Abietic Acid as a Novel Agent against Ocular Biofilms: An In Vitro and Preliminary In Vivo Investigation.

Biofilm-related ocular infections can lead to vision loss and are difficult to treat with antibiotics due to challenges with application and increasing microbial resistance. In turn, the design and testing of new synthetic drugs is a time- and cost-consuming process. Therefore, in this work, for the first time, we assessed the in vitro efficacy of the plant-based abietic acid molecule, both alone and when introduced to a polymeric cellulose carrier, against biofilms formed by Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in standard laboratory settings as well as in a self-designed setting using the topologically challenging surface of the artificial eye. These analyses were performed using the standard microdilution method, the biofilm-oriented antiseptic test (BOAT), a modified disk-diffusion method, and eyeball models. Additionally, we assessed the cytotoxicity of abietic acid against eukaryotic cell lines and its anti-staphylococcal efficacy in an in vivo model using Galleria mellonella larvae. We found that abietic acid was more effective against Staphylococcus than Pseudomonas (from two to four times, depending on the test applied) and that it was generally more effective against the tested bacteria (up to four times) than against the fungus C. albicans at concentrations non-cytotoxic to the eukaryotic cell lines and to G. mellonella (256 and 512 µg/mL, respectively). In the in vivo infection model, abietic acid effectively prevented the spread of staphylococcus throughout the larvae organisms, decreasing their lethality by up to 50%. These initial results obtained indicate promising features of abietic acid, which may potentially be applied to treat ocular infections caused by pathogenic biofilms, with higher efficiency manifested against bacterial than fungal biofilms.

The chronic wound milieu changes essential oils' antibiofilm activity-an in vitro and larval model study.

Essential Oils (EOs) are currently being researched as potential antibiofilm agents to combat infections related to chronic wound biofilms. As documented in the literature, EOs' in vitro antibacterial properties are often assessed using standard microbiological media and conditions that do not accurately reflect the actual environment of a chronic wound. To address this issue, In vitro Wound Milieu (IVWM) medium, which closely resembles the environment of a chronic wound, was applied for culturing S. aureus biofilms (n = 12) in this research. Biofilms cultivated in the standard Tryptic Soy Broth (TSB) medium served as a control for the experiment. Key biofilm features were analyzed and compared. Subsequently, staphylococci were exposed to the activity of thyme or rosemary EOs (T-EO and R-EO, respectively). As proof of concept, the cytotoxicity of T-EO and its antimicrobial in vivo activity were assessed using a G. mellonella larvae model. Key features of biofilm-forming cells were lower in the IVWM than in the TSB medium: biomass (up to 8 times), metabolic activity (up to 9 times), cell number (up to 100 times), and the live/dead cells ratio. Conversely, biofilm thickness was higher (up to 25%) in IVWM. These differences translated into varied responses of the biofilms to EOs exposure. The application of T-EO led to a greater reduction (up to 2 times) in 67% of biofilm-forming strains in IVWM compared to the TSB medium. Conversely, exposure to R-EO resulted in a higher reduction (up to 2.6 times) of 83% of biofilm-forming strains in TSB than in IVWM. The application of T-EO was not only non-toxic to G. mellonella larvae but also increased the survival of larvae infected with staphylococci (from 48 to 85%). Our findings suggest that EOs not only show promise as agents for treating biofilm-related wound infections but also that providing conditions reflecting the specific niche of the human body is of paramount importance in influencing the results obtained. However, before clinical application, challenges related to the methods of assessing their activity, microbial intra-species variability, and different levels of activity of various EOs should be analyzed and standardized.

Culture Shock: An Investigation into the Tolerance of Pathogenic Biofilms to Antiseptics in Environments Resembling the Chronic Wound Milieu.

