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Molecular characterization of Cryptosporidium and Giardia in children with diarrhoea in slum areas of Nairobi, KenyaMbae, Cacilia Kathure January 2013 (has links)
Intestinal parasitic infections are endemic worldwide and have been described as constituting the greatest single worldwide cause of illness. The distribution of and factors associated with intestinal parasitic infections are poorly defined in high risk vulnerable populations such as those living in urban informal settlements. Species of Cryptosporidium and Giardia are the most common non-bacterial causes of diarrhoea in children and the HIV infected individuals, yet data on their role remains scanty or non existent in Kenya. This study investigated the occurance of Cryptosporidium and Giardia species, genotypes and subtypes in children, both hospitalized and living in an informal settlement in Nairobi. Methods: This was a prospective cross- sectional study in which faecal samples were collected from HIV infected and uninfected children aged five years and below presenting with diarrhoea at selected outpatient clinics in Mukuru informal settlements, or admitted to paediatric wards to the Mbagathi District Hospital. A total of 2112 samples were collected between January 2010 and December 2011, analysed through formal ether concentration and Modified Ziehl Neelsen (MZN) staining, and screened for presence of intestinal parasites by microscopy. Those found positive for Cryptosporidium were further analysed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (pCR-RFLP) of the 18srRNA gene for species identification and PCR-sequencing of the 60 kDa glycoprotein (GP60) gene, while those Giardia positive were analysed through PCR-RFLP ii ------- of the Glutamate dehydrogenase (GDH) gene, assemblage specific primers targeting the Triose phosphate isomerase (Tpi) and PCR-sequencing of P giardin gene for identification of assemblages and sub-assemblages. Results: In total 54112112 (25.6%) children were positive for at least one intestinal parasite, with the common parasites being: Entamoeba his-to[ytica, 225 (36.7%), Cryptosporidium spp. 187, (30.5%) and Giardia intestinalis, 98 (16%).The prevalence of intestinal parasites infection was higher among children from outpatient clinics 43211577(27.4%) than among inpatients; 109/535 (20.1%) p<O.OOl. Infection with Cryptosporidium spp. was higher among inpatients than outpatients (15.3% vs. 6.7% respectively p<O.OOI). HIV-infected participants were more likely to be infected with any parasite than HIV- uninfected participants, (Adjusted Odds Ratio (AOR), 2.04, 95% Cl, 1.55-2.67, p<O.OOI), and with Cryptosporidium spp. (AOR, 2.96, 95% Cl 2.07-4.21, p<O.OOl). Cryptosporidiosis was more common among inpatients than outpatients (AOR, 1.91, 95% Cl, 1.33-2.73, p<O.OOI). C. hominis was the most common species of Cryptosporidium identified in 1251151(82.8%) the study subjects. Other species identified were C. parvum 18/151(11.9%), C. felis and C. meleagridis were identified in 4 and 2 isolates respectively. Cryptosporidium infections were found to be significantly associated with HIV infections and inpatients. Wide genetic variation was observed within C. ~ominis, with identification of 5 subtype families; la, lb, Id, le and If and several subtypes. Only subtype family IIc was identified within C. parvum. There was no association between species and mv status, or patient type. Of the 72 Giardia samples genotyped, 64 (88.9%) contained assemblage B and only one (1.4%) had assemblage A, while mixed infections with A an? B were observed in 7 (9.7%) of the isolates. Assemblage A isolate was identified as sub-assemblage AIl. Within iii r , '. assemblage B, sub- assemblage Bm was present in 6 isolates, BIV in 14 isolates and mixed infections with Bm and BIV was observed in 28 patients. Conclusion: The data suggests that C. hominis is the most common species associated with diarrhoea in the study population. There was high genetic variability in the C. hominis isolates with 22 different subtypes identified, with some of them b~ing reported for the first time, whereas it was low within C. parvum with only one subtype family ne identified, thus, suggesting predominance of anthroponotic transmission. For Giardia spp. assemblage B was more common than assemblage A, and occurrence of mixed infections between assemblages and sub-assemblages was noted. The data provides infonnation for the first time in Kenya about the genotypes of Giardia spp. circulating in the study population. The results enable better understanding of prevalence and distribution of various genotypes and sub types of Cryptosporidium and Giardia spp. in Kenya. This information will be important in the design of further studies of these pathogens in Kenya and other low-income countries, and develop interventions for control and prevention of these infections.
