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Shiga toxin-producing Escherichia coli (STEC) poses a significant public health risk due to its zoonotic potential and association with severe human diseases, such as hemorrhagic colitis and hemolytic uremic syndrome. Ruminants are recognized as primary reservoirs for STEC, but swine also contribute to the epidemiology of this pathogen, highlighting the need for effective prevention strategies across species. Notably, a subgroup of STEC that produces Shiga toxin type 2e (Stx2e) causes edema disease (ED) in newborn piglets, economically affecting pig production. This study evaluates the immunogenicity of a chimeric protein-based vaccine candidate against STEC in pregnant sows and the subsequent transfer of immunity to their offspring. This vaccine candidate, which includes chimeric proteins displaying selected epitopes from the proteins Cah, OmpT, and Hes, was previously proven to be immunogenic in pregnant cows. Our analysis revealed a broad diversity of STEC serotypes within swine populations, with the cah and ompT genes being prevalent, validating them as suitable antigens for vaccine development. Although the hes gene was detected less frequently, the presence of at least one of these three genes in a significant proportion of STEC suggests the potential of this vaccine to target a wide range of strains. The vaccination of pregnant sows led to an increase in specific IgG and IgA antibodies against the chimeric proteins, indicating successful immunization. Additionally, our results demonstrated the effective passive transfer of maternal antibodies to piglets, providing them with immediate, albeit temporary, humoral immunity against STEC. These humoral responses demonstrate the immunogenicity of the vaccine candidate and are preliminary indicators of its potential efficacy. However, further research is needed to conclusively evaluate its impact on STEC colonization and shedding. This study highlights the potential of maternal vaccination to protect piglets from ED and contributes to the development of vaccination strategies to reduce the prevalence of STEC in various animal reservoirs.
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We examined the antibacterial efficacy of streptomycin, hibiscus acid, and their combination against multidrug-resistant Shiga-toxin-producing Escherichia coli (STEC) and Salmonella Typhimurium in mice. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for streptomycin, hibiscus acid, and their combination against STEC and Salmonella. Fifteen sets of six mice in each set were utilised: six groups were orally exposed to 4 log10 colony forming units (CFUs) of S. Typhimurium and another six to STEC, and three acted as the controls. Six hours post-inoculation, specific groups of mice received either oral solutions containing hibiscus acid at 5 and 7 mg/ml; streptomycin at 50 and 450 µg/ml; hibiscus acid/streptomycin (5 mg/ml hibiscus acid and 50 µg/ml streptomycin); or isotonic saline. The study determined the MIC and MBC of 7 mg/ml of hibiscus acid; 300 and 450 µg/ml of streptomycin; and two concentrations of hibiscus/streptomycin (3 mg/ml / 20 µg/ml and 5 mg/ml / 50 µg/ml). Interestingly, the mice that were infected and subsequently treated with hibiscus acid at 7 mg/ml alone or in conjunction with streptomycin did not have either STEC or Salmonella in their faecal samples, and none of the mice died. In contrast, the untreated mice and those exclusively treated with streptomycin had the pathogens present in their stool, leading to the mortality of all the subjects.
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Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.
