Aquatic ecosystems in anthropogenically impacted areas are important reservoirs of antibiotic resistance genes (ARGs) of allochthonous origin. However, the dynamics of the different ARGs within the bacterial communities of lakes and rivers, as well as the factors that drive their selection, are not completely understood. In this study, we analysed the fate of the bacterial resistome (total content of ARGs and of metal resistance genes, MRGs) for a period of six months (summer-winter) in a continuum lake-river-lake system (Lake Varese, River Bardello, Lake Maggiore) in Northern Italy, by shotgun metagenomics. The metagenomic data were then compared with chemical, physical and microbiological data, to infer the role of anthropogenic pressure in the different sampling stations. ARGs and MRGs were more abundant and diverse in the River Bardello, characterised by the highest anthropogenic pollution. The date of sampling influenced ARGs and MRGs, with higher abundances in summer (August) than in fall or in winter, when the impact of the treated wastewater discharge in the river was limited by a higher water flow from Lake Varese. ARG and MRG abundances were significantly correlated and they co-occurred in the main network analysis modules with potential pathogenic bacteria. Different levels of anthropogenic impact selectively promoted specific ARGs while others, generally abundant in waters, were not affected by anthropogenic pressure. Reducing the level of anthropogenic pressure resulted in a rapid decrease of most ARGs. From our results, the role of anthropogenic pressure in promoting the spread of specific antibiotic resistances and of potential pathogens in aquatic ecosystem becomes clear. Finally we highlight the strict correlation between ARGs and MRGs suggesting their potential co-selection in stressed aquatic bacterial communities.

Benthic foraminifera are an important component of the marine biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically, these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but does not allow discrimination between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a new and useful approach to identify living cells possessing an active metabolism. Our work is the first test of the suitability of the FISH technique, based on fluorescent probes targeting the 18S rRNA, to detect live benthic foraminifera. The protocol was applied on Ammonia group and Miliolids, as well as on agglutinated polythalamous (i.e., Leptohalysis scottii and Eggerella scabra) and soft-shelled monothalamous (i.e., Psammophaga sp. and saccamminid morphotypes) taxa. The results from FISH analyses were compared with those obtained, on the same specimens assayed with FISH, from microscopic analysis of the cytoplasm colour, presence of pigments and pseudopodial activity. Our results indicate that FISH targets only metabolically active foraminifera, and allows discerning from low to high cellular activity, validating the hypothesis that the intensity of the fluorescent signal emitted by the probe is dependent upon the physiological status of cells. These findings support the usefulness of this molecular approach as a key tool for obtaining information on the physiology of living foraminifera, both in field and experimental settings.

Wastewater treatment plants are widely recognized as hotspots for antibiotic resistance. Although activated sludge processes are not designed to lower the abundance of pathogens or antibiotic resistance genes (ARGs), their effects on the pathobiome and antibiotic resistome require in-depth investigation. To this end, we collected wastewater samples before and after activated sludge process from five municipal wastewater treatment plants, each characterized by different inlet composition and treatment capacity. We extracted both intracellular and extracellular DNA and performed shotgun sequencing to characterize the bacterial community, pathobiome, and antibiotic resistome. Our aim was to assess the effects of activated sludge processes on bacterial community composition, the abundance of potentially pathogenic bacteria and high-risk ARGs, and the potential horizontal mobility of detected ARGs. Our results showed that activated sludge processes significantly reduced the abundance of potentially pathogenic bacteria and several high-risk ARGs. Notably, while the prevalence of plasmid-associated ARGs decreased following treatment, ARG-carrying contigs assigned to bacteriophages increased, particularly in extracellular DNA samples. Overall, activated sludge processes demonstrated a beneficial microbiological effect by lowering potentially pathogenic bacteria. However, the enrichment of viral particles carrying ARGs highlights a potential risk for ARG spreading during the following processes. These findings underscore the importance of analyzing both intracellular and extracellular DNA to fully understand the role of activated sludge in mitigating antibiotic resistance and pathogens in wastewater.

