Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: An assessment of R10 flowcells and V14 chemistry.

Oxford Nanopore Technologies (ONT) sequencing is a promising technology. We assessed the performance of the new ONT R10 flowcells and V14 rapid sequencing chemistry for Mtb whole genome sequencing of Mycobacterium tuberculosis (Mtb) DNA extracted from clinical primary liquid cultures (CPLCs). Using the recommended protocols for MinION Mk1C, R10.4.1 MinION flowcells, and the ONT Rapid Sequencing Kit V14 on six CPLC samples, we obtained a pooled library yield of 10.9 ng/µl, generated 1.94 Gb of sequenced bases and 214k reads after 48h in a first sequencing run. Only half (49%) of all generated reads met the Phred Quality score threshold (>8). To assess if the low data output and sequence quality were due to impurities present in DNA extracted directly from CPLCs, we added a pre-library preparation bead-clean-up step and included purified DNA obtained from an Mtb subculture as a control sample in a second sequencing run. The library yield for DNA extracted from four CPLCs and one Mtb subculture (control) was similar (10.0 ng/µl), 2.38 Gb of bases and 822k reads were produced. The quality was slightly better with 66% of the produced reads having a Phred Quality >8. A third run of DNA from six CPLCs with bead clean-up pre-processing produced a low library yield (±1 Gb of bases, 166k reads) of low quality (51% of reads with a Phred Quality score >8). A median depth of coverage above 10× was only achieved for five of 17 (29%) sequenced libraries. Compared to Illumina WGS of the same samples, accurate lineage predictions and full drug resistance profiles from the generated ONT data could not be determined by TBProfiler. Further optimization of the V14 ONT rapid sequencing chemistry and library preparation protocol is needed for clinical Mtb WGS applications.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA.

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing.

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique.

Site-directed mutagenesis of Mycobacterium tuberculosis and functional validation to investigate potential bedaquiline resistance-causing mutations.

Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25-0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.

Genomic Diversity of the Rarely Observed Genotype of the Mycobacterium tuberculosis Central Asian (CAS) Lineage 3 from North Brazil.

Mycobacterium tuberculosis (Mtb) Central Asian Strain (CAS) Lineage 3 (L3) genotype is predominantly found in East-Africa, Central-Asia, Western-Asia, and South-Asia; however, a new spoligotyping CAS/SIT2545 was found in northern regions of Brazil. We aimed to characterize and describe the genetic diversity and perform a phylogenetic assessment of this novel genotype. We performed 24-MIRU-VNTR loci and Whole-genome sequencing (WGS) of six Brazilian isolates previously spoligotyped. The libraries were prepared using a Nextera-XT kit and sequenced in a NextSeq 550 Illumina instrument. We performed lineage assignment and genomic characterization. From publicly available genomes of Mtb L3 and other lineages, we created a robust dataset to run the MTBSeq pipeline and perform a phylogenetic analysis. MIRU-VNTR and WGS confirmed CAS/SIT2545 belongs to L3. Out of 1691 genomes, 1350 (79.83%) passed in quality control (genomic coverage > 95%). Strain 431 differed in 52 single nucleotide variants (SNV), confirming it does not belong to the same transmission chain. The eight genomes from a global dataset clustered closer to Brazilian strains differed in >52 SNVs. We hypothesized L3 and L1 were introduced in Brazilian Northern in the same historical event; however, there is a need for additional studies exploring the genetic diversity of Mtb Brazilian Northern.

Whole genome sequencing improves the discrimination between Mycobacterium bovis strains on the southern border of Kruger National Park, South Africa.

Background: Mycobacterium bovis forms part of the Mycobacterium tuberculosis complex and has an extensive host range and zoonotic potential. Various genotyping methods (e.g., spoligotyping) have been used to describe the molecular epidemiology of M. bovis. Advances in whole genome sequencing (WGS) have increased resolution to enable detection of genomic variants to the level of single nucleotide polymorphisms. This is especially relevant to One Health research on tuberculosis which benefits by being able to use WGS to identify epidemiologically linked cases, especially recent transmission. The use of WGS in molecular epidemiology has been extensively used in humans and cattle but is limited in wildlife. This approach appears to overcome the limitations of conventional genotyping methods due to lack of genetic diversity in M. bovis. Methods: This pilot study investigated the spoligotype and WGS of M. bovis isolates (n = 7) from wildlife in Marloth Park (MP) and compared these with WGS data from other South African M. bovis isolates. In addition, the greater resolution of WGS was used to explore the phylogenetic relatedness of M. bovis isolates in neighbouring wildlife populations. Results: The phylogenetic analyses showed the closest relatives to the seven isolates from MP were isolates from wildlife in Kruger National Park (KNP), which shares a border with MP. However, WGS data indicated that the KNP and MP isolates formed two distinct clades, even though they had similar spoligotypes and identical in silico genetic regions of difference profiles. Conclusions: Mycobacterium bovis isolates from MP were hypothesized to be directly linked to KNP wildlife, based on spoligotyping. However, WGS indicated more complex epidemiology. The presence of two distinct clades which were genetically distinct (SNP distance of 19-47) and suggested multiple transmission events. Therefore, WGS provided new insight into the molecular epidemiology of the M. bovis isolates from MP and their relationship to isolates from KNP. This approach will facilitate greater understanding of M. bovis transmission at wildlife-livestock-human interfaces and advances One Health research on tuberculosis, especially across different host species.

The Application of a Commercially Available Citrus-Based Extract Mitigates Moderate NaCl-Stress in Arabidopsis thaliana Plants.

AIMS: The aim of this study was to assess the effect of BC204 as a plant biostimulant on Arabidopsis thaliana plants under normal and NaCl-stressed conditions. METHODS: For this study, ex vitro and in vitro growth experiments were conducted to assess the effect of both NaCl and BC204 on basic physiological parameters such as biomass, chlorophyll, proline, malondialdehyde, stomatal conductivity, Fv/Fm and the expression of four NaCl-responsive genes. RESULTS: This study provides preliminary evidence that BC204 mitigates salt stress in Arabidopsis thaliana. BC204 treatment increased chlorophyll content, fresh and dry weights, whilst reducing proline, anthocyanin and malondialdehyde content in the presence of 10 dS·m-1 electroconductivity (EC) salt stress. Stomatal conductivity was also reduced by BC204 and NaCl in source leaves. In addition, BC204 had a significant effect on the expression of salinity-related genes, stimulating the expression of salinity-related genes RD29A and SOS1 independently of NaCl-stress. CONCLUSIONS: BC204 stimulated plant growth under normal growth conditions by increasing above-ground shoot tissue and root and shoot growth in vitro. BC204 also increased chlorophyll content while reducing stomatal conductivity. BC204 furthermore mitigated moderate to severe salt stress (10-20 dS·m-1) in A. thaliana. Under salt stress conditions, BC204 reduced the levels of proline, anthocyanin and malondialdehyde. The exact mechanism by which this occurs is unknown, but the results in this study suggest that BC204 may act as a priming agent, stimulating the expression of genes such as SOS1 and RD29A.