Antibiotic resistance poses a significant global health threat, necessitating the development of novel strategies to combat resistant bacterial strains. This study employs in silico design and molecular docking techniques to develop novel benzimidazole derivatives targeting the AcrB efflux pump and NDM-1 carbapenemase, two critical mechanisms of antibiotic resistance. Using a suite of computational tools including Chemdraw, Spartan14, Chimera, Autodock Vina, and Biovia Discovery Studio, we designed and analyzed five benzimidazole derivatives (B1-B5). Biovia Discovery Studio was specifically utilized for the visualization and analysis of molecular interactions, providing crucial insights into the binding modes and types of interactions involved. The compounds were evaluated for drug-likeness using ADMETLab2.0 and toxicity using Protox3. The results show that all compounds comply with Lipinski's rule of five and exhibit high GI absorption, indicating favorable pharmacokinetic properties. Toxicity analysis revealed low to moderate toxicity, with compound B4 showing the most favorable safety profile. Molecular docking studies, complemented by interaction visualizations, demonstrated strong binding affinities to both target proteins, with some compounds outperforming native ligands. Notably, compounds B5 and B1 showed the strongest interactions with NDM-1 Carbapenemase, while B3 and B4 exhibited comparable or better binding to the AcrB Efflux Pump than the native ligand. The visualization of these interactions revealed key binding features, including hydrogen bonds and hydrophobic interactions, providing a molecular basis for the observed affinities. These findings suggest the potential of these benzimidazole derivatives as dual-action inhibitors, simultaneously targeting two critical resistance mechanisms. This study highlights the significance of in silico methods and interaction visualization in drug discovery, and presents promising candidates for further development in combating antibiotic resistance.

Introduction: The inadequate use of antibiotics in humans, animals, and agriculture has been associated with the emergence and dissemination of antibiotic-resistant bacteria. To successfully tackle the public health problem of antibiotic resistance (ABR), it is necessary to take a One Health approach.

Methods: Focus groups were carried out among Portuguese veterinarians whose practice was in production animals (bovine, swine, ovine, and caprine). The topic guide was constructed based on the information obtained from a previous systematic review. Qualitative analysis was performed using NVivo software.

Results: Six focus groups were conducted, with a total of 19 veterinarians. From this study, four main themes emerged: (1) a lack of control of antibiotic use/drug residues in food-producing animals: surveillance/monitoring should be improved by investing in human resources/tools. The illegal access to drugs should be more strictly controlled, as it undermines antibiotic stewardship; (2) challenges in guideline adherence: guidelines should be simplified and adjusted for ABR and production animals. Authorization/access to vaccines used in other countries is difficult and time-consuming; (3) limited ABR knowledge: fear of animal loss drives antibiotic overuse/misuse; (4) client resource constraints: poor husbandry and delayed veterinary intervention, combined with veterinarians' workload and stress, contribute to inappropriate antibiotic use.

Conclusions: To promote responsible antibiotic use in veterinary medicine, we need better monitoring systems, simplified guidelines, and increased vaccine access. Educational interventions for veterinarians and clients are essential, and collaboration among all stakeholders is vital for enhancing governance and ensuring sustainable antimicrobial practices.

Funding: Project 2022.04568.PTDC, supported by the Foundation for Science and Technology (FCT) in its state budget component (OE).

Resistance to conventional antibiotics used in medicine is rising dangerously high worldwide, and could lead to more than 10 billion deaths per year by 2050 according to the World Health Organisation (WHO). New types of antibacterial molecules have to be developed and should be i) nontoxic to humans, ii) active against bacteria already resistant to conventional antibiotics, and iii) unable or less likely to cause resistance compared to conventional antibiotics. Natural plant flavonoids are known to be effective molecules, but their activity and selectivity have to be increased. Among them, aurones have attracted attention due to their good antimicrobial activity associated with limited toxicity. Various aurone derivatives were chemically synthetized and tested for their biological activity on microorganisms (bacteria and fungi) and for their toxicity on human cells. Whereas some derivatives were found to be inactive or active only on some strains, others were found to have a larger spectrum of activity with Minimum Inhibitory Concentration (MIC) values as low as 0.78 μM. Limited toxicity on human cells was found, giving high therapeutic indexes (> 100 and up to 600) for the best candidate. The data show that 5-acetamidoaurones are far more active and safer compared with 5-aminoaurones, and that benzyloxy and isopropyl substitutions at the B ring are the most promising strategy in the exploration of new antimicrobial aurones.

