DataSheet_1_Antibacterial and antibiofilm activity of halogenated phenylboronic acids against Vibrio parahaemolyticus and Vibrio harveyi.docx

Vibrios are associated with live seafood because they are part of the indigenous marine microflora. In Asia, foodborne infections caused by Vibrio spp. are common. In recent years, V. parahaemolyticus has become the leading cause of all reported food poisoning outbreaks. Therefore, the halogenated acid and its 33 derivatives were investigated for their antibacterial efficacy against V. parahaemolyticus. The compounds 3,5-diiodo-2-methoxyphenylboronic acid (DIMPBA) and 2-fluoro-5-iodophenylboronic acid (FIPBA) exhibited antibacterial and antibiofilm activity. DIMPBA and FIPBA had minimum inhibitory concentrations of 100 μg/mL for the planktonic cell growth and prevented biofilm formation in a dose-dependent manner. Both iodo-boric acids could diminish the several virulence factors influencing the motility, agglutination of fimbria, hydrophobicity, and indole synthesis. Consequently, these two active halogenated acids hampered the proliferation of the planktonic and biofilm cells. Moreover, these compounds have the potential to effectively inhibit the presence of biofilm formation on the surface of both squid and shrimp models.</p

Data_Sheet_1_Antibiofilm and Antimicrobial Activities of Chloroindoles Against Uropathogenic Escherichia coli.docx

Uropathogenic Escherichia coli (UPEC) is a nosocomial pathogen associated with urinary tract infections and expresses several virulence factors that cause recurring infections and cystitis of the bladder, which can lead to pyelonephritis. UPEC uses different types of extracellular appendages like fimbriae and pili that aid colonization and adherence to bladder epithelium and can form persistent biofilm-like bacterial communities that aid its survival after the deployment of host immune responses. We investigated the antibiofilm, antimicrobial, and antivirulence properties of three indole derivatives namely, 4-chloroindole, 5-chloroindole, and 5-chloro 2-methyl indole. All the three chloroindoles had MICs of 75 μg/ml and inhibited biofilm formation by an average of 67% at 20 μg/ml. In addition, they inhibited swarming and swimming motilities, which are essential for dissemination from bacterial communities and colonization, reduced cell surface hydrophobicity, and inhibited indole production and curli formation. Gene expression analysis showed all three chloroindoles significantly downregulated the expressions of virulence genes associated with adhesion, stress regulation, and toxin production. A 3D-QSAR analysis revealed substitutions at the fourth and fifth positions of the indole moiety favored antimicrobial activity. Furthermore, these chloroindoles potently inhibited biofilm formation in other nosocomial pathogens and polymicrobial consortia.</p

Data_Sheet_1_Inhibition of growth, biofilm formation, virulence, and surface attachment of Agrobacterium tumefaciens by cinnamaldehyde derivatives.PDF

Agrobacterium tumefaciens, a soil-borne, saprophytic plant pathogen that colonizes plant surfaces and induces tumors in a wide range of dicotyledonous plants by transferring and expressing its T-DNA genes. The limited availabilities and efficacies of current treatments necessitate the exploration of new anti-Agrobacterium agents. We examined the effects of trans-cinnamaldehyde (t-CNMA) and its derivatives on the cell surface hydrophobicity, exopolysaccharide and exo-protease production, swimming motility on agar, and biofilm forming ability of A. tumefaciens. Based on initial biofilm inhibition results and minimum inhibitory concentration (MIC) data, 4-nitro, 4-chloro, and 4-fluoro CNMAs were further tested. 4-Nitro, 4-chloro, and 4-fluoro CNMA at ≥150 μg/ml significantly inhibited biofilm formation by 94–99%. Similarly, biofilm formation on polystyrene or nylon was substantially reduced by 4-nitro and 4-chloro CNMAs as determined by optical microscopy and scanning electron microscopy (SEM) and 3-D spectrum plots. 4-Nitro and 4-chloro CNMAs induced cell shortening and concentration- and time-dependently reduced cell growth. Virulence factors were significantly and dose-dependently suppressed by 4-nitro and 4-chloro CNMAs (P ≤ 0.05). Gene expressional changes were greater after 4-nitro CNMA than t-CNMA treatment, as determined by qRT-PCR. Furthermore, some genes essential for biofilm formation, motility, and virulence genes significantly downregulated by 4-nitro CNMA. Seed germination of Raphanus sativus was not hindered by 4-nitro or 4-fluoro CNMA at concentrations ≤200 μg/ml, but root surface biofilm formation was severely inhibited. This study is the first to report the anti-Agrobacterium biofilm and anti-virulence effects of 4-nitro, 4-chloro, and 4-fluoro CNMAs and t-CNMA and indicates that they should be considered starting points for the development of anti-Agrobacterium agents.</p

