Minimum Biofilm Inhibitory Concentration Research Articles

Increased susceptibility to azithromycin of Pseudomonas aeruginosa biofilms using RPMI 1640 testing media.

Azithromycin (AZM) is efficient for treatment of chronic Pseudomonas aeruginosa biofilm lung infections, despite of resistance in conventional susceptibility testing. It has been shown that planktonic P. aeruginosa are more susceptible to AZM when tested in RPMI 1640 medium. The aim of the study was to test the susceptibility to AZM of P. aeruginosa biofilms in LB vs RPMI 1640 media. We investigated the effect of AZM on planktonic and biofilms of (WT) P. aeruginosa (PAO1), the hypermutable (ΔmutS) and the antibiotic-resistant phenotype(ΔnfxB) mutants. The effect of AZM on young and mature biofilms was investigated in the modified Calgary Biofilm Device by estimation of the minimal biofilm inhibitory concentration (MBIC). The AZM MBIC90 in LB/RPMI1640 on young biofilms treated for 24 h was 16/4 μg/mL for PAO1, 32/8 μg/mL for ΔmutS, and 256/16 μg/mL for ΔnfxB, while in mature biofilms was 256/2 μg/mL for PAO1 and ΔmutS and 16/1 μg/mL for ΔnfxB. The effect of AZM was improved when the treatment was prolonged to 72 h, supporting the intracellular accumulation of AZM. An increased susceptibility of P. aeruginosa biofilms to AZM was observed in RPMI 1640 than in LB medium. Our results might improve susceptibility testing and dosing of AZM for treatment of biofilm infections.

Liposomal Rifabutin-A Promising Antibiotic Repurposing Strategy against Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 μg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 μg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

Potential of diallyl sulfide from bulbs of Allium parvum to inhibit the growth and biofilm formation in Malassezia furfur MTCC 1374

The bulbs of Allium parvum are used as a flavoring agent in diet and are conventionally assumed to treat skin infections. The current study evaluates the phytochemicals from organic extract of A. parvum and their antifungal activity against biofilm-forming dermatophytic fungi Malassezia furfur MTCC 1374, yeast found in the normal flora of the skin, causes superficial infections, systemic infections like pulmonary infections, fungemia, etc., and implant-associated infection by biofilm formation. Biofilm formation enables the fungi to escape from antifungal agents and renders resistance to antifungal agents. The phytochemical assay for the organic extract of the onion bulb documented that A. parvum contains phenol (58 + 0.1 mg GAE/g), flavonoids (112 + 0.12 mg QE/g), saponins (1.45 + 0.1 mg AE/g), and tannins (4.1 + 0.03 g TA/g). The FT-IR and GC-MS analysis revealed the presence of specific compounds, alkaloids such as glycosides and quinolones, plant flavonoids such as 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-ester, phenolics, saponins such as butanoic acid and gluconic acid, and organic sulfur compounds such as diallyl sulfide, thiopyran tetrahydro-ester, allyl methyl trisulfide, and allyl methyl di sulfide. These compounds inhibited the growth of M. furfur at a minimum inhibitory concentration (MIC) value of 6.25 mg/ml and its biofilm at a minimum biofilm inhibitory concentration (MBIC) value of 12.5 mg/ml. Further studies required to explore the possible mechanism in controlling the biofilm, so, as to recommend as fungal control agent in medical sector.

The Strain-Dependent Antimicrobial and Antibiofilm effect of Cis and Trans-Vaccenic Acid against Pseudomonas Aeruginosa

This study, it was aimed to investigate the antibacterial and antibiofilm activity of cis and trans-vaccenic acid against Pseudomonas aeruginosa. In the study, four different P. aeruginosa strains were used. Antibacterial activity was determined by microdilution and growth curve. The antibiofilm activity was determined by crystal violet assay. In addition, the effect of vaccenic acids on pyocyanin production was investigated. The minimum inhibitory concentration (MIC) of cis and trans-vaccenic acid against all strains was determined as 128-256 μg/mL, and the minimum biofilm inhibitory concentration (MBIC) value was 8-512 μg/mL. While vaccenic acids reduced cell growth in three strains, they also significantly inhibited pyocyanin production. In one strain, it inhibited biofilm formation without affecting cell growth. As a result, the presence of antibacterial and antibiofilm activity of cis and trans-vaccenic acid against P. aeruginosa was determined as potential agents in the fight against this bacteria.

