Evaluation of three different molecular markers for the detection of Staphylococcus aureus by polymerase chain reaction
Introduction
Staphylococcus aureus is one of the most commonly found pathogenic bacteria and is hard to eliminate from the human environment (Perez-Roth et al., 2001). It is responsible for many nosocomial infections, besides being the main causative agent of food intoxication by virtue of its variety of enterotoxins (Iandolo, 1989). Routine detection of S. aureus in food is usually carried out by traditional methods based on morphological and biochemical characterization. These methods are time consuming and tedious. In addition, misclassifications with automated susceptibility testing systems or commercially available latex agglutination kits have been reported by several workers (Ruane et al., 1986; Schwarzkopf et al., 1993; Wilkerson et al., 1997; Ribeiro et al., 1999). Consequently, there is a need for methods to specifically discriminate S. aureus from other staphylococci as quickly as possible (Roberson et al., 1992; Guzman et al., 1992). Microbial genomes are being sequenced at a staggering rate. Approximately, 10% of the genes of a species in a genus are unique to each organism, and we are now beginning to appreciate the genetic diversity among bacterial strains (Versalovic and Lupski, 2002). This forms the basis for genotypic identification of microorganisms including bacterial and fungal pathogens. PCR techniques have provided increased sensitivity, allowed for more rapid processing times, and enhanced the likelihood of detecting bacterial pathogens. In the last 15 years, several detection methods have been proposed for foodborne pathogens to replace the time-consuming classical techniques (Candrian, 1995; Hill, 1996; Olsen, 2000). Specific primers for the detection of S. aureus have been directed to the nuc gene encoding thermostable nuclease (Wilson et al., 1991; Brakstad, 1992), enterotoxin genes (Wilson et al., 1991; Johnson et al., 1991; Tsen and Chen, 1992; Mantynen et al., 1997; Becker et al., 1998), tst gene (shock syndrome) genes coding for exfoliative toxin A and B (eta and etb, respectively) (Johnson et al., 1991) the 16–23 rDNA spacer region (Saruta et al., 1997), the 23S rDNA (Straub et al., 1999) and femA gene (Vannuffel et al., 1995; Mehrotra et al., 2000). In addition to the analysis of foods, PCR has also been successfully applied to detection and identification of pathogenic organisms in clinical and environmental samples (White et al., 1992; Simon, 1999; Olsen, 2000).
Several studies have recently focused on detection of S. aureus in milk and milk products including the use of quantitative real-time PCR (Hein et al., 2001; Alarcon et al., 2006). However, due to variability in selectivity of different primers (Klaassen et al., 2003), it is imperative to target new genes for the detection of S. aureus, so that infallible assays are developed for its detection and identification. Validation is an important requirement for the development of a PCR-based detection system. The present study describes the comparison of PCR protocols based on two new target genes fmhA and catalase, and a previously used target gene, femA (Vannuffel et al., 1995; Mehrotra et al., 2000) and consequently the development of a validated and reliable PCR assay for the rapid detection of S. aureus. The femA gene encodes a factor, which is essential for methicillin resistance and is universally present in all S. aureus isolates (Johnson et al., 1995). Catalase is a haem-containing enzyme involved in dismutation of hydrogen peroxide generated during cellular metabolism to water and molecular oxygen (Loewen, 1992). The function of the FmhA protein is not clear, however, it has significant identities to FemA (Tschierske et al., 1999).
Section snippets
Bacterial strains
A total of 107 bacterial strains were used in this study (Table 1, Table 2). The cultures were grown on Tryptone Soya Agar (TSA) (HiMedia, India) at 37 °C and also maintained in glycerol (50%) normal saline (0.85% NaCl, w/v) at −70 °C.
DNA isolation for PCR
Templates were prepared from pure cultures of S. aureus and other bacterial species by thermal extraction. Strains were grown overnight at 37 °C on Tryptone Soya Agar or with shaking in 3 ml of Brain Heart Infusion Broth (HiMedia, India). Cell pellet from 1 ml of the
Results
PCR protocols were standardized with the purified genomic DNA of S. aureus ATCC 29313. All the four pairs of primers produced the specific amplification products under the same reaction parameters. The PCR products were sequenced and the nucleotide sequences, thus obtained, revealed that the right targets were amplified. A panel of 55 cultures (Table 1) were used in the exclusivity test reactions. Three of the reaction assays did not yield any amplifications with any of the exclusivity test
Discussion
Bacterial food-borne pathogens are an important food safety issue worldwide. It is estimated that in the USA, food-borne illnesses affect 6–80 million people each year, causing up to 9000 deaths, and cost about 5 billion US dollars (Altekruse et al., 1997; Buzby and Roberts, 1997). The development of rapid, sensitive and infallible methods of detecting food-borne pathogens has received much impetus in the recent years owing to an increased public awareness of the health hazards associated with
Conclusions
The assays described in this study may be employed for the quality control of milk and other food samples as well as detection of this pathogen in clinical and environmental settings using appropriate template preparation protocols. This study indicates that femA, fmhA and catalase genes of S. aureus are universally present in this organism and their unique DNA sequences allows for targeting this pathogen for its detection by PCR. Further, the oligonucleotide primers, cat, fem and fmh1 designed
Acknowledgements
The authors are thankful to the Council of Scientific and Industrial Research, New Delhi, India for supporting this research. We are also thankful to the Department of Biotechnology, New Delhi, India and Dr. Rama Murthy, NICED, Kolkata for supporting and participating in the validation studies, respectively.
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