Background

There is increasing evidence that severe pneumococcal disease is associated with coinfection with respiratory syncytial virus (RSV). Our previous work demonstrated a direct interaction between these pathogens, with RSV glycoprotein (RSV-G) binding penicillin binding protein (PBP) 1a of S. pneumoniae. This interaction led to enhanced virulence in a murine pneumonia model. This study aimed to determine the mechanism behind this, by examining the direct effects of RSV-G on PBP1a activity and bacterial division.

Methods

Clinical pneumococcal strains were screened for their ability to bind a purified recombinant RSV-G using immunoblot assays, fluorescence and electron microscopy. The effect on PBP1a activity was determined using enzymatic assays and by testing antibiotic susceptibility.

Results

Using fluorescent amino acid pulse labelling we observed RSV-G binding to the site of active pneumococcal cell wall synthesis. Following RSV-G exposure, pneumococci had morphological defects and increased lysis compared to controls, but growth rate was not reduced. PBP1a glycosyltransferase activity was not inhibited by RSV-G, while some pneumococci exhibited enhanced penicillin sensitivity.

Conclusions

RSV-G results in increased pneumococcal cell wall abnormalities and cell lysis. This may enhance pneumococcal virulence through the increased release of pneumolysin toxin. The implicated role of PBPs in RSV-G binding may be relevant to beta-lactam antibiotic usage.

Background

Pneumococcal conjugate vaccines have been very successful, but their use has increased infections by non-vaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). If pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire.

Methods

Chemical structures of capsular PS from an oral streptococcus strain, SK95, and a pneumococcal strain, D39, were determined by 2-dimensional NMR. An acapsular pneumococcus strain was transformed with cps from SK95 and D39, which were compared for their virulence by measuring non-specific phagocytic killing and their ability to kill mice.

Results

SK95 and D39 both produce structurally identical type 2 capsules. Baby rabbit serum (BRS) at 10% non-specifically killed >50% of SK95; however, 12·5% BRS killed <20% of D39. Non-encapsulated pneumococci became resistant to BRS as D39 following transformation with SK95 cps. Similarly, SK95 is avirulent in well-established in vivo mouse model. When the acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice.

Conclusions

Oral streptococcus cps can make acapsular pneumococcus virulent and inter-species cps transfer should be considered a potential mechanism for serotype replacement. Our findings highlight potential limitations of current WHO criterion for studying serotypes of pneumococci carried without first isolating bacteria.

Background

Streptococcus pneumoniae (Spn) strains rapidly kills Staphylococcus aureus (Sau) by producing membrane-permeable hydrogen peroxide (H₂O₂). The exact mechanism by which the H₂O₂-mediated killing occurs is not well understood.

Methods

An in vitro model that mimicked Spn-Sau contact during colonization of the upper airways and whole genome sequencing was conducted. Different Spn H₂O₂ mutants were constructed to confirm the Sau killing mechanism.

Results

Sau killing required outcompeting densities of Spn. A collection of MRSA/MSSA strains showed a linear sensitivity (R²=0.95) for Spn killing but the same strains had different susceptibilities when challenged against pure H₂O₂. WGS of these MRSA/MSSA strains revealed no association between clonal complex and susceptibility, or resistance, to Spn, or H₂O₂,respectively. A sublethal dose (~1 mM) of pure H₂O₂ when incubated with TIGR4DspxB eradicated cultures of Sau strains suggesting that Spn converts H₂O₂ to the hydroxyl radical (^·OH). Accordingly, Sau killing was completely blocked by incubating with scavengers of ^·OH radicals, DMSO, or thiourea.

Conclusions

Spn produces H₂O₂ which is rapidly converted to a more potent oxidant, the ^·OH radical. Hydroxyl radicals does not affect Spn viability but rapidly intoxicate Sau strains. The target(s) of the ^·OH radicals represents an exciting new alternative for the development of therapeutics against Sau infections.

Background

Detection of multiple pneumococcal serotypes in nasopharyngeal swabs (NPS) by latex agglutination, either by the WHO protocol or sweep serotyping may yield incomplete serum factor reactions. We investigated the impact of combining latex sweep serotyping with Quellung to improve the specificity of reactions while detecting multiple pneumococcal serotypes.

Methods

We randomly selected 103 STGG-NPS samples cultured and serotyped by latex agglutination of different morphological colonies (WHO protocol) in two cross-sectional carriage studies. We performed Quellung capsular serotyping on 1, 2 or 3 morphologically distinct pneumococcal colonies from the culture plates and then swept the same plates.

