Search Results (170)

Bacterial persistence has been extensively studied as a possible explanation for strain survival under stress; however, in Enterococcus spp., this ability is still an understudied phenomenon. In this study, 40 Enterococcus spp. isolates of human clinical (n = 10), veterinary commensal (n = 10), veterinary clinical (n = 10) and veterinary environmental (hospital surfaces) (n = 10) origins, were exposed to a high concentration of ciprofloxacin. Time–kill curves were established, after which antimicrobial susceptibility profiles were reassessed. Subsequently, the only presumptive persister was selected for Whole-Genome Sequencing, together with one isolate showing no evidence of persister formation. Comparative genomic analyses were conducted to identify genetic variations between exposed and non-exposed isolates and to explore potential genetic determinants associated with persistence. Observed genetic features present in the persister isolate included toxin–antitoxin systems, a cold-shock protein and the tyrosine-type recombinase/integrase XerC, which may represent putative candidates for further investigation. Interestingly, the majority of toxin–antitoxin system-associated genes were found in plasmids. This study represents an important step towards a better understanding of persistence development in Enterococcus spp.; however, validation using other methodologies such as RNA-sequencing is an important next step. Full article

Background: The root of Schisandra propinqua (Wall.) Baill. var. sinensis Oliv is a traditional ethnomedicine in China; it was widely used to treat abscesses, sores, carbuncles, rheumatism, and so on. The purpose of this study was to elucidate the bacteriostatic mechanism of the ethyl acetate extract from the root of Schisandra propinqua (Wall.) Baill. var. Sinensis Oliv (Xiao Xue Teng) against Staphylococcus aureus ATCC 25923 (S. aureus ATCC 25923). Methods: Bioactive bacteriostatic constituents in Xiao Xue Teng were identified through Hybrid Quadrupole-TOF LC/MS/MS. The minimum inhibitory concentration (MIC) of Xiao Xue Teng against S. aureus ATCC 25923 was determined using the microbroth dilution method. A time–kill curve analysis was used to evaluate the bacteriostatic effects. SDS-PAGE coupled with nano-liquid NanoLC-ESI-MS/MS, real-time PCR, and scanning electron microscopy (SEM) was used to study the bacteriostatic mechanism of Xiao Xue Teng against S. aureus ATCC 25923. Results: The MIC of Xiao Xue Teng against S. aureus ATCC 25923 was determined to be 15.625 µg/mL. The translation initiation factor (IF-2) and elongation factor (EF-Tu) were significantly decreased in S. aureus ATCC 25923 after treatment with Xiao Xue Teng, while the proteins SodA and AhpC were obviously increased. The intracellular levels of total reactive oxygen species (ROS) and hydrogen peroxide (H₂O₂) were significantly increased (p < 0.01) after the treatment with Xiao Xue Teng. Concurrently, the activities of SOD, CAT and GSH-Px were significantly increased (p < 0.01). Moreover, cellular swelling and shrinkage were observed using SEM. Conclusions: The bacteriostatic mechanism of Xiao Xue Teng against S. aureus ATCC 25923 was related to eliciting oxidative stress, inhibiting protein synthesis and enhancing cytoplasmic membrane permeability. Full article

