Introduction: The use of antibiotics in intensive animal farming is a significant contributor to antimicrobial resistance (AMR), which is a global public health concern. However, the role of autochthonous Portuguese laying hens as reservoirs of antibiotic-resistant Escherichia coli remains underexplored. These breeds, raised in extensive systems with minimal antibiotic use, provide a unique opportunity to study AMR under lower selective pressure. This study investigated the role of autochthonous Portuguese laying hens (Preta Lusitânica, Amarela, Branca, and Pedrês Portuguesa) as carriers of antibiotic-resistant E. coli, using cloacal and eggshell swabs to assess their antimicrobial resistance profiles.

Methods: A total of 64 cloacal and 64 eggshell swabs were collected from 31 selected flocks (2 per flock, 16 per breed), yielding 67 E. coli isolates (33 from cloaca, 34 from eggshell). The isolates’ susceptibility against 10 antibiotics was assessed following EUCAST/CLSI guidelines.

Results: All cloacal isolates (100%) and 94% of eggshells isolates exhibited resistance to gentamicin. Additional resistance in cloacal isolates was observed to tetracycline (29%, Branca, Pedrês Portuguesa, andAmarela), ampicillin (14%, Branca, Preta Lusitânica, and Amarela), trimethoprim+sulfamethoxazole (14%, Branca, Pedrês Portuguesa, and Amarela), and amikacin (7%, Branca and Amarela). In eggshell isolates, resistance was observed to tetracycline (38%, across all breeds), ampicillin (15%, Pedrês Portuguesa and Amarela), amikacin (6%, Preta Lusitânica), and trimethoprim+sulfamethoxazole (3%, Branca). Resistance to at least one antibiotic was found in 100% of cloacal isolates and 97% of eggshell isolates. Multidrug resistance was identified in 14% of cloacal isolates (Branca and Amarela) and 18% of eggshell isolates (Preta Lusitânica, Amarela, and Pedrês Portuguesa).

Conclusions: As sustainable poultry farming gains importance, this pioneering study provides valuable insights into the role of autochthonous hens in AMR transmission, emphasizing their impact on food safety and global AMR mitigation. Additionally, it explores whether native hens’ by-products contribute to the spread of antibiotic-resistant E. coli through the food chain.

Acknowledgements: SALMYTH_GI2-CESPU_2022; FCT/MCTES – UIDB/50006/2020 and UIDP/50006/2020.

Staphylococcus aureus is a significant pathogen that can be transmitted through the food chain, often linked to antimicrobial resistance (AMR) and virulence factors, which contribute to its persistence and pathogenicity. Biofilm formation enhances its survival under adverse conditions and contributes to AMR dissemination. This study aimed to evaluate the antimicrobial resistance profile and biofilm formation of S. aureus strains isolated from meat samples and its implications for food safety. Seventy-five meat samples, including fresh meats and meat-based products, were collected in Northern Portugal. S. aureus strains were isolated and antimicrobial resistance was determined using the Kirby–Bauer disk diffusion method, against 14 antimicrobial agents. Resistance and virulence genes were evaluated by PCR. All samples were tested regarding their biofilm formation using the microtiter biofilm assay at 24 and 48 hours. Biofilm biomass quantification was performed via Crystal Violet staining. The results were normalized against the reference strain S. aureus ATCC® 25923 to ensure consistent comparison S. aureus isolates were resistant to penicillin (52.6%), tetracycline (44.4%), chloramphenicol (36.8%) and tobramycin (26.3%). Resistance genes found were: tetK (31.58%), cat_pc223 (21.05%) and blaZ and ant(a’)-Ia (15.79%). All isolates demonstrated the ability to form biofilms at both 24 and 48 hours. Biofilm production was lower at 24 h, with a percentage mean of 114.34% ± 12.51, when compared to 48 hours, where it increased to 115.04% ± 22.43. However, these differences were not statistically significant (p > 0.05). Additionally, our isolates showed a higher biofilm production capacity when compared to the reference strain used. These findings highlight the public health risks posed by food-adapted S. aureus, particularly their role in promoting bacterial survival through biofilm formation, and AMR in food environments, emphasizing the need for targeted strategies to mitigate these risks in the food industry.