Inhibition of <i>Candida albicans</i> and <i>Staphylococcus aureus</i> biofilms by centipede oil and linoleic acid

Microbial biofilms are associated with persistent infections because of their high tolerance to antimicrobial agents and host defenses. The effects of centipede oil from Scolopendra subspinipes mutilans and its main components were investigated to identify non-toxic biofilm inhibitors. Centipede oil and linoleic acid at 20 µg ml−1 markedly inhibited biofilm formation by two fluconazole-resistant Candida albicans strains and three Staphylococcus aureus strains without affecting their planktonic cell growth. Also, both centipede oil and linoleic acid inhibited hyphal growth and cell aggregation by C. albicans. In addition, centipede oil and linoleic acid showed anti-biofilm activities against mixed C. albicans and S. aureus biofilms. Transcriptomic analysis showed that centipede oil and linoleic acid downregulated the expressions of several hypha/biofilm-related genes in C. albicans and α-hemolysin in S. aureus. Furthermore, both compounds effectively reduced C. albicans virulence in a nematode infection model with minimal toxicity.</p

Data_Sheet_1_Antivirulence activities of retinoic acids against Staphylococcus aureus.docx

Multidrug-resistant bacteria such as Staphylococcus aureus constitute a global health problem. Gram-positive S. aureus secretes various toxins associated with its pathogenesis, and its biofilm formation plays an important role in antibiotic tolerance and virulence. Hence, we investigated if the metabolites of vitamin A1 might diminish S. aureus biofilm formation and toxin production. Of the three retinoic acids examined, 13-cis-retinoic acid at 10 μg/mL significantly decreased S. aureus biofilm formation without affecting its planktonic cell growth (MIC >400 μg/mL) and also inhibited biofilm formation by Staphylococcus epidermidis (MIC >400 μg/mL), but less affected biofilm formation by a uropathogenic Escherichia coli strain, a Vibrio strain, or a fungal Candida strain. Notably, 13-cis-retinoic acid and all-trans-retinoic acid significantly inhibited the hemolytic activity and staphyloxanthin production by S. aureus. Furthermore, transcriptional analysis disclosed that 13-cis-retinoic acid repressed the expressions of virulence- and biofilm-related genes, such as the two-component arlRS system, α-hemolysin hla, nuclease (nuc1 and nuc2), and psmα (phenol soluble modulins α) in S. aureus. In addition, plant and nematode toxicity assays showed that 13-cis-retinoic acid was only mildly toxic at concentrations many folds higher than its effective antibiofilm concentrations. These findings suggest that metabolites of vitamin A1, particularly 13-cis-retinoic acid, might be useful for suppressing biofilm formation and the virulence characteristics of S. aureus.</p

Appraisal of Chitosan-Gum Arabic-Coated Bipolymeric Nanocarriers for Efficient Dye Removal and Eradication of the Plant Pathogen Botrytis cinerea

The treatment of textile wastewater comprising many dyes as contaminants endures an essential task for environmental remediation. In addition, combating antifungal multidrug resistance (MDR) is an intimidating task, specifically owing to the limited options of alternative drugs with multitarget drug mechanisms. Incorporating natural polymeric biomaterials for drug delivery provides desirable properties for drug molecules, effectively eradicating MDR fungal growth. The current study fabricated the bipolymeric drug delivery system using chitosan-gum arabic-coated liposome 5ID nanoparticles (CS-GA-5ID-LP-NPs). This study focused on improving the solubility and sustained release profile of 5I-1H-indole (5ID). These NPs were characterized and tested mechanically as a dye adsorbent as well as their antifungal potencies against the plant pathogen, Botrytis cinerea. CS-GA-5ID-LP-NPs showed 71.23% congo red dye removal compared to crystal violet and phenol red from water and effectively had an antifungal effect on B. cinerea at 25 μg/mL MIC concentrations. The mechanism of the inhibition of B. cinerea via CS-GA-5ID-LP-NPs was attributed to stabilized microtubule polymerization in silico and in vitro. This study opens a new avenue for designing polymeric NPs as adsorbents and antifungal agents for environmental and agriculture remediation

Red wines and flavonoids diminish <i>Staphylococcus aureus</i> virulence with anti-biofilm and anti-hemolytic activities

<div><p>The emergence of antibiotic resistant <i>Staphylococcus aureus</i> presents a worldwide problem that requires non-antibiotic strategies. This study investigated the anti-biofilm and anti-hemolytic activities of four red wines and two white wines against three <i>S. aureus</i> strains. All red wines at 0.5–2% significantly inhibited <i>S. aureus</i> biofilm formation and hemolysis by <i>S. aureus</i>, whereas the two white wines had no effect. Furthermore, at these concentrations, red wines did not affect bacterial growth. Analyses of hemolysis and active component identification in red wines revealed that the anti-biofilm compounds and anti-hemolytic compounds largely responsible were tannic acid, <i>trans</i>-resveratrol, and several flavonoids. In addition, red wines attenuated <i>S. aureus</i> virulence <i>in vivo</i> in the nematode <i>Caenorhabditis elegans</i>, which is killed by <i>S. aureus</i>. These findings show that red wines and their compounds warrant further attention in antivirulence strategies against persistent <i>S. aureus</i> infection.</p></div

