Comparison of culture and molecular techniques for microbial community characterization in infected necrotizing
Erin M Hanna1, Timothy J Hamp2, Iain H McKillop1
1Department of Surgery, Carolinas Medical Center, Charlotte, North Carolina.
Background:
Infected necrotizing pancreatitis is associated with significant morbidity and mortality. Peripancreatic fluid cultures may fail to identify all the infecting organisms. The aim of this study was to compare the bacterial biome of peripancreatic fluid from infected necrotizing pancreatitis patients using 16S ribosomal RNA (rRNA) DNA deep sequencing and quantitative polymerase chain reaction (qPCR) targeting the 16S rRNA gene versus standard laboratory culture.
Materials And Methods:
Peripancreatic fluid was collected during operative or radiologic intervention and samples sent for culture. In parallel, microbial DNA was extracted, qPCR targeting the 16S rRNA gene and 16S rRNA PCR amplification followed by Illumina deep sequencing were performed.
Results:
Using culture techniques, the bacterial strains most frequently identified were gram-negative rods (Escherichia coli, Klebsiella pneumoniae) and Enterococcus. Samples in which culture results were negative had copy numbers of the 16S rRNA gene close to background in qPCR analysis. For samples with high bacterial load, sequencing results were in some cases in good agreement with culture data, whereas in others there were disagreements, likely due to differences in taxonomic classification, cultivability, and differing susceptibility to background contamination. Sequencing results appeared generally unreliable in cases of negative culture where little microbial DNA was input into qPCR sequencing reactions.
Conclusions:
Both sequencing and culture data display their own sources of bias and potential error. Consideration of data from multiple techniques will yield a more accurate view of bacterial infections than can be achieved by any single technique.
Insights
Comparing bacterial identification in infected necrotizing pancreatitis, 16S rRNA gene sequencing and quantitative PCR (qPCR) offer complementary insights to standard cultures, revealing biases in each method for a comprehensive view of infection.
Area of Science:
- Microbiology
- Molecular Biology
- Gastroenterology
Background:
- Infected necrotizing pancreatitis presents high morbidity and mortality.
- Standard peripancreatic fluid cultures may miss causative organisms.
- Advanced molecular techniques are needed for accurate microbial identification.
Purpose of the Study:
- To compare bacterial identification in infected necrotizing pancreatitis using 16S rRNA DNA deep sequencing and quantitative PCR (qPCR) against standard culture.
- To evaluate the accuracy and limitations of each method in detecting the bacterial biome.
Main Methods:
- Peripancreatic fluid samples collected during interventions.
- Standard bacterial culture performed.
- Microbial DNA extraction, qPCR targeting 16S rRNA gene, and 16S rRNA gene deep sequencing (Illumina) conducted in parallel.
Main Results:
- Culture identified common bacteria like E. coli, K. pneumoniae, and Enterococcus.
- Sequencing showed agreement with culture in high bacterial load samples but discrepancies due to taxonomic classification and contamination.
- Sequencing results were unreliable in culture-negative samples with low microbial DNA input.
Conclusions:
- Both culture and molecular methods (sequencing, qPCR) have inherent biases and limitations.
- Integrating data from multiple techniques provides a more accurate understanding of bacterial infections in pancreatitis.
- A multi-technique approach is crucial for comprehensive diagnosis and management.
Related Concept Videos
01:29Modern Molecular Taxonomy
01:28Methods of Classification and Identification
01:20Applications of Molecular Taxonomy
01:30Methods to Assess Microbial Populations
01:19Methods to Assess Microbial Communities