Preformed Biofilms Research Articles

Berberine-fluconazole microparticle-based combination therapy to treat candidiasis infections.

This study aims to incorporate alginate microparticles containing berberine and fluconazole into two different types of pharmaceutical formulations, to subsequently evaluate the antifungal activity against Candida albicans. Alginate microparticles containing BBR (berberine) and FLU (fluconazole) were produced by the spray-drying technique, characterized and incorporated in two pharmaceutical formulations, a vaginal cream and artificial saliva. Broth microdilution, checkerboard, time-kill curve, and scanning electron microscopy were carried out to determine the antifungal effects of BBR and FLU against C. albicans. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of free BBR were 125μg ml-1. Synergism between BBR and FLU was demonstrated by a fractional inhibitory concentration index (FICI)=0.0762. The time-kill curve for the combination BBR+FLU showed a more pronounced decrease in fungal growth in comparison to free drugs, and an antibiofilm effect of BBR occurred in the formation and preformed biofilm. Alginate microparticles containing BBR and FLU were obtained and incorporated in a vaginal cream and artificial saliva. Both formulations showed good stability, antifungal effects, and organoleptic characteristics, which suggest that BBR-FLU microparticles in formulations have potential as antifungal therapy.

Detection of 2-ethyl-1-hexanol and its modulating effect in biofilm of Fusarium oxysporum.

In immunocompetent individuals, Fusarium spp. stands out as the causative agent of onychomycosis, among the non-dermatophyte molds. Despite evidence indicating that Fusarium oxysporum organizes itself in the form of a biofilm causing onychomycosis, there is little literature on the etiopathogenesis of the biofilm on the nail, specifically the signaling molecules present, known as quorum sensing (QS). Thus, this study detected the presence of a molecule related to QS from the exvivo biofilm of F. oxysporum on human nail and investigated its effect on preformed biofilm invitro. The detection and physicochemical characterization of a QS molecule, from the extracellular matrix (ECM), was carried out by Fourier transform infrared (FTIR) spectroscopy with an attenuated total reflectance (ATR) accessory and by headspace gas chromatography coupled to mass spectrometry (GC-MS) analyses. Determination of viable cells, cell activity, total biomass, ECM components and scanning electron microscopy (SEM) were performed to evaluate the influence of the QS molecule on the invitro biofilm of F. oxysporum. The beginning, in the exvivo biofilm of F. oxysporum on human nails, the volatile organic compound 2-ethyl-1-hexanol (2EH) was detected as a component of QS. Thereafter invitro analyses, synthetic 2EH was able to modulate the biofilm by stimulating its filament, increasing total biomass and ECM production in terms of total carbohydrates, but with a reduction in total proteins and nucleic acids. We thus evidence, for the first time, the presence of 2EH in the biofilm of F. oxysporum, developed on the human nail, and the invitro action of this compound as a QS molecule.

Candida krusei M4CK Produces a Bioemulsifier That Acts on Melaleuca Essential Oil and Aids in Its Antibacterial and Antibiofilm Activity.

Surface-active compounds (SACs) of microbial origin are an active group of biomolecules with potential use in the formulation of emulsions. In this sense, the present study aimed to isolate and select yeasts from fruits that could produce SACs for essential oil emulsions. The Candida krusei M4CK was isolated from the Byrsonima crassifolia fruit to make SACs. This emulsification activity (E24) was equal to or greater 50% in all carbon sources, such as olive oil, sunflower oil, kerosene, hexane, and hexadecane. E24 followed exponential growth according to the growth phase. The stability of emulsions was maintained over a wide range of temperatures, pH, and salinity. The OMBE4CK (melaleuca essential oil emulsion) had better and more significant inhibitory potential for biofilm reduction formation. In addition, bioemulsifier BE4CK alone on Escherichia coli and Pseudomonas aeruginosa biofilm showed few effective results, while there was a significant eradication for Staphylococcus aureus biofilms. The biofilms formed by S. aureus were eradicated in all concentrations of OMBE4CK. At the same time, the preformed biofilm by E. coli and P. aeruginosa were removed entirely at concentrations of 25 mg/mL, 12.5 mg/mL, and 6.25 mg/mL. The results show that the bioemulsifier BE4CK may represent a new potential for antibiofilm application.

