二級出水中環丙沙星耐藥菌光輻照滅活研究

石 娜,孫迎雪,齊 菲,胡春芳 (北京工商大學環境科學與工程系,北京 100048)

二級出水中環丙沙星耐藥菌光輻照滅活研究

石 娜,孫迎雪*,齊 菲,胡春芳 (北京工商大學環境科學與工程系,北京 100048)

針對二級出水中的一株環丙沙星耐藥菌,研究了菌株生長特性和光輻照對其滅活、復活及耐藥性的影響.結果表明,該菌株對青霉素、氨芐西林、磺胺甲惡唑、四環素和利福平均具有耐受性,在環丙沙星存在的條件下,其最大比生長速率由0.63h-1降低到0.51h-1.光輻照對環丙沙星耐藥菌的滅活率基本隨光照強度和輻照時間的增加而升高,且基本符合零級或一級化學反應動力學.可見光(100/300/500W汞燈和1000W氙燈(＞400nm))輻照60min,環丙沙星耐藥菌滅活率達到0.25～0.39log.100/300W汞燈和1000W氙燈(＞400nm)輻照下環丙沙星耐藥菌的變化符合零級反應動力學,滅活速率為10196.43～11345.24CFU/(mL·min),500W汞燈(＞400nm)輻照下環丙沙星耐藥菌的變化符合一級反應動力學,滅活速率為 0.01min-1.可見光+UVA(100/300/500W 汞燈和 1000W 氙燈(＞300nm))輻照 60min,環丙沙星耐藥菌滅活率達到 0.30～5.63log.100W 汞燈(＞300nm)輻照下環丙沙星耐藥菌的變化符合一級反應動力學,滅活速率為0.01min-1,300W汞燈(＞300nm)輻照下環丙沙星耐藥菌的變化符合零級反應動力學,滅活速率為2572.02CFU/(mL·min).未完全滅活的耐藥菌存在復活,其48h光、暗復活率達到-3.9%～123.4%.在光輻照過程中,只有1000W可見光+UVA輻照影響環丙沙星耐藥菌的耐藥性,輻照60min,其抑菌圈直徑由11.0mm下降到8.0mm.

環丙沙星耐藥菌；可見光；UVA；滅活率；環丙沙星耐藥性

抗生素的濫用導致抗生素耐藥菌的產生和傳播日益嚴重[1-2].據調查,2013年我國抗生素使用量高達 162000t,其中,喹諾酮類抗生素環丙沙星使用量達到5340t[3].抗生素在生物體內不能完全代謝,會以原有的形式或代謝產物的形式排出體外,最終進入城市污水處理廠,而污水處理廠的生物處理和氯消毒、紫外消毒等傳統消毒工藝對抗生素和抗生素耐藥菌的去除效果有限[1,4],進而導致抗生素和抗生素耐藥菌進入水環境[5-6],威脅水生態環境和人類健康.

太陽光具有滅活地表水中微生物的能力,這也是地表水中抗生素耐藥菌降解的重要機制之一[1,7-9].有研究發現,太陽光輻照水體可滅活幾個數量級的大腸桿菌[10].細菌細胞中的光敏化合物卟啉經400～430nm可見光輻照后會返回基態與氧結合,并轉移能量,產生單線態氧、超氧自由基、羥基自由基和過氧化氫等活性氧(ROS)破壞細胞[11].由于太陽光中的短波紫外線(UVC)在經過地球表面同溫層時被臭氧層吸收,不能到達地球表面,中波紫外線(UVB)在自然水體中很快衰減,因此,較長波段的光的潛在價值更加重要[12].

本研究選取環丙沙星耐藥菌作為研究對象,重點考察可見光和可見光+UVA光輻照對該菌株的滅活、復活的控制效果以及對耐藥性的影響,解析光輻照的機理,以期為城市水環境中抗生素耐藥菌的控制提供基礎數據.

