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1.
J Med Microbiol ; 69(3): 487-491, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31935188

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens that cause symptoms of severe gastrointestinal disease, including haemolytic uraemic syndrome (HUS), in humans. Currently in England, STEC serotypes other than O157:H7 are not cultured at the local hospital laboratories. The aim of this study was to evaluate the utility of CHROMagar STEC for the direct detection of STEC from faecal specimens in a diagnostic setting, compared to the current reference laboratory method using PCR targeting the Shiga-toxin gene (stx) to test multiple colonies cultured on MacConkey agar. Of the 292 consecutive faecal specimens submitted to the Gastrointestinal Bacterial Reference Unit that tested positive for stx by PCR, STEC could not be cultured on MacConkey agar or CHROMagar STEC from 87/292 (29.8 %). Of the 205 that were cultured, 106 (51.7 %) were detected on both MacConkey agar and CHROMagar STEC and 99 (48.3 %) were detected on MacConkey agar only. All 106 (100 %) isolates that grew on CHROMagar STEC had the ter gene cassette, known to be associated with resistance to tellurite, compared to 13/99 (13.1 %) that were not detected on CHROMagar STEC. CHROMagar STEC supported the growth of 36/40 (90 %) isolates harbouring stx2a or stx2d, the subtypes most frequently associated with progression to HUS. Of the 92 isolates harbouring eae, an important STEC virulence marker, 77 (83.7 %) grew on CHROMagar STEC. CHROMagar STEC is a useful selective media for the rapid, near-patient detection of STEC that have the potential to cause HUS.


Asunto(s)
Infecciones por Escherichia coli/diagnóstico , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Agar , Compuestos Cromogénicos , Infecciones por Escherichia coli/microbiología , Heces/microbiología , Humanos , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética
2.
Int J Food Microbiol ; 319: 108499, 2020 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-31954209

RESUMEN

Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of E.coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx+, eae+ genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx+, eae+ genotype.


Asunto(s)
Adhesinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Carne Roja/microbiología , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Animales , Bovinos , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Virulencia
3.
Food Microbiol ; 84: 103273, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31421766

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) are important pathogens transmitted by food that may cause severe illness in human beings. Thus, systems for STEC detection in food should have increasingly higher sensitivity and specificity. Here we compared six commercial systems for non-O157 STEC detection in meat and vegetables and determined their sensitivity, specificity and repeatability. A total of 46 samples (meat n = 23; chard n = 23) were experimentally contaminated with strains O26:H11, O45:H-, O103:H2, O111:NM, O121:H19 and O145:NM isolated in Argentina. Strain detection was confirmed by isolation according to ISO 13136:2012. Detection of the stx and eae genes in meat samples was highly satisfactory with all commercial kits, but only five had 100% sensitivity and specificity in chard. Of four kits evaluated for serogroup detection, three had 100% sensitivity and specificity, and one had 93.7% sensitivity and 100% specificity. All kits were adequate to analyze meat but not vegetable samples, and were not therefore validated for the latter matrix. The challenge for microbiology laboratories is to identify the advantages and disadvantages of the available kits for STEC detection in food based on a clear knowledge of the particular needs of each laboratory.


Asunto(s)
Contaminación de Alimentos/análisis , Microbiología de Alimentos/métodos , Carne/microbiología , Serotipificación/normas , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Verduras/microbiología , Adhesinas Bacterianas/genética , Microbiología de Alimentos/normas , Juego de Reactivos para Diagnóstico/normas , Sensibilidad y Especificidad , Serogrupo , Serotipificación/métodos , Toxina Shiga/genética
4.
APMIS ; 127(10): 671-680, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31344276

RESUMEN

Regardless of the communal impact of Shiga toxins, till today neither a specific treatment nor licensed vaccine is available. Lactococcus lactis (L. lactis), generally regarded as safe organism, is well known to provide a valuable approach regarding the oral delivery of vaccines. This study was undertaken to evaluate the protective efficacy of Stx2a1 expressed in nisin-inducible L. lactis, against Shiga toxins (Stx1, Stx2) in mouse model. Oral immunization of BALB/c mice with LL-Stx2a1 elicited significant serum antibody titer with elevated fecal and serum IgA, along with minimized intestinal and kidney damage resulting in survival of immunized animals at 84% and 100% when challenged with 10 × LD50 of Escherichia coli O157 and Shigella dysenteriae toxins, respectively. HeLa cells incubated with immune sera and toxin mixture revealed high neutralizing capacity with 90% cell survivability against both the toxins. Mice immunized passively with both toxins and antibody mixture survived the observation period of 15 days, and the controls administered with sham sera and toxins were succumbed to death within 3 days. Our results revealed protective efficacy and toxin neutralization ability of LL-Stx2a1, proposing it as an oral vaccine candidate against Shiga toxicity mediated by E. coli O157 and S. dysenteriae.


