本院所分離之所有鮑氏不動桿菌對carbapenem 的抗藥性從 2005 年之 2.6%上升到 2006 年的 29.6%，引起院內感染事件的鮑 氏不動桿菌對carbapenem 的抗藥性也從 2006 年的 36%上升到 2007 年的 58%。除此之外疾病管制局所發佈 2007 年全國醫學中 心及區域醫院加護病房院內感染事件之致病菌中鮑氏不動桿菌 已經超越綠膿桿菌成為第一名，這些結果顯示抗carbapenem 鮑氏 不動桿菌近幾年快速增加，且不僅是本院的問題，也是本國其它 醫院的問題。
我們的研究也發現，抗imipenem 的鮑氏不動桿菌對其它常規 檢驗之抗生素也多呈抗藥性，包括ampicillin/sulbactam，最低抑 菌濃度的檢驗也顯示相同的結果，因此在治療由抗 carbapenem 引 起的感染事件，需考慮合併治療或使用較高感受性之藥物，如 colistin 或 tigecycline。
我們使用自動化微生物分析儀(Phoenix)進行 colistin 最低抑菌 濃度之測試，顯示colistin 有 100%感受性(MIC 範圍 0.5-1 μg/ml)。使用 E-test 法檢驗 tigecycline 的最低抑菌濃度，最低抑菌 濃度範圍0.38-16 μg/ml，感受度有 82.2%。因此這兩個藥物應可
考慮為抗carbapenem 鮑氏不動桿菌引起感染時之治療藥物。
研究的28 株對 carbapenem 有抗藥性之鮑氏不動桿菌都具有 有 blaOXA-66(100%)，26 隻菌株具有 blaOXA23（92.9%）。ISAba1 分別在25 株具有 blaOXA-23及28 株具有 blaOXA-66基因的上游被發 現。另外8 株對 imipenem 具感受性之鮑氏不動桿菌，發現各有 6 隻菌株具有 blaOXA-23或 blaOXA-66，在這些對 imipenem 具感受性之 鮑氏不動桿菌中，只有一株細菌 blaOXA-23前有 ISAba1 存在，有五 株細菌 blaOXA-66前有 ISAba1 存在，blaOXA-23前 ISAba1 存在情形 在對imipenem 具感受性及不具感受性兩者之間有明顯差異，這 或許代表OXA-23 在鮑氏不動桿菌對 carbapenem 的抗藥性有較大 影響，但此推論需進一步之研究證實。
脈衝式膠體電泳法進行基因分型，總共有 16 個基因分型。依 據Bionumeric 軟體的分析結果，可將這些菌株分成 4 個群集。其 中cluster A 有 14 株（50%），cluster C 有 8 株 (28.6%)。這個研 究結果顯示在本院流行的抗carbapenem 鮑氏不動桿菌主要是由 同時能產生OXA-23 及 OXA-66 流行菌株 cluster A 及 C 所組成。
建議
1. 進行環境調查，瞭解散播的來源，並進一步採取相關的感染 管制措施，以判斷環境調查的正確性及感染管制措施的有效 性，來阻止此抗藥性菌株的進一步散播。
2. 進行 OXA-23 及 OXA-66 之間交互作用的研究，以了解不同 OXA-type carbapenemase 對 carbapenem 抗藥性的影響。
3. 進行其它抗 carbapenem 機轉的研究，釐清各種抗藥性機轉之 散佈情形及影響程度。
4. 進行跨院際研究，瞭解抗 carbapenem 鮑氏不動桿菌及 OXA-23 carbapenemase 在台灣散佈之情形。
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盧孝國，黃玉成. Carbapenemase的探討。 感染控制雜誌 2000年第11卷第6期
童綜合醫院2003到2007年鮑氏不動桿菌
PFGE AscI profiles
表 1： 童綜合醫院2002到2007年鮑氏不動桿菌imipenem 抗藥性趨勢 年份 總分離菌株數 對Imipenem具抗
藥性之菌株數
抗imipenem比率 (%)
P value for trend test
2002 840 19 2.2
2003 672 7 1.0
2004 1058 11 1.0
2005 829 22 2.6
2006 751 222 29.6
2007 928 437 47.1
<0.001
表 2 童綜合醫院 2007年1到9月全部476株鮑氏不動桿菌分離部位之
縮寫：Pip/Tazobactam: piperacillin/tazobactam; SAM: ampicillin/sulbactam;
TMP/SMZ: trimethoprim/sulfamethazole.
註：aN代表菌株數目; b代表佔476株細菌中的百分比；c紙錠擴散法的檢驗結果。
表 3. 童綜合醫院 2007年01到09月不重複175株抗imipenem鮑氏不
縮寫： SAM: ampicillin/sulbactam; Pip/Tazobactam: piperacillin/tazobactam;
TMP/SMZ: trimethoprim/sulfamethazole.
