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Pertussis, an acute respiratory infection caused by Bordetella pertussis, has recently experienced a dramatic increase in incidence and associated deaths in China, drawing significant clinical attention. This article retrospectively analyzes national data on pertussis incidence and mortality from 2010 to 2024, exploring potential factors contributing to this trend. It also discusses strategies for enhancing vaccination programs, improving early diagnosis and treatment, and optimizing the clinical management of high-risk infants, with the aim of addressing the challenges posed by the current pertussis epidemic.

In Slovenia, primary acellular pertussis vaccines (ACVs) containing pertactin (PRN) were mostly used during 1999-2016; ACVs without PRN were introduced in 2017. Among 123 Bordetella pertussis strains collected during 2002-2020, a total of 48 were PRN-deficient; 44 were collected after 2017. Changes to ACVs could increase PRN-deficient B. pertussis and infections.

Objectives: China has experienced a notable upsurge in pertussis cases post-COVID-19, alongside an age shift to older children, increased vaccine escape, and a notable rise in the prevalence of macrolide-resistant Bordetella pertussis. Here, we present a genomic epidemiological investigation of these events., Methods: We performed a retrospective observational study using culture-positive B pertussis isolated in Shanghai, China, from 2016 to 2024. We analysed strain and pertussis epidemiology dynamics by integrating whole-genome sequencing of 723 strains with antimicrobial susceptibility, transcriptomic profile, and clinical data. We compared the genome sequences of Shanghai strains with 6450 Chinese and global strains., Results: From pre-COVID-19 (before December 2019) to post-COVID-19, patients shifted from predominantly infants (90%, 397/442) to a higher proportion of infections in older children (infant: 16%, 132/844), with the share of vaccinated individuals surging from 31% (107/340) to 88% (664/756). The macrolide-resistant Bordetella pertussis prevalence increased from 60% (267/447) to 98% (830/845). The emergence and expansion of a ptxP3-lineage macrolide-resistant clone, MR-MT28, which is uniquely capable of causing substantial infections among older children and vaccinated individuals, was temporally strongly associated with the pertussis upsurge and epidemiological transition. Although MR-MT28 showed increased expression of genes encoding pertussis toxin, it was associated with significantly milder clinical symptoms and a lower hospitalization rate. MR-MT28 likely originated in China around 2016, after acquiring several key mutations, including a novel prn150 allele, and has been detected across multiple regions in China. In addition, 26% (50/195) of MR-MT28 has evolved into predicted Pertactin (PRN)-deficient strains, with an IS481 insertion being the predominant mechanism., Discussion: We report that the post-COVID-19 upsurge of pertussis in China is associated with ptxP3-MR-MT28, and provide evidence that pathogen evolution is likely the primary factor driving + pertussis upsurge, age shift, and vaccine escape. MR-MT28 poses a high risk of global spread and warrants global surveillance., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Pertussis, which is caused by Bordetella pertussis, has plagued humans for at least 800 years, is highly infectious and can be fatal in the unvaccinated, especially very young infants. Although the rollout of whole-cell pertussis (wP) vaccines in the 1940s and 1950s was associated with a drastic drop in incidence, concerns regarding the reactogenicity of wP vaccines led to the development of a new generation of safer, acellular (aP) vaccines that have been adopted mainly in high-income countries. Over the past 20 years, some countries that boast high aP coverage have experienced a resurgence in pertussis, which has led to substantial debate over the basic immunology, epidemiology and evolutionary biology of the bacterium. Controversy surrounds the duration of natural immunity and vaccine-derived immunity, the ability of vaccines to prevent transmission and severe disease, and the impact of evolution on evading vaccine immunity. Resolving these issues is made challenging by incomplete detection of pertussis cases, the absence of a serological marker of immunity, modest sequencing of the bacterial genome and heterogeneity in diagnostic methods of surveillance. In this Review, we lay out the complexities of contemporary pertussis and, where possible, propose a parsimonious explanation for apparently incongruous observations., (© 2024. Springer Nature Limited.)

