Theresa.J.Ochoa@uth.tmc.edu / Article / BACKGROUND: Diarrheagenic Escherichia coli strains are being recognized as important pediatric enteropathogens worldwide. However, it is unclear whether there are differences in age-related susceptibility to specific strains, especially among infants. METHODS: We conducted a passive surveillance cohort study of diarrhea that involved 1034 children aged 2-12 months in Lima, Peru. Control stool samples were collected from randomly selected children without diarrhea. All samples were analyzed for common enteric pathogens and for diarrheagenic E. coli with use of multiplex real-time polymerase chain reaction. RESULTS: The most frequently isolated pathogens in 1065 diarrheal episodes were diarrheagenic E. coli strains (31%), including enteroaggregative (15.1%) and enteropathogenic E. coli (7.6%). Diarrheagenic E. coli, Campylobacter species, and rotavirus were more frequently isolated from infants aged >or=6 months. Among older infants, diffusely adherent E. coli and enterotoxigenic E. coli were more frequently isolated from diarrheal samples than from control samples (P <.05). Children aged >or=6 months who were infected with enterotoxigenic E. coli had a 4.56-fold increased risk of diarrhea (95% confidence interval, 1.20-17.28), compared with younger children. Persistent diarrhea was more common in infants aged <6 months (13.5% vs 3.6%; P <.001). Among children with diarrheagenic E. coli-positive samples, coinfections with other pathogens were more common in children with diarrhea than in control children (40.1% vs 15.6%; P <.001). CONCLUSIONS: Diarrheagenic E. coli strains were more frequently isolated in samples from older infants. In this setting with high frequency of pathogen exposure and high frequency of breastfeeding, we hypothesize that the major age-related differences result from decreased exposure to milk-related protective factors and from increased exposure to contaminated food and water.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Diarrheagenic Escherichia coli (E. coli) strains are common etiological agents of diarrhea. Diarrheagenic E. coli are classified into enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC or enterohemorrhagic E. coli [EHEC]), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffuse-adherent E. coli (DAEC), and adherent invasive E. coli (AIEC). In addition to encoding toxins that cause diarrhea, diarrheagenic E. coli have evolved numerous strategies to interfere with host defenses. In the first project, we identified an ETEC-secreted factor (ESF) that blocked TNF-induced NF-[kappa]B activation. One of the consequences of TNF-induced NF-[kappa]B activation is the production of pro-inflammatory cytokines that help to eliminate pathogens. Modulation of NF-[kappa]B signaling may promote ETEC colonization of the host small intestine. In this study, we fractionated ETEC supernatants and identified flagellin as necessary and sufficient for blocking the degradation of the NF-[kappa]B inhibitor I[kappa]B[alpha] in response to TNF[alpha]. In the second project, we attempted to identify an ETEC cAMP importer. ETEC diarrhea leads to cAMP release into the lumen of the small intestine. cAMP is a key secondary messenger that regulates ETEC adhesin expression. We hypothesized that a cAMP importer is present in ETEC, accounting for its hypersensitivity to extracellular cAMP. We used Tn5 transposome-mediated mutagenesis to construct a mutant library and screen for cAMP-hyporesponsive mutants. However, none of the 17,956 mutants we screened were cAMP-hyporesponsive. In the third project, we focused on gut microbiota and the T3SS effector NleH. We used the mouse-specific pathogen C. rodentium and transplanted performed microbiota between different mouse strains. We evaluated microbiota populations as a function of infection with WT and [Delta]nleH C. rodentium strains before and after microbiota transplantation. Microbiota transfer altered the resistance to WT C. rodentium infection in C57BL/10ScNJ mice and the NleH effector promoted host resistance to C. rodentium.

