Additive effect of the probiotics Lactobacillus exopolysaccharides and the Satureja calamintha extracts on enteropathogenic Escherichia coli adhesion

Abstract This study assessed the inhibitory potential of the probiotics Lactobacillus (LB) exopolysaccharides (EPS) with or without extracts of Satureja calamintha on enteropathogenic Escherichia coli (EPEc) responsible for gastroenteritis. Methanolic and hydromethanolic extracts were prepared by cold maceration and subjected to phytochemical screening. The compounds of the extracts were determined with the colorimetric assays and identified using high-performance liquid chromatography coupled with diode array detector (HPLC-DAD). Antioxidant activities of the extracts were also evaluated by using 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging. Antibacterial effect on EPEc was evaluated by using both agar disc diffusion and microdilution methods. The in vitro test of auto-aggregation was investigated. Microbiological analysis showed that 63% of the isolated LB were producing EPS, with the amount ranging from 8.21 to 43.13 mg/L. Chemical analysis of the extracts revealed the presence of polyphenols and flavonoids, more abundant in the hydromethanolic extract, which presented the highest content with 2.11 mg EGA/g of polyphenol and 1.64 mg EC/g of flavonoids and 1.71 mg EGA/g of polyphenol and 1.15 mg EC/g of flavonoids in the methanolic extract. Hydromethanolic extracts and EPS exhibited a more important activity than did the methanolic extract against EPEc. The combined action of EPS and extracts reduced the aggregation ability of EPEc and decreased the rate of their adhesion.

Detection of the mcr-1 gene in Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains isolated from broilers / Detecção do gene mcr-1 em estirpes de Escherichia coli Enteropatogênicas (EPEC) e Shigatoxigênicas (STEC) isoladas de frangos de corte

Enteropathogenic Escherichia coli (EPEC) and Shigatoxigenic E. coli (STEC) strains are among the major pathotypes found in poultry and their products, which are capable of causing human enteric infections. Colistin has been claimed the drug of choice against diseases caused by multidrug-resistant Gram-negative bacteria (MDRGN) in humans. The mcr-1 gene was the first plasmidial gene that has been described to be responsible for colistin resistance and has also been detected in birds and poultry products. Our study aimed to detect the mcr-1 gene in enteropathogenic strains of E. coli in order to evaluate the resistance to colistin in broilers. The material was obtained from 240 cloacal samples and 60 broiler carcasses. The strains were isolated by the conventional bacteriological method and by the virulence genes, which characterize the enteropathogenic strains and resistance, and the samples were detected by polymerase chain reaction (PCR). Of the 213 isolated strains of E. coli, 57 (26.76%) were characterized as atypical EPEC and 35 (16.43%) as STEC. The mcr-1 gene was found in 3.5% (2/57) of the EPEC strains and 5.7% (2/35) of the STEC strains. In this study, it was possible to confirm that the mcr-1 resistance gene is already circulating in the broiler flocks studied and may be associated with the pathogenic strains.(AU)

Escherichia coli Enteropatogênica (EPEC) e Shigatoxigênica (STEC) estão entres os principais patotipos encontrados em aves e produtos avícolas que são capazes de causar doença entérica no homem. A colistina tem sido preconizada como droga de escolha para o tratamento de doenças causadas por bactérias Gram-negativas multirresistentes em humanos. O gene mcr-1 foi o primeiro gene plasmidial a ser descrito como responsável pela resistência a colistina e tem sido descrito em aves e produtos avícolas. Este estudo tem como objetivo a detecção do gene mcr-1 em estirpes de E. coli enteropatogênicas a fim de avaliar a resistência a colistina em frangos de corte. O material foi obtido a partir de 240 amostras cloacais e 60 carcaças de frango de corte. As estirpes foram isoladas pelo método bacteriológico convencional e os genes de virulência, que caracterizam as estirpes enteropatogênicas, e resistência foram detectados pela reação em cadeia pela polimerase (PCR). Das 213 estirpes de E. coli isoladas, 57 (26,76%) foram caracterizadas como EPEC atípica e 35 (16,43%) como STEC. O gene mcr-1 foi encontrado em 3,5% (2/57) das estirpes EPEC e 5,7% (2/35) das estirpes STEC. Neste estudo foi possível confirmar que o gene de resistência mcr-1 já está em circulação nos lotes de frango de corte estudados e pode estar associado às estirpes patogênicas.(AU)

Shigatoxigenic and atypical enteropathogenic Escherichia coli in fish for human consumption

ABSTRACT Shigatoxigenic and enteropathogenic Escherichia coli with virulence and multidrug resistance profile were isolated from Nile tilapia. This study finding is of great importance to public health because they help understand this pathogen epidemiology in fish and demonstrate how these animals can transmit E. coli related diseases to humans.

