Biofilm Inhibitory Effect Research Articles

Inhibitory effects of simeprevir on Staphylococcusepidermidis and itsbiofilm in vitro.

Staphylococcus epidermidis (S. epidermidis) is a Gram-positive opportunistic pathogen that often causes hospital infections. With the abuse of antibiotics, the resistance of S. epidermidis gradually increases, and drug repurposing has become a research hotspot in the treating of refractory drug-resistant bacterial infections. This study aims to study the antimicrobial and antibiofilm effects of simeprevir, an antiviral hepatitis drug, on S. epidermidis in vitro. The micro-dilution assay was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of simeprevir against S. epidermidis. Crystal violet staining assay was used to detect the biofilm inhibitory effect of simeprevir. The antimicrobial activity of simeprevir against S. epidermidis and its biofilm were explored by SYTO9/PI fluorescent staining. The combined effect between simeprevir and gentamycin was assessed by checkerboard assay and was confirmed by time-inhibition assay. Simeprevir showed significant antimicrobial effects against S. epidermidis type strains and clinical isolates with the MIC and MBC at 2-16 μg/mL and 4-32 μg/mL, respectively. The antimicrobial effects of simeprevir were confirmed by SYTO9/PI staining. Simeprevir at MIC could significantly inhibit and break the biofilm on cover slides. Similarly, simeprevir also significantly inhibit the biofilm formation on the surface of urine catheters either in TSB [from (0.700±0.020) to (0.050±0.004)] (t=54.03, P<0.001), or horse serum [from (1.00±0.02) to (0.13±0.01)] (t=82.78, P<0.001). Synergistic antimicrobial effect was found between simeprevir and gentamycin against S. epidermidis with the fractional inhibitory concentration index of 0.5. Simeprevir shows antimicrobial effect and anti-biofilm activities against S. epidermidis.

Evaluation of antibacterial and antibiofilm properties of Minthostachys verticillata essential oils against bovine Staphylococcus aureus strains

Staphylococcus aureus is the main important mastitis pathogen. Treatment strategies using medicinal plants are novel alternatives. The aim of the present study was to evaluate the antibacterial activity and the biofilm inhibitory effects in vitro of two Minthostachys verticillata essential oils, EO1 and EO2, collected from different regions of Argentina against bovine Staphylococcus aureus. Chemical composition and cytotoxic effects were determined. Furthermore, antimicrobial activity of EO1 was evaluated in vivo as topical application emulsion in dairy cows after milking. Chemical profile of both oils showed different percentages among the major chemical compounds. In addition, MTT reduction method showed that oils were not toxic up to the concentration of 250 μg/ml. Results obtained revealed that oils were able to inhibit the growth and reduce biofilm production, from 36.4% to 96.06%. EO1 inhibited bacterial growth at lower concentrations compared to EO2 and it was selected for the emulsion preparation. Cows were treated with EO1 emulsion, at 4 and 6 mg/ml and it was able to reduce the bacterial load, showing an antibacterial effect. Both oils exhibited antibacterial and anti-biofilm activities against test bacteria showing that Minstosthachys verticillata essential oil may have potential application as bactericidal agent against S. aureus.

The Antimicrobial Peptide Octopromycin Suppresses Biofilm Formation and Quorum Sensing in Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic bacterial pathogen that causes severe infections in immunocompromised individuals. A. baumannii forms biofilm and produces extracellular matrix, which supports bacteria to survive under harsh conditions and be resistant to antibacterial treatments. In the present study, we investigated the biofilm and quorum-sensing inhibitory effects of antimicrobial peptide, octopromycin in A. baumannii. Field emission-scanning electron microscopy results clearly showed significantly reduced biofilm mass and caused a collapse in biofilm architecture at the minimum inhibitory concentration (50 µg/mL) and minimum bactericidal concentration (200 µg/mL) of octopromycin. Antibiotic-resistant persister cells of A. baumannii were successfully killed by octopromycin treatment, and it inhibited violacein production in Chromobacterium violaceum in a concentration-dependent manner. Octopromycin also inhibited alginate production, surface movements (swarming and swimming), and twitching motility of A. baumannnii, confirming its anti-quorum-sensing activity. Multiple metabolic pathways, two-component regulation systems, quorum-sensing, and antibiotic synthesis-related pathways in A. baumannii biofilms were strongly affected by octopromycin treatment. The collective findings indicate that the antibacterial peptide octopromycin may control A. baumannii biofilms through multi-target interactions. Octopromycin could be a desirable therapeutic option for the prevention and control of A. baumannii infections.

