{'fraction_non_alphanumeric': 0.032672469992713886, 'fraction_numerical': 0.020669555547033786, 'mean_word_length': 5.390100465572163, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 5, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Genomic library of Bordetella

Background The re-emergence of whooping cough and geographic disparities in vaccine escape or antimicrobial resistance dynamics, underline the importance of a unified definition of Bordetella pertussis strains. Understanding of the evolutionary adaptations of Bordetella pathogens to humans and animals requires comparative studies with environmental bordetellae. Methods We have set-up a unified library of Bordetella genomes by merging previously existing Oxford and Pasteur databases, importing genomes from public repositories, and developing harmonized genotyping schemes. We developed a genus-wide cgMLST genotyping scheme and incorporated a previous B. pertussis cgMLST scheme. Specific schemes were developed to define antigenic, virulence and macrolide resistance profiles. Genomic sequencing of 83 French B. bronchiseptica isolates and of B. tumulicola, B. muralis and B. tumbae type strains was performed. Results The public library currently includes 2,581 Bordetella isolates and their provenance data, and 2,084 genomes. The “classical Bordetella” (B. bronchiseptica, B. parapertussis and B. pertussis), which form a single genomic species (B. bronchiseptica genomic species, BbGS), were overrepresented (n=2,382). The phylogenetic analysis of Bordetella genomes associated the three novel species B. tumulicola, B. muralis and B. tumbae in a clade with B. petrii and revealed 18 yet undescribed species. A sister lineage of the classical bordetellae, provisionally named Bbs lineage II, was uncovered and may represent a novel species (average nucleotide identity with BbGS strains: ∼95%). It comprised strain HT200 from India, two strains of ‘genogroup 6’ from the USA and six clinical isolates from France; this lineage lacked ptxP and its fim2 gene was divergent. Within B. pertussis, vaccine antigen sequence types marked important phylogenetic subdivisions, and macrolide resistance markers (23S_rRNA allele 13 and fhaB3) confirmed the current restriction of this phenotype in China with few exceptions. Conclusions The genomic platform provides an expandable resource for unified genotyping of Bordetella strains and will facilitate collective evolutionary and epidemiological understanding of the re-emergence of whooping cough and other Bordetella infections. Data summary Bordetella genomes list and accession numbers: Supplementary Table S4 Bordetella genus phylogeny dataset (92 isolates): https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst_bordetella_isolates&page=query&project_list=23&submit=1 B. bronchiseptica phylogeny dataset (213 isolates): https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst_bordetella_isolates&page=query&project_list=24&submit=1 B. pertussis phylogeny (124 isolates): https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst_bordetella_isolates&page=query&project_list=25&submit=1 iTOL interactive trees: https://itol.embl.de/shared/1l7Fw0AvKOoCF

Besides the large-scale evolution of Bordetellae, the population dynamics within Bp are an important topic of epidemiological surveillance, in light of vaccine-escape evolution and the possible emergence and global dissemination of antimicrobial resistance [23][24][25].
Major evolutionary events in vaccine antigen-related genomic features have been described, including within the promoter region of the pertussis toxin gene cluster (ptxP) and in the fimbriae gene fim3 [23]. In most countries using acellular vaccines, isolates characterized by ptxP3 and either fim3-1 or fim3-2 alleles are currently largely predominant, whereas isolates of the ancestral ptxP1 genotype still predominate elsewhere, for example in China [24,26]. In addition, in countries that use acellular vaccines, an increasing proportion of Bp isolates are deficient for the production of the immunodominant surface protein pertactin, raising questions on future vaccine effectiveness [27]. The nomenclature of genotypic markers, sublineages and strain subtypes needs unification to facilitate collective studies of the global epidemiology and population dynamics in Bp.
Harmonization of the genotype nomenclature of bacterial pathogens may be achieved by the gene-by-gene approach called MLST (for multilocus sequence typing), in which allele numbers are uniquely attributed to each locus sequence variant. Bioinformatics platforms that centralize allele definitions and allow curation and open access to genotype nomenclature are critical resources for unified pathogen subtype definitions [28]. Until now, there have been two dedicated Bordetella genome sequence databases: Oxford University's PubMLST (unpublished) and Institut Pasteur's BIGSdb [29] platforms. This duality has led to nomenclatural confusion and suboptimal services to the user's community. With the rapid developments of genomic epidemiology and biodiversity surveys, a common and optimized resource for Bordetella evolutionary studies and sequence variants naming is needed. Here, we aimed to set-up such a unified genomic platform and expand its features to enable addressing genus-wide evolutionary genomics questions and global tracking of important genotypes within individual species, and particularly B. pertussis.