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Infecções por Escherichia coli , Plasmídeos , Escherichia coli Shiga Toxigênica , Fatores de Virulência , Plasmídeos/genética , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Animais , Bovinos , Fatores de Virulência/genética , Humanos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Virulência/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Aptidão Genética , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologiaRESUMO
Shiga-toxin-producing Escherichia coli (STEC) is associated with diarrhea and hemolytic uremic syndrome (HUS). STEC infections in Costa Rica are rarely reported in children. We gathered all the records of STEC infections in children documented at the National Children's Hospital, a tertiary referral hospital, from 2015 to 2020. Clinical, microbiological, and genomic information were analyzed and summarized. A total of 3,768 diarrheal episodes were reviewed. Among them, 31 STEC were characterized (29 fecal, 1 urine, and 1 bloodstream infection). The prevalence of diarrheal disease due to STEC was estimated at 0.8% (n = 29/3,768), and HUS development was 6.4% (n = 2/31). The stx1 gene was found in 77% (n = 24/31) of STEC strains. In silico genomic predictions revealed a predominant prevalence of serotype O118/O152:H2, accompanied by a cluster exhibiting allele differences ranging from 33 to 8, using a core-genome multilocus sequence typing (cgMLST) approach. This is the first study using a genomic approach for STEC infections in Costa Rica.IMPORTANCEThis study provides a comprehensive description of clinical, microbiological, genomic, and demographic data from patients who attended the only pediatric hospital in Costa Rica with Shiga-toxin-producing Escherichia coli (STEC) infections. Despite the low prevalence of STEC infections, we found a predominant serotype O118/O152:H2, highlighting the pivotal role of genomics in understanding the epidemiology of public health threats such as STEC. Employing a genomic approach for this pathogen for the first time in Costa Rica, we identified a higher prevalence of STEC in children under 2 years old, especially those with gastrointestinal comorbidities, residing in densely populated regions. Limitations such as potential geographic bias and lack of strains due to direct molecular diagnostics are acknowledged, emphasizing the need for continued surveillance to uncover the true extent of circulating serotypes and potential outbreaks in Costa Rica.
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Infecções por Escherichia coli , Síndrome Hemolítico-Urêmica , Escherichia coli Shiga Toxigênica , Criança , Humanos , Lactente , Escherichia coli Shiga Toxigênica/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Estudos Retrospectivos , Centros de Atenção Terciária , Costa Rica/epidemiologia , Diarreia/epidemiologia , Diarreia/microbiologia , Síndrome Hemolítico-Urêmica/complicações , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , GenômicaRESUMO
Although it has been hypothesized that the acquisition of plasmids—especially those bearing virulence factors and antimicrobial resistance genes—increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from − 4.7 to 5.2% per generation. This suggests that there are few biological barriers—or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model—against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.
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Introduction. Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with clinical cases of diarrhea in humans. Its main virulence factors are the Shiga toxins (Stx1 and Stx2). Cattle are the main reservoir of STEC, and many outbreaks in humans have been related to the consumption of undercooked ground beef contaminated with this pathogen. Objective. To determine the prevalence of STEC in ground beef commercialized in all the butcher shops of a township in the department of Quindío and to characterize the virulence genes of the strains found. Materials and methods. Thirty ground beef samples were taken in three different times; stx genes and other STEC virulence factors (eae, ehxA, saa) were detected by multiplex PCR. Results. The overall prevalence of STEC was 33.33 % (10/30 positive samples). We isolated eight non-O157 (LEE-negative) strains with four different genetic profiles: stx 2 / stx 2-ehxA-saa / stx 1-stx 2-ehxA-saa / stx 1-saa. Conclusion. This is the first report on the prevalence of STEC in ground beef in a township in the department of Quindío.
Introducción. Escherichia coli productora de toxina Shiga (STEC) es un agente patógeno de origen alimentario asociado a casos clínicos de diarrea en humanos; sus principales factores de virulencia son las toxinas Shiga (Stx1 y Stx2). El principal reservorio de STEC es el ganado bovino y muchos brotes en humanos se han relacionado con el consumo de carne mal cocida contaminada con este agente patógeno. Objetivo. El objetivo de este trabajo fue determinar la prevalencia de STEC en carne molida comercializada en todas las carnicerías de un municipio del departamento del Quindío y caracterizar los genes de virulencia de las cepas encontradas. Materiales y métodos. Se tomaron 30 muestras de carne molida en tres momentos diferentes; se detectaron los genes stx y otros factores de virulencia de STEC (eae, ehxA, saa) mediante PCR Multiplex. Resultados. Los resultados mostraron una prevalencia global de STEC del 33,33 % (10/30 muestras positivas). En total se aislaron ocho cepas STEC no-O157 (LEE-negativas) con cuatro perfiles genéticos diferentes: stx 2 / stx 2-ehxA-saa / stx 1-stx 2-ehxA-saa / stx 1-saa. Conclusión. Este es el primer reporte que muestra la prevalencia de STEC en carne molida en un municipio del departamento del Quindío.
