Evaluation of three different molecular markers for the detection of Staphylococcus aureus by polymerase chain reaction

Abstract

The aim of this study was to target three genes of Staphylococcus aureus-fmhA (coding for a factor of unknown function), catalase and femA (coding for a factor essential for methicillin resistance) to establish and validate a PCR assay for the detection of this pathogen. Two pairs of primers were designed for fmhA and one pair each for catalase and femA genes. The PCR assays were standardized and found to give specific amplicons under similar reaction parameters. Target specificity of the primers was confirmed by DNA sequencing of the amplicons. While the initial inclusivity and exclusivity test reactions were in agreement in case of three of the primer pairs, one pair based on fmhA gene produced a non-specific product with a template DNA used in exclusivity test reactions. Forty-five strains of S. aureus were subjected to these PCR assays for their evaluation. Three among the four pairs of primers, one against each gene detected all the 45 strains precisely whereas one of the PCR assays using primers targeting the fmhA gene did not generate the specific amplicon with several of the strains. Seven unidentified strains of Gram-positive cocci subjected to these PCR assays produced negative results for each culture. Six of the strains were identified as Staphylococcus haemolyticus and one strain as Staphylococcus arlettae by 16S ribosomal gene analyses. All the three assay systems showed a detection limit of 100 cells per 20 μl reaction assay. For validation of these assay systems, 80 coded samples of 11% skimmed milk spiked with different pathogens were received from NICED (National Institute of Cholera and Enteric Diseases), Kolkata and subjected to these PCR assays. All the three assays could detect S. aureus correctly in two of the samples. Amongst 150 raw milk samples, 36 (24%) were found positive for S. aureus. We conclude that fmhA, catalase and femA genes are conserved in S. aureus and, therefore, could be used as specific targets for its detection and identification by PCR. The protocols developed herein could be used for rapid and specific detection of this pathogen in food, clinical and environmental samples, especially milk.

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).