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Resumen Objetivo: Aislar STEC en las heces del ganado bovino en el municipio de Ulloa, Valle del Cauca y detectar factores de virulencia asociados con la patogénesis. Materiales y métodos: Se tomaron 21 muestras provenientes de bovinos, las cuales fueron tomadas directamente del recto del animal mediante hisopos. Las muestras se procesaron hasta obtener colonias puras a las cuales se les evaluó la presencia de los genes stx1, stx2, eae, saa y hlyA mediante PCR y posteriormente, se evaluó el efecto citotóxico de las muestras positivas sobre células Vero (ATCC-CCL-81.4). Resultados: De las 21 muestras de heces de bovinos,12 presentaron bacterias con uno o ambos genes stx. Se obtuvieron 106 aislamientos totales de STEC y se observaron diferencias en cuanto a la presencia y ausencia de los genes de virulencia evaluados en los aislamientos de cada bovino, obteniendo cinco combinaciones de genes. 48 aislamientos presentaron únicamente el gen stx2 y 58 presentaron tanto el gen stx1 como el gen stx2; de los 106 aislamientos, se detectaron 44 con el gen hlyA y 57 con el gen saa. Conclusiones: Todos los sobrenadantes de STEC analizados mostraron actividad citotóxica sobre las células Vero, mientras que en ausencia de STEC las células formaron monocapa después de 48 h de incubación. Este trabajo es el primer reporte en Colombia que aporta información sobre la presencia de STEC en el ganado bovino, la presencia de factores de virulencia y el potencial efecto citotóxico que poseen estas cepas nativas.
Abstract Objective: To isolate STEC in stool samples from cattle in Ulloa, Valle del Cauca, and to detect virulence factors associated with its pathogenesis. Materials and methods: We took 21 samples from cattle, which were taken directly from the rectum of the animal using swabs. The samples were processed until obtaining pure colonies and evaluated for the presence of the stx1, stx2, eae, saa and hlyA genes by PCR. Afterward, the cytotoxic effect of positive samples were evaluated on Vero cells (ATCC-CCL- 81.4). Results: We observed that from the 21 stools samples, 12 presented bacteria with one or both stx genes. A total of 106 isolates of STEC were obtained and differences among each other were observed regarding the presence and absence of the virulence genes, obtaining five combinations of genes. We found that 48 isolates presented only stx2 gene and 58 presented both the stx1 and stx2 gene. Regarding the other virulence genes, the hlyA gene was detected in 44 isolates and the saa gene was detected in 57 isolates. Conclusions: All the STEC supernatants showed cytotoxic activity on Vero cells, while in its absence the cells formed monolayer after 48 h of incubation. This work is the first report in Colombia that provides information about the presence of STEC in stool cattle, virulence genes and its potential cytotoxic effect in native strains.
Subject(s)
Animals , Cattle , Shiga Toxin , Escherichia coli , Shiga-Toxigenic Escherichia coli , Feces , Livestock , Bacteria , Virulence , Polymerase Chain ReactionABSTRACT
Objective:To understand the molecular characteristics of Escherichia coli producing Shiga toxin 2e subtype isolated from different sources in China. Methods:Three human-derived, 13 animal-derived and eight food-derived stx2e-positive Escherichia coli strains which were isolated during 2012 to 2018 were analyzed by antimicrobial susceptibility testing and whole genome sequencing. The stx subtype, serotype, multi-locus sequence type, virulence genes and antimicrobial resistance genes of each strain were determined by whole genome sequences. The phylogenetic relationship and genetic composition of Shiga-toxin prophage were explored. Results:Twenty-four stx2e-STEC strains were typed into 19 O∶H serotypes and 19 sequence types (STs). Each strain carried at least one kind of antimicrobial resistance gene and 19 out of 24 strains were resistant to at least one kind of antimicrobials. Three human-derived strains were heterogenous in serotypes and STs, but there were several animal and food-derived strains shared the same serotype or ST with human strains and showed close relationship in the phylogenetic analysis. The sequences of stx2e among all strains were highly conserved (similarity >99.7%), but there were significant differences in the size and the gene composition of Shiga toxin prophage genome. Conclusions:This is report about the characteristics of rare human-derived Stx2e-STEC strains in China. Comparing human isolates with animal-and food-derived strains, it indicates that Stx2e-STEC strains are highly genetic diversity and have the potential to infect humans.
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Resumen Escherichia coli productora de toxina Shiga (STEC) es un patógeno de importancia alimentaria en los humanos, el bovino es su principal reservorio. El objetivo de este estudio fue determinar la portación de STEC en bovinos del Paraguay y analizar el perfil de virulencia y los serotipos de los aislados reunidos. Se estudiaron 197 muestras de hisopado rectal de bovinos y un promedio de 5 a 50 colonias por bovino positivo a genes stx 1 /stx 2. Se amplificaron por PCR los genes stx 1, stx 2, saa, ehxA y eae. El 84,8% de los bovinos resultaron portadores de STEC. Los perfiles de virulencia predominantes fueron stx 2 y stx 2 /saa/ehxA. La serotipificación se realizó por reacciones de aglutinación en 60 aislamientos seleccionados, se encontró un aislamiento del serogrupo O103, capaz de producir infecciones en humanos. Este trabajo muestra los primeros datos de portación de STEC de ganado bovino paraguayo y señala la necesidad de efectuar otros estudios con mayor cobertura territorial, para lograr una visión completa de este fenómeno.
Abstract Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen in humans, with cattle being the main reservoir. The objective of this study was to determine the carrying of STEC in Paraguayan bovines and to analyze the virulence profile and serotypes of these isolates. A total of 197 samples of bovine fecal samples and an average of 5 to 50 colonies from stx 1 /stx 2 positive samples were studied. The stx 1 , stx 2 , saa, ehxA and eae genes were amplified by PCR. 84.8% of the cattle were carriers of STEC. The predominant virulence profiles were stx 2 and stx 2 /saa/ehxA. The serotyping was performed by agglutination reactions for 60 selected isolates, resulting in isolation of serogroup O103, which could produce infections in humans. This work shows the first data of STEC carriers in Paraguayan cattle, and indicates the need for other studies with greater territorial coverage for a complete vision of this phenomenon.