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Escherichia coli Shiga Toxigênica , PrevalênciaRESUMO
BACKGROUND: Thrombotic Microangiopathy (TMA) is a syndrome characterized by the presence of anemia, thrombocytopenia and organ damage and has multiple etiologies. The primary aim is to develop an algorithm to classify TMA (TMA-INSIGHT score). METHODS: This was a single-center retrospective cohort study including hospitalized patients with TMA at a single center. We included all consecutive patients diagnosed with TMA between 2012 and 2021. TMA was defined based on the presence of anemia (hemoglobin level < 10 g/dL) and thrombocytopenia (platelet count < 150,000/µL), signs of hemolysis, and organ damage. We classified patients in eight categories: infections; Malignant Hypertension; Transplant; Malignancy; Pregnancy; Thrombotic Thrombocytopenic Purpura (TTP); Shiga toxin-mediated hemolytic uremic syndrome (STEC-SHU) and Complement Mediated TMA (aHUS). We fitted a model to classify patients using clinical characteristics, biochemical exams, and mean arterial pressure at presentation. RESULTS: We retrospectively retrieved TMA phenotypes using automatic strategies in electronic health records in almost 10 years (n = 2407). Secondary TMA was found in 97.5% of the patients. Primary TMA was found in 2.47% of the patients (TTP and aHUS). The best model was LightGBM with accuracy of 0.979, and multiclass ROC-AUC of 0.966. The predictions had higher accuracy in most TMA classes, although the confidence was lower in aHUS and STEC-HUS cases. CONCLUSION: Secondary conditions were the most common etiologies of TMA. We retrieved comorbidities, associated conditions, and mean arterial pressure to fit a model to predict TMA and define TMA phenotypic characteristics. This is the first multiclass model to predict TMA including primary and secondary conditions.
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AIMS: The aim of the present work was to characterize the Lactiplantibacillus sp. LP5 strain, isolated from pork production, and identify bacteriocin-like inhibitory substances produced by this strain. METHODS AND RESULTS: In this study, LP5 was identified by species-specific PCR and 16S rRNA sequencing. Additionally, bacterial growth kinetics, antimicrobial activity, the detection of genes related to plantaricin production, and the genetic expression of plantaricins were determined. Lactiplantibacillus sp. LP5 was identified as Lactiplantibacillus plantarum. The well-diffusion test using cell-free supernatants (CFS), neutralized CFS, CFS treated with catalase, and CFS treated with proteinase K showed that inhibitory effects on a Shiga toxin-producing Escherichia coli (STEC) strain were produced by bacteriocins. The PCR technique allowed the detection of genes encoding E/F plantaricins, as well as J/K and whole genome sequencing, and bacteriocin mining analysis allowed us to confirm the presence of these plantaricins. CONCLUSIONS: We can conclude that the inhibitory effect of L. plantarum LP5 isolated from pigs against the STEC EDL933 strain could be associated with the bacteriocins production and represents a potential use as a probiotic strain.
