Strengthening Bordetella pertussis genomic surveillance by direct sequencing of residual positive specimens.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.

Intramuscular vaccination against SARS-CoV-2 transiently induces neutralizing IgG rather than IgA in the saliva.

The mucosal immunity is crucial for restricting SARS-CoV-2 at its entry site. Intramuscularly applied vaccines against SARS-CoV-2 stimulate high levels of neutralizing Abs in serum, but the impact of these intramuscular vaccinations on features of mucosal immunity is less clear. Here, we analyzed kinetic and functional properties of anti-SARS-CoV-2 Abs in the saliva after vaccination with BNT162b2. We analyzed a total of 24 healthy donors longitudinally for up to 16 months. We found that specific IgG appeared in the saliva after the second vaccination, declined thereafter and reappeared after the third vaccination. Adjusting serum and saliva for the same IgG concentration revealed a strong correlation between the reactivity in these two compartments. Reactivity to VoCs correlated strongly as seen by ELISAs against RBD variants and by live-virus neutralizing assays against replication-competent viruses. For further functional analysis, we purified IgG and IgA from serum and saliva. In vaccinated donors we found neutralizing activity towards authentic virus in the IgG, but not in the IgA fraction of the saliva. In contrast, IgA with neutralizing activity appeared in the saliva only after breakthrough infection. In serum, we found neutralizing activity in both the IgA and IgG fractions. Together, we show that intramuscular mRNA vaccination transiently induces a mucosal immunity that is mediated by IgG and thus differs from the mucosal immunity after infection. Waning of specific mucosal IgG might be linked to susceptibility for breakthrough infection.

Genome-based prediction of cross-protective, HLA-DR-presented epitopes as putative vaccine antigens for multiple Bordetella species.

IMPORTANCE: Pertussis, caused by Bordetella pertussis, can cause debilitating respiratory symptoms, so whole-cell pertussis vaccines (wPVs) were introduced in the 1940s. However, reactogenicity of wPV necessitated the development of acellular pertussis vaccines (aPVs) that were introduced in the 1990s. Since then, until the COVID-19 pandemic began, reported pertussis incidence was increasing, suggesting that aPVs do not induce long-lasting immunity and may not effectively prevent transmission. Additionally, aPVs do not provide protection against other Bordetella species that are observed during outbreaks. The significance of this work is in determining potential new vaccine antigens for multiple Bordetella species that are predicted to elicit long-term immune responses. Genome-based approaches have aided the development of novel vaccines; here, these methods identified Bordetella vaccine candidates that may be cross-protective and predicted to induce strong memory responses. These targets can lead to an improved vaccine with a strong safety profile while also strengthening the longevity of the immune response.

Genomic characterization of Bordetella pertussis in South Africa, 2015-2019.

Pertussis remains a public health concern in South Africa, with an increase in reported cases and outbreaks in recent years. Whole genome sequencing was performed on 32 Bordetella pertussis isolates sourced from three different surveillance programmes in South Africa between 2015 and 2019. Genome sequences were characterized using multilocus sequence typing, vaccine antigen genes (ptxP, ptxA, ptxB, prn and fimH) and overall genome structure. All isolates were sequence type 2 and harboured the pertussis toxin promoter allele ptxP3. The dominant genotype was ptxP3-ptxA1-ptxB2-prn2-fimH2 (31/32, 96.9 %), with no pertactin-deficient or other mutations in vaccine antigen genes identified. Amongst 21 isolates yielding closed genome assemblies, eight distinct genome structures were detected, with 61.9 % (13/21) of the isolates exhibiting three predominant structures. Increases in case numbers are probably not due to evolutionary changes in the genome but possibly due to other factors such as the cyclical nature of B. pertussis disease, waning immunity due to the use of acellular vaccines and/or population immunity gaps.

Genomic characterization of cocirculating Corynebacterium diphtheriae and non-diphtheritic Corynebacterium species among forcibly displaced Myanmar nationals, 2017-2019.

Respiratory diphtheria is a serious infection caused by toxigenic Corynebacterium diphtheriae, and disease transmission mainly occurs through respiratory droplets. Between 2017 and 2019, a large diphtheria outbreak among forcibly displaced Myanmar nationals densely settled in Bangladesh was investigated. Here we utilized whole-genome sequencing (WGS) to characterize recovered isolates of C. diphtheriae and two co-circulating non-diphtheritic Corynebacterium (NDC) species - C. pseudodiphtheriticum and C. propinquum. C. diphtheriae isolates recovered from all 53 positive cases in this study were identified as toxigenic biovar mitis, exhibiting intermediate resistance to penicillin, and formed four phylogenetic clusters circulating among multiple refugee camps. Additional sequenced isolates collected from two patients showed co-colonization with non-toxigenic C. diphtheriae biovar gravis, one of which exhibited decreased susceptibility to the first-line antibiotics and harboured a novel 23-kb multidrug resistance plasmid. Results of phylogenetic reconstruction and virulence-related gene contents of the recovered NDC isolates indicated they were likely commensal organisms, though 80.4 %(45/56) were not susceptible to erythromycin, and most showed high minimum inhibition concentrations against azithromycin. These results demonstrate the high resolution with which WGS can aid molecular investigation of diphtheria outbreaks, through the quantification of bacterial genetic relatedness, as well as the detection of virulence factors and antibiotic resistance markers among case isolates.

Benchmark datasets for SARS-CoV-2 surveillance bioinformatics.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has spread globally and is being surveilled with an international genome sequencing effort. Surveillance consists of sample acquisition, library preparation, and whole genome sequencing. This has necessitated a classification scheme detailing Variants of Concern (VOC) and Variants of Interest (VOI), and the rapid expansion of bioinformatics tools for sequence analysis. These bioinformatic tools are means for major actionable results: maintaining quality assurance and checks, defining population structure, performing genomic epidemiology, and inferring lineage to allow reliable and actionable identification and classification. Additionally, the pandemic has required public health laboratories to reach high throughput proficiency in sequencing library preparation and downstream data analysis rapidly. However, both processes can be limited by a lack of a standardized sequence dataset. Methods: We identified six SARS-CoV-2 sequence datasets from recent publications, public databases and internal resources. In addition, we created a method to mine public databases to identify representative genomes for these datasets. Using this novel method, we identified several genomes as either VOI/VOC representatives or non-VOI/VOC representatives. To describe each dataset, we utilized a previously published datasets format, which describes accession information and whole dataset information. Additionally, a script from the same publication has been enhanced to download and verify all data from this study. Results: The benchmark datasets focus on the two most widely used sequencing platforms: long read sequencing data from the Oxford Nanopore Technologies platform and short read sequencing data from the Illumina platform. There are six datasets: three were derived from recent publications; two were derived from data mining public databases to answer common questions not covered by published datasets; one unique dataset representing common sequence failures was obtained by rigorously scrutinizing data that did not pass quality checks. The dataset summary table, data mining script and quality control (QC) values for all sequence data are publicly available on GitHub: https://github.com/CDCgov/datasets-sars-cov-2. Discussion: The datasets presented here were generated to help public health laboratories build sequencing and bioinformatics capacity, benchmark different workflows and pipelines, and calibrate QC thresholds to ensure sequencing quality. Together, improvements in these areas support accurate and timely outbreak investigation and surveillance, providing actionable data for pandemic management. Furthermore, these publicly available and standardized benchmark data will facilitate the development and adjudication of new pipelines.

Complete Genome Sequences of Four Macrolide-Resistant Nondiphtheritic Corynebacterium Isolates.

This report describes the complete genome sequences of four isolates of the nondiphtheritic Corynebacterium (NDC) species Corynebacterium pseudodiphtheriticum and Corynebacterium propinquum, recovered during investigation of a large diphtheria outbreak in Bangladesh. These data will assist in better delineating the boundary between these related species and understanding their virulence potential.

Toxigenic Corynebacterium diphtheriae Infection in Cat, Texas, USA.

We report a toxigenic strain of Corynebacterium diphtheriae isolated from an oozing dermal wound in a pet cat in Texas, USA. We also describe the epidemiologic public health efforts conducted to identify potential sources of infection and mitigate its spread and the molecular and genetic studies performed to identify the bacterium.

Does the cytokine adsorber CytoSorb® reduce vancomycin exposure in critically ill patients with sepsis or septic shock? a prospective observational study.