Antibiofilm mechanism of dielectric barrier discharge cold plasma against Pichia manshurica

Biofilm spoilage has become one of the most common concerns in fermented foods. The objective of the present study was to explore the biofilm-inhibitory effect and antibiofilm mechanism of dielectric barrier discharge (DBD) cold plasma on Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods). After plasma treatment (80 kV, 50 Hz) for 4.5 min and 7.5 min, viable counts decreased by 2.38 and 5.11 lg CFU/mL, respectively, and the biofilm-forming rate decreased to 73% and 48% of the control group, respectively, in microbiological media. In addition, DBD caused severe morphological damage, cell membrane permeabilization and metabolic changes. A number of amino acid metabolic pathways, the tricarboxylic acid cycle (TCA), and the synthesis of extracellular polymeric substance (EPS) were destroyed. The cell damage and changes in multiple metabolic activities led to significant inhibitory effects on Pichia manshurica and its biofilm-forming ability. Industrial relevanceThis study found that DBD can significantly inhibit the biofilm-forming ability of Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods) by causing severe cell damage and metabolic changes. The exploration of the biofilm inhibitory effect of DBD on Pichia manshurica and its mechanism can provide a theoretical basis for the application of DBD technology in the inhibition of biofilm spoilage in foods.

Vancomycin Loaded Amino-Functionalized MCM-48 Mesoporous Silica Nanoparticles as a Promising Drug Carrier in Bone Substitutes for Bacterial Infection Management.

Orthopedic infections due to biofilm formation in biomaterial-based implants have become challenging in bone tissue engineering. In the present study, in vitro antibacterial analysis of amino-functionalized MCM-48 mesoporous silica nanoparticles (AF-MSNs) loaded with vancomycin is analyzed for its potential as a drug carrier for the sustained/controlled release of vancomycin against Staphylococcus aureus. The effective incorporation of vancomycin into the inner core of AF-MSNs was observed by alternation in the absorption frequencies obtained by Fourier transform infrared spectroscopy (FTIR). Dynamic light scattering (DLS) and high resolution-transmission electron microscopy (HR-TEM) results show that all the AF-MSNs had homogeneous spherical shapes with a mean diameter of 165.2 ± 1.25nm, and there is a slight change in the hydrodynamic diameter after vancomycin loading. Furthermore, the zeta potential of all the AF-MSNs (+ 30.5 ± 0.54mV) and AF-MSN/VA (+ 33.3 ± 0.56mV) were positively charged due to effective functionalization with 3-aminopropyl triethoxysilane (APTES). Furthermore, cytotoxicity results show that the AF-MSNs have better biocompatibility than non-functionalized MSNs (p < 0.05), and results prove AF-MSNs loaded with vancomycin show better antibacterial effect against S. aureus than non-functionalized MSNs. Results confirm that bacterial membrane integrity was affected by treatment with AF-MSNs and AF-MSN/VA by staining the treated cells with FDA/PI. Field emission scanning electron microscopy (FESEM) analysis confirmed the shrinkage of bacterial cells and membrane disintegration. Furthermore, these results demonstrate that amino-functionalized MSNs loaded with vancomycin significantly increased the anti-biofilm and biofilm inhibitory effect and can be incorporated with biomaterial-based bone substitutes and bone cement to prevent orthopedic infections post-implantation.

Inhibitory effect of lactobacilli supernatants on biofilm and filamentation of Candida albicans, Candida tropicalis, and Candida parapsilosis.

