Search Results (2,719)

Background/Objectives: Antimicrobial resistance is a major public health concern requiring integrated surveillance across human, animal, and environmental sectors. Enterococcus spp. are widely distributed opportunistic bacteria with the capacity to acquire and disseminate resistance and virulence determinants. This study aimed to characterize species distribution, phenotypic antimicrobial resistance, resistance genes, and virulence-associated traits in Enterococcus spp. from One Health sources. Methods: Enterococci were recovered from 66 samples collected in Lisbon, Portugal, between late 2022 and early 2024, including healthy and sick humans, healthy and sick animals, canteen food, surface water, and public transport surfaces. RAPD-PCR was used to assess genetic diversity among 90 isolates and select 74 representative enterococci. Species identification, resistance gene screening, and virulence gene detection were performed by PCR. Antimicrobial susceptibility was assessed by disk diffusion against 12 antibiotics using CLSI criteria. Vancomycin resistance was further evaluated by agar dilution and Etest when applicable. Results: Enterococcus faecalis predominated, representing 63/74 isolates (85.1%), followed by Enterococcus faecium (5/74, 6.8%) and other Enterococcus spp. (6/74, 8.1%). Antibiotic resistance was detected across all One Health sectors. Sick Human isolates showed higher resistance than Healthy Human isolates. E. faecium showed higher resistance than E. faecalis, and all E. faecium isolates across sectors were multidrug-resistant. Rifampicin resistance was frequent, as was quinupristin-dalfopristin resistance (excluding intrinsically resistant E. faecalis), while linezolid resistance was not detected. Resistance and virulence determinants were distributed across sectors. Conclusions: Enterococci from human, animal, and environmental sources carried antimicrobial resistance and virulence-associated traits. These findings support integrated One Health surveillance to monitor resistant enterococci across interconnected reservoirs. Full article

Enterococci are environmentally persistent bacteria that are relevant to both water quality and the spread of antimicrobial resistance. This study aimed to phenotypically and genotypically characterize Enterococcus spp. isolated from freshwater (lakes and rivers) in north–central Poland, with a focus on antimicrobial susceptibility, virulence genes, and biofilm-forming capacity. Surface water samples were collected during the vegetation seasons of 2022 and 2023. Enterococci were isolated by culture-based methods and confirmed by MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was performed using the disk diffusion method, virulence-associated genes were detected by multiplex PCR, and biofilm formation was evaluated using a crystal violet assay. In total, 96 Enterococcus spp. isolates representing 12 species were identified from 328 freshwater samples, with E. faecalis (24.0%) and E. hirae (21.9%) being the most frequently isolated. Thirty-one isolates (32.3%) were resistant to at least one antibiotic, and two isolates were classified as multidrug-resistant. The most prevalent virulence genes were gelE, srtA, and hyl. The ace gene was detected exclusively in E. faecalis. Most isolates were non-biofilm producers, while biofilm formation was confirmed in 8 strains (6 weak and 2 moderate producers), with no strong biofilm-forming strains detected. These findings suggest that freshwater ecosystems may constitute important reservoirs of antimicrobial-resistant and virulence-associated Enterococcus spp., underscoring the importance of continuous surveillance within the One Health framework. Full article

