title: The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients.
abstract: BACKGROUND
  The application of molecular based diagnostics in sepsis has had limited success to date. Molecular community profiling methods have indicated that polymicrobial infections are more common than suggested by standard clinical culture. A molecular profiling approach was developed to investigate the propensity for polymicrobial infections in patients predicted to have bacterial sepsis.
  
  
  RESULTS
  Disruption of blood cells with saponin and hypotonic shock enabled the recovery of microbial cells with no significant changes in microbial growth when compared to CFU/ml values immediately prior to the addition of saponin. DNA extraction included a cell-wall digestion step with both lysozyme and mutanolysin, which increased the recovery of terminal restriction fragments by 2.4 fold from diverse organisms. Efficiencies of recovery and limits of detection using Illumina sequencing of the 16S rRNA V3 region were determined for both viable cells and DNA using mock bacterial communities inoculated into whole blood. Bacteria from pre-defined communities could be recovered following lysis and removal of host cells with >97% recovery of total DNA present. Applying the molecular profiling methodology to three septic patients in the intensive care unit revealed microbial DNA from blood had consistent alignment with cultured organisms from the primary infection site providing evidence for a bloodstream infection in the absence of a clinical lab positive blood culture result in two of the three cases. In addition, the molecular profiling indicated greater diversity was present in the primary infection sample when compared to clinical diagnostic culture.
  
  
  CONCLUSIONS
  A method for analyzing bacterial DNA from whole blood was developed in order to characterize the bacterial DNA profile of sepsis infections. Preliminary results indicated that sepsis infections were polymicrobial in nature with the bacterial DNA recovered suggesting a more complex etiology when compared to blood culture data.
authors.affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada. mmfaria@ucalgary.ca.|Department of Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada. mmfaria@ucalgary.ca.|Farncombe Family Digestive Health Research Institute, Departments of Medicine and Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, 1280 Main Street, HSC 3 N 8 F, Hamilton, ON, L8S 4 K1, Canada. mmfaria@ucalgary.ca.
authors.forename: M M P
authors.initials: MM
authors.lastname: Faria
authors.affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada. john.conly@albertahealthservices.ca.|Department of Medicine, University of Calgary, Calgary, AB, T2N 4 N1, Canada. john.conly@albertahealthservices.ca.|Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, T2N 4 N1, Canada. john.conly@albertahealthservices.ca.|Department of Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada. john.conly@albertahealthservices.ca.
authors.forename: J M
authors.initials: JM
authors.lastname: Conly
authors.affiliation: Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada. surette@mcmaster.ca.|Department of Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada. surette@mcmaster.ca.|Farncombe Family Digestive Health Research Institute, Departments of Medicine and Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, 1280 Main Street, HSC 3 N 8 F, Hamilton, ON, L8S 4 K1, Canada. surette@mcmaster.ca.|Department of Medicine, McMaster University, Hamilton, ON, L8S 4 K1, Canada. surette@mcmaster.ca.|Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, L8S 4 K1, Canada. surette@mcmaster.ca.
authors.forename: M G
authors.initials: MG
authors.lastname: Surette
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D001769:Blood / Q000382:microbiology*; D015331:Cohort Studies; D060085:Coinfection / Q000175:diagnosis* / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D018805:Sepsis / Q000175:diagnosis* / Q000382:microbiology; D055815:Young Adult
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S