Wastewater collection
Hospital wastewater samples were collected at the central complex of Sahlgrenska University Hospital in Gothenburg, the largest hospital in Sweden, with approximately 500 beds, with wards bringing their wastewater to the sampling point. Two 24-hour composite samples, HWW1 and HWW2, were collected on January 7 and 21, 2022, respectively, with eight additional samples collected on January 9, 2025, May 13, 2025, August 12, 2025, August 26, 2025, September 16, 2025, September 23, 2025, October 2, 2025, and 08 October 2025. Composite samples were collected over each 24 hour period (approximately 8:00 a.m. to 8:00 a.m.), with approximately 31 mL sampled every 9 minutes (160 subsamples per day), collected in 5 L refrigerated containers. Fresh samples were filtered sequentially (pore size of 1.0, 0.45, and 0.22 µm) to remove particles and bacteria. The sterility of each sample was confirmed by incubating 90% wastewater with 10% (v/v) lysogeny broth (LB) at 37 °C for 24 h (to mimic subsequent testing conditions) and the optical density (OD) was measured at 600 nm. Filtered samples were stored in 50 mL Falcon tube aliquots at −20 °C (2,022 samples) and −80 °C (2,025 samples) until further analyses.
Wider sampling of wastewater from hospitals and wastewater treatment plants in the Västra Götaland region (Sahlgrenska, Östra, Mölndal, Kungälv, Södra Älvsborgs sjukhus (SÄS), Norra Älvsborgs Sjukhus (NÄL) and Skaraborgs Sjukhus (SkaS)) and Uppsala (Akademiska) followed a similar sampling. protocol but with the critical difference that the samples were frozen without prior filtering (filtered only after thawing). The approximate number of beds for each hospital providing wastewater at the sampling points can be found in Supplementary Data 14. Composite 24-hour samples (approximately 8:00-8:00) were collected on a minimum of 4 different days between September 1, 2024 and November 30, 2024, with a minimum of 4 days between sampling days (Supplementary Data 9). A 160 ml subsample from each site was mixed with 40 ml of 100% glycerol (20% final concentration) and stored at -20°C for culture-based analyzes (without filtering) and DNA extraction for sequencing. Additionally, municipal wastewater treatment plants receiving untreated wastewater from these hospitals were sampled during the same period (Supplementary Data 9). Figure 1 shows the locations of hospitals and municipal sites. Ryaverket receives wastewater from four hospitals (Sahlgrenska, Östra, Mölndal and Kungälv). Figure 1 was produced using R (v. 4.4.0) with the packages sf (v, 1.1.0), dplyr (v. 1.1.4), ggplot2 (v. 4.0.0) and ggrepel (v. 0.9.6).
Composite samples (24 h) from two hospitals in Stockholm (Södersjukhuset and Huddinge Sjukhus) and their corresponding municipal wastewater treatment plant (two tributary streams and one effluent stream) were collected in the same way as in the above extensive sampling campaign in December 2022 and February 2023.
Antibiotic usage data
Region Västra Götaland kindly provided registry data on each dose of antibiotic administered, orally and intravenously, in all departments contributing to wastewater at each hospital wastewater sampling point (aggregated data only). Neonatal services were excluded because urine and stools are routinely collected in diapers. Emergency departments were also excluded due to a different system of recording utilization. Usage data were collected for each of the 3 days preceding each of the wastewater sampling dates, recognizing that the time from administration to excretion via urine and feces will vary both between antibiotics and between patients.
Antibiotic concentrations in collected hazardous waste
Based on usage data, we selected antibiotics for quantification with the aim of identifying which antibiotics present in HWW could potentially cause selection, as described in the “Results” section. Antibiotics were analyzed in triplicate by liquid chromatography (LC) coupled with triple quadrupole mass spectrometry (MS).16. Specifically, LC-MS grade methanol and acetonitrile (Lichrosolv – hypergrade) were used and purified water was prepared using a Milli-Q Advantage system. Mobile chromatographic phases were prepared using formic acid (Sigma-Aldrich, Steinheim, Germany) at a concentration of 0.1%. All standards, including isotope-labeled standards, are listed in Supplementary Data 15. Prior to solid-phase extraction and LC-MS measurement, samples were thawed and spiked with 5 ng of each labeled and surrogate internal standard. LC-MS analysis was performed on a UHPLC system connected to a TSQ quantitative triple quadrupole MS (Thermo Scientific, San Jose, USA) equipped with a heated electrospray ionization ion source operating in positive mode. The MS parameters used for the included chemicals are presented in Supplementary Data 15.
