Potential natural antimicrobial and antibiofilm properties of Piper betle L. against Staphylococcus pseudintermedius and methicillin-resistant strains.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper betle L. has potent of antimicrobial activity and is widely used as a traditional remedy to treat skin infections. However, no clear evidence exists concerning antimicrobial and antibiofilm activity against Staphylococcus pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) opportunistic pathogens that cause wound infections and pyoderma in canines and zoonotic disease. AIM OF THE STUDY: The antimicrobial and antibiofilm activities of P. betle extract were assessed against S. pseudintermedius and MRSP strains. MATERIALS AND METHODS: Ethanol leaf extract of P. betle was investigated for its antibacterial effect on S. pseudintermedius and MRSP by broth microdilution and time-kill assays. Biofilm inhibition and production assays were performed to evaluate antibiofilm and biofilm eradication effects, respectively. Biofilm-associated gene expression was further studied using real-time polymerase chain reaction (PCR). The possible interaction between IcaA and major compounds in P. betle was analyzed by molecular docking. RESULTS: The extract showed minimum inhibitory concentration (MIC) at 250 µg/mL. Growth inhibition of P. betle at 1 MIC against the bacteria was initially observed after treatment for 4 h. All isolates were completely killed after 18 h exposure to the extract. Minimum biofilm inhibitory concentrations (MBICs) of the extract against the tested isolates ranged 1/2 MIC to 1 MIC, while minimum biofilm eradication concentration (MBEC) of P. betle was initialed at 8 MIC. Quantitative inhibition and eradication effects were observed in representative strains. The extract at 1/2 MIC and 1 MIC values inhibited biofilm formation up to 100%, with bacterial biofilm removed at up to 94.21% by 4 MIC of the extract. The extract downregulated the expression of the icaA gene among biofilm-producing isolates. The most abundant compounds, 4-allyl-1,2-diacetoxybenzene and eugenol showed a strong affinity with IcaA protein at -5.65 and -5.31 kcal/mol, respectively. CONCLUSIONS: P. betle extract demonstrated the antibacterial, antibiofilm, and biofilm-removal activity against S. pseudintermedius and MRSP. Downregulation of the icaA gene expression and protein interaction were possible modes of action of the extract that impacted biofilm production. This extract showed promise as an alternative treatment for S. pseudintermedius infection, especially drug-resistant and biofilm-associated cases.

Efficacy of Thai Plant Extracts for Antibacterial and Anti-Biofilm Activities against Pathogenic Bacteria.

The emergence of drug-resistant bacteria has impacted the outcome of current therapeutics as a threat to global healthcare; novel medicines are urgently needed. Thirteen medicinal plants were collected in Northeastern Thailand, and their crude ethanolic extracts were evaluated for antibacterial activities against Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 using the broth micro-dilution method. Piper betle leaf ethanolic extract showed optimal activity against both representative bacterial strains. Activity was also observed against clinical isolates of methicillin-resistant S. aureus (MRSA) and E. coli, with minimal inhibitory concentration (MIC) ranging from 0.31 mg/mL to 2.5 mg/mL and minimal bactericidal concentration (MBC) ranging from 0.62 mg/mL to 2.5 mg/mL. A time-kill study revealed that the extract activity was time- and dose-dependent, and also bactericidal on the tested bacteria. P. betle extract inhibited biofilm formation and promoted biofilm eradication in both S. aureus and E. coli. 4-Allyl-1,2-diacetoxybenzene and eugenol were identified as the most abundant compounds in the extract and may play major roles in the anti-bacterial and anti-biofilm activity. Results suggest that ethanolic P. betle leaf extract shows promise as an alternative method for the prevention of bacterial diseases.

Rhodomyrtone decreases Staphylococcus aureus SigB activity during exponentially growing phase and inhibits haemolytic activity within membrane vesicles.

Sigma factor B (SigB) controls the expression of Staphylococcus aureus genes including virulence factors and plays a role in the bacterial secretion system through membrane vesicle production. Inhibition of SigB could attenuate SigB dependent virulence and secretion system. The objective of this study was to determine the effects of rhodomyrtone on SigB and virulence factors related to SigB. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of rhodomyrtone against 67 clinical methicillin-resistant S. aureus isolates were 0.25-8 µg/ml, which were similar to those of vancomycin. Using luciferase gene fused to SigB dependent promoters of asp23, five time reduction in SigB activity was observed when the bacteria were treated with rhodomyrtone for 3 h. Rhodomyrtone significantly reduced SigB activity in a concentration dependent manner in exponentially growing cells (P < 0.05). In addition, sigB mutant was more sensitive towards increasing concentrations of rhodomyrtone than the wild type and yabJ-spoVG mutant. Rhodomyrtone at 0.625 µg/ml reduced the growth of sigB mutant by approximately 99%, compared with the yabJ-spoVG mutant and the wild type. Membrane vesicles were significantly reduced in the bacterial cells when treated with 0.5 × MIC rhodomyrtone (P < 0.05). Decreased haemolytic activity was detected within rhodomyrtone-treated membrane vesicles. The results indicated that rhodomyrtone inhibited S. aureus SigB activity during exponentially growing phase and inhibited haemolytic activity within membrane vesicles.

Responses in the expression of extracellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone.

Rhodomyrtone from a medicinal plant species, Rhodomyrtus tomentosa, is a challenged effective agent against Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). The present study was undertaken to provide insight into MRSA extracellular protein expression following rhodomyrtone treatment. Secreteomic approach was performed on a representative clinical MRSA isolate exposing to subinhibitory concentration rhodomyrtone (0.174 µg/ml). The identified extracellular proteins of a response of MRSA to rhodomyrtone treated condition were both suppressed and overexpressed. Staphylococcal antigenic proteins, immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA) involved in cell wall hydrolysis were downregulated after the treatment. The results suggested that rhodomyrtone may interfere with WalK/WalR (YycG/YycF) system. Other enzymes such as lipase precursor and another lipase, glycerophosphoryl diester phosphodiesterase, were absent. In contrast, cytoplasmic proteins such as SpoVG and glycerol phosphate lipoteichoic acid synthase, and ribosomal proteins were found in the treated sample. Appearance of several cytoplasmic proteins in the treated culture supernatant revealed that the bacterial cell wall biosynthesis was disturbed. This finding provides a proteomic mapping of extracellular proteins after rhodomytone treatment. Extensive investigation is required for this natural compound as it has a great potency as an alternative anti-MRSA drug.

Production and purification of Burkholderia pseudomallei BipD protein.

Type III secretion system (TTSS) clusters contain virulent genes of both animal and plant Gram-negative bacteria. The full length (933 bp) gene of bipD, a member of TTSS cluster of Burkholderia pseudomallei, was isolated by PCR amplification from B. pseudomallei DNA and cloned into pGEX 4T-1. GST-BipD fusion protein and BipD protein obtained by removal of GST using thrombin were employed to detect the presence of B. pseudomallei antibodies in sera obtained from melioidosis and non-melioidosis patients by immunoblotting. Sensitivity and specificity of BipD protein was 100% and 91.1%, respectively, whereas that of fusion protein was 77.8% and 90.0%, respectively.