Biofilm Eradication Research Articles

Drug Resistance in Biofilm and Planktonic Cells of Achromobacter spp., Burkholderia spp., and Stenotrophomonas maltophilia Clinical Isolates.

Background: Biofilm production in nonfermenting Gram-negative bacteria influences drug resistance. The aim of this work was to evaluate the effect of different antibiotics on biofilm eradication of clinical isolates of Achromobacter, Burkholderia, and Stenotrophomonas maltophilia. Methods: Clinical isolates were identified by matrix-assisted laser desorption ionization-time of flightmass spectrometry in a third-level hospital in Monterrey, Mexico. Crystal violet staining was used to determine biofilm production. Drug susceptibility testing was determined by broth microdilution in planktonic cells and biofilm cells. Results: Resistance in planktonic cells was moderate to trimethoprim-sulfamethoxazole, and low to chloramphenicol, minocycline, levofloxacin (S. maltophilia and Burkholderia), ceftazidime, and meropenem (Burkholderia and Achromobacter). Biofilm eradication required higher drug concentrations of ceftazidime, chloramphenicol, levofloxacin, and trimethoprim-sulfamethoxazole than planktonic cells (p < 0.05). Levofloxacin showed biofilm eradication activity in S. maltophilia, minocycline and meropenem in Burkholderia, and meropenem in Achromobacter. Conclusions: Drug resistance increased due to biofilm production for some antibiotics, particularly ceftazidime and trimethoprim-sulfamethoxazole for all three pathogens, chloramphenicol for S. maltophilia and Burkholderia, and levofloxacin for Burkholderia. Some antibiotics could be used for the treatment of biofilm-associated infections in our population, such as levofloxacin for S. maltophilia, minocycline and meropenem for Burkholderia, and meropenem for Achromobacter.

The Impact of Silver Nanoparticles’ Size on Biofilm Eradication

IntroductionEfficient intracanal disinfection is required for a successful regenerative endodontic treatment. Thus, this study aimed to identify the silver nanoparticles’ (NPs) size (AgNPs) with the highest antibiofilm efficacy when mixed with calcium hydroxide [Ca(OH)2] to eradicate an in vitro endodontic biofilm. MethodsThe various sizes of AgNPs and mixtures were characterized by scanning electron microscopy, transmission electron microscopy, and ultraviolet-visible spectroscopy. A total of 168 dentin root segments were prepared, sterilized, and inoculated for 3 weeks with Actinomyces naeslundii and Fusobacterium nucleatum. Samples were randomly allocated to 4 experimental groups (n = 28/group): 2 nm AgNPs + 35% Ca(OH)2, 5 nm AgNPs + 35% Ca(OH)2, 10 nm AgNPs + 35% Ca(OH)2, and 35% Ca(OH)2 alone. Samples exposed to saline and triple antibiotic paste (TAP) acted as negative and positive control groups, respectively. After 1 and 2 weeks, samples were stained with LIVE/DEAD BacLight dye and examined under a confocal laser scanning microscope to determine the proportion of dead bacteria. ResultsThe characterization procedure revealed a spherical NP's structure with minor aggregations. Except for Ca(OH)2 group, all groups had significantly higher antibiofilm efficacy at 2 weeks. Both the 10 nm mixture (99.5%) and TAP (99.2%) exhibited the highest antibiofilm efficacy at 2 weeks and were not significantly different from one another (P > .05). No significant difference was noted between the 2 and 5 nm mixtures at 1 week (81% and 84%) and 2 weeks (89% and 91%). ConclusionThe 10 nm AgNPs (0.02%) + 35% Ca(OH)2 mixture exhibited the highest antibiofilm efficacy at 2 weeks compared to all other mixtures at both observation periods. Interestingly, the 10 nm mixture performed similarly to TAP at 2 weeks. Excluding Ca(OH)2 group, longer application significantly improved the antibiofilm efficacy of all tested medicaments. Clinical RelevanceThe 10 nm AgNPs + 35% Ca(OH)2 mixture revealed promising results as an intracanal medicament in the regenerative endodontic treatment protocol.

Antibiotic-Polyphosphate Nanocomplexes: A Promising System for Effective Biofilm Eradication.

