Lineage structure of Streptococcus pneumoniae may be driven by immune selection on the groEL heat-shock protein.

Sci Rep. 2017 Aug 22;7(1):9023. doi: 10.1038/s41598-017-08990-z.

Lineage structure of Streptococcus pneumoniae may be driven by immune selection on the groEL heat-shock protein.

Lourenço J1, Watkins ER2, Obolski U2, Peacock SJ2, Morris C3, Maiden MCJ2, Gupta S2.

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Populations of Streptococcus pneumoniae (SP) are typically structured into groups of closely related organisms or lineages, but it is not clear whether they are maintained by selection or neutral processes. Here, we attempt to address this question by applying a machine learning technique to SP whole genomes. Our results indicate that lineages evolved through immune selection on the groEL chaperone protein. The groEL protein is part of the groESL operon and enables a large range of proteins to fold correctly within the physical environment of the nasopharynx, thereby explaining why lineage structure is so stable within SP despite high levels of genetic transfer. SP is also antigenically diverse, exhibiting a variety of distinct capsular serotypes. Associations exist between lineage and capsular serotype but these can be easily perturbed, such as by vaccination. Overall, our analyses indicate that the evolution of SP can be conceptualized as the rearrangement of modular functional units occurring on several different timescales under different pressures: some patterns have locked in early (such as the epistatic interactions between groESL and a constellation of other genes) and preserve the differentiation of lineages, while others (such as the associations between capsular serotype and lineage) remain in continuous flux.

PMID: 28831154 DOI: 10.1038/s41598-017-08990-z

Capsule Type and Amount Affect Shedding and Transmission of Streptococcus pneumoniae.

MBio. 2017 Aug 22;8(4). pii: e00989-17. doi: 10.1128/mBio.00989-17.

Capsule Type and Amount Affect Shedding and Transmission of Streptococcus pneumoniae.

Zafar MA1, Hamaguchi S1, Zangari T1, Cammer M2, Weiser JN3.

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The capsular polysaccharide (CPS) of Streptococcus pneumoniae is characterized by its diversity, as it has over 95 known serotypes, and the variation in its thickness as it surrounds an organism. While within-host effects of CPS have been studied in detail, there is no information about its contribution to host-to-host transmission. In this study, we used an infant mouse model of intralitter transmission, together with isogenic capsule switch and cps promoter switch constructs, to explore the effects of CPS type and amount. The determining factor in the transmission rate in this model is the number of pneumococci shed in nasal secretions by colonized hosts. Two of seven capsule switch constructs showed reduced shedding. These constructs were unimpaired in colonization and expressed capsules similar in size to those of the wild-type strain. A cps promoter switch mutant expressing ~50% of wild-type amounts of CPS also displayed reduced shedding without a defect in colonization. Since shedding from the mucosal surface may require escape from mucus entrapment, a mucin-binding assay was used to compare capsule switch and cps promoter switch mutants. The CPS type or amount constructs that shed poorly were bound more robustly by immobilized mucin. These capsule switch and cps promoter switch constructs with increased mucin-binding affinity and reduced shedding also had lower rates of pup-to-pup transmission. Our results demonstrate that CPS type and amount affect transmission dynamics and may contribute to the marked differences in prevalence among pneumococcal types.IMPORTANCEStreptococcus pneumoniae, a leading cause of morbidity and mortality, is readily transmitted, especially among young children. Its structurally and antigenically diverse capsular polysaccharide is the target of currently licensed pneumococcal vaccines. Epidemiology studies show that only a subset of the >95 distinct serotypes are prevalent in the human population, suggesting that certain capsular polysaccharide types might be more likely to be transmitted within the community. Herein, we used an infant mouse model to show that both capsule type and amount are important determinants in the spread of pneumococci from host to host. Transmission rates correlate with those capsule types that are better at escaping mucus entrapment, a key step in exiting the host upper respiratory tract. Hence, our study provides a better mechanistic understanding of why certain pneumococcal serotypes are more common in the human population.

Copyright © 2017 Zafar et al.


