Porphyromonas gingivalis exacerbates ulcerative colitis via Porphyromonas gingivalis peptidylarginine deiminase / 国际口腔科学杂志·英文版

Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.

Presencia de Escherichia coli intracelular en mucosa intestinal de pacientes con Enfermedad Inflamatoria Intestinal y su asociación con características clínicas y el uso de corticosteroides / Presence of intracellular Escherichia coli in patients with inflammatory bowel disease

Background: Different strains of invasive Escherichia coli (E. coli), isolated from intestinal mucosa of patients, are related to the pathogenesis of inflammatory bowel diseases (IBD). Aim: To evaluate an association between intracellular E. coli and IBD; its clinical characteristics and use of steroids. Material and Methods: Sixty one patients with Crohn's disease and 83 with ulcerative colitis were studied. To determine the intracellular E. coli content, colonoscopy biopsies of these patients and 29 control subjects were processed using the gentamicin protection assay. Differences in the bacterial content between patient groups were evaluated using Mann-Whitney test, while the association between presence of E. coli with endoscopic activity, location/extension and use of corticosteroid as anti-inflammatory treatment were evaluated with Fisher's exact test or Chi-square test. Results: E. coli strains were detected in 36.1, 39.3 and 10.3% of patients with ulcerative colitis, Crohn's disease and controls, respectively. The number of bacteria per biopsy in Crohn's disease and ulcerative colitis was significantly higher than in controls (p < 0.01 between patients and controls). In ulcerative colitis, significant associations were found between the presence of bacteria and disease location and use of corticosteroids. In Crohn's disease, no association was found. Conclusions: IBD are associated with the presence of intracellular E. coli strains in the intestinal mucosa, suggesting an alteration in the microbiota or loss of integrity of the epithelial barrier. The association of intracellular E. coli with clinical features and the use of corticosteroids in ulcerative colitis suggests that different factors could promote colonization or proliferation of these bacteria.

Presence of Mycobacterium avium subsp. paratuberculosis (MAP) in Brazilian patients with inflammatory bowel diseases and in controls / Presença de Mycobacterium avium subsp. paratuberculosis (MAP) em pacientes brasileiros com doença inflamatória intestinal e em controles

CONTEXT AND OBJECTIVE: Mycobacterium avium subsp. paratuberculosis (MAP) has attracted the interest of researchers because of similarities between paratuberculosis and Crohn's disease (CD). The aim of this study was to evaluate the frequency of MAP through cultures, histology and polymerase chain reaction (PCR) on intestinal biopsies from Brazilian CD patients. Quantitative real time PCR (qRT-PCR) was performed on positive samples. DESIGN AND SETTING: Analytical cross-sectional study with control group at two federal universities. METHODS: Fresh samples were collected from 25 patients; five with CD, eight with ulcerative colitis (UC) and 12 controls with non-inflammatory bowel disease (nIBD). Formalin-fixed paraffin-embedded (FFPE) samples from 143 patients were also collected: 44 CD, 49 UC and 56 nIBD. RESULTS: None of the fresh samples was positive for MAP. Five FFPE samples (one CD, two UC and two nIBD) and three fresh samples (one in each group) were positive through IS900-PCR. qRT-PCR was performed on these eight samples. Among the FFPE samples, there were 192.12 copies/μl in the CD group, 72.28 copies/μl in UC and 81.43 copies/μl in nIBD. Among the fresh samples, there were 432.99 copies/μl, 167.92 copies/μl and 249.73 copies/μl in the CD, UC and nIBD groups, respectively. The highest bacterial load was in the CD group. CONCLUSION: This study does not provide evidence for a role of MAP in the etiology of CD, although MAP DNA was detected in all three patient groups. This is the first report of MAP presence in human intestinal biopsies in Brazil.