The eradication of bacterial biofilms poses an enormous challenge owing to the inherently low antibiotic susceptibility of the resident microbiota. The complexation of antibiotics with polyphosphate can substantially improve antimicrobial performance. Nanoparticular complexes of the model drug colistin and polyphosphate (CP-NPs) were developed and characterized in terms of their particle size and morphology, polydispersity index (PDI), zeta potential, and cytotoxicity. Enzyme-triggered monophosphate and colistin release from the CP-NPs was evaluated in the presence of alkaline phosphatase (AP). Subsequently, antimicrobial efficacy was assessed by inhibition experiments on planktonic cultures, as well as time-kill assays on biofilms formed by the model organism Micrococcus luteus. The CP-NPs exhibited a spherical morphology with particle sizes <200 nm, PDI <0.25, and negative zeta potential. They showed reduced cytotoxicity toward two human cell lines and significantly decreased hemotoxicity compared with native colistin. Release experiments with AP verified the enzymatic cleavage of polyphosphate and subsequent release of monophosphate and colistin from CP-NPs. Although CP-NPs were ineffective against planktonic M. luteus cultures, they showed major activity against bacterial biofilms, outperforming native colistin treatment. Strongly elevated AP levels in the biofilm state were identified as a potential key factor for the observed findings. Accordingly, polyphosphate-based nanocomplexes represent a promising tool to tackle bacterial biofilm.

Activity of Fluoroquinolones and Proton Pump Inhibitors against Resistant Oral Bacterial Biofilms, in silico and in vitro Analysis.

Oral bacterial infections are a great health concern worldwide especially in diabetic patients. Emergence of antimicrobial resistance with reference to biofilms in oral cavity is of great concern. We investigated antibiotics combination with proton pump inhibitors against oral clinical isolates. The strains were identified as Staphylococcus epidermidis and Staphylococcus aureus by the 16S rRNA gene sequencing. In molecular docking, ciprofloxacin, levofloxacin, and omeprazole best fit to active pockets of transcriptional regulators 4BXI and 3QP1. None of the proton pump inhibitors were active against S. epidermidis, whereas omeprazole showed significant inhibition (MIC 3.9 μg/ml). Fluoroquinolones were active against both S. epidermidis and S. aureus. In combination analysis, a marked decrease in minimum inhibitory concentration was noticed with omeprazole (MIC 0.12 μg/ml). In antiquorum sensing experiments, a significant inhibitory zone was shown for all fluoroquinolones (14-20 mm), whereas among proton pump inhibitors, only omeprazole (12 ± 0.12 mm) was active against Chromobacterium violaceum. In combination analysis, a moderate increase in antiquorum sensing activity was recorded for ciprofloxacin, ofloxacin, and proton pump inhibitors. Further, significant S. aureus biofilm eradication was recorded using of ciprofloxacin, levofloxacin, and omeprazole combination (78 ± 2.1%). The time-kill kinetic studies indicated a bactericidal effect by ciprofloxacin: levofloxacin: omeprazole combination over 24 hrs. It was concluded that fluoroquinolone combined with omeprazole could be an effective treatment option for eradicating oral bacterial biofilms.

Disease-Inspired Design of Biomimetic Tannic Acid-Based Hybrid Nanocarriers for Enhancing the Treatment of Bacterial-Induced Sepsis.

This study explored the development of novel biomimetic tannic acid-based hybrid nanocarriers (HNs) for targeted delivery of ciprofloxacin (CIP-loaded TAH-NPs) against bacterial-induced sepsis. The prepared CIP-loaded TAH-NPs exhibited appropriate physicochemical characteristics and demonstrated biocompatibility and nonhemolytic properties. Computational simulations and microscale thermophoresis studies validated the strong binding affinity of tannic acid (TA) and its nanoformulation to human Toll-like receptor 4, surpassing that of the natural substrate lipopolysaccharide (LPS), suggesting a potential competitive inhibition against LPS-induced inflammatory responses. CIP released from TAH-NPs displayed a sustained release profile over 72 h. The in vitro antibacterial activity studies revealed that CIP-loaded TAH-NPs exhibited enhanced antibacterial efficacy and efflux pump inhibitory activity. Specifically, they showed a 3-fold increase in biofilm eradication activity against MRSA and a 2-fold increase against P. aeruginosa compared to bare CIP. Time-killing assays demonstrated complete bacterial clearance within 8 h of treatment with CIP-loaded TAH-NPs. In vitro DPPH scavenging and anti-inflammatory investigations confirmed the ability of the prepared hybrid nanosystem to neutralize reactive oxygen species (ROS) and modulate LPS-induced inflammatory responses. Collectively, these results suggest that CIP-loaded TAH-NPs may serve as an innovative nanocarrier for the effective and targeted delivery of antibiotics against bacterial-induced sepsis.

