O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

O55 Polysaccharides Are Good Antigen Targets for the Formulation of Vaccines against O55 STEC and Capsulated aEPEC Strains.

The serogroup O55 of E. coli is composed of strains whose mechanisms of virulence are different from each other. Since the O55 polysaccharides are present in all E. coli O55 strains, and so are the polymers that compose the capsule of O55 atypical enteropathogenic E. coli (aEPEC), it was investigated whether anti-O55 antibodies were able to help the innate immune system to eliminate capsulated aEPEC and Shiga toxin-producing E. coli (STEC) belonging to the serogroup O55. The results demonstrate that the capsule of EPEC was able to inhibit the deposition of C3b on the bacterial surface and, as a consequence, their lysis by the alternative pathway of the complement system. However, in the presence of antibodies, the ability of the complement to lyse these pathogens was restored. It was also observed that macrophages were able to ingest EPEC and STEC, but they were only able to kill the ingested pathogens in the presence of antibodies. Anti-O55 antibodies were also able to inhibit aEPEC and STEC O55 adherence to human epithelial cells. In summary, the results demonstrated that the O55 polysaccharides have the potential to induce an effective humoral immune response against STEC and EPEC, indicating that they are good antigen targets to be used in vaccine formulations against these pathogens.

O55 polysaccharides are good antigen targets for the formulation of vaccines against O55 STEC and capsulated aEPEC strains

The serogroup O55 of E. coli is composed of strains whose mechanisms of virulence are different from each other. Since the O55 polysaccharides are present in all E. coli O55 strains, and so are the polymers that compose the capsule of O55 atypical enteropathogenic E. coli (aEPEC), it was investigated whether anti-O55 antibodies were able to help the innate immune system to eliminate capsulated aEPEC and Shiga toxin-producing E. coli (STEC) belonging to the serogroup O55. The results demonstrate that the capsule of EPEC was able to inhibit the deposition of C3b on the bacterial surface and, as a consequence, their lysis by the alternative pathway of the complement system. However, in the presence of antibodies, the ability of the complement to lyse these pathogens was restored. It was also observed that macrophages were able to ingest EPEC and STEC, but they were only able to kill the ingested pathogens in the presence of antibodies. Anti-O55 antibodies were also able to inhibit aEPEC and STEC O55 adherence to human epithelial cells. In summary, the results demonstrated that the O55 polysaccharides have the potential to induce an effective humoral immune response against STEC and EPEC, indicating that they are good antigen targets to be used in vaccine formulations against these pathogens.

Virulence Profiling and Molecular Typing of Shiga Toxin-Producing E. coli (STEC) from Human Sources in Brazil.

Since no recent data characterizing Shiga toxin-producing E. coli (STEC) from human infections in Brazil are available, the present study aimed to investigate serotypes, stx genotypes, and accessory virulence genes, and also to perform pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) of 43 STEC strains recovered from 2007 to 2017. Twenty-one distinct serotypes were found, with serotype O111:H8 being the most common. However, serotypes less frequently reported in human diseases were also found and included a hybrid STEC/ETEC O100:H25 clone. The majority of the strains carried stx1a as the sole stx genotype and were positive for the eae gene. Regarding the occurrence of 28 additional virulence genes associated with plasmids and pathogenicity islands, a diversity of profiles was found especially among the eae-harboring strains, which had combinations of markers composed of up to 12 distinct genes. Although PFGE analysis demonstrated genetic diversity between serotypes such as O157:H7, O111:H8, O26:H11, O118:H16, and O123:H2, high genetic relatedness was found for strains of serotypes O24:H4 and O145:H34. MLST allowed the identification of 17 distinct sequence types (STs) with ST 16 and 21 being the most common ones. Thirty-five percent of the strains studied were not typeable by the currently used MLST approach, suggesting new STs. Although STEC O111:H8 remains the leading serotype in Brazil, a diversity of other serotypes, some carrying virulence genes and belonging to STs incriminated as causing severe disease, were found in this study. Further studies are needed to determine whether they have any epidemiological relevance.

Frequência laboratorial e resistência antimicrobiana de enteropatógenos bacterianso isolados em crianças na primeira infância na região do ABC Paulista, 2015 - 2016 / Laboratory frequency and antimicrobial resistance of the enteropathogens bacteria isolated from infants at early childhood in the region of ABC Paulista, 2015 - 2016

O objetivo deste estudo foi avaliar o perfil dos enteropatógenos bacterianos isolados em crianças menores de 5 anos durante casos de diarreia em instituições de 4 municípios do Estado de São Paulo, durante 2015 e 2016. A coleta das fezes foi realizada em 107 crianças, 78 (72,9%) crianças com diarreia e 29 (27,1%) crianças sem diarreia. A metodologia foi coprocultura, identificação bacteriana e teste de sensibilidade aos antimicrobianos. Quarenta e seis das 107 (43%) amostras clínicas apresentaram crescimento de enteropatógenos. Amostras de Escherichia coli enteropatogênicas (EPEC), Escherichia coli enteroagregativas (EAEC) e Salmonella enterica subsp houtenae foram as mais frequentemente isoladas entre as crianças. Do total de crianças estudadas, três delas apresentaram co-infecção por 2 agentes etiológicos diferentes: EPEC/EAEC e Salmonella enterica subsp houtenae/EAEC. A maior ocorrência entre os 49 agentes etiológicos isolados foi EPEC (24/49, 49%), seguido de EAEC (14/49, 28,6%). Duas amostras de EPEC pertencentes ao sorotipo O109:H21 foram sensíveis aos antimicrobianos testados, enquanto outras duas pertencentes ao sorotipo O156:H1 foram resistentes a gentamicina e a amicacina e estreptomicina, respectivamente. Duas amostras de EAEC pertencentes ao mesmo sorotipo O80:H10 e duas EAEC O15:H2 apresentaram multirresistência, pelo menos, ao ácido nalidíxico, sulfametoxazol e tetraciclina. Podemos sugerir que crianças frequentadoras de três instituições diferentes, que apresentaram agregados de casos de diarreia sugestivo de surto, eram portadoras de clones bacterianos de amostras de EPEC ou EAEC, por pertencerem ao mesmo sorotipo e com semelhante perfil de sensibilidade. Nossos resultados são preocupantes e mostram que a vigilância epidemiológica antimicrobiana constante deve ser garantida para o monitoramento do surgimento de clones resistentes e para estabelecer estratégias para a prevenção e c ontrole de surtos e epidemias

This study aimed at evaluating the profile of enteropathogens isolated from children under 5 years of age, during the occurrence of cases of diarrhe a in the institutions of four municipalities in the State of São Paulo in 2015 and 2016. Feces samples were collected from 107 children, 78 (72.9%) with diarrhea and 29 (27.1%) without. The employed methodologies were copro-culture, bacterial identification and antimicrobial susceptibility testing. Forty-six (46%) of 107 clinical samples presented growth of enteropathogens. Enteropathogenic Escherichia coli (EPEC), enteroaggregative Escherichia coli (EAEC) and Salmonella enterica subsp houtenae were the mostly frequent isolated from children. Of the total number of the studied children, three of them presented co-infection with two etiological agents: EPEC/EAEC and Salmonella enterica subsp houtenae/EAEC. The highest occurrence among the isolated etiologic agents was EPEC (24/49, 49%), followed by EAEC (14/49, 28.6%). Two EPEC strains belonged to the O109:H21 serotype were sensitive to the tested antimicrobials, whereas two belonging to the O156:H1 serotype were resistant to gentamicin and amicacin and streptomycin, respectively. Two EAEC strains of the same serotype O80:H10 and two EAEC O15:H2 presented multi-resistance at least to nalidixic acid, sulfamethoxazole and tetracycline. It may suggest that the children attending three different institutions, who had clusters of cases of diarrhoea carried the bacterial clones of EPEC or EAEC strains, because they belonged to the same serotype and show a similar sensitivity profile. The results found in the present study are worrying and they show that the constant antimicrobial epidemiological surveillance should be ensured for monitoring the emergence of resistant clones, and for establishing strategies for preventing and controlling the outbreaks and epidemics.

