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Introduction Congenital Central Hypoventilation Syndrome (CCHS) is a rare disease due to a severely impaired central control of breathing and dysfunction of the autonomic nervous system. Ophthalmologic abnormalities are common in patients with CCHS and include horizontal strabismus, pupil and iris abnormalities and ptosis. We report a unique case of CCHS in association with monocular elevation deficit (MED) in a boy diagnosed with CCHS at birth.Case description We report a case of a boy with a confirmed diagnosis of CCHS (complete sequencing of the paired-like homeobox 2b (PHOX2B) gene) after presenting little respiratory effort and cyanosis at birth. The ophthalmological examination shows an impaired elevation of the left eye, both in adduction and abduction, associated with mild and variable left ptosis. His mother has observed that the left eyelid elevates when the child feeds. A deviation in the primary gaze position or a chin-up position are not present. The funduscopic examination is normal. Given that deviation is limited to upgaze, the ptosis is mild and the patient's age, observation is decided.Conclusions Ophthalmologic abnormalities are common in patients with CCHS and include horizontal strabismus, pupil and iris abnormalities and ptosis. To the best of our knowledge, this is the first report of MED in association with CCHS. Further studies are needed to determine if an association between MED and CCHS exists or is just a casual finding in this case.

La sección de Vía Aérea de la Sociedad Española De Anestesiología, Reanimación y Terapéutica del Dolor (SEDAR), la Sociedad Española de Medicina de Urgencias y Emergencias (SEMES) y la Sociedad Española de Otorrinolaringología y Cirugía de Cabeza y Cuello (SEORL-CCC) presentan la Guía para el manejo integral de la vía aérea difícil en el paciente adulto. Este documento proporciona recomendaciones respaldadas por la evidencia científica actual, herramientas teórico-educativas y herramientas de implementación, fundamentalmente ayudas cognitivas, aplicables al tratamiento de la vía aérea en el campo de la anestesiología, cuidados críticos, urgencias y medicina prehospitalaria. Sus principios están focalizados en los factores humanos, los procesos cognitivos para la toma de decisiones en situaciones críticas y la optimización en la progresión de la aplicación de estrategias para preservar una adecuada oxigenación alveolar con el objeto de mejorar la seguridad y la calidad asistencial.

St Alban’s English College in Valladolid, established at the height of the Catholic Reformation for the training of English secular clergy under the rule of Spanish Jesuits, underwent an alteration in its management after the expulsion of the religious order from Spain in 1767. As part of this process, numerous valuable archival records were produced which have not, thus far, been studied. This article analyses a portion of these documents: the surviving manuscript inventories of the library. It also considers the series of governmental orders issued by the Spanish authorities as part of the process of expulsion and examines how these orders shaped the production of the library inventories. It offers an overview of the contents of the catalogues, with descriptions of some of those specific book entries that make these inventories unique. The study of these archival documents provides insight into, and understanding of, a key moment in the College history: its shift from Spanish Jesuit control to an English secular one and the difficulties that the Spanish authorities faced because of this change in the College’s national identity.

Shiga toxin-producing Escherichia coli(STEC) are a heterogeneous group of foodborne pathogens causing a broad spectrum of human disease, from uncomplicated diarrhea to hemolytic uremic syndrome (HUS). In this study, we report an HUS case associated with an O59:NM[H19] strain, harboring stx2a, iha, lpfAO26, lpfAO113genes associated with STEC, and aatA, aap, pic, sigA, agg4Agenes associated with enteroaggregative E. coli(EAEC), named Stx-EAEC. The strain showed low toxicity on Vero cells, and was resistant to streptomycin and trimethoprim/sulfonamides. The child carried the bacteria for more than 100 days. Since the large outbreak associated with Stx-EAEC O104:H4, many strains with similar profiles have been described. In Germany, an O59:NM[H19] strain, with comparable characteristics to the Argentine strain, was isolated from a bloody diarrhea case. In Argentina, this is the first report of an HUS case associated with a Stx-EAEC infection, and represents a new challenge for the surveillance system.

