Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3\u03a6G Generates a Small Colony Drug-Resistant Phenotype

Alan J Weaver,Richard E Lee,Abigail Van Vuren,Brian Tripet,Valérie Copié,Martin Teintze,Rakesh Rakesh,Tami R Peters

doi:10.1038/s41598-018-28283-3

Alan J Weaver, Richard E Lee + Show 6 more

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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.

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We have reported that the polyguanide compound trishexylaminomelamine trisphenylguanide (THAM3ΦG) exhibits promising effectiveness as a novel antibacterial against S. aureus, including the methicillin-resistant strain USA300 (MRSA)[20]
This resistance likely occurs through at least two mechanisms. One of these is the development of an small colony variants (SCV) phenotype with a slower growth, and this correlates with THAM-3ΦG appearing to be active only against actively dividing cells (Fig. 2)
Additional selection with higher concentrations of THAM-3ΦG increased the minimal inhibitory concentrations (MICs) 16-fold, and this highly resistant SCV population was stable in the absence of selection, suggesting a second resistance mechanism resulting from another mutation(s)

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Introduction

We have reported that the polyguanide compound trishexylaminomelamine trisphenylguanide (THAM3ΦG) exhibits promising effectiveness as a novel antibacterial against S. aureus, including the methicillin-resistant strain USA300 (MRSA)[20]. THAM-3ΦG was initially selected from a library of guanide and biguanide-containing www.nature.com/scientificreports/. Compounds that were screened due to structural similarity to biguanides, such as chlorhexidine and alexidine, which have been used for decades in topical applications to treat both Gram-positive and Gram-negative bacterial infections, with minimal development of antibiotic resistance in the pathogens[21]. Concerns about bacterial resistance to these compounds are emerging[22,23]. THAM-3ΦG was found to be selectively active against S. aureus, including MRSA (MIC 2 mg/L). We sought to investigate the potential for MRSA to develop resistance against THAM-3ΦG and to characterize the phenotype of resulting antibacterial-resistant S. aureus isolates

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