Search Results (1,863)

Multidrug-resistant (MDR) strains of Klebsiella pneumoniae present an acute threat as they continue to disseminate globally. Phage therapy has shown promise as a powerful approach to combat MDR infections, but narrow phage host ranges make development of broad acting therapeutics more challenging. The goal of this effort was to use in vitro directed evolution (the “Appelmans protocol”) to isolate K. pneumoniae phages with broader host ranges for improved therapeutic cocktails. Five myophages in the genus Jiaodavirus (family Straboviridae) with complementary activity were mixed and passaged against a panel of 11 bacterial strains including a permissive host and phage-resistant clinical isolates. Following multiple rounds of training, we collected phage variants displaying altered specificity or expanded host ranges compared with parental phages when tested against a 100 strain diversity panel of K. pneumoniae. Some phage variants gained the ability to lyse previously phage-resistant strains but lost activity towards previously phage-susceptible strains, while several variants had expanded activity. Whole-genome sequencing identified mutations and recombination events impacting genes associated with host tropism including tail fiber genes that most likely underlie the observed changes in host ranges. Evolved phages with broader activity are promising candidates for improved K. pneumoniae therapeutic phage cocktails. Full article

Background/Objectives: The rapid emergence of multidrug-resistant bacterial infections demands innovative non-antibiotic therapeutic strategies. Dual-modal photoresponse therapy integrating photodynamic (PDT) and photothermal (PTT) effects offers a promising rapid antibacterial approach, yet designing single-material systems with synergistic enhancement remains challenging. This study aims to develop uniform Cu-based metal–organic framework micrometer cubes (Cu-BN) for efficient PDT/PTT synergy. Methods: Cu-BN cubes were synthesized via a one-step hydrothermal method using Cu(NO₃)₂ and 2-amino-p-benzoic acid. The material’s dual-mode responsiveness to visible light (420 nm) and near-infrared light (808 nm) was characterized through UV–Vis spectroscopy, photothermal profiling, and reactive oxygen species (ROS) generation assays. Antibacterial efficacy against multidrug-resistant Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was quantified via colony counting under dual-light irradiation. Results: Under synergistic 420 + 808 nm irradiation for 15 min, Cu-BN (200 μg/mL) achieved rapid eradication of multidrug-resistant E. coli (99.94%) and S. aureus (99.83%). The material reached 58.6 °C under dual-light exposure, significantly exceeding single-light performance. Photodynamic analysis confirmed a 78.7% singlet oxygen (¹O₂) conversion rate. This enhancement stems from PTT-induced membrane permeabilization accelerating ROS diffusion, while PDT-generated ROS sensitized bacteria to thermal damage. Conclusions: This integrated design enables spatiotemporal PDT/PTT synergy within a single Cu-BN system, establishing a new paradigm for rapid-acting, broad-spectrum non-antibiotic antimicrobials. The work provides critical insights for developing light-responsive biomaterials against drug-resistant infections. Full article

This review explores the biofilm architecture and drug resistance of Enterococcus faecalis in clinical and environmental settings. The biofilm in E. faecalis is a heterogeneous, three-dimensional, mushroom-like or multilayered structure, characteristically forming diplococci or short chains interspersed with water channels for nutrient exchange and waste removal. Exopolysaccharides, proteins, lipids, and extracellular DNA create a protective matrix. Persister cells within the biofilm contribute to antibiotic resistance and survival. The heterogeneous architecture of the E. faecalis biofilm contains both dense clusters and loosely packed regions that vary in thickness, ranging from 10 to 100 µm, depending on the environmental conditions. The pathogenicity of the E. faecalis biofilm is mediated through complex interactions between genes and virulence factors such as DNA release, cytolysin, pili, secreted antigen A, and microbial surface components that recognize adhesive matrix molecules, often involving a key protein called enterococcal surface protein (Esp). Clinically, it is implicated in a range of nosocomial infections, including urinary tract infections, endocarditis, and surgical wound infections. The biofilm serves as a nidus for bacterial dissemination and as a reservoir for antimicrobial resistance. The effectiveness of first-line antibiotics (ampicillin, vancomycin, and aminoglycosides) is diminished due to reduced penetration, altered metabolism, increased tolerance, and intrinsic and acquired resistance. Alternative strategies for biofilm disruption, such as combination therapy (ampicillin with aminoglycosides), as well as newer approaches, including antimicrobial peptides, quorum-sensing inhibitors, and biofilm-disrupting agents (DNase or dispersin B), are also being explored to improve treatment outcomes. Environmentally, E. faecalis biofilms contribute to contamination in water systems, food production facilities, and healthcare environments. They persist in harsh conditions, facilitating the spread of multidrug-resistant strains and increasing the risk of transmission to humans and animals. Therefore, understanding the biofilm architecture and drug resistance is essential for developing effective strategies to mitigate their clinical and environmental impact. Full article