Asunto(s)
Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Escherichia coli O157/inmunología , Envenenamiento/prevención & control , Toxina Shiga/inmunología , Toxina Shiga/toxicidad , Shigella dysenteriae/inmunología , Administración Oral , Animales , Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/sangre , Antitoxinas/administración & dosificación , Antitoxinas/sangre , Vacunas Bacterianas/genética , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Portadores de Fármacos/administración & dosificación , Escherichia coli O157/genética , Vectores Genéticos/administración & dosificación , Células HeLa , Humanos , Lactococcus lactis/genética , Ratones , Ratones Endogámicos BALB C , Toxina Shiga/genética , Shigella dysenteriae/genética , Análisis de Supervivencia , Resultado del Tratamiento , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología
5.
Indian J Med Res ; 149(3): 412-417, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-31249208

RESUMEN

Background & objectives: : Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: : Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: : The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 µg/cassette, respectively. Interpretation & conclusions: : The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.


Asunto(s)
Disentería Bacilar/diagnóstico , Síndrome Hemolítico-Urémico/diagnóstico , Toxina Shiga/aislamiento & purificación , Shigella dysenteriae/genética , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Artritis Reactiva/diagnóstico , Artritis Reactiva/genética , Artritis Reactiva/microbiología , Disentería Bacilar/genética , Disentería Bacilar/microbiología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Síndrome Hemolítico-Urémico/genética , Síndrome Hemolítico-Urémico/microbiología , Humanos , Ratones , Toxina Shiga/genética , Shigella dysenteriae/patogenicidad
6.
Microb Genom ; 5(5)2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31107203

RESUMEN

Shiga-toxin-producing Escherichia coli (STEC) infection is an important global cause of foodborne disease. To date however, genomics-based studies of STEC have been predominately focused upon STEC collected in the Northern Hemisphere. Here, we demonstrate the population structure of 485 STEC isolates in Australia, and show that several clonal groups (CGs) common to Australia were infrequently detected in a representative selection of contemporary STEC genomes from around the globe. Further, phylogenetic analysis demonstrated that lineage II of the global O157:H7 STEC was most prevalent in Australia, and was characterized by a frameshift mutation in flgF, resulting in the H-non-motile phenotype. Strong concordance between in silico and phenotypic serotyping was observed, along with concordance between in silico and conventional detection of stx genes. These data represent the most comprehensive STEC analysis from the Southern Hemisphere, and provide a framework for future national genomics-based surveillance of STEC in Australia.


Asunto(s)
Infecciones por Escherichia coli/microbiología , Genoma Bacteriano , Epidemiología Molecular , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Australia/epidemiología , Simulación por Computador , Farmacorresistencia Bacteriana/genética , Infecciones por Escherichia coli/epidemiología , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Evolución Molecular , Humanos , Tipificación de Secuencias Multilocus , Mutación , Fenotipo , Filogenia , Salud Pública , Serotipificación , Escherichia coli Shiga-Toxigénica/clasificación , Secuenciación Completa del Genoma
7.
PLoS One ; 14(4): e0214620, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30934002

RESUMEN

Illnesses caused by Shiga toxin-producing Escherichia coli (STECs) can be life threatening, such as hemolytic uremic syndrome (HUS). The STECs most frequently identified by USDA's Microbiological Data Program (MDP) carried toxin gene subtypes stx1a and/or stx2a. Here we described the genome sequences of 331 STECs isolated from foods regulated by the FDA 2010-2017, and determined their genomic identity, serotype, sequence type, virulence potential, and prevalence of antimicrobial resistance. Isolates were selected from the MDP archive, routine food testing by FDA field labs (ORA), and food testing by a contract company. Only 276 (83%) strains were confirmed as STECs by in silico analysis. Foods from which STECs were recovered included cilantro (6%), spinach (25%), lettuce (11%), and flour (9%). Phylogenetic analysis using core genome MLST revealed these STEC genomes were highly variable, with some clustering associated with ST types and serotypes. We detected 95 different sequence types (ST); several ST were previously associated with HUS: ST21 and ST29 (O26:H11), ST11 (O157:H7), ST33 (O91:H14), ST17 (O103:H2), and ST16 (O111:H-). in silico virulome analyses showed ~ 51% of these strains were potentially pathogenic [besides stx gene they also carried eae (25%) or 26% saa (26%)]. Virulence gene prevalence was also determined: stx1 only (19%); stx2 only (66%); and stx1/sxt2 (15%). Our data form a new WGS dataset that can be used to support food safety investigations and monitor the recurrence/emergence of E. coli in foods.


