Tackling strong biofilm and multi-virulent vancomycin-resistant Staphylococcus aureus via natural alkaloid-based porous nanoparticles: perspective towards near future eradication

Abstract

Introduction: As a growing direction, nano-based therapy has become a successful paradigm used to address the phytogenic delivery-related problems in overcoming multivirulent vancomycin-resistant Staphylococcus aureus (VRSA) infection.

Methods: Hence, our aim was to develop and assess a novel nanocarrier system (mesoporous silica nanoparticles, MPS-NPs) for free berberine (Free-BR) as an antimicrobial alkaloid against strong biofilm-producing and multi-virulent VRSA strains using in vitro and in vivo mouse model.

Results and discussion: Our outcomes demonstrated vancomycin resistance in 13.7% of Staphylococcus aureus (S. aureus) strains categorized as VRSA. Notably, strong biofilm formation was observed in 69.2% of VRSA strains that were all positive for icaA gene. All strong biofilm-producing VRSA strains harbored a minimum of two virulence genes comprising clfA and icaA with 44.4% of them possessing all five virulence genes (icaA, tst, clfA, hla, and pvl), and 88.9% being multi-virulent. The study findings affirmed excellent in vitro antimicrobial and antibiofilm properties of BR-loaded MPS-NPs. Real-time quantitative reverse transcription PCR (qRT-PCR) assay displayed the downregulating role of BR-loaded MPS-NPs on strong biofilm-producing and multi-virulent VRSA strains virulence and agr genes in both in vitro and in vivo mice models. Additionally, BR-loaded MPS-NPs supplementation has a promising role in attenuating the upregulated expression of pro-inflammatory cytokines' genes in VRSA-infected mice with attenuation in pro-apoptotic genes expression resulting in reduced VRSA-induced apoptosis. In essence, the current study recommends the future scope of using BR-loaded MPS-NPs as auspicious alternatives for antimicrobials with tremendous antimicrobial, antibiofilm, anti-quorum sensing (QS), and anti-virulence effectiveness against problematic strong biofilm-producing and multi-virulent VRSA-associated infections.

Keywords: QS; VRSA; biofilm; inflammation; multi-virulent; nanoparticles; natural alkaloids.

Figure 1

Scanning electron microscopy characterization (A) and release (B) of berberine loaded mesoporous silica nanoparticles.

Figure 2

Distribution of vancomycin resistant Staphylococcus aureus (VRSA), strong biofilm VRSA producers and van genes in human pus and mastitis milk samples.

Figure 3

Prevalence of icaA, tst, clfA, hla, pvl and agr genes among strong biofilm producing VRSA strains recovered from human pus and mastitis milk samples.

Figure 4

Relative mRNA expression levels of agrI (A), agrIII (B), hla (C), icaA (D), clfA (E), tst (F) and pvl (G) genes expression among four strong biofilm producing and multi-virulent vancomycin-resistant Staphylococcus aureus (VRSA) strains exposed to SICs of Free berberine (Free-BR) and prepared berberine conjugated mesoporous silica nanoparticles (BR loaded MPS-NPs) comparing with unexposed ones (control) with an estimated value of 1. The values expressed the means of three experiments ± standard error (error bar). ^a–c Means within the same column carrying different superscripts are significantly different at p < 0.05.

Figure 5

Relative mRNA expression levels of pro-inflammatory cytokine genes; interleukin (IL)- 6, IL-1β and tumor necrosis factor-alpha (TNF-α) in vancomycin resistant Staphylococcus aureus (VRSA) challenged mice and VRSA challenged mice and treated with Free-BR or BR loaded MPS-NPs at 5 and 10 days post infection (dpi). VRSA: mice challenged with vancomycin resistant Staphylococcus aureus; VRSA+Free-BR: mice challenged with vancomycin resistant Staphylococcus aureus and treated with Free berberine; VRSA+BR loaded MPS-NPs: mice challenged with vancomycin resistant Staphylococcus aureus and treated with berberine-loaded mesoporous silica nanoparticles. ^a–c Means within the same column carrying different superscripts are significantly different at p < 0.05.

Figure 6

Relative mRNA expression levels of pro-apoptotic genes; cyclooxygenase-2 (COX-2), caspase-3, inducible nitric oxide synthase (iNOS), and Bcl-2-associated X protein (BAX) in vancomycin resistant Staphylococcus aureus (VRSA) challenged mice and VRSA challenged mice and treated with Free-BR or BR loaded MPS-NPs at 5 and 10 days post infection (dpi). VRSA: mice challenged with vancomycin resistant Staphylococcus aureus;VRSA+Free-BR: mice challenged with vancomycin resistant Staphylococcus aureus and treated with Free berberine; VRSA+BR loaded MPS-NPs: mice challenged with vancomycin resistant Staphylococcus aureus and treated with berberine-loaded mesoporous silica nanoparticles. ^a–c Means within the same column carrying different superscripts are significantly different at p < 0.05.

Figure 7

Relative mRNA expression levels of agrI, agrIII, hla, icaA, clfA, tst and pvl genes expression at 10 days post experimental infection with strong biofilm producing and multi-virulent vancomycin resistant Staphylococcus aureus (VRSA) strain in VRSA challenged mice and VRSA challenged mice and treated with Free-BR or BR loaded MPS-NPs. VRSA: mice challenged with vancomycin resistant Staphylococcus aureus; VRSA+Free-BR: mice challenged with vancomycin resistant Staphylococcus aureus and treated with Free berberine; VRSA+BR loaded MPS-NPs: mice challenged with vancomycin resistant Staphylococcus aureus and treated with berberine-loaded mesoporous silica nanoparticles. The values expressed the mean of three independent experiments ± standard error (error bar).