Crystal Violet Assay Research Articles

Cytocompatibility, Antibacterial, and Anti-Biofilm Efficacy of Grape Seed Extract and Quercetin Hydrogels Against a Mature Endodontic Biofilm Ex Vivo Model

Background/Objectives: To assess the cytocompatibility, antibacterial and anti-biofilm efficacy of grape seed extract (GSE) and quercetin hydrogels versus calcium hydroxide (CH) as intracanal medications (ICMs) against an endodontic ex vivo biofilm model. Methods: Single-rooted teeth (n = 50) were prepared and sterilized before being infected with E. faecalis to develop a mature biofilm. They were divided into five equal groups according to the ICM used: G1: medicated with CH paste, G2: medicated with GSE hydrogel, G3: medicated with quercetin hydrogel, G4: positive control group that was infected and not medicated, and G5: negative control group that was neither infected nor medicated. After 1 week, the ICM was removed, and the root canals were cultured to assess the antibacterial efficacy by counting the colony-forming units and the anti-biofilm efficacy by the crystal violet assay. Dead/live bacterial viability was assessed by CFLSM examination, while the cytocompatibility was assessed using the MTT assay. Results: CH had the best antibacterial efficacy, followed by GSE and quercetin hydrogels (p < 0.001). Regarding the anti-biofilm efficacy, GSE was superior, followed by quercetin and CH (p < 0.001). CFLSM examination showed CH and GSE hydrogel to be highly effective in comparison to the positive control (p < 0.0001), with no statistical difference between them (p > 0.05). CH showed significantly higher cell viability percentages using a 500 μg/mL, while quercetin and GSE started to show cell viability > 70% at concentrations of 125 μg/mL and 62.5 μg/mL. Conclusions: CH fulfilled the ideal requirements of ICM as being both antibacterial and non-cytotoxic compared to the other materials tested.

Synergistic effect of antibiotics, α-linolenic acid and solvent type against Staphylococcus aureus biofilm formation.

A promising approach to the treatment of bacterial infections involves inhibiting the quorum sensing (QS) mechanism to prevent the formation and growth of bacterial biofilm. While antibiotics are used to kill remaining bacteria, QS inhibitors (QSIs) allow for antibiotic doses to be reduced. This study focuses on evaluating the synergy between gentamicin sulphate (GEN), tobramycin (TOB), or azithromycin (AZM) with linolenic acid (LNA) against the formation of an early Staphylococcus aureus biofilm. Minimum biofilm inhibitory concentration (MBIC) was determined using the resazurin reduction assay for all antibiotics and LNA. The reduction of biofilm mass was assessed using the crystal violet (CV) assay. We have also evaluated the effect of dimethyl sulfoxide with TWEEN (DMSO_T) on early biofilm formation. Synergy was determined by metabolic activity assay and fractional biofilm inhibitory concentration (FBIC). DMSO_T at a concentration of 1% enhanced early biofilm formation, but also decreased the doses of antibiotic needed to reduce the biofilm by up to 8 times. Adding LNA at a concentration of 32µg/ml or 64µg/ml allowed up to a 32-fold reduction of antibiotic doses for GEN and TOB and a 4-fold reduction for AZM. LNA's use in combination with various antibiotics could reduce their doses and help fight drug-resistant bacteria in the biofilm.

Biofilm formation and drug susceptibility of biofilm Candida spp. clinically isolated from nasopharyngeal cancer patients in Vietnam

Background and Objectives: The biofilm formation has been widely recognized as one of the main mechanisms of antimi- crobial resistance development in microorganisms. However, few studies are focusing on this phenomenon in Candida spp. in clinical settings, especially on immuno-compromised patients. Materials and Methods: In this study, both the rate of biofilm formation in those patients and its drug susceptibility in initial and mature biofilm were assessed using crystal violet assay and dilution method. Results: The results demonstrated that the biofilm formation rate was similar between albicans and non-albicans Candida. However, the biofilm formation capacity was more pronounced in non-albicans Candida, especially, C. glabrata. As expect- ed, there was a significant relationship between biofilm formation and drug resistance. In addition, our study reconfirmed that the age of high concentration of antifungal agents only affected Candida before its biofilm formation regardless of its biofilm formation capacity. In the contrary, once the biofilm was formed even elevated drug concentrations did not show sufficient efficacy, highlighting a need for high dosage at the early stage of treatment for those patients. Conclusion: The results of this study highlighted the importance of using appropriate antifungal agents for Candida treatment before the formation of biofilm.