A qualidade das águas marinhas destinadas a recreação de contato primário pode ser afetada por fontes de poluição pontuais e não pontuais. Escherichia coli é membro comensal da microbiota intestinal de humanos e animais endotérmicos e quando presente no ambiente marinho indica contaminação fecal recente. Embora a maioria das cepas desta espécie não seja patogênica para o homem, existem isolados virulentos e/ ou resistentes aos antibióticos. E.coli pode ser caracterizada em grupos filogenéticos - GF (A, B1, B2, C, D, E, F e clado I) e em categorias diarreiogênicas (ETEC, EPEC, EHEC, EIEC, EAEC e DAEC). Este trabalho objetivou caracterizar 99 isolados de E.coli obtidos de amostras água do mar de três regiões costeiras do estado de São Paulo com diferentes níveis de contaminação [Ubatuba (UBA), oligotrófico (n=20); Baixada Santista (BS), eutrófico (n=30) e canal de São Sebastião (CSS), mesotrófico (n=49)], frente à susceptibilidade aos antibióticos, GF, genes associados à virulência (GAV) e assim estimar os riscos microbiológicos à balneabilidade dos locais de coleta. A susceptibilidade a ampicilina (AMP), amoxicilina- ácido clavulânico (AMC), amicacina (AMI), cefotaxima (CTX), cefuroxima (CRX), ciprofloxacino (CIP), cloranfenicol (CLO), imipenema (IPM), piperacilina-tazobactam (PPT), sulfametoxazol-trimetropima (SUT) e tetraciclina (TET) foi determinada pelo método de disco difusão. A classificação em GF foi realizada por PCR utilizando duas metodologias (CLERMONT et al., 2000 e 2011). Os GAV (stx1, stx2, eae, bfpA, aggR, elt, esth, estp, invE e astA) foram pesquisados por PCR. Os resultados foram utilizados na avaliação do risco microbiológico por QMRA. Dezenove isolados foram resistentes a AMP, sendo UBA (3%), BS (33%) e CSS (16%). Dezessete isolados foram resistentes a TET: UBA (20%), BS (23%) e CSS (12%); 14 isolados foram resistentes a SUT: UBA e CSS (10%) e BS (23%). As frequências (%) dos GF (Clermont ET AL., 2000) comensais A e B1 foram, respectivamente (55 e 15 em UBA; 63 e 13 na BS; 69 e 8 no CSS); já as frequências (%) dos GF virulentos B2 e D foram, respectivamente, 5 e 25 em UBA; 7 e 16 na BS; 4 e 18 no CSS. As frequências (%) dos GF (CLERMONT et al., 2013) comensais A, B1 e C foram: respectivamente: (15, 15 e 5 em UBA; 20, 3 e 3 na BS; 2, 10 e 2 no CSS), já os grupos mais virulentos (B2, D, E e F) foram, respectivamente: (15, 15, 0 e 10 em UBA; 13, 27, 0 e 10 na BS; 10, 33, 8 e 18 no CSS); as frequências do clado I em UBA, BS e CSS foram respectivamente: (15, 23 e 12). Os GAV detectados em UBA foram: astA (15%) e bfpA (5%); BS: esth (3%) e astA (30%) e no CSS: stx1 (2%), eae (4%), estp (2%) e astA (47). Embora não tenha sido detectado risco à balneabilidade, observou-se isolados resistentes e GF virulentos em todos os pontos de coleta, dessa forma, esses ambientes devem ser conservados para assegurar a saúde ambiental e humana, além disso a QMRA não representou o risco total de todos micro-organismos e patógenos oportunistas presentes nessas regiões. / The quality of seawater intended for bathing can be affected by sources of point and non-point pollution. In this pollutants may be Escherichia coli. E.coli is a commensal member of the intestinal tract of humans and endothermic animals, and when present in the marine environment indicates recent fecal contamination. Although most strains are harmless, there are virulent and/or resistant to antibiotics. E. coli can be evaluated by phylogenetic groups - PG (A, B1, B2, C, D, E, F, clade 1) and diarrheagenic categories (ETEC, EPEC, EHEC, EIEC, EAEC and DAEC). The aim of the present study was to characterize 99 E. coli isolated from seawater samples from three coastal regions of the state of São Paulo with different levels of contamination [Ubatuba (UBA), oligotrophic (n=20); Santos (BS), eutrophic (n=30) and São Sebastião Channel (CSS), mesotrophic (n=49)], in regard to antibiotics susceptibility, PG, virulence genes (VGs) associated with diarrheagenic pathotypes and to estimate the microbiological risks to bathing. The susceptibility