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Escherichia coli productora de toxina Shiga (STEC) O157:H7 es el serotipo más frecuentemente identificado como agente causal de colitis hemorrágica y síndrome urémico hemolítico (SUH), aunque se han descripto más de 100 serotipos con potencial patogénico similar. El objetivo del trabajo fue describir casos de enfermedad humana asociados a la infección por STEC O121:H19, atendidos en la ciudad de Mar del Plata y establecer la relación genética de los aislamientos mediante técnicas de epidemiología molecular. Se observó un amplio espectro en la severidad clínica de los ocho casos estudiados: dos fueron asintomáticos (contactos de SUH), un paciente tuvo diarrea sanguinolenta, y cinco presentaron SUH. Uno de los pacientes con SUH falleció. Las cepas O121:H19 portadoras del genotipo stx2a/eae/ehxA fueron sensibles a los antibióticos ensayados y presentaron por electroforesis en gel de campo pulsado (Xbal-PFGE) distintos patrones de macrorrestricción, con similitud del 84,25%. El patrón AREXKX01.0072, detectado en un SUH y en su contacto, es nuevo en la Base de Datos Nacional de STEC no-O157 de la Argentina. La utilización de métodos estandarizados de detección y tipificación de STEC permite a los laboratorios de referencia monitorear la frecuencia temporal y la distribución geográfica de las cepas circulantes para la prevención y control de estos patógenos asociados a enfermedad humana.
Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the most frequent serotype identified as causative agent of sporadic cases and outbreaks of diarrhea with or without blood, hemorrhagic colitis and hemolytic uremic syndrome (HUS), although more than 100 serotypes have been described of similar pathogenic potential. The aim of the study was to describe cases of human disease associated with STEC O121:H19 infections, assisted in Mar del Plata City, and to establish the genetic relationship of the isolates by molecular epidemiology techniques. A wide spectrum was observed in the clinical severity of the eight cases studied: two were asymptomatic (contacts of HUS), one patient had bloody diarrhea, and five cases presented HUS. One HUS case died. All STEC O121:H19 strains carried the stx2a/eae/ehxA genotype, were sensitive to all antibiotics tested and showed different macrorestriction patterns by pulsed-field gel electrophoresis (Xbal-PFGE), with 84.25% similarity. The pattern AREXKX01.0072, detected in a HUS case and in his contact, is new in the Argentine National Database of non-O157 STEC. The use of standardized methods for detection and typing of STEC allows reference laboratories to monitor the temporal frequency and geographical distribution of circulating strains for the prevention and control of these pathogens associated with human diseases.
Escherichia coli produtora de toxina Shiga (STEC) O157:H7 é o sorotipo mais frequentemente identificado como o agente causador de colite hemorrágica e síndrome hemolítica urêmica (SHU), embora tenham sido descritas mais de 100 sorotipos com potencial patogênico semelhantes. O objectivo foi o de descrever os casos de doença humana associadas com a infecção por STEC O121:H19, assistido, na cidade de Mar del Plata e estabelecer relação genética de isolados utilizando epidemiologia molecular. Um amplo espectro foi observado na severidade clínica dos oito casos estudados, dois eram assintomáticos (contacto SHU), uma paciente teve diarreia com sangue, e cinco tiveram SHU. Um caso de SHU faleceu. As cepas O121:H19 portaram o genótipo stx2a/eae/ehxA, foram sensíveis aos antibióticos testados e apresentaram, por eletroforese em gel de campo pulsado (Xbal-PFGE), diferentes padrões de macrorestrição, com similaridade de 84,25%. O padrão AREXKX01.0072 detectado em SHU e em seu contato, é novo para a Base de Dados Nacional de STEC não-O157 na Argentina. O uso de métodos padrão de detecção e tipagem de STEC permite os laboratórios de referência monitorar frequência temporal e distribuição geográfica de estirpes circulantes para a prevenção e controlo destes agentes patogénicos associados com a doença humana.
Subject(s)
Shiga Toxin/analysis , Hemolytic-Uremic Syndrome , Molecular Epidemiology , Shiga Toxin/urine , Escherichia coli/virology , Hemolytic-Uremic Syndrome/ethnology , MicrobiologyABSTRACT
Shiga toxin (Stx), which can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), is an important virulence factor of Shigella spp. and certain strains of Escherichia coli. Stx, enco-ded by λ-like phage, blocks protein synthesis through removal of an adenine residue from the 28S rRNA. Stx can also induce apoptosis through multiple pathways. Humans may suffer from diarrhea, hemorrhagic colitis ( HC) and hemolytic uremic syndrome ( HUS) and even death when infected with Shiga toxin-producing bac-teria. At present, there is no specific treatment for diseases caused by Stx. In recent years, the application of Stx in cancer therapy and imaging has aroused great interest. This review provided a brief overview of Stx in its nomenclature, typing, structure, genetics, pathogenesis and application perspectives.
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Objective To understand the molecular characteristics of human-derived non-O157 Shiga toxin-producing Escherichia coil (STEC) strains circulating in five regions of China.Methods Twenty-seven non-O157 STEC strains isolated in five geographic regions were investigated by serotyping, stx1/stx2 subtyping and PCR screening for adhesion and other virulence genes.A multilocus sequence typing (MLST) scheme provided by E.coil MLST database were performed to amplify and sequence seven housekeeping genes (adk, icd, fumC, rgyrB, purA, mdh and recA) in those strains.Results Twenty-seven non-O157 STEC strains were typed into 16 O∶H serotypes.Among those strains, 11 harbored stx1a, 12 harbored stx1c, two harbored stx2e and the other three strains respectively harbored stx1a+stx2b, stx2d and stx2g.Positive rates of eae, efa1, saa, paa, toxB, astA and ehxA genes were 18.5%, 18.5%, 29.6%, 22.2%, 11.1%, 11.1% and 25.9%, respectively.The 27 strains were typed into 16 different sequence types (STs) based upon MLST.Conclusion Human-derived non-O157 STEC strains circulating in five regions of China are heterogeneous in their serotypes, stx1/stx2 subtypes and virulence gene profiles.