Streptococcus pneumoniae; bacterial shedding; capsular polysaccharide; host-pathogen interactions; influenza A; pneumococcus; transmission

PMID: 28830943 DOI: 10.1128/mBio.00989-17

The Transcriptome of Streptococcus pneumoniae Induced by Local and Global Changes in Supercoiling.

Front Microbiol. 2017 Jul 31;8:1447. doi: 10.3389/fmicb.2017.01447. eCollection 2017.

The Transcriptome of Streptococcus pneumoniae Induced by Local and Global Changes in Supercoiling.

de la Campa AG1,2, Ferrándiz MJ1, Martín-Galiano AJ1, García MT3, Tirado-Vélez JM1.

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The bacterial chromosome is compacted in a manner optimal for DNA transactions to occur. The degree of compaction results from the level of DNA-supercoiling and the presence of nucleoid-binding proteins. DNA-supercoiling is homeostatically maintained by the opposing activities of relaxing DNA topoisomerases and negative supercoil-inducing DNA gyrase. DNA-supercoiling acts as a general cis regulator of transcription, which can be superimposed upon other types of more specific trans regulatory mechanism. Transcriptomic studies on the human pathogen Streptococcus pneumoniae, which has a relatively small genome (∼2 Mb) and few nucleoid-binding proteins, have been performed under conditions of local and global changes in supercoiling. The response to local changes induced by fluoroquinolone antibiotics, which target DNA gyrase subunit A and/or topoisomerase IV, involves an increase in oxygen radicals which reduces cell viability, while the induction of global supercoiling changes by novobiocin (a DNA gyrase subunit B inhibitor), or by seconeolitsine (a topoisomerase I inhibitor), has revealed the existence of topological domains that specifically respond to such changes. The control of DNA-supercoiling in S. pneumoniae occurs mainly via the regulation of topoisomerase gene transcription: relaxation triggers the up-regulation of gyrase and the down-regulation of topoisomerases I and IV, while hypernegative supercoiling down-regulates the expression of topoisomerase I. Relaxation affects 13% of the genome, with the majority of the genes affected located in 15 domains. Hypernegative supercoiling affects 10% of the genome, with one quarter of the genes affected located in 12 domains. However, all the above domains overlap, suggesting that the chromosome is organized into topological domains with fixed locations. Based on its response to relaxation, the pneumococcal chromosome can be said to be organized into five types of domain: up-regulated, down-regulated, position-conserved non-regulated, position-variable non-regulated, and AT-rich. The AT content is higher in the up-regulated than in the down-regulated domains. Genes within the different domains share structural and functional characteristics. It would seem that a topology-driven selection pressure has defined the chromosomal location of the metabolism, virulence and competence genes, which suggests the existence of topological rules that aim to improve bacterial fitness.


DNA supercoiling; DNA topoisomerases; fluoroquinolones; global transcription; interactome; novobiocin; seconeolitsine; topological domains

PMID: 28824578 PMCID: PMC5534458 DOI: 10.3389/fmicb.2017.01447

Acanthamoeba castellanii interactions with Streptococcus pneumoniae and Streptococcus pyogenes.

Exp Parasitol. 2017 Aug 17. pii: S0014-4894(17)30278-3. doi: 10.1016/j.exppara.2017.08.005. [Epub ahead of print]

Acanthamoeba castellanii interactions with Streptococcus pneumoniae and Streptococcus pyogenes.

Siddiqui R1, Yee Ong TY1, Jung SY2, Khan NA3.

