Introduction: Bordetella pertussis is an important human pathogen that causes whooping cough (pertussis), an endemic illness responsible of significant morbidity and mortality, especially in infants and children. Worldwide, there are an estimated of 16 million cases of pertussis, resulting in about 195,000 child deaths per year. In Peru, pertussis is a major health problem that has been on the increase despite immunization efforts. The objective of this study was to determine the prevalence of B. pertussis among children under five years of age suspected to have whopping cough in Cajamarca, Peru. Methodology: Children diagnosed with whooping cough admitted to the Hospital Regional de Cajamarca from August 2010 to July 2013 were included. Nasopharyngeal samples were obtained for B. pertussis culture and polymerase chain reaction (PCR) detection. Results: In 133 children, the pertussis toxin and IS481 gene were detected in 38.35% (51/133) of the cases by PCR, while only 9.02% (12/133) of the Bordetella cultures were positive. The most frequent symptoms in patients with positive B. pertussis were paroxysm of coughing 68.63% (35/51), cyanosis 56.86% (29/51), respiratory distress 43.14% (22/51), and fever 39.22% (20/51). Pneumonia and acute bronchial obstructive syndrome were present in 17.65% (9/51) and 13.72% (7/51) of the cases, respectively. Conclusions: B. pertussis is responsible for an important proportion of whooping cough in hospitalized children in Cajamarca. Epidemiologic surveillance programs for B. pertussis are essential in Peru, especially in children who could most benefit from the vaccine.

Das Tex Protein aus Bordetella pertussis definiert eine neue Familie hoch konservierter Proteine in Eubakterien. Ursprünglich wurde das tex Gen aufgrund seines Einflusses auf die Toxinexpression in bestimmten regulatorischen Mutanten von B. pertussis gefunden (Fuchs et al. (1996), J Bacteriol 178, 4445-52). Wie hier gezeigt wird, sind Leserahmen für entsprechende Proteine bei den Eubakterien ubiquitär und mehrheitlich zu über 69 Prozent konserviert. Eine Ausnahme bilden einige wenige Taxa mit bekanntermaßen reduzierten Genomen, bei denen das Gen wahrscheinlich verloren gegangen ist, wie zum Beispiel verschiedene Mycoplasma spp. oder der obligate Blattlaus- (Aphiden-) Symbiont Buchnera aphidicola. In Eukaryonten und Archaeen konnte ein zu tex homologes Gen bisher nicht gefunden werden. Die Funktion von Tex in der Bakterienzelle ist unklar. Während das Gen in B. pertussis essenziell ist und auch nicht überexprimiert werden kann, sind Deletionsmutanten in Neisseria meningitidis und Escherichia coli phänotypisch nicht von den entsprechenden Wildtypen unterscheidbar. Ausgiebige Wachstumsstudien mit einer E. coli tex-Mutante unter verschiedenen Wachstums- und Stressbedingungen ergaben ebenfalls keinen Hinweis auf eine Bedeutung von Tex, die die außerordentliche Konservierung des Proteins erklären könnte. Das Protein zeigt in seinem N-Terminus ausgeprägte Ähnlichkeit mit dem Mannitol-Repressor (MtlR) von Escherichia coli und besitzt eine C-terminale S1-Domäne. Da die meisten der Proteine mit S1-Domänen als RNA-bindende Proteine gelten, wurde die Fähigkeit von Tex untersucht, mit Nukleinsäuren zu interagieren. In Festphasen-Bindeassays mit an Magnetkügelchen immobilisiertem Tex Protein aus E. coli konnte eine spezifische Bindung an RNA-Gesamtpräparationen gezeigt werden. DNA wurde hingegen nicht gebunden. Durch Verkürzung des N-Terminus geht die präferenzielle Bindung an RNA jedoch verloren und die Bindung von DNA erfolgt mit der gleichen Effizienz wie die von RNA. Festphasen-Bindeassays wurden weiterhin dazu benutzt, mögliche spezifische Interaktionspartner von Tex aus RNA-Gesamtpräparationen zu finden. Tatsächlich konnten über diesen Ansatz die regulatorische RNA CsrB und die ribosomale 16S RNA als spezifische Liganden isoliert werden. Über die biologische Relevanz dieser Interaktion kann zum gegenwärtigen Zeitpunkt allerdings noch keine Aussage gemacht werden. / The Bordetella pertussis