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Molecular Based Techniques to Find the Specificity and Sensitivity of the C. Perfringens

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Paper name: Frontiers of food microbiology

Table of Contents
Clostridium perfringens types
2. Discussion
Molecular detection techniques
1. PCR (polymerase chain reaction)
2. Multiple Locus Variable–Number Tandem Repeat Analysis (MLVA)
3. Conclusion
4. References


Clostridium perfringens is a spore-forming, rod -shaped Gram-positive, anaerobic, non-motile bacterium. It is a large, regular, round, slightly opaque and shiny colonies on the surface of agar plates. They are classified within the phylum Firmicutes (Mohamed E. Mohamed Mohamed E. Mohamed1 1*, 2010Yonogi et al., 2016). C. perfringens are isolated from a wide range of environments such as gastrointestinal tract of humans and domestic animals, chickens, clinical samples, soil, marine and fresh water residues (Park et al., 2016). The ubiquitous nature of this bacterium and its spores are constant problem for the food industry and when large amounts of foods are prepared (Kopliku et al., 2015). They are special problem for meat industries. Even though the food cooks under enough heat, it is not always possible to supply heavy heat all the time. For instance, in food serving place like restaurant, hospitals and homes, Clostridium perfringens is a common source for food poisoning because meat containing dishes stores with insufficient cooling after cooking and reheats it with moderate heat. Most strains grow between 15°C to 50°C with an optimum temperature of 45°C. The generation time (Gt) is below 20 mins for most of the strains at temperatures between 33°C to 49°C, and Gof 8 min also reported as minimum range  (Sigrid Brynestad & Granum, 2001).
More than 13 different toxins can be produced by C. perfringens, however production of four major lethal toxins are produced by type isolates  (A–E), and three of those can be seen on plasmids (Sigrid Brynestad & Granum, 2001). Anaerobic bacterium is a group of organisms, which places a major threat for public health as well as for food spoilage due to lack of oxygen. Clostridium perfringens is an anaerobic bacterium, which can contaminate food due to the formation of spores. During sporulation of vegetative cells, Clostridium perfringens produces enterotoxin in the intestine of the host and layers of spore coat will forms. It has a unique four step membrane action, which binds to receptors on epithelial cells of intestine and exerts its intestinal reaction, such as diarrhoea and cramping symptoms on abdomens associated with C. perfringens food poisoning (CHARLES L. DUNCAN, DOROTHY H. STRONG, & SEBAL, 1971).
Keywords: clostridium perfringens, ubiquitous nature, intestine, enterotoxin, lethal toxins

Clostridium perfringens types

The species of Clostridium perfringens are divided into five types (A to E) based on four major lethal toxins production (Table1). This type is attained based on the neutralization of fatal toxins with type-specific antisera using test animals as they are antigenic in property. Type A is the most familiar type in this species, which are inconsistent in their toxigenic properties and has confusing pathogenic properties. The type A enterotoxin-positive (CPE+) strains carry the genes of enterotoxin (cpe) cause many foodborne illnesses, erratic diarrhoea, and antimicrobial drug-associated diarrhoea (Freedman et al., 2015Sabry, Abd El-Moein, Hamza, & Abdel Kader, 2016) .They cause diseases due to the production of enterotoxin (CPE), which produced in small intestine after the ingestion with at least 10cells. It causes mainly gas gangrene disease in humans, diarrhoea and Necrotic enteritis in animals. Gas gangrene is not only due to the production of α toxins but also due to the total production of toxins products (NIILO, 1980). Type A disease are self-limiting and will last only for 24hrs.Type B strain bacteria mainly cause disease on animals, which occurs when the infection in intestine leads to enteritis. Enteritis is a disease resulted by the production of toxin in intestine produced by type B bacteria, simply says “inflammation of the intestine”. Type B bacteria will produce 3 major toxins.
Type C bacteria always connect with the individual who has low of proteolytic enzyme in their intestine which is mainly due to less protein intake. The symptoms of this bacteria start with awful abdominal pain and diarrhoea sometimes even vomiting and leads to necrotic inflammation in the small intestine (Sigrid Brynestad & Granum, 2001). Type D is another type of Clostridium perfringens bacterial strain, which will cause disease called enterotoxaemia in animals occurs due to abnormal proliferation strain in the intestinal tract. Type E mainly produces alpha and iota toxins in animals and described as a haemorrhagic enteritis often cause deadly syndrome in animals (NIILO, 1980).

