Pulmonary infection caused by nontypeable Haemophilus influenzae (NTHi) is a significant cause of morbidity and mortality in both industrialised and developing countries. Previous work from this group resulted in the development of a respiratory model in rodents which has precipitated studies into the pathogenesis of infection by NTHi and investigation of the humoral and cellular mechanisms by which the bacteria are cleared from the lung. Comparison of mucosally immunised with non-immunised animals has demonstrated that not only are bacteria cleared more rapidly from the lungs, but there is a more rapid response and resolution of inflammatory factors in the mucosally immunised animals following challenge with NTHi. This inflammatory response is partially regulated by the ability of the mucosally immunised animals to rapidly produce, then control the production of tumour necrosis factor (TNF)-a. The TNF-a is produced by both macrophages and type I pneumocytes in the alveoli and also by the endothelial cells lining the blood vessels in the lungs. Immunocytochemical studies have identified cellular subsets accumulating in the lung at various time points following infection. Marked differences in cellular infiltration into the lung tissue were noted between immunised and non-immunised animals after challenge with NTHi. Immunised animals demonstrated an early influx of macrophages, CD8+ T cells and Y8+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes and finally an increased number of both B cells and CD4+ T cells. In contrast, non-immunised animals did not demonstrate any proliferation nor extravasation of lymphocytes or increased expression of MHC-II before total bacterial clearance had occurred. Polymorphonuclear leukocyte infiltration occurred in the non-immunised animals, however at a later time than that seen in immunised animals. Challenging rodents to establish persistent infection highlighted the inappropriately aggressive white blood cell response to an initial challenge when bacteria may be masked by other substances, followed by the inability to amplify the polymorphonuclear leukocyte response on repeated challenge with NTHi. This hyporesponsiveness in the macrophage population, shown by lack of detectable TNF-a production, concomitant with low numbers of NTHi resulted in a continuously high number of macrophages in the alveoli and the possibility of increased damage to the lung tissue. The requirement for cell surface TNF-a and CD8+ T cells to enhance the clearance of NTHi from the lungs further strengthens previous in vitro and in vivo findings of the possible significance of cellular invasion as a mechanism of pathogenicity for NTHi. This thesis has contributed to the understanding of both the immune response to and the pathogenicity mechanisms of pulmonary infection with NTHi. Kinetic studies identifying cellular responses and cytokine levels have emphasised the ability of mucosal immunisation to increase the rate of immune response and resolution of inflammation to NTHi infection in the lung. Observations demonstrating a requirement for macrophages and CD8+ T cells in mechanisms associated with enhancing NTHi clearance from the lung will lead to further investigations.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-07-29T17:20:06Z No. of bitstreams: 1 Tese_Med_Josilene Borges Torres Lima.pdf: 812403 bytes, checksum: f3a47d8a2a2a13648f9abf0ee0d4b9aa (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-08-22T12:47:57Z (GMT) No. of bitstreams: 1 Tese_Med_Josilene Borges Torres Lima.pdf: 812403 bytes, checksum: f3a47d8a2a2a13648f9abf0ee0d4b9aa (MD5) / Made available in DSpace on 2016-08-22T12:47:57Z (GMT). No. of bitstreams: 1 Tese_Med_Josilene Borges Torres Lima.pdf: 812403 bytes, checksum: f3a47d8a2a2a13648f9abf0ee0d4b9aa (MD5) / A introdução de vacinas conjugadas contra o Haemophilus influenzae sorotipo “b” (Hib) foi um avanço de grande impacto na Saúde Pública, praticamente eliminando a meningite por Hib nos países onde foi implementada. O presente trabalho descreveu a meningite por H. influenzae em Salvador, Bahia, estudando o impacto da vacina, e caracterizou os isolados do sorotipo não “b”, identificados durante vigilância ativa populacional. A população estudada foi composta de pacientes identificados por cultura positiva para H. influenzae, e os dados clínicos obtidos através de entrevista e revisão de prontuário. O sorotipo dos isolados foi determinado utilizando antisoros, e reação em cadeia pela polimerase (PCR). A tipagem molecular foi realizada pelas técnicas de eletroforese em campo pulsátil e multilocus sequence typing; e para identificação de mecanismo de virulência, foi utilizada PCR e sequenciamento da região de deleção IS1016-bexA. Para cálculos de incidência foram utilizados números de casos da região Metropolitana de Salvador, e a população do censo do ano de 2000. Após 5 anos de introdução da vacina anti-Hib, a incidência da meningite pelo H. influenzae teve uma redução de 97,6% na população total, e de 97,5% em menores de cinco anos. Em 11 anos de vigilância, foram identificados 40 casos de meningite por isolados do sorotipo não b, sendo 80% após o uso da vacina. Na caracterização molecular, os isolados do sorotipo “a” foram agrupados em dois clones, sendo que um deles apresentava o fator de virulência, a deleção parcial IS1016-bexA, o qual foi associado com elevada mortalidade (OR 10,8 e IC 0,7-345). Estudos de epidemiologia molecular são importantes para estimar a eficácia da vacina, bem como monitorar a emergência de isolados de outros sorotipos para avaliar a necessidade de intervenção na composição da vacina.

