Major Antigen Genotypes and Molecular Epidemiological Analysis of Bordetella Pertussis Isolated in Shenzhen


 BackgroundAlthough the global epidemic of pertussis has been controlled through the expanded Programme on Immunization (EPI), the incidence of pertussis has increased significantly in recent years, with a "resurgence" of pertussis occurring in developed countries with high immunization coverage. The incidence of pertussis in Shenzhen, was about 2.02/100,000, far exceeding that of the whole province and the whole country (both < 1/100,000). At the same time, more and more studies have shown that there is antigenic drift in Bortella pertussis, which may be associated with the increased incidence. 50 strains of Bordetella pertussis isolated from 387 suspected cases were collected in Shenzhen in 2018 for genotype distributions and molecular epidemiological characteristics analysis. MethodsThere were 387 suspected cases of pertussis enrolled at surveillance sites in Shenzhen from June to August 2018. Nasopharyngeal swabs of suspicious cases were collected for separation and culture, and the positive strains were identified by real-time PCR. The immunization histories of patients were analyzed to investigate the relationship between pertussis vaccination and infection. The major antigen genes of the isolated positive strains, including ptxA, ptxC, ptxP, prn, fim2, and fim3, were analyzed by second-generation sequencing. The homology and phylogenetic analysis of these genes was performed using the public genome sequence downloaded from GenBank. Results50 strains of Bordetella pertussis were successfully isolated from nasopharyngeal swabs of 387 suspected cases, with a positive rate of 12.9%, including 28 males and 22 females, accounting for 56.0% and 44.0% respectively. It is worth noting that 38 were under one-year-old among the positive patients, accounting for 76.0%. Among the cases with a history of vaccination, 71.4% of positive patients did not complete the basic vaccination process of the DTaP at the time of onset. Three major antigen genotypes different from CS and Tohama I vaccine strains were identified, and they had distant genetic relationships and 62.0% of which was prn2/ptxC2/ptxP3/ptxA1/fim3-1/fim2-1. ConclusionsThe positive rate of cases under one-year-old was significantly higher than that of other age groups and should be monitored. The major antigenic genes of the Bordetella pertussis strains isolated in Shenzhen were different from those of common vaccine strains. This study explained the resurgence of whooping cough from certain angles, including immunization strategy, vaccination time and genome variation of strains, which is beneficial to prevent pertussis infections.