Uropathogenic Escherichia coli (UPEC) that form biofilms exhibit high-level antibiotic resistance, which poses substantial challenges to current therapeutic strategies for urinary tract infection (UTI). There is an urgent need for strategies specifically targeting UPEC biofilms. This study investigated the effects of the n-butanol extract of Polygonum capitatum (BPC) on UPEC strains, focusing on its antibacterial activity, biofilm formation, bacterial motility, adhesion capacity, and cell membrane integrity. The disk diffusion method, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays demonstrated that BPC exhibited potent antibacterial activity against both reference and clinically isolated UPEC strains. Time–kill curve assays further confirmed that BPC inhibits bacterial growth in a time-dependent manner. BPC inhibited UPEC biofilm formation in a dose-dependent manner, significantly reducing biofilm formation in both reference and clinical UPEC strains. Furthermore, BPC disrupted cell membrane integrity in UPEC strain CFT073, resulting in the leakage of alkaline phosphatase (AKP), β-galactosidase, and intracellular proteins. BPC treatment also significantly reduced bacterial surface hydrophobicity, impaired swimming and swarming motility, and diminished adhesion and invasion capabilities. A total of 32 active compounds, predominantly flavonoids, were identified in BPC by UHPLC-Q-orbitrap MS/MS. Molecular docking studies revealed that several compounds in BPC, such as quercetin-3,4′-O-di-beta-glucoside, exhibited strong binding affinity to AKP and β-galactosidase, further supporting its potential to disrupt membrane integrity and inhibit biofilm formation. Thus, BPC exerts anti-UPEC effects through biofilm disruption and multi-targeted anti-virulence mechanisms, highlighting its potential as a novel therapeutic or adjunctive agent for UTI, particularly against recalcitrant biofilm-associated infections. The mode of action of BPC provides a scientific basis for developing new anti-infective strategies as alternatives to conventional antibiotics. Full article

Background: Healthcare-associated infections (HAIs) caused by biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis represent a major public health challenge due to their high resistance and involvement in skin, wound, and soft-tissue infections. In this context, silver nanoparticles (AgNPs) incorporated into Gluconacetobacter sp. bacterial cellulose hydrogel emerge as a promising alternative therapeutic strategy. Methods: AgNPs and hydrogels were synthesized and characterized using physicochemical and morphological analyses. Antibacterial activity was assessed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) following CLSI guidelines, as well as by time–kill curve assays. Antibiofilm activity was evaluated through the determination of minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) using crystal violet staining, complemented by scanning electron microscopy (SEM) and Congo red agar method. Results: The hydrogel exhibited a three-dimensional microfibrillar structure characteristic of bacterial cellulose, while AgNPs showed rod-shaped, oval, and triangular morphologies, with particle sizes of 35 and 59 nm and positive zeta potentials. MIC and MBC values ranged from 6.25 to 50 µg/mL across all tested formulations and strains. Time–kill assays demonstrated significant bacterial population reductions after 6 to 9 h of exposure. MBIC values ranged from 0.78 to 50 µg/mL, whereas MBEC values ranged from 1.56 to >100 µg/mL. SEM analyses confirmed biofilm disruption, cell eradication, and a reduction in extracellular polysaccharides, particularly for AgNPs incorporated into the hydrogel. Conclusions: Overall, the results highlight the strong antibacterial and enhanced antibiofilm potential of AgNP-loaded bacterial cellulose hydrogel against S. aureus and S. epidermidis, supporting its potential application in infection treatment. Full article

Infections caused by carbapenem-resistant Pseudomonas aeruginosa (CRPA), especially chronic infections associated with biofilm formation, have become a major clinical challenge. Phage therapy has received much attention as an alternative strategy, but temperate phages have limited direct application due to their lysogenicity. The aim of this study was to explore the synergistic therapeutic effect of a novel temperate phage combined with antibiotics. A temperate Pseudomonas phage YF1204 was isolated from the patient’s bronchoalveolar lavage fluid and systematically characterized by whole-genome sequencing, transmission electron microscopy, and host range analysis. The synergistic antibacterial and anti-biofilm effects of phage with amikacin (AK) were evaluated by using the checkerboard test, a time-killing curve based on optical density (OD600) and crystal violet staining, and the cytocompatibility was analyzed by using the CCK-8 method. The results showed that phage YF1204 belonged to the Siphoviridae family and had typical temperate phage genome characteristics (containing integrase gene). It also showed lytic activity against 41.4% (87/210) of the clinical isolates, especially against carbapenem-resistant strains. When YF1204 was combined with AK, it reduced the minimum inhibitory concentration (MIC) of AK by 2- to 8-fold across all tested strains, respectively. Moreover, the inhibitory effect against CRPA was significantly enhanced (achieving suppression indexes about 80% ) and biofilm formation was inhibited with an inhibition ratio of 48.75%. Cell experiments showed that YF1204 had no significant toxicity to THP-1 cells. The combination of YF1204 and AK exhibited significant synergistic bactericidal and anti-biofilm activities, providing a novel therapeutic strategy with translational potential for CRPA-induced refractory infections. Full article

