二級(jí)出水中環(huán)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(xué)環(huán)境科學(xué)與工程系,北京 100048)

二級(jí)出水中環(huán)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(xué)環(huán)境科學(xué)與工程系,北京 100048)

針對(duì)二級(jí)出水中的一株環(huán)丙沙星耐藥菌,研究了菌株生長(zhǎng)特性和光輻照對(duì)其滅活、復(fù)活及耐藥性的影響.結(jié)果表明,該菌株對(duì)青霉素、氨芐西林、磺胺甲惡唑、四環(huán)素和利福平均具有耐受性,在環(huán)丙沙星存在的條件下,其最大比生長(zhǎng)速率由0.63h-1降低到0.51h-1.光輻照對(duì)環(huán)丙沙星耐藥菌的滅活率基本隨光照強(qiáng)度和輻照時(shí)間的增加而升高,且基本符合零級(jí)或一級(jí)化學(xué)反應(yīng)動(dòng)力學(xué).可見光(100/300/500W汞燈和1000W氙燈(＞400nm))輻照60min,環(huán)丙沙星耐藥菌滅活率達(dá)到0.25～0.39log.100/300W汞燈和1000W氙燈(＞400nm)輻照下環(huán)丙沙星耐藥菌的變化符合零級(jí)反應(yīng)動(dòng)力學(xué),滅活速率為10196.43～11345.24CFU/(mL·min),500W汞燈(＞400nm)輻照下環(huán)丙沙星耐藥菌的變化符合一級(jí)反應(yīng)動(dòng)力學(xué),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(huán)丙沙星耐藥菌滅活率達(dá)到 0.30～5.63log.100W 汞燈(＞300nm)輻照下環(huán)丙沙星耐藥菌的變化符合一級(jí)反應(yīng)動(dòng)力學(xué),滅活速率為0.01min-1,300W汞燈(＞300nm)輻照下環(huán)丙沙星耐藥菌的變化符合零級(jí)反應(yīng)動(dòng)力學(xué),滅活速率為2572.02CFU/(mL·min).未完全滅活的耐藥菌存在復(fù)活,其48h光、暗復(fù)活率達(dá)到-3.9%～123.4%.在光輻照過程中,只有1000W可見光+UVA輻照影響環(huán)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(huán)丙沙星耐藥菌；可見光；UVA；滅活率；環(huán)丙沙星耐藥性

抗生素的濫用導(dǎo)致抗生素耐藥菌的產(chǎn)生和傳播日益嚴(yán)重[1-2].據(jù)調(diào)查,2013年我國(guó)抗生素使用量高達(dá) 162000t,其中,喹諾酮類抗生素環(huán)丙沙星使用量達(dá)到5340t[3].抗生素在生物體內(nèi)不能完全代謝,會(huì)以原有的形式或代謝產(chǎn)物的形式排出體外,最終進(jìn)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(tǒng)消毒工藝對(duì)抗生素和抗生素耐藥菌的去除效果有限[1,4],進(jìn)而導(dǎo)致抗生素和抗生素耐藥菌進(jìn)入水環(huán)境[5-6],威脅水生態(tài)環(huán)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(jī)制之一[1,7-9].有研究發(fā)現(xiàn),太陽光輻照水體可滅活幾個(gè)數(shù)量級(jí)的大腸桿菌[10].細(xì)菌細(xì)胞中的光敏化合物卟啉經(jīng)400～430nm可見光輻照后會(huì)返回基態(tài)與氧結(jié)合,并轉(zhuǎn)移能量,產(chǎn)生單線態(tài)氧、超氧自由基、羥基自由基和過氧化氫等活性氧(ROS)破壞細(xì)胞[11].由于太陽光中的短波紫外線(UVC)在經(jīng)過地球表面同溫層時(shí)被臭氧層吸收,不能到達(dá)地球表面,中波紫外線(UVB)在自然水體中很快衰減,因此,較長(zhǎng)波段的光的潛在價(jià)值更加重要[12].

本研究選取環(huán)丙沙星耐藥菌作為研究對(duì)象,重點(diǎn)考察可見光和可見光+UVA光輻照對(duì)該菌株的滅活、復(fù)活的控制效果以及對(duì)耐藥性的影響,解析光輻照的機(jī)理,以期為城市水環(huán)境中抗生素耐藥菌的控制提供基礎(chǔ)數(shù)據(jù).

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(jí)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(jí)出水)篩選對(duì)環(huán)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(guī)定的最低抑菌濃度(MIC)[13].以大腸埃希菌ATCC 25922[13]作為質(zhì)控菌.

1.2 耐藥菌生長(zhǎng)特性

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

1.3 耐藥菌生長(zhǎng)動(dòng)力學(xué)參數(shù)確定

用S-Gompertz模型[16]擬合環(huán)丙沙星耐藥菌的生長(zhǎng)曲線,獲得環(huán)丙沙星耐藥菌的最大生長(zhǎng)量、最大比生長(zhǎng)速率和遲滯時(shí)間.具體數(shù)學(xué)表達(dá)式如下:

式中:N為某生長(zhǎng)時(shí)間t下細(xì)菌懸濁液在600nm波長(zhǎng)下的吸光度值,cm-1;N0為細(xì)菌懸濁液在600nm波長(zhǎng)下的初始吸光度值(低于檢測(cè)限,則初始值定為 0.001),cm-1;Nm為細(xì)菌的最大生長(zhǎng)量,即細(xì)菌懸濁液在 600nm波長(zhǎng)下的最大吸光度值,cm-1;μm為細(xì)菌的最大比生長(zhǎng)速率,h-1;λ為遲滯時(shí)間,h;t為生長(zhǎng)時(shí)間,h.