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Anti-Infecciosos , Bacteriocinas , Animais , Suínos , RNA Ribossômico 16S/genética , Bacteriocinas/genética , Bacteriocinas/farmacologia , Endopeptidase K , Expressão GênicaRESUMO
Shiga toxin-producing E. coli (STEC) are diarrheagenic strains that can cause bloody diarrhea and hemolytic-uremic syndrome. Their main virulence factor, the Shiga toxin (Stx), is encoded by phages integrated into the bacterial chromosome. Stx phages are widely diverse and carry many genes with limited or unknown function. As the toxin subtype Stx2a is associated with highly pathogenic strains, this study was mainly focused on the characterization of the stx flanking region of Stx2a phages. Of particular interest was a sialate O-acetylesterase (NanS-p), which has been described previously to be encoded downstream stx in some phage genomes and may confer a growth advantage for STEC. Complete DNA sequences of Stx2a phages and prophages were retrieved from the GenBank database, and the genomic regions from anti-terminator Q to holin S genes were bioinformatically analyzed. Predicted NanSp sequences from phages encoding other Stx subtypes were also studied. Additionally, expression of nanS-p was quantified by qPCR in strains selected from our laboratory collection. The analysis of Stx2a phage genomes showed that all carried the Q, stx2a, nanS-p and S genes, but with allele diversity and other sequence differences. In particular, sequence differences were detected in each of the three domains of NanS-p esterases encoded by Stx2a phages and other Stx phages; however, nanS-p was not identified in the Stx2e, Stx2f and Stx2g phages analyzed. The expression of nanS-p increased in most stx2a-positive strains under phage inducing conditions, as was previously shown for stx2a. As the present work showed diversity at the Q-S region among Stx phages, and particularly in the encoded NanS-p enzyme, future studies will be necessary to evaluate if NanS-p variants differ in their activity and to assess the impact of the absence of nanS-p in certain Stx phages.
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Background: Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that causes gastrointestinal infections, ranging from acute diarrhea and dysentery to life-threatening diseases such as Hemolytic Uremic Syndrome. Currently, a vaccine to prevent STEC infection is an unmet medical need. Results: We developed a chimeric protein-based vaccine targeting seven virulence factors of STEC, including the Stx2B subunit, Tir, Intimin, EspA, Cah, OmpT, and AggA proteins. Immunization of mice with this vaccine candidate elicited significant humoral and cellular immune responses against STEC. High levels of specific IgG antibodies were found in the serum and feces of immunized mice. However, specific IgA antibodies were not detected in either serum or feces. Furthermore, a significantly higher percentage of antigen-specific CD4+ T cells producing IFN-γ, IL-4, and IL-17 was observed in the spleens of immunized mice. Notably, the immunized mice showed decreased shedding of STEC O157:H7 and STEC O91:H21 strains and were protected against weight loss during experimental infection. Additionally, infection with the STEC O91:H21 strain resulted in kidney damage in control unimmunized mice; however, the extent of damage was slightly lower in immunized mice. Our findings suggest that IgG antibodies induced by this vaccine candidate may have a role in inhibiting bacterial adhesion and complement-mediated killing. Conclusion: This study provides evidence that IgG responses are involved in the host defense against STEC. However, our results do not rule out that other classes of antibodies also participate in the protection against this pathogen. Additional work is needed to improve the protection conferred by our vaccine candidate and to elucidate the relevant immune responses that lead to complete protection against this pathogen.
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Infecções por Escherichia coli , Escherichia coli Shiga Toxigênica , Vacinas , Animais , Camundongos , Imunoglobulina G , Formação de Anticorpos , Proteínas Recombinantes de FusãoRESUMO
In Uruguay, the mortality of dairy calves due to infectious diseases is high. Escherichia coli is a natural inhabitant of the intestinal microbiota, but can cause several infections. The aim of the work was to characterize E. coli isolates from intestinal and extraintestinal origin of dead newborn calves. Using PCR, virulence gene characteristics of pathogenic E. coli were searched. The pathogenic E. coli were molecularly characterized and the phylogroup, serogroup and the Stx subtype were determined. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method and plasmid-mediated quinolone resistance (PMQR) genes with PCR. Finally, clonal relationships were inferred using PFGE. Gene characteristics of the Shiga toxin-producing E. coli (STEC), Enteropathogenic E. coli (EPEC) and Necrotoxigenic E. coli (NTEC) were identified. The prevalence of the iucD, afa8E, f17, papC, stx1, eae and ehxA genes was high and no f5, f41, saa, sfaDE, cdtIV, lt, sta or stx2 were detected. The prevalence of STEC gene stx1 in the dead calves stood out and was higher compared with previous studies conducted in live calves, and STEC LEE+ (Enterohemorrhagic E. coli (EHEC)) isolates with stx1/eae/ehxA genotypes were more frequently identified in the intestinal than in the extraintestinal environment. E. coli isolates were assigned to phylogroups A, B1, D and E, and some belonged to the O111 serogroup. stx1a and stx1c subtypes were determined in STEC. A high prevalence of multi-resistance among STEC and qnrB genes was determined. The PFGE showed a high diversity of pathogenic strains with similar genetic profiles. It can be speculated that EHEC (stx1/eae/ehxA) could play an important role in mortality. The afa8E, f17G1 and papC genes could also have a role in calf mortality. Multidrug resistance defies disease treatment and increases the risk of death, while the potential transmissibility of genes to other species constitutes a threat to public health.
