Hesperidin inhibits biofilm formation, virulence and staphyloxanthin synthesis in methicillin resistant Staphylococcus aureus by targeting SarA and CrtM: an in vitro and in silico approach.

Methicillin resistant Staphylococcus aureus is considered multidrug resistant bacterium due to developing biofilm formation associated with antimicrobial resistance mechanisms. Therefore, inhibition of biofilm formation is an alternative therapeutic action to control MRSA infections. The present study revealed the non-antibacterial biofilm inhibitory potential of hesperidin against ATCC strain and clinical isolates of S. aureus. Hesperidin is a flavanone glycoside commonly found in citrus fruit. Hesperidin has been shown to exhibits numerous pharmacological activities. The present study aimed to evaluate the antibiofilm and antivirulence potential of hesperidin against MRSA. Results showed that hesperidin treatment significantly impedes lipase, hemolysin, autolysin, autoaggregation and staphyloxanthin production. Reductions of staphyloxanthin production possibly increase the MRSA susceptibility rate to H2O2 oxidative stress condition. In gene expression study revealed that hesperidin treatment downregulated the biofilm-associated gene (sarA), polysaccharide intracellular adhesion gene (icaA and icaD), autolysin (altA), fibronectin-binding protein (fnbA and fnbB) and staphyloxanthin production (crtM). Molecular docking analysis predicted the ability of hesperidin to interact with SarA and CrtM proteins involved in biofilm formation and staphyloxanthin production in MRSA.

3, 5-Di-tert-butylphenol combat against Streptococcus mutans by impeding acidogenicity, acidurance and biofilm formation.

Streptococcus mutans is a common pathogen present in the oral cavity and it causes dental caries for all aged groups of people, in particular, children. S. mutans have several virulence factors such as acidogenecity, aciduricity, adhesion and biofilm formation. These virulence factors are working together and lead to the development of caries in the tooth surface. The present study aimed to investigate the anticariogenic potential of 3, 5-di-tert-butylphenol (3, 5-DTBP) against S. mutans. 3, 5-DTBP biofilm inhibitory concentration (BIC) was found at 100 µg/ml concentration without any lethal effect on the growth. Moreover, 3, 5-DTBP significantly reduced water soluble and water insoluble glucans production, in concurrence with downregulation of gtfBC genes. Moreover, acidogenicity associated virulence factors such as lactate dehydrogenase and enolase enzymatic production was arrested upon 3, 5-DTBP treatment. In addition, 3, 5-DTBP greatly reduced acidtolerance ability through impedes of F1F0-ATPase. Gene expression analysis unveiled the downregulation of gtfB, gtfC, gtfD, vicRK, comDE, gbpB, smu0630 and relA upon 3, 5-DTBP treatment. The present study paves the way for exhibiting 3, 5-DTBP as a promising therapeutic agent to control S. mutans infections.

5-Hydroxymethylfurfural inhibits Acinetobacter baumannii biofilms: an in vitro study.

The present study was aimed to investigate the antibiofilm activity of 5-hydroxymethylfurfural against Acinetobacter baumanni and Vellar estuary isolates v3 (Acinetobacter nosocomialis). The biofilm inhibitory concentration (BIC) of 5HMF against A. baumannii and v3 (A. nosocomialis) was found to be 100 µg/ml) exhibited non-bactericidal concentration-dependent antibiofilm activities against Acinetobacter species. The present study found that 5HMF treatment is very effective in the initial stage of A. baumannii biofilms and it significantly disrupted the mature biofilms. Moreover, 5HMF treatment inhibited the extracellular polymeric substances (EPS), including polysaccharides and proteins production. Results from gene expression and in vitro assays further demonstrated the 5HMF treatment downregulated the expression of bfmR, bap, csuA/B, ompA and katE virulence genes, which consistently affects biofilm formation and its mediated virulence property. The present study suggests that 5HMF unveil its antibiofilm activity by interfering initial biofilm formation and suppressing the virulence regulator genes in A. baumannii. Further studies are required to explore the 5HMF mode of action responsible for the antibiofilm activity.

Eucalyptol inhibits biofilm formation of Streptococcus pyogenes and its mediated virulence factors.