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Objective@#To determine the serotypes and drug resistance profiles of Shiga toxin-producing Escherichia coli (STEC) in animal stools from the Weishan area in Shandong Province, China. To provide the basis for further study.@*Methods@#Five hundred animal stool samples (from pigs, cattle, sheep, dogs and birds) were collected from the Weishan area and STEC strains were isolated from these samples. Strains were serotyped by a serum agglutination test, and their drug resistance profiles were determined through antimicrobial sensitivity experiments. In this study, PCR was used to detect tetracycline resistance genes (tetA, tetB, tetC, tetD) and beta-lactam resistance genes (blaSHV-1, blaCTX-M, blaTEM).@*Results@#Sixteen strains of STEC were isolated from animal stool samples. Thirteen strains were isolated from pig stool samples, two from bovine stool samples and one from a sheep stool sample. Two of the strains were identified as E. coli O157:H7, and other 14 strains were non-O157 STEC of different serotypes. Antimicrobial sensitivity experiments showed that 15 of the strains were multidrug resistant. The rates of resistance were as follows: nalidixic acid (12/16 strains), sulfisoxazole (11/16), trimethoprim and sulphame-thoxazole (11/16), doxycycline (9/16), azithromycin (9/16), tetracycline (9/16), chloramphenicol (8/16) and streptomycin (8/16). Therefore, nalidixic acid showed the highest rate of resistance among the strains, followed by trimethoprim and sulphame-thoxazole, and sulfisoxazole. Resistance to cefepime or imipenem was not detected. In total, three types of drug resistance genes (tetA, tetB and tetC) were detected among the 16 strains.@*Conclusion@#The results showed that STEC strains isolated from animals in the Weishan area were of a range of serotypes. The 16 strains of STEC isolated from animal stools in this area were resistant to a number of antibiotics, with many strains displaying multidrug resistance.
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Purpose: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food‑borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. Materials and Methods: Two hundred water samples collected during (2011–2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups‑specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. Results: Thirty‑three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy‑two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups’ activities. Eighty‑nine percent of the strains showed resistance against tetracycline. Conclusion: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.
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Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.
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Objective To analyze the subtypes of eae genes in various non-O157 Shiga toxin-pro-ducing Escherichia coli ( STEC) strains isolated in China.Methods The complete nucleotide sequences of 10 eae genes were amplified by PCR and sequenced.The BLASTn software was used to analyze the se-quences for eae gene subtyping.A phylogenetic tree was constructed based on the10 ea e gene sequences to-gether with the gene sequences of 30 different subtypes in GenBank and those of STEC strains of 7 prevalent serotypes (O157 ∶H7, O26 ∶H11, O103 ∶H2, O111 ∶H8, O145 ∶H28, O45 ∶H2 and O121 ∶H19) using MEGA 5.0.Multilocus sequence typing (MLST) was performed on the 10 STEC strains with reference to the Escherichia coli ( E.coli) MLST website ( http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) for the typing of multiple loci.A minimum spanning tree ( MST) was constructed using the BioNumerics software to inves-tigate the phylogenetic relationships between the 10 eae gene-positive STEC strains in this study and hemolyt-ic uremic syndrome-associated enterohemorrhagic E.coli ( HUSEC) strains as well as all human STEC strains of O157, O26, O45, O103, O111, O121 and O145 serotypes submitted to the E.coli MLST website data-base.Results The complete nucleotide sequences of eae genes in 10 non-O157 STEC strains were 2.8 kb in length and belonged to 3 known subtypes.The predominant subtype wasβ1, accounting for 60%of the 10 STEC strains (6/10), followed byθandγ1 subtypes with two strains in each type.The eae gene sequences in certain strains were identical to those of the prevalent serotypes.Seven sequence types ( STs) were identi-fied from the 10 STEC strains carrying eae gene.Conclusion The eae genes harbored by the non-O157 STEC strains isolated from different specimens in China were diverse and had close phylogenetic relationships with the highly pathogenic and prevalent STEC strains.This study implied that the STEC strains harboring eae gene had high pathogenic potential.