to ampicillin (AMP), amoxicillin-clavulanic acid (AMC), amikacin (AMI), cefotaxime (CTX), cefuroxime (CRX), ciprofloxacin (CIP), chloramphenicol (CLO), imipenem (IPM), piperacillin-tazobactam (PPT), trimethoprim-sulfamethoxazole (SUT) and tetracycline (TET) was determined by disk diffusion. The classification in regard to GF was performed by PCR using two methodologies (2000 and 2013). The VGs (stx1, stx2, eae, bfpA, aggR, elt, esth, estp, invE and astA) were screened by PCR and the results were used to evaluate the microbiological risk by QMRA. Nineteen isolates were resistant to AMP: UBA (3%), BS (33%) and CSS (16%). Seventeen isolates were resistant to TET: UBA (20%), BS (23%) and CSS (12%); 14 isolates were resistant to the SUT: UBA and CSS (10%) and BS (23%). By the methodology of 2000, the frequencies (%) of commensals PG and B1 were, respectively, (55 and 15 in UBA; 63 and 13 in BS; 69 and 8 in CSS); while the frequencies (%) of virulent PG, B2 and D were, respectively (5 and 25 in UBA; 7 and 16 in BS; 4 and 18 in CSS). By the methodology of 2013, the frequencies (%) of PG commensals A, B1 and C were, respectively: (15, 15 and 5 in UBA; 20, 3 and 3 in BS; 2, 10 and 2 in CSS), on the other hand, the most virulent groups (B2, D, E and F) were, respectively: (15, 15, 0 and 10 in UBA; 13, 27, 0 and 10 in BS; 10, 33, 8 and 18 in CSS); clade 1 frequencies in UBA, BS and CSS were, respectively (15, 23 and 12). The VGs were detected in UBA; astA (15%) and bfpA (5%); BS: esth (3%) and astA (30%) and CSS: stx1 (2%), eae (4%), estp (2%) and astA (47). Although no microbiological hazard was detected to bathing, was observed resistant isolates and virulent PG, thereby these environments should be preserved to ensure the environmental and human health, moreover the QMRA did not represent the overall risk of all microorganisms and opportunistic pathogens present in these regions.

De todas as influências exógenas que possam alterar a microbiota intestinal, os antimicrobianos são capazes de causar as mais rápidas e drásticas mudanças. O impacto da exposição aos antimicrobianos na microbiota intestinal causa diminuição no número de microrganismos ou mesmo supressão, dependendo do antimicrobiano utilizado, da dose e do tempo de exposição. Assim, o objetivo deste estudo foi analisar de forma comparativa alguns microrganismos que compõem a microbiota fecal de crianças com e sem antibioticoterapia em idade escolar; bem como avaliar a susceptibilidade aos antimicrobianos e os genes de resistência envolvidos. Foram coletadas amostras fecais não diarreicas de 30 crianças sem antibiótico (controle) e 31 de crianças com antibioticoterapia. Na análise quantitativa foi observada redução no número de cópias por g/fezes de: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii e do filo Firmicutes nas amostras das crianças com antibióticos em relação ao grupo controle, exceto para Lactobacillus spp. e P. distasonis que apresentaram quantificação maior no grupo antibióticos quando comparados com o controle. E. coli foi isolada em 26 (86,7%) crianças controles e em 23 (74,2%) tratadas com antibióticos. A resistência foi verificada para diversas drogas no grupo controle exceto para ciprofloxacina, meropenem e tigeciclina; entretanto o grupo com antibioticoterapia apresentou elevada resistência para todas as drogas avaliadas, caracterizando os isolados desse estudo como MDR. Todos os isolados do grupo controle e antibióticos albergaram diversos genes de resistência, entretanto o gene blaKPC foi o único não detectado nos isolados do grupo controle. Desta forma, nossos dados demonstram que a antibioticoteria causa alterações qualitativas e quantitativas na microbiota intestinal; além disso, a elevada resistência as diversas classes de antimicrobianos das cepas de E. coli, bem como a presença de diversos genes de resistência ressalta a importância de cepas comensais serem MDR e albergarem esses genes. / Of all the exogenous influences that may alter the intestinal microbiota, antimicrobial agents are able to cause the more