Search Results (619)

Biofilm formation is a complex phenomenon employed by microbes to counteract antimicrobials. Biofilm-associated infections are a challenging threat to modern medicine. Antimicrobial peptides (AMPs) are recognized as some of the most promising therapeutics to tackle biofilm-producing and multidrug-resistant (MDR) pathogens. However, stability, toxicity, and potency are key issues in the case of naturally occurring AMPs. Next-generation antibiofilm tools, such as synthetic or engineered AMPs, have emerged as a potent therapeutic choice. Synthetic peptides offer structural simplicity, versatility for chemical modification, and increased stability, which makes them capable of effectively disrupting both the biofilm matrix and the bacterial membrane. For engineered peptides, rational sequence modification, hybridization, and computational design are used to overcome limitations related to selectivity, biofilm-specific targeting and regulatory pathway modulation. This review provides a critical evaluation of synthetic and engineered AMPs from various perspectives, such as design strategies, antibiofilm action mechanisms, therapeutic performance, and translational potential. This study sheds light on current advances and emerging technologies, including AI-guided peptide optimization and multifunctional peptide platforms, and thereby sets the stage for the rational development of peptide-based therapeutics aimed at overcoming biofilm-mediated antimicrobial resistance (AMR). Full article

Background/Objectives: Antimicrobial resistance (AMR) in companion animals is an emerging public health threat due to zoonotic potential and limited therapeutic options. Dogs with otitis externa may harbor multidrug-resistant (MDR) bacteria, including Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis), and Enterobacter cloacae complex (E. cloacae complex), some producing extended-spectrum beta-lactamase (ESBL) or AmpC β-lactamases. This study aimed to assess the prevalence, AMR patterns, MDR occurrence, β-lactamase production, and co-infection profiles of these pathogens in canine otitis externa. Methods: Ear canal samples were collected from 592 dogs presenting clinical signs of otitis externa, with one sample per dog included in the analysis. Samples were collected from veterinary clinics in Timiș County, Romania, from 2022 to 2025. Samples were cultured on blood agar and MacConkey agar, followed by biochemical testing and MALDI-TOF mass spectrometry for bacterial identification. Antimicrobial susceptibility testing against 15 agents across six classes was performed using the VITEK^® 2 system. MDR and β-lactamase production (ESBL, AmpC) were determined according to CLSI 2018 veterinary guidelines. Co-isolation with bacterial and fungal species were recorded. Results: E. coli, P. mirabilis, and E. cloacae complex were isolated in 9.12%, 6.25%, and 1.2% of cases, respectively. E. coli exhibited the highest resistance to aminoglycosides (tobramycin 72.2%, gentamicin 61.1%) and full susceptibility to carbapenems. P. mirabilis showed the highest resistance to ampicillin (54%) and trimethoprim + sulfamethoxazole (46%), with complete susceptibility to carbapenems and fluoroquinolones. E. cloacae complex displayed universal resistance to cephalosporins but remained susceptible to non-cephalosporin β-lactams (piperacillin–tazobactam), carbapenems and aminoglycosides. MDR prevalence was 35.2% for E. coli, 18.9% for P. mirabilis, and 14.3% for the E. cloacae complex. ESBL production was detected in 13% of E. coli and 8.1% of P. mirabilis isolates, while all E. cloacae complex isolates were AmpC-positive. Co-isolations were common, primarily involving Staphylococcus pseudintermedius (S. pseudintermedius) and Malassezia pachydermatis (M. pachydermatis). Conclusions: MDR and β-lactamase-producing bacteria were identified in dogs with otitis externa, emphasizing the importance of routine antimicrobial susceptibility testing, targeted therapy based on local resistance profiles, and continuous AMR surveillance to prevent treatment failure and mitigate zoonotic risk. Full article