1 材料與方法

1.1 耐藥菌的分離與純化

采用膜過濾法和平板劃線分離法從二級出水中(水樣取自甘肅省白銀市某城市污水處理廠A2/O工藝二級出水)篩選對環丙沙星抗生素有耐藥性的大腸桿菌菌株[6].添加抗生素的濃度參考CLSI規定的最低抑菌濃度(MIC)[13].以大腸埃希菌ATCC 25922[13]作為質控菌.

1.2 耐藥菌生長特性

將單菌落接種于含 4mg/L環丙沙星的胰蛋白胨大豆肉湯培養基(TSB)中,培養10～12h作為種子培養液(OD600＞1),再將種子液接種于含有不同濃度(0、4、8mg/L)環丙沙星的 TSB中培養,同時制作空白培養基,每 2h測定 OD600,測定至12h,20h后,每1h測定OD600,測定至24h.

1.3 耐藥菌生長動力學參數確定

用S-Gompertz模型[16]擬合環丙沙星耐藥菌的生長曲線,獲得環丙沙星耐藥菌的最大生長量、最大比生長速率和遲滯時間.具體數學表達式如下:

式中:N為某生長時間t下細菌懸濁液在600nm波長下的吸光度值,cm-1;N0為細菌懸濁液在600nm波長下的初始吸光度值(低于檢測限,則初始值定為 0.001),cm-1;Nm為細菌的最大生長量,即細菌懸濁液在 600nm波長下的最大吸光度值,cm-1;μm為細菌的最大比生長速率,h-1;λ為遲滯時間,h;t為生長時間,h.

1.4 耐藥菌對典型抗生素的耐受性分析

采用紙片擴散法考察環丙沙星耐藥菌對青霉素、氨芐西林、氯霉素、四環素、磺胺甲惡唑和利福平 6種典型抗生素的耐受性[13].(35±2)℃孵育 16～18h,根據抑菌圈直徑的大小,判斷細菌對6種抗生素的耐受性.

1.5 光輻照控制

1.5.1 樣品制備 從平板培養基上挑選單菌落接種至含 4mg/L環丙沙星的營養肉湯培養基中,37℃過夜培養,然后取 20mL的過夜培養菌液,4500g離心5min,棄清液,再用無菌水充分懸浮菌體沉淀,洗去培養基和抗生素,4500g離心5min,反復2次,最后將菌體懸浮于無菌水中,用麥氏比濁法獲得1.5×108CFU/mL(0.5麥氏),隨后加入到無菌水中,獲得預期濃度的反應樣品.

1.5.2 光輻照反應 使用光化學反應儀(XPA-7,南京胥江機電廠)進行光輻照實驗,反應儀中心部位配有 100/300/500W 汞燈(265.2～579nm)或1000W 氙燈(200～1200nm),另外,將汞燈和氙燈分別配合300或400nm濾波片使用產生不同的輻照條件.利用可見光光輻照計(FZ-A,北京師范大學機電廠)和紫外光輻照計(UVA,北京師范大學機電廠)測定光輻照強度.將準備好的菌液加入到已滅菌的反應試管,然后放入光化學反應儀外圍的固定裝置中,并進行磁力攪拌,開始光輻照實驗.

1.5.3 光復活和暗修復 將1.5.2中光輻照后的最終樣品轉移至滅菌的燒杯中,用于光復活和暗修復試驗.試驗條件分別為實驗室日光燈下和黑暗條件下磁力攪拌,24和48h測定細菌濃度[14].

1.5.4 耐藥菌的檢測和滅活率計算 采用平板計數法測定樣品中的環丙沙星耐藥菌.37℃培養24h,計菌落數,用單位體積水樣的菌落形成單位(CFU/mL)表示.

式中:N0為光輻照前樣品中環丙沙星耐藥菌的菌落數;Nt為光輻照后樣品中環丙沙星耐藥菌的菌落數.

1.5.5 耐藥性分析 采用藥敏紙片擴散法考察環丙沙星耐藥菌耐藥性的變化[13].同1.4.

