Search Results (839)

Background/Objectives: This study characterized the phenotypic and genotypic profiles of antimicrobial resistance in 104 Escherichia coli isolates obtained from 22 samples of artisanal Minas Frescal cheese from the Federal District, Brazil. Methods: The antimicrobial susceptibility of E. coli isolates was assessed using the disk diffusion method and antimicrobial resistance genes were detected using polymerase chain reaction methods with specific primers. Results: The highest rates of phenotypic antimicrobial resistance were observed for sulfonamides (85.58%, 89/104) and tetracyclines (38.46%, 40/104). In the genotypic profiles, most E. coli isolates carried the sulfonamide resistance genes sul1/sul2 (62.50%, 65/104), tetracycline resistance genes tetA/tetB (65.38%, 68/104), and β-lactam resistance genes blaCTX-M/blaTEM/blaSHV (55.77%, 58/104). Most E. coli strains that presented sulfonamide resistance genes carried the sul1 gene (49.04%, 51/104) and were phenotypically sulfonamide-resistant strains (59.61%, 62/104). Regarding the E. coli strains that carried tetracycline resistance genes, the majority harbored both tetA and tetB genes (34.61%, 36/104), with 35.56% (37/104) being phenotypically resistant and 29.80% (31/104) being phenotypically susceptible. For E. coli strains that presented β-lactam resistance genes, the most frequently detected gene was blaCTX-M (21.15%, 22/104) and, notably, most E. coli strains (43.26%, 45/104) were phenotypically susceptible. The cat1 and clmA genes (associated with phenicol resistance) were detected in 22.12% of the E. coli isolates (23/104), with only two strains (1.92%) being phenotypically resistant to chloramphenicol. Conclusion: The high prevalence of E. coli carrying antimicrobial resistance genes in artisanal cheese raises public health concerns regarding the dissemination of potentially pathogenic antimicrobial-resistant microorganisms through the food chain. Full article

Wastewater treatment plants (WWTPs) are recognized hotspots for the convergence and dissemination of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) into the environment. Among ARB, carbapenem-resistant Enterobacterales (CR-E) and extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae (ESBL-Ec/Kp) are of particular concern due to their clinical relevance. We characterized 30 CR-E and 176 ESBL-Ec/Kp isolates (two of them were both ESBL-producing and carbapenem-resistant) recovered from influent, intermediate, effluent, sludge, and downstream river samples collected from two WWTPs in northern Spain. Isolates were evaluated for resistance phenotypes against 12 antimicrobials, and β-lactamase-encoding genes were assessed by PCR and sequencing. Notably, among CR-E isolates, bla_KPC-2 was the most prevalent (93%), followed by bla_OXA-48-like, detected in two isolates from non-treated and pasteurized sludge; both isolates also carried bla_CTX-M-15, a finding not previously reported specifically in sludge samples. Among ESBL-Ec/Kp, a broad diversity of ESBL genes was identified, including bla_{CTX-M group 1} (variants 1, 3, 15, 32, 55), bla_{CTX-M group 9} (variants 14, 27, 65, 97), bla_SHV-12 and bla_TEM-169. The most prevalent ESBL gene was bla_CTX-M-15 (48.3%), followed by bla_CTX-M-14, bla_CTX-M-32, and bla_SHV-12, detected in 10.8%, 8.5%, and 6.8% of isolates, respectively. CR-E and ESBL-Ec/Kp were found in all sample types and were still detectable at terminal stages, indicating persistence throughout treatment. These findings support the need to improve and optimize current wastewater treatment methods and underscore the importance of integrating culture-based and molecular methods into routine WWTP monitoring for early detection of microbiological hazards, although further research is still needed. Full article