Made available in DSpace on 2014-08-26T17:15:20Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 145.pdf: 1867505 bytes, checksum: 87dab03551d06be6de32b042de770c29 (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / O presente estudo incluiu 235 cepas, procedentes dos estados de Santa Catarina (SC), Rio de Janeiro (RJ) e Pemambuco (PE). Verificamos que o Hib antes da vacinação predominava em mais de 90 por cento dos casos, sendo os outros tipos sorológicos raros. Após a vacinação, cai o tipo,b, crescem os tipos não b, em meningites, septicemias e infecções respiratórias. Os biotipos I e II foram os mais isolados. Houve variação da sensibilidade aos antimicrobianos testados (ampicilina, amoxicilina-ácido clavulânico, ceftriaxona, rifampicina, cloranfenicol e sulfametoxazol-trimetoprim), sobretudo na associação SMX- TMP cuja resistência dos Hi aumentou de 32,6 por cento para 65,8 por cento entre os dois períodos; enquanto se mantiveram taxas semelhantes de resistência para a ampicilina (17,5 por cento e 15,8 por cento) e a presença de cepas produtoras de p-lactamase; a sensibilidade à amoxicilina-ácido clavulânico e ceftriaxona foi de 100 por cento. Alteração na resistência ao cloranfenicol de 19,4 para 12,5 por cento e da rifampicina de 8,2 para 9,7 por cento.A tipagem sorológica realizada pelo método da SAL, teve bons resultados (96,2 por cento) e a molecular, pela PCR, diferenciou as cepas NT das capsuladas b -.Embora a estrutura populacional dos Hi tenha sido inicialmente descrita como clonal, os resultados obtidos através da técnica de ERIC- PCR, revelaram diversidade genética nas cepas estudadas. As do sorotipo b, revelaram 4 clones com diferentes características epidemiológicas. A diversidade genética foi maior nas Hinb e HiNT. Dos estados estudados, PE teve a mais alta diversidade genética (6 clones em 15 cepas) seguido do RJ (3 clones em 23 cepas) e SC (2 clones em 13 cepas). Concluímos, portanto, que há necessidade do monitoramento das cepas circulantes de Hi no país observando as possíveis alterações na prevalência dos sorotipos atuais e a real estimativa do impacto da vacina contra o Hib atualmente utilizada no Brasil. / The bacterial genus Haemophilus is included in the family Pasteurellaceae and the mostimportant specie causing infectious diseases in humans is the Hi. It shows capsularserotypes (a-f) as well as NT strains. A great number of different acute infectious diseasesare caused by this organism, especially affecting the CNS, as well as RD, occurringfrequently in children, especially with the Hib. Since 1988 disease associated with Hib hasbeen vaccine-preventable, by means of the first conjugate vaccine showing great efficacy.The vaccine is composed by the PRP and a carrier protein. However, the Hib is stillconsidered one of the most important human pathogens, which leaded us to developdifferent research lines, with isolates from different source of infections, after theintroduction of the conjugate vaccine against Hib by PNI/MS in 1999. We used in ourstudy 235 Hi isolates, from the brazilian states of Santa Catarina, Rio de Janeiro andPernambuco. Our results, show that the rate of type b infections before vaccination wasmore than 90%, whereas other serological types were rare. After the advent of the vaccine,we observe a decrease of type b and an increase of non-b isolation. Biotypes I and II arestill the most frequently isolated. The antimicrobial susceptibility found against ampicillin,amoxicillin-clavulanate, ceftriaxone, rifampicin, chloranphenicol and TMP-SMX, showed arecent increasing resistance against antibiotics commonly used to treat RD, especiallyamong the ones administrated by oral route, showing a resistance rate from 32.6% to 65.8%between the two periods studied. Ampicillin, showed resistance rates of 17.5% and 15.8%in the two periods studied and the presence of â-lactamase producing strains. Strainsevaluated against amoxicillin/clavulanate and ceftriaxone, were 100% sensible.Chloranphenicol showed a decrease in the resistance rates of the strains isolated during thepost-vaccination period, while rifampicin, showed an increase in the resistance rate from8.2 to 9.7%. The seroaglutination method used for capsular typing, showed good results,however, mutant b capsulated strains (b-), were only detected by the PCR, using specificprimers for the capsule region. The population structure of Hi which has been described asclonal, revealed a great genetic diversity by ERIC-PCR. Therefore we can conclude thatthere is a need of systematic tracking of circulating Hi strains in the country, continuouslywatching possible epidemiological changes and the evaluation of the impact of the vaccineagainst Hib used in Brazil to date.