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Among the genus Streptococcus, S. pyogenes and S. pneumoniae are the major causes of pharyngitis, impetigo, pneumonia and meningitis in humans. Streptococcus spp. are facultative anaerobes that are nutritionally fastidious, yet survive in the environment and target the predisposed population. Antibacterial disinfectants have been partially effective only, indicating the need for novel preventative measures and to understand mechanisms of bacterial resistance. Acanthamoeba is a free-living protist that is known to harbour microbial pathogens, provide shelter, and assist in their transmission to susceptible population. The overall aim of this study was to determine whether S. pyogenes and S. pneumoniae can interact with A. castellanii by associating, invading, and surviving inside trophozoites and cysts. It was observed that both S. pyogenes and S. pneumoniae were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. Notably, S. pyogenes and S. pneumoniae survived encystation process, avoided phagocytosis, multiplied, and exhibited higher recovery from the mature cysts, compared with the trophozoite stage (approximately 2 bacteria per amoebae ratio for cyst stage versus 0.02 bacteria per amoeba ration for trophozoite stage). As Acanthamoeba cysts are resilient and can disperse through the air, A. castellanii can act as a vector in providing shelter, facilitating growth and possibly genetic exchanges. In addition, these interactions may contribute to S. pyogenes and S. pneumoniae survival in harsh environments, and transmission to susceptible population and possibly affecting their virulence. Future studies will determine the molecular mechanisms associated with Acanthamoeba interactions with Streptococcus and the evolution of pathogenic bacteria and in turn expedite discovery of novel therapeutic and/or preventative measures.

Copyright © 2017. Published by Elsevier Inc.


Acanthamoeba; Association; Encystation; Invasion; Streptococcus; Survival

PMID: 28823705 DOI: 10.1016/j.exppara.2017.08.005

Cytosolic phospholipase A2α promotes pulmonary inflammation and systemic disease during Streptococcus pneumoniae infection.

Infect Immun. 2017 Aug 14. pii: IAI.00280-17. doi: 10.1128/IAI.00280-17. [Epub ahead of print]

Cytosolic phospholipase A2α promotes pulmonary inflammation and systemic disease during Streptococcus pneumoniae infection.

Bhowmick R1, Clark S2, Bonventre JV3, Leong JM4, McCormick BA5.

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Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells or PMNs) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A3 (HXA3) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A2 (PLA2) promotes the release of AA, we investigated the role of PLA2 in local and systemic disease during S. pneumoniae infection. The Group IVA cytosolic isoform of PLA2 (cPLA2α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae-induced PMN transepithelial migration in vitro Genetic ablation of the cPLA2 isoform cPLA2α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA2α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild type mice. Our data suggest that cPLA2α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniaelung challenge.

Copyright © 2017 American Society for Microbiology.

PMID: 28808157 DOI: 10.1128/IAI.00280-17

Streptococcus pneumoniae and lytic antibiotic therapy: are we adding insult to injury during invasive pneumococcal disease and sepsis?

J Med Microbiol. 2017 Aug 10. doi: 10.1099/jmm.0.000545. [Epub ahead of print]

Streptococcus pneumoniae and lytic antibiotic therapy: are we adding insult to injury during invasive pneumococcal disease and sepsis?

Brown LA1, Mitchell AM2, Mitchell TJ2.

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Streptococcus pneumoniae (S. pneumoniae), otherwise known as 'the pneumococcus', is a fascinating microbe that continues to pose a significant problem to public health. Currently there are no specific National Institute for Clinical Excellence (NICE) or British Thoracic Society (BTS) clinical guidelines referring to the treatment of invasive pneumococcal infection. NICE clinical guidelines suggest the use of lytic β-lactam antibiotic regimens for the management of community-acquired pneumonia and bacterial meningitis; infections for which S. pneumoniae is a likely causative organism. Lytic antibiotics have been shown to increase the release of pneumolysin (the highly inflammatory and damaging toxin of the pneumococcus), thus theoretically increasing host damage, which may lead to a decline of clinical outcomes in vulnerable patients. In light of this information, should the use of non-lytic antibiotics, such as quinolones, rifamycins and macrolides, be considered for the treatment of invasive pneumococcal disease?

PMID: 28792379 DOI: 10.1099/jmm.0.000545

The FAD synthetase from the human pathogen Streptococcus pneumoniae: a bifunctional enzyme exhibiting activity-dependent redox requirements.

Sci Rep. 2017 Aug 8;7(1):7609. doi: 10.1038/s41598-017-07716-5.

The FAD synthetase from the human pathogen Streptococcus pneumoniae: a bifunctional enzyme exhibiting activity-dependent redox requirements.

