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The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real- time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10−1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase- producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M- 15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacterales are a critical global health problem and wastewater treatment plants (WWTPs) can promote their spread into the environment ; yet their efficacy is not well characterized. Here, we have used conventional culturing to monitor coliform bacteria and quantitative PCR to monitor 2 ESBL and 5 carbapenemase (CP) genes and 4 enteric opportunistic pathogens (EOPs) in the influent and effluent of 7 Croatian WWTPs in two seasons. In general, levels of total, cefotaxime and carbapenem-resistant coliforms were significantly reduced but not eliminated by conventional treatment in most WWTPs. Most WWTPs efficiently removed EOPs such as K. pneumoniae and A. baumannii, while E. coli and Enterococcus spp. were reduced but still present in relatively high concentrations in the effluent. ESBL genes (blaTEM and blaCTX-M-32) were only slightly reduced or enriched after treatment. CP genes, blaKPC-3, blaNDM and blaOXA-48-like, were sporadically detected, while blaIMP and blaVIM were frequently enriched during treatment and correlated with plant size, number or size of hospitals in the catchment area, and COD effluent concentration. Our results suggest that improvements in wastewater treatment technologies are needed to minimize the risk of environmental contamination with top priority EOPs and ARGs and the resulting public health.

Antibiotic resistance is an emerging global health crisis, driven largely by overuse and misuse of antibiotics. However, there are examples in which the production of these antimicrobial agents has polluted the environment with active antibiotic residues, selecting for antibiotic resistant bacteria and the genes they carry. In this work, we have used shotgun metagenomics to investigate the taxonomic structure and resistance gene composition of sludge communities in a treatment plant in Croatia receiving wastewater from production of the macrolide antibiotic azithromycin. We found that the total abundance of antibiotic resistance genes was three times higher in sludge from the treatment plant receiving wastewater from pharmaceutical production than in municipal sludge from a sewage treatment plant in Zagreb. Surprisingly, macrolide resistance genes did not have higher abundances in the industrial sludge, but genes associated with mobile genetic elements such as integrons had. We conclude that at high concentrations of antibiotics, selection may favor taxonomic shifts towards intrinsically resistant species or strains harboring chromosomal resistance mutations rather than acquisition of mobile resistance determinants. Our results underscore the need for regulatory action also within Europe to avoid release of antibiotics into the environment.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum β-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (

Environments polluted with excessively high levels of antibiotics released from manufacturing sites can act as a source of transferable antibiotic resistance (AR) genes to human commensal and pathogenic bacteria. The aim of this study was to evaluate AR of bacteria isolated from the Sava river sediments (Croatia) at the discharge site of effluents from azithromycin production compared to those from the upstream site and isolates collected in Croatian hospitals. A total of 228 environmental strains of azithromycin-resistant bacteria were isolated and identified, with 124 from the discharge site and 104 from the upstream site. In addition, a total of 90 clinical, azithromycin-resistant streptococcal and staphylococcal isolates obtained from the Croatian Reference Center for Antibiotic Resistance Surveillance were analyzed. PCR screening of isolates on 11 relevant macrolide- resistance genes (MRGs) showed that discharge isolates had greater detection frequencies for 4 gene targets (ermB, msrE, mphE and ermF) compared to upstream isolates. Among clinical isolates, the most frequently detected gene was ermB, followed by msrD, mefE and mefC. The discharge site demonstrated a greater abundance of isolates with co-occurrence of two different MRGs (predominantly msrE-mphE) than the upstream site, but a lower abundance than the clinical sources (most commonly msrD-mefE). The simultaneous presence of three or even four MRGs was specific for the discharge and clinical isolates, but not for the upstream isolates. When MRG results were sorted by gene mechanism, the ribosomal methylation (erm) and protection genes (msr) were the most frequently detected among both the discharge and the clinical isolates. Following sequencing, high nucleotide sequence similarity was observed between ermB in the discharge isolates and the clinical streptococcal isolates, suggesting a possible transfer of the ermB gene between bacteria of clinical and environmental origin. Our study highlights the importance of environmental bacterial populations as reservoirs for clinically relevant macrolide- resistance genes.

