Isolates with low-level vancomycin resistance associated with persistent methicillin-resistant Staphylococcus aureus bacteremia

Abstract

Low-level vancomycin-resistant Staphylococcus aureus (vancomycin-intermediate S. aureus [VISA] and heterogenous VISA [hVISA]) is increasingly reported and leads to glycopeptide treatment failure. Various phenotypic features have been reported for these isolates, but the genetic changes leading to hVISA and VISA have yet to be clearly determined. We assessed phenotypic, antibiotic resistance, and genomic changes by using genomic DNA microarray comparison and sequencing of selected loci in five pairs of clinical hVISA/VISA strains and the initial methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained prior to vancomycin therapy. The isolates were from adult patients in Australia and New Zealand who had persistent MRSA bacteremia (>7 days) while receiving vancomycin therapy. In all cases, the initial isolates were found to be fully vancomycin-susceptible Staphylococcus aureus (VSSA). The hVISA/VISA phenotype was associated with increased cell wall thickness, reduced autolytic activity in four of five hVISA/VISA strains, and a striking reduction in biofilm formation compared to the parent strains in all pairs. All five pairs appeared to be isogenic, and genomic DNA microarray comparison suggested that major genetic changes are not required for the development of the resistant phenotype in these strains. No sequence differences were found in the agr locus or the tcaRA genes for any pair, but a marked reduction in RNAIII expression was found in four pairs. In summary, hVISA/VISA arises from fully VSSA during persistent infection that fails to respond to glycopeptide therapy and is associated with significant phenotypic changes, including a marked reduction in biofilm-forming ability. These clinically derived pairs of isolates will be a useful resource to elucidate the genetic mechanism of resistance in hVISA/VISA strains.

FIG. 1.

Vancomycin population analysis profiles of initial patient isolates prior to vancomycin therapy (JKD6000, JKD6009, JKD6021, JKD6052, and JKD6004) and later blood culture isolates after persistent bacteremia despite vancomycin therapy (JKD6001, JKD6008, JKD6023, JKD6051, and JKD6005). Also included are the hVISA reference strain Mu3 (ATCC 700698) and VSSA strain ATCC 25923.

FIG. 2.

Pulsed-field gel electrophoresis of SmaI-restricted genomic DNA for five VSSA and hVISA/VISA pairs. Banding patterns are identical for strains JKD6004 and JKD6005 (lanes 3 and 4), JKD6008 and JKD6009 (lanes 5 and 6), and JKD6051 and JKD6052 (lanes 9 and 10). A two-band difference was noted for strain pairs JKD6000 and JKD6001 (lanes 1 and 2) and JKD6021 and JKD6023 (lanes 7 and 8). C, control strain (NCTC8325).

FIG. 3.

Transmission electron microscopy of parent and hVISA/VISA strains. Magnification, ×30,000. The values given under each image are the mean and 95% confidence interval of the cell wall thickness of the cells in nanometers.

FIG. 4.

Autolytic assay results for five VSSA and hVISA/VISA pairs. The results are expressed as the averages of two independent experiments.

FIG. 5.

Relative RNAIII expression of hVISA/VISA strains compared to that of the parent strains, determined by quantitative real-time PCR and normalized to 16S rRNA expression.

FIG. 6.

(A) Analysis of biofilm formation by five VSSA and hVISA/VISA pairs, using a 24-h polystyrene microtiter plate adherence assay. (B) Quantification of biofilm formation determined by crystal violet staining and reading with an enzyme-linked immunosorbent assay plate reader (OD₅₇₀). Results are expressed as the averages of five independent experiments.