The drinking water cycle consists of the stages of untreated water, potable water, and sewage. Escherichia coli is considered an indicator of the faecal contamination of water, since it is a common bacterium of the intestinal flora of humans and warm-blooded animals and is a carrier of many antibiotic-resistance genes. The aim of this investigation was to assess the level of drug resistance of coliforms and E. coli isolates in samples of drinking water submitted from various sites of Central South Greece during the period 2018–2022. The highest resistance rates among both E.coli and coliform isolates were observed against ampicillin. The analysis of drug resistance conducted with reference to antibiotic groups indicated that most AMR and/or MDR isolates of E. coli or coliforms exhibited resistance against group A (ampicillin and amoxicillin/clavulanic acid). The most frequent phylogroup of the E. coli isolates was B1 followed by groups A and B2. The genus assignment for the coliform isolates other than E. coli was Enterobacter, Citrobacter, Klebsiella, and Serratia. In conclusion, various bacteria can be transferred from one stage of the drinking-water cycle to the next, either through the normal operation of the cycle or due to system failures, with the consequence that even drinking water contains various bacteria, pathogenic or non-pathogenic.

The increasing prevalence of antibiotic resistance is a major global healthcare concern, leading to higher mortality rates and rising treatment costs. This crisis is largely attributed to the excessive and improper use of antibiotics, which accelerates bacterial resistance. With diminishing pharmaceutical investment in novel antibiotics, there is an urgent need to explore alternative antimicrobial therapies. Snake venoms represent a promising avenue due to their complex protein compositions, which include phospholipases Aâ‚‚ (PLAâ‚‚s), L-amino acid oxidases (LAAOs), cathelicidins (CAMPs), and other protein toxins with antibacterial properties.

In this study, we evaluated the antimicrobial activity of crude venoms from seven snake species against three bacterial strains—Escherichia coli, Staphylococcus epidermidis, and Neisseria subflava—using spot assays, the well diffusion method, and microdilution assays to determine antimicrobial activity and minimum inhibitory concentrations (MICs) in both solid and liquid media.

On solid media, complete inhibition of E. coli, S. epidermidis, and N. subflava was observed with Macrovipera lebetina lebetina venom at 375 μg/50 μL and Bitis gabonica (Gaboon viper) venom at 675 μg/50 μL. Additionally, Naja nigricincta venom exhibited complete inhibition of N. subflava at 325 μg/50 μL. In liquid media, complete inhibition of all three bacterial strains occurred with M. lebetina lebetina venom at 500 μg/μL, while B. gabonica venom inhibited N. subflava at 1350 μg/μL.

These findings highlight the potential of snake venoms as novel antimicrobial agents. However, further research is necessary to assess their clinical applicability and safety. Continued investigation into venom-derived compounds may pave the way for innovative therapeutic strategies to combat antibiotic-resistant bacterial infections.

Introduction: Foodborne diseases associated with resistant and multidrug-resistant (MDR) bacteria represent a significant public health concern, with food-producing animals being a major source of these infections. The aim of this study was to identify and to characterise E. coli, Salmonella spp., and Shigella spp. isolated from turkeys and chickens of Portuguese origin produced for human consumption.

Methods: Between 2023 and 2024, 201 faecal samples (108 turkeys; 93 chickens), collected in various slaughterhouses, and 66 raw meat samples (33 each of turkeys and chickens), purchased in different retail stores, were analysed. Isolation was performed in selective and non-selective media. All isolates underwent to antimicrobial susceptibility testing and whole-genome sequencing.