rapid and dramatic changes. The impact of exposure to antimicrobial agents on intestinal microbiota causes a decrease in the number of certain genera and species, depending on the antimicrobial agent used, dose and duration of exposure. Thus, the aim of this study was to analyze comparatively some microorganisms that composing the fecal microbiota of children with and without antibiotic therapy in school age; and evaluates the antimicrobial susceptibility and resistance genes involved. Stool samples (not diarrhea) were collected of 30 children without antibiotic (control) and 31 children with antibiotic therapy. In quantitative analysis was observed decrease in the number of copies per g/feces: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii and the phylum Firmicutes in samples of children with antibiotic therapy in relation to control group, except Lactobacillus spp. and P. distasonis that showed a higher quantification in the antibiotics group when compared with control group. E. coli was isolated in 26 (86.7 %) children controls and in 23 (74.2 %) children treated with antibiotics. The resistance was verified for several drugs in the control group except for ciprofloxacin, meropenem and tigecycline; however the group with antibiotic therapy showed high resistance to all drugs evaluated, characterizing isolates of this study as MDR. All isolates from control group and antibiotics harbored several resistance genes, however blaKPC gene was the only one not detected in isolates from the control group. Thus, our data demonstrate that the antibiotic therapy cause qualitative or quantitative changes in intestinal microbiota leading to a decrease in the diversity and the elimination of microorganisms; in addition, the high resistance the various classes of antimicrobial of the strains of E. coli, as well as the presence of several genes of resistance highlights the importance of commensal strains are MDR and harboring these genes.

De todas as influências exógenas que possam alterar a microbiota intestinal, os antimicrobianos são capazes de causar as mais rápidas e drásticas mudanças. O impacto da exposição aos antimicrobianos na microbiota intestinal causa diminuição no número de microrganismos ou mesmo supressão, dependendo do antimicrobiano utilizado, da dose e do tempo de exposição. Assim, o objetivo deste estudo foi analisar de forma comparativa alguns microrganismos que compõem a microbiota fecal de crianças com e sem antibioticoterapia em idade escolar; bem como avaliar a susceptibilidade aos antimicrobianos e os genes de resistência envolvidos. Foram coletadas amostras fecais não diarreicas de 30 crianças sem antibiótico (controle) e 31 de crianças com antibioticoterapia. Na análise quantitativa foi observada redução no número de cópias por g/fezes de: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii e do filo Firmicutes nas amostras das crianças com antibióticos em relação ao grupo controle, exceto para Lactobacillus spp. e P. distasonis que apresentaram quantificação maior no grupo antibióticos quando comparados com o controle. E. coli foi isolada em 26 (86,7%) crianças controles e em 23 (74,2%) tratadas com antibióticos. A resistência foi verificada para diversas drogas no grupo controle exceto para ciprofloxacina, meropenem e tigeciclina; entretanto o grupo com antibioticoterapia apresentou elevada resistência para todas as drogas avaliadas, caracterizando os isolados desse estudo como MDR. Todos os isolados do grupo controle e antibióticos albergaram diversos genes de resistência, entretanto o gene blaKPC foi o único não detectado nos isolados do grupo controle. Desta forma, nossos dados demonstram que a antibioticoteria causa alterações qualitativas e quantitativas na microbiota intestinal; além disso, a elevada resistência as diversas classes de antimicrobianos das cepas de E. coli, bem como a presença de diversos genes de resistência ressalta a importância de cepas comensais serem MDR e albergarem esses genes. / Of all the exogenous influences that may alter the intestinal