Effect of Camel Peptide on the Biofilm of Staphylococcus epidermidis and Staphylococcus haemolyticus Formed on Orthopedic Implants.

The increasing bacterial drug resistance and the associated challenges in the treatment of infections warrant the search for alternative therapeutic methods. Hope is placed in antimicrobial peptides, which have a broad spectrum of action and are effective against strains which are resistant to conventional antibiotics. Antimicrobial peptides are also tested for their efficacy in the treatment of infections associated with the formation of biofilm. The aim of the present study was to examine the effect of Camel peptide on S. epidermidis and S. haemolyticus adhesion to and formation of biofilm on steel cortical bone screws and also on the process of reducing mature biofilm in orthopedic implants. The tests were performed on steel implants for osteosynthesis. The MIC value and MBEC values of the peptide were determined using the microdilution method in microtiter plates. The effect of the peptide on adhesion and biofilm formation, as well as on the activity on the preformed biofilm, was evaluated using quantitative methods and confocal microscopy. The presented research results indicate that the peptide exhibits very good antimicrobial properties against the analyzed strains. Concentrations above MIC reduced biofilm in the range of 90-99%.

Aerococcus viridans Phage Lysin AVPL Had Lytic Activity against Streptococcus suis in a Mouse Bacteremia Model

Streptococcus suis (S. suis) is a swine pathogen that can cause sepsis, meningitis, endocarditis, and other infectious diseases; it is also a zoonotic pathogen that has caused a global surge in fatal human infections. The widespread prevalence of multidrug-resistant S. suis strains and the decline in novel antibiotic candidates have necessitated the development of alternative antimicrobial agents. In this study, AVPL, the Aerococcus viridans (A. viridans) phage lysin, was found to exhibit efficient bactericidal activity and broad lytic activity against multiple serotypes of S. suis. A final concentration of 300 μg/mL AVPL reduced S. suis counts by 4–4.5 log10 within 1 h in vitro. Importantly, AVPL effectively inhibited 48 h S. suis biofilm formation and disrupted preformed biofilms. In a mouse model, 300 μg/mouse AVPL protected 100% of mice from infection following the administration of lethal doses of multidrug-resistant S. suis type 2 (SS2) strain SC19, reduced the bacterial load in different organs, and effectively alleviated inflammation and histopathological damage in infected mice. These data suggest that AVPL is a valuable candidate antimicrobial agent for treating S. suis infections.

Synergistic and Additive Effects of Menadione in Combination with Antibiotics on Multidrug-Resistant Staphylococcus aureus: Insights from Structure-Function Analysis of Naphthoquinones.

Antimicrobial resistance (AMR) interferes with the effective treatment of infections and increases the risk of microbial spread and infection-related illness and death. The synergistic activities of combinations of antimicrobial compounds offer satisfactory approaches to some extent. Structurally diverse naphthoquinones (NQs) including menadione (-CH3 group at C2) exhibit substantial antimicrobial activities against multidrug-resistant (MDR) pathogens. We explored the combinations of menadione with antibiotic ciprofloxacin or ampicillin against Staphylococcus aureus and its biofilms. We found an additive (0.5<FICI 1.0) and synergistic (FICI≤0.5) effect of menadione with ciprofloxacin and ampicillin, respectively. High reactive oxygen species (ROS) activity and inhibition of biofilm formation (>90 %) were also observed. However, preformed biofilms were not affected. Dent formation was also evident in S. aureus treated with the test compounds. The structure-function relationship (SFR) of NQs was used to determine and predict their activity pattern against pathogens. Analysis of 10 structurally distinct NQs revealed that the compounds with -Cl, -Br, -CH3 , or -OH groups displayed the lowest MICs (32-256 μg/mL). Furthermore, 1,4-NQs possessing a halogen or -CH3 moiety showed elevated ROS activity, whereas molecules with an -OH group affected cell integrity. Improved activity of antimicrobial combinations and SFR approaches are significant in antimicrobial therapies.

