Validation of Fourier Transform Infrared Spectroscopy for Serotyping of Streptococcus pneumoniae.

Fourier transform infrared (FT-IR) spectroscopy (IR Biotyper; Bruker) allows highly discriminatory fingerprinting of closely related bacterial strains. In this study, FT-IR spectroscopy-based capsular typing of Streptococcus pneumoniae was validated as a rapid, cost-effective, and medium-throughput alternative to the classical phenotypic techniques. A training set of 233 strains was defined, comprising 34 different serotypes and including all 24 vaccine types (VTs) and 10 non-vaccine types (NVTs). The acquired spectra were used to (i) create a dendrogram where strains clustered together according to their serotypes and (ii) train an artificial neural network (ANN) model to predict unknown pneumococcal serotypes. During validation using 153 additional strains, we reached 98.0% accuracy for determining serotypes represented in the training set. Next, the performance of the IR Biotyper was assessed using 124 strains representing 59 non-training set serotypes. In this setting, 42 of 59 serotypes (71.1%) could be accurately categorized as being non-training set serotypes. Furthermore, it was observed that comparability of spectra was affected by the source of the Columbia medium used to grow the pneumococci and that this complicated the robustness and standardization potential of FT-IR spectroscopy. A rigorous laboratory workflow in combination with specific ANN models that account for environmental noise parameters can be applied to overcome this issue in the near future. The IR Biotyper has the potential to be used as a fast, cost-effective, and accurate phenotypic serotyping tool for S. pneumoniae.

Examining the Distribution and Impact of Single-Nucleotide Polymorphisms in the Capsular Locus of Streptococcus pneumoniae Serotype 19A.

Streptococcus pneumoniae serotype 19A prevalence has increased after the implementation of the PCV7 and PCV10 vaccines. In this study, we have provided, with high accuracy, the genetic diversity of the 19A serotype in a cohort of Dutch invasive pneumococcal disease patients and asymptomatic carriers obtained in the period from 2004 to 2016. The whole genomes of the 338 pneumococcal isolates in this cohort were sequenced and their capsule (cps) loci compared to examine their diversity and determine the impact on the production of capsular polysaccharide (CPS) sugar precursors and CPS shedding. We discovered 79 types with a unique cps locus sequence. Most variation was observed in the rmlB and rmlD genes of the TDP-Rha synthesis pathway and in the wzg gene, which is of unknown function. Interestingly, gene variation in the cps locus was conserved in multiple alleles. Using RmlB and RmlD protein models, we predict that enzymatic function is not affected by the single-nucleotide polymorphisms as identified. To determine if RmlB and RmlD function was affected, we analyzed nucleotide sugar levels using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). CPS precursors differed between 19A cps locus subtypes, including TDP-Rha, but no clear correlation was observed. Also, significant differences in multiple nucleotide sugar levels were observed between phylogenetically branched groups. Because of indications of a role for Wzg in capsule shedding, we analyzed if this was affected. No clear indication of a direct role in shedding was found. We thus describe genotypic variety in rmlB, rmlD, and wzg in serotype 19A in the Netherlands, for which we have not discovered an associated phenotype.

Pathogenicity of anti-ganglioside antibodies in the Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a postinfectious inflammatory polyradiculo-neuropathy characterized by flaccid paralysis. Antibodies directed against glycolipid structures (gangliosides), which are highly expressed in the peripheral nervous system, are frequently detected in sera from GBS patients. These antibodies interfere with nerve conduction and have been shown to activate phagocytes via IgG receptors (FcgammaR). These findings support an important role of glycolipid-specific antibodies in the pathogenesis of GBS.

FcgammaR polymorphisms: Implications for function, disease susceptibility and immunotherapy.

Leukocyte Fcgamma receptors (FcgammaR) confer potent cellular effector functions to the specificity of IgG. FcgammaR-induced leukocyte functions, including antibody-dependent cellular cytotoxicity, phagocytosis, superoxide generation, degranulation, cytokine production and regulation of antibody production, are essential for host defense and immune regulation. The efficacy of IgG-induced FcgammaR function displays inter-individual heterogeneity due to genetic polymorphisms of three FcgammaR subclasses, FcgammaRIIa (CD32a), FcgammaRIIIa (CD16a), and FcgammaRIIIb (CD16b). FcgammaR polymorphisms have been associated with infectious and autoimmune disease, or with disease severity. FcgammaR polymorphisms may furthermore serve as markers for therapeutic efficacy and side-effects of treatment with monoclonal antibodies. In this review, FcgammaR function and the relevance of FcgammaR polymorphisms as prognostic markers for inflammatory disease and antibody-based immunotherapy are discussed.

Effective phagocytosis and killing of Candida albicans via targeting FcgammaRI (CD64) or FcalphaRI (CD89) on neutrophils.

Invasive fungal infections are an increasing problem for immunocompromised patients. As an approach to improve targeting of polymorphonuclear leukocytes (PMNL) toward Candida albicans, the effect of bispecific antibodies (BsAbs) directed against C. albicans and either FcalphaRI or FcgammaRI was evaluated. Control PMNL and in vivo granulocyte colony-stimulating factor (G-CSF)-primed PMNL served as effector cells. A new radiometric killing assay for measuring candidacidal activity was developed to facilitate quantification of PMNL-mediated killing of C. albicans. BsAbs directed to either FcgammaRI (CD64) or FcalphaRI (CD89) on human PMNL effectively enhanced both phagocytosis and killing of C. albicans in vitro. Fungicidal activity triggered via FcgammaRI required in vivo priming with G-CSF, whereas FcalphaRI-mediated activity was not dependent on this growth factor. Furthermore, PMNL from human FcgammaRI-transgenic mice effectively phagocytosed and eliminated C. albicans in the presence of BsAbs. These results document the capacity of FcR-directed BsAbs and G-CSF to trigger antifungal immune responses.