The steady increase in antimicrobial resistance to existent antibiotics is a growing threat. The ribosome remains one of the most convenient targets for antibiotics due to its conservation, multiple copies of the rRNA genes, and universal central biochemical role. Multiple classes of ribosomal antibiotics bind to several conservative sites at or close to the crutial functional centers in this huge target. Antimicrobial resistance is provided by genes, inactivating or pumping these antibiotics out of the cell or modifying the binding sites, which are ancient products of evolution. A promising strategy to overcome evolutionarily acquired resistance is the identification of compounds with novel binding sites on the ribosome.

There are several reliable datasets about antibiotics affecting the ribosome translocation, a multistep stochastic movement that is catalysed by elongation factor G (EF-G), which transfers the ribosome from the A/A,P/P- to the P/P,E/E-state. For example, octapeptides argyrins trap the EF-G in the conformation corresponding to the pre-translocational state [1]. Even for aminoglycosides, trapping the ribosome in one of its intermediate states was recently considered to be the main mechanism of action [2]. Thus, there can be more than two sites in which drug binding can arrest the translocation process.

In order to identify another potential site for trapping ribosome translocation, we performed all-atom MD simulations of the pre- and post-translocational states of the 70S ribosome. In the obtained trajectories, our comparative and cross-correlation analyses revealed reproducible differences in non-covalent interactions (hydrogen bonds and stacking interactions) in elongation factor G and intersubunit bridges. A time series analysis of occurencies of these interactions detected "hotspots" of multiple conserted interactions that define one of the intermediate states of translocation.

All the MD simulations were performed using the Lomonosov-II supercomputer of Lomonosov Moscow State University's supercomputer center.

1. Proc Natl Acad Sci USA. 2022 119(19):e2114214119.

2. Nucleic Acids Res. 2021 49(12):6880–6892

Abstract: Background and Objectives: Diabetes is linked to a higher risk of urinary tract infections (UTIs) in women, often leading to recurrent antibiotic treatment. Frequent antibiotic use for UTIs can contribute to antimicrobial resistance (AMR), a critical public health threat that increases treatment failure. This study investigated the prevalence of AMR and its associated factors among women with UTIs, comparing those with and without diabetes. Results: The study population had a mean age of 52 years (SD = 23) for women without diabetes and 68 years (SD = 14) for those with diabetes. Resistance was highest for cefazolin and levofloxacin in the Access and Watch antibiotic groups, while ciprofloxacin was the most frequently prescribed antibiotic. AMR prevalence was 35.7% among women with diabetes and 21.3% among those without. After adjustment, AMR was significantly associated with both uncomplicated diabetes (OR 1.14, 95% CI 1.08–1.21) and complicated diabetes (OR 1.54, 95% CI 1.45–1.64), as well as with higher numbers of prescribed antibiotics (OR 277.39,95% CI 253.79–303.17). Methods: Using a cross-sectional cohort from the Physionet database, we analyzed data on 116,902 female participants treated for UTIs, including their antibiotic exposure, diabetes status, comorbidities, and hospital admission details. Antimicrobials were classified per the WHO’s AWaRe criteria. The primary outcome was AMR identified in urine cultures, and the association with diabetes status was evaluated using multivariable logistic regression. Conclusions: Our findings highlight the need for focused antimicrobial stewardship in women with diabetes to reduce AMR rates in this vulnerable group.

In recent years, antibiotic resistance in groundwater has become a significant environmental and public health concern, with limited research on the issue. This review aims to synthesize existing research on the occurrence, distribution, and factors influencing the presence of antibiotics and antibiotic-resistant bacteria (ARB) in groundwater. Identifying and understanding the sources and pathways for antibiotics and ARB is critical in groundwater, as it is a vital source of drinking water for 2.2 billion people worldwide and is recurrently linked to significant outbreaks of infection. This review includes studies from the years 2004 to 2025 and it revealed that groundwater samples contained isolates that were resistant to antibiotics. Most of the studies on occurrence of antibiotics resistance in groundwater was carried out in China. Our meta-analysis revealed that sulfonamides, tetracyclines, and fluoroquinolones, are frequently detected in groundwater sources although the occurrence is less compared other water matrices. The highest mean value of CIP in groundwater was reported to be 28.13 mg/L . Reactivity, Hydrogeological and Physicochemical factors are vital factors involved in the transport and fate of ARB in groundwater, whereas environmental factors, such as groundwater flow, aquifer characteristics, and proximity to pollution sources, contribute to the variability of ARB contamination levels. This review highlights the urgent need for enhanced monitoring, improved wastewater management, and effective strategies to mitigate the infiltration of antibiotics and ARB into groundwater to protect both environmental and public health.

