Biofilm Assay Research Articles

Discovery of novel secondary metabolites from the basidiomycete Lentinus cf. sajor-caju and their inhibitory effects on Staphylococcus aureus biofilms

Three novel derivatives of microporenic acid, microporenic acids H–J, were identified from submerged cultures of a Lentinus species obtained from a basidiome collected during a field trip in the tropical rainforest in Western Kenya. Their structures were elucidated via HR-ESIMS spectra and 1D/2D NMR spectroscopic analyses, as well as by comparison with known derivatives. Applying biofilm assays based on crystal violet staining and confocal microscopy, two of these compounds, microporenic acids H and I, demonstrated the ability to inhibit biofilm formation of the opportunistic pathogen Staphylococcus aureus. Thereby, they were effective in a concentration range that did not affect planktonic growth. Additionally, microporenic acid I enhanced the anti-biofilm activity of the antibiotics vancomycin and gentamicin when used in combination. This opens up possibilities for the use of these compounds in combination therapy to prevent the formation of S. aureus biofilms.

Chemical composition, antifungal, antibiofilm, and molecular docking studies of Syzygium dyerianum essential oil.

The current study describes the chemical composition, antifungal, antibiofilm, antibacterial and molecular docking studies of Syzygium dyerianum growing in Malaysia. The essential oil was obtained through hydrodistillation and characterized using gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The antifungal and antibacterial activities were developed using the broth microdilution assay, whereas the effect on the microbial biofilms was determined using a semi-quantitative static biofilm assay. A total of 31 components were identified, which represent 99.5 % of the essential oil. The results revealed that the essential oil consisted mainly of β-pinene (15.6 %), α-terpineol (13.3 %), α-pinene (11.1 %), caryophyllene oxide (8.8 %), limonene (8.1 %), borneol (6.0 %) and viridiflorol (5.1 %). The results of the microdilution method showed that essential oil exhibited activity against Candida albicans and Streptococcus mutans with minimal inhibitory concentration values of 125 and 250 μg/mL, respectively. Furthermore, essential oil decreased the biofilm of C.albicans and S.mutans by 20.11±0.27 % and 32.10±4.81 % when treated with 250 μg/mL. The best docking energy was observed with viridiflorol (-29.7 kJ/mol). This study highlights that essential oil can potentially be a natural antifungal, antibacterial, and antibiofilm agent that could be applied in the pharmaceutical and food industries.

Detection of the early putative biofilm marker pgaB among the MDR strains of A.baumannii

BackgroundpgaB associated biofilm formation in Acinetobacter baumannii enhances the virulence in concert with the high propensity of drug resistance. This research is thus aimed to characterize the pgaB gene molecularly and to examine its co-occurrence with MDR. MethodologyMDR strains of A. baumannii (N = 73) were selected to detect the formation of biofilms. Genomic DNA was extracted further and screened for pgaB followed by amplicon sequencing from the representative strains. Frequency of its distribution in different groups of drug resistant strains at a significant p-value of <0.05 was further checked. ResultsThe biofilm assay showed high, low and negative biofilm formers in 58.9%, 31.5% and 0.9% of the strains respectively. The pgaB gene was detected in 14 strains of MDR A. baumannii (19.17%). Co-occurrence of pgaB gene was seen in all the strains that showed resistance to β-lactam inhibitors, cephems, carbapenems, fluoroquinolones and folates followed by 96% for the aminoglycosides and 25% in the efflux pump groups. ConclusionThe study findings showed the occurrence of biofilms associated with pgaB in MDR A. baumannii strains. The results also suggest to track its role in varying the pattern of drug resistance with further experimentation.

Effect of Potassium Permanganate on Staphylococcal Isolates Derived from the Skin of Patients with Atopic Dermatitis.

