Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2018.tde-10042018-141217
Document
Author
Full name
Emanuel Capistrano Costa Junior
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2017
Supervisor
Committee
Tamashiro, Edwin (President)
Santana, Rodrigo de Carvalho
Massuda, Eduardo Tanaka
Romano, Fabrizio Ricci
Sakano, Eulália
Title in Portuguese
Avaliação da associação entre biofilmes bacterianos, bactérias intracelulares e superantÃgenos estafilocócicos em pacientes com rinossinusite crônica
Keywords in Portuguese
Bactéria intracelular
Biofilme
Pólipo nasal
Rinossinusite crônica
Staphylococcus aureus
SuperantÃgeno
Abstract in Portuguese
Introdução: Embora a fisiopatogenia da rinossinusite crônica (RSC) ainda não esteja totalmente elucidada, em virtude da sua heterogeneidade e multifatorialidade, existe um crescente corpo de evidências apontando que as bactérias exerçam um papel significativo na gênese ou perpetuação da inflamação crônica. Uma das possÃveis formas de atuação são os biofilmes bacterianos, comumente encontrados em pacientes com RSC e que estão relacionados com má evolução clÃnica. Ainda, existem evidências de que algumas espécies bacterianas, especialmente o Staphylococcus aureus (S. aureus), são capazes de invadir as células epiteliais e permanecerem viáveis em seu interior. Por fim, tem se demonstrado que pacientes RSC com pólipo nasal (RSCcPN) revelam alta associação com a presença de superantÃgenos estafilocócicos na mucosa respiratória, responsáveis pela estimulação acentuada de respostas inflamatórias locais. Apesar de essas diferentes formas bacterianas estarem bem descritas na RSC, não se sabe ainda com clareza como elas estão associadas nesses indivÃduos. Objetivos: Avaliar a associação entre a presença de biofilmes, bactérias intracelulares e superantÃgenos estafilocócicos em pacientes com RSC (com e sem pólipo nasal), comparados com o grupo controle. CasuÃstica e Métodos: Avaliou-se a prevalência de biofilmes bacterianos, bactérias intracelulares e presença de superantÃgenos bacterianos em indivÃduos com RSCcPN, sem pólipo nasal (RSCsPN) e controles, analisando a associação de distribuição de prevalência desses diferentes grupos (teste exato de Fisher, nÃvel de significância quando p<0,05). Os biofilmes foram definidos por caracterÃsticas morfológicas à microscopia eletrônica de varredura (MEV), as bactérias intracelulares foram analisadas por microscopia eletrônica de transmissão (MET) e hibridização fluorescente in situ (FISH) para S. aureus, e superantÃgenos de S. aureus A-E foram quantificados pela técnica de ELISA (Enzime Linked Imunosorbent Assay). Foram incluÃdos 90 indivÃduos, divididos em três grupos: 1) 38 pacientes com RSCcPN, 2) 26 com RSCsPN e 3) 26 controles. Resultados: Quarenta e dois por cento dos pacientes com RSCcPN (16/38), assim como os com RSCsPN (11/26) apresentaram amostras positivas para biofilmes bacterianos, mas não observou essa positividade no grupo controle (0/26). A análise para bactérias intracelulares demonstrou a presença em 31,5% de pacientes com RSCcPN (12/38), 19,2% em RSCsPN (5/26) e 0% nos controles (0/26). No estudo por FISH, 58% dos pacientes com RSCcPN (18/31) apresentaram positividade para S. aureus intracelular, seguido de 54% nos com RSCsPN (13/24) e em nenhum caso dos 24 analisados do grupo controle. Na avaliação por ELISA, apenas um paciente com RSCcPN foi positivo para a presença de superantÃgenos estafilocócicos. A avaliação da associação de biofilme bacteriano na superfÃcie mucosa à MEV com bactéria intracelular à MET e com S. aureus intracelular por FISH nos dois diferentes grupos de RSC com e sem pólipo nasal, não mostrou diferença estatisticamente significativa. Conclusão: Foi observada uma maior prevalêcia de biofilmes e bactérias intracelulares em indivÃduos com RSC com ou sem pólipo nasal, comparado a Resumo controles. Não houve diferença significativa dentre os grupos de RSC, com e sem pólipo nasal para a presença de biofilmes e bactérias intracelulares. Não houve associação entre a presença de biofilme e bactéria intracelular em pacientes com RSC. Os achados do presente estudo indicam que tanto biofilmes na superfÃcie mucosa quanto microrganismos intracelulares podem estar envolvidos na fisiopatogenia da RSC.
