A practical three-dimensional checkerboard method for triple combinations: Ceftriaxone with cinnamaldehyde and carvacrol in Klebsiella pneumoniae ATCC 10031.

Antimicrobial photodynamic inactivation of Pseudomonas aeruginosa biofilms by a multifunctional triple combination of natural photosensitisers, gentamicin, and colistin.

The dimorphic fungus Talaromyces marneffei causes talaromycosis, a life-threatening fungal disease with limited treatment options. Olorofim, a first-in-class orotomide antifungal that targets pyrimidine synthesis essential for fungal growth, has low minimum inhibitory concentration (MIC) against T. marneffei and clinical efficacy against other invasive fungal diseases. Here, we tested the hypothesis that olorofim synergistically enhances amphotericin B (AmB), a potent membrane-targeting antifungal, against T. marneffei in 55 clinical isolates using a validated colorimetric checkerboard assay. The MIC was defined as the lowest drug concentration inhibiting ≥ 95% of fungal growth. Drug interactions were assessed using the fractional inhibitory concentration index (FICI), which defines ≤0.5 as synergy, 0.5 < FICI ≤ 4.0 as indifference, and FICI > 4 as antagonism. We found that interactions between AmB and olorofim were indifferent across all 55 isolates (0.5 < FICI ≤ 1.03). Time-kill assays showed an expected concentration-dependent fungicidal activity for AmB, but a concentration-independent fungistatic activity for olorofim against T. marneffei. Combinations of AmB and olorofim were also indifferent in time-kill experiments. Although synergy was not observed, and olorofim is unlikely to enhance AmB induction therapy, olorofim may have a role in the consolidation and maintenance therapy of talaromycosis.

Honokiol enhances the antibacterial efficacy of doxycycline against Vibrio parahaemolyticus by membrane disruption and confers protective efficacy in Pacific white shrimp (Penaeus vannamei).

The need to develop alternative treatment approaches to acne vulgaris and other skin infections is becoming increasingly urgent due to the rising prevalence of antibiotic resistance in Cutibacterium acnes (a key pathogen related to such conditions). Nanoparticles (NPs) have shown great potential as antimicrobial agents that could improve the effectiveness of traditional antibiotics when administered together. This research examines antibacterial activity of silver NPs (Ag-NPs) and zinc oxide NPs (ZnO-NPs), both independently and when used collaboratively with common anti-acne antibiotics used to treat C. acnes. Ag-NPs and ZnO-NPs were biosynthesized using aqueous Peganum harmala extract as a reducing and stabilizing agent. The synthesized nanoparticles were characterized using UV-Vis spectroscopy, X-ray diffraction (XRD), scanning/transmission electron microscopy (SEM/TEM), and dynamic light scattering (DLS). This research examined two C. acnes clinical isolates (P1 and P2) and one reference strain (NCTC747) were used in this research. Broth microdilution assays were employed to determine minimum inhibitory concentrations (MICs). Checkerboard assays and fractional inhibitory concentration index (FICI) analysis were performed to explore synergy between antibiotics and NPs. A successful formation of spherical Ag-NPs and hexagonal ZnO-NPs has been confirmed, with average crystallite sizes of approximately 13-14nm and 53-55nm, respectively. Both Ag-NPs and ZnO-NPs were found to exhibit strong antibiotic activity. The MICs of the tested antibiotics were significantly reduced when used in combination with AgNPs. The reduction was up to 2-8 fold compared to antibiotic monotherapy. Moreover, most Antibiotic-Ag-NPs combinations exhibited synergistic interactions in the FICI analysis (FICI ≤ 0.5). Meanwhile, there are increased moderate effects resulted from the ZnO-NP combinations. The Antibiotic MIC generally values reduced two-to-four-fold. The relevant FICI values suggested that the interactions were primarily additive (FICI ≈ 0.75), although there was some evidence of strain-dependent variations. A two-way ANOVA test showed that the MIC values reductions for both antibiotics and nanoparticles were statistically significant (p < 0.001). The potential of biosynthesized NPs as adjunct therapeutic agents for the management of acne-associated infections was suggested by their promising antibacterial and antibiotic-enhancing effects against C acnes.