In atopic dermatitis (AD), Staphylococcus aureus frequently colonizes lesions, leading to superinfections that can then lead to exacerbations. The presence of biofilm-producing isolates has been associated with worsening of the disease. Potassium permanganate is used as a topical treatment of infected eczema, blistering conditions, and wounds. Little is known of its effects against microbes in AD skin. The aim of this study was to explore antibacterial and antibiofilm properties of potassium permanganate against staphylococcal isolates derived from AD skin. Viable count and radial diffusion assays were used to investigate antibacterial effects of potassium permanganate against planktonic staphylococcal isolates. The antibiofilm effects were assessed using biofilm assays and scanning electron microscopy. The Staphylococcus aureus isolates were completely killed when exposed to 0.05% of potassium permanganate. In concentrations of 0.01%, potassium permanganate inhibited bacterial biofilm formation. Eradication of established staphylococcal biofilm was observed in concentrations of 1%. Electron microscopy revealed dense formations of coccoidal structures in growth control and looser formations of deformed bacteria when exposed to potassium permanganate. This suggests antibacterial and antibiofilm effects of potassium permanganate against staphylococcal isolates derived from AD skin, when tested in vitro, and a potential role in the treatment of superinfected AD skin.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli.

One of the causes of antibiotic resistance is the reduced accumulation of antibiotics in bacterial cells through pumping out the drugs. Silybin, a key component of the Silybum marianum plant, exhibits various beneficial properties, including anti-bacterial, anti-inflammatory, antioxidant, and hepatoprotective effects. Clinical isolates of E. coli were procured from 17 Shahrivar Children's Hospital in Rasht, Guilan, located in northern Iran. Their susceptibility to six antibiotics was assessed using disc diffusion and broth dilution (MIC) methods. The antibacterial effects of silybin-loaded polymersome nanoparticles (SPNs) were investigated with broth dilution (MIC) and biofilm assays. Molecular docking was utilized to evaluate silybin's (the antibacterial component) binding affinity to efflux pumps, porins, and their regulatory elements. Additionally, qRT-PCR analysis explored the expression patterns of acrA, acrB, tolC, ompC, and ompF genes in both SPNs (sub-MIC) and ciprofloxacin (sub-MIC)-treated and untreated E. coli isolates. The combined use of SPNs and ciprofloxacin exhibited a notable reduction in bacterial growth and biofilm formation, in ciprofloxacin-resistant isolates. The study identified eight overlapping binding sites of the AcrABZ-TolC efflux pump in association with silybin, demonstrating a binding affinity ranging from -7.688 to -10.33 Kcal/mol. Furthermore, the qRT-PCR analysis showed that silybin upregulated AcrAB-TolC efflux pump genes and downregulated ompC and ompF porin genes in combination with ciprofloxacin in transcriptional level in uropathogenic E. coli. Silybin, a safe herbal compound, exhibits potential in inhibiting antibiotic resistance within bacterial isolates, potentially through the regulation of gene expression and plausible binding to target proteins.

Characterization of AI-2/LuxS quorum sensing system in biofilm formation, pathogenesis of Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

LACTIC ACID BACTERIA AND FERMENTED MAIZE SUPERNATANT (Omidun) HAVE ANTI-BIOFILM PROPERTIES AGAINST STAPHYLOCOCCI AND ENTEROAGGREGATIVE Escherichia coli STRAINS

Bacterial infections caused by biofilm forming organisms are of public health concern due to their propensity to contribute to persistent chronic diseases, chiefly because of their ability to resist antibiotics and host immune functions. Probiotics are considered useful therapeutic option in combating pathogenic biofilms. This study evaluates the anti-biofilm properties of potential probiotic Lactic Acid Bacteria (LAB) and fermented maize supernatant (Omidun) against selected biofilm-forming pathogens. Crystal violet biofilm assay was used to determine LAB and Omidun biofilm inhibition and dispersion in selected pathogens (Pseudomonas aeruginosa, Coagulase-negative staphylococci (CoNS), S. aureu and Enteroaggregative Escherichia coli) at different concentration (1%, 10%, 50%, 100%) of neutralized and non-neutralized cell free supernatant (CFS). Percentage biofilm inhibitions and dispersions were evaluated, and data were analysed with ANOVA. Omidun and LAB showed promising biofilm inhibitory and dispersive effect against the selected pathogens. L. plantarum showed the greatest biofilm inhibitory effect (P. aeruginosa: 7.85%, CoNS: 27.75%, S. aureus: 66.90%, EAEC: 39.73%) and dispersive effect (P. aeruginosa: 15.94%, CoNS: 23.27%, S. aureus: 24.90%, EAEC: 32.09%) against the selected pathogens while Omidun showed the least biofilm inhibitory and dispersive effect against the selected pathogens. There was no significance difference in the percentage of biofilm inhibition and dispersion produced under different concentrations, neutralized and non-neutralized state. Pseudomonas aeruginosa was the most resistant pathogen while Enteroaggregative Escherichia coli (EAEC) was the most susceptible. Inhibition and dispersion of biofilm can be mediated by LAB and Omidun, these effects appear to be independent of the produced organic acids