The perianal skin is a unique “skin–gut” boundary that serves as a critical hotspot for the exchange and evolution of antibiotic resistance genes (ARGs). However, its role in the dissemination of antimicrobial resistance (AMR) has often been underestimated. To characterize the resistance patterns in the perianal skin environment of patients with perianal diseases and to investigate the drivers of AMR in this niche, a total of 51 bacterial isolates were selected from a historical strain bank containing isolates originally collected from patients with perianal diseases. All the isolates originated from the skin site and were subjected to antimicrobial susceptibility testing, whole-genome sequencing, and co-occurrence network analysis. The analysis revealed a highly structured resistance pattern, dominated by two distinct modules: one representing a classic Staphylococcal resistance platform centered around mecA and the bla operon, and a broad-spectrum multidrug resistance module in Gram-negative bacteria centered around tet(A) and predominantly carried by IncFIB and other IncF family plasmids. Further analysis pinpointed IncFIB-type plasmids as potent vehicles driving the efficient dissemination of the latter resistance module. Moreover, numerous unexplained resistance phenotypes were observed in a subset of isolates, indicating the potential presence of emerging and uncharacterized AMR threats. These findings establish the perianal skin as a complex reservoir of multidrug resistance genes and a hub for mobile genetic element exchange, highlighting the necessity of enhanced surveillance and targeted interventions in this clinically important ecological niche. Full article

The emergence and spread of antimicrobial resistance among pathogens are significantly reducing available therapeutic options, highlighting the urgent need for novel and complementary treatment strategies. Antimicrobial photodynamic therapy (aPDT) is a promising alternative approach that can overcome antimicrobial resistance through a multitarget mechanism of action, exerting direct bactericidal and fungicidal effects with minimal risk of resistance development. Although aPDT has shown efficacy against a variety of pathogens, data on its activity against large collections of clinical multidrug-resistant strains are still limited. In this study, we assessed the antimicrobial activity of the photosensitizer RLP068/Cl combined with a red light-emitting LED source at 630 nm (Molteni Farmaceutici, Italy) against a large panel of Gram-negative and Gram-positive bacterial strains harboring relevant resistance traits and Candida species. Our results demonstrated the significant microbicidal activity of RLP068/Cl against all of the tested strains regardless of their resistance phenotype, with particularly prominent activity against Gram-positive bacteria (range of bactericidal concentrations 0.05–0.1 µM), which required significantly lower exposure to photosensitizer compared to Candida and Gram-negative species (range 5–20 µM). Overall, these findings support the potential use of RLP068/Cl-mediated aPDT as an effective therapeutic strategy for the management of localized infections caused by MDR organisms, particularly when conventional therapeutic options are limited. Full article

