Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

ISfinder () is a dedicated database for bacterial insertion sequences (ISs). It has superseded the Stanford reference center. One of its functions is to assign IS names and to provide a focal point for a coherent nomenclature. It is also the repository for ISs. Each new IS is indexed together with information such as its DNA sequence and open reading frames or potential coding sequences, the sequence of the ends of the element and target sites, its origin and distribution together with a bibliography where available. Another objective is to continuously monitor ISs to provide updated comprehensive groupings or families and to provide some insight into their phylogenies. The site also contains extensive background information on ISs and transposons in general. Online tools are gradually being added. At present an online Blast facility against the entire bank is available. But additional features will include alignment capability, PsiBLAST and HMM profiles. ISfinder also includes a section on bacterial genomes and is involved in annotating the IS content of these genomes. Finally, this database is currently recommended by several microbiology journals for registration of new IS elements before their publication.

Abstract The Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca) is a curated resource providing reference DNA and protein sequences, detection models and bioinformatics tools on the molecular basis of bacterial antimicrobial resistance (AMR). CARD focuses on providing high-quality reference data and molecular sequences within a controlled vocabulary, the Antibiotic Resistance Ontology (ARO), designed by the CARD biocuration team to integrate with software development efforts for resistome analysis and prediction, such as CARD’s Resistance Gene Identifier (RGI) software. Since 2017, CARD has expanded through extensive curation of reference sequences, revision of the ontological structure, curation of over 500 new AMR detection models, development of a new classification paradigm and expansion of analytical tools. Most notably, a new Resistomes & Variants module provides analysis and statistical summary of in silico predicted resistance variants from 82 pathogens and over 100 000 genomes. By adding these resistance variants to CARD, we are able to summarize predicted resistance using the information included in CARD, identify trends in AMR mobility and determine previously undescribed and novel resistance variants. Here, we describe updates and recent expansions to CARD and its biocuration process, including new resources for community biocuration of AMR molecular reference data.