microbiota, antimicrobial agents are able to cause the more rapid and dramatic changes. The impact of exposure to antimicrobial agents on intestinal microbiota causes a decrease in the number of certain genera and species, depending on the antimicrobial agent used, dose and duration of exposure. Thus, the aim of this study was to analyze comparatively some microorganisms that composing the fecal microbiota of children with and without antibiotic therapy in school age; and evaluates the antimicrobial susceptibility and resistance genes involved. Stool samples (not diarrhea) were collected of 30 children without antibiotic (control) and 31 children with antibiotic therapy. In quantitative analysis was observed decrease in the number of copies per g/feces: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii and the phylum Firmicutes in samples of children with antibiotic therapy in relation to control group, except Lactobacillus spp. and P. distasonis that showed a higher quantification in the antibiotics group when compared with control group. E. coli was isolated in 26 (86.7 %) children controls and in 23 (74.2 %) children treated with antibiotics. The resistance was verified for several drugs in the control group except for ciprofloxacin, meropenem and tigecycline; however the group with antibiotic therapy showed high resistance to all drugs evaluated, characterizing isolates of this study as MDR. All isolates from control group and antibiotics harbored several resistance genes, however blaKPC gene was the only one not detected in isolates from the control group. Thus, our data demonstrate that the antibiotic therapy cause qualitative or quantitative changes in intestinal microbiota leading to a decrease in the diversity and the elimination of microorganisms; in addition, the high resistance the various classes of antimicrobial of the strains of E. coli, as well as the presence of several genes of resistance highlights the importance of commensal strains are MDR and harboring these genes.

A qualidade das águas marinhas destinadas a recreação de contato primário pode ser afetada por fontes de poluição pontuais e não pontuais. Escherichia coli é membro comensal da microbiota intestinal de humanos e animais endotérmicos e quando presente no ambiente marinho indica contaminação fecal recente. Embora a maioria das cepas desta espécie não seja patogênica para o homem, existem isolados virulentos e/ ou resistentes aos antibióticos. E.coli pode ser caracterizada em grupos filogenéticos - GF (A, B1, B2, C, D, E, F e clado I) e em categorias diarreiogênicas (ETEC, EPEC, EHEC, EIEC, EAEC e DAEC). Este trabalho objetivou caracterizar 99 isolados de E.coli obtidos de amostras água do mar de três regiões costeiras do estado de São Paulo com diferentes níveis de contaminação [Ubatuba (UBA), oligotrófico (n=20); Baixada Santista (BS), eutrófico (n=30) e canal de São Sebastião (CSS), mesotrófico (n=49)], frente à susceptibilidade aos antibióticos, GF, genes associados à virulência (GAV) e assim estimar os riscos microbiológicos à balneabilidade dos locais de coleta. A susceptibilidade a ampicilina (AMP), amoxicilina- ácido clavulânico (AMC), amicacina (AMI), cefotaxima (CTX), cefuroxima (CRX), ciprofloxacino (CIP), cloranfenicol (CLO), imipenema (IPM), piperacilina-tazobactam (PPT), sulfametoxazol-trimetropima (SUT) e tetraciclina (TET) foi determinada pelo método de disco difusão. A classificação em GF foi realizada por PCR utilizando duas metodologias (CLERMONT et al., 2000 e 2011). Os GAV (stx1, stx2, eae, bfpA, aggR, elt, esth, estp, invE e astA) foram pesquisados por PCR. Os resultados foram utilizados na avaliação do risco microbiológico por QMRA. Dezenove isolados foram resistentes a AMP, sendo UBA (3%), BS (33%) e CSS (16%). Dezessete isolados foram resistentes a TET: UBA (20%), BS (23%) e CSS (12%); 14 isolados foram resistentes a SUT: UBA e CSS (10%) e BS (23%). As frequências (%) dos GF (Clermont ET AL., 2000) comensais A e B1 foram, respectivamente (55 e 15 em UBA; 63 e 13 na BS; 69 e 8 no