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The molecular mechanism underlying the development of vancomycin-intermediate Staphylococcus aureus (VISA) remains unclear. The abuses of antibacterial compounds lead to a change in the bacterial susceptibility patterns. Therefore, we examined the effect of Chlorhexidine (CHX) on in vitro development of VISA and reported CHX-selected VISA mutant Tm1 with phenotypic features similar to the clinical VISA isolates. WalKR, VraTSR, and GraSR are the most common regulatory systems involved in VISA evaluation. The expression of these systems, as