In vitro efficacy of lavender oil, otological gel and gentamicin to eradicate biofilm produced by Pseudomonas aeruginosa.

Otitis externa (OE) is one of the most frequently diagnosed dermatological diseases in dogs, having a multifactorial aetiology. Among the bacterial agents associated with canine OE, Pseudomonas aeruginosa is of special concern owing to its frequent multidrug resistance profile and ability to form biofilms related to the infection's chronicity and recurrence. The main objective of this study was to evaluate and compare the antibiofilm activity of two innovative antimicrobials-an otological gel containing a synthetic antimicrobial peptide and Lavandula angustifolia essential oil-with gentamicin (a conventional antibiotic) using biofilm-producing P. aeruginosa isolates obtained from dogs with OE. The biofilm eradication capacity of gentamicin, otological gel and lavender oil was determined against a collection of 12 P. aeruginosa biofilm-producers among 35 clinical isolates obtained from the ear canals of dogs with OE. Also, the antimicrobial activity of the otological gel against P. aeruginosa biofilms was assessed in an invitro model of dog cerumen. Lavender oil showed the best effectiveness after 30 min of contact, eradicating 58.3% (seven of 12) of the isolates, and gentamicin showed full eradication (12 of 12) after 24 h. The otological gel acted more slowly than the lavender oil; yet at 24 h, the antibiofilm capacity of both compounds was similar, with no significant difference between them. It also was found that triglycerides from synthetic cerumen earwax had antipseudomonal activity and, when combined with the otological gel, led to the full eradication of P. aeruginosa. The results of this invitro study indicate that lavender oil and the otological gel may be effective topical treatments for canine OE promoted by P. aeruginosa biofilm-producers, as alternatives to gentamicin.

Spirulina platensis as a super prebiotic to enhance the antibacterial effect of Lactobacillus casei

Microalgae are diminutive unicellular magnificent creatures that exist in the microscopic form. Within this classification, two notable subtypes, namely Spirulina platensis and Chlorella vulgaris, have garnered significant attention. S. platensis is mostly known for its therapeutic benefits due to proteins, vitamins, essential amino acids, minerals, and essential fatty acids abundance.The subsequent work aimed to investigate the prebiotic effect of S. platensis enhancing Lactobacillus casei microbiome growth rate and antibacterial effect. Different concentrations of S. platensis were prepared (0, 0.5, 5, and 50 mg/mL) then co-cultured with L. casei. Live probiotic enumeration, molecular characterization and scanning electron microscopy studies were performed. Data revealed that 5 mg/mL of S. platensis significantly enhanced the probiotic live count and consequently the antibacterial activity. The antibacterial and antibiofilm effects of L. casei/S. platensis mixture showed complete inhibition to the growth of bacterial cells after 4- and 8-h incubation with Staphylococcus epidermidis 12228 and Escherichia coli 25922, respectively. Moreover, minimal biofilm eradication concentration (MBEC) value of 16 µg/mL was recorded for L. casei/S. platensis mixture

Effective biofilm eradication in MRSA isolates with aminoglycoside-modifying enzyme genes using high-concentration and prolonged gentamicin treatment.