Expression and characterization of novel chimeric endolysin CHAPk-SH3bk against biofilm-forming methicillin-resistant Staphylococcus aureus

The continuous evolution of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to the misuse of antibiotics lays out the need for the development of new antimicrobials with higher activity and lower resistance. In this study, we have expressed novel chimeric endolysin CHAPk-SH3bk derived from LysK to investigate its antibacterial activity against planktonic and biofilm-forming MRSA. The molecular docking and MD simulation results identified critical amino acids (ASP47, ASP56, ARG71, and Gly74) of CHAPk domain responsible for its catalytic activity. Chimeric endolysin CHAPk-SH3bk showed an effective binding to peptidoglycan fragment using 14 hydrogen bonds. The in-vitro antibacterial assays displayed higher activity of CHAPk against planktonic MRSA with 2-log10 reduction in 2 h. Both CHAPk and CHAPk-SH3bk displayed bactericidal activity against MRSA with ∼4log10 and ∼3.5log10 reduction in 24 h. Biofilm reduction activity displayed CHAPk-SH3bk reduced 33 % and 60 % of hospital-associated ATCC®BAA-44™ and bovine origin SA1 respectively. The CHAPk treatment reduced 47 % of the preformed biofilm formed by bovine-origin MRSA SA1. This study indicates an effective reduction of preformed MRSA biofilms of human and animal origin using novel chimeric construct CHAPk-SH3bk. Stating that the combination and shuffling of different domains of phage endolysin potentially increase its bacteriolytic effectiveness against MRSA.

Bioactive Compounds from an Endophytic Pezicula sp. Showing Antagonistic Effects against the Ash Dieback Pathogen.

A fungal endophyte originating from the Canary Islands was identified as a potent antagonist against the fungal phytopathogen Hymenoscyphus fraxineus, which causes the devastating ash dieback disease. This endophyte was tentatively identified as Pezicula cf. ericae, using molecular barcoding. Isolation of secondary metabolites by preparative high-performance liquid chromatography (HPLC) yielded the known compounds CJ-17,572 (1), mycorrhizin A (3) and cryptosporioptides A-C (4-6), besides a new N-acetylated dihydroxyphenylalanin derivative 2, named peziculastatin. Planar structures were elucidated by NMR and HRMS data, while the relative stereochemistry of 2 was assigned by H,H and C,H coupling constants. The assignment of the unknown stereochemistry of CJ-17,572 (1) was hampered by the broadening of NMR signals. Nevertheless, after semisynthetic conversion of 1 into its methyl derivatives 7 and 8, presumably preventing tautomeric effects, the relative configuration could be assigned, whereas comparison of ECD data to those of related compounds determined the absolute configuration. Metabolites 1 and 3 showed significant antifungal effects in vitro against H. fraxineus. Furthermore, 4-6 exhibited significant dispersive effects on preformed biofilms of S. aureus at concentrations up to 2 µg/mL, while the biofilm formation of C. albicans was also inhibited. Thus, cryptosporioptides might constitute a potential source for the development of novel antibiofilm agents.

Selected flavonoids exhibit antibiofilm and antibacterial effects against Vibrio by disrupting membrane integrity, virulence and metabolic activities

