by Dr Dagmara Lewandowska

Peri-implantitis and mucositis are common inflammatory microbial-biofilm diseases affecting tissues surrounding dental implants. Understanding the human oral micro­biome may hold the key to developing tailored and highly effective personalized treatments for these diseases. Liquid-handling automation, combined with magnetic bead separation, enables reliable, high-throughput next-generation sequencing library preparation for this crucial microbiome research.

The human microbiome is a complex community of microorganisms, and changes to its composition can disrupt key bodily functions, causing or aggravating various health conditions. Understanding the delicate interplay between these microorganisms and their gene expression mechanisms may, therefore, hold the key to developing tailored and highly effective personalized treatments for numerous diseases, including cancers, cardiovascular diseases, respiratory conditions and gastrointestinal disorders. Interest in the potential health impact of imbalances in the microbiome has snowballed in recent years, and many research groups around the world – such as Italian company PreBiomics S.r.l (https://www.prebiomics.com/) – are focused on decoding the human body’s unique microbial fingerprint through genetic analysis. PreBiomics was founded in 2017, and initially focused on developing its flagship microbiome-based kit, the PreBiomics Implant Test (https://shorturl.at/WyM7q), which supports dental professionals in the prevention, early diagnosis and personalized treatment of oral peri-implant diseases [1]. Peri-implantitis and mucositis are common inflammatory microbial-biofilm diseases that affect the soft and hard tissues surrounding dental implants, affecting over 20% and 50% of patients, respectively [2, 3].

Recent discoveries have shed much light on the causes of these conditions, providing vital information to support doctors in preventing, detecting and managing their patients’ conditions. It has been observed that shifts in the microbial composition of the plaque microbiome enable pathogenic bacterial species to become more prevalent. The exact composition of the microbiome associated with peri-implantitis is highly site specific, meaning it differs even within the same patient between affected and healthy implant sites [3]. However, a complex of specific pathogenic species has been found to be strongly associated with peri-implantitis [3]. This group of bacterial species is termed the peri-implantitisrelated complex (PiRC™), a microbial signature that includes the key colonizers Porphyromonas gingivalis and Fusobacterium nucleatum. The organisms within this complex are primary colonizers of the subgingival microbiota, and their proliferation can lead to inflammation. In turn, chronic inflammation creates an environment conducive to further microbial colonization and infection. Inflammation may compromise the health of the implant and cause significant bone loss in the jaw if left untreated. Bone loss can result in the failure of the implant, requiring further surgical interventions or even removal of the implant altogether. The presence of infection and chronic inflammation associated with peri-implant diseases may also contribute to other systemic health conditions, such as cardiovascular disease or diabetes [2].

INTEGRA Biosciences’ ASSIST PLUS pipetting robots
Used by PreBiomics in its high-throughput NGS laboratory to introduce automation into daily liquid-handling tasks.

Metagenomic analysis for accurate diagnosis and personalized treatment

Probiotics and localized administration of antimicrobials can promote the proliferation of health-related microbial species while eliminating harmful bacteria, helping to prevent and treat peri-implant diseases. The effectiveness of these treatments depends on the unique composition of the plaque microbiome, and so stratifying patients based on their specific microbiome profiles may help in developing tailored treatment plans and reducing the risk of disease recurrence.

Identifying the make-up of the bacterial pathogens present in the plaque microbiota requires highly specific and robust metagenomic analysis. These studies allow the accurate detection and characterization of known and unknown microorganisms in a sample – including bacteria, viruses and fungi – to provide a comprehensive picture of the oral microbial community. This information is vital for diagnosing potential diseases, and for the development of targeted medical treatments and long-term management plans.

Machine learning models based on shotgun metagenomic sequencing can be used to detect microbial signatures – such as the PiRC – enabling peri-implant health and disease to be accurately assessed with high diagnostic power (AUC >0.9). Unlike traditional 16S rRNA sequencing, metagenomic sequencing provides species- and strain-level resolution. This improves diagnostic specificity, which is crucial for PiRC detection, as only certain subspecies of F. nucleatum are associated with disease. Shotgun metagenomics also provides information on the functional potential of each species, which is essential for understanding their role in disease progression [3]. Long-term monitoring of microbial profiles can even be used to support the early detection of changes in the plaque microbiome over time, allowing the implementation of additional interventions to prevent disease progression and further complications [2]. For instance, sequencing-based machine learning models can be used to predict whether mucositis will progress to peri-implantitis, making it a valuable prognostic tool and informing timely intervention [3].

Accelerating microbiome analysis with liquid-handling automation

Microbiome analysis and research can be hindered by complex workflows and laborious manual sample preparation protocols prior to sequencing, limiting the number of labs that are able to perform completely unbiased and robust analyses. Carrying out next-generation sequencing (NGS) library preparation manually not only limits the sample throughput of a lab, but also introduces variability, impacting the reproducibility and reliability of results. Integrating automation can streamline NGS library preparation by reducing hands-on time and minimizing manual errors. Unfortunately, many labs cannot afford complex automation platforms with grippers and robotic arms.

