Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes

Abstract

Streptococcus pneumoniae is the leading cause of bacterial paediatric meningitis after the neonatal period worldwide, but the bacterial factors and pathophysiology that drive pneumococcal meningitis are not fully understood. In this work, we have identified differences in raffinose utilization by S. pneumoniae isolates of identical serotype and sequence type from the blood and cerebrospinal fluid (CSF) of a single pediatric patient with meningitis. The blood isolate displayed defective raffinose metabolism, reduced transcription of the raffinose utilization pathway genes, and an inability to grow in vitro when raffinose was the sole carbon source. The fitness of these strains was then assessed using a murine intranasal infection model. Compared with the CSF isolate, mice infected with the blood isolate displayed higher bacterial numbers in the nose, but this strain was unable to invade the ears of infected mice. A premature stop codon was identified in the aga gene in the raffinose locus, suggesting that this protein likely displays impaired alpha-galactosidase activity. These closely related strains were assessed by Illumina sequencing, which did not identify any single nucleotide polymorphisms (SNPs) between the two strains. However, these wider genomic analyses identified the presence of an alternative alpha-galactosidase gene that appeared to display altered sequence coverage between the strains, which may account for the observed differences in raffinose metabolic capacity. Together, these studies support previous findings that raffinose utilization capacity contributes to disease progression, and provide insight into a possible alternative means by which perturbation of this pathway may influence the behavior of pneumococci in the host environment, particularly in meningitis.

Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates

“…Previously, we have shown that while the CSF clinical S. pneumoniae isolate (60CSF) is found in the ears of infected mice 24 h post intranasal infection, the blood isolate (60B) was unable to survive in this niche ( Agnew et al., 2022 ). To determine whether the observed different virulence phenotypes of 60B and 60CSF in mice could be mediated by rearrangements in the spnIII Type I restriction-modification (RM) system, the spnIII allele distribution was assessed from pneumococcal DNA extracted from the nasopharynx of intranasally infected animals.…”

“…The wildtype S. pneumoniae strains used in this study ( Table 1 ) were provided to us by the German National Reference Center for Streptococci (Aachen) ( Agnew et al., 2022 ). Pneumococci serotype 15C blood and CSF isolates (designated 60B and 60CSF, respectively) were isolated in 2015 from a child, aged 2, admitted to hospital with meningitis.…”

“…The cells were routinely grown on Columbia agar supplemented with 5% (vol/vol) horse blood (BA), with or without gentamicin (5 µg/mL) or spectinomycin (200 μg/mL), at 37°C in 5% CO 2 overnight. Growth assays were carried out using pneumococci grown in a chemically defined media (CDM) which is composed of SILAC RPMI 1640 Flex Media supplemented with amino acids, vitamins, choline chloride and catalase (10 U/mL) as described previously ( Agnew et al., 2022 ), and either 0.5% (w/v) glucose or 0.5% (w/v) galactose.…”

“…Biofilm formation was measured in real-time with the real-time cell analyzer (RTCA) xCELLigence (Agilent Technologies Inc.) instrument as previously described ( Agnew et al., 2022 ; Tikhomirova et al., 2022 ). This instrument detects variations in the impedance signal (expressed as the arbitrary cell index, CI) as bacterial cells attach and form biofilms on the gold-microelectrodes present at the bottom of the E-plates (Agilent Technologies Inc.).…”

“…In the current study, we have characterized luxS in two S. pneumoniae clinical isolates obtained at the same time from the blood and cerebral spinal fluid (serotype 15C, ST 8711) of a single pediatric meningitis patient. Previously we have shown that these clinical isolates display different virulence phenotypes in a murine infection model ( Agnew et al., 2022 ). Here, we aimed to investigate the role of luxS in these two clinical isolates.…”