Carneiro L A M, Lins M C, Garcia F R A, Silva A P S, Mauller P M, Alves G B, Rosa A C P, Andrade J R C, Freitas-Almeida A C, Queiroz M L P
Disciplina de Microbiologia e Imunologia, Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87, fundos, 3 andar, 20551-030 Rio de Janeiro (RJ), Brazil.
Int J Food Microbiol. 2006 Apr 15;108(1):15-21. doi: 10.1016/j.ijfoodmicro.2005.10.010. Epub 2006 Feb 20.
Fifty-six Escherichia coli strains, serogrouped as EPEC, isolated from three different brands of pasteurised milk commercialised in Rio de Janeiro, Brazil, were tested for enteropathogenicity markers. Most of the strains (71.4%) were adherent to HEp-2 cells. The adherent strains were distributed among 7 EPEC serogroups (O26, O55, O111, O114, O125, O127, O128, O158). Although almost half of these strains (33.9%) presented unrecognisable adherence phenotypes, classical adherence patterns (localised-like, aggregative and diffuse adherence) described for E. coli and epidemiologically associated with diarrheagenic strains were observed. None of the strains showed typical localised adherence, usually associated with EPEC strains, but 4 of them displayed a localised-like adherence (LAL) phenotype, characterised by fewer and less compact microcolonies but that is still associated with diarrheagenic strains as well as strains of non-human origin. Indeed, 3 of these 4 strains were able to elicit the attaching-effacing lesion (FAS-positive), the central feature of EPEC pathogenesis, and hybridised with bfpA and eae DNA probes. The other LAL-positive strain hybridised with the bfpA probe but gave negative results for the eae probe and FAS assays. Interestingly, all LAL-positive strains produced amplicons of 200 bp in the PCR for bfpA, instead of the expected 326 bp fragment. PCR reactions for stx1 and stx2, two shiga-toxin-encoding genes, gave negative results. Typing of LEE-associated genes by PCR showed the profile eae (beta), tir (beta), espA (alpha) and espB (alpha) for one of the LAL-positive strain. The most prevalent adherence phenotype was the aggregative pattern which is observed in strains epidemiologically associated with persistent diarrhea. Additionally, one strain promoted complete detachment of the Hep-2 cell monolayer after 3 h of infection which might be related to the production of citotoxins, a feature that has been increasingly observed in clinical strains. The possession of EPEC-related O and H antigens is no longer deemed an essential characteristic of true pathogenic EPEC strains, emphasising the importance of routinely screen for virulence markers in E. coli strains isolated from foods. Our results are in accordance with data from the literature that demonstrate that environmental strains display atypical features but yet are capable of eliciting the classical A/E lesion and thus must be considered as potentially pathogenic. Further, our results demonstrate the potential of pasteurised milk as a vehicle for transmission of diarrheagenic E. coli in Brazil.
对从巴西里约热内卢商业化销售的三个不同品牌巴氏杀菌牛奶中分离出的56株血清型为肠致病性大肠杆菌（EPEC）的菌株进行了肠道致病性标志物检测。大多数菌株（71.4%）能黏附于HEP - 2细胞。黏附菌株分布在7个EPEC血清型（O26、O55、O111、O114、O125、O127、O128、O158）中。尽管这些菌株中近一半（33.9%）呈现出无法识别的黏附表型，但仍观察到了大肠杆菌中描述的经典黏附模式（局部样、聚集性和弥漫性黏附），且这些模式在流行病学上与致泻菌株相关。没有菌株表现出通常与EPEC菌株相关的典型局部黏附，但其中4株呈现出局部样黏附（LAL）表型，其特征是微菌落数量较少且不太紧密，但仍与致泻菌株以及非人类来源的菌株相关。实际上，这4株中的3株能够引发紧密黏附-抹平损伤（FAS阳性），这是EPEC发病机制的核心特征，并且能与bfpA和eae DNA探针杂交。另一株LAL阳性菌株与bfpA探针杂交，但eae探针和FAS检测结果为阴性。有趣的是，所有LAL阳性菌株在bfpA的PCR反应中产生的扩增子为200 bp，而非预期的326 bp片段。对stx1和stx2这两个志贺毒素编码基因进行PCR反应，结果均为阴性。通过PCR对LEE相关基因进行分型，其中一株LAL阳性菌株显示出eae（β）、tir（β）、espA（α）和espB（α）的图谱。最常见的黏附表型是聚集性模式，在流行病学上与持续性腹泻相关的菌株中可见。此外，一株菌株在感染3小时后导致HEP - 2细胞单层完全脱落，这可能与细胞毒素的产生有关，这一特征在临床菌株中越来越多地被观察到。拥有EPEC相关的O和H抗原不再被视为真正致病性EPEC菌株的必要特征，这强调了对从食品中分离出的大肠杆菌菌株常规筛查毒力标志物的重要性。我们的结果与文献数据一致，文献表明环境菌株表现出非典型特征，但仍能够引发经典的A/E损伤，因此必须被视为潜在致病性菌株。此外，我们的结果证明了巴氏杀菌牛奶在巴西作为致泻性大肠杆菌传播媒介的可能性。
