery, and consumption chain. There are over 200 foodborne hazards, including microbiological hazards such as bacteria, viruses, parasites, and chemical contaminants, that arise naturally or due to pollution, food processing, packaging, transportation, or storage [2]. Foodborne illnesses and outbreaks are among the leading causes of death globally [3,4]. According to studies, the magnitude of foodborne illnesses in 2010 included 600 million illnesses and 420,000 deaths worldwide [5,6]. Nausea, vomiting, retching, diarrhea, abdominal discomfort, prostration, abdominal cramps, fever, chills, headache, and arthralgia are just a few of the symptoms [7].
Clostridia spp. are anaerobic Gram-positive, spore-forming and non-motile bacteria commonly found in the intestinal tract of humans and animals and the soil [8]. Clostridium perfringens (C. perfringens) is the most widespread species and among the most common foodborne pathogens in industrial countries. There are several toxigenic types of C. perfringens, including A, B, C, D, and E. However, type A is primarily associated with foodborne illness [9]. C. perfringens food poisoning can occur when cooked meat is not adequately heated or refrigerated before serving. Illness often arises 8-15 h after consuming contaminated food. The symptoms include strong abdominal cramps, gas, and diarrhea (nausea and infrequent vomiting) [10]. Clostridioides difficile (C. difficile) is also associated with food poisoning, with symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis [11,12].
The recent rise and re-emergence of antimicrobial-resistant foodborne bacteria necessitate coordinated efforts, particularly in developing countries [13]. Antibacterial resistance can spread through the food chain, either directly or indirectly. Direct exposure happens when a human comes into contact with an animal or its blood, saliva, milk, sperm, feces, or urine. Indirect contact occurs, following consumption of infected food products such as eggs, meat, and dairy products [14,15].
Camel is a versatile animal that is used for milk, meat, wool, transportation, racing, tourism, agricultural work, and beauty contests. Camel meat is a major source of animal protein in many African and Asian countries, particularly in locations where the environment makes it difficult for other animals to produce. Camel meat is healthier than other meat animals since the carcass has less fat and has lower cholesterol levels in the fat. Camel meat also has a higher proportion of polyunsaturated fatty acids than beef [16,17].
In Saudi Arabia, a number of surveys have investigated the prevalence of camel meat contamination with some aerobic bacteria such as Staphylococcus aureus, E. coli, and Salmonella [18][19][20][21]. To the best of the authors' knowledge, no literature exists that investigates the anaerobic contamination of camel meat with bacteria. Therefore, the main goal of this study is to determine the occurrence, genotypes and antimicrobial resistance of C. perfringens and C. difficile in camel minced meat samples collected from small butcher shops and supermarkets in Al-Ahsa Governorate, Saudi Arabia.

Isolation and Identification of C. perfringens and C. difficile
Out of the 100 minced meat samples tested in the present study, 14% and 4% were contaminated with C. perfringens and C. difficile, respectively. More than 70% of the C. perfringens and C. difficile isolates were recovered from samples collected from butcher shops ( Table 1).
The number of C. perfringens in positive samples ranged from 200 to 2 × 10 3 CFU/g. Based on the 16S rRNA sequence analysis, the 14 C. perfringens and four C. difficile isolates were clustered with the reference C. perfringens (NR 121697, NR 113204, and NR 112169) and C. difficile (NR 112172), respectively, with a similarity level of 100% ( Figure 1).  The number of C. perfringens in positive samples ranged from 200 to 2x10 3 CFU/g. Based on the 16S rRNA sequence analysis, the 14 C. perfringens and four C. difficile isolates were clustered with the reference C. perfringens (NR 121697, NR 113204, and NR 112169) and C. difficile (NR 112172), respectively, with a similarity level of 100% ( Figure 1).

Figure 1.
Phylogenetic tree based on 16S rRNA sequences of C. perfringens and C. difficile isolates recovered from camel minced meat samples. The asterisk (*) refers to C. perfringens and C. difficile isolates recovered in this study.

Genotyping of C. perfringens and C. difficile Isolates
PCR genotyping revealed that C. perfringens isolated from camel minced meat was related to type A, B, C, and D, whereas C. difficile isolates were related to type A + B + and A − B + (Figure 2).

