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we studied 232 b. pertussis clinical isolates collected from january 2005 through june 2016 in japan .
pertactin production and mlva types ( mts ) of 111 isolates collected during 20052012 were previously determined by immunoblotting and mlva typing , respectively ( 5,8 ) . for our study , we extended these analyses to additional isolates collected during 20082016 ( n = 121 ) .
we determined the temporal trend in the frequency of pertactin - deficient isolates by 3-year periods ( figure , panel a ) .
percentages were 41% in 20052007 ( n = 39 isolates ) , 35% in 20082010 ( n = 43 ) , 25% in 20112013 ( n = 97 ) , and 8% in 20142016 ( n = 53 ) .
a significant decrease in the frequency of pertactin - deficient isolates was observed from 20052007 to 20142016 ( p<0.05 by fisher exact test ) .  
temporal trends in the frequency of pertactin - deficient isolates and changes in multi - locus variable - number tandem repeat analysis types ( mts ) in the bordetella pertussis population in japan .
pertactin production ( a ) and mts ( b ) were analyzed for 232 b. pertussis isolates collected from january 2005 through june 2016 .
the frequencies of pertactin - deficient isolates and 2 major mts ( mt27 and mt186 ) are shown by time period . for convenience ,
minor mts ( mt194 , mt224226 , and mt314316 ) are included as  others .  among the 232 b. pertussis isolates , 25 mts were identified ; mt27 and mt186 isolates were predominant , and other mt isolates were found at low frequencies ( technical appendix table 1 ) .
the frequency of mt27 isolates increased significantly over time ( figure 1 , panel b ) : 28% in 20052007 , 44% in 20082010 , 70% in 20112013 , and 77% in 20142016 . in contrast , the frequency of mt186 isolates decreased : 31% in 20052007 , 44% in 20082010 , 21% in 20112013 , and 6% in 20142016 .
we also observed a substitution of the major genotype in the b. pertussis population from mt186 to mt27 .
of 59 pertactin - deficient b. pertussis isolates collected during 20052016 , 45 ( 76.3% ) were mt186 isolates , whereas 2 ( 3.4% ) represented mt27 and 12 ( 20.3% ) other mt isolates ( mt194 , mt224226 , mt314316 ) ( table ) .
notably , 45 ( 83.3% ) of 54 mt186 isolates were pertactin - deficient , whereas only 2 ( 1.4% ) of 139 mt27 isolates were pertactin - deficient .
this finding indicates that pertactin - deficient isolates predominate among the mt186 strain but are rare among the mt27 strain . *
mt , types determined by using multilocus variable - number tandem - repeat analysis . 
for convenience , minor mts ( mt194 , mt224226 , and mt314316 ) are included in this category .
we previously showed that pertactin - deficient isolates in japan were generated by 2 different mutations : an 84-bp deletion of the prn gene signal sequence ( ss ) and an is481 insertion at nucleotide position 1598 in prn ( 15981599::is481 ) ( 5 ) .
thus , to confirm the molecular basis for the loss of pertactin production , pertactin - deficient isolates ( n = 26 ) that were newly identified in this study underwent pcr screening with 2 primer sets ( technical appendix table 2 ) .
we summarized the molecular mechanisms of loss of pertactin production in 59 pertactin - deficient isolates ( table ) ; the ss mutation was detected in 43 ( 72.9% ) mt186 isolates and in 11 ( 18.6% ) other mt isolates .
in contrast , the 15981599::is481 mutation was detected in 2 ( 3.4% ) mt186 isolates and 1 other mt isolate ( 1.7% , mt226 ) .
two mt27 isolates ( bp394 and bp533 ) do not have either of these mutations .
instead , bp533 isolates ( 1.7% ) have an is481 insertion at nucleotide position 245 ( 245246::is481 ; genbank accession no .
kc445198 ) , and bp394 ( 1.7% ) isolates exhibit transcriptional down - regulation of prn gene expression ( technical appendix figure 1 ) .