Toxino- types Major Toxins Minor Toxins Associated Diseases
α β ε ι CPE λ Θ δ Humans  Animals
A ++ + + Gangrene Gastrointestinal diseases Diarrhoea (foals, pigs.)
Necrotic enteritis in       fowl
B + + + + + + Dysentery in new-born lambs Haemorrhagic enteritis in neonatal calves and foals Enterotoxaemia in sheep
C + + + + Necrotic enteritis Necrotic enteritis in piglets, lambs, calves and foals Enterotoxaemia in sheep
D + + + + Enterotoxaemia in lambs, sheep, calves and goats
E + + + + Enterotoxaemia in calves

-no toxin production detected ; +1 toxin productions detected; ++ highest toxin producer
Table 1 – different types of Clostridium perfringens toxinotypes and associated diseases (Petit, Gibert, & Popoff, 1999)


The enterotoxins (CPE) is the major noxious factor that leads to food poisoning in humans and animals. CPE grows in the small intestine after the deglutition of 107 Clostridium perfringens cells. The sequence of CPE toxins is highly preserved in type A strains, whereas the deficient copies are bound with the iota toxins in the type E strains (Sigrid Brynestad & Granum, 2001).
Fig. 1. A  diagram illustrates the functional regions of CPE.
CPE is a solitary protein with 319 amino acid polypeptides of 3.5 kDa , which has an isoelectric point of 4.3 and with no notable similarities with other known proteins, but has an exception with a C. botulinum complexing protein for the limited homology .It can either carried by plasmid or by chromosomes of the proteins. Spores from the strains of the chromosomal cpe gene are more heat resistant than spores from the strains with gene in a plasmid (CHARLES L. DUNCAN et al., 1971). It has high biological activity due to limited trypsination and chymotrypsination, but they are both heat- and pH-labile in nature. The enterotoxins shows greater activity when aa 1-36 are detached .CPE has two domain structures. The aa 290–319 are C-terminal end of the protein, hold binding region which help the protein to binds with the intestinal protein receptor (Fig. 1). Antibodies that against this binding region will neutralize the cytotoxicity of CPE while without losing the activity during reaction the firsts 44 N-terminal aa region and three C-terminal aa can be removed. Amino acids 44 – 171 of CPE are responsible for the insertionin the membrane and cytotoxicity.

After the ingestion of 107– 10cells sporulation process will take place because of the spore forming ability of the gene. They oligomerizes to form pores that make easier for the passage of cation ions. Then eventually, the mother cell lyses and release out the enterotoxin in to the intestine (Sigrid Brynestad & Granum, 2001). Studies shows that CPE has two different complexes, at 4°C the CPE binds to claudin receptor and form first complexes, while the large complex does not bind at 4°C. The large complex forms only after the physical changes of small complex. It will merge with ca. 70-kDa protein for the formation of large complex, it is very hydrophobic. They develop porosity in the small molecules (Fig, 2).
Fig. 2. Flowchart describes the major steps involved in C. perfringens food-poisoning mechanisms.
Toxins produced by most of the strains  functions as extracellular hydrolytic enzyme or lytic enzyme. They are responsible for penetration of toxins into the host tissue as well for the discharge of metabolites with small molecule like sugars and amino acids. During infection those metabolites eventually move into the cells to supply enough nutrient sources (Hassan et al., 2015). C. perfringens toxins which is located mainly on chromosomes causes myonecrosis, which is a necrotic damage whereas toxins travels on plasmid causes intestinal as well as food borne diseases except enterotoxin which can be seen both on chromosome and plasmid (Popoff & Bouvet, 2013)