The global rise of antimicrobial resistance represents a critical challenge to public health, with Escherichia coli emerging as one of the most significant contributors due to its high adaptability and prevalence of extended-spectrum β-lactamase (ESBL) production. Outer membrane vesicles (OMVs), nanoscale structures released by Gram-negative bacteria, have recently been implicated in the dissemination of resistance determinants and direct antibiotic inactivation. This study investigated the role of OMVs derived from ESBL-producing E. coli in mediating resistance to ampicillin. Clinical strains harboring CTX-M-15 resistance genes were cultured under selective pressure, and OMVs were purified via size-exclusion chromatography. Characterization using tunable resistive pulse sensing (TRPS) and cryo-transmission electron microscopy confirmed vesicle integrity, with sizes ranging from 80 to 150 nm. DNA quantification and PCR analysis revealed the presence of CTX-M-15 genes within vesicles, which remained protected from DNase digestion, confirming encapsulation. Functional assays demonstrated β-lactamase activity within OMVs, with proteinase K treatment indicating localization primarily within vesicles rather than on their surface. Importantly, OMVs inactivated ampicillin in a dose-dependent manner, significantly reducing its efficacy against susceptible E. coli. Disc diffusion and microtiter plate assays confirmed that β-lactamase-positive OMVs protected susceptible strains from antibiotic killing, promoting bacterial survival and growth. This study uniquely demonstrates that OMVs from CTX-M-15–producing Escherichia coli carry both resistance genes and active β-lactamase enzymes, thereby facilitating both genetic dissemination and direct antibiotic inactivation. Targeting OMV biogenesis may represent a novel strategy to combat antimicrobial resistance. Full article

Disuse-induced bone loss during stall confinement and immobilization is a major concern in horses because it impairs recovery and increases susceptibility to further injury. Experimental models are needed to evaluate therapeutic options, but most available equine models rely on cast immobilization, which is technically demanding and may be associated with complications. This study aimed to assess a simpler and less restrictive model to induce a quantifiable decrease in bone density in horses. Six French Standardbred horses underwent eight weeks of stall confinement, with a wooden wedge fitted to one front foot to elevate the heels during the last four weeks. Bone density was assessed using computed tomography (CT) examinations of both forelimbs performed at the beginning (M0) and after the confinement period (M2). Serum markers of bone metabolism (CTX-I, osteocalcin, bone-specific alkaline phosphatase, and hydroxyproline) were analyzed monthly from baseline to 2 months post confinement. Statistical analysis used Wilcoxon signed-rank tests and mixed models as appropriate. Computed tomography revealed a significant decrease in bone density after confinement (p < 0.05), more pronounced distally in the wedge limb. CTX-I levels varied with physical activity. This model provides a practical and reproducible alternative to cast immobilization for inducing equine bone demineralization. Full article

The emergence of antimicrobial resistance (AMR) across the broiler production chain holds significant economic, animal, and public health implications. This study investigated phenotypic resistance to 13 antimicrobials and the presence of 35 antimicrobial resistance genes (ARGs) in Escherichia coli (n = 291) isolated across three broiler production chains (broiler breeder farms, hatcheries, commercial broiler farms, and retail meat shops). An extremely high phenotypic resistance (>70%) to doxycycline, ciprofloxacin, and cefpodoxime, and very high resistance (50–70%) to ampicillin, cefotaxime, gentamicin, and ceftazidime was observed. In addition, 97% of isolates were multidrug-resistant (resistant to ≥1 drug in ≥3 antimicrobial classes), 42% were extended-spectrum beta-lactamase (ESBL) producers, 65% were resistant to third-generation cephalosporins (3GCR), and 21% were resistant to colistin. The Poisson regression model revealed no significant difference in AMR among broiler production stages, except for colistin. Among 35 ARGs tested, 24 (67%) were detected at least once. The most prevalent were tetA, bla_TEM, qnrB, qnrS, and aac(6′)-Ib-cr, while qnrD, sul2, bla_OXA, and bla_CTX-M were detected at lower levels (1–7%). All five tested mcr genes (mcr-1 to mcr-5) were identified in commercial farms and retail shops. No bla_NDM, tetB, tetC, tetD, tetM, qnrC, aac(3)-IIa (aacC2), aph(3)-IIa (aphA2), or aac(6′)-Ib genes were found. A strong correlation was observed between AMR phenotypes and ARGs. High AMR among E. coli in broiler production poses significant One Health risks, with widespread MDR, ESBL production, and resistance to critically important antimicrobials. Prudent antimicrobial use, enhanced surveillance and education, farm biosecurity, and One Health strategies are crucial in mitigating these threats. Full article

