Rapid Bacterial and Fungal Pathogen Identification Using Sanger Sequencing

Introduction to Pathogen Identification Techniques

The rapid, efficient, and accurate identification of pathogenic microorganisms constitutes a fundamental task in clinical microbiology. This process provides invaluable etiological information that guides the selection of appropriate antibiotic therapies.

Phenotypic Methods for Microbial Identification

Phenotypic methods are microbial identification techniques based on the growth characteristics and biochemical properties of microorganisms. These methods involve the cultivation of microorganisms and the observation of their growth patterns and metabolic products under various environmental conditions. Although phenotypic methods are instrumental in determining microbial species, they require an extended duration for the identification of slow-growing bacteria, potentially spanning seven days or more. Moreover, the diagnostic accuracy of these methods is highly dependent on the quality of the specimen and the expertise of the microbiologist.

The rapid advancement of molecular biology techniques has provided alternative methods for the precise and efficient identification of microorganisms. Polymerase chain reaction (PCR) amplification, coupled with Sanger sequencing, has emerged as a valuable tool in clinical settings for species identification. This method offers advantages such as low cost and quick turnaround time, enabling the rapid identification of pathogens and elucidation of disease etiology.

Bacterial Identification via 16S rDNA Sequencing

In bacterial taxonomy, the detection of the 16S ribosomal RNA gene (16S rDNA gene) has been widely adopted for bacterial identification. Techniques such as PCR combined with Sanger sequencing or next-generation sequencing (NGS) are utilized to identify bacteria. This approach is particularly useful for identifying atypical bacteria that are challenging to culture using conventional microbiological methods. The 16S gene sequencing allows for the identification of approximately 90% of samples at the genus level and between 65% and 83% of samples at the species level^[1].

Recent advancements in 16S rDNA sequencing technology include improved resolution and reduced costs, enhancing its application in clinical settings. However, challenges remain, such as the inability to resolve species-level identification for some genera. Continued improvements in sequencing technology aim to address these limitations and expand the applicability of 16S rDNA sequencing.

Fungal Identification via Multiple Genetic Markers

Molecular identification of fungi involves several genetic markers, including 18S rDNA, 28S D1/D2, internal transcribed regions (ITS1-5.8S-ITS2), eukaryotic translation elongation factor alpha subunit (eEF1), RNA polymerase subunits I and II (RPB1 and RPB2), chitin synthase (CHS), and chitinase 18-5 (Chi18-5) [2, 3]. Currently, no single genomic marker has been identified that can consistently classify fungi at the species level. Therefore, to achieve a more specific identification, a combination of multiple genomic markers is often employed to capture conserved segments across species.

Comparison with Other Molecular Techniques

PCR and Next-Generation Sequencing (NGS)

While Sanger sequencing is a robust method for pathogen identification, it is often compared with next-generation sequencing (NGS) and other molecular techniques. NGS offers high-throughput sequencing capabilities and can analyze multiple genetic markers simultaneously, providing a broader spectrum of pathogen identification. However, NGS is typically more expensive and requires advanced computational tools for data analysis.

In contrast, Sanger sequencing provides a cost-effective and straightforward approach with high accuracy for specific genetic regions. It remains a preferred method in many clinical laboratories due to its established reliability and lower cost compared to NGS.

Cultivation Techniques

When compared to traditional microbial culture methods, Sanger sequencing demonstrates superior speed and accuracy. Microbial cultures can be time-consuming and may fail to grow certain pathogens, whereas Sanger sequencing directly detects genetic material, bypassing the need for culturing. This advantage is crucial for diagnosing infections caused by fastidious or slow-growing organisms that are challenging to culture.

Case Study

Sanger seduencing Implementation in Clinical Ill Patients for Bacterial and

Fungal Patogens Identification

The study presented examines the application of PCR and Sanger sequencing for the rapid diagnosis of bacterial and fungal pathogens in clinical settings [4].

This approach focuses on three primary aspects:

1. Identification of pathogens in critically ill patients through the detection of 16S and 18S/eEF1 gene sequences.

2. Evaluation of the correlation between Sanger sequencing results and conventional biomarkers such as procalcitonin, C-reactive protein, erythrocyte sedimentation rate, and neutrophil-to-lymphocyte ratio.

3. Comparison of molecular diagnostic methods with traditional culture techniques to analyze discrepancies in diagnostic outcomes.

Methodology

1. PCR and Gene Markers

The study involved 30 patients diagnosed with infectious diseases upon admission. Samples were collected, including whole blood, cerebrospinal fluid, bronchoalveolar lavage fluid, and ascitic fluid.

DNA Extraction and PCR Amplification:

PCR was performed to screen for the presence of pathogenic microbial DNA fragments. The specific gene markers used were:

16S rDNA genes (V3-V4 region): Expected product length 400 bp

• Forward primer: CCGTCAATTCCTTTGAGTT
• Reverse primer: CAGCAGCCGCGCTAATAC

eEF1: Expected product length 600 bp

• Forward primer: GAYTTCATCAAGAACATGA
• Reverse primer: GACGTTGAADCCRACRTTG

18S rDNA: Expected product length 150 bp

• Forward primer: GATCACACCGCCCGTC
• Reverse primer: TGATCCTTCTGCAGGTTCA

2. Sanger Sequencing

Amplified products were further analyzed using Sanger sequencing. Sequencing reactions utilized the Big Dye Terminator technique (Thermo Fisher Scientific) and were detected using the 3500 Genetic Analyzer (Applied Biosystems).