Background: Trimethoprim/sulfamethoxazole (TMP/SMZ), traditionally a second-line drug, is now proposed as the primary alternative treatment for macrolide-resistant Bordetella pertussis (B.pertussis). Thus, there is an urgent need to evaluate the phenotypic profile of B. pertussis regarding susceptibility to TMP/SMZ., Methods: Kirby-Bauer (KB) disk diffusion methods and E-test strips were employed to assess the in vitro susceptibility profiles of 128 B. pertussis strains isolated in China between 2017 and 2022 against TMP/SMZ. Additionally, whole genome sequencing was performed on one strain exhibiting a heterogeneous phenotype., Results: B. pertussis displayed three phenotypes to TMP/SMZ based on the first 10 days of culture: a predominant fuzzy-bordered phenotype (118/128, 92.2 %), a minority with a clear-bordered phenotype (8/128, 6.3 %), and a sporadic heterogeneous phenotype (2/128, 1.6 %) where TMP/SMZ heterozygous colonies emerged on the 9th day of incubation. The diameter of the inhibition zone around the paper disk will decrease as the culture time extends, while the MIC value will change in the opposite direction. Almost in all tests, the bacterial colony will cover the entire inhibitory zone after 10-14 days of incubation. Strains exhibiting heterogeneity were compared with the complete genomes of Tohama I and CS strains, and 191 and 179 point mutation sites were identified, respectively, with multiple variants associated with the expression of transcriptional regulators., Conclusion: This study highlights the diverse drug sensitivity phenotypes of B. pertussis to TMP/SMZ and the importance of uniform testing protocols. Continuous and dynamic monitoring of B. pertussis isolates for TMP/SMZ sensitivity, along with investigating its correlation with epidemiological changes, is essential for establishing a scientific foundation for the effective management and control of pertussis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: The cycle threshold (Ct) value is inversely proportional to the number of copies of the target region in a sample, suggesting that a low Ct value indicates a high pathogen load. The relationship between Ct value and clinical presentation in children with pertussis is not well-defined., Methods: We investigated the relationships between the Ct value of nasopharyngeal samples positive for Bordetella pertussis deoxyribonucleic acid via real-time polymerase chain reaction (PCR), collected from children on admission and their adult family members between May 2022 and March 2024 at Hangzhou Children's Hospital, China. The study focused on the correlation between Ct value and clinical presentation in children with pertussis., Results: The Ct value was positively correlated with age (r = 0.362, P = 0.001). The mean Ct value for children with pertussis was 28.0 (range: 22.0-32.0), which was lower than the 32.0 (range: 30.0-34.0) observed in adults. Ct value was inversely correlated with length of stay, an indicator of disease severity (r = -0.356, P = 0.001). Logistic regression analyses revealed that both Ct value (OR: 0.891, 95% CI: 0.799-0.993, P = 0.036) and white blood cell count (OR: 1.127, 95% CI: 1.005-1.263, P = 0.040) were independently associated with severity of pertussis., Conclusions: Real-time PCR Ct values at initial diagnosis for pertussis may potentially predict severe disease outcomes in children., (© 2024. The Author(s).)