Results: No cases of Shigella spp. were detected and Salmonella spp. (S. Newport) was isolated from one turkey faecal sample (0.9%). E. coli was detected in 97.5% of faeces (100% in turkeys; 94.6% in chickens) and in 92.4% of meat samples (90.9% in turkeys; 93.9% in chickens).

Virulence genes of enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC) and avian pathogenic E. coli (APEC) were identified in both animals. S. Newport was susceptible to all tested antibiotics. For E. coli, 78.1% faecal (74.1% in turkeys; 83.0% in chickens) and 78.7% (90.0% in turkeys; 67.7% in chickens) meat isolates were resistant to at least one antibiotic. The most common resistances were to ampicillin, tetracycline, and ciprofloxacin, and all isolates were susceptible to meropenem, cefoxitin and temocillin. A MDR profile was observed in 58.6% isolates (56.5% in turkeys; 61.7% from chickens), corresponding to 59.1% isolates from faeces and 56.8% from meat samples. Seven E. coli isolates (five from turkeys and two from chickens) were identified as extended-spectrum beta-lactamase (ESBL) producers (blaCTXM-15, blaCTX-M-55 and blaSHV-12), and eight isolates from turkeys carried the mcr-1.1 gene.

Conclusion: This study highlights the role of poultry slaughtered for human consumption and poultry meat as potential sources of human contamination with pathogenic and/or MDR isolates, underscoring the importance of the One Health concept.

Antibacterial resistance is one of the most significant global health threats, being, in 2019, directly responsible for 1.27 million global deaths and contributing to 4.85 million deaths. The complication of infection treatments leads to an urgent need for novel therapeutics strategies. Antimicrobial Photodynamic Therapy (aPDT) has demonstrated efficacy in eliminating various microorganisms and has become increasingly important as an effective strategy against multidrug-resistant strains. In this study, we assess the photodynamic efficiency of two different photosensitizers (PSs), Methylene Blue (MB) and a Phthalocyanine (ZnPc4+), {4, 40, 400, 4000-(29H, 31H-phthalocyanine-1,8,15,22-tetrayl-k4N29, N30, N31, N32) tetrakis [1- methylpyridiniumato 2-)]} zinc (4b) tetraiodide, for controlling skin infections by a clinical Pseudomonas aeruginosa strain. Both PSs were evaluated individually and in combination with Potassium Iodide (KI), a potentiator inorganic salt with photodynamic activity. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium and one of the most life-threatening pathogens that are frequently implicated in skin infections. For this study, in vitro assays were performed with planktonic cells and biofilms, along with ex vivo assays with porcine skin. In the in vitro assays with MB and with ZnPc4+ in combination with KI, the detection limit of the method that is used to quantify the bacteria was reached. When the PSs were conducted individually, MB was effective against planktonic cells and biofilms, but ZnPc4+ was only effective against planktonic cells. Regarding our ex vivo studies, only the MB without KI was effective against Pseudomonas aeruginosa. The addition of KI on porcine skin did not potentiate aPDT with MB and ZnPc4+. Although the PSs alone and in combination with KI on porcine skin did not achieve an inactivation as high as that in the in vitro assays, promising results were obtained with MB after two cycles of treatment. This emphasizes the need for further studies to identify novel strategies to optimize the best ex vivo conditions, namely, under red light and using more treatment cycles.

Introduction: Endophytes are microorganisms colonizing healthy plants tissue without causing any apparent symptoms and noticeable injury to the host. They are reported as reservoir of various classes of broad spectrum bioactive compounds. Hence this study was conducted to evaluate the antibacterial activity of endophytic bacteria isolated from Moringa oleifera leaves against Methicillin Resistant Staphylococcus aureus and multidrug-resistant Escherichia coli.

Methods: Endophytic bacteria were isolated from healthy leaves of Moringa oleifera and were identified based on their cultural, microscopic and biochemical characteristics. The endophytic bacteria were screened for antibacterial against Methicillin Resistant Staphylococcus aureus and multidrug-resistant Escherichia coli. Ethyl acetate extracts of metabolites produced by endophytic bacteria with activities were evaluated for antibacterial activity against MRSA and MDR E. coli using agar well diffusion assay with dimethyl sulfoxide as control. The Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of the ethyl acetate extracts were determined.