Title in English
Evaluation of the association between bacterial biofilms, intracellular bacteria and staphylococcal superantigens in patients with chronic rhinosinusitis
Keywords in English
Biofilm
Chronic rhinosinusitis
Intracellular bacteria
Nasal polyp
Staphylococcus aureus
Superantigen
Abstract in English
Introduction: Although the pathophysiology of chronic rhinosinusitis (CRS) has not yet been fully elucidated, due to its heterogeneity and multifactorial etiology, there is a growing body of evidence that bacteria play a significant role in the genesis or perpetuation of chronic inflammation. One of the possible forms of acting are bacterial biofilms, which are commonly found in patients with CRS, and are associated with poor clinical outcomes in these patients. In addition to biofilms, there are some evidence pointing out that some bacterial species, especially Staphylococcus aureus (S. aureus), are able to invade into epithelial cells and remain viable intracellulary. Finally, it has been demonstrated that patients with CRS with nasal polyps (CRSwNP) have a high association with the presence of staphylococcal superantigens in the respiratory mucosa, responsible for the stimulation of marked local inflammatory responses. Although these different bacterial forms are well described in CRS, it is still unclear how they are associated in these individuals. Objectives: To evaluate the correlation between the presence of biofilms, intracellular bacteria expression and S. aureus superantigens in CRS patients (with and without nasal polyposis) compared to a control group. Casuistic and Methods: We evaluated the prevalence of bacterial biofilms, intracellular bacteria and the presence of bacterial superantigens in individuals with CRSwNP, without nasal polyp (CRSsNP) and controls, evaluating the association of prevalence distribution of these different groups (Fisher exact test, level of significance set at p<0.05). The biofilms were defined by morphological characteristics by scanning electron microscopy, intracellular bacteria were analyzed by transmission electron microscopy and fluorescence in situ hybridization (FISH) for S. aureus, and S. aureus A-E superantigens were quantified by ELISA. Ninety individuals were included, divided into 38 patients with CRSwNP, 26 patients with CRSsNP and 26 control patients. Results: 42% of patients with CRSwNP (16/38) as well as those with CRSsNP (11/26) presented positive samples for bacterial biofilms, while none of the control patients (0/26) had positive samples. The analysis for intracellular bacteria showed the presence in 31.5% of patients with CRSwNP (12/38), 19.2% in CRSsNP (5/26) and 0% in control patients (0/26). In the FISH study, 58% of patients with CRSwNP (18/31) presented intracellular S. aureus positivity, followed by 54% in patients with CRSsNP (13/24) and in none of the 24 analyzed in the control group. In the ELISA evaluation, only one patient with CRSwNP was positive for the presence of staphylococcal superantigens. The evaluation of the association of bacterial biofilm on the mucosal surface (SEM) with intracellular bacteria (MET) and with intracellular S. aureus by FISH in the two different groups of CRS (with and without nasal polyps) did not show a statistically significant difference. Conclusion: We found a higher prevalence of biofilms and intracellular bacteria in individuals with CRS, either with and without nasal polyps. There was no significant difference between the groups of CRS, with and without nasal polyp, for the presence of biofilms or intracellular bacteria. There was no significant diference on the association of biofilms and intracellular bacteria on pacientes with CRS. Our data indicate that both biofilms on the mucosal surface and intracellular microorganisms may be involved in the pathophysiology of CRS.
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Publishing Date
2018-07-13