TY - JOUR
T1 - Molecular Characterization of Microbiota in Cerebrospinal Fluid From Patients With CSF Shunt Infections Using Whole Genome Amplification Followed by Shotgun Sequencing
AU - Cerebrospinal FLuId MicroBiota in Shunts Study (CLIMB) Group
AU - Hodor, Paul
AU - Pope, Christopher E.
AU - Whitlock, Kathryn B.
AU - Hoffman, Lucas R.
AU - Limbrick, David L.
AU - McDonald, Patrick J.
AU - Hauptman, Jason S.
AU - Ojemann, Jeffrey G.
AU - Simon, Tamara D.
N1 - Funding Information:
This study was supported by R01 NS095979. DL receives research funding and equipment for unrelated projects through Microbot Medical, Inc. and Medtronic, Inc. LH receives additional support from the National Institutes of Health via K24 HL141669.
Funding Information:
We would like to thank the children and families who participated in the study at SCH and PCH. We thank those who made CSF sample collection at SCH and PCH possible, including: Anne J. Blaschke and Jay Riva-Cambrin who led enrollment at PCH; the pediatric neurosurgeons and neurosurgical staff at SCH and PCH; Hydrocephalus Clinical Research Network coordinators Amy Anderson and Tracey Habrock-Bach who identified eligible children to our study staff; and study staff who enrolled patients and ensured appropriate collection and storage of CSF specimens including Marshal Werfelman at SCH and Chris Stockmann (deceased), Priscilla Cowan, Trenda Barney, and Abby Phillips at PCH.
Publisher Copyright:
© Copyright © 2021 Hodor, Pope, Whitlock, Hoffman, Limbrick, McDonald, Hauptman, Ojemann and Simon.
PY - 2021/8/20
Y1 - 2021/8/20
N2 - Understanding the etiology of cerebrospinal fluid (CSF) shunt infections and reinfections requires detailed characterization of associated microorganisms. Traditionally, identification of bacteria present in the CSF has relied on culture methods, but recent studies have used high throughput sequencing of 16S rRNA genes. Here we evaluated the method of shotgun DNA sequencing for its potential to provide additional genomic information. CSF samples were collected from 3 patients near the beginning and end of each of 2 infection episodes. Extracted total DNA was sequenced by: (1) whole genome amplification followed by shotgun sequencing (WGA) and (2) high-throughput sequencing of the 16S rRNA V4 region (16S). Taxonomic assignments of sequences from WGA and 16S were compared with one another and with conventional microbiological cultures. While classification of bacteria was consistent among the 3 approaches, WGA provided additional insights into sample microbiological composition, such as showing relative abundances of microbial versus human DNA, identifying samples of questionable quality, and detecting significant viral load in some samples. One sample yielded sufficient non-human reads to allow assembly of a high-quality Staphylococcus epidermidis genome, denoted CLIMB1, which we characterized in terms of its MLST profile, gene complement (including putative antimicrobial resistance genes), and similarity to other annotated S. epidermidis genomes. Our results demonstrate that WGA directly applied to CSF is a valuable tool for the identification and genomic characterization of dominant microorganisms in CSF shunt infections, which can facilitate molecular approaches for the development of better diagnostic and treatment methods.
AB - Understanding the etiology of cerebrospinal fluid (CSF) shunt infections and reinfections requires detailed characterization of associated microorganisms. Traditionally, identification of bacteria present in the CSF has relied on culture methods, but recent studies have used high throughput sequencing of 16S rRNA genes. Here we evaluated the method of shotgun DNA sequencing for its potential to provide additional genomic information. CSF samples were collected from 3 patients near the beginning and end of each of 2 infection episodes. Extracted total DNA was sequenced by: (1) whole genome amplification followed by shotgun sequencing (WGA) and (2) high-throughput sequencing of the 16S rRNA V4 region (16S). Taxonomic assignments of sequences from WGA and 16S were compared with one another and with conventional microbiological cultures. While classification of bacteria was consistent among the 3 approaches, WGA provided additional insights into sample microbiological composition, such as showing relative abundances of microbial versus human DNA, identifying samples of questionable quality, and detecting significant viral load in some samples. One sample yielded sufficient non-human reads to allow assembly of a high-quality Staphylococcus epidermidis genome, denoted CLIMB1, which we characterized in terms of its MLST profile, gene complement (including putative antimicrobial resistance genes), and similarity to other annotated S. epidermidis genomes. Our results demonstrate that WGA directly applied to CSF is a valuable tool for the identification and genomic characterization of dominant microorganisms in CSF shunt infections, which can facilitate molecular approaches for the development of better diagnostic and treatment methods.
KW - CSF shunt infection
KW - Staphylococcus epidermidis CLIMB1
KW - cerebrospinal fluid
KW - high throughput DNA sequencing
KW - microbiota
UR - http://www.scopus.com/inward/record.url?scp=85114286994&partnerID=8YFLogxK
U2 - 10.3389/fcimb.2021.699506
DO - 10.3389/fcimb.2021.699506
M3 - Article
C2 - 34490140
AN - SCOPUS:85114286994
SN - 2235-2988
VL - 11
JO - Frontiers in Cellular and Infection Microbiology
JF - Frontiers in Cellular and Infection Microbiology
M1 - 699506
ER -