Credible assessment methods must be applied to evaluate antiseptics' in vitro activity reliably. Studies indicate that the medium for biofilm culturing should resemble the conditions present at the site of infection. We cultured S. aureus, S. epidermidis, P. aeruginosa, C. albicans, and E. coli biofilms in IVWM (In Vitro Wound Milieu)-the medium reflecting wound milieu-and were compared to the ones cultured in the laboratory microbiological Mueller-Hinton (MH) medium. We analyzed and compared crucial biofilm characteristics and treated microbes with polyhexamethylene biguanide hydrochloride (PHMB), povidone-iodine (PVP-I), and super-oxidized solution with hypochlorites (SOHs). Biofilm biomass of S. aureus and S. epidermidis was higher in IVWM than in MH medium. Microbes cultured in IVWM exhibited greater metabolic activity and thickness than in MH medium. Biofilm of the majority of microbial species was more resistant to PHMB and PVP-I in the IVWM than in the MH medium. P. aeruginosa displayed a two-fold lower MBEC value of PHMB in the IVWM than in the MH medium. PHMB was more effective in the IVWM than in the MH medium against S. aureus biofilm cultured on a biocellulose carrier (instead of polystyrene). The applied improvement of the standard in vitro methodology allows us to predict the effects of treatment of non-healing wounds with specific antiseptics.

Antimicrobial and Cytotoxic Activities of Water-Soluble Isoxazole-Linked 1,3,4-Oxadiazole with Delocalized Charge: In Vitro and In Vivo Results.

The distinct structure of cationic organic compounds plays a pivotal role in enhancing their water solubility, which in turn influences their bioavailability. A representative of these compounds, which contains a delocalized charge, is 5-amino-2-(5-amino-3-methyl-1,2-oxazol-4-yl)-3-methyl-2,3-dihydro-1,3,4-oxadiazol-2-ylium bromide (ED). The high-water solubility of ED obviates the need for potentially harmful solvents during in vitro testing. The antibacterial and antifungal activities of the ED compound were assessed in vitro using the microtiter plate method and a biocellulose-based biofilm model. Additionally, its cytotoxic effects on wound bed fibroblasts and keratinocytes were examined. The antistaphylococcal activity of ED was also evaluated using an in vivo larvae model of Galleria mellonella. Results indicated that ED was more effective against Gram-positive bacteria than Gram-negative ones, exhibiting bactericidal properties. Furthermore, ED demonstrated greater efficacy against biofilms formed by Gram-positive bacteria. At bactericidal concentrations, ED was non-cytotoxic to fibroblasts and keratinocytes. In in vivo tests, ED was non-toxic to the larvae. When co-injected with a high load of S. aureus, it reduced the average larval mortality by approximately 40%. These findings suggest that ED holds promise for further evaluation as a potential treatment for biofilm-based wound infections, especially those caused by Gram-positive pathogens like S. aureus.

The In Vitro Impact of Isoxazole Derivatives on Pathogenic Biofilm and Cytotoxicity of Fibroblast Cell Line.

The microbial, biofilm-based infections of chronic wounds are one of the major challenges of contemporary medicine. The use of topically administered antiseptic agents is essential to treat wound-infecting microorganisms. Due to observed microbial tolerance/resistance against specific clinically-used antiseptics, the search for new, efficient agents is of pivotal meaning. Therefore, in this work, 15 isoxazole derivatives were scrutinized against leading biofilm wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa, and against Candida albicans fungus. For this purpose, the minimal inhibitory concentration, biofilm reduction in microtitrate plates, modified disk diffusion methods and antibiofilm dressing activity measurement methods were applied. Moreover, the cytotoxicity and cytocompatibility of derivatives was tested toward wound bed-forming cells, referred to as fibroblasts, using normative methods. Obtained results revealed that all isoxazole derivatives displayed antimicrobial activity and low cytotoxic effect, but antimicrobial activity of two derivatives, 2-(cyclohexylamino)-1-(5-nitrothiophen-2-yl)-2-oxoethyl 5-amino-3-methyl-1,2-oxazole-4-carboxylate (PUB9) and 2-(benzylamino)-1-(5-nitrothiophen-2-yl)-2-oxoethyl 5-amino-3-methyl-1,2-oxazole-4-carboxylate (PUB10), was noticeably higher compared to the other compounds analyzed, especially PUB9 with regard to Staphylococcus aureus, with a minimal inhibitory concentration more than x1000 lower compared to the remaining derivatives. The PUB9 and PUB10 derivatives were able to reduce more than 90% of biofilm-forming cells, regardless of the species, displaying at the same time none (PUB9) or moderate (PUB10) cytotoxicity against fibroblasts and high (PUB9) or moderate (PUB10) cytocompatibility against these wound cells. Therefore, taking into consideration the clinical demand for new antiseptic agents for non-healing wound treatment, PUB9 seems to be a promising candidate to be further tested in advanced animal models and later, if satisfactory results are obtained, in the clinical setting.