In vitro and computational investigation of antioxidant and anticancer properties of Streptomyces coeruleofuscus SCJ extract on MDA-MB-468 triple-negative breast cancer cells

This study aimed to explore the antioxidant potential of the ethyl acetate extract of Streptomyces coeruleofuscus SCJ strain, along with its inhibitory effects on the triple-negative human breast carcinoma cell line (MDA-MB-468). The ethyl acetate extract’s total phenolic and flavonoid contents were quantified, and its antioxidant activity was investigated using DPPH (1,1-Diphenyl-2-picrylhydrazyl), ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), and FRAP (Ferric Reducing Antioxidant Power) assays. Furthermore, the cytotoxic effect of the organic extract from Streptomyces coeruleofuscus SCJ on MDA-MB-468 cancer cells was assessed via the crystal violet assay. In tandem, a thorough computational investigation was conducted to explore the pharmacokinetic properties of the identified components of the extract, utilizing the SwissADME and pKCSM web servers. Additionally, the molecular interactions between these components and Estrogen Receptor Beta, identified as a potential target, were probed through molecular docking studies. The results revealed that ethyl acetate extract of SCJ strain exhibited remarkable antioxidant activity, with 39.899 ± 1.56% and 35.798 ± 0.082% scavenging activities against DPPH and ABTS, respectively, at 1 mg/mL. The extract also displayed significant ferric reducing power, with a concentration of 1.087 ± 0.026 mg ascorbic acid equivalents per mg of dry extract. Furthermore, a strong positive correlation (p < 0.0001) between the antioxidant activity, the polyphenol and the flavonoid contents. Regarding anticancer activity, the SCJ strain extract demonstrated significant anticancer activity against TNBC MDA-MB-468 cancer cells, with an inhibition percentage of 62.76 ± 0.62%, 62.67 ± 0.93%, and 58.07 ± 4.82% at 25, 50, and 100 µg/mL of the extract, respectively. The HPLC-UV/vis analysis revealed nine phenolic compounds: gallic acid, sinapic acid, p-coumaric acid, cinnamic acid, trans-fereulic acid, syringic acid, chloroqenic acid, ellagic acid, epicatechin. Streptomyces coeruleofuscus SCJ showed promise for drug discovery, exhibiting antioxidant and anticancer effects.

The Biofilm Removal and Bactericidal Effect of an 810-nm High-Power Laser on an Orthodontic Bracket Surface: An In Vitro Study.

Objective: The present study aimed to analyze the biofilm removal and bactericidal effect of laser treatment alone and laser combined with ultrasonic scaling on orthodontic brackets. It also assessed whether the use of a laser can improve the efficiency of biofilm removal and bactericidal effect compared with traditional ultrasonic instrumentation. Background Data: Streptococcus mutans (S. mutans) can lead to white spots and dental caries. Orthodontic brackets make teeth cleaning more difficult, and biofilms or bacteria on the surface of brackets worsen the oral environment, which may cause some oral diseases. Laser can be used for biofilm removal and killing bacteria on the surface of an object through thermal, photochemical, and pressure effects, which is widely used in the treatment of oral diseases. Methods: A total of 600 mandibular incisor brackets were collected for this study. Among these, 320 unused brackets were used for the S. mutans crystal violet assay (n = 160) and for S. mutans live/dead bacterial staining (n = 160). Another 280 brackets, obtained from patients who had undergone therapy for over two years, were used for the mature multispecies biofilms removal assay (n = 120) and multispecies bacterial live/dead bacterial staining (n = 160). Ultrasonic scaling, laser, and laser combined with ultrasonic scaling were applied to the labial surface of brackets covered by S. mutans biofilm or mature multispecies biofilms. Specifically, we used the following three methods: ultrasonic scaling for 10 sec without laser; 810-nm laser (Doctor Smile, Italy, LA5D0 001.1) with 0.3-mm spot size at total 21.2 kJ/cm2 for 10 sec; and 810-nm laser at total 10.6 kJ/cm2 for 5 sec, followed by ultrasonic scaling for 5 sec. The 810-nm diode laser removed biofilms with a power of 1.5 W and a power density of 2.12 kW/cm2. The S. mutans biofilm was examined using crystal violet assay, and scanning electron microscopy (SEM) was used for mature multispecies biofilms to evaluate the effect of the three methods on biofilm removal. Live/dead bacterial staining was used to examine the bactericidal effect on remaining biofilms by confocal laser scanning microscopy (CLSM). Results: For S. mutans biofilm, the optical density (OD) value and live/dead bacterial ratio in the laser and the laser combined with ultrasonic scaling groups were significantly lower than those in the ultrasonic scaling group (p < 0.05); moreover, the OD value and the live/dead bacterial ratio in laser treatment combined with ultrasonic scaling and laser treatment alone showed no significant difference (p > 0.05). For mature multispecies biofilms, the percentage of biofilm coverage after treatment was higher in the laser group than in the ultrasonic scaling group (p < 0.05) and lower in the laser combined with ultrasonic scaling group than in the ultrasonic scaling group (p < 0.05), and live/dead bacterial staining showed that laser treatment alone killed the most bacteria, followed by laser treatment combined with ultrasonic scaling, while ultrasonic scaling alone seldom killed bacteria. Conclusions: Laser treatment alone has a better bactericidal effect and can also remove more S. mutans biofilm than ultrasonic scaling alone, but it fails to remove more mature multispecies biofilms. Laser treatment combined with ultrasonic scaling can remove more S. mutans biofilm and mature multispecies biofilms than ultrasonic scaling alone and also has a better bactericidal effect than ultrasonic scaling alone on a bracket surface.

