Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing
https://doi.org/10.47665/tb.39.4.002
- Author:
John, D.V.
1
;
Aryalakshmi, B.
1
;
Deora, H.
2
;
Purushottam, M.
3
;
Raju, R.
1
;
Mahadevan, A.
4
;
Rao, M.B.
2
;
Patil, S.A.
1
Author Information
1. Department of Neuromicrobiology, National Institute of Mental Health and Neurosciences, Bangalore, India
2. Department of Neurosurgery, National Institute of Mental Health and Neurosciences, Bangalore, India
3. Molecular Genetics Laboratory, Department of Psychiatry, National Institute of Mental Health and Neurosciences, Bangalore, India
4. Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India
- Publication Type:Journal Article
- Keywords:
Culture-negative brain abscess;
16S/18S rRNA gene;
Sanger sequencing;
metagenomics sequencing, bioinformatics
- From:Tropical Biomedicine
2022;39(No.4):489-498
- CountryMalaysia
- Language:English
-
Abstract:
Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine
microbiological testing. Direct PCR of such samples enables the identification of microbes that may be
fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were
subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven
formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA
PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between
smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for
bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S
rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative
bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics
sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable
bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain
abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The
microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative
Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through
Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for
diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative
brain abscess samples by molecular methods could enable detection and/or treatment of the source
of infection.
- Full text:8.2022my1367.pdf