Lipopeptide biosurfactant as a potential root canal irrigation agent: Antimicrobial and anti-biofilm evaluation

ObjectivesLipopeptide Biosurfactant (LB) is a bacteria derived compound able to reduce surface tension between water and hydrophobic substances and exhibit antimicrobial and anti-biofilm properties. This study aimed to investigate the antimicrobial and anti-biofilm effect of a Lipopeptide Biosurfactant (LB) on Enterococcus faecalis, and its potential use in root canal treatment, either as a standalone irrigation solution or in conjunction with sodium hypochlorite (NaOCl). MethodsLB was extracted from Bacillus clausii isolate and the dry extract was diluted in deionized water. The antimicrobial effect of LB against planktonic E. faecalis was evaluated by determining the Minimal Inhibitory Concentration (MIC50). The anti-biofilm effect was evaluated by Minimal Biofilm Inhibitory Concentration (MBIC50) and Minimal Biofilm Eradication Concentration (MBEC50) assays on biofilm grown on dentin specimen surface. To evaluate the effectiveness of LB as a single irrigation solution and as a pre-irrigation prior to NaOCl, live and dead bacterial cells were quantified using Confocal Laser Scanning Microscopy (CLSM), and cell biomass was assessed. ResultsLB exhibited an MIC50 and MBIC50 of 100 ppm, with an MBEC50 of 1000 ppm, resulting in 52.94 % biofilm inhibition and 60.95 % biofilm eradication on dentin specimens. The effectiveness was concentration-dependent, at 500 ppm, LB demonstrated comparable antimicrobial efficacy to 2.5 % NaOCl. Pre-irrigation with LB resulted in lower biofilm biomass compared to NaOCl alone. ConclusionPre-irrigation with LB enhanced the antimicrobial effect when followed by NaOCl irrigation. Consequently, LB shows promise as both a standalone root canal irrigation solution and as an adjunct to NaOCl in root canal treatment. Clinical significanceThe study highlights the potential of Lipopeptide Biosurfactant (LB) as an environmentally friendly irrigation solution for root canal treatment, demonstrating potent antimicrobial and anti-biofilm properties against Enterococcus faecalis. LB exhibits concentration-dependent efficacy comparable to 2.5 % NaOCl and can be used as a standalone irrigation solution or in conjunction with NaOCl.

Selenium Nanoparticle Activity against S. mutans Biofilms as a Potential Treatment Alternative for Periodontitis.

The disruption of periodontal biofilms and prevailing antimicrobial resistance issues continue to pose a great challenge to the treatment of periodontitis. Here, we report on selenium nanoparticles (SeNPs) as a treatment alternative for periodontitis by determining their antibiofilm activity against S. mutans biofilms and the potential role of particle size in disrupting biofilms. SeNPs were synthesised via a reduction reaction. Various physicochemical characterisations were conducted on the NPs, including size and shape. The microbroth dilution method was used to conduct the biofilm and antibiofilm assay against S. mutans, which was analysed by absorbance. SeNPs displayed hydrodynamic sizes as low as 46 ± 4 nm at a volume ratio of 1:5 (sodium selenite/ascorbic acid) with good monodispersity and stability. Hydrodynamic sizes of SeNPs after resuspension in tryptic soy broth supplemented with 2.5% sucrose (TSB + 2.5% suc.) and incubated at 37 °C for 24 h, ranged from 112 to 263 nm, while the zeta potential values increased to greater than -11 mV. The biofilm assay indicated that S. mutans are weakly adherent, bordering on moderately adherent biofilm producers. The minimum biofilm inhibitory concentration (MBIC) was identified at 500 µg/mL. At a 1000 µg/mL concentration, SeNPs were able to inhibit S. mutan biofilms up to 99.87 ± 2.41% at a volume ratio of 1:1. No correlation was found between antibiofilm activity and particle size; however, antibiofilm activity was proven to be concentration-dependant. SeNPs demonstrate antibiofilm activity and may be useful for further development in treating periodontitis.