Results: Eleven endophytic bacteria were isolated from healthy leaves of Moringa oleifera. Four of the endophytes exhibited antibacterial activities against the test isolates. Ethyl acetate extracts of metabolites produced by endophytic bacteria showed a wide range of antibacterial activity with the inhibitory zone ranging from 11.0 to 27.0 mm for MRSA and 13.0 to 30.0 mm for MDR E. coli with MIC values between 50 mg/mL and 100 mg/mL. Dimethyl sulfoxide had no zone of inhibition against test isolates.

Conclusion: Ethyl acetate extracts of metabolites produced by endophytic bacteria isolated from leaves of Moringa oleifera exhibited antibacterial activity against antibiotic-resistant isolates and can serve as source of novel therapeutic agents.

Introduction: Cryptococcus (C.) neoformans has emerged as a pathogen of global importance and has been included in the critical group of the WHO fungal priority pathogens list. This inclusion is mainly due to drug resistance and ill-tolerance. This study sought to evaluate the impact of the anti-mitochondrial drug artemisinin on the growth, mitochondrial function, and virulence of cryptococcal cells. We theorise that the strictly aerobic cryptococcal cells will suffer deleterious effects when exposed to artemisinin, as they cannot switch to an alternate glycolytic fermentative pathway to generate energy.

Methods: A susceptibility assay was performed to assess the inhibitory properties of artemisinin of three C. neoformans isolates and H99, the reference laboratory strain. The ultrastructural changes were examined using SEM and TEM. Mitochondrial function was evaluated by monitoring the overproduction of ROS, cytochrome c release, and the activation of caspase 3, an early marker of apoptosis. Infection was established in Galleria mellonella, where infected larvae were monitored for cocoon formation. Expression of the IMPI gene in the larvae was also assessed.

Results: A 10-fold increase in artemisinin concentration led to growth inhibition in a dose-dependent manner. In this study, 0.1 mM was defined as the MIC_50. Treated cells showed elevated ROS levels and cytochrome c in the cytoplasm, indicating dysfunctional mitochondria. The activation of caspase 3 was notably higher in treated cells than in non-treated cells. The larvae with treated cells could form cocoons, while those infected with non-treated cells could not. Moreover, the expression levels of IMPI were significantly higher in treated cells than in non-treated cells, suggesting enhanced clearance of artemisinin-treated cells.

Conclusions: The preliminary data presented in this study present empirical evidence of artemisinin serving as a repurposed control agent that could inhibit cryptococcal growth and assist an infected animal in clearing infection.

Aquaculture is a key food production sector responsible for meeting the nutritional needs of a rapidly growing global population. However, the emergence of disease outbreaks has become a major challenge for the aquaculture industry, resulting in significant economic losses. The use of costly and toxic antibiotics for treatment has a negative impact on the aquatic environment. Consequently, there has been growing interest in probiotics as a non-antibiotic approach to managing disease outbreaks and improving fish performance. The use of the yeast probiotic Saccharomyces cerevisiae has shown remarkable benefits in aquaculture. In February 2025, a narrative search was conducted on the Web of Science (WoS) database for the period 2015-2025 to identify relevant studies investigating the beneficial effects of S. cerevisiae in aquatic species. After searching WoS, 644 articles were found, and the most relevant articles were reviewed, summarized, and discussed. The probiotic S. cerevisiae has shown a wide range of benefits, including an improved growth performance, improved feed efficiency, enhanced diversity of the gut microbiome, and a superior immune response. The implementation of S. cerevisiae has been a recent trend, and its efficacy in aquatic environments has been thoroughly investigated. This review aims to provide a valuable insight into S. cerevisiae as one of the most important aquaculture probiotics and highlights the need for more research to fully understand its benefits and mechanism of action.