建立從生乳中快速偵測B群鏈球菌的最適當方法

B 群鏈球菌（GBS, Group B Streptococcus, Streptococcus agalactiae，無乳鏈球菌，乙型鏈球菌）除了引發人類敗血症外，還會使乳牛感染乳房炎。過去，乳牛乳房炎的微生物檢測大多將生乳檢體直接塗抹在BAP及/或CNA平板上，若長出疑似GBS，則進一步操作傳統鑑定試驗，然而，此鑑定方法的過程耗時又耗力。吾等參考懷孕婦女產前GBS 的檢測方法，將約100 mL含GBS 的模擬生乳檢體經離心後，先以沉澱物接種本研究室的創新設計GBS carrot broth，增菌後再接種兩種選擇/區分性平板培養基(GBS carrotagar 與/ GBS detection agar)。利用添加純GBS 菌與混菌（GBS+其它菌）的模擬檢體，以過夜增菌及不經增菌接種法的效能進行比較。結果指出，純GBS 模擬檢體接種於GBS carrot broth 培養18、24 及48 小時後，若呈現胡蘿蔔色（即GBS 陽性）的最低菌量（偵測極限）分別為10^2、10 及1 CFU；而混菌模擬檢體者，則為10^3~10^6、10~10^4 及1~10^2 CFU/ mL。至於純GBS 模擬檢體使用過夜增菌及不經增菌的方法同時接種於GBS carrot agar 與/ GBS detection agar 二種平板培養基，其等的偵測極限均為10 CFU；而接種混菌模擬檢體，結果發現過夜增菌法的偵測GBS效能比不經增菌者佳（降低10^1~ 10^6 倍）。根據以上的發現，吾等建議檢驗室可將約100 mL生乳檢體離心後，將沉澱物回溶並移入GBS carrot broth 中，增菌約20 至24 小時後直接判讀，陰性者再接種GBS carrot agar與/ GBS detection agar（或配成分隔培養基），將可快速且正確地偵測出生乳中的GBS。

關鍵字

B群鏈球菌；乳牛乳房炎；偵測極限

並列摘要

Group B Streptococcus (GBS) can cause not only septicemia in humans, but also bovine mastitis. In the past, the microbiological diagnosis of bovine mastitis was conducted by inoculating raw milk from the cow being tested directly onto a blood agar plate (BAP) or Columbia CNA agar. If the colonies thus grown were suspected of being GBS, the traditional identification scheme was then used for GBS detection. However, this approach not only yields a low GBS isolation rate, but is also laborious and time-consuming. We therefore modified the diagnostic method used for isolating GBS from pregnant women so that it could be used in the isolation of GBS from raw milk. Two types of simulated raw milk specimens were prepared, one containing pure GBS and another containing mixed culture (more specifically, GBS and one of the other five mastitis-causing pathogens). In testing a given specimen, about 100 mL of the simulated raw milk specimen was collected and centrifuged, after which the precipitate thus obtained was then re-dissolved and used to inoculate a tube of enrichment medium, GBS carrot broth. After enrichment for different periods of time (18, 24 and 48 hours), the broths were then sub-cultured to two kinds of selective/ differential plated media, GBS carrot agar and / GBS detection agar. The efficiencies of GBS carrot broth, GBS carrot agar and / GBS detection agar were evaluated with either direct inoculation or after enrichment overnight. For the specimens containing the pure GBS culture, the results indicated that for the GBS carrot broth inoculated after the enrichment of individual specimens for 18, 24 and 48 hours individually, the detection limits (defined as the lowest GBS concentration that resulted in the carrot-pigmented formation in the test tube, i.e. GBS positive) were 10^2, 10, and 1 CFU, respectively, whereas for the specimens inoculated with mixed culture, the detection limits were 10^3~10^6, 10~10^4, and 1~10^2 CFU. When GBS carrot agar andβ/γ GBS detection agar were directly inoculated or inoculated after overnight enrichment, their detection limits were the same, 10 CFU. As regards the inoculation with mixed bacteria, the detection limits were better after the overnight enrichment rather than with direct inoculation (the detection limits were reduced by about 10~106 times). Based on the above findings, we propose that a laboratory can use about 100 ml of raw milk as a specimen for centrifugation, after which the precipitate obtained can then be re-dissolved and used to inoculate a tube of GBS carrot broth. After 20-24 hours of enrichment, direct interpretation of the GBS carrot broth can be made and, if necessary, the broth could be further subcultured onto GBS carrot agar and / GBS detection agar, which can be produced with a bi-plate. This approach will yield rapid and effective detection of GBS from raw milk.

並列關鍵字

Group B Streptocuccus ； raw milk ； GBS carrot broth ； optimal diagnostic method ； GBS carrot agar ； β/γ GBS detection agar

國際替代計量

建立從生乳中快速偵測B群鏈球菌的最適當方法

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