The Staphylococcus aureus ArlS Kinase Inhibitor Tilmicosin Has Potent Anti-Biofilm Activity in Both Static and Flow Conditions.

Staphylococcus aureus can form biofilms on biotic surfaces or implanted materials, leading to biofilm-associated diseases in humans and animals that are refractory to conventional antibiotic treatment. Recent studies indicate that the unique ArlRS regulatory system in S. aureus is a promising target for screening inhibitors that may eradicate formed biofilms, retard virulence and break antimicrobial resistance. In this study, by screening in the library of FDA-approved drugs, tilmicosin was found to inhibit ArlS histidine kinase activity (IC50 = 1.09 μM). By constructing a promoter-fluorescence reporter system, we found that tilmicosin at a concentration of 0.75 μM or 1.5 μM displayed strong inhibition on the expression of the ArlRS regulon genes spx and mgrA in the S. aureus USA300 strain. Microplate assay and confocal laser scanning microscopy showed that tilmicosin at a sub-minimal inhibitory concentration (MIC) had a potent inhibitory effect on biofilms formed by multiple S. aureus strains and a strong biofilm-forming strain of S. epidermidis. In addition, tilmicosin at three-fold of MIC disrupted USA300 mature biofilms and had a strong bactericidal effect on embedded bacteria. Furthermore, in a BioFlux flow biofilm assay, tilmicosin showed potent anti-biofilm activity and synergized with oxacillin against USA300.

Inhaled dry powder liposomal azithromycin for treatment of chronic lower respiratory tract infection

A dry powder inhaled liposomal azithromycin formulation was developed for the treatment of chronic respiratory diseases such as cystic fibrosis and bronchiectasis. Key properties including liposome size, charge and encapsulation efficiency powder size, shape, glass transition temperature (Tg), water content and in vitro respiratory deposition were determined. Antimicrobial activity against cystic fibrosis (CF) respiratory pathogens was determined by MIC, MBC and biofilm assays. Cytotoxicity and cellular uptake studies were performed using A549 cells. The average liposome size was 105 nm, charge was 55 mV and encapsulation efficiency was 75 %. The mean powder particle size d[v,50] of 4.54 µm and Mass Median Aerodynamic Diameter (MMAD) was 5.23 µm with a mean Tg of 76˚C and water content of 2.1 %. These excellent physicochemical characteristics were maintained over one year. Liposomal loaded azithromycin demonstrated enhanced activity against P. aeruginosa clinical isolates grown in biofilm. The formulation was rapidly delivered into bacterial cells with > 75 % uptake in 1 h. Rapid uptake into A549 cells via a cholesterol-dependent endocytosis pathway with no cytotoxic effects apparent. These data demonstrate that this formulation could offer benefits over current treatment regimens for people with chronic respiratory infection.

Effect of yeast extract on microbiologically influenced corrosion of X70 pipeline steel by Desulfovibrio bizertensis SY-1

Microbiologically influenced corrosion (MIC) is a complicated process that happens ubiquitously and quietly in many fields. As a useful nutritional ingredient in microbial culture media, yeast extract (YE) is a routinely added in the MIC field. However, how the YE participated in MIC is not fully clarified. In the present work, the effect of YE on the growth of sulfate reducing prokaryotes (SRP) Desulfovibrio bizertensis SY-1 and corrosion behavior of X70 pipeline steel were studied. It was found that the weight loss of steel coupons in sterile media was doubled when YE was removed from culture media. However, in the SRP assays without YE the number of planktonic cells decreased, but the attachment of bacteria on steel surfaces was enhanced significantly. Besides, the corrosion rate of steel in SRP assays increased fourfold after removing YE from culture media. MIC was not determined for assays with planktonic SRP but only for biofilm assays. The results confirm the effect of YE on D. bizertensis SY-1 growth and also the inhibitory role of YE on MIC.