The Assessment of Activity of Antiseptic Agents against Biofilm of Staphylococcus aureus Measured with the Use of Processed Microscopic Images.

Staphylococcal biofilms are major causative factors of non-healing wound infections. Their treatment algorithms recommend the use of locally applied antiseptic agents to counteract the spread of infection. The efficacy of antiseptics against biofilm is assessed in vitro by a set of standard quantitative and semi-quantitative methods. The development of software for image processing additionally allowed for the obtainment of quantitative data from microscopic images of biofilm dyed with propidium iodine and SYTO-9 reagents, differentiating dead cells from live ones. In this work, the method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, based on biofilms' processed images, was proposed and scrutinized with regard to clinically relevant antiseptics, polyhexanide, povidone-iodine and hypochlorite. The standard quantitative culturing method was applied to validate the obtained data from processed images. The results indicated significantly higher activity of polyhexanide and povidone-iodine than hypochlorite against staphylococcal biofilm. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool; providing reliable, quantitative data in this regard.

The Medium Composition Impacts Staphylococcus aureus Biofilm Formation and Susceptibility to Antibiotics Applied in the Treatment of Bone Infections.

The biofilm-associated infections of bones are life-threatening diseases, requiring application of dedicated antibiotics in order to counteract the tissue damage and spread of microorganisms. The in vitro analyses on biofilm formation and susceptibility to antibiotics are frequently carried out using methods that do not reflect conditions at the site of infection. To evaluate the influence of nutrient accessibility on Staphylococcus aureus biofilm development in vitro, a cohesive set of analyses in three different compositional media was performed. Next, the efficacy of four antibiotics used in bone infection treatment, including gentamycin, ciprofloxacin, levofloxacin, and vancomycin, against staphylococcal biofilm, was also assessed. The results show a significant reduction in the ability of biofilm to grow in a medium containing elements occurring in the serum, which also translated into the diversified changes in the efficacy of used antibiotics, compared to the setting in which conventional media were applied. The differences indicate the need for implementation of adequate in vitro models that closely mimic the infection site. The results of the present research may be considered an essential step toward the development of in vitro analyses aiming to accurately indicate the most suitable antibiotic to be applied against biofilm-related infections of bones.

Chemical Composition and Antibacterial Activity of Liquid and Volatile Phase of Essential Oils against Planktonic and Biofilm-Forming Cells of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening, hard-to-heal infections associated with the presence of a biofilm. Essential oils (EOs) are promising agents to combat pseudomonal infections because of the alleged antimicrobial activity of their volatile fractions and liquid forms. Therefore, the purpose of this paper was to evaluate the antibacterial efficacy of both volatile and liquid phases of seven EOs (thyme, tea tree, basil, rosemary, eucalyptus, menthol mint, lavender) against P. aeruginosa biofilm and planktonic cells with the use of a broad spectrum of analytical in vitro methods. According to the study results, the antibacterial activity of EOs in their liquid forms varied from that of the volatile fractions. Overall, liquid and volatile forms of rosemary EO and tea tree EO displayed significant antibiofilm effectiveness. The outcomes indicate that these particular EOs possess the potential to be used in the therapy of P. aeruginosa infections.

The High Impact of Staphylococcus aureus Biofilm Culture Medium on In Vitro Outcomes of Antimicrobial Activity of Wound Antiseptics and Antibiotic.

