Isolation, Genotyping and Antibiogram Profile of Clostridium perfringens Isolates Recovered from Freshwater Fish and Fish Products from Kolkata Region

1Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Izatnagar-243122, India. 2Veterinary Public Health Laboratory, ICAR-Indian Veterinary Research Institute, Belgachia road, Kolkata 700 037, India. 3Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, Kerala 680 651, India. 4Division of Livestock Products Technology, ICAR-Indian Veterinary Research Institute,Izatnagar-243122, India. 5Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar 243 122, India.

India is the second largest producer of fresh water fish in the world.Fisheries sector contributed about 0.9% to the National Gross Domestic Product (GDP) and 5.17% to the agricultural GDP (2014-15).West Bengal ranked second in the total production of fish after Andhra Pradesh; it has total fish production as increases from 1,447.26 x 10 3 tonnes (2007-08) to 1678.33 x 10 3 tonnes (2014-15) 1 .This aberrant growth in fish industry require careful monitoring since there is risk of bacterial contamination in fish, especially in the post harvesting storage period, a critical stage prior to human consumption.Whenever bacterially contaminated raw or partially cooked food is consumed by susceptible population, then there is a chance of detrimental impact on public health 2,3 .
Clostridium perfringens is one of the most important food-borne pathogen of humans and animals causing both histotoxic diseases and intestinal infections [4][5][6][7] .It is Gram-positive, anaerobic, straight rod, spore former, found in the soil, dust, sewage, marine sediments and in the gastro-intestinal tract of humans and animals [8][9][10] .Based on the production of four major lethal toxins i.e. alpha (α) beta (β), epsilon (ε) and iota (i), C. perfringens is divided into five major toxinotypes/biotypes (A-E) 6,10,11 .Apart from these, it also produces enterotoxin (CPE) and beta 2 toxin (CPB2) 12 .Symptoms of C. perfringens type A food poisoning include abdominal cramping, nausea and diarrhea, which usually begin 6-24 h after ingestion of contaminated food and then persist for 12-24 h 7,9 .C. perfringens associated symptoms are caused by an enterotoxin which is produced during sporulation of the organism in the small intestine following ingestion of large number of vegetative cells of enterotoxin positive C. perfringens 9,13 .
Isolates originating from humans with gastrointestinal diseases carry most commonly CPE and sometimes newly discovered CPB2 toxin 12,14 .Enterotoxigenic C. perfringens type A is also associated with antibiotic associated diarrhoea (AAD) and sporadic diarrhoea (SD) cases 15,16 .The emerging problem of antimicrobial resistance between pathogenic and commensal bacteria is becoming more intense by intervention of environmental resistance phenomenon of bugs 17,18 .Antibiotics represent one of the most successful forms of therapeutic regimens in medicine.But the overuse and misuse of antibiotics, mostly in developing countries, lead to growing number of antibiotic-resistant pathogens 19 .Antibiotic resistance, which is associated with increased morbidity and mortality rates as well as increased treatment costs, is considered to be one of the major global public health problems and its magnitude recently prompted a number of national and international bodies to take actions to protect the public health 20 .Problem of antimicrobial resistance in pathogenic and commensal bacteria is increasing by the adoption of mobile genetic elements 21 .Globally, there are reports of earlier studies on epidemiological characterisation of C. perfringens from fish samples 7,[22][23][24][25][26][27][28][29][30][31] , but in Indian scenario there are limited studies carried out earlier on given aspects 2,13,32 and there are no previous reports from the study area, therefore, it is necessary to carry out epidemiological studies in such prone areas to determine the types of C. perfringens prevalent in fish and fish products.Considering these facts, the present study was designed to isolate Clostridium perfringens from freshwater fish and fish products from Kolkata region of India, characterize these at molecular levels by determining their genotypes and study antibiotic sensitivity patterns.

Collection of samples
One hundred and two samples consisting of intestinal and gill samples from fresh water fish (n=69) and fish products including fish pickles, fish curry and fried fish (n=33) were collected from retail shops and restaurants from Kolkata city of India.All the collected samples were immediately transported to the laboratory in ice pack container and processed within 24 hours for isolation and molecular characterisation of C. perfringens using standard bacteriological and molecular procedures.The intestinal and gill samples from the fresh water fish were hygienically collected and processed for the bacteriological analysis.The fish products were aseptically collected in sterile container for the analysis.

Isolation of C. perfringens
All the samples (Intestine, gills and fish products) were inoculated aseptically in Robertson's cooked meat (RCM) medium supplemented with glucose, hemin and vitamin K broth (HiMedia, Mumbai, India) and heated at 75 0 C for 20 minute for removal of vegetative cells of other competing microorganisms, followed by incubation at 37 0 C for 24-48 hours under anaerobic condition in McIntosh anaerobic jar using gas pack (HiMedia, Mumbai, India).Enriched 100 µl inoculums were taken in sterile Petridish, followed by pouring of sterile molten sulfite polymyxin sulfadiazine (SPS) agar over the inoculum with proper mixing by rotating the plate in clockwise and anticlockwise direction.Plates were incubated at 37 0 C for 24 h under anaerobic condition 33 .

