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Doctoral Thesis
DOI
10.11606/T.10.2017.tde-29032017-161521
Document
Author
Full name
Rosely Gioia Martins Di Chiacchio
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2017
Supervisor
Committee
Gorniak, Silvana Lima (President)
Knöbl, Terezinha
Menão, Marcia Cristina
Piazza, Roxane Maria Fontes
Sá, Lilian Rose Marques de
Title in Portuguese
Caracterização fenotípica e genotípica de Escherichia coli diarreiogênicas isoladas de psitacídeos de pequeno porte criados como "pets": análise dos riscos zoonóticos
Keywords in Portuguese
Escherichia coli
EPEC
Psitacídeos
STEC
Zoonoses
Abstract in Portuguese
Dentre os psitacídeos criados como pets, destacam-se as calopsitas (Nymphicus hollandicus), os periquitos-australianos (Melopsittacus undulatus) e agapornis (Agapornis spp.). Os benefícios decorrentes da presença destas aves em ambiente doméstico são reconhecidos, porém pouco se conhece sobre os riscos de transmissão de zoonoses. E. coli pode ser isolada em diversos sítios do corpo humano e animal. Alguns sorotipos são considerados patogênicos e, nestes casos, a infecção pode estar associada à ocorrência de diversas manifestações clínicas. O presente estudo tem como objetivo a caracterização fenotípica e genotípica de Escherichia coli diarreiogênicas isoladas de pequenos psitacídeos mantidos como pets. 171 amostras de fezes de aves domiciliadas na cidade de São Paulo foram colhidas com auxílio de suabes estéreis, sendo 67 de calopsitas, 59 de periquitos australianos e 45 agapornis. Das 171 amostras, foram identificadas 42 colônias de Escherichia coli (22 de calopsitas, 10 de periquitos e 10 de agapornis), submetidas à extração de DNA e pesquisa dos genes eae, bfpA, stx1, stx2, LT, ST e aggR ; aaiA ; aatA ; aaiG pela Reac?a?o em Cadeia da Polimerase (PCR). Os resultados apontaram positividade em dez isolados: eae+, bfpA+, Stx2f+ de três calopsitas e cinco periquitos australianos (8/10); ST de uma calopsita (1/10) e eae+ de um periquito australiano (1/10). As oito estirpes de STEC/tEPEC foram classificadas como O137:HNM e nenhuma das estirpes exibiu um perfil de multirresistência. Apenas um isolado foi positivo para formação de biofilme (DO de 0,064), sendo classificado como fracamente aderente. A análise filogenética dos 4 grupos de Clermont et al. (2013) classificou três estirpes como B2, duas como F, e uma como Clado II. Duas estirpes foram classificadas como "não tipável". Os resultados para o AFLP foram: 7/8 estirpes (três de calopsitas e quatro de periquitos) foram agrupadas em um mesmo cluster, apresentando 90-100% de similaridade. Os resultados do sequenciamento de fragmentos internos de genes house-keeping - Multi-locus Sequence Typing (MLST) revelou que as oito estirpes pertenciam ao ST 3072. Neste estudo, nenhuma das estirpes apresentou atividade hemolítica, mas produziram toxinas in vitro. Aderiram in vitro em células HeLa e causaram lesões A/E em modelo de alça intestinal de coelho. As estirpes induziram processo inflamatório e aumento de muco intestinal. Os dados deste trabalho revelaram o potencial zoonótico dos híbridos STEC/tEPEC sorogrupo O137:HNM; ST 3072 isolados de psitacídeos de pequeno porte criados como "pet".
Title in English
Phenotypic and genetic characterization of diarrheogenic Escherichia coli isolated from small parrot created as pets: analysis of zoonotic risks
Keywords in English
Escherichia coli
EPEC
Psittacidae
STEC
Zoonoses
Abstract in English
Among the parrots (psittacidae family) kept as pets, the cockatiels (Nymphicus hollandicus), budgerigars (Melopsittacus undulatus) and lovebirds (Lovebirds spp.) stand out. Although the benefits from the presence of these birds in a household environment are acknowledged, little is known about the risks of transmission of zoonoses. E. coli can be isolated in various sites of the human and animal body. Some serotypes are considered as pathogenic, and in such cases, the infection may be associated with the occurrence of several clinical manifestations. The aim of the present study is to characterize, phenotypically and genotypically, diarrheagenic Escherichia coli, isolated from the small parrots kept as pets. One hundred and seventy-one fecal samples of birds living in households in the city of Sa?o Paulo were collected with the aid of sterile swabs, with 67 being from cockatiels, 59 from budgerigars, and 45 from lovebirds. From the 171 samples, 42 (22 from cockatiels, 10 from budgerigars and 10 from lovebirds) Escherichia coli colonies were identified, which had their DNA extracted, and the eae, bfpA, stx1, stx2, LT, ST and aggR; aaiA; aatA; aaiG genes were investigated, through Polymerase Chain Reaction (PCR). The results indicated positivity in ten isolates: eae+, bfpA+, Stx2f+ from three cockatiels and five budgerigars (8/10), ST from one cockatiel (1/10), and eae+ from one budgerigar (1/10). The eight STEC/tEPEC lineages were classified as O137:HNM, and none of the lineages showed a multiresistance antibiotic profile. Only one isolate was positive for biofilm formation (DO, 0.064), which was classified as weakly adherent. The phylogenetic analysis of the 4 groups by Clermont et al. (2013) classified three lineages as B2, two as F, and one as Clade II. Two lineages were classified as "non-typeable." The results for AFLP were: 7/8 lineages (three of cockatiels and four of budgerigars) were clustered together, with 90-100% of similarity being observed. The results of the internal fragment sequencing of house- keeping genes - Multi-locus Sequence Typing (MLST) revealed that the eight lineages belonged in to ST 3072. In this study, although none of the lineages showed hemolytic activity, they produced toxins in vitro. They adhered, in vitro, to HeLa cells and caused A/E lesions in an intestinal loop model of rabbits. The lineages induced an inflammatory process and an increase of intestinal mucus. This study data revealed the zoonotic potential of STEC/tEPEC hybrids, O137:HNM; ST 3072 serogroup, isolated from small-sized parrots kept as pets
 
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Publishing Date
2017-06-01
 
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