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Introduction: Shiga-toxin (Stx) producing Escherichia coli (STEC) O157:H7 is the most frequent serotype associated with hemolytic uremic syndrome (HUS) after gastrointestinal infections. Protection against HUS secondary to STEC infections has been experimentally assayed through the generation of different vaccine formulations. With focus on patients, the strategies have been mainly oriented to inhibit production of Stx or its neutralization. However, few approaches have been intended to block gastrointestinal phase of this disease, which is considered the first step in the pathogenic cascade of HUS. The aim of this work was to assay H7 flagellin as a mucosal vaccine candidate to prevent the systemic complications secondary to E. coli O157:H7 infections. Materials and methods: The cellular and humoral immune response after H7 nasal immunization in mice were studied by the analysis of systemic and intestinal specific antibody production, as well as cytokine production and lymphocyte proliferation against H7 flagellin ex vivo. Results: Immunized mice developed a strong and specific anti-H7 IgG and IgA response, at systemic and mucosal level, as well as a cellular Th1/Th2/Th17 response. H7 induced activation of bone marrow derived dendritic cells in vitro and a significant delayed-type hypersensitivity (DTH) response in immunized mice. Most relevant, immunized mice were completely protected against the challenge with an E. coli O157:H7 virulent strain in vivo, and surviving mice presented high titres of anti-H7 and Stx antibodies. Discussion: These results suggest that immunization avoids HUS outcome and allows to elicit a specific immune response against other virulence factors.
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Doenças Transmissíveis , Infecções por Escherichia coli , Escherichia coli O157 , Gastroenteropatias , Síndrome Hemolítico-Urêmica , Escherichia coli Shiga Toxigênica , Animais , Camundongos , Flagelina , Infecções por Escherichia coli/prevenção & controle , Imunização , Síndrome Hemolítico-Urêmica/prevenção & controleRESUMO
Shiga toxin producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are pathovars that affect mainly infants' health. Cattle are the main reservoir of STEC. Uremic hemolytic syndrome and diarrheas can be found at high rates in Tierra del Fuego (TDF). This study aimed to establish the prevalence of STEC and EPEC in cattle at slaughterhouses in TDF and to analyze the isolated strains. Out of 194 samples from two slaughterhouses, STEC prevalence was 15%, and EPEC prevalence was 5%. Twenty-seven STEC strains and one EPEC were isolated. The most prevalent STEC serotypes were O185:H19 (7), O185:H7 (6), and O178:H19 (5). There were no STEC eae + strains (AE-STEC) or serogroup O157 detected in this study. The prevalent genotype was stx2c (10/27) followed by stx1a/stx2hb (4/27). Fourteen percent of the strains presented at least one stx non-typeable subtype (4/27). Shiga toxin production was detected in 25/27 STEC strains. The prevalent module for the Locus of Adhesion and Autoaggregation (LAA) island was module III (7/27). EPEC strain was categorized as atypical and with the ability to cause A/E lesion. The ehxA gene was present in 16/28 strains, 12 of which were capable of producing hemolysis. No hybrid strains were detected in this work. Antimicrobial susceptibility tests showed that all strains were resistant to ampicillin and 20/28 were resistant to aminoglycosides. No statistical differences could be seen in the detection of STEC or EPEC either by slaughterhouse location or by production system (extensive grass or feedlot). The rate of STEC detection was lower than the one reported for the rest of Argentina. STEC/EPEC relation was 3 to 1. This is the first study on cattle from TDF as reservoir for strains that are potentially pathogenic to humans.
