1 Department of Food Science and Technology, Institute of Food Science, University of Natural Resources and Life Sciences, A-1190 Vienna, Austria.
2 Department of Food Science, Faculty of Agriculture, Ain Shams University, Cairo, Egypt.
J Food Prot. 2019 Jan;82(1):65-77. doi: 10.4315/0362-028X.JFP-18-036.
Cronobacter spp. are opportunistic human pathogens that cause serious diseases in neonates and immunocompromised people. Owing to their biofilm formation on various surfaces, both their detection and their removal from production plants constitute a major challenge. In this study, food samples were randomly collected in Austria and examined for the presence of Cronobacter spp. Presumptive isolates were identified by a polyphasic approach. Five percent of the samples were positive for C. sakazakii and 2.4% for C. dublinensis. Individual growth of the isolates was characterized based on lag time, growth rate, and generation time. During an incubation period of 6 to 72 h, biofilm formation of 11 selected isolates was quantified under model conditions by a crystal violet staining assay with 96-well plates with different carbon sources (lactose, glucose, maltose, sucrose, and sodium acetate) and NaCl levels and under variable temperature and pH conditions. Biofilm formation was more pronounced at lactose concentrations between 0.25 and 3% compared with 5% lactose, which lead to thinner layers. C. sakazakii isolate C7, isolated from infant milk powder, was the strongest biofilm producer at 10 mM Mg and 5 mM Mn, 0.5% sodium acetate, at pH levels between 7 and 9 at 37°C for 24 h. C. sakazakii strain C6 isolated from a plant air filter was identified as a moderate biofilm former and C. sakazakii strain DSM 4485, a clinical isolate, as a weak biofilm former. Based on PCR detection, genes bcsA, bcsB, and bcsG encoding for cellulose could be identified as markers for biofilm formation. Isolates carrying bcsA and bcsB showed significantly stronger biofilm formation than isolates without these genes ( P < 0.05), in strong correlation with the results obtained in the crystal violet assay. Further investigations using confocal laser scanning microscopy revealed that extracellular polymeric substances and glycocalyx secretions were the dominating components of the biofilms and that the viable fraction of bacteria in the biofilm decreased over time.
克罗诺杆菌属是一种机会性病原体，可导致新生儿和免疫功能低下人群发生严重疾病。由于其在各种表面形成生物膜，因此检测和从生产工厂中去除它们是一项重大挑战。在这项研究中，奥地利随机采集了食品样本，以检查是否存在克罗诺杆菌属。通过多相方法鉴定推定分离株。5%的样本呈阪崎克罗诺杆菌阳性，2.4%的样本呈都柏林克罗诺杆菌阳性。根据滞后时间、生长速率和世代时间，对分离株的个体生长进行了表征。在 6 至 72 小时的孵育期内，通过结晶紫染色法在 96 孔板上使用不同的碳源（乳糖、葡萄糖、麦芽糖、蔗糖和乙酸钠）和 NaCl 水平以及在不同温度和 pH 条件下，对 11 株选定分离株的生物膜形成进行了定量。与 5%乳糖相比，在 0.25%至 3%乳糖浓度下，生物膜形成更为明显，形成的层更薄。从婴儿配方奶粉中分离出的阪崎克罗诺杆菌 C7 分离株在 10 mM Mg 和 5 mM Mn、0.5%乙酸钠、pH 值为 7 至 9 和 37°C 的条件下培养 24 小时时，是最强的生物膜生产者。从工厂空气过滤器中分离出的阪崎克罗诺杆菌 C6 被鉴定为中度生物膜形成者，而临床分离株阪崎克罗诺杆菌 DSM 4485 则被鉴定为弱生物膜形成者。基于 PCR 检测，可以鉴定出编码纤维素的 bcsA、bcsB 和 bcsG 基因作为生物膜形成的标记。携带 bcsA 和 bcsB 的分离株的生物膜形成明显强于不携带这些基因的分离株（P<0.05），与结晶紫测定的结果强烈相关。使用共聚焦激光扫描显微镜的进一步研究表明，细胞外聚合物和糖萼分泌物是生物膜的主要成分，并且生物膜中细菌的存活部分随时间推移而减少。
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