註：a分離菌株數目; b在175株細菌裏所佔有的百分比
表 4. 童綜合醫院 2007年01到09月175株抗imipenem鮑氏不動桿菌
表 5. 二十八株抗carbapenem鮑氏不動桿菌分離病房單位分佈
病房單位/檢體類別 菌株數
加護病房 16
普通病房 7
慢性呼吸照護病房 3
急診室 1
門診 1
表 6. 二十八株抗carbapenem鮑氏不動桿菌分離檢體類別
檢體類別 菌株數
痰 11
尿液 6
膿 5
腹水 2
中心靜脈導管 2
關節腔引流管 1
血液 1
表 7. 二十八株抗carbapenem鮑氏不動桿菌對各類抗生素之最低抑菌
Amoxicillin/Clavulanate >16/8 >16/8 >16/8 0%
SAM >16/8 >16/8 ≤4/2 - >16/8 3.6%
縮寫： SAM: ampicillin/sulbactam; Pip/Tazobactam: piperacillin/tazobactam;
TMP/SMZ: trimethoprim/sulfamethazole.
依據美國臨床及實驗室標準協會（CLSI）2008年判讀標準，具感受性之最低抑 菌濃度（μg/ml）如下：Amikacin ≤16; gentamicin ≤4; imipenem ≤4; meropenem ≤4;
ceftazidime ≤8; cefotaxime ≤8; cefepime ≤8;SAM ≤8/4; pip/tazobactan ≤16/4;
TMP/SMZ ≤2/38; ciprofloxacin ≤1; levofloxacin ≤2; colistin ≤2; *tigecycline之最低 抑菌濃度檢測使用E-test紙條法，判讀標準依照美國食品藥物管理局發佈 tigecycline對腸道桿菌之判讀標準≤2μg/ml為具有感受性。
表 8. 二十八株抗carbapenem鮑氏不動桿菌對imipenem，colistin 及
縮寫：IPM imipenem; CS colistin; TGC tigecycline
註：a抗生素括弧內所標示為最低抑菌濃度之檢測方法。
依據美國臨床及實驗室標準協會（CLSI）2008年判讀標準，具感受性之最低抑菌濃度
（μg/ml）如下：imipenem ≤4; colistin ≤2; tigecycline之最低抑菌濃度檢測使用E-test紙條 法，判讀標準依照美國食品藥物管理局發佈tigecycline對腸道桿菌之判讀標準≤2μg/ml 為具有感受性。
表 9. 二十八株抗carbapenem鮑氏不動桿菌乙內醯胺酶等電點聚焦法分析
pI：等電點，isoelectric point
表 10. 二十八株抗carbapenem及八株不抗carbapenem的鮑氏不動桿菌 OXA-type carbapenemases分佈情形
抗carbapenem
（檢測菌株數：28）
不抗carbapenem
（檢測菌株數：8）
比例a
OXA-type 菌株數 菌株數 比例b
p value for X2 testc OXA-23 26 92.8% 6 75% 0.207
OXA-24 0 0% ND ND
OXA-51-like 28 100% 6 75% 0.044
OXA-58 0 0% ND ND
註：ND：not done，沒有進行此項檢測
註：a28株抗carbapenem的鮑氏不動桿菌OXA-23及OXA-66存在的百分比。b8株不抗 carbapenem的鮑氏不動桿菌OXA-23及OXA-66存在的百分比。
表 11. 二十八株抗imipenem及八株不抗imipenem的鮑氏不動桿菌ISAba1 存在blaOXA23 及 blaOXA66上游之情形
抗imipenem（總菌株數28） 不抗imipenem（總菌株數8）
ISAba1存在菌數/
ISAba1存在菌數/
有OXA-23或OXA-66 菌數
比例a 有OXA-23或OXA-66 菌數
比例b
P value for X2 testc
OXA-23 25/26 96.2% 1/6 16.7% <0.001 OXA-66 28/28 100% 5/6 83.3% 0.176
註：a28株抗carbapenem的鮑氏不動桿菌blaOXA23 及 blaOXA66上游存在ISAba1的百分比。
b8株不抗carbapenem的鮑氏不動桿菌blaOXA23 及 blaOXA66上游存在ISAba1的百分比。
表 12. 二十八株抗carbapenem鮑氏不動桿菌使用AscI切割後之脈衝式膠體
表 13. 二十八株抗imipenem鮑氏不動桿菌AscI 脈衝式膠體電泳分群及次
分群結果a
次分群 菌株數目 比例b
分群 (數目，比例)
A (14, 50%) A1 2 7.1%
A2 3 1.7%
A3 5 17.8%
A4 4 14.3%
B (1, 3.6%) Nil 1 3.6%
C (8, 28.6%) C1 2 7.1%
C2 4 14.3%
C3 2 7.1%
D (5, 17.8% ) Nil 5 17.8%
a相似度70%以上歸入同一分群（cluster），相似度85%以上歸入同一次分群。
b佔28株細菌的百分比。
附錄 附錄一、培養基
A. 液體培養基
1. Luria-Bertani broth (LB) Bacto-tryptone 4 g Bacto-yeast extract 2 g NaCl 4 g
加ddH2O 至400 ml，高溫滅菌 B. 固體培養基
1. Luria-Bertani agar plate Bacto-tryptone 4 g Bacto-yeast extract 2 g NaCl 4 g
Agar 6 g
加ddH2O 至400ml，高溫滅菌 2. Mueller-Hinton agar plate:
Beef extract 0.8 g
Acid hydrolysate ofcasein 7 g Agar 6 g
加ddH2O 至400ml，高溫滅菌 3. Tryptic Soy Broth (TSB)
Pancreatic digest of gelatin 4 g Lactose 2 g