El 27 de noviembre de 2008 ocurrió un brote de intoxicación alimentaria asociado al consumo de salpicón de ave en un jardín de infantes de Hurlingham, provincia de Buenos Aires. Treinta y siete niños y 10 adultos presentaron síntomas gastrointestinales. Cinco niños fueron internados con signos de deshidratación, y uno de ellos requirió cuidados intensivos. Se aisló Staphylococcus aureussubsp. aureusdel alimento involucrado, de 4/5 muestras de materia fecal de pacientes y de 3/5 manipuladores (nariz del manipulador 1, manos de manipuladores 2 y 3). Las cepas aisladas portaban los genes que codifican las enterotoxinas SEA y SED. Por electroforesis de campo pulsado con la enzima SmaI, los patrones de macrorrestricción presentaron 100% de similitud. La investigación oportuna del brote permitió identificar al agente causal de la intoxicación, determinar las fallas en la elaboración del alimento e implementar las medidas correctivas correspondientes.

The objectives of this study were: (1) to estimate STEC frequency in hide and carcass samples taken from beef slaughterhouses supplying the domestic market in Argentina, (2) to establish the pheno-genotypic characteristics of STEC and non-toxigenic Escherichia coliof serogroups O26, O45, O103, O121, O111, O145 or O157 isolated from the analyzed samples and, (3) to study their clonal relatedness. Sixty hides and 60 carcasses were analyzed. At the screening step, 48% of hide and 80% of carcass samples tested positive for the stxgene by endpoint PCR. The STEC isolation rate was 5% for hides and 8% for carcasses. The isolation rate of STEC-positive for O26, O45, O103, O111, O145 or O157 serogroups was 0% for hides and 2% for carcasses. With the purpose of studying the clonal relatedness of isolates, macrorestriction fragment analysis by pulsed-field gel electrophoresis was performed. The results indicated cross-contamination between hides and between carcasses of animals in the same lot and, that the origin of carcass contamination was their own hide, or the hides of other animals in the same lot. The high detection rate at the screening step, especially in carcasses, and the evidence of cross-contamination show the need to apply additional in-plant intervention strategies aimed at preventing carcass contamination.

Los bovinos son el principal reservorio de Escherichia coliproductor de toxina Shiga (STEC); las estrategias para evitar su transmisión se concentran en la planta de faena. El objetivo de este trabajo fue evaluar la calidad higiénico-sanitaria y la frecuencia de detección de STEC en medias reses bovinas de frigoríficos de tránsito provincial. Se procesaron 274 esponjados de media res; en 9 (3,3%) el recuento de E. coligenérico fue marginal, en 4 (1,4%) se aisló E. coliO157, de los cuales 2 fueron caracterizados como stx2c(vh-a)/eae/ehxA, y los otros 2 como no toxigénicos. A partir de una (0,4%) muestra se aisló E. colino-O157 ONT:H49, stx2a/ehxA/saa. En este trabajo la calidad del producto analizado indica que en la provincia de Tucumán se cumplen las buenas prácticas de manufactura en la faena de bovinos.

•A prospective study on STEC-bloody diarrhea was performed in Argentina between October 2018 and June 2019.•Twenty-nine (4.1%) of 714 bloody diarrhea patients aged 1-9 years were STEC-positive.•Four (13.8%) bloody diarrhea cases progressed to HUS 3 to 9 days after diarrhea onset.•The most common serotypes among BD and HUS cases were O157:H7 and O145:H28.•The prevalent genotypes among BD and HUS cases were stx2a-only or -associated.

