Cerebrospinal fluids (CSF) circulating human central nervous system (CNS) have long been considered aseptic in healthy individuals, because normally the blood-brain barrier protects against microbial invasions. However, this dogma has been questioned by several recent reports that colonized microbes were identified in human brains. To investigate whether CSF from healthy individuals without neurological diseases is colonized by a microbiome, we collected and analyzed a cohort of 23 CSF specimens from pregnant women with one-to-one matched contamination controls using metagenomic and metatranscriptomic next-generation sequencing. Metagenomic data analysis found no significant difference between CSF specimens and negative controls in terms of microbial species diversity. In addition, no active or viable microbiome were present in the CSF samples after being subtracted by microbes in negative controls and DNA extraction buffer. In conclusion, we found no strong evidence that colonized microbiome exist in the cerebrospinal fluids, but may harbor potential pathogens in some healthy individuals. ### Competing Interest Statement The authors have declared no competing interest. * CSF : Cerebrospinal fluids CNS : central nervous system mNGS : metagenomic next-generation sequencing QC : quality control CSF_DNA : CSF metagenomic CSF_RNA : CSF metatranscriptomic NMDS : Non-metric Multidimensional Scaling PCoA : principal coordinates analysis MDA : Multiple Displacement Amplification
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