Two variants among Haemophilus influenzae serotype b strains with distinct bcs4, hcsA and hcsB genes display differences in expression of the polysaccharide capsule

<p>Abstract</p> <p>Background</p> <p>Despite nearly complete vaccine coverage, a small number of fully vaccinated children in the Netherlands have experienced invasive disease caused by <it>Haemophilus influenzae </it>serotype b (Hib). This increase started in 2002, nine years after the introduction of nationwide vaccination in the Netherlands. The capsular polysaccharide of Hib is used as a conjugate vaccine to protect against Hib disease. To evaluate the possible rise of escape variants, explaining the increased number of vaccine failures we analyzed the composition of the capsular genes and the expressed polysaccharide of Dutch Hib strains collected before and after the introduction of Hib vaccination.</p> <p>Results</p> <p>The DNA sequences of the complete capsular gene clusters of 9 Dutch Hib strains were assessed and two variants, designated type I and type II were found. The two variants displayed considerable sequence divergence in the <it>hcsA </it>and <it>hcsB </it>genes, involved in transport of capsular polysaccharide to the cell surface. Application of <it>hcsA </it>type specific PCRs on 670 Hib strains collected from Dutch patients with invasive Hib disease showed that 5% of the strains collected before 1996 were type II. No endogenous type II Hib strains were isolated after 1995 and all type II strains were isolated from 0–4 year old, non-vaccinated children only. Analysis of a worldwide collection of Hib strains from the pre-vaccination era revealed considerable geographic differences in the distribution of the type I and type II strains with up to 73% of type II strains in the USA. NMR analysis of type I and type II capsule polysaccharides did not reveal structural differences. However, type I strains were shown to produce twice as much surface bound capsular polysaccharide.</p> <p>Conclusion</p> <p>Type II strains were only isolated during the pre-vaccination era from young, non-vaccinated individuals and displayed a lower expression of capsular polysaccharide than type I strains. The higher polysaccharide expression may have provided a selective advantage for type I strains resulting in the rapid elimination of type II from the Dutch Hib population after introduction of nationwide Hib vaccination. However, this phenomenon does not explain the increase in the number of Hib vaccine failures in the Netherlands.</p

Population Structure of Invasive Streptococcus pneumoniae in the Netherlands in the Pre-Vaccination Era Assessed by MLVA and Capsular Sequence Typing

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)

Horizontal Transfer of Segments of the 16S rRNA Genes between Species of the Streptococcus anginosus Group

The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes

Genotyping by Amplified Fragment Length Polymorphism Analysis Reveals Persistence and Recurrence of Infection with Streptococcus anginosus Group Organisms

Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius, commonly referred to as the Streptococcus anginosus group (SAG), are commensal organisms known for their propensity to cause purulent infections which are difficult to eradicate. In this study, we determined the genetic similarities between SAG isolates consecutively recovered from single patients to assess the duration of infection or colonization. A total of 97 SAG isolates recovered from 30 patients were included; 65 (67.0%) of the isolates were abscess related. The isolates were identified by the 16S rRNA reverse line blot hybridization assay as S. anginosus (n = 34), S. constellatus (n = 55), and S. intermedius (n = 8). Amplified fragment length polymorphism (AFLP) analysis of the SAG isolates produced discriminatory and reproducible patterns. Consecutive SAG isolates with identical AFLP types were found in 27 of 30 (90.0%) patients, and consecutive isolates with only a single AFLP type were demonstrated in 21 (70.0%) patients. The median delay between the times of recovery of the first and last isolates of identical AFLP types from each patient was 36 days, and this delay extended for more than 1 year in patients with both colonizing and abscess-related SAG isolates. In six bacteremic patients, paired blood and nonblood SAG isolates showed identical AFLP types

Bordetella pertussis epidemiology, transmission and evolution in the vaccine era.