Vibrio parahaemolyticus is a high-risk foodborne pathogen associated with raw or undercooked seafoods and its biofilm forming potential has become a threat to food safety and economic values. Hence, this study aims to examine the antibacterial and antibiofilm activities as well as virulence inhibitory effects of selected flavonoids against V. parahaemolyticus. Out of the sixteen flavonoid derivatives, 6-aminoflavone (6-AF), 3,2-dihydroxyflavone (3,2-DHF) and 2,2-dihydroxy-4-methoxybenzophenone (DHMB) were found as active biofilm inhibitors. 3,2-DHF and DHMB had minimum inhibitory concentrations of 20 and 50 μg/mL respectively against Vibrio planktonic cells and displayed superior antibacterial activities to standard controls. Also, they disrupted preformed biofilms and suppressed virulence properties including motilities, cell hydrophobicity and aggregation. They impaired iron acquisition mechanism and hemolysin production at sub-MICs as supported by transcriptomic studies. Interestingly, the flavonoids interfered with the metabolic activity, cell division and membrane permeability to exert antibiofilm and antibacterial activities. 6-AF and 3,2-DHF were non-toxic in the C. elegans model and showed excellent capacity to protect shrimps from biodeterioration. Furthermore, the flavonoids inhibited biofilm formation by V. harveyi, Staphylococcus aureus and Salmonella typhimurium and the mixed-species biofilm with Vibrio. This study discovered flavonoid derivatives, especially 3,2-DHF as potential bioactive compounds capable of offering protection from risks associated with biofilm formation by V. parahaemolyticus and other food pathogens.

Evaluation of anti-biofilm effect of antimicrobial sonodynamic therapy-based periodontal ligament stem cell-derived exosome-loaded kojic acid on Enterococcus faecalis biofilm.

Introduction. Antimicrobial sonodynamic therapy (aSDT) is an approach that uses ultrasound waves (UWs) and a sonosensitizer to generate reactive oxygen species (ROS) to damage microbial cells in biofilms. Using nano-carriers, such as exosomes (Exos), to deliver the sonosensitizer can potentially enhance the effectiveness of aSDT.Hypothesis/Gap Statement. aSDT can downregulate the expression of gelE and sprE genes, increasing the production of endogenous ROS and degradation of pre-formed Enterococcus faecalis biofilms.Aim. This study investigated the anti-biofilm effect of aSDT-based periodontal ligament stem cell-derived exosome-loaded kojic acid (KA@PDL-Exo) on pre-formed E. faecalis biofilms in root canals.Methodology. Following the isolation and characterization of PDL-Exo, KA@PDL-Exo was prepared and confirmed. The minimal biofilm inhibitory concentration (MBIC) of KA, PDL-Exo, KA@PDL-Exo and sodium hypochlorite (NaOCl) was determined, and their anti-biofilm effects were assessed with and without UWs. The binding affinity of KA with GelE and SprE proteins was evaluated using in silico molecular docking. Additionally, the study measured the generation of endogenous ROS and evaluated changes in the gene expression levels of gelE and sprE.Results. The results revealed a dose-dependent decrease in the viability of E. faecalis cells within biofilms. KA@PDL-Exo was the most effective, with an MBIC of 62.5 µg ml-1, while NaOCl, KA and PDL-Exo had MBIC values of 125, 250 and 500 µg ml-1, respectively. The use of KA@PDL-Exo-mediated aSDT resulted in a significant reduction of the E. faecalis biofilm (3.22±0.36 log10 c.f.u. ml-1; P<0.05). The molecular docking analysis revealed docking scores of -5.3 and -5.2 kcal mol-1 for GelE-KA an SprE-KA, respectively. The findings observed the most significant reduction in gene expression of gelE and sprE in the KA@PDL-Exo group, with a decrease of 7.9- and 9.3-fold, respectively, compared to the control group (P<0.05).Conclusion. The KA@PDL-Exo-mediated aSDT was able to significantly reduce the E. faecalis load in pre-formed biofilms, decrease the expression of gelE and srpE mRNA, and increase the generation of endogenous ROS. These findings imply that KA@PDL-Exo-mediated aSDT could be a promising anti-biofilm strategy that requires additional in vitro and in vivo investigations.

Recombinant Prevotella melaninogenica α-1,3 glucanase and Capnocytophaga ochracea α-1,6 glucanase as enzymatic tools for in vitro degradation of S. mutans biofilms.

