Antibiotic resistance (AR) remains one of the greatest threats to global health, and Aeromonas species have the potential to spread AR in the aquatic environment. The spread of resistance to antibiotics important to human health, such as third-generation cephalosporins (3GCs) and carbapenems, is of great concern. We isolated and identified 15 cefotaxime (3GC)- and 51 carbapenem-resistant Aeromonas spp. from untreated hospital and treated municipal wastewater in January 2020. The most common species were Aeromonas caviae (58%), A. hydrophila (17%), A. media (11%), and A. veronii (11%). Almost all isolates exhibited a multidrug-resistant phenotype and harboured a diverse plasmidome, with the plasmid replicons ColE, IncU, and IncR being the most frequently detected. The most prevalent carbapenemase gene was the plasmid-associated bla_KPC-2 and, for the first time, the bla_VIM-2, bla_OXA-48, and bla_IMP-13 genes were identified in Aeromonas spp. Among the 3GC-resistant isolates, the bla_GES-5 and bla_MOX genes were the most prevalent. Of the 10 isolates examined, three were capable of transferring carbapenem resistance to susceptible recipient E. coli. Our results suggest that conventionally treated municipal and untreated hospital wastewater is a reservoir for 3GC- and carbapenem-resistant, potentially harmful Aeromonas spp. that can be introduced into aquatic systems and pose a threat to both the environment and public health.

The role of marine environments in the global spread of antibiotic resistance still remains poorly understood, leaving gaps in the One Health-based research framework. Antibiotic resistance genes (ARGs) encoding resistance to five major antibiotic classes, including sulfonamides (sul1, sul2), tetracyclines (tetA, tetB), β-lactams (bla_CTX-M, bla_TEM bla_VIM), macrolides (ermB, mphA), aminoglycosides (aac3-2), and integrase gene (intl1) were quantified by RT-qPCR, and their distribution was investigated in relation to environmental parameters and the total bacterial community in bottom layer and surface waters of the central Adriatic (Mediterranean), over a 68 km line from the wastewater-impacted estuary to coastal and pristine open sea. Seasonal changes (higher in winter) were observed for antibiotic resistance frequency and the relative abundances of ARGs, which were generally higher in eutrophic coastal areas. In particular, intl1, followed by bla_TEM and bla_VIM, were strongly associated with anthropogenic influence and Gammaproteobacteria as their predominant carriers. Water column stratification and geographic location had a significant influence on ARGs distribution in the oligotrophic zone, where the bacterial community exhibited a seasonal shift from Gammaproteobacteria in winter to Marine group II in summer.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic–protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

Agricultural production without pollution is possible using bioactive plant compounds, which include essential oils as important substances of plant origin. The aim of this study was to evaluate the antiphytoviral and antibacterial potentials of lipid (essential oil, EO) and water (hydrosol, HY) extracts from five Veronica species (Plantaginaceae) obtained by Clevenger hydrodistillation (HD) and microwave-assisted extraction (MAE), with analysis by gas chromatography coupled with mass spectrometry. The antiphytoviral activities of both extracts were tested on local host plants infected with tobacco mosaic virus (TMV). The antibacterial potential was tested against ten strains of opportunistic pathogens using the broth microdilution test. Species V. chamaedrys EO-MAE extract, V. arvensis EO from both extractions and V. montana, V. serpyllifolia, and V. persica EO-HD extracts were more effective in inhibiting TMV infection. Furthermore, HY- HD extracts of V. arvensis, V. chamaedrys and V. persica showed significant antiphytoviral activity. HY fractions had no effect on bacterial growth, regardless of the Veronica species tested, likely due to the fact that the maximum concentrations of the HY fractions tested in this study were low (1.83 and 2.91 mg/mL). EOs showed significant antibacterial activity independent of the extraction method. Notably, V. chamaedrys EO-MAE fraction, showed significantly better activity against Listeria monocytogenes and Enterococcus faecalis. Also, the EO-HD fraction of V. arvensis showed slightly better antibacterial activity. By combining extracts and using different extraction methods, valuable bioproducts can be obtained from the investigated Veronica species for safe use in agricultural production and food conservation.