Background: Antimicrobial resistance (AMR) is a global healthcare threat. Traditional largely non-selective antibiotics produce side effects due to the natural host microbiome being modified creating a loss in homeostasis. In women, AMR is a cause of acute generational impact. For example, bacterial vaginosis (BV), the most common gynecological infection in reproductive-age women, is a serious public health concern due to its high rates of recurrence, secondary infections, and reproductive issues; and two currently prescribed antibiotics for BV do not fully resolve the symptoms. Objective: The strong need for innovative, potent, safe, and selective therapeutics has prompted a search for such bioactive molecules in milk. Resulting from 200 million years of evolutionary pressure, mammalian lactation not only nourishes infants, but it has also been under relentless Darwinian selective pressure to provide protection from a variety of infections. Methods: Computationally designed human-milk-inspired peptides (AMPs) were tested in standard microbicidal assays for activity against BV pathogens, and evaluated for stability and safety. Results: Several AMPs are bactericidal towards Gardnerella vaginalis, a major BV-associated pathogen, and other BV-associated pathogens. Some novel AMPs do not impact the viability of key lactobacilli linked to a healthy vaginal microbiome. These stable, membrane-acting cationic AMPs reduce inflammation during an infection assay and are safe in EpiVag organoid tissues. Conclusions: AMPs can address concerns like non-selectivity and antibiotic resistance—thereby addressing AMR. Lead AMPs from this study offer a promising solution for the development of novel therapeutics for the treatment of BV, which may reduce the burden of AMR. Full article

The appearance of antibiotic-resistant strains of Helicobacter pylori (H. pylori) is leading to a decreased eradication rate of H. pylori infection. There is an urgent need to find new agents with antimicrobial mechanisms different from those of antibiotics, with therapeutic potential to clear colonization of H. pylori in the stomach. Some antimicrobial peptides (AMPs) possess bactericidal activity by enhancing the permeability of the outer membrane and damaging the integrity of the cell membrane. Bacteria are not susceptible to drug resistance through this antimicrobial mechanism. In this study, 28 short peptides containing 12 amino acid residues were designed based on nine amino acid fragments (KRIVQRIKD) from human cathelicidin LL-37, which is stable in gastric juice, and 3 amino acids were added at the C-terminus of the peptide. These designed peptides were not digested and degraded by pepsin at low pH values. The peptides were predicted using the online tool platform. Then, the strongest antimicrobial peptide, named SAMP-12aa (KRIVQRIKDVIR), was screened from 28 short peptides. Further studies found that SAMP-12aa retained anti-H. pylori activity after incubation in simulated gastric juice. The MIC and MBC of SAMP-12aa were 8 μg/mL and 32 μg/mL, respectively. SAMP-12aa showed good bactericidal kinetics. SAMP-12aa was found to have cell selectivity, penetrating and damaging bacterial cell membranes and exhibiting almost no toxicity to human cells at a relatively high concentration (128 μg/mL). Regulatory T (Treg) cells express CD25^High with immunosuppressive activity that induces immune tolerance in response to H. pylori. Molecular docking prediction revealed that SAMP-12aa could target the active center of Foxp3. Flow cytometry analysis revealed that SAMP-12aa can inhibit Foxp3 activity and downregulate CD25 protein expression on CD4⁺ T cells, thereby reducing the development and differentiation of CD4⁺Foxp3⁺CD25^High Treg cells with immunosuppressive effects. Further research revealed that the levels of the cytokine interferon-γ (IFN-γ), which activates CD8⁺ T-cell activity, were significantly elevated, and the levels of transforming growth factor-β (TGF-β), which inhibits CD8⁺ T-cell activity, were significantly reduced. The results of this study reveal that SAMP-12aa not only possesses antibacterial activity but also has immunomodulatory effects. Full article

