Zhao Xingchen, Zervas Athanasios, Hendriks Marc, Rajkovic Andreja, van Overbeek Leo, Hendriksen Niels Bohse, Uyttendaele Mieke
Food Microbiology and Food Preservation Research Unit, Department of Food Technology, Safety and Health, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Department of Environmental Science, Aarhus University, Roskilde, Denmark.
Front Microbiol. 2022 Nov 30;13:1030921. doi: 10.3389/fmicb.2022.1030921. eCollection 2022.
(Bt), used as a biological control agent (BCA), can persist on plants, and from there can be introduced into the final food product. In routine food safety diagnostics, these Bt residues cannot be distinguished from natural populations of present in plants and all are enumerated as "presumptive ." In this study, information on eventual use of Bt biopesticides, brand, application times and intervals provided by three food processing companies in Belgium, were integrated with quantitative data on presumptive measured from fresh to frozen food products. This information together with data on genomic similarity obtained whole genome sequencing (WGS) and gene profiling using a quantitative real-time PCR (qPCR) assay, confirmed that six out of 11 Bt isolates originated from the applied Bt biocontrol products. These identified Bt strains were shown to carry enterotoxin genes () and express Hbl enterotoxin . It was also noted that these Bt biopesticide strains showed no growth at standard refrigeration temperatures and a low or moderate biofilm-forming ability and cytotoxic activity. Our results also showed that the use of Bt as a BCA on spinach plants in the field led to higher residual counts of Bt in spinach (fresh or frozen) in the food supply chain, but the residual counts exceeding at present commonly assumed safety limit of 10 CFU/g was only found in one fresh spinach sample. It is therefore recommended to establish a pre-harvest interval for Bt biopesticide application in the field to lower the likelihood of noncompliance to the generic safety limit. Furthermore, WGS was found to be the best way to identify Bt biopesticide isolates at the strain level for foodborne outbreaks and clinical surveillance. The developed qPCR assay for screening on the presence of genes in presumptive can be applied as a rapid routine test as an amendment to the already existing test on Bt crystal proteins determined phase-contrast microscopy.
苏云金芽孢杆菌（Bt）作为一种生物防治剂（BCA），可在植物上存活，并由此进入最终食品。在常规食品安全诊断中，这些Bt残留物无法与植物中天然存在的菌群区分开来，所有这些都被计为“推测性的”。在本研究中，比利时三家食品加工公司提供的关于Bt生物农药最终用途、品牌、施用时间和间隔的信息，与从新鲜食品到冷冻食品中测得的推测性芽孢杆菌的定量数据相结合。这些信息与通过全基因组测序（WGS）获得的基因组相似性数据以及使用定量实时PCR（qPCR）检测进行的基因谱分析数据一起，证实了11株Bt分离株中有6株源自所施用的Bt生物防治产品。这些鉴定出的Bt菌株被证明携带肠毒素基因（hbl）并表达Hbl肠毒素。还注意到，这些Bt生物农药菌株在标准冷藏温度下不生长，生物膜形成能力和细胞毒性活性较低或中等。我们的结果还表明，在田间菠菜植株上使用Bt作为生物防治剂会导致食品供应链中菠菜（新鲜或冷冻）中Bt的残留量增加，但目前仅在一个新鲜菠菜样品中发现残留量超过通常假定的10 CFU/g安全限值。因此，建议确定田间Bt生物农药施用的收获前间隔，以降低不符合一般安全限值的可能性。此外，发现全基因组测序是在菌株水平上鉴定食源性暴发和临床监测中Bt生物农药分离株的最佳方法。所开发的用于筛选推测性芽孢杆菌中hbl基因存在情况的qPCR检测，可作为一种快速常规检测方法，作为对已经存在的相差显微镜法检测Bt晶体蛋白的检测方法的补充。