Made available in DSpace on 2015-07-08T12:28:20Z (GMT). No. of bitstreams: 2 5.pdf: 1093384 bytes, checksum: 9432b61bcb4cfac37f05a1600b8f2fae (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Haemophilus influenzae pode ser encontrado, normalmente, na microbiota do trato respiratório, do trato gênito-urinário e da cavidade oral. Porém, essa espécie inclui um dos mais importantes patógenos bacterianos em infecções principalmente pediátricas. As cepas de Hi podem ser capsuladas, variando de a-f, ou não capsuladas (não tipáveis - NT). O tipo capsular b foi o mais frequente em infecções graves infantis até a utilização da vacina conjugada contra Hib, sendo ainda considerado patogênico. No Brasil, essa vacina foi introduzida no Programa Nacional de Imunização do Ministério da Saúde em agosto de 1999, e como em outros países, promoveu uma acentuada diminuição em doenças causadas por esse agente. No entanto, estudos realizados na era pós-vacinal têm mostrado que a incidência de doenças invasivas causadas por H. influenzae não b e NT têm aumentado, inclusive no Brasil. O objetivo desse trabalho foi obter informações sobre as cepas de Hi circulantes no município do Rio de Janeiro. Utilizou-se 96 amostras de quadros infeciosos (46 invasivas e 50 não invasivas), isoladas no período pós-vacinal (2000-2012). Em relação à idade, dos 54 pacientes que tiveram esse dado, 32 tiveram doenças invasivas e 22 não invasivas. Em doenças invasivas, houve o predomínio de crianças < 5 anos. Enquanto que nas não invasivas os adultos > 70 anos predominaram. Entre as cepas obtidas, 15 foram capsuladas e 81 não capsuladas. A maioria das cepas capsuladas foi proveniente de sítios invasivos, cuja faixa etária predominante foi de < 5 anos. O tipo capsular mais isolado foi o b, seguido do a e f. As cepas Hib, predominaram no início do período de estudo, enquanto os outros sorotipos predominaram no final. No presente trabalho, não encontramos cepas mutantes deficientes de cápsula tanto Hib-, quanto Hia- / Haemophilus influenzae can be found usually in the microbiota of the respiratory tract, genitourinary tract and oral cavity. However, this species includes one of the most important bacterial pathogens mainly in pediatric infections. The strains can be capsulated Hi, (serotypes a-f), or not capsulated (nontypeable - NT). The type b capsular was the most frequent serious infection in children until the use of conjugate vaccine against Hib and is still considered pathogenic. In Brazil, this vaccine was introduced in the National Immunization Program of the Ministry of Health in August 1999, and as in other countries, promoted a significant decrease of disease caused by this agent. However, studies during the post-vaccine period have shown that the incidence of invasive disease caused by H. influenzae b and NT have increased, including in Brazil. The aim of this study was to obtain information on the Hi strains circulating in the municipality of Rio de Janeiro. We used 96 samples (46 invasive and 50 noninvasive), isolated during the post-vaccination period (2000-2012). Concerning the age of the 54 patients who had this data, 32 had invasive and 22 noninvasive disease. In invasive disease, there was a predominance of children <5 years. While in the noninvasive group, adults > 70 years predominated. Among the strains obtained, 15 were capsulated and 81 non-capsulated. Most capsulated strains originated from invasive sites whose predominant age group was <5 years. The most frequent capsular type was b, followed by a and f. Hib strains predominated at the beginning of the study period, while the other serotypes prevailed in the end. In this study, we did not found mutant strains deficient in both capsule Hib- and Hia-. NT strains accounted for the vast majority of isolates in this study, 32 strains isolated from invasive sites and 49 sites noninvasive, and were obtained from patients of all age groups. The capsulated strains were predominantly biotype I and II, while non-typeable strains were most II and III. In this study, only the NT strains were resistant to two drugs: ampicillin and trimethoprim - sulfamethoxazole. These were mostly non-invasive. Thus, none of the capsulated isolates were resistant. The PFGE patterns for the 96 strains were quite different, however eight NT strains belonged to the same genotype. Capsulated strains of the same serotype were similar, getting most isolates grouped in the same cluster. We therefore conclude that it is necessary to monitor the Hi strains circulating in Rio de Janeiro, because of the geographic and economic importance of this municipality. Such conduct should be extended to the whole country in order to understand the possible changes of serotypes today, which will certainly guide for the design of new vaccines, improvement of existing ones and the use of antibiotics, resulting in a public health impact.