Image_1_Fatty Acids as Aminoglycoside Antibiotic Adjuvants Against Staphylococcus aureus.pdf

Fatty acids have diverse functions in the vast majority of cells. At high doses, they act as antimicrobials while, at low doses, they exhibit antibiofilm and antivirulence activities. In this study, the synergistic antibacterial and antibiofilm activities of 30 fatty acids and 11 antibiotics were investigated against methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains. Of the 15 saturated and 15 unsaturated fatty acids examined, 16 enhanced the antibacterial activity of tobramycin. Combinatorial treatment with myristoleic acid (the most active) at 10 μg/ml and tobramycin at 10 μg/ml decreased cell survival by >4 log as compared with tobramycin treatment alone. Notably, aminoglycoside antibiotics, such as tobramycin, kanamycin, gentamicin, and streptomycin exhibited antimicrobial synergy with myristoleic acid. Co-treatment with myristoleic acid and antibiotics markedly decreased biofilm formation. Interestingly, co-treatment with tobramycin and myristoleic acid induced a reduction in S. aureus cell size. These results suggest that fatty acids, particularly myristoleic acid, can be used as aminoglycoside antibiotic adjuvants against recalcitrant S. aureus infections.</p

Data_Sheet_1_Appraisal of Cinnamaldehyde Analogs as Dual-Acting Antibiofilm and Anthelmintic Agents.docx

Cinnamaldehyde has a broad range of biological activities, which include antibiofilm and anthelmintic activities. The ever-growing problem of drug resistance and limited treatment options have created an urgent demand for natural molecules with antibiofilm and anthelmintic properties. Hence, we hypothesized that molecules with a scaffold structurally similar to that of cinnamaldehyde might act as dual inhibitors against fungal biofilms and helminths. In this regard, eleven cinnamaldehyde analogs were tested to determine their effects on fungal Candida albicans biofilm and nematode Caenorhabditis elegans. α-Methyl and trans-4-methyl cinnamaldehydes efficiently inhibited C. albicans biofilm formation (>90% inhibition at 50 μg/mL) with minimum inhibitory concentrations (MICs) of ≥ 200 μg/mL and 4-bromo and 4-chloro cinnamaldehydes exhibited anthelmintic property at 20 μg/mL against C. elegans. α-Methyl and trans-4-methyl cinnamaldehydes inhibited hyphal growth and cell aggregation. Scanning electron microscopy was employed to determine the surface architecture of C. albicans biofilm and cuticle of C. elegans, and confocal laser scanning microscopy was used to determine biofilm characteristics. The perturbation in gene expression of C. albicans was investigated using qRT-PCR analysis and α-methyl and trans-4-methyl cinnamaldehydes exhibited down-regulation of ECE1, IFD6, RBT5, UCF1, and UME6 and up-regulation of CHT4 and YWP1. Additionally, molecular interaction of these two molecules with UCF1 and YWP1 were revealed by molecular docking simulation. Our observations collectively suggest α-methyl and trans-4-methyl cinnamaldehydes are potent biofilm inhibitors and that 4-bromo and 4-chloro cinnamaldehydes are anthelmintic agents. Efforts are required to determine the range of potential therapeutic applications of cinnamaldehyde analogs.</p

The anti-biofilm and anti-virulence activities of <i>trans-</i>resveratrol and oxyresveratrol against uropathogenic <i>Escherichia coli</i>

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections, which are one of the most common infectious disease types in humans. UPEC infections involve bacterial cell adhesion to bladder epithelial cells, and UPEC can also form biofilms on indwelling catheters that are often tolerant to common antibiotics. In this study, the anti-biofilm activities of t-stilbene, stilbestrol, t-resveratrol, oxyresveratrol, ε-viniferin, suffruticosol A, and vitisin A were investigated against UPEC. t-Resveratrol, oxyresveratrol, and ε-viniferin, suffruticosol A, and vitisin A significantly inhibited UPEC biofilm formation at subinhibitory concentrations (10–50 μg ml−1). These findings were supported by observations that t-resveratrol and oxyresveratrol reduced fimbriae production and the swarming motility in UPEC. Furthermore, t-resveratrol and oxyresveratrol markedly diminished the hemagglutinating ability of UPEC, and enhanced UPEC killing by human whole blood. The findings show that t-resveratrol, oxyresveratrol, and resveratrol oligomers warrant further attention as antivirulence strategies against persistent UPEC infections.</p