Probiotic Lactobacillus strains had been investigated for the potential to protect against infection caused by the major fungal pathogen of human, Candida albicans. Besides antifungal activity, lactobacilli demonstrated a promising inhibitory effect on biofilm formation and filamentation of C. albicans. On the other hand, two commonly isolated non-albicans Candida species, C. tropicalis and C. parapsilosis, have similar characteristics in filamentation and biofilm formation with C. albicans. However, there is scant information of the effect of lactobacilli on the two species. In this study, biofilm inhibitory effects of L. rhamnosus ATCC 53103, L. plantarum ATCC 8014, and L. acidophilus ATCC 4356 were tested on the reference strain C. albicans SC5314 and six bloodstream isolated clinical strains, two each of C. albicans, C. tropicalis, and C. parapsilosis. Cell-free culture supernatants (CFSs) of L. rhamnosus and L. plantarum significantly inhibited in vitro biofilm growth of C. albicans and C. tropicalis. L. acidophilus, conversely, had little effect on C. albicans and C. tropicalis but was more effective on inhibiting C. parapsilosis biofilms. Neutralized L. rhamnosus CFS at pH 7 retained the inhibitory effect, suggesting that exometabolites other than lactic acid produced by the Lactobacillus strain might be accounted for the effect. Furthermore, we evaluated the inhibitory effects of L. rhamnosus and L. plantarum CFSs on the filamentation of C. albicans and C. tropicalis strains. Significantly less Candida filaments were observed after co-incubating with CFSs under hyphae-inducing conditions. Expressions of six biofilm-related genes (ALS1, ALS3, BCR1, EFG1, TEC1, and UME6 in C. albicans and corresponding orthologs in C. tropicalis) in biofilms co-incubated with CFSs were analyzed using quantitative real-time PCR. When compared to untreated control, the expressions of ALS1, ALS3, EFG1, and TEC1 genes were downregulated in C. albicans biofilm. In C. tropicalis biofilms, ALS3 and UME6 were downregulated while TEC1 was upregulated. Taken together, the L. rhamnosus and L. plantarum strains demonstrated an inhibitory effect, which is likely mediated by the metabolites secreted into culture medium, on filamentation and biofilm formation of C. albicans and C. tropicalis. Our finding suggested an alternative to antifungals for controlling Candida biofilm.

Cytotoxic, Antioxidant, Antibiofilm, and Antimicrobial Activities of Mushroom Species from Turkey.

Mushrooms, which have been collected to meet the nutritional needs of the world for many years, have gained medical importance thanks to the bioactive compounds they produce. Thanks to studies carried out to determine mushroom diversity, the number of species identified is increasing year by year. Accordingly, in recent years, studies conducted to determine the biological activities of metabolites produced by fungi have been increasing. The present study was conducted to determine the cytotoxic, antioxidant, antibiofilm and antimicrobial activities of the seven different mushroom species (Craterellus cornucopioides, Hymenopellis radicata, Lepista nuda, Pisolithus arhizus, Ramaria flava, Schizophyllum commune, and Tricholoma ustale) collected from Tokat and Yozgat regions located in northern and central Turkey. Laboratory studies have demonstrated that mushrooms used in this study have different degrees of antibiofilm, antimicrobial, antioxidant and cytotoxic activities. At the end of the study, it is determined that C. cornucopioides and L. nuda species have the highest antimicrobial activity. In addition, mushroom species have biofilm inhibitory effects on indicator microorganisms at varying degrees ranging between 20.7 and 96.3%. As a result of antioxidant activity studies, it was determined that T. ustale has the highest free radical scavenging effect and P. arhizus, which has the highest polyphenol content, has the highest reducing power. Finally, it is determined that, among the mushrooms used in the present study, H. radicata showed higher selectivity on the MDA-MB-231 breast cancer cell line than on the normal cell line tested, while C. cornucopioides showed higher selectivity on the MCF-7 breast cancer cell line.

Antibacterial and antibiofilm activities of taxifolin against vancomycin-resistant S. aureus (VRSA)

Background and objective: The medicinal effects of flavonoids are widely described in the literature; however, their antimicrobial effects against antibiotic resistant bacteria are yet to be highlighted. This study was aimed at investigating the growth and biofilm inhibitory effects of taxifolin, a flavonoid, against vancomycin-resistant Staphylococcus aureus (VRSA). Methods: Seven VRSA isolates were used to assess the antimicrobial and antibiofilm influence of taxifolin. The agar-well diffusion method was used to determine the zones of inhibition caused by taxifolin, and resazurin-based microdilution technique was used to assess the minimum inhibitory concentration. Crystal violet staining technique was used to assess the biomass of biofilms formed by the microorganisms. GraphPad Prism software was used to present the data in figures. Results: Taxifolin inhibited bacterial growth in a dose-dependent fashion and reduced bacterial viability. It similarly attenuated the biofilm production activity of bacterial isolates in a dose-dependent manner. Conclusions: Current findings suggest the antibacterial and antibiofilm influence of taxifolin against VRSA in a dose-dependent manner.

Effect of Ruta graveolens Extract on the Major Virulence Factors in Methicillin Resistant Staphylococcus aureus.

