Microbial barcodes: The future of object provenance

• Object provenance – determining an object’s origin and history – is a fundamental challenge for human health and commerce.
• To address this problem, Nathan McDonald at the US Army DEVCOM Chemical Biological Center has optimised bio-engineered microbes containing DNA ‘barcodes’ paired with a sensitive CRISPR/Cas detection assay called SHERLOCK.
• His team demonstrated that barcoded microbes can be detected after one month on surfaces including carpet, sand, tile, linoleum and concrete, even after vacuuming or mopping.
• This system has multiple real-world applications in human health, commerce, food safety, and forensics.

A surprising amount of time and energy goes into answering the deceptively simple question of ‘where did this come from?’ – known as object provenance. From tracking agricultural and manufactured goods in our complex, globalised supply chains to determining who, or what, passed through particular locations, object provenance is a fundamental challenge for human health and commerce.

Barcoded microbial spores can be introduced to locations or objects of interest, creating a microbial ‘fingerprint’ for cheap and sensitive object provenance.

One example of this is in combating foodborne illness – a global problem, with an estimated 48 million cases a year in the USA alone. The first step in dealing with foodborne illness is to determine what the contaminants are and where they were introduced into the supply chain. Due to the complexity of modern markets, however, this can take weeks, causing massive costs and potentially leading to further cases of illness.

Object provenance is also a key consideration in forensic investigations and law enforcement. Alongside video surveillance and fingerprinting, technologies that enable the determination of whether an object or person was at a particular place are highly valuable.

Microbial fingerprints

One method for determining object provenance in forensic investigations is to analyse the microbes present on an object. Microbes are found in all environments, and any object will gradually adopt the naturally occurring microbes present in the environments it is exposed to. In principle, sequencing these microbial samples can provide a cheap and sensitive way to determine where the object has been. However, there are a number of hurdles with this approach, including the requirement for expensive and time-consuming surveys of natural microbial communities in locations of interest, the fact that many environments share very similar microbial populations, and that microbial communities in one location can vary over time. This is where a new study, led by Nathan McDonald at the US Army DEVCOM Chemical Biological Center, comes in.

Building on previous work by Professor Michael Springer at Harvard, McDonald and colleagues tested the use of synthetic, non-viable microbial spores containing ‘DNA barcodes’ to determine object provenance. Using modern molecular biology techniques, the researchers were able to take advantage of the natural information-encoding property of DNA to include DNA barcodes in microbial genomes – short artificial DNA sequences that can be read and identified by standard sequencing technologies. Barcoded microbial spores (BMS) with specific barcodes can then be introduced to locations or objects of interest, creating a microbial ‘fingerprint’ for cheap and sensitive object provenance.

A case for SHERLOCK

The team identified several key criteria for BMS to be viable in the real world: the microbes must be bio-contained (not viable in the wild), they must persist in the target environment and label objects passing through, and the decoding of the barcodes should be fast, sensitive, and specific. To address persistence, the researchers cultured metabolically dormant microbial spores, a natural part of some microbes’ life cycle, which allows them to persist in harsh environments for long periods without growth. The spores were made non-viable by removing key genes required for cell division and germination. Finally, for rapid detection, the researchers combined the BMS with SHERLOCK – a sensitive detection assay that uses cutting-edge CRISPR/Cas technology to produce a visible signal if a specific DNA sequence (or barcode) is present in a sample. SHERLOCK can be thought of as the scanner for the barcoded spores.

With the engineered BMS in hand and SHERLOCK on the case, McDonald and his team performed rigorous testing of the system in simulated home environments designed to mimic real-world scenarios. They started by spraying mixtures of BMS on carpet and linoleum. They monitored the detection of the spores over four weeks, both before and after vacuuming or mopping. Some of the BMS strains were easily detectable on both surfaces, even after four weeks and four rounds of vacuuming or mopping – these strains were selected for further testing. For the second round of testing, the best-performing BMS were released on sand, concrete, loose tiles, and carpet, and once again monitored over four weeks. Spores were still detectable after this period on all surfaces except concrete.

Inconsistencies in the BMS detection data led the team to hypothesise that variability was coming not from the spores themselves but from the sampling or sample processing methods. To address this, three sampling methods (swabs, wipes, or tape) were tested, alongside three sample processing protocols. Over the course of four weeks, they determined that both swabs and wipes worked well, and a commercial DNA extraction kit provided more consistent and sensitive detection of BMS.

This work shows that barcoded spores can be easily transferred and detected across multiple surface types over a period of at least one month.

Finally, the researchers wanted to test the applicability of the system to object provenance. Spores were sprayed on squares of carpet and concrete, then objects made of three common materials – cardboard, Styrofoam, and plastic – were placed on the squares for five minutes. The objects were moved to fresh carpet or concrete for another five minutes, and then again for 30 days; samples were taken following each transfer. BMS were readily detected even after 30 days on both cardboard and Styrofoam when using swabs and wipes. While BMS were not easily detected on the plastic object itself, it did transfer BMS to fresh carpet or concrete squares. Together, these results demonstrate the utility of BMS and SHERLOCK in addressing object provenance.

Case closed?

McDonald’s research demonstrates some of the revolutionary advancements made in molecular and synthetic biology utilising CRISPR/Cas systems, and how they can be applied in the fields of detection and diagnostics. This work shows that barcoded spores can be easily transferred and detected across multiple surface types over a period of at least one month, enabling use cases in object provenance. In addition, the optimisations developed in the study to both sample collection and preparation provide increased sensitivity for SHERLOCK and improve the transferability of this CRISPR/Cas technology from the lab to real-world applications. It looks like the cases of the future may again be solved by Sherlock, but this one won’t be smoking a pipe!