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.

Intralesional interferon alpha-2b as a novel treatment for periocular squamous cell carcinoma in horses.

OBJECTIVE: To determine the safety and efficacy of perilesional human recombinant interferon alpha-2b (IFNα2b) for treatment of periocular squamous cell carcinoma (PSCC) in horses. ANIMALS STUDIED: Eleven horses (12 eyes) with PSCC were enrolled in this prospective clinical study with owner consent. PROCEDURES: Systemically healthy horses were included in the study following confirmation of PSCC via biopsy. Every two weeks for a maximum of six treatments, horses were sedated and perilesional injection of IFNα2b (10 million IU) was performed. Tumors were measured prior to each injection and at one, three, and 12 months after treatment completion. A greater than 50% reduction in tumor size was considered positive response to treatment (i.e., partial or complete response). Development of anti-IFNα2b antibodies was assessed using serum samples obtained after treatment initiation and compared with treatment responses. Antibody concentrations were analyzed using a mixed model. Statistical significance was considered p < 0.05. RESULTS: Each horse received four to six perilesional injections of IFNα2b. Five of 12 eyes (4/11 horses) responded to treatment. Two of five eyes showed complete resolution of gross PSCC. No systemic adverse effects were seen. Local swelling occurred during treatment protocol in 6/11 horses but resolved without intervention. All horses developed serum anti-IFNα2b antibodies. There was no evidence of statistical difference in antibody concentration between responders and non-responders. CONCLUSIONS: Perilesional administration of IFNα2b was found to be well-tolerated in horses with PSCC, and induced tumor regression in 42% of treated eyes. Treatment failure appears unrelated to the development of IFNα2b antibodies.

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 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.

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.

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.

Investigation of a Large Diphtheria Outbreak and Cocirculation of Corynebacterium pseudodiphtheriticum Among Forcibly Displaced Myanmar Nationals, 2017-2019.

BACKGROUND: Diphtheria, a life-threatening respiratory disease, is caused mainly by toxin-producing strains of Corynebacterium diphtheriae, while nontoxigenic corynebacteria (eg, Corynebacterium pseudodiphtheriticum) rarely causes diphtheria-like illness. Recently, global diphtheria outbreaks have resulted from breakdown of health care infrastructures, particularly in countries experiencing political conflict. This report summarizes a laboratory and epidemiological investigation of a diphtheria outbreak among forcibly displaced Myanmar nationals in Bangladesh. METHODS: Specimens and clinical information were collected from patients presenting at diphtheria treatment centers. Swabs were tested for toxin gene (tox)-bearing C. diphtheriae by real-time polymerase chain reaction (RT-PCR) and culture. The isolation of another Corynebacterium species prompted further laboratory investigation. RESULTS: Among 382 patients, 153 (40%) tested tox positive for C. diphtheriae by RT-PCR; 31 (20%) PCR-positive swabs were culture confirmed. RT-PCR revealed 78% (298/382) of patients tested positive for C. pseudodiphtheriticum. Of patients positive for only C. diphtheriae, 63% (17/27) had severe disease compared to 55% (69/126) positive for both Corynebacterium species, and 38% (66/172) for only C. pseudodiphtheriticum. CONCLUSIONS: We report confirmation of a diphtheria outbreak and identification of a cocirculating Corynebacterium species. The high proportion of C. pseudodiphtheriticum codetection may explain why many suspected patients testing negative for C. diphtheriae presented with diphtheria-like symptoms.

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.

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.

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.

Complete Genome Sequences of Four Bordetella pertussis Vaccine Reference Strains from Serum Institute of India.

Serum Institute of India is among the world's largest vaccine producers. Here, we report the complete genome sequences for four Bordetella pertussis strains used by Serum Institute of India in the production of whole-cell pertussis vaccines.

Epidemiology of Pertussis Among Young Pakistani Infants: A Community-Based Prospective Surveillance Study.