Asunto(s)
Infecciones por Escherichia coli/microbiología , Microbiología de Alimentos , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/genética , Virulencia/genética , Técnicas de Tipificación Bacteriana , Infecciones por Escherichia coli/epidemiología , Alimentos/clasificación , Contaminación de Alimentos/análisis , Contaminación de Alimentos/legislación & jurisprudencia , Contaminación de Alimentos/estadística & datos numéricos , Microbiología de Alimentos/estadística & datos numéricos , Inocuidad de los Alimentos , Regulación Gubernamental , Análisis de Peligros y Puntos de Control Críticos , Síndrome Hemolítico-Urémico/microbiología , Humanos , Legislación Alimentaria , Tipificación de Secuencias Multilocus , Filogenia , Toxina Shiga/clasificación , Transcriptoma , Estados Unidos/epidemiología , United States Food and Drug Administration/legislación & jurisprudencia
8.
Comp Immunol Microbiol Infect Dis ; 63: 117-126, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30961806

RESUMEN

The aims of this study were to investigate the prevalence, antibiotic resistance, presence of class 1 and 2 integrons, Extended Spectrum ß-Lactamases (ESBL) genes, phylogenetic group and epidemiological relationships of EPEC, ETEC and EHEC pathotypes isolated from patients with diarrhea and farm animals in south east region of Iran. A total of 671 diarrheagenic E. coli (DEC) were collected from stool samples of 395 patients with diarrhea and 276 farm cattles and goats. Presence of EPEC, ETEC and EHEC were identified using multiplex-PCR employing primers targeted the shiga toxin (stx), intimin (eae), bundle forming pili (bfp), and enterotoxins (lt and st) genes. The highest proportion of the patients (64%) were children under age 1-15 year (p ≤ 0.05). Among the isolates, atypical EPEC was detected in 26 patients and 14 animal stool samples, while typical EPEC was found in 2 cattles. ETEC isolates were detected in stools of 13 patients and 4 EHEC was identified in 3 goats and one cattle. The isolates were checked for susceptibility to 14 antibiotics. 50% (n = 13) of EPEC and 61.5% (n =8) of ETEC showed multi-drug resistance (MDR) profiles and one EPEC was found to be extensive drug resistant (XDR). In contrast, EHEC isolates were susceptible to the majority of antimicrobial agents. The MDR isolates were positive for blaTEM and blaCTX-M ESBL genes and carried class 1 integrons. Further study on the biofilm formation indicated that, 3 out of 4 EHEC isolates showed strong biofilm, while other pathotypes had either moderate, weak or no biofilm activity. Majority of EPEC isolates were belonged to phylogenetic group B1, all except one ETEC were classified as phylogenetic group A and two EHEC were belonged to phylogroup D, respectively. A multilocus variable tandem repeat analysis (MLVA) exhibited 22 distinct patterns. In conclusion, MLVA data showed high clonal diversity. Presence of EHEC in animal origins pose public health concern in this region.


Asunto(s)
Diarrea/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli Enterohemorrágica/aislamiento & purificación , Escherichia coli Enteropatógena/aislamiento & purificación , Escherichia coli Enterotoxigénica/aislamiento & purificación , Infecciones por Escherichia coli/veterinaria , Adhesinas Bacterianas/genética , Adolescente , Animales , Animales Domésticos/microbiología , Antibacterianos/uso terapéutico , Bovinos , Enfermedades de los Bovinos , Niño , Preescolar , Escherichia coli Enterohemorrágica/efectos de los fármacos , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enteropatógena/efectos de los fármacos , Escherichia coli Enteropatógena/genética , Escherichia coli Enterotoxigénica/efectos de los fármacos , Escherichia coli Enterotoxigénica/genética , Enterotoxinas/genética , Infecciones por Escherichia coli/tratamiento farmacológico , Proteínas de Escherichia coli/genética , Fimbrias Bacterianas/genética , Enfermedades de las Cabras , Cabras , Humanos , Lactante , Integrones/genética , Irán , Pruebas de Sensibilidad Microbiana , Tipificación Molecular , Toxina Shiga/genética , beta-Lactamasas/genética
9.
mBio ; 10(2)2019 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-30992350

RESUMEN

Pulmonary exacerbations are the leading cause of death in cystic fibrosis (CF) patients. To track microbial dynamics during acute exacerbations, a CF rapid response (CFRR) strategy was developed. The CFRR relies on viromics, metagenomics, metatranscriptomics, and metabolomics data to rapidly monitor active members of the viral and microbial community during acute CF exacerbations. To highlight CFRR, a case study of a CF patient is presented, in which an abrupt decline in lung function characterized a fatal exacerbation. The microbial community in the patient's lungs was closely monitored through the multi-omics strategy, which led to the identification of pathogenic shigatoxigenic Escherichia coli (STEC) expressing Shiga toxin. This case study illustrates the potential for the CFRR to deconstruct complicated disease dynamics and provide clinicians with alternative treatments to improve the outcomes of pulmonary exacerbations and expand the life spans of individuals with CF.IMPORTANCE Proper management of polymicrobial infections in patients with cystic fibrosis (CF) has extended their life span. Information about the composition and dynamics of each patient's microbial community aids in the selection of appropriate treatment of pulmonary exacerbations. We propose the cystic fibrosis rapid response (CFRR) as a fast approach to determine viral and microbial community composition and activity during CF pulmonary exacerbations. The CFRR potential is illustrated with a case study in which a cystic fibrosis fatal exacerbation was characterized by the presence of shigatoxigenic Escherichia coli The incorporation of the CFRR within the CF clinic could increase the life span and quality of life of CF patients.