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) from companion dogs is an emerging One Health concern because dogs may serve as asymptomatic reservoirs of multidrug-resistant (MDR) and potentially pathogenic bacteria that can be transmitted to humans. This study aimed to determine antimicrobial susceptibility profiles, characterize antimicrobial resistance and virulence associated genes, and identify nucleotide substitutions in resistance determinants of E. coli isolated from healthy dogs in Thailand. Fecal samples (n = 200; 100 samples per province) were collected from healthy dogs attending a total of 50 small animal clinics located in Nakhon Si Thammarat and Chiang Mai provinces between January and March 2026. E. coli isolates were identified by culture and polymerase chain reaction (PCR) confirmation. Antimicrobial susceptibility was evaluated against 12 antimicrobial agents using the disk diffusion method according to CLSI guidelines. Thirty randomly selected isolates were further screened for resistance and virulence-associated genes by PCR, and the tetA gene was sequenced to identify nucleotide substitutions. Differences in gene distributions between provinces were assessed using Fisher’s exact test. A total of 66 E. coli isolates were recovered. High resistance rates were observed for ampicillin (100%), piperacillin (84.85%), ceftriaxone (60.61%), tetracycline (56.06%), and aztreonam (46.97%). Multidrug resistance was identified in 47% of the isolates. The 30 isolates exhibiting the highest levels of antimicrobial resistance were selected for molecular characterization. Among these, the β-lactam resistance gene bla_TEM was significantly more prevalent in Nakhon Si Thammarat than in Chiang Mai (60% vs. 0%; p = 0.022). The virulence gene stx2 was also detected significantly more frequently in isolates from Nakhon Si Thammarat (93.33% vs. 26.67%; p < 0.001). Sequence analysis of tetA revealed multiple nucleotide substitutions in two isolates, suggesting ongoing genetic variation within tetracycline resistance determinants that may contribute to the evolution and persistence of antimicrobial resistance. These findings demonstrate that healthy dogs in Thailand can act as reservoirs of MDR and potentially virulent E. coli. The observed geographic variation in resistance and virulence gene distributions highlights the importance of antimicrobial stewardship and continuous molecular surveillance in companion animals within a One Health framework. Full article

Serratia marcescens is a nosocomial pathogen that has acquired resistance to multiple antibiotics, necessitating alternative antimicrobial strategies. The aim of this study was to isolate and characterize a novel phage (ØAS2) against Serratia marcescens and evaluate its biocontrol potential in dairy matrices. In this study, a lytic bacteriophage specific to S. marcescens, designated ØAS2, was isolated from sewage samples collected in Assiut, Egypt. Phage ØAS2 was characterized using plaque assays, transmission electron microscopy (TEM), host range determination, pH and thermal stability tests, and one-step growth curve analysis. Its ability to inhibit bacterial growth and disrupt biofilms was also evaluated in vitro. TEM revealed that ØAS2 possesses an icosahedral head approximately 47.2 nm in diameter and a very short tail, consistent with the morphology of a member of the class Caudoviricetes that exhibits podovirus-like morphology. The phage exhibited a broad host range, infecting various Serratia strains as well as other Gram-negative bacteria, including Klebsiella spp., Escherichia coli, Salmonella typhi, and Shigella spp. ØAS2 was thermostable up to 60 °C and showed maximum activity at pH 8. One-step growth curve analysis revealed a short latent period of 10 min and a burst size of 115 PFU per infected cell. ØAS2 effectively inhibited the growth of S. marcescens SM02 in vitro and significantly reduced preformed biofilms at different multiplicities of infection (MOIs). When applied to skim milk and fresh soft cheese at various MOIs (Multiplicities of Infection), the phage successfully controlled bacterial contamination under refrigerated storage (7 °C for 7–10 days). At MOI 5.0, phage ØAS2 reduced biofilm biomass by 25.6%, planktonic growth by 85.7%, and achieved a reduction of 2.1 log₁₀ CFU/mL in skim milk. These findings indicate that ØAS2 is a promising biocontrol candidate for managing S. marcescens spoilage in dairy products. Full article

Teixobactin is a promising antibiotic that targets cell wall biosynthesis in Gram-positive bacteria and displays a low propensity for resistance; however, its structural complexity presents challenges for analogue development and optimisation. In this study, we investigated the effects of macrocycle size and replacement of the native depsipeptide linkage with an amide bond on antibacterial activity using a simplified Leu₁₀-teixobactin scaffold. An amide-based macrocyclisation strategy was developed for efficient lactam formation using readily accessible amino acid building blocks, avoiding reliance on synthetically demanding modified diamino acids employed in other approaches. Two complementary synthetic routes provided access to a series of ten analogues, comprising linear and macrocyclised variants with systematic variation at position 8. Antibacterial activity was evaluated against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant clinical isolates. While linear analogues exhibited weak or no measurable antibacterial activity, macrocyclised analogues retained measurable antibacterial activity, indicating that macrocyclisation is essential within this scaffold, whereas moderate expansion of the macrocycle was tolerated. The structure–activity relationships identified here demonstrate the suitability of a simplified Leu₁₀-teixobactin framework and provide a platform for further optimisation of teixobactin-inspired antibiotics. Full article

Agastache foeniculum (Pursh) Kuntze, a member of the Lamiaceae family, is a phytochemically rich yet underexplored species with potential biomedical applications. This study aimed to provide an organ-specific chemical characterization of its secondary metabolites and to evaluate the antimicrobial potential of ethanolic extracts derived separately from its leaves and flowers. Using UHPLC-MS/MS, we identified a total of 54 compounds, including phenolic acids and flavonoids. In total, 35 compounds in the flower extract and 38 in the leaf extract were reported here for the first time. The antimicrobial activity of both extracts was tested against six multidrug-resistant (MDR) clinical bacterial isolates (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Citrobacter freundii, Achromobacter xylosoxidans, and Acinetobacter baumannii) using seven concentration levels (12.5–87.5%). While the leaf extract showed limited antibacterial effects, the flower extract demonstrated stronger, concentration-dependent inhibitory effects. At concentrations of 62.5% and above, it markedly reduced viable bacterial counts in all tested MDR strains. These findings highlight the importance of organ-specific phytochemical analysis in medicinal plants and suggest that A. foeniculum, particularly its flowers, may serve as a promising source of bioactive compounds for further antimicrobial research and development. Full article

Pseudomonas aeruginosa is an opportunistic pathogen causing healthcare-associated infections. Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative bacteria. Resistance arises through mutations in two-component systems (TCS) regulating the arn operon. Data on colistin resistance in P. aeruginosa from Pakistan remain limited. A total of 3189 clinical samples (urine, blood, sputum, pus, wound swabs) were cultured. P. aeruginosa was identified by Gram staining, biochemical tests (catalase, oxidase, API 20E), and oprL gene amplification. Antibiotic susceptibility was determined by disk diffusion and MIC strips. Resistance genes (PhoP, PhoQ, PmrA, PmrB, mcr-1, oprD) were detected by PCR and Sanger sequencing. Wild-type protein structures were retrieved from PDB; mutant structures were predicted using AlphaFold3. ANP (phosphoaminophosphonic acid-adenylate ester) was docked using MOE 2019.0102. Of 3189 samples, 384 (12.0%) yielded P. aeruginosa. Wound/pus (38.0%) and surgical wards (30.0%) were the predominant sources. Colistin and polymyxin B showed 99.0% susceptibility (MIC₅₀/MIC₉₀ = 1 µg/mL). High resistance was observed for Piperacillin–Tazobactam (96.4%), Aztreonam (70.6%), and Gentamicin (64.2%). oprD was the most prevalent gene (87.5%), followed by PmrB (54.0%), PhoQ (44.0%), PhoP (36.0%), PmrA (18.0%), and mcr-1 (8.0%). Docking revealed the strongest binding in wild-type PhoQ (1ID0; −12.0 kcal/mol, LYS392), wild-type PmrB (2JSO; −9.8 kcal/mol, ASP37), and wild-type PhoP (2PKX; −9.1 kcal/mol, LYS87/ARG111). Mutant proteins showed reduced binding affinities and dispersed interaction networks. Mutant PhoP formed 16 contacts (strongest −4.3 kcal/mol) versus wild-type PhoP with 13 contacts (−9.1 kcal/mol). Colistin remains highly effective against P. aeruginosa in this setting (99.0% susceptibility). The presence of mcr-1 (8.0%) and high oprD prevalence (87.5%) require continued surveillance. Mutations in TCS proteins reduce ANP binding affinity and alter interaction specificity, suggesting that ATP-competitive inhibitors targeting these kinases merit further investigation and experimental validation. Full article