2 結果與討論

2.1 環丙沙星耐藥菌的生長特性

環丙沙星耐藥菌的生長特性能夠反映其在不同環境中的增殖特性[15].從表1可以看出,環丙沙星耐藥菌的最大生長量(以 OD600計)在 2.58～2.89之間,且隨抗生素濃度的增加而升高,在8mg/L環丙沙星條件下最大生長量最高為 2.89,這說明高濃度抗生素條件下菌株利用碳源和合成菌體的能力更強、效率更高[15].環丙沙星耐藥菌的最大比生長速率在 0.51～0.63h-1之間,抗生素濃度增加,最大比生長速率從 0.63h-1下降到0.51h-1.在有抗生素的條件下該菌株的遲滯時間增加,0、4、8mg/L環丙沙星條件下菌株的遲滯時間分別為2.06、2.52、2.12h.

表1 不同環丙沙星濃度下耐藥菌的最大生長量(Nm)、最大比生長速率(μm)和遲滯時間(λ)Table 1 Maximum increment (Nm), maximum specific growth rate (μm) and lag time (λ) of ciprofloxacinresistant bacteria versus different concentration of ciprofloxacin

2.2 環丙沙星耐藥菌對不同抗生素的耐受性

從表2可以看出,環丙沙星耐藥菌對青霉素、氨芐西林、磺胺甲惡唑未形成明顯抑菌圈,耐受能力很強;對四環素和利福平的抑菌圈直徑均為10.5mm,具有一定的耐受性;對氯霉素的抑菌圈直徑為 15mm,藥敏性為中介.這說明污水中環丙沙星耐藥菌對 β-內酰胺類抗生素的耐受能力普遍較高,同時,該菌株對磺胺甲惡唑耐受性也較強,對四環素和利福平的耐受性次之,對氯霉素的耐受能力最弱,這可能與污水處理廠附近居民的抗生素使用類型和使用頻率等有關.文獻中報道的從污水中分離的環丙沙星多重耐藥菌多對 β-內酰胺類抗生素、四環素類抗生素和磺胺類抗生素有耐藥性,這與本文的研究結果類似[6,17].

表2 環丙沙星耐藥菌對6種抗生素的耐受性Table 2 Resistance of ciprofloxacin-resistant bacteria to six antibiotics

2.3 可見光對環丙沙星耐藥菌的影響

100、300、500W汞燈和1000W氙燈配合400nm截止濾波片產生的可見光的光強分別是18.9、27.3、40.2和115.8mW/cm2.從圖1(a)可以看出,環丙沙星耐藥菌隨可見光輻照時間的增加而減少.可見光輻照60min時,環丙沙星耐藥菌由1.30×106～1.36×106CFU/mL 降低到 5.35×105～7.6×105CFU/mL,說明可見光對環丙沙星耐藥菌具有一定的滅活作用.早在1930年,Gates發現只要增加光通量,波長在400nm以上的可見光與紫外光一樣可以滅活細菌[18],當 DNA損傷率超過修復率,細胞即發生死亡[18-19].目前,可見光殺菌的研究光源多為單波長可見光,相關研究發現,可見光輻照是否具有殺菌效果與波長有很大關系,如405、435、470、525、610、630、810和880nm波長的可見光具有殺菌功能[20-25],405nm 可見光可以破壞耐藥基因[26],但 625nm 可見光不能殺菌[22].本文中環丙沙星耐藥菌的滅活應該是具有殺菌功能的不同單波段可見光共同作用的結果.

圖1 可見光輻照對環丙沙星耐藥菌的影響Fig.1 Effect of visible light irradiation onciprofloxacin-resistant bacteria

