RESUMO
Introduction. The different pathotypes of Escherichia coli can produce a large number of human diseases. Surveillance is complex since their differentiation is not easy. In particular, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157â:âH7 consists of stool culture of a diarrhoeal sample on enriched and/or selective media and identification of presumptive colonies and confirmation, which require a certain level of training and are time-consuming and expensive.Hypothesis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a quick and easy way to obtain the protein spectrum of a microorganism, identify the genus and species, and detect potential biomarker peaks of certain characteristics.Aim. To verify the usefulness of MALDI-TOF MS to rapidly identify and differentiate STEC O157â:âH7 from other E. coli pathotypes.Methodology. The direct method was employed, and the information obtained using Microflex LT platform-based analysis from 60 clinical isolates (training set) was used to detect differences between the peptide fingerprints of STEC O157â:âH7 and other E. coli strains. The protein profiles detected laid the foundations for the development and evaluation of machine learning predictive models in this study.Results. The detection of potential biomarkers in combination with machine learning predictive models in a new set of 142 samples, called 'test set', achieved 99.3â% (141/142) correct classification, allowing us to distinguish between the isolates of STEC O157â:âH7 and the other E. coli group. Great similarity was also observed with respect to this last group and the Shigella species when applying the potential biomarkers algorithm, allowing differentiation from STEC O157â:âH7Conclusion. Given that STEC O157â:âH7 is the main causal agent of haemolytic uremic syndrome, and based on the performance values obtained in the present study (sensitivity=98.5â% and specificity=100.0â%), the implementation of this technique provides a proof of principle for MALDI-TOF MS and machine learning to identify biomarkers to rapidly screen or confirm STEC O157â:âH7 versus other diarrhoeagenic E. coli in the future.
Assuntos
Infecções por Escherichia coli , Escherichia coli O157 , Escherichia coli Shiga Toxigênica , Humanos , Escherichia coli O157/metabolismo , Sorogrupo , Infecções por Escherichia coli/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Biomarcadores/metabolismoRESUMO
Los bovinos son el principal reservorio de Escherichia coli productor de toxina Shiga (STEC); las estrategias para evitar su transmisión se concentran en la planta de faena. El objetivo de este trabajo fue evaluar la calidad higiénico-sanitaria y la frecuencia de detección de STEC en medias reses bovinas de frigoríficos de tránsito provincial. Se procesaron 274 esponjados de media res; en 9 (3,3%) el recuento de E. coli genérico fue marginal, en 4 (1,4%) se aisló E. coli O157, de los cuales 2 fueron caracterizados como stx2c(vh-a)/eae/ehxA, y los otros 2 como no toxigénicos. A partir de una (0,4%) muestra se aisló E. coli no-O157 ONT:H49, stx2a/ehxA/saa. En este trabajo la calidad del producto analizado indica que en la provincia de Tucumán se cumplen las buenas prácticas de manufactura en la faena de bovinos.
Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), and the strategies to prevent the transmission of these microorganisms are concentrated in the slaughtering plant. The aim of this study was to evaluate the hygienic-sanitary quality and the frequency of detection of STEC in beef carcasses in abattoirs from Tucuman province. Two hundred and seventy four beef carcass sponges were processed; the count of generic E. coli was marginal in 9 (3,3%) of them. Escherichia coli O157 was isolated in 4 (1,4%) samples; 2 of which were characterized as stx2c(vh-a)/eae/ehxA whereas the other 2 were non-toxigenic strains. Non-O157 E. coli ONT:H49, stx2a/ehxA/saa was isolated from 1 sample (0,4%). In this work the quality of the analyzed product indicates that the good practices of manufacture are fulfilled in slaughtering facilities in Tucumán province.