Molecular diagnosis of diarrheagenic Escherichia coli isolated from Psittaciformes of illegal wildlife trade / Diagnóstico molecular de Escherichia coli diarreiogênicas isoladas de Psittaciformes do tráfico ilegal de animais silvestres

Diarrheagenic Escherichia coli (DEC) are considered one of the major causes of human diarrhea in developing countries. Some studies have pointed wild birds as important reservoirs for these pathogens. However, scarce species from the Psittaciformes order have been investigated. This study aimed to evaluate the presence of DEC strains in Psittaciformes from illegal wildlife trade. A total of 78 E. coli strains isolated from cloacal swab samples of 167 Psittaciformes in the Ceará State, Brazil, were evaluated regarding the presence of the following DEC virulence genes by polymerase chain reaction (PCR): eaeA and bfpA genes (Enteropathogenic E. coli - EPEC); stx1 and stx2 (Shiga toxin-producing E. coli - STEC); estA and eltB (Enterotoxigenic E. coli - ETEC); ipaH (Enteroinvasive E. coli - EIEC); aatA and aaiC (Enteroaggregative E. coli - EAEC). Positive strains for eaeA and bfpA genes were considered typical EPEC, while strain positive exclusively for the eaeA gene were classified as atypical EPEC. The eaeA gene was identified in 20 E. coli strains and bfpA in 22 isolates. In addition, 11 and 9 belonged to tEPEC and aEPEC, respectively. No strain was positive for stx1 or stx2. A total of 47 (60.3%) strains and a total of 136 birds (81.4%) were negative for the remaining DEC pathotypes investigated. In conclusion, psittacine from illegal wildlife trade in Ceará State, Brazil, presented a relevant prevalence of typical and atypical EPEC, potentially playing a role as reservoirs of DEC strains in the environment. Thus, proper control measures must be adopted to block the spread of these pathogens.(AU)

Escherichia coli diarreiogênicas (DEC) são consideradas uma das causas mais importantes de diarreia em países em desenvolvimento. Alguns estudos têm apontado aves silvestres como importantes reservatórios destes patógenos, entretanto, poucas espécies da ordem Psittaciformes têm sido investigada. O objetivo deste estudo foi analisar a presença de cepas de E. coli diarreiogênicas em Psittaciformes do tráfico de animais silvestres. Um total de 78 amostras de E. coli isoladas de suabes cloacais provenientes de 167 de Psittaciformes do Ceará, Brasil, foram avaliadas quanto a presença dos seguintes genes de virulência DEC por meio de reação em cadeia de polimerase (PCR): eaeA e bfpA (E. coli Enteropatogênica - EPEC); stx1 e stx2 (E. coli produtora de Shiga - STEC); estA e eltB (E. coli Enterotoxigênica - ETEC); ipaH (E. coli Enteroinvasiva - EIEC); aatA e aaiC (E. coli Enteroagregativa - EAEC). As cepas positivas para os genes eaeA e bfpA foram consideradas EPEC típicas, enquanto que as positivas exclusivamente para o gene eaeA foram classificadas como EPEC atípicas. O gene eaeA foi identificado em 20 cepas de E. coli e o gene bfpA em 22 dos isolados. Adicionalmente, 11 e 9 cepas foram classificadas como EPEC típicas e atípicas, respectivamente. Nenhuma cepa foi positiva para os genes stx1 e stx2. Um total de 47 cepas (60,3%) e um total de 136 aves (81,4%) foram negativas para os demais patotipos DEC pesquisados. Em conclusão, psitacídeos provenientes do tráfico de aves silvestres do estado do Ceará, Brasil, apresentaram relevante prevalência de EPEC típicas e atípicas, potencialmente participando como reservatórios de cepas DEC no ambiente. Portanto, medidas de controle devem ser adotadas para inibir a disseminação destes patógenos.(AU)

Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC)