Evaluation of Microbiological Susceptibility and Long-term Adhesive Properties to Dentin of Primers with Terminalia catappa Linn.

To investigate the antibacterial effects of Terminalia catappa Linn (TCL) leaf extracts at different concentrations and the effects of these extracts used as primers on the long-term adhesive properties of two universal adhesives. After extract preparation, the antimicrobial and antibacterial activities of TCL against Streptococcus mutans (UA 159) were assessed in microdilution assays to provide the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Additionally, to provide quantitative data on the ability of TCL extract to reduce cell viability, colony forming units (CFU) were counted. To examine adhesive properties, 288 human molars were randomly assigned to 32 experimental conditions (n = 9) according to the following variables: (1) treatment agent: negative control (untreated surface), and primers at concentrations of 1xMIC, 5xMIC, and 10xMIC; (2) adhesives: Scotchbond Universal (SBU) and Futurabond Universal (FBU); (3) adhesive strategy: etch-and-rinse (ER) or self-etch (SE); and (4) storage time: 24 h or after 2 years. Primers were applied for 60 s, upon which the teeth were incrementally restored and sectioned into adhesive-dentin bonded sticks. These were tested for microtensile bond strength (μTBS) and nanoleakage (NL) after 24-h and 2-year water storage, as well as in-situ degree of conversion (DC) at 24 h. The chemical profile of the hybrid layer was determined via micro-Raman spectroscopy. Biofilm assay data were analyzed using the Kruskal-Wallis test; the pH of culture media and the chemical profile were analyzed by one-way ANOVA. The adhesive properties (µTBS, NL, DC) were evaluated using a four-way ANOVA and Tukey's test. Significance was set at 5%. Similar values of MIC and MBC were observed (2 mg/ml), showing bactericidal potential. CFU analysis demonstrated that concentrations of 5xMIC and 10xMIC significantly inhibited biofilm formation (p < 0.001). The application of the TCL primer at all concentrations significantly increased the immediate μTBS and DC, and decreased the immediate NL values when compared to the control group (p < 0.05), regardless of the adhesive and adhesive strategies. Despite an increase in the NL values for all groups after 2 years (p > 0.05), in groups where the TCL primer was applied, the μTBS remained constant after 2 years for both adhesives, while a decrease in the μTBS was observed in the control groups (p < 0.05). Usually, 10xMIC showed better results than 1xMIC and 5xMIC (p < 0.05). The application of TCL promoted cross-linking; cross-linking rates increased proportionally to the concentration of TCL (p < 0.05). Primers containing TCL promoted bactericidal and bacteriostatic action, as well as cross-linking with dentin, while maintaining the adhesive properties of the adhesive-dentin interface after 2 years of water storage.

Efficacy of EDTA-NS irrigation in eradicating Staphylococcus aureus biofilm-associated infection.

