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Asset Tag: Microbiology

July 19, 2019

Comparative genomic analyses of Clavibacter michiganensis subsp. insidiosus and pathogenicity on Medicago truncatula.

Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,930 genes, with 401 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.

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July 19, 2019

Pacific Biosciences sequencing and IMGT/HighV-QUEST analysis of full-length single chain fragment variable from an in vivo selected phage-display combinatorial Library.

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires bothin vivoandin vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800?bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from anin vivoselection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of thein vivoselection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during thein vivopanning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767?bp, 53 of them covering the whole insert of 977?bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939?bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.

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July 19, 2019

Methylation in Mycobacterium tuberculosis is lineage specific with associated mutations present globally.

DNA methylation is an epigenetic modification of the genome involved in regulating crucial cellular processes, including transcription and chromosome stability. Advances in PacBio sequencing technologies can be used to robustly reveal methylation sites. The methylome of the Mycobacterium tuberculosis complex is poorly understood but may be involved in virulence, hypoxic survival and the emergence of drug resistance. In the most extensive study to date, we characterise the methylome across the 4 major lineages of M. tuberculosis and 2 lineages of M. africanum, the leading causes of tuberculosis disease in humans. We reveal lineage-specific methylated motifs and strain-specific mutations that are abundant globally and likely to explain loss of function in the respective methyltransferases. Our work provides a set of sixteen new complete reference genomes for the Mycobacterium tuberculosis complex, including complete lineage 5 genomes. Insights into lineage-specific methylomes will further elucidate underlying biological mechanisms and other important phenotypes of the epi-genome.

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July 19, 2019

Genomic analysis of hospital plumbing reveals diverse reservoir of bacterial plasmids conferring carbapenem resistance.

The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization ofblaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections withblaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission ofLeclerciasp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings.IMPORTANCECarbapenemase-producing organisms (CPOs) are a global concern because of the morbidity and mortality associated with these resistant Gram-negative bacteria. Horizontal plasmid transfer spreads the resistance mechanism to new bacteria, and understanding the plasmid ecology of the hospital environment can assist in the design of control strategies to prevent nosocomial infections. A 5-year genomic and epidemiological survey was undertaken to study the CPOs in the patient-accessible environment, as well as in the plumbing system removed from the patient. This comprehensive survey revealed a vast, unappreciated reservoir of CPOs in wastewater, which was in contrast to the low positivity rate in both the patient population and the patient-accessible environment. While there were few patient-environmental isolate associations, there were plasmid backbones common to both populations. These results are relevant to all hospitals for which CPO colonization may not yet be defined through extensive surveillance.

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July 19, 2019

A high-throughput approach for identification of nontuberculous mycobacteria in drinking water reveals relationship between water age and Mycobacterium avium.

Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTMrpoBgenes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age (i.e., combined distribution system residence time and home plumbing stagnation time) was associated with a greater relative abundance ofMycobacterium aviumsubsp.avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, includingMycobacterium abscessusandMycobacterium chelonaeOverall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM.IMPORTANCEAn extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTMrpoBgenes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). The extraction procedure recovered, on average, eight times as much NTM DNA and three times as much total DNA from DW as two widely used commercial DNA extraction kits. The combined DNA extraction and sequencing approach allowed high-throughput screening of DW samples to identify NTM, revealing that the relative abundance ofMycobacterium aviumsubsp.aviumincreased with water age. Furthermore, the two-step barcoding approach developed as part of the PacBio sequencing method makes this procedure highly adaptable, allowing it to be used for other target genes and species. Copyright © 2018 Haig et al.

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July 19, 2019

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.© 2018, Fallon et al.

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July 19, 2019

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species.

The fungal genus ofAspergillusis highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverseAspergillusspecies (A. campestris,A. novofumigatus,A. ochraceoroseus, andA. steynii) have been whole-genome PacBio sequenced to provide genetic references in threeAspergillussections.A. taichungensisandA. candidusalso were sequenced for SM elucidation. ThirteenAspergillusgenomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular,A. novofumigatuswas compared with the pathogenic speciesA. fumigatusThis suggests thatA. novofumigatuscan produce most of the same allergens, virulence, and pathogenicity factors asA. fumigatus, suggesting thatA. novofumigatuscould be as pathogenic asA. fumigatusFurthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol inA. ochraceoroseus,A. campestris, andA. steynii, respectively, and novofumigatonin,ent-cycloechinulin, andepi-aszonalenins inA. novofumigatusOur study delivers six fungal genomes, showing the large diversity found in theAspergillusgenus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports ofA. novofumigatuspathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

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July 19, 2019

Phasevarions of bacterial pathogens: Methylomics sheds new light on old enemies.