ABSTRACT Psittacine birds have been identified as reservoirs of diarrheagenic Escherichia coli, a subset of pathogens associated with mortality of children in tropical countries. The role of other orders of birds as source of infection is unclear. The aim of this study was to perform the molecular diagnosis of infection with diarrheagenic E. coli in 10 different orders of captive wild birds in the state of São Paulo, Brazil. Fecal samples were analyzed from 516 birds belonging to 10 orders: Accipitriformes, Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Pelecaniformes, Piciformes, Psittaciformes and Strigiformes. After isolation, 401 E. coli strains were subjected to multiplex PCR system with amplification of genes eae and bfp (EPEC), stx1 and stx2 for STEC. The results of these tests revealed 23/401 (5.74%) positive strains for eae gene, 16/401 positive strains for the bfp gene (3.99%) and 3/401 positive for stx2 gene (0.75%) distributed among the orders of Psittaciformes, Strigiformes and Columbiformes. None of strains were positive for stx1 gene. These data reveal the infection by STEC, typical and atypical EPEC in captive birds. The frequency of these pathotypes is low and restricted to few orders, but the data suggest the potential public health risk that these birds represent as reservoirs of diarrheagenic E. coli.

Aislamiento de Escherichia coli enteropatógeno 0157: H16 de un caso de diarrea infantil y sus contactos familiares en La Pampa, Argentina / Isolation of enteropathogenic Eschjerichia coli0157:H16 identified in a diarrhea case in a chil and his household contacts in La Pampa Province, Argentina

Escherichia coli enteropatógeno (EPEC) es uno de los principales agentes de diarrea infantil aguda en los países en desarrollo. Se clasifica en típico (tEPEC) y atípico (aEPEC) sobre la base de la presencia del factor bfp, asociado a la adherencia y codificado en el plásmido pEAF. Se describe el aislamiento de E. coli O157:H16, de la categoría aEPEC, en un caso de diarrea sanguinolenta infantil y en sus contactos familiares. De las muestras de materia fecal del niño, de la madre, del padre y de la hermana se aisló E. coli O157:H16 eae-??-positivo, sorbitol-positivo, ß-glucuronidasa-positivo, sensible a los antimicrobianos ensayados, y negativo para los factores stx1, stx2, ehxA y bfp. Por XbaI-PFGE, todos los aislamientos presentaron el patrón de macrorrestricción AREXHX01.1040, con 100% de similitud. Es importante la vigilancia epidemiológica de los casos de diarrea asociados a E. coli O157 y sus contactos familiares, y la incorporación de técnicas para detectar los distintos patotipos de E. coli

Enteropathogenic Escherichia coli (EPEC) is a major causative agent of acute diarrhea in children in developing countries. This pathotype is divided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the bfp virulence factor associated with adhesion, encoded in the pEAF plasmid. In the present study, the isolation of aEPEC O157:H16 from a bloody diarrhea case in a child and his household contacts (mother, father and sister) is described. The strain was characterized as E. coli O157:H16 eae-??-positive, sorbitol fermenter with ß-glucuronidase activity, susceptible to all antimicrobials tested, and negative for virulence factors stx1, stx2, ehxA and bfp. XbaI-PFGE performed on all isolates showed the AREXHX01.1040 macrorestriction pattern, with 100% similarity. These results highlight the importance of epidemiological surveillance of E. coli O157-associated diarrhea cases identified in children and their family contacts, as well as the incorporation of molecular techniques that allow the detection of the different E. coli pathotypes

Identification of Enteroaggregative Escherichia coli in Mongolia / Монголын Анагаах Ухаан

