Isolation and Molecular Characterization of C. perfringens from Meat Samples

The objective of the study was to observe the presence of C. perfringens in various meat samples and characterization of the isolates with molecular typing. The samples were collected from different retail outlets in Jabalpur city and processed aseptically. The samples were enriched in alternative thioglycollate medium followed by selective isolation in tryptose sulfite cycloserine agar. Out of 200 samples screened (50 each of raw buffen, chevon, chicken and fish meat), C. perfringens were isolated from 46 samples (23%). The highest presence of C. perfringens was found in buffen (28%), followed by fish (24%) and chevon and chicken (20% each). Lecithinase activity was shown by all the isolates of buffen, chevon and chicken, while, 11 (91.66%) isolates from fish were positive for lecithinase activity. Likewise, 36 (78.28%) isolates of C. perfringens displayed stormy fermentation in IMM within 14 – 18 hrs. The molecular study showed that all the isolates 46(100%) were found to harbor the 16S rRNA and lecithinase (cpa) gene, while none of the isolates displayed enterotoxin (cpe) gene. The presence of C. perfringens in raw meat indicated that the meat wasn’t processed hygienically and poor sanitary conditions in retail outlets. Therefore, consumption of such meat may produce infectious disease and is a public health threat as many food poisoning strains of C.perfringens are heat resistant.

Meat and meat products are important source of high quality proteins with high biological value and essential amino acids that are required for body growth. Meat is the richest source of vitamin B, particularly thiamine, riboflavin and niacin. It also contains a good amount of fat soluble vitamin A, phosphorous, iron and copper (Sherikar et al., 2013). India stands 5 th in global production (6.3 million tons) and accounts for 3% of the total world meat production (220 million tons) (GOI, 2016).
Meat is often contaminated due to unhygienic production resulting in spoilage and food borne diseases. Food borne diseases occurs through consumption of raw meat or improperly cooked or contaminated with pathogens from sources such as water, raw ingredients, environment, food handlers and during processing of meat (Biddle et al., 1993). Worldwide, food borne diseases are a major public health burden leading to high morbidity and mortality. The global burden of infectious diarrhea involves 3 -5 billion cases and nearly 1.8 million deaths annually, mainly young children, caused by contaminated food and water (NCDC, 2017). These deaths are due to contamination with virus, bacteria, parasites, toxins etc. The common bacteria found in meat and leads to infections are Salmonella, Campylobacter, S. aureus, C. perfringens, pathogenic E. coli etc (WHO, 2012).
C. perfringens is a Gram positive, anaerobe, sporeforming, mesophilic rod and is widely distributed in nature. It is generally associated with different forms of enterotoxaemia in animals and gas gangrene in animals and man (Singh and Bist, 2013;Das et al., 2014;Yadav et al., 2016). Based on their ability to produce certain exotoxins, C. perfringens are categorized in five types: type A, B, C, D and E. The food poisoning strains belong to type A, and are generally heat resistant. These strains of C. perfringens exist in soil, water, foods, dust, spices and the intestinal tract of humans and other animals. The bacteria enter into meat directly from intestinal tracts of slaughtered animals or by the subsequent contamination of slaughtered meat from containers, handlers, or dust. It can withstand the environmental conditions of drying, heating and toxic compounds because of its spore forming ability. It is also a leading cause of food poisoning in many developed counties (Jay et al., 2005;Gurmu et al., 2013;Yadav et al., 2016).
The foods involved in C. perfringens outbreaks are often due to consumption of preparations of meat or its by-products kept for longer period of time without refrigeration. When such preparations are rewarmed after cooking, the heat of such foods is presumably inadequate to destroy the heat resistant endospores, thereby providing endospores favorable conditions to germinate and grow (Jay et al., 2005). The presence of C. perfringens in raw meat is a direct source of contamination and thus may precipitate food borne illness. Thus, the present study was aimed to investigate the presence of C. perfringens in different meat samples and their molecular characterization.

