Development of PCR Assay for Identification of Staphylococcus Aureus and their Enterotoxins from Spoiled Food Samples

Author Name(s): Noor Nihad Abdul Hussein, Mondeddu Kiran Kumar, Dr. Varun Kumar Sharma
Author Email: noor.n.ameen@gmail.com

Abstract

Food-borne diseases due to microbial pathogens, have become a major issue of concern as they represent serious threat to the health of millions of people world – wide. Serious outbreaks of food borne disease have been documented on every continent in the past decades, illustrating both the public health and social significance of these diseases. [3, 4] Food Contamination is of major health hazards in India leading to food poisoning. The food borne diseases due to food contamination or spoilage is one of the important problems to be addressed. Staphylococcus aureus has been found to be one of the major cause of food poisoning among all the other food pathogens. S .aureus produces very important virulence factors including Staphylococcus enterotoxins (SEs) which are the main causes of diarrhoea, vomiting and other symptoms associated with food poisoning.[2, 5, 8] Therefore to minimize infection in food and water, the etiological agents and harmful toxins produced by them must be identified. Early identification will be helpful in minimizing food borne infections which helps in the prevention of diarrheal diseases. The conventional methods used for identification of S .aureus are limited to their Biological characterization only that are not only time consuming rather have limited reliability.[1, 6] Currently the molecular techniques based on PCR amplification of 16S rRNA of S.aureus for rapid and specific detection is widely used approach. This study focuses on rapid detection of S.aureus strains obtained from various food samples by development of a specific PCR Assay including a novel primer set and standardized PCR Conditions. A multiplex PCR assay has also been developed for specific Staphylococcal enterotoxins genes (SEA, SEB, SEC, SED, and SEE) produced by different isolates. Out of the 59 isolates of Staphylococcus aureus obtained from various food samples, found positive for enterotoxin infection, 15, 21, 6, 12 and 5 were found to be producing SEA, SEB, SEC, SED and SEE respectively further employing that these strains were capable of producing only one type of enterotoxin. The developed multiplex polymerase chain reaction assays will be useful for rapid detection of S. aureus and respective enterotoxins being produced from foods, clinical samples and environmental surveys. This may lead to early diagnosis of infection and help in timely prophylaxis.

Introduction

With the rapid increase in the out breaks of food poisoning Surveillance of food borne diseases is of an increasingly high priority in the public health agenda worldwide. Among all, Staphylococcus aureus is one of the most common clinical and food borne pathogen. It is reported that more than 70% of S. aureus strains produced one or more enterotoxins.[7] Staphylococcus aureus is a major human pathogen and causes a variety of food borne infections, nosocomial and community acquired infections. [11]Because of their thermal stability they remain a great hazard even in heat processed foods. Multiplex PCR were developed to detect S. aureus toxin for SEA, SEB, SEC, SED and SEE. All these toxins show emetic activity on humans. The 16S rRNA gene of Staphylococcus contain DNA sequence that are highly conserved at genus level but variable among other bacterial genera. The primer pair used in a study has been shown to be conserved in 19 Staphylococcus species and subspecies [2, 3] therefore combined detection of 16S rRNA and all the five Staphylococcal enterotoxin genes may be a reliable set of markers for the detection of Staphylococcus aureus. Primers designed for 16S rRNA gene for Staphylococcus will be a useful tool for identification and confirmation of Staphylococcus species

Conclusion

Food borne infection is currently widespread throughout the world and has evoked a prompt interest and concern for the rapid detection of toxigenic strains of Staphylococcus aureus. The most impressive advantages of PCR based detection method in comparison to the standard microbiological detection method are its speed, sensitivity and specificity and accuracy of the obtained results. Molecular based methods have well-grounded potential to overcome insufficiencies of identification procedures associated with the results based on biochemical characteristics. The purpose of our study was characterization of recovered strains phenotypically by conventional methods and genotypically by PCR for direct detection of S. aureus 16SrRNA gene and staphylococcus enterotoxin genes for SEA, SEB, SEC, SED and SEE. Out of the 59 isolates of Staphylococcus aureus obtained from various food samples, found positive for enterotoxin infection, 15, 21, 6, 12 and 5 were found to be producing SEA, SEB, SEC, SED and SEE respectively further employing that these strains were capable of producing only one type of enterotoxin. Developed PCR techniques on further investigations focus on the application to large scale of clinical samples so that this technique can be used in detecting toxigenic strain of Staphylococcus aureus for public safety concern in foods. Hence the developed mPCR method will help in faster diagnosis of Staphylococcus aureus food poisoning.

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