This study investigated the chemical composition and antibacterial and antibiofilm activities of Cymbopogon flexuosus essential oil (CFEO) against multidrug-resistant (MDR) bacteria within a One Health framework. The bacterial panel comprised four clinical isolates (n = 1 per source): a methicillin-resistant Mammaliicoccus sciuri (MR-M. sciuri) from bovine mastitis, a colistin-resistant ESBL-producing Escherichia coli (E. coli) from avian colibacillosis, and MR-Staphylococcus haemolyticus (MR-S. haemolyticus) and MDR Staphylococcus aureus (MDR S. aureus) from healthy farm personnel in contact with diseased animals. GC-MS analysis was performed to study the chemical composition of the CFEO. Antibacterial activities were evaluated via disk diffusion and broth microdilution assays. Disk diffusion was used exclusively as a qualitative screening tool to identify potential interactions; synergy was assessed definitively through the checkerboard assay by calculating the Fractional Inhibitory Concentration Index (FICI). The antibiofilm activity of CFEO was quantified via Congo red agar, crystal violet staining, and light microscopy. Molecular docking simulation was performed to assess the binding interactions of the CFEO constituents with key bacterial proteins. The major constituents of CFEO were geranial (α-citral; 32.98%), neral (β-citral; 28.62%), β-terpinene (11.50%), geraniol (5.42%), nerol acetate (3.40%), and linalool (2.31%). The inhibition zones ranged from 38 ± 2.00 to 56 ± 1.00mm, while the MIC values spanned from 8 to 8192µg/mL. CFEO demonstrated a synergistic effect with oxacillin against MR-M. sciuri (FICI = 0.28). Sub-MIC concentrations of CFEO significantly disrupted the biofilms of M. sciuri and E. coli. Bioinformatics analysis via molecular docking revealed favorable binding affinities between major compounds of CFEO and key bacterial proteins, including PBP2a, SarA, and AgrA. This study highlights the in vitro efficacy of CFEO against MDR, MR and biofilm-forming bacteria circulating at the animal-human interface, as well as its synergistic potential when combined with oxacillin against MR-M. sciuri. Molecular docking analyses suggest that the major compounds of CFEO may act as promising adjuvants in the development of new therapeutic strategies against MDR bacteria within a One Health framework.

Because antimicrobial resistance is one of the most pressing global health challenges of our time, it is crucial to find and develop new antimicrobial compounds, especially molecules with new targets and mechanisms of action. Among those, trans-translation, the main quality control system responsible for rescuing bacterial ribosomes present in non-stop complexes, is an appealing target. The objective of this study was to use in vitro and in vivo screening assays to evaluate organic compounds from the French Essential Chemical Library (Chimiothèque Nationale Essentielle, CNE) as trans-translation inhibitors. In vitro cell-free assays and whole-cell in vivo trans-translation assays were performed to screen compounds from the chemical library. MIC and chequerboard assays were performed to assess the compounds' inhibition of bacterial cell growth in ESKAPE pathogens, alone or in combination with current antibiotics. Our initial results revealed a new family of γ-carboline compounds capable of inhibiting trans-translation in both in vitro and in vivo Escherichia coli-based assays, without affecting canonical translation. One molecule from the γ-carboline family, namely compound 404, presented high activity and specificity. These findings raise the prospect of further optimization of γ-carboline compounds' activity in order to use them in combination with currently available antibiotics to combat resistant pathogenic bacteria.

Enterococcus faecalis is a common cause of healthcare-associated infections. They characteristically exhibit reduced susceptibility to penicillins and elevated MICs for aminoglycosides, limiting these drugs as single-agent therapies. Combinations of cell wall synthesis inhibitors and aminoglycosides have a synergistic effect, resulting in bactericidal activity. The mechanism behind this synergism is not fully understood. The present study was performed to explore the relationship between synergistic activity between cell wall-active agents/aminoglycoside combinations and cell membrane energetics in E. faecalis. Analysis was performed using reference broth microdilution MIC testing, checkerboard assays, time-kill assays and fluorescent microscopy. We observed that cell wall remodelling agents promoted aminoglycoside uptake facilitated by antibiotic-induced decreases in the intracellular pH (pHi). Drugs that inhibit cell wall synthesis induce a decrease in the pHi, suggesting that cell wall remodelling is linked to ion transport and cytoplasmic acidification. Our study supports a model in which inhibition of peptidoglycan synthesis produces membrane stress-associated intracellular acidification that promotes ΔpH-dependent aminoglycoside uptake in E. faecalis.