Continuing our work in the field of synthesis and research of amino acids, their derivatives, and cyclization products, in this work, we synthesized various 3-(6-bromo-2-oxo-1,3-benzoxazol-3(2H)-yl)propanoic acid derivatives and investigated their antimicrobial activity. A total of eighteen synthesized chemical compounds (No. 1–18), including several structural analogues (e.g., 3a, 3b, 4a–4e, 8a–8m, 9a–9d), were evaluated for their antibacterial properties. The antibacterial activity was assessed using the Kirby–Bauer disk diffusion method, and inhibition zone diameters (mm) were measured against five representative bacterial strains: S. aureus, MRSA, B. subtilis, E. coli, and P. aeruginosa. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the most active synthesized compounds were determined against representative Gram-positive and Gram-negative bacterial strains, including S. aureus, MRSA, B. subtilis, and E. coli. Overall, these results indicate that the tested compounds display selective antibacterial activity, mainly against Gram-positive bacteria, with compound 12 emerging as the most promising derivative in the series. The antibacterial activities of several synthesized compounds were systematically evaluated against S. aureus and MRSA over a 24 h incubation period, with optical density measured at ten time points. Bacterial growth was monitored spectrophotometrically at 600 nm (OD₆₀₀) at 1, 2, 3, 4, 5, 6, 7, 8, 20, and 24 h, enabling a detailed assessment of growth kinetics and the temporal dynamics of inhibition. The effect of compound 11 on the growth kinetics of S. aureus was evaluated by quantifying viable bacterial counts (log₁₀ CFU/mL) over a 6 h incubation period, and the results are presented in the time–kill curve. Compound 11 was selected for this experiment because it exhibited the most pronounced antibacterial activity against S. aureus in the disk diffusion assay. The cytotoxicity of compounds 9a, 11, 12, and 13 was evaluated at concentrations ranging from 125 to 1.95 µg/mL. The results showed a clear, concentration-dependent decrease in cytotoxicity for all tested compounds. The molecular structure of compound 3a was confirmed by a single-crystal X-ray diffraction. Full article

Essential oils (EOs), complex volatile compounds synthesized by plants, represent a vital class of natural products that are increasingly significant in scientific research due to their diverse biological properties and broad-spectrum medicinal applications. This study provides a comprehensive overview of EOs, commencing with a historical perspective and detailing their applications. It systematically catalogs their primary botanical sources, with specific examples of the most common and important plant families, including Lamiaceae (e.g., sage, oregano, thyme), Verbenaceae (vervain), Magnoliaceae (magnolia), Rutaceae (lemon), Myrtaceae (eucalyptus) and Lauraceae (cinnamon). A key focus is their antifungal activity, including the bioactive constituents involved and their mechanisms of action, with particular emphasis on their defense against pathogenic postharvest fungi. This includes an analysis of the key bioactive constituents responsible for these bioeffects and an exploration of their possible mechanisms of action against phytopathogenic fungi, with particular emphasis on postharvest pathogens infecting several crops. The discussion further highlights the role of EOs as sustainable alternatives to synthetic fungicides for controlling plant diseases that avoid the negative ecological and public health impacts associated with conventional agrochemicals. The study addresses these objectives by describing methods for testing antimicrobial efficacy, including kill-time studies, LD₅₀ determination, growth-curve analysis, the poisoned food technique, Spore-germination assays, and metabolic CO₂ measurement. The current review also highlights some recent studies reviewing the in vitro and in vivo antifungal performance of specific EOs against postharvest diseases. Full article

