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Additional file 1: Fig. S1. Size and mass distribution of extracted DNA after purification. Measurement was conducted on an Agilent high sensitivity TapeStation. This analysis demonstrated that 13.4% of extracted DNA has fragment smaller than 4851 bp, likely resulted from mechanical cell lysis of bead-beating in the extraction process. Fig. S2. VirSorter predicted numerous phages in the raw or assembled nanopore reads. (a). Both raw and assembled nanopore reads produced thousands of viral contigs. Cat1: VirSorter category 1 (most confident phage read/contig); Cat2: Virsorter category 2: (intermediate confidence phage read/contig); cat4: VirSorter category 4 (confident prophages). (b) Taxonomy of the best hits for each mobileOG present on the VirSorter-classified reads. (c) The majority of mobileOG-db hits (290 of 312) had identity values less than 60% and bitscore values under 100. (d) Only four shared mobileOGs were detected (mobileOG_000109022: clpB; chaperone, mobileOG_000314698: insF, integrase; mobileOG_000363469: intF, integrase; mobileOG_000712576: Lambda like terminase). Fig. S3. Alignment accuracy (%) of identified ARG sequences in nanopore reads to their reference genes. The alignment accuracy shows the percentage of base pairs in a nanopore read matching the reference ARG. Asterisks indicate significant difference (p

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 ��-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 (