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Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria.

ABSTRACT: Mixed infection with multiple species of nontuberculous mycobacteria (NTM) is difficult to identify and to treat. Current conventional molecular-based methods for identifying mixed infections are limited due to low specificity. Here, we evaluated the utility of whole-genome sequencing (WGS) analysis to detect and identify mixed NTM infections. Analytical tools used included PubMLST, MetaPhlAn3, Kraken2, Mykrobe-Predictor and analysis of heterozygous SNP frequencies. The ability of each to identify mixed infections of NTM species was compared. Sensitivity was tested using 101 samples (sequence sets) including 100 in-silico simulated mixed samples with various proportions of known NTM species and one sample of known mixed NTM species from a public database. Single-species NTM control samples (155 WGS samples from public databases and 15 samples from simulated reads) were tested for specificity. Kraken2 exhibited 100% sensitivity and 98.23% specificity for detection and identification of mixed NTM species with accurate estimation of relative abundance of each species in the mixture. PubMLST (99% and 96.47%) and MetaPhlAn3 (95.04% and 83.52%) had slightly lower sensitivity and specificity. Mykrobe-Predictor had the lowest sensitivity (57.42%). Analysis of read frequencies supporting single nucleotide polymorphisms (SNPs) could not detect mixed NTM samples. Clinical NTM samples (n = 16), suspected on the basis of a 16S-23S rRNA gene sequence-based line-probe assay (LPA) to contain more than one NTM species, were investigated using WGS-analysis tools. This identified only a small proportion (37.5%, 6/16 samples) of the samples as mixed infections and exhibited only partial agreement with LPA results. LPAs seem to be inadequate for detecting mixed NTM species infection. This study demonstrated that WGS-analysis tools can be used for diagnosis of mixed infections with different species of NTM.

SUBMITTER: Khieu V

PROVIDER: S-EPMC8308675 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In this study, 28 "historical" clinical freeze-dried nontuberculous mycobacterial isolates collected from 1948 to 1957, were analyzed by investigating their viability and performing whole genome sequencing (WGS) on DNA extracted (i) directly from freeze-dried cells versus (ii) after culturing, to determine cell properties and DNA quality after centuries of freeze-dried storage. The isolated DNA was sequenced on the Illumina MiSeq platform and data quality evaluated analyzing the per-base quality scores of paired-end sequencing reads as well as the overall contiguity of resulting de novo assemblies. After 72 years in storage, all freeze-dried isolates were viable, and showed no signs of cell damage and limited signs of contamination when reculturing. They were recultured without problems and identified through WGS with only four of 13 parameters showing statistical significance based on sequence data obtained directly from the freeze-dried cells versus after reculturing, indicating no DNA degradation. Thus, mycobacteria can be whole genome sequenced successfully directly from freeze-dried material without prior recultivation, saving laboratory time and resources, and emphasizing the value of freeze-drying for long-term storage. Our study lays the groundwork for further genomic investigations of freeze-dried bacterial isolates, and the approximately 4,000 historical isolates in our collection will provide a unique opportunity to investigate mycobacterial DNA from a variety of NTM species unexposed to antimicrobials, some maybe still undescribed species. IMPORTANCE The genus Mycobacterium was described more than a century ago and new species are continuously identified and described. There is an ongoing discussion about an increase in the incidence of disease caused by nontuberculous mycobacteria (NTM). How the different bacteria looked before exposure to antibiotics can only be investigated by looking at strains from before the antibiotic era. Strains from that era will be stored in different ways, for example by freeze-drying. The question is how to investigate these strains, and if they are still viable, whether they need to be cultured, and if that changes the DNA. Here, we test all these parameters on freeze-dried strains and show that NGS can be applied directly without culturing.

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Whole-Genome Sequencing Analysis to Identify Infection with Multiple Species of Nontuberculous Mycobacteria.

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