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Pertussis, commonly known as whooping cough, is attributable to the Gram-negative bacteriumBordetella pertussis. Vaccination options againstB. pertussisinclude whole-cell pertussis (wP) and acellular pertussis (aP) vaccines. Acellular vaccines typically consist of purified proteins fromB. pertussis, especially filamentous hemagglutinin (Fha), pertussis toxin (Ptx), and pertactin (Prn), and often include fimbrial proteins (Fim2 and Fim3). During the 1990s, several countries recommended substituting the less reactive aP vaccines for wP vaccines (1). In China, the National Immunization Program has been administering the diphtheria–tetanus–wP vaccine since the 1980s. A shift to the diphtheria–tetanus–aP (DTaP) vaccine, which incorporates Ptx, Prn and Fha as bioactive components, occurred between 2007 and 2013 (2).
The resurgence of pertussis poses a significant global public health challenge, primarily due to vaccine escape and antigenic shifts inBordetella pertussis. There has been a noted divergence in the antigens ofB. pertussisbetween the strains circulating in the population and those present in vaccines (3–4). Strains exhibiting a novel promoter for Ptx (ptxP3) have been identified in various countries, including China (5–7). Significant virulence factors such as Prn and Fha, both integral components ofB. pertussisvaccines, have shown variations. The first report of Prn-deficientB. pertussisstrains occurred in the USA in 1994 (4), with similar strains later identified in China in 2019 (8). Instances of Fha-deficientB. pertussisstrains have also been documented. Additionally, there has been a rise in the occurrence of high-level macrolide-resistantB. pertussisstrains, particularly in China since 2013, which is linked to the A2037G mutation in the 23S rRNA gene (5–6).
Since 2022, two Beijing-based sentinel hospitals have implemented a pertussis surveillance study, monitoring cases suspected of pertussis and conducting laboratory tests forB. pertussisusing real-time PCR from January 2022 to December 2023. A total of 44B. pertussisstrains were isolated from nasopharyngeal swab specimens collected from 44 outpatients, consisting of 13 infants, 28 children, and 3 adults. These strains underwent genetic analysis through sequencing using the Illumina HiSeq 2500 system. This analysis focused on genes encoding eight vaccine-related antigens (ptxA, ptxC, ptxP3, prn, fim2, fim3, fhaB,andtcfA) and a type III secretion system gene (bscI), analyzed using BLAST with an E-value threshold of 1e−5. Allelic variations forptxA, ptxC, ptxP3, prn, fim2-1, fim3-1, fhaB, tcfA2,andbscIwere identified through comparison on the BIGSdb-Pasteur platform (https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst_bordetella_seqdef). All identifiedBordetella pertussisstrains shared the same antigenic profile:ptxA1, ptxC2, ptxP3, prn150, fim2-1, fim3-1, fhaB1, tcfA2,andbscI2. Notably, 14 strains had theprn150allele located on the same DNA sequence contig, whereas in the remaining 30 strains, this allele was split across two contigs due to a gene disruption at a site 240 bp upstream from theprnstart site, identified as a common Prn-deficiency mechanism (4). This disruption was characterized by a 6-bp (GCTAGA) overlap and was associated with a reversed insertion of IS481, hinted by a “CTAG” termination sequence (accession no. M22031)（Fig. 1）. This insertion's location and orientation were confirmed by third-generation sequencing for two strains, one with an intactprn150(BJSY2023BRK008) and one with a truncatedprn150(BJSY2022BRK001), performed on the PacBio platform. The completed genome of BJSY2022BRK001 confirmed reversed IS481 insertion inprn(Fig. 2). Additionally, two strains showed a Fha deficiency, evidenced by a deletion of a “G” at position 1087 in the homopolymeric G-tract offhaB, a mutation previously documented for causing this deficiency (3).
Figure 1.Comparison of the disruptedprngene with the completeprn150allele, highlighting six overlapping bases (GCTAGA) between the contigs. OP866997-prn150: intactprn150.
Figure 2.Circularized genomes featuring complete and truncatedprn150genes. The purple ring denoted BJSY2023BRK008, while the light blue ring signified BJSY2022BRK001. The diagram illustrated the gene structures of completeprn150andprn150with a reversed IS481 insertion.
AllBordetella pertussisstrains possessed identical 23S rRNA gene sequences and exhibited an A2037G mutation. Susceptibility testing conducted with E-test strips indicated that all strains were resistant to erythromycin and azithromycin, with minimum inhibitory concentrations (MIC) exceeding 256 μg/mL.