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Characterisation of the role and regulation of excreted proteins produced by Enterococcus facecalisWalker, Rachel Georgina January 2008 (has links)
The commensal bacterium Enterococcus faecalis has emerged as an opportunistic pathogen capable of causing a range of infections. Little is known about the role and regulation of excreted proteins in E. faecalis virulence. Cytolysin is regulated via a specific density regulation mechanism and expression of GelE and SprE is dependent on the quorum sensing regulatory system FsrABCD. Fsr is predicted to be a global regulator of protein synthesis but it is not known whether there are multiple hierarchical regulators that modulate Fsr expression, as seen in the homologous Agr system in Staphylococcus aureus. This study sought lo investigate the variability and regulation of excreted proteins in the lab strains OGIRF and JH2-2 and in a set of diverse clinical isolates.
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Development of new photosensitising drugs for antibacterial therapyO'Grady, Cassandra Clare January 2003 (has links)
Antimicrobial photodynamic therapy (APDT) is a novel treatment method for the destruction of microorganisms. APDT uses the combined effect of a photosensitising drug, light and molecular oxygen to bring about a toxic effect. The work presented in this study investigates the photodynamic antibacterial efficacy of a series of symmetrical cationic phenothiazinium salts. The photosensitisers investigated were lipophilic derivatives of methylene blue, where the methyl groups of methylene blue are replaced with longer alkyl chains from ethyl to n-hexyl.
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The role of LILR in micobacterial infectionHogan, Louise January 2013 (has links)
Lcukocyte Ig-like receptors (LILR) are a family of innate immune receptors whose members have been shown 10 recognise both self and bacterial ligands. They can exert powerful immunomodulatory effects on monocytic cells and by doing so influence the adaptive immune response. Monocytic cells arc a target for mycobacterial infection, and LILR expression patterns correspond to leprosy disease phenotype during M.leprae infection. This thesis was based on the hypothesis that LJLR activity may also be relevant in tuberculosis. The main aim was to investigate LlLR with respect to infection with Mycobacterium bovis and to compare this with Mycobacterium tuberculosis infection. By measuring the transcription of LILR using qPCR, receptor expression was shown to be altered following culture of monocytic cells with mycobacteria. Culture with M bovis demonstrated the broadest change to expression patterns, with four L1LR down-regulated (LlLRA2, LILRA3, LlLRA6, LILRB 1), and one receptor, L1LRBS, up-regulated. In the absence of any previous published studies regarding the expression, ligand or potential functions of LlLRB5, further analyses were performed for this receptor L1LRBS transfectants exhibited signalling in the presence of BCG, indicating a relevance to mycobacterial recognition. Flow cytometric analysis demonstrated LILRBS expression on low numbers of mDC but on high numbers of T cells. Furthermore, crosslinking LI LRB5 revealed the down-regulation of CD80 on antigen presenting cells, which may impact on T cell activation. Phenotypic alterations were also detected on T cells following LILRBS crosslinking. Finally, cattle, as the natural hosts of M. bovis, were hypothesised to encode and express LILR. Here the identification and classification of 16 novel bovine L1LR was described. Cattle appear to have retained both LILR and Paired Ig-Like Receptors (pm.), which are thought to have evolved from a common ancestral gene, and whilst the signalling domains are more restricted than their human counterparts, cattle possess six genomically encoded soluble receptors.
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Glycosylation of Staphylococcus aureus surface proteinsMcAulay, Kathrine January 2011 (has links)
Staphylococcus aureus is a major human pathogen, causing a wide spectrum of superficial and systemic infections. With the advent of methicillin resistant Staphylococcus aureus (M RSA) and the increasing spread of antibiotic resistance it has become clear that novel approaches must be taken to control S. aureus. Glycosylation, the modification of proteins with carbohydrate moieties, was long considered a process confined to eukaryotes; however, the last 3 decades have seen increasing awareness and characterisation of glycosylation systems in bacteria, including many pathogens In this study S. aureus strains were found to express several glycoproteins, covalently anchored to the cell wall peptidoglycan, including Cif A, Pis and putatively SdrC, SdrD and Clfb. CIf A glycosylation was readily detected and the glycosyltransferase enzymes responsible for Cif A modification (GtfA and GtID) were found to be encoded in a separate chromosomal location from cl(A. MALDI-MS/MS and GC-MS experiments revealed the presence of hexose and terminal HexNAc residues, consistent with an observed affinity for concanavalin A (ConA) and wheatgerm agglutinin (WGA) lectins. ConA affinity was then utilised to localise CIf A on the surface of S. aureus cells using a lectin-fluorophore conjugate, revealing a punctate presence of glycosylated protein around the cell wall. Glycosylation of CIf A was found to have no effect on fibrinogen (Fg) binding, however a role has been revealed in infection in a murine model of septic arthritis. A strain deficient in c/fA was highly attenuated in this model, whereas a strain deficient in both c/fA and gtfAB resulted in infection similar to the wild type. This data suggests a role for glycosylation in anti-virulence and thus has provided a novel insight into the nature of the host-pathogen interaction. Also, glycosylated CIf A was found to react with human sera at a significantly higher level than recombinant protein. The results highlight potential new avenues concerning immunological approaches to the control of S. aureus.
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The microbial ecology of necrotising enterocolitisHoy, Christine Muriel January 2000 (has links)
Abnormal gastrointestinal colonisation has been implicated in the development of necrotising enterocolitis (NEC). Organisms capable of rapid fermentation of excess carbohydrate in the small bowel were postulated to produce adverse intraluminal conditions leading to intestinal injury. Quantitative culture of duodenal aspirates from 122 very low birth weight newborns revealed a high prevalence of Gram-negative colonisation, with counts up to 10<sup>8</sup> cfu/g. Gram-negative colonisation occurred in infants who had been fed, increased with postnatal age and was associated with a longer stay on the neonatal unit. Molecular typing of <i>E. coli, Klebsiella </i>spp. and <i>Enterobacter</i> spp. demonstrated marked temporal clustering of indistinguishable strains. Colonisation with particular strains of <i>Enterobacteriaceae</i> was not associated with subsequent development of NEC. Faecal flora, from NEC cases and control infants, comprised predominantly aerobic Gram-negative organisms and enterococci, with anaerobes isolated infrequently. Prior to clinical onset of confirmed NEC, <i>Enterobacteriaceae</i> isolates remained at high levels or increased, while there was a significant fall in enterococci. This fall in enterococci was not apparent in infants with suspected NEC. Asymptomatic infants were colonised with <i>Enterobacteriaceae</i> isolates indistinguishable from NEC cases, but enterococci were also present in high numbers, often being the predominant strain. One <i>E. coli</i> from a post-mortem peritoneal sample, was fully induced for <span style='font-family: Symbol'>b-galactosidase expression. Isolates identical by PFGE, from the same infant and controls were not induced. Other strains, from NEC cases and control infants, showed no difference in <span style='font-family:Symbol'>b-galactosidase activity. NEC appears not to be associated with colonisation with particular strains of <i>Enterobacteriaceae</i>, but the decline in enterococci preceding NEC should be investigated.
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Within-host evolution of bacteriaPark, Jennifer M. January 2004 (has links)
The Moxon and Murphy (1978) model proposes that bacterial infection is initiated by invasion of a small group of closely related organisms at a single point. The organisms expand to produce a homogeneous population and over time diversity is generated. Subsequent selective pressures reduce the population to organisms with characteristics that assist survival in that particular niche. Diversity is produced by numerous mechanisms, and in an infection the ability to generate variation may be the most crucial characteristic of all. To test the original infection model, which was proposed from animal work, analysis of the diversity of bacterial isolates from three different patients was carried out. In this PhD the diversity of antibiotic resistance mechanisms in <i>Pseudomonas aeruginosa </i>serial isolates from a single patient was investigated. Also, the diversity of <i>Escherichia coli </i>populations from another patient was explored. Finally, the mutation frequency in these <i>E. coli</i> populations and also in a set of serial antibiotic resistant <i>E. coli</i> isolates (from a previous study; Low <i>et al.,</i> 2001) was examined. The data from the different isolate sets provided some evidence to support the Moxon and Murphy infection model. The <i>P. aeruginosa</i> data showed diversity in antibiotic susceptibility and <span style='font-family: Symbol'>b-lactamases. The results also indicated that the strains had not evolved sequentially suggesting the occurrence of separate infection pockets within the model. The <i>E. coli</i> population isolates showed strain and sub-strain variation, giving strong confirmation that diversification occurs within the body. Finally, the antibiotic resistant <i>E. coli</i> isolates showed increasing mutation frequency, and may provide an example of the generation of a mutator phenotype within the host under strong selective pressure.
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The nematode Caenorhabditis elegans as an alternative model for bacterial infectionKhechara, Martin Peter January 2004 (has links)
No description available.
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Involvement of toll like receptor 4 and serum proteins in the innate recognition of gram-negative bacteriaMouratis, Marios Angelos January 2008 (has links)
No description available.
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Interplay between quorum sensing and metabolism in Pseudomonas aeruginosaRuparell, Avika January 2012 (has links)
The important human pathogen Pseudomonas aeruginosa causes a broad-spectrum of diseases including life threatening infections. A cell density-dependent regulatory network termed quorum sensing (QS) is pivotal in the control of P. aeruginosa pathogenicity, and the signal molecules employed are N-acyl-L-homoserine lactones (AHLs) and the Pseudomonas quinolone signal (PQS). Production of these QS signal molecules (QSSMs) requires precursors including fatty acids, S-adenosyl-L-methionine (SAM) and aromatic amino acids. SAM is derived from the activated methyl cycle (AMC) which is an important pathway dedicated to the degradation of the toxic metabolite S-adenosyl-L-homocysteine (SAH). Through removing the genes encoding the AHL synthases, RhlI and LasI from the complex hierarchical system of P. aeruginosa by expressing them in the heterologous host, Escherichia coli, this study has measured the influence of AHL production upon bacterial metabolism. AHL profiles were broader than previously reported, correlated with a reduction in the intracellular concentrations of several metabolites, and were more pronounced in the E. coli strain producing the LasI synthase than the RhlI enzyme. Production of foreign QSSM synthases had a knock-on effect on the native E. coli QSSM, autoinducer-2 (AI-2). We hypothesize that AI-2 production was significantly reduced since it also requires AMC metabolites for its synthesis. The influence that these metabolic perturbations had on cell fitness was manifest through reduced growth in minimal media. Complementation of growth by exogenously added metabolites confirmed our hypothesis that QSSM synthesis creates a drain on metabolite levels with consequences for cell fitness. Site-directed mutagenesis of key catalytic residues in the QSSM synthases was performed to directly prove that the effects observed were due to the function of the synthases, and not the production of a heterologous protein. Moreover, complete profiling of P. aeruginosa PA01 AHL synthase mutants is unravelling the interrelationship between metabolism and cell-to-cell communication in P. aeruginosa.
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