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Background: Seed sprouts contaminated with pathogenic microorganisms, such as Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) present an unacceptable health risk to consumers. An outbreak that occurred in Australia during 2005 and 2006 due to the consumption of alfalfa sprouts contaminated with Salmonella Oranienburg resulted in 141 infected cases, and cost an estimated $1.19 million to the Australian community. In Japan in 1996, consumption of radish sprouts contaminated with STEC O157:H7 affected more than 10,000 individuals. The outbreak of E. coli O104:H4 linked to the consumption of fenugreek sprouts that occurred in Europe in 2011 was an unprecedented foodborne outbreak. More than 4,000 individuals were infected by STEC O104:H4. Among them, 908 developed haemorrhagic uraemic syndrome (HUS), and 50 died of STEC infection. This demonstrates the potential food safety risk arising from seed sprouts and that the consequences can be devastating. Food Standards Australia New Zealand (FSANZ) initiated the development of a primary production and processing standard for seed sprouts in 2009 to enhance the safety of seed sprouts produced and sold in Australia. After extensive consultations with the State and Territory food safety regulators, and a thorough investigation of the Australian industry practices in producing seed sprouts for human consumption, a technical paper was prepared to inform the design of potential risk mitigation measures for a national food safety standard on seed sprout production. This technical paper described the Australian seed sprout industry, depicted the steps involved in the production of seed sprouts for human consumption, and provided an analysis of potential food safety hazards that could occur during seed sprout production and processing. A food safety standard for the production and sale of seed sprouts in Australia was finalised in November 2011. This extended abstract describes the key aspects of the technical paper. Aims: To provide technical and scientific information to support risk management decisions aimed at maximizing the safety of seed sprouts produced for human consumption in Australia. Study Design: A through-chain qualitative food safety risk analysis. Place and Duration of Study: FSANZ, Canberra, Australia, between July 2009 and January 2010. Methodology: This through-chain risk analysis was prepared upon a comprehensive review of literature available at the time on: investigations of foodborne outbreaks associated with consumption of seed sprouts; surveys of microbial contamination of seed sprouts; specific publications on crop production, seed harvest, post-harvest processing and storage of seeds; production of seed sprouts; risk assessments on seed sprouts; and regulatory guidelines published by Australian and international food safety regulatory authorities on seed sprouts. Members of the FSANZ project team conducted field studies of sprout production, lucerne crop production, lucerne seed processing, wholesale and retail sale of seed sprouts. A survey was conducted on the variety, volume and value of sprouts produced, source and quantity of seeds used to produce sprouts for human consumption, trend of consumption of seed sprouts in Australia, as well as the size and the location of sprout producers in Australia. Stakeholders were consulted through a FSANZ standard development committee with participants from State and Territory food safety regulators, peak sprout producer industry bodies, seed producers and seed processors, major food retailers, and consumer representatives. The through-chain analysis of food safety hazards associated with the production and processing of seed sprouts was prepared in line with the principles of hazard analysis critical control points (HACCP). Results: Key pathogens of concern: Among the range of biological, chemical and physical food safety hazards that were likely to be associated with seed sprouts produced for human consumption, pathogenic microorganisms represent the highest risk to consumers. Outbreaks associated with the consumption of seed sprouts contaminated with pathogenic microorganisms were seen to be frequent events in developed economies despite food regulatory interventions. The key pathogenic microorganisms of concern were Salmonella spp. and STEC. Salmonella spp. were found to be the causative pathogen almost five times more frequently than STEC. Main varieties of seed sprouts causing foodborne illness: Among the 41 reported outbreaks that occurred worldwide between 1988 and 2007 involving consumption of seed sprouts contaminated with pathogenic microorganisms, alfalfa sprouts represented 68% of the outbreaks, followed by mingbean sprouts (22%), clover sprouts (5%), radish sprouts (2%) and clover sprouts (2%). Source of pathogenic microorganisms: FSANZ divided the production and supply of seed sprouts for human consumption into eleven consecutive steps, starting with seed production in the field and ending with transportation and distribution of seed sprouts to retail establishments. This was to enable a systematic identification of the food safety hazards, sources of the hazards, specific controls that could be applied to control or eliminate food safety hazards, and the associated requirements of food safety management practices including food safety knowledge and food safety skills. Contamination of seeds by pathogenic microorganisms such as Salmonella spp. and STEC can occur during seed production, seed harvest, seed processing, seed storage and transportation. The origin of these pathogenic microorganisms is animal faeces and manure present in the field where the crop is grown. Soil for growing the seed crop, water used for irrigation, and machinery used for crop management including the harvest of seeds, can be contaminated with pathogenic microorganisms and can transfer the contamination to seeds during crop production and seed harvest. Seed processing as a post-harvest step may also contribute to seed contamination. For example, blending different harvest lots of seeds for seed cleaning can spread what was originally a localised contamination into a larger volume of seeds. Rodent, insect and bird activities in seed processing and seed storage establishments can introduce and spread pathogenic microorganisms to seeds. Provided that seeds delivered to sprout production sites are free of pathogenic microorganisms, activities of rodents, insects, and infected workers in seed receipt, storage, sprout production, sprout storage and transportation at sprouting establishment can lead to contamination of seed sprouts by pathogenic microorganisms. So is the use of contaminated water for sprouting. Much of these are also applicable to retail handling and storage of seed sprouts. Investigations into the source of sprout contamination for outbreaks that occurred between 1988 and 2007 found that in almost every case the pathogenic microorganisms causing the outbreaks were present in the seeds used for sprout production. In approximately 20% of the outbreaks, contamination in sprouting establishments was also identified as a likely source of contamination. Identified risk mitigation measures: Based on an analysis of a wide range of possible recommendations aimed at improving the safety of seed sprouts, the though-chain analysis recommended the following good agricultural practices to be implemented in the primary production phase of seeds: · Environment - soil and environment where seeds are grown for the production of seed sprouts as a human food should be suitable. · Inputs - manure, biosolids and other natural fertilisers should only be used for the growth of seed crops when a high level of pathogen reduction has been achieved; equipment (bins, containers, silos, vehicles) and machinery are maintained and used in a manner that minimises and/or avoids contamination of seeds with pathogenic microorganisms. · Protection - grazing animals and wild animals are prevented from entering the field where seeds are grown; and seed crops are protected from contamination by human, animal, domestic, industry and agricultural wastes. · Segregation - seeds produced for the production of sprouts for human consumption are segregated from seeds produced for the production of animal feed and are clearly labelled. The through-chain analysis also recommended the following components to be included in a Food Safety Program that must be effectively implemented in sprout production establishments: · Environment – the sprouting facility (including the seed storage area) should not allow access of rodents, insects, pests or animals; sprouting facility and equipment are effectively cleaned and sanitised to ensure the environment is suitable for producing ready-to-eat foods. · Input – each seed lot is tested for the presence of microbial pathogens of concern and seeds should not be used unless the testing results are negative; solid medium supporting sprout growth and water for sprouting are treated to eliminate pathogenic microorganisms; seeds are disinfected prior to sprouting to eliminate microbial pathogens. · Separation – seed rinsing and microbiological decontamination, seed germination/sprouting, and storage of seed sprouts are physically separated from each other to prevent cross contamination. · Monitoring – implement appropriate sampling/testing programs to regularly monitor microbial pathogens during and at the end of production of seed sprouts. Implementation of food safety controls on farm presents many challenges. One of the main obstacles is the inability to control environmental factors under conventional farming practices. The environment under which seeds are produced for the production of seed sprouts for human consumption should exclude animal grazing and minimise and avoid pest and wildlife interference. The cost involved in growing seeds under these conditions can be prohibitive unless s
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Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 represents the major Shiga toxin-producing E. coli (STEC) strain related to large outbreaks and severe diseases such as hemorrhagic colitis (HC) and the potentially lethal hemolytic uremic syndrome (HUS). The aim of this study was to report the occurrence and molecular characterization of O157:H7 isolates obtained by rectal swab from 52 healthy dairy cattle belonging to 21 farms in Mid-West of Brazil. Detection of 16SrRNA, stx1, stx2, rfbO157, fliCh7, eae, ehxA, saa, cnf1, chuA, yjaA and TSPE4.C2 genes was performed by PCR. The isolates were further characterized by serotyping. Two hundred and sixty E. coli isolates were obtained, of which 126 were characterized as STEC. Two isolates from the same cow were identified as serotype O157:H7. Both isolates presented the stx2, eae, ehxA, saa and cnf1 virulence factor genes and the chuA gene in the phylogenetic classification (virulent group D), suggesting that they were clones. The prevalence of O157:H7 was found to be 1.92% (1/52 animals), demonstrating that healthy dairy cattle from farms in the Mid-West of Brazil are an important reservoir for highly pathogenic E. coli O157:H7.
