Pamela K. Cassiday

Global Population Structure and Evolution of Bordetella pertussis and Their Relationship with Vaccination

ABSTRACT Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape. Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape.

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

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.

Polymorphism in Bordetella pertussis Pertactin and Pertussis Toxin Virulence Factors in the United States, 1935–1999

To elucidate the potential role of the etiologic agent in recent increases of pertussis incidence in the United States, we studied the polymorphism in pertactin and pertussis toxin, which are Bordetella pertussis proteins important for pathogenesis and immunity. We sequenced regions of their genes (prn and ptx) in 152 B. pertussis strains isolated from 1935 through 1999 and identified 2 prn sequences: prn1 (old), observed continuously since 1935, and prn2 (new), not recognized until 1981 but seen in 97% of tested isolates in 1999. There were 3 ptx S1 subunit sequences: ptxS1D (old) was identified in 3 strains (1935 and 1939); ptxS1B (old) represented 87% of the strains recovered during 1935-1974; and ptxS1A (new) was the most prevalent during 1975-1987 and 1989-1999 (64% and 78%, respectively). Potential association between vaccination and the observed shift from old to new types requires further study. Our results provide the basis for prospectively monitoring for changes among circulating B. pertussis that might have epidemiologic relevance.

Pertactin-Negative Bordetella pertussis Strains: Evidence for a Possible Selective Advantage

BACKGROUND A recent increase in Bordetella pertussis without the pertactin protein, an acellular vaccine immunogen, has been reported in the United States. Determining whether pertactin-deficient (PRN(-)) B. pertussis is evading vaccine-induced immunity or altering the severity of illness is needed. METHODS We retrospectively assessed for associations between pertactin production and both clinical presentation and vaccine history. Cases with isolates collected between May 2011 and February 2013 from 8 states were included. We calculated unadjusted and adjusted odds ratios (ORs) using multivariable logistic regression analysis. RESULTS Among 753 isolates, 640 (85%) were PRN(-). The age distribution differed between cases caused by PRN(-) B. pertussis and cases caused by B. pertussis producing pertactin (PRN(+)) (P = .01). The proportion reporting individual pertussis symptoms was similar between the 2 groups, except a higher proportion of PRN(+) case-patients reported apnea (P = .005). Twenty-two case-patients were hospitalized; 6% in the PRN(+) group compared to 3% in the PRN(-) group (P = .11). Case-patients having received at least 1 pertussis vaccine dose had a higher odds of having PRN(-) B. pertussis compared with unvaccinated case-patients (adjusted OR = 2.2; 95% confidence interval [CI], 1.3-4.0). When restricted to case-patients at least 1 year of age and those age-appropriately vaccinated, the adjusted OR increased to 2.7 (95% CI, 1.2-6.1). CONCLUSIONS The significant association between vaccination and isolate pertactin production suggests that the likelihood of having reported disease caused by PRN(-) compared with PRN(+) strains is greater in vaccinated persons. Additional studies are needed to assess whether vaccine effectiveness is diminished against PRN(-) strains.

Multilocus Sequence Typing Identifies Evidence for Recombination and Two Distinct Lineages of Corynebacterium diphtheriae

ABSTRACT We describe the development of a multilocus sequence typing (MLST) scheme for Corynebacterium diphtheriae, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize C. diphtheriae, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of C. diphtheriae examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.

Changes in Predominance and Diversity of Genomic Subtypes of Bordetella pertussis Isolated in the United States, 1935 to 1999

Pulsed-field gel electrophoresis (PFGE) of Bordetella pertussis chromosomal DNA fragments generated by XbaI restriction has been used to subtype isolates for epidemiologic studies. To better understand the natural history of pertussis, we determined the PFGE profiles of 1,333 strains isolated in the United States from 1935 to 1999. Results showed a shift in prevalent profiles from the earliest to the latest study periods. In addition, genetic diversity decreased over time, and prevalent profiles were more highly related to each other than to less common profiles. These results provide the foundation for investigating the impact of prevention strategies, including the use of the acellular vaccines, on the currently circulating B. pertussis population.

Novel Corynebacterium diphtheriae in domestic cats.

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.

Development of a Real-Time Fluorescence PCR Assay for Rapid Detection of the Diphtheria Toxin Gene

ABSTRACT We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.

Molecular epidemiology of Bordetella pertussis by pulsed-field gel electrophoresis profile : Cincinnati, 1989-1996

Reported cases of pertussis have increased in the United States, with peaks occurring every few years. Bordetella pertussis isolates collected in Cincinnati from 1989 to 1996 were analyzed with pulsed-field gel electrophoresis (PFGE), to evaluate trends. Among 496 isolates, 30 PFGE profiles were identified; 32% were CYXXI-010, the profile that predominated each year. Eighteen profiles (198 strains) were identified in 1989-1992, 20 profiles (197 strains) were identified during the 1993 epidemic, and 11 profiles (101 strains) were identified in 1994-1996. From 1989 to 1996, among 42 patients, isolates from household members in 17 (89%) of 19 households had concordant PFGE profiles. There was no association between PFGE profile and seasonality, age, and hospitalization or pneumonia in infants <1 year old. The 1993 epidemic was associated primarily with an increased prevalence of PFGE profiles that circulated before and after 1993, which suggests that the epidemic was due to factors other than the emergence of a novel B. pertussis strain.

Molecular Epidemiology of the Pertussis Epidemic in Washington State in 2012

ABSTRACT Although pertussis disease is vaccine preventable, Washington State experienced a substantial rise in pertussis incidence beginning in 2011. By June 2012, the reported cases reached 2,520 (37.5 cases per 100,000 residents), a 1,300% increase compared with the same period in 2011. We assessed the molecular epidemiology of this statewide epidemic using 240 isolates collected from case patients reported from 19 of 39 Washington counties during 2012 to 2013. The typing methods included pulsed-field gel electrophoresis (PFGE), multilocus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), and pertactin gene (prn) mutational analysis. Using the scheme PFGE-MLVA-MLST-prn mutations-Prn deficiency, the 240 isolates comprised 65 distinct typing profiles. Thirty-one PFGE types were found, with the most common types, CDC013 (n = 51), CDC237 (n = 44), and CDC002 (n = 42), accounting for 57% of them. Eleven MLVA types were observed, mainly comprising type 27 (n = 183, 76%). Seven MLST types were identified, with the majority of the isolates typing as prn2-ptxP3-ptxA1-fim3-1 (n = 157, 65%). Four different prn mutations accounted for the 76% of isolates exhibiting pertactin deficiency. PFGE provided the highest discriminatory power (D = 0.87) and was found to be a more powerful typing method than MLVA and MLST combined (D = 0.67). This study provides evidence for the continued predominance of MLVA 27 and prn2-ptxP3-ptxA1 alleles, along with the reemergence of the fim3-1 allele. Our results indicate that the Bordetella pertussis population causing this epidemic was diverse, with a few molecular types predominating. The PFGE, MLVA, and MLST profiles were consistent with the predominate types circulating in the United States and other countries. For prn, several mutations were present in multiple molecular types.