Sucrose 2 g
Dipotassium phosphate 0.8 g Agar 5.4 g
Eosin Y 0.16 g
Methylene Blue 0.026 g
加ddH2O 至400ml，高溫滅菌 4. Eosin-methylene blue (EMB)
Pancreatic digest of casein 6.8 g Papaic digest of soybean meal 1.2 g Sodium chloride 2 g
Dipotassium phosphate 1 g Dextrose 1 g
加ddH2O 至400ml，高溫滅菌
附錄二、試劑與緩衝溶液 1. 抽取質體試劑:
Invitrogen – Quick Plasmid Miniprep Kit 2. 抽取染色體DNA 試劑:
GeneMark- Tissue & Cell Genomic DNA Purification Kit 3. 一般電泳試劑:
(1) 6X loading dye: 0.25% (w/v) bromophenol blue，0.25% (w/v) Xylene cyanol 及30% (w/v) Glycerol
(2) TAE buffer: 40mM Tris-acetate (pH 8.0)及2mM EDTA (pH 8.0) (3) Staining buffer: ethidium bromide (EtBr, o.5 μg/ml)
附錄三、PFGE 配製藥品
Cell suspension Buffer (100 mM Tris and 100 mM EDTA, pH 8.0) Preparation:
10 ml of 1M Tris , pH 8.0 20 ml of 0.5M EDTA, pH 8.0
Dilute to 100ml with sterile de-ionized water Store at room temperature
TE Buffer (10 mM Tris and 1 mM EDTA, pH 8.0) Preparation:
10 ml of 1M Tris , pH 8.0 20 ml of 0.5M EDTA, pH 8.0
Dilute to 1000ml with sterile de-ionized water Store at room temperature
Cell Lysis Buffer (50 mM Tris; 50 mM EDTA, pH 8.0 and 1% Sarcosine) Preparation:
25 ml of 1M Tris , pH 8.0 50 ml of 0.5M EDTA, pH 8.0
50 ml of 10% sodium lauroyl sarcosine, membrane-filtered OR
5g of Sarcosyl powder (N-Lauroyl-Sarcosine, Sodium salt) Dilute to 500 ml with sterile de-ionized water
10X TBE Buffer (5 mM Tris; 4.5 mM Boric acid; 0.05 mM EDTA, pH 8.0) Preparation:
121.14g of Tris base 55g of Boric acid
20ml of 0.5M EDTA pH 8.0
Dilute to 1000 ml with sterile de-ionized water 0.5X TBE (TRIS Borate EDTA)
Preparation:
100ml of 10X TBE
Diluet to 2000ml with distilled water
Ethidium Bromide (10mg/ml) stock solution Carcinogenic Preparation:
250mg Ethidium Bromide
Dilute to 25ml with distilled water Store at 4°C in a lightproof container
附錄四、 PFGE PPlug Washing by Peristaltic Pump Method Solution
Amount of Solution
Water Bath Temperature
minutes, then flush
( ≒2 min) DDW 500ml 50°C 2.5 Circular for 6
minutes, then flush
( 2 ≒ min) TE 500ml 50°C 3.0 Flushed ( ≒2
min) TE 500ml 50°C 2.5 Circular for 6
minutes, then flush
( 2 ≒ min) TE 500ml 50°C 2.5 Circular for 6
minutes, then flush
( 2 ≒ min) TE 500ml 2.5 Circular for 6
minutes, then flush
( 2 ≒ min) 50°C
* 20 screening Cap Column holds ≒ 200ml; actual amount flushed into waste container is equal 300ml.
The setting in the above table gives excellent plug washing results for 15 to 25 plugs caps containing 2 plugs from reusable plug mold or 6 plugs from
disposable plug mold. For 10 plugs or less smaller amounts of water, buffer and time may be able to be adjusted.
附錄五：本研究所使用之聚合酶連鎖反應啟動子
附錄五：本研究所使用之聚合酶連鎖反應啟動子