Biology subjects

Tomita, T.

Publications and source records attributed to Tomita, T..

35 records · Page 2Linked to original sources

bioRxiv2022

A red fluorescent genetically encoded biosensor for extracellular L-lactate

L-Lactate, traditionally recognized as a waste product of metabolism, is now appreciated as a key intercellular energy currency in mammals. To enable investigations of intercellular shuttling of L-lactate, we have previously reported eLACCO1.1, a green fluorescent genetically encoded biosensor for extracellular L-lactate. eLACCO1.1 enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue. However, eLACCO1.1 spectrally overlaps with commonly used optical biosensors and actuators, limiting its application for multiplexed imaging or combined use with optogenetic actuators. Here, we report a red fluorescent extracellular L-lactate biosensor, designated R-eLACCO2. R-eLACCO2 is the end-product of extensive directed evolution and exhibits a large fluorescence response to L-lactate with high molecular specificity in vitro. We demonstrate that R-eLACCO2 with optimized leader and anchor sequences shows a large fluorescence change in response to extracellular L-lactate on the membrane of live mammalian cells. R-eLACCO2 should enable multicolor imaging of extracellular L-lactate in combination with other fluorescent probes and optogenetic actuators.

bioengineering↗

bioRxiv2022

WDR82 mediated H3K4me3 is associated with tumor proliferation and therapeutic efficacy in pediatric high-grade gliomas

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. Reduction of H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and expression of genes associated with stem cell features, cell proliferation, the cell cycle and DNA damage repair. Reduction of WDR82 mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82 mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve prognosis for children with malignant gliomas.

cancer biology↗

bioRxiv2022

Emergence of hyper insecticide-resistant dengue vectors in Indochina Peninsula: threats of concomitant knockdown resistance mutations

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. Nevertheless, the development of pyrethroid resistance is a major obstacle to mosquito/disease control worldwide. Here, we focused on the mutations in the target site of pyrethroid insecticides, voltage-sensitive sodium channel (Vssc), and found that Ae. aegypti collected from Vietnam has the L982W allele in the Vssc at a high frequency (>79%). L982W mutation is located in the highly conserved region of Vssc that is associated with sodium-ion selectivity and permeation rate. Strains having the L982W allele showed similar or even higher levels of resistance to pyrethroids than those having V1016G, a typical knockdown resistance allele in Asia. Furthermore, concomitant mutations L982W+F1534C and V1016G+F1534C were confirmed, and strains having these multiple Vssc mutations exhibited incomparably higher levels of pyrethroid resistance than any other field population ever reported. Molecular modeling analysis confirmed that these concomitant mutant alleles could interfere with approaching pyrethroid to Vssc. Remarkably, >90% of Vssc of Ae. aegypti were occupied by these hyper insecticide-resistant haplotypes in Phnom Penh city, Cambodia. Analysis of whole Vssc coding genes suggested that Vsscs have evolved into stronger resistant forms efficiently through gene recombination events. At this point, L982W has never been detected in Vssc of Ae. aegypti from any other neighboring countries. We strongly emphasize the need to be vigilant about these strong resistance genes spreading to the world through Indochina Peninsula. Significance StatementThe high frequency (>78%) of the L982W allele was detected at the target site of the pyrethroid insecticide, the voltage-sensitive sodium channel (Vssc) of Aedes aegypti collected from Vietnam and Cambodia. Haplotypes having concomitant mutations L982W+F1534C and V1016G+F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these haplotypes exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W mutation has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

neuroscience↗

bioRxiv2021

Dense granule protein, GRA64 interacts with host cell ESCRT proteins during Toxoplasma gondii infection

