IS256-Mediated Overexpression of the WalKR Two-Component System Regulon Contributes to Reduced Vancomycin Susceptibility in a Staphylococcus aureus Clinical Isolate.

Makoto Kuroda,Hideaki Hanaki,Toshio Ohshima,Jun Ohsuga,Hidehito Matsui,Tsuyoshi Sekizuka

doi:10.3389/fmicb.2019.01882

Abstract

Vancomycin (VAN)-intermediate-resistant Staphylococcus aureus (VISA) is continually isolated globally, with a systematic review suggesting a prevalence of 2% in all blood culture samples. Most VISA strains exhibit common characteristics, such as a thickened cell wall, reduced autolysis, and attenuated virulence. Here, based on multi-omics approaches, we have characterized clinical VISA isolates obtained through prolonged antimicrobial treatment in a single patient. All VISA isolates were isogenic, based on multi-locus sequence typing (MLST) ST5, SCCmec type II (2A), and spa type t17639. Core-genome single nucleotide variations (SNVs) found among thirteen isolates during the patient’s hospitalization, indicated clonality, but not notable genetic features of the VISA phenotype. We determined the complete genome sequence of VAN-susceptible strain KG-03 (minimum inhibitory concentration [MIC] 0.5 μg/mL) and two VISA strains, KG-18 and KG-22 (MIC 8.0 and 4.0 μg/mL, respectively). Comparative genome analysis showed remarkable strain-specific IS256 insertions. RNA-Seq transcriptome analysis revealed IS256-mediated overexpression of the walKR two-component system in VISA KG-18, possibly leading to modulation of cell wall integrity (lytM and sceD) and surface charge (mprF and dltABCD). In addition, secretome analysis indicated that cell wall-anchored proteins (Protein A, SasG, and SdrD) were significantly decreased. KG-18 and KG-22 exhibit thickened cell wall, and are relatively resistant to lysostaphin, which cleaves a staphylococcus-unique pentaglycine chain in the peptidoglycan. We conclude that KG-18 achieved reduced susceptibility to VAN by IS256-mediated WalKR overexpression, leading to a markedly thickened cell wall for trapping free VAN molecules with redundant D-Ala-D-Ala targets. In addition, a positively charged surface with lysyl-phosphatidylglycerol and depolarization of wall teichoic acid could contribute to inhibiting cationic daptomycin and VAN antimicrobial activity. Comparative omics approaches in this study strongly suggest that fully complete and annotated genome sequences will be indispensable for characterizing overall VISA phenotype.

Highlights

Staphylococcus aureus, one of the major nosocomial and community-acquired pathogens, causes a variety of clinical problems, including infections of the skin and soft tissues (Lowy, 1998)
We further investigated the structural variations by IS256 insertion throughout the genome sequences
To determine the factors involved in vancomycin intermediate resistant (VISA) phenotype, we focused on three isolates (KG-03, KG-18, and KG-22), that harbored multiple strain-specific chromosomal IS256 insertions (Figure 1B)

Summary

Introduction

Staphylococcus aureus, one of the major nosocomial and community-acquired pathogens, causes a variety of clinical problems, including infections of the skin and soft tissues (Lowy, 1998). Since the 1960s, the prevalence of methicillin-resistant S. aureus (MRSA), which has been associated with higher rates of morbidity and mortality than methicillin-susceptible S. aureus (MSSA) (Kaye et al, 2004), has increased at a dramatic rate (Maree et al, 2007). Glycopeptides, such as vancomycin (VAN) and teicoplanin (TEIC), are primary and effective antimicrobial drugs for treating MRSA. A retrospective study suggests that reduced susceptibility to vancomycin dates back at least to 1987 in the United States (Jackson and Hicks, 1987)

Methods

Results

Discussion

Conclusion