Dental biofilms represent a serious oral health problem playing a key role in the development of caries and other oral diseases. In the present work, we cloned and expressed in E. coli two glucanases, Prevotella melaninogenica mutanase (PmGH87) and Capnocytophaga ochracea dextranase (CoGH66), and characterized them biochemically and biophysically. Their three-dimensional structures were elucidated and discussed. Furthermore, we tested the capacity of the enzymes to hydrolyze mutan and dextran to prevent formation of Streptococcus mutans biofilms, as well as to degrade pre- formed biofilms in low and abundant sugar conditions. The percentage of residual biofilm was calculated for each treatment group in relation to the control, as well as the degree of synergism. Our results suggest that both PmGH87 and CoGH66 are capable of inhibiting biofilm formation grown under limited or abundant sucrose conditions. Degradation of pre-formed biofilms experiments reveal a time-dependent effect for the treatment with each enzyme alone. In addition, a synergistic and dose-dependent effects of the combined enzymatic treatment with the enzymes were observed. For instance, the highest biomass degradation was 95.5% after 30min treatment for the biofilm grown in low sucrose concentration, and 93.8% after 2h treatment for the biofilm grown in sugar abundant condition. Strong synergistic effects were observed, with calculated degree of synergism of 5.54 and 3.18, respectively and their structural basis was discussed. Jointly, these data can pave the ground for the development of biomedical applications of the enzymes for controlling growth and promoting degradation of established oral biofilms.

In vitro and in vivo antimicrobial potential of lithium complex against multi-drug resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen, accountable for lethal nosocomial infections, owing to high pathogenicity and multi-drug resistance (MDR). Hence, the World Health Organization has prioritized this bacterium against which new pharmacophores are required urgently. Therefore, this study evaluates the antimicrobial potential of lithium complex based on salicylic acid, and 1,10-phenanthroline {[Li(phen)2sal]} against MDR A. baumannii (MDRAB) through growth inhibition, growth curve assay, biofilm inhibition, and eradication alongside microscopy. The mechanism of action was proposed by flow cytometry and proteomic analysis. Moreover, in vivo efficacy was assessed using an A. baumannii-induced pneumonia model in mice. Results showed that lithium complex significantly inhibited the bacterial growth at 16-32 µg/mL. Growth kinetics revealed that it retarded the bacterial growth after 2-3 h of treatment. Lithium complex significantly inhibited and reduced >70% of biofilms at the MIC level which is a key problem of indwelling and prosthetic devices. Light and atomic force microscopy further indicated disarticulated biofilm and ruptured cellular membrane post-lithium complex treatment. Proteomic analysis revealed that the compound exerted oxidative stress on A. baumannii which may contribute to cell rupturing. Data are available via ProteomeXchange with identifier PXD044293. Flow cytometry confirmed its role in cytoplasmic membrane disintegration. The lithium complex ameliorated the pneumonia symptoms at non-toxic doses of 20 and 40 mg/kg in the pre-clinical A. baumannii-induced pneumonia model as evident by the significant reduction in bacterial burden and parenchymal inflammation. Taken together, these findings suggest that the lithium complex can be a potential drug candidate against MDRAB.IMPORTANCEMulti-drug resistance (MDR) by virtue of evolving resistance and virulence mechanisms among A. baumannii is a global concern which is responsible for lethal hospital-acquired infections. Therefore, it is crucial to develop new therapeutics against it. Metal complexes are compact structures with diverse mechanisms that the pathogens cannot evade easily which make them a strong drug candidate. In this study, we assessed the in vitro and in vivo efficacy of lithium complex {[Li(phen)2 sal]} against biofilm-forming MDR A. baumannii. The lithium complex displayed strong antimicrobial activity and reduced the pre-formed mature biofilm which is key barrier for antimicrobial action. Moreover, it employs oxidative stress as one of its mode of actions and causes cellular rupturing. Lithium complex was non-toxic and was significantly effective to overcome pneumonia in mice model. These results highlight the untapped potential of metal complexes that can be explored and utilized for combating notorious A. baumannii infections.

Limosilactobacillus vaginalis Exerts Bifidogenic Effects: A Novel Postbiotic Strategy for Infant Prebiotic Supplementation.