Sebastián M1,2, Lira-Navarrete E2,3, Serrano A1,2,4, Marcuello C5,6, Velázquez-Campoy A1,2,7,8, Lostao A5,7, Hurtado-Guerrero R2,7, Medina M9,10, Martínez-Júlvez M11,12.

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Prokaryotic bifunctional FAD synthetases (FADSs) catalyze the biosynthesis of FMN and FAD, whereas in eukaryotes two enzymes are required for the same purpose. FMN and FAD are key cofactors to maintain the flavoproteome homeostasis in all type of organisms. Here we shed light to the properties of the hitherto unstudied bacterial FADS from the human pathogen Streptococcus pneumoniae (SpnFADS). As other members of the family, SpnFADS catalyzes the three typical activities of prokaryotic FADSs: riboflavin kinase (RFK), ATP:FMN:adenylyltransferase (FMNAT), and FAD pyrophosphorylase (FADpp). However, several SpnFADS biophysical properties differ from those of other family members. In particular; i) the RFK activity is not inhibited by the riboflavin (RF) substrate, ii) the FMNAT and FADSpp activities require flavin substrates in the reduced state, iii) binding of adenine nucleotide ligands is required for the binding of flavinic substrates/products and iv) the monomer is the preferred state. Collectively, our results add interesting mechanistic differences among the few prokaryotic bifunctional FADSs already characterized, which might reflect the adaptation of the enzyme to relatively different environments. In a health point of view, differences among FADS family members provide us with a framework to design selective compounds targeting these enzymes for the treatment of diverse infectious diseases.

PMID: 28790457 PMCID: PMC5548840 DOI: 10.1038/s41598-017-07716-5

Streptococcus pneumoniae serotype 19A: Worldwide epidemiology.

Expert Rev Vaccines. 2017 Aug 7. doi: 10.1080/14760584.2017.1362339. [Epub ahead of print]

Streptococcus pneumoniae serotype 19A: Worldwide epidemiology.

Isturiz R1, Sings HL1, Hilton B1, Arguedas A2, Reinert RR3, Jodar L1.

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Streptococcus pneumoniae causes mucosal and invasive diseases with high morbidity and mortality. Introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) into routine infant immunization programs worldwide resulted in serotype 19A becoming a leading cause of the remaining pneumococcal disease burden in vaccinated and unvaccinated individuals. This article reviews the impact of the latest generation PCVs (10-valent PCV, PCV10, and 13-valent PCV, PCV13) on serotype 19A. Areas covered: This article covers immune responses elicited by PCV7, PCV10 and PCV13 against serotype 19A and their impact on nasopharyngeal (NP) carriage and disease in vaccinated and unvaccinated populations using data from surveillance systems, randomized controlled trials, and observational studies. Expert commentary: As expected from a PCV containing serotype 19A, PCV13 elicits significantly higher functional immune responses against serotype 19A than PCV7 and PCV10. Higher responses are likely to be linked to both direct impact in vaccinated populations and reductions in 19A NP carriage in children, thus inducing herd protection and reducing 19A invasive pneumococcal disease (IPD) in nonvaccinated children and adults. In contrast, PCV7 and PCV10 have shown mixed evidence of direct short-lived cross-protection and little to no impact on 19A carriage, resulting in continued transmission and disease.


Antimicrobial resistance; Streptococcus pneumoniae; epidemiology; invasive pneumococcal disease; nasopharyngeal carriage; pneumococcal conjugate vaccines; serotype 19A

PMID: 28783380 DOI: 10.1080/14760584.2017.1362339

Current challenges in the accurate identification of Streptococcus pneumoniae and its serogroups/serotypes in the vaccine era.

J Microbiol Methods. 2017 Aug 2;141:48-54. doi: 10.1016/j.mimet.2017.07.015. [Epub ahead of print]

Current challenges in the accurate identification of Streptococcus pneumoniae and its serogroups/serotypes in the vaccine era.

Varghese R1, Jayaraman R1, Veeraraghavan B2.