Asunto(s)
Fibrosis Quística/complicaciones , Progresión de la Enfermedad , Infecciones por Escherichia coli/diagnóstico , Genómica , Pulmón/microbiología , Metabolómica , Adulto , Estudios de Casos y Controles , Coinfección/complicaciones , Fibrosis Quística/microbiología , Manejo de la Enfermedad , Resultado Fatal , Perfilación de la Expresión Génica , Humanos , Pulmón/fisiopatología , Masculino , Metaboloma , Metagenoma , Microbiota , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/patogenicidad
10.
BMC Genomics ; 20(1): 271, 2019 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-30953471

RESUMEN

BACKGROUND: Wild birds, in particular pigeons are considered a natural reservoir for stx2f-carrying E. coli. An extensive comparison of isolates from pigeons and humans from the same region is lacking, which hampers justifiable conclusions on the epidemiology of these pathogens. Over two hundred human and pigeon stx2f-carrying E. coli isolates predominantly from the Netherlands were analysed by whole genome sequencing and comparative genomic analysis including in silico MLST, serotyping, virulence genes typing and whole genome MLST (wgMLST). RESULTS: Serotypes and sequence types of stx2f-carrying E. coli showed a strong non-random distribution among the human and pigeon isolates with O63:H6/ST583, O113:H6/ST121 and O125:H6/ST583 overrepresented among the human isolates and not found among pigeons. Pigeon isolates were characterized by an overrepresentation of O4:H2/ST20 and O45:H2/ST20. Nearly all isolates harboured the locus of enterocyte effacement (LEE) but different eae and tir subtypes were non-randomly distributed among human and pigeon isolates. Phylogenetic core genome comparison demonstrated that the pigeon isolates and clinical isolates largely occurred in separated clusters. In addition, serotypes/STs exclusively found among humans generally were characterized by high level of clonality, smaller genome sizes and lack of several non-LEE-encoded virulence genes. A bundle-forming pilus operon, including bfpA, indicative for typical enteropathogenic E. coli (tEPEC) was demonstrated in 72.0% of the stx2f-carrying serotypes but with distinct operon types between the main pigeon and human isolate clusters. CONCLUSIONS: Comparative genomics revealed that isolates from mild human disease are dominated by serotypes not encountered in the pigeon reservoir. It is therefore unlikely that zoonotic transmission from this reservoir plays an important role in the contribution to the majority of human disease associated with stx2f-producing E. coli in the Netherlands. Unexpectedly, this study identified the common occurrence of STEC2f/tEPEC hybrid pathotype in various serotypes and STs. Further research should focus on the possible role of human-to-human transmission of Stx2f-producing E. coli.


Asunto(s)
Enfermedades de las Aves/epidemiología , Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/epidemiología , Proteínas de Escherichia coli/metabolismo , Genómica/métodos , Toxina Shiga/metabolismo , Factores de Virulencia/metabolismo , Animales , Columbidae , Escherichia coli Enteropatógena/clasificación , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Humanos , Filogenia , Toxina Shiga/genética , Factores de Virulencia/genética
11.
Food Microbiol ; 82: 474-481, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31027808

RESUMEN

A 2016/2017 outbreak of Shiga toxin-producing Escherichia coli (STEC) O121 in Canada, was linked to wheat flour, milled at a single facility on three consecutive days in October 2016. Most Probable Number (MPN) estimates of the concentration of STEC O121 in the recalled flour were made using the results of qualitative testing conducted during the outbreak investigation and from analysis of 5 × 2.5 g, 5 × 25 g and 5 × 100 g analytical units of the recalled flour. The STEC O121 levels were estimated at 0.15 to 0.43 MPN/100 g, with no significant difference between production days and the two MPN estimates. The microbiota of the recalled flour, and eight retail flour samples, was enumerated by aerobic colony count, MacConkey agar and E. coli/Coliform petrifilm. The composition of the microbiota to a genus level was determined by identifying individual colonies with a Bruker Biotyper. All retail flour samples were negative for STEC in 5 × 100 g analytical units. There was no evidence of higher levels of organisms associated with fecal contamination in the recalled flour. The low levels of STEC O121 in the recalled flour indicate that a robust sampling plan, with multiple analytical units for a total of several hundred grams, may be required to reliably detect STEC in flour at levels observed in this outbreak.