BACKGROUND: Hemadsorption of cytokines is used in critically ill patients with sepsis or septic shock. Concerns have been raised that the cytokine adsorber CytoSorb® unintentionally adsorbs vancomycin. This study aimed to quantify vancomycin elimination by CytoSorb®. METHODS: Critically ill patients with sepsis or septic shock receiving continuous renal replacement therapy and CytoSorb® treatment during a prospective observational study were included in the analysis. Vancomycin pharmacokinetics was characterized using population pharmacokinetic modeling. Adsorption of vancomycin by the CytoSorb® was investigated as linear or saturable process. The final model was used to derive dosing recommendations based on stochastic simulations. RESULTS: 20 CytoSorb® treatments in 7 patients (160 serum samples/24 during CytoSorb®-treatment, all continuous infusion) were included in the study. A classical one-compartment model, including effluent flow rate of the continuous hemodialysis as linear covariate on clearance, best described the measured concentrations (without CytoSorb®). Significant adsorption with a linear decrease during CytoSorb® treatment was identified (p < 0.0001) and revealed a maximum increase in vancomycin clearance of 291% (initially after CytoSorb® installation) and a maximum adsorption capacity of 572 mg. For a representative patient of our cohort a reduction of the area under the curve (AUC) by 93 mg/L*24 h during CytoSorb® treatment was observed. The additional administration of 500 mg vancomycin over 2 h during CytoSorb® attenuated the effect and revealed a negligible reduction of the AUC by 4 mg/L*24 h. CONCLUSION: We recommend the infusion of 500 mg vancomycin over 2 h during CytoSorb® treatment to avoid subtherapeutic concentrations. Trial registration NCT03985605. Registered 14 June 2019, https://clinicaltrials.gov/ct2/show/NCT03985605.

Towards comprehensive understanding of bacterial genetic diversity: large-scale amplifications in Bordetella pertussis and Mycobacterium tuberculosis.

Bacterial genetic diversity is often described solely using base-pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplication/amplifications are thought to be widespread among bacteria but due to their often-intractable size and instability, comprehensive studies of these mutations are rare. We define a methodology to investigate amplifications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for amplifications to occur. Analysis of data for 2430 B. pertussis isolates identified 272 putative amplifications, of which 94 % were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of amplifications, suggesting unstable and sporadic characteristics. Genome instability was further described in vitro using long-read sequencing via the Nanopore platform, which revealed that clonally derived laboratory cultures produced heterogenous populations rapidly. We extended this research to analyse a population of 1000 isolates of another important pathogen, Mycobacterium tuberculosis. We found 590 amplifications in M. tuberculosis, and like B. pertussis, these occurred primarily at hotspots. Genes amplified in B. pertussis include those involved in motility and respiration, whilst in M. tuberuclosis, functions included intracellular growth and regulation of virulence. Using publicly available short-read data we predicted previously unrecognized, large amplifications in B. pertussis and M. tuberculosis. This reveals the unrecognized and dynamic genetic diversity of B. pertussis and M. tuberculosis, highlighting the need for a more holistic understanding of bacterial genetics.

Performance testing of four automated coagulation analyzers in a university hospital setting with focus on global coagulation assays.

INTRODUCTION: Several automated coagulation analyzers are available for laboratory use. In a university hospital central laboratory, we compared four different instruments. The results for global coagulation assays are presented here. METHODS: ACL TOP 750 CTS (Instrumentation Laboratory), Atellica COAG 360 (COAG 360), BCS XP (both Siemens Healthineers), and cobas t 711 (Roche Diagnostics) were compared. For inter-instrument comparison, five basic coagulation parameters were analyzed in 476 patient plasma samples. Additional assessments included precision testing using commercial control samples and plasma pools, analysis time for a defined set of samples, sample capacity testing, minimum required sample volumes, and detection quality for hemolytic, icteric, or lipemic (HIL) interferences. RESULTS: Good concordance between different instruments was evident from Bland-Altman plots and comparison of data from each instrument with the overall median (τ≥0.8). Shortest analysis times were found for BCS XP and COAG 360, COAG 360 revealed highest sample capacity. Observed required sample volumes were broadly in line with manufacturer specifications and varied according to instrument configurations. HIL detection differed between instruments and was best with ACL TOP 750 CTS. CONCLUSION: The four analyzers showed similarly high levels of concordance, although some variations were apparent. The most significant differences between the instruments were found in analysis times and sample capacity. Analyzer capabilities must be considered when selecting laboratory equipment and defining algorithms for clinical practice.