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Streptococcus pneumoniae is a major cause of pneumonia, meningitis and other invasive diseases resulting in high mortality and morbidity among children under the age of five. Inaccurate identification of S. pneumoniae masks the exact estimation of disease burden and could delay treatment options. This is the common problem most frequently faced in developing countries due to several reasons that include poor infrastructure, insensitive operational procedures and lack of expertise. Inconsistent methods for phenotypic detection often delay the early identification and confirmation of S. pneumoniae. For serotyping S. pneumoniae, Quellung method is the gold standard which can be performed only on viable isolates, needs expertise and is expensive. Therefore, the data available on disease burden and serotype prevalence is not truly estimated in most of the developing countries, in turn, the use of available pneumococcal vaccines have been restricted. This current review deliberates an overview on advantages and limitations of routinely used phenotypic tests for S. pneumoniae identification. Also discussed in this review are the roles and current challenges faced by various molecular identification and serogroup/serotype identification methods of S. pneumoniae, including PCR, real time PCR, sequence analysis of different specific genes of S. pneumoniae, PCR combined with RFLP, MALDI-TOF, MLST, MLSA and WGS.

Copyright © 2017 Elsevier B.V. All rights reserved.


Accurate identification; Limitations; Molecular methods; S. pneumoniae; Serotyping

PMID: 28780272 DOI: 10.1016/j.mimet.2017.07.015

Brain-derived neurotrophic factor reduces inflammation and hippocampal apoptosis in experimental Streptococcus pneumoniae meningitis.

J Neuroinflammation. 2017 Aug 4;14(1):156. doi: 10.1186/s12974-017-0930-6.

Brain-derived neurotrophic factor reduces inflammation and hippocampal apoptosis in experimental Streptococcus pneumoniae meningitis.

Xu D1, Lian D1, Wu J1, Liu Y2, Zhu M3, Sun J3, He D1, Li L4.

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Streptococcus pneumoniae meningitis is a serious inflammatory disease of the central nervous system (CNS) and is associated with high morbidity and mortality rates. The inflammatory processes initiated by recognition of bacterial components contribute to apoptosis in the hippocampal dentate gyrus. Brain-derived neurotrophic factor (BDNF) has long been recommended for the treatment of CNS diseases due to its powerful neuro-survival properties, as well as its recently reported anti-inflammatory and anti-apoptotic effects in vitro and in vivo.


In this study, we investigated the effects of BDNF-related signaling on the inflammatory response and hippocampal apoptosis in experimental models of pneumococcal meningitis. Pretreatment with exogenous BDNF or the tropomyosin-receptor kinase B (TrkB) inhibitor k252a was performed to assess the activation or inhibition of the BDNF/TrkB-signaling axis prior to intracisternal infection with live S. pneumoniae. At 24 h post-infection, rats were assessed for clinical severity and sacrificed to harvest the brains. Paraffin-embedded brain sections underwent hematoxylin and eosin staining to evaluate pathological severity, and cytokine and chemokine levels in the hippocampus and cortex were evaluated by enzyme-linked immunosorbent assay. Additionally, apoptotic neurons were detected in the hippocampal dentate gyrus by terminal deoxynucleotidyl transferase dUTP-nick-end labeling, key molecules associated with the related signaling pathway were analyzed by real-time polymerase chain reaction and western blot, and the DNA-binding activity of nuclear factor kappa B (NF-κB) was measured by electrophoretic mobility shift assay.


Rats administered BDNF exhibited reduced clinical impairment, pathological severity, and hippocampal apoptosis. Furthermore, BDNF pretreatment suppressed the expression of inflammatory factors, including tumor necrosis factor α, interleukin (IL)-1β, and IL-6, and increased the expression of the anti-inflammatory factor IL-10. Moreover, BDNF pretreatment increased TrkB expression, activated downstream phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling, and inhibited the myeloid differentiation primary response gene 88 (MyD88)/NF-κB-signaling pathway.


These data suggested that BDNF administration exerted anti-inflammatory and anti-apoptotic effects on an experimental pneumococcal meningitis model via modulation of MyD88/NF-κB- and PI3K/AKT-signaling pathways. Our results indicated that treatment with exogenous BDNF might constitute a potential therapeutic strategy for the treatment of bacterial meningitis.