Background: Raw-meat-based diets (RMBDs) for companion animals have gained popularity but may serve as vehicles for antimicrobial-resistant (AMR) bacteria, posing risks to animal and public health. This study investigated the occurrence and risk factors of AMR in indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium) and Campylobacter spp. from commercial RMBD products. Methods: In 2023, 50 RMBD samples were collected in Belgium, representing 21 brands from five countries. After both selective and non-selective isolation and MALDI-TOF identification, antimicrobial susceptibility testing of the isolates was performed using broth microdilution. Results: From non-selective media, E. coli was found in 45 samples (90.0%), E. faecalis in 31 samples (62.0%), E. faecium in 23 samples (46.0%), and Campylobacter spp. in 3 samples (6.0%). Among these, one E. faecalis strain with acquired resistance to vancomycin and daptomycin was isolated. Multidrug resistance (MDR) was identified in 17 isolates from 15 samples (30.0%), including 14 MDR E. coli, 1 MDR E. faecalis, and 2 MDR E. faecium. From selective media, presumptive ESBL/AmpC-producing E. coli were detected in 17 samples (34.0%), and 5 E. faecium from linezolid-supplemented media were confirmed by the broth microdilution method. Samples from Belgian origin showed significantly higher E. faecium prevalence (76.5%) compared to Dutch samples (21.4%) (OR = 11.9, p < 0.001). Minor livestock sources were associated with increased MDR risk (OR = 5.52, p = 0.016). Conclusions: Commercial RMBDs in Belgium exhibit widespread bacterial contamination with concerning AMR patterns. These findings highlight the need for improved production standards in the RMBD industry and the need to raise awareness in pet owners. Full article

The Citrobacter freundii (C. freundii) complex represents an increasingly significant group of opportunistic pathogens within healthcare settings. This bacterial complex demonstrates remarkable genomic plasticity, characterized by extensive horizontal gene transfer capabilities that facilitate rapid acquisition of resistance determinants and virulence factors. Although originally considered environmental organisms with limited pathogenic potential, members of the C. freundii complex have emerged as important nosocomial pathogens responsible for urinary tract infections, bacteremia, wound infections, and neonatal meningitis. Importantly, their clinical significance lies less in unique disease manifestations and more in the moderate risk of resistance emergence during therapy with third-generation cephalosporins, driven by inducible chromosomal AmpC β-lactamase production. Beyond this intrinsic mechanism, the genomic adaptability of the C. freundii complex also enables acquisition of additional resistance determinants, including extended-spectrum β-lactamases (ESBLs) and carbapenemases, further limiting therapeutic options and complicating clinical management. Understanding the molecular mechanisms underlying genomic plasticity, virulence expression, and resistance development in the C. freundii complex is crucial for developing effective diagnostic strategies, infection control measures, and novel therapeutic approaches. This pathogen exemplifies the challenge of emerging multidrug-resistant bacteria in contemporary healthcare and underscores the need for continued surveillance and research. This narrative review provides current insights into the taxonomy, genomic plasticity, virulence, and mechanisms of antibiotic resistance. Full article

The multiple-transferable resistance protein (MtrR) is a transcriptional repressor of the mtrCDE-encoded drug efflux pump and Type IV pilus biosynthesis (pilM), and an activator of penicillin-binding protein 1 (ponA) expression in Neisseria gonorrhoeae. Previously published microarray data suggested that MtrR is also an activator of ltgA expression in the gonococcus. LtgA is a lytic transglycosylase responsible for approximately half of recycled peptidoglycan fragments and released peptidoglycan-derived cytotoxins, which cause ciliary damage and induce specific inflammatory responses. The fragments generated by LtgA during peptidoglycan remodeling can either be recognized by the permease AmpG for uptake into the bacterial cytoplasm and recycled for new cell wall growth and general metabolism or released into the external milieu. Therefore, we sought to define the capacity of MtrR to regulate LtgA expression in gonococci. We show that MtrR binds to the ltgA promoter region in a concentration-dependent manner, and that this binding results both in increased ltgA mRNA transcription and LtgA protein levels during exponential growth. Deletion of mtrR in N. gonorrhoeae decreased peptidoglycan monomer release from growing cells and increased autolysis. These results suggest that MtrR regulation of ltgA impacts peptidoglycan-derived cytotoxin release and autolysis in the gonococcus. This study suggests a central role of MtrR in coordinating aspects of the cellular envelope that may contribute to gonococcal pathogenesis. Full article