Escherichia colienteropatógeno (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. coliO157: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. coliO157:H16 eae-ɛ-positivo, sorbitol-positivo, β-glucuronidasa-positivo, sensible a los antimicrobianos ensayados, y negativo para los factores stx1, stx2, ehxAy 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. coliO157 y sus contactos familiares, y la incorporación de técnicas para detectar los distintos patotipos de E. coli.

En la provincia del Chaco, el agua subterránea representa una fuente alternativa, y muchas veces única, para el consumo humano; esta es utilizada en el 14% de los hogares. A pesar de que se reconoce el riesgo de la exposición al agua contaminada, la prevalencia de los diferentes patotipos de Escherichia colien ambientes acuáticos no ha sido bien caracterizada. E. colienteroagregativo (ECEA) es un patógeno emergente cuya importancia en la salud pública mundial se incrementó y quedó claramente establecida en los últimos años. El objetivo del presente trabajo fue detectar la presencia de ECEA típico mediante el reconocimiento de los factores de virulencia aap, AA probe y aggR por reacción en cadena de la polimerasa, en fuentes de agua subterráneas de la provincia del Chaco. Se identificó E. colien 36 (38,7%) de las 93 muestras estudiadas, provenientes de diferentes localidades. De esos 36 aislamientos, se identificaron 6 (16,7%) portadores de los genes de ECEA, lo que representa una prevalencia del 6,4% considerando las 93 fuentes de agua subterránea estudiadas. De esos 6 aislamientos, 3 eran portadores del gen aap, 2 del gen AA probey uno de la combinación aggR/aap. El presente trabajo representa el primer aporte en el estudio de la presencia y distribución de genes de virulencia de ECEA en fuentes de agua subterránea de la región.

Escherichia coliO157 es un patógeno emergente asociado a diarrea, colitis hemorrágica y síndrome urémico hemolítico. Los productos cárnicos constituyen una importante fuente de contaminación con este microorganismo. Los objetivos de este estudio fueron establecer la frecuencia de detección de E. coliO157 en productos cárnicos y media res en la provincia de Tucumán, caracterizar los factores de virulencia de los aislamientos obtenidos, establecer la relación clonal entre cepas regionales mediante electroforesis de campo pulsado y comparar con lo consignado en la base de datos nacional. Desde 2004 hasta 2013 se analizaron 169 muestras de carne picada, 35 embutidos y 216 esponjados de media res. Se identificaron 13 aislamientos de E. coliO157; 6 de ellos fueron O157:H7 productores de toxina Shiga y se caracterizaron como stx2c(vh-a)/eae/ehxA (n = 5) y stx2/eae/ehxA (n = 1); los 7 aislamientos de E. coliO157 no toxigénicos fueron O157:NT(n = 4),O157:NM (n = 1),O157:ND (n = 1) y O157:H16 (n = 1). Los patrones de PFGE fueron diferentes entre sí y de los registrados en la base de datos nacional. Se concluye que existe gran diversidad genética en los aislamientos de E. coliO157 circulantes en nuestra región.

ABSTRACTWhile the differential association of Escherichia coliO157 genotypes with animal and human hosts has recently been well documented, little is known about their distribution between countries and how this might affect regional disease rates. Here, we used a 48-plex single nucleotide polymorphism (SNP) assay to segregate 148 E. coliO157 isolates from Australia, Argentina, and the United States into 11 SNP lineages. We also investigated the relationship between SNP lineages, Shiga toxin (Stx) gene profiles, and total Stx production. E. coliO157 isolates clearly segregated into SNP lineages that were differentially associated with each country. Of the 11 SNP lineages, seven were detected among isolates from a single country, two were detected among isolates from all three countries, and another two were detected only among U.S. and Argentinean isolates. A number of Australian (30%) and Argentinean (14%) isolates were associated with novel, previously undescribed SNP lineages that were unique to each country. Isolates within SNP lineages that were strongly associated with the carriage of stx2aproduced comparatively more Stx on average than did those lacking the stx2asubtype. Furthermore, the proportion of isolates in stx2a-associated SNP lineages was significantly higher in Argentina and the United States than Australia (P< 0.05). This study provides evidence for the geographic divergence of E. coliO157 and for a prominent role of stx2ain total Stx production. These results also highlight the need for more comprehensive studies of the global distribution of E. coliO157 lineages and the impacts of regionally predominant E. coliO157 lineages on the prevalence and severity of disease.