High-resolution serum proteome trajectories in COVID-19 reveal patient-specific seroconversion.

A deeper understanding of COVID-19 on human molecular pathophysiology is urgently needed as a foundation for the discovery of new biomarkers and therapeutic targets. Here we applied mass spectrometry (MS)-based proteomics to measure serum proteomes of COVID-19 patients and symptomatic, but PCR-negative controls, in a time-resolved manner. In 262 controls and 458 longitudinal samples of 31 patients, hospitalized for COVID-19, a remarkable 26% of proteins changed significantly. Bioinformatics analyses revealed co-regulated groups and shared biological functions. Proteins of the innate immune system such as CRP, SAA1, CD14, LBP, and LGALS3BP decreased early in the time course. Regulators of coagulation (APOH, FN1, HRG, KNG1, PLG) and lipid homeostasis (APOA1, APOC1, APOC2, APOC3, PON1) increased over the course of the disease. A global correlation map provides a system-wide functional association between proteins, biological processes, and clinical chemistry parameters. Importantly, five SARS-CoV-2 immunoassays against antibodies revealed excellent correlations with an extensive range of immunoglobulin regions, which were quantified by MS-based proteomics. The high-resolution profile of all immunoglobulin regions showed individual-specific differences and commonalities of potential pathophysiological relevance.

SARS-CoV-2 antibody immunoassays in serial samples reveal earlier seroconversion in acutely ill COVID-19 patients developing ARDS.

OBJECTIVES: During the COVID-19 pandemic, SARS-CoV-2 antibody testing has been suggested for (1) screening populations for disease prevalence, (2) diagnostics, and (3) guiding therapeutic applications. Here, we conducted a detailed clinical evaluation of four Anti-SARS-CoV-2 immunoassays in samples from acutely ill COVID-19 patients and in two negative cohorts. METHODS: 443 serum specimens from serial sampling of 29 COVID-19 patients were used to determine clinical sensitivities. Patients were stratified for the presence of acute respiratory distress syndrome (ARDS). Individual serum specimens from a pre-COVID-19 cohort of 238 healthy subjects and from a PCR-negative clinical cohort of 257 patients were used to determine clinical specificities. All samples were measured side-by-side with the Anti-SARS-CoV-2-ELISA (IgG), Anti-SARS-CoV-2-ELISA (IgA) and Anti-SARS-CoV-2-NCP-ELISA (IgG) (Euroimmun AG, Lübeck, Germany) and the Elecsys Anti-SARS-CoV-2 ECLIA (Roche Diagnostics International, Rotkreuz, Switzerland). RESULTS: Median seroconversion occurred earlier in ARDS patients (8-9 days) than in non-ARDS patients (11-17 days), except for EUR N-IgG. Rates of positivity and mean signal ratios in the ARDS group were significantly higher than in the non-ARDS group. Sensitivities between the four tested immunoassays were equivalent. In the set of negative samples, the specificity of the Anti-SARS-CoV-2-ELISA (IgA) was lower (93.9%) compared to all other assays (≥98.8%) and the specificity of Anti-SARS-CoV-2-NCP-ELISA (IgG) was lower (98.8%) than that of Elecsys Anti-SARS-CoV-2 (100%). CONCLUSIONS: Serial sampling in COVID-19 patients revealed earlier seroconversion and higher signal ratios of SARS-CoV-2 antibodies as a potential risk marker for the development of ARDS, suggesting a utility for antibody testing in acutely diseased patients.

Genomic Surveillance and Improved Molecular Typing of Bordetella pertussis Using wgMLST.