DNA preparation and genomic sequencing
Genomic paired-ends reads were clipped and trimmed with AlienTrimmer [30], corrected with Musket [31], merged (if needed) with FLASH [32], and subjected to a digital normalization procedure with khmer [33]. For each sample, remaining processed reads were assembled and scaffolded with SPAdes [34].

Development of pan-genus Bordetella cgMLST scheme
A genus-wide core genome MLST scheme [35], called Bordetella cgMLST v1.0, was established alongside a genus-wide locus annotation system (BORD loci), using the principles published for the genus Neisseria [36]. Six finished and annotated Bordetella genomes were uploaded into the BIGSdb Oxford database [37]: Bordetella pertussis Tohama I [38]; Bordetella pertussis CS [39]; Bordetella bronchiseptica RB50 [38]; Bordetella parapertussis 12822 [38]; Bordetella petri 12804 [18]; and, Bordetella avium 197N [40]. Using the GenomeComparator module of BIGSdb, the greatest number of common genes (1469) was found when B. bronchiseptica RB50 was used as the reference genome (even though B. petri 12804 had the greatest number of annotated loci, 5023). Genes described as 'hypothetical' or 'putative' were removed, resulting in 1415 loci. All loci entered into the database were given a BORD number of the format BORD000000, where the last six digits corresponded to the BB0000 numbers used in the annotation of B. bronchiseptica RB50 [38]. Other annotations and known aliases were included in the locus descriptions for comparison. As with other genus-wide locus schemes, this scheme can be expanded simply by adding additional loci, to give an expandable catalogue of the genus pangenome.

Merging of the Oxford and Pasteur databases
Two BIGSdb databases were originally designed separately for distinct purposes: while Oxford's PubMLST database offered MLST, Bordetella cgMLST v1.0 (see above) and virulence genes schemes, the Pasteur database was designed for the sole initial purpose of Bordetella pertussis genotyping, with a Bp-specific cgMLST scheme and a Bp-virulence genes schemes [29].
To merge the data available in the two databases, we proceeded as follows. As per BIGSdb dual design, isolates genomes and provenance data were imported into the "isolates" database, whereas allelic definitions of MLST, cgMLST and virulence genes were imported into the "seqdef" database.
Regarding the isolates database, we first dumped the Oxford database and uploaded it on the Institut Pasteur server. Second, we imported Pasteur isolates into this new database. To facilitate the understanding of historical origins of each entry, isolates identification numbers (BIGSdb ID number) were defined as follows: isolates from the original Oxford database were numbered from 1 to 1,914 as in the original database (i.e., their identification numbers were untouched). Second, the isolates from the original Pasteur database were numbered from 10,000 to 12,214 (original Pasteur identification number + 10000). We also completed the comment fields for these isolates, with the old identification number being added with the suffix word "Pasteur". We added "putative duplicate" in this comment field if the corresponding isolate name was present in both original databases. We also added a "duplicate number" field in the isolates database. If two or more isolates were identified as the same strain, they were attributed the same duplicate number, consecutively. Duplicated data identification was based on a combined analysis of metadata and phylogeny tree reconstruction.
At time of merging and closure, the Oxford database comprised 1,914 isolates entries, 57 of which were private, whereas the Pasteur database comprised 2,180 entries, 2,009 of which were private. As of January 7 th , 2022, the platform resulting from the merger comprises 2,581 public isolates entries, and 4,853 isolates in total when considering private entries.

cgMLST schemes
Two core genome MLST schemes are available. The first scheme, Bordetella cgMLST v1.0 (hereafter referred to as cgMLST_genus), was initially defined and hosted in the Oxford platform and was designed to be applicable for the entire Bordetella genus. The second scheme, called cgMLST_pertussis, was originally hosted in the Pasteur database and was built for B. pertussis isolates genotyping only [29]. Note that only the latter scheme has attached cgST definitions, i.e., unique cgST identifiers are attached to each distinct allelic profile. Both cgMLST schemes are available in the merged library.

Genotyping schemes for vaccine antigens and virulence-associated genes
In both original databases, virulence-associated gene schemes had been defined separately.
We choose to keep all loci from the virulence scheme designed in Oxford and to add some additional relevant loci. These loci were then grouped into different schemes comprising To make analyses of virulence-associated and vaccine antigen genes more user-friendly, common gene names used in the literature were used as locus identifier, instead of the locus tags that were initially used in the Oxford database (e.g., BORD005020 was replaced by fim2 and BORD005021 by fim3). In addition, for five loci common to the original Pasteur and Oxford databases (i.e. fim2, fim3, ptxA, ptxP and tcfA), the allele numbering was re-defined so that the alleles of each locus would match the nomenclature found in the literature for B. pertussis alleles; the correspondence between the original allele numbering system and the new one is defined in Table S1. Subsequently, the numbering of alleles was simply incremented as new genomes scans led to novel allele definitions.