Introduction: Enteroaggregative Escherichia coli (EAEC) is an important agent of acute and persistent diarrhea worldwide. Few cases have been reported in healthy children. EAEC strains are characterized by aggregative adherence (AA) to HEp-2 cells, wherein bacteria are seen in “stacked brick” aggregates attaching to HEp-2 cells and usually to the glass surface between cells. Goal: To identify Enteroaggregative Escherihia coli using multiplex polymerase chain reaction (PCR) and HEp-2 adherence assay in Ulaanbaatar, Mongolia Materials and Methods: A total of 329 E. coli strains were isolated from stool with diarrhea in National Center for Communicable Diseases from July 2012 through September 2014. All specimens were processed by routine microbiological and biochemical tests in the bacteriological laboratories to identify Salmonella spp., Shigella spp. All specimens in our study were negative for these bacterial and parasitic pathogens. The biofilm formation was evaluated by the growth rate of E.coli on plastic surface. PCR assays were used to detect genes of five types of diarrheagenic E.coli (DEC). All of the DEC strains showed mannose-resistant adherence to HEp-2 cells, and aggregative adherence was predominant in these isolates. Bacterial susceptibility to antimicrobial agents determined by the Kirby Bauer disk diffusion method on Muller Hinton agar. Results: EAEC (31.9%) was the most prevalent by PCR and HEp-2 assay comparing with others. EAEC by multiplex PCR in samples (11, 3.3%), followed by enteropathogenic E.coli (EPEC) seen in 2.1%. Enterohemorrhagic E.coli (EHEC) and enteroinvasive E.coli (EIEC) were found in 7 (2.1%) and 1 (0.3%) of the samples. Enterotoxigenic E.coli (ETEC) and diffusely adhering E.coli were detected in 2 (0.6%), respectively. The evaluation of bacterial biofilm formation using 96 well plates showed 309 negative (93%), 15 weak biofilm (4.6%) and 8 moderate biofilm (2.4%) formation for E.coli and no strong biofilm forming strain was detected. Above 50% of antibiotic resistance was observed for ampicillin, trimethoprim/sulfamethoxazole, cefuroxime and cephalotin. Also, 95.4% of isolates were resistant to at least three different classes of antimicrobial agents and considered as multidrug resistance. Conclusion: EAEC is most prevalent pathogen among DEC in our samples. It is necessary to implement EAEC identifying method on Hep-2 assay in our laboratory practice.

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.

Caracterización de cepas de Escherichia coli enteropatogénico (EPEC) aisladas durante el proceso de faena de pollos / Characterization of enteropathogenic Escherichiia coli (EPEC) strains isolated during the chicken slaughtering process

En Argentina, Escherichia coli enteropatogénico (EPEC) es uno de los agentes más prevalentes aislados de niños con diarrea. Debido a que la contaminación con este patotipo en productos de pollo podría ocurrir durante el proceso de faena, nos planteamos como objetivo aislar y caracterizar EPEC de muestras de animales vivos (cloacas), carcasas evisceradas sin lavar, carcasas lavadas y agua del tanque de enfriamiento. Se caracterizaron 29 aislamientos de EPEC que presentaron una amplia variedad de serotipos, algunos de los cuales (O2:H40, O8:H19 y O108:H9) han sido informados en otras especies animales. También se encontró el serotipo O45:H8, aislado con anterioridad de niños con diarrea. Se detectaron aislamientos de los serotipos O2:H40, O108:H9 y O123:H32 en distintas etapas del proceso de faena, lo que sugiere que el procesamiento no se realiza en forma adecuada. Se torna necesario reforzar las medidas de control e higiene en las distintas etapas del proceso para disminuir la contaminación microbiana

In Argentina, EPEC is one of the most prevalent agents isolated from children with diarrhea. Because contamination with this pathotype could occur during slaughter, the aim of this study was to isolate and characterize EPEC strains obtained from live animals (cloacae), eviscerated carcasses, washed carcasses and water from chillers. Twenty nine isolates of atypical EPEC were characterized. These isolates presented a wide variety of serotypes, some of which (O2:H40, O8:H19 and O108:H9) had been reported in other animal species. Serotype O45:H8, previously isolated from children with diarrhea was also found. Isolates of serotypes O2:H40, O108:H9 and O123:H32 were detected at different stages of the slaughtering process, suggesting that the process is not adequately performed. This latter fact highlights the importance of reinforcing control and hygienic measures at different stages of the chicken slaughtering process in order to reduce microbial contamination

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.

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.

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.

Detection & characterization of Shiga toxin producing Escherichia coli (STEC) & enteropathogenic Escherichia coli (EPEC) in poultry birds with diarrhoea.