CONTEXTO E OBJETIVO: Mycobaterium avium subsp. paratuberculosis (MAP) tem atraído o interesse de pesquisadores devido às semelhanças entre a paratuberculose e a doença de Crohn (CD). Este estudo objetivou avaliar a frequência de MAP por meio de cultura, histologia e reação da polimerase em cadeia (PCR), em biópsias intestinais de pacientes brasileiros com CD. PCR quantitativa em tempo real (qRT-PCR) foi realizada nas amostras positivas. TIPO DE ESTUDO E LOCAL: Estudo transversal analítico com grupo controle realizado em duas universidades federais. MÉTODOS: Amostras frescas foram coletadas de 25 pacientes; cinco com CD, oito com colite ulcerativa (UC) e 12 controles sem doença inflamatória intestinal (nIBD). Também foram coletadas 149 amostras fixadas em parafina (FFPE): 44 CD, 49 UC e 56 nIBD. RESULTADOS: Nenhuma das amostras frescas foi positiva para MAP. Cinco amostras FFPE (uma CD, duas UC e duas nIBD) e três amostras frescas (uma de cada grupo) foram positivas por IS900-PCR. qRT-PCR foi realizada nessas oito amostras. Nas amostras FFPE, havia 192,12 cópias/μl no grupo CD, 72,28 cópias/μl no UC e 81,43 cópias/μl no nIBD. Nas amostras frescas, havia 432,99 cópias/μl, 167,92 cópias/μl e 249,73 cópias/μl nos grupos CD, UC e nIBD, respectivamente. A maior carga bacteriana foi encontrada no grupo CD. CONCLUSÃO: Este estudo não fornece evidências do papel de MAP na etiologia da CD, embora DNA de MAP tenha sido detectado em pacientes dos três grupos. Este é o primeiro relato da presença de MAP em biópsias intestinais humanas no Brasil.

Trasplante de microbiota fecal en paciente con colitis ulcerosa e infección refractaria por: revisión de la literatura a partir de un caso clínico / Fecal microbiota transplantation in patient with ulcerative colitis and refractory infection due to: review of the literature based on a clinical case

Infection (CDI) is increasing both in the hospital environment as in the outpatient setting, and is associated with prior use of antibiotics, hospitalizations and inflammatory bowel disease (IBD), among others. It is also characterized by a high rate of recurrence with the usual antibiotic treatment, which increases with greater number of episodes, reaching up to 65 percent. In this context, the transplantation of fecal microbiota (FMT) emerges as recurrent CDI therapy, achieving success rates exceeding 90 percent, including in IBD patients, with minimum rates of recurrence. To achieve such efficiency, the colonization by the donated microbiota in the recipient is critical. The role of FMT is still unclear in IBD therapy not associated with CDI. Although there are great differences in the methodology of FMT, the process has been standardized even creating banks of frozen fecal samples, without reducing its effectiveness. FMT is a safe procedure, without serious adverse events, and accepted by the potential beneficiary population. There are few reported cases of refractory CDI management with FMT. Since 2012, the FMT in CDI and IBD publications have increased significantly, but in our country there are only few reports of this therapeutic strategy. We present a patient with ulcerative colitis and conventional antimicrobial management resistant CDI, which was successfully treated with FMT in a public hospital in Chile.

La infección por Clostridium difficile (ICD) está en aumento tanto en el ambiente hospitalario como ambulatorio, y se asocia a uso previo de antibióticos, hospitalización y enfermedades inflamatorias intestinales (EII), entre otros. Se caracteriza además por su alta tasa de recurrencia con el tratamiento antimicrobiano habitual, que aumenta con el mayor número de episodios alcanzando hasta 65 por ciento. En este contexto, el trasplante de microbiota fecal (TMF) surge como terapia para la ICD recurrente, logrando tasas de éxito superiores a 90 por ciento, incluyendo pacientes con EII, con mínimas tasas de recurrencia. Para lograr esa eficacia, la colonización por la microbiota donada en el receptor es fundamental. Aún no está claro el rol del TMF en la terapia de EII no asociada a ICD. Aunque existe gran heterogeneidad en la metodología del TMF, el proceso se ha ido estandarizando incluso hasta llegar a la creación de bancos de muestra fecal congelada, sin disminuir su efectividad. El TMF es un procedimiento seguro, sin eventos adversos graves y aceptado por la población potencialmente beneficiaria de él. Existen pocos casos publicados de manejo de ICD refractaria con TMF. Desde el 2012 el número de publicaciones sobre TMF en ICD y en EII ha aumentado considerablemente, sin embargo, en nuestro país los reportes sobre esta estrategia terapéutica son escasos. Presentamos el caso de un paciente con colitis ulcerosa e ICD refractaria al manejo antimicrobiano habitual, que se trató exitosamente con TMF en un hospital público de Chile.

Colitis y trasplante de microflora intestinal: ¿cuándo plantearlo? / Colitis and fecal microbiota transplantation: who benefits?

