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Despite universal immunization programs, pertussis remains a major public health concern. This study aimed to describe the pertussis epidemiology in the Puglia region in 2006–2015 and to identify recent polymorphisms in Bordetella pertussis virulence-associated genes.The pertussis cases in 2006–2015 were identified from the National Hospital Discharge Database and the Information System of Infectious Diseases. Samples of pertussis cases in 2014–2016 that were confirmed by the Regional Reference Laboratory were subjected to ptxA, ptxP and prn gene sequencing and, in 10 cases, multiple-locus variable-number tandem repeat analysis.In Puglia in 2006–2015, the pertussis incidence rose from an average of 1.39/100,000 inhabitants in 2006–2013 to 2.56–2.54/100,000 in 2014–2015. In infants <1 year of age, the incidence rose from an average of 60.4/100,000 infants in 2006–2013 to 149.9/100,000 in 2015. Of the 661 cases recorded in 2006–2015, 80.3% required hospitalization; of these, 45.4% were <1 year of age. Of the 80 sequenced samples, the allelic profile ptxA1-ptxP3-prn2 was detected in 74. This variant was detected in both vaccinated and unvaccinated people. Six Bordetella pertussis samples were prn deficient. The multiple-locus variable-number tandem repeat analysis cases exhibited multiple-locus variable-number tandem repeat analysis-type 27.The pertussis incidence in Puglia has risen. The hypervirulent strain was also found in vaccinated people. This suggests bacterial adaptation to the vaccine and raises questions about acellular vaccine effectiveness. Prevention of infant pertussis cases is best achieved by immunizing the pregnant mother. Enhanced surveillance and systematic laboratory confirmation of pertussis should be improved in Italy.