Comparative study of volatile oil constituents, anti-microbial properties, and antibiofilm activities in Eucalyptus camaldulensis and Eucalyptus globulus: insights from central Saudi Arabia

Eucalyptus is a fragrant plant with a highly volatile oil content grown worldwide. This study investigated the phytochemical and biological features of two Eucalyptus species, E. camaldulensis and E. globulus, cultivated in Central Saudi Arabia. Phytochemical analysis revealed 17 compounds in E. camaldulensis and 13 in E. globulus, with α-pinene and 1,8-cineole being both plants’ most abundant volatile components. The antimicrobial activity revealed that Salmonella typhimurium ATCC 13311 and Candida albicans ATCC 10231 are the most susceptible to E. camaldulensis, with the zone of inhibition diameters (ZIDs) of 17.0 ± 0.2 mm and 22.0 ± 0.3 mm, respectively. In contrast, for E. globulus, methicillin-resistant Staphylococcus aureus and Candida albicans ATCC 10231 were the most susceptible, with ZIDs of 13.0 ± 0.2 mm and 20.0 ± 0.2 mm, respectively. E. camaldulensis had minimum inhibitory concentration (MIC) range from 1.56 to 12.5 μL/mL, minimum biocidal concentration (MBC) range from 3.125 to 25.0 μL/mL, minimum biofilm inhibitory concentration (MBIC) range from 3.125 to 25.0 μL/mL, and minimum biofilm eradication concentration (MBEC) from 6.25 to 50.0 μL/mL, while E. globulus had MIC range from 3.125 to 12.5 μL/mL, MBC range from 6.25 to 25.0 μL/mL, MBIC range from 6.25 to 25.0 μL/mL, and MBEC range from 12.5 to 50.0 μL/mL, for the tested organisms. E. camaldulensis demonstrated better antimicrobial activity than E. globulus against the tested organisms, making it a promising antimicrobial drug candidate. Further research is required to determine its effectiveness in treating different human infections.

Antibiofilm Activity of Curcumin and Piperine and Their Synergistic Effects with Antifungals against Candida albicans Clinical Isolates.

Candidiasis is the common name for diseases caused by yeast of the genus Candida. Candida albicans is one of the most implicated species in superficial and invasive candidiasis. Antifungals, polyenes, and azoles have been used to treat candidiasis. However, due to the development of antifungal resistance, research of natural substances with potential antifungal effects at low concentrations or combined is also a possibility. The broth microdilution method was used to evaluate the antifungal activity. The biofilm formation was assessed using the microtiter plate method. The antibiofilm activities were assessed using micro plaque tetrazolium salt assay (MTT). The combination effect of antifungal with natural substances was made using the checkerboard method. Among our isolates, clotrimazole was the most resistant, but amphotericin B was the most effective antifungal. The biofilm was formed by all isolates of C. albicans. Curcumin and piperine displayed antibiofilm activity with minimum biofilm inhibitory concentration (MBIC) and minimum eradicating concentration (MBEC) ranging from 64 to 1024 μg/mL and 256 to 2048 μg/mL. In combination, piperine presented double synergistic effects compared to curcumin with all antifungals tested. Curcumin shows more synergistic effect when combined with polyenes than with azoles. However, piperine shows a more synergistic effect when combined with azoles compared to polyenes. C. albicans was susceptible to curcumin and piperine both on planktonic cells and biofilm. The combination of curcumin and piperine with antifungals has shown synergistic effects against multiresistant clinical isolates of Candida albicans representing an alternative drug research for the treatment of clinical candidiasis.

Synergistic bactericidal effects of phage-enhanced antibiotic therapy against MRSA biofilms.

The prevalence of biofilm-associated medical device infections caused by methicillin-resistant Staphylococcus aureus (MRSA) presents a pressing medical challenge. The latest research demonstrates the potential of phage-antibiotic combinations (PACs) as a promising solution, notably in vitro antibiofilm efficacy. By adopting modified checkerboard and 24-h time-kill assays, the study investigated the synergistic action of phages combined with humanized-simulated doses of daptomycin (DAP) and ceftaroline (CPT). The results were promising: a combination of DAP, CPT, and either a 2-phage or 3-phage cocktail effectively exhibited bactericidal activity against both DAP non-susceptible vancomycin intermediate S. aureus MRSA and DAP-susceptible MRSA strains within 168-h biofilm models. Moreover, post-treatment evaluations revealed no discernible rise in antibiotic resistance or complete phage resistance. This pioneering work suggests the potential of PACs in addressing MRSA biofilm infections, setting the stage for further expansive research tailored to diverse bacterial strains and treatment durations.

Anti-bacterial and anti-biofilm activities of arachidonic acid against the cariogenic bacterium Streptococcus mutans.