Our analysis of 101 MRSA clinical isolates has provided valuable insights that could enhance treatment selection for biofilm infections in orthopedics. We found that high concentrations of gentamicin were effective against MRSA biofilms, and even prolonged exposure to concentrations lower than the minimum biofilm eradication concentration (MBEC) value could eliminate biofilms. The presence of the aac(6')-aph(2″) gene, an aminoglycoside resistance gene, was found to correlate with the minimum inhibitory concentration (MIC) and MBEC values of gentamicin, providing a potential predictive tool for treatment susceptibility. These results suggest that extended high concentrations of local gentamicin treatment could effectively eliminate MRSA biofilms in orthopedic infections. Furthermore, testing for gentamicin MIC or the possession of the aac(6')-aph(2″) gene could help select treatment, including topical gentamicin administration and surgical debridement.

A self-supplied hydrogen peroxide and nitric oxide-generating nanoplatform enhances the efficacy of chemodynamic therapy for biofilm eradication

Bacterial biofilms present a profound challenge to global public health, often resulting in persistent and recurrent infections that resist treatment. Chemodynamic therapy (CDT), leveraging the conversion of hydrogen peroxide (H2O2) to highly reactive hydroxyl radicals (•OH), has shown potential as an antibacterial approach. Nonetheless, CDT struggles to eliminate biofilms due to limited endogenous H2O2 and the protective extracellular polymeric substances (EPS) within biofilms. This study introduces a multifunctional nanoplatform designed to self-supply H2O2 and generate nitric oxide (NO) to overcome these hurdles. The nanoplatform comprises calcium peroxide (CaO2) for sustained H2O2 production, a copper-based metal–organic framework (HKUST-1) encapsulating CaO2, and l-arginine (l-Arg) as a natural NO donor. When exposed to the acidic microenvironment within biofilms, the HKUST-1 layer decomposes, releasing Cu2+ ions and l-Arg, and exposing the CaO2 core to initiate a cascade of reactions producing reactive species such as H2O2, •OH, and superoxide anions (•O2–). Subsequently, H2O2 catalyzes l-Arg to produce NO, which disperses the biofilm and reacts with •O2– to form peroxynitrite, synergistically eradicating bacteria with •OH. In vitro assays demonstrated the nanoplatform’s remarkable antibiofilm efficacy against both Gram-positive Methicillin-resistant Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, significantly reducing bacterial viability and EPS content. In vivo mouse model experiments validated the nanoplatform’s effectiveness in eliminating biofilms and promoting infected wound healing without adverse effects. This study represents a breakthrough in overcoming traditional CDT limitations by integrating self-supplied H2O2 with NO’s biofilm-disrupting capabilities, offering a promising therapeutic strategy for biofilm-associated infection.

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and hexachlorophene in Candida albicans: a drug repurposing approach.

Candida albicans has been listed in the critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing offers an efficient, rapid, and cost-effective solution to develop alternative therapeutics against pathogenic microbes. Alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against the wild type (reference strain) and clinical isolates of C. albicans. The minimum inhibitory concentrations (MIC50) of AXD and HCP against C. albicans ranged between 0.34 and 0.69 µM and 19.66-24.58 µM, respectively. The biofilm inhibitory and eradication concentration of AXD was reported comparatively lower than that of HCP for the strains used in the study. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Revitalizing copper's efficacy: Using carvacrol to overcome copper tolerance in Pectobacterium carotovorum subsp. carotovorum for enhanced prevention of soft rot disease during crop storage

The phytopathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) is a major causal agent of soft rot disease in various vegetables, leading to substantial pre- and post-harvest losses in agriculture. Moreover, the emergence of copper-tolerant Pcc strain is increasingly threatening the efficacy of copper sulfate treatments to control soft rot. Here, we investigated the impact of carvacrol, copper sulfate and their combination application on a copper-tolerant strain of Pcc (CTP). The results showed that the minimum inhibitory concentrations (MIC) for copper sulfate and carvacrol against CTP was 500 and 200 µL/L, respectively. Notably, the combination of these chemical agents significantly enhanced their antibacterial efficacy, reducing the MIC to 140 µL/L. In follow-up control tests using detached potatoes, Chinese cabbage leaves and onions, the carvacrol-copper combination reduced the severity of soft rot and this treatment strongly affected the biofilm produced by CTP, with minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) values of 250 and 500 µL/L, respectively. The scanning electronic microscopy (SEM) and confocal laser scanning microscopy (CLSM) results confirmed the structural deformations and disruptions of CTP's biofilm under the carvacrol-copper sulfate treatment combination. Furthermore, the transcriptomic analyses and RT-qPCR validation revealed the significant down-regulation of genes associated with CTP's motility, biofilm formation, and copper tolerance after its exposure to carvacrol. Altogether, our findings demonstrate that carvacrol impairs biofilm formation in CTP, thus reducing its copper tolerance and restoring its sensitivity to copper sulfate. This comprehensive study is the first to report on the potential of carvacrol to enhance copper sensitivity in CTP and diminish its copper tolerance, offering a novel approach to managing copper-tolerant phytopathogens in agriculture.