Escherichia coli enterohemorrágica (EHEC) sorotipo O157:H7 representa as principais cepas de E. coli produtoras de toxina Shiga (STEC) relatadas em grandes surtos e doenças graves, tais como colite hemorrágica (CH) e síndrome hemolítica urêmica (SHU), potencialmente letais. O objetivo deste estudo foi reportar a ocorrência e caracterização molecular de STEC 0157:H7 isoladas por swab retal de 52 bovinos saudáveis pertencentes a 21 rebanhos leiteiros do Centro-Oeste do Brasil. A detecção dos genes 16SrRNA, stx1, stx2, rfbO157, fliCh7, eae, ehxA, saa, cnf1, chuA, yjaA e TSPE4.C2 foi realizada por PCR. Os isolados foram ainda caracterizados por sorotipagem. Dos 260 isolados de E. coli obtidos, 126 foram caracterizados como STEC. Dois deles, oriundos do mesmo animal, foram caracterizados como pertencentes ao sorotipo O157:H7. Ambos apresentaram os genes de virulência stx2, eae, ehxA, saa e cnf1 e na caracterização filogenética, o gene chuA (grupo patogênico D), sugerindo que eles foram clones. A prevalência de O157:H7 foi de 1,92% (1/52 animais), demonstrando que os bovinos leiteiros saudáveis de fazendas do Centro-Oeste do Brasil são importantes reservatórios de E. coli O157:H7 altamente patogênicas.
Subject(s)
Animals , Cattle/microbiology , /isolation & purification , Shiga-Toxigenic Escherichia coli/isolation & purification , Polymerase Chain Reaction/veterinaryABSTRACT
This study aimed to verify the occurrence of Shiga toxin-producing Escherichia coli (STEC) strains in three distinct anatomic parts of the stable fly Stomoxys calcitrans by multiplex polymerase chain reaction (PCR Multiplex). According to the results obtained, E. coli was identified in 19.5% of the stable flies. Shiga toxin genes were detected in 13% of the E. coli isolated, most frequently from the surface, followed by abdominal digestive tract and mouth apparatus of insects, respectively. This is the first study to detect presence of STEC in Stomoxys calcitrans in Brazil; it has also revealed the potential role of stable flies as carriers of pathogenic bacterial agents.
Este estudo teve por objetivo avaliar a ocorrência de Escherichia coli Shiga-Toxigênica (STEC) em três diferentes partes anatômicas da mosca dos estábulos pela Reação de Polimerase em Cadeia Multiplex (PCR Multiplex). De acordo com os resultados obtidos, foi identificada E. coli em 19,5% das moscas dos estábulos colhidas. Foram detectados genes de produção de Shiga toxina em 13,63% das Escherichia coli isoladas, sendo mais frequente a superfície externa, seguido pelo trato digestivo abdominal e pelo aparelho bucal, respectivamente. Este foi o primeiro estudo no Brasil que detectou a presença de STEC em Stomoxys calcitrans e revelou o potencial papel da mosca dos estábulos em carrear um agente bacteriano patogênico.
Subject(s)
Animals , Multiplex Polymerase Chain Reaction/veterinary , Muscidae/microbiology , Shiga-Toxigenic Escherichia coli/isolation & purificationABSTRACT
Objective To investigate the tellurite resistance level,the presence of tellurite resistance (ter) gene cluster and their relationships in non-O157 Shiga toxin-producing Escherichia coli(STEC) isolates.Methods Tellurite resistance level was evaluated by plate dilution method and the ter gene cluster was tested by PCR.Results Only 5 of 39 non-O157 STEC isolates tested in this study were identified to have ter gene cluster,which showed relatively high levels of tellurite resistance ranging from 128 μg/ml to 512 μg/ml.In contrast,the other 34 isolates without ter gene cluster were sensitive to potassium tellurite and showed very low levels of tellurite resistance,the minimal inhibitory concentration (MIC) was ＜1 μg/ml for 29 isolates,8 μg/ml for 2 isolates and 2 μg/ml for 3 isolates.Conclusion Most non-O157 STEC isolates were sensitive to potassium tellurite.It could be concluded that much attention should be paid when screening the non-O157 STEC isolates using the selective medium supplemented with potassium tellurite.
