Nanopore-only assemblies for genomic surveillance of the global priority drug-resistant pathogen, <i>Klebsiella pneumoniae</i>

Foster-Nyarko, Ebenezer; Cottingham, Hugh; Wick, Ryan R.; Judd, Louise M.; Wyres, Kelly L.; Stanton, Thomas D.; Tsang, Kara K.; David, Sophia; Aanensen, David M.; Brisse, Sylvain; Holt, Kathryn E.

doi:10.1101/2022.06.30.498322

Cited by 3 publications

(1 citation statement)

References 79 publications

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“…Many downstream analyses do not require high-quality assemblies, e.g., one can identify the species of a genome or the presence/absence of a gene using a low-quality draft assembly [ 3 ]. There are, however, tasks that require extreme accuracy, e.g., estimating mutation rates and inferring transmission chains, where even a small number of errors can have consequences.…”

Section: Introductionmentioning

confidence: 99%

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

2023

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A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism’s genome—each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

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Section: Introductionmentioning

confidence: 99%

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

2023

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From farm to fork: persistence of clinically-relevant multidrug-resistant and copper-tolerantKlebsiella pneumoniaelong after colistin withdrawal in poultry production

Mourão

Ribeiro-Almeida

Novais

et al. 2023

Preprint

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The concern of colistin-resistant bacteria in animal-food-environmental-human ecosystems prompted the poultry sector to implement colistin restrictions and explore alternative trace metals/copper feed supplementation. The impact of these strategies on the selection and persistence of colistin-resistant Klebsiella pneumoniae (Kp) in the whole poultry-production chain needs clarification. We assessed colistin-resistant and copper-tolerant Kp occurrence in chicken raised with inorganic and organic copper-formulas from one-day-old chicks to meat (7 farms/2019-2020), after long-term colistin withdrawal (>2-years). Clonal diversity and Kp adaptive features were characterized by cultural, molecular, and whole-genome-sequencing (WGS) approaches. Most chicken-flocks (75%) carried Kp at early+pre-slaughter stages, with a significant decrease (p<0.05) in meat batches (17%) and sporadic water/feed contamination. High rates (>50%) of colistin-resistant/mcr-negative Kp were observed among faecal samples, independently of feed. Most samples carried multidrug-resistant (90%) and copper-tolerant isolates (81%; pco+sil/MICCuSO4 ≥16mM). WGS revealed accumulation of colistin resistance associated mutations and F-type multireplicon plasmids carrying antibiotic resistance and metal/copper-tolerance genes. The Kp population was polyclonal, with various lineages dispersed throughout poultry production. ST15-KL19, ST15-KL146 and ST392-KL27, and IncF plasmids were similar to those from global human clinical isolates, suggesting chicken-production as a reservoir/source of clinically-relevant Kp lineages and genes with potential risk to humans through food and/or environmental exposure. Despite long-term colistin ban limited mcr spread, it was ineffective in controlling colistin-resistant/mcr-negative Kp, regardless of feed. This study provides crucial insights into the persistence of clinically-relevant Kp in the poultry-production chain and highlights the need for continued surveillance and proactive food safety actions within a One-Health perspective.

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Integrating Nanopore MinION Sequencing into National Animal Health AMR Surveillance Programs: An Indonesian Pilot Study of Chicken Slaughterhouse Effluent and Rivers

Telussa,

Rahayu,

Yunindika

et al. 2024

Preprint

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Antimicrobial resistance (AMR) poses significant risks to human and animal health while the environment can contribute to its spread. National AMR surveillance programs are pivotal for assessing AMR prevalence, trends, and intervention outcomes, however, integrating advanced surveillance tools can be difficult. This pilot study, conducted by FAO ECTAD Indonesia and DGLAHS, Indonesian Ministry of Agriculture, evaluated the costs and benefits of integrating the Nanopore MinION, Illumina MiSeq, and Sensititre system into a culture-based slaughterhouse-river surveillance system. Water samples were collected from six chicken slaughterhouses and adjacent rivers (pre- and post-treatment effluent, upstream, downstream). Culture-based ESBL and generalE. coliconcentrations were estimated via the WHO Tricycle Protocol, while isolates (n=42) were sequenced (MinION, MiSeq) and antimicrobial susceptibility testing conducted (Sensititre). The Tricycle Protocol results provided estimates of effluent and river concentrations of ESBL and generalE. coliidentifying ESBL-to-generalE. coliratios of 13.8% and 6.2%, respectively. Compared to hybrid sequencing assemblies, MinION had a higher concordance than MiSeq for ARG identification (98%), virulence genes (96%), and locations for both (predominately plasmids). Furthermore, MinION concordance with Sensititre AST was 91%. Cost-benefit comparisons suggest sequencing can complement culture-based methods but is dependent on the value placed on the additional information gained.Highlights:This study demonstrates the integration of Nanopore MinION sequencing into a national AMR surveillance program, highlighting its potential for monitoring antibiotic-resistantE. coliin slaughterhouse effluent and rivers.Our findings show that Nanopore MinION sequencing exhibits high concordance with hybrid sequencing assemblies, validating its effectiveness for genomic surveillance in decentralized settings.The study evaluates the cost-benefit of using Nanopore MinION, Illumina MiSeq, and Sensititre AST, comparing information gained with cost.Graphical Abstract

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Nanopore-only assemblies for genomic surveillance of the global priority drug-resistant pathogen, Klebsiella pneumoniae

Cited by 3 publications

References 79 publications

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

From farm to fork: persistence of clinically-relevant multidrug-resistant and copper-tolerantKlebsiella pneumoniaelong after colistin withdrawal in poultry production

Integrating Nanopore MinION Sequencing into National Animal Health AMR Surveillance Programs: An Indonesian Pilot Study of Chicken Slaughterhouse Effluent and Rivers

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