The emerging threat of drug-resistant Candida albicans, especially in recurrent vulvovaginal candidiasis (VVC) biofilms, has revealed weaknesses in existing monotherapeutic antifungal treatments. In this study, we investigated the antifungal activity of a new dual-drug in situ mucoadhesive gel system containing acarbose (AC), a pseudo-oligosaccharide sugar and FDA-approved α-glucosidase inhibitor, together with fluconazole (FLC). Although AC showed mild antifungal activity by itself, when combined with FLC, it dramatically potentiated fungicidal activity, disrupted biofilms, blocked morphogenetic switching, and arrested post-treatment hyphal regrowth. In checkerboard assays and time-kill kinetics, AC-FLC combination was highly synergistic against Candida spp., including clinical isolates, with > 2-log₁₀ decrease in CFU/mL. The thermosensitive gel (PCH16), formulated using poloxamer 407 along with the carbohydrate-based polymers chitosan and HPMC, exhibited sustained drug release, favorable rheological properties, and strong mucoadhesion suitable for vaginal delivery. Notably, the formulation retained Lactobacillus species viability, suggesting microbiome compatibility. These results affirm the promise of repurposing acarbose as a novel, non-traditional antifungal adjuvant that, in co-administration with FLC in vaginal gel, represents an encouraging, localized, and microbiota-conserving therapy for treating recurrent and resistant VVC.

This study evaluates the in vitro antimicrobial activity of Corallopyronin A (CorA) against a diverse collection of Staphylococcus aureus and coagulase-negative staphylococci (CNS), comprising both laboratory strains and clinical isolates. The dataset includes methicillin-resistant and methicillin-susceptible strains, as well as small colony variants (SCVs), to assess its therapeutic potential in staphylococcal infections. A total of 116 staphylococcal strains, comprising clinical isolates and laboratory strains, were subjected to minimum inhibitory concentration (MIC) testing. Minimum bactericidal concentrations (MBCs) were determined for a subset of 70 strains. Time-kill assays were conducted for five S. aureus strains using 4 × MIC of CorA. Additionally, checkerboard assays were performed with 11 antibiotics to evaluate potential additive or synergistic interactions. CorA demonstrated potent antimicrobial activity with MIC values ranging from 0.125 to 2mg/L. The MIC90 was 0.5mg/L for S. aureus and 1mg/L for CNS. Methicillin-resistant strains exhibited significantly higher susceptibility than methicillin-sensitive strains. Time-kill assays revealed a reduction of 1.5-3 log10 CFU/mL in viable counts within 24h. Minimum bactericidal concentration testing showed bactericidal activity in a subset of strains, occurring in 71% of CNS isolates and 34% of S. aureus strains, while the remaining S. aureus and CNS isolates displayed a bacteriostatic response. Checkerboard assays indicated additive interactions with glycopeptides, including dalbavancin and oritavancin. CorA shows strong in vitro activity against a broad range of staphylococcal strains, particularly methicillin-resistant isolates. Its additive effects with clinically relevant antibiotics further support its potential in combination therapy for the treatment of resistant staphylococcal infections.

Additive larvicidal activity of albendazole combined with a hydroxyethylamine-derived compound against Toxocara canis larvae: An in vitro and in silico study.

Beyond the stomach: Repurposing bismuth tripotassium dicitrate as a novel strategy against multidrug-resistant Mycobacterium abscessus.