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Escherichia coli Enteropatogênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli Shiga Toxigênica , Animais , Bovinos , Humanos , Toxina Shiga , Proteínas de Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Infecções por Escherichia coli/microbiologia , Argentina/epidemiologiaRESUMO
Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes gastroenteritis and Hemolytic Uremic Syndrome. Cattle are the main animal reservoir, excreting the bacteria in their feces and contaminating the environment. In addition, meat can be contaminated by releasing the intestinal content during slaughtering. Here, we evaluated the safety and immunogenicity of a vaccine candidate against STEC that was formulated with two chimeric proteins (Chi1 and Chi2), which contain epitopes of the OmpT, Cah and Hes proteins. Thirty pregnant cows in their third trimester of gestation were included and distributed into six groups (n = 5 per group): four groups were administered intramuscularly with three doses of the formulation containing 40 µg or 100 µg of each protein plus the Quil-A or Montanide™ Gel adjuvants, while two control groups were administered with placebos. No local or systemic adverse effects were observed during the study, and hematological parameters and values of blood biochemical indicators were similar among all groups. Furthermore, all vaccine formulations triggered systemic anti-Chi1/Chi2 IgG antibody levels that were significantly higher than the control groups. However, specific IgA levels were generally low and without significant differences among groups. Notably, anti-Chi1/Chi2 IgG antibody levels in the serum of newborn calves fed with colostrum from their immunized dams were significantly higher compared to newborn calves fed with colostrum from control cows, suggesting a passive immunization through colostrum. These results demonstrate that this vaccine is safe and immunogenic when applied to pregnant cows during the third trimester of gestation.
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Infecções por Escherichia coli , Síndrome Hemolítico-Urêmica , Escherichia coli Shiga Toxigênica , Vacinas de Subunidades Antigênicas , Animais , Bovinos , Feminino , Gravidez , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/veterinária , Imunização Passiva , Imunoglobulina G , Vacinas de Subunidades Antigênicas/efeitos adversosRESUMO
BACKGROUND: Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolysis, thrombocytopenia, and thrombus formation leading to tissue injury. HUS is classified according to its etiology as post-diarrheal or atypical HUS. Differential diagnosis of both entities continues to be a challenge for pediatric physicians. METHODS: The aim was to improve the rapid etiological diagnosis of post-diarrheal HUS cases based on the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection by screening of stx1/stx2 and rfbO157 in cultured stools by multiplex PCR, and the additional detection of anti-lipopolysaccharide (anti-LPS) O157, O145, and O121 antibodies by Glyco-iELISA test. In addition, we studied patients' relatives to detect circulating pathogenic strains that could contribute to HUS diagnosis and/or lead to the implementation of measures to prevent dissemination of familial outbreaks. This study describes the diagnosis of 31 HUS patients admitted to Hospital Municipal de Niños Prof Dr Ramón Exeni during the 2017-2020 period. RESULTS: Stool PCR confirmed the diagnosis of STEC associated with HUS in 38.7% of patients (12/31), while anti-LPS serology did in 88.9% (24/27). In those patients in which both methods were carried out (n = 27), a strong association between the results obtained was found. We found that 30.4% of HUS patients had at least one relative positive for STEC. CONCLUSIONS: We could identify 96.3% (26/27) of HUS cases as secondary to STEC infections when both methods (genotyping and serology) were used. The results demonstrated a high circulation of STEC in HUS families and the prevalence of the STEC O157 serotype (83%) in our pediatric cohort. A higher-resolution version of the Graphical abstract is available as Supplementary information.