Macrolide resistance
The 23S rRNA allele table was incremented with a sequence defined as allele 13, which carries the only mutation described so far as being associated with macrolide resistance [41].
To facilitate the detection of putative macrolide resistant isolates and their lineage [41,42], we built a scheme named "macrolide resistance" that includes the following loci: 23S_rRNA, fhaB (full) and four fhaB fragments. As the fhaB locus, which has a full-length size of 10,773 bp, is often fragmented in the genomic assemblies, three smaller fragments were designed to cover evenly the fhaB sequence: fhaBx-1_3193, fhaBy-3190_7183, fhaBz-7180_10773; the ranges in these locus names correspond to the begin and end positions of the three fragments.
A fourth fragment was designed to cover the region comprising 2 SNPs present individually in fhaB2 and fhaB3 [24] : fhaB-2400_5550. The allele numbering for fhaB fragments was defined so that the alleles of each locus would match the nomenclature found in the literature, ie. alleles fhaB1 and fhaB2 correspond to those defined by van Loo [43], and fhaB3 to the mutation C53301T [24]. Allele fhaB3 of full size locus and restricted locus (fhaBy-3190_7183 and fhaB-2400_5550 )is associated with ptxP1 isolates originating from China which exhibit a macrolide resistance phenotype [24].

Genome scanning for defined loci
Reference genomes were selected from either type-strain, reference genomes from literature and/or RefSeq genomes or most complete genomes available for each species (e.g., typestrain 18323 and reference strain Tomaha for B. pertussis). All these genomes are grouped into the public project "Bordetella Genus Phylogeny". We selected two representatives for each species, when available. All these genomes are found in the "Bordetella phylogeny" public project in the BIGSdb genome library. For the cgMLST_genus scheme, missing alleles were captured by relaxing the scanning parameters to 70% identity and 70% alignment in order to capture alleles from all Bordetella species, using the reference strains. New captured alleles were defining as type alleles. Then, all future detection of new alleles were based on 90% identity, 90% alignment parameters using defined type alleles as queries. Criteria for loci associated with virulence were stricter: only new alleles sharing 90% identity, 90% alignment with a type allele and with a complete CDS were recorded (excluding the ptxP promoter locus and the fhaB fragment fhaB-2400_5500). These strict parameters were used to minimize the detection of paralogous sequences and to exclude decaying alleles.
For each species, we defined one or two reference strains taken either from literature or from the NCBI RefSeq database (Table S2). Type alleles were defined from each reference isolate in order to constrain the search space so as to ensure future consistency of allele definitions.
The definition of new alleles was subject to constraining parameters. To detect alleles, BLASTN thresholds were set to 90% of identity, 90% of alignment length coverage, and considering only the complete coding sequences (except for the ptxP locus promoter and the 23S rRNA gene, which are not protein CDSs). These strict parameters were used to minimize the detection of paralogous sequences.

Phylogenetic analyses
Phylogenetic analyses were performed based on concatenated alignments of individual gene loci defined in the database. We used an in-house script which combines IQtree (IQ-TREE 2.0.6, model substitution GTR+G+I) and Gubbins (2.4.1, default parameters) [44,45].
To include the breadth of currently sampled Bordetella diversity, we first downloaded all genomic sequences from public databases and ran a rapid distance based phylogenetic analysis. From this, we retained all representative genomes of unique branches and downsampled shallow branches that were represented multiple times. The resulting dataset included the putative novel species initially described as genogroups by Spilker

Genomic library contents
After merging Oxford and Pasteur databases, importing publicly available genomes, and adding three genomes of recently described species B. muralis (CIP111920 T ), B. tumulicola Bordetella species was B. ansorpii, as previously observed [4], and it was associated with genogroup 13.
The ANI metric is widely used as a guide to define novel bacterial species [49]. ANI values between representative genomes of the Bordetella genus (