ABSTRACTHuman infection with Shiga toxin-producing Escherichia coli(STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coliO157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. colilipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis.

AbstractBetween April 2009 and July 2011, 311 surface water samples in 48 cattle feedlots distributed in an area of about 67,000 km2were analyzed to examine the environmental dissemination of Escherichia coliO157:H7. Samples were taken inside and outside the pens, exposed and not exposed to runoff from corrals, near the feedlots. Two types of samples were defined: (1) exposed surface waters (ESW; n=251), downstream from cattle pens; and (2) nonexposed surface waters (NESW; n=60), upstream from cattle pens. By multiplex PCR, 177 (70.5%) ESW samples were rfbO157-positive, and 62 (24.7%) E. coliO157, and 32 (12.7%) Shiga toxin-producing E. coli(STEC) O157:H7 strains were isolated. In the NESW samples, 36 (60.0%) were rfbO157- positive, and 9 (15.0%) E. coliO157, and 6 (10.0%) STEC O157:H7 strains were isolated. These results showed that the environmental surface waters exposed to liquid discharges from intensive livestock operations tended to be contaminated with more STEC O157:H7 than NESW. However, no significant difference was found. This fact emphasizes the relevance of other horizontal routes of transmission, as the persistence of E. coliin the environment resulting from extensive livestock farming. By XbaI-PFGE, some patterns identified are included in the Argentine Database of E. coliO157, corresponding to strains isolated from hemolytic uremic syndrome and diarrhea cases, food, and animals, such as AREXHX01.0022, second prevalent pattern in Argentina, representing 5.5% of the total database. In the study area, characterized by the abundance of waterways, pathogens contained in feedlot runoff could reach recreational waters and also contaminate produce through irrigation, increasing the potential dissemination of STEC O157:H7 and the risk of human infections. The control of runoff systems from intensive livestock is necessary, but other alternatives should be explored to solve the problem of the presence of E. coliO157 in the aquatic rural environment.

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, stx1and stx2). A mPCR to detect the rfbO104, fliCH4and terD genes, and PCR assays for the detection of pCVD432 plasmid, aaiC and lpfO113genes were included. Biochemical and antimicrobial susceptibility assays as well as serotyping were performed. The identified E. colistrain was susceptible to all antimicrobials tested and harbored the aggR, aaiC, pCVD432 plasmid, lpfO113, rfbO104, fliCH4and 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. colipathotypes.

ABSTRACTEnterohemorrhagic Escherichia coli(EHEC) is a food-borne pathogen that produces Shiga toxin (Stx) and causes hemorrhagic colitis. Under some circumstances, Stx produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome. Although retinoids like vitamin A (VA) and retinoic acid (RA) are beneficial to gut integrity and the immune system, the effect of VA supplementation on gastrointestinal infections of different etiologies has been controversial. Thus, the aim of this work was to study the influence of different VA status on the outcome of an EHEC intestinal infection in mice. We report that VA deficiency worsened the intestinal damage during EHEC infection but simultaneously improved survival. Since death is associated mainly with Stx toxicity, Stx was intravenously inoculated to analyze whether retinoid levels affect Stx susceptibility. Interestingly, while VA-deficient (VA-D) mice were resistant to a lethal dose of Stx2, RA-supplemented mice were more susceptible to it. Given that peripheral blood polymorphonuclear cells (PMNs) are known to potentiate Stx2 toxicity, we studied the influence of retinoid levels on the absolute number and function of PMNs. We found that VA-D mice had decreased PMN numbers and a diminished capacity to produce reactive oxygen species, while RA supplementation had the opposite effect. These results are in line with the well-known function of retinoids in maintaining the homeostasis of the gut but support the idea that they have a proinflammatory effect by acting, in part, on the PMN population.

ABSTRACTHemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli(EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucellaspp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.

AbstractThe purposes of this study were to detect non-O157 Shiga toxin–producing Escherichia coli(STEC) in bovine rectums and water in a beef cattle farm in Argentina, and to determine the pathogenic potential of the circulating strains. During the study, 292 rectal swabs from healthy animals and 79 environmental water samples were collected. The rectal swabs and one loop of the Moore swabs, enriched in Escherichia colibroth for 24 h at 37°C, were streaked on MacConkey agar plates and incubated overnight at 37°C. The isolates were characterized by biochemical tests and serotyped. Nonmotile STEC strains were typed for their H-specific (fliC) antigens by polymerase chain reaction (PCR). Isolates were characterized by detection of stx1, stx2,and their variants, eae, ehxA,and saagenes. Macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) was performed using the PulseNet standardized protocol. From 371 samples analyzed, 36.6% of rectal swabs and 34.2% of water samples were non-O157 STEC-positive by PCR, and 110 strains from rectal swabs, but only three from water, were isolated. The strains were grouped into 24 different serotypes, from which, O103:[H2] (n=12), O136:H12 (n=8), O178:H19 (n=8), and O103:NM (n=5) were most prevalent, representing 29.2% of the isolates. Predominant genotypes were stx1/eae/ehxA(16.8%) and stx2/saa/ehxA (15.9%). PFGE analysis revealed 56 different patterns, with 65 strains grouped in 19 clusters of 100% similarity. Two STEC O124:H19 strains isolated from rectal swabs and water with a 5-month interval harbored the stx1/stx2/saa/ehxAgenotype, and showed an indistinguishable PFGE profile. By comparison, some XbaI-PFGE patterns identified in the present study were identical to the profiles of strains isolated from human, food, and animal sources included in the Argentine PulseNet database. By PCR, similar non-O157 detection rates were found in rectal swabs and water. However, the methodology for water samples needs to be improved, since only three strains from the total number of positive samples were recovered.

AbstractShiga toxin–producing Escherichia coli(STEC) cause nonbloody (NBD) and bloody diarrhea (BD), and hemolytic uremic syndrome (HUS). Cattle have been described as their main reservoir. STEC O157:H7 is recognized as the predominant serotype in clinical infections, but much less is known about the dominant subtypes in humans and animals or their genetic relatedness. The aims of this study were to compare the STEC O157 subtypes found in sporadic human infections with those in the bovine reservoir using stx-genotyping, phage typing, and XbaI–pulsed-field gel electrophoresis (PFGE), and correlate the subtypes with the severity of clinical manifestations. The 280 STEC O157:H7 strains collected included in this study were isolated from HUS (n=122), BD (n=69), and NBD (n=30) cases, and healthy carriers (n=5), and from bovines (n=54) in the abattoirs. The stx-genotyping showed that stx2/stx2c(vh-a)was predominant in human (76.1%) and in bovine strains (55.5%), whereas the second more important genotype was stx2(20.8%) in human and stx2c(vh-a)(16.7%) in cattle strains. In human strains, PT4 (37.6%), PT49 (24.3%), and PT2 (18.6%) were the most frequent PTs (80.5%). In bovine isolates, PT2 (26%), PT39 (16.7%), and PT4 and PT49 (11.1% each) were predominant. By XbaI-PFGE, all 280 strains yielded 148 patterns with 75% similarity, and 169 strains were grouped in 37 clusters. Identical PT-PFGE-stxprofile combinations were detected in strains of both origins: PT4-AREXH01.0011-stx2/stx2c(vh-a)(12 humans and one bovine), PT4-AREXH01.0543-stx2/stx2c(vh-a)(one human and four bovines), PT2-AREXH01.0076-stx2/stx2c(vh-a)(one human and four bovines), PT49-AREXH01.0175-stx2/stx2c(vh-a)(seven humans and one bovine), and PT49-AREXH01.0022-stx2/stx2c(vh-a)(seven humans and one bovine). No correlation was found among the stx-genotypes, the phage type, and the clinical symptoms.

ABSTRACTEnterohemorrhagic Escherichia coli(EHEC) O157:H7 and enteropathogenic E. coli(EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, TirEHEC O157is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspFU, which mimics the function of Nck. tccPis carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccPgene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, β-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccPand tccP2genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccPnegative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2gene that encodes a secreted protein that can complement an EHEC O157:H7 ΔtccPmutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, TirEHEC O157is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspFU, which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, {szligbeta}-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 tccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.

ABSTRACTThe distribution of eight putative adhesins that are not encoded in the locus for enterocyte effacement (LEE) in 139 Shiga toxin-producing Escherichia coli(STEC) of different serotypes was investigated by PCR. Five of the adhesins (Iha, Efa1, LPFO157/OI-141, LPFO157/OI-154, and LPFO113) are encoded in regions corresponding to genomic O islands of E. coliEDL933, while the other three adhesins have been reported to be encoded in the STEC megaplasmid of various serotypes (ToxB [O157:H7], Saa [O113:H21], and Sfp [O157:NM]). STEC strains were isolated from humans (n= 54), animals (n= 52), and food (n= 33). They were classified into five seropathotypes (A through E) based on the reported occurrence of STEC serotypes in human disease, in outbreaks, and in the hemolytic-uremic syndrome (M. A. Karmali, M. Mascarenhas, S. Shen, K. Ziebell, S. Johnson, R. Reid-Smith, J. Isaac-Renton, C. Clark, K. Rahn, and J. B. Kaper, J. Clin. Microbiol. 41:4930-4940, 2003). The most prevalent adhesin was that encoded by the ihagene (91%; 127 of 139 strains), which was distributed in all seropathotypes. toxBand efa1were present mainly in strains of seropathotypes A and B, which were LEE positive. saawas present only in strains of seropathotypes C, D, and E, which were LEE negative. Two fimbrial genes, lpfAO157/OI-141and lpfAO157/OI-154, were strongly associated with seropathotype A. The fimbrial gene lpfAO113was present in all seropathotypes except for seropathotype A, while sfpAwas not present in any of the strains studied. The distribution of STEC adhesins depends mainly on serotypes and not on the source of isolation. Seropathotype A, which is associated with severe disease and frequently is involved in outbreaks, possesses a unique adhesin profile which is not present in the other seropathotypes. The wide distribution of ihain STEC strains suggested that it could be a candidate for vaccine development.

ABSTRACTThe genetic diversity of Vibrio choleraeO1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpAappeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns. Among the last group, a set of isolates from the province Tucuma´n showed a single RAPD pattern and four closely related PFGE profiles. These strains, isolated from patients with diarrhea, did not produce the major V. choleraeO1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber. All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea. We propose “Tucuma´n variant” as the designation for this new cluster of cholera toxin-negative V. choleraeO1 strains.