Isolation
A total of 200 meat samples consisting 50 each of raw carabeef, chevon, chicken and fish were collected from different retail outlets in Jabalpur city. The meat samples were processed as early as possible in the laboratory. Isolation was performed with enrichment of sample in alternative thioglycollate broth followed by selective plating in tryptose sulphite cycloserine (TSC) as per the method described by Agarwal et al. (2003) and Singh et al. (2006). The meat samples were diluted (1:10) by taking 10 g sample in 90 ml normal saline followed by addition of diluted sample into alternative thioglycollate broth. The enriched sample was incubated at 37°C for overnight.
During selective plating, an agar over lay technique in tube was used using tryptose sulphite cycloserine (TSC) agar. Alternative thioglycollate enriched inoculum (0.1 ml) was taken in tube followed by addition of 10 ml of TSC agar. After 5 min, tube was overlayed with 2-3ml of TSC agar and incubated at 37°C for 10-12h after solidification. Presumptive C. perfrigens colonies (black colonies) from TSC was taken in alternative thioglycollate broth and incubated at 37°C for 10-15 hrs. The presumptive isolates of C. perfringens were microscopically and biochemically characterized on the basis of colony morphology, Gram's staining, motility test, lactose -gelatin liquefaction and nitrate reduction test according to method described by Agarwal et al. (2003).
The isolates were tested for lecithinase and lactose fermenting activity using TSC with egg yolk emulsion and iron milk medium, respectively. The lecithinase activity was observed by analyzing zone of opalescence around the colony. This was done by agar overlay method in petriplates under anaerobic condition. The anaerobic condition was created by candle jar method, in which a lighted candle was kept under jar and thus anaerobiasis was created (Agarwal et al., 2003;Ananthnarayan and Paniker, 2013).
Stormy fermentation was observed by adding inoculums of overnight grown alternative thioglycollate culture in iron milk medium followed by incubation at 46°C in a water bath for 14 -18 hrs. Stormy clot fermentation was observed as described by John et al. ( 1982).

Molecular Characterization
DNA was isolated from overnight grown culture in alternative thioglycollate using Quik-start protocol (Qiagen, Germany). In brief, 1.0 ml overnight culture of C. perfringens isolate was taken into eppendorf tube and centrifuged at 10000 rpm for 5 min. The cells were resuspend in 200 µl phosphate buffer saline (PBS) followed by addition of 20 µl proteinase K and 200 µl buffer AL. Mixing was done by vortexing and then 200 µl ethanol (96 -100%) was added. The mixture was transferred into a DNeasy mini spin column followed by centrifugation twice at 8000 rpm for 1 min. The flowthrough was discarded. DNA was eluted by adding 200 µl buffer AE to the center of spin column membrane and incubated for 1 min at room temperature (15-25°C) followed by centrifugation for 1 min at 8000 rpm.
The amplification cycles were carried out in thermocycler (Bio-Rad) as per the method described by Yadav et al. (2016) with primer sequences as depicted table 1. The amplification cycles started with initial denaturation at 95°C for 5 min followed by 35 cycles each having denaturation at 95°C for 1 min, annealing at 52°C for 16S rRNA and 54°C for cpa (lecithinase gene) for 1 min and extension at 72°C for 1 min with final extension for 5 min at 72°C. The cpe (enterotoxin) gene reactions were carried out at annealing temperature between 50 -60°C. At every screening a nucleic acid blank negative control were taken along with isolates to monitor the quality control of PCR conditions. On completion of PCR, amplified products were resolved in 1.5% agarose gel under UV transilluminator.

