| 摘要: | 近年來隨著抗生素的濫用，已使得臨床中感染具多重抗藥性病原菌的案例逐年升高。由於大部分的致病菌已知多是透過一般環境系統(特別是農業系統)的微生物而獲得抗性基因，因此環境中抗生素抗藥性的發展所引發的公衛問題實有必要深入了解。值得注意的是，重金屬已被視為極可能是維持ARGs在環境流布存續的關鍵因子之一，但此種非傳統抗生素抗藥性發展的途徑(即透過金屬的共選擇而誘發抗生素抗性的機制)，截至目前為止仍未得到廣泛的研究，尤其是重金屬的生物有效性於此途徑所扮演的角色。有鑑於此，本研究以不具金屬與抗生素抗性的野生型大腸桿菌和四環素(Tet)做為模式細菌和模式抗生素，並以台灣環境最常見的重金屬污染“銅”做為模式重金屬，從自由離子活性模型(FIAM)的角度切入，探究重金屬的生物有效性與其共選擇抗生素抗藥性之間的實際關聯程度。模式菌株在以葡萄糖爲碳源且組成明確的試驗培養基中進行暴露實驗，當中的總銅Cu(II)和Tet濃度分別保持在亞致死等級的EC50 (0.125 ppm)和EC75 (6.80 ppm)，且將培養基所含的氯化物調控在1至100 mM之間，從中建立自由銅離子(即游離、未被錯合的二價銅物種，Cu2+)的濃度梯度，使得“二價銅的生物利用度提高會加速與加強菌株對Tet抗藥性”的假說得以測試。研究結果表明，在為期14天的培養過程中，雖然僅暴露Tet的對照組會因持續來自抗生素的直接選擇壓力最終發展出對於Tet的抗性，但在Tet和Cu(II)交替暴露的實驗組別中，除了發現Cu(II)的加入讓菌株對Tet更快產生抗性、並增強其抗性程度外，也發現較高的Cu2+ 濃度的暴露可導致較快和較高的Tet抗性發展；而當0.1至10 uM的EDTA取代氯化物以塑造出同樣的Cu2+濃度梯度的暴露環境時，也可觀察到幾乎相同的Tet抗藥性發展，再次證實FIAM可解釋菌株對於二價銅的攝取模式。不僅如此，當菌株在兩週的期程中只接觸到Cu(II)的情況下，最終仍表現出類似交替暴露時的Tet抗性發展，實質驗證重金屬的共選擇效應。本研究進一步利用RT-qPCR的相對定量法，檢視菌株在暴露過程中對於Cu和Tet的抗性基因型表徵，其數據顯示雖然只有兩個Cu抗性(pcoA, cusA)與一個Tet抗性(tetA)基因被誘導，但這些基因的表達皆與Cu2+的暴露量呈顯著正相關，此與以生長為主的表現型表徵結果相呼應。整體而言，本研究的結果顯示不論從細胞還是分子層面的解析，都反映出金屬的生物有效性確實是環境中驅動重金屬共選擇抗生素抗藥性發展與持續的關鍵決定因子。;In recent years, the misuse of antibiotics has led to a yearly increase in clinical cases of infections caused by multidrug-resistant pathogens. Since most pathogenic bacteria are known to acquire resistance genes through environmental systems (particularly agricultural systems) where microorganisms prevail, understanding the development of antibiotic resistance in the environment has become a crucial public health issue. Notably, heavy metals are considered a key factor in the persistence and distribution of antibiotic resistance genes (ARGs) in the environment. However, this non-traditional pathway of antibiotic resistance development (i.e., co-selection through metals) has not been extensively studied, particularly regarding the role of metal bioavailability in this process. Therefore, this study employs a wild-type Escherichia coli strain (lacking resistance to metals and antibiotics) and tetracycline (Tet) as the model bacterium and antibiotic, respectively. Copper (Cu), the most common heavy metal pollutant in Taiwan, is used as the model metal. From the perspective of the Free Ion Activity Model (FIAM), this study investigates the relationship between metal bioavailability and the co-selection of antibiotic resistance. The model strain was exposed to a defined test medium using glucose as the carbon source, maintaining total Cu(II) and Tet concentrations at sub-lethal levels of EC50 (0.125 ppm) and EC75 (6.80 ppm), respectively. The chloride content in the medium was adjusted between 1 to 100 mM to establish a gradient of free copper ions (unbound divalent copper species, Cu2+). This allowed testing the hypothesis that increased bioavailability of divalent copper accelerates and enhances the strain’s resistance to Tet. Results indicated that during the 14-day culture period, the control group exposed only to Tet developed Tet resistance due to sustained selective pressure. However, in the Tet and Cu(II) alternating exposure groups, the addition of Cu(II) not only expedited the development of Tet resistance but also enhanced its degree. Higher Cu2+ concentrations led to faster and stronger Tet resistance development. When EDTA (0.1 to 10 µM) was used instead of chloride to create a similar Cu2+ concentration gradient, nearly identical Tet resistance development was observed, further validating that FIAM could explain the uptake patterns of divalent copper by the strain. Furthermore, the strain exhibited similar Tet resistance development after two weeks of Cu(II) exposure alone, confirming the co-selection effect of heavy metals.Using RT-qPCR for relative quantification, the study examined the genotypic expression of Cu and Tet resistance genes during exposure. Data showed that although only two Cu resistance genes (pcoA, cusA) and one Tet resistance gene (tetA) were induced, their expression significantly correlated with Cu2+ exposure levels, aligning with the phenotypic results focused on growth. Overall, the study’s findings from both cellular and molecular analyses indicate that metal bioavailability is indeed a critical determinant driving the co-selection and persistence of antibiotic resistance in the environment. |