Background: The emergence of extended-spectrum β-lactamase (ESBL) producing and colistin-resistant Escherichia coli in retail meat poses a significant public health risk. Method: A total of 180 retail meat samples (chicken parts, internals, processed products; lamb; beef; fish) were purchased from markets and butcher shops across Turkiye. Presumptive ESBL-producing isolates were screened on chromogenic agar and phenotypically confirmed. Species identity was verified by uspA PCR, and resistance genes (blaCTX-M, blaTEM, blaOXA, blaSHV, mcr-1, mcr-2, mcr-3) were analyzed. Colistin MICs were determined by broth microdilution, while antimicrobial susceptibility of ESBL-positive isolates was assessed by disk diffusion. Results: Overall, ESBL-producing E. coli were detected in 21.7% (n = 39) of the 180 meat samples analyzed, with the highest prevalence observed in chicken parts (26/40, 65.0%) and giblets (6/10, 60%). All ESBL-E. coli isolates harbored blaCTX-M, with blaCTX-M-1 identified as the sole variant. The blaTEM gene was detected in 61.5% (24/39) of ESBL-positive E. coli isolates. Colistin resistance was identified in six isolates (15.4%), all of which carried the mcr-1 gene. Additionally, one lamb minced meat isolate harbored the mcr-2 gene. Co-occurrence analysis revealed that the most frequent resistance gene combination among ESBL-producing isolates was blaCTX-M1 + blaTEM, detected predominantly in chicken meat samples, while mcr-1 was observed only in isolates harboring single or limited resistance genes, suggesting a distinct acquisition pattern. Conclusions: A high prevalence of blaCTX-M-1 and the co-occurrence of mcr genes were detected in E. coli isolates from retail meat, particularly poultry. The detection of mcr-1/mcr-2 co-carriage in lamb meat, though rare, highlights the need for broader surveillance. These findings underscore the need for integrated monitoring and prudent antimicrobial use in food animals. The use of antibiotics as growth promoters is prohibited in Türkiye, and therapeutic applications require a veterinary prescription; however, stronger enforcement remains essential to limit the dissemination of multidrug-resistant bacteria in the food chain. Full article

Oxidative stress brought on by prolonged glucocorticoid therapy reduces bone growth, structure, and mechanical qualities. Free radicals promote osteoclastic activity and are harmful to osteoblasts. As an antioxidant, tocotrienol offers protection against illnesses linked to free radicals. Annatto tocotrienol (ATT) is a tocopherol-free tocotrienol, and palm tocotrienol (PTT) is a tocotrienol mixture. Finding out how ATT and PTT protect against glucocorticoid-induced osteoporosis was the aim of this study. In this study, 32 mature male Sprague-Dawley rats were employed. Twenty-four rats were divided into three groups: Dex, Dex + PTT, and Dex + ATT, after being adrenalectomized. A sham surgery was performed on the remaining eight rats. The Dex group received oral vehicle palm olein (0.1 mL/kg/day) and intramuscular injection of dexamethasone (120 µg/kg/day). Dexamethasone 120 µg/kg/day was administered intramuscularly to the Dex + PTT and Dex + ATT group, and palm tocotrienol (PTT) and annatto tocotrienol (ATT) 60 mg/kg/day were added as a supplement. Vehicle palm olein was administered intramuscularly to the sham-operated rats, 0.05 mL/kg/day and 0.1 mL/kg/day orally. The treatments were administered for two months before the rats were euthanized. The biomechanical strength of the femoral bones was evaluated, and the structural characteristics of bone histomorphometry were examined. According to the findings, prolonged glucocorticoid therapy resulted in decreased superoxide dismutase (SOD) activity, increased lipid peroxidation, and bone carboxy-terminal collagen cross-linkages (CTX). Bone Volume/Tissue Volume (BV/TV) and Trabecular Number (Tb.N) were drastically reduced, which severely reduced bone biomechanical strength. There were also alterations in the bone formation and resorption gene expressions. Lipid peroxidation, CTX levels, and SOD activity were all considerably maintained at control levels by PTT and ATT intake. Additionally, it preserved the biomechanical strength and bone structure, as well as maintaining the gene expressions. According to the study’s findings, ATT and PTT may have anabolic and anti-resorptive properties and have the potential to be utilized as a prophylactic for individuals receiving long-term glucocorticoid therapy. Full article