Assuntos
Animais , Bovinos , Matadouros , Escherichia coli Shiga Toxigênica , Carne , Argentina , Escherichia coli O157 , Proteínas de Escherichia coli/análise , Infecções por Escherichia coli , Escherichia coli Shiga Toxigênica/isolamento & purificação , Carne/microbiologiaRESUMO
Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), and the strategies to prevent the transmission of these microorganisms are concentrated in the slaughtering plant. The aim of this study was to evaluate the hygienic-sanitary quality and the frequency of detection of STEC in beef carcasses in abattoirs from Tucuman province. Two hundred and seventy four beef carcass sponges were processed; the count of generic E. coli was marginal in 9 (3,3%) of them. Escherichia coli O157 was isolated in 4 (1,4%) samples; 2 of which were characterized as stx2c(vh-a)/eae/ehxA whereas the other 2 were non-toxigenic strains. Non-O157 E. coli ONT:H49, stx2a/ehxA/saa was isolated from 1 sample (0,4%). In this work the quality of the analyzed product indicates that the good practices of manufacture are fulfilled in slaughtering facilities in Tucumán province.
Assuntos
Matadouros , Carne , Escherichia coli Shiga Toxigênica , Animais , Argentina , Bovinos , Infecções por Escherichia coli , Escherichia coli O157 , Proteínas de Escherichia coli/análise , Carne/microbiologia , Escherichia coli Shiga Toxigênica/isolamento & purificaçãoRESUMO
Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with cases of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). E. coli O157:H7 is the dominant serotype in Argentina and also in Neuquén Province, in which HUS incidence is above the national average, with a maximum of 28.6 cases per 100,000 children less than 5 years old reported in 1998. The aim of this study was to characterize a collection of 70 STEC O157 strains isolated from patients with diarrhea and HUS treated in the province of Neuquén, Argentina, between 1998 and 2011. All strains harbored eae, ehxA, rfbO157, and fliCH7 genes, and stx2a/stx2c (78.7%) was the predominant genotype. A total of 64 (91.4%) STEC O157 strains belonged to the hypervirulent clade 8 tested using both 4 and 32 SNP typing schemes. The strains showed the highest values reported in the literature for 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. Clade 8 strains were strongly associated with two of them: ECSP_3286, factor encoding an outer membrane protein that facilitates the transport of the heme complex (P=0.001), and in particular extracellular factor ECSP_2870/2872, coding proteins related to adaptation to plant hosts (P=0.000004). The q933 allele, which has been related to high toxin production, was present in 97.1% of the strains studied for the anti-terminator Q gene. In summary, this study describes, for the first time in Argentina, the almost exclusive circulation of strains belonging to the hypervirulent clade 8, and also the presence of putative virulence factors in higher frequencies than those reported worldwide. These data may help to understand the causes of the particular epidemiological situation related to HUS in Neuquén Province.
Assuntos
Diarreia/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/classificação , Doenças Transmitidas por Alimentos/microbiologia , Síndrome Hemolítico-Urêmica/microbiologia , Argentina/epidemiologia , Análise por Conglomerados , Diarreia/epidemiologia , Infecções por Escherichia coli/epidemiologia , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Proteínas de Escherichia coli/genética , Doenças Transmitidas por Alimentos/epidemiologia , Genótipo , Síndrome Hemolítico-Urêmica/epidemiologia , Humanos , Epidemiologia Molecular , Tipagem Molecular , Fatores de Virulência/genéticaRESUMO
Escherichia coli productora de toxina Shiga (STEC), fundamentalmente del serotipo O157:H7, está asociada a la ocurrencia de casos esporádicos y brotes de diarrea sanguinolenta (DS) y síndrome urémico hemolítico (SUH). Otros serotipos STEC como O26:H11, O103:H2, O111:NM, O113:H21 y O145:NM también pueden causar enfermedad severa. En Argentina O157:H7 es el serotipo prevalente siguiéndole en frecuencia O145:NM. El objetivo del presente trabajo fue establecer la diversidad genética y la relación clonal de cepas de E. coli O145:NM/H27 aisladas en diferentes localidades de la provincia de Buenos Aires en el período 2004-2009. A 17 cepas aisladas de casos de SUH (9), DS (5) y de contactos asintomáticos (3) se les realizó PCR para detectar genes stx1, stx2, rfbO157, eae y ehxA y electroforesis en gel en campos pulsados (Xbal-PFGE) para establecer diversidad genética y relación clonal. Todos los aislamientos fueron caracterizados genotípicamente como stx2/eae/ehxA. Por %baI-PFGE se obtuvieron 14 patrones diferentes, identificándose un único Cluster. Mediante la sub-tipificación molecular se conformó la base de datos de E. coli O145:NM/ H27 aislados en la región que permitirá monitorear los perfiles %baI-PFGE a fin de identificar tempranamente la probable ocurrencia de un brote en la población y notificar a las autoridades para aplicar acciones de control.