Multilocus sequence typing (MLST) provides allele-based characterization of bacterial pathogens in a standardized framework. However, classical MLST schemes for Bordetella pertussis, the causative agent of whooping cough, seldom reveal diversity among the small number of gene targets and thereby fail to delineate population structure. To improve the discriminatory power of allele-based molecular typing of B. pertussis, we have developed a whole-genome MLST (wgMLST) scheme from 225 reference-quality genome assemblies. Iterative refinement and allele curation resulted in a scheme of 3,506 coding sequences and covering 81.4% of the B. pertussis genome. This wgMLST scheme was further evaluated with data from a convenience sample of 2,389 B. pertussis isolates sequenced on Illumina instruments, including isolates from known outbreaks and epidemics previously characterized by existing molecular assays, as well as replicates collected from individual patients. wgMLST demonstrated concordance with whole-genome single nucleotide polymorphism (SNP) profiles, accurately resolved outbreak and sporadic cases in a retrospective comparison, and clustered replicate isolates collected from individual patients during diagnostic confirmation. Additionally, a reanalysis of isolates from two statewide epidemics using wgMLST reconstructed the population structures of circulating strains with increased resolution, revealing new clusters of related cases. Comparison with an existing core genome (cgMLST) scheme highlights the stable gene content of this bacterium and forms the initial foundation for necessary standardization. These results demonstrate the utility of wgMLST for improving B. pertussis characterization and genomic surveillance during the current pertussis disease resurgence.

Genomic epidemiology of nontoxigenic Corynebacterium diphtheriae from King County, Washington State, USA between July 2018 and May 2019.

Between July 2018 and May 2019, Corynebacterium diphtheriae was isolated from eight patients with non-respiratory infections, seven of whom experienced homelessness and had stayed at shelters in King County, WA, USA. All isolates were microbiologically identified as nontoxigenic C. diphtheriae biovar mitis. Whole-genome sequencing confirmed that all case isolates were genetically related, associated with sequence type 445 and differing by fewer than 24 single-nucleotide polymorphisms (SNPs). Compared to publicly available C. diphtheriae genomic data, these WA isolates formed a discrete cluster with SNP variation consistent with previously reported outbreaks. Virulence-related gene content variation within the highly related WA cluster isolates was also observed. These results indicated that genome characterization can readily support epidemiology of nontoxigenic C. diphtheriae.

Immunothrombotic Dysregulation in COVID-19 Pneumonia Is Associated With Respiratory Failure and Coagulopathy.

BACKGROUND: Severe acute respiratory syndrome corona virus 2 infection causes severe pneumonia (coronavirus disease 2019 [COVID-19]), but the mechanisms of subsequent respiratory failure and complicating renal and myocardial involvement are poorly understood. In addition, a systemic prothrombotic phenotype has been reported in patients with COVID-19. METHODS: A total of 62 subjects were included in our study (n=38 patients with reverse transcriptase polymerase chain reaction-confirmed COVID-19 and n=24 non-COVID-19 controls). We performed histopathologic assessment of autopsy cases, surface marker-based phenotyping of neutrophils and platelets, and functional assays for platelet, neutrophil functions, and coagulation tests, as well. RESULTS: We provide evidence that organ involvement and prothrombotic features in COVID-19 are linked by immunothrombosis. We show that, in COVID-19, inflammatory microvascular thrombi are present in the lung, kidney, and heart, containing neutrophil extracellular traps associated with platelets and fibrin. Patients with COVID-19 also present with neutrophil-platelet aggregates and a distinct neutrophil and platelet activation pattern in blood, which changes with disease severity. Whereas cases of intermediate severity show an exhausted platelet and hyporeactive neutrophil phenotype, patients severely affected with COVID-19 are characterized by excessive platelet and neutrophil activation in comparison with healthy controls and non-COVID-19 pneumonia. Dysregulated immunothrombosis in severe acute respiratory syndrome corona virus 2 pneumonia is linked to both acute respiratory distress syndrome and systemic hypercoagulability. CONCLUSIONS: Taken together, our data point to immunothrombotic dysregulation as a key marker of disease severity in COVID-19. Further work is necessary to determine the role of immunothrombosis in COVID-19.

Detection and Characterization of Diphtheria Toxin Gene-Bearing Corynebacterium Species through a New Real-Time PCR Assay.

Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae, with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene (tox). Nontoxigenic tox-bearing (NTTB) Corynebacterium isolates have been described, but impact of these isolates on the accuracy of molecular diagnostics is not well characterized. Here, we describe a new triplex RT-PCR assay to detect tox and distinguish C. diphtheriae from the closely related species C. ulcerans and C. pseudotuberculosis Analytical sensitivity and specificity of the assay were assessed in comparison to culture using 690 previously characterized microbial isolates. The new triplex assay characterized Corynebacterium isolates accurately, with 100% analytical sensitivity for all targets. Analytical specificity with isolates was 94.1%, 100%, and 99.5% for tox, Diph_rpoB, and CUP_rpoB targets, respectively. Twenty-nine NTTB Corynebacterium isolates, representing 5.9% of 494 nontoxigenic isolates tested, were detected by RT-PCR. Whole-genome sequencing of NTTB isolates revealed varied mutations putatively underlying their lack of toxin production, as well as eight isolates with no mutation in tox or the promoter region. This new Corynebacterium RT-PCR method provides a rapid tool to screen isolates and identify probable diphtheria cases directly from specimens. However, the sporadic occurrence of NTTB isolates reinforces the viewpoint that diphtheria culture diagnostics continue to provide the most accurate case confirmation.

Conserved Patterns of Symmetric Inversion in the Genome Evolution of Bordetella Respiratory Pathogens.

Whooping cough (pertussis), primarily caused by Bordetella pertussis, has resurged in the United States, and circulating strains exhibit considerable chromosome structural fluidity in the form of rearrangement and deletion. The genus Bordetella includes additional pathogenic species infecting various animals, some even causing pertussis-like respiratory disease in humans; however, investigation of their genome evolution has been limited. We studied chromosome structure in complete genome sequences from 167 Bordetella species isolates, as well as 469 B. pertussis isolates, to gain a generalized understanding of rearrangement patterns among these related pathogens. Observed changes in gene order primarily resulted from large inversions and were only detected in species with genomes harboring multicopy insertion sequence (IS) elements, most notably B. holmesii and B. parapertussis While genomes of B. pertussis contain >240 copies of IS481, IS elements appear less numerous in other species and yield less chromosome structural diversity through rearrangement. These data were further used to predict all possible rearrangements between IS element copies present in Bordetella genomes, revealing that only a subset is observed among circulating strains. Therefore, while it appears that rearrangement occurs less frequently in other species than in B. pertussis, these clinically relevant respiratory pathogens likely experience similar mutation of gene order. The resulting chromosome structural fluidity presents both challenges and opportunity for the study of Bordetella respiratory pathogens.IMPORTANCE Bordetella pertussis is the primary agent of whooping cough (pertussis). The Bordetella genus includes additional pathogens of animals and humans, including some that cause pertussis-like respiratory illness. The chromosome of B. pertussis has previously been shown to exhibit considerable structural rearrangement, but insufficient data have prevented comparable investigation in related species. In this study, we analyze chromosome structure variation in several Bordetella species to gain a generalized understanding of rearrangement patterns in this genus. Just as in B. pertussis, we observed inversions in other species that likely result from common mutational processes. We used these data to further predict additional, unobserved inversions, suggesting that specific genome structures may be preferred in each species.

Genomic Survey of Bordetella pertussis Diversity, United States, 2000-2013.

We characterized 170 complete genome assemblies from clinical Bordetella pertussis isolates representing geographic and temporal diversity in the United States. These data capture genotypic shifts, including increased pertactin deficiency, occurring amid the current pertussis disease resurgence and provide a foundation for needed research to direct future public health control strategies.

imGLAD: accurate detection and quantification of target organisms in metagenomes.

Accurate detection of target microbial species in metagenomic datasets from environmental samples remains limited because the limit of detection of current methods is typically inaccessible and the frequency of false-positives, resulting from inadequate identification of regions of the genome that are either too highly conserved to be diagnostic (e.g., rRNA genes) or prone to frequent horizontal genetic exchange (e.g., mobile elements) remains unknown. To overcome these limitations, we introduce imGLAD, which aims to detect (target) genomic sequences in metagenomic datasets. imGLAD achieves high accuracy because it uses the sequence-discrete population concept for discriminating between metagenomic reads originating from the target organism compared to reads from co-occurring close relatives, masks regions of the genome that are not informative using the MyTaxa engine, and models both the sequencing breadth and depth to determine relative abundance and limit of detection. We validated imGLAD by analyzing metagenomic datasets derived from spinach leaves inoculated with the enteric pathogen Escherichia coli O157:H7 and showed that its limit of detection can be comparable to that of PCR-based approaches for these samples (∼1 cell/gram).