Biochemical and phenotypical analysis of C. perfringens
Samples revealing characteristic black coloured colonies on SPS agar plates were subjected to biochemical tests for identification of C. perfringens 34 .About 3-5 colonies from the culture plate for each sample were considered for Gram's staining, lactose fermentation, nitrate reduction, gelatin liquefaction, lecithinase and indole tests.

Reference strains
Reference strains of C. perfringens were procured from Biological Standardization Division, ICAR-IVRI, Izatnagar, India.

Molecular characterization / genotyping of C. perfringens by PCR
All the presumptively identified C. perfringens isolates recovered from different sources were subjected to polymerase chain reaction (PCR) assays for the detection of species specific 16S rRNA 35 and virulent toxin genes viz., cpa, cpb, εtx, ιtx 36 , cpb2 and cpe genes 37 .The oligonucleotides used in present study were synthesized through M/S Xcelris Labs Limited, Ahmadabad, Gujarat (India).The details of primers used in present study are given in Table 1 [38][39][40] .
The snap chill method 41 was used to extract DNA from the isolates.About 200 µl of overnight culture of C. perfringens was taken in the microcentrifuge tube and the cell suspension was centrifuged for 10 min at 14,000 × g.The pellet was suspended in 100 µl of nuclease free water (NFW) by vortexing.The microcentrifuge tube was placed in hot water bath for 15 min at 100°C and immediately chilled in ice.An aliquot of 5 µl of the supernatant was used as the template DNA in the PCR assays.
The optimized PCR protocol for all the target genes with 25 µl reaction mixture volume included 1 µl (10 pmol/µl) each of forward and reverse primer sequences, 0.5 µl of 10 mM deoxyribonucleotide triphosphates (dNTPs), 2.5 µl of 10 × PCR buffer [(100 mM Tris-HCl (pH 8.8 at 25°C), 500 mM KCl, 0.8% (v/v) Nonidet P40)], 2.0 µl MgCl 2 (25 mM), 0.20 µl of 5 U/µl of Taq DNA polymerase (Thermo scientific, USA), 5.0 µl DNA template and remaining NFW to make the final volume.The PCR reaction mixture was set for amplification in a thermocycler (Biometra Personal Cycler, Goettingen, Germany) with specific condition for each PCR assay.For 16S rRNA PCR assay, the initial denaturation condition was 94°C for 5 min.then amplified for 35 cycles (1.0 min at 94°C, 1.3 min.at 53°C, 1.3 min.at 72°C for denaturation, annealing and extension phases, respectively), followed by an additional period of extension for 10 min.at 72°C.Regarding multiplex PCR for cpa, cpb, εtx and ιtx genes, there was initial denaturation at 95°C for 5 min.then amplified for 30 cycles (1.0 min.at 94°C, 1.0 min.at 55°C, 1.5 min.at 72°C for denaturation, annealing and extension phases, respectively), followed by an additional period of extension for 10 min.at 72°C.Similarly for cpb2 and cpe genes, there was initial denaturation at 94°C for 4 min.then amplified for 35 cycles (1.0 min.at 94°C, 1.2 min.at 55°C, 1.2 min.at 72°C for denaturation, annealing and extension phases, respectively), followed by an additional period of extension for 10 min.at 72°C.Amplified PCR products (10 µl) were separated by electrophoresis for 45 to 60 min.at 80 V in a 1.2% (w/v) agarose gel with ethidium bromide (0.5 µg/ml) and a 100 bp DNA ladder (Thermo scientific, USA) was included in each agarose run.The resolution of amplified fragments in the gel was visualized by a UV trans-illuminator and digitally recorded by gel documentation system (UVP Gel Sequencing Software).Materials contaminated with ethidium bromide were disposed as per the local guidelines.

Isolation and identification of C. perfringens by cultural and biochemical methods
On cultural and biochemical analyses, a total of 24 isolates [17 from fresh water fish (n=69) and 07 from fish products (n=33)] were found to be positive for C. perfringens.

