Antibiofilm effects of N,O-acetals derived from 2-amino-1,4-naphthoquinone are associated with downregulation of important global virulence regulators in methicillin-resistant Staphylococcus aureus

Abstract

Despite the existing antibiotics, antimicrobial resistance is a major challenge. Consequently, the development of new drugs remains in great demand. Quinones is part of a broad group of molecules that present antibacterial activity besides other biological properties. The main purpose of this study was to evaluate the antibiofilm activities of synthetic N,O-acetals derived from 2-amino-1,4-naphthoquinone [7a: 2-(methoxymethyl)-amino-1,4-naphthoquinone; 7b: 2-(ethoxymethyl)-amino-1,4-naphthoquinone; and 7c: 2-(propynyloxymethyl)-amino-1,4-naphthoquinone] against methicillin-resistant Staphylococcus aureus (MRSA). The derivatives 7b and 7c, specially 7b, caused strong impact on biofilm accumulation. This inhibition was linked to decreased expression of the genes fnbA, spa, hla and psmα3. More importantly, this downregulation was paralleled by the modulation of global virulence regulators. The substitution of 2-ethoxymethyl (7b) in comparison with 2-propynyloxymethyl (7c) enhanced sarA-agr inhibition, decreased fnbA transcripts (positively regulated by sarA) and strongly impaired biofilm accumulation. Indeed, 7b triggered intensive autolysis and was able to eliminate vancomycin-persistent cells. Consequently, 7b is a promising molecule displaying not only antimicrobial effects, but also antibiofilm and antipersistence activities. Therefore, 7b is a good candidate for further studies involving the development of novel and more rational antimicrobials able to act in chronic and recalcitrant infections, associated with biofilm formation.

Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments

“…The influence of high bacterial load in environments such those found in biofilms on antimicrobial persistence in vitro and in vivo has been described by others ( Thulin et al, 2006 ; Rio-Marques et al, 2014 ; Karslake et al, 2016 ; Li et al, 2017 ; Vulin et al, 2018 ; Novais et al, 2020 ). Persisters have conventionally been detected by indirectly determining CFUs after treating the bacterial cells with a high concentration of an antibiotic, or from bacterial cells that do not grow in the presence of the antibiotic, but regrow under a microfluidic device after drug removal ( Orman and Brynildsen, 2015 ; Yu et al, 2019 ).…”

“…The model used in this study to generate persisters was based on previous work done with Staphylococcus aureus strains ( Novais et al, 2020 ). In this system (here called biofilm like-environment) high bacterial load is inoculated in order to allow the formation of an initial bacterial film on the smooth surface of a cellophane membrane placed onto agar media containing antibiotics to mimic bacterial agglomeration found in some environments such as those encountered in biofilms.…”

Occurrence of antimicrobial persistence in Streptococcus pyogenes

“…The in uence of bio lm development and high bacterial density on antimicrobial resistance in vitro and in vivo has been described by others (Thulin et al, 2006;Rio-Marques et al, 2014;Karslake et al, 2016;Li et al 2017;Novais et al, 2020). Despite the fact that it might be a reason for failures in the drug therapy, antimicrobial persistence remains unexplored in Streptococcus pyogenes.…”

“…The model used in this study to generate persistent cells was based on previous work done with Staphylococcus aureus strains (Novais et al, 2020). GAS strains (n=211) were grown in Todd Hewitt broth containing 0.5% (w/v) of yeast extract (THB-Y) at 37 o C/6 h in order to reach the exponential phase.…”

“…However, patterns of purely intracellular bacteria were observed in less than half of the biopsies analyzed (Thulin et al 2006). Some in vitro studies have demonstrated the effect of bio lm and high cell density (HCD) in the failure of antibiotics to eliminate E. coli, mycobacteria, methicillin-resistant Staphylococcus aureus (MRS) and also in GAS (Nielsen et al, 2011;Ferro et al, 2015;Fiedler et al, 2015;Coates et al, 2018;Novais et al, 2020). Based on these studies, we hypothesized that persistent cells may also occur in GAS strains under these conditions.…”