從圖1(b)可以看出,環丙沙星耐藥菌的滅活率隨光照強度增加而升高,光照強度越高,滅活效果越好.可見光輻照60min時,100、300、500和 1000W 可見光輻照下環丙沙星耐藥菌的滅活率分別為 0.25、0.31、0.34和 0.39log.1000與500W對環丙沙星耐藥菌的滅活效果相當,其滅活率分別是 0.34和 0.39log,而前者光照強度遠高于后者光照強度,這可能是因為不同光源發射的可見光各波段光強有所差別.另外,同一光照強度下,環丙沙星耐藥菌的滅活率隨輻照時間的增加而升高.光照強度在時間上的累積即為輻照劑量,可見光對細菌的影響與輻照劑量密切相關[21-22].例如,470nm 可見光輻照劑量達到10J/cm2以上才能滅活金黃色葡萄球菌[21]; 405nm藍光和880nm紅外光同時輻照可滅活金黃色葡萄球菌和銅綠假單胞菌,最佳輻照劑量20J/cm2分別可滅活金黃色葡萄球菌、銅綠假單胞菌72%和93.8%[25].

表3 可見光輻照0～60min環丙沙星耐藥菌滅活動力學擬合結果Table 3 Kinetic of visible light irradiation on ciprofloxacin-resistant bacteria

將可見光輻照 0～60min的環丙沙星耐藥菌濃度變化進行動力學方程擬合,如表3所示,得出100、300和1000W輻照下環丙沙星耐藥菌的變化符合零級反應動力學,反應速率隨光照強度的增加而升高,在1000W輻照下反應速率常數最大為 11345.24CFU/(mL·min),500W 輻照下環丙沙星耐藥菌的變化符合一級反應動力學,光輻照反應速率常數為0.01min-1.

2.4 可見光+UVA對環丙沙星耐藥菌的影響

100、300、500W汞燈和1000W氙燈配合300nm截止濾波片產生的可見光+UVA的光強分別是25.4、37.3、61.2和118.63mW/cm2,其中UVA光強分別是6.5、10.0、20.0和2.83mW/cm2.從圖2可以看出,光輻照60min時,環丙沙星耐藥菌由2.90×105～1.30×106CFU/mL降低到0～1.46× 105CFU/mL,滅活率達到0.30～5.63log.500W可見光+UVA對環丙沙星耐藥菌的滅活效果最好,反應20min,即輻照劑量為414J/cm2時,環丙沙星耐藥菌的滅活率達到5.63log,遠遠高于500W可見光輻照60min時的滅活率,這說明UVA在耐藥菌滅活過程中起主要作用.100、300W輻照下,可見光+UVA消毒效果略好于可見光消毒效果,這可能是因為低功率汞燈發射的 UVA較弱.而1000W輻照下,10min時可見光+UVA與可見光對環丙沙星耐藥菌的滅活率相同,隨后前者的滅活率低于后者,50min后,前者對環丙沙星耐藥菌的滅活率才迅速增加,60min時其滅活率達到1.60log,高于可見光輻照的滅活率 0.39log,這說明UVA在反應前期未對環丙沙星耐藥菌產生明顯的滅活作用,而在后期開始發揮顯著的消毒效果,這可能是UVA氧化損傷積累的結果.UVA對DNA的破壞分為直接損傷和氧化損傷,直接損傷是通過形成環丁烷嘧啶二聚體直接破壞DNA,氧化損傷包括I型和II型光氧化反應[27].

圖2 可見光+UVA(＞300nm)輻照對環丙沙星耐藥菌的影響Fig.2 Effect of visible light with UVA irradiation on ciprofloxacin-resistant bacteria

將可見光+UVA輻照 0～60min環丙沙星耐藥菌濃度的變化進行動力學方程擬合,如表4所示,得出 100W輻照下環丙沙星耐藥菌的變化符合一級反應動力學,光輻照反應速率常數為0.01min-1,300W 輻照下環丙沙星耐藥菌的變化符合零級反應動力學,光輻照反應速率常數為2572.02CFU/(mL·min),其他2種輻照條件下環丙沙星耐藥菌的變化既不符合零級反應動力學,也不符合一級反應動力學.

