Estandarización de una técnica de PCR múltiple para la detección de los serogrupos O157, O104 y "big six" de Escherichia coli productora de la toxina Shiga (STEC) / Standardization of a multiplex PCR technique for the detection of Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O104 and "big six"

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)

STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)

The 2011 German Enterohemorrhagic Escherichia Coli O104:H4 Outbreak-The Danger Is Still Out There.

Enterohemorrhagic Escherichia coli (EHEC) are Shiga toxin (Stx) producing bacteria causing a disease characterized by bloody (or non-bloody) diarrhea, which might progress to hemolytic uremic syndrome (HUS). EHEC O104:H4 caused the largest ever recorded EHEC outbreak in Germany in 2011, which in addition showed the so far highest incidence rate of EHEC-related HUS worldwide. The aggressive outbreak strain carries an unusual combination of virulence traits characteristic to both EHEC-a chromosomally integrated Stx-encoding bacteriophage, and enteroaggregative Escherichia coli-pAA plasmid-encoded aggregative adherence fimbriae mediating its tight adhesion to epithelia cells. There are currently still open questions regarding the 2011 EHEC outbreak, e.g., with respect to the exact molecular mechanisms resulting in the hypervirulence of the strain, the natural reservoir of EHEC O104:H4, and suitable therapeutic strategies. Nevertheless, our knowledge on these issues has substantially expanded since 2011. Here, we present an overview of the epidemiological, clinical, microbiological, and molecular biological data available on the 2011 German EHEC O104:H4 outbreak.

Pro-inflammatory capacity of Escherichia coli O104:H4 outbreak strain during colonization of intestinal epithelial cells from human and cattle.

In 2011, Germany was struck by the largest outbreak of hemolytic uremic syndrome. The highly virulent E. coli O104:H4 outbreak strain LB226692 possesses a blended virulence profile combining genetic patterns of human adapted enteroaggregative E. coli (EAEC), rarely detected in animal hosts before, and enterohemorrhagic E. coli (EHEC), a subpopulation of Shiga toxin (Stx)-producing E. coli (STEC) basically adapted to the ruminant host. This study aimed at appraising the relative level of adaptation of the EAEC/EHEC hybrid strain LB226692 to humans and cattle. Adherence and invasion of the hybrid strain to intestinal (jejunal and colonic) epithelial cells (IEC) of human and bovine origin was compared to that of E. coli strains representative of different pathovars and commensal E. coli by means of light and electron microscopy and culture. Strain-specific host gene transcription profiles of selected cytokines and chemokines as well as host-induced transcription of bacterial virulence genes were assessed. The release of Stx upon host cell contact was quantified. The outbreak strain's immunomodulation was assessed by cultivating primary bovine macrophages with conditioned supernatants from IEC infection studies with E. coli, serving as model for the innate immunity of the bovine gut. The outbreak strain adhered to IEC of both, human and bovine origin. Electron microscopy of infected cells revealed the strain's particular affinity to human small IEC, in contrast to few interactions with bovine small IEC. The outbreak strain possessed a high-level of adhesive power, similar to human-associated E. coli strains and in contrast to bovine-associated STEC strains. The outbreak strain displayed a non-invasive phenotype, in contrast to some bovine-associated E. coli strains, which were invasive. The outbreak strain provoked some pro-inflammatory activity in human cells, but to a lower extent as compared to other pathotypes. In contrasts to bovine-associated E. coli strains, the outbreak strain induced marked pro-inflammatory activity when interacting with bovine host cells directly (IEC) and indirectly (macrophages). Among stx2-positive strains, the human-pathogenic strains (LB226692 and EHEC strain 86-24) released higher amounts of Stx compared to bovine-associated STEC. The findings imply that the outbreak strain is rather adapted to humans than to cattle. However, the outbreak strain's potential to colonize IEC of both host species and the rather mixed reaction patterns observed for all strains under study indicate, that even STEC strains with an unusual genotype as the EHEC O104:H4 outbreak strain, i.e. with an EAEC genetic background, may be able to conquer other reservoir hosts.

Comparative virulence characterization of the Shiga toxin phage-cured Escherichia coli O104:H4 and enteroaggregative Escherichia coli.

