First report of the prevalence of Shiga toxinproducing Escherichia coli in ground beef in Quindío, Colombia

Introduction. Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with clinical cases of diarrhea in humans. Its main virulence factors are the Shiga toxins (Stx1 and Stx2). Cattle are the main reservoir of STEC, and many outbreaks in humans have been related to the consumption of undercooked ground beef contaminated with this pathogen. Objective. To determine the prevalence of STEC in ground beef commercialized in all the butcher shops of a township in the department of Quindío and to characterize the virulence genes of the strains found. Materials and methods. Thirty ground beef samples were taken in three different times; stx genes and other STEC virulence factors (eae, ehxA, saa) were detected by multiplex PCR. Results. The overall prevalence of STEC was 33.33 % (10/30 positive samples). We isolated eight non-O157 (LEE-negative) strains with four different genetic profiles: stx 2 / stx 2-ehxA-saa / stx 1-stx 2-ehxA-saa / stx 1-saa. Conclusion. This is the first report on the prevalence of STEC in ground beef in a township in the department of Quindío.

Introducción. Escherichia coli productora de toxina Shiga (STEC) es un agente patógeno de origen alimentario asociado a casos clínicos de diarrea en humanos; sus principales factores de virulencia son las toxinas Shiga (Stx1 y Stx2). El principal reservorio de STEC es el ganado bovino y muchos brotes en humanos se han relacionado con el consumo de carne mal cocida contaminada con este agente patógeno. Objetivo. El objetivo de este trabajo fue determinar la prevalencia de STEC en carne molida comercializada en todas las carnicerías de un municipio del departamento del Quindío y caracterizar los genes de virulencia de las cepas encontradas. Materiales y métodos. Se tomaron 30 muestras de carne molida en tres momentos diferentes; se detectaron los genes stx y otros factores de virulencia de STEC (eae, ehxA, saa) mediante PCR Multiplex. Resultados. Los resultados mostraron una prevalencia global de STEC del 33,33 % (10/30 muestras positivas). En total se aislaron ocho cepas STEC no-O157 (LEE-negativas) con cuatro perfiles genéticos diferentes: stx 2 / stx 2-ehxA-saa / stx 1-stx 2-ehxA-saa / stx 1-saa. Conclusión. Este es el primer reporte que muestra la prevalencia de STEC en carne molida en un municipio del departamento del Quindío.

Caracterización genotípica y biofilms de E. coli shigatoxigénica aisladas de casos clínicos humanos / Genotypic characterization and biofilms of shigatoxigenic E. coli isolated from human clinical cases

Resumen Escherichia coli shigatoxigénica (STEC) está involucrada en el desarrollo del síndrome urémico hemolítico, entre otras enfermedades que son de gran importancia para la salud pública e inocuidad alimentaria a nivel mundial. La capacidad de STEC de formar biofilms en los alimentos y en diferentes superficies podría conducir a la contaminación cruzada por el desprendimiento de las células bacterianas. El objetivo del presente trabajo fue detectar la presencia de genes que codifican factores de adherencia mediante la técnica de PCR y determinar la capacidad de formación de biofilms por medio de cultivo en microplacas de poliestireno de 96 pocillos y la técnica de cristal violeta, en cepas de STEC aisladas de muestras clínicas humanas en la ciudad de Mar del Plata, Argentina. El perfil de genes de adherencia más frecuente fue efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas las cepas de STEC formaron biofilms con valores de densidad óptica entre 0,209 y 3,251 y el 54,4% (31/57) de las mismas fueron clasificadas como fuertes formadoras de biofilms. La capacidad de formación de biofilms de STEC constituye un riesgo evidente en la transmisión de este patógeno al ser humano a tener en cuenta para su vigilancia y control.

Abstract Shigatoxigenic Escherichia coli (STEC) is involved in the development of hemolytic uremic syndrome, among other diseases that are relevant to public health and food safety worldwide. The ability of STEC to form biofilms in food and on different surfaces could lead to cross-contamination by shedding bacterial cells. The aim of this work was to detect the presence of genes encoding adherence factors by the PCR technique and to determine the biofilm formation ability by culture in 96-well polystyrene microplates and the crystal violet technique, in STEC strains isolated from human clinical samples in Mar del Plata city, Argentina. The most frequent adherence gene profile was efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43.9%). All STEC strains formed biofilms with optical density values between 0.209 and 3.251. Also, the 54.4% (31/57) of STEC strains were classified as strong biofilm formers. The ability of STEC to form biofilms constitutes an evident risk in the transmission of this pathogen to humans, which must be taken into account for its surveillance and control.

Resumo A Escherichia coli shigatoxigênica (STEC) está envolvida no desenvolvimento da síndrome hemolítica urêmica, entre outras doenças relevantes para a saúde pública e segurança alimentar em todo o mundo. A capacidade do STEC de formar biofilmes nos alimentos e em diferentes superfícies poderia levar à contaminação cruzada através do desprendimento de células bacterianas. O objetivo do presente trabalho foi detectar a presença de genes que codificam fatores de aderência através da técnica PCR e determinar a capacidade de formação de biofilme por cultura em microplacas de poliestireno de 96 poços e da técnica de cristal violeta, em cepas STEC isoladas de amostras clínicas humanas na cidade de Mar del Plata, Argentina. O perfil de genes de aderência mais frequente foi efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas as cepas de STEC formaram biofilmes com valores de densidade ótica entre 0,209 e 3,251. Também, os 54,4% (31/57) das estirpes STEC foram classificados como fortes formadores de biofilmes. A habilidade de formação de biofilmes de STEC constitui um risco evidente na transmissão deste patógeno ao humano, que deve ser levado em consideração para sua vigilância e controle.

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)

Desarrollo de un producto anti-toxina shiga para la prevención del síndrome urémico hemolítico / Development of a product anti-Shiga toxin for prevention of the hemolytic uremic syndrome

El síndrome urémico hemolítico (SUH) típico es una enfermedad huérfana causada por cepas de Escherichia coli productoras de toxina Shiga (Stx) y caracterizada por daño renal agudo, anemia hemolítica microangiopática y plaquetopenia. Es endémico en Argentina, el país con mayor incidencia de SUH en el mundo. Debido al rol fundamental de la Stx en su patogenia, se puede considerar que, como otras toxemias conocidas, el SUH podría ser tratado con anticuerpos. Este trabajo describe el desarrollo de un nuevo tratamiento capaz de neutralizar el efecto tóxico de distintas variantes de la Stx. El tratamiento consiste en fragmentos F(ab')2 provenientes de un antisuero equino cuya eficacia y potencia contra Stx1 y Stx2 se comprobó en diferentes modelos preclínicos. El producto mostró ser seguro en animales, presentó la farmacocinética descripta para compuestos similares y se pudo establecer una posible ventana terapéutica para su adecuada administración. En conjunto, los resultados preclínicos obtenidos validan la realización de un estudio clínico de primer uso en humanos. En dicho estudio, que se realizará en el Hospital Italiano de Buenos Aires, se analizará la seguridad y la farmacocinética del producto en voluntarios adultos sanos. Estos resultados sentarán las bases para la realización del estudio clínico fase II en pacientes pediátricos con infección por cepas de E. coli productoras de Stx.

The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) -producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.

Characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli in animal feces, from Shandong Province / 中华预防医学杂志

Objective@#To understand the infection status, characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli (STEC) in animal feces in Shandong Province.@*Methods@#From 2015 to 2016, convient sampling method was used to collect 1 022 fresh feces of animals in Weishan county and Laizhou city, and 24 non-O157 STEC were isolated. The serotypes of non-O157 STEC strains were confirmed through serum agglutination test. The susceptibility was explored through the antimicrobial sensitivity experiments. ESBLs activity was confirmed by double-disc diffusion. PCR method was used to detect the resistance genes. PFGE typing was operated to assess the relatedness and variability of the strains. The multi-locus sequence typing (MLST) was adopted to get the allelic profile and ST sequence of strains. Analysis was made on the evolutionary relationship between different ST groups was made through CLC Sequence Viewer and Counting Express.@*Results@#A total of 24 non-O157 STEC were isolated from animal feces. 23 strains were from pig feces, and 1 strain was from cow feces, and the serotypes were more dispersed. All of the 24 strains carried stx2 genes. The highest resistance rate was sulfamethoxazole(22 strains), the mount of cotrimoxazole and nalidixic acid was 18 strains, chloramphenicol was 13 strains, tetracycline was 19, and there was a phenomenon of multiple drug resistance. The drug resistance spectrum was sulfamethoxazole tetracycline-compound novammin-naphthidine-chloramphenicol. All strains were sensitive to cefepime and imipenem. The ESBLs confirmatory test showed that 4 strains of non O157 STEC produced beta lactamase. PCR detected 7 resistance genes, and 4 tetracycline resistance genes (Tet A, Tet B, tetC and tetD) were detected. The beta lactamase resistance genes (blaSHV-1, bla CTX-M, bla TEM) were all negative. 24 strains were divided into 15 PFGE types, and their clustering results were more dispersed and no dominant PFGE type. There were 11 kinds of MLST types, most of them are ST540 and ST5133 types, each of which was 4 strains, and clustered into 1 MLST genomes.@*Conclusion@#The serotypes of non-O157 STEC in animal feces O157 STEC were dispersed, and the resistant rate to common antibiotic was high. MLST typing results presents obvious polymorphism. Surveillance and manage ment of these strains should be strengthened.

Pathogenic and phylogenetic characteristics of non-O157 Shiga toxin-producing Escherichia coli isolates from retail meats in South Korea

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx₁, stx(2c), stx(2e), or stx₁ + stx(2b)) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

Prevalence of Salmonella enterica and Shiga toxin-producing Escherichia coli in zoo animals from Chile

Salmonella (S.) enterica and Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens. Here, we report the prevalence of S. enterica and STEC in feces of 316 zoo animals belonging to 61 species from Chile. S. enterica and STEC strains were detected in 7.5% and 4.4% of animals, respectively. All Salmonella isolates corresponded to the serotype Enteritidis. To the best of our knowledge, this is the first report of S. Enteritidis in the culpeo fox (Lycalopex culpaeus), black-capped capuchin (Sapajus apella) and Peruvian pelican (Pelecanus thagus) and the first STEC report in Thomson's gazelle (Eudorcas thomsonii).

Escherichia coli, produtoras de Shigatoxinas detectadas em fezes de bovinos leiteiros / Shigatoxin-producing Escherichia coli detected in dairy cattle feces

Este trabalho teve como objetivo determinar a prevalência de Escherichia coli produtoras de Shigatoxinas (STEC) e E. coli dos sorogrupos O157, O111 e O113 em rebanhos leiteiros do Município de Jaboticabal, SP, Brasil. A presença de sequências gênicas stx1, stx2e eae foi detectada pela reação em cadeia da polimerase (PCR) em amostras de fezes. Todas as amostras stx e eae positivas foram submetidas a uma nova reação de PCR para detecção das sequências rfb O157, O111 e O113. Observou-se uma alta prevalência (72,16%) de sequências stx nas fezes dos bovinos, sendo que o perfil genotípico encontrado com maior frequência foi o stx1 associado à stx2. Os coeficientes de prevalência das sequências rfb O157, O111 e O113 foram, respectivamente, 14,77%, 0,2% e 30,83%. Animais de todos os rebanhos (100%) apresentaram em suas fezes STEC e E. coli O113 e os sorogrupos O157 e O111 foram observados em 60,0% e 10,0% dos rebanhos, respectivamente. Concluiu-se que a alta prevalência de STEC detectada em rebanho leiteiro evidenciada nas fezes de bovinos desempenham um papel importante na contaminação ambiental e podem oferecer risco de agravo à saúde pública.

The purpose of this study was to determine the prevalence of Shigatoxigenic Escherichia coli (STEC) and serogroups O157, O111 and O113 in dairy cattle from Jaboticabal, state of São Paulo, Brazil. Feces samples were collected from 10 herds and assessed for the presence of the virulence genes stx1, stx2 and eae by polymerase chain reaction (PCR). All samples positive for stx and eae were submitted to a second PCR reaction targeting the sequences rfb O157, rfb O111 and rfb O113. A high prevalence of stx (72.16%) was detected in the fecal samples, the most frequent being stx1 associated to stx2. The prevalence of sequences rfb O157, rfb O111 and rfb O113 was 14.77%, 0.2% and 30.83%, respectively. STEC and serogroup O113 was identified in all herds (100%), and serogroups O157 and O111 were observed in 60% and 10% of the herds. In conclusion, the high STEC prevalence detected in dairy herds evidences that bovine feces might play an important role as a contamination source in the region of Jaboticabal.

