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1.
Methods Mol Biol ; 2291: 1-17, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704747

RESUMO

Shiga toxin-producing Escherichia coli (STEC) are human pathogens causing severe diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The prompt diagnosis of STEC infection is of primary importance to drive the most appropriate patient's management procedures. The methods to diagnose STEC infections include both direct isolation of the STEC from stool samples and the identification of indirect evidences based on molecular, phenotypic, and serological applications. Here, the procedures in use at the Italian Reference Laboratory for E. coli infections are described.


Assuntos
Colite , Hemorragia Gastrointestinal , Síndrome Hemolítico-Urêmica , Escherichia coli Shiga Toxigênica , Colite/diagnóstico , Colite/microbiologia , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/microbiologia , Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação
2.
Methods Mol Biol ; 2291: 381-397, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704765

RESUMO

Shiga toxin-producing E. coli (STEC) is a common foodborne pathogen in developed countries. STEC generates "attaching and effacing" (AE) lesions on colonic epithelium, characterized by effacement of microvilli and the formation of actin "pedestals" beneath intimately attached bacteria. In addition, STEC are lysogenized with a phage that, upon induction, can produce potent Shiga toxins (Stx), potentially leading to both hemorrhagic colitis and hemolytic uremic syndrome. Investigation of the pathogenesis of this disease has been challenging because STEC does not readily colonize conventional mice.Citrobacter rodentium (CR) is a related mouse pathogen that also generates AE lesions. Whereas CR does not produce Stx, a murine model for STEC utilizes CR lysogenized with an E. coli-derived Stx phage, generating CR(Φstx), which both colonizes conventional mice and readily gives rise to systemic disease. We present here key methods for the use of CR(Φstx) infection as a highly predictable murine model for infection and disease by STEC. Importantly, we detail CR(Φstx) inoculation by feeding, determination of pathogen colonization, production of phage and toxin, and assessment of intestinal and renal pathology. These methods provide a framework for studying STEC-mediated systemic disease that may aid in the development of efficacious therapeutics.


Assuntos
Bacteriófagos , Citrobacter rodentium , Colite , Hemorragia Gastrointestinal , Síndrome Hemolítico-Urêmica , Mucosa Intestinal , Lisogenia , Toxinas Shiga , Escherichia coli Shiga Toxigênica , Animais , Bacteriófagos/genética , Bacteriófagos/metabolismo , Citrobacter rodentium/genética , Citrobacter rodentium/metabolismo , Citrobacter rodentium/patogenicidade , Citrobacter rodentium/virologia , Colite/genética , Colite/metabolismo , Colite/microbiologia , Modelos Animais de Doenças , Hemorragia Gastrointestinal/genética , Hemorragia Gastrointestinal/metabolismo , Hemorragia Gastrointestinal/microbiologia , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/microbiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Toxinas Shiga/biossíntese , Toxinas Shiga/genética
3.
Proc Natl Acad Sci U S A ; 117(40): 25055-25065, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968018

RESUMO

Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR' late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR' immediately downstream of PR', generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5' UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.


Assuntos
Escherichia coli Êntero-Hemorrágica/genética , Síndrome Hemolítico-Urêmica/genética , Pequeno RNA não Traduzido/genética , Toxina Shiga/genética , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Bacteriófago lambda/patogenicidade , Escherichia coli Êntero-Hemorrágica/patogenicidade , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Síndrome Hemolítico-Urêmica/microbiologia , Fator Proteico 1 do Hospedeiro/genética , Humanos , Lisogenia/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Fator sigma/genética
4.
PLoS One ; 15(8): e0232305, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785271