To investigate the efficacy of ethylenediaminetetraacetic acid-normal saline (EDTA-NS) in dispersing biofilms and reducing bacterial infections. EDTA-NS solutions were irrigated at different durations (1, 5, 10, and 30 minutes) and concentrations (1, 2, 5, 10, and 50 mM) to disrupt Staphylococcus aureus biofilms on Matrigel-coated glass and two materials widely used in orthopaedic implants (Ti-6Al-4V and highly cross-linked polyethylene (HXLPE)). To assess the efficacy of biofilm dispersion, crystal violet staining biofilm assay and colony counting after sonification and culturing were performed. The results were further confirmed and visualized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). We then investigated the efficacies of EDTA-NS irrigation in vivo in rat and pig models of biofilm-associated infection. When 10 mM or higher EDTA-NS concentrations were used for ten minutes, over 99% of S. aureus biofilm formed on all three types of materials was eradicated in terms of absorbance measured at 595 nm and colony-forming units (CFUs) after culturing. Consistently, SEM and CSLM scanning demonstrated that less adherence of S. aureus could be observed on all three types of materials after 10 mM EDTA-NS irrigation for ten minutes. In the rat model, compared with NS irrigation combined with rifampin (Ti-6Al-4V wire-implanted rats: 60% bacteria survived; HXLPE particle-implanted rats: 63.3% bacteria survived), EDTA-NS irrigation combined with rifampin produced the highest removal rate (Ti-6Al-4V wire-implanted rats: 3.33% bacteria survived; HXLPE particle-implanted rats: 6.67% bacteria survived). In the pig model, compared with NS irrigation combined with rifampin (Ti-6Al-4V plates: 75% bacteria survived; HXLPE bearings: 87.5% bacteria survived), we observed a similar level of biofilm disruption on Ti-6Al-4V plates (25% bacteria survived) and HXLPE bearings (37.5% bacteria survived) after EDTA-NS irrigation combined with rifampin. The in vivo study revealed that the biomass of S. aureus biofilm was significantly reduced when treated with rifampin following irrigation and debridement, as indicated by both the biofilm bacterial burden and crystal violet staining. EDTA-NS irrigation (10 mM/10 min) combined with rifampin effectively removes S. aureus biofilm-associated infections both in vitro and in vivo. EDTA-NS irrigation with or without antibiotics is effective in eradicating S. aureus biofilm-associated infection both ex and in vivo.

Naringin and graphene oxide incorporated Moringa oleifera gum/poly(vinyl) alcohol patch for enhanced wound healing

Patients and healthcare systems stand to gain much from the use of substances that can accelerate wound healing. In this research work, a polymeric patch was fabricated using polymers like poly (vinyl alcohol) (PVA) and Moringa oleifera gum (MO) incorporated with graphene oxide (GO) and naringin (Nar) (drug). This study determined the impact of using PVA/MO/GO/Nar polymeric patch on wound healing via in vitro and in vivo investigations. Graphene oxide was synthesized by modified Hummer's method. The synthesized sample was characterized using XRD, FT-IR, RAMAN Spectroscopy, FESEM and HRTEM. Antibacterial analysis of the GO on four different bacteria was studied through well diffusion, colony count, growth curve and biofilm assay. Biocompatibility was analysed by haemolysis assay. The morphology, antibacterial activity, haemolysis assay, swelling, degradation, porosity, water vapour transmission rate, drug release, blood pump model, in-vitro scratch assay and MTT assay were analysed for the fabricated polymeric patches under in-vitro condition. The PVA/MO/GO/Nar patch has shown enhanced wound healing in in-vivo wound healing experiments on albino Wistar rats.

Regulatory role of Mss11 in Candida glabrata virulence: adhesion and biofilm formation.

Candida glabrata has emerged as a fungal pathogen with high infection and mortality rates, and its primary virulence factors are related to adhesion and biofilm formation. These virulence factors in C.glabrata are primarily mediated by epithelial adhesins (Epas), most of which are encoded in subtelomeric regions and regulated by subtelomeric silencing mechanisms. The transcription factor Mss11, known for its regulatory role in adhesion, biofilm formation, and filamentous growth in Saccharomyces cerevisiae and Candida albicans, has also been implicated in the expression of EPA6, suggesting its potential influence on C.glabrata virulence. The present study aims to determine the regulatory role of Mss11 in the virulence of C. glabrata. In this work, a Δmss11 null mutant and its complemented strain were constructed from a C.glabrata standard strain. The impact of the transcription factor Mss11 on the virulence of C.glabrata was investigated through a series of phenotypic experiments, including the microbial adhesion to hydrocarbons (MATH) test, adherence assay, biofilm assay, scanning electron microscopy and Galleria mellonella virulence assay. Furthermore, transcriptome sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and chromatin immunoprecipitation sequencing (ChIP-seq) were employed to investigate the molecular mechanisms behind the regulation of Mss11. In C.glabrata, the loss of MSS11 led to a significant reduction in several virulence factors including cell surface hydrophobicity, epithelial cell adhesion, and biofilm formation. These observations were consistent with the decreased virulence of the Δmss11 mutant observed in the Galleria mellonella infection model. Further exploration demonstrated that Mss11 modulates C. glabrata virulence by regulating EPA1 and EPA6 expression. It binds to the upstream regions of EPA1 and EPA6, as well as the promoter regions of the subtelomeric silencing-related genes SIR4, RIF1, and RAP1, indicating the dual regulatory role of Mss11. Mss11 plays a crucial role in C. glabrata adhesion and biofilm formation, and thus has a broad influence on virulence. This regulation is achieved by regulating the expression of EPA1 and EPA6 through both promoter-specific regulation and subtelomeric silencing.