表4 可見光+UVA輻照0～60min環丙沙星耐藥菌滅活動力學擬合結果Table 4 Kinetic of visible light with UVA irradiation on ciprofloxacin-resistant bacteria

2.5 光復活和暗修復

從圖3可以看出,除500W可見光+UVA輻照的環丙沙星耐藥菌完全滅活后無復活外,其他條件輻照后的環丙沙星耐藥菌在光照和黑暗條件下復活率隨時間的增加而升高,復活率從 24h的-74.5～89.0%增加到48h的-3.9～123.4%.從圖3(a)光復活情況可以看出,可見光輻照后的環丙沙星耐藥菌的光復活率隨光照強度的增加而降低,48h光復活后, 100W可見光輻照后的耐藥菌復活率最高為96.0%,1000W可見光輻照后的耐藥菌復活率最低為-3.9%;可見光+UVA輻照后,除 500W 外,24h時環丙沙星耐藥菌仍在持續消減,48h時光復活率隨光照強度的增加而降低,其中100W可見光+UVA光輻照后的耐藥菌復活率最高為123.4%, 1000W可見光+UVA光輻照后的耐藥菌復活率最低為 27.4%.從圖 3(b)暗修復情況可以看出,在可見光中,300W 輻照后的環丙沙星耐藥菌復活率最低,24和48h的復活率分別為-29.9%和67.9%,1000W輻照后的環丙沙星耐藥菌復活率最高,24和48h的復活率分別為73%和90.8%;可見光+UVA輻照后,除 500W 外,在24h,100W輻照后的環丙沙星耐藥菌復活率最低為-74.5%,在48h,1000W輻照后的環丙沙星耐藥菌復活率最低為37.7%.

光輻照后環丙沙星耐藥菌持續消減可能與殘余消毒效果有關.Xiong和 Hu研究了 UVA/ TiO2體系中大腸埃希菌ATCC 700891在30、60、90min間歇照射后0～240min的暗修復情況,發現UVA/TiO2體系在黑暗條件下具有一定的殘余消毒效果,且光照時間越長,殘余消毒效果越顯著[14].殘余消毒效果可能與光輻照過程中產生的穩定氧化劑如 H2O2有關[28],H2O2可在水中持續存在數小時,起到抑制細菌再生的作用[29].

圖3 光輻照后環丙沙星耐藥菌的光復活(a)和暗修復(b)效果(“+”表示環丙沙星耐藥菌無存活)Fig.3 The photo reactivation (a) and dark repair (b) of the ciprofloxacin-resistant bacteria after light irradiation (“+”represented neither photo reactivation nor dark repair )

2.6 環丙沙星耐藥菌耐藥性的去除

圖4 可見光+UVA對環丙沙星耐藥菌耐藥性的影響Fig.4 Effect of visible light with UVA on the ciprofloxacin-resistant bacteria

抗生素耐藥性是指細菌在抗生素存在條件下的生存和生長能力[30].根據美國臨床和實驗室標準協會制定的抗菌藥物敏感性試驗執行標準,腸桿科細菌對環丙沙星耐受性的判定標準是:抑菌圈直徑 D≥21mm為敏感,抑菌圈直徑 D=16～20mm為中介,抑菌圈直徑 D≤15mm為耐藥[13].抑菌圈直徑越大,抗生素耐藥性越低.結果表明,1000W可見光+UVA輻照的環丙沙星耐藥菌抑菌圈直徑總體呈下降趨勢,輻照 60min,抑菌圈直徑由11.0mm下降到8.0mm,即環丙沙星耐藥菌耐藥性增強,如圖4所示,其他光輻照條件均不影響環丙沙星耐藥菌的耐藥性,其抑菌圈直徑大小一直維持在 11.0mm,這與有關太陽光輻照對多重耐藥菌耐藥性影響的研究結果有所不同, Rizzo等[1]研究發現太陽光輻照 180min,多重耐藥性大腸桿菌對環丙沙星的MIC降低33%.

