RESUMO
The use of molecular diagnostics for pathogen detection in epidemiological studies have allowed us to get a wider view of the pathogens associated with diarrhea, but the presence of enteropathogens in asymptomatic individuals has raised several challenges in understanding the etiology of diarrhea, and the use of these platforms in clinical diagnosis as well. To characterize the presence of the most relevant bacterial enteropathogens in diarrheal episodes, we evaluated here the prevalence of diarrheagenic E. coli pathotypes, Salmonella spp., and Yersinia enterocolitica in stool samples of children with and without diarrhea using real-time quantitative PCR (qPCR). We found that the presence of genetic markers associated with bacterial pathogens was significantly higher in stool samples from the diarrhea group compared to the control (P < 0.001). Bacterial loads in samples positive for eae and aggR markers were also determined. Compared to samples from asymptomatic children, a significantly higher number of copies of the eae gene were found in diarrhea samples. Also, the presence of genetic markers associated with STEC strains with clinical significance was evaluated in eae-positive samples by high-throughput real-time PCR. The data presented herein demonstrated that asymptomatic children of an urban area in Brazil might be enteropathogen reservoirs, especially for STEC.
Assuntos
Infecções por Escherichia coli , Escherichia coli , Brasil/epidemiologia , Criança , Diarreia/epidemiologia , Infecções por Escherichia coli/epidemiologia , Fezes , Humanos , Lactente , Prevalência , VirulênciaRESUMO
The use of molecular diagnostics for pathogen detection in epidemiological studies have allowed us to get a wider view of the pathogens associated with diarrhea, but the presence of enteropathogens in asymptomatic individuals has raised several challenges in understanding the etiology of diarrhea, and the use of these platforms in clinical diagnosis as well. To characterize the presence of the most relevant bacterial enteropathogens in diarrheal episodes, we evaluated here the prevalence of diarrheagenic E. coli pathotypes, Salmonella spp., and Yersinia enterocolitica in stool samples of children with and without diarrhea using real-time quantitative PCR (qPCR). We found that the presence of genetic markers associated with bacterial pathogens was significantly higher in stool samples from the diarrhea group compared to the control (P < 0.001). Bacterial loads in samples positive for eae and aggR markers were also determined. Compared to samples from asymptomatic children, a significantly higher number of copies of the eae gene were found in diarrhea samples. Also, the presence of genetic markers associated with STEC strains with clinical significance was evaluated in eae-positive samples by high-throughput real-time PCR. The data presented herein demonstrated that asymptomatic children of an urban area in Brazil might be enteropathogen reservoirs, especially for STEC.
RESUMO
ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.
Assuntos
Humanos , Animais , Doenças das Aves Domésticas/microbiologia , Aderência Bacteriana , Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Clostridium perfringens/fisiologia , Células Epiteliais/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Vero , Chlorocebus aethiops , Galinhas , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/genéticaRESUMO
ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and - and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.(AU)
Assuntos
Clostridium perfringens/isolamento & purificação , Células Epiteliais/química , Enterite/classificaçãoRESUMO
OBJECTIVE: In this study, the presence of the prtC and fimA genes involved in the pathogenicity of oral Porphyromonas spp. isolated from dogs with periodontitis and healthy, as well as their genetic diversity was investigated. DESIGN: Thirty-two Beagle dogs, 24 with periodontitis and 8 healthy were evaluated. Subgingival samples from only one gingival site of both groups were collected. Bacteria grown in anaerobiosis were identified by RAPID ID 32A kits. From each strain the respective DNA was obtained and used to genotyping by conventional PCR and AP-PCR. RESULTS: Dogs with periodontitis harbored 28 P. gulae, 2 P. creviocaricanis, 1 P. cangingivalis and 7 P. macacae; and from healthy dogs, 11 P. gulae and 5 P. circumdentaria. In P. gulae isolated from periodontal dogs the gene prtC was observed in 19 (67.85%) and in 7 (63.63%) from healthy dogs. P. gulae strains from periodontal dogs harbored either the gene fimA I or fimA II; while strains from healthy dogs harbored the gene fimA I, fimA II, fimA III or fimA IV, as well as 1 P. circumdentaria the gene fimA II. By AP-PCR strains were grouped in different clusters suggesting heterogeneity of these microorganisms. CONCLUSIONS: The results presented herein inform that Porphyromonas spp. isolated from dogs with and without periodontitis harbored the prtC and fimA genes and it could be a role in the establishment of the infectious process.
Assuntos
Periodontite/microbiologia , Porphyromonas/genética , Porphyromonas/patogenicidade , Virulência/genética , Animais , Cães , Genótipo , Reação em Cadeia da Polimerase , Porphyromonas/isolamento & purificaçãoRESUMO
Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.