Background & objectives Limited information is available on shiga toxin producing Escherichia coli (STEC) in animals and birds from India. An outbreak of acute diarrhoea in poultry birds at Aizawl, Mizoram was investigated for detection and characterization of STEC and enteropathogenic E. coli (EPEC). Methods E. coli was isolated and identified from rectal swabs, intestinal contents, heart blood and spleen of 19 poultry birds that died due to acute diarrhoea during the outbreak. Phenotypic characterization was done by standard bacteriological and biochemical techniques. All the isolates were serotyped based on their somatic antigens. Virulence genes (stx1, stx2, eaeA and hlyA) were detected by multiplex PCR assay. Results A total of 42 E. coli isolates were obtained, of which 24 belonged to 3 serogroups (O64, O89 and O91) and the remaining 18 were untypable (UT). Altogether, 14 (33.33%) isolates carried at least 1 virulence gene, of which 10 (23.81%) and 4 (9.52%) were recorded as STEC and EPEC, respectively. Of the 10 STEC isolates, one carried only stx2, one carried stx2 and hlyA, four carried stx1, stx2 and hlyA, two carried stx1, eaeA and hlyA genes and two carried stx1 and eaeA. Of the four EPEC isolates, two carried eaeA and hlyA, one carried only eaeA gene and 1 carried only hlyA gene. Interpretation & conclusions This is the first report on the involvement of STEC in poultry in India.

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.

Atividade antibacteriana do óleo essencial do manjericão frente a sorogrupos de Escherichia coli enteropatogênica isolados de alfaces / Antibacterial activity of essential basil oil against serogroups of enteropathogenic Escherichia coli isolated from lettuce

Considerando-se a crescente participação dos vegetais na transmissão de microrganismos, incluindo as Escherichia coli enteropatogênicas multirresistentes às drogas convencionais, a busca por novos compostos com propriedades antimicrobianas a partir de fontes naturais, tais como os óleos essenciais, tem se intensificado nos últimos anos. Esta pesquisa objetivou avaliar a atividade antibacteriana do óleo essencial do manjericão (Ocimum basilicum Linn.) frente a sorogrupos de Escherichia coli enteropatogênicas (EPEC) isolados de alfaces (Lactuca sativa), utilizando-se o Método de Difusão em Disco (MDD). Os resultados evidenciaram que todas as cepas de E. coli EPEC testadas apresentaram sensibilidade à ação do óleo essencial, sugerindo que este possa ser uma fonte de compostos com potencial terapêutico no combate a bactérias patogênicas.

Considering the increase of the involvement of vegetables in the transmission of microorganism including the enteropathogenic Escherichia coli multiresistance to conventional drugs, the search for new antimicrobial compounds from natural sources, such as the essential oils, has been intensified in the last years. This research aimed to evaluate the antibacterial activity of the essential oil from basil (Ocimum basilicum Linn.) front to sorogroups of enteropathogenic Escherichia coli (EPEC) isolated in lettuces (Lactuca sativa) using the Diffusion in Disk Method. The results showed that, all the tested strains of E. coli EPEC presented sensitivity to the essential oil's action, suggesting that it can be a source of compounds with therapeutic potential in the fighting to bacteria pathogenic.

Comparación entre el diagnóstico serológico y el diagnóstico por Reacción en Cadena de la Polimerasa (PCR) para Escherichia coli Enteropatogénica (EPEC) / Comparison of Enteropathogenic Escherichia Coli (EPEC) diagnosis by serology and by Polymerase Chain Reaction (PCR)

Introdución. En los laboratorios clínicos la identificación de EPEC se basa en la determinación de serotipos específicos por técnicas de aglutinación utilizando antisueros O y H. Actualmente la identificación del gen de intimina (eaeA) por PCR es el método diagnóstico de elección para EPEC. Objetivos. Comparar el diagnóstico por serología con el diagnóstico por PCR de cepasde EPEC. Materiales y Métodos. Se recolectaron cepas identificadas como EPEC en base al antígeno O, de 4 laboratorios clínicos de Lima, procedentes de muestras de diarrea de niños menores de 5 años. En estas cepas se buscaron genes relacionados a virulencia mediante un PCR múltiple a tiempo real para las E. coli diarreogénicas. Resultados. Se recolectaron 113 cepas; 82% de niños menores de 2 años. Únicamente15 cepas (13.3%) presentaron el gen de intimina con un diagnóstico confirmatorio de EPEC. Adicionalmente se encontraron 3 cepas enterotoxigénicas (ETEC), 3 productoras de shiga-toxina (STEC), 1 entero agregativa (EAEC) y 1 enteroinvasiva (EIEC). Conclusiones. Para la identificación correcta de EPEC se debe usar el PCR. Sin embargo, los métodos moleculares aún no están fácilmente disponibles en los laboratorios clínicos a nivel mundial.