The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In attempts to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a trans-membrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrate that GRA64 appears to interact with certain components of the host Endosomal Sorting Complexes Required for Transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence in mice nor encystation as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of {Delta}gra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection. IMPORTANCEToxoplasma gondii is a widespread foodborne parasite that causes congenital disease and life-threatening complications in immune compromised individuals. Part of this parasites success lies in its ability to infect diverse organisms and host cells, as well as to persist as a latent infection within parasite constructed structures called tissue cysts. In this study, we characterized a protein secreted by T. gondii into its parasitophorous vacuole during intracellular infection, which we dub GRA64. On the vacuole, this protein is exposed to the host cell and interacts with specific host ESCRT proteins. Parasites lacking the GRA64 protein exhibit ultrastructural changes in tissue cysts during chronic infection. This study lays the foundation for future studies on the mechanics and consequences of host ESCRT-parasite protein interactions.

microbiology↗

bioRxiv2021

Arnold tongue entrainment reveals dynamicalprinciples of the embryonic segmentation clock

Living systems exhibit an unmatched complexity, due to countless, entangled interactions across scales. Here we aim to understand a complex system, i.e. segmentation timing in mouse embryos, without a reference to these detailed interactions. To this end, we develop a coarse-grained approach, in which theory guides the experimental identification of the segmentation clock entrainment responses. We demonstrate period- and phase-locking of the segmentation clock across a wide range of entrainment parameters, including higher-order coupling. These quantifications allow to derive the phase response curve (PRC) and Arnold tongues of the segmentation clock, revealing its essential dynamical properties. Our results indicate that the somite segmentation clock has characteristics reminiscent of a highly non-linear oscillator close to an infinite period bifurcation and suggests the presence of long-term feedbacks. Combined, this coarse-grained theoretical-experimental approach reveals how we can derive simple, essential features of a highly complex dynamical system, providing precise experimental control over the pace and rhythm of the somite segmentation clock.

developmental biology↗

bioRxiv2021

BORCS6 is involved in the enlargement of lung lamellar bodies in Lrrk2 knockout mice

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson disease. It has been shown that Lrrk2 knockout (KO) rodents have enlarged lamellar bodies (LBs) in their alveolar epithelial type II cells, although the underlying mechanisms remain unclear. Here we performed proteomic analyses on LBs isolated from Lrrk2 KO mice and found that the LB proteome is substantially different in Lrrk2 KO mice compared with wild-type mice. In Lrrk2 KO LBs, several Rab proteins were increased, and subunit proteins of BLOC-1-related complex (BORC) were decreased. The amount of surfactant protein C was significantly decreased in the bronchoalveolar lavage fluid obtained from Lrrk2 KO mice, suggesting that LB exocytosis is impaired in Lrrk2 KO mice. We also found that the enlargement of LBs is recapitulated in A549 cells upon KO of LRRK2 or by treating cells with LRRK2 inhibitors. Using this model, we show that KO of BORCS6, a BORC subunit gene, but not other BORC genes, causes LB enlargement. Our findings implicate the LRRK2-BORCS6 pathway in the maintenance of LB morphology.

cell biology↗

bioRxiv2020

Seeded assembly in vitro does not replicate the structures of α-synuclein filaments from multiple system atrophy

The propagation of conformational strains by templated seeding is central to the prion concept. Seeded assembly of -synuclein into filaments is believed to underlie the prion-like spreading of protein inclusions in a number of human neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). We previously determined the atomic structures of -synuclein filaments from the putamen of five individuals with MSA. Here, we used filament preparations from three of these brains for the in vitro seeded assembly of recombinant human -synuclein. We find that the structures of the seeded assemblies differ from those of the seeds, suggesting that additional, as yet unknown, factors play a role in the propagation of pathology. Identification of these factors will be essential for understanding the prion-like spreading of -synuclein proteinopathies.

molecular biology↗

bioRxiv2020

Suppression of amyloid-β secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid

Conjugated linoleic acid (CLA) comprises several geometric and positional isomers of the parental linoleic acid (LA). Two of the isomers, cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA) exert various biological activities. However, the effect of CLA on generation of neurotoxic amyloid-{beta} (A{beta}) protein remains unclear. We found that c9,t11CLA significantly suppressed generation of A{beta} in primary cultures of mouse neurons. CLA treatment did not affect the levels of {beta}-site APP-cleaving enzyme 1 (BACE1), a component of active {gamma}-secretase complex presenilin 1 amino-terminal fragment (PS1 NTF), or A{beta} protein precursor (APP) in cultured neurons. BACE1 activity in lysate of neurons treated with c9,t11 CLA, but not t10,c12 CLA, decreased slightly, although c9,t11 CLA did not directly affect the activity of recombinant BACE1. Interestingly, localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, where APP is predominantly cleaved by BACE1. c9,t11 CLA and t10,c12 CLA appeared to be incorporated into membrane phospholipids, as the level of CLA-containing lysophosphatidylcholine (CLA-LPC) increased dramatically in neurons incubated with CLA. Taken together, our findings indicate that accumulation of c9,t11 CLA-LPC, but not t10,c12 CLA-LPC, in neuronal membranes suppresses amyloidogenic cleavage of APP, thereby contributing to preservation of brain neurons by suppressing neurotoxic A{beta} production in aged subjects.

biochemistry↗

bioRxiv2020

Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

ABSTRACTToxoplasma gondii is a highly successful parasite that infects a significant portion of the human population. As an intracellular parasite, T. gondii thrives within many different cell types due to its residence in the parasitophorous vacuole, a specialized and heavily modified compartment in which parasites divide. Within this vacuole, numerous secreted proteins facilitate functions that optimize intracellular survival. We characterized one such protein, TgPPM3C, which is predicted to contain a domain belonging to the PP2C class of serine/threonine phosphatases and is secreted by both tachyzoites and differentiating bradyzoites into the vacuolar lumen. Genetic deletion of TgPPM3C established that parasites lacking this predicted phosphatase exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. A label-free phosphoproteomic approach was utilized to identify putative TgPPM3C substrates and demonstrated several secreted proteins with altered phosphorylation status in the absence of TgPPM3C. Altered phosphorylation status was seen in MYR1, a protein essential to the process of protein effector export from the parasitophorous vacuole into the host cell, and in GRA16 and GRA28, two exported effector proteins. Defects were seen in the export of GRA16 and GRA28, but not the effector TgIST, in the TgPPM3C knockout strain. Parasites lacking TgPPM3C also exhibited defects in host c-Myc induction, a process influenced by effector export. Phosphomimetic mutations of GRA16 serine residues recapitulated export defects, implicating de-phosphorylation as an important process in facilitating the export of GRA16. These findings provide an example of the emerging critical role that phosphatases play in regulating the complex environment of the T. gondii parasitophorous vacuole.View Full Text

microbiology↗

bioRxiv2020

Structures of α-synuclein filaments from multiple system atrophy

Synucleinopathies are human neurodegenerative diseases that include multiple system atrophy (MSA), Parkinsons disease, Parkinsons disease dementia (PDD) and dementia with Lewy bodies (DLB) (1). Existing treatments are at best symptomatic. These diseases are characterised by the presence in brain cells of filamentous inclusions of -synuclein, the formation of which is believed to cause disease (2, 3). However, the structures of -synuclein filaments from human brain are not known. Here we show, using electron cryo-microscopy, that -synuclein inclusions from MSA are made of two types of filaments, each of which consists of two different protofilaments. Non-proteinaceous molecules are present at the protofilament interfaces. By two-dimensional class averaging, we show that -synuclein filaments from the brains of patients with MSA and DLB are different, suggesting that distinct conformers (or strains) characterise synucleinopathies. As was the case of tau assemblies (4-9), the structures of -synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, with implications for understanding the mechanisms of aggregate propagation and neurodegeneration in human brain. These findings have diagnostic and potential therapeutic relevance, especially in view of the unmet clinical need to be able to image filamentous -synuclein inclusions in human brain.