Genotyping of C. perfringens and C. difficile Isolates
PCR genotyping revealed that C. perfringens isolated from camel minced meat was related to type A, B, C, and D, whereas C. difficile isolates were related to type A + B + and A − B + (Figure 2).  Table 2 shows the distribution of C. perfringens and C. difficile types and toxin genes in camel minced meat samples. Out of the 14 C. perfringens isolates, nine (64.3%) were type A, one (7.1%) was type B, three (21.5%) were type C, and one (7.1%) was type D. C. perfringens type A with only cpa + gene was found in five (55.6%) isolates and cpa + associated cpe + genes were found in four (44.4%) isolates. However, out of the four C. difficile isolates, three (75%) were type A + B + and one (25%) was type A − B + .  Table 2 shows the distribution of C. perfringens and C. difficile types and toxin genes in camel minced meat samples. Out of the 14 C. perfringens isolates, nine (64.3%) were type A, one (7.1%) was type B, three (21.5%) were type C, and one (7.1%) was type D. C. perfringens type A with only cpa + gene was found in five (55.6%) isolates and cpa + associated cpe + genes were found in four (44.4%) isolates. However, out of the four C. difficile isolates, three (75%) were type A + B + and one (25%) was type A − B + .

Detection of C. perfringens and C. difficile Toxin by ELISA
None of the C. perfringens or C. difficile toxins were detected in the supernatant of homogenized meat samples using ELISA. The alpha toxin of C. perfringens was detected by ELISA in culture supernatants from all C. perfringens isolates, but the beta and epsilon toxins were detected in culture supernatants from four and one C. perfringens isolates, respectively. C. difficile alpha and beta toxins were detected in the culture supernatant of three isolates, but only beta toxin was detected in one isolate's culture supernatant. Enterotoxin was found in the culture supernatant of four C. perfringens types A and one C. perfringens type C. Table 2. Distribution of C. perfringens and C. difficile types and toxin genes in camel minced meat samples.

Antimicrobial Susceptibility
The distribution of minimum inhibitory concentration (MIC) values of different antimicrobials against the 14 C. perfringens and four C. difficile isolates recovered from camel minced meat is shown in Figure 2. C. perfringens and C. difficile isolates had a high frequency of tetracycline (TET) resistance (56% and 75%, respectively), followed by ceftriaxone (CRO) resistance (50% and 50%, respectively). All C. perfringens isolates were amoxicillinclavulanate (AMC) and moxifloxacin (MXF) sensitive; however, all C. difficile isolates were only AMC sensitive. The frequency of antimicrobial resistance of C. perfringens and C. difficile genotypes isolated from camel minced meat is shown in Figure 3. Multidrug resistance was observed in three (21.4%) C. perfringens and one (25%) C. difficile isolates. The mean multiple antibiotic resistance (MAR) index for resistant C. perfringens isolates was 0.30 (range 0.14-0.57), and C. difficile isolates was 0.29 (range 0.14-0.43) ( Table 3).

