Umpiérrez Ana, Bado Inés, Oliver Martín, Acquistapace Sofía, Etcheverría Analía, Padola Nora Lía, Vignoli Rafael, Zunino Pablo
Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable.
Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República.
Microbes Environ. 2017 Sep 27;32(3):275-282. doi: 10.1264/jsme2.ME17046. Epub 2017 Sep 12.
Escherichia coli is one of the main etiological agents of neonatal calf diarrhea (NCD). The objective of this study was to assess the presence of virulence genes, genetic diversity, and antibiotic resistance mechanisms in E. coli associated with NCD in Uruguay. PCR was used to assess the presence of intimin, Shiga-like toxin, and stable and labile enterotoxin genes. Resistance to fluoroquinolones and oxyimino-cephalosporins was estimated on Müller-Hinton agar plates. Further antibiotic disc-diffusion tests were performed to assess bacterial multi-resistance. The presence of PMQR, ESBL, MCR-1, and integron genes was evaluated. Isolates were typed using ERIC-PCR, and 20 were selected for MLST, adhesion to Hep-2 cells, in vitro biofilm formation, and eukaryotic cytotoxicity. The prevalence of ETEC genes was lower than 3% in each case (estA and elt). Six isolates were EPEC (eae+) and 2 were EHEC/STEC (eae+/stx1+). The results of a diversity analysis showed high genetic heterogenicity among isolates. Additionally, different sequence types, including ST10, ST21, and ST69, were assigned to selected isolates. Thirty-six percent (96/264) of the isolates were fluoroquinolone-resistant, with 61/96 (63.5%) being multidrug-resistant. Additionally, 6 were oxyimino-cephalosporin-resistant. The qnrB, qnrS1, and bla genes were detected, whereas no isolates carried the mcr-1 gene. Isolates had the ability to adhere to Hep-2 cells and form biofilms. Only 1 isolate expressed toxins in vitro. E. coli from NCD cases in Uruguay are very diverse, potentially virulent, and may interact with eukaryotic cells. Zoonotic potential, together with resistance traits and the presence of horizontal transfer mechanisms, may play a significant role in infections caused by these microorganisms.
大肠杆菌是新生犊牛腹泻(NCD)的主要病原体之一。本研究的目的是评估乌拉圭与NCD相关的大肠杆菌中毒力基因的存在情况、遗传多样性和抗生素耐药机制。采用聚合酶链反应(PCR)评估紧密黏附素、志贺样毒素以及稳定和不稳定肠毒素基因的存在情况。在 Müller-Hinton 琼脂平板上评估对氟喹诺酮类和氧亚氨基头孢菌素的耐药性。进一步进行抗生素纸片扩散试验以评估细菌多重耐药性。评估质粒介导的喹诺酮类耐药基因(PMQR)、超广谱β-内酰胺酶(ESBL)、耐多粘菌素基因(MCR-1)和整合子基因的存在情况。使用肠杆菌基因间重复共有序列聚合酶链反应(ERIC-PCR)对分离株进行分型,并选择20株进行多位点序列分型(MLST)、对 Hep-2 细胞的黏附、体外生物膜形成和真核细胞毒性检测。在每种情况下,产肠毒素大肠杆菌(ETEC)基因的流行率均低于3%(estA 和 elt)。6株分离株为肠致病性大肠杆菌(EPEC,eae+),2株为肠出血性大肠杆菌/产志贺毒素大肠杆菌(EHEC/STEC,eae+/stx1+)。多样性分析结果显示分离株之间存在高度的遗传异质性。此外,选定的分离株被归为不同的序列类型,包括 ST10、ST21 和 ST69。36%(96/264)的分离株对氟喹诺酮耐药,其中61/96(63.5%)为多重耐药。此外,有6株对氧亚氨基头孢菌素耐药。检测到 qnrB、qnrS1 和 bla 基因,但没有分离株携带 mcr-1 基因。分离株具有黏附 Hep-2 细胞和形成生物膜的能力。只有1株分离株在体外表达毒素。乌拉圭 NCD 病例中的大肠杆菌非常多样,具有潜在的毒力,并且可能与真核细胞相互作用。人畜共患病潜力,连同耐药特性和水平转移机制的存在,可能在这些微生物引起的感染中起重要作用。