Search Results (210)

Background/Objectives: Listeria monocytogenes is a foodborne pathogen of major public health concern due to its ability to invade host cells and cause severe illness. This study aimed to develop and validate a multi-tiered screening pipeline to identify Bacillus strains with probiotic potential against L. monocytogenes. Methods: A total of 26 Bacillus isolates were screened for antimicrobial activity, gastrointestinal resilience, and host cell adhesion. A fluorescence-based infection assay using mCherry-expressing HCT 116 cells was used to assess cytoprotection against L. monocytogenes NCTC 7973. Eight strains significantly improved host cell viability and were validated by quantification of intracellular CFU. Two top candidates were tested in a murine model of listeriosis. The genome of the lead strain was sequenced to evaluate safety and biosynthetic potential. Results: B. subtilis CECT 8266 completely inhibited intracellular replication of L. monocytogenes in HCT 116 cells, reducing bacterial recovery to undetectable levels. In vivo, it decreased splenic bacterial burden by approximately 6-fold. Genomic analysis revealed eight bacteriocin biosynthetic clusters and silent antibiotic resistance genes within predicted genomic islands, as determined by CARD and Alien Hunter analysis. The strain also demonstrated bile and acid tolerance, as well as strong adhesion to epithelial cells. Conclusions: The proposed pipeline enables efficient identification of probiotic Bacillus strains with intracellular protective activity. B. subtilis CECT 8266 is a promising candidate for translational applications in food safety or health due to its efficacy, resilience, and safety profile. Full article

Pasteurella multocida (Pm) is a zoonotic pathogen that poses a significant threat to animal health and causes substantial economic losses, further aggravated by rising tetracycline resistance. To restore the efficacy of tetracyclines to Pm, we evaluated the synergistic antibacterial activity of doxycycline combined with metformin, an FDA-approved antidiabetic agent. Among several non-antibiotic adjuvant candidates, metformin exhibited the most potent in vitro synergy with doxycycline, especially against capsular serogroup A strain (PmA). The combination demonstrated minimal cytotoxicity and hemolysis in both mammalian and avian cells and effectively inhibited resistance development under doxycycline pressure. At 50 mg/kg each, the combination of metformin and doxycycline significantly reduced mortality in mice and ducks acutely infected with PmA (from 100% to 60%), decreased pulmonary bacterial burdens, and alleviated tissue inflammation and damage. Mechanistic validation confirmed that metformin enhances membrane permeability in Pm without compromising membrane integrity, dissipates membrane potential, increases intracellular doxycycline accumulation, and downregulates the transcription of the tetracycline efflux gene tet(B). Morphological analyses further revealed pronounced membrane deformation and possible leakage of intracellular contents. These findings highlight metformin as a potent, low-toxicity tetracycline adjuvant with cross-species efficacy, offering a promising therapeutic approach for managing tetracycline-resistant Pm infections. Full article

Background: The global spread of antibiotic-resistant bacteria presents a major public health challenge and necessitates the development of innovative antimicrobial agents. Artificial intelligence (AI)-driven drug discovery has recently enabled the repurposing of existing compounds with novel therapeutic potential. Halicin, originally developed as an anti-diabetic molecule, has been identified through AI screening as a promising antibiotic candidate due to its broad-spectrum activity, including efficacy against multidrug-resistant pathogens. Methods: In this study, the antibacterial activity of halicin was evaluated against a range of clinically relevant multidrug-resistant bacterial strains. Bacterial isolates were first characterized using the agar disk diffusion method with a panel of 22 conventional antibiotics to confirm resistance profiles. The minimum inhibitory concentration (MIC) of halicin was then determined for selected isolates, including Escherichia coli ATCC^® 25922™ and Staphylococcus aureus ATCC^® 29213™, using broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Halicin demonstrated notable antibacterial activity, with MIC values of 16 μg/mL and 32 μg/mL against E. coli ATCC^® 25922™ and S. aureus ATCC^® 29213™, respectively. A dose-dependent inhibition of bacterial growth was observed for the majority of tested isolates, except for Pseudomonas aeruginosa, which exhibited intrinsic resistance. This lack of susceptibility is likely related to reduced outer membrane permeability, limiting the intracellular accumulation of halicin. Conclusions: Our findings support the potential of halicin as a novel antimicrobial agent for the treatment of infections caused by antibiotic-resistant bacteria. However, further investigations, including pharmacokinetic, pharmacodynamic, and toxicity studies, are essential to assess its clinical safety and therapeutic applicability. Full article