SHIGA TOXIN-PRODUCING Escherichia coli (STEC), especially the serotype 0157-.H7, is associated with the occurrence of sporadic cases and outbreaks of bloody diarrhea (DS) and hemolytic uremic syndrome (HUS). Other STEC serotypes such as 026:H11, 0103:H2, 0111:NM, 0113:H21, and 0145:NM can also cause severe human illness. In Argentina 0157:H7 is prevalent, followed in frequency by serotype 0145:NM. The aim of this study was to determine the genetic diversity and clonal relationship of E. coM 0145:NM/ H27 strains isolated in different locations of the Buenos Aires province, in the period 2004-2009. A total of 17 strains isolated from HUS (9), DS (5) and asymptomatic contacts (3) were characterizea by POR to detect stXj, stx2, rfbO157, eae and ehxA genes, and pulsed-field gel electrophoresis (Xbal-PFGE) to establish the genetic diversity and the clonal relationship. All isolates were characterized as stx2/eae/ehxA. By Xba/-PFGE, 14 different patterns were established, with a single cluster identified. A regional database of E. coii 0145:NM/H27 was created, which will monitor the /-PFGE profiles of the circulating strains in order to identify the probable occurrence of an outbreak in the community and report to the authorities to implement control measures.
Escherichia coii produtora de toxina Shiga (STEO), especialmente do sorotipo 0157:H7, é associado com a ocorrència de casos esporádicos e surtos de diarreia sanguinolenta (DS) e a síndrome urèmica hemolítica (HUS). 0utros sorotipos STEO como 026H11, 0103H2, 0111NM, 0113H21, e 0145NM também podem causar doengas severas. Na Argentina, é o sorotipo prevalente seguindo em frequència 0145-.NM. 0 objetivo deste estudo foi determinar a diversidade genética e a relagäo clonal de cepas de E. coii 0145:NM/H27 isoladas em diferentes locais da provincia de Buenos Aires, no período 2004-2009. Foi realizado POR em um total de 17 cepas isoladas de casos de HUS (9), DS (5) e de contatos assintomáticos (3) para detectar genes stx1, stx2, rfbO157, eae e ehxA e eletroforese em gel em campos pulsados (Xba/ -PFGE) para estabelecer diversidade genética e a relagäo clonal. Todos os isolamentos foram caracterizados genotipicamente como stx2/eae/ehxA. Por Xba/-PFGE foram obtidos 14 padröes diferentes, identificando um só cluster. Através da subtipificagäo molecular foi conformada a base de dados de E. coii 0145:NM/H27 isolados na regiäo, que irá permitir monitorar os perfis Xba/-PFGE visando identificar precocemente a provável ocorrència de um surto na populagäo e notificar às autoridades para implementar medidas de controle.