從圖5可以看出,100、300W可見光和可見光+UVA輻照后的環丙沙星耐藥菌在光復活和暗修復后,抑菌圈直徑不變.除 48h光復活,500W可見光輻照后的環丙沙星耐藥菌在復活后抑菌圈直徑由11.0mm增加到21.0～22.0mm,即環丙沙星耐藥菌的耐藥性有所降低.1000W可見光輻照后的環丙沙星耐藥菌只在暗修復 48h時,抑菌圈直徑由11mm減小到10.5mm.而在可見光+UVA中,只有1000W可見光+UVA輻照影響環丙沙星耐藥菌在光復活和暗修復后抑菌圈直徑,分別從11.0mm減小到10.0mm和9.3mm,即環丙沙星耐藥菌耐藥性增強,這說明環丙沙星耐藥菌對UVA具有一定的耐受性.Huang等發現紫外(UV254)消毒可使四環素耐藥菌的半抑制濃度(IC50)降低40%,即四環素耐藥菌對 UV254無耐受性[31].可見抗生素耐藥菌對不同波長光的耐受性不同.

圖5 光復活(a)和暗修復(b)后環丙沙星耐藥菌抑菌圈直徑的變化Fig.5 The Inhibitory circle diameter of ciprofloxacin-resistant bacteria after photo reactivation (a) and dark repair (b)*, P＜0.1; **, P＜0.05; ***, P＜0.01

3 結論

3.1 抗生素對抗生素耐藥菌的生長具有一定的影響.該環丙沙星耐藥菌對青霉素、氨芐西林、磺胺甲惡唑、四環素和利福平均具有耐受性.

3.2 可見光和可見光+UVA對環丙沙星耐藥菌的滅活率基本隨光照強度和輻照時間的增加而升高.可見光中,1000W 輻照下環丙沙星耐藥菌的滅活效果最好,輻照 60min(輻照劑量為73.44J/cm2),滅活率達到0.39log;可見光+UVA中, 500W 輻照下環丙沙星耐藥菌的滅活效果最好,輻照 20min(輻照劑量為 414J/cm2),滅活率達到5.63log.

3.3 不同光輻照條件對環丙沙星耐藥菌的光復活和暗修復影響不同.500W 可見光+UVA 輻照的環丙沙星耐藥菌完全滅活后無復活,未完全滅活的環丙沙星耐藥菌復活率達到-3.9%～123.4%. 3.4 在光輻照過程中,只有1000W可見光+UVA輻照影響環丙沙星耐藥菌的耐藥性;光復活和暗修復后,100、300W可見光和可見光+UVA輻照后的環丙沙星耐藥菌的抑菌圈直徑不變,其他條件輻照后的耐藥菌抑菌圈直徑發生改變.

[1] Rizzo L, Fiorentino A, Anselmo A. Effect of solar radiation on multidrug resistant E. coli strains and antibiotic mixture photodegradation in wastewater polluted stream [J]. Science of the Total Environment, 2012,427-428:263-268.

[2] Dunlop P S, Civola M, Rizzo L, et al. Effect of photocatalysis on the transfer of antibiotic resistance genesin urban wastewater [J]. Catalysis Today, 2015,240:55-60.

[3] Zhang Q Q, Ying G G, Pan C G, et al. Comprehensive evaluation of antibiotics emission and fat in the river basins of China: Source analysis, multimedia modeling, and linkage to bacterialresistance [J]. Environmental Science & Technology, 2015,49(11):6772-6782.

[4] Munir M, Wong K, Irene X. Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan [J]. Water Research, 2011,45(2):681-693.

[5] Karaolia P, Michael I, Fernandez I G, et al. Reduction of clarithromycin and sulfamethoxazole-resistant Enterococcus by pilot-scale solar-driven Fenton oxidation [J]. Science of the Total Environment, 2014,468-469:19-27.

[6] 盧 誠,張 俊,王 釗,等.河北潘家口水庫氯霉素類抗生素檢測及風險評估 [J]. 中國環境科學, 2016,36(6):1843-1849.

[7] Boehm A B, Yamahara K M, Love D C, et al. Covariation and photoinactivation of traditional and novel indicator organisms and human viruses at a sewage-impacted marine beach [J]. Environmental Science & Technology, 2009,43(21):8046-8052.

[8] Davies C M, Evison L M. Sunlight and the survival of enteric bacteria in natural-waters [J]. Journal Applied Microbiology, 1991,70(3):265-274.