2. Discussion

Molecular detection techniques

  1. PCR (polymerase chain reaction)

A polymerase chain reaction is a rapid enumeration technique which can used to identify the genes which is encoded by the major 5 toxins of C. perfringens. This method is widely used to detect the sensitivity and specificity of the genes (Uzal, Plumb, Blackall, & Kelly, 1997) used PCR technique to find out genes which produces major toxins of C. perfringens in goats. The Uzal, Plumb et al. (1997) illustrate that positive result was found when the known toxinotypes were used and negative result obtained for known non-target clostridia. Hence it proved that PCR technique is highly specific in result.
(Uzal et al., 1997)The fecal sample of goat which contain C. perfringens types A to E were used as a template in the PCR and found amplicons as band on agarose gel nearly the 247 bp (α-primers),1025 bp (β-primers), 403bp (epsilon primers), and 298bp (iota primers) of the DNA marker. They used two PCR techniques in this research PCR-100 (100µl final volume), PCR-20 (20µl final volume). The reaction was done by DNA Thermal Cycler. To find out the specificity of the toxins, strains of C. perfringens types A to E were performed by both PCR techniques such as, PCR-100 and PCR-20. All five strains tested by each primer, whereas the strains that produces more than one toxin were also tested by all primers in single reaction. In single reaction the reaction volume was constant at 100µl or 20µl along with reducing the amount of water used in the process. The result showed that PCR detection technique are highly specific in finding targeted genes. For finding the sensitivity of the test, sterilized faecal sample from goat were penetrated with different amount of C. perfringens types A-E and tested by both techniques PCR-100 and PCR-20, continued by doing anaerobic plate count with 18h thioglycolate culture. When the test done directly by PCR enriching of the culture, the positive result was achieved all faecal sample with the count of bacteria 1-1.5×10to 1-1.5×10cfu g-1. The positive result of the faecal sample without enriching the culture in concentration of 1-1.5×10cfu g-1 (Uzal et al., 1997).

  1. Real time polymerase chain reaction

Real-time PCR is a type of PCR and a molecular based detection technique. This method is widely used in food industries due to its speed outbreaks and it allows specific detection of genes which produces determined toxins with an improved sensitivity. In Real-time PCR, the risk of cross contamination is very less. Loh, Liu et al. (2008) used Rt PCR for the detection C. perfringens during gastroenteritis in military camp. They collected 12 different stool samples from the affected patients. DNA was then extracted from the culture using methods of the QIAamp DNA mini-kit. The amount of C. perfringens was relatively high in number (excess of 106 spores/g stool) when the C. perfringens detected using the technique Rt PCR. Positive findings of specificity for C. perfringens were completed within 4hr time using preliminary Real time PCR assay (Loh et al., 2008).
Hernández, López-Enríquez, and Rodríguez-Lázaro (2017) tested and developed a new real time PCR assay for the detection of specious specificity and to find out the false negative results, which occurs frequently during the high-level food samples PCR inhibition. In this research they targeted mainly on phospholipase C (plc) gene and included internal amplification control (IAC), which is a 118bp DNA fragment which included with 70bp of listeriosis-positive regulatory (prfA) gene from the CECT 911 strain of Listeria monocytogenes which is bound with specific reverse and forward primers CPplc. DNA was extracted from eight clostridium strains and 85 non-clostridium strains including isolates of 53 C. perfringens strains and 27 non-C. perfringens strains using CTAB based standard protocol. All C. perfringens isolates showed positive result and strains which did not derived from C. perfringens showed negative result. The overall efficiency (E) and linearity (R2) was 0.858 and 0.991 respectively (Hernández et al., 2017)
Chon (2012)used this method for the detection of C. perfringens form meat ang vegetable sample by comparing with cultural method. They used five C. perfringens and 11 non-C. perfringens samples for this study. DNA were extracted from the culture and done the sensitivity and specificity test of the sample using Rt PCR. When compared with cultural method and Rt PCR techniques no negative control showed as positive reaction as well as no positive strains showed negative reactions. In sensitivity test from food sample, culture method detected strains of 52 positive whereas Real-time PCR detected 51 positive strains, but the study reported that culture method showed extremely wane result for the sample from vegetable when it compares with Real-time PCR technique (Chon, 2012).
1.2 Multiplex PCR
This is also a molecular based detection technique and it is another type of PCR. The main advantage of using multiplex PCR is accuracy, sensitivity and specificity. They are fast and specific for the detection of low copy number of genes. Multiplex PCR is highly useful for the quick detection of C. perfringens toxin genes concurrently in one step PCR.
As in the studies for detecting and typing of C. perfringens from the different chicken type  (Guran & Oksuztepe, 2013), they are planned to detect cpa ,cpb,elx,Ia,cpe and cpb2 toxin genes, isolated from different parts of the chicken.  They have collected 200 samples of parts of chicken from different retail shop which are contaminated by C. perfringens and found 558 positive strains from the isolates. DNA was extracted from the culture and undergone multiplex PCR reaction. In 558 positive strains, 545 were determined as type A, it is because cpa toxin is a common toxin in all C. perfringens types and its very dominant when it compare with other toxins