Helicobacter pylori is a gastric pathogen implicated in peptic ulcer disease and gastric cancer. The increasing prevalence of antibiotic-resistant strains underscores the urgent need for alternative therapeutic strategies. In this study, we investigated the chemical composition and antibacterial activity of an aqueous extract from Caulerpa lentillifera (sea grape), a farm-cultivated edible green seaweed collected from Krabi Province, Thailand. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) revealed that the extract was enriched in bioactive nucleosides and phenolic compounds. In vitro assays demonstrated dose-dependent inhibition of H. pylori growth following exposure to sea grape extract. Furthermore, untargeted intracellular metabolomic profiling of H. pylori cells treated with the extract uncovered significant perturbations in central carbon and nitrogen metabolism, including pathways associated with the tricarboxylic acid (TCA) cycle, one-carbon metabolism, and alanine, aspartate, and glutamate metabolism. Pyrimidine biosynthesis was selectively upregulated, indicating a potential stress-induced shift toward nucleotide salvage and DNA repair. Of particular note, succinate levels were markedly reduced despite accumulation of other TCA intermediates, suggesting disruption of electron transport-linked respiration. These findings suggest that bioactive metabolites from C. lentillifera impair essential metabolic processes in H. pylori, highlighting its potential as a natural source of antimicrobial agents targeting bacterial physiology. Full article

The escalating crisis of antimicrobial resistance in aquaculture, driven by the indiscriminate use of antibiotics, underscores the urgent need to develop novel anti-infective agents. This study addresses this requirement by investigating cysteine-rich antimicrobial peptides (AMPs) in understudied crustacean species. A cysteine-rich AMP, designated PpRcys1, was identified and characterized from the genome of Pollicipes pollicipes. PpRcys1 comprises 104 amino acids, with 85 residues forming the mature peptide region, and exhibits random coils, a CSαβ-fold, and one β-sheet. Our findings demonstrated that recombinant PpRcys1 (rPpRcys1) possesses broad-spectrum antimicrobial activity against three Gram-positive bacteria (Staphylococcus aureus, Bacillus sp. T2, and Streptococcus agalactiae) and four Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, Vibrio alginolyticus, and Acinetobacter sp. L3), with minimum inhibitory concentrations ranging from 8 to 32 μM. It exerts antimicrobial effects by inducing membrane disruption without impacting bacterial protease activity, DNA migration, or respiratory chain reductase activity. Further investigation is warranted to determine whether it can target and interfere with intracellular bacterial processes. Our discovery and characterization of this novel AMP provide a promising foundation for its development as an alternative to antibiotics. Full article

Listeriosis is a disease associated with the consumption of food contaminated with Listeria monocytogenes. Probiotic lactic acid bacteria (LAB) or their postbiotics have been of interest for their anti-listerial effect. This study focused on isolating LAB from artisanal cheeses and characterizing their potential as probiotics. Twelve LAB isolates exhibiting typical LAB traits were evaluated for their ability to survive in simulated gastric juice, hydrolyze bile salts, auto-aggregate, hydrophobicity, and antagonistic activity against L. monocytogenes. The four most promising LAB strains demonstrated anti-listerial probiotic potential and were identified as Latilactobacillus (Lat.) curvatus SC076 and Lactiplantibacillus (Lact.) paraplantarum SC291, SC093, and SC425. The antimicrobial activity of these strains was mainly attributed to bacteriocin-like substances and organic acids. While three Lact. paraplantarum strains were resistant to ampicillin, Lat. curvatus was sensitive to all tested antibiotics. All selected strains exhibited no hemolytic, gelatinase, and lecithinase activity. Exposure to LAB supernatants resulted in a significant reduction in the adhesion and intracellular count of L. monocytogenes in Caco-2 cells, with Lat. curvatus SC076 showing the most significant effect. Based on its probiotic characteristics, Lat. curvatus SC076 is a promising candidate for functional foods, pending further in vivo studies to assess its potential in the food industry. Full article