Antisalmonella Activities of Essential Oil of Psidium guajava Leaves: An In vitro and In vivo Study

Background: Life-threatening typhoidal salmonella infection is becoming more difficult to treat due to rising resistance to commonly used antibiotics. This resistance is aided by the biofilm-forming ability of the bacteria. This study investigated the in vitro and in vivo antisalmonella activities of essential oil of Psidium guajava leaves. Methods: Essential oil was extracted from the leaves of Psidium guajava with solvents. The sensitivity of Salmonella Typhi to the essential oil of Psidium guajava (EOPG) was assessed via the agar well diffusion method. Minimum inhibitory, bactericidal and biofilm-inhibitory concentrations (MIC, MBC and MBIC respectively) were measured using micro-broth dilution and crystal violet assay respectively. The effect of EOPG on the bacterial cell membrane was evaluated by measuring the efflux of potassium ions, inorganic phosphate and pyruvic acid. Bioautography and GC-MS were employed to identify the constituents of the active fraction of the EOPG. Effects of EOPG on the level of bacteremia and bone marrow infection in male Wistar rats were determined by plating caudal blood on salmonella-shigella agar. Selected serum cytokines were measured using ELISA kits. Effect on hepatic DNA was measured by comet assay while histological evaluation of the liver of infected rats was done by Hematoxylin and Eosin staining. Results: Essential oil of Psidium guajava leaves (EOPG) inhibited the growth of S. Typhi with a zone of inhibition of 13±0.2mm compared to 31±0.3mm recorded by ciprofloxacin. Minimum inhibitory, minimum bactericidal and minimum biofilm inhibitory concentrations were 25, &gt;50 and 12.5 mg/mL respectively. Exposure to EOPG caused efflux of potassium ions, inorganic phosphate and pyruvic acid in S. Typhi. Oral administration of 50 mg/mL of EOPG reduced bacterial load in the blood and bone marrow of S. Typhi-infected rats significantly (P&lt;0.05) when compared to untreated animals. Infection-induced reduction of serum IL-1β and rise in serum IFN-γ were ameliorated considerably in treated animals. The hepatic DNA of infected animals were protected from breakage in the group administered 50 mg/mL/day of EOPG compared with untreated, ciprofloxacin-treated as well as those administered 100 mg/mL/day of EOPG. Conclusion: EOPG has both in vitro and in vivo anti-salmonella activities, this could be exploited in developing new drug leads for treatment of typhoid fever

Transcriptomic Analysis of the Effect of Glabridin on Biofilm Formation in Staphylococcus Aureus.

Staphylococcus aureus (S. aureus) is among the major skin infection-causing pathogens in animals and humans. Its ability to form biofilms has become a foremost cause of bacterial infections and the extensive spread of drug resistance, which poses a great difficulty in clinical treatment. Glabridin (Glb), an extract of licorice with antibacterial and anti-infective properties, has a partially understood biofilm-inhibitory mechanism. This study investigated the inhibitory and antibiofilm activities of subinhibitory concentrations of Glb against S. aureus. The crystal violet assay revealed that Glb significantly suppressed biofilm expression. Scanning electron microscopy observations unveiled that Glb reduced S. aureus adhesion and accumulation by disrupting the spatial structure of the biofilm. In vitro extracellular DNA (eDNA) inhibition assays demonstrated that Glb inhibited biofilm formation by S. aureus by suppressing eDNA secretion. In total, 184 differentially expressed genes were obtained through transcriptomic (RNA-seq) sequencing, of which 81 and 103 genes were upregulated and downregulated, respectively. Glb regulated the transcript levels of biofilm-related genes through the phosphatase transfer system, two-component regulatory system, and nitrogen metabolism. The qPCR analysis was performed to confirm whether Glb interfered with the expression of regulatory genes involved in S. aureus biofilm formation (SarA, ArlR, FnbA, ClfA, icaD, and icaR) as well as the virulence gene Hla. In conclusion, this study demonstrates that Glb has a significant inhibitory effect on biofilm activity and is expected to be a good antibiofilm drug.

Zidovudine in synergistic combination with nitrofurantoin or omadacycline: in vitro and in murine urinary tract or lung infection evaluation against multidrug-resistant Klebsiella pneumoniae.