Objective: To evaluate the role of Bordetella pertussis (B. pertussis), B. parapertussis, B. holmesii, and B. bronchiseptica on pertussis resurgence in China, particularly the sharp rise since the latest winter., Methods: Nasopharyngeal swabs collected from children with pertussis-like illness from January 2018 to March 2024 were cultured to detect B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica, and tested for all of these except for B. bronchiseptica using a pooled real-time polymerase chain reaction (PCR) kit targeting insertion sequences ptxS1, IS481, IS1001, and hIS1001., Results: Out of the collected 7732 nasopharyngeal swabs, 1531 cases tested positive for B. pertussis (19.8%, 1531/7732), and 10 cases were positive for B. parapertussis (0.1%, 10/7732). B. holmesii and B.bronchiseptica were not detected. The number of specimens and the detection rate of B. pertussis were 1709 and 26.9% (459/1709) in 2018, 1936 and 20.7% (400/1936) in 2019, which sharply declined to 308 and 11.4% (35/308) in 2020, 306 and 4.2% (13/306) in 2021, and then notably increased to 754 and 17.6% (133/754) in 2022, 1842 and 16.0% (295/1842) in 2023, 877 and 22.3% (196/877) in the first quarter of 2024. The proportion of children aged 3 to less than 6 years (preschool age) and 6 to 16 years (school age) in pertussis cases increased significantly during the study period, especially the proportion of school-aged children increased from 2.0% (9/459) in 2018 to 40.8% (80/196) in 2024., Conclusions: B. pertussis was the predominant pathogen among children with pertussis-like illness in China, with sporadic detection of B. parapertussis and no detection of B. holmesii or B.bronchiseptica. The preschool and school-age children are increasingly prevalent in B. pertussis infection cases, which may be associated with the latest rapid escalation of pertussis outbreak., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bordetella pertussis is a Gram-negative bacterium that is the causative agent of the respiratory disease known as pertussis. Since the switch to the acellular vaccines of DTaP and Tap, pertussis cases in the US have risen and cyclically fallen. We have observed that mRNA pertussis vaccines are immunogenic and protective in mice. Here, we further evaluated the pertussis toxoid mRNA antigen and refined the formulation based on optimal pertussis toxin neutralization in vivo . We next evaluated the mRNA pertussis vaccine in Sprague-Dawley rats using an aerosol B. pertussis challenge model paired with whole-body plethysmography to monitor coughing and respiratory function. Female Sprague-Dawley rats were primed and boosted with either commercially available vaccines (DTaP or wP-DTP), an mRNA-DTP vaccine, or mock-vaccinated. The mRNA-DTP vaccine was immunogenic in rats and induced antigen-specific IgG antibodies comparable to DTaP. Rats were then aerosol challenged with a streptomycin-resistant emerging clinical isolate D420Sm1. Bacterial burden was assessed at days 1 and 9 post-challenge, and the mRNA vaccine reduced burden equal to both DTaP and wP-DTP. Whole-body plethysmography revealed that mRNA-DTP vaccinated rats were well protected against coughing which was comparable to the non-challenged group. These data suggest that an mRNA-DTP vaccine is immunogenic in rats and provides protection against aerosolized B. pertussis challenge in Sprague-Dawley rats., Competing Interests: The authors declare no conflict of interest.

In the United States, the general laboratory method for diagnosing pertussis, caused by Bordetella pertussis, is real-time PCR (rt-PCR) targeting insertion sequence 481 (IS 481 ). Other Bordetella species ( parapertussis, holmesii , and bronchiseptica ) can also cause a pertussis-like syndrome, and some commercial laboratory assays include the insertion sequence 1001 (pIS 1001 ) that can detect B. parapertussis / B. bronchiseptica (BppBb ). Because IS 481 exists in B. pertussis and B. holmesii , current commercial assays cannot differentiate these two species. We used a multiplex rt-PCR assay containing species-specific targets to Bordetella to evaluate clinical specimens detected as B. pertussis / B. holmesii ( BpBh ) or BppBb by commercial laboratories. A sample of 3,984 clinical specimens positive for IS 481 or pIS 1001 from two commercial laboratories during 2012-2019 were re-tested at CDC. Agreement of Bordetella species between the CDC and commercial laboratory assays, and the proportion of commercial laboratory specimens that were non- B . pertussis by CDC's assay was assessed. Overall agreement in Bordetella species detection and identification between the CDC and commercial lab assays was 85%. Agreement for identifying B. pertussis was 87% for 3,663 BpBh specimens and 98% for identifying B. parapertussis in 310 BppBb specimens. CDC's assay detected B. holmesii in 55/3,984 (1.4%) specimens. Most discrepant results (410/490, 82%) were BpBh specimens interpreted as indeterminate B. pertussis at CDC. We found a small portion of B. holmesii in a sample of IS 481 -positive clinical specimens originally identified by commercial laboratory rt-PCR assays, suggesting that commercial PCR assays are a reliable diagnostic tool for correctly identifying Bordetella species in most patients with suspected pertussis., Importance: When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS 481 -based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii . The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS 481 -positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States., Competing Interests: Contributing author Dr. Clemente Montero is an employee of Quest Diagnostics, and contributing author Dr. Melinda B. Nye is an employee of Labcorp.