Ältere Menschen sind gegenüber invasiven Infektionen und Sepsis besonders vulnerabel mit ungünstiger Prognose. Staphylococcus aureus und Haemophilus influenzae können beide invasive Infektionen verursachen. Oft geht eine asymptomatische Besiedelung einer Infektion voraus und ist ein Risikofaktor für eine invasive Infektion. Daher wurde eine bizentrische Querschnittstudie in den Regionen Aachen und Würzburg durchgeführt, um die Prävalenz von H. influenzae, S. aureus und MRSA (Methicillin resistenter S. aureus) bei asymptomatischen Senioren zu bestimmen, wie auch Risikofaktoren für eine Besiedelung. Von Oktober 2012 bis Mai 2013 wurden 677 Erwachsenen im Alter von 65 Jahren oder älter eingeschlossen, die zu Hause oder in Seniorenheimen lebten. Die Prävalenz von H. influenzae bei älteren Menschen war mit einer Trägerrate von nur 1,9% ([95% CI: 1,0 - 3,3%]; 13/677) sehr niedrig. Trägerisolate waren überwiegend nicht typisierbare H. influenzae, zeigten eine hohe clonale Diversität und waren alle Ampicillin-sensibel. Die Prävalenz von S. aureus war mit 28,5% ([95% CI: 25,1 - 32,1%]; 193/677) hoch, wie für die deutsche Allgemeinbevölkerung bekannt, während MRSA bei weniger als 1% der Teilnehmer gefunden wurde (0,7% [95% CI: 0,2 - 1,7%]; 5/677). Die Prävalenz von H. influenzae, S. aureus und MRSA unterschied sich nicht signifikant zwischen selbständig zu Hause lebenden Senioren und Pflegeheimbewohnern. Ältere, selbständig lebende Menschen mit höherem Bildungsniveau hatten signifikant höhere Kolonisierungsraten mit S. aureus (adjusted OR: 1,905 [95% CI: 1,248 - 2,908]; p = 0,003). Bei Pflegeheimbewohnern war eine Kolonisierung signifikant mit Verheiratet sein assoziiert (adjusted OR: 3,367 [95% CI: 1,502 - 7,546]; p = 0,003). Diese Ergebnisse unterstreichen die Bedeutung von sozio-demographischen Faktoren für eine Kolonisierung mit S. aureus und schließen eine Lücke bei epidemiologischen Daten zu H. influenzae. / Elderly people are especially vulnerable to invasive infections and sepsis with often poor outcome. Staphyloccus aureus and Haemophilus influenzae both can cause invasive infections. Asymptomatic colonization often precedes infection and poses a risk for invasive infection. Therefore, a bi-centric cross-sectional carrier study was conducted in the regions of Aachen and Wuerzburg, Germany, to determine the prevalence of H. influenzae, S. aureus and MRSA (methicillin resistant S. aureus) in asymptomatic elderly people and to identify risk factors for colonization. From October 2012 to May 2013 677 adults aged 65 years and older were included, living at home or in nursing homes. In contrast to children and younger adults the prevalence of H. influenzae was very low among elderly people with a carriage rate of only 1.9% ([95% CI: 1.0 - 3.3%]; 13/677). Carrier isolates were predominantly non typeable H. influenzae, showed a high clonal diversity and were all susceptible to ampicillin. The prevalence of S. aureus was expectedly high as known for the German general population (28.5% [95% CI: 25.1 - 32.1%]; 193/677), while MRSA was found in less than 1% of the individuals (0.7% [95% CI: 0.2 - 1.7%]; 5/677). The prevalence of H. influenzae, S. aureus und MRSA did not differ significantly between community dwellers and nursing home residents. Elderly community-dwellers with higher education level had significantly higher colonization rates with S. aureus (adjusted OR: 1.905 [95% CI: 1.248 - 2.908]; p = 0.003). Among nursing home residents, colonization was significantly associated with being married (adjusted OR: 3.367 [1.502 - 7.546]; p = 0.003). These results underline the importance of socio-demographic factors for colonization with S. aureus and close a gap in epidemiological data on H. influenzae.