3. Data Analysis

Sequence data were analyzed using Geneious Prime v2019.2.3 (https://www.geneious.com/) and compared against the GenBank database (https://www.ncbi.nlm.nih.gov/).

Figure 5. Study Workflow for Identifying Bacterial and Fungal Pathogens Using Sanger Sequencing

Results

1. Quality Assessment of Sanger Sequencing

Quality assessment of Sanger sequencing data involved semi-quantitative PCR of the 16S and 18S/eEF1 gene markers. Sequencing quality was evaluated based on base call accuracy, peak height distribution, secondary peak appearance, and overall sequence quality (Figure 1a). Statistical analysis revealed no significant differences in read quality between genes (Figure 1e).

Figure 1: Quality analysis of Sanger sequencing reads for all genomic markers used in the study. (a) Representative electropherogram; (b) Nucleotide quality analysis for 16S; (c) 18S; (d) eEF1 sequences; (e) Quality score distribution for nucleotide positions in 16S, 18S, and eEF1 sequences.

2. Clinical Gene Detection Results

Sequence analysis of bacterial 16S and fungal 18S/eEF1 genes from clinical samples revealed:

46.6% of samples were positive for fungal infections.

23.3% of samples were positive for bacterial infections.

23.3% of samples were positive for both bacterial and fungal infections.

6.7% of samples were negative for any detected pathogens.

Figure 2: (b) Comparison of Sanger sequencing and culture results; (c) Classification of pathogens identified in clinical samples using PCR+Sanger sequencing.

3. Comparison: Sanger Sequencing vs. Traditional Culture Methods

Sanger sequencing demonstrated a lower correlation with microbial culture results; many cultures failed to grow and did not align with sequencing findings. Contrarily, Sanger sequencing results directly corresponded with the clinical symptoms of the patients. A statistically significant difference was observed between Sanger sequencing and culture results (p = 9.47×10−8). Out of 30 cases, 28 (93.33%) infections were identified using Sanger sequencing, compared to 18 (60%) identified via culture methods (Figure 2b).

Conclusion

The use of PCR in conjunction with Sanger sequencing for the analysis of 16S and 18S/eEF1 gene markers significantly optimizes the time required to obtain results for bacterial or fungal infections. Sanger sequencing provides a reliable method for pathogen identification, which aids in preventing disease progression and enables the formulation of more targeted therapeutic strategies, ultimately improving clinical outcomes.

Future Directions in Pathogen Identification

Integration with Emerging Technologies

The future of pathogen identification lies in the integration of Sanger sequencing with emerging technologies. Advances in microfluidics, automated sample processing, and real-time sequencing technologies promise to enhance the efficiency and accessibility of molecular diagnostics. Combining Sanger sequencing with these innovations could lead to even faster and more accurate pathogen identification in clinical settings.

Development of Novel Genetic Markers

Ongoing research aims to identify novel genetic markers that improve pathogen detection and differentiation. The development of new markers and sequencing techniques will further enhance the resolution of pathogen identification and expand the applicability of Sanger sequencing to a broader range of infectious agents.

Personalized Medicine and Targeted Therapies

The advancement of pathogen identification techniques is poised to significantly bolster the field of personalized medicine. Specifically, the precise identification capabilities of Sanger sequencing facilitate the development of targeted therapies that are customized to combat specific infections. This precision medicine approach not only holds the potential to enhance treatment efficacy but also aims to mitigate the ongoing issue of antibiotic resistance by reducing the dependence on broad-spectrum antibiotics.

Conclusion

Sanger sequencing remains a valuable tool for the rapid and accurate identification of bacterial and fungal pathogens. Its ability to provide detailed genetic information complements traditional methods and offers significant advantages in clinical diagnostics. As advancements in molecular biology and technology continue to evolve, Sanger sequencing, in conjunction with other emerging techniques, will play a pivotal role in enhancing pathogen detection and guiding effective treatment strategies.

For comprehensive and reliable pathogen identification services, CD Genomics remains at the forefront of integrating advanced molecular techniques to support research applications.

References

1. Drancourt, M., Bollet, C., Carlioz, A., et al. (2000) 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates. Journal of Clinical Microbiology, 38, 3623-3630.
2. Kidd, S., Halliday, C., Alexiou, H. and Ellis, D. (2016) Descriptions of Medical Fungi. 3rd Edition, CutCut Digital, Paris.
3. Balajee, S.A., Houbraken, J., Verweij, P.E., et al. (2007) Aspergillus Species Identification in the Clinical Setting. Studies in Mycology, 59, 39-46.
4. Fong-Flores, V. , Murillo-Gallo, A. , Contreras-Ojeda, L. and Barraza, A. (2022) Sanger Sequencing Implementation in Clinically Ill Patients for Bacterial and Fungal Pathogens Identification. Advances in Infectious Diseases, 12, 363-382.