CSS); já as frequências (%) dos GF virulentos B2 e D foram, respectivamente, 5 e 25 em UBA; 7 e 16 na BS; 4 e 18 no CSS. As frequências (%) dos GF (CLERMONT et al., 2013) comensais A, B1 e C foram: respectivamente: (15, 15 e 5 em UBA; 20, 3 e 3 na BS; 2, 10 e 2 no CSS), já os grupos mais virulentos (B2, D, E e F) foram, respectivamente: (15, 15, 0 e 10 em UBA; 13, 27, 0 e 10 na BS; 10, 33, 8 e 18 no CSS); as frequências do clado I em UBA, BS e CSS foram respectivamente: (15, 23 e 12). Os GAV detectados em UBA foram: astA (15%) e bfpA (5%); BS: esth (3%) e astA (30%) e no CSS: stx1 (2%), eae (4%), estp (2%) e astA (47). Embora não tenha sido detectado risco à balneabilidade, observou-se isolados resistentes e GF virulentos em todos os pontos de coleta, dessa forma, esses ambientes devem ser conservados para assegurar a saúde ambiental e humana, além disso a QMRA não representou o risco total de todos micro-organismos e patógenos oportunistas presentes nessas regiões. / The quality of seawater intended for bathing can be affected by sources of point and non-point pollution. In this pollutants may be Escherichia coli. E.coli is a commensal member of the intestinal tract of humans and endothermic animals, and when present in the marine environment indicates recent fecal contamination. Although most strains are harmless, there are virulent and/or resistant to antibiotics. E. coli can be evaluated by phylogenetic groups - PG (A, B1, B2, C, D, E, F, clade 1) and diarrheagenic categories (ETEC, EPEC, EHEC, EIEC, EAEC and DAEC). The aim of the present study was to characterize 99 E. coli isolated from seawater samples from three coastal regions of the state of São Paulo with different levels of contamination [Ubatuba (UBA), oligotrophic (n=20); Santos (BS), eutrophic (n=30) and São Sebastião Channel (CSS), mesotrophic (n=49)], in regard to antibiotics susceptibility, PG, virulence genes (VGs) associated with diarrheagenic pathotypes and to estimate the microbiological risks to bathing. The susceptibility to ampicillin (AMP), amoxicillin-clavulanic acid (AMC), amikacin (AMI), cefotaxime (CTX), cefuroxime (CRX), ciprofloxacin (CIP), chloramphenicol (CLO), imipenem (IPM), piperacillin-tazobactam (PPT), trimethoprim-sulfamethoxazole (SUT) and tetracycline (TET) was determined by disk diffusion. The classification in regard to GF was performed by PCR using two methodologies (2000 and 2013). The VGs (stx1, stx2, eae, bfpA, aggR, elt, esth, estp, invE and astA) were screened by PCR and the results were used to evaluate the microbiological risk by QMRA. Nineteen isolates were resistant to AMP: UBA (3%), BS (33%) and CSS (16%). Seventeen isolates were resistant to TET: UBA (20%), BS (23%) and CSS (12%); 14 isolates were resistant to the SUT: UBA and CSS (10%) and BS (23%). By the methodology of 2000, the frequencies (%) of commensals PG and B1 were, respectively, (55 and 15 in UBA; 63 and 13 in BS; 69 and 8 in CSS); while the frequencies (%) of virulent PG, B2 and D were, respectively (5 and 25 in UBA; 7 and 16 in BS; 4 and 18 in CSS). By the methodology of 2013, the frequencies (%) of PG commensals A, B1 and C were, respectively: (15, 15 and 5 in UBA; 20, 3 and 3 in BS; 2, 10 and 2 in CSS), on the other hand, the most virulent groups (B2, D, E and F) were, respectively: (15, 15, 0 and 10 in UBA; 13, 27, 0 and 10 in BS; 10, 33, 8 and 18 in CSS); clade 1 frequencies in UBA, BS and CSS were, respectively (15, 23 and 12). The VGs were detected in UBA; astA (15%) and bfpA (5%); BS: esth (3%) and astA (30%) and CSS: stx1 (2%), eae (4%), estp (2%) and astA (47). Although no microbiological hazard was detected to bathing, was observed resistant isolates and virulent PG, thereby these environments should be preserved to ensure the environmental and human health, moreover the QMRA did not represent the overall risk of all microorganisms and opportunistic pathogens present in these regions.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

MSc (Microbiology) / Department of Microbiology / See the attached abstract below