Environmental discharges of very high (mg/L) antibiotic levels from pharmaceutical production contributed to the selection, spread and persistence of antibiotic resistance. However, the effects of less antibiotic-polluted effluents (mu g/L) from drug-formulation on exposed aquatic microbial communities are still scarce. Here we analyzed formulation effluents and sediments from the receiving creek collected at the discharge site (DW0), upstream (UP) and 3000 m downstream of discharge (DW3000) during winter and summer season. Chemical analyses indicated the largest amounts of trimethoprim (up to 5.08 mg/kg) and azithromycin (up to 039 mg/kg) at DW0, but sulfonamides accumulated at DW3000 (total up to 1.17 mg/kg). Quantitative PCR revealed significantly increased relative abundance of various antibiotic resistance genes (ARGs) against beta-lactams, macrolides, sulfonamides, trimethoprim and tetracyclines in sediments from DW0, despite relatively high background levels of some ARGs already at UP site. However, only sulfonamide (sul2) and macrolide ARG subtypes (mphG and msrE) were still elevated at DW3000 compared to UP. Sequencing of 165 rRNA genes revealed pronounced changes in the sediment bacterial community composition from both DW sites compared to UP site, regardless of the season. Numerous taxa with increased relative abundance at DW0 decreased to background levels at DW3000, suggesting die-off or lack of transport of effluent-originating bacteria. In contrast, various taxa that were more abundant in sediments than in effluents increased in relative abundance at DW3000 but not at DWO, possibly due to selection imposed by high sulfonamide levels. Network analysis revealed strong correlation between some clinically relevant ARGs (e.g. bla(GEs), bki(OXA), ennB, tet39,sul2) and taxa with elevated abundance at DW sites, and known to harbour opportunistic pathogens, such as Acinetobacter, Arcobacter,Aeromonas and Slietvanella. Our results demonstrate the necessity for improved management of pharmaceutical and rural waste disposal for mitigating the increasing problems with antibiotic resistance.

High antibiotic releases from manufacturing facilities have been identified as a risk factor for antibiotic resistance development in bacterial pathogens. However, the role of antibiotic pollution in selection and transferability of antibiotic resistance genes (ARGs) is still limited. In this study, we analyzed effluents from azithromycin-synthesis and veterinary-drug formulation facilities as well as sediments from receiving river and creek taken at the effluent discharge sites, upstream and downstream of discharge. Culturing showed that the effluent discharge significantly increased the proportion of antibiotic resistant bacteria in exposed sediments compared to the upstream ones. Quantitative real-time PCR revealed that effluents from both industries contained high and similar relative abundances of resistance genes [sul1, sul2, qacE/qacEΔ1, tet(A)], class 1 integrons (intI1) and IncP-1 plasmids (korB). Consequently, these genes significantly increased in relative abundances in receiving sediments, with more pronounced effects being observed for river than for creek sediments due to lower background levels of the investigated genes in the river. In addition, effluent discharge considerably increased transfer frequencies of captured ARGs from exposed sediments into Escherichia coli CV601 recipient as shown by biparental mating experiments. Most plasmids exogenously captured from effluent and polluted sediments belonged to the broad host range IncP-1ε plasmid group, conferred multiple antibiotic resistance and harbored class 1 integrons. Discharge of pharmaceutical waste from antibiotic manufacturing sites thus poses a risk for development and dissemination of multi-resistant bacteria, including pathogens. Keywords: Plasmid capture, Antibiotic pollution, Manufacturing, Antibiotic resistance, intI1, IncP-1