[9] Sinton L W, Hall C H, Lynch P A, et al. Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters [J]. Applied and Environmental Microbiology, 2002,68(3):1122-1131.

[10] Maraccini P A, Wenk J, Boehm A B. Photoinactivation of eight health-relevant bacterial species: Determining the importance of the exogenous indirect mechanism [J]. Environmental Science & Technology, 2016,50:5050-5059.

[11] Ghate V, Leong A L, Kumar A, et al. Enhancing the antibacterial effect of 461 and 521nm light emitting diodes on selected foodborne pathogens in trypticase soy broth by acidic and alkaline pH conditions [J]. Food Microbiology, 2015,48:49-57.

[12] Kadir K, Nelson K L. Sunlight mediated inactivation mechanisms of Enterococcus faecalis and Escherichia coli in clear water versus waste stabilization pond water [J]. Water Research, 2014, 50:307-317.

[13] CLSI. Clinical and Laboratory Standards Institute. http://www. clsi.org/.

[14] Xiong P, Hu J Y. Inactivation/reactivation of antibiotic-resistant bacteria by a novel UVA/LED/TiO2system [J]. Water Research, 2013,47:4547-4555.

[15] 席勁瑛,黃晶晶,胡洪營,等.污水處理廠二級出水中四環素抗性菌的生長特性與耐藥性 [J]. 環境科學, 2014,121(7):1723-1729.

[16] Zwietering M H, Jongenburger I, Rombouts F M, et al. Modeling of the bacteria growth curve [J]. Applied and Evironmental Microbiology, 1990,56(6):1875-1881.

[17] Ferro G, Fiorentino A, Alferez M C, et al. Urban wastewater disinfection for agricultural reuse: effect of solar driven AOPs in thei nactivation of a multidrug resistant E. coli strain [J]. Applied Catalysis B: Environmental, 2015,178:65-73.

[18] Gates F L. A study of the bactericidal action of ultraviolet light. III: The absorption of ultraviolet light by bacteria [J]. The Journal Genral Physiology, 1930,14(1):31-42.

[19] Gad F, Zahra T, Hasan T, et al. Effects of growth phase and extracellular slime on photodynamic inactivation of grampositive pathogenic bacteria [J]. Antimirobial Agents and Chemotherapy, 2004,48(6):2173-2178.

[20] Wilson M. Lethal photosensitization of oral bacteria and its potential in the photodynamic therapy of oral infections [J]. Photochemistry and Photobiology, 2004,3(5):412-418.

[21] Guffey J S, Wilborn J. In vitro bactericidal effect of 405nm and 470nm Blue Light [J]. Phtomedicine and Laser Surgery, 2006, 24(6):684-688.

[22] Kim S, Kim J, Lim W, et al. In vitro bactericidal effect of 625, 525 and 425nm wavelength (red, green, and blue) light-emitting diode irradiation [J]. Photochemistry and Photobiology, 2013, 31(11):554-562.

[23] Nussbaum E L, Lilge L, Mazzulli T. Effects of low level laser therapy (LLLT) of 810nm upon in vitro growth of bacteria: relevance of irradiance and radiant exposure [J]. Journal Clinical Laser Medicine and Surgery, 2003,21(5):283-290.

[24] Nussbaum E L, Lilge L, Mazzulli T. Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50J/cm2on three species of bacteria in vitro [J]. Journal Clinical Laser Medicine and Surgery, 2002,20(6):325-333.

[25] Guffey J S, Wilborn J. Effects of combined 405nm and 880nm light on Staphylococcus aureus and Pseudomonas aeruginosa in vitro [J]. Photomedicine and Laser Surgery, 2006,24(6):680-683.

[26] Enwemeka C S, Williams D, Enwemeka S K, et al. Blue 470nm light kills Methicillin-resistant Staphylococcus aureus (MRSA) in vitro [J]. Photomedicine and Laser Surgery, 2009,27(2):221-226.