Tex protein defines a new family of highly conserved eubacterial proteins. The tex gene was originally found due to its negative influence on toxin expression when over-expressed slightly in certain regulatory mutants of B. pertussis (Fuchs et al. (1996), J Bacteriol 178, 4445-52). As shown in this work apparently homologous reading frames are ubiquitous within the Eubacteria. The majority of these mostly putative proteins are more than 69 per cent conserved. Only some of the genomes of some obligate intracellular pathogens, e. g. various Mycoplasma ssp., or the aphid symbiont Buchnera aphidicola known for their reduced gene numbers were found to lack a tex gene and probably have lost it during phylogeny. The actual function of Tex within the bacterial cell is as yet mysterious as the gene can neither be deleted nor can it be highly over-expressed in B. pertussis. However, deletion mutants in Neisseria gonorrhoeae and Escherichia coli do not show any phenotype different to the respective wild type. Extensive growth studies on an E. coli tex-mutant did not reveal any clue to the reason for the extraordinary conservation of the protein. The N-terminus of Tex shows extensive similarity to the mannitol repressor (MtlR) of E. coli whereas the C-terminus contains an S1 domain. Because most S1 domain containing proteins have been shown to bind RNA, the capacity of Tex to interact with nucleic acids was investigated. In solid phase binding assays with purified Tex protein from E. coli immobilized to magnetic beads the protein showed specific binding of RNA but not DNA. NÓterminal deletions of different length abolished this preference for RNA and both types of nucleic acid bound equally well. Again a solid phase binding assay was used to enrich for specific ligands of Tex from total cellular RNA preparations. This approach identified the regulatory CsrB RNA and ribosomal 16S RNA as preferential binding targets. However, it is not yet possible to comment on the biological relevance of these findings.

Objectives To report the incidence, epidemiology, and clinical features of Bordetella pertussis in Peruvian infants under 1 year old. Patients and methods A prospective cross-sectional study was conducted in five hospitals in Peru from January 2010 to July 2012. A total of 392 infants under 1 year old were admitted with a clinical diagnosis of whooping cough and tested for B. pertussis by PCR. Results The pertussis toxin and IS481 genes were detected in 39.54% (155/392) of the cases. Infants aged less than 3 months were the most affected, with a prevalence of 73.55% (114/155). The most common household contact was the mother, identified in 20% (31/155) of cases. Paroxysm of coughing (89.03%, 138/155), cyanosis (68.39%, 106/155), respiratory distress (67.09%, 104/155), and breastfeeding difficulties (39.35%, 61/155) were the most frequent symptoms reported. Conclusion An increase in pertussis cases has been reported in recent years in Peru, despite national immunization efforts. Surveillance with PCR for B. pertussis is essential, especially in infants less than 1 year old, in whom a higher rate of disease-related complications and higher mortality have been reported. / This 312 work was supported by Sanofi Aventis del Peru. Conflict 313 of interest: On behalf of all authors, the corresponding author 314 states that there are no conflicts of interest or funding related 315 to this study

Bordetellen sind Gram-negative Kokkobazillen, die phylogenetisch zu den β-Proteobakterien zählen und in der Familie der Alcaligenaceae eingeordnet sind. Der bedeutendste Vertreter der Gattung, die nach heutigem Kenntnisstand neun Arten umfasst, ist Bordetella pertussis, der Erreger des Keuchhustens. Der Keim ist obligat humanpathogen und besitzt zahlreiche Virulenzfaktoren, um die Epithelzellen des Respirationstraktes zu besiedeln und zu zerstören, wodurch es zu dem charakteristischen Krankheitsverlauf kommt. Neben B. pertussis werden noch B. bronchiseptica und B. parapertussis dem sogenannten