Asunto(s)
Brotes de Enfermedades , Infecciones por Escherichia coli/epidemiología , Harina/microbiología , Microbiología de Alimentos/métodos , Enfermedades Transmitidas por los Alimentos/epidemiología , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Triticum , Técnicas Bacteriológicas , Canadá/epidemiología , Infecciones por Escherichia coli/microbiología , Harina/análisis , Enfermedades Transmitidas por los Alimentos/microbiología , Microbiota , Toxina Shiga/genética , Toxina Shiga/metabolismo , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/metabolismo
12.
Lett Appl Microbiol ; 68(6): 546-552, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30801745

RESUMEN

Domestic ruminants are regarded as the major reservoir of Shiga toxin-producing Escherichia coli (STEC) closely related to human infection. A total of 363 ovine carcasses were swabbed in an Algiers city slaughterhouse for research on STEC. First of all, screening of the STECs was carried out by a multiplex PCR searching for the genes coding for the virulence factors stx1 , stx2 and eae. This step was followed by STEC isolation and serotyping. The presence of stx+ /stx+ eae+ genes was shown in 116 sheep carcasses (31·95%). From the 116 positive samples, 20 bacterial strains (17·24%) were isolated. Nineteen strains belonged to the species E. coli (STEC), and 1 belonged to Citrobacter braakii (eae+ stx1 + ). During this study, the presence of potentially pathogenic STEC for humans on the surface of sheep carcasses was confirmed. Corrective measures should be considered at the slaughterhouse level to avoid outbreaks of STEC in Algeria. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR screening revealed the significant presence of the genetic markers of Shiga toxin-producing Escherichia coli (STEC) (stx+ /stx+ eae+ ) on the surfaces of sheep carcasses. Citrobacter braakii (stx1 + eae+ ) was isolated for the first time in this study. The risk of foodborne diseases due to STEC must be taken into account in Algeria. To prevent the emergence of epidemic outbreaks among children and older by people, preventive measures should be taken.


Asunto(s)
Infecciones por Escherichia coli/veterinaria , Ovinos/microbiología , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Factores de Virulencia/genética , Mataderos , Argelia , Animales , Niño , Brotes de Enfermedades/prevención & control , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/prevención & control , Humanos , Reacción en Cadena de la Polimerasa , Serotipificación , Toxina Shiga/metabolismo
13.
Appl Environ Microbiol ; 85(7)2019 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-30709824

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni are notable health hazards associated with the consumption of raw milk. These bacteria may colonize the intestines of asymptomatic cattle and enter bulk tank milk via fecal contamination during milking. We studied the frequency of STEC O157:H7 and C. jejuni contamination in tank milk (n = 785) and the in-line milk filters of milking machines (n = 631) versus the frequency of isolation from cattle feces (n = 257) on three Finnish dairy farms for 1 year. Despite simultaneous isolation of STEC O157:H7 (17%) or C. jejuni (53%) from cattle, these bacteria were rarely isolated from milk filters (2% or <1%, respectively) and milk (0%). As revealed by phylogenomics, one STEC O157:H7 strain at a time was detected on each farm and persisted for ≤12 months despite rigorous hygienic measures. C. jejuni strains of a generalist sequence type (ST-883 and ST-1080) persisted in the herds for ≥11 months, and several other C. jejuni types were detected sporadically. The stx gene carried by STEC was detected more frequently from milk filters (37%) than from milk (7%), suggesting that milk filters are more suitable sampling targets for monitoring than milk. A questionnaire of on-farm practices suggested lower stx contamination of milk when major cleansing in the barn, culling, or pasturing of dairy cows was applied, while a higher average outdoor temperature was associated with higher stx contamination. Because pathogen contamination occurred despite good hygiene and because pathogen detection from milk and milk filters proved challenging, we recommend heat treatment for raw milk before consumption.IMPORTANCE The increased popularity of raw milk consumption has created demand for relaxing legislation, despite the risk of contamination by pathogenic bacteria, notably STEC and C. jejuni However, the epidemiology of these milk-borne pathogens on the herd level is still poorly understood, and data are lacking on the frequency of milk contamination on farms with cattle shedding these bacteria in their feces. This study suggests (i) that STEC contamination in milk can be reduced, but not prevented, by on-farm hygienic measures while fecal shedding is observable, (ii) that milk filters are more suitable sampling targets for monitoring than milk although pathogen detection from both sample matrices may be challenging, and (iii) that STEC and C. jejuni genotypes may persist in cattle herds for several months. The results can be utilized in developing and targeting pathogen monitoring and risk management on the farm level and contributed to the revision of Finnish legislation in 2017.