There is, therefore, a clear need for smaller, more affordable solutions to make efficient and reliable NGS library preparation available to a wider range of labs. Cost-effective automated platforms, such as the ASSIST PLUS pipetting robot (https://shorturl.at/gf58N), can be equipped with electronic multichannel pipettes and single channel pipetting modules to perform various liquid-handling tasks with precision and consistency. These affordable solutions can play a pivotal role in enabling high-throughput NGS with reproducible and reliable results.

PreBiomics opened its new NGS laboratory in 2023, and chose to install several of these pipetting platforms to enable straightforward automation of the team’s daily liquid-handling tasks. The aim of automating certain time-consuming components of the NGS workflow was to give staff more walk-away time and greatly speed up repetitive tasks at every stage of the process, from sample reformatting through to normalization and pooling of NGS libraries, and even PCR reaction set-up. This ultimately helps the team to enhance its sample processing capacity to deal with a growing workload, as well as building flexibility into workflows for its diversifying metagenomic services portfolio. Reducing hands-on time also enables team members to focus on other, more complex and urgent tasks in the lab, such as data analysis.

Optimizing size selection with advanced magnetic bead technologies

Magnetic beads specifically designed for NGS size selection, such as MAGFLO™ NGS* magnetic beads (https://shorturl.at/SjIcx), provide a reliable solution for the purification of nucleic acid fragments with a consistent size distribution, and have become the standard in NGS library preparation processes. Recent advances in magnetic separation technologies have significantly enhanced the reproducibility of bead handling steps in NGS library preparation workflows. For example, the powerful magnets in MAG and HEATMAG modules (https://shorturl.at/sM1Dj) move up and down automatically, eliminating the need for manual plate handling. These innovative technologies enable the collection of magnetic beads together in one spot, reducing bead carry-over and simplifying later pipetting steps. Some magnetic modules, like the HEATMAG, also feature built-in heating capabilities, helping to improve the elution process and support efficient lysis during nucleic acid extraction.

PreBiomics recently conducted an evaluation comparing the performance of MAGFLO NGS* beads to industry-standard beads for size selection and library clean-up. For this beta testing trial, liquid handling was performed using an ASSIST PLUS equipped with a 12 channel electronic pipette. The magnetic separation steps were carried out twice for side-by-side comparison: once manually with a standard magnetic plate and once automated with a MAG module. Sequencing results revealed that the combination of MAGFLO NGS* beads and the MAG module outperformed other methods, including the industry-standard beads and standard magnetic plates. The set-up also produced a higher number of reads with less variation between read values, and demonstrated greater reproducibility and reduced variability compared to NGS library preparation and size selection performed with other beads and magnets. The workflow also yielded the highest library concentrations and the most consistent concentration values across all tested configurations, further emphasizing its reliability and reproducibility [4].

Summary

Fluctuations in the composition of the plaque microbiome following dental implant surgery can lead to oral peri-implant diseases. Confidently identifying the microbial signature of the plaque microbiome through genomic analysis is crucial for ensuring personalized, effective treatment with probiotics and antimicrobials. However, microbiome analysis can be hindered by complex workflows and laborious, manual sample preparation, which introduces variation and negatively impacts the reliability of results. Affordable liquid-handling instruments and recent innovations in magnetic separation technologies enable efficient and reliable NGS library preparation for more reproducible sequencing results. This combination creates a streamlined and extremely versatile system for high-throughput microbiome analysis, and can be tailored to meet various future testing demands, empowering researchers to advance the field of personalized medicine.

* At the time of publication, MAGFLO NGS magnetic beads are currently available in Austria, Denmark, Germany, Sweden, Switzerland and the United Kingdom. Consult our website (https://www.integra-biosciences.com/global/en/ngspcr-purification/magflotm-ngs) to check the up-to-date availability of MAGFLO NGS magnetic beads in your country at the time of reading this article.

The author

Dagmara Lewandowska PhD
Product Manager – Reagents
INTEGRA Biosciences, Zizers, Switzerland

Email: dagmara.lewandowska@integra-biosciences.com

 References
1. PreBiomics implant test (webpage). PreBiomics (https://www.prebiomics.com/prebiomics-implant-test/).
2. Bazzani D, Heidrich V, Manghi P et al. Favorable subgingival plaque microbiome shifts are associated with clinical treatment for peri-implant diseases. NPJ Biofilms Microbiomes 2024;10(1):12 (https://doi.org/10.1038/s41522-024-00482-z).
3. Ghensi P, Manghi P, Zolfo M et al. Strong oral plaque microbiome signatures for dental implant diseases identified by strain-resolution metagenomics. NPJ Biofilms Microbiomes 2020;6(1):47 (https://doi.org/10.1038/s41522-020-00155-7).
4. Exploring the human microbiome with automated NGS library prep (webpage). INTEGRA Biosciences 17 September 2024 (https://www.integra-biosciences.com/global/en/stories/exploring-human-microbiome-automated-ngs-library-prep).