Assuntos
Eletroforese , Eletroforese em Gel Bidimensional , Escherichia coli , Variação Genética , Argentina , Técnicas MicrobiológicasRESUMO
Escherichia coli productora de toxina Shiga (STEC), fundamentalmente del serotipo O157:H7, está asociada a la ocurrencia de casos esporádicos y brotes de diarrea sanguinolenta (DS) y síndrome urémico hemolítico (SUH). Otros serotipos STEC como O26:H11, O103:H2, O111:NM, O113:H21 y O145:NM también pueden causar enfermedad severa. En Argentina O157:H7 es el serotipo prevalente siguiéndole en frecuencia O145:NM. El objetivo del presente trabajo fue establecer la diversidad genética y la relación clonal de cepas de E. coli O145:NM/H27 aisladas en diferentes localidades de la provincia de Buenos Aires en el período 2004-2009. A 17 cepas aisladas de casos de SUH (9), DS (5) y de contactos asintomáticos (3) se les realizó PCR para detectar genes stx1, stx2, rfbO157, eae y ehxA y electroforesis en gel en campos pulsados (Xbal-PFGE) para establecer diversidad genética y relación clonal. Todos los aislamientos fueron caracterizados genotípicamente como stx2/eae/ehxA. Por %baI-PFGE se obtuvieron 14 patrones diferentes, identificándose un único Cluster. Mediante la sub-tipificación molecular se conformó la base de datos de E. coli O145:NM/ H27 aislados en la región que permitirá monitorear los perfiles %baI-PFGE a fin de identificar tempranamente la probable ocurrencia de un brote en la población y notificar a las autoridades para aplicar acciones de control.(AU)
SHIGA TOXIN-PRODUCING Escherichia coli (STEC), especially the serotype 0157-.H7, is associated with the occurrence of sporadic cases and outbreaks of bloody diarrhea (DS) and hemolytic uremic syndrome (HUS). Other STEC serotypes such as 026:H11, 0103:H2, 0111:NM, 0113:H21, and 0145:NM can also cause severe human illness. In Argentina 0157:H7 is prevalent, followed in frequency by serotype 0145:NM. The aim of this study was to determine the genetic diversity and clonal relationship of E. coM 0145:NM/ H27 strains isolated in different locations of the Buenos Aires province, in the period 2004-2009. A total of 17 strains isolated from HUS (9), DS (5) and asymptomatic contacts (3) were characterizea by POR to detect stXj, stx2, rfbO157, eae and ehxA genes, and pulsed-field gel electrophoresis (Xbal-PFGE) to establish the genetic diversity and the clonal relationship. All isolates were characterized as stx2/eae/ehxA. By Xba\-PFGE, 14 different patterns were established, with a single cluster identified. A regional database of E. coii 0145:NM/H27 was created, which will monitor the \-PFGE profiles of the circulating strains in order to identify the probable occurrence of an outbreak in the community and report to the authorities to implement control measures.(AU)
Escherichia coii produtora de toxina Shiga (STEO), especialmente do sorotipo 0157:H7, é associado com a ocorrÞncia de casos esporádicos e surtos de diarreia sanguinolenta (DS) e a síndrome urÞmica hemolítica (HUS). 0utros sorotipos STEO como 026H11, 0103H2, 0111NM, 0113H21, e 0145NM também podem causar doengas severas. Na Argentina, é o sorotipo prevalente seguindo em frequÞncia 0145-.NM. 0 objetivo deste estudo foi determinar a diversidade genética e a relagõo clonal de cepas de E. coii 0145:NM/H27 isoladas em diferentes locais da provincia de Buenos Aires, no período 2004-2009. Foi realizado POR em um total de 17 cepas isoladas de casos de HUS (9), DS (5) e de contatos assintomáticos (3) para detectar genes stx1, stx2, rfbO157, eae e ehxA e eletroforese em gel em campos pulsados (Xba\ -PFGE) para estabelecer diversidade genética e a relagõo clonal. Todos os isolamentos foram caracterizados genotipicamente como stx2/eae/ehxA. Por Xba\-PFGE foram obtidos 14 padr÷es diferentes, identificando um só cluster. Através da subtipificagõo molecular foi conformada a base de dados de E. coii 0145:NM/H27 isolados na regiõo, que irá permitir monitorar os perfis Xba\-PFGE visando identificar precocemente a provável ocorrÞncia de um surto na populagõo e notificar Os autoridades para implementar medidas de controle.(AU)