RESUMO

Shiga toxin-producing Escherichia coli (STEC) that cause severe disease predominantly carry the toxin gene variant stx2a. However, the role of Shiga toxin in the ruminant reservoirs of this zoonotic pathogen is poorly understood and strains that cause severe disease in humans (HUSEC) likely constitute a small and atypical subset of the overall STEC flora. The aim of this study was to investigate the presence of stx2a in samples from cattle and to isolate and characterize stx2a-positive E. coli. In nationwide surveys in Sweden and Norway samples were collected from individual cattle or from cattle herds, respectively. Samples were tested for Shiga toxin genes by real-time PCR and amplicon sequencing and stx2a-positive isolates were whole genome sequenced. Among faecal samples from Sweden, stx1 was detected in 37%, stx2 in 53% and stx2a in 5% and in skin (ear) samples in 64%, 79% and 2% respectively. In Norway, 79% of the herds were positive for stx1, 93% for stx2 and 17% for stx2a. Based on amplicon sequencing the most common stx2 types in samples from Swedish cattle were stx2a and stx2d. Multilocus sequence typing (MLST) of 39 stx2a-positive isolates collected from both countries revealed substantial diversity with 19 different sequence types. Only a few classical LEE-positive strains similar to HUSEC were found among the stx2a-positive isolates, notably a single O121:H19 and an O26:H11. Lineages known to include LEE-negative HUSEC were also recovered including, such as O113:H21 (sequence type ST-223), O130:H11 (ST-297), and O101:H33 (ST-330). We conclude that E. coli encoding stx2a in cattle are ranging from strains similar to HUSEC to unknown STEC variants. Comparison of isolates from human HUS cases to related STEC from the ruminant reservoirs can help identify combinations of virulence attributes necessary to cause HUS, as well as provide a better understanding of the routes of infection for rare and emerging pathogenic STEC.


Assuntos
Bovinos/microbiologia , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/genética , Animais , Reservatórios de Doenças/microbiologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Variação Genética , Genoma Bacteriano , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Tipagem de Sequências Multilocus , Noruega/epidemiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Escherichia coli Shiga Toxigênica/citologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Suécia/epidemiologia , Virulência/genética , Zoonoses/epidemiologia , Zoonoses/microbiologia
5.
Ir Med J ; 113(1): 5, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-32298569

RESUMO

Aims To describe laboratory data on clinical human Verotoxigenic E. coli (VTEC) strains causing haemolytic uraemic syndrome (HUS) and to characterise the VTEC strains, thus contributing to risk mitigation to decrease HUS incidence in Ireland. Methods Laboratory characterisation was performed on isolates from 52 VTEC-associated HUS cases identified in the National clinical VTEC Reference Laboratory (NRL-VTEC) for the years 2012-2014. Data were analysed with respect to age, gender, serogroup and verotoxin type and subtype. Results 52/83 (62.6%) culture positive HUS cases were identified from laboratory data; 30 (57.7%) cases occurred in females. Seven HUS-associated serogroups and eleven patterns of verotoxin subtypes are described. Conclusion Ireland has the highest incidence of VTEC infection in Europe and a variety of VTEC serogroups causing clinical infection, suggesting any viable VTEC may potentially cause HUS. A broad diagnostic approach, to detect uncommon serotypes, should be considered when analysing clinical and food samples for VTEC.


Assuntos
Escherichia coli , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Toxinas Shiga , Humanos , Incidência , Irlanda/epidemiologia
6.
Lett Appl Microbiol ; 70(6): 440-446, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32270510

RESUMO

Typical haemolytic uraemic syndrome (STEC-HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), is a serious, life-threating disease that mainly affects children. Bacteriological and genetic tests are commonly used in the routine laboratory diagnosis of STEC-HUS; however, serological methods have emerged as useful and reliable diagnostic tools, especially when bacterial isolation fails. In this study, we present the results of the serological investigation of 72 paediatric patients suspected for HUS, hospitalized during 2011-2019 at the Department of Pediatrics and Nephrology of Children's Hospitals in Poland. During the routine laboratory investigation STEC strains were isolated only from nine stool samples. However, serological investigations confirmed 45 cases of STEC infections in children with HUS. In this study, 22 (48·9%) paediatric patients were infected by E. coli serotype O26, 11 (24·4%) by serotype O145, 9 (20·0%) by serotype O157, and 3 (6·7%) by E. coli serotype O111. In the majority of these patients, in addition to a high level of IgA, IgG and IgM antibodies to lipopolysaccharide of particular E. coli serotypes, antibodies to recombinant proteins Tir, Stx2b and intimin were detected. Our results confirm that serological tests are useful in the diagnosis of STEC-HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that serological analysis greatly complements bacterial isolation and helps in the diagnosis and confirmation of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) infections. Serological tests can be performed to qualify the patient for the typical haemolytic uraemic syndrome (STEC-HUS). In Poland, STEC-HUS in children is mostly caused by the E. coli serotype O26, which indicates that there is an increasing number of non-O157 STEC infections associated with human diseases in Europe.