Escherichia coli O104:H4 (E. coli O104:H4), which caused in 2011 a massive foodborne outbreak in Germany, is characterized by an unusual combination of virulence traits. E. coli O104:H4 contains a prophage-encoded Shiga toxin (Stx) gene, which is the cardinal virulence factor of enterohemorrhagic E. coli (EHEC). However, the outbreak strain shares highest DNA sequence similarity with enteroaggregative E. coli (EAEC) and displays the EAEC-characteristic tight adherence to epithelial cells. The virulence potential of the underlying EAEC background has not been investigated and it is therefore not clear whether E. coli O104:H4 displays distinct virulence characteristics in comparison to prototypical EAEC. In this study, we performed a detailed comparative phenotypic characterization of the Stx phage-cured E. coli O104:H4 strain C227-11φcu, the closely related EAEC strain 55989 and two other well-characterized EAEC strains 042 and 17-2 with focus on virulence traits. C227-11φcu displayed superior aggregative adherence phenotype to cultured HCT-8 epithelial cells, adhering with 3-6 times more bacteria per epithelial cells than the tested EAEC strains. Otherwise, C227-11φcu showed similar virulence characteristics to its closest relative 55989, i.e. strong acid resistance, good biofilm formation and cytotoxic culture supernatants. Furthermore, C227-11φcu was characterized by significantly weaker motility and pro-inflammatory properties than 55989 and 042, nevertheless stronger than 17-2. Taken together, C227-11φcu displayed mostly robust, but not outstanding virulence characteristics in comparison to the tested EAEC. Therefore, it appears likely that the combination of Stx production and EAEC characteristics in general, rather than an exceptionally potent EAEC background resulted in the unusual virulence of the E. coli O104:H4. Thus, the emergence of such hypervirulent strains in the future might be more likely than previously anticipated.

DNA microarray-based assessment of virulence potential of Shiga toxin gene-carrying Escherichia coli O104:H7 isolated from feedlot cattle feces.

Escherichia coli O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing E. coli (STEC) O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-E. coli Identification (ECID) DNA microarray to determine their virulence profiles and compare them to the human strains (clinical) of O104:H7, STEC O104:H4 (German outbreak strain), and O104:H21 (milk-associated Montana outbreak strain). Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r) were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (eae). The bovine strains were positive for Shiga toxin 1 subtype c (stx1c), enterohemolysin (ehxA), tellurite resistance gene (terD), IrgA homolog protein (iha), type 1 fimbriae (fimH), and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86-0.98) to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665) was more closely related to the bovine O104:H7 strains (r = 0.81-0.85) than the other four human clinical O104:H7 strains (r = 0.75-0.79). Montana outbreak strain (O104:H21) was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine E. coli O104 strains carried genes characteristic of E. coli O104:H4 German outbreak strain and unlike other human strains were also negative for Shiga toxin 2. Because cattle E. coli O104:H7 strains possess stx1c and genes that code for enterohemolysin and a variety of adhesins, the serotype has the potential to be a diarrheagenic foodborne pathogen in humans.

Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli.

Escherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.

Intermediate Follow-up of Pediatric Patients With Hemolytic Uremic Syndrome During the 2011 Outbreak Caused by E. coli O104:H4.

BACKGROUND.: In 2011 Escherichia coli O104:H4 caused an outbreak with >800 cases of hemolytic uremic syndrome (HUS) in Germany, including 90 children. Data on the intermediate outcome in children after HUS due to E. coli O104:H4 have been lacking. METHODS.: Follow-up data were gathered retrospectively from the medical records of patients who had been included in the German Pediatric HUS Registry during the 2011 outbreak. RESULTS.: Seventy-two of the 89 (81%) patients were included after a median follow-up of 3.0 (0.9-4.7) years. Hypertension and proteinuria were present in 19% and 28% of these patients, respectively. Of 4 patients with chronic kidney disease (CKD) > stage 2 at short-term follow-up, 1 had a normalized estimated glomerular filtration rate, and 3 (4%) had persistent CKD > stage 2. In 1 of these patients, CKD improved from stage 4 to 3; 1 who had CKD stage 5 at presentation received kidney transplantation; and 1 patient required further hemodialysis during follow-up. One patient (1.4%) still had major neurological symptoms at the latest follow-up. Dialysis during the acute phase (P = .01), dialysis duration (P = .01), and the duration of oligo-/anuria (P = .005) were associated with the development of renal sequelae. Patients treated with eculizumab (n = 11) and/or plasmapheresis (n = 13) during the acute phase of HUS had comparable outcomes. CONCLUSIONS.: The overall outcome of pediatric patients after HUS due to E. coli O104:H4 was equivalent to previous reports on HUS due to other types of Shiga toxin-producing E. coli (STEC). Regular follow-up visits in patients are recommended after STEC-HUS.