Pertussis is an acute respiratory infectious disease caused by the gram-negative bacterium Bordetella pertussis [1]. The typical symptoms of pertussis include paroxysmal coughing with an inspiratory whoop, post-tussive vomiting, cyanosis, and persistent coryzal symptoms [2]. Pertussis is mainly transmitted by droplets, and infection sources are mainly early patients and carriers. People are generally susceptible to pertussis, especially infants under one year old [3]. The whole-cell pertussis vaccine (WPV) was invented in 1914, then combined with tetanus and diphtheria toxoids in the 1940s to become widely used diphtheria-tetanus-pertussis DTP vaccine, which is about 80% effective in preventing severe illness and death from pertussis. Since then, a successive decline in the incidence of the disease has been observed [4]. However, due to the high frequency of adverse events following WPV immunization, many parents refused to vaccinate their children and lawsuits against the vaccine manufacturers forced many of them to stop producing the vaccine [5,6]. Therefore, the pertussis vaccine currently used in the world is mainly Acellular Pertussis Vaccine (APV). Studies have con rmed that compared with WPV, APV can signi cantly reduce the adverse effects on the body [1]. But in the past decade, pertussis incidence has risen again, and the so-called whooping-cough epidemics have reappeared [7,8]. According to the World Health Organization (WHO), as many as 195,000 children worldwide died of pertussis and its complications in 2008, with 90% of cases occurring in less developed and developing countries [9]. Even in developed countries or countries with high pertussis vaccination rates, the incidence of pertussis has been on the rise in recent years [10]. In 2012, there was an outbreak of pertussis in Washington and other states in the United States, with the highest reported incidence (37.5/100,000) since 1942. It is worth noting that 43% of patients had at least 4 doses of immunological history of acellular DPT vaccine (DTaP) [11]. APV vaccine in China mainly contains puri ed pertussis toxoid (PT) and lamentous hemagglutinin (FHA), and some imported vaccines have additional pertussis adhesion protein (Pertactin, PRN) or mbriae (FIM) antigens [12,13]. The production process of APV can be divided into two types: 1.
Co-puri cation of chemically detoxi ed PT and FHA; 2. Separate puri cation of PT, FHA and 1~3 types of pertactin PRN). Historically, countries around the world have used modi ed intra-cerebral challenge assay (MICA), the only effective method that has obtained clinically veri ed protection on the evaluation of pertussis vaccine, to evaluate the effectiveness of WPV and its stock solution.
In recent years, the incidence of pertussis in adolescent and adult has been increasing year by year, but the clinical symptoms are not typical and the epidemiological characteristics have also changed [14][15][16].
Vaccines are the main way to prevent pertussis, which have been widely used for immunization in since 1960s [17]. Shenzhen adopted whole-cell vaccines before 2008, and gradually replaced it with acellular vaccines from 2008 to 2010. Since 2010, acellular vaccines have been used throughout the city.
According to the national planned immunization program, the age of vaccination is 3, 4, 5, and 18 months after birth. The vaccination rate has kept above 99% in recent years. However, according to the statistics of the China Information System for Disease Control and Prevention, the number of reported cases of pertussis in Shenzhen is rising sharply. Studies have shown that pathogens have undergone adaptive changes under the pressure of immune protection pressure induced by the body after vaccination. The PT, PRN, FIM and other genes encoding vaccine-related antigens of current popular strains are different from vaccine strains [18,19]. Thereby, they could evade the body's immune protection response.
To further explain the possible impact of the genetic difference on the immune effects, and to better understand the pathogenic characteristics, evolutionary characteristics, and molecular epidemiological rules of isolated Bordetella pertussis in China, we sequenced 50 strains of Bordetella pertussis isolated from Shenzhen in 2018 to analyze their population structure and sequence characteristics of major antigen gene.

Material And Methods
Research object. 387 suspected pertussis cases from the outpatient department of Shenzhen Children's Hospital from June to August 2018. Culture. The samples were cultured on the carbon agar plate at 37°C for 3 days. Round, moist, protruding, transparent or translucent suspected colonies were taken for pure culture and then identi ed by biochemical reaction and agglutination tests. Simultaneously, DNA was extracted from bacterial precipitation and the pertussis-speci c genes IS481 and ptxA were detected by Real-time PCR method. Genome sequencing and de novo assembly. The genomic DNA of Bordetella pertussis was extracted using the whole-genome DNA extraction kits according to the manufacturer's instructions. The library was constructed using the Illumina HiSeq 4000 system (Illumina, San Diego, CA, USA) at the Beijing Genomics Institute (Shenzhen, China). DNA sample preparation kit (Illumina, San Diego, CA, USA), and whole gene sequencing and preliminary evaluation ltering was conducted using the Illuminas HiSeq X-Ten platform (BGI, Shenzhen, China). The original data was further ltered by SOAPnuke to obtain valid data via quality control. De novo assembly of the genome was performed by SPAdes gene assembly software (V3.9.1). AY464179.1, m3-3 type:AY464180.1, m3-4 type: AY464181.1). The homologous sequences of virulence factors in the genome sequence of Shenzhen strains were extracted using Blastn, and the sequence alignment and phylogenetic analysis on the homologous genes of ptxA, ptxC, ptxP, prn, m2 and m3 sequences was performed using Mega 7.0 software.

Main reagents and instruments
Single nucleotide polymorphism (SNP) extraction and phylogenetic typing. 842 public genome sequences were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/1008/, as of September 2020). Snippy software (https://github.com/tseemann/snippy/) was used to detect the core genome SNP sites of the public sequence and the Shenzhen strain sequence, and the genome sequence of Tohama I strain (GenBank number: BX470248.1) was used as a reference. TRF software (https://tandem.bu.edu/trf/trf.html) and BLASTn were used to identify the recombination region of the reference genome and to remove the SNP sites located in the repeating region. Based on non-repetitive regions of SNP sites, the maximum likelihood phylogenetic tree was built using IQ-Tree software.

