# Genome characteristics of Bordetella pertussis isolates from Tunisia

Ikram Ben Fraj, Valérie Bouchez, Hanen Smaoui, Amel Kechrid, Sylvain Brisse

► To cite this version:

Ikram Ben Fraj, Valérie Bouchez, Hanen Smaoui, Amel Kechrid, Sylvain Brisse. Genome character-
istics of Bordetella pertussis isolates from Tunisia. Journal of Medical Microbiology, 2019, 68 (9),
pp.1320-1323. 10.1099/jmm.0.001042. pasteur-03221002

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Genome characteristics of *Bordetella pertussis* isolates from Tunisia

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Ikram Ben Fraj¹, Valérie Bouchez²,³, Hanen Smaoui¹, Amel Kechrid¹ and Sylvain Brisse²,³,*

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¹ University of Tunis El Manar, Children's Hospital of Tunis, Laboratory of Microbiology,

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UR12ES01, Tunis, Tunisia

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²Institut Pasteur, Biodiversity and Epidemiology of Bacterial Pathogens, Paris, France

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³National Reference Center for Whooping Cough and other Bordetella infections, Paris,

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France

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* Corresponding author: Sylvain Brisse. Biodiversity and Epidemiology of Bacterial

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Pathogens, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris, France.

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E-mail: sylvain.brisse@pasteur.fr; Phone +33 1 45 68 83 34

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**Running title:** *Bordetella pertussis* isolates from Tunisia

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**Keywords:** *Bordetella pertussis*, Tunisia, genomic epidemiology, phylogeny

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**Accession numbers**

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The genomic sequence data generated in this work were submitted to the European

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Nucleotide Archive and are available from the International Nucleotide Sequence Database

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Collaboration (NCBI/ENA/DDBJ) databases under project accession number PRJEB27412

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and run data accession numbers ERS2572942 to ERS2572951.
21 **Abstract**

22 **Purpose:** The recent increase in pertussis cases observed in some countries may have several
23 causes, including the evolution of *Bordetella pertussis* populations towards escape of vaccine-

24 induced immunity. Most genomic studies of *B. pertussis* isolates performed so far are from
25 countries that use acellular vaccines. The objective was to analyze genomic sequences of
26 isolates collected during the 2014 whooping cough epidemic in Tunisia, a country where

27 whole cell vaccines are used. **Methodology:** Ten Tunisian isolates and four vaccine strains
28 were sequenced and compared to 169 isolates from countries where acellular vaccines are
29 used. **Results:** Phylogenetic analysis showed that Tunisian isolates are diverse, demonstrating

30 a multi-strain 2014 epidemic peak, and are intermixed with those circulating in other world
31 regions, showing inter-country transmission. Consistently, Tunisian isolates have antigen
32 variant composition observed in other world regions. No pertactin-deficient strain was

33 observed. **Conclusion:** The Tunisian *B. pertussis* population appears to be largely connected
34 with populations from other countries.
## 35 Introduction

Pertussis is a highly contagious infectious disease, caused by the bacterium *Bordetella pertussis* and more rarely by *B. parapertussis*. Although the infection is largely controlled by vaccination, a recent increase in pertussis incidence has been observed in several countries [1]. Possible explanations of this increase include sub-optimal vaccine coverage, improved surveillance and diagnosis of the disease, waning of vaccine-induced immunity, and genetic variations in circulating *B. pertussis* isolates that escape vaccine-induced immunity [2–4].

In Tunisia, pertussis is still present as a cyclical disease despite high (98%) primo-vaccination coverage [5], and two epidemic peaks were observed in 2009 and 2014. A 9-year surveillance study (2007-2016) among 1,844 infants and children reported 306 (16.6%) *Bordetella* infection that were confirmed by real time PCR [6, 7]. While initial vaccination of Tunisian children is performed at 2, 3 and 6 months of age, a booster dose is received at the age of 18 months. Since April 2011, a pentavalent vaccine (Pentavac, Serum Institute of India), which includes a whole cell pertussis component, is used in Tunisia.