B. bronchiseptica-Cluster zugeteilt. Alle Vertreter des B. bronchiseptica-Clusters sind in der Lage, bei verschiedenen Wirtsspezies respiratorische Erkrankungen mit unterschiedlichem Schweregrad auszulösen. Dabei weist B. bronchiseptica ein breiteres Wirtsspektrum auf und kann Atemwegserkrankungen in einer Vielzahl von Säugetieren auslösen, wohingegen B. parapertussis vornehmlich Schafe und Menschen infiziert und bei letzteren eine schwächere Form des Keuchhustens bewirkt. Das Hfq-Protein wurde ursprünglich als Wirtsfaktor identifiziert, welcher für die Replikation des RNA-Phagen Qβ in Escherichia coli benötigt wird (host factor for Qβ oder HF-1). Es ist in Struktur und Funktion homolog zu den Sm-Proteinen aus Eukaryoten, die am Splicing von mRNAs involviert sind. Die Beteiligung des Hfq-Proteins an regulatorischen Vorgängen, die durch kleine nicht-kodierende RNAs (sRNAs) vermittelt werden, wurde erstmals in einer Studie zum Mechanismus der rpoS-Regulation durch die kleine regulatorische RNA OxyS ersichtlich. Seitdem konnte für eine Vielzahl an sRNAs gezeigt werden, dass sie an Hfq gebunden vorliegen und die Hilfe des Proteins bei der post-transkriptionellen Kontrolle ihrer Ziel-mRNAs benötigen. In dieser Hinsicht übernimmt Hfq die Rolle eines RNA-Chaperons, indem es trans-kodierte sRNAs stabilisiert und die Basenpaarung mit ihren Ziel-mRNAs fördert. Dabei beeinflusst die Bindung der sRNA-Regulatoren an ihre Ziel-mRNAs deren Translation, sowohl aktivierend als auch inhibierend. Bislang wurden Hfq-Homologe in der Hälfte aller sequenzierten Gram-positiven und Gram-negativen Bakterienarten gefunden. Eine BLAST-Analyse ergab, dass B. pertussis und B. bronchiseptica Homologe zum Hfq-Protein aufweisen und diese in der veröffentlichten Genomsequenz bereits als Hfq-Protein annotiert sind. Fokus dieser Arbeit war weitestgehend, die Funktion des Hfq-Proteins in B. pertussis und vergleichend in B. bronchiseptica zu charakterisieren. Mittels Primer Extension-Analyse konnte zunächst der Startpunkt des hfq-Transkripts in B. pertussis und B. bronchiseptica unter logarithmischen Wachstumsbedingungen bestimmt werden. Dieser Startpunkt war zudem unter stationären Wachstumsbedingungen und nach Hitzestress aktiv, was in Diskrepanz zur Beobachtung in E. coli steht. Ferner konnte festgestellt werden, dass die hfq-Transkription nach Induktion verschiedener Stressformen in beiden Organismen erhöht war. Nach Generierung der jeweiligen Δhfq-Mutanten in beiden Organismen wurden diese charakterisiert. Die B. pertussis Δhfq-Mutante zeigte ein deutliches Wachstumsdefizit gegenüber dem Wildtyp, im Gegensatz zu B. bronchiseptica Δhfq, die sich im Wachstum wie der Wildtyp verhielt. Beide Mutanten zeigten sich sensitiver gegenüber H2O2-Stress als der Wildtyp, nicht jedoch gegenüber weiteren oxidativen Stressbedingungen oder Membranstress induzierenden Substanzen. Die Δhfq-Mutante in B. pertussis war zudem in ihrer Fähigkeit zur Biofilmbildung beeinträchtigt, was jedoch nicht für B. bronchiseptica Δhfq galt. Da Hfq an sRNA-mRNA-Interaktionen, welche die Translation der mRNAs beeinflussen, beteiligt ist, sollte über 2D-Gelelektrophorese das Hfq-regulierte Proteom in B. pertussis und B. bronchiseptica bestimmt werden. Auffällig war, dass viele periplasmatische Transport-bindeproteine von der Δhfq-Mutation betroffen waren. Es zeigten sich aber auch Stoffwechselenzyme und wichtige Housekeeping-Faktoren, wie z. B. der Elongationsfaktor EF-Tu und das Chaperon GroEL, in der Δhfq-Mutante dereguliert. Generell scheint das Hfq-regulierte Proteom in B. pertussis und B. bronchiseptica nur einen kleinen Teil des gesamten Proteoms auszumachen. Zudem ist das Hfq-regulierte Proteom variabel zwischen verschiedenen Wachstumsbedingungen, aber auch zwischen den beiden Organismen trotz der engen Verwandtschaft. Die Expression ausgewählter Virulenzfaktoren zeigte keinen Unterschied zwischen Δhfq-Mutante und B. pertussis-Wildtyp. / Bordetellae are Gram-negative coccobacilli phylogenetically belonging to the β-group of proteobacteria and