The Antimicrobial Efficacy of Topically Applied Mafenide Acetate, Citric Acid and Wound Irrigation Solutions Lavanox and Prontosan against Pseudomonas aeruginosa.

Since burn wound infections caused by Pseudomonas aeruginosa (PA) lead to major complications and sepsis, this study evaluates the antimicrobial efficacy of the wound irrigation solutions Prontosan (PRT), Lavanox (LAV), citric acid (CA) and mafenide acetate (MA) using microbiology assays and an ex vivo skin wound model. In suspension assays, all the solutions showed significant reductions in bacterial number (log10 reduction: CA 5.77; LAV 4.91; PRT 4.74; MA 1.23). The biofilm assay revealed that PRT and LAV reduced biofilm formation by ~25% after a 15 min treatment, while PRT was most effective after a 24 h treatment (~68%). The number of PA in biofilms measured directly after a 15 min treatment was reduced most effectively with CA and LAV (log10 reductions ~2.5), whereas after a 24 h treatment, all solutions achieved only 1.36-1.65 log10 reductions. In the skin wound model, PRT and LAV provided the highest bacterial reduction after a 15 min treatment (log10 reduction 1.8-1.9), while MA was more effective after a 22 h treatment (log10 reduction 3.6). The results demonstrated the antimicrobial efficacy of all solutions against PA. Further investigation is needed to explore the potential clinical applications of a combination or alternating use of these solutions for infection prophylaxis and treatment of wound infections caused by PA.

Heparinoid enhances the efficacy of a bactericidal agent by preventing Cutibacterium acnes biofilm formation via quorum sensing inhibition.

Cutibacterium acnes is an opportunistic pathogen in acne vulgaris. C. acnes produces autoinducer-2 (AI-2）, a signaling molecule used for communication known as quorum sensing (QS）. In C. acnes, QS reportedly upregulates biofilm formation leading to resistance against bactericidal agents. In this study, we analyzed how heparinoid affected QS and biofilm formation of the opportunistic pathogen C. acnes. We also verified whether heparinoid would suppress biofilm formation and enhance the efficacy of the bactericidal agent 4-isopropyl-3-methylphenol (IPMP) against C. acnes biofilms. We ran an AI-2 bioassay using Vibrio harveyi ATCC BBA-1121. Heparinoid exhibited inhibitory activity against AI-2 at concentrations of 0.003-0.005%, suggesting an AI-2 analog-derived or C. acnes culture supernatant-derived inhibition of the AI-2 activity. To evaluate how heparinoid suppresses biofilm formation in C. acnes, we completed a biofilm assay in 96-well plates. We also evaluated the bactericidal activity of IPMP against the C. acnes biofilm prepared with or without heparinoid. Heparinoid inhibited C. acnes biofilm formation and IPMP bactericidal efficacy increased upon heparinoid-mediated suppression of biofilm formation. In this study, we clarified that heparinoid inhibits the AI-2-mediated QS of C. acnes, thereby suppressing biofilm formation and increasing IPMP bactericidal efficacy, potentially suppressing acne vulgaris.

Penurunan Aktivitas Biofilm Strain MRSA 22156 oleh Tanaman Saga (Abrus precatorius L.)