Palindromic antimicrobial peptides (PAMs) constitute versatile scaffolds for the design and optimization of anticancer agents with applications in therapy, diagnosis, and/or monitoring. In the present study, fluorolabeled peptides derived from the palindromic sequence RWQWRWQWR containing fluorescent probes, such as 2-Aminobenzoyl, 5(6)-Carboxyfluorescein, and Rhodamine B, were obtained. RP-HPLC analysis revealed that the palindromic peptide conjugated to Rhodamine B (RhB-RWQWRWQWR) exhibited the presence of isomers, likely corresponding to the open-ring and spiro-lactam forms of the fluorescent probe. This equilibrium is dependent on the peptide sequence, as the RP-HPLC analysis of dimeric peptide (RhB-RRWQWR-hF-KKLG)₂K-Ahx did not reveal the presence of isomers. The antibacterial activity of the fluorescent peptides depends on the probe attached to the sequence and the bacterial strain tested. Notably, some fluorescent peptides showed activity against reference strains as well as sensitive, resistant, and multidrug-resistant clinical isolates of E. coli, S. aureus, and E. faecalis. Fluorolabeled peptides 1-Abz (MIC = 62 µM), RhB-1 (MIC = 62 µM), and Abz-1 (MIC = 31 µM) exhibited significant activity against clinical isolates of E. coli, S. aureus, and E. faecalis, respectively. The RhB-1 (IC₅₀ = 61 µM), Abz-1 (IC₅₀ = 87 µM), and RhB-2 (IC₅₀ = 35 µM) peptides exhibited a rapid, significant, and concentration-dependent cytotoxic effect on HeLa cells, accompanied by morphological changes characteristic of apoptosis. RhB-1 (IC₅₀ = 18 µM) peptide also exhibited significant cytotoxic activity against breast cancer cells MCF-7. These conjugates remain valuable for elucidating the possible mechanisms of action of these novel anticancer peptides. Rhodamine-labeled peptides displayed cytotoxicity comparable to that of their unlabeled analogues, suggesting that cellular internalization constitutes a critical early step in their mechanism of action. These findings suggest that cell death induced by both unlabeled and fluorolabeled peptides proceeds predominantly via apoptosis and is likely contingent upon peptide internalization. Functionalization at the N-terminal end of the palindromic sequence can be evaluated to develop systems for transporting non-protein molecules into cancer cells. Full article

The escalating global challenges of antimicrobial resistance (AMR) and cancer necessitate innovative therapeutic solutions from natural sources. This study investigated the multifaceted therapeutic potential of pigment-enriched plant extracts. We screened diverse plant extracts for antimicrobial and antibiofilm activity against multidrug-resistant bacteria and fungi. Hibiscus sabdariffa emerged as the most promising, demonstrating potent broad-spectrum antimicrobial and significant antibiofilm activity. Sub-inhibitory concentrations of H. sabdariffa robustly downregulated essential bacterial virulence genes and suppressed aflatoxin gene expression. Comprehensive chemical profiling via HPLC identified major anthocyanin glucosides, while GC-MS revealed diverse non-pigment bioactive compounds, including fatty acids and alcohols. Molecular docking suggested favorable interactions of key identified compounds (Cyanidin-3-O-glucoside and 1-Deoxy-d-arabitol) with E. coli outer membrane protein A (OmpA), indicating potential antiadhesive and antimicrobial mechanisms. Furthermore, H. sabdariffa exhibited selective cytotoxicity against MCF-7 breast cancer cells. These findings establish H. sabdariffa pigment-enriched extract as a highly promising, multi-functional source of novel therapeutics, highlighting its potential for simultaneously addressing drug resistance and cancer challenges through an integrated chemical, biological, and computational approach. Full article