Antifungal activity of Cinnamaldehyde derivatives against fluconazole-resistant Candida albicans

BackgroundCandida albicans is an opportunistic pathogen commonly found in human mucous membranes. In light of the escalating challenge posed by antibiotic resistance of C. albicans strains worldwide, it is an urgently necessary to explore alternative therapeutic options. ObjectiveThis study aims to assess the efficacy of two Cinnamaldehyde derivatives, 2-Cl Cinnamaldehyde (2-Cl CA) and 4-Cl Cinnamaldehyde (4-Cl CA), against C. albicans through both in vitro experiments and in vivo murine models and to evaluate their potential as new drug candidates for treating C. albicans. Methods and resultsThe minimum inhibitory concentrations (MICs) of Cinnamaldehyde 2-Cl and 4-Cl benzene ring derivatives against C. albicans were 25 μg/mL. Time-killing experiments revealed that both Cinnamaldehyde derivatives exhibited fungicidal activity against C. albicans at concentrations of 5 MIC and 10 MIC. In the checkerboard experiment, 4-Cl CA did not show any antagonistic effect when combined with first-line antifungal drugs. Instead, it exhibited additive effects in combination with nystatin. Both 2-Cl and 4-Cl CA demonstrated inhibitory activity against C. albicans biofilm formation, especially at 8 MIC and 16 MIC concentrations. In C. albicans biofilm eradication experiments, although high drug concentrations of 2-Cl and 4-Cl CA were unable to eradicate the biofilm completely, they were still effective in killing C. albicans cells within the biofilm. Moreover, sub-inhibitory concentrations of 4-Cl CA (ranging from 5 to 20 μg/mL) significantly inhibited cell aggregation and hyphal formation. Furthermore, 4-Cl CA effectively inhibited intracellular C. albicans infection in macrophages. Lastly, the effectiveness of 4-Cl CA was evaluated in a mouse model of hematogenous disseminated candidiasis caused by C. albicans, which revealed that 4-Cl CA significantly reduced fungal burden and improved mouse survival compared to the untreated controls. ConclusionThe 4-Cl CA exhibited inhibitory effects against C. albicans through both in vivo and in vitro models, demonstrating its therapeutic potential as a promising new drug candidate for treating drug-resistant candidiasis albicans.

Anti-biofilm and anti-quorum sensing activities of extract, fractions and compounds from the leaves of Cassia alata L. against yeast pathogens

