Microbial Resources Division, Institute of Bioresources and Sustainable Development, Takyelpat, Imphal 795001, India; Department of Biotechnology, Gauhati University, Guwahati 781014, India.
Department of Biotechnology, Gauhati University, Guwahati 781014, India.
Int J Food Microbiol. 2019 May 2;296:21-30. doi: 10.1016/j.ijfoodmicro.2019.02.012. Epub 2019 Feb 21.
The microbial risk involved with natural food fermentation is largely unknown. Here, we report the prevalence of enteric bacterial pathogens in the traditional fermented foods marketed in Northeast region of India. A total of 682 samples of 39 food types (broadly categorized into fermented soybean, bamboo shoot, fish, milk and pork products) collected over four different seasons from seven states of India were analyzed in this study. Cultivation-independent analysis by MiSeq amplicon sequencing of V4-V5 region of the 16S rRNA gene showed the bacterial community structure in the foods. Among the WHO prioritized foodborne bacterial pathogens, we detected the prevalence of phylotypes related to Clostridium botulinum, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Listeria monocytogenes, and Escherichia coli in these ethnic foods. We also observed the occurrence of other well known human enteric pathogens like Proteus mirabilis, Clostridium difficile, and Yersinia enterocolitica. Further pathogen-specific qPCR assays confirmed a higher population (>10 cells/g) of B. cereus, P. mirabilis, and a C. botulinum related phylotype in the fermented soybean, fish, and pork products. We noticed a general trend of higher pathogen occurrence during the colder months without any seasonal variation of total bacterial load in the fermented foods. Further qPCR analysis on toxigenic and pathogenic potential, and toxins production by immunoassays showed that all the soybean samples and the isolated B. cereus cultures were positive for diarrheal toxins (Nhe and Hb1), and nearly half of the samples were positive for emetic toxin (cereulide). Similarly, the food samples and associated swarming P. mirabilis cultures were positive with the pathogenic factors like hemolysin (hpm), urease (ure) and multidrug resistance. However, we could not confirm the presence of botulinum neurotoxin (toxins A, B, E, and F) in the C. botulinum positive food samples. This is the first baseline data of the enteric bacterial pathogens prevalent in the traditional fermented foods of India, which will support the sustained effort of WHO to estimate the global foodborne disease burden. The unusual presence of P. mirabilis in the fermented foods marketed in the Indian region with high incidence of urolithiasis cases is a concern. Our study emphasizes the need of the hour to have a coordinated action to control and prevent the spread of enteric bacterial pathogens through fermented foods marketed in India. Moreover, replacing the indigenous process with a defined starter culture based controlled fermentation will enhance the safety of Indian fermented foods.
天然食品发酵所涉及的微生物风险在很大程度上是未知的。在这里，我们报告了在印度东北地区销售的传统发酵食品中肠细菌病原体的流行情况。本研究共分析了来自印度七个州的四个不同季节采集的 39 种食品类型（广义上分为发酵大豆、竹笋、鱼类、牛奶和猪肉产品）的 682 个样本。通过对 16S rRNA 基因的 V4-V5 区进行 MiSeq 扩增子测序的非培养分析，显示了食品中的细菌群落结构。在世界卫生组织优先考虑的食源性细菌病原体中，我们在这些民族食品中检测到了与肉毒梭菌、蜡样芽孢杆菌、金黄色葡萄球菌、产气荚膜梭菌、单核细胞增生李斯特菌和大肠杆菌相关的生物型的流行。我们还观察到了其他著名的人类肠道病原体，如奇异变形杆菌、艰难梭菌和小肠结肠炎耶尔森菌的发生。进一步的病原体特异性 qPCR 检测证实，发酵大豆、鱼类和猪肉产品中蜡样芽孢杆菌、奇异变形杆菌和肉毒梭菌相关生物型的种群（>10 个细胞/g）较高。我们注意到，在较冷的月份，病原体的发生更为普遍，而发酵食品中的总细菌负荷没有任何季节性变化。进一步的 qPCR 分析显示，毒素产生和免疫分析的毒力和致病性潜力表明，所有大豆样品和分离的蜡样芽孢杆菌培养物均对腹泻毒素（Nhe 和 Hb1）呈阳性，近一半的样品对呕吐毒素（肠毒素）呈阳性。同样，食品样品和相关的 swarm P. mirabilis 培养物也对溶血素（hpm）、脲酶（ure）和多药耐药性等致病因子呈阳性。然而，我们无法确认在 C. botulinum 阳性食品样品中存在肉毒神经毒素（毒素 A、B、E 和 F）。这是印度传统发酵食品中肠细菌病原体流行的第一份基线数据，这将支持世界卫生组织努力估计全球食源性疾病负担。奇异变形杆菌在印度地区销售的发酵食品中的异常存在，而该地区的尿路结石病例发病率很高，这令人担忧。我们的研究强调，需要采取协调一致的行动来控制和预防通过印度销售的发酵食品传播肠细菌病原体。此外，用基于明确的起始培养物的受控发酵取代本土工艺，将提高印度发酵食品的安全性。