Candida biofilm-associated infections pose a major therapeutic challenge due to their high antifungal tolerance, species-specific pathogenicity, and rising resistance to conventional drugs. Essential oils (EOs) have emerged as promising natural antifungal agents, yet comparative studies evaluating whole oils and their major constituents across biofilms of different maturation stages remain limited. This study investigated the antibiofilm effects of tea tree, ceylon cinnamon bark, thyme, clove, and peppermint EOs, as well as their major components, against Candida albicans, C. glabrata, C. krusei, and C. tropicalis. Minimal inhibitory concentration was tested with microdilution assay. Biofilm inhibition was assessed at four maturation time points (4, 12, 24, and 48 h), and synergistic interactions were examined using checkerboard assays and Fractional Inhibitory Concentration Index (FICI) calculations. Tea tree oil was the most potent across all species and stages (e.g., 82.3% inhibition at 4 h vs. 40.0% at 48 h), followed by cinnamon bark and thyme oil. Whole EOs consistently outperformed their isolated components, indicating synergistic multi-component effects. Species-specific susceptibility patterns were apparent: C. krusei and C. glabrata were the most sensitive, whereas C. albicans exhibited the highest tolerance. Checkerboard assays identified strong synergism for tea tree-cinnamon oil against C. albicans and C. krusei (FICI 0.375) and for tea tree-thyme oil against C. tropicalis (FICI 0.49). These optimized EO combinations achieved exceptional inhibition of 24-hour biofilms (89-94%). Overall, EOs - particularly tea tree and its species-tailored combinations - represent promising candidates for disrupting Candida biofilms and may serve as effective components of future antifungal treatments.

Prosapogenin CP4 and ursolic acid from Eriocapitella rivularis inhibit fluconazole-resistant Candida albicans synergistically by regulating Ras/cAMP/PKA pathway.

Dental caries is closely associated with microbiome dysbiosis. Incorporating antimicrobial agents can enhance the efficacy of fluoride toothpaste. Our previous studies showed that caffeic acid phenethyl ester (CAPE), derived from propolis, effectively inhibited cariogenic bacteria. To formulate a novel CAPE-containing fluoridated toothpaste and establish a multistage evaluation system assessing its caries-controlling efficacy. The CAPE toothpaste's physicochemical properties were characterized. Its in vitro antimicrobial activity against Streptococcus mutans was examined using quantitative suspension and checkerboard microdilution assays. In vivo anticaries efficacy and biosafety were evaluated in a rat caries model (n = 9/group) comparing 5 groups: untreated control (group NC), base toothpaste without CAPE (group B, 600 ppm F-), CAPE-fluoride toothpaste (group C, 0.16 mg/mL CAPE + 600 ppm F-), fluoride control (group F, DARLIE®, 600 ppm F-), and propolis nonfluoride control (group P, Red Seal®). Caries severity was scored using the Keyes method. Finally, in situ enamel repair (n = 24 enamel blocks) and plaque microbiome modulation (n = 6 samples) were assessed in a 7-d clinical study (ChiCTR2400089643) with 4 groups (groups NC, C, F, and P). The novel formulation showed stable physicochemical properties. Group C reduced S. mutans by 1.5-log10 within 3 min (P = 0.0062 vs NC), and CAPE plus fluoride exhibited additive antibacterial properties (fractional inhibitory concentration index = 0.75). In rats, group C showed the lowest smooth-surface score (21.0 ± 1.7, P < 0.005 vs. all) and reduced sulcal lesion severity (slight dentinal lesions: P = 0.0181 vs. NC and P = 0.0318 vs. F). In situ, group C achieved 27.58% surface microhardness recovery (P < 0.01 vs. all), with significant reductions in mineral loss and lesion depth (P < 0.001 vs. all). Microbiome analysis revealed a preserved microbial diversity, increased Streptococcus oralis, and reduced cariogenic populations. The novel CAPE-containing fluoridated toothpaste effectively inhibited the onset and development of caries. Wider-ranging and longer-term clinical investigations are still needed.

Primin inhibits New Delhi metallo-β-lactamase-5 and restores meropenem activity in Escherichia coli.

Toluidine blue O-mediated antimicrobial photodynamic inactivation reinforced with naringenin against Streptococcus mutans biofilms.