RESUMO
STEC strains can infect extra-intestinal sites such as the human urinary tract and sometimes cause severe complications. We report two cases of urinary tract infection caused by STEC in two elderly women with comorbidities. Although both strains belonged to the O157:H7 serotype and carried genes associated with severe illness, none of the patients developed hemolytic uremic syndrome (HUS). These findings provide additional evidence for the presence of these agents in our country and in the region, and highlight the need to maintain an active surveillance system of HUS cases, placing special emphasis on the study of other sites of infection in patients with non-diarrheal HUS.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Infecções Urinárias/microbiologia , Adesinas Bacterianas/genética , Idoso , Idoso de 80 Anos ou mais , Argentina/epidemiologia , Técnicas de Tipagem Bacteriana , Cistite/microbiologia , Diarreia/complicações , Infecções por Escherichia coli/epidemiologia , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Feminino , Proteínas de Fímbrias/genética , Humanos , Falência Renal Crônica/complicações , Testes de Sensibilidade Microbiana , Toxinas Shiga/genética , Infecções Urinárias/complicaçõesRESUMO
The purposes of this study were to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) in bovine rectums and water in a beef cattle farm in Argentina, and to determine the pathogenic potential of the circulating strains. During the study, 292 rectal swabs from healthy animals and 79 environmental water samples were collected. The rectal swabs and one loop of the Moore swabs, enriched in Escherichia coli broth for 24 h at 37°C, were streaked on MacConkey agar plates and incubated overnight at 37°C. The isolates were characterized by biochemical tests and serotyped. Nonmotile STEC strains were typed for their H-specific (fliC) antigens by polymerase chain reaction (PCR). Isolates were characterized by detection of stx1, stx2, and their variants, eae, ehxA, and saa genes. Macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) was performed using the PulseNet standardized protocol. From 371 samples analyzed, 36.6% of rectal swabs and 34.2% of water samples were non-O157 STEC-positive by PCR, and 110 strains from rectal swabs, but only three from water, were isolated. The strains were grouped into 24 different serotypes, from which, O103:[H2] (n = 12), O136:H12 (n = 8), O178:H19 (n = 8), and O103:NM (n = 5) were most prevalent, representing 29.2% of the isolates. Predominant genotypes were stx1/eae/ehxA (16.8%) and stx2/saa/ehxA (15.9%). PFGE analysis revealed 56 different patterns, with 65 strains grouped in 19 clusters of 100% similarity. Two STEC O124:H19 strains isolated from rectal swabs and water with a 5-month interval harbored the stx1/stx2/saa/ehxA genotype, and showed an indistinguishable PFGE profile. By comparison, some XbaI-PFGE patterns identified in the present study were identical to the profiles of strains isolated from human, food, and animal sources included in the Argentine PulseNet database. By PCR, similar non-O157 detection rates were found in rectal swabs and water. However, the methodology for water samples needs to be improved, since only three strains from the total number of positive samples were recovered.
