Development and validation of novel methods for the study of Staphylococcus aureus PVL strains

Since the initial association of the Panton Valentine leucocidin (PVL) toxin with highly virulent strains of Staphylococcus aureus found in the community, a firm epidemiological link has been established between the PVL encoding genes and community-acquired strains of both meticillin resistant (CA-MRSA) and susceptible (CA-MSSA) Staphylococcus aureus. While most research has predominantly concentrated on the genotyping of CA-MRSA strains, PVL-MSSA appear to pose an increasing public health risk. Currently though, there exists a dearth of epidemiological data on PVL-MSSA strains, particularly with regard to the lukS and lukF genes which encode the PVL toxin. This first aim of the present study therefore was to explore the genetic diversity of a group of PVL-MSSA clinical local isolates in order to contribute to the limited current data and provide insight into the evolution and emergence of PVL-MRSA isolates. In addition, as current typing systems are cumbersome, time consuming and expensive, this present study was also aimed at the development of a rapid high resolution melt (HRM) typing system for the characterisation of PVL-positive isolates. The PVL toxin is encoded for by two highly conserved adjacent genes (lukF and lukS) which are co-transcribed. Variations in these genes correlate with a strain’s genotype. Therefore, the present study set out to genotype isolates based on the four major SNPs at positions 527 and 663 of the lukS gene and 1396 and 1729 of the lukF gene. The final aim of the present study was the development of an enzyme linked immunosorbent assay (ELISA) system (for the detection and quantification of both PVL and alpha haemolysin) that has potential application in clinical diagnosis and as a research tool. Characterisation of a collection of UK PVL-MSSA isolates by MLST and spa typing which is presented in the present study, showed a genetic similarity to circulating PVL-MRSA strains, with 94.7% of the isolates belonging to CA-MRSA related genetic backgrounds (ST1, ST22, ST30, ST772 and ST88). Three novel spa types (t6642, t6643 and t6769) and a novel ST (ST1518) were however detected in this population. Furthermore, the presence of identical PVL phages and haplotypes in the PVL-MSSA isolates to those previously described in PVL-MRSA isolates point strongly at the role these strains may play as precursors of emerging lineages of clinical significance. The HRM assay developed in this study was able to accurately genotype PVL-positive isolates based on the double allelic variations in both the lukS and lukF genes. The high degree of sensitivity of this technique was clearly demonstrated by its ability to differentiate between the lukS A527/G663 and G527/T663 genotypes which theoretically should have the same melt temperature. Despite the issues in data interpretation, which arose following attempts to improve the discrimination of this technique by the addition of a third locus (spa), the technique still showed potential as a useful tool for the rapid genotyping of PVL-positive isolates. While HRM was useful in rapidly detecting and genotyping PVL-positive isolates, the actual level of protein production of both PVL and HLA toxins could only be determined following the development and validation in the present study of a simple competitive ELISA platform which exploited the high affinity biotin/streptavidin interaction to improve sensitivity. This technique would be especially useful in settings which lack the specialised equipment required for genetic studies like HRM. In summary, in addition to contributing to the limited epidemiological information about PVL-MSSA strains and demonstrating a clear role for these strains in the evolution of PVL-MRSA strains, the present study has developed two distinct methods to aid the study of S. aureus PVL producing strains which are becoming a significant healthcare problem worldwide. The present study will contribute to our understanding of these strains and to the development of intervention strategies to curb their spread and threat.