therein to the family of Alcaligenaceae. The most prominent member of the genus comprising nine species so far is Bordetella pertussis, the etiological agent of whooping cough. This organism is an obligatory human pathogen and expresses a variety of virulence factors in order to colonize and destroy the epithelial cells of the respiratory tract causing the characteristic symptoms of the disease. In addition to B. pertussis, B. bronchiseptica and B. parapertussis are assigned to the so-called B. bronchiseptica-cluster. All members of the B. bronchiseptica-cluster have the ability to cause respiratory symptoms with varying severity. B. bronchiseptica exhibits a broad host range causing respiratory symptoms in a variety of mammals, whereas B. parapertussis infects sheep and humans causing a milder form of whooping cough in the latter. The Hfq protein was originally identified as a host factor necessary for the replication of the RNA-phage Qβ in Escherichia coli (host factor for Qβ or HF-1). It is functionally and structurally homologous to Sm-proteins involved in splicing of mRNAs in eukaryotes. The involvement of Hfq in regulatory processes caused by small non-coding RNAs (sRNAs) was first recognized in a study on the mechanism of rpoS-regulation by the small regulatory RNA OxyS. Since then a variety of sRNAs were shown to be bound to Hfq and require its help for post-transcriptional control of their target-mRNAs. In this regard, Hfq functions as an RNA-chaperone by stabilizing trans-encoded sRNAs and their basepairing to target-mRNAs. Binding of the sRNA-regulators to their target-mRNAs thereby either activates or inhibits their translation. To date Hfq homologues were identified in half of all sequenced Gram-positive and Gram-negative bacterial species. BLAST analysis revealed that B. pertussis and B. bronchiseptica possess an Hfq homologue which has already been annotated as such in the published genome sequence. The main focus of this work was to characterize the function of the Hfq protein in B. pertussis as well as in B. bronchiseptica. By primer extension analysis we could identify the start of the hfq-transcript in B. pertussis and B. bronchiseptica under logarithmic growth conditions. This transcriptional start site was also active under stationary growth conditions and after heat shock which is discrepant from the observations in E. coli. Furthermore, it could be shown that the hfq-transcription was elevated in both B. pertussis and B. bronchiseptica under various stress conditions. Δhfq-mutants were established and characterized in both organisms. The Δhfq-mutant of B. pertussis exhibited a pronounced growth deficit in comparison to the wildtype whereas the Δhfq-mutant of B. bronchiseptica showed the same growth properties as the wildtype. Both Δhfq-mutants expressed a higher sensitivity to stress caused by H2O2 compared to the wildtype. However, there was no increased sensitivity of the Δhfq-mutants to other oxidative stress agents or membrane stress inducing agents. Furthermore, the Δhfq-mutant of B. pertussis but not the Δhfq-mutant of B. bronchiseptica was impaired in its ability to form biofilms. Since Hfq is involved in sRNA-mRNA-interactions affecting the efficient translation of mRNAs, the Hfq-regulated proteome of B. pertussis and B. bronchiseptica was determined by 2D-gelelectrophoresis. Strikingly, a variety of periplasmic binding proteins involved in transport were affected by the Δhfq-mutation. In addition, enzymes of various metabolic pathways and important housekeeping factors, such as elongation factor EF-Tu and the protein chaperone GroEL, were deregulated in the Δhfq-mutant. The Hfq-regulated proteome comprises generally only a small part of the complete proteome in B. pertussis and B. bronchiseptica. Furthermore, this Hfq-regulated proteome differs between certain growth conditions as well as between the two closely related organisms. No difference could be observed in the expression of selected virulence factors between B. pertussis Δhfq and wildtype.

