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Prevalence and molecular characterization of pertactin-deficient Bordetella pertussis in the United States - PubMed

doi: 10.1128/CVI.00717-13. Epub 2013 Nov 20.

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Prevalence and molecular characterization of pertactin-deficient Bordetella pertussis in the United States

L C Pawloski et al. Clin Vaccine Immunol. 2014 Feb.

Abstract

Pertussis has shown a striking resurgence in the United States, with a return to record numbers of reported cases as last observed in the 1950s. Bordetella pertussis isolates lacking pertactin, a key antigen component of the acellular pertussis vaccine, have been observed, suggesting that B. pertussis is losing pertactin in response to vaccine immunity. Screening of 1,300 isolates from outbreak and surveillance studies (historical isolates collected from 1935 up to 2009, isolates from the 2010 California pertussis outbreak, U.S. isolates from routine surveillance between 2010-2012, and isolates from the 2012 Washington pertussis outbreak) by conventional PCR and later by Western blotting and prn sequencing analyses ultimately identified 306 pertactin-deficient isolates. Of these pertactin-deficient strains, 276 were identified as having an IS481 in the prn gene (prnIS481 positive). The first prnIS481-positive isolate was found in 1994, and the next prnIS481-positive isolates were not detected until 2010. The prevalence of pertactin-deficient isolates increased substantially to more than 50% of collected isolates in 2012. Sequence analysis of pertactin-deficient isolates revealed various types of mutations in the prn gene, including two deletions, single nucleotide substitutions resulting in a stop codon, an inversion in the promoter, and a single nucleotide insertion resulting in a frameshift mutation. All but one mutation type were found in prn2 alleles. CDC 013 was a predominant pulsed-field gel electrophoresis (PFGE) profile in the pertactin-positive isolates (203/994) but was found in only 5% (16/306) of the pertactin-deficient isolates. Interestingly, PFGE profiles CDC 002 and CDC 237 represented 55% (167/306) of the identified pertactin-deficient isolates. These results indicate that there has been a recent dramatic increase in pertactin-deficient B. pertussis isolates throughout the United States.

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Figures

FIG 1
FIG 1

Flow chart of the progression of pertactin deficiency identification, from the conventional PCR screening of the IS481 insertion in the prn gene to Western blot (WB) analysis and sequencing. IS481 positive, IS481 insertion in prn; IS481 negative, IS481 insertion in prn not detected; WB+, pertactin protein present by Western blotting; WB−, pertactin protein absent by Western blotting; WT, wild type; STOP, stop codon.

FIG 2
FIG 2

(A) Proportion of prnIS481-positive B. pertussis isolates, identified by PCR and sequencing, from the 2012 Washington outbreak, 2010-2012 U.S. collection, 2010 California outbreak, and 1935-2009 U.S. historic collection, stratified by origin and time period. (B) Proportion of prnIS481-positive B. pertussis isolates from the U.S. 2010-2012 collection, stratified by year.

FIG 3
FIG 3

Location of mutations in the prn gene, identified through PCR, Western blotting, and sequencing analyses. Mutations included 3 IS481 insertions, 2 premature stop codons (☆), a nucleotide insertion (G), a signal sequence deletion (ΔSS), a large deletion in the 5′ region and upstream (Δ2.6 kb), and a large inversion of the promoter region (Inv). IS481 insertions were found in both directions, where indicated, according to alignment with GenBank accession number M22031. Mutations marked with an asterisk (*) were identified previously. AA, amino acid; VR, variable region.

FIG 4
FIG 4

PFGE profiles of the 1,300 B. pertussis isolates analyzed for pertactin deficiency, stratified by origin and time period. The top three predominant profiles are indicated per group; all remaining profiles are identified as “All others.” PRN+, B. pertussis isolates that do not contain genetic mutations and/or express pertactin; PRN−, B. pertussis isolates that are pertactin deficient as tested by PCR screening, Western blot analysis, and prn sequencing; n, number of isolates.

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