The secreted Ipa complex of Shigella flexneri promotes entry into mammalian cells.

The bacterial pathogen Shigella flexneri causes bacillary dysentery in humans by invading coloncytes. Upon contact with epithelial cells, S. flexneri elicits localized plasma membrane projections sustained by long actin filaments which engulf the microorganism. The products necessary for Shigella entry include three secretory proteins: IpaB, IpaC, and IpaD. Extracellular IpaB and IpaC associate in a soluble complex, the Ipa complex. We have immunopurified this Ipa complex on latex beads and found that they were efficiently internalized into HeLa cells. Like S. flexneri entry, uptake of the beads bearing the Ipa complex was associated with membrane projections and polymerization of actin at the site of cell-bead interaction and was dependent on small Rho GTPases. These results indicate that a secreted factor can promote S. flexneri entry into epithelial cells.

Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility.

Shigella flexneri is a Gram-negative bacterial pathogen that can grow directly in the cytoplasm of infected host cells and uses a form of actin-based motility for intra- and intercellular spread. Moving intracellular bacteria are associated with a polarized "comet tail" composed of actin filaments. IcsA, a 120-kDa outer membrane protein necessary for actin-based motility, is located at a single pole on the surface of the organism, at the junction with the actin tail. Here, we demonstrate that stable expression of IcsA on the surface of Escherichia coli is sufficient to allow actin-dependent movement of E. coli in cytoplasmic extracts, at rates comparable to the movement of S. flexneri in infected cells. Thus, IcsA is the sole Shigella-specific factor required for actin-based motility. Continuous protein synthesis and polarized distribution of the protein are not necessary for actin tail formation or movement. Listeria monocytogenes is an unrelated bacterial pathogen that exhibits similar actin-based intracytoplasmic motility. Actin filament dynamics in the comet tails associated with the two different organisms are essentially identical, which indicates that they have independently evolved mechanisms to interact with the same components of the host cytoskeleton.

Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells.

Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S.flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.

Isolation and identification of Salmonella [and] Shigella /

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Center for Disease Control shigella surveillance.

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Coma caused by Shigella toxin-producing Escherichia coli in the absence of haemorrhagic uraemic syndrome (HUS)

Shigella toxin-producing Escherichia coli (STEC) is well known for its complications such as haemolytic uraemic syndrome (HUS), but neurological symptoms have also been reported. While most cases of infection with STEC occur with concurrent HUS, we describe a patient with severe neurological symptoms in the absence of HUS.

Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

In a study conducted in Joao Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them. (C) 2010 Elsevier Inc. All rights reserved.

Bacterial triggers and autoimmune rheumatic diseases

Autoimmune rheumatic diseases are generally considered as a multifactorial aetiology, mainly genetic susceptibility combined with environmental triggers of which bacteria are considered one of the most prominent. Among the rheumatic diseases where bacterial agents are more clearly involved as triggers are: reactive arthritis (ReA), rheumatic fever (RF) and Lyme disease. The role of bacterial infections in inducing other seronegative spondyloarthritis and antiphospholipid antibody syndrome has been hypothesized but is still not proven. The classic form of ReA is associated with the presence of HLA-B27 and is triggered by the urethritis or enteritis causing pathogens Chlamydia trachomatis and the enterobacteria Salmonella, Shigella, and Yersinia, respectively. But several other pathogens such as Brucella, Leptospira, Mycobacteria, Neisseria, Staphylococcus and Streptococcus have also been reported to cause ReA. RF is due to an autoimmune reaction triggered by an untreated throat infection by Streptococcus pyogenes in susceptible individuals. Carditis is the most serious manifestation of RF and HLA-DR7 is predominantly observed in the development of valvular lesions. Lyme disease is a tick-transmitted disease caused by the spirochete Borrelia burgdorferi. Knowledge is limited about how this spirochete interacts with human tissues and cells. Some data report that Borrelia burgdorferi can manipulate resident cells towards a pro- but also anti-inflammatory reaction and persist over a long period of time inside the human body or even inside human cells.

