N-Nonyloxypentyl-l-Deoxynojirimycin Inhibits Growth, Biofilm Formation and Virulence Factors Expression of Staphylococcus aureus

Abstract

Staphylococcus aureus is one of the major causes of hospital- and community-associated bacterial infections throughout the world, which are difficult to treat due to the rising number of drug-resistant strains. New molecules displaying potent activity against this bacterium are urgently needed. In this study, d- and l-deoxynojirimycin (DNJ) and a small library of their N-alkyl derivatives were screened against S. aureus ATCC 29213, with the aim to identify novel candidates with inhibitory potential. Among them, N-nonyloxypentyl-l-DNJ (l-NPDNJ) proved to be the most active compound against S. aureus ATCC 29213 and its clinical isolates, with the minimum inhibitory concentration (MIC) value of 128 μg/mL. l-NPDNJ also displayed an additive effect with gentamicin and oxacillin against the gentamicin- and methicillin-resistant S. aureus isolate 00717. Sub-MIC values of l-NPDNJ affected S. aureus biofilm development in a dose-dependent manner, inducing a strong reduction in biofilm biomass. Moreover, real-time reverse transcriptase PCR analysis revealed that l-NPDNJ effectively inhibited at sub-MIC values the transcription of the spa, hla, hlb and sea virulence genes, as well as the agrA and saeR response regulator genes.

Keywords: Staphylococcus aureus; anti-virulence agents; antibiofilm agents; antimicrobial agents; deoxynojirimycin (DNJ); iminosugars.

Figure 1

(A) Iminosugar-based drugs on the market. (B) Iminosugars showing antibacterial activity.

Figure 2

d- and l-deoxyiminosugars studied in this work. DNJ, deoxynojirimycin; NBDNJ, N-butyl DNJ; NNDNJ, N-nonyl DNJ; NPDNJ, N-nonyloxypentyl DNJ; AMP-DNM, N-[5-(adamantan-1-ylmethoxy) pentyl] -1-DNJ.

Figure 3

Killing kinetics for S. aureus ATCC 29213 following treatment with the l-NPDNJ. Growth kinetics were monitored following exposure to l-NPDNJ at 1× MIC, 2× MIC and 4× MIC.

Figure 4

Inhibition of S. aureus ATCC29213 biofilm formation with l-NPDNJ. (A) Biofilm was quantified after crystal violet staining. Values are presented as means ±SDs. Asterisks indicate statistically significant differences between treated and untreated biofilms (* p < 0.05, ** p < 0.01, *** p < 0.001). (B) Confocal laser scanning microscopy (CLSM) analysis of the biofilm formed in the absence (upper panel) or presence of l-NPDNJ at the concentrations of 32 (middle panel) and 64 μg/mL (inferior panel).

Figure 5

Transcriptional profiling of virulence factor genes in S. aureus ATCC 29213 after treatment with l-NPDNJ determined by qRT-PCR. Data were normalized to rpoB expression. Fold-changes were calculated using treated versus untreated S. aureus cells. Asterisks indicate statistically significant differences between treated and untreated S. aureus cells (* p < 0.05, ** p < 0.01, *** p < 0.001). capC, capsule biosynthesis protein C; fnbA, fibronectin-binding protein A; fnbB, fibronectin-binding protein B; spa, surface protein A; icaA, intercellular adhesion protein A; sarA, staphylococcal accessory regulator A; sigB, RNA polymerase sigma factor B; saeR, response regulator SaeR; arlR, response regulator ArlR; agrA, accessory gene regulator A; hld, delta-hemolysin; hla, alpha-haemolysin; hlb, beta-haemolysin; lukD, pore-forming leucocidin; sea, staphylococcal enterotoxin A; psmα, alpha phenol-soluble modulin.