Introduction. The identification of EPEC in clinical laboratories is based on the determination of the serotypes by agglutination with O and H antiserum. Currently the proper diagnosis of EPEC should be done by the identification of the intimin gen (eaeA) by PCR. Objectives. To compare the diagnosis of EPEC by serotypin and by PCR. Materials and Methods. We collected EPEC strains, identify by their O antigen, from 4 clinical laboratories in Lima from diarrheal samples in children less than 5 years of age. In those strains we have searched for virulence genes by a real time multiplex PCR for the diarrheagenic E. coli. Results: We collected 113 strains; 82% from children less than 2 years of age. Only 15 strains (13.3%) had the intimin gene and therefore a confirmatory diagnosis of EPEC. In addition we found 3 enterotoxigenic (ETEC), 3 shiga toxin-producing (STEC), 1 enteroagreggative (EAEC) and 1 enteroinvasive (EIEC) strains. Conclusions. PCR should be use for the proper identification of EPEC. However, molecular methods are still not easily available in clinical laboratories worldwide.

Anticorpos anti-intimina: análise da reatividade dos anticorpos policlonal e monoclonal, clonagem e expressão do fragmento variável de cadeia simples (scFv) do anticorpo monoclonal / Anti-intimin antibodies: polyclonal and monoclonal reactivity analyzes, cloning and expression of single chain fragment variable (scFv) of monoclonal antibodies

Intimina é o principal fator de virulência na patogênese de Escherichia coli enteropatogênica (EPEC) e de Escherichia coli enterohemorrágica (EHEC). A detecçãode EHEC e EPEC típica ou atípica é de fundamental impotância na definição da conduta terapêutica das infecções promovidas por E. coli, que ainda são a principal causa de diarreia aguda em crianças e adultos em muitos paises desenvolvidos e em desenvolvimento. Anticorpos são ferramentas importantes na detecção de diversos patógenos. Neste trabalho avaliou-se a sensibilidade e especificidade dos anticorpos policlonal e monoclonal anti-intimina frente a isolados de EPEC e EHEC por immunoblotting. Os anticorpos apresentam 100% de especificidade e a sensibilidade foi de 97%, 92% e 78%, quando se utilizou a fração enriquecida em IgG do soro de coelho, antissoro de rato e anticorpo monoclonal, respectivamente. Esse anticorpo monoclonal anti-intimina foi caracterizado como IgG2b e 1 μg desse corpo reconheceu 0,6 μg de intimina purificada com uma constante de dissociação de 1.3 x 10&sup-8; M. A menor reatividade do anticorpo monoclonal em relação aos anticorpos policlonais levou-nos à clonagem e expressão do fragmento variável de cadeia simples desse anticorpo (scFv). Para isto, o mRNA do hibridoma anti-intimina foi extraído, reversamente transcrito para cDNA e amplicadas as cadeias leve e pesada da fração variável do anticorpo, utilizando iniciadores aleatórios comerciais. As cadeias amplificadas foram ligadas ao vetor pGEM-T Easy e sequenciadas. Iniciadores específicos foram desenhados e utilizados em uma estratégia de amplificação e união das cadeias, formando o scFv, que por sua vez foi clonado no vetor de expressao pAE. Linhagem E. coli BL 21(DE3)pLys foi transformada com o plasmídeo pAE-scFv anti-intimina e submetida a indução proteíca. O scFv anti-intimina foi expresso de forma insolúvel, solubilizado, purificado e submetido ao ensaio de refolding. O rendimento obtido foi de 1 mg de proteina por 100mL...