Bacterial transformation is defined as a mechanism of genetic exchange whereby a population of bacteria can obtain genetic informa-tion as a result of cellular uptake and integration of extracellular deoxyribonucleic acid released from other bacteria by natural or in-duced lysis. In order for a transformable strain of bacteria to take up DNA and to undergo transformation, the cells must be in a physiolog-ical state called competence. A cell is referred to as competent if it has the ability to bind extracellular DNA irreversibly and subsequently to integrate and express the DNA.

Die Inzidenz invasiver H. influenzae-Infektionen in Deutschland steigt seit Jahren an. Die akkurate Identifizierung und Resistenztestung dieses Erregers sind von großer klinischer und epidemiologischer Bedeutung. Daher wurden im Rahmen der vorliegenden Promotionsarbeit umfangreiche Untersuchungen zur Diagnostik und zur Epidemiologie von Antibiotikaresistenzen bei H. influenzae durchgeführt. Es konnte gezeigt werden, dass die in der Routinediagnostik mittlerweile weit verbreitete MALDI-TOF-MS-Diagnostik durch das VITEK MS IVD nur eingeschränkt zur sicheren Unterscheidung von H. influenzae und H. haemolyticus einsetzbar ist. H. influenzae-Isolate erkannte das System mit einer Genauigkeit von 100 %. Bei H. haemolyticus-Isolaten wurden dagegen 42 % der untersuchten Stämme fälschlicherweise als H. influenzae erkannt. Dieser Fragestellung wurde mit der bisher umfangreichsten molekularbiologisch charakterisierten Studienpopulation beider Bakterienspezies nachgegangen. Die kalkulierte antibiotische Therapie einer Sepsis oder Meningitis erfolgt häufig mit Carbapenemen, die leitliniengerechte Therapie invasiver H. influenzae-Infektionen mit Drittgenerations-Cephalosporinen. Imipenem und Cefotaxim gehören zu den Hauptvertretern dieser Gruppen. Bezüglich der Antibiotikaresistenztestung wurde erstmalig für H. influenzae herausgefunden, dass die routinemäßig verwendete Gradientenagardiffusion (GAD) bei der Testung von Cefotaxim im Vergleich zum Goldstandard Bouillon-Mikrodilution gleichwertig und bei Imipenem sogar sensitiver in der Detektion von Heteroresistenzen ist. Die Epidemiologie dieser Resistenzen wurde in dieser Arbeit erstmalig für Deutschland systematisch erfasst, indem alle verfügbaren invasiven Isolate gemeldeter H. influenzae-Infektionen der Jahre 2016 (Imipenem) beziehungsweise 2016-2019 (Cefotaxim) untersucht wurden. Es wurde eine hohe Prävalenz einer Imipenem-Resistenz von 13,5 % festgestellt. Die Prävalenz einer Cefotaxim-Resistenz lag bei 0,9 %. Zur molekularen Typisierung wurde bei den Imipenem-resistenten Isolaten eine Multilocus-Sequenztypisierung, bei den Cefotaxim-resistenten Stämmen eine Sequenzierung des vollständigen Genoms durchgeführt. Hierbei wurde eine hohe genetische Diversität der Stämme festgestellt, was die Schlussfolgerung zulässt, dass resistente Mutanten sporadisch entstehen. Die Untersuchung möglicher spatio-temporaler Cluster führte zum Nachweis einer sehr selten vorkommenden Übertragung eines Imipenem-resistenten Stamms. Durch die Sequenzierung von Resistenzgenen wurde die Epidemiologie und Relevanz bekannter Aminosäuresubstitutionen beleuchtet. Unter anderem wurde für die PBP3-Substitutionen L389F und Y557H eine hochsignifikante Korrelation mit dem Auftreten von Cefotaxim-Resistenzen nachgewiesen. Die gewonnenen Genomdaten bieten die Grundlage für die Forschung an weiteren Antibiotikaresistenzdeterminanten von H. influenzae. / Haemophilus influenzae is a fastidious, facultative anaerobic, Gram-negative bacillus that colonizes the respiratory tract and can cause respiratory and invasive infection such as meningitis and sepsis. Invasive H. influenzae infections are potentially life-threatening and incidence rates have been increasing for years. Therefore, fast and accurate diagnostics, reliable testing of antibiotic resistance and a successful antibiotic treatment is of great importance. Therefore, the objective of the first part of this thesis was to evaluate the diagnostic and discriminative potential of the MALDI-TOF-MS system VITEK® MS regarding H. influenzae and H. haemolyticus. H. influenzae can cause invasive infections, whereas H. haemolyticus is mostly apathogenic. The system showed excellent accuracy for the identification of H. influenzae isolates, as 100 % of the 236 isolates were correctly identified. When testing 50 H. haemolyticus strains, however, the system showed significant limitations, since 42 % of these strains were misidentified as H. influenzae. According to the current German guidelines for the treatment of sepsis and meningitis, treatment of invasive H. influenzae infections is carried out using carbapenems, such as imipenem, or third-generation cephalosporins, such as cefotaxime. Therefore, the prevalence of antibiotic resistances to these substances was investigated and possible resistance mechanisms were examined. The two antibiotic susceptibility testing methods Gradient agar diffusion (GAD) and broth microdilution (BMD) were compared. As a result, for the determination of