Background: Various predisposing factors contribute to the emergence and dissemination of the multidrug-resistant (MDR) phenotype in Escherichia coli and Klebsiella pneumoniae. Understanding these factors is crucial for guiding appropriate antimicrobial therapy and infection control strategies. This study investigated the predisposing factors contributing to the MDR characteristics of E. coli and K. pneumoniae isolated in a community hospital in northeastern Thailand. Methods: This case–control study utilized retrospective data from bacterial culture, as well as demographic, clinical, and antibiotic susceptibility records collected during 5 years (January 2016–December 2020). E. coli and K. pneumoniae isolates were analyzed from various clinical samples, including blood, urine, pus, sputum, and other body fluids. Data were analyzed using descriptive statistics and univariate logistic regression. Results: In total, 660 clinical isolates were analyzed (421 E. coli and 239 K. pneumoniae). Blood was the most common source of the detection of E. coli (63.0%) and sputum was the most common source of K. pneumoniae (51.0%). The median ages of patients were 67 and 63 years for E. coli and K. pneumoniae, respectively. E. coli cases were significantly associated with prior antibiotic use (OR = 1.79, 95% CI: 1.17–2.74 p = 0.008). MDR was observed in 50.1% of E. coli and 29.7% of K. pneumoniae (p < 0.001). E. coli compared to K. pneumoniae had lower resistance to third-gen cephalosporins (64.9% versus 95.8%) and carbapenems (8.0% versus 6.9%). ICU admission was the only factor significantly associated with MDR E. coli (OR = 2.40, 95% CI: 1.11–5.20 p = 0.026). No significant differences were observed in gender, age, or comorbidities between MDR cases. Antibiotic usage patterns also differed, with E. coli more likely to receive third-gen cephalosporins compared to carbapenems (OR = 3.02, 95% CI:1.18–7.74 p = 0.021). Conclusions: The use of third-generation cephalosporin may drive MDR E. coli more than K. pneumoniae. Prior antibiotic exposure was linked to E. coli bloodstream infections, while MDR E. coli showed greater clinical severity. These findings highlighted the need for improved antibiotic stewardship in rural hospitals. Full article

Nosocomial infections caused by ESKAPE pathogens represent a significant burden to global health. These pathogens may exhibit multidrug resistance (MDR) mechanisms, of which mechanisms such as efflux pumps and biofilm formation are gaining significant importance. Multidrug resistance mechanisms in ESKAPE pathogens have led to an increase in the effective costs in health care and a higher risk of mortality in hospitalized patients. These pathogens utilize antimicrobial efflux pump mechanisms and bacterial biofilm-forming capabilities to escape the bactericidal action of antimicrobials. ESKAPE bacteria forming colonies demonstrate increased expression of efflux pump-encoding genes. Efflux pumps not only expel antimicrobial agents but also contribute to biofilm formation by bacteria through (1) transport of molecules and transcription factors involved in biofilm quorum sensing, (2) bacterial fimbriae structure transport for biofilm adhesion to surfaces, and (3) regulation of a transmembrane gradient to survive the difficult conditions of biofilm microenvironments. The synergistic role of these mechanisms complicates treatment outcomes. Given the mechanistic link between biofilms and efflux pumps, therapeutic strategies should focus on targeting anti-biofilm mechanisms alongside efflux pump inactivation with efflux pump inhibitors. This review explores the molecular interplay between efflux pumps and biofilm formation, emphasizing potential therapeutic strategies such as efflux pump inhibitors (EPIs) and biofilm-targeting agents. Full article

Background: Antibacterial resistance (ABR) poses a major challenge to global health, with methicillin-resistant Staphylococcus aureus (MRSA) being one of the prominent multidrug-resistant strains. MRSA has developed resistance through the expression of Penicillin-Binding Protein 2a (PBP2a), a key transpeptidase enzyme involved in bacterial cell wall biosynthesis. Objectives: The objective was to design and characterize a novel small-molecule inhibitor targeting PBP2a as a strategy to combat MRSA. Methods: We synthesized a new indole triazole conjugate (ITC) using eco-friendly and click chemistry approaches. In vitro antibacterial tests were performed against a panel of strains to evaluate the ITC antibacterial potential. Further, a series of in silico evaluations like molecular docking, MD simulations, free energy landscape (FEL), and principal component analysis (PCA) using the crystal structure of PBP2a (PDB ID: 4CJN), in order to predict the mechanism of action, binding mode, structural stability, and energetic profile of the 4CJN-ITC complex. Results: The compound ITC exhibited noteworthy antibacterial activity, which effectively inhibited the selected strains. Binding score and energy calculations demonstrated high affinity of ITC for the allosteric site of PBP2a and significant interactions responsible for complex stability during MD simulations. Further, FEL and PCA provided insights into the conformational behavior of ITC. These results gave the structural clues for the inhibitory action of ITC on the PBP2a. Conclusions: The integrated in vitro and in silico studies corroborate the potential of ITC as a promising developmental lead targeting PBP2a in MRSA. This study demonstrates the potential usage of rational drug design approaches in addressing therapeutic needs related to ABR. Full article

