Culture-independent metagenomic diagnostics

Full title

Culture-independent metagenomic diagnostics for genomic surveillance and infection control of pathogenic bacteria in clinical settings.

Aim

Using the PacBio sequel II, we aim to develop a metagenomics pipeline that can be used as a diagnostic tool in a clinical setting, removing the need to perform bacterial culture.

1.  Sequencing polymicrobial communities directly from clinical samples
2.  Assemble multiple complete bacterial genomes from the metagenomic output
3. Use bioinformatic processes to develop a pipeline to adequately analyse the genome for clinical relevance, such as:

• identifying intra-host and environmental reservoirs of pathogens associated with recurrent infections and clinical outbreaks,
• recover complete plasmids with the ability to characterize their respective mobile antimicrobial resistant genes, independent of the genome, and
• track their movement in a clinical environment. All of which significantly progress the development and implementation of personalized precision medicine.

Brief project outline

Metagenomics allows construction of accurate representations of microbial communities directly from mixed samples. Thereby eliminating biases introduced by culture or gene-centric diagnostic techniques. However, metagenomic assembled genomes (MAGs) are fragmented, incomplete and prone to contamination, limiting their use for clinical diagnostics. Long read metagenomics constitutes a potential paradigm shift in how we diagnose and respond to hospital associated infections, offering the potential to recover complete genomes from each organism within a polymicrobial community.

Both clinical (blood, stool) and environmental (swabs) will directly undergo direct DNA extraction and human DNA depletion with the ZYMO HostZERO Microbial DNA Kit. Samples will then be subject to long read sequencing on the PacBio sequel II. Using existing bioinformatics software and processes such as SMRT Analysis, PacBio Devnet and Lathe, we aim to construct a reliable metagenomics pipeline to assemble complete bacterial epi-metagenomes from clinical samples. The complete bacterial metagenomes can then be analysed to: i) identify intra-host and environmental reservoirs of pathogens associated with recurrent infections and clinical outbreaks; ii) recover complete plasmids with the ability to characterize their respective mobile antimicrobial resistant genes, independent of the genome, and iii) confirm their distribution and track their movement in a clinical environment. Designing and assessing these methods will determine the feasibility of using culture-independent, host-associated, clinical samples in a diagnostic and infection control capacity. A clinical metagenomic diagnostic pipeline will significantly progress the development and implementation of personalized precision medicine, with this study highlighting the possibilities. Implementing culture-independent sequencing as a clinical diagnostic tool will have strong implications on how clinicians approach outbreaks, infection control and prevention, as well as the treatment of patients suffering from debilitating recurrent infections. Furthermore, the pipeline will provide a framework for the utilisation of personalized precision medicine. Overall, resulting in fewer pathogenic outbreaks, reducing the economic burden on patients and healthcare providers, all while improving patient treatment options and outcomes.

Genomics-based innovative aspect of proposal

Innovating a culture-independent non-bias metagenomic DNA extraction for use on clinical samples, followed by accurate assembly and identification of the resulting complete bacterial genomes using the PacBio Sequel II; will allow for the novel use of metagenomics in precision medicine.

The sequencing technology of the PacBio Sequel II is cutting edge and is expected to provide the data required to implement this clinical-based metagenomic innovation.

The proposed methods are currently beyond the capabilities of traditional molecular microbiology and genomics in the clinical setting and will transform infectious disease treatment and control when perfected. At present, infectious disease identification and surveillance is maintained by standard microbiological techniques such as 24hr organism culture, automated identification, and limited panels of phenotypic antimicrobial resistance testing. While clinical genomics is only applied to single organism, pure isolates infrequently, excluding important information such as antimicrobial resistance genes, the potential for outbreaks and other underlying organisms within a population that can be lost during culturing methods.

Once the advantages of our approach have been verified, current clinical genomic practices can be phased out and replaced with the protocols developed here. Replacement of these existing surveillance and diagnostic practices is where we will see the most from this research. It is expected that the methods developed here will outperform current practices in terms of terms of turn-around-time, accuracy and clinical relevance resulting in significant cost savings and improved patient outcomes.

Developing and facilitating culture-independent metagenomic surveillance and diagnostics will put UQCCR at the forefront of clinical genomics and precision medicine both nationally and internationally, as it is still out of reach for many institutes and hospitals.

Broad applicability of the technique

The protocols developed in this project will be directly applicable to clinical scientists, infectious diseases clinicians, medical microbiologists and infection control personal. These include a large number of infectious disease researcher at UQ (http://www.aidrc.org.au), infection control personnel at Brisbane metropolitan hospitals e.g. Royal Brisbane, Princess Alexandra Hospital.

We expect that protocols developed here will be suitable for inclusion within the portfolio of a UQ sequencing centre. Bioinformatics pipelines, visualisation tools and reporting software will be freely available via github.

Implementation into clinical practice can be facilitated through collaboration with Queensland Genomics (QG, https://queenslandgenomics.org/). QG is a multi-million-dollar investment by The Queensland Government to facilitate the integration of genomics into clinical practice. Leveraging the networks and infrastructure established by Queensland genomics (CIs Forde, Paterson and Harris) will allow long read metagenomics to be offered to Queensland health facilities.