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.

Keywords: antimicrobial resistance; culture-independent diagnostic test; food safety; metagenomics; molecular epidemiology; next-generation sequencing; public health; targeted-amplicon.

FIGURE 1

Cycle of how CIDT upsurge in clinical laboratories may have compound effects on the food industry. Increased usage of culture-independent syndromic panels to diagnose cases of acute gastroenteritis is expected to result in fewer microbial cultures referred to public health laboratories. Fewer isolates at public health laboratories will limit surveillance abilities with negative impacts to (i) monitoring trends in AMR, (ii) detection and response to food safety incidents and outbreaks, and (iii) source attribution. Consequently, the food supply in Canada and potentially other countries, may be exposed to an increase in foodborne illness. In turn, the repercussions of this cycle in the absence of reflex cultures could have serious implications at all levels of foodborne disease management.

FIGURE 2

Schematic representation of a sample workflow with timelines from patient presentation at a primary health care facility through the diagnostic process and public health surveillance in the case of a reportable disease. Depending on the facility and its use of CIDT, the biological specimen will be submitted for culture-based assay and biochemical testing; CIDT testing including molecular or serological assays; or NGS-CIDTs when all other assays have failed. In the case of a CIDT workflow, a positive assay from the clinical specimen is discarded leading to the inability to forward an isolate or specimen for public health surveillance. The prospect of NGS-CIDT use in clinical laboratories has the advantage of replacing a variety of diagnostic tests. The application of NGS-CIDT may ultimately negate the need to retain clinical specimens and isolates for public health laboratory surveillance. In the context of NGS-CIDT, while we provide a general timeframe, it should be stressed that sequencing and analysis times are highly variable and dependent on the sequencing platform and bioinformatics analysis. Importantly, while this workflow is illustrated to reflect foodborne diseases, it is easily applied to other infectious diseases, though in the case of culture-based methods, timelines would be drastically different for some pathogens.

FIGURE 3

Timelines for the detection of foodborne pathogens using past, current or future techniques implemented in food microbiology laboratories. Foodborne pathogen detection historically and at present requires prior suspicion of the suspected etiological agent. Food samples using either past or current techniques are worked up via one of two methods dependent on the culturability of the presumptive pathogen. Time to results is variable with timeframes ranging from two to more than 7 days. We propose that metagenomics and targeted-amplicon assays will be executed in food microbiology laboratories in the near future. For non-culturable pathogens such as norovirus, metagenomics approaches have already been developed to recover the pathogens (Nasheri et al., 2017) in as few as 3 days. As metagenomics and comparable detection techniques are adapted for routine use in food microbiology laboratories for both culturable and non-culturable pathogens, this data will shorten the time to foodborne pathogen identification and secondly, to issue a recall if necessary. As these methods become more common in food microbiology laboratories, sample preparation technologies will change to help shorten and possibly eliminate the need for enrichment to increase the desired target. It will be important, however, to simultaneously perform reflex culturing of the pathogen for archiving and potential litigations associated with the investigation.