Salmonella is one of the main causes of food-borne illness worldwide. In most cases, Salmonella contamination can be traced back to food processing plants and/or to cross-contamination during food preparation. To avoid food-borne diseases, food processing plants use sanitizers and biocidal to reduce bacterial contaminants below acceptable levels. Despite these preventive actions, Salmonella can survive and consequently affect human health. This study investigates the adaptive capacity of the main Salmonella enterica serotypes isolated from the poultry production line, focusing on their replication, antimicrobial resistance, and biofilm formation under stressors such as acidic conditions, oxidative environment, and high osmolarity. Using growth curve analysis, crystal violet staining, and microscopy, we assessed replication, biofilm formation, and antimicrobial resistance under acidic, oxidative, and osmotic stress conditions. Disinfectant tolerance was evaluated by determining the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of sodium hypochlorite. The antibiotic resistance was assessed using the Kirby–Bauer method. The results indicate that, in general, acidic and osmotic stress reduce the growth of Salmonella. However, no significant differences were observed specifically for serotypes Infantis, Heidelberg, and Corvallis. The S. Infantis isolates were the strongest biofilm producers and showed the highest prevalence of multidrug resistance (71%). Interestingly, S. Infantis forming biofilms required up to 8-fold higher concentrations of sodium hypochlorite for eradication. Furthermore, osmotic and oxidative stress significantly induced biofilm production in industrial S. Infantis isolates compared to a reference strain. Understanding how Salmonella responds to industrial stressors is vital for designing strategies to control the proliferation of these highly adapted, multi-resistant pathogens. Full article

The rise in multidrug-resistant bacterial strains and persistent infections such as Lyme disease caused by Borrelia burgdorferi highlights the need for novel antimicrobial agents. The present study explores the antioxidant, antibacterial, and cytotoxic properties of extracts from submerged mycelial biomass of Fomitopsis pinicola, cultivated in synthetic and lignocellulosic media. Four extracts were obtained using hot water and 80% ethanol. The provided analysis of extracts confirmed the presence of various bioactive compounds, including flavonoids, alkaloids, and polyphenols. All extracts showed dose-dependent antioxidant activity (IC₅₀: 1.9–6.7 mg/mL). Antibacterial tests revealed that Klebsiella pneumoniae was most sensitive, with the L2 extract producing the largest inhibition zone (15.33 ± 0.47 mm), while the strongest bactericidal effect was observed against Acinetobacter baumannii (MBC as low as 0.5 mg/mL for L1). Notably, all extracts significantly reduced the viability of stationary-phase B. burgdorferi cells, with L2 reducing viability to 42 ± 2% at 5 mg/mL, and decreased biofilm mass, especially with S2. Cytotoxicity assays showed minimal effects on NIH 3T3 cells, with slight toxicity in HEK 293 cells for S2 and L1. These results suggest that F. pinicola extracts, particularly ethanolic L2 and S2, may offer promising natural antimicrobial and antioxidant agents for managing resistant infections. Full article