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Síndrome Hemolítico-Urêmica Atípica , Infecções por Escherichia coli , Escherichia coli Shiga Toxigênica , Criança , Humanos , Diarreia/diagnóstico , Diarreia/etiologia , Diarreia/epidemiologia , Sorogrupo , Lipopolissacarídeos , Anticorpos Antibacterianos , Infecções por Escherichia coli/complicações , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/epidemiologiaRESUMO
Shiga toxin 2 (Stx2) from enterohemorrhagic Escherichia coli (EHEC) produces hemorrhagic colitis, hemolytic uremic syndrome (HUS), and acute encephalopathy. The mortality rate in HUS increases significantly when the central nervous system (CNS) is involved. Besides, EHEC also releases lipopolysaccharide (LPS). Many reports have described cognitive dysfunctions in HUS patients, the hippocampus being one of the brain areas targeted by EHEC infection. In this context, a translational murine model of encephalopathy was employed to establish the deleterious effects of Stx2 and the contribution of LPS in the hippocampus. The purpose of this work is to elucidate the signaling pathways that may activate the inflammatory processes triggered by Stx2, which produces cognitive alterations at the level of the hippocampus. Results demonstrate that Stx2 produced depression-like behavior, pro-inflammatory cytokine release, and NF-kB activation independent of the ERK1/2 signaling pathway, while co-administration of Stx2 and LPS reduced memory index. On the other hand, LPS activated NF-kB dependent on ERK1/2 signaling pathway. Cotreatment of Stx2 with LPS aggravated the pathologic state, while dexamethasone treatment succeeded in preventing behavioral alterations. Our present work suggests that the use of drugs such as corticosteroids or NF-kB signaling inhibitors may serve as neuroprotectors from EHEC infection.
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Encefalopatias , Disfunção Cognitiva , Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Síndrome Hemolítico-Urêmica , Camundongos , Humanos , Animais , Toxina Shiga II/toxicidade , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases , NF-kappa B , Encéfalo/patologia , Infecções por Escherichia coli/complicações , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/patologia , Hipocampo/patologia , CogniçãoRESUMO
The human colonic mucus is mainly composed of mucins, which are highly glycosylated proteins. The normal commensal colonic microbiota has mucolytic activity and is capable of releasing the monosaccharides contained in mucins, which can then be used as carbon sources by pathogens such as Enterohemorrhagic Escherichia coli (EHEC). EHEC can regulate the expression of some of its virulence factors through environmental sensing of mucus-derived sugars, but its implications regarding its main virulence factor, Shiga toxin type 2 (Stx2), among others, remain unknown. In the present work, we have studied the effects of five of the most abundant mucolytic activity-derived sugars, Fucose (L-Fucose), Galactose (D-Galactose), N-Gal (N-acetyl-galactosamine), NANA (N-Acetyl-Neuraminic Acid) and NAG (N-Acetyl-D-Glucosamine) on EHEC growth, adhesion to epithelial colonic cells (HCT-8), and Stx2 production and translocation across a polarized HCT-8 monolayer. We found that bacterial growth was maximum when using NAG and NANA compared to Galactose, Fucose or N-Gal, and that EHEC adhesion was inhibited regardless of the metabolite used. On the other hand, Stx2 production was enhanced when using NAG and inhibited with the rest of the metabolites, whilst Stx2 translocation was only enhanced when using NANA, and this increase occurred only through the transcellular route. Overall, this study provides insights on the influence of the commensal microbiota on the pathogenicity of E. coli O157:H7, helping to identify favorable intestinal environments for the development of severe disease.