ENTEROAGGREGATIVE ESCHERICHIA COLI O104 FROM THAI AND IMPORTED MALAYSIAN RAW BEEF.

Local Thai and imported Malaysian beef in southern Thailand area carry several Shiga toxin-producing Escherichia coli (STEC) serotypes. STEC O104 is an important pathogen capable of causing outbreaks with considerable morbidity and mortality. This study investigated the presence of E. coli O104 from local Thai and imported Malaysian beef obtained from markets in Hat Yai City, Songkhla Province during August 2015 - February 2016. Thirty-one E. coli O104 strains were isolated from 12 beef samples (16% and 23% Thai and imported Malaysian, respectively). Thirty strains possessed aggA (coding for a major component of AAF/I fimbriae), a gene associated with enteroaggregative E. coli (EAEC) pathotype, and all strains carried fimH (encoding Type 1 fimbriae). Thirty strains belonged to phylogenetic group B1 and one strain (from Malaysian beef) to group A. Agglutination of yeast cells was observed among 29 E. coli O104 strains. Investigation of stx2 phage occupancy loci demonstrated that sbcB was occupied in 12 strains. Antimicrobial susceptibility assay revealed that 7 strains were resistant to at least one antimicrobial agent and two were multi-drug resistant. One strain carried extended spectrum ß-lactamase gene blaCTX-M and three carried blaTEM. PFGE-generated DNA profiling showed identical DNA pattern between that of one EAEC O104 strain from Thai beef and another from Malaysian beef, indicating that these two strains originated from the same clone. This is the first report in Thailand describing the presence of EAEC O104 from both Thai and imported Malaysian beef and their transfer between both countries. Thorough surveillance of this pathogen in fresh meats and vegetables should help to prevent any possible outbreak of E. coli O104.

Primer aislamiento de Escherichia coli enteroagregativo 0104: H4 de un caso de diarrea en Argentina / First isolation of enteroaggregative Escherichia coli 0104:H4 from a diarrhea case in Argentina

Se describe el primer aislamiento de una cepa de Escherichia coli enteroagregativo (EAEC) O104:H4 de un caso de diarrea aguda en Argentina. Se realizaron dos PCR múltiples como tamizaje: mPCR1 para los genes eae, lt y st, y mPCR2 para los genes IpaH, aggR, stx1y stx2. Se incluyó una mPCR para detectar los genes rfbO104, fliCH4 y terD, además de PCR simples para los genes del plásmido pCVD432, aaiC y lpfO113. Se realizaron ensayos bioquímicos, de sensibilidad a los antimicrobianos y de serotipificación. La cepa de E. coli identificada fue sensible a todos los antimicrobianos ensayados y presentó los genes aggR, aaiC, plásmido pCVD432, lpfO113, rfbO104, fliCH4 y terD. Si bien EAEC O104:H4 es un serotipo poco común, se han comunicado casos esporádicos, pero la preocupación global aumentó después del brote masivo ocurrido en Europa en 2011. El hallazgo de EAEC O104:H4 refuerza la necesidad de mejorar las metodologías para la detección de todos los patotipos de E. coli en Argentina

We describe the first isolation of an enteroaggregative Escherichia coli (EAEC) O104:H4 strain associated with an acute diarrhea case in Argentina. Two multiplex PCRs (mPCR) were performed as screening of genes mPCR1 (eae, lt, and st) and mPCR2 (IpaH, aggR, stx1 and stx2). A mPCR to detect the rfbO104, fliCH4 and terD genes, and PCR assays for the detection of pCVD432 plasmid, aaiC and lpfO113 genes were included. Biochemical and antimicrobial susceptibility assays as well as serotyping were performed. The identified E. coli strain was susceptible to all antimicrobials tested and harbored the aggR, aaiC, pCVD432 plasmid, lpfO113, rfbO104, fliCH4 and terD genes. Although serotype EAEC O104:H4 rarely spreads and sporadic cases have been reported, global concern increased after the large-scale outbreak in Europe in 2011. The finding of EAEC O104:H4 reinforces the need for improved methodologies for the detection of all E. coli pathotypes