Dataset Information

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

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.

SUBMITTER: Berbers B

PROVIDER: S-EPMC7062872 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Berbers Bas B Saltykova Assia A Garcia-Graells Cristina C Philipp Patrick P Arella Fabrice F Marchal Kathleen K Winand Raf R Vanneste Kevin K Roosens Nancy H C NHC De Keersmaecker Sigrid C J SCJ

Scientific reports 20200309 1

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 fe ...[more]

PMID: 32152350

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Project description:Recently, the presence of an unauthorized genetically modified (GM) Bacillus subtilis bacterium overproducing vitamin B2 in a feed additive was notified by the Rapid Alert System for Food and Feed (RASFF). This has demonstrated that a contamination by a GM micro-organism (GMM) may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of RASFF. As no sequence information of this GMM nor any specific detection or identification method was available, Next GenerationSequencing (NGS) was used to generate sequence information. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. This hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qPCRdetection method.Data generated by NGS were exploited using a simple BLAST approach. A TaqMan® qPCR method was developed and tested on isolated bacterial strains and on the feed additive directly.In this study, a very simple strategy based on the common BLAST tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the B. subtilis data generated by NGS. The results were used to design and assess a new TaqMan® qPCR method, specifically detecting this GM vitamin B2 overproducing bacterium. The method complies with EU critical performance parameters for specificity, sensitivity, PCR efficiency and repeatability. The VitB2-UGM method also could detect the B. subtilis strain in genomic DNA extracted from the feed additive, without prior culturing step.The proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized GM bacterium in food and feed additives by enforcement laboratories. Moreover, this work can be seen as a case study to substantiate how the use of NGS data can offer an added value to easily gain access to sequence information needed to develop qPCR methods to detect unknown andunauthorized GMO in food and feed.

Project description:Genetic manipulation of bacterial spores of the genus Bacillus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores, coupled with their desiccation resistance, makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes, one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of GMO spores by utilizing the concept of a thymineless death, a phenomenon first reported 6 decades ago. Using Bacillus subtilis, we have inserted chimeric genes in the two thymidylate synthase genes, thyA and thyB, using a two-step process. Insertion is made first at thyA and then at thyB whereby resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine), and in its absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system, we have successfully created a number of spore vaccines as well as spores displaying active enzymes.IMPORTANCE Genetic manipulation of bacterial spores offers a number of exciting possibilities for public and animal health, including their use as heat-stable vehicles for delivering vaccines or enzymes. Despite this, one remaining problem is the fate of recombinant spores released into the environment where they could survive in a dormant form indefinitely. We describe a solution whereby, following genetic manipulation, the bacterium is rendered dependent on thymine. As a consequence, spores if released would produce bacteria unable to survive, and they would exhibit a thymineless death due to rapid cessation of metabolism. The method we describe has been validated using a number of exemplars and solves a critical problem for containing spores of GMOs in the environment.

Project description:BACKGROUND:Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT:Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION:We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.

Project description:Evolution of resistance to genetically modified Bacillus thuringiensis (Bt) crops in pest populations is a major threat to the sustainability of the technology. Incidents of field resistance that have led to control problems of Bt crops or significantly reduced susceptibility of individual Bt proteins in pyramided plants have increased dramatically across the world, especially in recent years. Analysis of globally published data showed that 61.5% and 60.0% of the cases of resistance with major alleles that allowed homozygous resistant genotypes to survival on Bt crops were functionally non-recessive and did not involve fitness costs, respectively. Dominance levels (DFLs) measured on Bt plants ranged from -0.02 to 1.56 with a mean (± sem) of 0.35 ± 0.13 for the 13 cases of single-gene resistance to Bt plants that have been evaluated. Among these, all six cases with field control problems were functionally non-recessive with a mean DFL of 0.63 ± 0.24, which was significantly greater than the DFL (0.11 ± 0.07) of the seven cases without field resistance. In addition, index of fitness costs (IFC) of major resistance was calculated for each case based on the fitness of resistant (R'R') and heterozygous (R'S') genotypes on non-Bt plants divided by the fitness of their susceptible (S'S') counterparts. The estimated IFCs for 15 cases of single-gene resistance were similar for R'R' and R'S', and for the cases with and without field resistance; and the values averaged 1.10 ± 0.12 for R'R' and 1.20 ± 0.18 for R'S'. Limited published data suggest that resistance of insects to dual/multiple-gene Bt crops is likely to be more recessive than the related single-gene resistance, but their IFCs are similar. The quantitative analysis of the global data documents that the prevalence of non-recessive resistance has played an essential role in the widespread evolution of resistance to Bt crops, while the lack of fitness costs is apparently not as critical as the non-recessive resistance. The results suggest that planting of 'high dose' traits is an effective method for Bt crop IRM and more comprehensive management strategies that are also effective for functionally non-recessive resistance should be deployed.

Dataset Information

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

Publications

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

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