Assuntos
Anticorpos Antibacterianos/sangue , Infecções por Escherichia coli/diagnóstico , Síndrome Hemolítico-Urêmica/microbiologia , Lipopolissacarídeos/imunologia , Toxina Shiga/imunologia , Escherichia coli Shiga Toxigênica/imunologia , Adesinas Bacterianas/genética , Formação de Anticorpos , Criança , Pré-Escolar , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Europa (Continente) , Feminino , Síndrome Hemolítico-Urêmica/diagnóstico , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Masculino , Polônia , Proteínas Recombinantes/genética , Sorogrupo , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/genética
7.
Eur J Clin Microbiol Infect Dis ; 39(3): 427-432, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31782026

RESUMO

The objective is to establish the frequency of STEC infections in household contacts of HUS patients. We studied 292 household contacts of 82 HUS patients attended from 2010 to 2018. In HUS cases, diagnostic criteria were (1) isolation and characterization of STEC strains, (2) detection of free fecal Shiga toxin (FFStx), and (3) detection of anti-O serogroup-specific antibodies. Contacts were studied by screening of stx genes by polymerase chain reaction and/or STEC isolation from stool samples. Clonal relation of STEC strains was established by pulsed-field gel electrophoresis (PFGE). Frequencies of HUS patients without STEC isolation with STEC-positive contacts were determined. Serotypes and stx-genotypes in patients and contacts were analyzed. Thirty (36.6%) HUS patients had 36 STEC-positive contacts. Fourteen (38.8%) were children, 20 adults, and 2 dogs. One sibling developed HUS, 6 contacts had gastrointestinal symptoms, and the rest were asymptomatic. In 5 of 30 HUS patients, STEC infection could not be confirmed, and 2 cases were diagnosed only by FFStx detection. Of the remaining 23 HUS patients, 16 had E. coli O157 and 7 E. coli O145 infection. Serotype and/or stx-genotype concordance was established in 19 (83%) of 23 HUS patients and their contacts. Five HUS cases and their contacts studied by PFGE showed macrorestriction patterns with more than 90% similarity. Nearly one third of HUS patients had STEC-positive family contacts, and one third of them were children. Early identification is important to prevent ongoing contamination among family and institutional contacts and to facilitate prompt detection of HUS in STEC-positive contacts.


Assuntos
Família , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Escherichia coli Shiga Toxigênica , Adolescente , Adulto , Fatores Etários , Idoso , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Fezes/microbiologia , Feminino , Genótipo , Síndrome Hemolítico-Urêmica/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Adulto Jovem
8.
Eur J Clin Microbiol Infect Dis ; 39(3): 539-547, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31776873

RESUMO

To describe an operating protocol for bloody diarrhea (BD) in a pediatric population as a rapid response to a public health threat represented by an excess of pediatric HUS cases in the Apulia region (Southern Italy) starting from 2013. The protocol was set up with the goal of correct clinical management of Shiga toxin-producing Escherichia coli (STEC) infections, reductions in subsequent cases of hemolytic uremic syndrome (HUS), and improved short- and long-term disease outcomes. The protocol consisted of rapid hospitalization of children with bloody diarrhea (BD), hematochemical laboratory tests every 12-24 hours, and prompt laboratory diagnosis of STEC. No antibiotics were recommended until diagnosis. Children positive for STEC infections underwent early vigorous volume expansion. In June-December 2018, 438 children with BD were hospitalized, of which 53 (12.1%) had a STEC infection. The most common serogroups were O26 (36.1%), O111 (23.0%), and O157 (14.8%). Thirty-one samples carried the stx2 gene. Four cases evolved into HUS (7.5%), all with favorable outcome despite neurological involvement in two cases. Prompt and accurate laboratory diagnosis of STEC infections is of the utmost importance in patients with BD for correct clinical management. The strict adherence to the protocol could reduce the progression rate of STEC infections to HUS and prevents complications. Enhanced BD surveillance may help reduce cases of pediatric HUS in Southern Italy.