Phylogenetic diversity within Bordetella bronchiseptica
A phylogenetic tree of Bbs isolates, including 83 isolates from France sequenced for this study, was inferred from the cgMLST_genus gene sequences (Figure 2 & Figure S2) and revealed a primary separation of Bbs diversity into two deep lineages. We here define the minor one as lineage II of B. bronchiseptica. Lineage II comprised nine isolates, one of which is HT200 from a natural spring water in India and was previously recognized as being atypically distant from other Bbs isolates [50]. Bbs lineage II isolates from the USA (AU22978 et AU3139) were previously described as genogroup 6 and had been collected from patients with cystic fibrosis [21]. The remaining lineage II isolates were six human clinical isolates from France, collected from adults (mean age = 71. The independent origins of Bpp hu and Bpp ov have been debated [1,6]. We note that Bpp5, the reference strain of ovine B. parapertussis, was associated with the earliest branching Bbs sublineage I-1, rendering B. parapertussis paraphyletic (Figures 2 & S2). A separate phylogenetic analysis performed using the cgMLST_pertussis scheme, instead of the genuswide scheme, provided a nearly identical phylogenetic tree of Bbs isolates (data not shown).
However, we recommend using the cgMLST_genus scheme for Bbs phylogenetic analyses, as a lower number of uncalled alleles was observed than for the cgMLST_pertussis scheme ( Figure S3).

Genotyping of vaccine antigen and virulence-associated genes of Bordetella bronchiseptica
The allele diversity and presence/absence of vaccine antigens or virulence-associated loci were investigated within Bbs. For convenience, the loci were grouped into schemes labeled as Bp-vaccine antigens, Autotransporters, Phase genes, Other Toxins and T3SS.
T3SS genes are known to be present within the BbGS [6]. For most other loci, alleles were specific for either Bbs lineage I or II, or for unique sublineages within lineage I (see details in the Supplementary text and in Figure S5).
Consistent with its large (~5%) genomic divergence from the BbGS, alleles observed within B. bronchiseptica lineage II were unique for this lineage. In addition, even though only few isolates have been sampled so far for this lineage, we noted a high diversity of alleles indicating that it forms a genetically diverse group, suggesting phenotypic variability. Some loci including fim2, ptxP, dnt, tcfA, vag8, prn or bipA had no allele tagged according to our stringent criteria. When relaxing these criteria, we noted that ptxP, prn and dnt were absent in Bbs lineage II, whereas fim2, bipA and tcfA presented partial matches (< 90%) with existing alleles in almost all isolates.

Allele diversity and macrolide resistance in B. pertussis
A phylogeny of Bp isolates, which were selected with a focus on macrolide resistance, was built using the 2,038 loci of cgMLST_pertussis scheme (Figure 3). Three isolates of the early ptxP2 branch correspond to Bp lineage II-a as defined by Bart et al. [23]. The remaining isolates fell in the ptxP1 and ptxP3 branches, which represent the main subdivisions of Bp lineage IIb.
Bp isolates were screened with the macrolide resistance scheme to detect alleles associated with this phenotype. Allele 13 of the 23S_rRNA locus is specific for erythromycin resistant B. pertussis isolates, whereas alleles 1, 2 and 20 correspond to susceptible ones. Allele 13 was found in 46 of 51 isolates from China [24] and in two isolates previously described as erythromycin resistant: one from USA (ATCC:BAA-1335D-5, [42]) and one from France (FR4991, [41]). When checking our entire library, which contained 1,567 Bp isolates, no additional resistant isolate was found. In a previous study, Xu et al. [24] showed that allele fhaB3 characterized by the mutation C5330T was found in a unique branch of the We also defined a Bp vaccine antigens scheme, which groups the loci ptxP, ptxABCDE, fim2, fim3 and fhaB2400_5550. Bp vaccine antigens sequence types (BPagST) were defined for this scheme, corresponding to the unique combinations of alleles at the individual loci. These STs were distributed along the Bp tree (Figure 3). For example, BPagST34 marked the sublineage with allele fim3-4, whereas BPagST9 corresponded to most isolates of the fim3-2 branch.
BPagST37 (allele 3 for fhaB2400_5550 locus) corresponded to macrolide resistant fhaB3 lineage of Xu et al. [24]. The macrolide-resistant isolate from France was characterized by BPagST68, whereas no BPagST could be determined for the macrolide resistant isolate from the USA, because its allele at the fim3 locus was not defined due to a frameshift. We suggest that the BPagST sequence types will be useful for future tracking of specific sublineages of

Bp.
Loci variation within other schemes (Other toxins, Autotransporters, T3SS and Phase biology schemes) are detailed in the Supplementary Text.