the cefotaxime MIC, the two methods showed an excellent correlation, whereas for imipenem there were significant differences in the measured MIC values. Since strains tested by GAD often showed double or fuzzy inhibition zones, heteroresistances may be more apparent using this method and GAD may be more sensitive at detecting imipenem resistance. The prevalence of imipenem resistance was determined for the year 2016. The analysis of 474 different invasive isolates showed a high prevalence of 5.5 %. If including all resistant isolates according to GAD, the prevalence would be even as high as 13.5 %. MLST was performed on all isolates to investigate the genetic relationship. As a result, however, some sequence types were observed more frequently, it revealed a significant diversity. Both the analysis of ftsI and acrR showed previously described amino acid substitutions. Cefotaxime resistance was investigated for all 2432 invasive H. influenzae for the years 2016-2019. The low prevalence of 0.9 % shows that cefotaxime is still well suited for the treatment of invasive H. influenzae infections. For the investigation of the genetic relationship and possible causes of cefotaxime resistance whole genome sequencing (WGS) was performed on all resistant isolates. The strains showed high genetic diversity and the geographic analysis also showed that the resistant strains were evenly spread throughout the population in Germany. This led to the conclusion that cefotaxime resistance is more likely caused by sporadic mutation events rather than by specific clones spreading in certain areas. The analysis of the ftsI gene showed that the amino acid substitutions L389F and Y557H are significantly associated with elevated cefotaxime MICs. This dissertation provides comprehensive data regarding diagnostics, antimicrobial susceptibility testing and the epidemiology of antibiotic resistances of invasive H. influenzae. It could be shown that the VITEK MS IVD, although established in the routine diagnostics, can only be used to a limited extent for reliably differentiating H. influenzae and H. haemolyticus isolates. Regarding antibiotic susceptibility testing, it was found that GAD showed similar results compared to the gold standard BMD when testing cefotaxime. When testing imipenem, this method was even more sensitive in detecting heteroresistances compared to the gold standard. For the first time, the epidemiology of antibiotic resistance against cefotaxime and imipenem was carried out using a large set of precisely defined invasive H. influenzae isolates to obtain representative data for the prevalence of imipenem and cefotaxime resistance in Germany. By investigating amino acid substitutions in the ftsI and acrR gene the epidemiology and relevance of these substitutions could be shown. A well-founded statement on the relationship of the resistant strains could be made using the state-of-the-art typing methods MLST and whole genome sequencing. The genome data also offers the possibility of examining other genes of these strains in more detail.

Haemophilus influenzae, as well as other members of the genus which require V-factor, display a unique growth requirement for intact NAD. This organism, the primary cause of bacterial meningitis, is incapable of synthesis of pyridine nucleotides from the usual precursors. An externally directed nucleotide pyrophosphatase was extracted from the organism and purified 700-fold using ammonium sulfate precipitation, ion-exchange and affinity chromatography. The enzyme was determined to be a periplasmic glycoprotein consisting of a single polypeptide of M_r= 65,000. The enzyme had a pH optimum over the range .pH 8.0-9.0 and was not activated by the addition of mono or divalent cations, nor was it inhibited by EDTA. The enzyme was observed to have a broad substrate specificity and functioned in a manner indicative of negative cooperativity with all substrates except several modified in the adenine ring. The most effective inducer of negative cooperativity was NAD as indicated by its Hill coefficient of 0.26. The enzyme was inhibited by adenine nucleotides _ and 5'-AMP, at 20 μM, abolished the negative cooperativity of the enzyme. The enzyme was determined to possess excitation and emission maxima at 286 and 337 nm, respectively, indicative of the presence of tryptophan. The fluorescence of the enzyme was quenched by addition of aliquots of adenine nucleotides. The quenching occurred in a biphasic manner. The enzyme was inactivated by 2,3- butanedione and by Woodward's Reagent K. Studies of the ability of compounds to serve as V-factor revealed that nicotinamide mononucleotide (NM), NAD, as well as analogs of NAD, served as V-factor. The ability of compounds to inhibit growth was also accessed, and the growth of the organism was seen to be inhibited by adenine nucleotides as well as other compounds. The inhibition of growth of Haemophilus influenzae has important clinical implications which are discussed, as well as a model of the NAD metabolism of the organism which is presented. / Ph. D.