Discussion
Foodborne pathogens cause a wide range of diseases, with serious consequences for human health and the economy. C. perfringens is one of the most common foodborne pathogens that contaminate many types of retail meat products and cause food poisoning in humans and domestic animals [22]. Several studies have investigated the occurrence and genotypes of C. perfringens in retail food, including chicken [23], beef [24], and sheep [25] meats. However, few studies have determined C. perfringens genotypes and antimicrobial resistance in camel meat [26,27].
In the present study, C. perfringens was isolated from 14% of the tested minced meat samples. This finding is consistent with previous studies that reported the occurrence of C. perfringens in 33.7% [27] and 2.7% [26] of tested camel meat samples and swabs, respectively. On the other hand, previous studies have reported the occurrence of C. perfringens in 18% of tested minced meat in Turkey [28]; 21.2% of beef slaughtering and butchering processes in China [29]; 44.3% of goat, sheep, and cattle meat in Pakistan [30]; and 23.5% of raw processed meat in South Africa [31].
Clostridium perfringens types A, B, C, and D have been isolated in this study and type A (64.3%) was the most prevalent, followed by type C (21.5%). Several studies that investigated the distribution of C. perfringens types in retail food samples have reported that C. perfringens type A was the most common among the types recovered from camel [26,27], beef [24] and sheep [25] meats. However, a recent study in Korea detected only C. perfringens type A (33%) in tested chicken and beef meat samples [32]. In another study conducted in sheep meat, C. perfringens type A was not detected, and types B, C, and D were isolated [33]. All types of C. perfringens are commensal in the gastrointestinal tract of animals, therefore, contamination during slaughter and butchering could be the primary source of C. perfringens in meats [34,35].
Toxin genes (cpa, cpb, cpe, and etx) were found on the C. perfringens isolates recovered in our study. Although all C. perfringens isolates were found to be associated with cpa + gene, only five (35.7%) isolates were associated with the enterotoxins (cpe + ) gene, which is responsible for nearly all C. perfringens food poisoning outbreaks [25,36,37]. Similarly, previous studies found that all C. perfringens isolates from meat samples were positive for cap + gene [38,39], suggesting that it might be a universal gene in C. perfringens isolated from meat samples [40]. Moreover, the cpe + gene has been detected in 1.4% [25], 5% [41], and 27.2% [42] of C. perfringens isolated from raw and processed meats. In contrast, a recent study did not find cpe + gene in any of the C. perfringens isolates recovered from meat samples in Korea [32].
Clostridium difficile infections have been increased globally in the last two decades, causing severe intestinal infections in humans [43,44]. Thus, the occurrence of C. difficile has been detected in 4% of camel minced meat used in this study, and all isolates were identified as toxigenic. This finding is consistent with previous studies that isolated C. difficile from 1.9% of ground beef samples in France [45], 5% of ground beef and hamburger samples in Sweden [46], and 6.3% of lamb meat samples in the Netherlands [47]. However, C. difficile was not detected in ground beef in Switzerland [48] and Austria [49]. Food animals are known carriers of C. difficile [50], and multiple reports show C. difficile shedding in animals at slaughter [51,52]. Contamination of meats may occur due to gut content leakage during evisceration or due to the accumulation of spores within the slaughterhouse environment [53]. The discovery of genetically identical C. difficile strains in food, livestock, and humans has raised awareness for C. difficile potential as an unspecific foodborne agent [54,55].
ELISA can detect C. perfringens and C. difficile toxins. In the current investigation, neither C. perfringens nor C. difficile toxins were found in camel minced meat. In comparison, C. perfringens toxins were found in 13% of minced meat samples in Turkey [28]. Although C. perfringens and C. difficile were isolated from some samples, they were toxin negative, which could be related to the fact that some clostridia are incapable of making toxins [25] or the toxin concentration in the samples may be below the ELISA detection limit (5 ng/mL).
In Saudi Arabia, antimicrobials have been used in livestock to promote growth and prevent several infectious diseases. However, their use has paradoxically increased the bacterial resistance to antimicrobials [56,57]. In this study, C. perfringens and C. difficile isolates exhibited a high resistance rate to TET (56% and 75%, respectively). However, all isolates were susceptible to AMC. Similarly, previous reports have shown that C. perfringens and C. difficile isolates are highly resistant to TET [39,57,58] and are susceptible to betalactams [58,59]. In South Korea, more than 93% of C. perfringens isolates from beef, poultry, duck, and pork meats were tetracycline resistant [60]. Furthermore, many investigations have found that beta-lactam antibiotics, such as ampicillin, can inhibit C. perfringens isolated from beef, poultry, and pork meats from the United States, Belgium, Scandinavia, and India [61,62].
Multidrug resistance has been reported in C. perfringens [62,63] and C. difficile [64,65] isolates from retail meats. In the present study, 21.4% of C. perfringens and 25% of C. difficile isolates from camel minced meat showed multidrug resistance, higher than the prevalence of multidrug-resistant observed in other studies [11,32,61]. Overuse of antimicrobials (over-the-counter antibiotics without a prescription), use of TET as a growth promoter, and extensive international travel to Saudi Arabia [66] are all possible explanations for the high resistance rate found in this study. In addition, the prevalence and/or emergence of multidrug-resistant C. perfringens has been increasing and poses a tremendous public health concern [67]. A recent study has reported that multidrug resistance bacteria in retail meats originates primarily in veterinary healthcare settings or on farms where animals are administered antibiotics in their feeds or treat diseases [68].
The limitation of this study is the small number of minced meat samples collected. However, the collected samples represent only one district and do not represent different regions of Saudi Arabia. Despite this limitation, the study identified that camel meat was contaminated with C. perfringens and C. difficile and further studies are warranted to determine their prevalence and zoonotic potential.

Sample Collection
A total of 100 camel minced meat samples were randomly collected from small butcher's shops (n = 50) and supermarkets (n = 50) in Al-Ahsa Governorate, Saudi Arabia, from September 2019 to June 2020. Approximately 100 gm of minced meat was collected aseptically in sterile plastic bags and stored at 4 • C until processing within 12-24 h. For sample processing, 25 gm of each sample was aseptically placed in a sterile plastic bag containing 225 mL sterile peptone solution (Oxoid, Basingstoke, Hampshire, UK) and homogenized using a stomacher at low speed for 3 min.

Isolation and Counting of C. perfringens
A plate count of viable C. perfringens was performed according to Rhodehamel and Harmon [10]. Briefly, homogenized meat samples were serially diluted (10 −1 to 10 −6 ) using a sterile peptone diluent. Dilutions were thoroughly mixed by gentle shaking before each transfer. An aliquot of 0.1 mL of each dilution was inoculated into Tryptose-sulfitecycloserine (TSC) agar (HiMedia, Mumbai, India) containing egg yolk emulsion (Oxoid, Basingstoke, Hampshire, UK), then spread over agar surface with sterile glass rod spreader. All plates were incubated at 37 • C under anaerobic conditions for 24 h. Plates showing 20-200 black colonies surrounded by white zone were selected for counting.
For isolation, five suspected colonies from each plate were selected and inoculated into a freshly prepared thioglycollate broth, then incubated under anaerobic conditions at 37 • C for 24 h. For each sample, a loopful was then sub-cultured into 5% sheep blood agar and incubated anaerobically for purification. Colonies were identified based on Gram staining and hemolysis on blood agar. Colonies showing Gram-positive rods and hemolysis on agar were selected for further automated biochemical identification by VITEK-2 compact system using VITEK-2 ANC card for Anaerobes (BioMerieux, Marcyl'Etoile, France). A reference strain of C. perfringens ATCC 19574 was used as a control. Purified isolates were sub-cultured into fluid thioglycollate broth and incubated for 24 h under anaerobic conditions to further detect C. perfringens toxins in the culture supernatant.

Isolation of C. difficile
The broth enrichment method was used to isolate C. difficile [69]. Briefly, 25 gm of minced meat was thoroughly homogenized with 25 mL phosphate-buffered saline (pH 7.4) in a sterile container. One mL of the homogenate was inoculated into 9 mL C. difficile moxalactam and norfloxacin broth (CDMN) (Oxoid, Basingstoke, Hampshire, UK), then incubated under anaerobic conditions at 37 • C for 48 h. An aliquot of the broth was subjected to alcohol shock by adding an equal volume of anhydrous ethanol for 1 h in a sterile tube. The tubes were then centrifuged at 1900 g for 10 min. Pellets were inoculated into CDMN agar and incubated anaerobically at 37 • C for 48 h. Five suspicious colonies were sub-cultured onto 5% sheep blood agar for purification. Isolates were identified based on Gram staining, colony morphology, and production of proline aminopeptidase. Further biochemical identification was performed by VITEK-2 compact system using VITEK-2 ANC card for Anaerobes (BioMerieux, Marcyl'Etoile, France). A reference strain of C. difficile ATCC 43596 was used as a control. Recovered isolates were sub-cultured in TY medium (3% w/v tryptose, 2% w/v yeast extract, and 0.1% w/v thioglycollate) for 48 h at 37 • C for detection of C. difficile toxins.