Pseudomonas aeruginosa, a notorious pathogen, is responsible for severe human infections such as respiratory tract infections, soft tissue infections, and urinary tract infections. Beta-lactams are the cornerstone of treatment for these infections; however, the rise of beta-lactamase enzymes has steadily eroded their efficacy. Among these, Bel-1, a Class A extended-spectrum beta-lactamase (ESBL), stands out due to its unusual features resembling Class A carbapenemases, including a distinct Cys69-Cys238 disulfide bond. Despite this, Bel-1 primarily exhibits ESBL-like activity. To uncover its evolutionary trajectory towards carbapenemase activity, we conducted a detailed biochemical characterization of Bel-1 with various beta-lactam drugs. Interestingly, oxacillin inhibited Bel-1, consistent with its ESBL nature. Using isothermal titration calorimetry (ITC), we studied oxacillin binding and further explored substrate interactions by employing a deacylation-deficient mutant (E166A), enabling substrate-binding analysis without catalytic turnover. We crystallized the wild-type Bel-1 (PDB: 8JK3) and its E166A mutant (PDB: 8JOI), performing thermal melting and molecular dynamics simulations at various temperatures to examine the role of E166 in enzyme stability. These experiments revealed E166's crucial contribution to maintaining enzyme stability and its involvement in deacylation water positioning within the active site. While deacylation water was present in wild-type Bel-1, it was absent in the E166A mutant, underscoring E166's role in substrate processing. This study provides significant insights into Bel-1's behavior, delineating its distinctions from Class A carbapenemases and advancing our understanding of beta-lactamase evolution and function.

Introduction: Among the multiple mechanisms of acquired drug resistance in coagulase-negative staphylococci (CoNS), methicillin resistance appears to be the most important from a clinical and epidemiological perspective. According to the Clinical and Laboratory Standard Institute (CLSI) guidelines, the detection of mec genes is the gold standard for identification of staphylococcal methicillin resistance. In this study, we tested the efficiency of routine methods in the detection of methicillin-resistant coagulase-negative staphylococci strains (MRCoNS).

Methods: Methicillin resistance was identified using cefoxitin (30 μg) and oxacillin (1 μg) by the disk diffusion method (DDM) on Mueller–Hinton agar (Becton Dickinson, Franklin Lakes, NJ, USA) per CLSI recommendations. Oxacillin MICs (Minimal Inhibitory Concentrations) were determined by the agar dilution method (ADM) according to CLSI recommendations [M07-A10]. To detect the mecA gene, mecA primers were designed. For mecA-negative CoNS strains, the mecC and mecB genes were tested by simplex PCR.

Results: The sensitivity of the disk diffusion method to oxacillin compared to the detection of the mecA gene was 100%, while the serial dilution method for oxacillin had a lower value (87.1%). A lower sensitivity was obtained for the disk diffusion method for cefoxitin (69.7%), while the specificity of the disk diffusion method for cefoxitin was the highest, at 95.4%. A slightly lower specificity was obtained for oxacillin by the serial dilution and disk diffusion methods, at 90.6% and 87.1%, respectively. According to CLSI guidelines, the reference for phenotypic methods was the detection of mec genes by PCR.

Conclusions: Oxacillin-based methods were highly sensitive in detecting mecA-positive CoNS strains. Due to the heterogeneity of MRCoNS, confirmation of methicillin resistance by different methods seems crucial. This can prevent the misidentification of MRCoNS and failed antibiotic therapy.

Introduction. Phage–antibiotic synergy (PAS) is a novel concept that highlights the combined use of bacteriophages and antibiotics in the fight against bacterial infections. PAS shows promise in overcoming antimicrobial resistance, as phages can enhance the bacterial sensitivity to antibiotics and influence biofilms, resistance mechanisms, and the metabolic activity of microorganisms. In addition to these positive effects, an antagonistic effect has also been described in the interaction between bacteriophages and certain types of antimicrobial agents. Understanding the dynamics of this interaction is crucial for the development of innovative therapeutic strategies against multidrug-resistant pathogens.

