Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses.

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Transcription dosage compensation does not occur in Down syndrome.

BACKGROUND: The increase in DNA copy number in Down syndrome (DS; caused by trisomy 21) has led to the DNA dosage hypothesis, which posits that the level of gene expression is proportional to the gene's DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0×). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in trisomy 21, providing support to the DNA dosage hypothesis. RESULTS: In our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation, even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family with an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. Furthermore, we link the limited apparent dosage compensation to expected allelic variation in transcription levels. CONCLUSIONS: Transcription dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.

Simulation study and comparative evaluation of viral contiguous sequence identification tools.

BACKGROUND: Viruses, including bacteriophages, are important components of environmental and human associated microbial communities. Viruses can act as extracellular reservoirs of bacterial genes, can mediate microbiome dynamics, and can influence the virulence of clinical pathogens. Various targeted metagenomic analysis techniques detect viral sequences, but these methods often exclude large and genome integrated viruses. In this study, we evaluate and compare the ability of nine state-of-the-art bioinformatic tools, including Vibrant, VirSorter, VirSorter2, VirFinder, DeepVirFinder, MetaPhinder, Kraken 2, Phybrid, and a BLAST search using identified proteins from the Earth Virome Pipeline to identify viral contiguous sequences (contigs) across simulated metagenomes with different read distributions, taxonomic compositions, and complexities. RESULTS: Of the tools tested in this study, VirSorter achieved the best F1 score while Vibrant had the highest average F1 score at predicting integrated prophages. Though less balanced in its precision and recall, Kraken2 had the highest average precision by a substantial margin. We introduced the machine learning tool, Phybrid, which demonstrated an improvement in average F1 score over tools such as MetaPhinder. The tool utilizes machine learning with both gene content and nucleotide features. The addition of nucleotide features improves the precision and recall compared to the gene content features alone.Viral identification by all tools was not impacted by underlying read distribution but did improve with contig length. Tool performance was inversely related to taxonomic complexity and varied by the phage host. For instance, Rhizobium and Enterococcus phages were identified consistently by the tools; whereas, Neisseria prophage sequences were commonly missed in this study. CONCLUSION: This study benchmarked the performance of nine state-of-the-art bioinformatic tools to identify viral contigs across different simulation conditions. This study explored the ability of the tools to identify integrated prophage elements traditionally excluded from targeted sequencing approaches. Our comprehensive analysis of viral identification tools to assess their performance in a variety of situations provides valuable insights to viral researchers looking to mine viral elements from publicly available metagenomic data.

Complete genome assembly of Hawai'i environmental nontuberculous mycobacteria reveals unexpected co-isolation with methylobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous environmental opportunistic pathogens that can cause chronic lung disease. Within the United States, Hawai'i has the highest incidence of NTM lung disease, though the precise reasons are yet to be fully elucidated. One possibility is the high prevalence of NTM in the Hawai'i environment acting as a potential reservoir for opportunistic NTM infections. Through our previous initiatives to collect and characterize NTM in Hawai'i, community scientists of Hawai'i have collected thousands of environmental samples for sequencing. Here, these community scientists were invited for the first time into a high school lab in O'ahu for a genomic sequencing workshop, where participants sequenced four of the collected isolate genomic samples using the Oxford Nanopore Technologies MinION sequencer. Participants generated high quality long read data that when combined with short read Illumina data yielded complete bacterial genomic assemblies suitable for in-depth analysis. The gene annotation analysis identified a suite of genes that might help NTM thrive in the Hawai'i environment. Further, we found evidence of co-occurring methylobacteria, revealed from the sequencing data, suggesting that in some cases methylobacteria and NTM may coexist in the same niche, challenging previously accepted paradigms. The sequencing efforts presented here generated novel insights regarding the potential survival strategies and microbial interactions of NTM in the geographic hot spot of Hawai'i. We highlight the contributions of community scientists and present an activity that can be reimplemented as a workshop or classroom activity by other research groups to engage their local communities.

Intraspecies plasmid and genomic variation of Mycobacterium kubicae revealed by the complete genome sequences of two clinical isolates.

Mycobacterium kubicae is 1 of nearly 200 species of nontuberculous mycobacteria (NTM), environmental micro-organisms that in some situations can infect humans and cause severe lung, skin and soft tissue infections. Although numerous studies have investigated the genetic variation among prevalent clinical NTM species, including Mycobacterium abscessus and Mycobacterium avium, many of the less common but clinically relevant NTM species, including M. kubicae, still lack complete genomes to serve as a comparative reference. Well-characterized representative genomes for each NTM species are important both for investigating the pathogenic potential of NTM, as well as for use in diagnostic methods, even for species that less frequently cause human disease. Here, we report the complete genomes of two M. kubicae strains, isolated from two unrelated patients. Hybrid short-read and long-read sequencing and assembly, using sequence reads from Illumina and Oxford Nanopore Technologies platforms, were utilized to resolve the chromosome and plasmid sequences of each isolate. The genome of NJH_MKUB1 had 5135 coding sequences (CDSs), a circular chromosome of length 5.3 Mb and two plasmids. The genome of NJH_MKUB2 had 5957 CDSs, a circular chromosome of 6.0 Mb and five plasmids. We compared our completed genomic assemblies to four recently released draft genomes of M. kubicae in order to better understand intraspecies genomic conservation and variability. We also identified genes implicated in drug resistance, virulence and persistence in the M. kubicae chromosome and plasmids. Virulence factors encoded in the genome and in the plasmids of M. kubicae provide a foundation for investigating how opportunistic environmental NTM may cause disease.