Assuntos
Administração de Caso , Protocolos Clínicos , Diarreia/diagnóstico , Diarreia/microbiologia , Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/microbiologia , Escherichia coli Shiga Toxigênica , Adolescente , Algoritmos , Criança , Pré-Escolar , Diarreia/terapia , Gerenciamento Clínico , Surtos de Doenças , Feminino , Síndrome Hemolítico-Urêmica/terapia , Hospitalização , Humanos , Lactente , Recém-Nascido , Itália , Masculino , Sorogrupo , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética
9.
Pediatr Int ; 62(3): 371-378, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31758824

RESUMO

BACKGROUND: Transforming growth factor ß1 (TGF-ß1) is the main profibrotic cytokine. Its urinary excretion reflects intrarenal production; thus, we conjectured that it is elevated during hemolytic uremic syndrome related to Shiga-toxin-producing Escherichia coli (STEC-HUS). In this pilot study, we explored the ability of baseline TGF-ß1 excretion (exposure variable) to predict renal prognosis at 6 months (outcome variable). In a secondary investigation, we compared changes in cytokine levels during the study period between patients with opposite renal outcomes. METHODS: Urinary TGF-ß1 concentrations were measured prospectively in 24 children with STEC-HUS on admission, and at 15, 30, 60, 90, and 180 days. Normal values were obtained from 20 healthy subjects. RESULTS: Baseline TGF-ß1 concentrations predicted renal outcomes with an area under the curve of 1 (95%CI 0.85-1; sensitivity 100%, specificity 100%) with the best cutoff level >293.7 pg/mg uCr. All patients with high TGF-ß1 levels developed persistent renal impairment, unlike none with low concentrations (4/4 vs. 20/0 respectively, P = 0.0001). The latter had higher cytokine levels (P < 0.05) at each time point without reaching normal concentrations (<45 pg/mg uCr). CONCLUSIONS: Baseline urinary TGF-ß1 levels accurately predicted short-term renal outcomes in STEC-HUS children, and cytokine excretion during the first 6 months after diagnosis was higher among those with worse evolution. Larger studies are needed to validate these findings.


Assuntos
Síndrome Hemolítico-Urêmica/microbiologia , Escherichia coli Shiga Toxigênica/patogenicidade , Fator de Crescimento Transformador beta1/urina , Adolescente , Biomarcadores/urina , Criança , Pré-Escolar , Feminino , Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/urina , Humanos , Lactente , Rim/patologia , Masculino , Projetos Piloto , Prognóstico , Estudos Prospectivos , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
10.
PLoS One ; 14(12): e0220483, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31881024

RESUMO

E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.


Assuntos
Síndrome Hemolítico-Urêmica/metabolismo , Lectina de Ligação a Manose/metabolismo , Trombose/metabolismo , Lesão Renal Aguda/metabolismo , Animais , Plaquetas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Rim/metabolismo , Glomérulos Renais/metabolismo , Lectina de Ligação a Manose/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Toxina Shiga/metabolismo , Toxina Shiga II/metabolismo , Tromboembolia/metabolismo
11.
Sci Rep ; 9(1): 17014, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31745113

RESUMO

Enterohemorrhagic E. coli (EHEC) is a major cause of large outbreaks worldwide associated with hemorrhagic colitis and hemolytic uremic syndrome. While vaccine development is warranted, a licensed vaccine, specific for human use, against EHEC is not yet available. In this study, the reverse vaccinology approach combined with genomic, transcriptional and molecular epidemiology data was applied on the EHEC O157:H7 genome to select new potential vaccine candidates. Twenty-four potential protein antigens were identified and one of them (MC001) was successfully expressed onto Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing this vaccine candidate was immunogenic, raising a specific antibody response. Immunization with the MC001 candidate was able to reduce the bacterial load of EHEC O157:H7 strain in feces, colon and caecum tissues after murine infection. MC001 is homologue to lipid A deacylase enzyme (LpxR), and to our knowledge, this is the first study describing it as a potential vaccine candidate. Gene distribution and sequence variability analysis showed that MC001 is present and conserved in EHEC and in enteropathogenic E. coli (EPEC) strains. Given the high genetic variability among and within E. coli pathotypes, the identification of such conserved antigen suggests that its inclusion in a vaccine might represent a solution against major intestinal pathogenic strains.