Methods. To determine the effect of different antibiotics on bacteriophage reproduction, a modification of the disk-diffusion method was used. For this, a daily culture of host bacteria and the corresponding model phage strain were added to a melted and cooled medium. After the medium solidified, antibiotic disks were placed on its surface. The effect of antibiotics on bacteriophage reproduction was assessed after 24 hours by determining the inhibition or enhancement of plaque formation at the boundary of the bacterial culture growth inhibition zone. The bacterial test culture used was Staphylococcus aureus DSM 799, and the model bacteriophage was phage St12f.

Results. The experimental results showed that different classes of antibiotics affected the reproduction of the bacteriophage St12f in different ways. Three types of effects were observed: synergy (the enhancement of plaque formation in the zone with sublethal doses of antibiotics), antagonism (reductions in the number and size of plaques), and neutral action. Specifically, synergistic effects were observed with penicillins, cephalosporins, and fluoroquinolones; antagonistic effects were noted with certain antibiotics from the groups of macrolides, tetracyclines, oxazolidinones, rifamycins, sulfonamides, and phenicols. Aminoglycosides exhibited neutral effects.

Conclusions. We believe that studying phage–antibiotic interactions should become an essential component of the combined use of phages and antibiotics for the treatment of various bacterial infections.

Bacterial infections are the second leading cause of mortality worldwide and represent a major global health challenge. In 2023, approximately 1.25 million deaths were linked to tuberculosis, making it one of the deadliest infectious diseases in the world. In recent years, non-tuberculous mycobacteria (NTM) have also become a public health issue and are closely monitored by researchers. Currently, their incidence exceeds that of tuberculosis in North America and Europe, with 1.0 to 1.8 cases per 100,000 people. NTM are opportunistic bacteria and commonly found in natural or urban water sources. Some species are known as pulmonary pathogens and mainly infect people with compromised immune systems or pre-existing lung diseases such as cystic fibrosis, bronchiectasis, or chronic obstructive pulmonary disease. NTM are classified into two main groups : slow-growing species such as MAC complex (with M. avium, M. intracellulare, and M. chimaera mainly), M. xenopi and M. kansasii, and rapid-growing species such as M. abscessus complex (smooth and rough) and M. fortuitum. Current NTM infection treatments involve a long treatment (18 to 24 months) and rely on a combination of three antibiotics, generally including a macrolide. However, the effectiveness of these treatments remains limited, with a moderate cure rate, some side effects, and an increasing resistance to macrolides. Some developed or commercial antimicrobial agents, containing a quinoline ring, have shown potent antimycobacterial activity against NTM, such as bedaquiline (BQ), mefloquine (MQ), and Labio-17. Based on MQ’s pharmacophore, the main objective of this work was to develop new 2,8-bis(trifluoromethyl)quinoline-4-carboxamides as new antimycobacterial agents. Here, we present the design, synthesis, and first biological results of new MQ-based compounds.

Antimicrobial resistance (AMR) is recognized as a global public health threat. Resistant bacteria have been disseminated in ecosystems with AMR in isolates from wildlife which were not directly exposed to antimicrobials has been reported. The aim of the present study was to detect AMR in Enterobacteriales isolates recovered from wild red foxes (Vulpes vulpes) in Latvia. Identified microorganisms were confirmed with matrix-assisted laser desorption/ionization (MALDI-TOF MS), and antimicrobial resistance was detected with the minimum inhibitory concentration (MIC) test. Antimicrobial resistance genes were identified with whole-genome sequencing (WGS). A total of 27 isolates of Enterobacteriales, including Enterobacter cloacea (1), E. asburiae (1), Enterobacter coli (21), Hafnia alvei (3) and Salmonella Typhimurium (1), were identified in 32 red fox faecal samples. Among the isolates, 19 (70%) did not exhibit resistance to any of the tested antimicrobials. None of the strains exhibited multidrug resistance. Resistances to ampicillin (4/27, 15%), colistin (3/27, 11%), azithromycin (2/27, 7%), ceftazidime (2/27, 7%) and cefotaxime (2/27, 7%) were the most frequently reported; the antimicrobial resistance pattern differed between Enterobacteriales species. S. Typhimurium was susceptible against all antibiotics tested. The most prevalent AMR genes were acrF, blaEC and mdtM. One E. coli (1/21, 5%) isolate was confirmed as extended-spectrum beta-lactamase producing E. coli (blaTEM-1). The present study highlights the presence of AMR in Enterobacteriales in foxes from areas without direct exposure to anthropogenic factors.