A phylogenetic tree was constructed based on core SNPs from 148B. pertussisgenomes. This set includes genomes from all strains analyzed in this study, two reference genomes (Tohama I, NC002929.2 and CS, CP086368), and an additional 102 genomes sourced from the NCBI database (BioProject no. PRJNA908268). The analysis revealed no specific linkage among the 44 isolated strains (Figure 3).
Figure 3.Phylogenetic tree based on core SNPs fromBordetella pertussis. The genome highlighted with a light blue background was sequenced in this study, and red fonts were Prn-deficient strains. Genomes marked with a red and green hexagon represented vaccine and Fha-deficient strains, respectively. The strains isolated during this study were distributed across various clusters within the tree.
Abbreviation: SNP=Single Nucleotide Polymorphism. -
Pertussis, a disease preventable by vaccination, has seen an increase in global incidence even with high vaccination coverage; for instance, coverage for the diphtheria-tetanus-pertussis (DTaP) vaccine in China is reported at 99% (7). The DTaP vaccines used in China compriseBordetella pertussisstrains with the antigenic composition ofptxA2/ptxC1/ptxP1/prn1/fim2-1/fim3-1/fhaB1/tcfA2. Recent studies have indicated shifts in the primary vaccine antigens ofB. pertussiscompared to the vaccine strains. Li et al. identified the predominant virulence-associated genotype in northern China asptxA1/ptxC1/ptxP1/prn1/fim2-1/fim3A/tcfA2, noting a rare occurrence of theptxP3strains, all of which were sensitive to erythromycin (6). Fu et al. discovered that 41.1% of the highly virulentB. pertussisstrains carriedptxP3/prn2/ptxC2and were all susceptible to macrolides, whereas the remaining 58.9% were less virulent, carryingptxP1/prn1/ptxC1(5). All identified strains displayed the antigenic configurationptxA1/ptxC2/ptxP3/prn150/fim2-1/fim3-1/fhaB1/tcfA2andbscI2, markedly diverging from the vaccine strain. Notably, all isolated strains containedptxP3/bscI2, differing from previous studies that associatedptxP3strains withbscI3(9). Strains withptxP3are reported to produce more Ptx than those withptxP1, potentially indicating increased virulence (10). However, there are differing opinions on whetherptxP1strains might cause more severe diseases thanptxP3strains (5). Further research is necessary to elucidate the mechanisms driving the rapid proliferation ofptxP3strains in recent years.
Various mechanisms can result in Prn deficiency, such as mutations, deletions, and insertions within theprngene. Cai et al. observed Prn-deficient strains in 11.7% of samples from Shanghai, characterized by nucleotide deletions and stop codon mutations inprn(7). The insertion of IS481 was identified as the predominant cause ofprndisruption (4). Another study reported that 100% of Prn-deficient isolates had Prn inactivated by IS481 insertion, which occurred independently across differentB. pertussislineages (4). In our research, we detected a high prevalence (68.2%, 30/44) of Prn-deficientB. pertussisstrains resulting from IS481 insertion, representing the first documentation of such a significant occurrence.
Fha is a significant virulence factor ofB. pertussis. In this study, we identified two strains deficient in Fha and one strain that was deficient in both Prn and Fha, marking the first reported cases of Fha-deficient strains in China. The emergence of strains with deficiencies in multiple immunogens poses an increasing public health concern, highlighting the necessity for ongoing surveillance ofB. pertussisstrains in China.
AllB. pertussisstrains examined in this study displayed resistance to macrolides, aligning with results from a recent study (7). The increasing prevalence of macrolide-resistantB. pertussisstrains presents considerable challenges to clinical treatment.
The increasing dispersion ofptxP3strains, which are potentially more virulent and exhibit higher resistance to macrolides, poses substantial challenges for treating pertussis. Given the widespread prevalence of Prn-deficiency and the emergence of Fha-deficientB. pertussisstrains, the development of vaccines by numerous local manufacturers is complicated, as Prn and Fha are key antigens in these vaccines. Selecting the appropriate pertussis vaccine for inclusion in China’s National Immunization Program is crucial to enhancing the efficacy of pertussis prevention and control strategies.
The analysis in this study encompassed a limited number of strains, underscoring the need for ongoing surveillance of pertussis pathogens. In addition, only the genotypes of vaccine-associated antigens of isolated strains were analyzed in this study. The subsequent research on the function and pathogenic mechanism of antigen proteins is crucial for the evaluation of vaccine effectiveness and the development of vaccines.
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No conflicts of interest.
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The staff of the participating hospitals and colleagues at Shunyi CDC.
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