Pesquisa de enterobacteriáceas e enterovírus em crianças normais e com quadros diarréicos agudos

Num grupo de 263 crianças de 0 a 24 meses de idade, atendidas de outubro de 1963 a setembro de 1964 no Centro de Saúde da Lapa, na Capital de São Paulo, foram feitos exames de fezes para a pesquisa de Escherichia coli, do grupo da gastroenterite infantil, Shigella, Salmonella, poliovírus, vírus Coxsackie e vírus ECHO. O agente mais freqüentemente isolado foi o vírus da poliomielite (15,97% de positividade), seguindo-se-lhe a Escherichia coli G.E.I. (10,65%), Shigella (9,51%), vírus Coxsackie (7,22%), Salmonella em 3,04% dos casos e vírus ECHO no mesmo percentual. Em 16,22% das 111 que apresentaram resultado positivo, o exame revelou a presença de dois ou mais agentes. Os agentes acima referidos foram isolados de 42,21% da totalidade das crianças examinadas. Dentre as 167 que apresentavam diarréia, a porcentagem de positividade chegou a 47,90%, e nas 96 que nao a referiam, desceu a 32,29%. Sòmente em relação às shigelas foi possível evidenciar associação entre isolamento de microrganismos e quadro diarréico. Para a E. coli 0111, salmonelas e vírus Coxsackie A, os resultados foram apenas sugestivos desta associação, e para os demais agentes não houve evidência alguma favorável à mesma.

Identificação de cepas de Escherichia coli enteropatogênicas em amostras de fezes, por reação de imunofluorescência direta

Foi realizado diagnóstico etiológico de casos de diarréia aguda em 121 pacientes internados na Clínica Pediátrica do Hospital da Santa Casa de São Paulo, Brasil. Foram utilizados os métodos bacteriológico clássico e de reação de imunofluorescência direta para a identificação de cepas de Escherichia coli enteropatogênicas: para estudo da sensibilidade das cepas de Escherichia coli isoladas, a diferentes antibióticos, foi usado o método de Concentração Inibitória Minima (CIM). Dos 56 casos positivos, 89,3% correspondiam a diferentes sorotipos enteropatogênicos de Escherichia coli, quando utilizada a técnica de imunofluorescência direta. O método bacteriológico clássico revelou ainda, nos 121 casos examinados, 4 cepas de Salmonella e 2 de Shigella. No estudo da CIM verificou-se maior sensibilidade das cepas de Escherichia coli enteropatogênicas estudadas à Gentamicina e Amikacina, do que aos outros antibióticos.

Hygiene habits and carriers in families with a child who has had typhoid fever

The relationship between asymptomatic shedding of bacterial enteropathogens and the hygiene habits of families who have had a child with typhoid fever (TF) are investigated. The sample was made up of 80 families: 40 families in which one child had had TF (Group A) and 40 in which no children or either of the parents had had a history of TF (Group B). In each group 20 families belonged to a low socieconomic status (SES) and 20 to a high SES. A structured interview was used to evaluate the SES and the hygiene habits of the child; observations were made to measure the hygiene habits of the family (toilet, kitchen and food preparation) and bacteriological studies (fecal samples and hand markers). Results show that carriers were more frequent in Group A than in Group B. The bacterial species found were significantly more numerous in Group A than in Group B (fecal samples: E. coli, the classic serotypes, Shigella ssp, and hand markers: E. coli). Families of Group A had higher carriage rates than those of Group B. Finally there exists a significantly higher association between inadequate hygiene habits and carrier families. These results show the need to teach specific habits of proper hygiene to the entire population, because the fact of belonging to the high SES does not in itself preclude inadequate hygiene habits.

