Reemergence of Bordetella parapertussis, United States, 2019-2023.

To determine changes in Bordetella pertussis and B. parapertussis detection rates, we analyzed 1.43 million respiratory multiplex PCR test results from US facilities from 2019 through mid-2023. From mid-2022 through mid-2023, Bordetella spp. detection increased 8.5-fold; 95% of detections were B. parapertussis. While B. parapertussis rates increased, B. pertussis rates decreased.

Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Bordetella filamentous hemagglutinin and adenylate cyclase toxin interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells.

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

Pertussis outbreak in children hospitalized in Rabat (Morocco).

INTRODUCTION: Cyclical pertussis epidemics primarily affect young infants. This study aims to estimate pertussis prevalence during the ongoing 2023 outbreak at our institution, focusing on affected age groups and clinical presentations. MATERIEL AND METHODS: This retrospective study includes patients admitted to Rabat University Hospital Center from 1st January 2021 to 30th June 2023. Symptomatic patients underwent Multiplex Respiratory Panel PCR testing for respiratory infections. The analysis included cases where RT-PCR identified Bordetella spp., with data analysed using SPSS 15.0. RESULTS: Pertussis cases sharply increased from December 2022, constituting 85.4 % of positive samples. Most cases (78.2 %) occurred in infants under 3 months, presenting symptoms such as coughing (94.5 %) and dyspnoea (94.5 %). Pertussis was suspected in 60 % of RT-PCR confirmed cases. B. pertussis DNA was identified in 81.8 % of cases and B. parapertussis DNA in 18.2 % of cases. CONCLUSION: The study exposes a significant pertussis outbreak affecting predominantly young infants.

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.

Combined transcriptomic and ChIPseq analyses of the Bordetella pertussis RisA regulon.

The regulation of Bordetella pertussis virulence is mediated by the two-component system BvgA/S, which activates the transcription of virulence-activated genes (vags). In the avirulent phase, the vags are not expressed, but instead, virulence-repressed genes (vrgs) are expressed, under the control of another two-component system, RisA/K. Here, we combined transcriptomic and chromatin immunoprecipitation sequencing (ChIPseq) data to examine the RisA/K regulon. We performed RNAseq analyses of RisA-deficient and RisA-phosphoablative B. pertussis mutants cultivated in virulent and avirulent conditions. We confirmed that the expression of most vrgs is regulated by phosphorylated RisA. However, the expression of some, including those involved in flagellum biosynthesis and chemotaxis, requires RisA independently of phosphorylation. Many RisA-regulated genes encode proteins with regulatory functions, suggesting multiple RisA regulation cascades. By ChIPseq analyses, we identified 430 RisA-binding sites, 208 within promoter regions, 201 within open reading frames, and 21 in non-coding regions. RisA binding was demonstrated in the promoter regions of most vrgs and, surprisingly, of some vags, as well as for other genes not identified as vags or vrgs. Unexpectedly, many genes, including some vags, like prn, brpL, bipA, and cyaA, contain a BvgA-binding site and a RisA-binding site, which increases the complexity of the RisAK/BvgAS network in B. pertussis virulence regulation.IMPORTANCEThe expression of virulence-activated genes (vags) of Bordetella pertussis, the etiological agent of whooping cough, is under the transcriptional control of the two-component system BvgA/S, which allows the bacterium to switch between virulent and avirulent phases. In addition, the more recently identified two-component system RisA/K is required for the expression of B. pertussis genes, collectively named vrgs, that are repressed during the virulent phase but activated during the avirulent phase. We have characterized the RisA/K regulon by combined transcriptomic and chromatin immunoprecipitation sequencing analyses. We identified more than 400 RisA-binding sites. Many of them are localized in promoter regions, especially vrgs, but some were found within open reading frames and in non-coding regions. Surprisingly, RisA-binding sites were also found in promoter regions of some vags, illustrating the previously underappreciated complexity of virulence regulation in B. pertussis.

Bordetella pertussis isolates in Finland after acellular vaccination: serotype change and biofilm formation.