Molecular Identification and Genotyping
Genomic DND was extracted and purified from all C. perfringens and C. difficile isolates using QIAamp DNA mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. C. perfringens toxin genes including alpha (cpa), beta (cpb), epsilon (etx), and enterotoxin (cpe) toxin genes, were amplified by real-time PCR using specific primers and probes previously designed by Gurjar, et al. [71]. The 20 µL uniplex reaction mix containing 8 µL PCR grade water, 4 µL of 5X FastStart DNA Master Plus (Roche Diagnostics, Mannheim, Germany), 1 µL of forward and reverse primers, 1 µL of Taqman hybridization probe, and 5 µL DNA template. Cycling conditions comprised initial denaturation at 95 • C for 10 min followed by 40 cycles of denaturation (95 • C for 30 s) and annealing and extension (55 • C for 1 min) using Light cycler 2.0 (Roche Applied Science, Penzberg, Germany). Negative results were considered when no amplification was recorded or when Ct value was higher than 36 cycle.
C. difficile toxins A (tcdA) and B (tcdB) were amplified using a commercial kit (RealStar Clostridium difficile PCR Kit, Altona Diagnostics, Hamburg, Germany) according to the manufacturer's instructions in Light cycler 2.0.

Detection of C. perfringens and C. difficile Toxin by ELISA
Minced meat samples were homogenized and centrifuged at 17,096 g for 10 min, and the supernatant was filtrated through a sterile 0.45 µm syringe filter in a sterile tube. Sandwich ELISA kits (Multiscreen AgELISA Enterotoxemia, Bio-X Diagnostics, Jemelle, Belgium) were used to detect C. perfringens toxins (alpha, beta and epsilon) according to the manufacturer's instructions. However, for detection of enterotoxin, the overnight growth of C. perfringens in cooked meat media was heat-inactivated at 75 • C for 20 min then subcultured in modified Duncan and Strong Medium [72] and incubated at 37 • C for 24 h. Cells were removed by centrifugation, and the enterotoxin was detected in the supernatant by a commercial latex test (PET-RPLA Toxin Detection Kit, Oxoid, Basingstoke, Hampshire, UK) as per the manufacturer's instructions. Commercial ELISA Kits (Ridascreen Clostridium difficile Toxin A/B, R Biopharm AG, Germany) were used to detect C. difficile toxin A/B following the manufacturer's guidelines.

Antimicrobial Susceptibility Testing
The MIC was determined by broth microdilution methods. Seven different antimicrobials (penicillin (PEN, ≥2 µg/mL), amoxicillin-clavulanate (AMC, ≥16 µg/mL), ceftriaxone (CRO, ≥64 µg/mL), moxifloxacin (MXF, ≥8 µg/mL), clindamycin (CLI, ≥8 µg/mL), metronidazole (MTZ, ≥32 µg/mL), and tetracycline (TET, ≥16 µg/mL)) from seven different antimicrobial classes were used to assess the antimicrobial susceptibility of both C. perfringens and C. difficile. Antimicrobial solutions were prepared, and a double-fold dilution in brucella broth (0.125-256 µg/mL) was performed in a sterile microtiter plate. A fresh culture from overnight growth was adjusted to 0.5 McFarland standard (10 6 CFU/mL) and added to each dilution before being incubated at 37 • C for 48 h under anaerobic conditions. The MIC values were determined according to the Clinical Laboratory Standards Institute [73,74]. Sterile brucella broth and C. perfringens ATCC 19574 cultures were included in each run as negative and positive controls to assess the method reliability. The MAR index was calculated (number of antimicrobials that isolate showed resistance/total number of antimicrobials that isolate had been evaluated for susceptibility) [75]. However, multidrug resistance was defined as resistance to at least three antimicrobial classes [76].

Data Analysis
Collected data were visualized with R software (R Core Team, 2019; version 3.5.3), and the "Complex-Heatmap" R package was used to build heatmap [77]. Phylogenetic analysis was performed using MEGA (version 11) software. Multiple sequence alignments were then performed by ClustalW, and the neighbour-joining method with 1000 bootstrap was used to establish the phylogenetic tree.

Conclusions
Results of this work show that camel minced meat was contaminated with C. perfringens and C. difficile. Moreover, our results provide further evidence on the emergence of multidrug-resistant strains. Thus, food safety standards and frequent inspections of abattoirs, small butcher shops, and supermarkets should be enforced. Furthermore, proactive antimicrobial agent control measures should be developed to limit the spread of multidrug-resistant strains.  Institutional Review Board Statement: The Taif University Ethics Committee has approved the study protocol (TURSP-2020-262).
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.