Infant microbiota shaping strictly influences newborns' well-being and long-term health, and babies born by cesarean-section and formula-fed generally show low microbial gut diversity and are more prone to develop various disorders. The supplementation with beneficial microbes of vaginal origin or derivatives (postbiotics, including heat-inactivated cells) represents a valid strategy to drive the correct gut microbiota shaping. Here, we explored for the first time the bifidogenic activity of a heat-killed vaginal strain (Limosilactobacillus vaginalis BC17), in addition to the assessment of its safety. L. vaginalis BC17 whole genome was sequenced by Nanopore technology and highlighted the absence of antibiotic resistance genes and virulence factors, indicating the strain safety profile for human health. MIC values confirmed that L. vaginalis BC17 is susceptible to widely employed antibiotics. Heat-killed BC17 cells significantly enhanced the planktonic growth of Bifidobacterium spp. For the first time, stimulating effects were observed also toward biofilm formation of bifidobacteria and their pre-formed biofilms. Conversely, heat-killed BC17 cells exerted antibacterial and anti-biofilms activities against Gram-positive and Gram-negative pathogens. Lyophilized heat-killed BC17 cells were formulated in a sunflower oil suspension (1010 heat-killed cell/g) intended for infant oral intake. This possessed optimal technological (i.e., re-dispersibility and stability) and functional properties (i.e., bifidogenic activity) that were maintained even after pre-digestion in acidic conditions.

Molybdenum oxide nanotube caps decorated with ultrafine Ag nanoparticles: Synthesis and antimicrobial activity

In the contemporary era, microorganisms, spanning bacteria and viruses, are increasingly acknowledged as emerging contaminants in the environment, presenting significant risks to public health. Nevertheless, conventional methods for disinfecting these microorganisms are often ineffective. Additionally, they come with disadvantages such as high energy usage, negative environmental consequences, increased expenses, and the generation of harmful byproducts. The development of next-generation antifungal and antibacterial agents is dependent on newly synthesized nanomaterials with inherent antimicrobial behavior. In this study, we report an arc-discharge method to synthesize MoOx nanosheets and microbelts, followed by decorating them with ultrafine Ag nanoparticles (NPs). Scanning and transmission electron microscopies show that Ag NPs formation on the Molybdenum oxide nanostructures rolls them into nanotube caps (NTCs), revealing inner and outer diameters of approximately 19.8 nm and 105.5 nm, respectively. Additionally, the Ag NPs are ultrafine, with sizes in the range of 5–8 nm. Results show that the prepared NTCs exhibit dose-dependent sensitivity to both planktonic and biofilm cells of Escherichia coli and Candida albicans. The anti-biofilm activity in terms of biofilm inhibition ranged from 19.7 to 77.2% and 11.3–82.3%, while removal of more than 70% and 90% of preformed biofilms was achieved for E. coli and C. albicans, respectively, showing good potential for antimicrobial coating. Initial MoOx exhibits positive potential, while Ag-decorated Molybdenum oxide NTCs show dual potential effects (positive for Molybdenum oxide NTCs and negative for Ag NPs. Molybdenum oxide NTCs, with their strong positive potential, efficiently attract microbes due to their negatively charged cell surfaces, facilitating the antimicrobial effect of Ag NPs, leading to cell damage and death. These findings suggest that the synthesized NPs could serve as a suitable coating for biomedical applications.

Antibacterial and antibiofilm activity of nanocomposite (BJ-Ag0.6@GO0.4) synthesized from animal-derived liquid waste against ESKAPE pathogen

Herein, Ag nanoparticles were prepared by fish bile juice as a reducing and capping agent. Then Ag/ graphene oxide (GO) nanocomposite was fabricated. The nanocomposite (BJ-Ag0.6 @GO0.4) showed remarkable antibacterial activities against Gram-positive and Gram-negative pathogenic bacteria. The MIC values of the nanocomposite were recorded as 10 and 8 µg/mL for S.aureus (MTCC 1430) and P. auregenosa (MTCC-1688), respectively. Antibacterial mechanism study of the synthesized nanocomposite excavates that, BJ-Ag0.6 @GO0.4 caused extensive membrane damage and cytoplasmic leakage, leading to cell’s metabolic arrest. Increased ROS generation is observed, causing disruption in the antioxidant defense mechanism of the bacterial cell. The synthesized nanocomposite also showed excellent destruction of preformed biofilm up to 96.52% and 61.23% for S.aureus (MTCC 1430) and P. auregenosa (MTCC-1688), respectively, at MIC concentration of the nanocomposite.

