Date Published: July 16, 2015
Publisher: Public Library of Science
Author(s): Birgit Wagner, Helen Melzer, Georg Freymüller, Sabine Stumvoll, Pamela Rendi-Wagner, Maria Paulke-Korinek, Andreas Repa, Frits R. Mooi, Herwig Kollaritsch, Helmut Mittermayer, Harald H. Kessler, Gerold Stanek, Ralf Steinborn, Michael Duchêne, Ursula Wiedermann, Igor Mokrousov.
In Austria, vaccination coverage against Bordetella pertussis infections during infancy is estimated at around 90%. Within the last years, however, the number of pertussis cases has increased steadily, not only in children but also in adolescents and adults, indicating both insufficient herd immunity and vaccine coverage. Waning immunity in the host and/or adaptation of the bacterium to the immunised hosts could contribute to the observed re-emergence of pertussis. In this study we therefore addressed the genetic variability in B. pertussis strains from several Austrian cities. Between the years 2002 and 2008, 110 samples were collected from Vienna (n = 32), Linz (n = 63) and Graz (n = 15) by nasopharyngeal swabs. DNA was extracted from the swabs, and bacterial sequence polymorphisms were examined by MLVA (multiple-locus variable number of tandem repeat analysis) (n = 77), by PCR amplification and conventional Sanger sequencing of the polymorphic regions of the prn (pertactin) gene (n = 110), and by amplification refractory mutation system quantitative PCR (ARMS-qPCR) (n = 110) to directly address polymorphisms in the genes encoding two pertussis toxin subunits (ptxA and ptxB), a fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), and the virulence sensor protein (bvgS). Finally, the ptxP promoter region was screened by ARMS-qPCR for the presence of the ptxP3 allele, which has been associated with elevated production of pertussis toxin. The MLVA analysis revealed the highest level of polymorphisms with an absence of MLVA Type 29, which is found outside Austria. Only Prn subtypes Prn1/7, Prn2 and Prn3 were found with a predominance of the non-vaccine type Prn2. The analysis of the ptxA, ptxB, fimD, tcfA and bvgS polymorphisms showed a genotype mixed between the vaccine strain Tohama I and a clinical isolate from 2006 (L517). The major part of the samples (93%) displayed the ptxP3 allele. The consequences for the vaccination strategy are discussed.
Bordetella pertussis, the causative agent of whooping cough, is still responsible for significant morbidity and mortality in many countries of the world. Mainly in resource-poor regions, every year 195,000 children, mostly unimmunised, are estimated to die from pertussis .
B. pertussis samples (n = 110) from patients aged from 1 month to 47 years were collected from 2002 to 2008 in three Austrian cities (Vienna, Linz, and Graz). The first part, collected from 2002 to 2006 (n = 77) was characterised by MLVA typing , and all samples (n = 110) were examined by sequencing of the prn gene polymorphic region-1 , and ARMS-qPCR  of target SNPs in the genes coding for pertussis toxin (ptxA and ptxB), fimbrial adhesin (fimD), tracheal colonisation factor (tcfA), the virulence sensor protein (bvgS) , and the pertussis toxin promoter region ptxP . The detailed results can be found in S1 Table.
In Austria, similar to many other countries with routine vaccination programs against pertussis, the disease is far from eradication, and the number of cases has been increasing since the year 1995. To explain this rising prevalence, one of the current hypotheses is that under vaccine pressure B. pertussis may adapt slowly via the selection of mutant strains. The aim of this study was to examine DNA polymorphisms in B. pertussis strains found in the Austrian population.