Background/Objectives: Multidrug-resistant (MDR) strains of Streptococcus suis are increasingly prevalent and present significant challenges in clinical management. Given that the development of new antibiotics is a resource-intensive process and time-consuming, there is an urgent need for alternative therapeutic strategies to address resistance in the short term. One promising approach is the use of combination therapy, which involves pairing potent antibiotics with agents that may be less effective on their own, to enhance therapeutic efficacy and potentially overcome resistance mechanisms. This study aimed to investigate the in vitro antibacterial activity of combining two classes of antibiotics with distinct mechanisms of action—cell wall synthesis inhibitors and protein synthesis inhibitors—against MDR S. suis strains isolated from diseased pigs. Methods: A total of 36 MDR S. suis strains were tested using a microbroth dilution checkerboard assay to determine the minimum inhibitory concentration (MIC) of four cell wall synthesis inhibitors —amoxicillin/clavulanic acid (AMC), ampicillin (AMP), penicillin G (PEN), and vancomycin (VAN)— in combination with four protein synthesis inhibitors —gentamicin (GEN), neomycin (NEO), tilmicosin (TMS), and tylosin (TYL). Time–kill curve assays were conducted to evaluate the in vitro bactericidal activity of synergistic antibiotic combinations (PEN–GEN and AMP–NEO) against Beta-lactam-resistant and Beta-lactam-susceptible MDR S. suis strains. Results: Checkerboard analysis revealed that penicillin-gentamicin combination exhibited the most effective synergistic activity against the MDR S. suis strains (10/19, 52.6%), with ∑FIC values of 0.25–1.06 and MIC reductions from resistant to susceptible levels. Time-kill assays further confirmed the synergistic bactericidal effect of the combination, demonstrating complete bacterial clearance within 6–9 h, markedly rapid bacterial killing compared to monotherapy. Conclusions: This study demonstrates that antibiotic combinations, particularly Beta-lactams combined with aminoglycosides, show synergistic activity against pig-isolated S. suis MDR strains. The PEN-GEN combination exhibited strong synergistic and bactericidal effects, supporting combination therapy as a potential strategy to address antimicrobial resistance. Further evaluation in diverse strain backgrounds and prudent antibiotic use are essential to confirm efficacy and limit the emergence of antibiotic resistance. Full article

Phage therapy is the use of bacterial viruses, or bacteriophages, as antibacterial agents. It has been in use for over 100 years and is becoming increasingly common clinically. The first steps of phage therapy include identification of bacteria to be targeted and then obtaining phages with appropriate host ranges. This is followed by various approaches to in vitro phage characterization. Increasingly common for phage phenotypic characterization is the use of kinetic microtiter plate readers. They can both decrease workloads and increase throughput, especially relative to analyses that require plating on agar-based media. These colorimetric/turbidimetric/optical density approaches primarily assess phage-induced culture-wide bacterial lysis, in the shorter term, or instead the phage potential to suppress phage-resistance evolution over longer time frames. Considered here are methods relevant to phage characterization especially for phage-therapy purposes. Discussed are turbidity-reduction assays, determinations of phage antibacterial virulence, and related time-kill curve analysis. All are or can be optical density-based approaches to assessing phage-based bacterial reduction. Emphasis is placed on consideration of the utilities, limitations, and intersections of these similar methods. Emphasized is that the start of “Deviation”—where phage-treated culture turbidity diverges from phage-free controls—may represent a superior endpoint for such optical density-based bacterial-reduction protocols. Full article

Background/Objectives: Stenotrophomonas maltophilia (SM) is a significant cause of hospital-acquired infections in immunocompromised and critical care patients. This study investigates the impact of combining ceftazidime/avibactam (CZA) with conventional antibiotics on SM obtained from various sources in planktonic and biofilm cell cultures. Methods: Using broth microdilution, the MICs of different antibiotics, including CZA, were determined on 37 SM strains. CZA’s bactericidal and synergistic effectiveness were examined through in vitro time–kill curve tests with tigecycline (TGC), chloramphenicol (CHL), levofloxacin (LVX), colistin (CS), and amikacin (AMK). In addition, synergistic activity was investigated against SM biofilm cell cultures, and antibiotic Mutant Prevention Concentrations (MPCs) were tested against SM isolates. Results: Compared to ceftazidime (CAZ), CZA was four times more efficient against 37 SM strains. Unlike TGC and CHL, CS, AMK, and CZA had 2–4 times higher MBCs than MICs. All studied antibiotics were bactericidal at 1× or 4× MIC doses against SM bacteria, except for CZA. The combinations of CZA with LVX and CZA with AMK or CS demonstrated synergistic effects in four out of seven (57%) strains and in three out of seven (43%) strains, respectively, when tested at doses equivalent to the MIC. Moreover, all antibiotic combinations with CZA showed a synergistic effect at dosages four times the MIC. Additionally, CZA and the tested drugs synergistically inhibited SM biofilm formation, and MPC values were 8–16 times the MIC. Conclusions: The results of this study indicate that combining CZA with LVX and CS was more effective against SM strains. These combinations might provide alternatives for treating SM pathogens in both planktonic and biofilm cell cultures. Full article