Rising Antibiotic Resistance has put the world in real threat. Methicillin resistant Staphylococcus aureus (MRSA), is a predominant cause of suppurative chronic skin and soft-tissue infections. Novel insights have focused the light on plant extracts. In this study, Ruta graveolens ethanolic active extract was tested for its potential anti-virulence activities in MRSA. A total of 100 MRSA strains causing skin and soft tissue infections were isolated and antibiotic susceptibility testing was done. Ability to form biofilm was tested phenotypically. Furthermore, the antimicrobial activity of Ruta graveolens was evaluated followed by detection of its Minimum inhibitory concentration (MIC). The inhibitory activity of this extract on biofilm formation was investigated. Afterwards, we investigated its effect on the transcription of biofilm-related genes and mecA gene. All tested isolates were sensitive to Vancomycin and Linezolid while high resistance was noted with both Fusidic acid (83%) and Gentamicin (68%). (83%) of the isolates were biofilm producers. Ruta graveolens extract showed strong antimicrobial activity against the MRSA strains with MIC 0.78 mg/mL. At subinhibitory concentration (1/2 MIC), the extract had high biofilm inhibitory effects with mean inhibition (70%). Moreover, transcriptional analysis results showed that the mean percentages of inhibition in expression of mecA, icaA and icaD genes were 52.3%, 34.8% and 33.7%, respectively, in which all showed statistically significant difference (p ≤ 0.05). The current study proposes the ability of Ruta graveolens extract to reduce the biofilm formation and antibiotic resistance of MRSA through downregulation of some biofilm forming genes and mecA gene which confers resistance to B-lactam antibiotics. This may decrease our reliance on antibiotics and improve our ability to effectively treat biofilm-related skin and soft-tissue infections caused by MRSA.

Efficacy of Silver Coated Foley’s Catheter in Comparison with Latex Foley’s Catheter for Prevention of Antimicrobial Resistance and Catheter Associated Urinary Tract Infections in Obstructive Urolithiasis of Male Buffalo Calves

Background: Foley’s catheters are widely used for treating obstructive urolithiasis and are often associated with high rate of urinary tract infections. The urinary catheters act as a platform for colonization and biofilm formation by pathogens, leads to increasing the risk of catheter associated bacterial infections. Methods: The present study was aimed to address the antimicrobial activity and biofilm inhibition effect of silver Foley’s catheter in male buffalo calves suffering from obstructive urolithiasis. The study was conducted on twelve male buffalo calves suffering from obstructive urolithiasis, randomly divided into two groups irrespective of their age viz., group I (latex Foley’s catheter treatment) and group II (silver hydrogel coated Foley’s catheter treatment). Cultural examination and antibiotic sensitivity of cultured bacteria from the urine passed through fixed urinary catheter was performed at 0th, 7th, 14th and 28th postoperative days in both the groups. Result: There was significant reduction in growth of bacteria like Streptococcus, Staphylococcus and E. coli bacterial colony formation on blood agar and MacConkey lactose agar, respectively in silver catheter group as compare to latex catheter group from day 0 to day 28th. The zone of inhibition was more pronounced for both antibiotics viz., Cefotaxime and Ceftriaxone (upto 24 mm) in urine samples collected from silver hydrogel coated Foley’s catheter as compared to latex Foley’s catheter indicating its biofilm inhibition effect on bacteria. Silver-hydrogel Foley’s Catheter are more cost-effective as compare to latex Foley’s catheter.

Synthesis, Characterization, Antibiofilm and Anticancer Activity of New Ruthenium Complexes with 2,2'-bipyridine-4,4'-dicarboxamide

Abstract: New ruthenium complexes bearing bipyridine ligands with different substituents (propyl, hexyl, isobutyl, and benzyl) were synthesized and characterized by MS, NMR, FTIR, and UV/Visible spectroscopy. Moreover, their cytotoxic, anti-carcinogenic, and anti-biofilm activities were evaluated. The electrochemical properties of the complexes have been investigated by cyclic voltammetry. The HOMO and LUMO energy levels of RuL1-RuL4 were found to be (-5.45 eV)-(-5.46 eV) and (-2.98 eV)-(-3.01 eV), respectively. Cytotoxic activities of ruthenium complexes were investigated in Caco-2, HepG2, and HEK293 cells. It was found that RuL3 showed a cytotoxic effect on cancer cells without affecting non-cancerous cells at applied doses. The presence of the benzyl group may increase the cytotoxic effect of RuL3 compared to other derivatives that contain the alkyl group. The apoptotic effect of the RuL3 derivative was determined by using Arthur image-based cytometer. It found that RuL3 induced apoptosis in Caco-2 (5-fold) and HepG2 (2-fold) cancer cells, respectively. All ruthenium complexes inhibited Staphylococcus aureus ATCC 29213 biofilm, but RuL3 had a more pronounced effect. Moreover, RuL3 had biofilm inhibition and biofilm degradation effect, while RuL1 and RuL4 demonstrated only biofilm inhibition. The fluorescent microscopy analysis confirmed the antibiofilm effect of ruthenium complexes. All of these results clearly showed that RuL3 showed cytotoxic and apoptotic effects on cancer cells.

