Increased IS256 Transposition Associated with Vancomycin Intermediate Staphylococcus aureus Mutants.

DI GREGORIO S; CUIROLO A; FERNÁNDEZ S; VERLAINE O; AMOROSO A; JORIS B; MOLLERACH M

Conferencia; 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy; 2013

American Society for Microbiology

Background: This work describes the characterization of four MRSA isolates: two recovered from a single patient suffering bone and joint infection, before and after vancomycin (VAN) treatment (D1 and D2 respectively), and two D2 laboratory derived mutants (D23C9 and D2P11) selected by independent serial passages in the presence of increasing concentrations of VAN. Methods: VAN intermediate S. aureus (VISA) and heterogeneous (hVISA) were defined by population analysis profile method (PAP-AUC). The isolates were characterized by using: transmission electron microscopy (TEM), peptidoglycan composition by RP-HPLC and mass spectroscometry of digested muropeptides, autolysis rate, and agr locus functionality by delta-heamolysis assay. Genotyping included PFGE, MLST, spa, SCCmec, agr typing and southern hybridization of PFGE with IS256 and agrAC specific probes. Results: Both clinical isolates were defined as hVISA, while both mutants were categorized as VISA. SCCmec was not typeable but Tn4001 was detected upstream the mecA-
mecR1 genes. Clonality was confirmed by PFGE. The isolates belong to ST100, t002, and agr group II, but D23C9 was agr not typeable by conventional PCR typing. An increase in the number of IS256 copies was demonstrated in both independent mutants, but a shift in the agrAC band was detected in D23C9, who didn´t show delta-haemolytic activity when the agr locus functionality was evaluated; additionally the disruption of agrB gene by IS256 insertion was demonstrated by sequencing. Increased cell wall thickness was observed by TEM. Muropeptide composition of D2, D23C9, and D2P11 showed an increase in a monomeric pentaglycin-dissacharide pentapeptide associated with a decrease of its tetrameric form when compared to D1. Moreover, D23C9 exhibited enhanced autolysis while D2P11 showed reduced autolytic activity. Conclusions: A decrease in peptidoglycan crosslinking degree along with an increased cell wall thickness was confirmed in these strains. We report an augment in the number of copies of IS256 associated with the selection of VISA strains, indicating that VAN pressure could enhance transposition frequency, being responsible for the eventual loss of agr function. Our results showed the selection of different VISA phenotypes from a single genetic background