Background: Natural antimicrobial peptides (AMPs) present a promising solution to address the global threat of drug-resistant infections; however, their clinical translation is challenged by limitations in stability, cytotoxicity, and production costs. Methods: In the present study, a linear Battacin-derived peptide (DDLFD) was modified at the N-terminus with lipid chains, cinnamic acid, or lipoic acid. The lipoic acid-modified variant was further crosslinked by UV irradiation to form stable nanoparticles. The antibacterial performance against planktonic and biofilm bacteria was systematically evaluated in vitro. Results: The results demonstrated that lauric acid-modified pentapeptide (C₁₂-5) and crosslinked lipoic acid-modified pentapeptide (cLA-5) exhibited potent and rapid-acting effects against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Moreover, they showed enhanced efficacy in eradicating bacterial biofilms. Biosafety assessments based on hemolysis and cytotoxicity assays indicated favorable biocompatibility profiles of cLA-5. Mechanistic investigations confirmed that the modified pentapeptides retained a membrane-targeting mode of action characteristic of natural AMPs, involving membrane depolarization and increased permeability. This physical mechanism effectively prevented the development of resistance in sequential passaging assays and showed strong synergistic effects with ciprofloxacin against ciprofloxacin-resistant strains, effectively restoring their antibiotic susceptibility. Conclusions: Together, these findings underscore the strategic potential of rational structural modification, especially the crosslinked nanostructure, in advancing engineered AMPs toward clinical application. Full article

Background/Objectives: Salmonella Heidelberg (SH) is a globally distributed pathogen associated with gastrointestinal disease in humans and animals and frequently affects poultry. Among the classic strategies used in vaccine development, evolutionary engineering enables the generation of attenuated bacterial strains through exposure to selective pressures such as antibiotics. In this study, spontaneous antibiotic-resistant mutant strains of SH were generated by exposure to high concentrations of streptomycin and rifampicin, after which their phenotypic and genotypic characteristics were evaluated. Methods: The wild-type strain SA628 wt was subjected to continuous and discontinuous selection under antibiotic pressure. Phenotypic characterization included biochemical profiling and antibiotic susceptibility testing. Whole-genome sequencing was performed to identify genetic changes affecting virulence- and resistance-associated genes, plasmid content, and point mutations using variant calling approaches. The potential functional relationships of the mutated genes were further analyzed through genetic network analysis. Results: The mutant strains SA628 mut1 and SA628 mut3 were obtained through discontinuous selection, whereas strain SA628 mut2 was generated under continuous selection. Phenotypically, all the mutant strains exhibited resistance to streptomycin, whereas SA628 mut2 and SA628 mut3 also exhibited resistance to rifampicin. Genomic analyses revealed mutations in rpoS, ascD, ynfE, rpoB, and cyaA associated with discontinuous selection and in iscU, ybiO, rpoB, and rsmG associated with continuous selection. Network analysis indicated that these genes are functionally connected within regulatory and metabolic interaction networks, including global transcriptional regulation, anaerobic metabolism, cAMP-mediated signaling, translation, and iron–sulfur cluster biogenesis. Conclusions: Collectively, these findings suggest that antibiotic-driven selection promotes coordinated genetic changes affecting stress responses and metabolism, which may contribute to reduced virulence. This work provides insights into bacterial adaptation under antibiotic stress and supports the potential use of evolutionary engineering for the development of attenuated strains. Full article

Rapid detection of β-lactamase activity is becoming increasingly important as β-lactam resistance spreads at an alarming rate and conventional diagnostics often require several hours to deliver actionable results. Over the past decade, methods based on luminescence, bioluminescence, chemiluminescence, and fluorescence have become powerful tools for the functional assessment of resistance resulting from β-lactamase activity. These approaches provide highly sensitive, activity-based readouts, often within minutes, and frequently rely on simple optical instrumentation. In this review, we summarize recent developments in luminescent probe design between 2015 and 2025, with emphasis on reaction mechanisms, analytical performance, and the ability of these systems to discriminate between different β-lactamases, including narrow-spectrum enzymes, AmpC, ESBL, and carbapenemases. We also discuss their applications in bacterial cultures, clinical isolates, complex biological matrices and, in some cases, in vivo models. While luminescent assays are not yet positioned to replace standard susceptibility testing, they offer a practical and increasingly robust complement to culture-based and molecular methods. The emerging trends highlighted here, such as self-immobilizing fluorogenic probes, chemiluminescent relay systems, nanomaterial-based sensors and AI-assisted mobile platforms, suggest that luminescent β-lactamase detection could play a meaningful role in future rapid diagnostics and resistance surveillance. 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

