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.

Surveillance for enterotoxigenic & enteropathogenic Escherichia coli isolates from animal source foods in Northwest Iran

Background & objectives: Diarrhoeagenic Escherichia coli strains are common agents of diarrhoea particularly in developing countries. Food products of animal origin are considered as common carriers of E. coli. This study was undertaken to identify enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) pathotypes in animal-source foods (ASF). Methods: A total of 222 ASF samples were investigated. Based on the culture and biochemical tests, 109 E. coli isolates were identified. Duplex-polymerase chain reaction assay was used to detect ETEC and EPEC. The target genes selected for each category were the lt and st for the ETEC, and eae and bfp for the EPEC isolates. Results: The occurrence of E. coli in dairy and meat products was 45 and 52.5 per cent, respectively. Among the E. coli isolates, two ETEC, one typical EPEC and three atypical EPEC were detected in meat samples, whereas only one typical EPEC and one atypical EPEC were detected in dairy samples. Interpretation & conclusions: Our results showed presence of ETEC and EPEC strains in ASFs. The milk without pasteurization and traditional dairy products produced in unhygienic conditions are most likely the main sources of E. coli pathotypes and other zoonotic pathogens and thus can be considered a potential hazard to the health of the community.

Occurrence of Escherichia coli virulence genes in feces of wild birds from Central Italy

Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic, enterohemorrhagic and Shiga-toxins producing Escherichia coli (E. coli), enteropathogenic E. coli, enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular, eaeA encoding intimin, hlyA encoding for hemolysin, stx1 and stx2 genes encoding Shiga-toxins 1 and 2, respectively, were investigated. Results: Overall, 21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail, 12(9.91%) samples were positive for eaeA, 10(8.26%) for stx1, 4(3.31%) for hylA and 1(0.83%) for stx2. An owl (Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However, virulence genes characterizing EPEC, and EHEC strains were mainly found among seagulls, waterfowl and feral pigeons. Conclusions: Seagulls, waterfowl and feral pigeons, which frequently reach and contaminate rural, urban and peri-urban areas with their droppings, may be important sources of E. coli infection for other animals and humans.

Occurrence of Escherichia coli virulence genes in feces of wild birds from Central Italy

Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic, enterohemorrhagic and Shiga-toxins producing Escherichia coli (E. coli), enteropathogenic E. coli, enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular, eaeA encoding intimin, hlyA encoding for hemolysin, stx1 and stx2 genes encoding Shiga-toxins 1 and 2, respectively, were investigated. Results: Overall, 21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail, 12(9.91%) samples were positive for eaeA, 10(8.26%) for stx1, 4(3.31%) for hylA and 1(0.83%) for stx2. An owl (Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However, virulence genes characterizing EPEC, and EHEC strains were mainly found among seagulls, waterfowl and feral pigeons. Conclusions: Seagulls, waterfowl and feral pigeons, which frequently reach and contaminate rural, urban and peri-urban areas with their droppings, may be important sources of E. coli infection for other animals and humans.

Preparation and effect evaluation of the adjuvant vaccine of enteropathogenic Escherichia coli in Tibetan piglets / 中国人兽共患病学报

To study the immune protection of the inactivated vaccine against the enteropathogenic E.coli in Tibetan pigs,the strains isolated from the dead pig was identified by biochemistry and PCR methods.After that,the biological adjuvant vaccine was prepared by following procession.Firstly,selected enteropathogenic E.coli strain was cultured.Then,we harvested the bacteria and inactived it to prepare the antigen.Finally,we added the recombined cholera toxin B subunit as the biological adjuvant,added the mannose in solution 3 ％-5％ (W/V),distributed in ampoule,and freeze-dried.The performances of the vaccine was evaluated by administration for the nine groups of KM mice in oral and intramuscular immuno strategies,respectively.Results demonstrated that the effect of intramuscular injection of low dose containing adjuvant group were better than those without adjuvant group.The oral group contained both high dose of adjuvant group and low dose effect of immune adjuvant group were better than that of high and low dose not containing adjuvant group,and high dose of immune effect was better than low dose immune effect.The antibody titers proved that immunization for 4 times was much better than those immunization for times less than that.The data showed the vaccine was high protection against Tibetan Pig enteropathogenic E.coli challenge,especially the high dose of adjuvant vaccine was 100％ protection rate against enteropathogenic E.coli when orally immunization for 4 times in mice.