BackgroundCassia alata or Senna alata, also known as “ringworm bush” because of its very effective fungicidal properties, is commonly used in African traditional medicine to treat fungal infections. Despite extensive phytochemical and pharmacological studies previously reported on C. alata, the antibiofilm activity against pathogenic yeast as well as the related anti-quorum sensing mechanism of some active constituents has not yet been elucidated. The aim of the study was to isolate the bioactive constituents from the methanol extract of the leaves of C. alata (CAExt) using antibiofilm-guided fractionation against yeast fungal pathogens and then to investigate the anti-quorum sensing activity of the active constituents by assessing their ability to inhibit violacein production in Chromobacterium violaceum. MethodsChromatographic methods were used to isolate the constituents of CAExt, and spectroscopic methods were used to elucidate the chemical structures of the isolated compounds. The broth microdilution assay was used to evaluate the antifungal activity against Candida albicans and C. parapsilosis, while crystal violet staining was used for the inhibition of biofilm formation and the disruption of preformed biofilm. The biosensor strain C. violaceum ATCC 12472 was used to investigate the anti-quorum sensing activity of the most active constituents. ResultsThe crude extract exhibited biofilm inhibition and eradication activities against the tested pathogenic yeast. The biofilm inhibition percentages ranged from 53.22 % to 75.38 %, while the biofilm eradication percentages ranged from 23.21 % to 64.25 %. The ethyl acetate fraction demonstrated high biofilm inhibition and eradication activities against the tested microorganisms. The biofilm inhibition percentages ranged from 58.19 % to 79.30 %, while the biofilm eradication percentages ranged from 34.105 % to 69.54 %. The purification of subfractions led to the identification of six compounds: stigmasterol (1), sitosterol (2), lupeol (3), emodin (4), kaempferol (5) and stigmasterol glycoside (6), two of which (4 and 5) showed potent biofilm inhibition and eradication activities. Both compounds demonstrated significantly lower MBIC50 values of 70.81 µg/mL and 65.65 µg/mL against Candida albicans and MBEC50 values of 63.65 µg/mL and 82.66, respectively, against C. albicans and C. parapsilosis. The crude extract and compounds (4) and (5) also demonstrated quorum sensing inhibitory activity, as indicated by the MQSIC value of 1024 µg/mL for the crude extract and 128 µg/mL for the two compounds. Moreover, compounds (4) and (5) displayed significant inhibitory effects on violacein production, as indicated by their low IC50 values of 28.08 µg/mL and 26.44 µg/mL, respectively. ConclusionsData obtained in this study not only support the traditional use of C. alata in the treatment of fungal infections but also reveal C. alata extract, as well as the two isolated bioactive compounds emodin (4) and kaempferol (5), as a potential source for developing antibiofilm alternative agents against biofilm-associated yeast infections. List of compounds studiedstigmasterol (1), sitosterol (2), lupeol (3), emodin (4), kaempferol (5), stigmasterol glycoside (6)

Molecular-level regulating intersystem crossing of polyphenols: Engineering high-efficiency phytochemical photosensitizer for MRSA elimination

Natural polyphenols-mediated green photosterilization is promising but remains challenging in practical applications due to their unsatisfactory performance with insufficient intrinsic energy level and ROS generation. In this work, the molecular-level engineering strategy of natural polyphenols (quercetin, QC) by p-Methoxybezaldehyde (MB)-induced nucleophilic substitution is constructed for enhanced photodynamic MRSA therapy. Both the experimental and theoretical results disclose that the unprecedented H-aggregates of QC-MB supramolecular realized accelerated ISC with minimizing the energy level difference (ΔEst), achieving enhanced photodynamic performance with 2.2-fold enhancement on 1O2 quantum yield (75 %) compared to free quercetin (33 %). The well-designed photosensitizer realizes efficient MRSA elimination: with enhanced killing efficacy from 30.44 % to 99.95 % in vitro and effective eradication of mature biofilms. Mechanism study and gene transcription analysis reveal that the combined therapy of optical stimuli with QC-MB achieves the dual mechanism of ROS attack and virulence regulation against MRSA infection during the whole infection process. Moreover, the excellent biocompatibility and anti-infectious in vivo demonstrate the engineered QC-MB as a promising strategy for MRSA therapy.

Antibacterial and Antibiofilm Effects of L-Carnitine-Fumarate on Oral Streptococcal Strains Streptococcus mutans and Streptococcus sobrinus.