ABSTRACT
Background: The consumption of uncooked comminuted fermented meat (UCFM) contaminated with Shiga toxin-producing Escherichia coli (STEC) poses a public health risk. The severity of such a health risk can be demonstrated by an outbreak that occurred in South Australia in 1995, where the consumption of Mettwurst contaminated with E. coli O111:NM resulted in the death of one child, haemolytic uraemic syndrome in twenty-two children, and permanent adverse health effects in at least six children and one 60 years old consumer. The Australian meat industry incurred an estimated loss of more than $A 400 million. In response to the outbreak, the Australian Government introduced an emergency measure in 1996 to ensure the safety of UCFM products. A key performance criterion prescribed in the emergency measure – that the UCFM production process must reduce the number of E. coli organisms by 99.9% (a 3-log10 reduction) or greater – could not be effectively implemented by the industry or enforced by the health authorities. This was largely due to a lack of an objective means to determine compliance. Food Standards Australia New Zealand (FSANZ) undertook a review of the emergency measure between 2001 and 2003. This paper describes the risk analysis FSANZ undertook to improve the effectiveness of food safety regulation in this area. Aims: To develop a set of outcome-based regulatory measures to replace a prescriptive requirement of a 3-log10 reduction of E. coli, designed to minimise STEC contamination in UCFM. Study design: Food safety risk analysis. Place and Duration of Study: FSANZ, Canberra, Australia, between November 2001 and July 2003. Methodology: The ability of Australian UCFM manufacturers to effectively implement the processing requirement of a 3-log10 reduction in E. coli concentration was assessed using an Excel® based predictive model developed by the University of Tasmania that estimates the inactivation of generic E. coli during the UCFM manufacturing process. Temperature and time parameters of fermentation and maturation applied to the production of UCFM for sale in Australia were collected during 2002 and 2003 and applied to the predictive model. Outcome-based regulatory measures to minimise STEC contamination in UCFM were developed based on (1) the conclusions of a quantitative microbiological risk assessment (based on point estimates), (2) close consultation with the Australian UCFM sector and food regulation enforcement authorities, and (3) a regulatory impact assessment. Tools to facilitate effective implementation of the outcome-based regulatory measures were developed between 2004 and 2005 with the assistance of a national expert advisory panel on UCFM safety. This panel was comprised of food safety and technical experts from the Australian smallgoods sector and state enforcement authorities. Results: Assessment of 96 production protocols used by Australian UCFM manufacturers in April 2002 using the predictive E. coli inactivation model showed that only 19% of the protocols were capable of achieving greater than or equal to a 3-log10 reduction of E. coli. Up to 51% of the protocols assessed achieved less than 2-log10 reduction of E. coli. The remaining protocols were capable of achieving a maximum reduction of E. coli between 2 and less than 3-log10. Among the 96 production protocols assessed, the highest level of inactivation of E. coli potentially achievable was 9.08 log10 and the lowest was 0.13 log10. A relatively long period of maturation and a relatively high temperature during the maturation phase contributed to the bulk of E. coli inactivation achieved during the manufacture of UCFM. Production protocols resubmitted from UCFM manufacturers in the state of Victoria, following the initial assessment, showed a steady improvement of capability in achieving greater than or equal to a 3-log10 reduction of E. coli. This was achieved by making adjustments to the time and temperature parameters of the production processes. Despite these adjustments, 34% of the resubmitted protocols failed to meet the requirement of reduction of E. coli by 3-log10. Consultations with technical experts of the Australian smallgoods sector and enforcement authorities identified several additional issues with the 3-log10 reduction requirement. These included: • the rationale behind of the need for a 3-log10 reduction of E. coli when safe UCFM products can be produced using deep muscle meat and when subject to close adherence to operational hygiene, knowing the fact that the extent of STEC contamination in deep muscle meat is very low; • doubts on the adequacy of a 3-log10 reduction of E. coli when raw ingredients used to produce UCFM contain excessively high numbers of STEC; • enforcement authorities did not have the tools to verify whether manufacturers of UCFM had achieved a 3-log10 reduction of E. coli; and • the science underpinning the mandatory requirement of a 3-log10 reduction of E. coli in manufacturing UCFM was difficult to comprehend by members of the industry, let alone their ability to demonstrate compliance against the requirement. A microbiological risk assessment was undertaken by FSANZ to provide a scientific basis for the identification and development of effective outcome-based regulatory measures to minimise STEC contamination in UCFM products. The main conclusions of the risk assessment were that: • the ingestion of as few as 1 STEC could lead to severe adverse health consequences in susceptible individuals; • children under the age of 6 are more likely to develop severe complications from STEC infections; • based on the available data at the time, it was estimated that a mean of 0.15 STEC/100 g was present in approximately 7.2% of the UCFM manufactured in Australia; and • under this level of STEC contamination, it was estimated that the likelihood of encountering 1 STEC organism by UCFM consumers under the age of 6 years old would be approximately 1 in 174 UCFM meals. If UCFM was produced under minimum (time and temperature) processing conditions, this likelihood would shift to approximately 1 in 3 UCFM meals. The above findings of the risk assessment established the basis for further regulatory interventions in UCFM production. The implementation of hazard analysis critical control point (HACCP) based food safety programs, together with a number of specific requirements, was identified as the preferred option to replace the prescriptive processing requirement of a 3-log10 reduction of E. coli. This risk management decision took into consideration of the issues identified during the consultations with the Australian smallgoods sector and enforcement authorities, and the factors of: • a mandatory requirement for having HACCP based food safety programs developed and implemented by the UCFM sector would impose minimal compliance costs because HACCP-based food safety systems have been introduced into the Australian UCFM sector on a voluntary basis since 1998; and • the policy of the Council of Australian Governments requires national food standards to be outcome based. Together with the requirement of having HACCP based food safety programs implemented, the outcome-based