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Clostridioides difficileis a ubiquitous, diarrhoeagenic pathogen often associated with healthcare-acquired infections that can cause a range of symptoms from mild, self-limiting disease to toxic megacolon and death. Since the early 2000s, a large proportion ofC. difficilecases have been attributed to the ribotype 027 (RT027) lineage, which is associated with sequence type 1 (ST1) in theC. difficilemultilocus sequence typing scheme. The spread of ST1 has been attributed, in part, to resistance to fluoroquinolones used to treat unrelated infections, which creates conditions ideal forC. difficilecolonization and proliferation. In this study, we analysed 27 isolates from a healthcare network in northern Arizona, USA, and 1352 publicly available ST1 genomes to place locally sampled isolates into a global context. Whole genome, single nucleotide polymorphism analysis demonstrated that at least six separate introductions of ST1 were observed in healthcare facilities in northern Arizona over an 18-month sampling period. A reconstruction of transmission networks identified potential nosocomial transmission of isolates, which were only identified via whole genome sequence analysis. Antibiotic resistance heterogeneity was observed among ST1 genomes, including variability in resistance profiles among locally sampled ST1 isolates. To investigate why ST1 genomes are so common globally and in northern Arizona, we compared all high-qualityC. difficilegenomes and identified that ST1 genomes have gained and lost a number of genomic regions compared to all otherC. difficilegenomes; analyses of other toxigenicC. difficilesequence types demonstrate that this loss may be anomalous and could be related to niche specialization. These results suggest that a combination of antimicrobial resistance and gain and loss of specific genes may explain the prominent association of this sequence type withC. difficileinfection cases worldwide. The degree of genetic variability in ST1 suggests that classifying all ST1 genomes into a quinolone-resistant hypervirulent clone category may not be appropriate. Whole genome sequencing of clinicalC. difficileisolates provides a high-resolution surveillance strategy for monitoring persistence and transmission ofC. difficileand for assessing the performance of infection prevention and control strategies.

Background 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. Methods and findings 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. Conclusions 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, Rebecca E. Colman and colleagues assess performance, cost, and throughput of a new sequencer designed for detecting drug-resistant tuberculosis strains., Author summary Why was this study done? M. tuberculosis is the leading cause of death from a single infectious agent. Diagnosis of drug-resistant M. tuberculosis is imperative for reducing tuberculosis (TB) mortality. Next-generation sequencing (NGS) can provide comprehensive genetic information on drug resistance for supporting rapid clinical decision-making but currently can only be performed at the reference-laboratory level. Illumina’s solid-state benchtop sequencer iSeq100 adds diversity to the intended use settings in which NGS can be practically utilized for drug-resistant TB detection, but it has not yet been demonstrated that existing TB NGS workflows will perform on the new instrument. What did the researchers do and find? The study was focused on the performance of existing TB whole-genome sequencing (WGS) and targeted NGS approaches that work on the Illumina MiSeq to determine if they could be also utilized on the iSeq100 platforms. WGS library preparations were performed on three strains, and the same libraries were run on two MiSeq instruments and one early-access iSeq100 instrument. The percentage of genome covered was similar across the instruments and the three isolates, ranging from 99.20% to 99.92% of the genome covered. Targeted NGS preparations were performed on 46 strains and run on two MiSeq instruments and one iSeq100 instrument with 99.6% (CI. 98.0%–99.9%) agreement on drug resistance–associated SNPs between iSeq100 and MiSeq data sets. Because of batching-based pricing constraints for TB sequencing, the sequencing cost per sample is higher on the iSeq100 platform than for the same coverage on the MiSeq when batched optimally. However, the iSeq100 allows for a more open-access workflow due to a lower number of samples being in one sequencing batch. This relationship is the same for WGS and targeted NGS, although the scale is different depending on the sequencing approach chosen. What do these findings mean? In settings of individualized TB care, lower throughput will drive a different need for batching, and the iSeq100, with its lower-throughput capabilities, has an advantage over MiSeq. However, 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, the MiSeq will make the most sense.

Modern genome analysis and phylogenomic methods have increased the number of fungal species, as well as enhanced appreciation of the degree of diversity within the fungal kingdom. In this context, we describe a new Parengyodontium species, P. americanum, which is phylogenetically related to the opportunistic human fungal pathogen P. album. Five unusual fungal isolates were recovered from five unique and confirmed coccidioidomycosis patients, and these isolates were subsequently submitted to detailed molecular and morphological identification procedures to determine identity. Molecular and morphological diagnostic analyses showed that the isolates belong to the Cordycipitaceae. Subsequently, three representative genomes were sequenced and annotated, and a new species, P. americanum, was identified. Using various genomic analyses, gene family expansions related to novel compounds and potential for ability to grow in diverse habitats are predicted. A general description of the genomic composition of this newly described species and comparison of genome content with Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris shows a shared core genome of 6371 genes, and 148 genes that appear to be specific for P. americanum. This work provides the framework for future investigations of this interesting fungal species.

RationaleMinority drug-resistant Mycobacterium tuberculosis subpopulations can be associated with phenotypic resistance but are poorly detected by Sanger sequencing or commercial molecular diagnostic assays.ObjectivesTo determine the role of targeted next-generation sequencing in resolving these minor variant subpopulations.MethodsWe used single molecule overlapping reads (SMOR), a targeted next-generation sequencing approach that dramatically reduces sequencing error, to analyze primary cultured isolates phenotypically resistant to rifampin, fluoroquinolones, or aminoglycosides, but for which Sanger sequencing found no resistance-associated variants (RAVs) within respective resistance-determining regions (study group). Isolates also underwent single-colony selection on antibiotic-containing agar, blinded to sequencing results. As a positive control, isolates with multiple colocalizing chromatogram peaks were also analyzed (control group).Measurements and main resultsAmong 61 primary culture isolates (25 study group and 36 control group), SMOR described 66 (49%) and 45 (33%) of 135 total heteroresistant RAVs at frequencies less than 5% and less than 1% of the total mycobacterial population, respectively. In the study group, SMOR detected minor resistant variant subpopulations in 80% (n = 20/25) of isolates with no Sanger-identified RAVs (median subpopulation size, 1.0%; interquartile range, 0.2-3.9%). Single-colony selection on drug-containing media corroborated SMOR results for 90% (n = 18/20) of RAV-containing specimens, and the absence of RAVs in 60% (n = 3/5) of isolates. Overall, Sanger sequencing was concordant with SMOR for 77% (n = 53/69) of macroheteroresistant (5-95% total population), but only 5% of microheteroresistant (

Whole genome sequencing (WGS) of bacteria is becoming standard practice in many laboratories. Applications for WGS analysis include phylogeography and molecular epidemiology, using single nucleotide polymorphisms (SNPs) as the unit of evolution. The Northern Arizona SNP Pipeline (NASP) was developed as a reproducible pipeline that scales well with the large amount of WGS data typically used in comparative genomics applications. In this study, we demonstrate how NASP compares to other tools in the analysis of two real bacterial genomics datasets and one simulated dataset. Our results demonstrate that NASP produces comparable, and often better, results to 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.

The Staphylococcus aureus clonal lineage CC45 is a predominant colonizer of healthy individuals in northern Europe and constitutes a highly basal cluster of the S. aureus population. Here, we report the complete genome sequence of S. aureus strain CA-347 (NRS648), a representative of the methicillin-resistant USA600 clone predominantly found in the United States.