Limited treatment options and multidrug-resistant (MDR) Klebsiella pneumoniae present a significant therapeutic challenge, underscoring the need for novel approaches. Drug repurposing is a promising tool for augmenting the activity of many antibiotics. This study aimed to identify novel synergistic drug combinations against K. pneumoniae based on drug repurposing. We used the clinically isolated GN 172867 MDR strain of K. pneumoniae to determine the reversal resistance activity of zidovudine (AZT). The combined effects of AZT and various antibiotics, including nitrofurantoin (NIT) and omadacycline (OMC), were examined using the checkerboard method, growth curves, and crystal violet assays to assess biofilms. An in vitro combination activity testing was carried out in 12 isolates of K. pneumoniae. In vivo murine urinary tract and lung infection models were used to evaluate the therapeutic effects of AZT + NIT and AZT + OMC, respectively. The fractional inhibitory concentration index and growth curve demonstrated that AZT synergized with NIT or OMC against K. pneumoniae strains. In addition, AZT + NIT inhibited biofilm formation and cleared mature biofilms. In vivo, compared with untreated GN 172867-infected mice, AZT + NIT and AZT + OMC treatment decreased colony counts in multiple tissues (P < 0.05) and pathological scores in the bladder and kidneys (P < 0.05) and increased the survival rate by 60% (P < 0.05). This study evaluated the combination of AZT and antibiotics to treat drug-resistant K. pneumoniae infections and found novel drug combinations for the treatment of acute urinary tract infections. These findings suggest that AZT may exert significant anti-resistance activity.

Transfer of beef bacterial communities onto food-contact surfaces.

Food spoilage and pathogenic bacteria on food-contact surfaces, especially biofilm-forming strains, can transfer to meats during processing. The objectives of this study were to survey the bacterial communities of beef cuts that transfer onto two commonly used food-contact surfaces, stainless steel (SS) and high-density polyethylene (HDPE) and identify potentially biofilm-forming strains. Top round, flank, chuck, and ground beef were purchased from 3 retail stores. SS and HDPE coupons (approximately 2cm × 5cm) were placed on beef portions (3h, 10°C), after which, the coupons were submerged halfway in PBS (24h, 10°C). Bacteria from the beef cuts and coupon surfaces (n = 3) were collected, plated on tryptic soy agar plates and incubated (5 days, 25°C). Bacterial isolates were identified by 16S rRNA gene amplicon sequencing and assayed for biofilm formation using a crystal violet binding (CV) assay (72h, 10°C). Additionally, beef and coupon samples were collected for bacterial community analysis by 16S rRNA gene amplicon sequencing. Sixty-one of 972 beef isolates, 29 of 204 HDPE isolates, and 30 of 211 SS isolates were strong biofilm-formers (Absorbance>1.000 at 590 nm in the CV assay). Strong-binding isolates identified were of the genera Pseudomonas, Acinetobacter, Psychrobacter, Carnobacterium, and Brochothrix. Coupon bacterial communities among stores and cuts were distinct (p < 0.001, PERMANOVA), but there was no distinction between the communities found on HDPE or SS coupons (p > 0.050, PERMANOVA). The bacterial communities identified on the coupons may help determine the communities capable of transferring and colonizing onto surfaces, which can subsequently cross-contaminate foods.

The Influence of Streptococcus mutans Biofilm Formation in a Polymicrobial Environment (Streptococcus gordonii & Porphyromonas gingivalis).

Biofilms play a vital role in the occurrence or worsening of an infectious disease. Streptococcus mutans is a bacterium with the ability to form biofilms that plays a key role in the development of infectious diseases such as dental caries. The formation of biofilms in S. mutans is mediated by quorum sensing. Inhibiting quorum sensing can be considered as one of the approaches to prevent caries. This study aims to investigate the ability of Streptococcus gordonii and Porphyromonas gingivalis bacteria to inhibit the formation of S. mutans biofilm. This research was conducted to analyze bacterial biofilm formation and metabolism. The bacteria used are S. mutans (serotype C), S. gordonii (ATCC 5165), and P. gingivalis (ATCC 33277). Biofilm formation was analyzed using the crystal violet assay. Bacterial metabolism was analyzed using the methylthiazol tetrazolium (MTT) assay. The results of the crystal violet assay indicate a decrease in biofilm formation in S. mutans when in the presence of S. gordonii and S. mutans in the presence of P. gingivalis. The results of the MTT assay show no significant change in the bacterial metabolism of S. mutans in the presence of S. gordonii and S. mutans in the presence of P. gingivalis. However, S. mutans with the presence of S. gordonii and P. gingivalis show an increase in biofilm formation and bacterial metabolism. S. gordonii and P. gingivalis are each capable of inhibiting the formation of S. mutans biofilm in a polymicrobial environment.