Assuntos
Hidrolases de Éster Carboxílico/imunologia , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157/imunologia , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/microbiologia , Camundongos , Camundongos Endogâmicos BALB C
13.
Sci Rep ; 9(1): 14362, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591425

RESUMO

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Síndrome Hemolítico-Urêmica/tratamento farmacológico , Receptores Purinérgicos P2X1/genética , Toxina Shiga/genética , Trifosfato de Adenosina/metabolismo , Animais , Benzenossulfonatos/farmacologia , Plaquetas/microbiologia , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Células HeLa , Síndrome Hemolítico-Urêmica/genética , Síndrome Hemolítico-Urêmica/microbiologia , Síndrome Hemolítico-Urêmica/patologia , Humanos , Camundongos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Toxina Shiga/antagonistas & inibidores
14.
Toxins (Basel) ; 11(10)2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31652648

RESUMO

Shiga toxin-producing Escherichia coli (STEC) infections are the product of the interaction between bacteria, phages, animals, humans, and the environment. In the late 1980s, Alberta had one of the highest incidences of STEC infections in North America. Herein, we revisit and contextualize the epidemiology of STEC O157 human infections in Alberta for the period 2009-2016. STEC O157 infections were concentrated in large urban centers, but also in rural areas with high cattle density. Hospitalization was often required when the Shiga toxin genotype stx2a stx2c was involved, however, only those aged 60 years or older and infection during spring months (April to June) independently predicted that need. Since the late 1980s, the rate of STEC O157-associated hemolytic uremic syndrome (HUS) in Alberta has remained unchanged at 5.1%, despite a marked drop in the overall incidence of the infection. While Shiga toxin genotypes stx1a stx2c and stx2a stx2c seemed associated with HUS, only those aged under 10 years and infection during spring months were independently predictive of that complication. The complexity of the current epidemiology of STEC O157 in Alberta highlights the need for a One Health approach for further progress to be made in mitigating STEC morbidity.


Assuntos
Infecções por Escherichia coli/epidemiologia , Escherichia coli O157 , Síndrome Hemolítico-Urêmica/epidemiologia , Adolescente , Adulto , Alberta/epidemiologia , Criança , Pré-Escolar , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Feminino , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Virulência/genética , Adulto Jovem
15.
Toxins (Basel) ; 11(10)2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635282

RESUMO

Escherichia coli O157:H7 is the predominant cause of diarrhea-associated hemolytic uremic syndrome (HUS) worldwide. Its cardinal virulence traits are Shiga toxins, which are encoded by stx genes, the most common of which are stx1a, stx2a, and stx2c. The toxins these genes encode differ in their in vitro and experimental phenotypes, but the human population-level impact of these differences is poorly understood. Using Shiga toxin-encoding bacteriophage insertion typing and real-time polymerase chain reaction, we genotyped isolates from 936 E. coli O157:H7 cases and verified HUS status via chart review. We compared the HUS risk between isolates with stx2a and those with stx2a and another gene and estimated additive interaction of the stx genes. Adjusted for age and symptoms, the HUS incidence of E. coli O157:H7 containing stx2a alone was 4.4% greater (95% confidence interval (CI) -0.3%, 9.1%) than when it occurred with stx1a. When stx1a and stx2a occur together, the risk of HUS was 27.1% lower (95% CI -87.8%, -2.3%) than would be expected if interaction were not present. At the population level, temporal or geographic shifts toward these genotypes should be monitored, and stx genotype may be an important consideration in clinically predicting HUS among E. coli O157:H7 cases.


Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Síndrome Hemolítico-Urêmica/microbiologia , Toxina Shiga/genética , Virulência/genética , Adolescente , Adulto , Criança , Pré-Escolar , Infecções por Escherichia coli/terapia , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/patogenicidade , Genótipo , Síndrome Hemolítico-Urêmica/terapia , Humanos , Pessoa de Meia-Idade , Terapia de Substituição Renal , Risco , Adulto Jovem
17.
Pediatrics ; 144(4)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31519792

RESUMO

BACKGROUND: In 2017, we conducted a multistate investigation to determine the source of an outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infections, which occurred primarily in children. METHODS: We defined a case as infection with an outbreak strain of STEC O157:H7 with illness onset between January 1, 2017, and April 30, 2017. Case patients were interviewed to identify common exposures. Traceback and facility investigations were conducted; food samples were tested for STEC. RESULTS: We identified 32 cases from 12 states. Twenty-six (81%) cases occurred in children <18 years old; 8 children developed hemolytic uremic syndrome. Twenty-five (78%) case patients ate the same brand of soy nut butter or attended facilities that served it. We identified 3 illness subclusters, including a child care center where person-to-person transmission may have occurred. Testing isolated an outbreak strain from 11 soy nut butter samples. Investigations identified violations of good manufacturing practices at the soy nut butter manufacturing facility with opportunities for product contamination, although the specific route of contamination was undetermined. CONCLUSIONS: This investigation identified soy nut butter as the source of a multistate outbreak of STEC infections affecting mainly children. The ensuing recall of all soy nut butter products the facility manufactured, totaling >1.2 million lb, likely prevented additional illnesses. Prompt diagnosis of STEC infections and appropriate specimen collection aids in outbreak detection. Child care providers should follow appropriate hygiene practices to prevent secondary spread of enteric illness in child care settings. Firms should manufacture ready-to-eat foods in a manner that minimizes the risk of contamination.


Assuntos
Surtos de Doenças/estatística & dados numéricos , Infecções por Escherichia coli/epidemiologia , Escherichia coli O157 , Doenças Transmitidas por Alimentos/epidemiologia , Escherichia coli Shiga Toxigênica , Alimentos de Soja/microbiologia , Adolescente , Idoso , Criança , Creches/estatística & dados numéricos , Pré-Escolar , Infecções por Escherichia coli/microbiologia , Fast Foods/efeitos adversos , Fast Foods/microbiologia , Feminino , Manipulação de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Lactente , Masculino , Recall e Retirada de Produto , Alimentos de Soja/efeitos adversos , Estados Unidos/epidemiologia
18.
J Clin Microbiol ; 57(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31366691

RESUMO

Shiga toxin-producing Escherichia coli (STEC) and the STEC subgroup enterohemorrhagic E. coli cause intestinal infections with symptoms ranging from watery diarrhea to hemolytic-uremic syndrome (HUS). A key tool for the epidemiological differentiation of STEC is serotyping. The serotype in combination with the main virulence determinants gives important insight into the virulence potential of a strain. However, a large fraction of STEC strains found in human disease, including strains causing HUS, belongs to less frequently detected STEC serovars or their O/H antigens are unknown or even untypeable. Recent implementation of whole-genome sequence (WGS) analysis, in principle, allows the deduction of serovar and virulence gene information. Therefore, here we compared classical serovar and PCR-based virulence marker detection with WGS-based methods for 232 STEC strains, focusing on less frequently detected STEC serovars and nontypeable strains. We found that the results of WGS-based extraction showed a very high degree of overlap with those of the more classical methods. Specifically, the rate of concordance was 97% for O antigens (OAGs) and 99% for H antigens (HAGs) of typeable strains and >99% for stx 1, stx 2, or eaeA for all strains. Ninety-eight percent of nontypeable OAGs and 100% of nontypeable HAGs were defined by WGS analysis. In addition, the novel methods enabled a more complete analysis of strains causing severe clinical symptoms and the description of four novel STEC OAG loci. In conclusion, WGS is a promising tool for gaining serovar and virulence gene information, especially from a public health perspective.