Multidrug-resistant (MDR) pathogens present a significant threat to hematopoietic stem cell transplant (HSCT) recipients; despite their critical implications, regional data on their infection patterns remain scarce. This study aimed to characterize the incidence, pathogen and antimicrobial resistance distribution of clinically confirmed bacterial infections among HSCT recipients. A retrospective analysis was conducted at King Hussein Cancer Center, Jordan (2018–2022). MDR pathogens were defined per CDC criteria. During the study period, 1157 HSCT procedures were performed. A total of 327 patients developed clinically documented bacterial infections, yielding an overall cumulative incidence of 28.3%, with a higher burden in the pediatric cohort (34.7%), including exclusive identification of Klebsiella oxytoca in pediatrics (2.3%). Gram-negative bacteria dominated, with Escherichia coli (50.5%) and Klebsiella pneumoniae (22.0%) being most common. Extended-spectrum beta-lactamase (ESBL) production was the dominant resistance mechanism (71.3%), followed by carbapenem-resistant Enterobacteriaceae (CRE; 14.1%), methicillin-resistant Staphylococcus aureus (MRSA; 8.6%), and carbapenem-resistant Pseudomonas aeruginosa (CRPA; 7.0%). The urogenital (39.1%) and bloodstream (31.2%) were the most infected sites. Significant site-specific associations were noted for ESBL production, MDR-Acinetobacter baumannii (p < 0.001) and MRSA (p = 0.007). Temporal analysis revealed a convergent MDR peak in 2021. Our findings offer critical insights into MDR pathogen incidence in HSCT recipients in the Middle East, informing improved infection management and intensified antimicrobial stewardship in this high-risk population. Full article

Antimicrobial resistance is increasing, necessitating the development of novel and efficacious therapies. Plants contain phytochemicals, some of which may possess antibacterial properties. This research employed broth dilution experiments to investigate the antibacterial efficacy of fifteen phytochemicals identified in medicinal plant extracts. The sum of fractional inhibitory concentration of phytochemicals in conjunction with reference antibiotics were also analysed. The inhibitory effects of phytochemicals against β-lactamase were evaluated to explore their potential mechanisms of action. The phytochemicals were evaluated for toxicity on human dermal fibroblast cells. Gallic acid and luteolin significantly inhibited Staphylococcus aureus and the methicillin-resistant S. aureus (MRSA) strain, with minimum inhibitory concentration (MICs) of 62.5 µg/mL. Gallic acid also demonstrated restricted efficacy against Gram-negative species, with MICs ranging from 312.5 to 1250 µg/mL. Gram-negative bacteria exhibited no response to luteolin. Ellagic acid, catechin, naringenin, and quercetin exhibited moderate antibacterial efficacy against the tested pathogens (625–2500 µg/mL MIC). Corilagin exhibited significant antibacterial activity against S. aureus and MRSA, with a MIC of 7.81 µg/mL. Corilagin also exhibited notable efficacy against Bacillus cereus, Shigella flexneri, and Klebsiella pneumoniae, with MICs ranging from 62.5 to 250 µg/mL. Fractional inhibitory concentration studies revealed a synergistic effect between amoxicillin and corilagin against B. cereus. Additionally, catechin, luteolin, and quercetin synergised penicillin G against S. aureus. Quercetin potentiated the activity of β-lactams (amoxicillin, penicillin G, and oxacillin) against MRSA. Notably, these antibiotics were ineffective against MRSA alone. Isobologram analysis revealed potentiation between penicillin G and quercetin against MRSA at all tested ratios. The β-lactamase inhibitory activity of the phytochemicals was evaluated using a commercial screening kit, and the percentage of relative inhibition was determined. Quercetin and luteolin both inhibited β-lactamase, achieving relative inhibition rates of 77–100% across two time intervals. All phytochemicals were nontoxic against human dermal fibroblasts. Indeed, quercetin enhanced cell survival by 200%. Full article