Discussion
We have set-up a unified genomic platform, available at https://bigsdb.pasteur.fr/bordetella/, by merging the data from the two previously existing PubMLST and Pasteur BIGSdb-Bordetella databases. This merger has enabled allelic nomenclature unification and has led to reassemble on the same platform, genotyping schemes that will facilitate the study of Bordetella diversity at different phylogenetic breadths. We have also expanded the genomic library with public sequences and sequenced genomes from the French NRC. The resulting unified platform for Bordetella genotyping will ease the future curation and expansion of Bordetella genomic data by a community of users and curators.
Two complementary cgMLST schemes are available on the genomic platform. While the cgMLST_genus scheme should be preferred for genus-wide phylogenetic analyses and within individual species including B. bronchiseptica, the cgMLST_pertussis scheme comprises more loci that are conserved within Bp, and its added discriminatory power will be useful for strain identification and comparison within Bp. Recently, a whole-genome MLST (wgMLST) scheme comprising even more loci (n =3,506; 1,822 of which are common with our cgMLST_pertussis scheme) was proposed for Bp subtyping, and concordant results were obtained for classification of outbreak and sporadic isolates from the USA using the cgMLST and wgMLST schemes [55]. As the later provides slightly higher discrimination among Bp isolates than the cgMLST_pertussis scheme, it might provide valuable additional genotyping information for local transmission investigations, and may be incorporated into the unified platform in the future.
We provided examples of applications of the genomic library and its associated genotyping schemes, from the analysis of the genus-level diversity of species, to strain genotyping within Bp is the medically most important Bordetella species, being the most frequent cause of important infections in humans. The unified Bordetella genomic platform provides specific tools for characterizing genes linked to Bp vaccine antigens and virulence. These were organized into several genotyping schemes that might be used separately for specific biological or epidemiological questions. Where available, we defined allele identifiers in accordance with their literature numbering. This will disambiguate, in particular, the genotyping of vaccine antigens within Bp. The population of Bp has been noted to evolve in response to the selective pressure exerted by vaccination, in particular in countries using the acellular vaccines [23,59]. The proposed vaccine schemes will help keeping track of the circulating strains and defining the prevalence of particular sublineages, including those that are deficient for pertactin production [27,60,61].
We also created a genotyping scheme dedicated to macrolide resistance, the possible dissemination of which is an important point of public health concern. To enable easy identification of the macrolide-resistant sublineage that is currently highly prevalent in China, we defined this scheme as combining the causative allele 23S rRNA with the associated marker fhaB3. This scheme thus provides a convenient way to distinguish 'out of China' dissemination of the fhaB3 sublineage, from parallel evolution of macrolide resistance in other locations.
An important current limitation of the genomic library is its contents imbalance towards genomic sequences from acellular vaccine-using high-income countries, as observed for other pathogens [62]: our database contained only few genomes from Africa (0.2 %), Asia (1.9 %) or Oceania (2.1 %) as compared to those from North America (13.6 %) or Europe (58.7 %).
In the future, efforts should be made to generate and incorporate more genomic sequences from world regions that are currently underrepresented.
In conclusion, the unified Bordetella genomic library was built with the intention to facilitate and harmonize the genotyping of strains of this important bacterial genus, and should represent a useful tool even for genomic epidemiology consortia unfamiliar with genomic analyses. It provides a uniformization of genetic variants designations, which will clarify the communication on genotypes and will enable a collective understanding on the biodiversity and epidemiology of Bordetella. We provide a timely solution for genomic studies of Bordetella pathogens, and in particular Bp, the reemergence of which is partly caused by evolutionary and epidemiological dynamics of public health concern, including vaccine escape and antibiotic resistance emergence.

Conflicts of interest
The authors declare that there are no conflicts of interest.

Funding information
This work was supported financially by the French Government's Investissement d'Avenir

Acknowledgments
We thank Julien Guglielmini (Institut Pasteur) for help with the BIGSdb-Pasteur database configuration. The phylogenetic tree was obtained based on the concatenated multiple sequence alignments of the 1,415 core gene sequences from the cgMLST_genus scheme; recombination was accounted for using Gubbins. The tree was rooted on the branch leading to strain FB-8, which was the earliest branching genome in a phylogenetic analysis that included the external group Ralstonia solanacearum strain IBSBF1503 ( Figure S4). Leaves are labelled with the identifier of the strain in the BIGSdb database, followed by the strain name. isolates; see Figure S5 for the corresponding phylogram. For each isolate, the host is represented using a leaf symbol, where available (see key in Figure S5). The numbers corresponding to T3SS sequence types are indicated along the external circle around the tree;

Figure legends
only the identifiers of main T3STs are indicated.