There is growing understanding that the environment plays an important role both in the transmission of antibiotic resistant pathogens and in their evolution. Accordingly, researchers and stakeholders world-wide seek to further explore the mechanisms and drivers involved, quantify risks and identify suitable interventions. There is a clear value in establishing research needs and coordinating efforts within and across nations in order to best tackle this global challenge. At an international workshop in late September 2017, scientists from 14 countries with expertise on the environmental dimensions of antibiotic resistance gathered to define critical knowledge gaps. Four key areas were identified where research is urgently needed: 1) the relative contributions of different sources of antibiotics and antibiotic resistant bacteria into the environment; 2) the role of the environment, and particularly anthropogenic inputs, in the evolution of resistance; 3) the overall human and animal health impacts caused by exposure to environmental resistant bacteria; and 4) the efficacy and feasibility of different technological, social, economic and behavioral interventions to mitigate environmental antibiotic resistance.1.

Over the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context. Up to 89% of identified open reading frames encode known resistance genes, or variations thereof (> 1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1–3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe. Our findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.

Introduction: The occurrence of antibiotic resistance in faecal bacteria in sewage is likely to reflect the current local clinical resistance situation. Aim: This observational study investigated the relationship between Escherichia coli resistance rates in sewage and clinical samples representing the same human populations. Methods: E. coli were isolated from eight hospital (n = 721 isolates) and six municipal (n = 531 isolates) sewage samples, over 1 year in Gothenburg, Sweden. An inexpensive broth screening method was validated against disk diffusion and applied to determine resistance against 11 antibiotics in sewage isolates. Resistance data on E. coli isolated from clinical samples from corresponding local hospital and primary care patients were collected during the same year and compared with those of the sewage isolates by linear regression. Results: E. coli resistance rates derived from hospital sewage and hospital patients strongly correlated (r² = 0.95 for urine and 0.89 for blood samples), as did resistance rates in E. coli from municipal sewage and primary care urine samples (r² = 0.82). Resistance rates in hospital sewage isolates were close to those in hospital clinical isolates while resistance rates in municipal sewage isolates were about half of those measured in primary care isolates. Resistance rates in municipal sewage isolates were more stable between sampling occasions than those from hospital sewage. Conclusion: Our findings provide support for development of a low-cost, sewage-based surveillance system for antibiotic resistance in E. coli, which could complement current monitoring systems and provide clinically relevant antibiotic resistance data for countries and regions where surveillance is lacking.

Antibiotic resistance surveillance data is lacking in many parts of the world, limiting effective therapy and management of resistance development. Analysis of urban wastewater, which contains bacteria from thousands of individuals, opens up possibilities to generate informative surveillance data in a standardized and resource-efficient way. Here, we evaluate the relationship between antibiotic resistance prevalence in E. coli from wastewater and clinical samples by studying countries with different resistance situations as assessed by traditional clinical surveillance. Composite, influent wastewater samples were collected over 24 h from treatment plants serving major cities in ten European countries. Using a broth screening method, resistance to six antibiotic classes was analyzed for 2507 E. coli isolates (n = 247-252 per country). Resistance prevalence in wastewater E. coli was compared to that in clinical E. coli reported by the European Antibiotic Resistance Surveillance Network. Resistance prevalence was lower in wastewater than clinical E. coli but followed similar geographic trends. Significant relationships were found for resistance to aminopenicillins (R² = 0.72, p = 0.0019) and fluoroquinolones (R² = 0.62, p = 0.0072), but not for aminoglycosides (R² = 0.13, p = 0.31) and third-generation cephalosporins (R² = 0.00, p = 0.99) where regression analyses were based on considerably fewer resistant isolates. When all four antibiotic classes were taken into account, the relationship was strong (R² = 0.85, p < 0.0001). Carbapenem resistance was rare in both wastewater and clinical isolates. Wastewater monitoring shows promise as method for generating surveillance data reflecting the clinical prevalence of antibiotic resistant bacteria. Such data may become especially valuable in regions where clinical surveillance is currently limited.

Metallo-β-lactamases are bacterial enzymes that provide resistance to carbapenems, the most potent class of antibiotics. These enzymes are commonly encoded on mobile genetic elements, which, together with their broad substrate spectrum and lack of clinically useful inhibitors, make them a particularly problematic class of antibiotic resistance determinants. We hypothesized that there is a large and unexplored reservoir of unknown metallo-β-lactamases, some of which may spread to pathogens, thereby threatening public health. The aim of this study was to identify novel metallo-β-lactamases of class B1, the most clinically important subclass of these enzymes. Based on a new computational method using an optimized hidden Markov model, we analyzed over 10,000 bacterial genomes and plasmids together with more than 5 terabases of metagenomic data to identify novel metallo-β-lactamase genes. In total, 76 novel genes were predicted, forming 59 previously undescribed metallo-β-lactamase gene families. The ability to hydrolyze imipenem in an Escherichia coli host was experimentally confirmed for 18 of the 21 tested genes. Two of the novel B1 metallo-β-lactamase genes contained atypical zinc-binding motifs in their active sites, which were previously undescribed for metallo-β-lactamases. Phylogenetic analysis showed that B1 metallo-β-lactamases could be divided into five major groups based on their evolutionary origin. Our results also show that, except for one, all of the previously characterized mobile B1 β-lactamases are likely to have originated from chromosomal genes present in Shewanella spp. and other Proteobacterial species. This study more than doubles the number of known B1 metallo-β-lactamases. The findings have further elucidated the diversity and evolutionary history of this important class of antibiotic resistance genes and prepare us for some of the challenges that may be faced in clinics in the future.