Análise do perfil de virulência e epidemiologia molecular de Escherichia coli enteroagregativa (EAEC) isoladas de casos esporádicos de diarreia no Brasil: um estudo retrospectivo de 2010 a 2016
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Data
2020-02-18
Autores
Dias, Regiane Chrysostomo Bitencort
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Universidade Estadual Paulista (Unesp)
Resumo
A Escherichia coli enteroagregativa (EAEC) é um importante agente causador de diarreia aguda e persistente em crianças e adultos em todo o mundo. EAEC é definida como isolados de E. coli que produzem o padrão de aderência agregativa (AA) em células epiteliais (HeLa e/ou HEp-2) cultivadas in vitro. O patotipo EAEC pode ser dividido em típico e atípico com base na presença do gene aggR, que codifica um ativador transcricional, presente apenas no primeiro grupo. O objetivo deste estudo foi caracterizar uma coleção de isolados de EAEC obtidos de pacientes com diarreia durante um período de 7 anos de vigilância epidemiológica (2010-2016). Um total de 220 isolados de EAEC (194 típicas e 26 atípicas) foi classificado nos distintos grupos filogenéticos de E. coli, e caracterizados quanto aos sorotipos (O:H), padrão de aderência produzidos em células HeLa, sensibilidade aos antimicrobianos, e a presença de 25 genes responsáveis por codificarem fatores de virulência no patotipo EAEC. A maioria dos isolados de EAEC foi classificada nos grupos filogenéticos A (44,1%; 97/220) e B1 (21,4%; 47/220). Em relação ao padrão de aderência, observamos que 92,7% (204/220) produziram o padrão AA. Além disso, foram identificados nove isolados (4,1%; 9/220) que produziram a aderência em cadeia (CLA), dos quais seis produziram concomitantemente o padrão AA, além de isolados de EAEC que produzem um padrão de aderência indefinido (1,4%; 3/220) e isolados não aderentes (3,6%; 8/220). Foi identificado somente 0,9% (2/220) de multirresistência entre os isolados de EAEC. Os genes responsáveis por codificar a subunidade principal das cinco fimbrias de adesão agregativa (AAF) foram detectados em 55,7% (108/194) dos isolados de EAEC típica estudados, sendo os genes agg4A e agg5A os mais prevalentes, e igualmente detectados em 16,5% (32/194). Entre os 25 genes que codificam fatores de virulência investigados, aap (95,5%; 210/220), aar (81,8%; 180/220), orf3 (89,5%; 197/220) e pic (57,7%; 127/220) foram os mais frequentes, enquanto doze genes (agg4A, agg5A, pic, aaiA, aaiC, aaiG, aap, orf3, aar, air, capU e shf) foram estatisticamente associados ao subgrupo das EAEC típicas (P<0,05). Os genes afpB, afpD, afpP e afpA2, localizados no operon afp (aggregate-forming pili), bem como seu regulador afpR, foram detectados em 57,7% (15/26) dos isolados de EAEC atípica, mostrando uma associação significativa com esse subgrupo de EAEC (P<0,001). Em conclusão, nosso estudo demonstrou que apesar da heterogeneidade encontrada entre os isolados de EAEC estudados, os genes localizados no operon afp foram estatisticamente associados aos isolados de EAEC atípica, apontando esse conjunto de genes como possíveis novos marcadores de diagnóstico para aumentar a eficiência na identificação desse subgrupo de EAEC.
Enteroaggregative Escherichia coli (EAEC) is an important agent that causes acute and persistent diarrhea in children and adults worldwide. EAEC is defined as E. coli isolates that produce the aggregative adherence pattern (AA) on epithelial cells (HeLa and/or HEp-2) cultured in vitro. The EAEC pathotype can be divided in typical and atypical based on the presence of the aggR gene, which encodes a transcriptional activator, in the former group. The aim of this study was to characterize a collection of EAEC isolates obtained from diarrheal patients over a 7-year period of surveillance (2010-2016). A total of 220 EAEC isolates (194 typical and 26 atypical) were evaluated regarding the phylogenetic classification, serotypes, adherence pattern produced on HeLa cells, susceptibility to antimicrobial drugs and the presence of 25 virulence factor-encoding genes. The majority of the EAEC isolates were assigned to phylogroups A (44.1%; 97/220) and B1 (21.4%; 47/220). Regarding the adherence pattern, was observed that 92.7% (204/220) produced AA. Moreover, we identified nine isolates (4.1%; 9/220) that produced the chain-like adherence (CLA), with six of them producing concomitantly the AA pattern, besides EAEC isolates producing an undefined adherence (1.4%; 3/220) and isolates non-adherent (3.6%; 8/220). Only 0.9% (2/220) of the EAEC isolates studied presented the multidrug resistance phenotype. The genes encoding for the major pilin subunit of all five previously described aggregative adherence fimbriae (AAF) were detected among the EAEC isolates studied (55.7%; 108/194), with agg4A and agg5A being the most frequent, and equally detected in 16.5% (32/194) of the typical EAEC isolates. Among the 25 virulence factor-encoding genes investigated here, aap (95.5%; 210/220), aar (81.8%; 180/220), orf3 (89.5%; 197/220), and pic (57.7%; 127/220) were the most prevalent, while twelve genes (agg4A, agg5A, pic, aap, aaiA, aaiC, aaiG, orf3, aar, air, capU and shf) were statistically more frequent in typical than atypical EAEC (P<0.05). The afpB, afpD, afpP and afpA2 genes, present in the afp operon, and its afpR regulator, were detected in 57.7% (15/26) of the atypical EAEC isolates, showing a statistically significant association with this EAEC subgroup (P<0.001). In conclusion, our study demonstrated that despite the heterogeneity found among the EAEC isolates studied, the genes from the afp operon were statistically associated with the atypical EAEC isolates, pointing this set of genes as a putative new diagnostic marker to be use, in order to increase the efficiency in the identification of this EAEC subgroup.
Enteroaggregative Escherichia coli (EAEC) is an important agent that causes acute and persistent diarrhea in children and adults worldwide. EAEC is defined as E. coli isolates that produce the aggregative adherence pattern (AA) on epithelial cells (HeLa and/or HEp-2) cultured in vitro. The EAEC pathotype can be divided in typical and atypical based on the presence of the aggR gene, which encodes a transcriptional activator, in the former group. The aim of this study was to characterize a collection of EAEC isolates obtained from diarrheal patients over a 7-year period of surveillance (2010-2016). A total of 220 EAEC isolates (194 typical and 26 atypical) were evaluated regarding the phylogenetic classification, serotypes, adherence pattern produced on HeLa cells, susceptibility to antimicrobial drugs and the presence of 25 virulence factor-encoding genes. The majority of the EAEC isolates were assigned to phylogroups A (44.1%; 97/220) and B1 (21.4%; 47/220). Regarding the adherence pattern, was observed that 92.7% (204/220) produced AA. Moreover, we identified nine isolates (4.1%; 9/220) that produced the chain-like adherence (CLA), with six of them producing concomitantly the AA pattern, besides EAEC isolates producing an undefined adherence (1.4%; 3/220) and isolates non-adherent (3.6%; 8/220). Only 0.9% (2/220) of the EAEC isolates studied presented the multidrug resistance phenotype. The genes encoding for the major pilin subunit of all five previously described aggregative adherence fimbriae (AAF) were detected among the EAEC isolates studied (55.7%; 108/194), with agg4A and agg5A being the most frequent, and equally detected in 16.5% (32/194) of the typical EAEC isolates. Among the 25 virulence factor-encoding genes investigated here, aap (95.5%; 210/220), aar (81.8%; 180/220), orf3 (89.5%; 197/220), and pic (57.7%; 127/220) were the most prevalent, while twelve genes (agg4A, agg5A, pic, aap, aaiA, aaiC, aaiG, orf3, aar, air, capU and shf) were statistically more frequent in typical than atypical EAEC (P<0.05). The afpB, afpD, afpP and afpA2 genes, present in the afp operon, and its afpR regulator, were detected in 57.7% (15/26) of the atypical EAEC isolates, showing a statistically significant association with this EAEC subgroup (P<0.001). In conclusion, our study demonstrated that despite the heterogeneity found among the EAEC isolates studied, the genes from the afp operon were statistically associated with the atypical EAEC isolates, pointing this set of genes as a putative new diagnostic marker to be use, in order to increase the efficiency in the identification of this EAEC subgroup.
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Palavras-chave
EAEC, Escherichia coli enteroagregativa, Aderência agregativa, Diarreia, Adesinas, Fatores de virulência, Resistência antimicrobiana, Enteroaggregative Escherichia coli, Aggregative adherence, Diarrhea, Adhesins, Virulence factors, Antimicrobial resistance