Background: Intracellular bacteria frequently result in chronic and recurrent infections. MRSA is one of the most prevalent facultative intracellular bacteria in clinical infections. The drug resistance of MRSA and the difficulty of most antibiotics in entering cells result in a suboptimal clinical efficacy of antibiotics in the treatment of intracellular MRSA. Bacteriophages represent a promising alternative therapy in the context of the current antimicrobial resistance crisis. Nevertheless, the low efficiency of phage entry into cells and their rapid inactivation remain challenges in the treatment of intracellular MRSA using phages. The utilization of functionalized carriers for the delivery of phages into cells and their protection represents a feasible strategy. Methods: In this study, a new MRSA bacteriophage (vB_SauS_PHM) was isolated from hospital sewage, exhibiting the characteristics of short incubation period, large lytic amount, and good environmental tolerance. Subsequently, vB_SauS_PHM was encapsulated by TAT peptide-functionalized liposomes through microfluidic technology and size-exclusion chromatography (SEC), forming a phage delivery system, designated TAT-Lip@PHM. Results: The encapsulation rate of the phage by TAT-Lip@PHM was 20.3%, and the cell entry efficiency was ≥90% after 8 h. The 24 h eradication rate of 300 μg/mL TAT-Lip@PHM against intracellular MRSA was 94.05% (superior to the 21.24% and 44.90% of vB_SauS_PHM and Lip@PHM, respectively), while the mammalian cell activity was >85% after 24 h incubation. Conclusions: The TAT-Lip@PHM effectively delivered the phage into the cell and showed an excellent killing effect on intracellular MRSA with low cytotoxicity. This work provides a technical reference for the application of phages in the treatment of intracellular bacterial infection. Full article

Background: The well-studied 18-residue-long proline-rich antimicrobial designer peptide Api137 utilizes at least two lethal intracellular mechanisms that target the bacterial 70S ribosome. First, Api137 stalls the ribosome by binding to the peptidyl-transferase center, trapping the release factor, and inhibiting protein expression. Second, Api137 disrupts the assembly of the large 50S subunit of the ribosome, resulting in partially assembled pre-50S dead-end particles that are unable to form the functional 70S ribosome. Methods: All six proline residues in Api137 were substituted with 4S- and 4R-fluoro-l-proline (Fpr), which promote the cis- and trans-conformer ratio of the preceding Xaa-Pro-bond, respectively. The effect on the antibacterial activity was studied using Escherichia coli. The underlying mechanisms were investigated by studying 70S ribosome binding, inhibition of in vitro translation, and ribosome profile analysis. Results: Interestingly, the analogs were equipotent to Api137, except for the 4S-Fpr11 and 4S-Fpr16 analogs, which were four times more or less active, respectively. The most active 4S-Fpr11 analog competed the least with Api137 for its ribosome binding site, suggesting a shifted binding site. Both Fpr14 and the 4S-Fpr16 analogs disturbed 50S subunit assembly less than Api137 or not at all. The strongest effect was observed with the 4R-Fpr16 analog resulting in the lowest 70S ribosome content and the highest pre-50S particle content. This peptide also showed the strongest competition with Api137 for its binding site. However, its antibacterial activity was similar to that of Api137, possibly due to its slower cellular uptake. Conclusions: Api137 inhibits protein translation and disrupts 50S assembly, which can be adjusted by substituting specific proline residues with fluoroproline. 4R-Fpr16 potently inhibits ribosome assembly and offers a novel, unexploited clinical mechanism for future antibiotic development. Full article

The scope of the antibacterial effects of plasma-activated water (PAW) is not yet fully comprehended. We investigated the activity of PAW produced by the in-house 3-pin atmospheric pressure plasma jet against carbapenem-resistant Klebsiella pneumoniae and vancomycin-resistant Enterococcus faecalis, with a focus on PAW’s potential to promote susceptibility to conventional antibiotics in these bacteria. Bacterial inactivation was determined by the colony count after 15 and 60 min PAW treatments. Minimum inhibitory concentrations (MICs) measured following repeated exposures to PAW across multiple generations of bacteria enabled the assessment of changes in susceptibility to antibiotics. The PAW’s efficacy was also analyzed through the detection of intracellular reactive oxygen and nitrogen species in treated bacteria. Time-dependent significant inactivation efficiency against K. pneumoniae was observed (log reduction 6.92 ± 0.24 after 60 min exposure), while effects on E. faecalis were limited. PAW demonstrated potential to decrease the MICs of crucial antibiotics. Namely, a 50 to 62.5% decrease in the MICs of colistin against K. pneumoniae and a 25% reduction in the MICs of vancomycin against enterococci were recorded. We found a significant increase in the superoxide anion concentration in K. pneumoniae and E. faecalis cells after PAW treatments. This study indicates that PAW’s inactivating efficacy coupled with the capacity for the potentiation of antibiotic effects is a promising combination against multidrug-resistant bacteria. Full article

