Improved Quantification of Intracellular Bacterial Load in Osteomyelitis Using Digital Droplet PCR

Core Concepts

The colony-forming-unit (CFU) method is unreliable for quantifying intracellular bacterial load in osteomyelitis, as it fails to capture non-culturable bacteria. A workflow combining direct DNA lysis and digital droplet PCR provides a more accurate and rapid assessment of bacterial burden in both in vitro and clinical bone infection samples.

Abstract

The study addresses the limitations of the standard CFU method for quantifying intracellular bacterial load in osteomyelitis. It introduces a workflow that combines direct DNA lysis and digital droplet PCR (ddPCR) to provide a more accurate and rapid assessment of bacterial burden.
Key highlights:

The CFU method significantly underestimates the actual bacterial load in an in vitro osteocyte-Staphylococcus aureus co-culture model, with up to 106-fold differences compared to ddPCR quantification.
The direct DNA lysis approach using a lysis buffer (Direct buffer) results in higher and more consistent bacterial genome copy numbers compared to a standard DNA extraction kit.
ddPCR enables absolute quantification of bacterial genome copies without the need for a standard curve, improving reproducibility.
The workflow is applied to clinical bone samples from culture-negative periprosthetic joint infection (PJI) cases, identifying the presence of coagulase-negative Staphylococcus species and quantifying their bacterial load.
The use of targeted PCR amplification and Oxford Nanopore sequencing allows for rapid pathogen identification in clinical samples, with potential for point-of-care diagnostics.
The authors conclude that the combined direct lysis and ddPCR approach provides a more reliable and efficient method for quantifying intracellular bacterial burden in osteomyelitis compared to the standard CFU technique.

Stats

Genome copy numbers quantified by ddPCR were 5-fold higher in SaOS2-OY samples and 100-fold higher in S. aureus samples when using the Direct buffer compared to a standard DNA extraction kit.
In the high virulence S. aureus strain SK2-infected group, the genome copy numbers were maintained at 107-108 cells/well by 5 days post-infection, while the low virulence strain SK3 showed a decrease to ~106 cells/well.
The three culture-negative PJI bone samples contained between 2 × 104 and 1 × 106 bacterial genomic copies per million human genomic copies.

Quotes

"Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens."
"Together, our results indicated the dramatic change in bacterial culturability when comparing growth in ideal microbial suspension culture conditions and the growth limiting intracellular environment."
"For the purposes of unknown pathogen diagnosis in clinical cases, the exact bacterial species readout is required from sequencing the generated amplicons."

Key Insights Distilled From

Beyond the Colony-Forming-Unit: Rapid Bacterial Evaluation in Osteomyelitis

by Sun,Q., Huyn... at www.biorxiv.org 11-22-2023

https://www.biorxiv.org/content/10.1101/2023.11.21.568051v3

Deeper Inquiries

How could the workflow be further optimized to enable rapid, point-of-care diagnosis of osteomyelitis in a clinical setting?

To optimize the workflow for rapid, point-of-care diagnosis of osteomyelitis in a clinical setting, several enhancements can be considered:

Automation: Implementing automated processes for DNA extraction, PCR amplification, and ddPCR analysis can significantly reduce turnaround time and human error. This can be achieved through the use of robotic systems that streamline the workflow and ensure consistency in sample processing.

Miniaturization: Developing microfluidic devices or lab-on-a-chip technology for DNA extraction and amplification can reduce reagent consumption, sample volume requirements, and overall processing time. This can enable faster and more efficient analysis of clinical samples at the point of care.

Integration of Portable Sequencing Technologies: Incorporating portable sequencing technologies, such as nanopore sequencing, directly into the workflow can provide real-time pathogen identification without the need for complex laboratory equipment. This can further expedite the diagnostic process and facilitate immediate treatment decisions.

Validation and Clinical Trials: Conducting extensive validation studies and clinical trials to assess the accuracy, sensitivity, and specificity of the optimized workflow in diverse clinical settings is crucial. This will ensure the reliability and effectiveness of the point-of-care diagnostic approach for osteomyelitis.

User-Friendly Interface: Designing a user-friendly interface for data analysis and result interpretation can enhance the usability of the workflow by healthcare professionals with varying levels of expertise. Providing clear and concise output reports can facilitate quick decision-making in clinical practice.

What are the potential limitations or challenges in applying this approach to a broader range of clinical samples beyond bone tissue?

While the described approach shows promise for diagnosing osteomyelitis, there are several limitations and challenges in applying this method to a broader range of clinical samples beyond bone tissue:

Sample Complexity: Clinical samples from different anatomical sites or biological fluids may contain a diverse range of microbial species, varying in abundance and composition. Adapting the workflow to handle this complexity and ensure accurate pathogen identification can be challenging.

Host DNA Contamination: Clinical samples often contain a significant amount of host DNA, which can interfere with the amplification and detection of bacterial DNA. Developing strategies to selectively amplify bacterial targets while minimizing host DNA interference is essential for accurate diagnosis.

Pathogen Diversity: Infectious diseases can be caused by a wide range of pathogens, including bacteria, viruses, fungi, and parasites. Adapting the workflow to detect and quantify different types of pathogens in clinical samples requires specific target sequences and optimized amplification protocols for each pathogen group.

Sample Preparation Variability: Clinical samples may vary in quality, quantity, and preservation methods, leading to variability in DNA extraction efficiency and PCR amplification success. Standardizing sample preparation protocols and quality control measures is crucial to ensure consistent results across different sample types.

Regulatory Approval: Introducing a new diagnostic workflow for clinical use requires regulatory approval and validation to meet quality standards and ensure patient safety. Navigating the regulatory pathway and obtaining necessary approvals can be a time-consuming and resource-intensive process.

What other host-pathogen interaction models could benefit from the improved bacterial quantification method described in this study?

The improved bacterial quantification method described in this study can benefit various host-pathogen interaction models beyond osteomyelitis, including:

Biofilm Infections: Studying bacterial biofilms formed on medical devices or tissues can benefit from accurate quantification of bacterial load within the biofilm structure. The method's ability to quantify bacterial genome copies without the need for standard curves can enhance biofilm research.

Chronic Wound Infections: Understanding the dynamics of bacterial colonization and persistence in chronic wounds is crucial for effective treatment. The precise quantification of bacterial burden using ddPCR can provide insights into the pathogenesis of chronic wound infections.

Respiratory Infections: Models of respiratory infections, such as pneumonia or tuberculosis, can benefit from the workflow's ability to quantify bacterial load in host cells or clinical samples. This can aid in monitoring disease progression and treatment response in respiratory infections.

Gastrointestinal Infections: Investigating host-pathogen interactions in gastrointestinal infections, including bacterial pathogens like Helicobacter pylori or Clostridium difficile, can be enhanced by the accurate quantification of bacterial genome copies. This method can help elucidate the role of bacteria in gastrointestinal diseases.

Intracellular Pathogen Infections: Models of intracellular pathogen infections, such as those caused by Mycobacterium tuberculosis or Chlamydia trachomatis, can benefit from the workflow's ability to quantify bacterial persistence within host cells. This can provide insights into the mechanisms of intracellular pathogen survival and host immune response.