Between 9 October and 12 November 1996, an outbreak of bloody diarrhea occurred in the neonatal nursery ward of the Policlínico Neuquén, in Neuquén, a city in the southwestern region of Argentina. Seven patients of the intermediate care unit were affected. Isolates of Escherichia coli O18ac:H31 that were non-lactose and -sorbitol fermenting were recovered from outbreak cases. Although the strains were negative for a number of virulence factors typically found in diarrheagenic groups of E. coli, all isolates from the present neonatal outbreak possessed the enterohemolysin gene, ehl1. All isolates showed resistance to the antibiotics ampicillin and chloramphenicol. These isolates showed a low adherence property in HeLa cells without any recognizable pattern. In order to evaluate the outbreak dissemination in the neonatology ward, a prevalence study was conducted on 13 November. Stool specimens were obtained from 16 neonates hospitalized in the sector and from 33 medical staff members. E. coli isolates with identical biochemical characteristics of the outbreak strain were recovered from 11 of 16 inpatients and from 4 of 33 staff members during the prevalence study. A total of 15 E. coli strains recovered both from the outbreak and the prevalence study were processed by random amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE). By RAPD-PCR 14 of 15 strains showed patterns with 85 to 100% similarity, and by PFGE these strains were identical, each showing a unique pattern with 15 bands between 40 and 400 kb. One strain isolated from a nurse during the prevalence study presented a pattern not related to the others, and this was characterized as E. coli O81:HNM resistant to ampicillin only and negative for all the virulence factors studied. This outbreak occurred despite strict regulations in place to prevent cross-infection in the hospital. Postoutbreak prevalence studies were performed weekly thereafter without detecting new cases.

ABSTRACTBetween 9 October and 12 November 1996, an outbreak of bloody diarrhea occurred in the neonatal nursery ward of the Policli´nico Neuque´n, in Neuque´n, a city in the southwestern region of Argentina. Seven patients of the intermediate care unit were affected. Isolates of Escherichia coliO18ac:H31 that were non-lactose and -sorbitol fermenting were recovered from outbreak cases. Although the strains were negative for a number of virulence factors typically found in diarrheagenic groups of E. coli, all isolates from the present neonatal outbreak possessed the enterohemolysin gene, ehl1. All isolates showed resistance to the antibiotics ampicillin and chloramphenicol. These isolates showed a low adherence property in HeLa cells without any recognizable pattern. In order to evaluate the outbreak dissemination in the neonatology ward, a prevalence study was conducted on 13 November. Stool specimens were obtained from 16 neonates hospitalized in the sector and from 33 medical staff members. E. coliisolates with identical biochemical characteristics of the outbreak strain were recovered from 11 of 16 inpatients and from 4 of 33 staff members during the prevalence study. A total of 15 E. colistrains recovered both from the outbreak and the prevalence study were processed by random amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE). By RAPD-PCR 14 of 15 strains showed patterns with 85 to 100% similarity, and by PFGE these strains were identical, each showing a unique pattern with 15 bands between 40 and 400 kb. One strain isolated from a nurse during the prevalence study presented a pattern not related to the others, and this was characterized as E. coliO81:HNM resistant to ampicillin only and negative for all the virulence factors studied. This outbreak occurred despite strict regulations in place to prevent cross-infection in the hospital. Postoutbreak prevalence studies were performed weekly thereafter without detecting new cases.

Between February and May 2000, 279 meat samples were collected from 136 retail stores in Gualeguaychú City, Argentina. Samples were assayed for Escherichia coliO157:H7 by selective enrichment in modified EC broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto both sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and a chromogenic medium. Eleven E. coliO157:H7 isolates were detected in 6 (3.8%) of 160 ground beef samples, in 4 (4.8%) of 83 fresh sausages, and in 1 (3.3%) of 30 dry sausages. E. coliO157:H7 was not isolated from five hamburger patties or one barbecue-type fresh sausage assayed. The isolates were tested for virulence-related genes. Ten additional Shiga toxin-producing E. coli(STEC) O157:H7 isolates of food origin, recovered from different locations in Argentina, were included for comparison purposes. All 21 isolates harbored both eaeand EHEC-hlyAgenes, and 12 (57.1%) encoded stx2/stx2vh-a. The isolates were of phage types 87 (seven strains), 14 (four strains), 4 (three strains), and 26 (one strain). Six strains were nontypable by phage typing. Pulsed-field gel electrophoresis (PFGE) revealed 19 XbaI-PFGE profiles. Fifteen (71%) strains were grouped in four clusters, which shared more than 80% of DNA restriction fragments. The enrichment culture method with IMS was a sensitive procedure to detect E. coliO157:H7 strains in retail meats. Some of the isolates from different stores presented a high clonal relatedness, as determined by XbaI-PFGE and phage typing, and harbored the virulence factors associated with human illness.

