Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus

Abstract

Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.

Impairment of the Cell Wall Ligase, LytR-CpsA-Psr Protein (LcpC), in Methicillin Resistant Staphylococcus aureus Reduces Its Resistance to Antibiotics and Infection in a Mouse Model of Sepsis

“…Mu50 is a vancomycinintermediate S. aureus (VISA), while BA01611 is a vancomycinsensitive S. aureus. Both Mu50 and Mu3 contain a mutation in another LCP member, lcpA, and both are VISA (Katayama et al, 2016). Introduction of mutated lcpA into vancomycin-susceptible S. aureus (VSSA) strain N315 IP increased the level of VISA (Katayama et al, 2016).…”

“…Both Mu50 and Mu3 contain a mutation in another LCP member, lcpA, and both are VISA (Katayama et al, 2016). Introduction of mutated lcpA into vancomycin-susceptible S. aureus (VSSA) strain N315 IP increased the level of VISA (Katayama et al, 2016). Thus, it seems that both normal LcpA and LcpC proteins could keep VISA strains sensitive to teicoplanin.…”

“…Complete loss of WTA occurred when all three lcp genes were deleted, significantly reducing the antimicrobial resistance (Dengler et al, 2012) and impairing the cell division process (Chan et al, 2013). The deficiency of LcpA reduced the resistance to oxacillin (Schaefer et al, 2017) in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3 (Katayama et al, 2016).…”

Role of the msaABCR Operon in Cell Wall Biosynthesis, Autolysis, Integrity, and Antibiotic Resistance in Staphylococcus aureus

“…VISA strains are phenotypically and genotypically different from vancomycinsusceptible strains (VSSA) strains. Increased cell wall thickness (44), reduced autolysis (45), altered cell WTA content (46,47), and decreased virulence (48) are examples of altered phenotypes associated with VISA strains, which have been explained in detail in reviews (49,50). Differentially expressed or mutated genes involved in Staphylococcus metabolism (47,51), cell wall synthetic pathways, and murein hydrolases are examples of altered genotypes in VISA strains (47,49,50,52).…”

Polycyclic Polyprenylated Acylphloroglucinols: An Emerging Class of Non‐Peptide‐Based MRSA‐ and VRE‐Active Antibiotics

“…However,n ot only the resistance to b-lactam antibiotics is problematic in the case of S. aureus, but also the emergence of vancomycin-intermediate-resistant (VISA) and vancomycinresistant Staphylococcus aureus (VRSA) strains. [3][4][5][6][7][8][9][10][11][12] Apart from VRSA, the vancomycin-resistant Enterococcus (VRE) strains Enterococcus faecium and Enterococcus faecalis harboring the vanHAX gene are ag reat burden to the healthcare system. [13] Thus far, there are only two drugs approved by the FDAt ot reat specifically vancomycinresistant enterococcal infections.L inezolid, an oxazolidinone-based antibiotic is known to inhibit VRE isolates with am inimal inhibitory concentration (MIC) value of 0.75 to 4 mgmL À1 whereas quinupristin/dalfopristin is only active on Enterococcus faecium strains,w ith comparable MIC values reported in the literature (Figure 1).…”