Introduction. Streptococcus pyogenes is a diverse virulent synthesis pathogen responsible for invasive systemic infections. Establishment of antibiotic resistance in the pathogen has produced a need for new antibiofilm agents to control the biofilm formation and reduce biofilm-associated resistance development.Aim. The present study investigates the in vitro antibiofilm activity of eucalyptol against S. pyogenes.Methodology. The antibiofilm potential of eucalyptol was assessed using a microdilution method and their biofilm inhibition efficacy was visualized by microscopic analysis. The biochemical assays were performed to assess the influence of eucalyptol on virulence productions. Real-time PCR analysis was performed to evaluate the expression profile of the virulence genes.Results. Eucalyptol showed significant antibiofilm potential in a dose-dependent manner without affecting bacterial growth. Eucalyptol at 300 µg ml-1 (biofilm inhibitory concentration) significantly inhibited the initial stage of biofilm formation in S. pyogenes. However, eucalyptol failed to diminish the mature biofilms of S. pyogenes at biofilm inhibitory concentration and it effectively reduced the biofilm formation on stainless steel, titanium, and silicone surfaces. The biochemical assay results revealed that eucalyptol greatly affects the cell-surface hydrophobicity, auto-aggregation, extracellular protease, haemolysis and hyaluronic acid synthesis. Further, the gene-expression analysis results showed significant downregulation of virulence gene expression upon eucalyptol treatment.Conclusion. The present study suggests that eucalyptol applies its antibiofilm assets by intruding the initial biofilm formation of S. pyogenes. Supplementary studies are needed to understand the mode of action involved in biofilm inhibition.

Quebrachitol inhibits biofilm formation and virulence production against methicillin-resistant Staphylococcus aureus.

The present study evaluated the quebrachitol (QBC) antibiofilm and antivirulence potential against methicillin-resistant Staphylococcus aureus (MRSA). QBC inhibited MRSA biofilm formation at concentration dependent manner without affecting the bacterial growth. Then, QBC biofilm efficacy was confirmed with light and confocal laser scanning microscopy analysis. QBC treatment significantly inhibited the biofilm formation on stainless steel, titanium and silicone surfaces. Besides, QBC treatment significantly reduced the MRSA virulence productions such as lipase and hemolysis. Moreover, it reduced MRSA survival rate in the presence of hydrogen peroxide. QBC treatment inhibited the MRSA adherence on hydrophobic, hydrophilic, collagen coating and fibrinogen coating surfaces. As well as it significantly reduced the autolysin and bacterial aggregation progress. The real-time PCR analysis revealed the ability of QBC downregulated the virulence genes expression including global regulator sarA, agr and polysaccharide intracellular adhesion (PIA) encode ica. The cumulative results of the present study suggest that QBC as a potential agent to combat against MRSA pathogenesis.

