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Characterization of in vitro biofilm-associated pneumococcal phase variants of a clinically relevant serotype 3 clone
M Catherine McEllistrem1, Jennifer V Ransford, Saleem A Khan
1Division of Infectious Diseases, University of Pittsburgh School of Medicine, PA 15213-2582, USA. mcellistremc@dom.pitt.edu
Abstract:
An increasing proportion of children with acute otitis media due to Streptococcus pneumoniae have serotype 3 infections since licensure of the seven-valent pneumococcal conjugate vaccine. These serotype 3 strains are genetically related by molecular subtyping. During otitis media with effusion and recurrent otitis media, biofilms commonly develop. Pneumococcal in vitro biofilms are comprised of phase variants that differ in colony morphology. By using a representative strain of the mucoid serotype 3 clone, rough phase variants with a diverse array of mutations were detected in biofilms formed in vitro. Most phase variants had mutations in the cps3D gene, the first gene of the capsular operon. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D gene, one had an SNP in the -10 promoter, and three had large deletions in the cps3D gene. Reversion to the mucoid phenotype was associated with reversion of the mutation in the cps3D gene. Unlike the phase variants detected in the nasopharynx, which have at least 20% of the parental amount of capsule, the in vitro biofilm-associated phase variants had < or =12% of the parental amount of capsule, as determined by capsule enzyme-linked immunosorbent assays. Using real-time reverse transcription-PCR, we determined that capsule expression in the phase variants was likely regulated at multiple levels. These in vitro phase variation data, which underscore the plasticity of the pneumococcus, need to be confirmed with in vivo analyses of the middle ear mucosa during otitis media.
Insights
Streptococcus pneumoniae serotype 3 strains causing otitis media exhibit phase variation in biofilms. Mutations in the cps3D gene lead to reduced capsule expression, impacting biofilm formation in children.
Area of Science:
- Microbiology
- Genetics
- Vaccinology
Background:
- Increasing incidence of Streptococcus pneumoniae serotype 3 in pediatric otitis media post-vaccination.
- Biofilm formation is common in otitis media with effusion and recurrent otitis media.
- Pneumococcal biofilms contain phase variants with altered colony morphology.
Purpose of the Study:
- Investigate phase variation in Streptococcus pneumoniae serotype 3 within in vitro biofilms.
- Identify genetic mutations associated with phase variation in biofilm-associated pneumococci.
- Characterize capsule expression levels in biofilm-derived phase variants.
Main Methods:
- Utilized a representative mucoid serotype 3 clone for in vitro biofilm formation.
- Employed molecular subtyping and sequencing to detect mutations in phase variants.
- Quantified capsule expression using enzyme-linked immunosorbent assays (ELISA).
- Assessed capsule gene expression using real-time reverse transcription-PCR (RT-PCR).
Main Results:
- Identified rough phase variants in vitro with diverse mutations, primarily in the cps3D gene.
- Observed single nucleotide polymorphisms (SNPs) and large deletions in the cps3D gene.
- Phase variants exhibited significantly reduced capsule expression (<12% of parental levels).
- Reversion to the mucoid phenotype correlated with reversion of cps3D mutations.
Conclusions:
- Pneumococcal serotype 3 demonstrates significant genetic plasticity within in vitro biofilms.
- Mutations in the cps3D gene are key drivers of reduced capsule expression and phase variation.
- These findings highlight the need for in vivo studies to confirm in vitro observations in otitis media.
