A novel multiplex PCR-based tool of typing strains of Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and morbidity and mortality rates associated with this pathogen have increased markedly in recent years. The prevalence of MRSA is no longer confined to hospital patients since MRSA infections have been increasingly reported in the community. More recently, community-acquired MRSA strains have become more prevalent and their infections are no longer confined to the community but have started to replace hospital-acquired MRSA in some health care settings. MRSA strains are generally resistant to several classes of antibiotics and are therefore difficult and costly to treat. Consequently, an understanding of the epidemiological characteristics of S. aureus is an essential tool for the management of its infections in both the hospital and community setting. The purpose of any epidemiology study, such as the investigation of an outbreak, is to identify the potential source(s) of an infection and to monitor their dissemination. The early identification of an outbreak, making use of a rapid, precise and simple MRSA typing technique, can lead to prompt and effective precautions that avoid further spread of the infection. Pulsed-field gel electrophoresis (PFGE) is considered the gold standard for MRSA typing and has been recently supported by multi-locus sequence typing (MLST). However, technical limitations restrict the use of PFGE and MLST in the majority of routine hospital laboratories: they are timeconsuming, expensive, require specific expertise and specialist equipment. In this study a novel typing technique was developed for S. aureus, based on single nucleotide polymorphism (SNP) variations in and around SmaI-restriction sites (CCCGGG), following the analysis of eighteen S. aureus strains that have had their genomes sequenced. The developed SmaI-multiplex PCR genotyping method combines the high discriminatory power and reproducibility of PFGE, with the simplicity of a multiplex PCR-based technique that can be performed in a routine clinical laboratory. The validity of SmaI-multiplex PCR was carefully assessed in comparison with PFGE and MLST against many sequenced S. aureus strains and showed high discriminatory power and reproducibility. There was also high level of concordance in the clustering of strains analyzed by each of the techniques. The SmaI-multiplex PCR was ultimately evaluated against a large number of clinical S. aureus outbreak strains and was shown to be a useful tool for providing reliable epidemiological information for the investigation of clinical staphylococcal outbreaks. The newly developed technology is suitable for high throughput sample analysis, is relatively cheap and provides reliable and comparable genotyping data. At the same time, the SmaI-multiplex PCR meets most of the criteria of practical typing method: it is simple, inexpensive, highly discriminatory and does not require sophisticated equipment or expertise. Consequently, SmaImultiplex PCR could be used routinely in any clinical microbiology laboratory since it relies on standard clinical laboratory apparatus (i.e. PCR machine and agarose gel electrophoresis). SmaI-multiplex PCR proved to be more discriminatory than MLST/SCCmec typing, but less discriminatory than PFGE. Currently the SmaImultiplex PCR protocol takes between 4 to 6 hours; however, it would be possible to adapt this technology towards an automated genotyping assay using RT-multiplex PCR. This would reduce the processing time to less than 60 minutes. Since individual targets are identifiable on the basis of the size of their amplicons, the RT-PCR output could be processed directly via dedicated analytical software.