  1. Multiple Locus Variable–Number Tandem Repeat Analysis (MLVA)

This molecular based detection method is widely used for strain typing and population genetics studies of pathogenic microorganism and this method is based on  PCR amplification of variable tandem repeats (VNTRs). Strain typing is important to understand the epidemiology of the C. perfringens infections and to study ecology and evolution of the organism. MLVA has proved its efficiency in typeability, discriminate power, cost of the media, accessibility, and ease in the performance.
Variable tandem repeats are a polymorphic DNA segment from multiple genomic loci. They identified five different variable tandem repeat loci and four of them are encoded with protein genes. To analyse typeability, reproducibility, and discriminate power of the research and the authors research around 112 C. perfringens isolates from samples. Sawires and Songer (2005) studies showed the determination of direct relationship between the analysed number of loci and number of MLVA genotypes. “The numerical index of discrimination for all five VNTRs loci is 0.995”. it means that if two different samples isolated from population, they belong to different MLVA genotypes in 99.5% occasions. The main application of MLVA is for all the five loci, no strain showed null alleles (Sawires & Songer, 2005)
The reports from Korea (HAN SANG YOO, SANG UN LEE, & PAR, 1996)  reports that the most prevalent type of C. perfringens in calves and chicken was type A. The result also shows thatsamples isolated from piglet contain alpha and beta toxin which indicates that type C are present in piglet which causes major causative disease, necrotic enteritis in human as well in animals including piglets. When doing research with hospitalized Antibiotic-Associated Disease (AAD) patients, the prevalence of C. perfringens CPent was less than 1% when measured by ELISA technique (Heimesaat, Granzow, Leidinger, & Liesenfeld, 2005). Whereas two studies from UK reported the range of 8-15% CPent from more than 200 AAD. but the ELISA technique is not efficient because If the sensitivity is too low, there are chances to show some false negative results. Also, it can affect the stability of the CPent which can lead to inactivation. When targeting alpha toxinogenic and enterotoxinogenic C. perfringens in the fecal of healthy Japanese infants and adults, alpha toxin observed significantly high proportion in infants when compared with adult (Kopliku et al., 2015). In summarise, all the researchers observed that C. perfringens toxinotypes A is the most ubiquitous causative factors in all cases.
In the test by Uzal et al. (1997)(Uzal, Plumb et al. 1997) showed sensitivity and specificity of the toxins detection was competent , but the process is not applicable for food outbreaks, because incubation time for sample preparation alone is 12hr. Hernández et al. (2017)did a research to quantify the C. perfringens from milk using Real Time PCR techniques. Although Hernández et al. (2017) were able to find key findings and high sensitivity in the research, the critiquing part in the paper was not mentioning the incubation time for sample preparation. Even though  the presence of C. perfringens reported within 4hr for food outbreak in military camp by Loh et al. (2008), it took 1day to find out the quantitative amount of spore in the organism.

3.   Conclusion

       Molecular based techniques such as PCR, MLVA, Multiplex PCR is an excellent tool to find the specificity and sensitivity of the C. perfringens. This is rapid enumeration technique to find the presence of disease causing organisms from different sources like food, soil, gastrointestinal of human and animals. The main advantages of using such molecular based techniques is its speed outbreak, low risk of cross contamination, highly sensitive and specific. Although these techniques are efficient, the major drawback in this tool is that at some point of time it shows false negative result due to the presence of PCR inhibitors in the sample. This technique may hold a key as an effective preventive measure against C. perfringens which is the major causative factor for food poisoning.