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ / Transferência placentária e colostral de anticorpos reativos a Escherichia coli enteropatogênica com expressão das intiminas α, ß, or γ

Abstract Objective: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. Methods: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, β, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. Results: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. Conclusions: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Resumo Objetivo: As intiminas são adesinas proteicas de Escherichia coli enteropatogênicas (EPEC) e enterro-hemorrágicas (EHEC) capazes de induzir as lesões attaching and effacing nos enterócitos. Anticorpos anti-intiminas são importantes para a proteção contra infecções por EPEC e EHEC porque esses anticorpos inibem a adesão bacteriana e impedem o passo inicial do mecanismo patogênico dessas bactérias. Nós estudamos a transferência de anticorpos maternos anti-intiminas de mães brasileiras saudáveis para os seus recém-nascidos através da placenta e do colostro. Métodos: Anticorpos séricos da classe IgG e secretórios da classe IgA (SIgA) reativos com as porções conservada (cons) e variáveis das intiminas α (vα), β (vβ) e γ (vγ) foram analisados pelo teste de ELISA no sangue e no colostro de 45 parturientes saudáveis e no sangue de cordão umbilical dos seus respectivos recém-nascidos. Resultados: As concentrações de anticorpos reativos com intimina vα foram significativamente mais baixas que as dos anticorpos anti-vγ e anti-cons nas amostras de colostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos, mas as concentrações de anti-cons foram significativamente mais altas tanto nas mães como nos recém-nascidos do que os anticorpos reativos com as regiões variáveis das intiminas. O padrão de transferência de IgG das mães para os recém-nascidos foi muito semelhante para todos os anticorpos anti-intiminas. Os valores de porcentagem de transferência foram semelhantes à transferência de IgG total. Conclusões: Anticorpos anti-intimina são transferidos das mães para os recém-nascidos pela placenta e corroboram a proteção contra infecções por Escherichia coli diarreiogênicas (DEC) conferida pelo aleitamento materno.

Antimicrobial susceptibility profile of enterotoxigenic and enteropathogenic Escherichia coli isolates obtained from fecal specimens of children with acute diarrhea / Perfil de suscetibilidade a drogas antimicrobianas de amostras de Escherichia coli enterotoxigênica e enteropatogênica isoladas de espécimes fecais de crianças com doença diarreica aguda

ABSTRACT Among the diseases which etiopathogenesis is associated with Escherichia coli, acute diarrhea stands out. Studies on the characterization of the antimicrobial susceptibility profile contribute to the selection of appropriate empirical antimicrobial therapy. In this study, the antimicrobial susceptibility profile of 98 enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) strains isolated from fecal specimens of children with acute diarrhea was evaluated. The resistance rates to ampicillin, sulfamethoxazole/trimethoprim, amoxicillin/clavulanate, and nalidixic acid were high, ranging from 34.7% to 10.2%. The result of this research recommends the use of cefotaxime and ceftriaxone for the empirical treatment of children with acute diarrhea which the etiology suggested is ETEC or EPEC.

RESUMO Entre as doenças cuja etiopatogenia está associada à Escherichia coli, destaca-se a doença diarreica aguda. Estudos que visam à caracterização do perfil de suscetibilidade antimicrobiana contribuem para o delineamento de antibioticoterapia empírica eficaz. Neste estudo, foi avaliado o perfil de suscetibilidade a antimicrobianos de 98 amostras de E. coli enterotoxigênica (ETEC) e E. coli enteropatogênica (EPEC) isoladas de crianças com doença diarreica. As frequências de resistência a ampicilina, sulfametoxazol-trimetoprima, amoxicilina-clavulanato e ácido nalidíxico foram elevadas, variando entre 34,7% e 10,2%. Esta pesquisa recomenda o emprego de cefotaxima e ceftriaxona para o tratamento empírico de crianças com quadro de diarreia cuja etiologia sugerida seja ETEC ou EPEC.