Study of the antimicrobial activity of cationic carbosilane dendrimers against clinical strains of multidrug-resistant bacteria and their biofilms.

Antimicrobial Resistance is a serious public health problem, which is aggravated by the ability of the microorganisms to form biofilms. Therefore, new therapeutic strategies need to be found, one of them being the use of cationic dendritic systems (dendrimers and dendrons). The aim of this study is to analyze the in vitro antimicrobial efficacy of six cationic carbosilane (CBS) dendrimers and one dendron with peripheral ammonium groups against multidrug-resistant bacteria, some of them isolated hospital strains, and their biofilms. For this purpose, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and minimum eradication biofilm concentration (MBEC) studies were carried out. In addition, the cytotoxicity on Hela cells of those compounds that proved to be the most effective was analyzed. All the tested compounds showed in vitro activity against the planktonic forms of methicillin-resistant Staphylococcus aureus and only the dendrimers BDSQ017, BDAC-001 and BDLS-001 and the dendron BDEF-130 against their biofilms. On the other hand, only the dendrimers BDAC 001, BDLS-001 and BDJS-049 and the dendron BDEF-130 were antibacterial in vitro against the planktonic forms of multidrug-resistant Pseudomonas aeruginosa, but they lacked activity against their preformed biofilms. In addition, the dendrimers BDAC-001, BDLS-001 and BDSQ-017 and the dendron BDEF-130 exhibited a good profile of cytotoxicity in vitro. Our study demonstrates the possibility of using the four compounds mentioned above as possible topical antimicrobials against the clinical and reference strains of multidrug-resistant bacteria.

Enhanced Antimicrobial Screening Sensitivity Enabled the Identification of an Ultrashort Peptide KR-8 for Engineering of LL-37mini to Combat Drug-Resistant Pathogens.

Identification of novel antibiotics is of top importance because of the threat of antibiotic-resistant pathogens. Antimicrobial screening in Mueller-Hinton broth is frequently the first step in antimicrobial discovery. Although widely utilized, this medium is not ideal as it could mask activity of candidates such as human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus (MRSA). This study identified a sensitive medium where LL-37 displayed excellent activity against numerous pathogens, including MRSA. Our screen of ultrashort overlapping LL-37 peptides in this medium led to the identification of KR-8, four residues shorter than KR-12. Hence, our screen condition may increase positive compound hits during antimicrobial screening. KR-8 provided an appealing template for us to design LL-37mini, which was potent against MRSA, Escherichia coli, and Pseudomonas aeruginosa but not toxic to mammalian cells. LL-37mini also inhibited bacterial attachment and biofilm formation and disrupted preformed biofilms in vitro and killed MRSA in murine wound biofilms in vivo. Consistent with membrane targeting, MRSA failed to develop resistance to LL-37mini in a multiple-passage experiment. Because LL-37mini can be made cost effectively, it can be developed into new antibiofilm and antimicrobial agents.

