Molecular characterisation of enteroinvasive Escherichia coli O136:K78 isolates from patients of a diarrhoea outbreak in China.

Purpose: A diarrhoea outbreak occurred in a kindergarten, which caused 21 relevant infected cases. Our object was to confirm the pathogens and their molecular characterisation. Materials and Methods: Faecal samples from 21 patients were collected on the 3rd day after their symptom onset, and a regular epidemiological investigation was conducted. Bacterial isolation was performed in accordance with standard laboratory protocol, serological and molecular characterisations were determined by serum agglutination test and real‑time polymerase chain reaction (PCR) method, respectively. The pulsed field gel electrophoresis (PFGE) and 16S rRNAs were conducted to determine the homology. Results: Eleven enteroinvasive Escherichia coli (EIEC) O136:K78 strains were isolated. The serum agglutination test showed that all strains’ serotypes were E. coli (EIEC) O136:K78. Real‑time PCR showed that 10 (91%) strains carried the invasion plasmid antigen H gene (ipaH), carried by all four Shigella species and EIEC. The strain that didn’t carry the ipaH gene had different biochemical reactions of L‑lizyna and L‑rhamnose with the other strains. The complete 16S rRNA sequences showed 98.4% identity between ipaH‑negative isolate and the others, and the PFGE indicated that the ipaH‑negative isolate was not homological with other isolates in this diarrhoea outbreak. Conclusions: The diarrhoea outbreak was caused by E. coli (EIEC) O136:K78.

O papel da flagelina e do sistema de secreção de Escherichia coli enteroinvasora na resposta imune inata dos macrófagos / The role of flagellin and secretion system of enteroinvasive Escherichia coli in the immune response innate macrophages

Escherichia coli enteroinvasora (EIEC) é um dos agentes etiológicos da disenteria bacilar. Seu processo fisiopatológico é desencadeado pela expressão de fatores de virulência, que proporcionam sua invasão e sobrevivência nas células do hospedeiro, ativando o sistema imune inato e adaptativo da mucosa intestinal. Trabalhos recentes têm salientado a importância do sistema de secreção e da flagelina bacteriana como agonista de receptores da imuninade inata dos macrófagos, em especial alguns dos receptores do tipo NLR. Uma vez que esta espécie de E. coli também é capaz de expressar flagelina e fazer a montagem completa do flagelo e do sistema de secreção do tipo III, a nossa proposta foi avaliar o papel da flagelina e do sistema de secreção de EIEC na resposta imune dos macrófagos murinos. Para isso, utilizamos três cepas de EIEC: a cepa selvagem; a cepa mutante no gene responsável pela síntese da flagelina; e a cepa sem o plasmídio de virulência plnv, deficiente no sistema de secreção, para a infecção de macrófagos peritoniais de camundongos C57BI/6, caspase-1-/-, IPAF-/- e ASC-/-. Neste estudo foi possível observar que o escape bacteriano e a morte dos macrófagos infectados por EIEC, assim como a ativação da caspase-1 e posterior secreção de IL-1ß é independente da flagelina bacteriana, mas dependente do sistema de secreção, além disso, a ativação da caspase-1 de macrófagos infectados por EIEC é dependente do receptor IPAF e parcialmente da proteína adaptadora ASC. Assim, no nosso modelo, a ativação da caspase-1 dos macrófagos infectados por EIEC parece estar envolvida com o processamento e secreção de IL-1ß e, possivelmente na secreção de IL-18, mas não na morte celular. No modelo de infecção in vivo, o sistema de secreção bacteriano foi importante para a sobrevivência bacteriana no hospedeiro, assim como para a indução de uma resposta inflamatória no local da infecção. Ainda, a caspase-1 parece ter um papel importante para o controle da infecção in vivo por EIEC, podendo assim contribuir para uma resposta imune protetora do hospedeiro

Enteroinvasive Escherichia coli (EIEC) is one of the etiologic agents responsible for bacillary dysentery. The pathophysiological process induced by this bacteria is triggered by the expression of virulence factors that provide the invasion and survival in host cells, resulting in activation of innate and adaptive immune system present on intestinal mucosa. Recent studies have emphasized the importance of the secretion system and bacterial flagellin as agonist of innate immune receptors present in macrophage, especially NLR (Nod like receptors). Then, our proposal was evaluate the role of flagellin (f1iC) and secretion system of EIEC in the induction of immune response of murine macrophages using the EIEC strains wild type (WT), mutant flagellin gene (f1iC), and a strain deficient in secretion system (DSS) for infection of peritoneal macrophages of C57Bl/6, caspase-1-/-, IPAF-/- and ASC-/-- mice. In this study we observed that the bacterial escape and death of infected macrophages with EIEC, the caspase-1 activation and subsequent IL-1ß secretion is independent of bacterial flagellin, but dependent of secretion system, moreover, the caspase-1 activation in infected macrophages is IPAF-dependent and partially dependent of the adapter protein ASC. Thus, in our model, the caspase-1 activation in EIEC infected macrophages seems to be involved with the processing and secretion of IL-1ß and possibly with the secretion of IL-18, but not involved with cell death. In the infection model in vivo, bacterial secretion system was important for bacterial survival in the host, as well as for the inflammatory response induction at the infection site. In addition, caspase-1 seems to have an important role to the control of in vivo infection by EIEC and can contribute to a protective immune response of the host

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

In vitro modulation of adherence and invasion ability of enteroinvasive escherichia coli by different viruses.