Background/Objectives: Vancomycin-resistant enterococci (VRE) are significant nosocomial pathogens worldwide, potentially transmitted by food-producing animals and related products. This study investigates the epidemiological role of bovine raw milk in the transmission of VRE to humans. Methods: Bulk milk samples were screened for van gene presence using a multiplex PCR. Mastitogenic enterococci isolated from individual milk samples were tested for antimicrobial susceptibility using the broth microdilution method. Strains not susceptible to vancomycin were whole genome sequenced. Results: Overall, vanC genes were detected in 299/1026 (29.14%) bulk milk samples. Specifically, vanC1 was found in 204 samples (19.88%) and vanC2/3 in 57 samples (5.56%), with both detected simultaneously in 38 samples (3.70%). Clinically significant vanA and vanB genes were not identified. A total of 163 mastitogenic Enterococcus strains were isolated from individual milk samples. Eight different Enterococcus species were detected, with E. faecium (104/163, 63.80%) and E. faecalis (34/163, 20.86%) being the most common. Multidrug resistance was observed in 106/163 (65.03%) isolates. The most common resistance frequencies were to ciprofloxacin and erythromycin (102/163, 62.58% both), followed by quinupristin/dalfopristin (93/163, 57.06%), linezolid (65/163, 39.88%), tetracycline (58/163, 35.58%), daptomycin (46/163, 28.22%), chloramphenicol (33/163, 20.25%), ampicillin, tigecycline, and high-dosage gentamycin (8/163, 4.91% all). Resistance to teicoplanin was not observed. Two vancomycin non-susceptible strains were identified: one vanC2/3 E. casseliflavus and one vanC1 E. gallinarum. Whole genome sequencing confirmed the presence of the complete vanC gene cluster and several virulence genes in both strains. Conclusions: Our findings suggest that while raw milk is unlikely to be a source of vancomycin resistance genes of highest clinical importance (vanA or vanB), it may contribute to the spread of vanC enterococci, which are increasingly associated with human infections.

Drinking water supplies and water basins used for fisheries represent two essential water sources for humans. Despite the growing accessibility of metagenomic approaches, their routine use for water quality monitoring is still limited. Many key water resources have yet to be fully characterized in terms of microbiome, pathobiome, and antimicrobial resistome. In this study, surface water samples were collected over one year from the artificial Lake Bidighinzu (drinking water supply) and the coastal lagoon Cabras (fisheries) located in the western Mediterranean area. Samples were analyzed for physical and chemical properties, and 16S rRNA gene amplicon and shotgun sequencing were used to characterize bacterial communities, pathobiomes, and antimicrobial resistomes. Physical and chemical properties were generally similar between sites, except for higher salinity in Cabras Lagoon. In Cabras Lagoon, richness of the bacterial community and pathobiome was generally higher in the largest trophic fraction (>20 μm), while in both sites the abundance of potentially pathogenic bacteria (PPB) increased at this fraction. PPB, including ESKAPE pathogens, were more abundant in Lake Bidighinzu. The overall antimicrobial resistome was similar across sites, with high-risk antimicrobial resistance genes (ARGs) such as emrB prevalent. Lake Bidighinzu also had more contigs where ARGs co-occurred with mobile genetic elements. This study highlights microbiological risks in two aquatic systems, particularly Lake Bidighinzu, and underscores the need to integrate metagenomic approaches, possibly with cultivation-based methods, to monitor water quality and assess health risks in drinking water supplies and fisheries.

Aquaculture practices generate nutrient-rich effluents with associated microbiological hazards, such as pathogens and antimicrobial resistance genes (ARGs). Despite their growing popularity as nature-based solutions, little is known about how constructed wetlands (CWs) affect the dynamics of microbial communities at the field scale. By combining flow cytometry, 16S rRNA gene sequencing, shotgun metagenomics, and metabolic potential assays, we investigated the structural and functional responses of the aquatic microbial community following the recurrent exposure to CW-treated effluents from an intensive marine fish farm (Orbetello lagoon, Italy). While the CW promoted abundant, metabolically active, and functionally redundant microbial communities, the phylogenetic composition diverged primarily between water and sediments. Microbial profiles in CW outlet waters converged towards those of the lagoon baselines, suggesting gradual ecological recovery. The CW attenuated the occurrence of potential pathogens (e.g., Francisella spp., Campylobacter spp.) and limited ARG dissemination, though sediments remained reservoirs of microbial and genetic signatures. Functional profiles, dominated by chemoheterotrophy, denitrification, and sulfur respiration, remained stable across environments, reflecting microbial resilience. Our results highlight CWs as effective, field-proven solutions to mitigate aquaculture wastewater impacts while preserving core ecosystem services.