Metagenomics: The Next Culture-Independent Game Changer

Abstract

A trend towards the abandonment of obtaining pure culture isolates in frontline laboratories is at a crossroads with the ability of public health agencies to perform their basic mandate of foodborne disease surveillance and response. The implementation of culture-independent diagnostic tests (CIDTs) including nucleic acid and antigen-based assays for acute gastroenteritis is leaving public health agencies without laboratory evidence to link clinical cases to each other and to food or environmental substances. This limits the efficacy of public health epidemiology and surveillance as well as outbreak detection and investigation. Foodborne outbreaks have the potential to remain undetected or have insufficient evidence to support source attribution and may inadvertently increase the incidence of foodborne diseases. Next-generation sequencing of pure culture isolates in clinical microbiology laboratories has the potential to revolutionize the fields of food safety and public health. Metagenomics and other ‘omics’ disciplines could provide the solution to a cultureless future in clinical microbiology, food safety and public health. Data mining of information obtained from metagenomics assays can be particularly useful for the identification of clinical causative agents or foodborne contamination, detection of AMR and/or virulence factors, in addition to providing high-resolution subtyping data. Thus, metagenomics assays may provide a universal test for clinical diagnostics, foodborne pathogen detection, subtyping and investigation. This information has the potential to reform the field of enteric disease diagnostics and surveillance and also infectious diseases as a whole. The aim of this review will be to present the current state of CIDTs in diagnostic and public health laboratories as they relate to foodborne illness and food safety. Moreover, we will also discuss the diagnostic and subtyping utility and concomitant bias limitations of metagenomics and comparable detection techniques in clinical microbiology, food and public health laboratories. Early advances in the discipline of metagenomics, however, have indicated noteworthy challenges. Through forthcoming improvements in sequencing technology and analytical pipelines among others, we anticipate that within the next decade, detection and characterization of pathogens via metagenomics-based workflows will be implemented in routine usage in diagnostic and public health laboratories.

Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next‐generation sequencing strategy

“…Culture independent techniques such as 16S rRNA profiling or shotgun metagenomics widely employed in microbiome studies would also not be of great help to this purpose as they lack phylogenetic resolution (e.g. 16S rRNA gene‐based analysis) and/or might be hampered by large amounts of host DNA (Nhung et al ., ; Forbes et al ., ). For instance, C .…”

The challenges of designing a benchmark strategy for bioinformatics pipelines in the identification of antimicrobial resistance determinants using next generation sequencing technologies

“…It is clear that this covers only a fraction of the work necessary to fully implement this technology in a regulatory context, which will also need to cover additional steps such as the sampling, library preparation, sequencing run, and interpretation of the AMR profiles (see Figure 1). However, this resource would facilitate the implementation of the NGS technology in routine laboratory analyses by: Ensuring confidence in the implementation of the bioinformatics component of the procedure, a step currently identified as limiting in the field 2, 8– 10 .Allowing evaluation and comparisons of new/existing bioinformatics strategies, resources and tools.Contributing to the validation of specific pipelines and the proficiency testing of testing facilities."Future-proofing" bioinformatics pipelines to updates and replacement of the tools and resources used in their different steps. …”

“…These experiments will need to be properly characterised in terms of the "true" conclusions the benchmarked pipeline is expected to reach. In addition, although there can be actions taken to ensure that most of the host DNA is filtered from the dataset, real experiments from a human source could lead to privacy problems, while samples from food should ensure the absence of information on patented genetically modified food potentially present in the sample 8, 45 . Careful filtering against a standard “exclusion database”, or other adequate strategies, may be necessary to solve this issue - however, the risk is that the filtered dataset is no longer representative of a real experiment, which would contain a fraction of human reads.…”

Medical Management and Diagnostic Approaches

“…Other molecular methodologies such as nextgeneration sequencing, which allows for the simultaneous screening of hundreds of agents, will surely add to the arsenal of molecular techniques available to the diagnostician in the near future (Chiu, 2013;Deurenberg et al, 2017;Forbes et al, 2017;Kim et al, 2018;Simner et al, 2018). Molecular techniques are also applicable in many other areas of rat medicine and biology, including the detection of contaminants in tissue culture material and monitoring of genetic purity of inbred strains or genetically engineered rats (Bryda and Riley, 2008;Shimoyama et al, 2017).…”