Combining short and long read sequencing to characterize antimicrobial resistance genes on plasmids applied to an unauthorized genetically modified Bacillus

Bas Berbers,Sigrid C J De Keersmaecker,Kevin Vanneste,Raf Winand,Patrick Philipp,Nancy H C Roosens,Fabrice Arella,Assia Saltykova,Kathleen Marchal,Cristina Garcia-Graells

doi:10.1038/s41598-020-61158-0

Bas Berbers, Sigrid C J De Keersmaecker + Show 8 more

Open Access

https://doi.org/10.1038/s41598-020-61158-0

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Journal: Scientific Reports	Publication Date: Mar 9, 2020
Citations: 58	License type: open-access

Affiliation: Sciensano (Belgium), Ghent University

Abstract

Antimicrobial resistance (AMR) is a major public health threat. Plasmids are able to transfer AMR genes among bacterial isolates. Whole genome sequencing (WGS) is a powerful tool to monitor AMR determinants. However, plasmids are difficult to reconstruct from WGS data. This study aimed to improve the characterization, including the localization of AMR genes using short and long read WGS strategies. We used a genetically modified (GM) Bacillus subtilis isolated as unexpected contamination in a feed additive, and therefore considered unauthorized (RASFF 2014.1249), as a case study. In GM organisms, AMR genes are used as selection markers. Because of the concern of spread of these AMR genes when present on mobile genetic elements, it is crucial to characterize their location. Our approach resulted in an assembly of one chromosome and one plasmid, each with several AMR determinants of which five are against critically important antibiotics. Interestingly, we found several plasmids, containing AMR genes, integrated in the chromosome in a repetitive region of at least 53 kb. Our findings would have been impossible using short reads only. We illustrated the added value of long read sequencing in addressing the challenges of plasmid reconstruction within the context of evaluating the risk of AMR spread.

Highlights

Antimicrobial resistance (AMR) genes are naturally present in bacteria, where they function as a defense mechanism
We used an unauthorized genetically modified (GM) B. subtilis (2014-3557, Rapid Alert System for Food and Feed (RASFF) 2014.1249) as a case study to deliver a proof of concept for a whole genome sequencing (WGS) strategy to fully characterize the present AMR genes and their genomic location
Before applications of genetically modified microorganisms (GMMs) can be brought to the European market, the microorganism used as producer organism should be characterized and this evaluation should be presented to European Food Safety Authority (EFSA) for authorization[14]

Summary

Introduction

Antimicrobial resistance (AMR) genes are naturally present in bacteria, where they function as a defense mechanism. The rather high error rate of ONT9 would benefit from the combination of the long reads with the accuracy of short read sequencing[10] This kind of hybrid assembly approach was shown previously to be effective in reconstructing accurate, contiguous genomes, including plasmids containing AMR genes in (pathogenic) wild-type bacteria[10,11,12,13]. As there is no dossier submitted to EU for this use in the context of these regulations, this GMM is per se unauthorized and zero tolerance, including for its associated recombinant DNA, must be applied. Besides this regulatory aspect, AMR gene transmission poses health and environmental concerns. The presence of full AMR genes in food/feed additives cannot only lead to direct transmission along the food chain to pathogens, but AMR genes can spread to environmental reservoirs[24,25,26]

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