Results
Laboratory test results and epidemiological characteristics analysis. 50 strains of Bordetella pertussis were obtained from nasopharyngeal swabs of 387 children with bedside inoculation, with a positive rate of 12.9% after bedside inoculation. The epidemiological characteristics of patients were shown in Table 1. For the vaccination status of children with pertussis, 8 children did not provide detailed vaccination information. Of the remaining 42 children, 11 cases did not vaccinate the pertussis vaccine (26.2%), 19 cases did not complete the basic immunization process (45.2%), 5 cases completed the basic immunization process (11.9%), and 7 have completed the booster immunization process (16.7%). Taken together, 71.4% of children did not complete the basic pertussis vaccination program at the time of onset.

Genome evolution analysis.
Genetic analysis was performed on 50 isolates and 842 pertussis strains with known sequences worldwide, and the results were shown in Figure 5. The Shenzhen isolates in this study were distributed in three evolutionary branches (phylogenetic group, PG). Genotype of prn2/ptxA1/ptxP3/ptxC2/ m3-1/ m2-1 is concentrated in PG1, genotype of prn3/ptxA1/ptxP1/ptxC1/ m3-1/ m2-1 is located in PG2, and genotype prn1/ptxA1/ptxP1/ptxC1/ m3-1/ m2-1 are concentrated in PG3. The isolates from PG3 are closely related to the epidemic strains in northern China in recent years, but far from foreign strains. All isolates were genetically distant from vaccine strains CS and Tohama I.

