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Updated: Oct 19, 2025
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Next-generation sequencing (NGS) to determine microbiome of herniated intervertebral disc
Nelson Astur1, Brenno Ferreira Bento Maciel2, Andre Mario Doi3
1Programa Locomotor, Hospital Israelita Albert Einstein, Av Albert Einstein 627, Sao Paulo, SP, Brazil, 05652-900.
Background Context:
There is apparent causality between chronic infection of the intervertebral disc and its degenerative process. Although disc is considered a sterile tissue, collected samples of uninfected patients sent to culture testing resulted positive.
Purpose:
The purpose of this study was to analyze the microbiome of the intervertebral disc by using and validating the next-generation sequencing (NGS) molecular test, controlled with tissue culture and clinical presentation of patients.
Study Design/Setting:
Prospective study of consecutive patients in a hospital.
Patient Sample:
Patients with lumbar disc herniation undergoing open microdiscectomy aging 18 to 65 years.
Outcome Measures:
NGS, tissue culture METHODS: Subjects undergoing open decompression surgery for lumbar disc herniation were consecutively included and clinically followed for one year. Three samples of the excised herniated disc fragment were sent to tissue culture and another sample of the disc was sent to NGS test for microbiome analysis. Control samples of the ligamentum flavum and deep muscle were collected and sent to culture.
Results:
A total of 17 patients were included. All patients presented negative cultures of the removed disc samples, as well as negative cultures of muscle and yellow ligament. None of the patients evolved to clinical infection one year after surgery, nor presented significant alteration of laboratory markers. NGS mapped a mean of 14,645 (range 6,540 to 27,176) DNA sequences for each disc sample of each patient. There were a total of 45 different bacteria genera remnants with different amount of DNA sequences detected. There was a mean of 8 (range 3-17) different bacterial elements in each sample of intervertebral disc. Three bacteria were present in all disc samples (Herbaspirillum, Ralstonia, and Burkolderia). Although there were a considerable mean number of bacterial sequences mapped in each disc sample, the amount of sequences related to bacteria was low. Cutibacterium acnes elements was not found in any disc microbiome analysis.
Conclusions:
NGS has been proven to adequately determine bacterial DNA presence within the intervertebral disc. C. acnes was not isolated in culture neither in microbiome analysis of patients with lumbar disc herniation. We cannot confirm disc sterility since, even if it does not cause infection, there is bacterial or remnant DNA in herniated discs.