Leaves of Cucumis callosus, traditionally employed in Ayurvedic medicine for the treatment of urinary disorders, were investigated in depth for their therapeutic potential against bacterially induced urinary tract infection (UTI) for the first time. The present work is the first to explore the antibacterial activity of C. callosus leaf fractions with an integrative in silico, in vitro, and in vivo approach. Through bioassay-guided fractionation, the chloroform fraction (F1) was identified as the most active, exhibiting potent activity against Uropathogenic Escherichia spp. species. Liquid chromatography–mass spectrometry (LC-MS) analysis of F1 revealed the presence of bioactive compounds, including stigmasterol, 1,2,3,4-tetrahydroisoquinoline, lactose, hydroxy(mesityl)acetic acid, and 2,4-di-tert-butylphenol. Molecular docking studies validated the strong binding affinities of these compounds for bacterial resistance enzymes, including AmpC β-lactamase and carbapenemases, thereby providing plausible mechanisms of antimicrobial action. In vivo studies carried out on female rats infected with Escherichia spp. species revealed a dose-dependent reduction in bacterial load, with a significant decrease in urinary tract inflammation upon F1 administration. Histopathological evaluation confirmed the protective effect, with reduced epithelial damage and inflammation in bladder tissues. These findings indicate significant antibacterial and tissue-protective effects of the C. callosus leaf fraction F1, supporting its ethnomedicinal use and establishing it as a promising phytotherapeutic agent for the treatment of urinary tract infections. Full article

Equinin B (GQCQRKCLGHCSKKCPKHPQCRKRCIRRCFGYCL), a marine peptide from Actinia equina exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria. To identify a smaller active region and explore tunable properties, three peptide fragments were synthesized: GQCQRKCLGHCS (EB1), KKCPKHPQCRK (EB2), and RCIRRCFGYCL (EB3), yielding peptides with key AMP-like properties, including the most positively charged and most hydrophobic regions. Only the 11-residue C-terminal fragment showed selective activity against Gram-positive bacteria, including Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus hirae, while remaining inactive against Escherichia coli. Peptide modifications, achieved by replacing cysteine residues with arginine, generally did not enhance activity, but in the C-terminal fragment EB3 they reduced hemolytic activity and increased bacterial specificity. Membrane depolarization assays confirmed that the unmodified fragment EB3 strongly compromises bacterial membranes, whereas the modified variant showed minimal depolarization, highlighting its markedly reduced membrane-perturbing potential. In silico modelling revealed that the EB3 can adopt multiple membrane-disruption modes, from transient shallow pores to carpet-like mechanisms, while the cysteine-to-arginine variant interacts mainly via partial insertion anchored by arginine residues. Phenylalanine appears to interact with the membrane, and reducing hydrophobicity by its removal abolished antibacterial activity. These findings highlight the 11-residue C-terminal fragment as a tunable, membrane-targeting motif with mechanistic novelty, offering a blueprint for developing safer, selective antimicrobial peptides with reduced cytotoxicity. Full article

Drug-resistant bacteria cause millions of global infections each year, and the development of alternative antimicrobial drugs has become a serious undertaking. Currently, peptides with antimicrobial activity represent potential candidates for new antibiotic discovery as they are less likely to cause drug resistance in bacteria. In this study, bifunctional peptides with potent trypsin-inhibitory activity and antimicrobial activity were obtained by rational computation-based structural modifications to a novel Bowman–Birk-type inhibitor (BBI) peptide. The analogues not only displayed potent bacterial killing ability against two drug-resistant bacteria strains of E. coli but also an excellent safety profile, as assessed by low haemolytic activity and low anti-proliferation activity on HaCaT cells. Throughout the molecular dynamics simulations, the peptides exhibited stable adsorption onto the mixed POPE/POPG membrane; most amino acid residues of the AMPs remained bound to the membrane surface, with a few amino acid residues partially penetrating the membrane interior. This showed that the electrostatic interactions were the dominant driving force mediating the peptide–membrane associations. In addition, the tested peptides displayed a degree of stability in the presence of salt ions, serum, and trypsin. These modified peptides thus possess potential as clinical antibacterial agents, and the strategies used in structural modification may also provide a different path to developing new antimicrobial peptides. Full article