Objetivos generales de la tesis: Con el desarrollo de esta propuesta se espera contribuir al conocimiento que sirva de base para el diseño de una vacuna más efectiva contra pertussis, no sólo en términos generales, sino en lo que se refiere a su efectividad en Argentina, determinando la definición de la/s cepas / componentes a incluir en una nueva formulación. Objetivos específicos de la tesis: En este marco conceptual y tomando como hipótesis la divergencia de la población bacteriana circulante respecto a las cepas vacunales hoy en uso, se proponen los siguientes objetivos: 1-Caracterizar mediante la aplicación de técnicas de genómica funcional los aislamientos de B. pertussis locales. Identificar de nuevos inmunógenos. Los aislamientos clínicos de nuestra colección han sido previamente agrupados en base a sus divergencias a nivel de las secuencias que codifican para antígenos específicos, así como en su genoma completo a través de los ensayos de PFGE y los años en que fueron aislados. Proponemos abordar la búsqueda de diferencias específicas, a nivel de expresión génica, entre los aislamientos circulantes y las cepas que hoy se usan en la producción de vacunas. Para ello hemos emplearemos herramientas de genómica funcional, proteómica e inmunoblot; para identificar potenciales candidatos vacunales a incorporar en una formulación acelular. 2-Analizar la relevancia de la divergencia genética entre la Población Bacteriana Circulante (PBC) y las cepas vacunales respecto a la protección contra pertussis. Para abordar este punto emplearemos el modelo animal de desafío intranasal. Consideramos que la información obtenida marcará la necesidad o no de incluir determinadas variantes polimórficas para obtener una nueva vacuna más efectiva. El abordaje de este aspecto se espera también contribuya al conocimiento general de la adaptación y evolución bacteriana bajo la presión de selección ejercida por las diferentes estrategias de vacunación (celular/acelular). / Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).

No description available.

University, Diss., 2006--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

Würzburg, Univ., Diss., 2001. / Erscheinungsjahr an der Haupttitelstelle: 2001

Objective: Describe the prevalence of Bordetella pertussis via PCR in children under 5 years old hospitalized as probable cases of pertussis and report the most common clinical features among them. Results: A positive PCR result for B. pertussis was observed in 20.5% of our samples (18/88), one-third of them were from infants between 2 and 3 months old. The most common symptoms were paroxysms of coughing (88.9%), difficulty breathing (72.2%), cyanosis (77.8%) and fever (50%). The mother was the most common symptomatic carrier (27.8%), followed by uncles/aunts (22.2%) among children with pertussis. / This work was supported by fourth research incentive of the Universidad Peruana de Ciencias Aplicadas (UPC), Lima‑Peru. / Revisión por pares

Made available in DSpace on 2016-03-28T12:41:00Z (GMT). No. of bitstreams: 2 diego_cambuy_ioc_mest_2014.pdf: 2689625 bytes, checksum: 9a0d86cf1fe73771c946b0bd687aec4b (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A coqueluche, ou pertússis, é uma doença do trato respiratório causada principalmente pela bactéria Bordetella pertussis. Após 50 anos de vacinação, pertussis reemergiu, passando a ser a doença imunoprevinível mais frequente mesmo em países desenvolvidos. Várias são as hipóteses para a reemergência de pertússis, uma delas é a adaptação do patógeno frente à vacinação. Linhagens contemporâneas de B. pertussis diferem de linhagens do período pré-vacinal, especialmente em genes codificadores de proteínas usadas na produção de vacinas acelular. Esta re-emergência também tem sido observada no Brasil, assim, realizamos a caracterização genética por MLST baseado nesses genes, de 26 isolados B. pertussis de surtos de três regiões brasileiras (Norte, Sul e Nordeste). Foram identificados dois perfis alélicos, em 24 isolados: prn2-ptxS1A-fim3B-ptxP3, de surtos (2008-2013) de Alagoas, Pernambuco e Rio Grande do Sul - e o perfil prn2-ptxS1A-fim3A-ptxP3 , em dois