The staphylococcal biofilm-based infections of wounds still pose a significant therapeutical challenge. Treated improperly, they increase the risk of limb amputation or even death of the patient. The present algorithms of infected wound treatment include, among others, the application of antiseptic substances. In vitro wound biofilm models are applied in order to scrutinize their activity. In the present work, using a spectrum of techniques, we showed how the change of a single variable (medium composition) in the standard in vitro model translates not only to shift in staphylococcal biofilm features but also to the change of efficacy of clinically applied wound antimicrobials such as octenidine, polyhexamethylene biguanide, chlorhexidine, hypochlorite solutions, and locally applied gentamycin. The data presented in this study may be of a pivotal nature, taking into consideration the fact that results of in vitro analyses are frequently used to propagate application of specific antimicrobials in hospitals and ambulatory care units.

Modifications of bacterial cellulose in wound care.

Wound infection may occur in acute and chronic wounds, wounds resulting from surgery or traffic accidents, and burns. Regardless of the extent and cause of the wound, prompt treatment is essential in reducing the patient's pain and limiting the spread of contamination. Improper wound care and associated chronic diseases may hinder the therapeutic success. Bacterial cellulose (BC) is highly biocompatible and has no cytotoxic effect on cells engaged in wound healing, such as fibroblasts and keratinocytes. Its high hydration level guarantees the maintenance of a moist wound environment. High mechanical strength, flexibility and resistance to damage make BC a promising material for dressings. Unfortunately, it does not display an inhibitory effect on bacterial growth. Introducing antimicrobial agents into the structure of BC has been a subject of many studies. This paper aims to present the latest reports on the possibility of the absorption of bacteriostatic and bactericidal agents in BC, such as metal particles, essential oils, antibiotics, antiseptics, and wound irrigation solutions. Moreover, the modifications in BC culture and post-production treatments in order to improve its physical properties are discussed.

The Antimicrobial and Antibiofilm In Vitro Activity of Liquid and Vapour Phases of Selected Essential Oils against Staphylococcus aureus.

The high resistance of staphylococcal biofilm against antibiotics and developing resistance against antiseptics induces a search for novel antimicrobial compounds. Due to acknowledged and/or alleged antimicrobial activity of EOs, their application seems to be a promising direction to follow. Nevertheless, the high complexity of EOs composition and differences in laboratory protocols of the antimicrobial activity assessment hinders the exact estimation of EOs effectiveness. To overcome these disadvantages, in the present work we analysed the effectiveness of volatile and liquid forms of seven EOs (derived from thyme, tea tree, basil, rosemary, eucalyptus, lavender, and menthol mint) against 16 staphylococcal biofilm-forming strains using cohesive set of in vitro techniques, including gas chromatography-mass spectrometry, inverted Petri dish, modified disk-diffusion assay, microdilution techniques, antibiofilm dressing activity measurement, AntiBioVol protocol, fluorescence/confocal microscopy, and dynamic light scattering. Depending on the requirements of the technique, EOs were applied in emulsified or non-emulsified form. The obtained results revealed that application of different in vitro techniques allows us to get a comprehensive set of data and to gain insight into the analysed phenomena. In the course of our investigation, liquid and volatile fractions of thyme EO displayed the highest antibiofilm activity. Liquid fractions of rosemary oil were the second most active against S. aureus. Vapour phases of tea tree and lavender oils exhibited the weakest anti-staphylococcal activity. The size of emulsified droplets was the lowest for T-EO and the highest for L-EO. Bearing in mind the limitations of the in vitro study, results from presented analysis may be of pivotal meaning for the potential application of thymol as a antimicrobial agent used to fight against staphylococcal biofilm-based infections.

Screening Papaveraceae as Novel Antibiofilm Natural-Based Agents.