Haemophilus influenzae, the primary causative factor in bacterial meningitis, displays a unique growth requirement for intact NAD. Selective inhibitors of the pyridine nucleotide-requiring enzymes from H. influenzae could have a pronounced effect on growth of the organism. Haemophilus malate dehydrogenase was purified 109-fold with a 26% recovery through a 4-step procedure involving salt fractionation, and hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of M,= 61,000. Initial velocity, product, and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme. Several NAD analogs structurally altered in either the pyridine or purine moiety functioned as coenzymes in the reaction catalyzed. Selective interactions occurring at the coenzyme binding sites were investigated. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate moiety of the coenzyme. Positive chain length effects in the coenzyme-competitive inhibition by aliphatic carboxylic acids indicated the presence of a hydrophobic region close to the pyrophosphate region at the coenzyme binding site. Several structural analogs of NAD and malate were evaluated as selective inhibitors of the enzyme. The enzyme was inactivated by incubation with diethylpyrocarbonate whereas no inactivation was observed with sulfhydryl reagents. Haemophilus influenzae 6-phosphogluconate dehydrogenase was purified 308-fold with a 16% recovery through a 4-step chromatographic procedure involving a PhenylSepharose hydrophobic column, and affinity chromatography on Matrex gel Green A, Matrex gel Red A, and 2',5’ADP-Sepharose resin. The purified enzyme was demonstrated to be a dimer of M,= 70,000. Initial velocity studies of 6-phosphogluconate oxidation indicated a sequential reaction mechanism. Although certain product and dead-end inhibition studies were consistent with an ordered mechanism, the direct binding of 6-phosphogluconate in protection experiments did not support a strictly ordered reaction sequence. Inhibition by adenosine derivatives indicated that the 2’-phosphate is important in binding to the coenzyme binding site of the enzyme. The 3-acetylpyridine analogs of NAD and NADP which support growth of H. influenzae were demonstrated to function as coenzymes with the two dehydrogenases studied. The most effective inhibitors of the purified malate dehydrogenase and 6-phosphogluconate dehydrogenase were observed to inhibit the growth of Haemophilus influenzae. However, the most potent inhibition of growth by 3-aminopyridine analogs of NAD and NADP could not be explained on the basis of interactions of these analogs with the two dehydrogenases studied. / Ph. D.

Objetivos: Avaliar o impacto global, direto, indireto e a tendência da duração de proteção da vacinação contra o Haemophilus influenzae tipo b (Hib), no estado de São Paulo (ESP) e no município de São Paulo (MSP), na população de 0 - 59 meses, comparando os períodos pré-vacinal (1996 - 1998) e pós-vacinal (2001 - 2009). Métodos: estudo com componente descritivo e de cunho analítico, retrospectivo. A população de estudo incluiu os menores de cinco anos residentes no ESP e no MSP. Adotou-se como definição de caso confirmado o menor de cinco anos identificado como positivo para o Hib em cultura e/ou contraimunoeletroforese e/ou látex e/ou RT-PCR, em amostra de LCR e sangue, e/ou vínculo epidemiológico. Os dados foram obtidos a partir do SINAN, SIGH-Web Instituto Adolfo Lutz e Fundação IBGE. As variáveis de estudo incluíram as demográficas, clínicas e relativas ao agente, apresentadas em séries temporais e períodos estabelecidos para parametrização e comparabilidade. O parâmetro das avaliações de impacto foi a magnitude da variação da incidência de meningite causada pelo Hib. Para cada estimativa de impacto construiu-se um Intervalo de Confiança (IC) de 95 por cento a partir do cálculo de Risco Relativo (RR). As estimativas do risco relativo (RR) e os respectivos intervalos de 95 por cento de confiança foram analisados utilizando-se o software R. Resultados: nos períodos considerados, foram descritos 1.561 casos confirmados de meningites por Hib no ESP, sendo 27,16 por cento (424/1.561) no MSP, e 80,78 por cento (1.261/1.561) dos casos foram registrados em menores de cinco anos. A maioria dos casos foi confirmada por cultura, com percentual médio de 65 por cento no ESP e 66 por cento no MSP. As taxas médias de incidência de meningites por Hib mais significativas no período pré-vacinal verificaram-se