[27] Giannakis. Solar disinfection is an augmentable, in situ-generated photo-Fenton reaction-Part 1: A review of the mechanisms and the fundamental aspects of the process [J]. Applied Catalysis B: Environmental, 2016,199(15):199-223.

[28] Rincón A G, Pulgarin C. Bactericidal action of illuminated TiO2on pure Escherichia coli and natural bacterial consortia: post-irradiation events in the dark and assessment of the effective disinfection time [J]. Applied Catalysis B: Environmental, 2004, 49(2):99-112.

[29] Shang C, Cheung L M, Ho C M, et al. Repression of photoreactivation and dark repair of coliform bacteria by TiO2-modified UV-C disinfection [J]. Applied Catalysis B: Environmental, 2009,89(3/4):536-542.

[30] Pruden. Balancing water sustainability and public health goals in the face of growing concerns about antibiotic resistance [J]. Environmental Science & Technology, 2014,48:5-14.

[31] Huang J J, Hu H Y, Tang F, et al. Inactivation and reactivation of antibiotic-resistant bacteria by chlorination in secondary effluents of a municipal wastewater treatment plant [J]. Water Research, 2011,45(5):2775-2781.

Inactivate ciprofloxacin-resistant bacteria in secondary treated effluent by light irradiation.

SHI Na, SUN Ying-xue*, Qi Fei, Hu Chun-fang (Deparment of Environmental Science and Engineering, Beijing Technology and Business University, Beijing 100048, China). China Environmental Science, 2017,37(7)：2599～2606

A ciprofloxacin-resistant bacterium strain was isolated from secondary treated effluent, and effects of light irradiation for disinfection of ciprofloxacin-resistant bacterium strain were investigated. The ciprofloxacin-resistant bacterium strain presented resistance to penicillin, ampilicillin, sulfamethoxazole, tetracyline and rifampicin. In the presence of ciprofloxacin, the maximum specific growth rate of the strain decreased from 0.63h-1to 0.51h-1. The inactivation ratio of ciprofloxacin-resistant bacterium strain raised with increasing of light intensity and irradiation time, and the inactivated reaction followed either the zero order or first order kinetics. By irradiation of visible light (100/300/500W mercury lamp and 1000W xenon lamp (＞400nm)) for 60min, the inactivation ratio of ciprofloxacin-resistant bacterium strain reached 0.25～0.39log. The inactivated reaction by 100/300W mercury lamp and 1000W xenon lamp(＞400nm) irradiation fited in with zero order kinetics, and the reaction rate constant was 10196.43～11345.24CFU/(mL·min). The inactivated reaction by 500W mercury lamp(＞400nm) followed first order kinetics, and the reaction rate constant was 0.01min-1. By irradiation of visible light with UVA (100/300/500W mercury lamp and 1000W xenon lamp (＞300nm)) for 60min, the inactivation ratio of ciprofloxacin-resistant bacteria reached 0.30～5.63log. The inactivated reaction by 100W mercury lamp(＞300nm) followed first order kinetics, and the reaction rate constant was 0.01min-1. The inactivated reaction by 300W mercury lamp(＞300nm) irradiation fited in with zero order kinetics, and the reaction rate constant was 2572.02CFU/(mL·min). Both of photo reactivation and dark repair took place when ciprofloxacin-resistant bacteria were not completely inactivated. The reactivation ratio reached -3.9～123.4% after photo reactivation of 48h and dark repair. During light irradiation, the ciprofloxacin resistance of the strain was only affected by 1000W visible light with UVA irradiation. By irradiation for 60min, its inhibition diameter decreased from 11.0mm to 8.0mm.

ciprofloxacin-resistant bacteria；visible light；UVA；inactivation ratio；ciprofloxacin resistance

X703.1,X172

A

1000-6923(2017)07-2599-08

石 娜(1988-),女,遼寧遼陽人,碩士,主要研究方向為水污染控制理論與技術.

2016-11-28

北京市屬高等學校高層次人才引進與培養計劃項目(CIT&TCD201304032)

* 責任作者, 副教授, sunyx@th.btbu.edu.cn