*B. pertussis* is an highly monomorphic bacterium [4, 8]. Therefore, molecular typing of *B. pertussis* isolates requires the full resolution of whole genome sequencing (WGS) for accurate phylogenetic comparisons and to follow the spread of sublineages, as other methods largely fail to resolve relationships at strain level [4, 9–11]. Inter-country transmission of *B. pertussis* isolates was demonstrated based on genomic-scale genotyping of a global dataset of isolates collected before 2011 [4] and of isolates collected more recently in countries using acellular vaccines [9, 11, 12]. However, only few data are currently available on the genomic sublineages of *B. pertussis* isolates collected in countries using whole cell vaccines [13, 14]. Therefore, whether transmission occurs between these countries and those using acellular vaccines is not enough documented, limiting our understanding of the drivers of *B. pertussis* diversity across countries with distinct vaccination strategies.
This study aimed to determine the genomic characteristics of Tunisian *B. pertussis* clinical isolates and to compare them with isolates from other world regions and with strains used to prepare the whooping cough vaccine used in Tunisia.

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## Methods

**B. pertussis** isolates and genomes. Tunisian *Bordetella pertussis* clinical isolates were recovered during the epidemic peak of 2014. Their phenotypic characteristics and antigen genotyping of genes *ptxP*, *prn*, *ptxA*, *fim2* and *fim3* were described earlier [7]. Genomic DNA was isolated and purified from subcultured isolates [7] using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche, Germany) according to the manufacturer's instructions. Sequencing libraries were prepared using the Nextera XT DNA Sample Preparation Kit (Illumina, USA). Whole genome sequencing was performed on the Illumina® NextSeq® 500 system (Illumina, USA) using a 2x150 paired-end protocol by the Mutualized Platform for Microbiology at Institut Pasteur of Paris. AlienTrimmer v0.4.0, Musket v1.1 and KhmerStream v1.1 software packages were used for paired-end reads clipping, trimming and sequencing errors correction, respectively. SPAdes/3.9.0 was used for genome assembly. Genome sequences were deposited in the European Nucleotide Archive and their accession numbers are available in Table 1.

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**cgMLST and phylogenetic analysis.** Genome sequence data were analyzed using a gene-by-gene approach known as core genome MLST (cgMLST) [11, 15]. cgMLST was performed using the BIGSdb platform of Institut Pasteur (http://bigsdb.pasteur.fr/bordetella) as previously described [11]. Briefly, genome assemblies were compared using BLASTN to the reference alleles of 2,038 predefined gene loci. When novel alleles were discovered in the
84 genomes of the Tunisian isolates, they were imported into the reference allele database and an
85 allelic number was assigned. Isolates from other world regions were investigated in a previous
86 study and corresponded to isolates from France, UK and USA [11]. Genomic sequences of
87 vaccine strains from Serum Institute of India J445 (GCA_001831395), J446
88 (GCA_001831415), J447 (GCA_001831435) and J448 (GCA_001831455) were also
89 included. We used IQ-TREE v1.5.4 to infer a maximum likelihood phylogenetic tree based on
90 concatenated alignments of the sequences from each of the 2,038 cgMLST loci.

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## 92 Results and Discussion

93 Ten isolates collected during the epidemic of 2014 at the Children's Hospital of Tunis, the
94 only pediatric university hospital in Tunisia, were analyzed by genome sequencing. The
95 characteristics of genomes are summarized in **Table 1**. Assemblies characteristics were
96 similar for the ten isolates: about 300 contigs were obtained for each genome, with a mean
97 length of 3,867,263 bp. **Figure 1** presents the genomic sequence-based phylogenetic tree of
98 Tunisian *B. pertussis* isolates and their positioning with respect to isolates from other world
99 regions. Tunisian isolates were all distributed in the *ptxP3* clade but were genetically diverse,
100 with eight of them falling within the *fim3-2* clade and two within the *fim3-1* clade. TN0003
101 and TN0005 were closely related, with only 4 different cgMLST genes among them, whereas
102 they were more distant from other isolates (11 to 14 different loci). Likewise, isolates
103 TN0007, TN0001, TN0006 and TN0011 were closely related to each other within the *fim3-2*
104 branch, displaying only 1 or 2 different loci among themselves. These two groups of
105 genetically very similar Tunisian isolates may correspond to local chains of transmission [11].
106 Nevertheless, the 10 Tunisian isolates were mixed phylogenetically with isolates from other
107 countries, indicating several independent transmissions between Tunisia and other countries.
108 The closest neighbors of the Tunisian strains in this dataset were either from France, UK or
the USA, and diverged from them by 3 to 8 loci (Table 2). This close genetic relatedness between Tunisian isolates and isolates from other geographic origins is consistent with the high global genetic homogeneity of *B. pertussis* and is likely favored by the increased international exchanges with Tunisia. Hence, despite the monomorphic nature of *B. pertussis*, the genome sequencing approach shows genetic heterogeneity among the Tunisian isolates included in this study, demonstrating that the epidemic peak of 2014 was not due to the spread of a single strain but instead, was caused by the simultaneous infection by various strains as observed previously [9, 11].