nos menores de um ano (30,56/105- ESP; 32,06/105 - MSP), considerada a faixa etária de maior risco de adoecimento. Após a introdução da vacina contra o Hib em 1999 (menores de dois anos), as taxas de incidência de meningites por Hib declinaram de forma sustentável nos períodos subsequentes analisados. A incidência de meningite por Hib durante o período pós-vacinal variou de 4,02/105- 1,68/105 nos menores de um ano, no ESP e MSP respectivamente e, de forma similar, de 1,43/105 1,01/105 nos menores de cinco anos. Nos menores de 7 - 23 meses (impacto direto), o percentual de redução foi de 95,11 por cento [66,43 - 99,29] no ESP e 95,91 por cento [70,63 - 99,43] no MSP. O impacto global observado nos menores de cinco anos foi 88,19 por cento [26,58 - 98,10] no ESP e 91,06 por cento [33,99 - 98,79] no MSP. Os dados de vigilância mostram que os casos de meningites por Hib continuam ocorrendo, porém em níveis baixos, ao longo de 10 anos após a introdução do esquema de três doses primárias da vacina conjugada específica. Conclusão: a partir deste racional pode-se inferir a utilidade prática e econômica a favor desta modalidade programática adotada no território paulista, com a evidência de redução relativa de meningites por Hib. / Objectives: To evaluate global impact, direct and indirect, as well as the tendency of the duration of vaccine protection against Haemophilus influenzae type b (Hib) in the state of São Paulo (ESP) and in the city of São Paulo (MSP), amongst the population between 0-59 months of age during the periods pre-vaccine (1996-1998) and post vaccine (2001-2009). Methods: a retrospective study with a descriptive component and with analytic venue. Studied population included children under five years old, dwelling in ESP and MSP. Criteria adopted as definition of confirmed case was child under five years of age identified as positive for Hib in culture and/or counterimmunelectroforesis and/or latex and/or RT/PCR, in LCR sample and blood. and/or epidemiologic link. Data were obtained from the SINAN, SIGH-Web Instituto Adolfo Lutz and IBGE Foundation. Variables under study included socio-demographic and clinical ones, and those related to the agent; they were presented in temporal series and periods established in order to allow parametric and comparison. Impact evaluation was established upon the variation of incidence magnitude of meningitis caused by Hib. For each impact estimate a Confidence Interval (IC) of 95 per cent from the calculus of Relative Risk (RR). Estimates of relative risk (RR) and the respective intervals of 95 per cent confidence were analyzed employing the R software. Results: During the analyzed periods 1561 confirmed cases of meningitis caused by Hib were described in the state of São Paulo, 27.16 per cent of which (424/1561) in MSP; 80.78 per cent (1261/1561) of the registered cases occurred in children under five years of age. The majority of the cases were confirmed by culture, with an average percentage of 65 per cent in ESP and 66 per cent in MSP. More significant average rates of meningitis per Hib during the pre-vaccine period were registered in children under one year of age (30.56/105- ESP; 32.06/105 - MSP), considered the bracket under higher risk of disease. After the introduction of the vaccine against Hib, in 1999 (for children under two years of age), the rates of meningitis incidence per Hib decreased in sustainable fashion for the subsequent periods analyzed. The incidence of meningitis per Hib during the post vaccine period varied from 4.02/105 - 1.68/105 for children under one year old, in ESP and MSP, respectively and, in similar way, from 1.43/105 to 1.01/105 for those under five years old. For children between 7-23 months old (direct impact), the percentage of reduction was of 95.11 per cent [66.43 - 99.29] in ESP and 95.91 per cent [70.63 - 99.43] in MSP, whereas the global impact observed in children under five years old was 88.19 per cent [26.58 - 98.10] in ESP and 91.06 per cent [33.99 98.79] in MSP. Surveillance data show that cases of meningitis by Hib continue to occur but in low levels along 10 years after the introduction of the three doses primary scheme of administration of the specific conjugated vaccine. Conclusion: from this rationale, it is possible to infer the practical and economic utility favoring this programmatic modality adopted in São Paulo, with the evidence of the relative reduction of meningitis caused by Hib.