Johanna M. C. Jefferies

Identification of Invasive Serotype 1 Pneumococcal Isolates That Express Nonhemolytic Pneumolysin

ABSTRACT Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing “wild-type” pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.

Genetic Analysis of Diverse Disease-Causing Pneumococci Indicates High Levels of Diversity within Serotypes and Capsule Switching

ABSTRACT We have used multilocus sequence typing (MLST) and serotyping to build a phylogenetic framework for pneumococcal disease isolates in Scotland that provides a snapshot of the relationships between capsular type and genotype. The results show that while the MLST type correlates with the serotype, isolates within a serotype can belong to a number of individual clonal complexes or sequence types (STs). We also show that isolates of the same ST can express different capsular polysaccharides, i.e., display capsular switching, and that this phenomenon is observed both for capsular types commonly isolated from patients with invasive disease and for serogroups less commonly isolated from patients with invasive disease but which may commonly be carried asymptomatically in the human nasopharynx.

Presence of Nonhemolytic Pneumolysin in Serotypes of Streptococcus pneumoniae Associated with Disease Outbreaks

Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.

Genomic Diversity between Strains of the Same Serotype and Multilocus Sequence Type among Pneumococcal Clinical Isolates

ABSTRACT The important human pathogen Streptococcus pneumoniae is known to be a genetically diverse species. We have used comparative genome hybridization (CGH) microarray analysis to investigate this diversity in a collection of clinical isolates including several capsule serotype 14 pneumococci, a dominant serotype among disease isolates. We have identified three new regions of diversity among pneumococcal isolates and, importantly, clearly demonstrate genetic differences between strains of the same multilocus sequence type (ST) and capsule serotype. CGH may therefore, under certain circumstances, prove to be a valuable tool to supplement current typing methods. Finally, we show that these clonal strains with the same serotype and ST behave differently in an animal model. Strains of the same ST and serotype therefore have important genetic and phenotypic differences.

Automated pneumococcal MLST using liquid-handling robotics and a capillary DNA sequencer

Multilocus sequence typing (MLST) is used by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) as a routine method for the characterization of certain bacterial pathogens. The SMPRL recently started performing MLST on strains of Streptococcus pneumoniae, and here we describe a fully automated method for MLST using a 96-well-format liquid-handling robot and a 96-capillary automated DNA sequencer.

Increase in serotype 6C pneumococcal carriage, United Kingdom.

To the Editor: Streptococcus pneumoniae is a major human pathogen. In 2007, Park et al. identified a novel serotype, 6C (1), which emerged from serotype 6A. A study of children in the Netherlands who had not previously received a pneumococcal vaccine found low prevalence of this newly identified serotype before the heptavalent pneumococcal conjugate vaccine Prevnar/Prevenar (PCV7) (Wyeth, Taplow, UK) was introduced (2). Studies have shown cross-protection between vaccine serotype 6B and vaccine-related serotype 6A. However, PCV7 elicits no cross-protection against serotype 6C. The potential exists for the emergence of nonvaccine serotypes or novel clones. These serotypes and clones may be better adapted to colonize the nasopharynx, evade the human immune response, and cause disease. A recent study showed an increase in prevalence of serotype 6C pneumococci in children and a corresponding decrease in serotype 6A after introduction of PCV7 (3). We studied the underlying genetic basis for expansion of serotype 6C. Initial data from an ongoing study of pneumococcal carriage are presented. This study was reviewed and approved by the Southampton and South West Hampshire Research Ethics Committee (B) (reference 06/Q1704/105). A total of 697 nasopharyngeal swab specimens were collected from unselected (not selected by a method) children Appendix Figure Appendix Figure. Appendix Figure. Genotypes of serotype 6C pneumococci isolated from children in 2006–2007 (year 1) and 2007–2008 (year 2), United Kingdom. ST, sequence type. In the first year of this study, we identified 3 (3.1%) serotype 6C pneumococci belonging to 3 sequence types (STs): ST65, ST1714, and ST1692 (Appendix Figure). ST1714 and ST 1692 shared a common clonal complex. Only ST 65 was shared between serotype 6C and serotype 6A. In the second year, we identified 14 (13.6%) serotype 6C pneumococci belonging to 6 STs (Appendix Figure). Two of these STs, of the same ST, were from siblings. Three of them (ST1692 [n = 8], ST1714 [n = 2], and ST395 [n = 1]) were members of a common clonal complex with a predicted founder of ST395. Each of the remaining 3 STs (ST398, ST1862, and ST3460) was isolated only once. One serotype 6A isolate of ST1692 was also observed. No serotype 6C ST65 was observed in the second year. We isolated more serotype 6C pneumococci in year 2 than in year 1 (p Our study showed a large increase in ST1692 in serotype 6C pneumococci during the implementation of PCV7 and an increase in serotype 6C. Depending on the extent of cross-protection between vaccine-related serotypes, introduction of conjugate vaccines could induce clearance or emergence of vaccine-related serotypes. This introduction could also contribute to their substitution with novel or existing serotypes that are better adapted to the ecologic niche. However, our data may only be relevant to carried pneumococci and not reflected in pneumococcal disease epidemiology. Nevertheless, the increase in serotype 6C pneumococci in the United Kingdom, which is supported by a similar observation in the United States (3), highlights the potential for emergence of serotypes not included in the current study and newly developed pneumococcal conjugate vaccines.