OBJECTIVES: In Finland, whole cell pertussis vaccine (wP) was introduced in 1952 and was replaced by acellular pertussis vaccine (aP) without fimbrial (FIM) antigen in 2005. We aimed to analyse the changes in serotypes of circulating Bordetella pertussis before and after acellular vaccination and to explore the relationship between biofilm formation and serotype diversity after the introduction of aP vaccine. METHODS: Serotyping of 1399 B. pertussis isolates collected at the Finnish National Reference Laboratory for Pertussis and Diphtheria in Turku, Finland, from 1974 to 2023 was performed by slide agglutination or indirect ELISA. Of 278 isolates collected after 2005, 53 were selected, genotyped for fim3 and fim2 alleles, and tested for biofilm formation. The selection criteria included maintaining a relatively equal distribution of isolates per time interval, ensuring approximately a 50:50 ratio of FIM2 (N = 26) and FIM3 (N = 27) serotypes. The reference strain Tohama I was used as a control. RESULTS: During the wP era, the majority of circulating B. pertussis exhibited the FIM2 serotype. However, FIM3 strains have appeared since 1999 and become prevalent. After the implementation of aP vaccines, the distribution of serotypes has exhibited substantial variability. FIM3 isolates displayed an enhanced biofilm formation compared to FIM2 isolates (Geometric mean value (95% CI): 0.90 (0.79-1.03) vs. 0.75 (0.65-0.85); p < 0.05). Of the 27 FIM3 isolates, 8 harboured fim3-1 and 19 fim3-2 alleles. FIM3 isolates with fim3-2 allele were significantly associated with increased biofilm formation when compared to those with fim3-1 (1.07 (0.96-1.19) vs. 0.61 (0.52-0.72); p < 0.0001). CONCLUSION: Following the implementation of aP vaccines, the distribution of serotypes in Finland has exhibited substantial variability. FIM3 isolates with the fim3-2 allele displayed an enhanced biofilm formation capability compared to FIM2 isolates.

[Impact of vaccination on the evolution of Bordetella pertussis]. / Impact de la vaccination sur l'évolution de Bordetella pertussis.

Vaccines against pertussis, or whooping cough, have been commercialized and used in most countries worldwide for decades. The history of these vaccines is distinctive, marked by the transition from whole-cell vaccines to acellular vaccines in many developed countries over the last two decades. This particular history has had a significant impact on the evolution of Bordetella pertussis, the etiological agent of whooping cough. Both genetic and phenotypic changes appeared, with the emergence of novel alleles for antigens targeted by the vaccines and changes in the expression of these antigens. The main consequence of these changes is the resurgence of whooping cough in many countries and the appearance of strains capable of evading vaccine-induced immunity. The emergence of novel strains under vaccine pressure underscores the importance of considering biological evolution in the conception of new vaccines and vaccine strategies.

Title: Impact de la vaccination sur l'évolution de Bordetella pertussis. Abstract: Les vaccins contre la coqueluche sont commercialisés et utilisés dans la plupart des pays du monde depuis déjà plusieurs décennies. L'histoire des vaccins anticoquelucheux est particulière, les traditionnels vaccins cellulaires ayant été remplacés par des vaccins acellulaires dans de nombreux pays développés au cours des deux décennies passées. Selon leur composition, ces vaccins ont un effet important sur l'évolution de l'agent étiologique de la coqueluche, la bactérie Bordetella pertussis. Cette évolution se traduit par des changements génétiques et phénotypiques chez la bactérie, et a eu comme conséquences la résurgence de la coqueluche dans de nombreux pays ainsi que l'apparition de nouvelles souches échappant aux vaccins. L'émergence de nouvelles souches par pression vaccinale illustre ainsi l'importance de la prise en compte de la biologie de l'évolution de la bactérie dans l'élaboration de nouveaux vaccins.

Whole-genome comparison of two same-genotype macrolide-resistant Bordetella pertussis isolates collected in Japan.