Evaluation of riboflavin, nanocurcumin, and hydrogen peroxide under light conditions: reduction of mature dental biofilms and enamel mineral loss

BackgroundBiofilms are a potential harbor for many microorganisms. The aim of this study was to test the efficacy of riboflavin (Rib), nano-micelle curcumin (NC), and hydrogen peroxide (HP), alone and in combination with the respective light (light-emitting diode (LED) or 980 nm diode laser) on the reduction of Streptococcus mutans and Lactobacillus acidophilus dual-species biofilms and their effect on the enamel mineral loss. Materials and methodsThe biofilms were formed on saliva-coated enamel slabs. Then, the biofilms were treated with antimicrobial photodynamic therapy (PDT) based on LED, Rib, and NC photosensitizers and with HP also based on a 980 nm diode laser (n = 8 per group). A crystal violet assay was performed to determine the reduction of the dual-species biofilms. The enamel slabs were analyzed for calcium and phosphorus content by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). ResultsWhile HP-PDT showed a reduction of 37% (p < 0.001), PDT with NC resulted in an even greater reduction of dual-species biofilms (40%, p < 0.001) than HP- and Rib-mediated PDT. In the EDX test, no significant difference was found between the control group and the treatment groups. ConclusionsThe use of natural photosensitizers such as NC in PDT has an effect that may be potentially important in reducing caries-causing bacteria.

Investigation of cockleshell waste as a natural biocarrier and its performance efficiency for blackwater treatment in a free-floating plant-constructed wetland

ABSTRACT This study investigated the potential of using waste cockleshell (CS) as a natural biocarrier for blackwater treatment. The CS were analysed for surface porosity, area, volume, elements, and crystal structure. Then, the pilot-scale study confirmed biofilm growth on the CS surface after 29 days, using a crystal violet assay and scanning electron microscopy (SEM) analysis. The free-floating plant-constructed wetland (FFP-CW) was optimally designed with a redox gradient: an aerobic-aquatic zone for nitrification and an anoxic gravel-filled zone for denitrification. The effect of integrating CS biocarriers and active aeration in FFP-CW (aquatic zone) treatment efficiency was investigated. The FFP-CW treatment efficiency for nitrate, chemical oxygen demand (COD), and total ammonia nitrogen (TAN) increased by 9.62%, 6.8%, and 4.83%, respectively, after integrating CS biocarriers of 0.55% filling ratio. Furthermore, the optimal dosage of 192.51 mol of oxygen in FFP-CW increased in TAN and COD removal by 31.11% and 16.54%, respectively. The X-ray diffractometry of CS unravelled aragonite and calcite crystalline forms at 2θ range from 10 to 70°C. The stability of CS biocarriers during 427 days of treatment was monitored using SEM-energy-dispersive X-ray spectroscopy to assess the physical and chemical changes. Thus, CS holds the potential to be a sustainable biocarrier.

Enhancing antimicrobial efficacy against Pseudomonas aeruginosa biofilm through carbon dot-mediated photodynamic inactivation

Pseudomonas aeruginosa biofilms shield the bacteria from antibiotics and the body’s defenses, often leading to chronic infections that are challenging to treat. This study aimed to assess the impact of sub-lethal doses of antimicrobial photodynamic inactivation (sAPDI) utilizing carbon dots (CDs) derived from gentamicin and imipenem on biofilm formation and the expression of genes (pelA and pslA) associated with P. aeruginosa biofilm formation.The anti-biofilm effects of sAPDI were evaluated by exposing P. aeruginosa to sub-minimum biofilm inhibitory concentrations (sub-MBIC) of CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN, combined with sub-lethal UVA light irradiation. Biofilm formation ability was assessed by crystal violet (CV) assay and enumeration method. Additionally, the impact of sAPDI on the expression of pelF and pslA genes was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR).Compared to the control group, the sAPDI treatment with CDsGEN-NH2, CDsIMP-NH2, CDsGEN-IMP, and CDsIMP-GEN resulted in a significant reduction in biofilm activity of P. aeruginosa ATCC 27853 (P < 0.0001). The CV assay method demonstrated reductions in optical density of 83.70%, 81.08%, 89.33%, and 75.71%, while the CFU counting method showed reductions of 4.03, 3.76, 4.39, and 3.21 Log10 CFU/mL. qRT-PCR analysis revealed decreased expression of the pelA and pslA genes in P. aeruginosa ATCC 27853 following sAPDI treatment compared to the control group (P < 0.05).The results indicate that sAPDI using CDs derived from gentamicin and imipenem can decrease the biofilm formation of P. aeruginosa and the expression of the pelA and pslA genes associated with its biofilm formation.

Antifouling activity of Malaysian green seaweed Ulva lactuca and its isolated non-polar compound

Marine natural products especially seaweeds have gained much attention to combat biofouling. Ulva lactuca was determined for its antifouling activity and characterized the isolated non-polar metabolite involved. The methanolic crude extract (MCE) of U. lactuca was screened using crystal violet assay against biofilm-forming bacteria Pseudomonas aeruginosa and was further tested on laboratory and field tests. Then, it was fractionated and isolated using Liquid-Liquid Fractionation (LLE) and Column Chromatography (CC). The isolated compound was characterized using Liquid Chromatography-Mass Spectrometry (LC-MS), Nuclear Magnetic Resonance (NMR), and Fourier Transform-Infrared Spectroscopy (FTIR). The current study showed that the growth of biofilm produced by P. aeruginosa was inhibited by MCE at concentrations of 0.0156 mg/mL. The laboratory test indicated UL5% demonstrated a higher bacterial reduction of bacterial colonies with 1.903 × 106 CFU/mL better than blank paint. According to the field test, crude panels of UL5% were successful in reducing the settlement of fouling organisms due to less macrofouler growth compared to blank paint. The isolated compound A4 was identified as hexadecanoic acid (C16H32O2) through NMR with a molecular mass of 256 g/mol detected using LC-MS. The characterization through FTIR obtained functional groups consisting of CH3, CH2, C=O, and OH. Therefore, U. lactuca produced hexadecanoic acid as one of the promising compounds from the seaweed group as an eco-friendly antifouling agent.

Evaluating the Efficacy of Actinidia deliciosa (Kiwi Fruit) Extract in Inhibiting Pseudomonas aeruginosa Biofilm Formation: An In Vitro Study With Therapeutic Implications.

Background Gram-negative Pseudomonas aeruginosa is a common bacteria that is well-known for its capacity to build biofilms, which are organized cell communities encased in a self-produced polymeric matrix. Treating infections becomes more challenging due to biofilms' capacity to provide immunity and resistance to antibiotics. The search for novel anti-biofilm agents has gained significant momentum, and the diverse range of bioactive compounds found in natural products offers a promising avenue. Rich in vitamins, antioxidants, and various phytochemicals, Actinidia deliciosa (kiwi fruit) has demonstrated potential as an antibacterial agent. Aim of the study This study aims to assess the efficacy of A. deliciosa extract in inhibiting biofilm formation by P. aeruginosa in vitro, providing valuable insights into its potential as a natural therapeutic agent for preventing recurrent bacterial infections. Materials and methodology The antibacterial and antibiofilm properties of A. deliciosa (kiwi fruit) methanolic extract were assessed in this study against P. aeruginosa (PAO1). The fruit was gathered, examined by a botanist for authenticity, and then cold macerated in methanol to create an extract. A two-fold broth dilution procedure was used to calculate the minimum inhibitory concentration (MIC), and agar well-diffusion was used to evaluate the antimicrobial activity. Pyocyanin pigment quantification was carried out after the extract was applied, and the antibiofilm impact was evaluated using a crystal violet assay. GraphPad Prism (GraphPad Software, San Diego, CA) was used for statistical analysis. Results Based on our findings, A. deliciosa was shown to have significant antibacterial and antibiofilm properties against P. aeruginosa (PAO1). At 5 mg/mL, the methanolic extract of A. deliciosa exhibited antibacterial activity with an 8 mm zone of inhibition and suppressed PAO1 growth. At 2.5 mg/mL and 1.25 mg/mL doses, PAO1 significantly decreased the production of biofilms by 60% and 29%, respectively. In addition, pyocyanin pigment synthesis was decreased by 30% and 9.25%, respectively, at sub-MIC doses of 2.5 mg/mL and 1.25 mg/mL. When evaluated at 2.5 mg/mL, the extract did not, however, appreciably affect bacterial growth. Conclusion This study enhances the understanding of antibiotic resistance, alternative treatments, and pathogenic microbes. The in vitro findings suggest that A. deliciosa fruit extract may inhibit pyocyanin production in PAO1. Further research with different formulations is recommended to explore its anti-biofilm properties and potential pharmacological applications.