Antibiotic-resistant bacteria are widely distributed and threaten public health. Photocatalytic antimicrobial technology can effectively inactivate multidrug-resistant bacteria without readily inducing resistance. We previously showed that oxygen-rich vacancy Bi₂MoO₆ (OBM) exhibits excellent activity against methicillin-resistant Staphylococcus aureus (MRSA), but the underlying molecular mechanisms remain poorly understood. Here, we employed integrated transcriptomics and metabolomics, with qRT-PCR validation, to systematically elucidate the antibacterial mechanism of OBM against MRSA. OBM treatment induced profound transcriptional and metabolic alterations: 231 differentially expressed genes and 206 differentially abundant metabolites were identified. Functional enrichment analysis revealed cooperative involvement in multiple critical pathways, including inhibition of amino acid biosynthesis and protein translation, disruption of cell wall and membrane integrity, induction of oxidative stress, collapse of energy metabolism (suppression of oxidative phosphorylation and impaired ATP synthesis), and imbalance in nucleotide metabolism (down-regulation of DNA helicase and mismatch repair genes, dysregulation of purine/pyrimidine metabolism). These findings demonstrate that OBM photocatalytically inactivates MRSA through a multi-target systemic attack at both the transcriptional and metabolic levels, providing a novel theoretical foundation for the development of photocatalytic materials aimed at controlling MRSA and other drug-resistant bacteria. Full article

Background/Objectives: Gram-negative neonatal sepsis remains a cause of morbidity and mortality in preterm infants, yet the relationship between early clinical severity and long-term neurodevelopmental outcomes is incompletely defined. This study aimed to characterize Gram-negative sepsis in preterm infants and to evaluate its short-term and 18–24-month neurodevelopmental consequences. Methods: We conducted a retrospective observational cohort study of preterm infants admitted to a tertiary neonatal intensive care unit between 1 January 2022 and 31 December 2023. Infants with culture-proven Gram-negative neonatal sepsis, including both early-onset sepsis (EOS) and late-onset sepsis (LOS), were included. Clinical, microbiological, therapeutic, and laboratory data were collected, and survivors were assessed at 18–24 months’ corrected age using the Bayley Scales of Infant and Toddler Development. Results: Among infants with culture-proven Gram-negative sepsis, late-onset cases were more frequent than early-onset cases, and Klebsiella pneumoniae was the most common pathogen (38.0%). Multidrug-resistant organisms were associated with 52.0% of infections. In-hospital mortality was 26.0%. Major short-term complications included intraventricular hemorrhage (24.0%), severe intraventricular hemorrhage (20.0%), necrotizing enterocolitis (12.0%), bronchopulmonary dysplasia (20.0%), and meningitis (10.0%). Among survivors who underwent neurodevelopmental assessment, neurodevelopmental impairment was observed in 38.0%, most frequently affecting the language (22.5%) and cognitive (20.0%) domains. Infants with neurodevelopmental impairment had significantly lower gestational age and birth weight and higher inflammatory biomarker levels. In multivariable analyses, lower gestational age emerged as the strongest independent predictor of both mortality (adjusted OR 0.19, 95% CI 0.04–0.99) and neurodevelopmental impairment (adjusted OR 0.12, 95% CI 0.02–0.71). Conclusions: Gram-negative neonatal sepsis in preterm infants was associated with substantial mortality, severe neonatal complications, and a high burden of later neurodevelopmental impairment. Lower gestational age was independently associated with adverse short- and long-term outcomes. These findings support early recognition, targeted antimicrobial therapy, and structured neurodevelopmental follow-up in this high-risk population. Full article