A wide variety of bacterial pathogens express phase-variable DNA methyltransferases that control expression of multiple genes via epigenetic mechanisms. These randomly switching regulons – phasevarions – regulate genes involved in pathogenesis, host adaptation, and antibiotic resistance. Individual phase-variable genes can be identified in silico as they contain easily recognized features such as simple sequence repeats (SSRs) or inverted repeats (IRs) that mediate the random switching of expression. Conversely, phasevarion-controlled genes do not contain any easily identifiable features. The study of DNA methyltransferase specificity using Single-Molecule, Real-Time (SMRT) sequencing and methylome analysis has rapidly advanced the analysis of phasevarions by allowing methylomics to be combined with whole-transcriptome/proteome analysis to comprehensively characterize these systems in a number of important bacterial pathogens. Copyright © 2018 Elsevier Ltd. All rights reserved.

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July 19, 2019

Resolving the complete genome of Kuenenia stuttgartiensis from a membrane bioreactor enrichment using Single-Molecule Real-Time sequencing.

Anaerobic ammonium-oxidizing (anammox) bacteria are a group of strictly anaerobic chemolithoautotrophic microorganisms. They are capable of oxidizing ammonium to nitrogen gas using nitrite as a terminal electron acceptor, thereby facilitating the release of fixed nitrogen into the atmosphere. The anammox process is thought to exert a profound impact on the global nitrogen cycle and has been harnessed as an environment-friendly method for nitrogen removal from wastewater. In this study, we present the first closed genome sequence of an anammox bacterium, Kuenenia stuttgartiensis MBR1. It was obtained through Single-Molecule Real-Time (SMRT) sequencing of an enrichment culture constituting a mixture of at least two highly similar Kuenenia strains. The genome of the novel MBR1 strain is different from the previously reported Kuenenia KUST reference genome as it contains numerous structural variations and unique genomic regions. We find new proteins, such as a type 3b (sulf)hydrogenase and an additional copy of the hydrazine synthase gene cluster. Moreover, multiple copies of ammonium transporters and proteins regulating nitrogen uptake were identified, suggesting functional differences in metabolism. This assembly, including the genome-wide methylation profile, provides a new foundation for comparative and functional studies aiming to elucidate the biochemical and metabolic processes of these organisms.

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July 19, 2019

Biomonitoring for traditional herbal medicinal products using DNA metabarcoding and single molecule, real-time sequencing.

Global concerns have been paid to the potential hazard of traditional herbal medicinal products (THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, real-time (SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan (JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing (CCS) reads belonging to the ITS2 and psbA-trnH regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.

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July 19, 2019

Herbivorous turtle ants obtain essential nutrients from a conserved nitrogen-recycling gut microbiome.

Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of herbivory across the ants have raised expectations that nutritional symbionts have shaped their diversification. Direct evidence for N provisioning by internally housed symbionts is rare in animals; among the ants, it has been documented for just one lineage. In this study we dissect functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous Cephalotes ants through in vivo experiments, metagenomics, and in vitro assays. Gut bacteria recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes species encode the pathways directing these activities, and several recycle N in vitro. These findings point to a highly efficient N economy, and a nutritional mutualism preserved for millions of years through the derived behaviors and gut anatomy of Cephalotes ants.

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July 19, 2019

Survey on the use of whole-genome sequencing for infectious diseases surveillance: Rapid expansion of European national capacities, 2015-2016.