Asunto(s)
Campylobacter jejuni/aislamiento & purificación , Heces/microbiología , Microbiología de Alimentos , Leche/microbiología , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Animales , Campylobacter jejuni/clasificación , Campylobacter jejuni/genética , Bovinos , Industria Lechera/instrumentación , Industria Lechera/métodos , Escherichia coli O157/genética , Escherichia coli O157/aislamiento & purificación , Granjas , Femenino , Finlandia , Genómica , Genotipo , Estudios Longitudinales , Tipificación de Secuencias Multilocus , Filogenia , Factores de Riesgo , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/genética , Secuenciación Completa del Genoma
14.
Lett Appl Microbiol ; 68(2): 112-119, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30411807

RESUMEN

Shiga toxin-producing Escherichia coli strains (STEC) are food-borne pathogens. While E. coli O157:H7 is commonly associated with cattle, less is known about the prevalence of non-O157 STEC serogroups in bovines. This study evaluated the prevalence and virulence status of O157:H7 and six E. coli O-serogroups (O26, O103, O45, O145, O121, O111) in New Zealand dairy farms using molecular as well as culture-based methods. Fresh farm dairy effluent (FDE) (n = 36) and composite calf faeces (n = 12) were collected over three samplings from 12 dairy farms. All seven target serogroups were detected through molecular techniques. Of the 202 isolates which were serologically confirmed following traditional culturing and immunomagnetic separation (IMS), O103, O26, O45 and O121 were the most common serogroups, being found in 81, 47, 42 and 32% of the FDE and in 17, 33, 25 and 9% of the calf faeces respectively. The majority (157/202) of the isolates were negative for stx and eae virulence genes. The prevalence of the seven target STEC was low, and only nine O26 isolates (4%) were recovered from four of the farms. The study has highlighted the need for improving the isolation of Top 7 STEC from the stx-negative populations present in fresh dairy effluent and calf faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens that can cause severe illness in humans. Cattle are asymptomatic reservoirs for STEC, and transmission to humans can be by consumption of food products or water contaminated with cattle faeces. Our study investigated the prevalence of O157:H7 and six E. coli serogroups of STEC (O26, O103, O45, O145, O121, O111) over time in the dairy reservoir and increases the knowledge and understanding of these pathogens on pasture-based farms. Such information is required to develop risk-assessment models aiming at limiting transmission of these STEC to human.


Asunto(s)
Adhesinas Bacterianas/genética , Infecciones por Escherichia coli/veterinaria , Escherichia coli O157/aislamiento & purificación , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/genética , Toxina Shiga/genética , Animales , Bovinos , Industria Lechera , Escherichia coli O157/clasificación , Granjas , Heces/microbiología , Enfermedades Transmitidas por los Alimentos/microbiología , Humanos , Separación Inmunomagnética , Nueva Zelanda , Prevalencia , Serogrupo , Virulencia
15.
Emerg Microbes Infect ; 7(1): 203, 2018 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-30514915

RESUMEN

A large German outbreak in 2011 was caused by a locus of enterocyte effacement (LEE)-negative enterohemorrhagic E. coli (EHEC) strain of the serotype O104:H4. This strain harbors markers that are characteristic of both EHEC and enteroaggregative E. coli (EAEC), including aggregative adhesion fimbriae (AAF) genes. Such rare EHEC/EAEC hybrids are highly pathogenic due to their possession of a combination of genes promoting severe toxicity and aggregative adhesion. We previously identified novel EHEC/EAEC hybrids and observed that one strain exhibited aggregative adherence but had no AAF genes. In this study, a genome sequence analysis showed that this strain belongs to the genoserotype O23:H8, MLST ST26, and harbors a 5.2 Mb chromosome and three plasmids. One plasmid carries some EAEC marker genes, such as aatA and genes with limited protein homology (11-61%) to those encoding the bundle-forming pilus (BFP) of enteropathogenic E. coli. Due to significant protein homology distance to known pili, we designated these as aggregate-forming pili (AFP)-encoding genes and the respective plasmid as pAFP. The afp operon was arranged similarly to the operon of BFP genes but contained an additional gene, afpA2, which is homologous to afpA. The deletion of the afp operon, afpA, or a nearby gene (afpR) encoding an AraC-like regulator, but not afpA2, led to a loss of pilin production, piliation, bacterial autoaggregation, and importantly, a >80% reduction in adhesion and cytotoxicity toward epithelial cells. Gene sets similar to the afp operon were identified in a variety of aatA-positive but AAF-negative intestinal pathogenic E. coli. In summary, we characterized widely distributed and novel fimbriae that are essential for aggregative adherence and cytotoxicity in a LEE-negative Shiga-toxigenic hybrid.