Background: The global emergence of antibiotic resistance highlights the urgent need for novel therapeutic strategies, including adjuvants and potentiating compounds, against multidrug-resistant bacteria. Pseudomonas aeruginosa is classified by the World Health Organization (WHO) as a critical priority pathogen due to its high resistance potential and its ability to cause severe nosocomial infections. Beauvericin (BEA), a frequently detected mycotoxin, has been reported to exhibit various bioactive properties, including potential antibacterial and potentiating effects. Methods: The interaction between BEA and a last-resort antibiotic, colistin (COL), was evaluated in seven multidrug-resistant P. aeruginosa isolates using a microplate-based growth assay after preliminary MIC tests. Results: BEA at non-inhibitory concentrations (2.5–10 µg/mL) significantly enhanced the antibacterial activity of COL (1 and 2 µg/mL) in six out of seven isolates, resulting in a marked reduction in residual bacterial growth. Conclusions: BEA exhibited no measurable antibacterial activity at the concentrations used in the combination experiments but acted as a strain-dependent potentiator of colistin activity against multidrug-resistant P. aeruginosa. The observed enhancement of colistin-mediated growth inhibition supports the potential of BEA as an antibiotic adjuvant at clinically relevant colistin concentrations and provides a basis for further mechanistic investigation. Full article

Salmonella remains one of the most critical zoonotic pathogens in the poultry sector, linked to animal disease, foodborne illness, and the global crisis of antimicrobial resistance (AMR). Poultry acts as a major reservoir, enabling Salmonella transmission from hatchery to retail products through horizontal, vertical, and environmental routes. Despite the use of biosecurity, vaccination, antibiotics, and chemical decontamination, effective and sustainable control across the poultry value chain remains difficult, particularly in the face of rising multidrug-resistant strains and growing consumer concerns over chemical residues. Bacteriophages (phages), viruses that selectively infect and lyse bacteria, have emerged as a promising biological alternative for Salmonella control. Although many studies have reported the effectiveness of phages against bacterial species, including Salmonella, in the poultry industry, reports on their full potential to combat antimicrobial-resistant Salmonella across the entire poultry value chain remain limited. Therefore, this review synthesizes current evidence on the application of phages throughout the poultry value chain, including on-farm interventions, processing plant decontamination, and food packaging and storage. Findings from the reviewed articles indicate over a 90% reduction in Salmonella spp. in poultry farms and post-harvest meat, along with lower mortality in phage-treated groups compared to untreated groups; however, these outcomes depend on several factors (e.g., phage strains, concentrations, application methods, and environmental conditions). Laboratory, pilot, and field studies consistently demonstrate that phage preparations, especially when formulated as cocktails or combined with complementary interventions, can achieve substantial reductions in Salmonella, including antibiotic-resistant serovars, in live birds, eggs, poultry environments, and meat products. Unlike antibiotics and chemical sanitizers, phages act with high specificity, preserving beneficial microbiota and maintaining the sensory and nutritional quality of poultry products. Their safety has been supported by toxicological and genomic assessments, and several phage-based products have obtained regulatory approval, including Generally Recognized as Safe (GRAS) status for food applications in the United States. By integrating efficacy, safety, regulatory, and practical deployment data, this review highlights bacteriophages as a scientifically validated and One Health–aligned tool capable of reducing Salmonella transmission from farm to fork across the poultry value chain, thereby laying the foundation for their future adoption in the poultry industry. Phage-based interventions offer a sustainable pathway to enhance food safety, limit antimicrobial resistance (AMR) dissemination, and strengthen consumer confidence in poultry products. However, the major limitation is the emergence of phage-resistant bacterial strains, as well as the potential involvement of some phages in the transfer of resistance and virulence genes, which could raise public concern. Nevertheless, the use of phage cocktails and whole-genome sequencing, involving tools such as ResFinder and virulence finder, can facilitate the selection of safe phages for application. Full article

Essential oils (EOs) from wild Calamintha nepeta (Portugal) populations collected in Portugal (Évora) were investigated in order to evaluate the impact of Mediterranean seasonal conditions on their phytochemical composition and biological activity. Essential oil GC-FID and GC-MS analyses revealed distinct seasonal chemotypes, with spring samples dominated by isopulegone/pulegone, whereas autumn samples contained higher proportions of isomenthone and menthol. Antioxidant activity was assessed through lipid peroxidation inhibition, DPPH radical scavenging and ferric reducing power assays, while antibacterial activity was evaluated against multidrug-resistant (MDR) clinical isolates. Seasonal differences were reflected in both EO chemical composition and bioactivity. Autumn samples displayed greater antioxidant potential, with Y1A showing the highest inhibition of lipid peroxidation (IC₅₀ = 0.85 mg/mL) and Y2A exhibiting the highest ferric reducing power. Conversely, spring samples were more active against MDR bacteria. Among them, Y1S showed the broadest antimicrobial spectrum, with MIC values ranging from 465 to 1767 μg/mL. The unusually wet spring season coincided with marked isopulegone accumulation (≈50%), while warmer autumn conditions favoured higher levels of isomenthone and menthol in the EOs. These findings highlight the importance of seasonal environmental conditions in determining the phytochemical profile and bioactive potential of C. nepeta EOs, providing valuable insights for their standardisation and valorisation in pharmaceutical, food and conservation-related applications. Full article

Background/Objectives: Antimicrobial resistance and bacterial persistence underscore the need to develop new chemotypes with multifunctional antibacterial mechanisms. This study aimed to design, synthesize, and evaluate curcumin-inspired 3,5-diarylidene-4-piperidones as versatile small molecules exhibiting antibacterial, antibiofilm, anti-efflux, DNA gyrase-inhibitory, and antiproliferative properties. Methods: A targeted series of triazole-conjugated 3,5-diarylidene-4-piperidones was synthesized through copper-catalyzed azide-alkyne cycloaddition click chemistry and subsequently characterized using standard spectroscopic techniques. The compounds were assessed for antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli. Selected active compounds underwent further evaluation for DNA gyrase inhibition, antibiofilm activity against multidrug-resistant S. aureus ATCC 33591, ethidium bromide accumulation, and antiproliferative effects on HCT116 and MCF7 cancer cells, with RPE1 cells serving as a control to evaluate cytotoxicity in normal cells. Additionally, computational studies, including QSAR analysis and molecular docking, were conducted to bolster structure–activity relationships and provide mechanistic insights. Results: Several derivatives demonstrated selective antibacterial activity against Gram-positive bacteria, particularly S. aureus, while exhibiting limited or no efficacy against E. coli. Compounds 7n and 7l emerged as the most potent against S. aureus, with minimum inhibitory concentrations (MICs) of 7.8 and 8.2 μM, respectively. Notably, compound 7l inhibited S. aureus DNA gyrase supercoiling, displaying an IC₅₀ of 3.20 μM, comparable to ciprofloxacin. Compound 7e exhibited the strongest antibiofilm activity against multidrug-resistant S. aureus, whereas compound 7a resulted in the highest accumulation of ethidium bromide, indicating robust anti-efflux activity. Antiproliferative assays revealed that select halogenated derivatives were effective against HCT116 and MCF7 cells, while the most promising antibacterial compounds exhibited minimal cytotoxicity toward RPE1 cells. Quantitative structure–activity relationship (QSAR) and docking studies supported the observed structure–activity relationships and suggested potential interactions with the ATPase binding site of DNA gyrase B. Conclusions: Triazole-conjugated 3,5-diarylidene-4-piperidones are promising multifunctional scaffolds with selective anti-S. aureus activity, antibiofilm and anti-efflux properties, and, for compound 7l, potent DNA gyrase inhibition. These findings support further optimization of this chemotype as a platform for developing antibacterial agents with polymechanistic activity. Full article