食品中克羅諾桿菌屬（阪崎腸桿菌）檢驗方法之評估   全文下載

Evaluation of Test Methods for Detection of Cronobacter (Enterobacter sakazakii) in Foods

克羅諾桿菌屬（Cronobacter）舊稱阪崎腸桿菌（Enterobacter sakazakii），為革蘭氏陰性桿菌，屬於腸桿菌科（Enterobacteriacae）的一屬，嬰兒如食用受此菌污染的奶粉會引起腦膜炎及敗血症。有關克羅諾桿菌屬的檢驗方法，衛生福利部（下稱衛福部）已於104年公告最確數計數法，考量實驗室執行檢驗之實務需求，本研究乃參酌現行公告檢驗方法、美國FDA/BAM（Bacteriological Analytical Manual）及ISO 22964相關檢驗方法進行食品中克羅諾桿菌屬定性方法之培養基效能評估。在檢驗流程方面，考量檢體取樣量及操作方便性，採用FDA/BAM之檢驗流程；在鑑別培養基方面，則針對R&F ESPM（FDA/BAM方法）、ESA（現行公告及FDA/BAM方法）、CCI（ISO方法）3種培養基進行靈敏度及專一性評估。結果發現以克羅諾桿菌屬51株進行測試，3者靈敏度均為100%，無偽陰性；以非目標菌44株進行測試，其中僅有1株普通變形桿菌（Proteus vulgaris）於R&F ESPM培養基呈現偽陽性，專一性為97.7%，而ESA及CCI培養基專一性為100%。在培養基菌數回收試驗，3種鑑別培養基的菌數回收率均大於70%，而以市售含益生菌奶粉為基質進行克羅諾桿菌屬之添加試驗結果發現，3者克羅諾桿菌屬之檢驗值均與原添加菌量相當。另以市售產品15件，進行克羅諾桿菌屬定性方法實測評估，其中有2件非屬提供6個月以下嬰兒食用之食品檢出克羅諾桿菌屬。根據本研究評估結果，擬參考FDA/BAM檢驗流程及ISO方法鑑別培養基修訂公告檢驗方法。

Cronobacter, formerly known as Enterobacter sakazakii, is a genus of Gram-negative bacteria of the family Enterobacteriaceae. Powdered infant formula (PIF) contaminated with Cronobacter sakazakii has been confirmed to be related with neonatal meningitis and septicemia. In Taiwan, Ministry of Health and Welfare (MOHW) has promulgated a method of the most probable number (MPN) to estimate Cronobacter in foods in 2015. In response to the needs of laboratory inspection, a qualitative method to test the presence of Cronobacter in the food is required. Therefore, this study aimed to evaluate the performance of several media used in qualitative methods for detecting Cronobacter established by the MOHW, US Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) and International Standard for Organization (ISO) 22964. Considering the sample size and the convenience of operation, the inspection processes of FDA/BAM were adopted. Three commercial chromogenic media were compared for the sensitivity and specificity in detecting Cronobacter: R&F Enterobacter sakazakii chromogenic plating medium (R&F ESPM) (method of FDA/BAM), Brilliance Enterobacter sakazakii Agar (ESA) (method of MOHW and FDA/BAM), and Chromogenic Cronobacter isolation agar (CCI) (method of ISO). The sensitivity was 100% for all types of chromogenic plates in detecting 51 strains of Cronobacter. Only 1 strain of Proteus vulgaris was shown false positive in R&F ESPM, so that the specificity was 97.7% for R&F ESPM and 100% for ESA and CCI, as evaluated with 44 non-targeted bacteria. The recovery rates were higher than 70% for all types of chromogenic plates. The effect of food matrix was evaluated by spiking probiotic milk powder with Cronobacter and the final colony counts out of three commercial chromogenic media were comparable to the numbers of spiked bacteria. Finally, 15 food products were tested, and Cronobacter was detected in two products which were not food sold exclusively for infants less than 6 months of age. Based on the results of this study, the official test method will be revised toward the use of FDA/BAM method and ISO method chromogenic media.