In this study, lettuce roots and leaves were contaminated with enterohemorrhagic Escherichia coli O157:H− strain 431/97 under greenhouse conditions. Furthermore, the internalization of strain 431/97 in lettuce roots and leaves was examined. To track the inoculated bacteria during the experiments and to differentiate them from the autochthonous microbiota, a combined protocol including matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and PCR was established. At different time points after inoculation of Batavia lettuce with 5.5 × 106 cfu/mL (high inoculation level) or 3.0 × 101 cfu/mL (low inoculation level) E. coli O157:H− strain 431/97 as well as sterile 0.9% (w/v) sodium chloride solution (negative control), samples from the root and the leaf were taken and surface disinfected with gentamicin. After homogenization, microorganisms were isolated from the samples and analyzed by MALDI-TOF MS. Analysis of the root samples resulted in bacterial counts of 1.0 × 102–1.0 × 106 cfu/0.25 g depending on the inoculated viable counts and the incubation period. In the leaf samples, strain 431/97 was not detected. The investigation of the viable cell counts of E. coli O157:H− 431/97 following irrigation of the leaves resulted in bacterial counts of 102 cfu/0.25 g for the disinfected leaf samples. Thus, the established protocol is suitable for detecting the investigated strain under greenhouse conditions in plant infection experiments. This strain may indeed survive in the soil, but did not enter the plant via the root in detectable numbers. Contrarily, viable counts exceeding the generally accepted infective dose of less than 100 cells for enterohemorrhagic E. coli were determined internalized after irrigation of the leaves. As this may pose a risk for the consumer, the present study provides a valuable set of tools for further research.

中文翻译：

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MALDI-TOF质谱和特异性PCR在跟踪巴达维亚生菜中的O157：H ^- 431/97大肠杆菌中的应用

在这项研究中，莴苣的根和叶在温室条件下被肠出血性大肠杆菌O157：H- 431/97株污染。此外，检查了莴苣根和叶中431/97菌株的内在化。为了在实验过程中跟踪接种的细菌并将其与自生微生物区分开来，建立了包括基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和PCR的组合方案。在接种巴达维亚生菜后的不同时间点，接种量为5.5×106 cfu / mL（高接种水平）或3.0×101 cfu / mL（低接种水平）大肠杆菌O157：H- 431/97菌株以及无菌的0.9％ （w / v）氯化钠溶液（阴性对照），从根和叶中取样并用庆大霉素进行表面消毒。匀浆后，从样品中分离出微生物并通过MALDI-TOF MS进行分析。根样品的分析结果表明细菌计数为1.0×102–1.0×106 cfu / 0.25 g，具体取决于接种的活菌数和潜伏期。在叶片样品中，未检测到菌株431/97。灌溉后，对大肠杆菌O157：H- 431/97的活细胞计数进行调查，结果得出消毒叶样本的细菌计数为102 cfu / 0.25 g。因此，建立的协议适合在植物感染实验中在温室条件下检测所研究的菌株。该菌株确实可以在土壤中存活，但并未以可检测的数量通过根进入植物。相反，在冲洗叶子后，确定超过肠道出血性大肠杆菌的少于100个细胞的普遍接受的感染剂量的存活计数。由于这可能给消费者带来风险，因此本研究提供了一套有价值的工具，可供进一步研究。