Streptococcus mutans is a major pathogenic habitant of oral caries. Owing to its physiological and biochemical features, it prevails in the form of plaque biofilm together with another important mutans streptococci species, Streptococcus sobrinus. Both species are considered as initiators of cavity lesions, and biofilm is essential to the dental caries process. Compared with the planktonic populations, the biofilm form has higher resistance to environmental conditions and antibiotics. Dental plaques also secure the long-term survival of microorganisms and protection from any stress conditions. To address the need for new antibiofilm agents, we have focused on L-carnitine-fumarate, a fumarate-conjugated quaternary ammonium compound. Using the macro-broth susceptibility testing method, we established its MIC value as 6.0 mg/mL. The MBC value, determined from the broth dilution minimum inhibitory concentration test by sub-culturing it to BHI agar plates, was established as 7.0 mg/mL. Antibiofilm efficacy was tested in 96-well plates coated with saliva using BHI broth supplemented with 1% sucrose as a standard approach. The obtained results allowed us to assess the MIBC as 7.5 mg/mL and the MBBC value as 10.0 mg/mL. The latter concentration also caused approximately 20% eradication of pre-existing biofilm. EPS-rich matrix, forming the core of the biofilm and enabling a confined acidic microenvironment, was also examined and confirmed the effectiveness of 10.0 mg/mL L-carnitine-fumarate concentration in inhibiting EPS formation. Furthermore, the anti-adherent and anti-aciduric impacts of L-carnitine-fumarate were investigated and revealed significant inhibitory effects at sub-MIC concentrations. The influence of L-carnitine-fumarate on the phosphotransferase system was investigated as well. Our results provide a new insight into the antibacterial potential of L-carnitine-fumarate as a valuable compound to be considered for alternative or adjunct anti-caries and antibiofilm preventive approaches.

Synergistic combination of baicalein and rifampicin against Staphylococcus aureus biofilms.

Staphylococcus aureus, a Gram-positive bacterium, is a predominant pathogen associated with various infections. The rapid emergence of antibiotic resistance has intensified the challenge of managing fracture-related infections in severe osteoporotic patients. Rifampicin, a potent antimicrobial agent employed against fracture and implant-related infections, necessitates combination therapies due to its susceptibility to antibiotic resistance. In this study, we explored the potential of baicalein, a bioactive flavonoid from Oroxylum indicum and Scutellaria baicalensis, in combination with rifampicin against S. aureus biofilms invitro. The minimum inhibitory concentration of baicalein and rifampicin were determined as 500 μg/mL and 12.5 ng/mL respectively. The synergistic activity of baicalein and rifampicin was determined by the fractional inhibitory concentration index (FICI) using checkerboard assay. The results showed the FICI of baicalein and rifampicin was lesser than 0.5, demonstrating synergistic effect. Furthermore, the efficacy of baicalein and rifampicin, both individually and in combination, was evaluated for biofilm inhibition and eradication. Scanning electron microscopy and confocal laser microscopy also confirmed that the synergistic combinations effectively removed most of the biofilms and partially killed pre-formed biofilms. In conclusion, the findings demonstrate that baicalein is as effective as rifampicin in inhibiting and eradicating S. aureus biofilms. Their combination exhibits synergistic effect, enhancing their bactericidal effect in completely eradicating S. aureus biofilms. The findings of this research underscore the research potential of combining baicalein and rifampicin as a novel therapeutic strategy against S. aureus biofilms, offering a promising direction for future research in the treatment of fracture-related S. aureus infections.

Anti-virulence potential of iclaprim, a novel folic acid synthesis inhibitor, against Staphylococcus aureus

Infections caused by Staphylococcus aureus pose a significant global public problem. Therefore, new antibiotics and therapeutic strategies are needed to combat this pathogen. This investigation delves into the effects of iclaprim, a newly discovered inhibitor of folic acid synthesis, on S. aureus virulence. The phenotypic and genotypic effects of iclaprim were thoroughly examined in relation to virulence factors, biofilm formation, and dispersal, as well as partial virulence-encoding genes associated with exoproteins, adherence, and regulation in S. aureus MW2, N315, and ATCC 25923. Then, the in vivo effectiveness of iclaprim on S. aureus pathogenicity was explored by a Galleria mellonella larvae infection model. The use of iclaprim at sub-inhibitory concentrations (sub-MICs) resulted in a reduction of α-hemolysin (Hla) production and a differential effect on the activity of coagulase in S. aureus strains. The results of biofilm formation and eradication assay showed that iclaprim was highly effective in depolymerizing the mature biofilm of S. aureus strains at concentrations of 1 MIC or greater, however, inhibited the biofilm-forming ability of only strains N315 and ATCC 25923 at sub-MICs. Interestingly, treatment of strains with sub-MICs of iclaprim resulted in significant stimulation or suppression of most virulence-encoding genes expression. Iclaprim did not affect the production of δ-hemolysin or staphylococcal protein A (SpA), nor did it impact the total activity of proteases, nucleases, and lipases. In vivo testing showed that sub-MICs of iclaprim significantly improves infected larvae survival. The present study offered valuable insights towards a better understating of the influence of iclaprim on different strains of S. aureus. The findings suggest that iclaprim may have potential as an anti-virulence and antibiofilm agent, thus potentially mitigating the pathogenicity of S. aureus and improving clinical outcomes associated with infections caused by this pathogen.Key points• Iclaprim effectively inhibits α-hemolysin production and biofilm formation in a strain-dependent manner and was an excellent depolymerizing agent of mature biofilm• Iclaprim affected the mRNA expression of virulence-encoding genes associated with exoproteins, adherence, and regulation• In vivo study in G. mellonella larvae challenged with S. aureus exhibited that iclaprim improves larvae survival