Preparation of rutin-loaded mesoporous silica nanoparticles and evaluation of its physicochemical, anticancer, and antibacterial properties.

The studies have shown that rutin has great potential as an anticancer and antimicrobial plant base agent; nevertheless, poor bioavailability and low aqueous solubility of rutin limit its application. One of the beneficial routes to increase the solubility and bioavailability of rutin is the development of nanoparticulate material. This study aimed to assess the anticancer and antibacterial effects of rutin-loaded mesoporous silica nanoparticles (RUT-MSNs). RUT-MSNs were prepared and physicochemically characterized. The cytotoxicity of RUT-MSNs on the HN5 cells as head and neck cancer cells was evaluated. The expression level of apoptosis-related genes such as Bcl-2 and Bax genes were evaluated. In addition, ROS production of RUT-MSNs treated cells was assessed. In addition, minimum inhibitory concentration (MIC), biofilm, and attachment inhibitory effects of RUT-MSNs compared with free rutin were assessed against different bacterial strains. Transmission electron microscopy (TEM) showed mesoporous rod-shaped nanoparticles with an average particle size of less than 100nm. RUT-MSNs displayed the cytotoxic effect with IC50 of 20.23 µM in 48h of incubation time (p < 0.05). The elevation in the ratio of Bax/Bcl-2 was displayed within the IC50 concentration of RUT-MSNs in 48h (p < 0.05). The antibacterial action of rutin was improved by loading rutin in MSNs to the nano-sized range in the MIC test. The anticancer and antibacterial effects of RUT-MSNs were considerably more than rutin. RUT-MSNs inhibited the growth of HN5 cells by inducing apoptosis and producing ROS. These results suggest that RUT-MSNs may be useful in the treatment of cancers and infections.

Anti-quorum sensing and biofilm inhibitory effect of some medicinal plants against gram-negative bacterial pathogens: in vitro and in silico investigations

Multidrug resistance (MDR) in pathogenic bacteria have become a major clinical issue. Quorum sensing regulated bacterial virulence is a promising key drug target for MDR infections. Therefore, the aim of the present work was to assess the anti-quorum sensing properties of selected medicinal plants against bacterial pathogens as well in silico interaction of selected bioactive phytocompounds with QS and biofilm-associated proteins. Based on the ethnopharmacological usage, 18 plants were selected using methanolic extract against Chromobacterium violaceum 12472. The most active extract (Acacia nilotica) was fractionated in increasing polarity solvents (n-hexane, chloroform and ethyl acetate) and tested for anti-QS activity. The most active fraction i.e. ethyl acetate fraction was evaluated for their activity at sub-MICs against QS-associated virulence factors of Pseudomonas aeruginosa PAO1 and Serretia marcescens MTCC 97. Microtiter plate assay and light microscopy was used to determine inhibition of biofilm. Phytochemicals of the ethyl acetate fraction were analysed by GC/MS and LC/MS. Phytocompounds were docked with QS (LasI, LasR, CviR, and rhlR) and biofilm proteins (PilY1 and PilT) using Auto dock vina. The MIC of ethyl acetate fraction determined was 250, 500, and 1000 μg/ml against C. violaceum 12472, P. aeruginosa PAO1, and S. marcescens MTCC97 respectively. At sub-MICs QS regulated virulence factors production and inhibited biofilms broadly (more than 50 percent). GC/MS detected the major bioactive compound benzoic acid, 3,4,5-trihydroxy-, methyl ester (61.24 %) and LC-MS detected Retronecine for the first time in A. nilotica pods. In silico, dehydroabietic acid occupied the same cavity as its antagonist in the CviR ligand binding domain. Also, betulin and epicatechin gallate interact with biofilm proteins PilY1 and PilT, preventing biofilm formation. The findings suggest that the phytochemicals of A. nilotica pod could be exploited as an anti-QS agent against Gram-negative pathogens. To discover therapeutic efficacy of standardised bioactive extract/phytochemicals must be tested under in vivo condition.

P068 Inhibition of Candida glabrata biofilm using the combination of a novel antifungal cyclic lipopeptide and azole: An in vitro synergistic approach

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMThe general feature of the biofilm is a densely packed microbial community with an extracellular matrix composed of polysaccharides and proteins. Candida glabrata was reported as the most biofilm-forming species responsible for causing virulent Candida infections among Candida non-albicans (CNA). Candida glabrata is the most azole-resistant species of all Candida species. The early and maturing biofilms enable the Candida cells to overcome the effects of azoles and exhibit higher drug resistance. Since simple azole mono-therapy rarely eradicates recalcitrant Candida biofilms, removal of the infected device becomes necessary for curing the infection.ObjectiveThe objective was to assess the biofilm inhibition effect of antifungal cyclic lipopeptide which has been purified by a multi-step process from the cell-free supernatant of Bacillus subtilis, and to analyze the synergistic activity of the purified lipopeptide lead compound designated as AF4 with the standard antifungal fluconazole using different concentrations.MethodsBriefly, cell-free supernatant was extracted using a solvent mixture and silica gel-based adsorption chromatography. The potential antifungal cyclic lipopeptide variant AF4 was extracted from the soil isolate B. subtilis by a multi-step purification process that involves reversed-phase HPLC and exhibited a wide-spectrum of anti-Candida activity. The XTT reduction assay was used for metabolically active cells after treatment with a combination of drugs, and crystal violet (CV) assay was performed to quantify the cell biomass. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to analyze the biofilm morphology and architecture of the AF4 lipopeptide at (2xMIC) and fluconazole (8x-16xMIC)-treated biofilms. For confocal microscopy-based biofilm visualization, a combination of two different dyes was used. Extracellular polymeric substances were stained by concanavalin A-Alexa Fluor 488 conjugate and, the FUN- 1 fluorescent dye was used for staining the live biofilm-forming Candida cells. After capturing the Z-stack images in CLSM, the biomass (μm3/μm2), average thickness (μm), and roughness of the biofilm were analyzed by Comstat 2.1 software.ResultsThe AFST analysis showed that the MIC of the lead antifungal lipopeptide was 8 μg/mL against the C. glabrata planktonic cells. Adding the antifungal compound at higher MICs such as by 2-folds exhibited strong antibiofilm effect that significantly enhanced the synergistic antifungal activity of the fluconazole against C. glabrata biofilm formation. Synergistic effect of fluconazole at 8x-16xMIC with 2xMIC of the lead compound AF4 on C. glabrata 24 h maturing biofilm had increased significantly as revealed by the XTT and CV assays. The results confirmed ˃ 50% inhibition of the biofilm in both 6 h developmental and 24 h maturing biofilms in 8xMIC fluconazole plus 2xMIC AF4 while compared with fluconazole alone at the same concentration. The results from the XTT and, CV assays and SEM and CLSM (Fig. 1) suggest that the combinatorial treatments of the lead antifungal lipopeptide and fluconazole at varying concentrations have the potential of eradicating or nearly eradicating the maturing biofilm of CAN.ConclusionsThe combinatorial study was found effective in combating the biofilm formation. A correlation between metabolic activity and antifungal resistance in maturing biofilms has been found.

D-Cysteine Functionalized Superhydrophobic Nanocomposite Coating with Multiple-Action Antibacterial Property and Enhanced Mechanical Durability

In this study, a durable superhydrophobic antibacterial coating was developed by a facile spraying method. A mixture of bisphenol A diglycidyl ether (BADGE) and hydrophobic SiO2 nanoparticles was sprayed on carbon steel to provide a superhydrophobic substrate. D-cysteine (D-cys) functionalized SiO2/dopamine/silver nanoparticles with multilayer core-shell structure were then sprayed on superhydrophobic substrate to enhance antibacterial performance. The results of morphology observation and X-ray photoelectron spectroscopy (XPS) indicated the successful preparation of antibacterial nanoparticles and presented the hierarchical micro/nanostructures of coating surface. The as-prepared coating exhibited superhydrophobicity, with the water contact angle of ~153°. The coating was endowed with good mechanical durability, which maintained the water contact angle of ~150° after 180 cycles in tape-peel tests. The results of electrochemical impedance spectroscopy showed satisfactory corrosion resistance of this coating during 3 days of immersion in 3.5% NaCl solution. Furthermore, the coating showed excellent antibacterial performances against Escherichia coli and Pseudomonas aeruginosa, which benefited from the synergistic actions of low wettability of superhydrophobic surface, bactericidal behavior of Ag nanoparticles, and biofilm inhibition effect of D-cys.