Development and validation of shiga toxin-producing escherichia coli immunodiagnostic assay

Shiga toxin (Stx)–producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are important pathogens involved in diarrhea, which may be complicated by hemorrhagic colitis and hemolytic uremic syndrome, the leading cause of acute renal failure in children. Early diagnosis is essential for clinical management, as an antibiotic treatment in STEC infections is not recommended. Previously obtained antibodies against Stx1 and Stx2 toxins were employed to evaluate the sensitivity and specificity of the latex Agglutination test (LAT), lateral flow assay (LFA), and capture ELISA (cEIA) for STEC detection. The LAT (mAb Stx1 plus mAb stx2) showed 99% sensitivity and 97% specificity. Individually, Stx1 antibodies showed 95.5% and 94% sensitivity and a specificity of 97% and 99% in the cEIA and LFA assay, respectively. Stx2 antibodies showed a sensitivity of 92% in both assays and a specificity of 100% and 98% in the cEIA and LFA assay, respectively. These results allow us to conclude that we have robust tools for the diagnosis of STEC infections.

Development and validation of shiga toxin-producing escherichia coli immunodiagnostic assay

Shiga toxin (Stx)–producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are important pathogens involved in diarrhea, which may be complicated by hemorrhagic colitis and hemolytic uremic syndrome, the leading cause of acute renal failure in children. Early diagnosis is essential for clinical management, as an antibiotic treatment in STEC infections is not recommended. Previously obtained antibodies against Stx1 and Stx2 toxins were employed to evaluate the sensitivity and specificity of the latex Agglutination test (LAT), lateral flow assay (LFA), and capture ELISA (cEIA) for STEC detection. The LAT (mAb Stx1 plus mAb stx2) showed 99% sensitivity and 97% specificity. Individually, Stx1 antibodies showed 95.5% and 94% sensitivity and a specificity of 97% and 99% in the cEIA and LFA assay, respectively. Stx2 antibodies showed a sensitivity of 92% in both assays and a specificity of 100% and 98% in the cEIA and LFA assay, respectively. These results allow us to conclude that we have robust tools for the diagnosis of STEC infections.

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl.

Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.

Escherichia coli O26 and O113:H21 on Carcasses and Beef from a Slaughterhouse Located in Mato Grosso, Brazil.

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.

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl

Abstract Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.

Novel hybrid of Typical Enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) emerging from pet birds

Exotic psittacine birds have been implicated as reservoir of diarrheagenic Escherichia coli (E. coli), including enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Here, we present a genotypic and phenotypic characterization of typical EPEC/STEC hybrid strains isolated from exotic psittacine birds. The strains were positive for eae, bfpA, and stx2f genes, belong to serotype O137:H6 and ST2678. Two strains were subject to whole genome sequencing, confirming the presence of the virulence factors of both E. coli pathotypes. Phenotypical in vitro tests confirmed their ability to adhere to HeLa cells and cause cytotoxicity to Vero cells. The rabbit ileal loop assays showed the attaching and effacing lesion, in addition to inflammatory process and overproduction of intestinal mucus. This is the first report of hybrid typical EPEC/STEC (O137:H6/ST2678) strains isolated from companion psittacine birds and the results suggest zoonotic risks.

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl

Abstract Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.

Novel hybrid of Typical Enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) emerging from pet birds

Exotic psittacine birds have been implicated as reservoir of diarrheagenic Escherichia coli (E. coli), including enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Here, we present a genotypic and phenotypic characterization of typical EPEC/STEC hybrid strains isolated from exotic psittacine birds. The strains were positive for eae, bfpA, and stx2f genes, belong to serotype O137:H6 and ST2678. Two strains were subject to whole genome sequencing, confirming the presence of the virulence factors of both E. coli pathotypes. Phenotypical in vitro tests confirmed their ability to adhere to HeLa cells and cause cytotoxicity to Vero cells. The rabbit ileal loop assays showed the attaching and effacing lesion, in addition to inflammatory process and overproduction of intestinal mucus. This is the first report of hybrid typical EPEC/STEC (O137:H6/ST2678) strains isolated from companion psittacine birds and the results suggest zoonotic risks.

Population Structure Analysis of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates from Brazil Reveals Predominance of Clonal Complexes 1, 15, and 79.

The population structure of 71 carbapenem-resistantAcinetobacter baumanniiclinical isolates from several hospitals in Brazil was investigated by ApaI pulsed-field gel electrophoresis,blaOXA-51-like subtyping, and multilocus sequence typing (Institute Pasteur scheme). In addition to the predominance of strains carryingblaOXA-23, we detected the presence ofblaOXA-72andblaOXA-231 We observed a predominance of clonal complex 1 (CC1), CC15, and CC79 and representative strains of the worldwide-disseminated international clone I.