PclA, a pneumococcal collagen-like protein with selected strain distribution, contributes to adherence and invasion of host cells

Analysis of Streptococcus pneumoniae sequenced genomes revealed a region present only in selected strains consisting of two ORFs: a putative cell wall anchored protein and a putative transcriptional regulator. The cell wall anchored protein contains large regions of collagen-like repeats, the number of which varies between strains. We have therefore named this protein PclA for pneumococcal collagen-like protein A. The second gene, spr1404, encodes a putative transcriptional regulator. We examined the strain distribution of these two genes among a collection of clinical isolates from invasive pneumococcal disease and found them to be present in 39% of the strains examined. Strains were either positive for both genes or lacked both, with the two genes always present together in the same location of the genome. RT-PCR analysis revealed that pclA is transcribed in vitro, even in the absence of spr1404. Single deletion mutants lacking either gene were not attenuated in a mouse model of invasive pneumonia. However, the pclA mutant was defective in adherence and invasion of host cells in vitro.

Identification of a Secreted Cholesterol-Dependent Cytolysin (Mitilysin) from Streptococcus mitis

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.

13-valent pneumococcal conjugate vaccine (PCV13)

The thirteen valent pneumococcal conjugate vaccine (PCV13, Prevenar 13Ô) is the broader coverage successor to the highly effective seven valent vaccine (PCV7, PrevenarÔ) which has reduced rates of pneumococcal disease in many countries. Despite the success of PCV7, pneumococcal disease due to non-PCV7 serotypes remains a threat in many settings, in particular many developing countries with a high burden of pneumococcal disease where serotype 1 and 5 are among the most common serotypes. Disease due to certain non-PCV7 serotypes, in particular serotype 19A has also begun to increase in incidence in countries with widespread use of PCV7. PCV13 consists of thirteen pneumococcal capsular polysaccharides individually conjugated to the diphtheria-derived protein carrier CRM197. In addition to serotypes 4, 6B, 9V, 14, 18C, 19F and 23F included in PCV7, PCV13 also includes serotypes 1, 3, 5, 6A, 7F and 19A. PCV13 was licensed on the basis of non-inferiority trials and has proved to be at least as safe and effective as PCV7. PCV13 replaced PCV7 in the childhood immunisation schedules of the USA and UK in 2010 and is being rolled out to an increasing number of developing countries during 2011. Here we review the current literature regarding this vaccine, describing safety, efficacy, global serotype coverage and use and future directions.

Low-dose nitric oxide as targeted anti-biofilm adjunctive therapy to treat chronic Pseudomonas aeruginosa infection in cystic fibrosis

Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases.