Background: Group B streptococcus (GBS) is a leading cause of invasive bacterial disease in neonates. The possibility of maternal immunization with GBS-vaccines is being explored. Vaccine candidates include serotype-specific polysaccharide-protein conjugates and GBS surface proteins, including pilus island proteins. In this project, we aimed to undertake capsular serotype identification, pilus island identification and genotypic characterization of GBS isolates associated with colonization in mothernewborn dyads and invasive disease in infants. Methods: Colonizing GBS isolates were identified by vaginal swabbing of mothers (n=541) during active labor and from skin of their newborns post-delivery (n=395). Invasive GBS isolates from infants (n=284) were identified through laboratory-based surveillance. GBS serotyping was done by latex agglutination. Serologically nontypeable isolates were typed by a serotype-specific PCR method. The pilus islands from 541 colonizing isolates and 284 invasive isolates were characterized by real-time PCR targeting the ancillary protein 1 and 2. We undertook sequence typing based on the three most heterogeneous genes (adhP, atr and glnA) of multilocus sequence typing (MLST) on GBS isolates identified in young-infants with invasive disease (n=283) and those associated with maternal (n=525) and newborn colonization at birth (n=369). A total of 121 colonizing and 131 invasive disease GBS isolates that were representative of 55 and 35 clusters respectively were analyzed by the remaining four MLST genes. The gbs2018 locus was characterized by DNA sequencing.

The upper respiratory tract (URT) of humans is complex and interconnected region and comprises several major ecosystems including the oral cavity, oropharynx, nasal cavity, sinuses, nasopharynx and middle ear. Most of the anatomical locations within the URT are colonised with a normal bacterial microbiota, within which are often organisms having the potential to cause disease. The diseases of the URT are both varied and frequent in their occurrence, and conditions such as otitis media, rhinosinusitis and pharyngitis are sources of morbidity and mortality in adults and children in both developing and developed countries. The study of diseases of the URT has traditionally been based on application of culture-based methods in which the infection-implicated organisms are first grown in vitro and then studied further. Ongoing advances in DNA-based techniques have led to the development of new molecular tools for the study of infectious diseases. One such technique is PCR-denaturing gradient gel electrophoresis (PCR-DGGE). This is a PCR-based tool that allows the investigation of microbial communities independent of culture. Although this technique has been applied extensively in the study of the gastrointestinal tract, the vagina and endodontic infections in humans, there have been few reports of its application to URT infections. PCR-DGGE was applied in the present study to investigate (a) the bacteria present in the middle ear of children suffering from otitis media with effusion (OME), (b) the microbiota associated with the sinuses in patients with chronic rhinosinusitis (CRS) and (c) perioperative changes in the bacterial population of the middle meatus of patients undergoing nasal or sinus surgery. The analysis of the middle ear fluid samples indicated an increased role in OME for the newly-discovered pathogen Alloiococcus otitidis and also the possible involvement of certain coryneform bacteria and coagulase-negative staphylococci in the aetiology of this condition. PCR-DGGE analysis of patients with CRS revealed a polymicrobial disease with considerable variability in the predominant species detected when multiple, serial samples were evaluated. The perioperative audit showed that when good clinical practice is adhered to, there was no apparent introduction of potentially-harmful organisms into the middle meatus. Streptococcus salivarius is a common, commensal inhabitant of the oral cavity of humans and has also been shown to inhabit the nasopharynx of infants. S. salivarius is also a well known producer of bacteriocins with activity directed against Streptococcus pyogenes. One such strain, S. salivarius K12, is now marketed in New Zealand as the probiotic, K12 Throat Guard[TM]. In the present study, S. salivarius K12 was compared with two additional strongly-inhibitory S. salivarius (strains T18A and T30A) for activity against the common causative pathogens of otitis media. A paediatric formulation of strain K12 was also tested in a pilot clinical trial for its ability to colonise the URT of young children. Although the levels of colonisation of these subjects was not as high as typically obtained with use of the K12 Throat Guard[TM] formulation, it was considered that further development of the paediatric formulation is warranted, particularly with respect to use of a different pre-treatment regimen. In other studies, the molecular basis for the unusual in vitro inhibitory activity of S. salivarius strain T30A was investigated. Although this still remains unresolved, other observations made during the course of this study have led to the introduction of a schema for the division of inhibitory S. salivarius into three groups based on (a) their sensitivity to the lantibiotic salivaricin A and (b) the structure of their salivaricin A genetic locus. This grouping is analogous to the "rock-paper-scissors" system previously described for colicin-producing strains of E. coli. Streptococcus pneumoniae is a major human pathogen responsible for a variety of diseases in humans. There have been very few reports of bacteriocin production by S. pneumoniae when compared to other streptococcal species. In the present study a putative cluster of bacteriocins encoded by the blp locus has been investigated. The distribution of the individual blp determinants within this locus was evaluated in a collection of S. pneumoniae strains using PCR. The blp genes were detected in 92% of 57 tested strains and a variant form (termed the B-form) of the cluster was identified that appeared to have arisen due to a genetic recombination event. In this case an approximately 250 bp portion of the blpMNO cluster appears to have recombined into blpK of the blpIJK cluster. Attempts were made to express the putative bacteriocin peptide genes in an Escherichia coli expression system. Failure to achieve expression was taken to indicate that these bacteriocin-like peptides may be toxic for the host producer cells under these test conditions. Future attempts to achieve expression of the Blp peptides, could explore the use of different fusion proteins, a Gram-positive expression host or a cell-free protein expression system.

Thesis (Ph.D.)--University of Wollongong, 2006. / Typescript. Includes bibliographical references: leaf 148-160.

Thesis (Ph. D.)--University of Sydney, 2004. / Title from title screen (viewed 8 May 2008). A reference list of published articles is provided in Appendices. Includes copies of seven published papers, co-authored by Fanrong Kong. Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Dept. of Medicine. Includes bibliographical references. Also available in print form.

Thesis (doctoral)--Göteborgs Universitet, 1982. / Extra t.p. with thesis statement inserted. Includes the author's published papers. Includes bibliographical references.

Thesis (Ph. D. in Mathematics)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

Streptococcus pyogenes infections can give rise to a diverse array of long-term secondary sequelae, including those in the brain characterized by both motor and neuropsychiatric disorders: Sydenham’s chorea and Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus infections (PANDAS). These conditions are thought to be mediated by neuroinflammatory responses and autoantibody entry into the brain, but the mechanisms are not well understood.Previous work by our laboratory has demonstrated that recurrent intranasal S. pyogenes (Group A Streptococcus, or GAS) inoculations in mice cause infiltration of CD4 T cells into the anterior brain, disruption of the blood-brain barrier (BBB), increased numbers of activated myeloid cells and degradation of excitatory synapses leading to neural circuitry deficits. However, the molecular mechanisms underlying these phenotypes have not been fully explored. To understand how the neurovasculature and myeloid cells respond to recurrent GAS infections at the transcriptome level, I profiled cells from mouse olfactory bulb (OB) and nasal lymphoid tissue by single-cell RNA sequencing (scRNAseq). I found marked shifts in both endothelial cell and microglia populations at the transcriptome level after GAS infections, including downregulation of BBB-associated transcripts by endothelial cells (ECs), and increased production of inflammatory cytokines and chemokines, type I interferon response, and antigen presentation genes by microglia (Chapter 3). I validated several differentially expressed genes using flow cytometry, immunosorbant assays, RNA fluorescence in situ hybridization (FISH), and multiplexed error-robust FISH (MERFISH). Single-cell spatial transcriptomics of the OB revealed regional heterogeneity among microglial responses to GAS, possibly driven by proximity to infiltrating T cells. Analysis of transgenic CX3CR1/TMEM119 dual myeloid reporter mice confirmed that perivascular and meningeal macrophage numbers increase in response to GAS, but, unlike in other neuroinflammatory diseases, few macrophages infiltrate the brain parenchyma. Our laboratory has previously shown that Th17 cells are critical for BBB damage and activated microglia in response to repeated intranasal GAS infections, but the contribution of T helper (Th) 17 cell-derived cytokines in this process, as well as the transcriptional effects of Th17 cells on endothelial cells and microglia are unknown. To expand on these findings, I performed scRNAseq on retinoic acid-related orphan receptor γt (RORγt) mutant mice (Chapter 4) which showed a significant rescue in BBB-associated genes (e.g. Mfsd2a, Itm2a and Itih5) in endothelial cells. Chemokine production and type I interferon gene expression by microglia was also significantly rescued in RORγt mutants; surprisingly antigen presentation by microglia in response to GAS was exacerbated, at both the gene and protein level. Interleukin (IL)-17A is a major cytokine produced by Th17 cells. To examine the role of IL-17A in disease pathogenesis, I treated wild-type mice with an IL-17A neutralizing antibody during the course of GAS infections (Chapter 4). This treatment was sufficient to recapitulate the transcriptional effects on microglia and endothelial cells, as well as rescue BBB permeability previously found in RORγt mutants, indicating that IL-17A may play a critical role in transcriptional responses of endothelial cells and microglia to recurrent GAS infections in vivo. However, IL-17A did not disrupt tight junctions or induce transcytosis on ECs in vitro, suggesting that its effects on ECs in vivo are indirect. Th17 cells are capable of considerable phenotypic plasticity in response to chronic inflammation. To understand this process during recurrent GAS infections, I performed a time course analysis of CD4 T cell subsets after two, three, four and five infections (Chapter 5). This analysis revealed that proportions of “pathogenic” interferon γ-expressing Th17 cells increased over time, as did the number of CD4 T cells expressing granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with pleiotropic effects on autoimmunity. Moreover, I determined that RORγt mutants have decreased proportions of GM-CSF+ CD4 T cells in their nasal mucosa, raising the question of whether GM-CSF may also contribute to CNS pathology (BBB permeability or microglial activation) in addition to IL-17A. To address this question, I generated mice deficient for GM-CSF in T cells and found that conditional deletion of GM-CSF in CD4+ cells partly rescued type I interferon and antigen presentation responses in microglia by scRNAseq, but did not rescue BBB leakage, suggesting that GM-CSF and IL-17A have distinct roles in the neurovascular and neuroinflammatory responses to GAS. To relate the findings in mice to the human disease, in Chapter 6 we performed cytokine profiling in sera from PANDAS/PANS patients at the acute phase of the disease using a multiplex bead-based immunoassay. We found that many chemokines and cytokines produced by activated microglia or macrophages in the mouse model were also highly elevated in the sera of PANDAS/PANS patients. These findings suggest an important link to the human disorder both to understand disease mechanisms in humans and to use them as future clinical biomarkers for diagnosis and treatment monitoring.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Pneumonia is considered to be one of the leading causes of death worldwide. The outcome depends on both, proper antibiotic treatment and the effectivity of the immune response of the host. However, due to the complexity of the immunologic cascade initiated during infection, the latter cannot be predicted easily. We construct a biomathematical model of the murine immune response during infection with pneumococcus aiming at predicting the outcome of antibiotic treatment. The model consists of a number of non-linear ordinary differential equations describing dynamics of pneumococcal population, the inflammatory cytokine IL-6, neutrophils and macrophages fighting the infection and destruction of alveolar tissue due to pneumococcus. Equations were derived by translating known biological mechanisms and assuming certain response kinetics. Antibiotic therapy is modelled by a transient depletion of bacteria. Unknown model parameters were determined by fitting the predictions of the model to data sets derived from mice experiments of pneumococcal lung infection with and without antibiotic treatment. Time series of pneumococcal population, debris, neutrophils, activated epithelial cells, macrophages, monocytes and IL-6 serum concentrations were available for this purpose. The antibiotics Ampicillin and Moxifloxacin were considered. Parameter fittings resulted in a good agreement of model and data for all experimental scenarios. Identifiability of parameters is also estimated. The model can be used to predict the performance of alternative schedules of antibiotic treatment. We conclude that we established a biomathematical model of pneumococcal lung infection in mice allowing predictions regarding the outcome of different schedules of antibiotic treatment. We aim at translating the model to the human situation in the near future.

A collection of 29 clinical streptococcal isolates obtained from the University of KwaZulu-Natal, Medical School, Durban Metro area (South Africa) were studied to establish their penicillin G susceptibility patterns often refered to as minimal inhibitory concentration (MIC) and to determine the genetic diversity among them using two genotyping methods, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) analysis. All isolates with MIC less than or equal to 0.12 µg/ml were considered susceptible, intermediate resistant if MIC was between 0.25 µg/ml and 4 µg/ml and resistant if greater than 4 µg/ml, The percentage of isolates with resistance was relatively high (75.9%), only 10.3% of isolates showed intermediate resistance and 13.8% of the isolates were completely susceptible to penicillin G. Some of the resistant isolates were highly resistant reaching penicillin G MIC levels of 5000 µ/ml. They were speculated to contain Path altered penicillin binding proteins and high level of crosslinking cell wall induced by the gene products of the MurMN operon. RAPD analysis was performed using three primers, MBPZ-1, MBPZ-2, and MBPZ-3, respectively. RAPD analysis allowed for the identification of 27 RAPD types with MBPZ-1 and MBPZ-3 and 26 RAPD types with MBPZ-2. Ninety-eight percent of these isolates were clustered into two groups, group I and group II, with 90% to 100% dissimilarity among them. Fifty two percent of the isolates of MBPZ-1 group I were in MBPZ-2 group I, 72% isolates of MBPZ-1 group I were in MBPZ-3 group I, and 72% of the isolates of MBPZ-2 group I were in MBPZ-3 group 1. This shows the discriminatory ability of the primers used in this study. Despite clustering of isolates, relatively high diversity was seen. PFGE analysis of macrorestriction fragments obtained after digestion of chromosomal DNA by restriction enzyme, SmaI showed 24 PFGE patterns. The 24 PFGE patterns were divided into three groups (I, II and III) of isolates, with an average of 85% dissimilarity (15% homology) among them. At 25% homology, four clusters, A (13 isolates), B (9 isolates), C (4 isolates), and D (4 isolates) were observed. Two pairs of isolates in group I, cluster A, showed 100% homology. This suggested that each represent the same strain. Four isolates of group I, cluster B, also exhibited 100% homology. This study showed that most of streptococcal isolates with the same penicillin G susceptibility patterns grouped together in a phylogenetic tree by both RAPD and PFGE analysis. There was also some similarity between the results obtained by RAPD analysis and PFGE analysis. Seventeen and nine of the 29 isolates grouped into group I and group II, respectively, two pairs of isolates were indistinguishable, and two pairs of islates were closely related by both RAPD (using MBPZ-3) and PFGE analysis. Although, RAPD analysis is sensitive, specific, faster and cost effective, the ease with which PFGE analysis can be performed, high discriminatory power, reproducibility of the results, and the polymorphism seen in the patterns, suggests that PFGE method has the potential to be very useful for epidemiological evaluations of nosocomial streptococcal infections in KwaZulu-Natal. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.