The effect of photodynamic therapy using Radachlorin on biofilm-forming multidrug-resistant bacteria.

ObjectivesThis study aimed to test the effect of photodynamic therapy (PDT) on the inhibition and removal of biofilms containing multidrug-resistant Acinetobacter baumannii.Methods Using multidrug-resistant A. baumannii strains, an antibiotic susceptibility test was performed using the Gram-negative identification card of the Vitek 2 system (bioMérieux Inc., France), as well as an analysis of resistance genes, the effects of treatment with a light-emitting diode (LED) array using Radachlorin (RADA-PHARMA Co., Ltd., Russia), and transmission and scanning electron microscopy to confirm the biofilm-inhibitory effect of PDT.Results The antibiotic susceptibility test revealed multiple resistance to the antibiotics imipenem and meropenem in the carbapenem class. A class-D–type β-lactamase was found, and OXA-23 and OXA-51 were found in 100% of 15 A. baumannii strains. After PDT using Radachlorin, morphological observations revealed an abnormal structure due to the loss of the cell membrane and extensive morphological changes, including low intracellular visibility and small vacuoles attached to the cell membrane.Conclusion PDT involving a combination of LED and Radachlorin significantly eliminated the biofilm of multidrug-resistant A. baumannii. Observations made using electron microscopy showed that PDT combining LED and Radachlorin was effective. Additional studies on the effective elimination of biofilms containing multidrug-resistant bacteria are necessary, and we hope that a treatment method superior to sterilization with antibiotics will be developed in the future.

Screening the Tocriscreen™ bioactive compound library in search for inhibitors of Candida biofilm formation.

Biofilms formed by Candida species present a significant clinical problem due to the ineffectiveness of many conventional antifungal agents, in particular the azole class. We urgently require new and clinically approved antifungal agents quickly for treatment of critically ill patients. To improve efficiency in antifungal drug development, we utilized a library of 1280 biologically active molecules within the Tocriscreen 2.0 Micro library. Candida auris NCPF 8973 and Candida albicans SC5314 were initially screened for biofilm inhibitory activity using metabolic and biomass quantitative assessment methods, followed up by targeted evaluation of five selected hits. The initial screening (80% metabolic inhibition rate) revealed that there was 90 and 87 hits (approx. 7%) for C. albicans and C. auris, respectively. Additionally, all five compounds selected from the initial hits exhibited a biofilm inhibition effect against several key Candida species tested, including C. glabrata and C. krusei. Toyocamycin displayed the most potent activity at concentrations as low as 0.5 μg/mL, though was limited to inhibition. Darapladib demonstrated an efficacy for biofilm inhibition and treatment at a concentration range from 8 to 32 μg/mL and from 16 to 256 μg/mL, respectively. Combinational testing with conventional antifungals against C. albicans strains demonstrated a range of synergies for planktonic cells, and notably an anti‐biofilm synergy for darapladib and caspofungin. Together, these data provide new insights into antifungal management possibilities for Candida biofilms.

Biofilm and hyphal inhibitory synergistic effects of phytoactives piperine and cinnamaldehyde against Candida albicans.