As other European countries, France is experiencing a resurgence of pertussis in 2024. Between 1 January and 31 May 2024, 5,616 (24.9%) positive Bordetella pertussis qPCR tests were identified, following a 3-year period of almost null incidence. Of 67 cultured and whole genome sequenced B. pertussis isolates, 66 produced pertactin and 56 produced FIM2, in contrast to pre-COVID-19 years. One isolate of genotype Bp-AgST4 was resistant to macrolides. Pertussis resurgence may favour isolates that produce FIM2 and pertactin.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 McKay et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bordetella pertussis is the causative pathogen of whooping cough or pertussis, a contagious respiratory disease. Aside from serodiagnosis, laboratory confirmation of pertussis is done through PCR, as B. pertussis is difficult to culture. The ELITe InGenius instrument (ELITechGroup, France) with accompanying Bordetella ELITe MGB Kit was evaluated against a laboratory-developed assay. Both assays combine two screening (IS481, IS1001) and two confirmation targets (recA, ptxA-Pr or IS1002) for optimal sensitivity and specificity. The company's stated claims on sensitivity and reproducibility were confirmed. Accuracy testing showed full concordance between both assays for the screening targets. Minor discrepancies were seen for the B. pertussis confirmation target. Some cross-reactivity with other Bordetella species was observed for the IS481-target, however, none of these were confirmed in the ptxA-Pr target. These results show the suitability of the Bordetella ELITe MGB Kit for the detection and differentiation of B. pertussis, B. parapertussis and B. holmesii., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Helena Martini reports equipment, drugs, or supplies was provided by ELITechGroup BV. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Whopping cough (or Pertussis) is an acute infectious respiratory disease caused by Bordetella pertussis bacteria. The disease is highly transmissible and can be fatal in children under two years old. Since the introduction of vaccine immunization in 1940, Pertussis incidence decreased worldwide. In Brazil, the immunization was introduced in 1977 using the whole cell (wP) vaccine. Despite the high vaccination coverage, an unexpected increase in the number of observed Pertussis cases was observed in 2012. In this year, 2257 cases were reported exceeding the average incidence rate of <1000 cases per year until 2010. This outbreak reached a peak level in 2014 and ended in 2018 according to the Brazilian National Surveillance System (SINAN). To understand the relationship between the outbreak and the vaccination, bacterial isolates (n = 136) from the Brazilian Midwest region obtained during the outbreak were submitted to genotyping of two vaccine loci: ptxP and fim3. Most of isolates (102) were obtained from nursing children (29 days to 2 years old). Genotyping of 94 isolates revealed that fim3-24/ptxP-3 was the most prevalent genotype (68%) associated with the outbreak peak. Two additional genotypes were also observed: fim3-1/ptxP-3 (15%) and fim3-3/ptxP-3 (17%). Conversely, the fim3-1/ptxP-2 genotype, which is harbored by the strain used in the wP vaccine (Bp137), was not observed. These results showed that B. pertussis circulating strains in the outbreak analyzed were different from the strain used for Pertussis immunization in Brazil. These observations provide insights that could be used to target vaccination programs to prevent future whooping cough outbreaks in Brazil., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Background: In 2020, the Council of State and Territorial Epidemiologists (CSTE) pertussis case definition was modified; the main change was classifying polymerase chain reaction (PCR)-positive cases as confirmed, regardless of cough duration. Pertussis data reported through Enhanced Pertussis Surveillance (EPS) in 7 sites and the National Notifiable Diseases Surveillance System (NNDSS) were used to evaluate the impact of the new case definition., Methods: We compared the number of EPS cases with cough onset in 2020 to the number that would have been reported based on the prior (2014) CSTE case definition. To assess the impact of the change nationally, the proportion of EPS cases newly reportable under the 2020 CSTE case definition was applied to 2020 NNDSS data to estimate how many additional cases were captured nationally., Results: Among 442 confirmed and probable cases reported to EPS states in 2020, 42 (9.5%) were newly reportable according to the 2020 case definition. Applying this proportion to the 6124 confirmed and probable cases reported nationally in 2020, we estimated that the new definition added 582 cases. Had the case definition not changed, reported cases in 2020 would have decreased by 70% from 2019; the observed decrease was 67%., Conclusions: Despite a substantial decrease in reported pertussis cases in the setting of coronavirus disease 2019 (COVID-19), our data show that the 2020 pertussis case definition change resulted in additional case reporting compared with the previous case definition, providing greater opportunities for public health interventions such as prophylaxis of close contacts., Competing Interests: Potential conflicts of interest . The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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., Competing Interests: The authors declare no conflict of interest (such as defined by ASM policy).

