Correlation between ITS-RFLP & sequencing for Staphylococcus Aureus typing in Lebanon. (c2009)

Abstract

Staplylocol'l'UJ' aureus has been associated with a wide range of serious communityand hospital-related infections worldwide. The molecular characterization of S. aureus is of both clinical and infection control importance. In this context we studied the virulence determinants among 130 S. aureus isolates recovered at a major tertiary care medical center in Lebanon in relation to their exfoliative toxin A (eta) and B (etb) and arginine catabolite mobile element (ACME) using PCR. PCR-RFLP analysis and sequencing of the 16S-23S DNA spacer region was done on 15 S. auretfs isolates representing the major spa types to investigate the level of 16S-23S ITS (Internal Transcribed spacer) polymorphism and thus the feasibility to be used for subtyping the different isolates at the strain level. The prevalence of the exfoliative toxin genes was 10.8% for eta and 4.6% for etb. The incidence among MRSA vs. MSSA for eta was 11 % within both groups, whereas for etb it was 3% vs. 9%, with isolates positive for eta and etb being mainly recovered from wound (14% vs. 4%). ACME, which potentially enhances vimlence and the ability to colonize humans, was detected in only one MSSA isolate. PCRmediated amplification of the 16S-23S DNA spacer region revealed the presence of two ribotypes, but the sizes were not variable enough to discriminate between the different subtypes. Ribotype I yielded two amplification products with a size of 1500 and 1800 bp, while ribotype II yielded one with a size of 1800 bp. Ribotype I was the most common accounting for 87% of the isolates. Restriction fragment length polymorphism (RFLP) profIles of the amplified spacer region obtained using TaqI allowed further differentiation between the isolates and revealed five distinct RFLP haplotypes. Moreover, sequencing of 16S-23S ITS primary product (1800 bp) allowed further characterization of each strain and the construction of a phylogenetic tree revealed the presence of two clusters designated A and B. It's noteworthy that strains belonging to the same cluster belonged to the same ITS ribotype and had similar restriction patterns. This study revealed that the 16S-23S ITS PCR-RFLP combined with sequencing of the primary product was a successful technique that can be used to generate molecular fingerprints of S. aureus for the purposes of sub typing and identification to the strain level. Moreover, this is considered the first study to demonstrate the incidence of virulent genes, ACME and genetic diversity of S. aureus isolates in Lebanon.