Bordetella pertussis causes whooping cough in humans. Despite high routine vaccination coverage, pertussis has become the most prevalent vaccine-preventable disease in many industrialized countries, since the 1990s. While a plethora of candidate explanations for the resurgence remain hotly debated, two prevailing ideas focus on vaccine driven pathogen adaptation and on the switch to the latest generation vaccines. We use a suite of genomic and epidemiological approaches to characterize pertussis transmission in the Netherlands, between 1949 and 2017, investigate drivers of B. pertussis population diversity, and the role of allele shifts in the resurgence. Reconciling the epidemiological data with sequence data has allowed us to characterize a resurgence that is not accompanied with increased transmission but has unique functional genomic signatures of decreased diversity. Here we show a positive association between genetic diversity and incidence which validates the hypothesis that the resurgence is associated with the expansion of more pathogenic strains. We see a direct effect of vaccination on the replicative fitness of pathogens. Our models suggest, large outbreaks are unlikely to be due to immune evasion and the vaccine switch cannot be contributing to increased pertussis incidence. Therefore, efforts aimed at mitigating the resurgence in the population at large, and particularly in vulnerable infants, are more likely to succeed by maintaining routine vaccine coverage high

Lagging Immune Response to Serotype b (Hib) Conjugate Vaccine after the Primary Vaccination with Hib of Infants in The Netherlands.

In 1993, a Haemophilus influenzae serotype b (Hib) conjugate vaccine was introduced in the Dutch national immunization program, resulting in a sharp decrease in invasive Hib disease. We used a population-based set of serum samples collected in the Netherlands in 2006-2007 (Pienter-II, 5696 sera) to assess the concentration of antibodies to the capsular polysaccharide of Hib, and compared the results with those obtained from a similar set collected in 1995-1996 (Pienter-I, 7837 sera). Post-primary vaccination serum samples from children aged 6-11 months from the Pienter-II study contained approximately 4-fold lower anti-Hib antibody concentrations than samples from children from the Pienter-I study. No such difference was found in post-booster samples from children older than 11 months of age. In Pienter-II, the proportion of children aged 6-11 months with anti-Hib antibody concentrations below the putative protective concentration of 0.15 µg/mL was 30%, which is significantly higher than in the Pienter-I study (12%). Fewer children in the Pienter-II group developed antibodies able to kill Hib in a serum bactericidal assay compared to the Pienter-I children. The cause of the lagged response in Pienter-II children remain uncertain, but lack of natural boosting, interference by the acellular pertussis vaccine, combining vaccines and acceleration of the schedule may have contributed

Increase in Genetic Diversity of Haemophilus influenzae Serotype b (Hib) Strains after Introduction of Hib Vaccination in The Netherlands

Recently, there has been an increase in The Netherlands in the number of cases of invasive disease caused by Haemophilus influenzae serotype b (Hib). To study a possible change in the Hib population that could explain the rise in incidence, a multiple-locus variable number tandem repeats analysis (MLVA) was developed to genotype H. influenzae isolates. The MLVA enabled the differentiation of H. influenzae serotype b strains with higher discriminatory power than multilocus sequence typing (MLST). MLVA profiles of noncapsulated H. influenzae and H. influenzae serotype f strains were more heterogeneous than serotype b strains and were distinct from Hib, although some overlap occurred. The MLVA was used to genotype a collection of 520 H. influenzae serotype b strains isolated from patients in The Netherlands with invasive disease. The strains were collected from 1983 from 2002, covering a time period of 10 years before and 9 years after the introduction of the Hib vaccine in the Dutch national vaccination program. MLVA revealed a sharp increase in genetic diversity of Hib strains isolated from neonates to 4-year-old patients after 1993, when the Hib vaccine was introduced. Hib strains isolated from patients older than 4 years in age were genetically diverse, and no significant change in diversity was seen after the introduction of the vaccine. These observations suggest that after the introduction of the Hib vaccine young children no longer constitute the reservoir for Hib and that they are infected by adults carrying genetically diverse Hib strains

Serotype specific important residues in the <i>wciP</i> gene of serogroup 6.

<p>Serotype specific important residues in the <i>wciP</i> gene of serogroup 6.</p