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 (Δ BP1092 ), and a Δ BP1092 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.

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., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

The regulation of Bordetella pertussis virulence is mediated by the two-component system BvgA/S, which activates the transcription of virulence-activated genes ( vag s). In the avirulent phase, the vag s are not expressed, but instead, virulence-repressed genes ( vrg s) 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 vrg s 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 vrg s and, surprisingly, of some vag s, as well as for other genes not identified as vag s or vrg s. Unexpectedly, many genes, including some vag s, 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 ( vag s) 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 vrg s, 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 vrg s, 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 vag s, illustrating the previously underappreciated complexity of virulence regulation in B. pertussis ., Competing Interests: The authors declare no conflict of interest.

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 IS 481 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., Competing Interests: The authors declare no conflict of interest.

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., (© 2024 The Societies and John Wiley & Sons Australia, Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Koide et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (Copyright © 2023 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

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., (© 2024 médecine/sciences – Inserm.)

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 σ 54 , 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., (© 2023 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

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., Competing Interests: The authors declare no conflict of interest.

The present article offers a historical overview on pertussis (whooping cough) by analysing the ancient epidemic manifestations of the disease and the path towards the discovery of an effective vaccine against it. The original mentions of pertussis are examined with reference to Mediaeval Afghanistan and the famous AD 1578 Paris epidemic described by the French physician Guillaume de Baillou. The historical data are then matched with information derived from analyses of phylogenetic trees of B. pertussis. Finally, this article also highlights some recent challenges posed to public health by this infectious disease., Competing Interests: The authors declare no conflict of interest., (©2024 Pacini Editore SRL, Pisa, Italy.)

Bordetella pertussis, a slow-growing Gram-negative coccobacillus and the causative agent of whooping cough, is one of the leading causes of vaccine-preventable death and morbidity globally. A state of asymptomatic human carriage has not yet been demonstrated by population studies but is likely to be an important reservoir for community transmission of infection. Such a carriage state may be a target for future vaccine strategies. This chapter presents a short summary of the characteristics of B. pertussis, which should be taken into account when developing a human challenge model and any future experimental medicine interventions. Three studies involving deliberate infection with B. pertussis have been described to date. The first of these was a scientifically and ethically unacceptable paediatric challenge study involving four children in 1930. The second was an investigation of a putative live vaccine using a genetically modified and attenuated strain of B. pertussis. Finally, a systematically constructed human challenge model using a wild-type, potentially pathogenic strain has been established. The latter study has demonstrated that deliberate induction of asymptomatic colonisation in humans is safe and immunogenic, with colonised participants exhibiting seroconversion to pertussis antigens. It has also shown nasal wash to be a more sensitive method of detecting the presence of B. pertussis than either pernasal swab or throat swab, and that B. pertussis carriage can be cleared effectively with Azithromycin. The development of this wild-type B. pertussis human challenge model will allow the investigation of host-pathogen and facilitate future vaccine development., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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., Competing Interests: Authors M.P., D.I., N.D. and S.S. are employed by the GSK group of companies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

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 ptxP 3 -ptxA 1 -ptxB2-prn 2 -fimH 2 (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.

