Stenotrophomonas maltophilia多重抗藥機轉之分析

Mechanisms of Multiple Antibiotic Resistance in Stenotrophomonas maltophilia

陳惠鳳(Hui-Feng Chen)

指導教授 ： 張玲麗

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

Stenotrophomonas maltophilia為需氧的革蘭氏陰性桿菌，是一種院內感染的重要病原體 ，它可伺機感染一些免疫能力不良的病人，且對多種的抗生素具抗藥性，尤其β-lactams類抗生素,然而造成 S. maltophilia多種抗藥性之機制目前尚未被清楚的提出，所以本實驗欲探討臨床S. maltophilia菌株對多種抗生素具抗藥性的原因與機轉。由於革蘭氏陰性菌中多重抗藥性的產生與抗藥基因的散佈有關，而抗藥基因除了以質體散佈外，近幾年integron也被認為是造成抗藥基因散佈的原因之一，所以本實驗首先以快速質體抽取法及以PCR增幅的方法來篩選93株S. maltophilia質體與integron的存在情形。結果發現10株(11 %)具有質體，20株(22 %)具class 1 integron，核酸定序基因片匣發現主要攜帶抵抗gentamicin (aac6’-Ib)、amikacin (aacA4)及trimethoprim (dfrⅡ)之抗藥基因，但並沒有發現具有class 2 integron。因此，S. maltophilia存在integron之比例較一般革蘭氏陰性菌低，由以上的結果認為S. maltophilia具多重抗藥性主要不是由質體或integron傳遞所造成。
由於S. maltophilia對β-lactams類抗生素具高度之抗藥性，且有研究報告指出與L1及L2 β-lactamase有關，所以本實驗也利用PCR增幅的方法分析L1與L2 β-lactamase基因於S. maltophilia中存在之情形及對β-lactams藥物之影響，結果發現83 %的菌株具有L1 β-lactamase基因，且具有L1 β-lactamase基因之菌株，會顯著提高meropenem及cefoperazone之抗藥程度。另外，有97 %之菌株具有L2 β-lactamase基因。由以上的數據發現L1與L2 β-lactamase基因廣泛的存在於S. maltophilia中，也推論L1與L2 β-lactamase可能是S. maltophilia造成β-lactams具抗藥性之主要原因。
近幾年也有報告指出S. maltophilia之多重抗藥性與multidrug efflux pump有關，目前於S. maltophilia發現有SmeABC及SmeDEF兩種multidrug efflux pump，所以吾人也利用RT-PCR及real-time PCR的方法研究分析smeABC及smeDEF表現與否，與抗生素之抗藥程度的關聯性，結果發現以RT-PCR與real-time PCR兩種方法做出來的結果是一致的，約59 %之菌株smeABC過度表現，且顯著提高S. maltophilia對cefoperazone、carbenicillin、ciprofloxacin、gentamicin及tetracycline之抗藥程度。另外，約31 %之菌株smeDEF過度表現，並顯著增加S. maltophilia對meropenem、cefoperazone及ciprofloxacin之抗藥程度。
綜合以上所述，發現integron在造成S. maltophilia多重抗藥性上，不若腸內菌來的重要，但認為β-lactamase的產生及multidrug efflux pumps的存在與S. maltophilia之多重抗藥性是密不可分的，希望藉此抗藥機制的研究能找出有效控制S. maltophilia多重抗藥性的
方法。

關鍵字

Stenotrophomonas maltophilia ； 抗藥性 ； 抗藥基因

並列摘要

Stenotrophomonas maltophilia is an aerobic, non-fermentative gram-negative bacteria . This organism has increasingly emerged as an important nosocomial pathogen, particularly for immunocompromised patient. Although S. maltophilia are often highly resistant to multiple antibiotics, especially β-lactams. However, the mechanisms of resistance are generally poorly understood. Therefore, the mechanism of multidrug resistance in clinical isolates of S. maltophilia was investigated in this study。
Generally, multidrug resistance is associated with widespread of integron and plasmid among gram-negative bacteria. For this reason, we analyzed the presence of plasmid and integron in 93 clinical isolates of S. maltophilia by plasmid extraction and polymerase chain reaction (PCR) in this study . The plasmid was found in 10 isolates (11%). Class 1 integron was detected in 20 of 93 (22 %) isolates. DNA sequencing of the PCR products analysis revealed a predominance of cassettes that confer resistance to aminoglycoside and trimethoprim. But, class 2 integron have not been found in the isolates. These data indicate that integron and plasmid did not play major roles in multidrug resistance in S. maltophilia.
L1 and L2 β-lactamases, which allow many S. maltophilia isolates to be resistant to β-lactam drug have been reported. Since these of clinical isolates display high-level of β-lactam drug resistance, the presence of L1、L2 β-lactamase and its association with resistance to β-lactam drug were analyzed. The results showed that L1 β-lactamase was observed in 83 % of clinical isolates and was significantly associated with increased resistance level to meropenem and cefoperazone. L2 β-lactamase was found in 97 % of clinical isolates. These results demonstrated that L1 and L2 β-lactamase were broadly present in S. maltophilia and may play major roles in intrinsic resistance to β-lactam drug.
Recent evidence indicates that multidrug resistant（MDR）efflux pump may be another contributing factor to the intrinsic and acquired multidrug resistance of S. maltophilia. By using RT-PCR and real-time PCR, the importance of multidrug efflux systems, SmeABC and SmeDEF, to antibiotic resistance in S. maltophilia was assessed. Consistent results were observed from RT-PCR and real-time PCR. Fifty-nine % of the strain overexpress the smeABC gene, which significantly increased resistance levels to cefoperazone、carbenicillin、ciprofloxacin、gentamicin and tetracycline. Thirty-one % of the strains overexpress the smeDEF gene. A significant correlation between smeDEF expression and meropenem、cefoperazone and ciprofloxacin resistance was observed.
All of the results from this research elucidate that β-lactamase and MDR efflux pumps may contribute more important roles than integron in multidrug resistance in S. maltophilia. Further development of antibiotics that are not substrates for efflux systems and of antibiotic efflux pump inhibitor combination will be important.

並列關鍵字

Stenotrophomonas maltophilia ； integron ； β-lactamase ； multidrug efflux pump

參考文獻

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