neuroscience↗

bioRxiv2020

Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

Posttranslational modifications (PTMs) on histone tails spatiotemporally dictate mammalian neural stem cell (NSC) fate. Bone morphogenetic protein 4 (BMP4), a member of the transforming growth factor {beta} (TGF-{beta}) superfamily, suppresses NSC proliferation and fosters differentiation into astroglial cells. Whether PTMs mediate these effects of BMP4 is unknown. Here we demonstrate that BMP4 signaling causes a net reduction in cellular histone H3 lysine 4 trimethylation (H3K4me3), an active histone mark at promoters of genes associated with human NSC proliferation. We also show that H3K4me3 reduction by BMP4 is mediated by decreased expression of SETD1A and WDR82, two methyltransferase components of SETD1A-COMPASS. Down-regulation of these components decreases expression of key genes expressed in hNSCs, while ectopic expression via transfection dedifferentiates human astrocytes (HAs). These observations suggest that BMP4 influences NSC fate by regulating PTMs and altering chromatin structure. SIGNIFICANCE STATEMENTBMP4 is critical in determining hNSC fate. Whether histone posttranslational modifications (PTM) mediate the effects of BMP4 is unknown. Here we report that H3K4me3, brought about by its methyltransferases SETD1A and WDR82, at promoters of stem cell genes OCT4 and NESTIN is involved in human neural stem cell (hNSC) maintenance. BMP4 promotes hNSC astroglial differentiation in part through reduction of SETD1A and WDR82 and thus decreased frequency of H3K4me3 at the promoters of these genes. These results provide evidence that BMP4 promotes hNSC differentiation through a potential epigenetic mechanism and extend our understanding of the role of histone PTM in central nervous system development.

neuroscience↗

bioRxiv2019

Complete microbial genomes for public health in Australia and Southwest Pacific

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health lab activities. Here, we present complete genomes of pathogen strains of regional importance to the Southwest Pacific and Australia. These enrich the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health labs.\n\nAnnouncementWhole-genome sequence (WGS) data is increasingly important in public health microbiology (1-4). The data can be used to replicate many of the basic bacterial sub-typing approaches, as well as support epidemiological investigations, such as surveillance and outbreak investigation (5-7). The appeal of WGS data comes from the promise of a single workflow to process all microbial pathogens that can provide easily portable data that promotes deeper integration of surveillance and investigation efforts across jurisdictions. This promise is leading to a concerted effort to move microbial public health to a primarily genome-based workflow at numerous jurisdictions (8-10), including Australia (11).

genomics↗

bioRxiv2019

Genetic analysis of Aedes aegypti captured in two international airports serving the Greater Tokyo area during 2012-2015

Introduction of exotic diseases vectors into a new habitat can drastically change the local epidemiological situation. During 2012--2015, larvae and an adult of the yellow-fever mosquito, Aedes aegypti, were captured alive in two international airports serving to the Greater Tokyo Area, Japan. Because this species does not naturally distribute in this country, those mosquitoes were considered to be introduced from oversea via air-transportation. To infer the places of origin of those mosquitoes, we genotyped 12 microsatellite loci for which the most comprehensive population genetic reference is available. Although clustering by Bayesian and multivariate methods both suggested all those airport mosquitoes belong to Asia/Pacific population, they were not clustered into a single population. Also, there was variation in mitochondrial Cox1 haplotypes among mosquitoes collected in different incidents of discovery which indicated the existence of multiple maternal origins. Whereas we conclude there is little evidence to support overwintering of Ae. aegypti in the airports in this study, special attention is still desired to prevent the invasion of this prominent arbovirus vector.

genetics↗

bioRxiv2019

High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations

Insects voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein are known to confer insecticide resistance and are selected under insecticide pressure. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure of gene VGSC, it is usually challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes via annotated CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all homologous VGSC CDSs except tiny exons from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency by virtue of the high conservation of VGSC at the nucleotide level. Furthermore, we developed an automated bioinformatic analysis pipeline to genotype VGSC after capture sequencing--MoNaS (Mosquito Na+ channel mutation Search)--which conducts mapping of reads, variant calling, and variant annotation for nonsynonymous mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetics studies on resistance alleles with respect to the origin, selection, and migration of both clinically and agriculturally important insect pests.

