建立藥品中Burkholderia cepacia Complex的實用診斷流程

Recommended Procedure for Detection of Burkholderia cepacia Complex in Pharmaceuticals

梁多恩(Tabitha Liang) ； 歐柏廷(Po-Ting Ou) ； 蔡岳廷(Yueh-Ting Tsai) ； 蔡文城(Wen-cherng Tsai)

《檢驗及品保雜誌》 8卷3期 (2019/08) Pp. 93-103

https://doi.org/10.6573/JTQA.201908_8(3).01

摘要

洋蔥伯克氏複合群（Burkholderia cepacia complex，BCC）的成員早期被歸類為假單胞菌屬（Pseudomonas），但隨後分類學家將其成員分類成為洋蔥伯克氏菌屬（Burkholderia）之複合群，目前含有20個菌種。在2012年前6個月美國藥物召回事件之統計中，以BCC的污染占39%，其中以Burkholderia cepacia、B. multivorans、B. cenocepacia以及B. gladioli最為常見。為了瞭解這些菌的表現型特徵，本研究將四種菌的ATCC參考菌株分別接種於Burkholderia cepacia selective agar（洋蔥伯克氏菌選擇性培養基，BCSA）、Tryptic soy agar（胰蛋白大豆培養基，TSA）、Eosin Methylene Blue Agar（伊紅亞甲基藍培養基，EMB）以及MacConkey Agar（麥康凱培養基），培養48～72小時後進行菌落生長能力及特徵觀察。接著分別挑取BCSA或TSA的生長菌落接種Triple sugar iron agar（三糖鐵斜面，TSI）、lysine decarboxylase medium（賴胺酸脫羧酶試驗，LYS）、O/F（oxidative/fermentative）lactose及O/F maltose氧化試驗，並且操作氧化酶試驗（oxidase test）、革蘭氏染色（Gram stain）以及polymyxin B藥敏試驗（susceptibility test），結果指出四個菌種皆為革蘭氏陰性桿菌，於35±1℃培養24小時後，菌落的生長均不明顯，至少培養48～72小時才能顯現圓形的單一菌落生長特徵，其中，在BCSA平板，B. gladioli生長不佳，B. cepacia的菌落呈淡黃色，菌落周圍培養基會變黃；B. multivorans紫色，菌落周圍培養基為淡紫色；B. cenocepacia的菌落呈現淡粉紅色，周圍培養基為黃或粉紅色；培養72小時後，B. gladioli才在第一區長出極小的菌落。另外，四種菌在其測試的各種生化試驗（包括TSI、LYS、O/F lactose、O/F maltose 及oxidase）中分別顯示其獨特的反應。吾等根據這些項目的測試結果，建議藥品不合宜微生物中BCC檢測流程可包括（i）接種1 mL（或1 g）樣本於10 mL TSB；（ii）於35℃培養48～72小時後，將TSB培養液以四區劃線法移種至BCSA，然後培養48～72小時；（iii）挑取BCSA生長菌落操作革蘭氏染色及氧化酶試驗；（iv）接種TSI，培養18～24小時；以及（v）操作其它生化試驗（包括LYS, O/F lactose及O/F maltose）以便進一步鑑定。

關鍵字

洋蔥伯克氏菌複合群 ； BCC ； 洋蔥伯克氏菌選擇性培養基 ； BCC檢測流程

並列摘要

Burkholderia cepacia and its related species were considered as Pseudomonas species, but are now classified as Burkholderia cepacia complex (BCC), which includes approximately 20 species. During the first 6 months in 2012, BCC contamination accounted for 39% of pharmaceutical recalls. Burkholderia cepacia, B. multivorans, B. cenocepacia, and B. gladioli are the most common encounters in clinical specimens. To understand the growth characteristics of these four BCC members, we inoculated the standard (reference) ATCC strains of these species separately on Burkholderia cepacia selective agar (BCSA), tryptic soy agar (TSA), eosin methylene blue agar (EMB), and MacConkey agar and found that colonies of all four strains did not appear on these agar plates until 48-72 hours of incubation in a 35±1℃ ambient incubator. At 72 hours of culture on BCSA, the colonies of B. cepacia appeared yellowish with yellow surrounding areas; B. multivorans colonies were purple with light purp le surrounding areas; B. cenocepacia colonies were pinkish with yellow or pinkish surrounding areas; and B. gladioli colonies were pinpoint in size in the first streaking area of BCSA. The colonies on BCSA were then inoculated individually in triple sugar iron agar (TSI), lysine decarboxylase medium (LYS), oxidative/ fermentative (O/F) lactose medium, and O/F maltose medium. Oxidase test, Gram stain, and polymyxin B susceptibility test were then performed. Results showed that all four species/ strains were aerobic Gram-negative bacilli. Biochemical test results including those of TSI, LYS, O/F maltose, O/F lactose, and oxidase of these four organisms were distinct. Based on results of this study, we recommend the following procedures for detection of BCC in pharmaceuticals: (i) Inoculate 1 mL (or 1 g) of a sample in 10 mL TSB; (ii) After 48-72 hours of incubation at 35℃, inoculate the TSB culture on BCSA for 48-72 hours; (iii) Perform Gram staining and oxidase test; (iv) Inoculate to TSI and incubate for 18-24 hours; (v) Perform biochemical tests including LYS, O/F lactose and O/F maltose for further identification.

並列關鍵字

Burkholderia cepacia complex ； BCC ； Burkholderia cepacia selective agar ； procedure for detection of BCC