Two-dimensional molybdenum carbide (Mo_1.33CT_x MXene) with ordered vacancies is one of the most promising materials for electrochemical energy storage. However, the high defectivity and tendency to aggregate of nanosheets hinders the large-scale fabrication of highly efficient Mo_1.33CT_x -based electrodes. In this study, Mo_1.33CT_x/carbon nanotubes (CNTs) electrodes of varying thicknesses were fabricated using a scalable doctor blade technique. Their electrochemical performance was studied in H₂SO₄, H₃PO₄, LiCl and KCl electrolytes using cyclic voltammetry and galvanostatic charge–discharge methods. Electrodes with an active material mass loading of 1.6 mg/cm² exhibited specific capacitances of 352, 287, 172, and 107 F/g in H₂SO₄, H₃PO₄, LiCl, and KCl electrolytes, respectively, at a scan rate of 2 mV/s. Increasing the mass loading of the electrode material to 3.5 mg/cm² resulted in a specific capacitance of 349, 260, 162 and 98 F/g in the same electrolytes. The incorporation of CNTs enabled rapid electrolyte ion transport throughout the electrode bulk, maintaining high capacitance values even at high scan rates. These results open new avenues for the development of high-performance electrode materials for supercapacitors. Full article

Introduction: Klebsiella variicola, a member of Klebsiella pneumoniae complex, has emerged as an opportunistic pathogen for human infection; however, antimicrobial resistance and hypervirulent characteristics of K. variicola have rarely been investigated in South Korea. Methods: We analyzed 76 clinical K. variicola isolates collected from 12 hospitals between September 2022 and October 2023. Bacterial identification was performed by MALDI-TOF MS. Antimicrobial susceptibility was tested by disk diffusion tests. Resistance determinants and virulence traits were investigated, and whole-genome sequencing was performed for hypermucoviscous or carbapenem-resistant K. variicola isolates. Results: Most (89.5%, 68/76) were susceptible to all 18 antimicrobials tested in this study, and 3 isolates harbored bla_CTX-M-15. One isolate carried bla_KPC-2 on its IncX3 plasmid, which is closely related to carbapenem-resistant K. pneumoniae plasmids. Capsular typing revealed 51 wzi allelic types. Ten isolates showed mucoid phenotype, mainly with KL60 and KL61. Conclusions: This study reveals relatively low resistance rates in K. variicola strains but the presence of multidrug-resistant and hypermucoviscous K. variicola strains. In addition, the evidence of interspecies dissemination of bla_KPC-2 highlights the need for continuous genomic surveillance. Full article

Antimicrobial resistance poses a growing threat to public health, and integrated surveillance strategies across environmental compartments such as treated wastewater and biosolids can substantially improve monitoring efforts. A key challenge is the diversity of available protocols, which complicates comparability for the concentration and detection of antibiotic resistance genes (ARGs), particularly in complex matrices. In this study, we compared two commonly used concentration methods—filtration–centrifugation (FC) and aluminum-based precipitation (AP)—and two detection techniques, quantitative PCR (qPCR) and droplet digital PCR (ddPCR), for the quantification of four clinically relevant ARGs: tet(A), bla_CTX-M group 1, qnrB, and catI. Analyses were performed in both secondary treated wastewater and biosolid samples, including their purified bacteriophage-associated DNA fractions. Results showed that the AP method provided higher ARG concentrations than FC, particularly in wastewater samples. ddPCR demonstrated greater sensitivity than qPCR in wastewater, whereas in biosolids, both methods performed similarly, although ddPCR yielded weaker detection. Importantly, ARGs were detected in the phage fraction of both matrices, with ddPCR generally offering higher detection levels. These results provide comparative insights into established methodologies and highlight the value of selecting appropriate protocols based on matrix characteristics and surveillance objectives. Full article

