Integrating Scalable Genome Sequencing Into Microbiology Laboratories for Routine Antimicrobial Resistance Surveillance

Abstract

Antimicrobial resistance (AMR) is considered a global threat, and novel drug discovery needs to be complemented with systematic and standardized epidemiological surveillance. Surveillance data are currently generated using phenotypic characterization. However, due to poor scalability, this approach does little for true epidemiological investigations. There is a strong case for whole-genome sequencing (WGS) to enhance the phenotypic data. To establish global AMR surveillance using WGS, we developed a laboratory implementation approach that we applied within the NIHR Global Health Research Unit (GHRU) on Genomic Surveillance of Antimicrobial Resistance. In this paper, we outline the laboratory implementation at 4 units: Colombia, India, Nigeria, and the Philippines. The journey to embedding WGS capacity was split into 4 phases: Assessment, Assembly, Optimization, and Reassessment. We show that on-boarding WGS capabilities can greatly enhance the real-time processing power within regional and national AMR surveillance initiatives, despite the high initial investment in laboratory infrastructure and maintenance. Countries looking to introduce WGS as a surveillance tool could begin by sequencing select Global Antimicrobial Resistance Surveillance System (GLASS) priority pathogens that can demonstrate the standardization and impact genome sequencing has in tackling AMR.

Keywords: AMR surveillance; WGS; antimicrobial resistance; microbiology laboratory; whole-genome sequencing.

Figure 1.

A typical laboratory workflow to perform WGS starting with pure bacterial colony isolates. Isolates were grown in Sigma Aldrich’s BHI, lysogeny or TS broths, followed by genomic DNA isolation and quantification (using ThermoFisher Nanodrop for nucleic acid purity and ThermoFisher Qubit for absolute DNA concentration). Next, double-stranded libraries were constructed using a combination of fragmentation, adaptor ligation, and the addition of multiplexing oligos (or oligo barcodes). Finally, these libraries were sequenced on an Illumina (MiSeq) sequencer using SBS chemistry. To access the full list of SOPs for this workflow, see https://www.pathogensurveillance.net/resources/protocols/ [19]. Abbreviations: BHI, brain-heart infusion medium; PCR, polymerase chain reaction; QC, quality control; SBS, sequencing-by-synthesis; SOP, standard operating procedure; TS, tryptone soy; WGS, whole-genome sequencing; PF, pass filter.

Figure 2.

The WGS Implementation Journey (WIJ) Roadmap. A typical WIJ consisted of 4 distinct phases of activities—(1) Assessment: precise review of laboratory infrastructure using the WGS-ready checklist (Supplementary Information) followed by an IPR to evaluate competency in molecular methods (Supplementary Figure 1); Assembly: workflow mapping, procurement of WGS equipment and consumables (Supplementary Table 1), followed by a laboratory redesign if needed (Supplementary Figure 2) (based on whether a laboratory has the necessary equipment, the IPR can be fast-tracked); Optimization: hands-on WGS training workshops organized by experienced instructors; and Reassessment: automation and quality assurance of DNA, library preparation, and genome sequencing methods, making them more scalable, cost-effective, and sustainable. Subsequently, an EQA can be performed as an independent verification of process quality. As shown, EQA audits should not be fast-tracked and only be performed once laboratory users have had sufficient time to demonstrate proficiency at DNA preparation, library creation, and sequencing protocols. In the event that a participating laboratory does not pass an EQA, refresher training (or re-optimization) could be offered before the next assessment. Abbreviations: EQA, External Quality Assessment; IPR, Internal Process Review; WGS, whole-genome sequencing.

Figure 3.

Scalability of laboratory networks within a WGS-based AMR surveillance system. (a) Surveillance networks implementing AMR action plans consist of 4 broad tiers (with varying nomenclature globally). Collection site: Primary patient touchpoint (like hospitals, clinics, and diagnostic centers) involved only with collection of bacterial samples from patients, animals, and the environment, complete with geographic/temporal metadata and supplemented with bacterial species information. Sentinel site: Coordinator of several collection sites, with a robust capacity to perform bacterial identification and antimicrobial susceptibility profiling (in case their satellites lack this). These laboratories, in many cases, can expand capacity to perform downstream activities like DNA extraction before shipping isolates to an RRL/NRL (hub-and-spoke model). RRL: Operates similar to an NRL with a subnational jurisdiction. Performs confirmatory bacterial ID/AST on sentinel site referrals using higher throughput and automated methods and can perform bacterial DNA isolation protocols. If expanded to service a regional demographic, they can potentially transform into low- to medium-throughput WGS centers offering sequencing. NCL/NRL: The largest operation of its kind within a national surveillance setup. Fully equipped to perform large-scale confirmatory phenotypic bacterial/AMR typing on isolates identified and referred by regional satellites (like collection, sentinel, or even RRLs). Due to its size, infrastructure investment, on-hand expertise, and national influence, it is generally the entry point to introduce WGS for national needs. (b) To create a coordinated, sustainable flow of samples and data, countries looking to on-board WGS could consider the following network setup models—(left) centralized network: governed by a central, fully kitted, end-to-end genome sequencing center (NRL/NCL) surrounded by satellite collection and sentinel sites; (center) hub-and-spoke network: governed by a central genome sequencing center surrounded by varying degrees of laboratories, some with expandable capacity (shown above in panel a) to add growing value to the network; (right) decentralized network: the most advanced form of surveillance where each stakeholder site within a network has some level of WGS capacity on-site. Refer to Supplementary Table 3 for the advantages and potential drawbacks of implementing each of these models. Abbreviations: AMR, antimicrobial resistance; ID/AST, Identification/Antimicrobial Susceptibility Testing; WGS, whole-genome sequencing.