TY - JOUR
T1 - Shotgun sequencing of clinical biofilm following scanning electron microscopy identifies bacterial community composition
AU - Fritz, Blaine
AU - Stavnsbjerg, Camilla
AU - Markvart, Merete
AU - Damgaard, Peter De Barros
AU - Nielsen, Sofie Holtsmark
AU - Bjørndal, Lars
AU - Qvortrup, Klaus
AU - Bjarnsholt, Thomas
PY - 2019
Y1 - 2019
N2 - Bacterial biofilm infections often involve aggregates of bacteria heterogeneously distributed throughout a tissue or on a surface (such as an implanted medical device). Identification of a biofilm infection requires direct visualization via microscopy, followed by characterization of the microbial community by culturing or sequencing-based approaches. A sample, therefore, must be divided prior to analysis, often leading to inconsistent results. We demonstrate a combined approach, using scanning electron microscopy and next-generation shotgun sequencing, to visually identify a biofilm and characterize the microbial community, without dividing the sample. A clinical sample recovered from a patient following a dental root-filling procedure was prepared and visualized by scanning electron microscopy. DNA was then extracted from the sample several years later and analyzed by shotgun sequencing. The method was subsequently validated on in vitro cultures of Pseudomonas aeruginosa biofilm. Between 19 and 21 different genera and species were identified in the clinical sample with an estimated relative abundance greater than 1% by two different estimation approaches. Only eight genera identified were not associated with endodontic infections. This provides a proof-of-concept for a dual, microscopy and sequencing-based approach to identify and characterize bacterial biofilms, which could also easily be implemented in other scientific fields.
AB - Bacterial biofilm infections often involve aggregates of bacteria heterogeneously distributed throughout a tissue or on a surface (such as an implanted medical device). Identification of a biofilm infection requires direct visualization via microscopy, followed by characterization of the microbial community by culturing or sequencing-based approaches. A sample, therefore, must be divided prior to analysis, often leading to inconsistent results. We demonstrate a combined approach, using scanning electron microscopy and next-generation shotgun sequencing, to visually identify a biofilm and characterize the microbial community, without dividing the sample. A clinical sample recovered from a patient following a dental root-filling procedure was prepared and visualized by scanning electron microscopy. DNA was then extracted from the sample several years later and analyzed by shotgun sequencing. The method was subsequently validated on in vitro cultures of Pseudomonas aeruginosa biofilm. Between 19 and 21 different genera and species were identified in the clinical sample with an estimated relative abundance greater than 1% by two different estimation approaches. Only eight genera identified were not associated with endodontic infections. This provides a proof-of-concept for a dual, microscopy and sequencing-based approach to identify and characterize bacterial biofilms, which could also easily be implemented in other scientific fields.
KW - biofilm
KW - chronic infection
KW - implanted device
KW - metagenomics
KW - microbiome
KW - SEM
U2 - 10.1093/femspd/ftz013
DO - 10.1093/femspd/ftz013
M3 - Journal article
C2 - 30844070
AN - SCOPUS:85064169686
SN - 2049-632X
VL - 77
JO - Pathogens and Disease
JF - Pathogens and Disease
IS - 1
M1 - ftz013
ER -