Assuntos
Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Escherichia coli O157 , Proteínas de Escherichia coli , Muco , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Expectorantes/metabolismo , Fucose/metabolismo , Galactose , Microbioma Gastrointestinal , Humanos , Intestinos/metabolismo , Intestinos/microbiologia , Mucinas/metabolismo , Muco/imunologia , Muco/metabolismo , Toxina Shiga II/metabolismo , Virulência , Fatores de Virulência/metabolismoRESUMO
Background: Diarrheagenic E. coli (DEC) pathogenicity relies on the interaction of bacteria with the host's gut environment, which is regulated by the resident microbiota. Previously, we identified indicative bacterial species of gut microbiota in DEC-positive stool samples from children. Here, we evaluated the role of two indicative species, Citrobacter werkmanii (CW) and Escherichia albertii (EA), in the virulence of two DEC pathotypes, Shiga toxin-producing (STEC) and enteroaggregative (EAEC) Escherichia coli. Methods: We determined the effect of supernatants obtained from CW and EA cultures on the gene expression of STEC strain 86-24 and EAEC strain 042 by RNA-seq analysis. We evaluated IL-8 secretion from T84 cells infected with these DEC strains in the presence or absence of the supernatant from EA. The effect of the supernatant from EA on the growth and adherence of STEC and EAEC to cells was also evaluated. Finally, we studied the effect of the EA supernatant on the STEC-induced inflammation mediated by the long polar fimbriae (Lpf) in T84 cells and the expression of plasmid-encoded toxin (Pet) in EAEC. Results: RNA-seq analysis revealed that several virulence factors in STEC and EAEC were upregulated in the presence of supernatants from CW and EA. Interestingly, an increase in the secretion of IL-8 was observed in cells infected with STEC or EAEC in the presence of a supernatant from EA. Similar results were observed with the supernatants obtained from clinical strains of E. albertii. The supernatant from EA had no effect on the growth of STEC and EAEC, or on the ability of these DEC strains to adhere to cells. We found that Pet toxin in EAEC was upregulated in the presence of a supernatant from EA. In STEC, using mutant strains for Lpf fimbriae, our data suggested that these fimbriae might be participating in the increase in IL-8 induced by STEC in cells in the presence of a supernatant from EA. Conclusion: Supernatant obtained from an indicative species of DEC-positive diarrhea could modulate gene expression in STEC and EAEC, and IL-8 secretion induced by these bacteria. These data provide new insights into the effect of gut microbiota species in the pathogenicity of STEC and EAEC.
Assuntos
Infecções por Escherichia coli , Microbioma Gastrointestinal , Criança , Diarreia/microbiologia , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Humanos , Interleucina-8 , Toxina Shiga , VirulênciaRESUMO
Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in the pediatric population. The etiology of HUS is linked to Gram-negative, Shiga toxin (Stx)-producing enterohemorrhagic bacterial infections. While the effect of Stx is focused on endothelial damage of renal glomerulus, cytokines induced by Stx or bacterial lipopolysaccharide (LPS) and polymorphonuclear cells (PMNs) are involved in the development of the disease. PMN release neutrophil extracellular traps (NETs) to eliminate pathogens, although NETs favor platelets (Plts) adhesion/thrombus formation and can cause tissue damage within blood vessels. Since thrombus formation and occlusion of vessels are characteristic of HUS, PMN-Plts interaction in the context of Stx may promote netosis and contribute to the endothelial damage observed in HUS. The aim of this study was to determine the relevance of netosis induced by Stx in the context of LPS-sensitized Plts on endothelial damage. We observed that Stx2 induced a marked enhancement of netosis promoted by Plts after LPS stimulation. Several factors seemed to promote this phenomenon. Stx2 itself increased the expression of its receptor on Plts, increasing toxin binding. Stx2 also increased LPS binding to Plts. Moreover, Stx2 amplified LPS induced P-selectin expression on Plts and mixed PMN-Plts aggregates formation, which led to activation of PMN enhancing dramatically NETs formation. Finally, experiments revealed that endothelial cell damage mediated by PMN in the context of Plts treated with LPS and Stx2 was decreased when NETs were disrupted or when mixed aggregate formation was impeded using an anti-P-selectin antibody. Using a murine model of HUS, systemic endothelial damage/dysfunction was decreased when NETs were disrupted, or when Plts were depleted, indicating that the promotion of netosis by Plts in the context of LPS and Stx2 plays a fundamental role in endothelial toxicity. These results provide insights for the first time into the pivotal role of Plts as enhancers of endothelial damage through NETs promotion in the context of Stx and LPS. Consequently, therapies designed to reduce either the formation of PMN-Plts aggregates or NETs formation could lessen the consequences of endothelial damage in HUS.