Studies on the relationship between viral and bacterial infections showed that in the context of viral infections the immunity of host organism is reduced temporarily, increasing the incidence of bacterial infections, like faster bacterial colonization of immunocompromised bodies, by increasing the level of expression of epithelial cell receptor for bacterial adesins. Modulation of viruses infected host cells signaling may also induce changes in the cytoscheleton, which may result in the increase / decrease invasion capacity of bacterial cells. Enteroinvasive Escherichia coli causes intestinal infections exploiting host cell function, which include the invasion into non-phagocytic eukaryotic cells such as epithelial and endothelial cells and associated host cell actin cytoskeletal rearrangements. One of our aims was to investigate the in vitro adherence and invasion capacity induced by an diarhhoeal enteroinvasive Escherichia coli strain in the presence of different viral strains: Vaccinia virus (Poxviridae), measles virus (Paramyxoviridae II); echovirus 32 (Picornaviridae) and Herpes simplex virus 1 (Herpesviridae). The viral adsorption on HeLa cells was done for six hours at 370C, followed by the evaluation of bacterial adherence and invasion to HeLa cells performed by the adapted Cravioto’s method and gentamycin protection assay. Viral preinfection of the cellular substrate induced an increased bacterial adherence index, as well as changes in the adherence pattern from diffuse tu aggregative. In exchange, the general effect of viral infection on invasive bacterial capacity was the decrease of invasive ability. In conclusion, viral preinfection of the susceptible substrate influenced the adherence and invasion ability of enteroinvasive E. coli bacterial strain, as observed by the intensification of the adherence capacity, explaining the increased incidence of bacterial infections after viral infections, as well as faster bacterial colonization of immunocompromised hosts and by reducing the invasive capacity of epithelial cell by bacterial strains, pleading for increased incidence of extracellular pathogenic organisms in post-viral infections.

Molecular characterization of enteroinvasive Escherichia coli ipa genes by PCR-RFLP analysis

In this study, polymorphism in ipa genes was found in five out of nine EIEC serotypes studied. When SalI and HindII were used in RFLP-PCR assays many EIEC serotypes showed polymorphism in ipaB and ipaD. On the other hand, no polymorphism was observed in ipaA and ipaC in these strains. The polymorphism present in EIEC strains is serotype-dependent, since restriction patterns were conserved amongst strains belonging to the same serotype. When IpaB deduced amino acid sequences of S. flexneri M90T and FBC124-13 were compared, ten amino acids changes could be observed mainly in the amino-terminal region. The deduced EIEC IpaD amino-acid sequence presented 91% similarity with the Shigella strain. In this case, amino acid changes were spread out through the whole structure, except in the carboxyl-terminal region.

No presente estudo, foi encontrado polimorfismo no gene ipa em cinco sorotipos de EIEC, de nove estudados. Quando enzimas de restrição SalI e HindII foram utilizadas no ensaio de PCR-RFLP, amostras de EIEC apresentaram polimorfismo em ipaB e ipaD. Por outro lado, não foram observados polimorfismos nos genes ipaA e ipaC nestas cepas, quando diversas enzimas de restrição foram utilizadas. O polimorfismo presente em cepas de EIEC é sorotipo-dependente, uma vez que os padrões de restrição foram conservados entre as cepas pertencentes ao mesmo sorotipo. Quando a seqüência deduzida de aminoácidos de IpaB de S. flexneri M90T e FBC124-13 foram comparadas, mudanças foram observadas em dez aminoácidos na região amino-terminal. A seqüência deduzida de aminoácidos de IpaD de EIEC apresentou similaridade de 91% com a cepa de Shigella. Neste caso, mudanças de aminoácidos ocorreram em toda a estrutura da molécula de IpaD, exceto na região carboxi-terminal.

EVALUATION ON DETECTION OF SHIGELLA CULTURES WITH THE DIAGNOSTIC TYPING PHAGE SET FOR ENTEROBACTERIACEAE / 微生物学通报

In this paper , 2280 strains of suspicious Shigella c culture were detected by the diagnostic typing phage set for Enterobacteriaceae, the routine identification of them were carried oat at the same time . The results showed that 100% of shigella cultures were lysed with 10~3RTD and 99.9% lysed with 1RTD of phage Sh. 12.3% of 65 non-shigeUa cultures agglutinating with typing serum of shigeUa was lysed with 10~3RTD, 4.6% lysed with 1RTD of phage Sh. The determination of lytic-pattern of 2215 shigella culture indicated that only 3 strains of Boyd 5 of lytic-pattern 3 were not reported in literature, the rest strains were consistent to fomer studies. The nonshigella cultures lysed by Sh 10~3RTD could be excluded with 1RTD and its lytic-pattern. 20 strains of Enteroinvasive Escherichia coli possessing shigeUa-related antigens could be differentiated by Sh 10~3RTD. 1RTD and its lytic-pattern.