BACKGROUND: Pertussis remains a cause of morbidity and mortality among young infants. There are limited data on the pertussis disease burden in this age group from low- and lower-middle-income countries, including in South Asia. METHODS: We conducted an active community-based surveillance study from February 2015 to April 2016 among 2 cohorts of young infants in 4 low-income settlements in Karachi, Pakistan. Infants were enrolled either at birth (closed cohort) or at ages up to 10 weeks (open cohort) and followed until 18 weeks of age. Nasopharyngeal swab specimens were obtained from infants who met a standardized syndromic case definition and tested for Bordetella pertussis using real-time polymerase chain reaction. We determined the incidence of pertussis using a protocol-defined case definition, as well as the US Centers for Disease Control and Prevention (CDC) definitions for confirmed and probable pertussis. RESULTS: Of 2021 infants enrolled into the study, 8 infants met the protocol-defined pertussis case definition, for an incidence of 3.96 (95% confidence interval [CI], 1.84-7.50) cases per 1000 infants. Seven of the pertussis cases met the CDC pertussis case definition (5 confirmed, 2 probable), for incidences of CDC-defined confirmed pertussis of 2.47 (95% CI, .90-5.48) cases per 1000 infants, and probable pertussis of 0.99 (95% CI, .17-3.27) cases per 1000 infants. Three of the pertussis cases were severe according to the Modified Preziosi Scale score. CONCLUSIONS: In one of the first prospective surveillance studies of infant pertussis in a developing country, we identified a moderate burden of pertussis disease in early infancy in Pakistan.

Complete Genome Sequences of Four Different Bordetella sp. Isolates Causing Human Respiratory Infections.

Species of the genus Bordetella associate with various animal hosts, frequently causing respiratory disease. Bordetella pertussis is the primary agent of whooping cough and other Bordetella species can cause similar cough illness. Here, we report four complete genome sequences from isolates of different Bordetella species recovered from human respiratory infections.

Complete Genome Sequences of Bordetella pertussis Vaccine Reference Strains 134 and 10536.

Vaccine formulations and vaccination programs against whooping cough (pertussis) vary worldwide. Here, we report the complete genome sequences of two divergent Bordetella pertussis reference strains used in the production of pertussis vaccines.

Genome Structural Diversity among 31 Bordetella pertussis Isolates from Two Recent U.S. Whooping Cough Statewide Epidemics.

During 2010 and 2012, California and Vermont, respectively, experienced statewide epidemics of pertussis with differences seen in the demographic affected, case clinical presentation, and molecular epidemiology of the circulating strains. To overcome limitations of the current molecular typing methods for pertussis, we utilized whole-genome sequencing to gain a broader understanding of how current circulating strains are causing large epidemics. Through the use of combined next-generation sequencing technologies, this study compared de novo, single-contig genome assemblies from 31 out of 33 Bordetella pertussis isolates collected during two separate pertussis statewide epidemics and 2 resequenced vaccine strains. Final genome architecture assemblies were verified with whole-genome optical mapping. Sixteen distinct genome rearrangement profiles were observed in epidemic isolate genomes, all of which were distinct from the genome structures of the two resequenced vaccine strains. These rearrangements appear to be mediated by repetitive sequence elements, such as high-copy-number mobile genetic elements and rRNA operons. Additionally, novel and previously identified single nucleotide polymorphisms were detected in 10 virulence-related genes in the epidemic isolates. Whole-genome variation analysis identified state-specific variants, and coding regions bearing nonsynonymous mutations were classified into functional annotated orthologous groups. Comprehensive studies on whole genomes are needed to understand the resurgence of pertussis and develop novel tools to better characterize the molecular epidemiology of evolving B. pertussis populations. IMPORTANCE Pertussis, or whooping cough, is the most poorly controlled vaccine-preventable bacterial disease in the United States, which has experienced a resurgence for more than a decade. Once viewed as a monomorphic pathogen, B. pertussis strains circulating during epidemics exhibit diversity visible on a genome structural level, previously undetectable by traditional sequence analysis using short-read technologies. For the first time, we combine short- and long-read sequencing platforms with restriction optical mapping for single-contig, de novo assembly of 31 isolates to investigate two geographically and temporally independent U.S. pertussis epidemics. These complete genomes reshape our understanding of B. pertussis evolution and strengthen molecular epidemiology toward one day understanding the resurgence of pertussis.

Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin-deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.