In this study, sucrase A (SacA) from Bacillus subtilis was successfully displayed on the outer membrane of Escherichia coli via fusion with the AIDA-I autotransporter from E. coli. The pAIDA-sacA plasmid was constructed by fusing sacA with the ctxB signal sequence and the β-barrel domain of aida gene, enabling surface expression under both aerobic and anaerobic conditions. Functional expression of AIDA–SacA was confirmed by the appearance of reducing sugars in enzymatic assays of sucrose hydrolysis and by acid production on phenol red agar. Structural prediction suggested correct localisation of the catalytic domain on the extracellular surface. Enzymatic characterisation revealed that AIDA-SacA exhibits optimal activity at 40 °C and pH 7. The calculated Km for sucrose was 1.18 mM, while the corresponding Vmax was 2.32 U mL⁻¹. Thermal stability assays showed that the enzyme retained over 80% of its activity after 60 min at 45 °C, indicating notable resistance to moderate temperatures. Metal ion assays indicated that K⁺ enhanced enzymatic activity, while Zn²⁺, Cu²⁺, and Mg²⁺ were inhibitory. SDS-PAGE analysis confirmed the expression of the recombinant fusion protein, with a distinct band at approximately 114 kDa corresponding to the expected size. These results demonstrate the feasibility of employing the AIDA-I system for the surface display of SacA in E. coli, providing a functional platform for future applications in whole-cell biocatalysis. Full article

Anaerobic digestate from manure, a byproduct of biogas production, is increasingly used as an organic fertilizer in circular agriculture systems. This study assessed the microbiological impact of maize fertigation with anaerobic digestate, focusing on fecal indicators (Escherichia coli, Salmonella), antibiotic resistance genes (ARGs), and integrons. The trial was conducted in a commercial maize field, where on-site manure-based anaerobic digestate was applied via center-pivot irrigation. Leaf samples were collected two days (2 dai) and four weeks (4 wai) after the last fertigation. E. coli and Salmonella were assessed by culturable methods, while ARGs and integrons were analyzed by qPCR. Results showed that E. coli (3 MPN/g) and Salmonella were detected at 2 dai but were undetectable at 4 wai and in the control condition, suggesting transient contamination. The abundance of tetW was approximately tenfold higher in digestate-treated plants than in the control, while no consistent changes were observed for the other genes. Overall, fertigation with anaerobic digestate appears to pose minimal microbiological impact within the specific conditions of this study, although it may act as a source of specific resistance determinants. Although limited by the use of single treated and control plots, this study offers preliminary insight into microbial and resistance gene dynamics in the phyllosphere, providing a basis for future replicated hypothesis-driven studies. Full article

Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-E. coli) are widespread in the food chain, but nationwide surveillance in Indonesian broiler production is limited. This study investigated the occurrence, antimicrobial resistance, phylogenetic diversity, and molecular characteristics of ESBL-E. coli from broilers in Indonesia. A total of 2182 E. coli isolates from broiler cecal samples across three regions during the period 2018–2020 were analyzed. Antimicrobial susceptibility testing and ESBL phenotyping were performed following the CLSI guidelines. ESBL resistance genes and phylogenetic groups were detected using multiplex/quadruplex PCR. ESBL-E. coli (9.9%) was most frequently observed in the western (15.2%) region, followed by the central (8.0%) and eastern (7.2%) regions. A total of 85 resistance patterns were identified, with 98.5% exhibiting multidrug resistance. The bla_CTX-M gene was detected in 97.5% of isolates, predominantly bla_CTX-M-1 (97.5%), while bla_CTX-M-9 was found in 2.5%. The bla_TEM gene was present in 33.0% of ESBL isolates; however, bla_SHV and bla_OXA-1 were absent. Phylogenetic group A predominated (42.0%), followed by E (22.5%), B1 (20.5%), F (10.5%), C (2.5%), and D (2.0%). This study demonstrates a significant occurrence of ESBL-E. coli in Indonesian broilers with regional variation and bla_CTX-M predominance. The high rate of multidrug resistance poses a serious public health concern, emphasizing the urgent need for antimicrobial stewardship and enhanced surveillance programs. Full article