Membrane-targeted mechanism for amphiphilic vitamin C compounds as methicillin-resistant Staphylococcus aureus biofilm eradicating agents

Staphylococcus aureus infections and its biofilm removal is an important concern in health care management. Methicillin-resistant S. aureus is responsible for severe morbidity and mortality worldwide. The extensive use of disinfectants against biofilms has led to negative environmental impacts. Developing new and more potent biofilm eradication agents with minimal detrimental effects on human and environmental health is currently on the agenda. The alkyl esters of L-ascorbic acid (ASCn) are antioxidant amphiphiles, which show antimicrobial capacity against methicillin-sensitive and resistant S. aureus strains. ASC12 and ASC14 formulations are able to kill the persister cells of the deepest layers of the biofilm. We tested the hypothesis that the antimicrobial and antibiofilm capacity found for the ASCn emerges from a combined effect of its amphiphilic and their redox capacity. This mechanism appears related to: I) a larger diffusion capacity of the ASC12 micelles than ASC14 and ASC16 microstructures; II) the neutralization of the ASCn acid hydroxyl when the amphiphile reaches the surface of an anionic surface, followed by a rapid insertion; III) the disruption of cell membrane by alteration of membrane tension and structure and IV) ASCn accumulation in the cell membrane or biofilm extracellular matrix surfaces, reducing functional chemical groups and affecting its biological function.

The Antibacterial Potential of Brazilian Red Propolis against the Formation and Eradication of Biofilm of Helicobacter pylori.

Helicobacter pylori is associated with gastrointestinal diseases, and its treatment is challenging due to antibiotic-resistant strains, necessitating alternative therapies. Brazilian red propolis (BRP), known for its diverse bioactive compounds with pharmaceutical properties, was investigated for its anti-H. pylori activity, focusing on biofilm formation inhibition and eradication. BRP was tested against H. pylori (ATCC 43526) using several assays: time-kill, nucleotide leakage, biofilm formation inhibition (determining the minimum inhibitory concentration of biofilm of 50%-MICB50, and cell viability), and biofilm eradication (determining the minimum eradication concentration of biofilm of 99.9%-MBEC). Standardization of H. pylori biofilm formation was also conducted. In the time-kill assay, BRP at 50 µg/mL eliminated all H. pylori cells after 24 h. The nucleotide leakage assay showed no significant differences between control groups and BRP-treated groups at 25 µg/mL and 50 µg/mL. H. pylori formed biofilms in vitro at 109 CFU/mL after 72 h. The MICB50 of BRP was 15.6 µg/mL, and at 500, 1000, and 2000 µg/mL, BRP eradicated all bacterial cells. The MBEC was 2000 µg/mL. These findings suggest that BRP has promising anti-H. pylori activity, effectively inhibiting and eradicating biofilms. Further studies are necessary to elucidate BRP's mechanisms of action against H. pylori.

A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia.

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans into the patient's lower airways. Currently, there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates the biofilm formation of these pathogens in a host-mimicking environment and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilated airway mucus medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-scanning electron microscopy were employed to characterize the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved the biofilm eradication of all pathogens. Our in vitro endotracheal tube model informs on fundamental microbiology in the ventilator-associated pneumonia context and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants.