Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 862, 3 andar, São Paulo, 04023-062, Brazil.
BMC Microbiol. 2020 May 29;20(1):138. doi: 10.1186/s12866-020-01809-4.
In previous studies, we have shown that atypical enteropathogenic Escherichia coli (aEPEC) strains are important diarrheal pathogens among Brazilian children. In the characterization of a collection of 126 aEPEC strains, we identified 29 strains expressing the localized-like adherence (LAL) pattern on HEp-2 cells and harboring large plasmids in the range of 60 to 98 MDa. In this study, we examined 18 of these strains for their ability to transfer the LAL phenotype to a E. coli K-12 C600 strain.
In conjugation experiments, using eight strains which were resistant to one or more antimicrobials and positive for F-pili genes (traA), we were able to cotransfer antimicrobial resistance markers along with adhesion genes. By transforming E. coli DH5α with plasmid DNA from strains A46 (pIS46), A66 (pIS66) and A102 (pIS102), we were able to demonstrate that genes encoding ampicillin, tetracycline and LAL were encoded on a 98-MDa conjugative plasmid. To identify a gene responsible for LAL, we constructed a transposon mutant library of A102 strain. Among 18 mutants that did not adhere to HeLa cells, four carried insertions within fimbrial genes (fimA and traJ) and agglutinin genes (tia and hek). Using these Tn5 mutants as donors, we were able to obtain kanamycin-resistant E. coli MA3456 transconjugants. Sequence analysis of the plasmid genes revealed a region exhibit to 80 and 73% amino acid similarities to the agglutinins Tia and Hek, respectively.
In this study, we have identified three large conjugative plasmids, pIS46, pIS66 and pIS102, coding for antimicrobial resistance and localized-like adherence (LAL) to HeLa cells. In addition, we identified a tia/hek homolog encoded on the pIS102 plasmid, which seems to be involved in adhesion of A102 strain.
在之前的研究中，我们已经表明非典型肠致病性大肠杆菌（aEPEC）菌株是巴西儿童腹泻的重要病原体。在对 126 株 aEPEC 菌株的特征进行描述时，我们发现 29 株菌株在 HEp-2 细胞上表达局灶性粘附（LAL）模式，并携带 60 至 98MDa 范围内的大质粒。在这项研究中，我们检查了其中的 18 株菌株，以评估它们将 LAL 表型转移到大肠杆菌 K-12 C600 菌株的能力。
在使用对一种或多种抗生素具有抗性且 F- 菌毛基因（traA）阳性的 8 株菌株进行的结合实验中，我们能够同时转移抗生素耐药标记物和粘附基因。通过用来自菌株 A46（pIS46）、A66（pIS66）和 A102（pIS102）的质粒 DNA 转化大肠杆菌 DH5α，我们能够证明编码氨苄青霉素、四环素和 LAL 的基因位于 98-MDa 的可接合质粒上。为了鉴定编码 LAL 的基因，我们构建了 A102 菌株的转座子突变体文库。在 18 株不粘附 HeLa 细胞的突变体中，有 4 株携带菌毛基因（fimA 和 traJ）和凝集素基因（tia 和 hek）内的插入物。使用这些 Tn5 突变体作为供体，我们能够获得对卡那霉素有抗性的大肠杆菌 MA3456 转导子。质粒基因的序列分析显示，一个区域与凝集素 Tia 和 Hek 的氨基酸序列相似性分别达到 80%和 73%。
在这项研究中，我们已经鉴定了三个编码抗生素耐药性和局灶性粘附（LAL）的大型可接合质粒，即 pIS46、pIS66 和 pIS102。此外，我们在 pIS102 质粒上鉴定了一个 tia/hek 同源物，它似乎参与了 A102 菌株的粘附。
