Amplification of 16sr RNA for Staphylococcus Aureus Isolates From Food Samples

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Abstract

Staphylococcus aureus, a gram positive bacterium, is one of the most common clinical and food borne pathogens worldwide. S .aureus produces very important virulence factors including Staphylococcus enterotoxins (SEs) which are the main causes of diarrhea, vomiting and other symptoms associated with S.aureus infections. The conventional methods used for identification of S.aureus are time consuming and the reliability of these methods have been reported to be between 80-90%. Currently the molecular techniques based on PCR amplification of 16S rRNA of S.aureus for rapid and specific detection is widely used approach. In this study S.aureus strains are obtained from various food samples which are further confirmed by molecular characterization using 16S rRNA primer. The amplification of 1267 base pair fragments specific for 16SrRNA of S.aureus revealed positive amplification with all the isolates previously identified as S.aureus with bacteriological examination and with Staphylococcus enterotoxins genes (SEA, SEB, SEC, SED, and SEE). Two sets of primer pairs were used, the first was SauF 234 and the other was SauR 1501. The objective of the study was to examine the recovered strains phenotypically by conventional methods and genotypically by PCR for direct detection of S.aureus 16SrRNA gene which serves as internal control. This became the confirmation for the developed multiplex PCR for the identification of S.aureus isolated from various types of food samples. The developed multiplex polymerase chain reaction (PCR) method will be useful for the detection and identification of S.aureus from foods, clinical samples and environmental surveys.

Introduction

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 [1]. 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 this method 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

Staphylococcal infection is currently widespread throughout the world and has prompt interest and concern for the rapid detection of toxigenic strains of Staphylococcus aureus. Staphylococcus aureus is a major human pathogen and causes a variety of food borne infections, nosocomial and community acquired infections [18, 19, 20]. Molecular based methods have well grounded potential to overcome insufficiencies of identification procedures associated with the results based on biochemical characteristics. 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 a public safety concern in foods. The strains of S.aureus developing five toxins are further identified by SDS-PAGE and confirmed by immune blotting technique [21, 22]. Hence the developed mPCR method will help in faster diagnosis of Staphylococcus aureus food poisoning. The purpose ofthis study was firstly, characterization of recovered strains phenotypically by conventional methods and genotypically by PCR for direct detection of S.aureus 16SrRNA gene [23] and staphylococcus enterotoxin genes foe SEA, SEB, SEC, SED and SEE. The most impressive advantages of PCR based detection method in comparison to the standard microbiological detection method are admittedly considered speed, sensitivity and exactness of the obtained results.

Aknowledgement

I am grateful to my guide M.ShivaPrakash, Scientist-D , National institute of Nutrition (ICMR),Hyderabad for his valuable guidance and encouragement. I am also thankful to all my family members and friends for their constant support.

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