Whole-genome sequencing (WGS) of bacterial isolates has become standard practice in many laboratories. Applications for WGS analysis include phylogeography and molecular epidemiology, using single nucleotide polymorphisms (SNPs) as the unit of evolution. NASP was developed as a reproducible method that scales well with the hundreds to thousands of WGS data typically used in comparative genomics applications. In this study, we demonstrate how NASP compares with other tools in the analysis of two real bacterial genomics datasets and one simulated dataset. Our results demonstrate that NASP produces similar, and often better, results in comparison with other pipelines, but is much more flexible in terms of data input types, job management systems, diversity of supported tools and output formats. We also demonstrate differences in results based on the choice of the reference genome and choice of inferring phylogenies from concatenated SNPs or alignments including monomorphic positions. NASP represents a source-available, version-controlled, unit-tested method and can be obtained from tgennorth.github.io/NASP.

Multidrug-resistant Klebsiella pneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired bla_KPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258.

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics.

Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.

Accurate, comprehensive, and timely detection of drug-resistant tuberculosis (TB) is essential to inform patient treatment and enable public health surveillance. This is crucial for effective control of TB globally. Whole-genome sequencing (WGS) and targeted next-generation sequencing (NGS) approaches have potential as rapid in vitro diagnostics (IVDs), but the complexity of workflows, interpretation of results, high costs, and vulnerability of instrumentation have been barriers to broad uptake outside of reference laboratories, especially in low- and middle-income countries. A new, solid-state, tabletop sequencing instrument, Illumina iSeq100, has the potential to decentralize NGS for individual patient care. In this study, we evaluated WGS and targeted NGS for TB on both the new iSeq100 and the widely used MiSeq (both manufactured by Illumina) and compared sequencing performance, costs, and usability. We utilized DNA libraries produced from Mycobacterium tuberculosis clinical isolates for the evaluation. We conducted WGS on three strains and observed equivalent uniform genome coverage with both platforms and found the depth of coverage obtained was consistent with the expected data output. Utilizing the standardized, cloud-based ReSeqTB bioinformatics pipeline for variant analysis, we found the two platforms to have 94.0% (CI 93.1%–94.8%) agreement, in comparison to 97.6% (CI 97%–98.1%) agreement for the same libraries on two MiSeq instruments. For the targeted NGS approach, 46 M. tuberculosis–specific amplicon libraries had 99.6% (CI 98.0%–99.9%) agreement between the iSeq100 and MiSeq data sets in drug resistance–associated SNPs. The upfront capital costs are almost 5-fold lower for the iSeq100 ($19,900 USD) platform in comparison to the MiSeq ($99,000 USD); however, because of difference in the batching capabilities, the price per sample for WGS was higher on the iSeq100. For WGS of M. tuberculosis at the minimum depth of coverage of 30x, the cost per sample on the iSeq100 was $69.44 USD versus $28.21 USD on the MiSeq, assuming a 2 × 150 bp run on a v3 kit. In terms of ease of use, the sequencing workflow of iSeq100 has been optimized to only require 27 minutes total of hands-on time pre- and post-run, and the maintenance is simplified by a single-use cartridge–based fluidic system. As these are the first sequencing attempts on the iSeq100 for M. tuberculosis, the sequencing pool loading concentration still needs optimization, which will affect sequencing error and depth of coverage. Additionally, the costs are based on current equipment and reagent costs, which are subject to change. The iSeq100 instrument is capable of running existing TB WGS and targeted NGS library preparations with comparable accuracy to the MiSeq. The iSeq100 has reduced sequencing workflow hands-on time and is able to deliver sequencing results in <24 hours. Reduced capital and maintenance costs and lower-throughput capabilities also give the iSeq100 an advantage over MiSeq in settings of individualized care but not in high-throughput settings such as reference laboratories, where sample batching can be optimized to minimize cost at the expense of workflow complexity and time.

Multidrug-resistant tuberculosis (TB) presents a major public health dilemma. Heteroresistance, the coexistence of drug-resistant and drug-susceptible strains or of multiple drug-resistant strains with discrete haplotypes, may affect accurate diagnosis and the institution of effective treatment. Subculture, or passage of cells onto fresh growth medium, is utilized to preserve Mycobacterium tuberculosis cell lines and is universally employed in TB diagnostics. The impact of such passages, typically performed in the absence of drug, on drug-resistant subpopulations is hypothesized to vary according to the competitive costs of genotypic resistance-associated variants. We applied ultradeep next-generation sequencing to 61 phenotypically rifampin-monoresistant ( n = 17) and preextensively ( n = 41) and extensively ( n = 3) drug-resistant isolates with presumptive heteroresistance at two time points in serial subculture. We found significant dynamic loss of minor-variant resistant subpopulations across all analyzed resistance-determining regions, including eight isolates (13%) whose antibiogram data would have transitioned from resistant to susceptible for at least one drug through subculture. Surprisingly, some resistance-associated variants appeared to be selected for in subculture.