Assuntos
Aderência Bacteriana , Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Clostridium perfringens/fisiologia , Células Epiteliais/microbiologia , Doenças das Aves Domésticas/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Galinhas , Chlorocebus aethiops , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Humanos , Células VeroRESUMO
AIM: To evaluate whether Porphyromonas gingivalis-induced periodontitis aggravates the antigen-induced arthritis (AIA) model, and whether this effect is dependent on the Th17/IL-17 signalling pathway. MATERIALS AND METHODS: Antigen-induced arthritis was triggered by local injection of methylated bovine serum albumin into the knee joint of previously immunized C57BL/6 wild-type (WT) and IL-17 receptor A (IL-17RA)-knockout mice. Periodontal disease in naïve or arthritic mice was induced by oral infection with P. gingivalis. Animals were sacrificed 7, 15 and 30 days after infection. Alveolar bone loss, joint histopathology, articular hyperalgesia and joint cytokine production were assessed, in addition to the proportion of Th17 and Treg cells isolated from the inguinal lymph nodes. RESULTS: No influence of experimentally-induced arthritis was found on the alveolar bone resorption induced by P. gingivalis. However, mice with experimentally-induced arthritis that were exposed to P. gingivalis presented higher joint damage and Th17 frequencies when compared to non-infected mice. The aggravation of arthritis by periodontitis was accompanied by increased TNF and IL-17 production and articular neutrophil infiltration, whereas arthritis aggravation and changes in neutrophil infiltration were absent in IL-17RA-deficient mice. CONCLUSION: The effects of P. gingivalis-induced periodontitis on arthritis are dependent on Th17 expansion and IL-17RA signalling, which lead to increased neutrophil infiltration into the joints.
Assuntos
Artrite Experimental/imunologia , Periodontite/imunologia , Periodontite/microbiologia , Receptores de Interleucina-17/imunologia , Perda do Osso Alveolar/imunologia , Perda do Osso Alveolar/patologia , Animais , Artrite Experimental/patologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Periodontite/patologia , Porphyromonas gingivalis/imunologia , Distribuição Aleatória , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , DNA Bacteriano/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Infecções Bacterianas/microbiologia , Técnicas de Tipagem Bacteriana , Bacteroides/efeitos dos fármacos , Bacteroides/genética , Bacteroides/crescimento & desenvolvimento , Bacteroides/isolamento & purificação , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/genética , Bifidobacterium/crescimento & desenvolvimento , Bifidobacterium/isolamento & purificação , Estudos de Casos e Controles , Criança , Pré-Escolar , Clostridium/efeitos dos fármacos , Clostridium/genética , Clostridium/crescimento & desenvolvimento , Clostridium/isolamento & purificação , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Feminino , Firmicutes/efeitos dos fármacos , Firmicutes/genética , Firmicutes/crescimento & desenvolvimento , Firmicutes/isolamento & purificação , Microbioma Gastrointestinal/genética , Humanos , Lactobacillus/efeitos dos fármacos , Lactobacillus/genética , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/isolamento & purificação , Masculino , Methanobrevibacter/efeitos dos fármacos , Methanobrevibacter/genética , Methanobrevibacter/crescimento & desenvolvimento , Methanobrevibacter/isolamento & purificaçãoRESUMO
NOD2 is a member of the NLR family of proteins that participate in the activation of the innate immune response. RIP2 is a downstream kinase activated by both NOD1 and NOD2. There is scarcity of information regarding the relevance of NOD2 in periodontitis, a chronic inflammatory condition characterized by inflammatory bone resorption. We used NOD2-KO and RIP2-KO mice in a model of microbial-induced periodontitis. Heat-killed Aggregatibacter actinomycetemcomitans was injected in the gingival tissues three times/wk for 4 wk. Bone resorption was assessed by µCT analysis; osteoclasts were identified by immunohistochemical staining for TRAP and inflammation was assessed using a severity score system in H/E-stained sections. In vitro studies using primary macrophages assessed the response macrophages using qPCR-based array and multi-ligand ELISA. Bone resorption and osteoclastogenesis were significantly reduced in NOD2-KO mice. Severity of inflammation was not affected. qPCR-focused arrays and multi-ligand ELISA showed that expression of pro-inflammatory mediators was reduced in NOD2- and RIP2-deficient cells. RANKL-induced osteoclastogenesis was impaired in NOD2- and RIP2-deficient macrophages. We conclude that NOD2 is important for osteoclast differentiation and inflammatory bone resorption in vivo and also for the macrophage response to Gram-negative bacteria.
Assuntos
Reabsorção Óssea/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Macrófagos/fisiologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Osteogênese/imunologia , Periodontite/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD2/genética , Ligante RANK/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Colorectal carcinoma is considered the fourth leading cause of cancer deaths worldwide. Several microorganisms have been associated with carcinogenesis, including Enterococcus spp., Helicobacter pylori, enterotoxigenic Bacteroides fragilis, pathogenic E. coli strains and oral Fusobacterium. Here we qualitatively and quantitatively evaluated the presence of oral and intestinal microorganisms in the fecal microbiota of colorectal cancer patients and healthy controls. Seventeen patients (between 49 and 70 years-old) visiting the Cancer Institute of the Sao Paulo State were selected, 7 of whom were diagnosed with colorectal carcinoma. Bacterial detection was performed by qRT-PCR. Although all of the tested bacteria were detected in the majority of the fecal samples, quantitative differences between the Cancer Group and healthy controls were detected only for F. nucleatum and C. difficile. The three tested oral microorganisms were frequently observed, suggesting a need for furthers studies into a potential role for these bacteria during colorectal carcinoma pathogenesis. Despite the small number of patients included in this study, we were able to detect significantly more F. nucleatum and C. difficile in the Cancer Group patients compared to healthy controls, suggesting a possible role of these bacteria in colon carcinogenesis. This finding should be considered when screening for colorectal cancer.