Identificación y tipificación de biotipos y serotipos de Yersinia enterocolitica

OBJETIVO: Conocer la existencia de Yersinia enterocolitica en suínos visiblemente sanos y sacrificados para el consumo humano. MÉTODOS: Fueron estudiadas 100 muestras de tejido linfático obtenidas en el momento del sacrifício, en un matadero del Estado de México. Fueron realizados muestreos pilotos de 20 casos, de los cuales 20% fueron positivos, permitiendo obtener una muestra estudiada (n=100). Las muestras colectadas de tejido linfático fueron acondicionadas para el aislamiento de Yersinia enterocolitica en caldo de Rappaport y en medio de cultivo de Salmonella-Shigella y MacConkey. Las identificaciones fueron efectuadas por medio de pruebas bioquímicas y serológicas, utilizándose en el caso los antisueros O:3, O:8 y O:9 para la biotipificación correspondiente. RESULTADOS: Fueron obtenidos 22 aislamientos tipificándose 8 serotipos pertenecientes al O:3 y 8 al O:9 correspondientes al biotipo 1; y, en 6 muestras no fue posible la serotipificación. No se encontró en el total de los aislados el serotipo O:8. CONCLUSIONES: En base en la metodología, se registró la presencia de Y. enterocolitica y sus serogrupos en tejido linfático de porcinos por la primera vez en México; esto es importante porque el patógeno y sus serotipos aislados están comprometidos con mayor frecuencia con problemas de salud pública.

Species and serovars of enteropathogenic agents associated with acute diarrheal disease in Rosario, Argentina

We report the most frequent species and serovars of enteropathogenic organisms in Rosario from 1985 to 1993. Enteropathogenic Escherichia coli was the most prevalent agent affecting 144/570 (25.2%) children; 0111 represented 41.8%, 055: 13.6%, 0119: 12.7%. Among enterotoxigenic E. coli (ETEC) the most frequent were ETEC-ST 0128:H21 and 0153:H45. Shigella spp were isolated in 8.8%; S.flexneri: 7%, principally type 2 (59.5%); S. sonnei: 1.6%, and S. dysenteriae type 2: 0.2%. Campylobacter spp were found in 6.1% of patients; C.jejuni: 4.6%; C. coli: 1.4% and C. lari: 0.2%; except groups 0 13,50 and 0 4 (2 cases each), no predominant serogroups were found. Salmonella was isolated in 2.8% of cases, being the predominant serovar S. typhimurium until 1986, but a dramatically increase of cases due to S. enteritidis was observed since 1987. There was 1.9% of Aeromonas spp and 2 cases due to Vibrio cholerae non 0-1. No Yersinia was found. In patients with gastroenteritis due to Shigella, Campylobacter, Salmonella, or EPEC as the unique pathogen, leukocytes were observed in the faeces in 70%, 50%, 20%, and 10% of cases respectively.

EVALUATION OF A RAPID SCREENING ASSAY FOR BACTERIAL IDENTIFICATION (DOT-ELISA) IN FECAL SAMPLES FROM CHILDREN

With the objective of standardizing a Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA) to detect antigens of fecal bacterial enteropathogens, 250 children, aged under 36 months and of both sexes, were studied; of which 162 had acute gastroenteritis. The efficacy of a rapid screening assay for bacterial enteropathogens (enteropathogenic Escherichia coli "EPEC", enteroinvasive Escherichia coli "EIEC", Salmonella spp. and Shigella spp.) was evaluated. The fecal samples were also submitted to a traditional method of stool culture for comparison. The concordance index between the two techniques, calculated using the Kappa (k) index for the above mentioned bacterial strains was 0.8859, 0.9055, 0.7932 and 0.7829 respectively. These values express an almost perfect degree of concordance for the first two and substantial concordance for the latter two, thus enabling this technique to be applied in the early diagnosis of diarrhea in infants. With a view to increasing the sensitivity and specificity of this immunological test, a study was made of the antigenic preparations obtained from two types of treatment: 1) deproteinization by heating; 2) precipitation and concentration of the lipopolysaccharide antigen (LPS) using an ethanol-acetone solution, which was then heated in the presence of sodium EDTA

Etiology of acute diarrhea among children in Ribeirão Preto-SP, Brazil

To study the main enteropathogens causing diarrhea in the region of Ribeirão Preto regarding serogroups and serotypes, the feces of 1836 children under 10 years old, from both sexes, attack of acute gastroenteritis, were analysed during a period of 4 years in Adolfo Lutz Institute - Ribeirão Preto, SP. The pathogens identified by standard methods were the following: Escherichia coli, Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., and Cryptosporidium spp. Positive samples were 22.8% (419) with 1.7% association of pathogens. Larger isolates were mainly from children 0 to 11 months old. Enteropathogenic E. coli (EPEC) was most frequent (8.7%) with predominance of serogroup O119 (40.2%), followed by Shigella (6.2%), 63.6% of which S. sonnei.