regulatory measures specified validation and verification procedures to ensure that the number of E. coli in the final product complies with limits specified for UCFM in Standard 1.6.1 of the Australian and New Zealand Food Standards Code (n=5, c=1, m=3.6, M=9.2). UCFM manufacturers were also required to provide evidence to demonstrate that their production processes are capable of handling the variations in the level of E. coli contamination in the ingredients. The latter requirement puts UCFM manufacturers in charge of product safety by allowing the flexibility in raw material selection. In addition, it requires that appropriate adjustments in manufacturing parameters be made to cope with the extent of fluctuation of E. coli contamination in the raw materials, to ensure UCFM safety. To assist the UCFM sector to implement HACCP based food safety programs, a Protocol for Assessing HACCP Based Food Safety Programs in the UCFM Sector (the protocol) has been developed by FSANZ in association with experts in manufacturing smallgoods and enforcing food safety regulations. The protocol has been adopted by the state enforcement authorities for assessing UCFM manufacturers’ compliance against the requirement of implementation of HACCP based food safety programs. To raise the overall level of skills and knowledge on food safety in the UCFM sector, a set of Competency Criteria for UCFM Manufacturers on food safety skills and knowledge has been developed and incorporated into an industry training package. The package was developed jointly by FSANZ, experts in manufacturing smallgoods and enforcing food safety regulations, and the National Meat Industry Training Advisory Council. It targets those who intend to enter the UCFM manufacturing sector. This training package has been made available nationwide through technical and further education institutes. Conclusion: Careful considerations ought to be given to prescriptive requirements developed for food safety regulation to ensure that they are practical and can be effectively implemented by the food industry and verified by enforcement authorities. Critical production parameters, such as time and temperature, applied in food production, and appropriate tools such as predictive models for pathogen inactivation in food production can facilitate an objective assessment of processing requirements to ensure food safety. Implementation of outcome based food safety requirements, if supported by appropriate implementation tools, can lead to enhanced effectiveness in managing food safety. Acknowledgements and additional information: The authors wish to acknowledge the support and assistance provided to this study by the following organisations and individuals: Ms. Amanda Hill and Dr
ABSTRACT
We encountered a patient with hemolytic uremic syndrome (HUS) with persistent isolation of shiga toxin-producing Escherichia coli (STEC) for 3 weeks despite of having no clinical symptoms. STEC has been recognized as an important food-borne pathogen that causes severe diseases such as HUS. We characterized this STEC strain via a polymerase chain reaction, reverse-passive latex agglutination and the slide agglutination method. In this STEC strain, stx2 (shiga toxin), eaeA, tir, iha (adherence genes), espADB (type III secretion genes), and hlyA, ehxA, clyA (hemolysin genes) were present. The O antigen of the strain was non-typable.
Subject(s)
Child, Preschool , Female , Humans , Hemolytic-Uremic Syndrome/diagnosis , Shiga-Toxigenic Escherichia coli/isolation & purificationABSTRACT
Background & objectives: Verotoxigenic Escherichia coli are important serotypes of enterohaemorrhagic E. coli (EHEC) subgroup that cause attaching and effacing lesions in enterocytes by producing verotoxins or shiga-like toxins resulting in haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). The aim of this study was to detect these serotypes specially E. coli O157:H7 in stool samples of patients with diarrhoea and identification of virulence genes (STX1, STX2, Hly and EAE) in Shahrekord-Iran area using PCR technique. Methods: Two hundred diarrhoeal stool samples of patients were collected through 2007-2008. Microbiological and biochemical examinations were done to detect the E. coli. Serological tests carried out to identify the O157 or O157:H7 serotypes. Results: Of the 58 E. coli isolates, 16 (27.6%) were detected as STX1 carrying E. coli, four (6.9%) carrying STX2, eight (13.8%) carrying both STX1 and STX2, and 12 (20.7%) were Hly carrying E. coli, but none of the isolates contained EAE gene. None of the isolates were E. coli O157 or O157:H7 serotypes. Interpretation & conclusions: Our results revealed that verotoxigenic E. coli isolates other than O157 serotype were involved in causing diarrhoea in Shahrekord-Iran.
Subject(s)
Adhesins, Bacterial/genetics , Diarrhea/microbiology , Escherichia coli O157/genetics , Escherichia coli O157/isolation & purification , Escherichia coli O157/metabolism , Escherichia coli Proteins/genetics , Feces/microbiology , Female , Hemolysin Proteins/genetics , Humans , Iran , Male , Surveys and Questionnaires , Shiga Toxin 1/genetics , Shiga Toxin 2/genetics , Shiga Toxins/metabolismABSTRACT
As Enfermidades Transmitidas por Alimentos representam um crescente e relevante problema de saúde pública. Além do prejuízo social, a contaminação de alimentos com microrganismos patogênicos gera um enorme prejuízo econômico. Técnicas de Análise de Risco permitem mensurar de forma mais adequada o impacto dos microrganismos contaminantes de alimentos na saúde da população. Uma Análise de Riscos, associada a uma combinação patógeno-alimento, envolve três passos: avaliação do risco, gestão do risco e comunicação do risco. Uma das etapas da avaliação do risco é a avaliação da exposição, baseada em dados sobre freqüência e nível de contaminação dos alimentos pelo patógeno avaliado no alimento em questão, o nível atingido pelo patógeno no momento do consumo e os padrões de consumo. Os produtos cárneos são os principais alimentos responsáveis pela veiculação de patógenos ao homem e os microrganismos de maior relevância nestes produtos são Listeria monocytogenes, Salmonella spp., Campylobacter spp. e Escherichia coli produtora de toxina de Shiga. O objetivo do presente estudo foi levantar informações qualitativas e quantitativas desses quatro patógenos em produtos cárneos (salsicha bovina, lingüiça suína, carne bovina moída e coxa de frango) comercializados no município de São Paulo, de forma a contribuir com dados para futuras avaliações de risco em relação a estes microrganismos nestes produtos. Das 552 amostras de produtos cárneos analisadas, L. monocytogenes foi o patógeno isolado com maior freqüência, sendo detectado em 48,7% das amostras, seguido por Campylobacter spp. em 6,0% e Salmonella spp. em 5,8%. E. coli produtora de toxina de Shiga não foi detectada em nenhuma das amostras estudadas. Listeria monocytogenes foi detectada em todos os tipos de produtos cárneos estudados, com freqüências mais elevadas nas amostras de carne bovina moída (59,4%), seguido de coxa de frango (58,0%), lingüiça suína (39,8%) e salsicha bovina (37,7%). Na maioria das amostras...