Asunto(s)
Adhesión Bacteriana , Escherichia coli Enterohemorrágica/patogenicidad , Proteínas de Escherichia coli/genética , Fimbrias Bacterianas/genética , Toxina Shiga/genética , Técnicas de Tipificación Bacteriana , Escherichia coli Enterohemorrágica/metabolismo , Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Fimbrias Bacterianas/metabolismo , Genoma Bacteriano , Humanos , Tipificación de Secuencias Multilocus , Análisis de Secuencia de ADN , Serogrupo , Virulencia
16.
Microb Pathog ; 125: 463-467, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30300666

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) O91 has ranked in the top five of the non-O157 serogroups most frequently associated with human cases. In order to gain insight into the genetic diversity of O91 Latin American STEC strains, we analyzed their virulence properties and carried out a subtyping assay. A panel of 21 virulence genetic markers associated with human and animal infections was evaluated and the relatedness among strains was determined by a multiple-locus variable-number tandem repeats analysis (MLVA) comprising 9 VNTR loci. Twenty-two STEC O91 isolated from cattle and meat food and belonging to 5 serotypes (O91:H21, O91:H8, O91:H14, O91:H28, O91:H40) were studied. Eight virulence profiles were obtained for the O91 STEC strains: 4 for O91:H21 plus one for O91:H8, O91:H14, O91:H28 and O91:H40. All strains contained ehxA and lpfA0113 genes and only both stx1-positive strains lacked saa, which encodes the STEC autoagglutinating adhesin. Other genes involved in adhesion were detected: ehaA (91%), elfA and espP (86%), ecpA (82%) and, hcpA (77%). The gene encoding the cytolethal distending toxin type-V (CDT-V) was found only in O91:H8 and O91:H21, being present in the majority (89%) of strains of this last serotype. MLVA typing divided the total number of strains into 12 genotypes, and 9 of them were unique to a single strain. No association was observed between the virulence profiles and the source of the strains. Although they lack the eae gene, most of the strains have the genetic potential to adhere to host cells through other structures and possess cdt-V, which has been found in STEC strains involved in serious diseases. The MLVA showed clonal relatedness among strains isolated from cattle belonged to a same dairy farm and suggested that the same clone remains circulating throughout the year and, on the other hand, the need to increase the number of VNTR loci which could allow a higher discrimination among O91:H21 isolates.


Asunto(s)
Variación Genética , Productos Avícolas/microbiología , Carne Roja/microbiología , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Factores de Virulencia/genética , Animales , Bovinos , Genotipo , Repeticiones de Minisatélite , Tipificación Molecular , Reacción en Cadena de la Polimerasa , Aves de Corral , Serogrupo , Escherichia coli Shiga-Toxigénica/genética
17.
BMC Microbiol ; 18(1): 98, 2018 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-30170562

RESUMEN

BACKGROUND: In many Asian countries including Bangladesh E. coli O157 are prevalent in animal reservoirs and in the food chain, but the incidence of human infection due to E. coli O157 is rare. One of the reasons could be inability of the organism from animal origin to produce sufficient amount of Shiga toxin (Stx), which is the main virulence factor associated with the severe sequelae of infection. This study aimed to fill out this knowledge gap by investigating the toxigenic properties and characteristics of stx phage of E. coli O157 isolated from animal sources in Bangladesh. RESULTS: We analysed 47 stx2 positive E. coli O157 of food/animal origin for stx2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q933 and Q21) and stx2 upstream region. Of the 47 isolates, 46 were positive for both stx2a and stx2d while the remaining isolate was positive for stx2d only. Reverse Passive Latex Agglutination assay (RPLA) showed that 42/47 isolates produced little or no toxin, while 5 isolates produced a high titre of toxin (64 to 128). 39/47 isolates were positive for the Toxin Non-Producing (TNP) specific regions in the stx2 promoter. Additionally, all isolates were negative for antiterminator Q933while a majority of isolates were positive for Q21 gene suggesting the presence of defective stx phage. Of the yehV and wrbA phage insertion sites, yehV was found occupied in 11 isolates while wrbA site was intact in all the isolates. None of the isolates was positive for the virulence gene, cdt but all were positive for hlyA, katP, etpD and eae genes. Isolates that produced high titre Stx (n = 5) produced complete phage particles capable of infecting multiple bacterial hosts. One of these phages was shown to produce stable lysogens in host strains rendering the Stx2 producing ability. CONCLUSION: Despite low frequency in the tested isolates, E. coli O157 isolates in Bangladesh carry inducible stx phages and have the capacity to produce Stx2, indicating a potential risk of E. coli O157 infection in humans.