Background: To meet the urgent need for novel antibacterial agents that are active also against worrying superbugs, natural pentacyclic triterpenoids, including totally inactive betulin (BET) and betulinic acid (BA), as well as ursolic acid (UA), active on Gram-positive bacteria, have been chemically modified, achieving compounds 1–7. Methods: Triterpenoid derivatives 1–7 and all synthetic intermediates were characterized by chemometric-assisted FTIR and NMR spectroscopy, as well as by other analytical techniques, which confirmed their structure and high purity. Minimum inhibitory concentration values (MICs) of 1–7, BET, BA and UA were determined by the broth dilution method, using a selection of Gram-positive and Gram-negative clinically isolated superbugs. Results: Performed experiments evidenced that compounds 4–7 had potent antibacterial effects against Gram-positive methicillin-resistant Staphylococcus aureus and S. epidermidis (MRSA and MRSE), as well as against vancomycin-resistant Enterococcus faecalis and E. faecium (VRE). The antibacterial effects of 4–7 were due to the insertion of a triphenyl phosphonium (TPP) group and were higher than those reported so far for other BET, BA and UA derivatives, especially considering the complex pattern of resistance of the isolates used here and their clinical source. Conclusions: For the first time, by inserting TPP, a real activity (MICs 2–16 µg/mL) was conferred to inactive BET and BA (MICs > 1024 and 256 µg/mL). Moreover, the antibacterial effects of UA were improved 16- and 32-fold against MRSE and MRSA (MICs = 2 vs. 32 and 64 μg/mL). Future Perspectives: Based on these very promising microbiologic results, new experiments are currently underway with the best-performing compounds 5 and 7 (MICs = 2 μg/mL) on an enlarged number of Gram-positive isolates, to confirm their MICs. Moreover, investigations about their possible antibiofilm activity, time-killing curves and cytotoxicity on eukaryotic cells will be carried out to define their pharmacological behavior and clinical potential. Full article

The aim of this research was to examine the antibacterial activity of commercially available silver nanoparticles against foodborne bacteria and to evaluate the properties of pullulan films incorporating these nanoparticles, including their antibacterial activity and selected physical properties. First, the antibacterial activity of silver nanoparticles against foodborne bacteria was investigated. The following parameters were assessed to evaluate the antibacterial activity of silver nanoparticles: minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), percentage antibacterial activity, bacterial survival based on time–kill curves, leakage of DNA and intracellular proteins using spectrophotometric measurements, and changes in bacterial cell morphology using scanning and transmission electron microscopy (SEM and TEM). Pullulan films with silver nanoparticle content ranging from 2 to 32 µg/cm² were obtained. The films were evaluated for antibacterial activity and physical properties, including macroscopic and microstructural (SEM) observations, thickness, light barrier, and color. Silver nanoparticles at a concentration of 25 µg/mL achieved 100% inhibition of the test bacteria, with destruction of bacterial cells after 3 or 6 h of incubation, depending on the silver nanoparticle concentration. Incorporation of silver nanoparticles into pullulan films, even in lower amounts, resulted in an antibacterial effect. All films had a compact and uniform microstructure and were shiny and flexible. Analysis of variance showed a significant (p < 0.05) effect of the addition of silver nanoparticles on the thickness, transparency, and color of the films. The obtained pullulan films containing silver nanoparticles were characterized by strong inhibitory activity against foodborne bacteria, had a brown color of varying intensity, a uniform microstructure, a smooth surface, and were barriers to UV radiation and visible light. Full article