Escherichia coli verotoxigénica: modelo cuantitativo de exposición y escenarios de riesgos en canales bovinas en Argentina / Verotoxin-producing Escherichia coli: quantitative model of exposure and risk scenarios in cattle carcasses in Argentina

OBJETIVOS: Cuantificar la contaminación por Escherichia coli verotoxigénica asociada con el síndrome urémico hemolítico (ECvt-SUH) en las canales de ganado bovino y generar estimaciones de exposición en tres escenarios probables. MÉTODOS: Se modeló la frecuencia y la magnitud de la contaminación por ECvt-SUH desde la producción primaria hasta la salida de las canales del frigorífico, a partir de la información científica publicada y datos epidemiológicos y de expertos locales. Las distribuciones de la probabilidad que mejor describieron cada paso del proceso y los escenarios se incorporaron en el programa @Risk®, con simulaciones múltiples mediante el análisis Monte Carlo. Para el análisis de sensibilidad se aplicó la prueba de correlación de Pearson. RESULTADOS: La frecuencia estimada de canales con ECvt-SUH fue 0,37 (IC95 por ciento: 0,26 a 0,58) y la carga final de ECvt-SUH fue 0,47 log ufc/canal (IC95 por ciento: -2,46 a 3,62). Las variables más fuertemente relacionadas fueron: el sistema de engorde (r = -0,681) y la concentración teórica de ECvt-SUH en la piel de los bovinos (r = 0,702). La vacunación de los animales redujo en 54,1 por ciento la frecuencia de ECvt-SUH en las canales, aunque la carga final de ECvt-SUH no sufrió cambios significativos. El duchado de las canales redujo la carga final en 0,42 log ufc/canal con respecto al modelo basal, sin modificar la frecuencia. Un incremento en la proporción de animales engordados en corrales hasta 50-60 por ciento aumentaría un 15-23 por ciento la frecuencia de canales contaminadas con ECvt-SUH. CONCLUSIONES: La vacunación de los animales resultó el escenario más eficaz para reducir el ingreso de la bacteria en la cadena agroindustrial de la carne bovina. La intensificación de la producción ganadera incrementará el riesgo a la salud pública por una mayor exposición a ECvt-SUH.

OBJECTIVES: Quantify contamination by verotoxin-producing Escherichia coli associated with hemolytic uremic syndrome (VTEC-HUS) in cattle carcasses and generate estimates of exposure in three likely scenarios. METHODS: A model was constructed of the frequency and magnitude of VTEC-HUS contamination from primary production to the removal of the carcasses from cold storage, based on the published scientific information, epidemiological data, and information from local experts. The probability distributions that best described each step in the process and scenarios were input to the @Risk® program with multiple simulations using Monte Carlo analysis. Pearson´s correlation test was used for the sensitivity analysis. RESULTS: The estimated frequency of carcasses with VTEC-HUS was 0.37 (95 percent CI: 0.26 to 0.58) and the final load of VTEC-HUS was 0.47 log CFU/carcass (95 percent CI: -2.46 to 3.62). The most closely related variables were the fattening system (r = -0.681) and the theoretical concentration of VTEC-HUS on the cattle's skin (r = 0.702). Vaccinating the animals reduced the frequency of VTEC-HUS in the carcasses by 54.1 percent, although there were no significant changes in the final VTEC-HUS load. Washing the carcasses reduced the final load by 0.42 log CFU/carcass compared with the baseline model, without any change in the frequency. A 50 percent-60 percent increase in the percentage of animals fattened in pens would increase the frequency of carcasses contaminated with VTEC-HUS by 15 percent-23 percent. CONCLUSIONS: Vaccinating the animals was the most effective scenario for reducing introduction of the bacteria in the beef production chain. Intensifying livestock production will increase the public health risk due to greater exposure to VTEC-HUS.