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.

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.

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.

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.

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).

Widely Used Benzalkonium Chloride Disinfectants Can Promote Antibiotic Resistance.

While the misuse of antibiotics has clearly contributed to the emergence and proliferation of resistant bacterial pathogens, with major health consequences, it remains less clear if the widespread use of disinfectants, such as benzalkonium chlorides (BAC), a different class of biocides than antibiotics, has contributed to this problem. Here, we provide evidence that exposure to BAC coselects for antibiotic-resistant bacteria and describe the underlying genetic mechanisms. After inoculation with river sediment, BAC-fed bioreactors selected for several bacterial taxa, including the opportunistic pathogen Pseudomonas aeruginosa, that were more resistant to several antibiotics than their counterparts in a control (no BAC) bioreactor. A metagenomic analysis of the bioreactor microbial communities, confirmed by gene cloning experiments with the derived isolates, suggested that integrative and conjugative elements encoding a BAC efflux pump together with antibiotic resistance genes were responsible for these results. Furthermore, the exposure of the P. aeruginosa isolates to increasing concentrations of BAC selected for mutations in pmrB (polymyxin resistance) and physiological adaptations that contributed to a higher tolerance to polymyxin B and other antibiotics. The physiological adaptations included the overexpression of mexCD-oprJ multidrug efflux pump genes when BAC was added in the growth medium at subinhibitory concentrations. Collectively, our results demonstrated that disinfectants promote antibiotic resistance via several mechanisms and highlight the need to remediate (degrade) disinfectants in nontarget environments to further restrain the spread of antibiotic-resistant bacteria.IMPORTANCE Benzalkonium chlorides (BAC) are biocides broadly used in disinfectant solutions. Disinfectants are widely used in food processing lines, domestic households, and pharmaceutical products and are typically designed to have a different mode of action than antibiotics to avoid interfering with the use of the latter. Whether exposure to BAC makes bacteria more resistant to antibiotics remains an unresolved issue of obvious practical consequences for public health. Using an integrated approach that combines metagenomics of natural microbial communities with gene cloning experiments with isolates and experimental evolution assays, we show that the widely used benzalkonium chloride disinfectants promote clinically relevant antibiotic resistance. Therefore, more attention should be given to the usage of these disinfectants, and their fate in nontarget environments should be monitored more tightly.

Genomic and Transcriptomic Insights into How Bacteria Withstand High Concentrations of Benzalkonium Chloride Biocides.

Benzalkonium chlorides (BAC) are commonly used biocides in broad-spectrum disinfectant solutions. How microorganisms cope with BAC exposure remains poorly understood, despite its importance for disinfection and disinfectant-induced antibiotic resistance. To provide insights into these issues, we exposed two isolates of an opportunistic pathogen, Pseudomonas aeruginosa, to increasing concentrations of BAC. One isolate was preadapted to BAC, as it originated from a bioreactor fed with subinhibitory concentrations of BAC for 3 years, while the other originated from a bioreactor that received no BAC. Replicated populations of both isolates were able to survive high concentrations of BAC, up to 1,200 and 1,600 mg/liter for the non- and preadapted strains, respectively, exceeding typical application doses. Transcriptome sequencing (RNA-seq) analysis revealed upregulation of efflux pump genes and decreased expression of porins related to BAC transport as well as reduced growth rate. Increased expression of spermidine (a polycation) synthase genes and mutations in the pmrB (polymyxin resistance) gene, which cause a reduction in membrane negative charge, suggested that a major adaptation to exposure to the cationic surfactant BAC was to actively stabilize cell surface charge. Collectively, these results revealed that P. aeruginosa adapts to BAC exposure by a combination of mechanisms and provided genetic markers to monitor BAC-resistant organisms that may have applications in the practice of disinfection.IMPORTANCE BAC are widely used as biocides in disinfectant solutions, food-processing lines, domestic households, and health care facilities. Due to their wide use and mode of action, there has been rising concern that BAC may promote antibiotic resistance. Consistent with this idea, at least 40 outbreaks have been attributed to infection by disinfectant- and antibiotic-resistant pathogens such as P. aeruginosa However, the underlying molecular mechanisms that bacteria use to deal with BAC exposure remain poorly elucidated. Elucidating these mechanisms may be important for monitoring and limiting the spread of disinfectant-resistant pathogens. Using an integrated approach that combined genomics and transcriptomics with physiological characterization of BAC-adapted isolates, this study provided a comprehensive understanding of the BAC resistance mechanisms in P. aeruginosa Our findings also revealed potential genetic markers to detect and monitor the abundance of BAC-resistant pathogens across clinical or environmental settings. This work contributes new knowledge about high concentrations of benzalkonium chlorides disinfectants-resistance mechanisms at the whole-cell genomic and transcriptomic level.

Screening and Genomic Characterization of Filamentous Hemagglutinin-Deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure.

Complete Genome Sequences of Bordetella pertussis Isolates with Novel Pertactin-Deficient Deletions.

Clinical isolates of the respiratory pathogen Bordetella pertussis in the United States have become predominantly deficient for the acellular vaccine immunogen pertactin through various independent mutations. Here, we report the complete genome sequences for four B. pertussis isolates that harbor novel deletions responsible for pertactin deficiency.

Quantifying the Importance of the Rare Biosphere for Microbial Community Response to Organic Pollutants in a Freshwater Ecosystem.

A single liter of water contains hundreds, if not thousands, of bacterial and archaeal species, each of which typically makes up a very small fraction of the total microbial community (<0.1%), the so-called "rare biosphere." How often, and via what mechanisms, e.g., clonal amplification versus horizontal gene transfer, the rare taxa and genes contribute to microbial community response to environmental perturbations represent important unanswered questions toward better understanding the value and modeling of microbial diversity. We tested whether rare species frequently responded to changing environmental conditions by establishing 20-liter planktonic mesocosms with water from Lake Lanier (Georgia, USA) and perturbing them with organic compounds that are rarely detected in the lake, including 2,4-dichlorophenoxyacetic acid (2,4-D), 4-nitrophenol (4-NP), and caffeine. The populations of the degraders of these compounds were initially below the detection limit of quantitative PCR (qPCR) or metagenomic sequencing methods, but they increased substantially in abundance after perturbation. Sequencing of several degraders (isolates) and time-series metagenomic data sets revealed distinct cooccurring alleles of degradation genes, frequently carried on transmissible plasmids, especially for the 2,4-D mesocosms, and distinct species dominating the post-enrichment microbial communities from each replicated mesocosm. This diversity of species and genes also underlies distinct degradation profiles among replicated mesocosms. Collectively, these results supported the hypothesis that the rare biosphere can serve as a genetic reservoir, which can be frequently missed by metagenomics but enables community response to changing environmental conditions caused by organic pollutants, and they provided insights into the size of the pool of rare genes and species.IMPORTANCE A single liter of water or gram of soil contains hundreds of low-abundance bacterial and archaeal species, the so called rare biosphere. The value of this astonishing biodiversity for ecosystem functioning remains poorly understood, primarily due to the fact that microbial community analysis frequently focuses on abundant organisms. Using a combination of culture-dependent and culture-independent (metagenomics) techniques, we showed that rare taxa and genes commonly contribute to the microbial community response to organic pollutants. Our findings should have implications for future studies that aim to study the role of rare species in environmental processes, including environmental bioremediation efforts of oil spills or other contaminants.

The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement.

Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology.IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology.