Streptococcus pneumoniae is a commensal inhabitant of the human nasopharynx that can sometimes become pathogenic and cause diseases such as pneumonia, otitis media and meningitis. Carbohydrate metabolism is a critical component of S. pneumoniae virulence. Among the myriad of carbohydrate-specific pathways involved in the host-pneumococcus interaction, the N-glycan foraging pathway stands out because of its direct implication in numerous aspects of virulence such as fitness, adhesion/invasion and impairment of the host immune response. Much of the literature has been focussed on the importance of step-wise depolymerisation of N-glycans by the enzymes NanA, BgaA and StrH. However, the importance of the liberation of N-glycans from host glycoconjuguates and their intake by the bacterium has yet to be examined. We have identified a Carbohydrate Processing Locus (CPL) that is highly conserved throughout a large number of Firmicutes and whose individual components appear widespread in bacteria that we hypothesize is active on host N-glycans. This locus encodes for two putative α-mannosidases GH92 and GH38, a characterised α-mannosidase GH125, a putative β-hexosaminidase GH20C, a putative α-fucosidase GH29 and a ROK (Repressor, Open reading frame, Kinase) protein. The genomic context of CPL orthologues suggests that an endo-β-N-acetylglucosaminidase (EndoD) and an ABC transporter (ABCN-glycan) are functionally associated with this locus. Based on our bioinformatic analyses and known functions of these proteins we hypothesize that the CPL encodes a concerted pathway responsible for the liberation, transport, and processing of N-glycans. The objective of this research is to characterize the putative components of this pathway and assess their implication in virulence. Specific focus on ABCN-glycan demonstrated its specificity for a range of N-glycans liberated by EndoD, shedding light on a novel import system for branched N-glycans. Furthermore, we provided evidence that GH92 is an α-1,2-mannosidase that likely removes the terminal mannose residues found on high-mannose N-glycans. EndoD and GH92 are shown to participate in virulence in mice; however, their role in virulence has yet to be determined. This work will significantly advance the construction and validation of a model of N-glycan processing by S. pneumoniae. As the components of this model pathway are conserved amongst a wide variety of bacteria, this work is of fundamental relevance to understanding how microbes from various environments degrade and metabolize N-glycans. / Graduate

Antibiotic resistance is a serious global public health problem. ESBLs are enzymes that destroy expanded-spectrum beta-lactam antibiotics rendering these drugs ineffective. Infection with ESBL-producing K.pneumoniae are hard to treat and result in longer hospital stay and higher mortality rates. The Clinical Laboratory Standard Institute (CLSI) have standard methods for detection of ESBL producing strains of bacteria in infected patients to guide antibiotic therapy, reduce the risk of mortality and risk of transmission. The presence of K.pneumoniae and E.coli which produce ESBLs have been confirmed in natural environments such as soil and water but no standard methods exist to identify directly and quantify these bacteria to understand the risk of human exposure in these settings. The purpose of this research is to assess the ability of an agar dilution method, using a differential agar Bio-Rad Rapid E.coli 2 agar utilized in environmental water quality studies, to identify correctly ESBL-producing K.pneumoniae. The minimum inhibitory concentration (MIC) of ceftriaxone antibiotic for wild-type ESBL producing K.pneumoniae isolates were compared on Mueller-Hinton broth (MHB) and Bio-Rad Rapid E.coli 2 agar. Using the MIC values, the isolates were classified as susceptible, intermediate or resistant. The MIC of wild-type strains of K.pneumoniae were above 4μg/mL for both methods on all susceptibility tests performed. The results of this research suggest that Bio-Rad Agar dilution method performed well, correctly identifying these strains as resistant to ceftriaxone, an indication of ESBL production. The Bio-Rad agar dilution method can be considered as a viable standard method for direct identification of ESBL-producing K.pneumoniae in natural environments.

The sialidases are a group of glycosyl hydrolases that specifically remove terminal sialic acid (Neu5Ac) residues from various glycans. In the two common human pathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, these enzymes have been shown to be key virulence factors directly involved in bacterial colonization and infection. However, little is known about their detailed structural and mechanistic features and lack of this information significantly slows down the progress of new drug discovery targeting these enzymes. Therefore, we embarked structural and kinetic studies towards the three distinct sialidases (designated as NanA, NanB and NanC) from S. pneumoniae, as well as the putative sialidase (designated as PaNA) from P. aeruginosa. Full-length NanA failed to crystallize due to the presence of some natively disordered regions. The catalytic domain of NanA (CNanA) was therefore subcloned, which was crystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer with close contacts between the two monomers. The second pneumococcal sialidase NanB only shares 24% sequence identity with NanA. Crystal structure of NanB was also determined to 1.7 Å, which exhibits a multi-domain monomeric architecture. In general, the core catalytic domain of both CNanA and NanB adopts the classic six- bladed β-propeller fold (or called sialidase fold), with a set of highly conserved residues stacking around the proposed active sites. NanC is a close homologue of NanB, sharing over 50% sequence identity. However, NanC crystallization is not successful so far. To compare the three sialidases in more detail, a computational NanC model was made based on the structure of NanB. Mapping of the active sites of CNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as the probe. Although sharing many common features, NanA, NanB and NanC present different topologies around the catalytic centre, give these enzymes a high level of diversity in enzymatic kinetics, substrate specificity and catalytic properties. NMR studies show that NanA acts as a classic hydrolytic sialidase; while NanB is found to be an intermolecular trans-sialidase like the leech sialidase; NanC, however, handles multiple catalytic roles efficiently, which include releasing Neu5Ac2en from α2,3- sialyllactose and hydration of Neu5Ac2en to Neu5Ac with high efficiency. S. pneumoniae thus expresses NanA, NanB and NanC for disparate but cooperative roles. Such a working pattern of three sialidases in one microbe is unusual in nature, which might be essential for pneumococcal pathogenesis at various stages. Based on the crystal structures of CNanA and NanB, preliminary work towards S. pneumoniae sialidases inhibitor design is under way, in which, a variety of techniques, such as the fluorescence-based thermal shift assay, NMR spectroscopy, computational docking and X-ray crystallography, are incorporated in. The crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be a unique trimer in crystal that is associated, in part, by the immunoglobulin-like trimerization domain around a three-fold crystallographic axis. The core catalytic domain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidase activity was detected with this enzyme. In addition, two key catalytic residues including one of the arginine in the arginine triad and the acid/base catalyst aspartic acid are missing in PaNA. In silico docking suggests that Phe129 may confer substrate selectivity towards pseudaminic acid, which is a specific carbohydrate superficially similar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directed mutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNA into a poor sialidases. Moreover, the crystal structure of PaNA also indicates that His45, Tyr21 and Glu315 may form a charge relay to compensate the missing aspartic acid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315 to be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNA could be a pseudaminidase with structural and mechanistic variations. This enzyme, together some other uncharacterized fellow proteins, might form a novel subclass in the sialidase superfamily. The various findings in the current projects provide meaningful insights towards several sialidases that have been linked to bacterial virulence, which may contribute to a more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.

<p>The purpose of the study was to set up a method for cultivation of C. pneumoniae from infected mouse tissue in Hep2 cells. We also evaluated a new reagent, Chlamatis, which is considered to increase the detection sensitivity of the bacterium with both PCR and cultivation. All 10 serum samples treated with Chlamatis were negative for C. pneumoniae in PCR. The cultivation of tissue was found to work without problems. The yield of the bacteria was highest at the speed of 30 Hz using homogenization with TissueLyser. Mice infected with C. pneumoniae were killed at days 4, 8, 20 and 40. The highest yields of C. pneumoniae were found at days 4 and 8 using PCR with all infected mice. The results obtained with PCR and cultivation confirmed each other to a large extent. For heart tissue, PCR positive mice were found only at days 4 and 8. The number of PCR positive lung samples confirmed to a large extent the number found by cultivation, except for mice killed after 4 days and 40 days where the results differed slightly. This indicated that the optimization of the cultivation method was successful</p>

A Mycoplasma pneumoniae se le atribuye entre un 15 a 20% de las neumonías adquiridas en la comunidad en los adultos mayores. Este estudio, el primero realizado en Chile para adultos mayores, se hizo para conocer la frecuencia, mediante la técnica de Reacción de la Polimerasa en Cadena, de Mycoplasma pneumoniae en este grupo etario y para probar la sensibilidad y especificidad analítica de ésta usando los partidores para el gen de la adhesina P1 y el gen del rRNA de 16S en el diagnóstico de dicho microorganismo, datos que no han sido estandarizados, como sí lo son las pruebas diagnósticas de Serología. Sin embargo, las pruebas de Serología no cuentan con una sensibilidad lo suficientemente buena, por lo que es necesario aplicar técnicas más sensibles y rápidas para la detección de Mycoplasma pneumoniae como lo es la Reacción de la Polimerasa en Cadena. Para llevar a cabo el estudio se tomaron muestras de expectoración y de sangre a 84 adultos mayores con neumonía adquirida en la comunidad, pertenecientes al Hospital Clínico de la Universidad de Chile, entre Agosto de 2002 a Septiembre de 2004, a los cuales se les realizó la técnica de Reacción de la Polimerasa en Cadena para ambos genes y la técnica de Serología Inmunofluorescencia Indirecta para IgM e IgG. A partir de los datos obtenidos se determinó que el porcentaje de Mycoplasma pneumoniae presente en los pacientes con neumonía fue de 8.3%, cifra inferior a la citada por la literatura y a la obtenida mediante la complementación de las técnicas de Reacción de la Polimerasa en Cadena y serología. Entre ambos partidores no existen diferencias significativas en cuanto a su sensibilidad y especificidad analítica. No obstante, se requiere de más estudios que verifiquen la calidad de estas técnicas para que lleguen a estandarizarse y constituir así técnicas de diagnóstico rápido de Mycoplasma pneumoniae, lo cual contribuiría importantemente a la clínica.

Streptococci resident in the oral cavity have been linked to infective endocarditis (IE). While viridans streptococci are commonly studied and associated with IE, less research has been focused on Streptococcus pneumoniae. Two-component systems (TCSs), consisting of a histidine kinase (HK) protein and response regulator (RR) protein, are bacterial signaling systems that may mediate S. pneumoniae TIGR4 strain virulence in IE. To test this hypothesis, TCS RR mutants of TIGR4 were examined in vivo through use of rabbit models. There were 14 RR proteins identified and 13 RR mutants synthesized because SP_1227 was found to be essential. The requirement of the 13 RRs for S. pneumoniae growth in IE models was assessed by quantifying mutants after overnight inoculation in IE infected rabbits through use of real time PCR (qPCR), colony enumeration on antibiotic selection plates, and competitive index assays. Real time PCR pinpointed several candidate virulence factors. Candidate RR SP_0798 was selected to be further examined. In the in vivo model, mutant SP_0798 grew significantly less than our control mutant SP_1678, which encodes a hypothetical protein and grew at a comparable rate to wild-type TIGR4 strains. Literature and databases identified SP_0798 as the ciaR gene, which has roles in regulating many diverse cellular functions. Our data suggests that RR SP_0798 is a virulence factor of S. pneumoniae TIGR4 strain in IE. This research may place more emphasis on virulence factors and lead to novel methods to combat pneumococcal endocarditis.

Due to the development of multidrug resistance in Streptococcus pneumoniae, research has begun to define new drug targets for pneumonia therapy. Different research groups have identified a lipoprotein, PsaA that is important for pneumonia virulence. PsaA is a manganese transporter that is required for bacterial virulence and growth. We have employed computer modeling to virtually screen a small-molecule database for inhibition of PsaA function by targeting the metal binding pocket, performing receptor-based virtual screening and molecular docking and scoring to identify potential inhibitors of PsaA function. We have developed an assay for screening compounds, including the use of a PsaA mutant, testing of multiple compounds, and identification of compounds that inhibit Streptococcus pneumoniae growth at concentrations less than 20 μM. We experimentally tested the effect on Mn uptake and their PsaA dependence for 42 compounds, but these experiments suggested that these compounds were affecting bacterial growth by a different mechanism.

Introducción: El aumento progresivo de las tasas de resistencia en bacterias uropatógenas, así como su propagación se ha convertido en un gran problema a nivel mundial, cobrando mayor importancia la diseminación de aislamientos resistentes productores de ß-lactamasas de espectro extendido, por lo que son útiles la caracterización molecular de plásmidos y otros elementos genéticos de las bacterias para establecer la relación genética que existe entre ellas y tener datos epidemiológicos relevantes que nos puedan sugerir la dirección de propagación de este tipo de resistencia. Objetivo: Describir los perfiles plasmídicos en aislamientos de Escherichia coli y Klebsiella pneumoniae productoras de ß-lactamasas de espectro extendido recuperados de urocultivos en el servicio de Microbiología del Instituto Nacional de Salud del Niño (INSN). Diseño: Estudio descriptivo, observacional de corte transversal. Institución: Centro de Investigación de Bioquímica y Nutrición CIBN-UNMSM; Instituto Nacional de Salud del Niño Participantes: Aislamientos de Escherichia coli y Klebsiella pneumoniae durante Noviembre y Diciembre del 2012 en el servicio de Microbiología del Instituto Nacional de Salud del Niño (INSN). Intervenciones: Extracción de ADN plasmídico de E. coli y de K. pneumoniae, detección de perfiles plasmídicos mediante electroforesis en gel de agarosa al 0.8% y tinción con bromuro de etidio. Principales medidas de resultados: Análisis de los patrones de huella de los perfiles plasmídicos obtenidos usando el programa NTSYSpc 2.1 y el algoritmo de agrupamiento UPGMA. Resultados. En las muestras analizadas se detectaron diferentes patrones electroforéticos obteniéndose de 1 hasta 7 bandas por muestra y en total 15 bandas de distinto tamaño, las cuales variaron desde 1.5 kb hasta más de 20 kb. Teniendo en cuenta los patrones de similitud, los aislamientos estudiados se distribuyeron en diferentes agrupamientos o ramas. Conclusión: No se encontró una similitud genética marcada entre los aislamientos de origen comunitario y de origen hospitalario, por lo que no se podría establecer una relación significativa y determinar un origen común entre los mismos. / --- Background: The progressive increase in resistance rates in uropathogenic bacteria and their spread have become in a significant worldwide public health, becoming more important the spread of resistant isolates producing of extended spectrum ß-lactamases, which are useful the molecular characterization of plasmids and other genetic elements of the bacteria to establish the genetic relationship between them, to have relevant epidemiological data that we can suggest the direction of propagation of this type of resistance. Objetive: Determine the plasmid profiles in isolates of Escherichia coli and Klebsiella pneumoniae producing of extended spectrum ß-lactamases recovered from urine cultures in the service of Microbiology of National Institute of Child Health (NICH). Design: Descriptive, observational cross-sectional study. Institution: Biochemestry and Nutrition Investigation Center CIBN-UNMSM; National Institute of Child Health. Participants: Isolates of Escherichia coli and Klebsiella pneumoniae during November and December of 2012 in the service of Microbiology of National Institute of Child Health. Interventions: Extraction of plasmid DNA from E. coli and K. pneumoniae, detection of plasmid profiles by electrophoresis gel of 0.8% agarose and staining of DNA fragments was carried out using ethidium bromide and they were visualized by UV-Trans illumination, the analysis of fingerprint patterns of plasmid profiles obtained using the NTSYSpc 2.1 program and the UPGMA clustering algorithm. Results: Were detected different electrophoretic patternsin the samples, obtained from 1-7 bands per sample and a total of 15 bands of different sizes, which varied from 1.5 kb to more than 20 kb. Considering the patterns of similarity, the isolates analyzed were distributed into different clusters or branches. Conclusion: The determination of plasmid profiles proved to be a sensitive and reliable technique for the analysis of genetic diversity isolates of E. coli and K. pneumoniae from the National Institute of Child Health. Key words: Plasmid profiles, E. coli, K. pneumoniae, extended spectrum ß- lactamases.

Orientador: Edison Iglesias de Oliveira Vidal / Resumo: Introdução: O Streptococcus Pneumoniae é o agente infeccioso mais frequentemente associado à ocorrência de pneumonia bacteriana e a vacinação é considerada a principal estratégia para a prevenção dessa doença. De acordo com o Programa Nacional de Imunização (PNI) a vacina pneumocócica polissacarídica 23-valente (Pn23) não faz parte do calendário básico de vacinação e deve ser dispensada a indivíduos a partir de 2 anos de idade desde que portadores de um conjunto de doenças e condições de alto risco para infecções pneumocócicas. A forma de operacionalização atual do PNI em relação à Pn23 se dá de modo que essa vacina não se encontra disponível nas Unidades Básicas de Saúde (UBS) e sua liberação se dá mediante solicitação aos Centros de Referência em Imunobiológicos Especiais (CRIEs), através de uma ficha de Solicitação de Imunobiológicos Especiais (SIBE). Acredita-se que tal formatação da logística de dispensação da vacina constitui um elemento limitador do acesso da população adulta à mesma. O objetivo da presente pesquisa foi avaliar a efetividade de uma intervenção piloto no município de Jahu de caráter multifatorial sobre a frequência de dispensação da Pn23 para a população adulta com indicação clínica conforme definida pelo PNI. Métodos: A intervenção foi composta por um componente caracterizado pela descentralização do fluxo de dispensação da vacina, de modo que esta passasse a estar disponível diretamente nas UBS, como é feito com as demais vacinas do calendário básico ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Streptococcus Pneumoniae is the infectious agent most commonly associated with bacterial pneumonia and vaccination against it is considered the main strategy to prevent its occurrence. According to the Brazilian National Immunization Program (NIP) the 23-valent pneumococcal polysaccharide vaccine (Pn23) is not part of the country’s basic vaccination program and is recommended only for individuals aged 2 years and older who suffer from a variety of high-risk diseases and conditions for pneumococcal infections. The current operationalization of the NIP regarding the Pn23 vaccine determines that that vaccine is not available at Primary Healthcare Units (PHU) and that its distribution to those units is conditional to the receipt of a special vaccine request form by the regional Reference Centers for Special Immunobiologic Products (RCSIP). We believed that such centralized system of distribution of the Pn23 vaccine constituted a barrier for the eligible adult population to have access the vaccine. The aim of the present study was to assess the effectiveness of a multifactorial intervention on the frequency of use of the Pn23 vaccine among adults of the municipality of Jahu with a clinical indication for the vaccine according to the NIP. Methods: The intervention consisted of the decentralization of the Pn23 vaccine distribution so that doses of that vaccine were made available at each PHU as if it were part of the country’s basic vaccination program. Additionally, t... (Complete abstract click electronic access below) / Mestre

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Medicine. / The emergence of strains of S. pneumoniae resistant to penicillin and to other antibiotics, and the spread of that resistance over the world, have become major concerns and increase the need for epidemiological surveillance. The following typing methods have been used to detect strain variability in pneumococci: Serotyping, antibiotic susceptibility profiles, multilocus enzyme electrophoresis (MLEE), penicillin-binding protein (PBP) profiles, pulse-field gel electrophoresis (pFGE), and ribotyping. Serotyping, antibiograms, and MLEE only detect phenotypic variability. PBP gene profiles, PFGE, and ribotyping detect genotypic differences but these techniques are labour intensive and time consuming. Random amplified polymorphic DNA (RAPD) is a new technique that bas proved useful for typing bacteria, fungi, and parasites, but has not been. studied using pneumococci. Unlike conventional polymerase chain reaction (peR), RAPD utilizes single, short primers, usually 10 oligonucleotides in length. As the primer is short and low astringency annealing temperatures are used, there will be many complimentary sites scattered randomly throughout a bacterium's genome, When such sites occur a few hundred base pairs away from each other and on opposite DNA strands, the enclosed region can be amplified by peR This results in numerous discrete target fragments which can be separated by agarose-gel electrophoresis and ethidium bromide staining. RAPD requires no sequence information and it scans the whole genome rather than relying on hypervariability within one specific gene. The aims of this study were: to determine strain variability using RAPD, to determine the reproducibility ofRAPD, and to demonstrate intercontinental spread of a multiresistant pneumococcal strain. The following strains were evaluated: a) 10 strains from a day-care centre (DCC), the index case being a 3 year old girl 'with otitis media. An aunt from Spain had recently been staying with the family. The other strains were isolated from class mates and siblings of the index case.; b) 18 clinical isolates from Seoul, Korea; and c) 11 epidemiologically unrelated isolates from South Africa, including the reference strain, R6. Two DNA extraction methods were used. The first involving lysis with sodmm-dodecyl-sulphaze and proteinase K. Proteins were removed with phenol-chloroform, and the DNA precipitated with ethanol. The second method involved incubating the cells at 95 0C for 15 microlitres, followed by centrifugation. 2 microlitres of the supernatant was then used for each PCR reaction, Three primers were evaluated. After 01uimisation of the RAPDreaction for pneumococci, the final peR mixtures per 50 ul was: primer (4 plY1), template (0.5 ng), nuc1eotides (300 pMeach), magnesium (4 mM~, and Taq polymerase (2 U). 35 cycles were used with an annealing temperature of 35'C. Both DNA extraction methods: gave reproducible results but were not comparable to each other. All 10 strains from the DCC gave the same banding pattern as the Spanish done for all 3 primers. 7 of the Korean strains gave the same banding pattern as the Spanish clone using the first two primers, however one strain showed an additional band using the third primer. Of the remaining 22 strains, 21 different banding patterns were obtained. This study has shown that RAPD is a simple and rapid technique that can distinguish strain variation among pneumococci. The reproducibility is excellent within the same laboratory. Finally using RAPD. this study identified a Spanish multiresistant 23F clone in South Africa and Korea. / Andrew Chakane 2018