Brain-derived neurotrophic factor; Hippocampal apoptosis; Neuroinflammation; Streptococcus pneumoniae meningitis

PMID: 28778220 PMCID: PMC5545027 DOI: 10.1186/s12974-017-0930-6

Assessment of a novel bile solubility test and MALDI-TOF for the differentiation of Streptococcus pneumoniae from other mitis group streptococci.

Sci Rep. 2017 Aug 2;7(1):7167. doi: 10.1038/s41598-017-07772-x.

Assessment of a novel bile solubility test and MALDI-TOF for the differentiation of Streptococcus pneumoniae from other mitis group streptococci.

Slotved HC1, Facklam RR2, Fuursted K3.

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This study assesses a novel bile solubility test and MALDI-TOF for the differentiation of Streptococcus pneumoniae from other mitis group streptococci, including differentiation of S. pneumoniae from Streptococcus pseudopneumoniae. Eighty-four species verified mitis group isolates were subjected to our bile solubility test (which measures and calculates the differences of absorbance in the test tube containing 10% sodium deoxycholate versus a blank control tube, after incubation for 10 minutes at 36 °C using a spectrophotometer) and MALDI-TOF MS (both the standard result output and by visual spectra evaluation). Applying a calculated optimal cut-off absorbance-value of 2.1, differentiated S. pneumoniae from all but one other mitis group streptococci (one S. mitis isolate generated an OD-value above 2.1). MALDI-TOF score value identification identified correctly 46 S. pneumoniae and 4 S. pseudopneumoniae but misidentified 16 other mitis group strains. Visual spectra evaluation correctly identified all S. pneumoniae and S. pseudopneumoniae strains but misidentified 13 other mitis group strains. The bile solubility test based on spectrophotometric reading described in this study can differentiate S. pneumoniae from other Streptococcus species. Combining the bile solubility test and the MALDI-TOF spectra results provide a correct identification of all S. pneumoniae and S. pseudopneumoniae isolates.

PMID: 28769078 PMCID: PMC5540920 DOI: 10.1038/s41598-017-07772-x

Fluorescence Imaging of Streptococcus pneumoniae with the Helix pomatia agglutinin (HPA) As a Potential, Rapid Diagnostic Tool.

Front Microbiol. 2017 Jul 18;8:1333. doi: 10.3389/fmicb.2017.01333. eCollection 2017.

Fluorescence Imaging of Streptococcus pneumoniae with the Helix pomatia agglutinin (HPA) As a Potential, Rapid Diagnostic Tool.

Domenech M1,2, García E1,2.

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Streptococcus pneumoniae is a common human pathogen and a major causal agent of life-threatening infections that can either be respiratory or non-respiratory. It is well known that the Helix pomatia (edible snail) agglutinin (HPA) lectin shows specificity for terminal αGalNAc residues present, among other locations, in the Forssman pentasaccharide (αGalNAc1→3βGalNAc1→3αGal1→4βGal1→4βGlc). Based on experiments involving choline-independent mutants and different growth conditions, we propose here that HPA recognizes the αGalNAc terminal residues of the cell wall teichoic and lipoteichoic acids of S. pneumoniae. In addition, experimental evidence showing that pneumococci can be specifically labeled with HPA when growing as planktonic cultures as well as in mixed biofilms of S. pneumoniae and Haemophilus influenzae has been obtained. It should be underlined that pneumococci were HPA-labeled despite of the presence of a capsule. Although some non-pneumococcal species also bind the agglutinin, HPA-binding combined with fluorescence microscopy constitutes a suitable tool for identifying S. pneumoniae and, if used in conjunction with Gram staining and/or other suitable technique like antigen detection, it may potentially facilitate a fast and accurate diagnosis of pneumococcal infections.


Forssman antigen; Streptococcus pneumoniae; binding lectins; fluorescence microscopy; teichoic acids

PMID: 28769901 PMCID: PMC5513899 DOI: 10.3389/fmicb.2017.01333