The antimicrobial properties of herbs from Papaveraceae have been used in medicine for centuries. Nevertheless, mutual relationships between the individual bioactive substances contained in these plants remain poorly elucidated. In this work, phytochemical composition of extracts from the aerial and underground parts of five Papaveraceae species (Chelidonium majus L., Corydalis cava (L.) Schweigg. and Körte, C. cheilanthifolia Hemsl., C. pumila (Host) Rchb., and Fumaria vaillantii Loisel.) were examined using LC-ESI-MS/MS with a triple quadrupole analyzer. Large differences in the quality and quantity of all analyzed compounds were observed between species of different genera and also within one genus. Two groups of metabolites predominated in the phytochemical profiles. These were isoquinoline alkaloids and, in smaller amounts, non-phenolic carboxylic acids and phenolic compounds. In aerial and underground parts, 22 and 20 compounds were detected, respectively. These included: seven isoquinoline alkaloids: protopine, allocryptopine, coptisine, berberine, chelidonine, sanguinarine, and chelerythrine; five of their derivatives as well as non-alkaloids: malic acid, trans-aconitic acid, quinic acid, salicylic acid, trans-caffeic acid, p-coumaric acid, chlorogenic acid, quercetin, and kaempferol; and vanillin. The aerial parts were much richer in phenolic compounds regardless of the plant species. Characterized extracts were studied for their antimicrobial potential against planktonic and biofilm-producing cells of S. aureus, P. aeruginosa, and C. albicans. The impact of the extracts on cellular metabolic activity and biofilm biomass production was evaluated. Moreover, the antimicrobial activity of the extracts introduced to the polymeric carrier made of bacterial cellulose was assessed. Extracts of C. cheilanthifolia were found to be the most effective against all tested human pathogens. Multiple regression tests indicated a high antimicrobial impact of quercetin in extracts of aerial parts against planktonic cells of S. aureus, P. aeruginosa, and C. albicans, and no direct correlation between the composition of other bioactive substances and the results of antimicrobial activity were found. Conclusively, further investigations are required to identify the relations between recognized and unrecognized compounds within extracts and their biological properties.

Activity of Liquid and Volatile Fractions of Essential Oils against Biofilm Formed by Selected Reference Strains on Polystyrene and Hydroxyapatite Surfaces.

Biofilms are surface-attached, structured microbial communities displaying higher tolerance to antimicrobial agents in comparison to planktonic cells. An estimated 80% of all infections are thought to be biofilm-related. The drying pipeline of new antibiotics efficient against biofilm-forming pathogens urges the search for alternative routes of treatment. Essential Oils (EOs), extracted from medicinally important plants, are a reservoir of bioactive compounds that may serve as a foothold in investigating novel antibiofilm compounds. The aim of this study was to compare antimicrobial activity of liquid and volatile fractions of tested EOs against biofilm-forming pathogens using different techniques. In this research, we tested five EOs, extracted from Syzygium aromaticum L., Boswelia serrata Roxb., Juniperus virginiana L., Pelargonium graveolens L. and Melaleuca alternifolia Cheel., against planktonic and biofilm forms of five selected reference strains, namely Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. To obtain cohesive results, we applied four various methodological approaches: to assess the activity of the liquid fraction of EOs, disc diffusion and the microdilution method were applied; to test EOs' volatile fraction, the AntiBioVol assay and modified Antibiofilm Dressing Activity Measurement (A.D.A.M.) were used. The molecular composition and dynamics of antimicrobial substances released from specific EOs was measured using Gas Chromatography-Mass Spectrometry (GC-MS). The antimicrobial potency of EO's volatile fraction against biofilm formed by tested strains differed from that of the liquid fraction and was related to the molecular weight of volatile compounds. The liquid fraction of CW-EO and volatile fraction of F-EO acted in the strongest manner against biofilm of C. albicans. The addition of 0.5% Tween 20 to liquid phase, enhanced activity of G-EO against E. coli and K. pneumoniae biofilm. EO activity depended on the microbial species it was applied against and the chosen assessment methodology. While all tested EOs have shown a certain level of antimicrobial and antibiofilm effect, our results indicate that the choice of EO to be applied against a specific biofilm-forming pathogen requires careful consideration with regard to the above-listed aspects. Nevertheless, the results presented in this research contribute to the growing body of evidence indicating the beneficial effects of EOs, which may be applied to fight biofilm-forming pathogens.

In Vitro Efficacy of Bacterial Cellulose Dressings Chemisorbed with Antiseptics against Biofilm Formed by Pathogens Isolated from Chronic Wounds.

Local administration of antiseptics is required to prevent and fight against biofilm-based infections of chronic wounds. One of the methods used for delivering antiseptics to infected wounds is the application of dressings chemisorbed with antimicrobials. Dressings made of bacterial cellulose (BC) display several features, making them suitable for such a purpose. This work aimed to compare the activity of commonly used antiseptic molecules: octenidine, polyhexanide, povidone-iodine, chlorhexidine, ethacridine lactate, and hypochlorous solutions and to evaluate their usefulness as active substances of BC dressings against 48 bacterial strains (8 species) and 6 yeast strains (1 species). A silver dressing was applied as a control material of proven antimicrobial activity. The methodology applied included the assessment of minimal inhibitory concentrations (MIC) and minimal biofilm eradication concentration (MBEC), the modified disc-diffusion method, and the modified antibiofilm dressing activity measurement (A.D.A.M.) method. While in 96-well plate-based methods (MIC and MBEC assessment), the highest antimicrobial activity was recorded for chlorhexidine, in the modified disc-diffusion method and in the modified A.D.A.M test, povidone-iodine performed the best. In an in vitro setting simulating chronic wound conditions, BC dressings chemisorbed with polyhexanide, octenidine, or povidone-iodine displayed a similar or even higher antibiofilm activity than the control dressing containing silver molecules. If translated into clinical conditions, the obtained results suggest high applicability of BC dressings chemisorbed with antiseptics to eradicate biofilm from chronic wounds.

The In Vitro Ability of Klebsiella pneumoniae to Form Biofilm and the Potential of Various Compounds to Eradicate It from Urinary Catheters.

Urinary infections related to the presence of bacterial biofilm on catheters are responsible for loss of patients' health and, due to their high frequency of occurrence, generate a significant economic burden for hospitals. Klebsiella pneumoniae is a pathogen frequently isolated from this type of infection. In this study, using a cohesive set of techniques performed under stationary and flow conditions, we assessed the ability of 120 K. pneumoniae strains to form biofilm on various surfaces, including catheters, and evaluated the usefulness of clinically applied and experimental compounds to remove biofilm. The results of our study indicate the high impact of intraspecies variability with respect to K. pneumoniae biofilm formation and its susceptibility to antimicrobials and revealed the crucial role of mechanical flushing out of the biofilm from the catheter's surface with use of locally active antimicrobials. Therefore, our work, although of in vitro character, may be considered an important step in the direction of efficient reduction of K. pneumoniae biofilm-related hospital infections associated with the presence of urine catheters.

Phytochemical Composition and Antimicrobial Activity of Corydalis solida and Pseudofumaria lutea.

Corydalis and Pseudofumaria are two closely related genera from the Papaveraceae subfamily Fumarioideae with Corydalis solida (C. solida) and Pseudofumaria lutea (P. lutea) as two representative species. Phytochemical analysis revealed significant differences in the quality and quantity of isoquinoline alkaloids, phenolic compounds and non-phenolic carboxylic acids between aerial and underground parts of both species. Using the Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique, 21 compounds were identified: five protoberberine derivatives, three protopine derivatives, four phenanthridine derivatives, as well as three carboxylic acids, two hydroxycinnamic acids, one chlorogenic acid, one phenolic aldehyde, and two flavonoids. Moroever, significant differences in the content of individual compounds were observed between the two studied species. The phytochemical profile of C. solida showed a higher variety of compounds that were present in lower amounts, whereas P. lutea extracts contained fewer compounds but in larger quantities. Protopine was one of the most abundant constituents in C. solida (440-1125 µg/g d.w.) and in P. lutea (1036-1934 µg/g d.w.). Moreover, considerable amounts of coptisine (1526 µg/g) and quercetin (3247 µg/g) were detected in the aerial parts of P. lutea. Extracts from aerial and underground parts of both species were also examined for the antimicrobial potential against S. aureus, P. aeruginosa and C. albicans. P. lutea herb extract was the most effective (MIC at 0.39 mg/L) against all three pathogens.