The gastrointestinal microbiota acts as a metabolic organ that provides enzymatic pathways, contributes to the development and maintenance of local and systemic lymphoid organs, regulates the homeostasis of the intestinal epithelial barrier, modulates the systemic inflammatory and metabolic processes and activates the immune system, providing protection against bacterial and viral agents. Clostridium difficile diarrhea is the leading cause of nosocomial diarrhea with high morbidity and mortality rates. This occurs, among other causes, due to dysbiosis. Fecal microflora transplantation is an option, particularly in recurrent episodes. There are case reports on fecal microflora transplantation used for the treatment of inflammatory bowel disease, with promising results.

La microbiota gastrointestinal funciona como un órgano metabólico que provee de rutas enzimáticas no presentes en nuestro organismo; contribuye al desarrollo y mantenimiento de órganos linfoides locales y sistémicos, a la homeostasis de la barrera epitelial intestinal; modula los procesos inflamatorios sistémicos y metabólicos y activa el sistema inmunológico sitémico, brindando protección frente agresiones bacterianas y virales. La diarrea por Clostridium difficile es la principal causa de diarrea intrahospitalaria con una alta morbilidad y mortalidad. Esta se produce entre otras causas por una disbiosis. El trasplante de microflora fecal ha demostrado ser una opción terapéutica eficaz, especialmente en los episodios recurrentes. Existen reportes de casos con resultados promisorios en que se utiliza el trasplante de microflora fecal para el tratamiento de enfermedad inflamatoria intestinal crónica.

Infecciones en la patogenia de la enfermedad inflamatoria intestinal / Infections in the pathogeny of inflammatory bowel disease

La enfermedad inflamatoria intestinal (EII) es una entidad compleja. Su desarrollo requiere de la interacción entre factores ambientales y la flora gastrointestinal, en un individuo genéticamente susceptible. Nuestro aparato gastrointestinal es un extraordinario, complejo y dinámico modelo de simbiosis o mutualismo con la flora. Los factores ambientales como el tabaco, las infecciones gastrointestinales y los antiinflamatorios no esteroidales juegan probablemente un rol iniciador y/o modificador de la enfermedad. La mucosa intestinal tiene la difícil tarea de limitar la respuesta inflamatoria contra la flora y de mantener la habilidad de generar una respuesta inmune contra los microorganismos patógenos. Esto crea una relación de equilibrio dinámico y frágil que al alterarse cualquiera de sus componentes puede generar un proceso inflamatorio. Existe evidencia que las infecciones pueden tener un rol tanto en el inicio de la enfermedad como en las reagudizaciones de ésta. Es así como las infecciones bacterianas gastrointestinales agudas y la Escherichia coli adherente invasora confieren un riesgo para desarrollar una enfermedad inflamatoria intestinal. Para Mycobacterium avium subespecie paratuberculosis (MAP) sólo se ha establecido una asociación y no un rol patogénico. Por último ha aumentado la incidencia y morbimortalidad de la infección por Clostridium difficile en los pacientes con EII.

Infections in the pathogeny of inflammatory bowel disease Inflammatory bowel disease (IBD) is a complex entity. Its development requires the interaction between environmental factors and gastrointestinal flora in a genetically susceptible subject. Our gastrointestinal tract is an extraordinary, complex and dynamic model of symbiosis or mutualism with the flora. Environmental factors such as tobacco, gastrointestinal infections and non-steroidal anti-inflammatory drugs might play a role of starter and/or modifier of the disease. The intestinal mucosa has the difficult task of limiting the inflammatory response against the flora and of keeping the capability of generating an immune response against pathogenic microorganisms. This creates a dynamic equilibrium relation that is fragile, and that when any of its components is altered, it can cause an inflammatory process. There is evidence that infections can have a role both in the beginning and in the episodes of New-Rebounds of the disease. Therefore, acute gastrointestinal bacterial infections and adherent-invasive Escherichia coli pose a risk of developing an inflammatory bowel disease. In the case of Mycobacterium avium subspecies paratuberculosis (MAP) an association has been established, but not a pathogenic role. The incidence and morbimortality of Clostridium difficile infection has increased in patients with IBD.

Estimation of faecal carriage of Clostridium difficile in patients with ulcerative colitis using real time polymerase chain reaction.