Streptococcus mutans is a Gram-positive, facultative anaerobic bacterium, which causes dental caries after forming biofilms on the tooth surface while producing organic acids that demineralize enamel and dentin. We observed that the polyunsaturated arachidonic acid (AA) (ω-6; 20:4) had an anti-bacterial activity against S. mutans, which prompted us to investigate its mechanism of action. The minimum inhibitory concentration (MIC) of AA on S. mutans was 25 μg/ml in the presence of 5% CO2, while it was reduced to 6.25-12.5 μg/ml in the absence of CO2 supplementation. The anti-bacterial action was due to a combination of bactericidal and bacteriostatic effects. The minimum biofilm inhibitory concentration (MBIC) was the same as the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies showed decreased expression of biofilm-related genes, suggesting that AA also has a specific anti-biofilm effect. Flow cytometric analyses using potentiometric DiOC2(3) dye, fluorescent efflux pump substrates, and live/dead SYTO 9/propidium iodide staining showed that AA leads to immediate membrane hyperpolarization, altered membrane transport and efflux pump activities, and increased membrane permeability with subsequent membrane perforation. High-resolution scanning electron microscopy (HR-SEM) showed remnants of burst bacteria. Furthermore, flow cytometric analysis using the redox probe 2',7'-dichlorofluorescein diacetate (DCFHDA) showed that AA acts as an antioxidant in a dose-dependent manner. α-Tocopherol, an antioxidant that terminates the radical chain, counteracted the anti-bacterial activity of AA, suggesting that oxidation of AA in bacteria leads to the production of cytotoxic radicals that contribute to bacterial growth arrest and death. Importantly, AA was not toxic to normal Vero epithelial cells even at 100 μg/ml, and it did not cause hemolysis of erythrocytes. In conclusion, our study shows that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans.

Comparative Assessment of the Antibacterial and Antibiofilm Actions of Benzalkonium Chloride, Erythromycin, and L(+)-Lactic Acid against Raw Chicken Meat Campylobacter spp. Isolates.

Campylobacter spp. are significant zoonotic agents, which cause annually millions of human cases of foodborne gastroenteritis worldwide. Their inclusion in biofilms on abiotic surfaces seems to play a pivotal role in their survival outside of the host, growth, and spread. To successfully mitigate the risks that arise with these bacteria, it is crucial to decrease their prevalence within the food production chain (from farm to the table), alongside the successful treatment of the resulting illness, known as campylobacteriosis. For this, the use of various antimicrobial agents remains actively in the foreground. A general-purpose biocide and cationic surfactant (benzalkonium chloride; BAC), a widely used macrolide antibiotic (erythromycin; ERY), and a naturally occurring organic acid (L(+)-lactic acid; LA) were comparatively evaluated in this work for their potential to inhibit both the planktonic and biofilm growth of 12 selected Campylobacter spp. (of which, seven were C. jejuni and five were C. coli) raw chicken meat isolates, all grown in vitro as monocultures. The inhibitory action of LA was also studied against four mixed-culture Campylobacter biofilms (each composed of three different isolates). The results showed that the individual effectiveness of the agents varied significantly depending on the isolate, growth mode (planktonic, biofilm), intercellular interactions (monocultures, mixed cultures), and the growth medium used (with special focus on blood presence). Thus, BAC exhibited minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum biofilm inhibitory concentrations (MBICs) that ranged from 0.5 to 16 μg/mL. Interestingly enough, these values varied widely from 0.25 to 1024 μg/mL for ERY. Concerning LA, the MICs, MBCs, and MBICs varied from 1024 to 4096 μg/mL, with mixed-culture biofilm formation always being more difficult to suppress when compared to biofilm monocultures. In addition, it was evident that intercellular interactions encountered within mixed-culture Campylobacter biofilms significantly influenced both the population dynamics and the tolerance of each consortium member to acid exposure. Overall, the findings of this study provide useful information on the comparative effectiveness of three well-known antimicrobial agents for the control of Campylobacter spp. under various growth modes (i.e., planktonic, biofilm, monocultures, mixed cultures) that could potentially be encountered in food production and clinical settings.

A hope for ineffective antibiotics to return to treatment: investigating the anti-biofilm potential of melittin alone and in combination with penicillin and oxacillin against multidrug resistant-MRSA and -VRSA.

