title: Comparison of culture and molecular techniques for microbial community characterization in infected necrotizing pancreatitis.
abstract: 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.
  
  
  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.
authors: Department of Surgery, Carolinas Medical Center, Charlotte, North Carolina.
authors: Erin M
authors: EM
authors: Hanna
authors: Department of Bioinformatics and Genomics, UNC Charlotte, Charlotte, North Carolina.
authors: Timothy J
authors: TJ
authors: Hamp
authors: Department of Surgery, Carolinas Medical Center, Charlotte, North Carolina. Electronic address: iain.mckillop@carolinashealthcare.org.
authors: Iain H
authors: IH
authors: McKillop
authors: Department of Oral Medicine, Carolinas Medical Center, Charlotte, North Carolina.
authors: Farah
authors: F
authors: Bahrani-Mougeot
authors: Department of Surgery, Carolinas Medical Center, Charlotte, North Carolina.
authors: John B
authors: JB
authors: Martinie
authors: Department of Internal Medicine, Carolinas Medical Center, Charlotte, North Carolina.
authors: James M
authors: JM
authors: Horton
authors: Department of Surgery, Carolinas Medical Center, Charlotte, North Carolina.
authors: David
authors: D
authors: Sindram
authors: Department of Bioinformatics and Genomics, UNC Charlotte, Charlotte, North Carolina; Department of Bioinformatics and Genomics, Bioinformatics Services Division, UNC Charlotte, Kannapolis, North Carolina.
authors: Raad Z
authors: RZ
authors: Gharaibeh
authors: Department of Bioinformatics and Genomics, UNC Charlotte, Charlotte, North Carolina.
authors: Anthony A
authors: AA
authors: Fodor
authors: Department of Surgery, Carolinas Medical Center, Charlotte, North Carolina.
authors: David A
authors: DA
authors: Iannitti
mesh_terms: D000328:Adult; D000368:Aged; D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D001419:Bacteria / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000382:microbiology*; D003470:Culture Media; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D019283:Pancreatitis, Acute Necrotizing / Q000188:drug therapy / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA gene; Deep sequencing; Microbial identification; Necrotizing pancreatitis; Severe acute pancreatitis; qPCR