Background: The emergence of pan-drug-resistant (PDR) Klebsiella pneumoniae has compromised the efficacy of last-line agents, leaving few therapeutic options. Repurposing zidovudine, an FDA-approved thymidine analog with antibacterial activity, may enhance existing therapies, but pharmacodynamic data under clinically relevant conditions are scarce. This study addresses this gap using static and dynamic in vitro models. Materials/methods: A PDR strain of Klebsiella pneumoniae harboring bla_NDM-1, bla_CMY-6, bla_CTX-M-15, bla_SHV-2, and disrupted mgrB was used in this study. Minimum inhibitory concentrations (MICs) followed by static time-kills were performed to investigate the synergistic interplay between zidovudine and last-line antibiotics (ceftazidime/avibactam, polymyxin B). To simulate human pharmacokinetics, a hollow-fiber infection model (HFIM) was employed using steady-state concentrations of zidovudine (4 mg/L), polymyxin B (4 mg/L), and avibactam (22 mg/L). Structural and morphological effects on bacterial cells were examined via fluorescence microscopy following glutaraldehyde fixation. Results: In this study, the PDR K. pneumoniae showed a ~5-fold reduction in polymyxin MIC when combined with zidovudine (from >4 µg/mL to 0.25 µg/mL). Time-kill assays demonstrated ≥2.5 log₁₀ CFU/mL bacterial reduction with zidovudine-based combinations, whereas monotherapies failed to inhibit bacterial growth. In the HFIM, the triple combination achieved rapid bactericidal activity (>3 log₁₀ CFU/mL reduction within 4 h) and sustained killing (>5–6 log₁₀ reduction maintained through 216 h), with bacterial counts remaining below 1 CFU/mL. In contrast, dual combinations initially reduced bacterial burden (1–3 log₁₀ reduction) but failed to maintain suppression, with significant regrowth (>10¹⁰ CFU/mL) observed by 168 h. Microscopy corroborated these findings, revealing extensive cellular damage in the zidovudine-containing treatment arms. These HFIM results underscore the potential of zidovudine-based triple therapy in overcoming resistance to last-line antibiotics in K. pneumoniae. Conclusions: Our results provide promising unprecedented insight into novel zidovudine-based combination therapies against difficult-to-treat MBL Gram-negatives. The observed synergy in MIC reduction, rapid killing in time-kill assays, and near-complete eradication in the HFIM underscore the therapeutic potential of this triple combination. Future studies will focus on broadening the application of these novel combinations to other ‘superbugs’, such as highly resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa. Full article

Background: Hepatocellular carcinoma (HCC) is a highly lethal malignancy with limited therapeutic options. Arctigenin (ARC), a natural lignan derived from Saussurea medusa, exhibits anti-cancer activity, but its mechanism against HCC remain incompletely elucidated. Methods: This study integrated network pharmacology, molecular docking, molecular dynamics, in vitro, and in vivo experiments to investigate ARC’s anti-HCC effects. Results: Seventy-five potential targets shared between ARC and HCC were identified, with KEGG analysis highlighting the PI3K/AKT pathway as central. ARC showed strong binding to key proteins, and molecular dynamics indicated stable interactions with PIK3CA and GSK3B. In HepG2 cells, ARC inhibited proliferation in a dose- and time-dependent manner (IC50: 11.17 μM at 24 h, 4.888 μM at 48 h), induced apoptosis at high concentrations, suppressed PIK3CA phosphorylation, and increased GSK3B (Ser9) phosphorylation. In H22 tumor-bearing mice, ARC dose-dependently inhibited tumor growth (high dose: 50.6% vs. 63.0% for CTX) with minimal weight loss. Conclusions: These findings suggest ARC suppresses HCC by modulating the PI3K/AKT pathway, providing evidence for its development as a plant-derived therapeutic agent. Full article