Musa acuminata and its bioactive metabolite 5-Hydroxymethylfurfural mitigates quorum sensing (las and rhl) mediated biofilm and virulence production of nosocomial pathogen Pseudomonas aeruginosa in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Musa acuminata, a tropical plant belongs to the family Musaceae. The fruit peels of this plant have been well documented for their therapeutic value in Asia and Africa. It has also been previously reported for numerous biological applications such as antimicrobial, antioxidant, itching, psoriasis and anti-diarrheal activities. Moreover, M. acuminata peels have been well known for its anti-healing and antiseptic properties and most commonly used for healing wounds and heat burns in South Asian and African traditional medicines. AIM OF THE STUDY: To evaluate the QS-mediated antibiofilm and antivirulence potential of M. acuminata, and its bioactive metabolites 5-Hydroxymethylfurfural (5HMF) against Pseudomonas aeruginosa. MATERIALS AND METHODS: The M. acuminata peel methanol extract (MAM) was evaluated for its antibiofilm potential against P. aeruginosa with increasing concentration. Besides, biofilm related phenomenon's such as total biofilm proteins, microcolony formation exopolysaccharides (EPS) and cell surface hydrophobicity (CSH) productions were also examined to support the antibiofilm potential of MAM. Further, MAM was evaluated for its antivirulence efficacy against P. aeruginosa by assessing the protease, LasA protease, LasB elastase, pyocyanin, alginate and rhamnolipid productions at 400 µg ml-1 concentration. Transcriptional analysis of QS regulated virulence genes expression level was also done by real-time PCR analysis. Then, the MAM was subjected to column chromatography for further fractions and the bioactive compounds present in MAM were identified by gas chromatograph-mass spectrometry analysis. Further, the major compounds such as 5-hydroxymethylfurfural, vaccenic acid and pentanoic acid identified from active fraction of MAM were evaluated for their antibiofilm and antivirulence potential against P. aeruginosa. RESULTS: MAM significantly inhibited the biofilm formation in P. aeruginosa at 400 µg ml-1 concentration which also inhibited the production of biofilm proteins, biofilm adherence, EPS and CSH productions to the level of 79%, 82% and 77% respectively. Further, the antivirulence potential was confirmed through numerous virulence inhibition assays. The MAM at 400 µg ml-1 concentration inhibited the QS-mediated virulence production such as protease, LasA protease, LasB elastase, pyocyanin, alginate and rhamnolipid productions to the level of 77%, 75%, 68%, 80%, 78% and 69% respectively. Moreover, the results of qPCR analysis confirmed the downregulation of QS regulated virulence genes expression upon treatment with MAM. The chromatographic analysis revealed the presence of 5-Hydroxymethylfurfural (5HMF), vaccenic acid and pentanoic acid in MAM and the potential bioactive compounds with antibiofilm and antivirulence was identified as 5-hydroxymethylfurfural, without exerting any growth inhibition in P. aeruginosa. CONCLUSION: This study investigated the ideal antibiofilm and antivirulence potential of MAM and its bioactive compound 5HMF, and confirms the ethnopharmacological value of these peels against P. aeruginosa infections.

Antiquorum sensing and biofilm potential of 5- Hydroxymethylfurfural against Gram positive pathogens.

The present investigates the antiquorum sensing and biofilm inhibitory potential of 5 - hydroxymethylfurfural against Chromobacterium violaceum, Streptococcus pyogenes, Streptococcus mutans, Staphylococcus aureus and Staphylococcus epidermidis. 5HMF inhibits the quorum sensing mediated violacein pigment production by 87% at 100 µg/ml concentration. A 100 µg/ml concentration of the compound inhibits S. mutans and S. epidermidis biofilm formation by 86% and 79% whereas for S. pyogenes and S. aureus 125 µg/ml concentration inhibits biofilm formation by 83% and 82%. The Confocal images clearly indicate that 5HMF as a promising antibiofilm agent.

Limonene inhibits streptococcal biofilm formation by targeting surface-associated virulence factors.

The present study explores the efficacy of limonene, a cyclic terpene found in the rind of citrus fruits, for antibiofilm potential against species of the genus Streptococcus, which have been deeply studied worldwide owing to their multiple pathogenic efficacy. Limonene showed a concentration-dependent reduction in the biofilm formation of Streptococcus pyogenes (SF370), with minimal biofilm inhibitory concentration (MBIC) of 400 µg ml - 1. Limonene was found to possess about 75-95 % antibiofilm activity against all the pathogens tested, viz. Streptococcus pyogenes (SF370 and 5 clinical isolates), Streptococcus mutans (UA159) and Streptococcus mitis (ATCC 6249) at 400 µg ml - 1 concentration. Microscopic analysis of biofilm architecture revealed a quantitative breach in biofilm formation. Results of a surface-coating assay suggested that the possible mode of action of limonene could be by inhibiting bacterial adhesion to surfaces, thereby preventing the biofilm formation cascade. Susceptibility of limonene-treated Streptococcus pyogenes to healthy human blood goes in unison with gene expression studies in which the mga gene was found to be downregulated. Anti-cariogenic efficacy of limonene against Streptococcus mutans was confirmed, with inhibition of acid production and downregulation of the vicR gene. Downregulation of the covR, mga and vicR genes, which play a critical role in regulating surface-associated proteins in Streptococcus pyogenes and Streptococcus mutans, respectively, is yet further evidence to show that limonene targets surface-associated proteins. The results of physiological assays and gene expression studies clearly show that the surface-associated antagonistic mechanism of limonene also reduces surface-mediated virulence factors.