the expansion of pertactin - deficient b. pertussis isolates has been reported worldwide ( 38 ) . however , we observed a significant decrease in pertactin - deficient isolates within the b. pertussis population in japan , caused by a genotypic replacement from the pertactin - deficient mt186 strain to the pertactin - producing mt27 strain .
the most likely explanation for the prevalence of pertactin - deficient strains is vaccine - driven strain evolution , because pertactin is a component of acvs .
no pertactin - deficient isolates have been detected in denmark , where an acv that does not contain pertactin is used , and pertactin - deficient strains exhibit a selective advantage against acv - induced immunity ( 7,911 ) . in japan , 5 brands of the diphtheria  tetanus  acellular pertussis vaccine ( dtap ) had been used to control pertussis for many years .
these dtaps had different formulations of components ; only 3 contained the pertactin antigen ( 1 ) .
when the dtap vaccine was replaced , however , 2 brands of combined dtap  inactivated poliovirus ( dtap - ipv ) vaccine that did not contain pertactin were introduced in november 2012 .
therefore , most japanese children < 4 years of age do not have immunity to pertactin ; consequently , the selective pressure for pertactin - deficient strains in the host environment has recently been reduced .
this effect may be responsible for the recent decline in pertactin - deficient isolates and further supports the hypothesis that pertactin - deficient strains are selected on the basis of host immunity to pertactin .
notably , a new brand of dtap - ipv vaccine containing pertactin was also introduced in december 2015 in japan .
if the hypothesis of vaccine - driven evolution is correct , pertactin - deficient isolates should increase again in japan in the near future .
we demonstrated that genotypic replacement from mt186 to mt27 has taken place among recent b. pertussis isolates in japan : mt27 is a triple - locus variant of mt186 .
mt186 strains carry the pertussis - toxin promoter allele ptxp1 , whereas mt27 strains carry the allele ptxp3 ( 8) .
b. pertussis strains carrying ptxp3 ( i.e. , mt27 ) produce more of several virulence factors than do ptxp1 ( i.e. , mt186 ) strains ( 12,13 ) .
the population of mt27 strains carrying ptxp3 has increased worldwide ( 14,15 ) , although a low frequency of ptxp1 isolates was observed in japan ( 8) , suggesting that mt27 strains are associated with the recent pertussis resurgence .
it is possible , therefore , that the genotypic replacement in the b. pertussis population may have resulted from the expansion of the more virulent ptxp3 ( i.e. , mt27 ) strains .
in addition , given that pertactin - deficient mt27 isolates are rare , this genotypic replacement may have contributed to the recent decrease in pertactin - deficient isolates in japan . in japan , most pertactin - deficient isolates carry a deletion of the prn signal sequence ( ss ) , which has been found primarily in mt186 isolates carrying the prn1 allele ( technical appendix table 1 ) .
in other countries , a common prn mutation includes an insertion of is481 into the prn2 allele ( 4,6,7 ) . in this study , we identified 2 pertactin - deficient mt27 isolates carrying the prn2 allele , due to the is481 insertion ( 245246::is481 ) and the transcriptional down - regulation of the prn gene .
these pertactin - deficient isolates were previously identified in europe and the united states ( 6,7 ) .
one possible explanation for the appearance of pertactin - deficient mt27 isolates is that they were imported from other countries .
technical appendix .   table showing characteristics of bordetella pertussis isolates , japan , 20052016 , table showing primers used in the study of b. pertussis isolates , japan , 20082016 , and figure showing lack of prn transcript expression in pertactin - deficient b. pertussis isolate bp394 .
prevalence of pertactin - lacking bordetella pertussis isolates has been observed worldwide . in japan , however , we found that the frequency of pertactin - deficient isolates in 20142016 ( 8% ) was significantly lower than the frequency in 20052007 ( 41% ) , 20082010 ( 35% ) , and 20112013 ( 25% ) . 
 this reduction was closely associated with changes in genotypes .