Potential of silver nanoparticles synthesized from Justicia adhatoda metabolites for inhibiting biofilm on urinary catheters

In the present study, we investigated the anti-biofilm effect of urinary catheters fabricated with biogenic nanoparticles synthesized from metabolites of Justicia adhatoda under in vitro conditions against human pathogenic bacteria. Silver nanoparticles were synthesized in the reaction mixture composed of 2 % w/v of 0.1 M of precursor (silver nitrate) and 0.2 g of the metabolites obtained from ethanolic extract of Justicia adhatoda. Characterization of the nanoparticles was done by UV visible spectroscopy, fourier infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X ray diffraction (XRD) to confirm the structural and functional properties. Primary conformation of nanoparticles synthesis by UV visible spectroscopy revealed the notable absorption spectra at 425 nm with a wavelength shift around 450 nm, likely due to surface plasmon resonance excitation. SEM analysis showed spherical, monodisperse, nano scale particles with a size range of 50–60 nm. Crystaline phase of the synthesized nanoparticles was confirmed by x ray diffraction studies which showed the distinct peaks at (2θ) 27.90, 32.20, 46.30, 54.40, and 67.40, corresponding to (111), (200), (220), (222), and (311) planes of nano scale silver. The biocompatibility of these nanoparticles was assessed through zebrafish embryonic toxicity study which showed more than 90 % of embryos were alive and healthy. No marked changes on the blood cells also confirmed best hemocompatibility of the nanoparticles. Synthesized nanoparticles thus obtained were fabricated on the urinary catheter and the fabrication was confirmed by FTIR and SEM analysis. Notable changes in the absorption peaks, uniform coating and embedding of silver nanoparticles studied by FTIR and SEM analysis confirmed the fabrication of silver nanoparticles. The coated catheters demonstrated significant antibacterial activity against pathogenic bacterial strains, including E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Anti-biofilm studies, conducted using a modified microtiter plate crystal violet assay, revealed effective inhibition of both bacterial adhesion and biofilm development. 85 % of biofilm inhibition was recorded against both the tested strains. The coating method presented in this study shows promise for enhancing infection resistance in commonly used medical devices like urinary catheters, thus addressing device-associated infections.

Abstract C054: Current anticancer drugs differentially impact Black and White prostate cancer cell lines

Abstract Introduction/Background: In pre-clinical models of prostate cancer (PCa) such as the utilization of cell lines, it is important to address the limited diversity present. This acknowledgment is vital to facilitate equitable assessment of the efficacy and safety of drug development across various races and ethnicities. Of all commercially available PCa cell lines, 98% of them are of European origin. The genetic and biological disparities between PCa cell lines derived from men of European ancestry (MEA) and men of African ancestry (MAA) may underlie the observed enhanced responsiveness of tumours to existing anticancer agents in MEA compared to MAA. This difference is notable given that MAA are 2.5 times more likely to succumb to PCa. This study compared the cytotoxic effects of five anticancer drugs (Docetaxel, Cabazitaxel, Abiraterone, Olaparib, Enzalutamide) and the natural product betaine on MAA (ACRJ-PC28, MDA-PCA-2b) and MEA (DU-145, PC-3) PCa cell lines. Method: After a 72-hour treatment with the anti-cancer drugs and the natural product, the inhibitory effects of these anti-cancer agents on the four cell lines were investigated using three different assays. CellTiter Aqueous, Neutral Red, and Crystal Violet assays were utilized due to distinct growth patterns observed in the various cell lines. By comparing the optical density (OD) readings and examining the cellular morphology through microscopic visualization, we aimed to determine which assay(s) yielded the most reliable results in determining accurate IC50 values. Results: Drug selectivity was observed for Abiraterone and Olaparib that were most effective in ACRJ-PC28 (IC50 = 1.10 μM) and PC-3 (IC50 = 0.28 μM) cells compared to the other PCa cell lines. Enzalutamide differentially impacted MAA, (IC50= 206 μM for ACRJ-PC28 and 104 μM for MDA-PCA-2b) and MEA (IC50 = 37 μM for PC-3 and 48 μM for DU-145) PCa cell lines. However, no distinct impact was observed for Docetaxel (IC50s = 0.05 nM for ACRJ-PC28; 0.08 nM for MDA-PCA-2b, 0.07 nM for PC-3 and 0.08 nM for DU-145) and Cabazitaxel ((IC50s = 0.56 nM for ACRJ-PC28; 0.45 nM for MDA-PCA-2b, 0.43 nM for PC-3 and 0.38 nM for DU-145) as both drugs impacted the cell lines very potently in the nM range. Conclusion: Due to the heterogenous nature of cancers, especially in PCa, a single drug or drug class may not be the best course of treatment. Instead, a combination of drugs with different mechanisms may be more suitable for addressing the diversity in cancer types. This points to the evolving understanding of personalized cancer treatment, where a strategic integration of first, second, and third-line therapies can effectively address the genetic and epigenetic factors that are unique to individuals in cancer management. Citation Format: Simone Badal, Bor-Jang Hwang, Ashlianne Nelson, Kristoff Frank, Rory Thompson, Belinda Morison, Valerie Marah, Camille Ragin. Current anticancer drugs differentially impact Black and White prostate cancer cell lines [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C054.