The aim of this research is to determine the effect of using saga extract on bacterial biofilm growth, and to determine the most appropriate dose of saga extract in reducing MRSA 22156 biofilm activity. This research uses the Dowd method to determine the total flavonoid content of A. precatorius L leaves. Treatment reduction in MRSA 22156 biofilm growth using the Plate Biofilm Assay method with three repetitions. The results of the research show that total flavonoids can reduce the growth of MRSA 22156 biofilms. The use of total flavonoids from A. precatorius L. leaves in concentrations of 800 mgL-1 to 25 mgL-1 has been proven to have an effect on biofilm growth activity as indicated by the reduction in MRSA 22156 biofilm growth by 67.9% and 25%. The ethanol extract of A. precatorius L. has anti-biofilm activity in reducing the growth of Staphylococcus aureus bacteria that are resistant to Methicillin.

Efficacy of biofilm disrupters against Candida auris and other Candida species in monomicrobial and polymicrobial biofilms

AbstractCandida species are now considered global threats by the CDC and WHO. Candida auris specifically is on the critical pathogen threat list along with Candida albicans. In addition, it is not uncommon to find Candida spp. in a mixed culture with bacterial organisms, especially Staphylococcus aureus producing polymicrobial infections. To eradicate these organisms from the environment and from patient surfaces, surface agents such as chlorhexidine (CHD) and Puracyn are used. Biofilm disrupters (BDs) are novel agents with a broad spectrum of antimicrobial activity and have been used in the management of chronic wounds and to sterilise environmental surfaces for the past several years. The goal of this study was to evaluate BDs (BlastX, Torrent, NSSD) and CHD against Candida spp. and S. aureus using zone of inhibition assays, biofilm and time‐kill assays. All BDs and CHD inhibited C. auris growth effectively in a concentration‐dependent manner. Additionally, CHD and the BDs showed excellent antimicrobial activity within polymicrobial biofilms. A comparative analysis of the BDs and CHD against C. auris and C. albicans using biofilm kill‐curves showed at least 99.999% killing. All three BDs and CHD have excellent activity against different Candida species, including C. auris. However, one isolate of C. auris in a polymicrobial biofilm assay showed resistance/tolerance to CHD, but not to the BDs. The fungicidal activity of these novel agents will be valuable in eradicating surface colonisation of Candida spp, especially C. auris from colonised environmental surfaces and from wounds in colonised patients.

Whole-transcriptome analysis reveals mechanisms underlying antibacterial activity and biofilm inhibition by a malic acid combination (MAC) in Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is a highly prevalent bacterial species known for its ability to cause various infections and its remarkable adaptability and biofilm-forming capabilities. In earlier work, we conducted research involving the screening of 33 metabolites obtained from a commercial source against two prevalent bacterial strains, Escherichia coli and Staphylococcus aureus. Through screening assays, we discovered a novel malic acid combination (MAC) consisting of malic acid, citric acid, glycine, and hippuric acid, which displayed significant inhibitory effects. However, the precise underlying mechanism and the potential impact of the MAC on bacterial biofilm formation remain unknown and warrant further investigation. Methods To determine the antibacterial effectiveness of the MAC against Pseudomonas aeruginosa, we conducted minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques were employed to observe bacterial morphology and biofilm formation. We further performed a biofilm inhibition assay to assess the effect of the MAC on biofilm formation. Whole-transcriptome sequencing and bioinformatics analysis were employed to elucidate the antibacterial mechanism of the MAC. Additionally, the expression levels of differentially expressed genes were validated using the real-time PCR approach. Results Our findings demonstrated the antibacterial activity of the MAC against P. aeruginosa. SEM analysis revealed that the MAC can induce morphological changes in bacterial cells. The biofilm assay showed that the MAC could reduce biofilm formation. Whole-transcriptome analysis revealed 1093 differentially expressed genes consisting of 659 upregulated genes and 434 downregulated genes, in response to the MAC treatment. Mechanistically, the MAC inhibited P. aeruginosa growth by targeting metabolic processes, secretion system, signal transduction, and cell membrane functions, thereby potentially compromising the survival of this human pathogen. This study provides valuable insights into the antibacterial and antibiofilm activities of the MAC, a synergistic and cost-effective malic acid combination, which holds promise as a potential therapeutic drug cocktail for treating human infectious diseases in the future.