Intimin is the major virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli(EPEC) and enterohemorrhagic Escherichia coli(EHEC). The detection of EHEC and typical or atypical EPEC has fundamental importance in defining the therapeutic management of infections causes by E. Coli, which are still the leading cause of acute diarrhea in children and adults in many developed and developing countries. Antibodies are important tools in the detection of several pathogens. In this study it was evaluated the sensitivity and specificityit of polyclonal and monoclonal antibodies against intimin in the detection of EPEC AND EHEC by immunobloting. All employed antibodies showed 100% specifity and sensitivity was 97%, 92% and 78% for rabbit anti-intimin IgG-enriched fraction, rat antisera and monoclonal antibody, repectively. This anti-intimin monoclonal was characterized as IgG2b and 1 μg recognized 0.6 μg of purified intimin with a dissociation constant of 1.3 x 10&sup-8;M. The less extend reactivity of monoclonal led us to clone and express the single chain fragment variable of this antibody (scFV). Thus, the anti-intimin hybridoma mRNA was extrated, reverse transcribed to cDNA and the light and heavy chains of variable fragment of the antibody were amplified using commercial random primers. The chains were amplified, ligated to the pGEM-T Easy vector and the insert was sequenced. Specific primers were designed and used in strategy to amplify and link the chains, obtaining the scFv, wich was cloned into the pAE expression vector. E. coli BL21(DE3)plys was transformed with the pAE-scFv anti-intimin plasmid and subjected to induction to protein expression. The scFv anti-intimin, expressed in the insoluble fraction, was purified and submitted to refolding. The yield was 1 mg of protein per 100 mL of bacterial culture. To test the functionalityof scFv, ELISA and immunofluorescence assays were performed. The results showed 275 ng of scFv...

Genotypic and Phenotypic Characterization of Enteropathogenic Escherichia coli Isolated from Diarrheal Patients in Gwangju

Enteropathogenic Escherichia coli (EPEC) have been implicated in human diarrhea in several countries. Central to EPEC-mediated disease is its ability to cause intestinal lesions, known as attaching and effacing (A/E) lesion. We investigated 92 EPEC strains isolated from patients with diarrhea in Gwangju for their genotypic and phenotypic characteristics. Sixteen (17.4%) of all strains were found to be typical EPEC because they were bfpA gene positive by PCR. The most of typical EPEC isolates (87.5%) showed a localized adhesion (LA) pattern in Hep-2 cell adherence assay, whereas, only 11 atypical EPEC isolates (14.5%) were adhered to Hep-2 cells in a localized manner. Thirteen of the EPEC strains studied belonged to classical O-serogroups of EPEC and 7 isolates were classified as nonclassical EPEC serogroup and the other isolates could not be serotyped with our antisera. The subtypes of eae, tir, espA and espB genes which are major virulence genes concerned of A/E lesion on chromosome were analyzed by multiplex PCR for finding the original resource. The results showed that the composition of these genes subtypes was homogenous and heterogenous in 12 and 26 isolates, respectively. The others were non-determined type in terms of the gene subtype because of genetic diversity of intimin-coding eae genes. Our findings indicated that EPEC isolates from patients with diarrhea were diverse genetically and phenotypically, which require further study in regard to their virulence and epidemiological significance.

Phenotypic and Genotypic Characteristics of Enteropathogenic E. coli Isolated from Children with Diarrhea in Gwangju City

BACKGROUND: Enteropathgenic Escherichia coli (EPEC) commonly causes infantile diarrhea in the developing countries. This study aims to find out the phenotypic and genotypic characteristics of EPEC in children with diarrhea in Gwangju city. METHODS: We isolated 35 strains from the stool obtained from diarrheal patients and investigated the presence of various virulence genes, adherence patterns, hemolysis, and antibiotic resistance patterns. RESULTS: All isolates were negative for the EPEC adherence factor (EAF) plasmid, and 14 isolates were bfpA-positive by PCR. The eae, tir, espA, and espB genes were analyzed by multiplex PCR. When the results of the four multiplex PCRs were analysed, we observed that the rate of the presence of eaegamma-tiralpha-espAalpha-espBalpha was highest. The incidence of enteroaggregative E. coli heat-stable enterotoxin (east) was 17.1%. Analysis of Hep-2 cell adherence showed three adherence patterns:the localized adherence pattern, the diffuse adherence pattern, the localized adherence-like pattern. In hemolysin assay, four isolates produced enterohemolysin. The resistance rate of isolates against tetracycline, streptomycin, ampicillin, and rifampin was 56%, 39%, 34%, and 34%, respectively. All isolates were susceptible to ciprofloxacin and colistin. CONCLUSION: In our study, the rate of the presence of eaegamma-tiralpha-espAalpha-espBalpha was the highest. Analysis of Hep-2 cell adherence showed various adherence patterns. Seventy-five percent of isolates were resistant to at least one antibiotic and 28% were resistant to four or more antibiotics.