Competing Interests: Declaration of interests No interests are declared.

The number of cases of pertussis has been increasing since 2014 in China, with high prevalence of macrolide resistance in ptxP1 isolates and low prevalence in ptxP3 isolates. This study aimed to investigate the dynamic changes in the B. pertussis population from paediatric patients and household contacts in Shanghai between 2018 and 2022. Clinical data of laboratory-confirmed cases of pertussis were analysed, while isolates recovered from hospitalized children and household contacts were characterized by antimicrobial susceptibility testing, whole-genome sequencing, vaccine antigen gene typing and phylogenetical analysis. Among 640 laboratory-confirmed cases, 340 (53.1%) were fully vaccinated with DTaP and 114 (17.8%) were hospitalized for treatment. The frequency of erythromycin resistance in the 103 B. pertussis isolates from inpatients (n=73) and household contacts (n=30) was 78.6% (81/103), increasing from 65% (13/20) in 2018 to 100% (26/26) in 2022. The proportion of ptxP3 isolates increased from 35% (7/20) in 2018 to 100% (26/26) in 2022. Based on genomic analysis, a novel ptxP3 clone (MT28-Shanghai) belonging to sublineage IVd was discovered and dominated in 2021-2022, which was characterized with ptxP3, erythromycin resistance and prn150. Twelve (11.7%) predicted pertactin-deficient isolates were found; of these, nine were ptxP3 isolates and three were ptxP1 isolates. A complete shift from ptxP1 to ptxP3 in Shanghai, China, which may have been accelerated by the domination of a novel erythromycin-resistant MT28 clone, challenges the pertussis vaccines used at present in China., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giovannna Brito Carneiro reports financial support was provided by State of Sao Paulo Research Foundation (FAPESP). Julia Tavares Castro reports financial support was provided by State of Sao Paulo Research Foundation (FAPESP). Eliane Namie Miyaji reports financial support was provided by State of Sao Paulo Research Foundation (FAPESP). Eliane Namie Miyaji reports financial support was provided by Butantan Foundation. Maria Leonor Sarno Oliveira reports financial support was provided by State of Sao Paulo Research Foundation (FAPESP). Maria Leonor Sarno Oliveira reports financial support was provided by Butantan Foundation. Marilyne Davi reports financial support was provided by Centre National de la Recherche Scientifique (CNRS). Marilyne Davi reports financial support was provided by Institut Pasteur. Daniel Ladant reports financial support was provided by Centre National de la Recherche Scientifique (CNRS). Daniel Ladant reports financial support was provided by Institut Pasteur. Giovanna Brito Carneiro has patent pending to Licensee. Julia Tavares Castro has patent pending to Licensee. Marilyne Davi has patent pending to Licensee. Eliane Namie Miyaji has patent pending to Licensee. Daniel Ladant has patent pending to Licensee. Maria Leonor Sarno Oliveira has patent pending to Licensee., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Bordetella pertussis, the causative agent of whooping cough, can cause pertussis outbreaks in humans, especially in school-aged children. Here, we performed whole-genome sequencing of 51 B. pertussis isolates (epidemic strain MT27) collected from patients infected during 6 school-associated outbreaks lasting less than 4 months. We compared their genetic diversity with that of 28 sporadic isolates (non-outbreak MT27 isolates) based on single-nucleotide polymorphisms (SNPs). Our temporal SNP diversity analysis revealed a mean SNP accumulation rate (time-weighted average) of 0.21 SNPs/genome/year during the outbreaks. The outbreak isolates showed a mean of 0.74 SNP differences (median, 0; range, 0 to 5) between 238 isolate pairs, whereas the sporadic isolates had a mean of 16.12 SNP differences (median, 17; range 0 to 36) between 378 isolate pairs. A low SNP diversity was observed in the outbreak isolates. Receiver operating characteristic analysis demonstrated that the optimal cutoff value to distinguish between the outbreak and sporadic isolates was 3 SNPs (Youden's index of 0.90 with a true-positive rate of 0.97 and a false-positive rate of 0.07). Based on these results, we propose an epidemiological threshold of ≤3 SNPs per genome as a reliable marker of B. pertussis strain identity during pertussis outbreaks that span less than 4 months. IMPORTANCE Bordetella pertussis is a highly infectious bacterium that easily causes pertussis outbreaks in humans, especially in school-aged children. In detection and investigation of outbreaks, excluding non-outbreak isolates is important for understanding the bacterial transmission routes. Currently, whole-genome sequencing is widely used for outbreak investigations, and the genetic relatedness of outbreak isolates is assessed based on differences in the number of single-nucleotide polymorphisms (SNPs) in the genomes of different isolates. The optimal SNP threshold defining strain identity has been proposed for many bacterial pathogens, but not for B. pertussis. In this study, we performed whole-genome sequencing of 51 B. pertussis outbreak isolates and identified a genetic threshold of ≤3 SNPs per genome as a marker defining the strain identity during pertussis outbreaks. This study provides a useful marker for identifying and analyzing pertussis outbreaks and can serve as a basis for future epidemiological studies on pertussis., Competing Interests: The authors declare no conflict of interest.