Genotyping by PCR detection of virulence toxin genes
All the biochemically positive isolates were screened by PCR assays for detection of species specific 16S rRNA and virulence toxin genes viz., cpa, cpb2, cpe, cpb, εtx and ιtx.All the 24 (100%) presumptively confirmed isolates of C. perfringens [17 (70.83%) from fresh water fish and 07 (29.16%) from fish products] was also shown amplification of species specific 16S rRNA of product size 279 bp (Table 2, Fig 1) and α toxin gene of product size 402 bp (Table 2, Fig 2).However, 17 (70.83%)cpa positive isolates [12 (70.58%) from fresh water fish and 05 (71.42%) from fish products] were also positive for additional beta2 toxin gene (cpb2) of 567 bp length (Table 2, Fig 3).The other virulence gene specific primers of cpb, εtx, ιtx and cpe genes did not amplify any of the isolates.The virulence gene detection by PCR assay used in this study revealed that all the isolates were belonged to C. perfringens type A.

DISCUSSION
C. perfringens is considered as one of the most important food-borne pathogen and is ubiquitous in nature [6][7][8][9] , however, in Indian scenario there are limited studies occurred earlier on epidemiological and molecular aspects of C. perfringens in fresh water fish and fish products.In the present study, a total of 24 samples (23.52%) [17 (24.63%) from fresh water fish (n=69) and 07 (21.21%) from fish products (n=33)], were found to be positive for C. perfringens.It has been estimated that 17/24 isolates of C. perfringens (70.83%) were derived from the intestinal and gill samples of fresh water fish and 07/24 (29.17%) from fish products.
On PCR detection of species specific 16S rRNA and virulence toxin genes of the C. perfringens isolates it was found that all the isolates (24) were shown species specific amplicon of 16S rRNA and positive for α toxin gene (cpa).Apart from these, 17 cpa positive isolate (70.83%) [12 (70.58%) from fresh water fish and 05 (71.42%) from fish products], were also positive for beta2 toxin gene (cpb2).Previous studies have reported occurrence of C. perfringens from fish and sea food samples with a varying isolation rates of 1% to 84% from different countries 2,7,13,[22][23][24][25][26][27][28][29][30][31][32] .Cai et al. 27 reported that out of 75 isolates of C. perfringens from fresh water fish, 13 (17.3%)isolates were found to be positive for only α toxin gene, 58 (77.3%) isolates for α & β toxin genes positive (C.perfrigens type C) and 04 (5.3%) isolates for α, β and ε toxin genes positive (C.perfringens type B), while 47 (62.70%) isolates have additional beta 2 toxin gene, however, none of the isolates were found to be positive for cpe and other virulence toxin genes, since, the presence of cpe gene in C. perfringens is very uncommon, and only <5% of global C. perfringens type A isolates were found to be cpe gene positive 7,9 .In concurrence to present results, the cpe-negative C. perfringens type A from the fish sample and clinical cases of gastroenteritis was reported previously 2,13,31,32,43 .In another study, out of 34 isolates of C. perfringens from fish samples, 31 were found to be positive for C. perfringens type A (only cpa gene positive) and 3 isolates were cpa as well as cpe genes positive, but none of the C. perfringens type A isolates were found to be positive for cpb2 gene 2 .
In contradiction, present study reported the occurrence of cpb2 positive isolates in 70.83% of C. perfringens type A isolates.These findings highlights the high prevalence of cpb2 associated C. perfringens type A which is mainly associated with antibiotic associated diarrhoea (AAD) and sporadic diarrhoea (SD) cases 12 .
The antibiogram studies performed against isolates recovered from freshwater fish and fish products unveils an alarming public health concern due to higher resistance in recovered isolates.Highest resistance was observed against co-trimoxazole (88%), followed by ceftriaxone (87%), ceftazidime (53%), tetracycline (44%), norfloxacin (21%) and amikacin (11%), while all the isolates were found to be sensitive to ciprofloxacin and amoxicillin/clavulinic acid.These finding are found to be concurrent with earlier studies reporting a high emerging resistance toward various tested antibiotics [44][45][46][47][48][49][50][51][52] .These observations are major point of concern from public health perspectives, since most of the antibiotics drugs used in the present study are employed as a first line of treatment in diarrhoeal cases.

CONCLUSION
The present study suggested that PCR is a reliable molecular technique and useful tool for the detection of virulence genes typing of C. perfringens isolates recovered from fresh water fish and fish products.The presence of species specific 16S rRNA and cpa gene in all the isolates of C. perfringens suggest that C. perfringens type A (only have α toxin gene) is the most predominant type in fresh water fish and fish products in this study area.Primer specific to species specific 16S rRNA and cpa gene in PCR assays, used in present study, could be useful for the rapid identification of C. perfringens and provided a clues to the importance and improvement of the present method for the surveillance of C. perfringens from fresh water fish and fish products.On antibiogram profile, except for ciprofloxacin and amoxicillin/clavulinic acid, a high frequency of multiple drug resistance pattern was observed among the C. perfringens type A isolates.These emerging resistance trends are major point of concern from public health perspectives.

Table 1 .
Details of the primers for testing of samples for Clostridium perfringens by PCR

Table 2 .
Samples found positive for C. perfringens and their virulence genes by PCR