*B. pertussis* isolates can be characterized by their allelic profile, which recapitulates the allelic variants at virulence and antigen genes loci. As previously observed [7], the allelic profile of all Tunisian isolates is *ptxP3-ptxA1-prn2-fim2-1-fim3-2*. Notably, all Tunisian isolates had alleles *ptxA1* for the subunit 1 of pertussis toxin, and pertactin allele *prn2*, irrespective of the *fim3* clade they belong to, as observed for the majority of current *B. pertussis* isolates collected in countries using acellular *B. pertussis* vaccine [4, 11, 12]. An increase in reported *B. pertussis* isolates not producing pertactin has been observed in many countries using the acellular pertussis vaccine, including the USA [16], Japan [12] and European countries [17]. All Tunisian *B. pertussis* isolates were shown to produce pertactin [7], and the genomic sequence confirmed that they all had an intact *prn* gene. The low number of isolates from Tunisia that could be analyzed does not allow to exclude the presence of pertactin non-producing isolates in this country, but the results indicate that they are much less frequent than pertactin-producing ones. This situation therefore contrasts with countries where acellular vaccines are used since nearly two decades such as Japan, USA or some EU countries [17–19]. Therefore, the inter-country transmission of *B. pertussis* sublineages is not intense enough not erase the specificities of *B. pertussis* populations in countries with distinct vaccination strategies.
The four vaccine strains (J445 to J448) incorporated in the vaccine used in Tunisia appear to belong to early-branching clades (Figure 1), as does the Tohama reference strain previously shown to be distant from most *B. pertussis* isolates that currently cause infection [4, 20]. Genetic distances of the four vaccine strains to Tunisian isolates were higher for strain J446 (117 to 124 cgMLST gene mismatches) than for strain J445 (28 to 31 mismatches) and for strains J447 and J448 (17 to 24 mismatches). These genetic distances and the phylogenetic position of vaccine strains are consistent with the promoter sequence or antigenic variants of vaccine strains (J446: *ptxP2/ptxA4/prn7/fim2-2/fim3-1*; J445: *ptxP1/ptxA2/prn1/fim2-1/fim3-1*; J447 and J448: *ptxP1/ptxA1/prn1/fim2-1/fim3-1*), which do not match entirely with the profiles of most circulating isolates (*ptxP3/ptxA1/prn2/fim2-1/fim3-2*). Vaccine efficacy might be improved by the use of vaccine strains that have a better phylogenetic and antigenic match with the circulating isolates.

In conclusion, although based on a limited collection of isolates, this study provides a snapshot of the genomic make-up of Tunisian isolates. Despite the use of whole cell vaccines in Tunisia, *B. pertussis* isolates from this country are phylogenetically and antigenically closely related to those circulating in countries using acellular vaccines, whereas they are less related to vaccine strains. However, our sample of *B. pertussis* isolates did not reveal the presence of pertactin-deficient strain in Tunisia. The relative contributions of long-range transmission and local selection by vaccine-induced immunity in shaping *B. pertussis* populations is an important subject for future research. Surveillance of pertussis infections in Tunisia and other countries using whole-cell vaccines should be reinforced to inform control measures and vaccination policy.
156 Author statements

157 **Author contributions:** Conceptualization: IBF, AK, HS and SB; Experiments: IBF, VB; Data analysis: IBF, VB; Original draft preparation: IBF, VB, HS, SB; Review and editing: SB; Supervision: HS, SB; Funding: SB.

160 **Conflict of Interest:** The authors declare that there are no conflicts of interest.

161 **Funding information:** This research received no specific grant. It was supported financially by Institut Pasteur support to the research unit Biodiversity and Epidemiology of Bacterial Pathogens.