Coumarin derivatives as new anti-biofilm agents against Staphylococcus aureus

Staphylococcus aureus infections are the primary causes of morbidity, and mortality, particularly in immuno-compromised individuals. S. aureus associated infections are acquired from community, as well as hospital settings, and difficult to treat because of the emerging resistance against available antibiotics. One of the key factors of its resistance is the biofilm formation, which can be targeted to treat S. aureus-induced infections. Currently, there is no drug available that function by targeting the biofilm. This unmet need demands the discovery of drug candidates against S. aureus biofilm. The present study was designed to evaluate coumarin derivatives 1–21 against S. aureus biofilm. The 96-well plate crystal violet assay was employed for the quantification of biofilm. Results showed that the coumarin derivatives 2–4, 10, and 17 possess potent antibiofilm activity, with MBIC values between 25–100 μg/mL. The results were further confirmed through atomic force microscopy (AFM), scanning electron (SEM), and fluorescence microscopic studies. The quantitative RT-PCR analysis revealed the downregulation of biofilm associated genes, icaA and icaD. These coumarin derivatives were also found to be non-cytotoxic to fibroblasts. This study, therefore, identifies the antibiofilm potential of coumarin derivatives that will pave the way for further research on these derivatives.

HPLC-PDA Analysis of Polyacetylene Glucosides from Launaea capitata and Their Antibacterial and Antibiofilm Properties against Klebsiella pneumoniae.

Background/Objectives: Bacterial resistance and virulence are challenges in treating bacterial infections, especially in Klebsiella pneumoniae. Plants of the Launaea Cass. genus are used traditionally to address a variety of diseases, including infections, but the potential bioactive compounds are unknown. Our goals were to verify the potential contribution of two major polyacetylene glycosides isolated from our previous study, (3S,6E,12E)-6,12-tetradecadiene-8,10-diyne-1-ol 3-O-β-D-glucopyranoside (1) and bidensyneoside A (syn. gymnasterkoreaside A) [(3R,8E)-3-hydroxy-8-decene-4,6-diyn-1-yl β-D-glucopyranoside] (2), to the anti-infective properties of Launaea capitata and to develop a dependable HPLC method for their quantification; Methods: On a panel of K. pneumoniae clinical isolates, the antibacterial action of 1, 2, and the methanol extract of the whole L. capitata plant were evaluated by broth microdilution assay, while their antibiofilm action was evaluated by the crystal violet assay. qRT-PCR investigated luxS, mrkA, wzm, and wbbm genes that encode biofilm formation and quorum sensing (QS). The antibacterial activity of 1 was revealed by employing mice infection. Chromatographic separation was conducted using isocratic elution on a Hypersil BDS C18 column using a photodiode array (PDA) detector; Results: Compound 1 showed antibacterial activity with MIC values of 16-128 µg/mL. It remarkably reduced strong and moderate biofilm-forming bacterial isolates from 84.21% to 42.1% compared with the extract (68.42%) and 2 (78.95%). Compound 1 also downregulated the QS genes, luxS, mrkA, wzm, and wbbm, and exhibited in vivo antibacterial action through the enhancement of the histological construction of the liver and spleen, decreased TNF-α immunoreaction, bacterial burden, and the inflammatory mediators IL-1β and IL-6. A successful HPLC-PDA approach was developed to separate the binary mixture of 1 and 2 in less than 10 min with high sensitivity, with detection limits down to 0.518 and 0.095 µg/mL for 1 and 2, respectively; Conclusions: Compound 1 exhibited remarkable antibacterial and antibiofilm properties and may contribute to the anti-infectious traditional uses of L. capitata, meriting further clinical studies and serving as a reliable quality control biomarker for the plant.

Exploring the hepatoprotective properties of citronellol: In vitro and in silico studies on ethanol-induced damage in HepG2 cells.

Citronellol (CT) is a monoterpene alcohol present in the essential oil of plants of the genus Cymbopogon and exhibits diverse pharmacological activities. The aim of the current study was to investigate the hepatoprotective potential of CT against ethanol-induced toxicity in HepG2 cell lines. Silymarin (SIL) was used as a standard drug. MTT, crystal violet assay, DAPI, and PI staining were carried out to assess the effect of ethanol and CT on cell viability. RT-PCR determined the molecular mechanisms of hepatoprotective action of CT. CT ameliorated cell viability and restricted ethanol-induced cell death. DAPI and PI staining showed distinct differences in cell number and morphology. Less cell viability was observed in the diseased group obviously from strong PI staining when compared to the CT- and SIL-treated group. Moreover, CT showed downregulation of interleukin (IL-6), transforming growth factor-beta 1 (TGF-β1), collagen type 1 A 1 (COL1A1), matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and glutathione peroxidase-7 (GPX-7) levels. Molecular docking studies supported the biochemical findings. It is concluded that the cytoprotective activity of CT against ethanol-induced toxicity might be explained by its anti-inflammatory, immunomodulatory, and collagen-regulating effects.