Bordetella pertussis is the primary agent of the acute respiratory disease pertussis. It has been reported that the disease has recently become more common, especially in adults and adolescents, and adaptation of the pathogen is thought to have an important influence on the recurrence of the disease. This study aims to determine the effect of erythromycin, azithromycin, and trimethoprim-sulfamethoxazole used in the treatment of pertussis on the virulence gene expressions (prn, ptxS1, fhaB), biofilm-forming and growth of B. pertussis. In this study, the minimum inhibitory concentration (MIC) values of azithromycin and erythromycin in B. pertussis local strain Saadet were determined to be 0.09 μg/mL and 0.3 μg/mL, respectively. However, the Tohama-I and Saadet strains were resistant to trimethoprim-sulfamethoxazole (MIC>32 μg/mL). The biofilm-forming of the Saadet strain decreased with the increase in antibiotic doses. It was observed that 1/32MIC erythromycin and 1/32MIC azithromycin upregulated the expression of fhaB in Tohama-I, whereas the expression of ptxS1 and prn significantly decreased in sub-MICs of erythromycin. In the Saadet strain, only ptxS1 was highly expressed at 1/16MIC azithromycin and erythromycin (p > 0.05). This is the first study to investigate the effect of sub-MIC antibiotics on the expression of virulence genes and biofilm-forming of B. pertussis., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Bordetella pertussis causes pertussis, which is characterized by paroxysmal coughing. This disease is generally prevented through vaccination; however, the number of pertussis cases is increasing worldwide despite high vaccination coverage. We previously reported that an autotransporter of B. pertussis, virulence-associated gene 8 (Vag8), causes coughing in combination with pertussis toxin and lipooligosaccharide. Here, we show that immunization with Vag8 protected mice from coughing after B. pertussis infection and enhanced the efficacy of a current pertussis vaccine containing pertussis toxoid against the cough. Our findings indicate that Vag8 could be a vaccine antigen to prevent pertussis cough., (© 2023 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of competing interest ER, SF, SG, EA and NN are employed by Sanofi Pasteur and ER, SF and EA hold stock options., (Copyright © 2023. Published by Elsevier Ltd.)

Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.