genetics↗

bioRxiv2018

Comprehensive antibiotic-linked mutation assessment by Resistance Mutation Sequencing (RM-seq)

Acquired mutations are a major mechanism of bacterial antibiotic resistance generation and dissemination, and can arise during treatment of infections. Early detection of sub-populations of resistant bacteria harbouring defined resistance mutations could prevent inappropriate antibiotic prescription. Here we present RM-seq, a new amplicon-based DNA sequencing workflow based on single molecule barcoding coupled with deep-sequencing that enables the high-throughput characterisation and sensitive detection of resistance mutations from complex mixed populations of bacteria. We show that RM-seq reduces both background sequencing noise and PCR amplification bias and allows highly sensitive identification and accurate quantification of antibiotic resistant sub-populations, with relative allele frequencies as low as 10-4. We applied RM-seq to identify and quantify rifampicin resistance mutations in Staphylococcus aureus using pools of 10,000 in vitro selected clones and identified a large number of previously unknown resistance-associated mutations. Targeted mutagenesis and phenotypic resistance testing was used to validate the technique and demonstrate that RM-seq can be used to link subsets of mutations with clinical resistance breakpoints at high-throughput using large pools of in vitro selected resistant clones. Differential analysis of the abundance of resistance mutations after a selection bottleneck detected antimicrobial cross-resistance and collateral sensitivity-conferring mutations. Using a mouse infection model and human clinical samples, we also demonstrate that RM-seq can be effectively applied in vivo to track complex mixed populations of S. aureus and another major human pathogen, Mycobacterium tuberculosis during infections. RM-seq is a powerful new tool to both detect and functionally characterise mutational antibiotic resistance.

genomics↗

bioRxiv2017

Real-time genomic and epidemiological investigation of a multi-institution outbreak of KPC-producing Enterobacteriaceae: a translational study

BackgroundUntil recently, KPC-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.\n\nMethods and FindingsAll KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing (WGS) was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids. Initial investigations indicated the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012-2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.\n\nConclusionsA real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase producing Enterobacteriaceae management.

genomics↗

bioRxiv2017

A Supervised Statistical Learning Approach For Accurate Legionella pneumophila Source Attribution During Outbreaks

Public health agencies are increasingly relying on genomics during Legionnaires disease investigations. However, the causative bacterium (Legionella pneumophila) has an unusual population structure with extreme temporal and spatial genome sequence conservation. Furthermore, Legionnaires disease outbreaks can be caused by multiple L. pneumophila genotypes in a single source. These factors can confound cluster identification using standard phylogenomic methods. Here, we show that a statistical learning approach based on\n\nL. pneumophila core genome single nucleotide polymorphism (SNP) comparisons eliminates ambiguity for defining outbreak clusters and accurately predicts exposure sources for clinical cases. We illustrate the performance of our method by genome comparisons of 234 L. pneumophila isolates obtained from patients and cooling towers in Melbourne, Australia between 1994 and 2014. This collection included one of the largest reported Legionnaires disease outbreaks, involving 125 cases at an aquarium. Using only sequence data from L. pneumophila cooling tower isolates and including all core genome variation, we built a multivariate model using discriminant analysis of principal components (DAPC) to find cooling tower-specific genomic signatures, and then used it to predict the origin of clinical isolates. Model assignments were 93% congruent with epidemiological data, including the aquarium Legionnaires outbreak and three other unrelated outbreak investigations. We applied the same approach to a recently described investigation of Legionnaires disease within a UK hospital and observed model predictive ability of 86%. We have developed a promising means to breach L. pneumophila genetic diversity extremes and provide objective source attribution data for outbreak investigations.

microbiology↗