RESUMO
BACKGROUND: Oral fibroblast immunological responses to bacterial stimuli are well known. However, there are few studies about pulp fibroblasts from deciduous teeth (HDPF) responses, which are important for the treatment of pulp infections in children. The aim of this study was to evaluate expression and production of inflammatory cytokines and chemokines by HDPF when challenged with bacterial antigens normally present in advanced caries lesions. METHODS: Triplicate HDPF from 4 children (n = 4; 2 boys and 2 girls) were cultured by explant technique and challenged or not with Escherichia coli lipopolysaccharide/1 µg/mL (EcLPS) or Enterococcus faecalis lipoteichoic acid/1 µg/mL (EfLTA) for 6 and 24 h. Most of published studies employed immortalized cells, i.e., without checking possible gender and genetic variables. mRNA expression and protein production were evaluated by RT-qPCR and ELISA MILLIPLEX®, respectively, for Interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, Chemokine C-C motif ligand 2/monocyte chemoattractant protein 1 (CCL2/MCP-1), Chemokine C-C motif ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP1-α), Chemokine C-C motif ligand 5/ regulated on activation, normal T cell expressed and secreted (CCL5/RANTES), C-X-C motif chemokine 12/ stromal cell-derived factor 1 (CXCL12/SDF-1), Tumor Necrosis Factor-alpha (TNF-α), Interferon-gamma (IFN γ), Vascular Endothelial Growth Factor (VEGF), Colony stimulating factor 1 (CSF-1) and Macrophage colony-stimulating factor (M-CSF). RESULTS: EcLPS increased IL-1α, IL-1ß, IL-8, CCL2, CCL5, TNF-α and CSF-1 mRNA and protein levels while EfLTA was only able to positively regulate gene expression and protein production of IL-8. CONCLUSION: The results of the present study confirmed our hypothesis, since pulp fibroblasts from deciduous teeth are capable of increasing gene expression and protein production after being stimulated with EcLPS and EfLTA.
Assuntos
Polpa Dentária/patologia , Escherichia coli/fisiologia , Escherichia/fisiologia , Fibroblastos/fisiologia , Dente Decíduo/patologia , Células Cultivadas , Criança , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , MasculinoRESUMO
Escherichia albertii are emerging enteropathogens, whose identification is difficult, as they share biochemical characteristics and some virulence-related genes with diarrheagenic Escherichia coli (DEC). Studies on phylogeny, phenotypic characteristics and potential virulence factors of human E. albertii strains are scarce. In this study, we identified by multiplex PCR five E. albertii among 106 strains isolated from diarrheic children in São Paulo, Brazil, which were previously classified as atypical enteropathogenic E. coli. All strains were investigated regarding their phylogeny, biochemical properties, virulence-related properties, antimicrobial resistance and presence of putative virulence-related genes. All strains belonged to different E. albertii lineages and adhered to and produced attaching and effacing lesions on HeLa cells. Three strains invaded Caco-2 cells, but did not persist intracellularly, and three formed biofilms on polystyrene surfaces. All strains were resistant to few antibiotics and only one carried a self-transmissible resistance plasmid. Finally, among 38 DEC and 18 extraintestinal pathogenic E. coli (ExPEC) virulence-related genes searched, six and three were detected, respectively, with paa and cdtB being found in all strains. Despite the limited number of strains, this study provided additional knowledge on human E. albertii virulence potential, showing that they share important virulence factors with DEC and ExPEC.
Assuntos
Diarreia/epidemiologia , Diarreia/microbiologia , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Escherichia/fisiologia , Fenótipo , Antibacterianos/farmacologia , Biofilmes , Brasil/epidemiologia , Linhagem Celular , Criança , Pré-Escolar , Escherichia/classificação , Escherichia/isolamento & purificação , Escherichia/patogenicidade , Genótipo , Humanos , Mucosa Intestinal , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Filogenia , Sorogrupo , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic Escherichia coli (aEPEC), one of the diarrheagenic E. coli pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually E. albertii, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of E. albertii infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of E. albertii colonization. We also observed that an E. albertii strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how E. albertii might cross the intestinal epithelium by performing infections of M-like cells in vitro to identify the potential in vivo translocation route. Altogether, our approaches allowed us to draft a general E. albertii infection route from the colonization till the bacterial spreading in vivo.
Assuntos
Enterócitos/microbiologia , Escherichia/fisiologia , Mucosa Intestinal/microbiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Infecções por Enterobacteriaceae/microbiologia , Enterócitos/ultraestrutura , Escherichia/ultraestrutura , Feminino , Humanos , Mutação , Ratos , Sistemas de Secreção Tipo III/genética , VirulênciaRESUMO
AIMS: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo. METHODS AND RESULTS: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae. CONCLUSIONS: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.