Selection experiment with wastewater and individual antibiotics in complex communities
To assess the selectivity of wastewater or individual antibiotics, we used a previously described method using municipal wastewater microbial communities (n= 3) as inocula17.28. In particular, this is a test of selection potential and not a reconstruction of sewer ecology. Briefly, at the start of each selection experiment, a frozen batch of an inoculum was thawed to room temperature. Next, 0.1 mL aliquots were inoculated into 15 mL Falcon tubes containing 4.9 mL of a mixture consisting of 90% sterile filtered wastewater (or physiological saline as a control) and 10% LB. The tubes were incubated at 37°C and 170 rpm for 24 h. Five microliters of these cultures were serially passed daily for 72 h in a fresh tube containing the same ratio of wastewater (or physiological saline) and LB as described above. The screening potential of individual antibiotics was evaluated using the same setup. Antibiotics (Sigma Aldrich; Sweden) were tested at concentrations measured in wastewater and at threshold concentrations.
At the start of each experiment (0 h saline control) as well as after three passages, aliquots were cultured on antibiotic/non-antibiotic plates. Quantifying resistants and non-resistants E.coliCHROMagar™ ECC plates (CHROMagar™, France) were used with and without antibiotics supplemented at EUCAST 2022 clinical end points (8 mg/L and 2 mg/L amoxicillin and clavulanic acid (AMC); 4 mg/L ceftazidime (CAZ); 16 mg/L cefadroxil (CDR); 0.5 mg/L ciprofloxacin (CIP); 4 mg/L and 76 mg/L trimethoprim-sulfamethoxazole (SXT), respectively; 2 mg/L tobramycin (TOB) and 8 mg/L and 4 mg/L piperacillin and tazobactam, respectively. These antibiotics represent several major classes of antibiotics commonly used in hospitals, all with activity against; E.coli . In addition, resistance to all these antibiotics is present E.coliin wastewater treatment communities. It was therefore considered plausible that resistance to these classes could be selected, directly or indirectly (co-selection by other antibiotics). For the extensive sampling campaign, including analyzes of samples from Stockholm (ten hospitals), only resistance to CIP (0.5 mg/L) and CTX (2 mg/L) was assessed. For K. pneumoniaeagar plates were Simmons Citrate Agar, pH 7.2 plus inositol (SCAI) plates (CHROMagar™, France) and SCAI supplemented with the same antibiotics at the clinical endpoints specified for E.coli. CHROMagar™ ECC plates were incubated for 20-24 h at 37°C and typical blue colonies (E.coli ) were counted and reported in CFU/mL. SCAI plates were incubated for 44–48 h at 42°C, and typical yellow colonies were counted and reported as CFU/mL. Since SCAI plates are less specific for K. pneumoniae that CHROMagar™ is intended E.coli16.56colonies randomly selected and counted on each SCAI plate were confirmed to be K. pneumoniae using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF MS). The resistance ratio was calculated by determining the number of colonies on plates containing antibiotics (CFU/mL) compared to control plates without antibiotics. The percentage of resistance was calculated by multiplying this ratio by 100. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was used to compare wastewater with physiological saline obtained at 0 h and 72 h, with each type of resistance studied separately. The response variable in the ANOVA was the save10-transformed resistance percentage increased by 1, to account for cases where the number of colonies containing antibiotics could be 0. One-way ANOVA with Dunnett’s multiple comparisons test was performed in GraphPad Prism (v10.1.0(316)).
To go beyond classifying percent resistance above/below clinical breakpoints and to assess whether multidrug-resistant strains were selected, disk diffusion testing was performed on fifty-two K. pneumoniae isolates (26 from 0 h and 26 from 72 h) that were randomly collected from SCAI plates. Overnight cultures were prepared for each isolate and a solution corresponding to 0.5 McFarland’s standard was plated on Mueller–Hinton agar (MHA). Antibiotic disks (Oxoid, Sweden) were then placed on agar plates, which were then incubated at 37°C for 16 h. The diameter of the inhibition zone around each disc was measured and the results were interpreted according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2022).
Growth of the individual K. pneumoniae isolated in wastewater
To enable further comparison of the level of sensitivity of individual strains to antibiotics with their growth potential in hospital wastewater (HWW2), a wastewater collection K. pneumoniaeisolated (n= 117) with various resistance profiles was studied (Supplementary Data 7). The antibiotic sensitivity of each strain was assessed by the Etest® method. The strips (BioMérieux, Marcy-l’Etoile, France) contained amoxicillin/clavulanic acid (256 µg/mL),…
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