Despite widespread vaccination, Bordetella pertussis continues to cause pertussis infections worldwide, leaving infants at the highest risk of severe illness and death, while people around them are likely the main sources of infection and rapidly spread the disease. Rapid and less complex molecular testing for the specific and timely diagnosis of pertussis remains a challenge that could help to prevent the disease from worsening and prevent its transmission. We aimed to develop and validate a colorimetric loop-mediated isothermal amplification (LAMP) assay using a new target uvrD&#95;2 informed by the pangenome for the specific and early detection of B. pertussis. Compared to that of multitarget quantitative polymerase chain reaction (multitarget qPCR) using a large clinical DNA specimen (n = 600), the diagnostic sensitivity and specificity of the uvrD&#95;2 LAMP assay were 100.0% and 98.6%, respectively, with a 99.7% degree of agreement between the two assays. The novel colorimetric uvrD&#95;2 LAMP assay is highly sensitive and specific for detecting B. pertussis DNA in nasopharyngeal swabs and showed similar diagnostic accuracy to complex and high-cost multitarget qPCR, but it is faster, simpler, and inexpensive, which makes it very helpful for the reliable and timely diagnosis of pertussis in primary health care and resource-limited settings., (© 2023. The Author(s).)

Histamine plays pivotal role in normal physiology and dysregulated production of histamine or signaling through histamine receptors (HRH) can promote pathology. Previously, we showed that Bordetella pertussis or pertussis toxin can induce histamine sensitization in laboratory inbred mice and is genetically controlled by Hrh1/HRH1. HRH1 allotypes differ at three amino acid residues with P 263 -V 313 -L 331 and L 263 -M 313 -S 331 , imparting sensitization and resistance respectively. Unexpectedly, we found several wild-derived inbred strains that carry the resistant HRH1 allotype (L 263 -M 313 -S 331 ) but exhibit histamine sensitization. This suggests the existence of a locus modifying pertussis-dependent histamine sensitization. Congenic mapping identified the location of this modifier locus on mouse chromosome 6 within a functional linkage disequilibrium domain encoding multiple loci controlling sensitization to histamine. We utilized interval-specific single-nucleotide polymorphism (SNP) based association testing across laboratory and wild-derived inbred mouse strains and functional prioritization analyses to identify candidate genes for this modifier locus. Atg7, Plxnd1, Tmcc1, Mkrn2, Il17re, Pparg, Lhfpl4, Vgll4, Rho and Syn2 are candidate genes within this modifier locus, which we named Bphse, enhancer of Bordetella pertussis induced histamine sensitization. Taken together, these results identify, using the evolutionarily significant diversity of wild-derived inbred mice, additional genetic mechanisms controlling histamine sensitization., (© 2023. The Author(s).)

Objectives: To reveal the clinical and molecular characteristics of Bordetella pertussis (BP) prevalent in Shanghai, China., Methods: A total of 9430 children with suspected pertussis from 2021 to 2022 were included, and nasopharyngeal swab samples were collected for polymerase chain reaction detection, culture, antimicrobial susceptibility testing, and 23S rRNA gene A2047G detection. BP strains were typed using multilocus variable-number tandem-repeat analysis and virulence genotyping., Results: Of 9430 cases, 5.1% and 1.6% were confirmed by polymerase chain reaction and culture, respectively. Infants (aged <1 year) accounted for 24.7% and presented much more severe symptoms than noninfants. Pertussis was most frequently detected in infants aged 0-6 months (11.3∼14.0%) and children aged >6-10 years (10.8∼21.7%). Macrolide-resistant BP (MRBP) accounted for 89.3%, and all carried the A2047G mutation. There were six multilocus variable-number tandem-repeat analysis types (MTs), including MT28 (62.0%), MT195 (20%), MT27 (10.0%), MT104 (4.7%), MT55 (2.7%), and MT32 (0.7%). BP strains with pertussis toxin (ptx)P3/(pertactin) prn2/ptxC2/ptxA1/(fimbrial proteins) fim2-1/fim3-1, including MT27, MT28, and MT32, accounted for 72.7%, among which MT27 and MT32 were macrolide-sensitive BP, whereas most (94.6∼100%) of MT28 were MRBP. Strains harboring ptxP1/prn1/ptxC1/ptxA1/fim2-1/fim3-1, including MT55, MT104, and MT195, belonged to macrolide-sensitive BP., Conclusion: The emergence and spread of MT28 ptxP3-MRBP was first reported in China, highlighting the importance of continuous surveillance of ptxP3-MRBP to prevent its potential circulation., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)