Results

Sweep serotyping was more likely to detect multiple serotypes (Table 1) than the WHO protocol (p=0.004). A common serotype was identified in 95/103 (92.2%) NPS samples by the WHO, latex sweep and Quellung. Quellung gave more conclusive antisera reactions (Table 2) than the WHO protocol (p=0.007). No significant difference in conclusiveness of antisera reaction between Quellung and latex sweep (p=0.119).

Conclusions

Combining sweep serotyping and Quellung is a cost-effective way of detecting multiple pneumococcal serotype co-colonization and resolving issues related to antisera reactivity in latex agglutination. Inconclusive Quellung results occurred because we detected more serotypes by latex sweep than were selected according to different morphological colonies.

Background

Healthy children frequently carry Streptococcus pneumoniae and Haemophilus influenzae. The aim was to study the association of H.influenzae carriage with S.pneumoniae serotypes.

Methods

Nasopharyngeal swabs were collected from about 500 children, 1-6 years old, in March 2009, and then annually 2012-2018. S.pneumoniae and H.influenzae were identified from the swabs using conventional culture methods and colonization density estimated by semi-quantitative grading. S.pneumoniae were serotyped with latex agglutination and/or mPCR.

Results

In total, 4,060 samples were collected. S.pneumoniae were detected in 2,501 samples (61.6%), and H.influenzae in 2,736 (67.4%). 1,882 samples were positive for both species and neither species could be detected in 705 samples. S.pneumoniae only were found in 619 samples and H.influenzae only in 854 samples. The Phi Coefficient of Association was 0.21.

Of the 147 samples with S.pneumoniae serotype 23F, 124 (84.4%) also had H.influenzae compared to 1,882 of the 2,501 (75.2%) S.pneumoniae positive samples (p=0.008). Of the 156 samples with non-encapsulated S.pneumoniae, 97 (62.2%) also had H.influenzae (p=0.002).

The proportion of positive samples for one of the species increased with increasing density of the other species.

Conclusions

A weak association was seen with co-carriage of S.pneumoniae and H.influenzae. H.influenzae was more often associated with serotype 23F than with non-encapsulated S.pneumoniae.

Background

In older adults, pneumococcal disease is strongly associated with viral respiratory infections, though the impact of viruses on pneumococcal carriage remains poorly understood. Here, we have investigated effects of influenza-like illness (ILI) on pneumococcal carriage in older adults.

Methods

Pneumococcal presence was determined in saliva samples that were collected in the 2014/2015 influenza season from individuals aged ≥60 years, including 232 individuals with ILI sampled trice and 194 controls sampled twice. Pneumococcal carriage was detected with quantitative-PCRs targeting piaB and lytA genes in minimally-processed saliva and culture-enriched saliva. Bacterial and pneumococcal abundances were determined in minimally-processed saliva. Respiratory viruses were detected in nasopharyngeal and oropharyngeal samples using qPCR.

Results

In general, pneumococcal carriage was significantly associated with exposure to young children and rhinovirus infection. The prevalence of pneumococcal carriage was highest at ILI-onset (18%) and lowest among controls (11-13%). Compared with controls, both absolute and relative pneumococcal abundances were significantly elevated at ILI-onset and remained elevated 7-9 weeks later. Pneumococcal abundances were highest in individuals with carriage acquisition between ILI-onset and 2-3 weeks later.

Conclusions

Our findings support the notion that in older adults acute respiratory infections favor pneumococcal colonization of the upper respiratory tract and this impact persists beyond recovery from ILI.

Background

Interaction of Streptococcus pneumoniae and viruses in the nasopharynx is a known phenomenon. We have explored the possible role of pneumococcus colonization on Respiratory Syncytial Virus (RSV) infection.

Methods

Newborns from Bangladesh, India, and Pakistan were enrolled and followed until the age of 60 days through household visits. Sick infants were referred to the study hospital where study physicians collected nasopharyngeal swabs. Pneumococcus and RSV were detected using the Real-time Polymerase Chain Reaction technique.

Results

Between 2011 and 2014, study physicians collected nasopharyngeal specimens from 5,209 episodes of suspected sepsis cases. Pneumococcus was detected from 35.3% (1,843/5,209) and RSV from 7.7% (402/5209) specimens. RSV was detected among 14.3% cases where pneumococcus was colonized (264/1,843), a contrast to 4.1% (138/3,366) among pneumococcus negative cases. The risk of RSV infection among the children with pneumococcal colonization was 4.6 times higher (95% CI: 3.8-5.5) compared to the infants who were not colonized by pneumococcus.

Conclusions

The study showed a direct relation of pneumococcal colonization with RSV infection in young infants. It is important to do further studies in this regard to understand the interaction of pneumococcus and RSV and the possible role of specific pneumococcal serotypes.