Detection of heterogeneous vancomycin intermediate resistance in MRSA isolates from Latin America

Abstract

Background: Vancomycin is a common first-line option for MRSA infections. The heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) phenotype is associated with therapeutic failure. However, hVISA isolates are usually reported as vancomycin susceptible by routine susceptibility testing procedures.

Objectives: To detect and characterize the hVISA phenotype in MRSA isolates causing infections in nine Latin American countries.

Methods: We evaluated a total of 1189 vancomycin-susceptible MRSA isolates recovered during 2006-08 and 2011-14. After an initial screening of hVISA using glycopeptide-supplemented agar strategies, the detection of hVISA was performed by Etest (GRD) and Macro-method (MET). Isolates deemed to be hVISA were subjected to population analysis profile/AUC (PAP/AUC) and WGS for further characterization. Finally, we interrogated alterations in predicted proteins associated with the development of the VISA phenotype in both hVISA and vancomycin-susceptible S. aureus (VSSA) genomes.

Results: A total of 39 MRSA isolates (3.3%) were classified as hVISA (1.4% and 5.6% in MRSA recovered from 2006-08 and 2011-14, respectively). Most of the hVISA strains (95%) belonged to clonal complex (CC) 5. Only 6/39 hVISA isolates were categorized as hVISA by PAP/AUC, with 6 other isolates close (0.87-0.89) to the cut-off (0.9). The majority of the 39 hVISA isolates exhibited the Leu-14→Ile (90%) and VraT Glu-156→Gly (90%) amino acid substitutions in WalK. Additionally, we identified 10 substitutions present only in hVISA isolates, involving WalK, VraS, RpoB and RpoC proteins.

Conclusions: The hVISA phenotype exhibits low frequency in Latin America. Amino acid substitutions in proteins involved in cell envelope homeostasis and RNA synthesis were commonly identified. Our results suggest that Etest-based methods are an important alternative for the detection of hVISA clinical isolates.

Figure 1.

PAP of isolates confirmed as hVISA by PAP/AUC using vancomycin. cfu determinations at increasing vancomycin concentrations are shown. Results are representative of three independent experiments. Strains from the study with AUC ratio higher than 0.9 when compared with the AUC of S. aureus Mu3 are shown in black. Control strains of hVISA (ATCC 700698; Mu3) and VSSA (ATCC 29213) are represented by triangles and grey circles, respectively. Panels (a) and (b) include the results of strains collected during 2006–08 and 2011–14, respectively, which were performed by different methodologies.

Figure 2.

Phylogenetic relationships and changes in proteins associated with the hVISA phenotype. The genetic relationship was determined by SNP-based phylogenetic tree using the core genome. The clade of isolates belonging to CC5 is highlighted in grey. ^aBR (Brazilian clone); CH (Chilean/Cordobes clone); NY/JP (New York/Japan clone). ^bPAP/AUC ratio values higher than 0.9 when compared with the Mu3 strain are highlighted in dark grey and isolates with ratio values between 0.87 and 0.89 in light grey. ^cVancomycin MICs (mg/L) were determined by the agar dilution method. ^dDaptomycin MICs (mg/L) were determined by the Etest method. ^eShade intensity is proportional to the accumulation of changes in the predicted protein. Changes in bold were not present in VSSA isolates.