The combination of monoclonal antibodies with aminoglycosides represents a promising strategy to counter the increasing prevalence of aminoglycoside resistance in avian pathogenic E. coli (APEC). Although the diguanylate cyclase DgcE (UniProt: P38097) is known to regulate virulence through c-di-GMP signaling, its potential as a therapeutic target has not been investigated. In this study, five female BALB/c mice were immunized to generate a monoclonal antibody. We developed an IgM monoclonal antibody (E11G12) targeting a defined region of DgcE and evaluated its ability to potentiate gentamicin activity. Structural prediction and docking analyses suggested specific binding interactions within the targeted DgcE region. In vitro checkerboard assays demonstrated synergistic activity between E11G12 and gentamicin (FICI ≤ 0.5), and time-kill assays showed enhanced bactericidal activity compared to monotherapy. Treatment was associated with reduced intracellular c-di-GMP levels and increased gentamicin accumulation. These findings suggest that the E11G12-aminoglycoside combination exhibits synergistic anti-APEC activity, potentially through blocking virulence via DgcE inhibition and enhancing antibiotic efficacy in association with c-di-GMP suppression. This approach offers a novel therapeutic strategy against drug-resistant APEC infections.

Antimicrobial resistance (AMR) has resulted in the need for the development of alternative strategies for combating pathogens and growth promotion of poultry, including the use of plant-derived compounds such as hop (Humulus lupulus) isolates. The present study evaluates the correlation patterns of the biological activity of five major hop isolates (humulone, lupulone, isohumulone, xanthohumol, and isoxanthohumol) against Bacillus subtilis, Micrococcus luteus, and a chicken cell line UMNSAH/DF-1 using a two-dimensional checkerboard assay. Fractional inhibitory concentrations (∑FIC) were used to classify interactions as additive, synergistic, or antagonistic, and selectivity indices assessed antibacterial versus cytotoxic effects. On B. subtilis, combinations were predominantly additive (∑FIC = 0.89–1.15), whereas M. luteus, in contrast, showed variable interactions, including also synergistic (humulone + lupulone, ∑FIC = 0.43 ± 0.18) and antagonistic combinations (isohumulone + isoxanthohumol, ∑FIC = 1.53 ± 0.38), demonstrating the impact of the metabolic resilience of the target organism. Cytotoxicity in UMNSAH/DF-1 cells was largely additive, with synergistic effects observed only for isomerized compounds (∑FIC = 0.50 ± 0.23). Selectivity analysis highlighted humulone-lupulone combinations with selective indices of 3.2 for M. luteus and 1.3 for B. subtilis as most favorable, indicating moderate to good antibacterial selectivity. These results provide novel insights for selecting hop isolate combinations for the development of phytogenic feed additives (PFAs), emphasizing that both compound composition and target organism physiology critically shape efficacy and safety outcomes.

Staphylococcus (S.) pseudintermedius, as a commensal of the skin and mucosa, leads to a variety of diseases in dogs, most commonly skin and ear infections. The development of methicillin-resistant S. pseudintermedius (MRSP) is an emerging risk for animals and humans. The aim of this study was to test cetirizine and N-acetylcysteine as synergistic substances with cephalexin for treating S. pseudintermedius infections. Each of the five methicillin-sensitive S. pseudintermedius (MSSP) isolates and five MRSP isolates, and one control strain were tested. The minimal inhibitory concentration (MIC) of the substances was tested by broth microdilution assay. In a checkerboard assay, the MIC of cefalexin alone was compared to the MIC of the substances combined. The determined dose reduction index (DRI) shows the influence each substance had on the efficacy of cefalexin. Furthermore, the minimal bactericide concentration (MBC) of N-acetylcysteine (NAC) was identified, and a time kill assay was performed to determine its time-related efficacy on selected isolates. Cetirizine showed no inhibition on bacterial growth or influence on antibiotic efficacy. NAC inhibited bacterial growth at 2 mg/mL. A significant synergistic influence was shown against the MRSP (p < 0.001) and MSSP isolates (p < 0.01). The MBC of the MSSP isolates and control strain was 12.8 and 25.6 mg/mL for the MRSP isolates. The time kill assay showed that NAC is bactericidal within 120 s at the prior determined MBC concentrations. NAC showed an antibacterial effect alone and a synergistic influence on cefalexin's antibacterial properties. Thus, NAC shows promising efficacy in treating infections with S. pseudintermedius; according to the preliminary study conducted here, this effect may be independent of the resistance profile.