Whole-genome sequencing (WGS) has become an essential tool for public health surveillance and molecular epidemiology of infectious diseases and antimicrobial drug resistance. It provides precise geographical delineation of spread and enables incidence monitoring of pathogens at genotype level. Coupled with epidemiological and environmental investigations, it delivers ultimate resolution for tracing sources of epidemic infections. To ascertain the level of implementation of WGS-based typing for national public health surveillance and investigation of prioritized diseases in the European Union (EU)/European Economic Area (EEA), two surveys were conducted in 2015 and 2016. The surveys were designed to determine the national public health reference laboratories’ access to WGS and operational WGS-based typing capacity for national surveillance of selected foodborne pathogens, antimicrobial-resistant pathogens, and vaccine-preventable diseases identified as priorities for European genomic surveillance. Twenty-eight and twenty-nine out of the 30 EU/EEA countries participated in the survey in 2015 and 2016, respectively. National public health reference laboratories in 22 and 25 countries had access to WGS-based typing for public health applications in 2015 and 2016, respectively. Reported reasons for limited or no access were lack of funding, staff, and expertise. Illumina technology was the most frequently used followed by Ion Torrent technology. The access to bioinformatics expertise and competence for routine WGS data analysis was limited. By mid-2016, half of the EU/EEA countries were using WGS analysis either as first- or second-line typing method for surveillance of the pathogens and antibiotic resistance issues identified as EU priorities. The sampling frame as well as bioinformatics analysis varied by pathogen/resistance issue and country. Core genome multilocus allelic profiling, also called cgMLST, was the most frequently used annotation approach for typing bacterial genomes suggesting potential bioinformatics pipeline compatibility. Further capacity development for WGS-based typing is ongoing in many countries and upon consolidation and harmonization of methods should enable pan-EU data exchange for genomic surveillance in the medium-term subject to the development of suitable data management systems and appropriate agreements for data sharing.

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July 19, 2019

Genomic repeats, misassembly and reannotation: a case study with long-read resequencing of Porphyromonas gingivalis reference strains.

Without knowledge of their genomic sequences, it is impossible to make functional models of the bacteria that make up human and animal microbiota. Unfortunately, the vast majority of publicly available genomes are only working drafts, an incompleteness that causes numerous problems and constitutes a major obstacle to genotypic and phenotypic interpretation. In this work, we began with an example from the class Bacteroidia in the phylum Bacteroidetes, which is preponderant among human orodigestive microbiota. We successfully identify the genetic loci responsible for assembly breaks and misassemblies and demonstrate the importance and usefulness of long-read sequencing and curated reannotation.We showed that the fragmentation in Bacteroidia draft genomes assembled from massively parallel sequencing linearly correlates with genomic repeats of the same or greater size than the reads. We also demonstrated that some of these repeats, especially the long ones, correspond to misassembled loci in three reference Porphyromonas gingivalis genomes marked as circularized (thus complete or finished). We prove that even at modest coverage (30X), long-read resequencing together with PCR contiguity verification (rrn operons and an integrative and conjugative element or ICE) can be used to identify and correct the wrongly combined or assembled regions. Finally, although time-consuming and labor-intensive, consistent manual biocuration of three P. gingivalis strains allowed us to compare and correct the existing genomic annotations, resulting in a more accurate interpretation of the genomic differences among these strains.In this study, we demonstrate the usefulness and importance of long-read sequencing in verifying published genomes (even when complete) and generating assemblies for new bacterial strains/species with high genomic plasticity. We also show that when combined with biological validation processes and diligent biocurated annotation, this strategy helps reduce the propagation of errors in shared databases, thus limiting false conclusions based on incomplete or misleading information.

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July 19, 2019

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity.Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel.Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain’s genetic profile from pathogenic to environmental.

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July 19, 2019

Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats.

Bat white-nose syndrome (WNS), caused by the fungal pathogen Pseudogymnoascus destructans, has decimated North American hibernating bats since its emergence in 2006. Here, we utilize comparative genomics to examine the evolutionary history of this pathogen in comparison to six closely related nonpathogenic species. P. destructans displays a large reduction in carbohydrate-utilizing enzymes (CAZymes) and in the predicted secretome (~50%), and an increase in lineage-specific genes. The pathogen has lost a key enzyme, UVE1, in the alternate excision repair (AER) pathway, which is known to contribute to repair of DNA lesions induced by ultraviolet (UV) light. Consistent with a nonfunctional AER pathway, P. destructans is extremely sensitive to UV light, as well as the DNA alkylating agent methyl methanesulfonate (MMS). The differential susceptibility of P. destructans to UV light in comparison to other hibernacula-inhabiting fungi represents a potential “Achilles’ heel” of P. destructans that might be exploited for treatment of bats with WNS.

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