Oral candidiasis, the most common mycotic infection of the human oral cavity is non-life-threatening yet if untreated may advance as systemic infections. Ability of Candida albicans to adapt sessile lifestyle imparts resistance to drugs and host immunity. Consequently, due to limited effectiveness of conventional antifungal treatment, novel therapeutic strategies are required. In the present study, synergistic interaction of phytochemicals, piperine and cinnamaldehyde against the biofilm and hyphal of C. albicans was evaluated. Minimum inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) of piperine and cinnamaldehyde against C. albicans were analysed through microbroth dilution assay and crystal violet staining method, respectively. Combinatorial biofilm and hyphal inhibitory effect were investigated through checkerboard assay. In vitro results were validated through gene expression analysis. BIC of piperine and cinnamaldehyde was determined to be 32µg/mL and 64µg/mL, respectively. Interaction between these two phytocomponents was found to be synergistic and six different synergistic antibiofilm combinations were identified. Microscopic analysis of biofilm architecture also evidenced the biofilm and surface adherence inhibitory potential of piperine and cinnamaldehyde combinations. Phenotypic switching between yeast and hyphal morphological forms was influenced by synergistic combinations. qPCR analysis corroborated the results of in vitro activities. nrg1 and trp1, the negative transcriptional regulators of filamentous growth were upregulated whereas other genes that are involved in biofilm formation, filamentous growth, adhesion etc were found to be downregulated. These proficient phytochemical combinations provide a new therapeutic avenue for the treatment of biofilm associated oral candidiasis and to combat the recurrent infections due to antibiotic resistance.

Assessment of Antibacterial and Anti-biofilm Effects of Vitamin C Against Pseudomonas aeruginosa Clinical Isolates

There is a persistent need to look for alternative therapeutic modalities to help control the pandemic of antimicrobial resistance. Assessment of antibacterial and anti-biofilm effects of vitamin C (ascorbic acid) was the aim of the current study. The micro-dilution method determined the minimal inhibitory concentration (MIC) of ascorbic acid or antibiotics alone and in combinations against Pseudomonas aeruginosa (P. aeruginosa) clinical isolates. The micro-titer plate method monitored the effect of ascorbic acid on the biofilm-producing isolates of P. aeruginosa. The effect of ascorbic acid on the differential expression of different antibiotic-resistant genes and biofilm encoding genes of P. aeruginosa isolates were also tested using real-time polymerase chain reaction (PCR). For in vivo assessment of the antibacterial effects of ascorbic acid alone or combined with an antibiotic, rats were infected with P. aeruginosa clinical isolate followed by different treatment regimens. MICs of ascorbic acid among P. aeruginosa isolates were in the range of 156.2–1,250 μg/ml, while MIC50 and MIC90 were 312.5 and 625 μg/ml, respectively. At sub-inhibitory concentrations (19.5–312.5 μg/ml), ascorbic acid had 100% biofilm inhibitory effect. Furthermore, ascorbic acid-treated bacteria showed downregulation of genes underpinning biofilm formation and antibiotic resistance. In vivo assessment of vitamin C and ceftazidime in rats showed that administration of both at a lower dose for treatment of pseudomonas infection in rats had a synergistic and more powerful effect. Vitamin C shows excellent in vitro results as an antibacterial and anti-biofilm agent. Vitamin C should be routinely prescribed with antibiotics to treat bacterial infections in the clinical setting.

Antibiofilm effect of nisin alone and combined with food-grade oil components (thymol and eugenol) against Listeria monocytogenes cocktail culture on food and food-contact surfaces

Listeria monocytogenes is a prominent foodborne pathogen with potent biofilm-forming capability, which is to be crucial for its antimicrobial resistance and persistence. Therefore, using a combination of preservative components (hurdle technology) to control biofilms is a promising strategy. The purpose of this study was to investigate the antibiofilm efficacy of nisin alone and combined with thymol and eugenol against L. monocytogenes cocktail biofilm formed on abiotic (rubber and MBEC™ biofilm device) and biotic (lettuce) surfaces. Results showed that nisin at low concentrations (250 and 500 IU/mL) was not able to inhibit the biofilms to an acceptable level. Sub-MICs (0.5 × MIC) of thymol (0.02%) and eugenol (0.075%) did not significantly inhibit ( P > 0.05), but at high concentration (2 × MIC) inhibited significantly ( P < 0.05). When nisin (250 or 500 IU/mL) was combined with thymol at 0.5 × MIC (0.02%), 1 × MIC (0.04%), and 2 × MIC (0.08%) or eugenol at 0.5 × MIC (0.075%), 1 × MIC (0.15%), and 2 × MIC (0.30%), the inhibitory effect was remarkably superior to that of the single compounds. These results suggest the feasible benefits of combining nisin with food-grade essential oil components to inhibit L. monocytogenes biofilm formation in the food industry. • Nisin at low concentrations was unable to sufficiently inhibit the biofilms. • Biofilm inhibitory effect of nisin increased after combining with EO components. • Combination let to better biofilm inhibition than the single treatments. • Combining nisin with food-grade EO components could be a promising approach.