Efectos del medio de cultivo y de la metodología aplicada sobre la formación de biopelículas de 2 cepas de Escherichia coli diarreagénicas / Effects of the culture medium and the methodology applied on the biofilm formation of 2 diarrheagenic Escherichia coli strains

La capacidad de formar biopelículas de los microorganismos patógenos en gran variedad de ambientes, superficies y condiciones trae consigo un importante riesgo, tanto para la industria alimentaria como para la salud pública. Este trabajo tuvo como objetivo evaluar y comparar los efectos de la metodología empleada y de los medios de cultivo utilizados, sobre la capacidad de una cepa de Escherichia coli verotoxigénica no O157 y una enteropatogénica de formar biopelículas sobre una superficie de poliestireno. Se ensayaron 2 variantes metodológicas en cultivo estático y se utilizaron medios de cultivo con diferente composición. Los resultados mostraron que ambas cepas formaron una mayor cantidad de biopelícula en cultivo en LB suplementado con glucosa, con recambio del medio a las 24 h y la cuantificación de la biopelícula realizada a las 48 h de incubación. Dichas condiciones podrían ser utilizadas en futuros estudios sobre formación de biopelícula.

The ability to form biofilms of pathogenic microorganisms in a wide variety of environments, surfaces and conditions constitute an important risk, both for the food industry and for public health. The aim of this work was to evaluate and to compare the effects of the methodology applied and the culture medium used on the ability of a non-O157 verotoxigenic Escherichia coli strain and an enteropathogenic strain to form biofilm on polystyrene surface. Two methodological variants were tested in static culture and culture mediums with different composition were used. The results showed that both strains were able to form a greater biofilm under culture in LB supplemented with glucose, with medium replacement at 24 h and the quantification of the biofilm carried out at 48 h of incubation. These conditions could be used in future studies on biofilm formation.

Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli. Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli. Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene. EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg, aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea (P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

Typical and atypical enteropathogenic Escherichia coli bacterial translocation associated with tissue hypoperfusion in rats

Although enteropathogenic Escherichia coli (EPEC) are well-recognized diarrheal agents, their ability to translocate and cause extraintestinal alterations is not known. We investigated whether a typical EPEC (tEPEC) and an atypical EPEC (aEPEC) strain translocate and cause microcirculation injury under conditions of intestinal bacterial overgrowth. Bacterial translocation (BT) was induced in female Wistar-EPM rats (200-250 g) by oroduodenal catheterization and inoculation of 10 mL 10(10) colony forming unit (CFU)/mL, with the bacteria being confined between the duodenum and ileum with ligatures. After 2 h, mesenteric lymph nodes (MLN), liver and spleen were cultured for translocated bacteria and BT-related microcirculation changes were monitored in mesenteric and abdominal organs by intravital microscopy and laser Doppler flow, respectively. tEPEC (N = 11) and aEPEC (N = 11) were recovered from MLN (100 percent), spleen (36.4 and 45.5 percent), and liver (45.5 and 72.7 percent) of the animals, respectively. Recovery of the positive control E. coli R-6 (N = 6) was 100 percent for all compartments. Bacteria were not recovered from extraintestinal sites of controls inoculated with non-pathogenic E. coli strains HB101 (N = 6) and HS (N = 10), or saline. Mesenteric microcirculation injuries were detected with both EPEC strains, but only aEPEC was similar to E. coli R-6 with regard to systemic tissue hypoperfusion. In conclusion, overgrowth of certain aEPEC strains may lead to BT and impairment of the microcirculation in systemic organs.

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.

Anticorpos anti-intiminas α, β, e γ de Escherichia coli em soros e colostros de adultos saudáveis da Grande São Paulo / Antibodies reactive with α, β,and γ intimins of Escherichia coli in serum and colostrum samples of healthy adults living in São Paulo, Brazil

A diarréia é um importante problema de saúde pública no mundo inteiro e a Escherichía coli é um dos mais freqüentes microorganismos causadores desta doença. A Escherichia coli enteropatogênica (EPEC), um dos principais agentes etiológicos das diarréias infantis no nosso país, é genética e fenotipicamente relacionada com a E. colí enterohemorrágica (EHEC) que além de provocar diarréia é responsável por complicações como síndrome hemolítica urêmica (HUS) e colite hemorrágica (HC). Embora a EHEC seja considerada emergente pela OMS, no Brasil poucos casos de complicações como HUS e HC foram reportados. O mecanismo de patogenicidade comum entre EPEC e EHEC é conhecido como a lesão "attaching and effacing" nos microvilos do enterócito. Esta lesão é mediada por um conjunto de fatores de virulência, dentre eles a intimina. A intimina é uma proteína de membrana externa, responsável pelo íntimo contato da bactéria com o enterócito, possui uma região N-terminal que é altamente conservada e uma região C-terminal que é variável. De acordo com a região variável, existem vários subtipos de intimina, dentre eles as intiminas , α, β e γ...