The emergence of macrolide-resistant Bordetella pertussis (MRBP) is a significant problem because it reduces treatment options for pertussis and exacerbates the severity and spread of the disease. MRBP has been widely prevalent in mainland China since the 2010s and has been sporadically detected in other Asian countries. In Japan, two MRBP clinical strains were first isolated in Tokyo and Osaka between June and July 2018. The isolates BP616 in Osaka and BP625 in Tokyo harbored the same virulence-associated allelic genes (including ptxP1, ptxA1, prn1, fim3A, and fhaB3) and MT195 genotype and exhibited similar antimicrobial susceptibility profiles. However, despite their simultaneous occurrence, a distinguishable epidemiological link between these isolates could not be established. To gain further insight into the genetic relationship between these isolates in this study, we performed whole-genome analyses. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms revealed that the isolates belonged to one of the three clades of Chinese MRBP isolates, but there were 11 single-nucleotide polymorphism differences between BP616 and BP625. Genome structure analysis revealed two large inversions (202 and 523 kbp) and one small transposition (3.8 kbp) between the genomes. These findings indicate that the two Japanese MRBP isolates are closely related to Chinese MRBP isolates but are genomically distinct, suggesting that they were introduced into Japan from mainland China through different transmission routes.

Novel vaccine candidates of Bordetella pertussis identified by reverse vaccinology.

Whooping cough is a disease caused by Bordetella pertussis, whose morbidity has increased, motivating the improvement of current vaccines. Reverse vaccinology is a strategy that helps identify proteins with good characteristics fast and with fewer resources. In this work, we applied reverse vaccinology to study the B. pertussis proteome and pangenome with several in-silico tools. We analyzed the B. pertussis Tohama I proteome with NERVE software and compared 234 proteins with B. parapertussis, B. bronchiseptica, and B. holmessi. VaxiJen was used to calculate an antigenicity value; our threshold was 0.6, selecting 84 proteins. The candidates were depurated and grouped in eight family proteins to select representative candidates, according to bibliographic information and their immunological response predicted with ABCpred, Bcepred, IgPred, and C-ImmSim. Additionally, a pangenome study was conducted with 603 B. pertussis strains and PanRV software, identifying 3421 core proteins that were analyzed to select the best candidates. Finally, we selected 15 proteins from the proteome study and seven proteins from the pangenome analysis as good vaccine candidates.

Strain identity of Bordetella pertussis isolates from household members based on whole-genome sequencing.

We evaluated the genetic diversity of Bordetella pertussis, the causative agent of pertussis, within households by whole-genome sequencing. In pairwise comparisons of 23 isolates collected from 11 households, single-nucleotide polymorphism (SNP) analysis revealed extremely low SNP diversity (≤1 SNP) between isolate pairs: no SNPs were detected in 10 households and one SNP was obtained in the remaining household. This SNP was uncommon for B. pertussis and resulted in a nonsynonymous substitution (Ala303Thr) in nicotinate phosphoribosyltransferase. We demonstrated that the same strain is transmitted between household members and that B. pertussis is genomically stable during household transmission.

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.

RpoN (sigma factor 54) contributes to bacterial fitness during tracheal colonization of Bordetella bronchiseptica.

The Gram-negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high-throughput transposon sequencing (Tn-seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN-deficient mutant strain was defective in tracheal colonization when co-inoculated with the wild-type strain. rpoN encodes σ54 , a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions.

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation.

Previously developed whole-cell vaccines against Bordetella pertussis, the causative agent of whooping cough, appeared to be too reactogenic due to their endotoxin content. Reduction in endotoxicity can generally be achieved through structural modifications in the lipid A moiety of lipopolysaccharides (LPS). In this study, we found that dephosphorylation of lipid A in B. pertussis through the heterologous production of the phosphatase LpxE from Francisella novicida did, unexpectedly, not affect Toll-like receptor 4 (TLR4)-stimulating activity. We then focused on the inner core of LPS, whose synthesis has so far not been studied in B. pertussis. The kdtA and kdkA genes, responsible for the incorporation of a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue in the inner core and its phosphorylation, respectively, appeared to be essential. However, the Kdo-bound phosphate could be replaced by a second Kdo after the heterologous production of Escherichia coli kdtA. This structural change in the inner core affected outer-core and lipid A structures and also bacterial physiology, as reflected in cell filamentation and a switch in virulence phase. Furthermore, the eptB gene responsible for the non-stoichiometric substitution of Kdo-bound phosphate with phosphoethanolamine was identified and inactivated. Interestingly, the constructed inner-core modifications affected TLR4-stimulating activity. Whereas endotoxicity studies generally focus on the lipid A moiety, our data demonstrate that structural changes in the inner core can also affect TLR4-stimulating activity.