isolados de Pará/2004. Análises filogenéticas agruparam esses perfis com isolados do período pós vacinal de outras partes do globo. Deste conjunto, três do perfil mais frequente e um do perfil menos frequente, tiveram seus genomas sequenciados na plataforma GS 454 Junior. A comparação desses genomas com outros genomas de B. pertussis disponíveis em dados públicos não identificou SNPs ou genes únicos que caracterizassem os isolados do Brasil Este estudo desenvolveu uma metodologia que permitiu definir a posição da IS481 nos genomas, e uma delas corresponde a um gene relacionado a regulação da transcrição da família MarR, Análise filogenômica, baseada em 826 SNPs, demonstrou que os isolados recentes do Brasil da linhagem pandêmica que presente em todos os continentes, exceto a África. Foi observado também que as relações filogenéticas inferidas pelo MLST são semelhantes àquelas inferidas quando se utiliza o genoma completo, isso denota a pressão seletiva sobre esses genes. Sendo assim, a cepa utilizada na produção da vacina no Brasil, que apresenta o perfil alélico prn1-ptxS1D - fim3A-ptxP2, pode não ser capaz de gerar uma resposta imune protetora frente às linhagens circulantes no país. Este estudo traz, pela primeira vez, informações genéticas e genômicas de isolados de B. pertussis do Brasil, país que apresenta cobertura vacinal bastante heterogênea, que utiliza, oficialmente, a vacina celular, mas que, também, aplica a vacina acelular. As informações reveladas neste estudo podem auxiliar a tomada de ações para o controle de pertússis no Brasil, além do conhecimento sobre epidemiologia e evolução de B. pertussis / Pertussis more commonly referred as whooping cough is respiratory tract disease mainly caused by the bacteria B. pertussis. After 50 years of vaccination pert ussis remerged, becoming the most frequent vaccine preventable disease in developed countries. Many hypotheses have been proposed for the re - emergence of pertussis, one being the pathogen adaptation in a vaccinated environment. Current pertussis strains ar e different than those from the prevaccination era, especially in genes that code for proteins used in acelluar pertussis vaccines. This re - emergence is also observed in Brazil, therefore we characterized 26 isolates from 3 regions of Brazil (North,South,N ortheast) using an MLST approach based on these genes. We identified two allelic profiles, 24 isolates from the states of Rio Grande do Sul (2008 - 2009), Alagoas (2008 - 2009), Pernambuco (2013) and Pará (2004) presented the prn2 - ptxS1A - fim3B - ptxP3 allelic pr ofile, while 2 isolates from Pará (2004) presented the prn2 - ptxS1A - fim3A - ptxP3 allelic profile. Phylogenetic analysis branch these two allelic profiles along with other post vaccination isolates around the globe. Four isolates, three from the dominant prof ile and one from the less frequent profile, had their genomes completed sequenced on the GS 454 Junior Platform. We compared these genomes with others available in public databases and no SNP or unique genes were identified in the Brazilian genomes. This s tudy also developed a methodology that identifies the location of the repetitive region IS481, and what genes it interrupted. One of them was the MarR transcriptional regulator gene. Phylogenomic analysis based on 826 SNPs revealed that Brazilian B. pertus sis lineages are part of the current pandemic linage present in all continents, except Africa. We also observed that phylogenomic relationships are similar to MLST’s. Therefore, strain used for pertussis vaccine in Brazil, that presents the prn1 - ptxS1D - f im3A - ptxP2 allelic profile, might not be able to induce immune response to the current linage circulating in the country. This is the first study with genetic and genomic informations of B. pertussis isolates in Brazil, which is a country with heterogeneou s vaccine coverage and mixed and has both cellular and acellular vaccine administrated to the population. Information brought with this study can help the decision making on the control of pertussis in Brazil and gives new insights on the epidemiology and evolution of B. pertussis