Genomic evolution characteristics of pathogenicity islands of enteropatho-genic Escherichia coli Deng strain / 中国感染控制杂志

Objective To analyze the genomic evolution characteristics of pathogenicity islands (PAIs)in Deng strain of enteropathogenic Escherichia coli (E.coli,EPEC Deng).Methods EPEC Deng was isolated from infant stool specimen,serotypes were identified and antimicrobial susceptibility testing was performed;whole-genome se-quencing was performed by Illumina 2000 system,the locations of prophages(PPs)in the chromosome were detected using PHAST software,collinearity analysis was performed by MUMmer software,phylogenetic trees of homolo-gous gene were constructed in order to understand the evolutional rule of homology gene.PAIs prediction was per-formed using PAI finder software,the homologous evolutionary rule of PAIs core region(LEE)and core genes were clarified,genetic polymorphism was analyzed.Results The serotype of EPEC Deng strain was O119:H6,the strain was resistant to ciprofloxacin,levofloxacin,and ampicillin,but sensitive to other antimicrobial agents.The complete circular chromosome contained 5 025 482 bp with a GC content of 50.52 %,and the plasmid contained 207 564 bp with a GC content of 49.50%.A total of 17 PPs in the chromosomal genome were discovered,phyloge-netic trees analysis suggested that EPEC Deng strain was highly homologous with O26:H11 and O111 :H strains;PAIs and core genes were highly homologous with RDEC-1 and O26:H413/89-1 strains;genetic diversity analysis showed that the intimin (eae)and its receptor tir had high polymorphism,with the pi (π)value>0.10,the genes in type III secretion system was relatively stable.Conclusion The study clarified the genomic evolution characteris-tics of EPEC Deng genome and it’s PAIs,and is helpful for understanding genetic characteristics of native EPEC.

Avaliação do transporte transmembrânico de glicose, glutamina e alanil-glutamina em íleo decamundongos nutridos e desnutridos infectados por Escherichia coli enteropatogênica

A Escherichia coli enteropatogênica (EPEC) e a desnutrição são uma das maiores causadorasde morbidade e mortalidade infantil em países subdesenvolvidos. Este estudo propõe avaliar otransporte de Na+-glicose, Na+-glutamina e H+-alanil-glutamina através da medida da correntede curto circuito (CCC) e da resistência transepitelial (Rt) no íleo de camundongos nutridos edesnutridos infectados com EPEC. Camundongos albinos de 28 a 35 dias de idade foramsubmetidos a uma dieta padrão ou a uma dieta multideficiente em proteínas, lipídeos eminerais, a DBR. Parte desses animais foi submetida à infecção aguda por 3 horas de contatodireto com a bactéria em alça ligada. Para comparações, o mesmo foi feito utilizando abactéria comensal Escherichia coli HS...

Enteropathogenic Escherichia coli (EPEC) and malnutrition are a major cause of morbidityand mortality in developing countries. The purpose of this study was to evaluate the transportof Na+-glucose, Na+-glutamine and H+-alanyl-glutamine by measurement of short circuitcurrent (Isc) and transepithelial resistance (TR) in the ileum of nourished and malnourishedmice infected with EPEC. Albino mice of approximately 35 days of age were subjected to astandard diet or a diet deficient in proteins, lipids and minerals, the Regional Basic Diet(RBD). Of these animals was submitted to acute infection for 3 hours of direct contact withbacteria in isolated intestinal loop. For comparison, the same was done using the commensalbacterium Escherichia coli HS...

Enteropatógenos de importancia en salud pública / Relevant public health enteropathogens

La diarrea continúa siendo la tercera causa de muerte en niños menores de 5 años, pese a los avances recientes en el manejo y prevención de esta enfermedad. Es causada por múltiples patógenos, sin embargo, la prevalencia de cada uno varía según el grupo de edad, la zona geográfica y el escenario donde se registran los casos (comunitario vs hospitalario). Los patógenos de mayor relevancia en salud pública son aquellos asociados con mayor carga de enfermedad, severidad, complicaciones y mortalidad. En nuestro medio, el norovirus, Campylobacter y las E. coli diarrogénicas, son los patógenos más prevalentes a nivel comunitario en niños. En este artículo se revisa la epidemiología local y las potenciales áreas de desarrollo en cinco patógenos seleccionados: rotavirus, norovirus, E. coli productora de toxina Shiga (STEC), Shigella y Salmonella. De estos, el rotavirus es el más importante en la población pediátrica y el principal responsable de la mortalidad infantil por diarrea. La introducción de la vacunación contra rotavirus en nuestro país tendrá un importante impacto en la carga de enfermedad y la mortalidad por diarrea. Sin embargo, se requieren estudios de vigilancia para determinar el impacto de la vacunación y el cambio en la epidemiología de la diarrea en el Perú luego de la introducción de nuevas vacunas, así como la vigilancia de las tasas de resistencia antibiótica para las bacterias de importancia clínica.