Feline urinary tract infections (UTIs) present a common challenge in veterinary practice, underscoring the importance of understanding local bacterial pathogens and antimicrobial resistance (AMR). This study determined bacterial prevalence and antimicrobial susceptibility in cats at Kasetsart University’s Veterinary Teaching Hospital in Bangkok, Thailand. Of the 543 cystocentesis urine samples collected from 428 cats, 115 (21.2%) tested positive for bacterial cultures, leading to a diagnosis of UTIs in 95 cats (22.2%). The most prevalent isolates included Escherichia coli (24.8%), Staphylococcus species (19.2%), Proteus mirabilis (13.6%), Pseudomonas aeruginosa (12.0%), and Enterococcus species (12.0%). Staphylococcus felis (8.8%) and Staphylococcus pseudintermedius (5.6%) were the predominant Staphylococcus species. Rare pathogens such as Corynebacterium urealyticum and Lactococcus garvieae were also identified. Antimicrobial testing revealed alarming resistance, with 69.2% of isolates exhibiting multidrug resistance (MDR). Escherichia coli and Proteus mirabilis showed high resistance to amoxicillin/clavulanic acid (AMC) (45.2–70.6%) and sulfamethoxazole/trimethoprim (SXT) (51.6–52.9%). Enterococcus faecium exhibited 85.7% resistance to AMC. Methicillin resistance was identified in 41.7% of Staphylococcus isolates, particularly high in Staphylococcus epidermidis (75.0%) and Staphylococcus pseudintermedius (71.4%). High fluoroquinolone resistance among MDR isolates further exacerbates AMR concerns. These results indicate that MDR Gram-negative, Staphylococcus, and Enterococcus species complicate the empirical treatment of feline UTIs, highlighting significant implications for AMR in veterinary practice. Full article

The rise of multidrug-resistant Pseudomonas aeruginosa underscores the need for novel therapeutic targets beyond conventional peptidoglycan biosynthesis. Some bacterial strains bypass MurA inhibition by fosfomycin via a cell wall salvage pathway. This study targeted P. aeruginosa AmgK (PaAmgK) and MurU (PaMurU) to identify inhibitors that could complement fosfomycin therapy. A malachite-green-based dual-enzyme assay enabled efficient activity measurements and high-throughput chemical screening. Screening 232 compounds identified Congo red and CTAB as potent PaMurU inhibitors. A targeted mass spectrometric analysis confirmed the selective inhibition of PaMurU relative to that of PaAmgK. Molecular docking simulations indicate that Congo red preferentially interacts with PaMurU through electrostatic contacts, primarily involving the residues Arg28 and Arg202. The binding of Congo red to PaMurU was corroborated further using SUPR-differential scanning fluorimetry (SUPR-DSF), which revealed ligand-induced thermal destabilization. Ongoing X-ray crystallographic studies, in conjunction with site-directed mutagenesis and enzyme kinetic analyses, aim to elucidate the binding mode at an atomic resolution. Full article

Vibrio parahaemolyticus (V. parahaemolyticus) is a preeminent seafood-borne pathogen, imposing significant economic burdens on global aquaculture. The escalating prevalence of multidrug-resistant strains has accentuated the critical urgency for developing sustainable biocontrol strategies. In this study, a bacteriophage designated vB_VPAP_XY75 (XY75) was isolated and biologically characterized to establish an effective control against V. parahaemolyticus. XY75 exhibited remarkable specificity toward V. parahaemolyticus, effectively lysing 46.2% of the target strains while showing no lytic activity against non-target bacterial species. Morphological characterization confirmed its taxonomic assignment to the Myoviridae family, featuring an icosahedral head (40 ± 2 nm) and contractile tail (60 ± 2 nm). XY75 demonstrated strong environmental tolerance, remaining stable at pH 4–11 and temperatures as high as 50 °C. At an optimal multiplicity of infection (MOI = 0.01), XY75 achieved a peak titer of 8.1 × 10¹⁰ PFU/mL, a 5 min latent period, and burst size of 118 PFU/cell. Critically, XY75 reduced V. parahaemolyticus in salmon by more than 5.98 log CFU/g (99.9%) within 6 h at 4 °C, demonstrating exceptional cold tolerance and lytic activity. Genomic analysis confirmed that no virulence or antibiotic resistance genes were present. These results establish XY75 as a safe and efficacious biocontrol candidate for seafood preservation, with particular utility under refrigerated storage conditions. Full article