Biodiversity surveys through morphology provide invaluable data to inform biological monitoring efforts, involving specialised taxonomic skills that are not always available. The revolution brought by the advent of metabarcoding associated to massive sequencing is currently seen as a potential advance, even if different approaches may often provide different results. Here we test if reliable results from metabarcoding can be obtained by i) basing the analyses on a detailed knowledge of the local diversity from morphology, ii) applying tools from DNA taxonomy to create a local reference library, ii) developing custom primers, taking as example the crustacean zooplankton of a subalpine lake in Northern Italy, Lake Maggiore. We support the idea that occurrences from metabarcoding can be reliable, especially with targeted primers, but we confirm that read numbers from massive sequencing could not be related to abundance of individuals in our analyses. Data from metabarcoding can thus be used to reliably monitor species occurrence in the lake, but not changes in abundance.

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.

Wastewater treatment plants (WWTP) effluents can release microbiological pollutants, including the int I1 gene (integrases of class 1 integrons), which has been proposed as a target for monitoring anthropogenic pollution in surface waters. This gene has also a strong correlation with antibiotic resistance, making of it an important proxy to evaluate the level of genetic contamination in aquatic environments. he ecological factors that influence the abundance and dynamics of intI 1 within natural water bodies are largely unknown. To better understand the fate of class 1 integrons in aquatic systems, we resorted to classical limnological monitoring of intI 1 over multiple years. We also conducted experiments to elucidate the impact of Daphnia grazing on its abundance. The monitoring of different size fractions of the Lake Maggiore microbial community has shown a particle-bound life-style for intI 1-harbouring bacteria. Most of the bacteria hosting intI 1, originating from both a wastewater effluent that discharges intro Lake Maggiore and lake water itself, grow on particulate substrates in open waters, making them particularly vulnerable to grazing by large filter feeders such as Daphnia . Daphnia grazing is independent from the origin (lake water or wastewater) of the bacterial genera; it selectively removes bacteria that are present in aggregates or even filamentous forms from both origins. To understand if intI 1 is related to viable bacteria or just DNA residues, it is important to study the persistence of class 1 integrons with their gene cassettes, which often contain antibiotic resistance genes in freshwater ecosystems. Significance Statement: While faecal pollution of freshwaters is commonly monitored, genetic pollution through wastewater treatment plant outflows, such as antibiotic resistance genes, is difficult to monitor due to the diverse nature of genes present. The intI 1 gene is proposed as a proxy for anthropogenic pollution; however, there is a major lack of understanding regarding the persistence of this gene in freshwaters. In this study, we demonstrate that intI 1 in freshwaters is associated with both the natural microbial community and allochthonous microbes arriving from wastewater. Furthermore, we show that intI 1 harbouring bacteria preferentially reside in the aggregated microbial fraction and are easily removed by zooplankton grazing. This study is the first limnological investigation of this gene and highlights a significant gap in our knowledge regarding the ecology of class 1 integrons. Genetic pollution of surface waters is however a global problem and of very broad interest on the one hand, on the other hand, the question of the establishment of an allochthonous gene into a natural microbial community is also an interesting fundamental question in ecology, thus this study has both more applied and more fundamental aspects. Therefore, we consider it perfect for the readership of L&O.