The Importance of Shiga Toxin-Producing Escherichia coli O145:NM[H28]/H28 Infections in Argentina, 1998-2020.

Shiga toxin-producing Escherichia coli (STEC) is known as a pathogen associated with food-borne diseases. The STEC O145 serogroup has been related with acute watery diarrhea, bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Argentina has the highest rate of HUS worldwide with 70% of the cases associated with STEC infections. We aimed to describe the epidemiology and genetic diversity of STEC O145 strains isolated across Argentina between 1998-2020. The strains isolated from 543 cases of human disease and four cattle, were pheno-genotipically characterized. Sequencing of five strains was performed. The strains were serotyped as O145:NM[H28]/H28, O145:H25, and O145:HNT, and mainly characterized as O145:NM[H28]/stx2a/eae/ehxA (98.1%). The results obtained by sequencing were consistent with those obtained by traditional methods and additional genes involved in different mechanisms of the pathogen were observed. In this study, we confirmed that STEC O145 strains are the second serogroup after O157 and represent 20.3% of HUS cases in Argentina. The frequency of STEC O145 and other significant serogroups is of utmost importance for public health in the country. This study encourages the improvement of the surveillance system to prevent severe cases of human disease.

An Stx-EAEC O59:NM[H19] strain isolated from a hemolytic uremic syndrome case in Argentina / Cepa EAEC-Stx O59:NM[H19] aislada de un caso de síndrome urémico hemolítico en Argentina

Abstract 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 mstrain, harboring stx2a, iha, lpfAO26, lpfAO113 genes associated with STEC, and aatA, aap, pic, sigA, agg4A genes 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. © 2019 Published by Elsevier Espana, S.L.U. on behalf of Asociacion Argentina de Microbiolog´a. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/ licenses/by-nc-nd/4.0/).

Resumen Escherichia coli productor de la toxina Shiga (STEC) es un grupo heterogéneo de patógenos transmitidos por alimentos que causan un amplio espectro de enfermedades humanas, desde diarrea no complicada hasta síndrome urémico hemolítico (SUH). Nosotros informamos de un caso de SUH por O59:NM[H19], que portaba los genes stx2a, iha, lpfAo26, lpfAoii3 asociados con STEC, y los genes aatA, aap, pic, sigA, agg4A de E. coli enteroagregativo (EAEC), llamado EAEC-Stx. La cepa mostró baja citotoxicidad en las células Vero, y fue resistente a estreptomicina y trimetoprima/sulfonamidas. El niño excretó la bacteria durante más de 100 días. Desde el brote asociado con EAEC-Stx O104:H4, se describieron muchas cepas con perfiles similares. En Alemania se aisló una cepa O59:NM[H19] de una diarrea sanguinolenta, con características comparables a la cepa argentina. Este es el primer informe de un caso de SUH asociado a una infección por EAEC-Stx, y representa un nuevo desafío para el sistema de vigilancia. © 2019 Publicado por Elsevier Espana, S.L.U. en nombre de Asociación Argentina de Microbiología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons. org/licenses/by-nc-nd/4.0/).

An Stx-EAEC O59:NM[H19] strain isolated from a hemolytic uremic syndrome case in Argentina.

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, lpfAO113 genes associated with STEC, and aatA, aap, pic, sigA, agg4A genes 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.

Frecuencia, caracterización y análisis genotípico de Escherichia coli productor de toxina Shiga en frigoríficos de carne bovina de Argentina / Frequency, characterization and genotypic analysis of Shiga toxin-producing Escherichia coli in beef slaughterhouses of Argentina

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 coli of 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 stx gene 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 objetivos del presente estudio fueron tres: 1) estimar la frecuencia de Escherichia coli productor de toxina Shiga (STEC) en muestras de cuero y carcasa de bovinos en frigoríficos de consumo interno de Argentina; 2) realizar la caracterización feno-genotípica de las cepas STEC y de Escherichia coli no toxigénicas pertenecientes a los serogrupos O26, O45, 0103, O121, O145 u O157 aisladas a partir de las muestras analizadas; 3) establecer la relación clonal de ese conjunto de cepas. Se analizaron 60 cueros y 60 carcasas. En la etapa de tamizaje, el gen stx se detectó en el 48% de las muestras de cuero y en el 80% de las muestras de carcasa por una PCR de punto final. La frecuencia de recuperación de cepas STEC fue del 5% en cueros y del 8% en carcasas, y la de cepas STEC positivas para los serogrupos O26, O45, O103, O121, O111, O145 u O157 fue del 0% en los cueros y del 2% en las carcasas. La relación clonal de las cepas aisladas se investigó a través de electroforesis de campo pulsado y análisis de los patrones de macrorrestricción generados. Los resultados demostraron la existencia de contaminación cruzada entre cueros y carcasas de animales pertenecientes a un mismo lote, y también que el origen de la contaminación fue el propio cuero del animal o el cuero de otros animales pertenecientes al mismo lote. Los altos porcentajes de detección en la etapa de tamizaje, especialmente en carcasas, y la evidencia de contaminación cruzada ponen de manifiesto la necesidad de evaluar la implementación de estrategias de intervención tendientes a evitar la contaminación de carcasas.

Frequency, characterization and genotypic analysis of Shiga toxin-producing Escherichia coli in beef slaughterhouses of Argentina.

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 coli of 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 stx gene 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.

Putative virulence factors and clonal relationship of O174 Shiga toxin-producing Escherichia coli isolated from human, food and animal sources.

Shiga toxin-producing Escherichia coli (STEC) is th etiological agent of gastrointestinal diseases as haemorrhagic colitis and haemolytic-uraemic syndrome (HUS). Shiga toxin (Stx) is the main virulence factor. There are two types, Stx1 and Stx2, and several subtypes. Other virulence factors are involved in pathogenesis. While O157:H7 is the predominant serotype, at present non-O157 STEC strains are increasingly recognized as foodborne pathogens worldwide. In Argentina, STEC O174 stands out as an emerging pathogen and is one of the four most prevalent non-O157 STEC serogroups. The aim of this study was to characterize 41 O174:[H21, H28] STEC strains isolated from animals, food, and humans. Isolates were characterized by stx genotyping, adhesion factors (afaC, eae, iha, lpfO113, saa, and toxB), additional toxins (cdtV, ehxA, subA) and clonal relationship by pulsed-field gel electrophoresis (PFGE). Among 30 O174:H21 strains, the most prevalent stx subtype was stx2c (56.7%), and among 11 O174:H28 strains, the most prevalent was stx2a (90.9%). Regarding virulence factors, all strains were positive for afaC gene and negative for eae and toxB genes. In O174:H21, the frequency of additional factors was lpfO113 (96.6%), iha (83.3%), ehxA and saa (10%), and subA (3.3%), meanwhile in O174:H28 strains the frequency was iha and subA (100%), lpfO113 (90.9%), ehxA and saa (90.9%), and cdtV (9.09%). By Xbal-PFGE, 29 patterns were established with 64.01% similarity and three clusters were detected. Given the fact that the O174 serogroup is a local emergent, it is important to study the virulence profiles to understand its potential pathogenicity.

Whole-Genome Sequencing Applied to the Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli O157:H7 in Argentina.

Shiga toxin-producing Escherichia coli strains are worldwide associated with sporadic human infections and outbreaks. In this work, we report the availability of high-quality draft whole-genome sequences for 19 O157:H7 strains isolated in Argentina.

[Isolation of enteropathogenic Escherichia coli O157:H16 identified in a diarrhea case in a child and his household contacts in La Pampa Province, Argentina]. / Aislamiento de Escherichia coli enteropatógeno O157:H16 de un caso de diarrea infantil y sus contactos familiares en La Pampa, Argentina.

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-ɛ-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.

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

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

First isolation of enteroaggregative Escherichia coli O104:H4 from a diarrhea case in 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.

First isolation of enteroaggregative Escherichia coli O104:H4 from a diarrhea case in Argentina. / First isolation of enteroaggregative Escherichia coli O104:H4 from a diarrhea case in 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.

Subtyping of Escherichia coli O157:H7 strains isolated from human infections and healthy cattle in Argentina.

Shiga 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/stx(2c(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 stx(2c(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-stx profile combinations were detected in strains of both origins: PT4-AREXH01.0011-stx2/stx(2c(vh-a)) (12 humans and one bovine), PT4-AREXH01.0543-stx2/stx(2c(vh-a)) (one human and four bovines), PT2-AREXH01.0076-stx2/stx(2c(vh-a)) (one human and four bovines), PT49-AREXH01.0175-stx2/stx(2c(vh-a)) (seven humans and one bovine), and PT49-AREXH01.0022-stx2/stx(2c(vh-a)) (seven humans and one bovine). No correlation was found among the stx-genotypes, the phage type, and the clinical symptoms.