Molecular Evolution and Increasing Macrolide Resistance of Bordetella pertussis, Shanghai, China, 2016-2022.

Resurgence and spread of macrolide-resistant Bordetella pertussis (MRBP) threaten global public health. We collected 283 B. pertussis isolates during 2016-2022 in Shanghai, China, and conducted 23S rRNA gene A2047G mutation detection, multilocus variable-number tandem-repeat analysis, and virulence genotyping analysis. We performed whole-genome sequencing on representative strains. We detected pertussis primarily in infants (0-1 years of age) before 2020 and older children (>5-10 years of age) after 2020. The major genotypes were ptxP1/prn1/fhaB3/ptxA1/ptxC1/fim2-1/fim3-1 (48.7%) and ptxP3/prn2/fhaB1/ptxA1/ptxC2/fim2-1/fim3-1 (47.7%). MRBP increased remarkably from 2016 (36.4%) to 2022 (97.2%). All MRBPs before 2020 harbored ptxP1, and 51.4% belonged to multilocus variable-number tandem-repeat analysis type (MT) 195, whereas ptxP3-MRBP increased from 0% before 2020 to 66.7% after 2020, and all belonged to MT28. MT28 ptxP3-MRBP emerged only after 2020 and replaced the resident MT195 ptxP1-MRBP, revealing that 2020 was a watershed in the transformation of MRBP.

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.

The whole-cell proteome shows the characteristics of macrolides-resistant Bordetella pertussis in China linked to the biofilm formation.

The macrolides-resistant Bordetella pertussis (MR-Bp) isolates in China evolved from the ptxP1/fhaB3 allele and rapidly became predominant, suggestive of an adaptive transmission ability. This was different from the global prevalent ptxP3 strains, in which MR-Bp was rarely reported. The study aimed to determine the underlying mechanism responsible for fitness and resistance in these two strains. We identify proteomic differences between ptxP1/fhaB3 and ptxP3/fhaB1 strains using tandem mass tag (TMT)-based proteomics. We then performed in-depth bioinformatic analysis to determine differentially expressed genes (DEGs), followed by gene ontology (GO), and protein-protein interaction (PPI) network analysis. Further parallel reaction monitoring (PRM) analysis confirmed the expression of four target proteins. Finally, the crystal violet method was used to determine biofilm-forming ability. The results showed that the main significantly different proteins between the two represent isolates were related to biofilm formation. Furthermore, we have confirmed that ptxP1/fhaB3 showed hyperbiofilm formation in comparison with ptxP3/fhaB1. It is suggested that the resistance and adaptability of ptxP1/fhaB3 strains may be related to the formation of biofilm through proteomics. In a word, we determined the significantly different proteins between the ptxP1/fhaB3 and ptxP3/fhaB1 strains through whole-cell proteome, which were related to biofilm formation.

Development of a droplet digital PCR for pertussis toxin locus copy number determination in a genetically-modified Bordetella pertussis strain.

To improve pertussis toxin (PT) yield in B. pertussis strains for vaccine production a genetically-engineered strain (gdPT 191-134 strain) with a second copy of the genetically detoxified PT (gdPT) locus was developed. The consistency of the production and genetic stability of the strain when used for vaccine production must be established. We developed two simplex ddPCR assays with PCR systems for ptxA, the target gene present in two copies, and pgm, the reference gene present as a single copy. The ddPCR assay had sufficient precision to discriminate the copy number of the PT locus accurately in two B. pertussis strains: one copy in the parent, non-genetically-engineered strain and two copies in the gdPT 191-134 strain. Using the ddPCR assays, we were able to show that the ratio of the ptxA to pgm genes decreased during serial culture passages, due to the loss of PT locus, which in turn, resulted in lower levels of PT production over time. We were then able to assess culture conditions that improved the stability of the double locus, as shown by non-significant reduction in gdPT toxin yield.