The long regimen of drug therapy, the emergence of drug-resistance (DR), and infections with non-tuberculous mycobacteria (NTMs) are alarming challenges in controlling tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M.tb), necessitating the pursuit of new, broad-spectrum anti-mycobacterials. With more than two-thirds of the clinically useful antibiotics originating from the bacterial phylum Actinomycetota, and their enormous diversity in India, we explored atypical environments for new bacterial strains with potential anti-M.tb activity. In this study, we the examined the secondary metabolites of soil and endophytic bacterial isolates from the wetland niches of Manipur, India, and determined their anti-mycobacterial properties using viability assays. The ethyl acetate culture filtrate extracts of one of the isolates, named Streptomyces sp. SbAr007, showed broad-spectrum anti-mycobacterial activity against laboratory M.tb strains H37Ra and H37Rv, a clinical drug-resistant M.tb and non-tuberculous mycobacteria (NTM). The isolate was characterized for its phenotype and genetic identity, which indicated its closeness to Streptomyces samsunensis, Streptomyces malaysiensis, and Streptomyces solisilvae. Further, macrophage infection assays showed that the extracts could effectively control the intracellular mycobacterial growth but had negligible cytotoxicity to PBMCs from healthy donors. LC-MS identified an unusual combination of antibiotics in these culture filtrate extracts, which can be further explored for specific active molecules or as a formulation against DR-TB. Full article

The traditional marination process enhances food flavor and inhibits microbial growth. However, in hyperosmotic environments, microorganisms can activate stress responses to ensure survival, potentially compromising food safety. This study investigated the osmotolerance mechanisms of Salmonella Derby (S. Derby) by comparing a wild-type strain (S. D-WT) and an osmotolerant strain (S. D-OT) under NaCl-induced hyperosmotic stress. Both strains were subjected to 0.85%, 4%, and 16% NaCl for 0, 8, and 16 days, and their growth behavior, membrane integrity, intracellular osmoprotectant content, and transcription of related genes were evaluated. By day 16, both strains showed a growth delay of approximately 3 h. S. D-OT maintained better membrane integrity and exhibited higher intracellular levels of osmoprotectants (K⁺, trehalose, and proline), which aligned with the upregulation of the transcriptional levels of kdpC, kuP, rpoS, and proU. These findings indicated that S. D-OT achieved improved osmotic stress tolerance by regulating osmoprotectant synthesis and maintaining intracellular homeostasis. In contrast, S. D-WT displayed greater resistance to multiple antibiotics (gentamicin, ciprofloxacin, trimethoprim-sulfamethoxazole, and chloramphenicol) under 4% and 16% NaCl conditions, which may pose a higher food safety risk. Overall, this study provides insights for improving microbial control strategies in preserved foods and mitigating foodborne disease risks. Full article

The development of antibiotic-independent antimicrobial materials is critical for addressing bacterial resistance to conventional antibiotics. Currently, there is a lack of comprehensive understanding of ionic liquid-modified composites in antimicrobial applications. Here, we innovatively prepared GO-based composites modified with phosphonate ionic liquids via a series of surface functionalizations. The resulting antibacterial composites exhibit significant broad-spectrum activity against both Gram-negative and Gram-positive bacteria, including drug-resistant strains, with stronger efficacy against Gram-negative species. Additionally, the material features excellent long-term reusability and the ability to inhibit/destroy biofilms, which is vital for combating persistent infections. Mechanistic studies reveal its antibacterial effects through multiple pathways: disrupting bacterial membranes, inducing ROS, and inactivating intracellular substances—mechanisms less likely to promote resistance. Overall, these phosphonate ionic liquid-modified polycationic materials demonstrate substantial potential in treating bacterial infections, offering a promising strategy to tackle antibiotic resistance challenges. Full article

