Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype

Abstract

This study investigated resistance against trishexylaminomelamine trisphenylguanide (THAM-3ΦG), a novel antibacterial compound with selective microbicidal activity against Staphylococcus aureus. Resistance development was examined by culturing methicillin resistant S. aureus (MRSA) with sub-lethal doses of THAM-3ΦG. This quickly resulted in the formation of normal (WT) and small colonies (SC) of S. aureus exhibiting minimal inhibitory concentrations (MICs) 2× and 4× greater than the original MIC. Continuous cell passaging with increasing concentrations of THAM-3ΦG resulted in an exclusively SC phenotype with MIC >64 mg/L. Nuclear magnetic resonance (NMR)-based metabolomics and multivariate statistical analysis revealed three distinct metabolic profiles for THAM-3ΦG treated WT, untreated WT, and SC (both treated and untreated). The metabolome patterns of the SC sample groups match those reported for other small colony variants (SCV) of S. aureus. Supplementation of the SCV with menadione resulted in almost complete recovery of growth rate. This auxotrophism was corroborated by NMR analysis revealing the absence of menaquinone production in the SCV. In conclusion, MRSA rapidly acquires resistance to THAM-3ΦG through selection of a slow-growing menaquinone auxotroph. This study highlights the importance of evaluating and monitoring resistance to novel antibacterials during development.

Quantitative 1H NMR Metabolomics Reveal Distinct Metabolic Adaptations in Human Macrophages Following Differential Activation

“…Then, 1D 1 H NMR spectra acquisition was performed using the Bruker-supplied excitation sculpting (ES)-based ‘zgesgp’ pulse sequence [71,72], and NMR spectra were recorded with 256 scans and a 1 H spectral window of 9615.38 Hz. Free induction decays (FIDs) were collected with 32K data points and a dwell time interval of 52 µsec, amounting to a data acquisition time of 1.7 s. Recovery delay (D1) times between acquisitions were set to 1 s, resulting in an overall 2.7 s relaxation recovery delay between scans [73,74]. DSS chemical shift referencing and phase correction of 1D 1 H NMR spectra were conducted using Topspin software (Bruker version 3.2).…”

Pseudomonas aeruginosa Planktonic- and Biofilm-Conditioned Media Elicit Discrete Metabolic Responses in Human Macrophages

“…1D 1 H-NMR spectral acquisitions were performed using the Bruker-supplied ‘zgesgp’ pulse sequence [ 42 , 43 ]; NMR spectra were acquired with 256 scans and a 9615.38 Hz 1 H spectral window. Free induction decays were recorded with 32,000 data points and a dwell time interval of 52 microseconds, equating to a total data acquisition time of 1.7 s. Recovery delay times between data acquisitions were fixed at 1 s, amounting to an overall 2.7 s relaxation recovery delay in between scans [ 44 , 45 ]. Spectral phase correction and chemical shift referencing of DSS were performed using Topspin software (Bruker Scientific LLC, Billerica, MA, USA; version 3.6).…”

Virulence alterations in staphylococcus aureus upon treatment with the sub-inhibitory concentrations of antibiotics

“…Weaver et al . showed that S. aureus can rapidly increase resistance to rishexylaminomelamine–trisphenylguanide via the selection of a menaquinone-auxotroph SCV, which became an exclusively SCV phenotype after continuous cell passage in media with increasing drug concentrations [97] . By using skin fibroblast and lung epithelial cell infection models, Häffner et al .…”