Background: Given the increasing problem of antibiotic resistance in A. baumannii, this study examines in vitro how combinations of colistin, meropenem, and sulbactam influence the expression of genes associated with multiresistance in this pathogen. Methods: Three multidrug-resistant strains, isolated from clinical infections in Panama (2022–2023), were identified using Vitek 2 compact. Susceptibility by broth microdilution, qualitative synergy, time-kill curves, and gene expression analysis by quantitative PCR were performed. Results: Synergistic effects were observed for the colistin–meropenem combination in all three strains, while the sulbactam–colistin combination exhibit synergy only in one of the A. baumannii isolates. Time-kill assays revealed bactericidal effects for the colistin–meropenem and sulbactam–colistin combinations. qPCR analyses indicated that colistin, meropenem, and sulbactam modified the expression of the genes under study. Colistin–meropenem and meropenem–sulbactam combinations decreased the expression of blaADC and blaOXA-51, while sulbactam–colistin did not have a significant effect. carO expression levels were not reduced with any antibiotic combination, while adeB expression was reduced with all the combinations tested. omp33–36 expression varied depending on the antibiotic and strain. Conclusions: Therefore, this study offers a new perspective on how rational combinations of clinically used antibiotics have the potential to modulate gene expression and contribute to the control of MDR strains, indicating that high-dose combination therapy with sulbactam and colistin could offer improved efficacy in treating multidrug resistant Acinetobacter baumannii infections. Full article

Pathogenic bacterial infections pose serious health risks, underscoring the need for timely treatments. Manganese dioxide (MnO₂) nanoparticles (NPs) have attracted considerable attention owing to their outstanding chemical stability, favorable biocompatibility, high reactivity, and catalytic ability to decompose hydrogen peroxide, making them promising antibacterial agents. A clear understanding of their antibacterial mechanisms is essential for evaluating their therapeutic potential in clinical settings. In this study, MnO₂ NPs were synthesized by reacting potassium permanganate (KMnO₄) with poly(allylamine hydrochloride) (PAH), ensuring complete conversion to MnO₂ NPs. The resulting NPs were characterized for their physicochemical properties, and their antibacterial activity against E. coli and S. aureus was evaluated using growth curve assays and reactive oxygen species (ROS) quantification. Results indicated the killing efficiency of MnO₂ NPs increased with exposure time and concentration, reflecting high susceptibility of both bacterial strains. Scanning electron microscopy (SEM) analysis revealed that the interaction between MnO₂ NPs and bacterial cells caused significant disruption of cell wall integrity. This study provides a valuable platform for evaluating MnO₂ nanoparticles as antibacterial agents and for exploring their mechanisms in medical applications. Full article

Objective: This study aims to evaluate free vancomycin concentrations in tissues of septic patients that received empirical doses. Methods: A PBPK model was built in PK-Sim to simulate vancomycin concentrations in healthy volunteers and septic patients. Literature data were used to validate the model. A strain of MRSA (methicillin-resistant Staphylococcus aureus) was evaluated through time-kill curves. Based on the information obtained from the time-kill study, a PD model, including adaptive resistance, was developed using NONMEM. The PBPK and PD models were combined to evaluate the vancomycin effect in plasma and tissues against MRSA. Results: A PBPK model was successfully built for both healthy volunteers and septic patients. The tissue concentrations were found to be significantly lower than plasma concentrations. The studied strain of MRSA was found to have an MIC of 2 µg/mL, and the PD model described the EC50 as 1.05 µg/mL. The PBPK and PD models were successfully combined, and septic patients infected with MRSA strains with MIC of 2 µg/mL had effective treatment response. However, septic patients infected with MRSA strains with MICs of 4 µg/mL and 8 µg/mL did not have adequate response to vancomycin treatment. Conclusions: In septic patients, response was limited against resistant MRSA strains. These findings should be considered hypothesis-generating and interpreted with caution, underscoring the need for individualized approaches and rigorous monitoring. Full article