Asunto(s)
Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Escherichia coli O157/genética , Escherichia coli O157/virología , Microbiología de Alimentos , Toxina Shiga/genética , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/genética , Bangladesh , ADN Bacteriano/genética , ADN Viral , Países en Desarrollo , Escherichia coli O157/aislamiento & purificación , Proteínas de Escherichia coli/genética , Heces/microbiología , Variación Genética , Lisogenia , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Toxina Shiga II/genética , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Virulencia/genética
18.
Eur J Clin Microbiol Infect Dis ; 37(12): 2361-2370, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30267169

RESUMEN

The dynamics related to the loss of stx genes from Shiga toxin-producing Escherichia coli remain unclear. Current diagnostic procedures have shortcomings in the detection and identification of STEC. This is partly owing to the fact that stx genes may be lost during an infection or in the laboratory. The aim of the present study was to provide new insight into in vivo and in vitro stx loss in order to improve diagnostic procedures. Results from the study support the theory that loss of stx is a strain-related phenomenon and not induced by patient factors. It was observed that one strain could lose stx both in vivo and in vitro. Whole genome comparison of stx-positive and stx-negative isolates from the same patient revealed that different genomic rearrangements, such as complete or partial loss of the parent prophage, may be factors in the loss of stx. Of diagnostic interest, it was shown that patients can be co-infected with different E. coli pathotypes. Therefore, identification of eae-positive, but stx-negative isolates should not be interpreted as "Shiga toxin-lost" E. coli without further testing. Growth and recovery of STEC were supported by different selective agar media for different strains, arguing for inclusion of several media in STEC diagnostics.


Asunto(s)
Infecciones por Escherichia coli/diagnóstico , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Adulto , Anciano , Niño , Preescolar , Medios de Cultivo/química , Diarrea/microbiología , Heces/microbiología , Femenino , Humanos , Lactante , Masculino , Técnicas Microbiológicas , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Factores de Virulencia , Adulto Joven
19.
Biosensors (Basel) ; 8(3)2018 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-30110986

RESUMEN

The present study was aimed to develop "fluorine doped" tin oxide glass electrode with a MoSe2 nano-urchin based electrochemical biosensor for detection of Escherichia. coli Shiga toxin DNA. The study comprises two conductive electrodes, and the working electrodes were drop deposited using MoSe2 nano-urchin, and DNA sequences specific to Shiga toxin Escherichia. coli. Morphological characterizations were performed using Fourier transforms infrared spectrophotometer; X-ray diffraction technique and scanning electron microscopy. All measurements were done using methylene blue as an electrochemical indicator. The proposed electrochemical geno-sensor showed good linear detection range of 1 fM⁻100 µM with a low detection limit of 1 fM where the current response increased linearly with Escherichia. coli Shiga toxin dsDNA concentration with R2 = 0.99. Additionally, the real sample was spiked with the dsDNA that shows insignificant interference. The results revealed that the developed sensing platform significantly improved the sensitivity and can provide a promising platform for effective detection of biomolecules using minute samples due to its stability and sensitivity.


Asunto(s)
Técnicas Biosensibles/métodos , ADN Bacteriano/análisis , Técnicas Electroquímicas/métodos , Molibdeno/química , Nanoestructuras/química , Compuestos de Selenio/química , Toxina Shiga/genética , Técnicas Biosensibles/instrumentación , Técnicas Electroquímicas/instrumentación , Electrodos , Escherichia coli/química , Escherichia coli/genética , Azul de Metileno/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Compuestos de Estaño/química
20.
Foodborne Pathog Dis ; 15(10): 653-659, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30036077

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) is a group of emerging pathogens that can cause human diseases, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Monitoring slaughtering stages and checking contamination points are crucial for the production of safe food. In this context, the aim of this study was to verify contamination by STEC strains, to determine the contamination points and evaluate the resistance profile to 12 antimicrobials used in both veterinary and human medicine. A total of 80 samples were obtained from eight collection points (pen floor, rectum, hide, carcass swabs and esophagus, diaphragm, masseter, and retail beef tissue samples). The isolates were collected by dilution plating on MacConkey agar with sorbitol, cefixime, and tellurite and analyzed by multiplex polymerase chain reaction for virulence genes. Serotyping of non-O157 was performed, and testing for 12 antibiotics by disk diffusion was carried out. A total of 18 STEC strains were isolated, presenting different virulence profiles. Contamination by STEC was observed in the rectum (5/18), carcass surface (5/18), hide (3/18), diaphragm (2/18), retail beef (2/18), and masseter muscle (1/18). Pen floor swabs and esophagus tissues showed no STEC contamination. Moreover, three strains were identified as O26 and three as O113:H21 strains, which have been linked to HUS and HC outbreak cases in Brazil. All STEC isolates were susceptible to all evaluated antimicrobials, except streptomycin. The presence of STEC strains is a direct risk to the consumer, especially when isolated from retail beef, and contamination can occur during different slaughter stages. However, antimicrobial resistance profiles did not identify multidrug-resistant strains, limiting potential antimicrobial resistance transmission to other pathogens.


Asunto(s)
Contaminación de Alimentos/análisis , Carne Roja/microbiología , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Mataderos , Animales , Brasil , Bovinos , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa Multiplex , Serotipificación , Factores de Virulencia/genética
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