Carbapenemase-producing Klebsiella pneumoniae (CPKP) isolated from influent (I) and effluent (E) of two wastewater treatment plants, with (S1) or without (S2) hospital contribution, were investigated. The strains belonged to the Kp1 phylogroup, their highest frequency being observed in S1, followed by S2. The phenotypic and genotypic hypervirulence tests were negative for all the strains tested. At least one carbapenemase gene (CRG), belonging to the blaKPC, blaOXA-48, blaNDM and blaVIM families, was observed in 63% of CPKP, and more than half co-harboured two to four CRGs, in different combinations. Only five CRG variants were observed, regardless of wastewater type: blaKPC-2, blaNDM-1, blaNDM-6, blaVIM-2, and blaOXA-48. Sequence types ST258, ST101 and ST744 were common for both S1 and S2, while ST147, ST525 and ST2502 were found only in S1 and ST418 only in S2. The strains tested were multi-drug resistant (MDR), all being resistant to beta-lactams, cephalosporins, carbapenems, monobactams and fluoroquinolones, followed by various resistance profiles to aminoglycosides, trimethoprim-sulphamethoxazole, tigecycline, chloramphenicol and tetracycline. After principal component analysis, the isolates in S1 and S2 groups did not cluster independently, confirming that the antibiotic susceptibility patterns and gene-type profiles were both similar in the K. pneumoniae investigated, regardless of hospital contribution to the wastewater type.

As a consequence of global demographic challenges, both the artificial and the natural environment are increasingly impacted by contaminants of emerging concern, such as bacterial pathogens and their antibiotic resistance genes (ARGs). The aim of this study was to determine the extent to which anthropogenic contamination contributes to the spread of antibiotic resistant enterococci in aquatic compartments and to explore genetic relationships among Enterococcus strains. Antimicrobial susceptibility testing (ampicillin, imipenem, norfloxacin, gentamycin, vancomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole) of 574 isolates showed different rates of phenotypic resistance in bacteria from wastewaters (91.9–94.4%), hospital effluents (73.9%), surface waters (8.2–55.3%) and groundwater (35.1–59.1%). The level of multidrug resistance reached 44.6% in enterococci from hospital effluents. In all samples, except for hospital sewage, the predominant species were E. faecium and E. faecalis. In addition, E. avium, E. durans, E. gallinarum, E. aquimarinus and E. casseliflavus were identified. Enterococcus faecium strains carried the greatest variety of ARGs (bla_TEM-1, aac(6′)-Ie-aph(2″), aac(6′)-Im, vanA, vanB, ermB, mefA, tetB, tetC, tetL, tetM, sul1), while E. avium displayed the highest ARG frequency. Molecular typing using the ERIC2 primer revealed substantial genetic heterogeneity, but also clusters of enterococci from different aquatic compartments. Enterococcal migration under anthropogenic pressure leads to the dispersion of clinically relevant strains into the natural environment and water resources. In conclusion, ERIC-PCR fingerprinting in conjunction with ARG profiling is a useful tool for the molecular typing of clinical and environmental Enterococcus species. These results underline the need of safeguarding water quality as a strategy to limit the expansion and progression of the impending antibiotic-resistance crisis.

Introduction: Karstic springs are used worldwide by rural communities as sources of fresh water for humans and livestock. In Romania, one-third of the population has no direct access to a public water supply. The present study is part of a country-wide project to develop simple, quick and cheap methods for seasonal environmental and microbiological monitoring of karstic springs used as drinking water by rural populations. Critical steps for monitoring workflow consist of evaluating water quality and selecting suitable membrane filters to efficiently capture environmental DNA for further microbial diversity estimation using 16S rRNA gene-based metabarcoding. Methods: Several commercial membrane filters of different compositions and pore sizes were tested on the water sampled from three karstic springs in Romania, followed by water chemistry and whole community 16S rRNA gene-based metabarcoding analysis. Results: We found that different types of applied membrane filters provide varying recovery in diversity and abundance of both overall and pathogenic bacteria. Conclusions: The result of the experiment with different filters shows that mixed cellulose ester, cellulose acetate, and nitrate membranes of 0.20 and 0.22 µm are the best for amplicon-based metabarcoding monitoring of karst springs.

Antimicrobial resistance (AMR) is a critical threat to global health, with aquatic ecosystems playing a key role in the spread of antibiotic resistance genes (ARGs), especially those impacted by wastewater discharge. In this study, we investigate how water flow hydrodynamics influence ARG distribution and microbial community dynamics in river biofilms exposed to wastewater effluent. Experiments were performed under laboratory conditions using natural river biofilms in river flume systems that simulate static (S; stagnant pools or lake/pond-like water) and dynamic (D; flowing or river-like water) aquatic environments. High-throughput qPCR was used to analyze the presence and abundance of 36 marker genes for AMR, mobile genetic elements, and specific bacterial faecal pollution indicators. 16S rRNA gene sequencing tracked bacterial community dynamics over time. We observed distinct microbial community compositions in the S and D systems, with the D system exhibiting higher diversity. Across genes including intI1, mphA, aph3, aac(6′), blaCTX-M, ARG trajectories diverged by flow: D maintained or accumulated relative ARG abundances over time, whereas S exhibited progressive ARG loss. In contrast, the S system showed lower diversity and progressive ARG loss over time, emphasizing the role of water flow in maintaining ARG abundance within biofilm communities. Overall, these findings inform One Health and wastewater management by demonstrating that hydrodynamic conditions shape ARG trajectories, with dynamic flow promoting ARG persistence and slower loss compared to static conditions.