Complete Genome Sequences of Bordetella pertussis Isolates with Novel Pertactin-Deficient Deletions.

Clinical isolates of the respiratory pathogen Bordetella pertussis in the United States have become predominantly deficient for the acellular vaccine immunogen pertactin through various independent mutations. Here, we report the complete genome sequences for four B. pertussis isolates that harbor novel deletions responsible for pertactin deficiency.

GENOME ANNOUNCEMENT

Bordetella pertussis is the causative agent of whooping cough (pertussis), a vaccine-preventable respiratory disease with recent increased case reports in the United States and other developed countries (1). The majority of circulating isolates recovered in the United States do not produce the acellular vaccine immunogen pertactin (Prn) due to one of at least 16 independent mutations to the structural gene prn, including point mutations, promoter disruption, and mobile element disruption (2, 3). Here, we report the complete genome sequences of four such Prn-deficient clinical isolates which carry novel prn deletion mutations.

Whole-genome shotgun sequencing was performed using a combination of the PacBio RSII (Pacific Biosciences, Menlo Park, CA), MiSeq (Illumina, San Diego, CA), and Argus (OpGen, Gaithersburg, MA) platforms, as described previously (4). Briefly, genomic DNA libraries were prepared for PacBio sequencing using the SMRTbell template prep kit 1.0 and polymerase binding kit P6, while Illumina libraries were prepared using the NEBNext Ultra library prep kit (New England BioLabs, Ipswich, MA). De novo genome assembly of filtered reads was performed using the Hierarchical Genome Assembly Process (HGAP) version 3 (Pacific Biosciences) (5), with at least 100× coverage. The resulting consensus sequences were determined with Quiver version 1, manually checked for circularity, and then reordered to start at the coding region for glucose-inhibited cell division protein A (gidA), consistent with available genome sequences of B. pertussis. To ensure accuracy, assemblies were confirmed by comparison to BamHI and KpnI restriction digestion optical maps using the Argus system (OpGen) with MapSolver version 2.1.1 (OpGen) and further polished by mapping Illumina MiSeq PE-300 reads using CLC Genomics Workbench version 10.0.1 (CLC bio, Boston, MA). Final assemblies were annotated using the NCBI automated Prokaryotic Genome Annotation Pipeline (PGAP). Partial deletion of the prn coding region or upstream promoter sequence was confirmed by Sanger sequencing, as described previously (2).

Isolate characteristics and prn mutations are summarized in Table 1. In J625, the deletion spanned the promoter and 5′ end of prn and was replaced with a 355-bp sequence fragment that was identical to a region adjacent to an IS1663 element upstream. These results add to the diverse catalogue of observed mutations which confer Prn deficiency through independent parallel disruption of prn.

TABLE 1 

Characteristics of B. pertussis isolates and prn mutations

Isolate	U.S. state	Yr	prn mutationa	prn location (bp)b	Accession no.
H696	CA	2011	prn2::del(−1513, 145)	1008517–1011120	CP021402
J078c	MN	2013	prn2::del(−283, −40)	1079512–1082303	CP021401
J473c	NY	2016	prn2::del(666, 667)	2979800–2977055	CP021403
J625	VT	2016	prn2::del(−292, 1340)d	1085097–1086504	CP022362

Numbers in parentheses indicate the position of each mutation (start, stop) relative to the prn start codon.

Numbers in the prn location column indicate genomic coordinates of the prn gene.

Isolates collected through Enhanced Pertussis Surveillance (6).

Replaced with 355-bp fragment identical to 1074011 to 1073657.

Accession number(s).

The complete genome sequences have been deposited at DDBJ/EMBL/GenBank under the accession numbers listed in Table 1. The versions described in this paper are the first versions.