4.   References

Uncategorized References
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Chon, J.-W. (2012). Development of Real-Time PCR for the Detection of Clostridium perfringens in Meats and Vegetables. Journal of Microbiology and Biotechnology, 22(4), 530-534. doi:10.4014/jmb.1107.07064
Freedman, J. C., Theoret, J. R., Wisniewski, J. A., Uzal, F. A., Rood, J. I., & McClane, B. A. (2015). Clostridium perfringens type A-E toxin plasmids. Res Microbiol, 166(4), 264-279. doi:10.1016/j.resmic.2014.09.004
Guran, H. S., & Oksuztepe, G. (2013). Detection and typing of Clostridium perfringens from retail chicken meat parts. Lett Appl Microbiol, 57(1), 77-82. doi:10.1111/lam.12088
HAN SANG YOO, SANG UN LEE, K. Y. P., & PAR, Y. H. (1996). Molecular Typing and Epidemiological Survey of Prevalence of Clostridium perfringens Types by Multiplex PCR.
Hassan, K. A., Elbourne, L. D., Tetu, S. G., Melville, S. B., Rood, J. I., & Paulsen, I. T. (2015). Genomic analyses of Clostridium perfringens isolates from five toxinotypes. Res Microbiol, 166(4), 255-263.
Heimesaat, M. M., Granzow, K., Leidinger, H., & Liesenfeld, O. (2005). Prevalence of Clostridium difficile toxins A and B and Clostridium perfringens enterotoxin A in stool samples of patients with antibiotic-associated diarrhea. Infection, 33(5-6), 340-344. doi:10.1007/s15010-005-5067-3
Hernández, M., López-Enríquez, L., & Rodríguez-Lázaro, D. (2017). Quantitative Detection of Clostridium perfringens by Real-Time PCR in Raw Milk. Food Analytical Methods, 10(5), 1139-1147.
Kopliku, F. A., Schubert, A. M., Mogle, J., Schloss, P. D., Young, V. B., & Aronoff, D. M. (2015). Low prevalence of Clostridium septicum fecal carriage in an adult population. Anaerobe, 32, 34-36.
Loh, J. P., Liu, Y. C., Chew, S. W., Ong, E. S., Fam, J. M., Ng, Y. Y., . . . Ooi, E. E. (2008). The rapid identification of Clostridium perfringens as the possible aetiology of a diarrhoeal outbreak using PCR. Epidemiol Infect, 136(8), 1142-1146.
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NIILO, L. (1980). Clostridium perfringens in Animal Disease: A Review of Current Knowledge.
Park, M., Deck, J., Foley, S. L., Nayak, R., Songer, J. G., Seibel, J. R., . . . Rafii, F. (2016). Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids. Anaerobe, 38, 25-35. doi:10.1016/j.anaerobe.2015.11.003
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Popoff, M. R., & Bouvet, P. (2013). Genetic characteristics of toxigenic Clostridia and toxin gene evolution. Toxicon, 75, 63-89. doi:10.1016/j.toxicon.2013.05.003
Sabry, M., Abd El-Moein, K., Hamza, E., & Abdel Kader, F. (2016). Occurrence of Clostridium perfringens Types A, E, and C in Fresh Fish and Its Public Health Significance. J Food Prot, 79(6), 994-1000.
Sawires, Y. S., & Songer, J. G. (2005). Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens. Anaerobe, 11(5), 262-272. doi:10.1016/j.anaerobe.2005.03.004
Sigrid Brynestad, & Granum, P. E. (2001). Clostridium perfringens and foodborne infections.
Uzal, F., Plumb, J., Blackall, L., & Kelly, W. (1997). PCR detection of Clostridium perfringens producing different toxins in faeces of goats. Letters in applied microbiology, 25(5), 339-344.
Yonogi, S., Kanki, M., Ohnishi, T., Shiono, M., Iida, T., & Kumeda, Y. (2016). Development and application of a multiplex PCR assay for detection of the Clostridium perfringens enterotoxin-encoding genes cpe and becAB. J Microbiol Methods, 127, 172-175. doi:10.1016/j.mimet.2016.06.007

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