Shiga toxin-producing Escherichia coli(STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases. In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined. Net water movement (Jw) and short-circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2. Stx2 significantly inhibited the absorptive Jwwith no effect on the basal Iscafter 60 min of exposure. These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells. Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells. These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium.

ABSTRACTIn a follow-up study, enterotoxigenic Escherichia coli(ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P= 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P= 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P= 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P= 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81;P= 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P= 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.

AbstractShiga toxin-producing Escherichia coli(STEC) is an important foodborne pathogen, and ruminants are recognized as the main natural reservoir. The purposes of this study were to detect E. coliO157 in bovine feces and surface water in a beef cattle farm of Gualeguaychú, Argentina; to characterize the isolates; and to establish the clonal relatedness by pulsed-field gel electrophoresis. Between September 2005 and November 2006, 288 samples of bovine feces and 79 samples of water troughs were studied. E. coliO157 was detected by immunomagnetic separation and polymerase chain reaction as screening techniques. The rfbO157gene was detected in 3.8% of the 288 fecal samples and in 17.7% of the 79 water samples. The stxgene was detected in all rfbO157-positive fecal samples and in 5.1% of water samples. Eleven E. coliO157 strains isolated from bovine fecal samples and eight from water samples were characterized. The most frequent stxgenotype identified was stx1and stx2c(vh-a). Twelve (63.2%) strains harbored fliCH7, eae, and ehxA genes. Using pulsed-field gel electrophoresis with the enzyme XbaI, a total of eight patterns with at least 72.1% similarity were identified among the 19 strains. The patterns of 15 strains were grouped into four clusters: two of them included only bovine strains and the other two only aquatic strains. No genetic correlation was established between the bovine and water STEC strains detected. The prevalence of STEC O157:H7 established in the herd studied was higher than that previously reported for Argentine grazed cattle.

Shiga toxin (Stx)-producing Escherichia coli(STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype.

ABSTRACTA multiplex PCR assay for the identification of human diarrheagenic Escherichia coliwas developed. The targets selected for each category were eaefor enteropathogenic E. coli, stxfor Shiga toxin-producing E. coli, eltand estfor enterotoxigenic E. coli, ipaHfor enteroinvasive E. coli, and aggRfor enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. colistrain in a single reaction tube.

A polymerase chain reaction (PCR) to detect a region of the A1 cholera toxin gene was applied to the identification of 43 Vibrio choleraestrains isolated from the recent outbreak in Argentina. A good correlation was observed between the GM1‐enzyme‐linked immunosorbent assay (GM1‐ELISA) to detect the B subunit of the enterotoxin and PCR. However, a V. choleraenon‐01 strain that was negative by the ELISA test, was positive by the PCR assay for the Al region. A second PCR test to detect the A2‐B coding region was developed to solve this case. We propose that routine detection of toxigenic V. choleraeby PCR should include analysis of A2‐B coding region or the whole cholera toxin operon.

A polymerase chain reaction (PCR) to detect a region of the A1 cholera toxin gene was applied to the identification of 43 Vibrio choleraestrains isolated from the recent outbreak in Argentina. A good correlation was observed between the GM1-enzyme-linked immunosorbent assay (GM1-ELISA) to detect the B subunit of the enterotoxin and PCR. However, a V. choleraenon-01 strain that was negative by the ELISA test, was positive by the PCR assay for the Al region. A second PCR test to detect the A2-B coding region was developed to solve this case. We propose that routine detection of toxigenic V. choleraeby PCR should include analysis of A2-B coding region or the whole cholera toxin operon.