164 **Ethical approval:** not relevant

165 **Acknowledgements:** We thank Annie Landier and Sophie Guillot (National Reference Center for Whooping Cough and other Bordetella infections, Institut Pasteur) for their continuous implication in data collection, strain characterization and genomic sequencing.
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<table><caption>**Table 1:** Characteristics of Tunisian *Bordetella pertussis* genomes used in this study</caption><thead><tr><th>Isolate Name</th><th>Month of collection</th><th>Age of patient</th><th>Number of contigs</th><th>Genome size (nt)</th><th>N50 length (nt)</th><th>Genotype</th><th>Accession number (PRJEB27412)</th></tr></thead><tbody><tr><td>TN0001</td><td>April 2014</td><td>2 months</td><td>300</td><td>3,872,771</td><td>20,131</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572942</td></tr><tr><td>TN0002</td><td>April 2014</td><td>3 weeks</td><td>293</td><td>3,864,234</td><td>20,328</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572943</td></tr><tr><td>TN0003</td><td>April 2014</td><td>6 weeks</td><td>298</td><td>3,863,302</td><td>20,108</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-1</td><td>ERS2572944</td></tr><tr><td>TN0004</td><td>April 2014</td><td>3 weeks</td><td>301</td><td>3,878,136</td><td>19,862</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572945</td></tr><tr><td>TN0005</td><td>April 2014</td><td>1 month</td><td>290</td><td>3,868,375</td><td>20,544</td><td>ptxP3 ptxA1 prn3 fim2-1 fim3-1</td><td>ERS2572946</td></tr><tr><td>TN0006</td><td>May 2014</td><td>6 weeks</td><td>283</td><td>3,868,089</td><td>20,787</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572947</td></tr><tr><td>TN0007</td><td>May 2014</td><td>2 months</td><td>288</td><td>3,865,925</td><td>20,544</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572948</td></tr><tr><td>TN0008</td><td>May 2014</td><td>2 months</td><td>298</td><td>3,862,980</td><td>19,926</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572949</td></tr><tr><td>TN0009</td><td>May 2014</td><td>2 months</td><td>293</td><td>3,865,015</td><td>20,346</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572950</td></tr><tr><td>TN0011</td><td>May 2014</td><td>3 months</td><td>289</td><td>3,863,801</td><td>20,541</td><td>ptxP3 ptxA1 prn2 fim2-1 fim3-2</td><td>ERS2572951</td></tr></tbody></table>
<table><caption>**Table 2:** Genetically closest isolate(s) for each Tunisian *Bordetella pertussis* isolate</caption><thead><tr><td>Isolate Name</td><td>Closest isolate(s) by cgMLST distance</td><td>No. of cgMLST mismatches</td></tr></thead><tbody><tr><td>TN001</td><td>FR3903; FR3916</td><td>2</td></tr><tr><td>TN002</td><td>FR3903; FR3916</td><td>3</td></tr><tr><td>TN003</td><td>FR5438; FR5439</td><td>5</td></tr><tr><td>TN004</td><td>FR5791; P2M</td><td>3</td></tr><tr><td>TN005</td><td>I538; I539; I656</td><td>2</td></tr><tr><td>TN006</td><td>FR5859; FR5860; FR5942; FR6031; H379; H788; H918</td><td>2</td></tr><tr><td>TN007</td><td>FR3903; FR3916</td><td>2</td></tr><tr><td>TN008</td><td>FR3903; FR3916; FR5943; FR5793</td><td>2</td></tr><tr><td>TN009</td><td>FR5942; FR6031; H379; H788; H918; FR5859; FR5860</td><td>2</td></tr><tr><td>TN011</td><td>FR3903; FR3916</td><td>2</td></tr></tbody></table>
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**Figure 1:** Maximum-likelihood phylogenetic tree of Tunisian *Bordetella pertussis* isolates and isolates from other geographical origins based on the concatenated alignments of 2,038 cgMLST gene sequences. The tree was rooted with strain J446. Black branches correspond to the *fim3-1* clade and green branches to the *fim3-2* clade. The external circle indicates the geographic origin of isolates (long red bars, Tunisia; long blue bars: vaccine strains of Serum Institute of India; light green, France; light blue, United Kingdom; light pink, USA). The scale bar indicates the number of nucleotide substitutions per site.

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