Effluents from antibiotic manufacturing may contain high concentrations of antibiotics, which are the main driving force behind the selection and spread of antibiotic resistance genes in the environment. However, our knowledge about the impact of such effluent discharges on the antibiotic resistome and bacterial communities is still limited. To gain insight into this impact, we collected effluents from an azithromycin-manufacturing industry discharge site as well as upstream and downstream sediments from the receiving Sava river during both winter and summer season. Chemical analyses of sediment and effluent samples indicated that the effluent discharge significantly increased the amount of macrolide antibiotics, heavy metals and nutrients in the receiving river sediments. Quantitative PCR revealed a significant increase of relative abundances of macrolide-resistance genes and class 1 integrons in effluent-impacted sediments. Amplicon sequencing of 16S rRNA genes showed spatial and seasonal bacterial community shifts in the receiving sediments. Redundancy analysis and Mantel test indicated that macrolides and copper together with nutrients significantly correlated with community shift close to the effluent discharge site. The number of taxa that were significantly increased in relative abundance at the discharge site decreased rapidly at the downstream sites, showing the resilience of the indigenous sediment bacterial community. Seasonal changes in the chemical properties of the sediment along with changes in effluent community composition could be responsible for sediment community shifts between winter and summer. Altogether, this study showed that the discharge of pharmaceutical effluents altered physicochemical characteristics and bacterial community of receiving river sediments, which contributed to the enrichment of macrolide-resistance genes and integrons. Keywords: Macrolide resistance, Antibiotic manufacturing, Pollution, Sediment, Macrolides, Bacterial community

Environments polluted by direct discharges of effluents from antibiotic manufacturing are important reservoirs for antibiotic resistance genes (ARGs), which could potentially be transferred to human pathogens. However, our knowledge about the identity and diversity of ARGs in such polluted environments remains limited. We applied functional metagenomics to explore the resistome of two Croatian antibiotic manufacturing effluents and sediments collected upstream of and at the effluent discharge sites. Metagenomic libraries built from an azithromycin-production site were screened for resistance to macrolide antibiotics, whereas the libraries from a site producing veterinary antibiotics were screened for resistance to sulfonamides, tetracyclines, trimethoprim, and beta-lactams. Functional analysis of eight libraries identified a total of 82 unique, often clinically relevant ARGs, which were frequently found in clusters and flanked by mobile genetic elements. The majority of macrolide resistance genes identified from matrices exposed to high levels of macrolides were similar to known genes encoding ribosomal protection proteins, macrolide phosphotransferases, and transporters. Potentially novel macrolide resistance genes included one most similar to a 23S rRNA methyltransferase from Clostridium and another, derived from upstream unpolluted sediment, to a GTPase HflX from Emergencia. In libraries deriving from sediments exposed to lower levels of veterinary antibiotics, we found 8 potentially novel ARGs, including dihydrofolate reductases and beta-lactamases from classes A, B, and D. In addition, we detected 7 potentially novel ARGs in upstream sediment, including thymidylate synthases, dihydrofolate reductases, and class D beta- lactamase. Taken together, in addition to finding known gene types, we report the discovery of novel and diverse ARGs in antibiotic-polluted industrial effluents and sediments, providing a qualitative basis for monitoring the dispersal of ARGs from environmental hotspots such as discharge sites of pharmaceutical effluents.

Effluents from pharmaceutical industries are recognized as significant contributors to aquatic pollution with antibiotics. Although such pollution has been mostly reported in Asia, knowledge on industrial discharges in other regions of the world, including Europe, and on the effects associated with such exposures is still limited. Thus, we performed chemical, microbiological and ecotoxicological analyses of effluents from two Croatian pharmaceutical industries during four seasons. In treated effluents of the company synthesizing macrolide antibiotic azithromycin (AZI), the total concentration of AZI and two macrolide by-products from its synthesis was 1–3 orders of magnitude higher in winter and springtime (up to 10.5 mg/L) than during the other two seasons (up to 638 μg/L). Accordingly, the highest total concentrations (up to 30 μg/L) in the recipient river were measured in winter and spring. Effluents from second company formulating veterinary antibiotics contained fluoroquinolones, trimethoprim, sulfonamides and tetracyclines ranging from low μg/L to approx. 200 μg/L. Low concentrations of these antibiotics, from below the limit of quantification to approx. few μg/L, have also been measured in the recipient stream. High frequency of culturable bacteria resistant to AZI (up to 83%) or sulfamethazine (up to 90%) and oxytetracycline (up to 50%) were also found in studied effluents. Finally, we demonstrated that toxicity to algae and water fleas often exceeded the permitted values. Most highly contaminated effluents induced multiple abnormalities in zebrafish embryos. In conclusion, using a wide array of analyses we have demonstrated that discharges from pharmaceutical industries can pose a significant ecological and public health concern due to their toxicity to aquatic organisms and risks for promoting development and spread of antibiotic resistance.