Comprehensive evaluation of complex polymicrobial specimens using next generation sequencing and standard microbiological culture

Abstract

Optimal clinical decision-making depends on identification of clinically relevant organisms present in a sample. Standard microbiological culture may fail to identify unusual or fastidious organisms and can misrepresent relative abundance of sample constituents. Culture-independent methods have improved our ability to deconvolute polymicrobial patient samples. We used next-generation 16S rRNA gene sequencing (NGS16S) to determine how often cultivatable organisms in complex polymicrobial samples are not reported by standard culture. Twenty consecutive bronchoalveolar lavage (BAL) samples were plated to standard and additional media; bacteria were identified by NGS16S analysis of DNA extracted directly from samples or from washed culture plates. 96% of organisms identified were cultivable, but only 21% were reported by standard culture, indicating that standard work-up provides an incomplete assessment of microbial constituents. Direct NGS16S correlated well with standard culture, identifying the same predominant organism in 50% of samples. When predominant organisms differed, NGS16S most often detected anaerobes, whose growth is unsupported by standard culture conditions for this specimen. NGS16S identified more organisms per sample and allowed identification of fastidious organisms, while culture was better at capturing organisms when bacterial load was low, and allowed incidental recovery of non-bacterial pathogens. Molecular and culture-based methods together detect more organisms than either method alone.