Diarrhea remains the third leading cause of death in children under five years, despite recent advances in the management and prevention of this disease. It is caused by multiple pathogens, however, the prevalence of each varies by age group, geographical area and the scenario where cases (community vs hospital) are recorded. The most relevant pathogens in public health are those associated with the highest burden of disease, severity, complications and mortality. In our country, norovirus, Campylobacter and diarrheagenic E. coli are the most prevalent pathogens at the community level in children. In this paper we review the local epidemiology and potential areas of development in five selected pathogens: rotavirus, norovirus, Shiga toxin-producing E. coli (STEC), Shigella and Salmonella. Of these, rotavirus is the most important in the pediatric population and the main agent responsible for child mortality from diarrhea. The introduction of rotavirus vaccination in Peru will have a significant impact on disease burden and mortality from diarrhea. However, surveillance studies are needed to determine the impact of vaccination and changes in the epidemiology of diarrhea in Peru following the introduction of new vaccines, as well as antibiotic resistance surveillance of clinical relevant bacteria.

Distribution of non-LEE-encoded type 3 secretion system dependent effectors in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are important human gastroenteritis agents. The prevalence of six non-LEE genes encoding type 3 translocated effectors was investigated. The nleC, cif and nleB genes were more prevalent in typical than in atypical EPEC, although a higher diversity of genes combinations was observed in atypical EPEC.

Virulence profiles of Shiga toxin-producing Escherichia coli and other potentially diarrheagenic E.coli of bovine origin, in Mendoza, Argentina

This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.

Role of enteropathogenic escherichia coli in paediatric diarrhoeas in South India.

Background: Enteropathogenic Escherichia coli( EPEC) is a major cause of diarrhoea in children below 5 years of age. Serotyping is the classical method and PCR detection of virulence factors is a rapid way of detecting diarrhoeagenic Esch.coli. Objectives : To study the role of EPEC in Paediatric diarrhoea by both Serogrouping and Multiplex PCR assay and to analyse the antibiotic susceptibililty pattern of EPEC strains in our area. Materials and Methods : Prospective study of stool samples collected from children with diarrhoea and without diarrhoea who were below 5 years of age was conducted from May to November 2011. EPEC isolates were identified by Serogrouping. Escherichia coli isolates were subjected to Serogrouping and Multiplex PCR assay and those isolates which showed pathogenic genes were further serotyped. Antibiotic susceptibility pattern of EPEC isolates was determined by Clinical and Laboratory Standards Institute guidelines. Results : Among the Escherichia coli isolates in the diarrhoeal group, 36.8% were identified as EPEC by Serogrouping and 38.8% of them were found to possess EAEC genes by molecular characterisation. In the nondiarrhoeal Esch. coli strains , none agglutinated with EPEC polyvalent sera, 46.6% showed EAEC genes out of which 85.7% were of a single serotype O153. Among the Escherichia coli isolates which agglutinated with EPEC polyvalent antisera , 33.3% were positive for Enteroaggregative genes. Conclusion : EPEC is still an important pathogen in paediatric diarrhoeas . O serogrouping can still be relied upon for detection of EPEC. EAEC are present in classical ‘ O ‘ serogroups. Serotype O 153 has an increasing potential for asymptomatic carrier state in children below 5 years of age.

Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq.

Purpose: Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of diarrhoea. The study was carried out to investigate the occurrence of EPEC as a cause of infectious diarrhoea in children younger than 2 years of age and characterize their virulence genes. Materials and Methods: During the study period, a total of 656 faecal specimens from children with diarrhoea and 54 from healthy children were analyzed. E. coli isolates were serotypically identified with EPEC polyvalent and monovalent antisera. The isolated EPEC were examined for the presence of the attaching and effacing (eaeA), bundle-forming pilus (bfpA), Shiga like toxins (stx1 and stx2 ), enterohaemorrhagic E. coli enterohaemolysin (EHEC hlyA) and EPEC adherence factor (EAF) genes by the PCR assay. Results: The study has shown that 22 (3.4%) had diarrhoea due to EPEC, while no EPEC isolates were detected in asymptomatic children. The highest number of the EPEC isolated belonging to polyvalent 2. The primers encoding virulence genes were subjected to all the EPEC isolates. Only 9.1%, 27.3%, and 9.1% isolates gave positive re sults with intimin (eaeA), bfbA and (EAF) genes, respectively. None of the isolates were positive for stx 1, stx 2, and hlyA genes. Typical EPEC (eaeA +, bfpA +) was diagnosed in two isolates, while, atypical EPEC was manifested in four isolates. Conclusions: According to the results, the frequency of EPEC isolates in Najaf was lower than what has been suspected and the investigation including the use of molecular technique and serotyping, are necessary to allow precise identification and epidemiological study of these pathogens.