The emergence and rapid spread of multi-drug resistant (MDR) bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have posed a significant challenge to the medical community due to their ability to form biofilm and develop resistance to common antibiotics. Traditional antibiotics that were once effective in treating bacterial infections are now becoming increasingly ineffective, leading to severe consequences for patient outcomes. This concerning situation has called for urgent research to explore alternative treatment strategies. Recent studies have shown that antimicrobial peptides (AMPs) hold promise as effective agents against biofilm-associated drug-resistant infections as well as to enhance the efficacy of conventional antibiotics. Accordingly, we aimed to investigate the antimicrobial and antibiofilm effects of melittin AMP, both alone and in combination with penicillin and oxacillin, against biofilm-forming MDR-MRSA and -VRSA. In this study, we investigated the kinetics of biofilm formation and assessed various parameters related to the antimicrobial and antibiofilm efficacy of melittin and antibiotics, both alone and in combination, against MDR-MRSA and -VRSA. The antimicrobial parameters included the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Fractional Inhibitory Concentration Index (FICi), Fractional Bactericidal Concentration Index (FBCi), and the antibiofilm activity of melittin and antibiotics indicated by the Minimum Biofilm Inhibitory Concentration (MBIC), Minimal Biofilm Eradication Concentration (MBEC), Fractional Biofilm Inhibitory Concentration Index (FBICi), and Fractional Biofilm Eradication Concentration Index (FBECi). The MIC results showed that all S. aureus isolates were resistant to penicillin (≥0.25 μg/mL), and 66% of isolates were resistant to oxacillin. The geometric means of the MIC values for penicillin, oxacillin, and melittin were 19.02, 16, and 1.62 μg/ml, respectively, and the geometric means of the MBC values for penicillin, oxacillin, and melittin were 107.63, 49.35, and 5.45 μg/ml, respectively. The study revealed that the combination indexes of melittin-penicillin and melittin-oxacillin, as determined by FIC values against all isolates, were 0.37 and 0.03, respectively. Additionally, melittin-penicillin and melittin-oxacillin exhibited combination indexes based on FBC values against all isolates at 1.145 and 0.711, respectively. Besides, melittin inhibited the biofilm formation of all S. aureus isolates, with MBIC values ranging from 10 to 1.25 μg/mL, and MBEC values ranging from 40 to 10 μg/mL. Generally, the combination indexes of melittin-penicillin and melittin-oxacillin, determined using FBIC values against all isolates, were 0.23 and 0.177, respectively. Moreover, melittin-penicillin and melittin-oxacillin typically had combination indexes based on FBEC values against all isolates at 5 and 2.97, respectively. In conclusion, our study provides evidence that melittin is effective against both planktonik and biofilm forms of MRSA and VRSA and exhibits significant synergistic effects when combined with antibiotics. These results suggest that melittin and antibiotics could be a potential candidate for further investigation for in vivo infections caused by MDR S. aureus. Furthermore, melittin has the potential to restore the efficacy of penicillin and oxacillin antibiotics in the treatment of MDR infections. Applying AMPs, like melittin, to revive beta-lactam antibiotics against MRSA and VRSA is an innovative approach against antibiotic-resistant bacteria. Further research is needed to optimize dosage and understand melittin mechanism and interactions with beta-lactam antibiotics for successful clinical applications.

Poly-Gamma-Glutamic Acid Nanopolymer Effect against Bacterial Biofilms: In Vitro and In Vivo Study.