RESULTS AND DISCUSSION
The results of the isolation study indicated that out of 200 samples examined, 23% (46 isolates) had C. perfringens, with buffen (28%), fish (24%) and chevon & chicken (20% each) as depicted in table 2. The biochemical profiles of the isolates are shown in table 2. The molecular study showed that all the isolates were found to harbor the 16S rRNA ( Fig. 1) and lecithinase (cpa) gene (Fig. 2), while none of the isolates displayed enterotoxin (cpe) gene. The presence of microorganisms in all types of meat samples validates the earlier findings that the bacteria are widespread in nature including the intestinal tract of humans and animals, which makes the presence of pathogen in different foods inevitable, if the processing of meat is improper. Previous examinations also reported the occurrence of C. perfringens in various domestic animals viz. 30% to 60% in beef meat, 30% to 50% in pork and 40% to 80% in poultry (Ryan and Ray, 2004).  High presence have also been reported by various workers in our country viz. Singh et al. (2006) (Das and Jain, 2012). Likewise, examination of 400 food samples in Guwahati, displayed 33 food samples were positive for C. perfringens (Gurmu et al., 2013). A high occurrence of the pathogen (81.69%) was reported from 71 chicken meat samples of Bangalore, Karnataka by Prabhu et al. (2013). Similarly, an observation in abroad (Japan) revealed that out of 200 retail meat samples tested, 71% were found to be contaminated with C. perfringens with 33.30% in lamb and 100.0% in ground chicken and duck (Yasuhiro et al., 2008). Likewise, the screening of 200 meat product samples from supermarket and retail meat shops revealed 48.80% and 21.30% C. perfringens from ready to cook and ready to eat meat products, respectively (Elham and Nahla, 2011). Another examination revealed 2 and 1.42% C. perfringens from imported frozen chicken and imported frozen meat respectively, in Cairo and Giza cities (Elnawawi et al., 2012). A prevalence study (48.27%) was done by Mehtaz et al. (2013) in apparently healthy, clinically affected animals, meat and milk. An investigation in Lahore (Pakistan) revealed low prevalence of C. perfringens in chicken -6% (6 / 100), mutton -5% (5/ 100) and beef -1% (1 / 100) (Khan et al., 2014). During a study of 90 samples (45 heart -livers and 45 gizzards), Hanifehnezhad et al. (2015) observed 46.70% of heart -liver and 17.70% gizzard were contaminated with C. perfringens. In another study in Zaria, Tizhe et al. (2015) found 14 (3.50%) samples positive for C. perfringens out of 400 samples examined from seven different retail markets and abattoir.
The different studies carried out by various workers in our country and abroad showed the presence of C. perfringens in meat, indicated the poor hygienic conditions prevailing in the retail shops, abattoir, etc. The difference in study may be attributed to isolation protocols practiced in laboratories viz. pre-enrichment or enrichment that enhances the recovery of pathogens due to favorable conditions which in turn increases the growth and multiplication and thus, increases the occurrence (Ananthnarayan and Paniker, 2013). The use of antibiotics in selective media, besides decreasing the growth of other bacteria also inhibits the recovery of many strains of desired microorganisms and therefore, lowering the incidence of pathogen in different samples (Singh, 2010). The occurrence in present study in different meat sample didn't show wider variation, which revealed similar conditions in retail outlets.
C. perfringens possess an enzyme lecithinase/ phospholipase that break down lecithin to insoluble diglyceride and phosphorylcholine, which results in a white opaque zone around colony on egg yolk agar media. This activity is being exhibited by alpha toxin and is produced in-vitro by most of the strains of C. perfringens. In this study, all the isolates from buffen, chevon and chicken showed lecithinase activity while 11 (91.66%) isolates of fish meat were positive for lecithinase activity. Few strains of C. perfringens were unable to display lecithinase activity in the previous reported works (Sjelkvale et al., 1979;Singh et al., 2006;Agarwal et al., 2009;Yadav et al., 2016). The absence of lecithinase activity by C. perfringens in presence of an antitoxin is due to neutralization of lecithinase (Nagler's reaction). This phenomenon isn't common in other Clostridium spp viz. C. baratti, C. absonum, C. bifermantans, C. sordelli and C. novyi because these microorganisms showed lecithinase activity in presence of antitoxin (Ananthnarayan and Paniker, 2013).
C. perfringens ferments the lactose resulting in production of lactic acid and gas in iron milk medium (IMM). The acid coagulates the casein in milk resulting in clear watery fluid. The stormy clot formation is due to fracturing of curd into spongy mass which usually rises above medium surface. This is used for presumptive estimation of C. perfringens. For this, the bacteria are incubated in IMM at 45 -46°C for 14 -18 hrs. At 45 -46°C temperature, the growth of other bacteria is decreased but the multiplication of C. perfringens is increased due to reduction in generation time interval to 6 -10 min. Further, the selectivity can be enhanced by addition of neomycin, cycloserine, etc. which inhibits the growth of E. coli and Bacillus (John et al., 1982;Abeyta et al., 1983).
The molecular study is helpful in establishing genotypic and phenotypic co-relation of different virulence marker of the pathogens. The microorganism was identified with the help of 16S rRNA gene, which is commonly used in the identification of various food borne pathogens.
All the isolates displayed presence of lecithinase (cpa) gene, which is commonly found in C. perfringens type A, a common food borne pathogen. One of the isolates from fish genotypically showed lecithinase (cpa) gene, but didn't display phenotypic (lecithinase) activity on egg yolk agar. This indicated the lack of expression of lecithinase enzyme phenotypically. None of the samples was observed to have enterotoxin gene at annealing temperature between 50 -60°C. Earlier works revealed that only 5 -6% of C. perfringens food borne strains had enterotoxin gene, which lead to food poisoning incidences (Uzal et al., 2014;Nashwa et al., 2016). Most of the food poisoning C. perfringens strains are heat resistant and in type A category. In concurrence to present results, previous food sample study have also displayed the absence of enterotoxin gene from the isolates of C. perfringens and these isolates were reported from food samples and gastroenteritis cases (Yadav et al., 2016).
The observation of the raw meat samples showed the occurrence of C. perfringens in all the species, indicating poor implementation of hygienic measures in the retail premises. Therefore, consumption of such meat may cause gastrointestinal infections and thus lead to food poisoning incidence if not cooked and refrigerated properly.