Burkholderia pseudomallei biofilm resists Acanthamoeba sp. grazing and produces 8-O-4′-diferulic acid, a superoxide scavenging metabolite after passage through the amoeba

Burkholderia pseudomallei, an etiological agent of melioidosis is an environmental bacterium that can survive as an intracellular pathogen. The biofilm produced by B. pseudomallei is crucial for cellular pathogenesis of melioidosis. The purpose of this investigation is to explore the role of biofilm in survival of B. pseudomallei during encounters with Acanthamoeba sp. using B. pseudomallei H777 (a biofilm wild type), M10 (a biofilm defect mutant) and C17 (a biofilm-complemented strain). The results demonstrated similar adhesion to amoebae by both the biofilm wild type and biofilm mutant strains. There was higher initial internalisation, but the difference diminished after longer encounter with the amoeba. Interestingly, confocal laser scanning microscopy demonstrated that pre-formed biofilm of B. pseudomallei H777 and C17 were markedly more persistent in the face of Acanthamoeba sp. grazing than that of M10. Metabolomic analysis revealed a significant increased level of 8-O-4′-diferulic acid, a superoxide scavenger metabolite, in B. pseudomallei H777 serially passaged in Acanthamoeba sp. The interaction between B. pseudomallei with a free-living amoeba may indicate the evolutionary pathway that enables the bacterium to withstand superoxide radicals in intracellular environments. This study supports the hypothesis that B. pseudomallei biofilm persists under grazing by amoebae and suggests a strategy of metabolite production that turns this bacterium from saprophyte to intracellular pathogen.

Effect of florfenicol on Piscirickettsia salmonis biofilm formed in materials used in salmonid nets, nylon and high-density polyethylene.

Piscirickettsiosis is the most prevalent bacterial disease affecting seawater salmon in Chilean salmon industry. Antibiotic therapy is the first alternative to counteract infections caused by Piscirickettsia salmonis. The presence of bacterial biofilms on materials commonly used in salmon farming may be critical for understanding the bacterial persistence in the environment. In the present study, the CDC Biofilm Reactor® was used to investigate the effect of sub- and over-MIC of florfenicol on both the pre-formed biofilm and the biofilm formation by P. salmonis under the antibiotic stimuli on Nylon and high-density polyethylene (HDPE) surfaces. This study demonstrated that FLO, at sub- and over-MIC doses, decreases biofilm-embedded live bacteria in the P. salmonis isolates evaluated. However, it was shown that in the P. salmonis Ps007 strain the presence of sub-MIC of FLO reduced its biofilm formation on HDPE surfaces; however, biofilm persists on Nylon surfaces. These results demonstrated that P. salmonis isolates behave differently against FLO and also, depending on the surface materials. Therefore, it remains a challenge to find an effective strategy to control the biofilm formation of P. salmonis, and certainly other marine pathogens that affect the sustainability of the Chilean salmon industry.

The Antimicrobial, Antibiofilm and Anti-Inflammatory Activities of P13#1, a Cathelicidin-like Achiral Peptoid.

Cationic antimicrobial peptides (CAMPs) are powerful molecules with antimicrobial, antibiofilm and endotoxin-scavenging activities. These properties make CAMPs very attractive drugs in the face of the rapid increase in multidrug-resistant (MDR) pathogens, but they are limited by their susceptibility to proteolytic degradation. An intriguing solution to this issue could be the development of functional mimics of CAMPs with structures that enable the evasion of proteases. Peptoids (N-substituted glycine oligomers) are an important class of peptidomimetics with interesting benefits: easy synthetic access, intrinsic proteolytic stability and promising bioactivities. Here, we report the characterization of P13#1, a 13-residue peptoid specifically designed to mimic cathelicidins, the best-known and most widespread family of CAMPs. P13#1 showed all the biological activities typically associated with cathelicidins: bactericidal activity over a wide spectrum of strains, including several ESKAPE pathogens; the ability to act in combination with different classes of conventional antibiotics; antibiofilm activity against preformed biofilms of Pseudomonas aeruginosa, comparable to that of human cathelicidin LL-37; limited toxicity; and an ability to inhibit LPS-induced proinflammatory effects which is comparable to that of "the last resource" antibiotic colistin. We further studied the interaction of P13#1 with SDS, LPSs and bacterial cells by using a fluorescent version of P13#1. Finally, in a subcutaneous infection mouse model, it showed antimicrobial and anti-inflammatory activities comparable to ampicillin and gentamicin without apparent toxicity. The collected data indicate that P13#1 is an excellent candidate for the formulation of new antimicrobial therapies.