To manage the developing resistance of Alternaria spp. [the causal fungi of ginseng Alternaria leaf and stem blight (GALSB)] to QoIs fungicides, the toxicity and biochemical activity of pyrimidine nucleoside antibiotics (PNA) against Alternaria spp., cross-resistance between PNA and eight other fungicides currently used to control GALSB disease, and the efficacy of PNA for controlling GALSB in vitro and in vivo were investigated. The distributions of EC₅₀ values of PNA against the mycelial growth (115 isolates) and conidia germination (89 isolates) of A. alternata were unimodal, with mean EC₅₀ values of 10.192 ± 4.961 μg/mL and 0.828 ± 0.101 μg/mL, respectively. There were no significant correlations between the sensitivity of A. alternata to PNA and eight other fungicides (p < 0.05). PNA caused morphological changes in A. alternata mycelia and germ tubes, increased cell membrane permeability, and reduced intracellular DNA and protein levels. On detached ginseng leaves, 300 μg/mL PNA achieved mean protective and curative effects of 87.93% and 94.77% against A. alternata 7 days post-inoculation, outperforming that of 300 μg/mL kresoxim-methyl. Field trial results showed that PNA (180 g a.i./hm²) achieved mean efficacies of 85.63%, 84.07%, and 72.55% at three sites 7, 15, and 30 days after the last spray, which were 5.28–37.74% higher than those of control fungicides pyraclostrobin, azoxystrobin, and kresoxim-methyl at corresponding time points. Overall, our findings indicate that PNA are effective agents for the management of Alternaria spp. resistance to QoIs fungicides. Full article

This comprehensive review explores the advancements in the study of antimicrobial peptides (AMPs), highlighting their potential as promising alternatives to conventional antibiotics in the context of growing antibiotic resistance. AMPs are small molecular proteins found ubiquitously in nature, exhibiting broad-spectrum antimicrobial activity, including antibacterial, antiviral, and antifungal effects, and are vital components of the innate immune system. Due to their non-specific membrane-disrupting mechanism, AMPs are emerging as effective candidates for novel anti-infective agents. The integration of AMPs with biomaterials, such as nanoparticles, liposomes, polymers, and hydrogels, enhances their stability and efficacy while offering multifunctional therapeutic benefits. These combinations promote diverse antibacterial mechanisms, including membrane disruption, intracellular metabolic interference, cell wall modulation, and immune system activation. Despite challenges, such as toxicity, stability, and resistance, innovative strategies including computer-aided design and structural modification show promise in optimizing AMPs’ activity, targeting precision, and biocompatibility. The potential for AMPs in clinical applications remains highly promising, with significant opportunities for overcoming antimicrobial resistance through novel AMP-based therapeutic strategies. Full article

Multidrug-resistant tuberculosis (MDR-TB) is a significant public health challenge globally, exacerbated by the limited efficacy of existing therapeutic approaches, prolonged treatment duration, and severe side effects. As drug resistance continues to emerge, innovative drug delivery systems and treatment strategies are critical to combating this crisis. This review highlights the molecular mechanisms underlying resistance to drugs in Mycobacterium tuberculosis, such as genetic mutation, efflux pump activity, and biofilm formation, contributing to the persistence and difficulty in eradicating MDR-TB. Current treatment options, including second-line drugs, offer limited effectiveness, prompting the need for innovation of advanced therapies and drug delivery systems. The progression in drug discovery has resulted in the approval of innovative therapeutics, including bedaquiline and delamanid, amongst other promising candidates under investigation. However, overcoming the limitations of traditional drug delivery remains a significant challenge. Nanotechnology has emerged as a promising solution, with nanoparticle-based drug delivery systems offering improved bioavailability and targeted and controlled release delivery, particularly for pulmonary targeting and intracellular delivery to macrophages. Furthermore, the development of inhalable formulations and the potential of nanomedicines to bypass drug resistance mechanisms presents a novel approach to enhancing drug efficacy. Moreover, adjunctive therapies, including immune modulation and host-directed therapies, are being explored to improve treatment outcomes. Immunotherapies, such as cytokine modulation and novel TB vaccines, offer complementary strategies to the use of antibiotics in combating MDR-TB. Personalized medicine approaches, leveraging genomic profiling of both the pathogen and the host, offer promise in optimizing treatment regimens and minimizing drug resistance. This review underscores the importance of multidisciplinary approaches, combining drug discovery, advanced delivery system development, and immune modulation to address the complexities of treating MDR-TB. Continued innovation, global collaboration, and improved diagnostics are essential to developing practical, accessible, and affordable treatments for MDR-TB. Full article