Discussion
Among the genus of Bordetella, Bordetella pertussis is the only bacteria that can produce PT, which is the main virulence factor of Bordetella pertussis and consists of ve subunits. Among them, subunit A is the main functional subunit, which has immunoprotective properties and a variety of enzyme activities, and is expressed by ptxA gene [20]. The region between amino acid 65 and 233 is the conserved sequence of pertussis toxin, causing the host cell immune response and is the antigen recognition site of T cells. According to the difference of the 68th, 228th, and 232nd amino acids of the Subunit A, the ptxA gene is divided into four subtypes, 1, 2, 3 and 4 [21]. The ptxA gene subtypes of pertussis vaccine strains and strains isolated before or early in vaccination are reported to be ptxA2 or ptxA4 subtypes in many countries in the world. The reference strains Tohama I and CS in this study belong to ptxA2 subtype strain, while all of our 50 isolates had non-synonymous mutations (G to A), that is, from the original leucine (I) to methionine (M), and belong to the ptxA1 subtype strain. The protective effect of the vaccine produced by the vaccine containing the ptxA2 vaccine strain is weak against the strain containing the ptxA1 gene [22].
The promoter of upstream pertussis toxin gene (ptxP) region of the PT gene of Bordetella pertussis is composed of approximately 170 bases, compared with Bordetella parapertussis and Bordetella bronchitis, which is species-speci c characteristics [23]. This region contains binding sites for RNA polymerase and 6 binding sites of bvg A regulatory protein dimers, which can promote the transcriptional expression of PT by interacting with the regulatory protein bvg A. In recent years, researchers have discovered that Bordetella pertussisPtxP presents a certain polymorphism, and according to the mutation bases at speci c positions, Bordetella pertussisPtxP is divided into 11 subtypes. Dutch researchers have con rmed that strains containing ptxP3 can highly express PT and have gradually become the dominant epidemic strains in the Netherlands. It is speculated that the "pertussis recurrence" may be related to this. Therefore, the ptxP gene of Bordetella pertussis has attracted more and more attention and has been used as a molecular genetic marker for the evolution of Bordetella pertussis [10]. PtxP3 accounted for 62% of the 50 pertussis strains isolated in this study, which is consistent with the international trend.
PRN protein is a non-ciliary outer membrane protein produced by Bordetella pertussis, as an important virulence factor, PRN protein plays an important role in bacterial infection and adhesion to the epithelial cell membrane of the host respiratory system. PRN protein is also an important protective antigen that can induce humoral and cellular immune responses in mice. PRN protein has shown a good protection rate in the respiratory tract attack animal model test of Bordetella pertussis [20]. According to the difference in the structure of this region and amino acids at speci c positions, the prn gene is divided into prn1-12 subtypes [21,24]. Studies in some countries have con rmed that prn genotypes of strains isolated before or in the early stages of inoculation and pertussis vaccine strain are mainly prn1 genotypes. Since the 1980s, prn2 subtypes have appeared in some European countries. By 2000s, prn2 or prn3 had been the dominant genotypes of the isolates in the Netherlands, Finland, France, the United States, and other countries [25,26]. Bioinformatics analyses of the amino acid secondary structure deduced from the prn gene sequence of strains isolated in different ages show that the prn genotype structure of the strains isolated after the 2000s changed to a certain extent, and the hydrophilic region appeared, which led to the change of its immunogenicity. However, the previously isolated prn1 prn7 prn10 and prn11 are a completely hydrophobic region in this region 1 [27]. In this study, 62% of the isolates belonged to prn2 and 2% belonged to prn3, which was consistent with the evolution trend of prn genes in the world.
Clinical trials in some countries have reported that there is a good correlation between the anti-Fim protein antibody titer in the pertussis vaccine and the immune response in mice and the immune vaccine protection in the children. WHO guidelines for pertussis vaccine recommend that strains expressing of type 2 and 3 Fim protein antigens should be selected as the production strain in the pertussis vaccine production. As reported, these two mbriae proteins also have certain immunogenicity and have been used as components of acellular pertussis vaccine by some vaccine manufacturers. In this study, the isolates contained the same m2-1 and m3-1 genes as the vaccine strain, indicating that these two antigen-related genes are relatively conservative.
In this study, only 16.7% of the children had completed the full vaccination of pertussis vaccine, and about 71.4% of positive patients had not completed the basic process of APV vaccination at the time of onset, mainly because these children were relatively young at the age of onset and had not yet reached the planned immunization. The current pertussis immunization targets in China do not include pregnant women, resulting in infants receiving little pertussis antibody from the mother, and the rst dose of vaccination for the child is 3 months old, leading to a longer unprotected window a long-unprotected window for infants of younger months. These may be important reasons for the high incidence of pertussis in China. Another possible reason is that the effectiveness of the pertussis vaccine declines rapidly over time [28,29]. Studies have shown that the pertussis vaccine is highly effective within three years of vaccination, but then the immunity gradually weakens, with little protection after seven years [30,31].
Since the APV vaccine was widely used worldwide at the late 1990s, in addition to FHA, other components including PT, PRN, Fim2, and Fim3 showed different degrees of antigenic gene polymorphisms between vaccine strains and isolated epidemic strains, resulting in a signi cant change in the population of Bordetella pertussis. That explains why strains isolated in regions or countries with low vaccination coverage are similar to those circulating before the introduction of the vaccine. Therefore, molecular monitoring of epidemic strains is particularly important. This study recommends that bacterial typing methods (such as the MLVA method) should be used to add information on pertussis epidemic strains in different countries or regions to the established database, and to gradually establish a pertussis epidemic surveillance system that can be shared by global public health departments and researchers.

Conclusions
This study explained the resurgence of whooping cough from certain angles, including immunization strategy, vaccination time and genome variation of strains, which is bene cial to prevent pertussis infections.

Availability of data and materials
The data that support the ndings of this study have been deposited into CNGB Sequence Archive (CNSA) [32] of China National GeneBank DataBase (CNGBdb) [33] with accession number CNP0001528. The datasets generated for this study are available on request to the corresponding author.
Ethics approval and consent to participate Bordetella pertussis isolates from the Shenzhen Center for Disease Control and Prevention were deidenti ed and anonymized to protect patient privacy and con dentiality; therefore, ethical clearance was not required.

Consent for publication
Not applicable.