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Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution. A new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. The MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology.

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA).To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in The Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination.

Background Despite vaccination since the 1950s, pertussis has persisted and resurged. It remains a major cause of infant death worldwide and is the most prevalent vaccine-preventable disease in developed countries. The resurgence of pertussis has been associated with the expansion of Bordetella pertussis strains with a novel allele for the pertussis toxin (Ptx) promoter, ptxP3 , which have replaced resident ptxP1 strains. Compared to ptxP1 strains, ptxP3 produce more Ptx resulting in increased virulence and immune suppression. To elucidate how B. pertussis has adapted to vaccination, we compared genome sequences of two ptxP3 strains with four strains isolated before and after the introduction vaccination. Results The distribution of SNPs in regions involved in transcription and translation suggested that changes in gene regulation play an important role in adaptation. No evidence was found for acquisition of novel genes. Modern strains differed significantly from prevaccination strains, both phylogenetically and with respect to particular alleles. The ptxP3 strains were found to have diverged recently from modern ptxP1 strains. Differences between ptxP3 and modern ptxP1 strains included SNPs in a number of pathogenicity-associated genes. Further, both gene inactivation and reactivation was observed in ptxP3 strains relative to modern ptxP1 strains. Conclusions Our work suggests that B. pertussis adapted by successive accumulation of SNPs and by gene (in)activation. In particular changes in gene regulation may have played a role in adaptation.

Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite high vaccination coverage, pertussis has resurged and has become one of the most prevalent vaccine-preventable diseases in developed countries. We have proposed that both waning immunity and pathogen adaptation have contributed to the persistence and resurgence of pertussis. Allelic variation has been found in virulence-associated genes coding for the pertussis toxin A subunit (ptxA), pertactin (prn), serotype 2 fimbriae (fim2), serotype 3 fimbriae (fim3) and the promoter for pertussis toxin (ptxP). In this study, we investigated how more than 60 years of vaccination has affected the Dutch B. pertussis population by combining data from phylogeny, genomics and temporal trends in strain frequencies. Our main focus was on the ptxA, prn, fim3 and ptxP genes. However, we also compared the genomes of 11 Dutch strains belonging to successful lineages. Our results showed that, between 1949 and 2010, the Dutch B. pertussis population has undergone as least four selective sweeps that were associated with small mutations in ptxA, prn, fim3 and ptxP. Phylogenetic analysis revealed a stepwise adaptation in which mutations accumulated clonally. Genomic analysis revealed a number of additional mutations which may have a contributed to the selective sweeps. Five large deletions were identified which were fixed in the pathogen population. However, only one was linked to a selective sweep. No evidence was found for a role of gene acquisition in pathogen adaptation. Our results suggest that the B. pertussis gene repertoire is already well adapted to its current niche and required only fine tuning to persist in the face of vaccination. Further, this work shows that small mutations, even single SNPs, can drive large changes in the populations of bacterial pathogens within a time span of six to 19 years.

Background Bordetella pertussis is the causative agent of human whooping cough (pertussis) and is particularly severe in infants. Despite worldwide vaccinations, whooping cough remains a public health problem. A significant increase in the incidence of whooping cough has been observed in many countries since the 1990s. Several reasons for the re-emergence of this highly contagious disease have been suggested. A particularly intriguing possibility is based on evidence indicating that pathogen adaptation may play a role in this process. In an attempt to gain insight into the genomic make-up of B. pertussis over the last 60 years, we used an oligonucleotide DNA microarray to compare the genomic contents of a collection of 171 strains of B. pertussis isolates from different countries. Results The CGH microarray analysis estimated the core genome of B. pertussis , to consist of 3,281 CDSs that are conserved among all B. pertussis strains, and represent 84.8% of all CDSs found in the 171 B. pertussis strains. A total of 64 regions of difference consisting of one or more contiguous CDSs were identified among the variable genes. CGH data also revealed that the genome size of B. pertussis strains is decreasing progressively over the past 60 years. Phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the phylogenetic structure of the strains. B. pertussis strains with the same gene content were found in several different countries. However, geographic specificity of the B. pertussis strains was not observed. The gene content was determined to highly correlate with the ptxP -type of the strains. Conclusions An overview of genomic contents of a large collection of isolates from different countries allowed us to derive a core genome and a phylogenetic structure of B. pertussis . Our results show that B. pertussis is a dynamic organism that continues to evolve.

In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpson's diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in The Netherlands before the introduction of the 7-valent conjugate vaccine.

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.

To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). In this study, a single nucleotide polymorphism (SNP) typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in The Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis.

Despite more than 50 years of vaccination, whooping cough is still an endemic disease in the Netherlands with regular epidemic outbreaks. In the last 20 years, two periods of increased notifications were observed. The causes of the increased notifications in the first period, from 1983 to 1987, are contentious. At the time it was suggested to be a surveillance artifact, caused by changes in diagnostic procedures and increased awareness. An alternative explanation, a reduction in the vaccine dose, was downplayed at the time. The aim of this study was to reinvestigate the causes of the increased notifications by identifying changes in the Bordetella pertussis population. B. pertussis strains, isolated from 1965 to 1992, were characterized by means of fimbrial serotyping, multiple-locus sequence typing of virulence genes (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Shifts in fimbrial serotypes and MLVA types were associated with changes in vaccine dose and increased number of notifications. One to three years after lowering of the vaccine dose, the predominant fimbrial serotype changed from Fim3 to Fim2, and the reverse trend was observed when the vaccine dose was increased. Significantly, changes in fimbrial serotypes were evident at least seven years before the increase in notifications. Our results provide evidence that the change in vaccine dose affected host immunity and, consequently, contributed to an increase in pertussis morbidity. Further, we show that MLVA and fimbrial serotyping of strains can be used as early warning for pertussis epidemics.