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.

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2 percent) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66 percent) were typical (escv+, bfp+) and 14 (34 percent) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90 percent) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.

Infecção experimental com Escherichia coli enteropatogênicas, identificadas por PCR utilizando cápsulas com revestimento entérico em filhotes da raça boxer / Experimental infection with enteropathogenic Escherichia coli identified by PCR using enteric-coated capsules in boxer pups

PURPOSE: To verify the possibility of an experimental infection with enteropathogenic Escherichia coli and to confirm by PCR that the symptoms manifested after infection were due to the virulence factors of the studied bacteria. METHODS: Experimental units were 14 healthy pups of Boxer breed, aged 60 days. The animals were divided into three groups. One animal from each litter was included in a control group and the remaining animals were divided into two groups: one inoculated with strain 4083, and another one inoculated with strain SPA14. Gelatinous capsules coated with enteric-coating solution were used for the inoculation of strains. E. coli isolation from feces was performed for all tested animals, and the extracted DNA was subjected to Polymerase Chain Reaction (PCR). RESULTS: All infected animals presented diarrhea and had the gene eae amplified by PCR. CONCLUSION: The efficiency of PCR for the studied strains indicates that this technique can be recommended for the diagnosis of enteropathogenic Escherichia coli as a differential from other pathogens causing diarrhea. It may also be used in the future to verify whether other virulence factors (bfpA gene and EAF plasmid) persist after infection and to assess the pathogenicity of these bacteria.

OBJETIVO: Verificar a possibilidade de uma infecção experimental com Escherichia coli enteropatogênicas e confirmar por PCR que os sintomas manifestados após a infecção foram decorrentes dos fatores de virulência da bactéria estudada. MÉTODOS: As unidades experimentais foram 14 filhotes saudáveis com idade de 60 dias da raça Boxer. Os animais foram divididos em três grupos, sendo um controle de cada ninhada e o restante dividido em dois grupos, um de animais inoculados com a cepa 4083 e o outro de animais inoculados com a cepa SPA14. Para inoculação das cepas, utilizaram-se cápsulas gelatinosas revestidas com solução de revestimento entérico. O isolamento de E. coli das fezes foi realizado em todos os animais testados, e o DNA extraído foi submetido à técnica de PCR. RESULTADOS: Todos os animais infectados apresentaram diarréia e tiveram a gene eae amplificado por meio de PCR. CONCLUSÃO: Através da eficiência da PCR das amostras, a técnica seria recomendada para diagnóstico da Escherichia coli enteropatogênicas como diferencial de outros patógenos que causam diarréia, e, no futuro, verificar se outros fatores de virulência (gene bfpA e plasmídeo EAF) permaneceriam após a infecção, podendo avaliar a patogenicidade das EPEC.

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.

An aptamer-based biosensor for colorimetric detection of Enteropathogenic Escherichia coli / 中华检验医学杂志

Objective To develop and evaluate an aptamer based biosensor (aptasensor) for rapid colorimetric detection of enteropathogenic Escherichia coli (EPEC). Method The aptasensor was fabricated by modifying the truncated LPS-binding aptamer on the surface of nanoscale polydiacetylene vesicles using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between EPEC and aptamer at the interface of the vesicle led to blue-red transition of polydiacetylene which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR). Transmission electron microscopy (TEM) was used to confirm the specific interactions between EPEC and polydiacetylene vesicles. Result Truncated aptamer showed the similar LPS-binding activity. The aptasensor could detect the target bacteria in a range of 105-108 colony-forming units (CFU)/ml within less than 30 minutes and its specificity was 100% for detection of EPEC O111. The sensor reproducibiliry obtained at 106 CFU/ml was 6. 08% R. S. D. The results of TEM confirmed that the specific interactions between EPEC and polydiacetylene vesicles. Conclusion A new aptasensor was developed successfully for rapid colorimetric detection of EPEC.