In this study, a biodegradable poly-gamma-glutamic-acid nanopolymer (Ɣ-PGA NP) was investigated for its activity against clinical strains of Gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli), and reference strains of S. aureus ATCC 6538, S. pyogenes ATCC 19615 (Gram-positive), and Gram-negative E. coli ATCC 25922, and K. pneumoniae ATCC 13884 bacterial biofilms. The minimum inhibitory concentration (MIC) effect of Ɣ-PGA NP showed inhibitory effects of 0.2, 0.4, 1.6, and 3.2 μg/mL for S. pyogenes, S. aureus, E. coli, and K. pneumoniae, respectively. Also, MIC values were 1.6, 0.8, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Afterwards, MBEC (minimum biofilm eradication concentration) and MBIC (minimum biofilm inhibitory concentration) were investigated to detect Ɣ-PGA NPs efficiency against the biofilms. MBEC and MBIC increased with increasing Ɣ-PGA NPs concentration or time of exposure. Interestingly, MBIC values were at lower concentrations of Ɣ-PGA NPs than those of MBEC. Moreover, MBEC values showed that K. pneumoniae was more resistant to Ɣ-PGA NPs than E. coli, S. aureus, and S. pyogenes, and the same pattern was observed in the reference strains. The most effective results for MBEC were after 48 h, which were 1.6, 0.8, 0.4, and 0.2 µg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. Moreover, MBIC results were the most impactful after 24 h but some were the same after 48 h. MBIC values after 48 h were 0.2, 0.2, 0.2, and 0.1 μg/mL for K. pneumoniae, E. coli, S. aureus, and S. pyogenes, respectively. The most effective results for MBEC were after 24 h, which were 1.6, 0.8, 0.4, and 0.4 µg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Also, MBIC results were the most impactful after an exposure time of 12 h. MBIC values after exposure time of 12 h were 0.4, 0.4, 0.2, and 0.2 μg/mL for K. pneumoniae ATCC 13884, E. coli ATCC 25922, S. aureus ATCC 6538, and S. pyogenes ATCC 19615, respectively. Besides that, results were confirmed using confocal laser scanning microscopy (CLSM), which showed a decrease in the number of living cells to 80% and 60% for MBEC and MBIC, respectively, for all the clinical bacterial strains. Moreover, living bacterial cells decreased to 70% at MBEC while decreasing up to 50% at MBIC with all bacterial refence strains. These data justify the CFU quantification. After that, ImageJ software was used to count the attached cells after incubating with the NPs, which proved the variation in live cell count between the manual counting and image analysis methods. Also, a scanning electron microscope (SEM) was used to detect the biofilm architecture after incubation with the Ɣ-PGA NP. In in vivo wound healing experiments, treated wounds of mice showed faster healing (p < 0.00001) than both the untreated mice and those that were only wounded, as the bacterial count was eradicated. Briefly, the infected mice were treated faster (p < 0.0001) when infected with S. pyogenes > S. aureus > E. coli > K. pneumoniae. The same pattern was observed for mice infected with the reference strains. Wound lengths after 2 h showed slightly healing (p < 0.001) for the clinical strains, while treatment became more obvious after 72 h > 48 h > 24 h (p < 0.0001) as wounds began to heal after 24 h up to 72 h. For reference strains, wound lengths after 2 h started to heal up to 72 h.

Expanded library of novel 2,3-pyrrolidinedione analogues exhibit anti-biofilm activity

Herein we report a new library of 2,3-pyrrolidinedione analogues that expands on our previous report on the antimicrobial studies of this heterocyclic scaffold. The novel 2,3-pyrrolidinediones reported herein have been evaluated against S. aureus and methicillin-resistant S. aureus (MRSA) biofilms, and this work constitutes our first report on the antibiofilm properties of this class of compounds. The antibiofilm activity of these 2,3-pyrrolidinediones has been assessed through minimum biofilm eradication concentration (MBEC) and minimum biofilm inhibition concentration (MBIC) assays. The compounds displayed antibiofilm properties and represent intriguing scaffolds for further optimization and development.

Apple cider vinegar exhibits promising antibiofilm activity against multidrug-resistant Bacillus cereus isolated from meat and their products.

Bacillus cereus (B. cereus) biofilm is grown not only on medical devices but also on different substrata and is considered a potential hazard in the food industry. Quorum sensing plays a serious role in the synthesis of biofilm with its surrounding extracellular matrix enabling irreversible connection of the bacteria. The goal of the current investigation was to ascertain the prevalence, patterns of antimicrobial resistance, and capacity for B. cereus biofilm formation in meat and meat products in Egypt. In all, 150 meat and meat product samples were used in this study. For additional bacteriological analysis, the samples were moved to the Bacteriology Laboratory. Thereafter, the antimicrobial, antiquorum sensing, and antibiofilm potential of apple cider vinegar (ACV) on B. cereus were evaluated. Out of 150 samples, 34 (22.67%) tested positive for B. cereus. According to tests for antimicrobial susceptibility, every B. cereus isolates tested positive for colistin and ampicillin but negative for ciprofloxacin and imipenem. The ability to form biofilms was present in all 12 multidrug-resistant B. cereus isolates (n = 12); of these, 6 (50%), 3 (25%), and 3 (25%) isolates were weak, moderate, and strong biofilm producers, respectively. It is noteworthy that the ACV demonstrated significant inhibitory effects on B. cereus isolates, with minimum inhibitory concentrations varying between 2 and 8 μg/ml. Furthermore, after exposing biofilm-producing B. cereus isolates to the minimum biofilm inhibitory concentrations 50 of 4 μg/ml, it demonstrated good antibiofilm activity (>50% reduction of biofilm formation). Strong biofilm producers had down-regulated biofilm genes (tasA and sipW) and their regulator (plcR) compared to the control group, according to reverse transcriptase quantitative polymerase chain reaction analysis. Our study is the first report, that spotlights the ACV activity against B. cereus biofilm and its consequence as a strong antibacterial and antibiofilm agent in the food industry and human health risk.