Assuntos
Infecções por Escherichia coli/microbiologia , Genótipo , Síndrome Hemolítico-Urêmica/microbiologia , Antígenos O/genética , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética , Sequenciamento Completo do Genoma , Antígenos de Bactérias/genética , Biologia Computacional/métodos , Loci Gênicos , Genoma Bacteriano , Genômica/métodos , Técnicas de Genotipagem , Humanos , Filogenia , Vigilância em Saúde Pública , Sorotipagem , Virulência/genética
19.
Epidemiol Infect ; 147: e215, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-31364538

RESUMO

Shiga toxin-producing Escherichia coli (STEC) infection can cause serious illness including haemolytic uraemic syndrome. The role of socio-economic status (SES) in differential clinical presentation and exposure to potential risk factors amongst STEC cases has not previously been reported in England. We conducted an observational study using a dataset of all STEC cases identified in England, 2010-2015. Odds ratios for clinical characteristics of cases and foodborne, waterborne and environmental risk factors were estimated using logistic regression, stratified by SES, adjusting for baseline demographic factors. Incidence was higher in the highest SES group compared to the lowest (RR 1.54, 95% CI 1.19-2.00). Odds of Accident and Emergency attendance (OR 1.35, 95% CI 1.10-1.75) and hospitalisation (OR 1.71, 95% CI 1.36-2.15) because of illness were higher in the most disadvantaged compared to the least, suggesting potential lower ascertainment of milder cases or delayed care-seeking behaviour in disadvantaged groups. Advantaged individuals were significantly more likely to report salad/fruit/vegetable/herb consumption (OR 1.59, 95% CI 1.16-2.17), non-UK or UK travel (OR 1.76, 95% CI 1.40-2.27; OR 1.85, 95% CI 1.35-2.56) and environmental exposures (walking in a paddock, OR 1.82, 95% CI 1.22-2.70; soil contact, OR 1.52, 95% CI 2.13-1.09) suggesting other unmeasured risks, such as person-to-person transmission, could be more important in the most disadvantaged group.


Assuntos
Infecções por Escherichia coli/epidemiologia , Disparidades nos Níveis de Saúde , Síndrome Hemolítico-Urêmica/epidemiologia , Toxina Shiga/efeitos adversos , Escherichia coli Shiga Toxigênica/isolamento & purificação , Adulto , Análise de Variância , Bases de Dados Factuais , Diarreia/epidemiologia , Diarreia/microbiologia , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Feminino , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Incidência , Masculino , Análise Multivariada , Determinação de Necessidades de Cuidados de Saúde , Prevalência , Estudos Retrospectivos , Medição de Risco , Classe Social , Reino Unido/epidemiologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-31297339

RESUMO

The most frequent form of hemolytic-uremic syndrome (HUS) is associated with infections caused by Shiga-like toxin-producing Enterohaemorrhagic Escherichia coli (STEC). In rarer cases HUS can be triggered by Streptococcus pneumoniae. While production of Shiga-like toxins explains STEC-HUS, the mechanisms of pneumococcal HUS are less well-known. S. pneumoniae produces neuraminidases with activity against cell surface sialic acids that are critical for factor H-mediated complement regulation on cells and platelets. The aim of this study was to find out whether S. pneumoniae neuraminidase NanA could trigger complement activation and hemolysis in whole blood. We studied clinical S. pneumoniae isolates and two laboratory strains, a wild-type strain expressing NanA, and a NanA deletion mutant for their ability to remove sialic acids from various human cells and platelets. Red blood cell lysis and activation of complement was measured ex vivo by incubating whole blood with bacterial culture supernatants. We show here that NanA expressing S. pneumoniae strains and isolates are able to remove sialic acids from cells, and platelets. Removal of sialic acids by NanA increased complement activity in whole blood, while absence of NanA blocked complement triggering and hemolytic activity indicating that removal of sialic acids by NanA could potentially trigger pHUS.


Assuntos
Neuraminidase/sangue , Neuraminidase/metabolismo , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/genética , Plaquetas/metabolismo , Proteínas do Sistema Complemento/efeitos dos fármacos , Eritrócitos , Células HEK293 , Hemólise , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Inflamação , Neuraminidase/genética , Neuraminidase/farmacologia , Infecções Pneumocócicas/microbiologia , Deleção de Sequência , Ácidos Siálicos
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