BACKGROUND & OBJECTIVE: Ulcerative colitis (UC) is a disease of unknown aetiology in which exacerbations are sometimes linked to intestinal colonization by toxin-producing Clostridium difficile. We undertook this study to detect and quantitatively assess C. difficile in the stool of patients with UC using real time polymerase chain reaction (RT-PCR), and to compare it with healthy individuals. METHODS: A total of 37 consecutive patients with UC (26 male, mean age 41.3 yr) and 36 healthy adult volunteers (20 male, mean age 36.4), none of whom had received antibiotics within two months prior to faecal collection, were included in the study. Faecal DNA was extracted, quantitative PCR (qPCR) carried out using primers to amplify species-specific segments of 16S rDNA of C. difficile, and expressed as relative fold difference against amplification of highly conserved (universal) segments. Toxins A and B were assayed by ELISA. RESULTS: Quantitative PCR detected C. difficile sensitively, and spiking with increasing numbers of the organism resulted in linear increase in amplification (R(2)=0.974). C. difficile was detected by qPCR in faeces of 20 of 36 healthy volunteers and 34 of 37 patients with UC. Relatively greater amplification of C. difficile (fold difference) was noted in UC compared to controls (P<0.0001). There was no significant difference in C. difficile amplification between patients with proctitis, left sided colitis and pancolitis, or between active and quiescent colitis. Toxin was detected in the faeces of 8 of 37 patients with UC compared to 2 of 36 healthy volunteers. INTERPRETATION & CONCLUSION: Findings of this study showed overgrowth of C. difficile in the stool of Indian patients with UC. However, its relevance to disease pathogenesis and severity in a tropical country like India needs to be investigated further.

Estudo da microbiota intestinal em doentes com retocolite ulcerativa antes e após retocolectomia com anastomose de bolsa ileal ao canal anal / Intestinal microbiota in patients with ulcerative colitis before and after proctolectomy and ileal pouch-anal anastomosis

Este estudo tem como objetivo, descrever a microbiota intestinal de pacientes com retocolite ulcerativa grave, em tratamento clínico, antes e após retocolectomia com anastomose de bolsa ileal ao canal anal. Comparou-se a flora bacteriana do íleo terminal e do reto no pré-operatório com a flora encontrada na bolsa ileal após dois e oito meses do fechamento da ileostomia e com a flora do íleo terminal e do reto de um grupo controle. Observou-se que a Veillonella sp foi a bactéria mais freqüentemente encontrada em todos os grupos. Não houve diferenças significativas entre a flora intestinal do grupo controle e dos pacientes com retocolite / The aim of this study is to describe the intestinal microbiota of patients with severe ulcerative colitis, under clinical treatment, before and after proctocolectomy and ileal pouch-anal anastomosis. Intestinal flora of distal ileum and rectum before surgery was compared with the flora found in ileal pouch after two and eight months after ileostomy closure and with the flora of distal ileum and rectum of controls. Veillonella sp was the most frequent microorganism found in all groups. There were no significant differences between the intestinal microbiota found in controls and in patients...

Frequency and spectrum of microorganisms isolated from cases of chronic colitis

Forty patients, 24 males and 16 females aged 17-65 years, diagnosed clinically, sigmoidoscopically and histopathologically as chronic colitis cases [14 [35%] ulcerative colitis, 4 [10%] Crohn's disease and 22[55%] non specific colitis] were investigated by stool analysis, stool culture and biopsy culture. Chlamydia antigen was detected in biopsy specimens by direct immunofluorescence technique. Ten healthy subjects we included as controls. Hundred percent of cases of inflammatory bowel disease [IBD] were positive for bacterial infections compared to 73% in cases of nonspecific colitis [P<0.05] and infection rate was prominant in bilharzial cases 85%. Salmonella paratyphi [A] and Shigella dysentriae were isolated from both rectal biopsy and stool culture each in 2 cases only. The potentially pathogenic bacteria isolated from biopsy culture only were chlamydia trachomatis, Aeromonas hydrophlla, Pseudomonas aeruginosa, Staph. aureus and Diphtheroid. E. coli were isolated from 40 cases by stool culture and from 14 cases by biopsy culture compared to 4 and 20 cases for Klebsiella sp. In cases that were positive by both biopsy and stool cultures the organisms isolated were identical. There were 4 cases that were diagnosed macroscopically as suspecious ulcerative colitis and microscopically as non specific colitis, while by bacterial cultures each of the following organisms were isolated from one case; Klebsiella sp., E. coli, Chlamydia trachomatis and Pseudomonas aeruginosa. The results of the present work revealed that an appropriate diagnostic program of chronic colitis must include endoscopic, histopathologic and bioptic microbiological examinations in addition to stool culture and analysis to avoid inevitable misdiagnosis of IBD and there is a necessity for the cooperation between the endoscopists, histopathologists and microbiologists to reach a proper diagnosis and proper management