Crude Aceh patchouli alcohol (Pogostemon cablin Benth.) elucidated antibiofilm activity of against Staphylococcus aureus

The emergence of biofilm-producing bacteria has prompted the search for novel antibacterial agents, including plant-based compounds, such as patchouli oil (Pogostemon cablin Benth.). This study evaluated the phytochemical and antibiofilm activities of crude Aceh patchouli oil (CPO) against Staphylococcus aureus. This study employed a laboratory experimental design: two controls (growth and negative) and three varying concentrations (0.5%, 1%, and 2%) of CPO. Phytochemical analysis was conducted using gas chromatography-mass spectrometry, which revealed the presence of terpenes, sesquiterpenes, and fatty acids in CPO, with patchouli alcohol, azulene, and alpha-guaiene as the dominant compounds. CPO exhibited significant antibiofilm activity against S. aureus, with a minimum biofilm-inhibitory concentration of 0.5%. Notably, CPO was also effective in eradicating existing S. aureus biofilms at a concentration of 1%. The findings of this study suggest that CPO could be a promising candidate for the development of novel anti-staphylococcal agents.

Antibiofilm potential of nanonized eugenol against Pseudomonas aeruginosa.

The purpose of this study was to synthesize a nanoform of eugenol (an important phytochemical with various pharmacological potentials) and to investigate its antibiofilm efficacy on Pseudomonas aeruginosa biofilm. Colloidal suspension of eugenol-nanoparticles (ENPs) was synthesized by the simple ultrasonic cavitation method through the emulsification of hydrophobic eugenol into hydrophilic gelatin. Thus, the nanonization process made water-insoluble eugenol into water-soluble nano-eugenol, making the nanoform bioavailable. The size of the ENPs was 20-30 nm, entrapment efficiency of eugenol within gelatin was 80%, and release of eugenol from the gelatin cap was slow and sustained over 5 days. Concerning the clinically relevant pathogen P. aeruginosa, ENPs had higher antibiofilm (for both formation and eradication) activities than free eugenol. Minimal biofilm inhibitory concentration and minimal biofilm eradication concentration of ENP on P. aeruginosa biofilm were 2.0 and 4.0 mM, respectively. In addition, the measurement of P. aeruginosa biofilm biomass, biofilm thickness, amount of biofilm extra-polymeric substance, cell surface hydrophobicity, cell swarming and twitching efficiencies, cellular morphology, and biofilm formation in catheter demonstrated that the antibiofilm efficacy of nano-eugenol was 30%-40% higher than that of bulk eugenol. These results signify that future pharmacological and clinical studies are very much required to investigate whether ENPs can act as an effective drug against P. aeruginosa biofilm-mediated diseases. Thus, the problem of intrinsic antibiotic tolerance of biofilm-forming cells may be minimized by ENPs. Moreover, ENP may be used as a potential catheter-coating agent to inhibit pseudomonal colonization on catheter surfaces and, therefore, to reduce catheter-associated infections and complications.

Comparative evaluation of effect of nisin-incorporated ethylenediamine tetraacetic acid and MTAD on endodontic biofilm eradication, smear layer removal, and depth of sealer penetration.