Foodborne Diseases represent an increasingly important public health problem. Besides the social losses, contamination of food with pathogenic microorganisms generates an enormous economic damage. A more accurate measurement of the impact of microorganisms in food health can be achieved using Risk Analysis techniques. A risk analysis is composed by three elements: risk assessment, risk management and risk communication. One of the four steps of a risk assessment is the exposure assessment, based on data on frequency and level of contamination of a food by the pathogen under evaluation, levels of the pathogen in the food at the time of consumption and consumption patterns. Meat products are the main vehicles of pathogens to humans, where Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Shiga toxin-producing Escherichia coli are the most relevant pathogens. The aim of this study was to obtain qualitative and quantitative information on these four pathogens in four types of meat products (beef sausage, pork sausage, ground beef and chicken leg) marketed in the city of Sao Paulo in order to contribute with data for future risk assessments for these microorganisms in these products. L. monocytogenes is the most frequent pathogen in the 552 samples of meat products analyzed, being detected in 48.7% of the samples, followed by Campylobacter spp. 6.0% and Salmonella spp. 5.8%. Shiga toxin-producing E. coli was not detected in any sample. L. monocytogenes was detected in all types of meat products, with highest frequency in ground beef (59.4%), followed by chicken leg (58.0%), pork sausage (39.8%) and beef sausage (37.7%). In most samples (94.4%), the counts of L. monocytogenes were below 102 CFU/g. L. monocytogenes strains were widely distributed in the four groups of serotypes: 28.7% belonged to Group 1 (serotypes 1/2a and 3a), 21% to Group 2 (serotypes 1/2c and 3c), 17% to Group 3 (serotypes 1/2b, 3b and 7) and 13.8% to Group 4...
Subject(s)
Campylobacter , Escherichia coli , /analysis , Listeria monocytogenes , Meat Products/statistics & numerical data , Salmonella , Shiga Toxin/analysis , Bacteriology , Brazil , Chi-Square Distribution , Food MicrobiologyABSTRACT
Shiga toxin-producing Escherichia coli (STEC) can cause a broad spectrum of human illness ranging from symptom-free to hemolytic uremic syndrom (HUS). Associations between known or putative virulence factors of STEC and diseases in human were investigated. PCR analyses showed that 33 (78.6%) isolates carried an ehxA enterohemolysin gene and 6 (14.3%) isolates possessed an saa autoaggutinating adhesin gene, and 31 (73.8%) isolates carried an eae intimin gene (7 isolates with type beta, 16 with type gamma, and 3 with type epsilon). Twenty-nine (69%) isolates from patients carried eae+, ehxA+, saa- (genotype A) and 68 (86%) isolates from asymptomatic outbreaks and 4 (36%) isolates from bovine possessed eae-, ehxA+, saa+ (genotype C). Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. In HEp-2 cell adherence assay, isolates carrying eae gene exhibited a localized adherence phenotype, the other isolates carrying saa showed LC (loose clusters of bacteria) and IS (isolated bacteria). In conclusion, most STEC isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. But these results may be useful information for pathogenesis judgement of STEC.
Subject(s)
Animals , Cattle , Humans , Diarrhea , Disease Outbreaks , Enteropathogenic Escherichia coli , Escherichia coli Proteins , Feces , Hemolysin Proteins , Korea , Lifting , Molecular Biology , Phenotype , Plasmids , Polymerase Chain Reaction , Shiga-Toxigenic Escherichia coli , Virulence FactorsABSTRACT
Shiga toxin-producing Escherichia coli (STEC) strains are commensal bacteria in cattle and cause food borne disease in human. We analyzed the isolation rate of STEC in stool specimens of patients with diarrhea and in fecal samples of cattle in Gwangju, Korea. STEC strains were detected from 33 (0.19%) out of 17,148 patients with diarrhea while there has been a progressive increase in the incidence rate from 0.07% in 2004 to 0.33% in 2008. We investigated serotypes, shiga toxin genes, and antimicrobial resistance patterns of the 44 STEC isolates from human and cattle sources. The 33 STEC isolates from human belonged to 14 O serotypes including O157, O26 and O111. The 11 isolates from cattle belonged to 11 O serotypes. PCR detection for stx genes showed that 12 (27.3%) isolates carried stx1 genes, 20 (45.5%) possessed stx2 genes, and 12 (27.3%) carried both stx1 and stx2. Of the 33 STEC isolates from human, 25 strains (76%) were resistant to one or more antibiotics. High level of resistance to tetracycline (73%) was most common, followed by ticarcillin and ampicillin (64%). But none of the 33 isolates from human were resistant to amikacin, cefazolin, cefepime, cefotetan, cefotaxime, ciprofloxacin, or imipenem. The 5 strains (45%) of the 11 isolates from cattle were resistant to at least one or three antibiotics but most of the isolates were sensitive to the 16 antibiotics employed in this survey. In conclusion, toxin types and serotypes of STEC isolated from human and cattle were diverse, and non-O157 STEC was also observed to be a greater proportion of STEC isolates. According to a specific comparison solely based on the toxin types and serotypes, most of the STEC strains isolated from cattle feces in Gwangju, Korea showed characteristics different from those isolated from patients. Therefore, laboratory surveillance is required to detect and carefully monitor the potentially hypervirulent STEC not only in human and cattle but also in other animals.