Assuntos
Bovinos/microbiologia , Reto/microbiologia , Rios/microbiologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Microbiologia da Água , Animais , Argentina , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Fezes/química , Fezes/microbiologia , Genótipo , Família Multigênica , Fenótipo , Reação em Cadeia da Polimerase , Sorotipagem , Toxina Shiga I/genética , Toxina Shiga I/isolamento & purificação , Toxina Shiga II/genética , Toxina Shiga II/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidadeRESUMO
En Argentina se notifican más de 500 nuevos casos de síndrome urémico hemolítico (SUH) anuales. El objetivo del trabajo fue investigar epidemiológicamente casos de SUH y contactos de los que se aislaron cepas de STEC O145:NM que pertenecían a un mismo cluster. Para detectar STEC se realizó PCR-múltiple para amplificar genes de toxinas Shiga 1 y 2, y otros marcadores de virulencia como eae y ehxA. Se subtipificó STEC por separación por electroforesis de campos pulsados (XbaI-PFGE). Entre enero y febrero de 2006, en tres casos de SUH y un contacto familiar conviviente se identificó STEC O145:NM. Genotípicamente se caracterizaron como productores de stx2, eae+ y ehxA+. Todas las cepas presentaron el mismo patrón por XbaI-PFGE (AREXSX0 1.0207) y por B/nI-PFGE (AREXSA26.0018). Estas cepas pertenecieron a un mismo cluster, diseminado en distintos barrios de la ciudad de Mar del Plata. Los datos de la investigación epidemiológica fueron incompletos para establecer un nexo entre los casos. Sin embargo, no se descarta la posibilidad de ocurrencia de un brote difuso. Se destaca la importancia que tiene el sistema de vigilancia de laboratorio en tiempo real mediante PFGE como mecanismo de alerta que sirve para afianzar los resultados con los datos de epidemiología.
More than 500 new cases of hemolytic uremic syndrome (HUS) are annually reported in Argentina. The aim of this work was to carry out epidemiological studies on cases of HUS and their household contacts that were isolated from STEC O145 strains: NM belonging to the same cluster. In order to detect STEC, Multiplex PCR was performed to amplify Shiga toxin 1 and 2 genes and other virulence markers like eae and ehxA. STEC was subtypified by means of separation by pulsed field electrophoresis (PFGE-XbaI). Between January and February 2006, STEC O145:NM strains were identified in three cases of HUS and one household contact. Genotypically, they were characterized as producing stx2, eae+ and ehxA +. All strains showed the same pattern by PFGE-XbaI (AREXSX01.0207) and BlnI-PFGE (AREXSA26.0018). These strains belonged to the same cluster, scattered in different areas of the city of Mar del Plata. Data from epidemiological research were not enough to establish a link between the cases. However, the possibility of occurrence of a diffuse outbreak. is not ruled out The importance of a laboratory surveillance system in real time by PFGE is stressed, as a warning mechanism that serves to strengthen the results with epidemiologic data.
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Lactente , Pré-Escolar , Criança , Escherichia coli , Síndrome Hemolítico-Urêmica/microbiologia , Síndrome Hemolítico-Urêmica/epidemiologia , Argentina , Toxinas Shiga , Infecções por Escherichia coliRESUMO
Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen, and ruminants are recognized as the main natural reservoir. The purposes of this study were to detect E. coli O157 in bovine feces and surface water in a beef cattle farm of Gualeguaychú, Argentina; to characterize the isolates; and to establish the clonal relatedness by pulsed-field gel electrophoresis. Between September 2005 and November 2006, 288 samples of bovine feces and 79 samples of water troughs were studied. E. coli O157 was detected by immunomagnetic separation and polymerase chain reaction as screening techniques. The rfb(O157) gene was detected in 3.8% of the 288 fecal samples and in 17.7% of the 79 water samples. The stx gene was detected in all rfb(O157)-positive fecal samples and in 5.1% of water samples. Eleven E. coli O157 strains isolated from bovine fecal samples and eight from water samples were characterized. The most frequent stx genotype identified was stx(1) and stx(2c(vh-a)). Twelve (63.2%) strains harbored fliC(H7), eae, and ehxA genes. Using pulsed-field gel electrophoresis with the enzyme XbaI, a total of eight patterns with at least 72.1% similarity were identified among the 19 strains. The patterns of 15 strains were grouped into four clusters: two of them included only bovine strains and the other two only aquatic strains. No genetic correlation was established between the bovine and water STEC strains detected. The prevalence of STEC O157:H7 established in the herd studied was higher than that previously reported for Argentine grazed cattle.