To comparatively evaluate the nisin-incorporated ethylenediamine tetraacetic acid (N-EDTA) and MTAD on cytotoxicity, endodontic biofilm eradication potential, smear layer removal ability, and sealer penetration depth. N-EDTA was prepared and characterized using high-performance liquid chromatography (HPLC). Minimum inhibitory, minimum bactericidal, and minimum biofilm inhibitory concentration (MBC, MIC, and MBIC) were determined on Enterococcus faecalis (E. faecalis) strain. The cytocompatibility of N-EDTA and MTAD was evaluated using 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay. Dentin specimens (n = 88 for antibacterial analysis, n = 170 for sealer penetration depth) were prepared and subjected to the classical irrigating strategy and obturation, respectively. The scanning electron microscopic evaluation (SEM) was done for the evaluation of biofilm disruption and smear layer removal. Confocal laser scanning microscopy (CLSM) evaluation was done for determining percentage of bacterial viability and sealer penetration depth. Statistical analysis of one-way ANOVA and Tukey's HSD post hoc tests for bacterial viability and Kruskal-Wallis test and Mann-Whitney test for smear layer removal and depth of penetration were done with the significance level set at p < 0.05. MTAD and N-EDTA showed cytocompatibility without any statistical differences from each other. For N-EDTA, the MIC and MBC values were 12.5 μg/ml (1:8), and MBIC values were 36 μg/ml. Biofilm disruption and killed bacterial percentage of N-EDTA was statistically higher than MTAD, whereas both the materials showed similar efficacy in the removal of the smear layer and sealer penetration depth. N-EDTA had negligible cytotoxicity with similar smear layer removal ability, sealer penetration, and better antibiofilm potential than MTAD. N-EDTA can serve as a viable alternative endodontic irrigant.

Preparation, Physicochemical Characterization, Antimicrobial Effects, Biocompatibility and Cytotoxicity of Co-Loaded Meropenem and Vancomycin in Mesoporous Silica Nanoparticles.

Mesoporous silica nanoparticles (MSNPs) have been reported as an effective system to co-deliver a variety of different agents to enhance efficiency and improve biocompatibility. This study was aimed at the preparation, physicochemical characterization, antimicrobial effects, biocompatibility, and cytotoxicity of vancomycin and meropenem co-loaded in the mesoporous silica nanoparticles (Van/Mrp-MSNPs). The prepared nanoparticles were explored for their physicochemical features, antibacterial and antibiofilm effects, biocompatibility, and cytotoxicity. The minimum inhibitory concentrations (MICs) of the Van/Mrp-MSNPs (0.12-1 µg/mL) against Staphylococcus aureus isolates were observed to be lower than those of the same concentrations of vancomycin and meropenem. The minimum biofilm inhibitory concentration (MBIC) range of the Van/Mrp-MSNPs was 8-64 μg/mL, which was lower than the meropenem and vancomycin MBICs. The bacterial adherence was not significantly decreased upon exposure to levels lower than the MICs of the MSNPs and Van/Mrp-MSNPs. The viability of NIH/3T3 cells treated with serial concentrations of the MSNPs and Van/Mrp-MSNPs were 73-88% and 74-90%, respectively. The Van/Mrp-MSNPs displayed considerable inhibitory effects against MRSA, favorable biocompatibility, and low cytotoxicity. The Van/Mrp-MSNPs could be a potential system for the treatment of infections.

Identification of a Staphylococcus aureus amidase catalytic domain inhibitor to prevent biofilm formation by sequential virtual screening, molecular dynamics simulation and biological evaluation

Staphylococcus aureus (S. aureus) is one of the common causes of implant associated biofilm infections and their biofilms are resistant to antibiotics. S. aureus amidase (AM) protein, a cell wall hydrolase that cleaves the amide bond between N-acetylmuramic acid and L-alanine residue of the stem peptide, is several fold over-expressed under biofilm conditions. Previous studies demonstrated an autolysin mutant in S. aureus that lacks the AM protein, is highly impaired in biofilm development. We carried out a structure-based small molecule design using the crystal structure of AM protein catalytic domain to identify inhibitors that can block amidase activity and therefore inhibits S. aureus biofilm formation. Sequential virtual screening followed by pharmacokinetic analysis and bioassay studies filtered 25 small molecules from different databases. Two compounds from the SPECS database, SPECS-1 and SPECS-2, were selected based on the best docking score and minimum biofilm inhibitory concentration towards S. aureus biofilms. SPECS-1 and SPECS-2 were further tested for their structural/energetic stability in complex with the AM protein using molecular dynamics simulation and MM-GBSA techniques. In vitro, biofilm inhibition studies on different surfaces confirmed that treatment with SPECS-1 and SPECS-2 at a concentration of 250 μg/ml exhibited significant prevention and disruption of S. aureus biofilms. Finally, the in vitro anti-biofilm activities of these two compounds were validated against Methicillin-resistant S. aureus clinical isolates. We concluded that the discovered compounds SPECS-1 and SPECS-2 are safe and exhibit biofilm preventive and disruption activity for inhibiting the S. aureus biofilms and hence can be used to treat implant-associated biofilm infections.