1.4 耐藥菌對(duì)典型抗生素的耐受性分析

采用紙片擴(kuò)散法考察環(huán)丙沙星耐藥菌對(duì)青霉素、氨芐西林、氯霉素、四環(huán)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(jù)抑菌圈直徑的大小,判斷細(xì)菌對(duì)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(yǎng)基上挑選單菌落接種至含 4mg/L環(huán)丙沙星的營(yíng)養(yǎng)肉湯培養(yǎng)基中,37℃過夜培養(yǎng),然后取 20mL的過夜培養(yǎng)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(yǎng)基和抗生素,4500g離心5min,反復(fù)2次,最后將菌體懸浮于無菌水中,用麥?zhǔn)媳葷岱ǐ@得1.5×108CFU/mL(0.5麥?zhǔn)?,隨后加入到無菌水中,獲得預(yù)期濃度的反應(yīng)樣品.

1.5.2 光輻照反應(yīng) 使用光化學(xué)反應(yīng)儀(XPA-7,南京胥江機(jī)電廠)進(jìn)行光輻照實(shí)驗(yàn),反應(yīng)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(chǎn)生不同的輻照條件.利用可見光光輻照計(jì)(FZ-A,北京師范大學(xué)機(jī)電廠)和紫外光輻照計(jì)(UVA,北京師范大學(xué)機(jī)電廠)測(cè)定光輻照強(qiáng)度.將準(zhǔn)備好的菌液加入到已滅菌的反應(yīng)試管,然后放入光化學(xué)反應(yīng)儀外圍的固定裝置中,并進(jìn)行磁力攪拌,開始光輻照實(shí)驗(yàn).

1.5.3 光復(fù)活和暗修復(fù) 將1.5.2中光輻照后的最終樣品轉(zhuǎn)移至滅菌的燒杯中,用于光復(fù)活和暗修復(fù)試驗(yàn).試驗(yàn)條件分別為實(shí)驗(yàn)室日光燈下和黑暗條件下磁力攪拌,24和48h測(cè)定細(xì)菌濃度[14].

1.5.4 耐藥菌的檢測(cè)和滅活率計(jì)算 采用平板計(jì)數(shù)法測(cè)定樣品中的環(huán)丙沙星耐藥菌.37℃培養(yǎng)24h,計(jì)菌落數(shù),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(huán)丙沙星耐藥菌的菌落數(shù);Nt為光輻照后樣品中環(huán)丙沙星耐藥菌的菌落數(shù).

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

2 結(jié)果與討論

2.1 環(huán)丙沙星耐藥菌的生長(zhǎng)特性

環(huán)丙沙星耐藥菌的生長(zhǎng)特性能夠反映其在不同環(huán)境中的增殖特性[15].從表1可以看出,環(huán)丙沙星耐藥菌的最大生長(zhǎng)量(以 OD600計(jì))在 2.58～2.89之間,且隨抗生素濃度的增加而升高,在8mg/L環(huán)丙沙星條件下最大生長(zhǎng)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(qiáng)、效率更高[15].環(huán)丙沙星耐藥菌的最大比生長(zhǎng)速率在 0.51～0.63h-1之間,抗生素濃度增加,最大比生長(zhǎng)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(shí)間增加,0、4、8mg/L環(huán)丙沙星條件下菌株的遲滯時(shí)間分別為2.06、2.52、2.12h.

表1 不同環(huán)丙沙星濃度下耐藥菌的最大生長(zhǎng)量(Nm)、最大比生長(zhǎng)速率(μm)和遲滯時(shí)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(huán)丙沙星耐藥菌對(duì)不同抗生素的耐受性

從表2可以看出,環(huán)丙沙星耐藥菌對(duì)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(qiáng);對(duì)四環(huán)素和利福平的抑菌圈直徑均為10.5mm,具有一定的耐受性;對(duì)氯霉素的抑菌圈直徑為 15mm,藥敏性為中介.這說明污水中環(huán)丙沙星耐藥菌對(duì) β-內(nèi)酰胺類抗生素的耐受能力普遍較高,同時(shí),該菌株對(duì)磺胺甲惡唑耐受性也較強(qiáng),對(duì)四環(huán)素和利福平的耐受性次之,對(duì)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(guān).文獻(xiàn)中報(bào)道的從污水中分離的環(huán)丙沙星多重耐藥菌多對(duì) β-內(nèi)酰胺類抗生素、四環(huán)素類抗生素和磺胺類抗生素有耐藥性,這與本文的研究結(jié)果類似[6,17].

表2 環(huán)丙沙星耐藥菌對(duì)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(duì)環(huán)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(chǎn)生的可見光的光強(qiáng)分別是18.9、27.3、40.2和115.8mW/cm2.從圖1(a)可以看出,環(huán)丙沙星耐藥菌隨可見光輻照時(shí)間的增加而減少.可見光輻照60min時(shí),環(huán)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(duì)環(huán)丙沙星耐藥菌具有一定的滅活作用.早在1930年,Gates發(fā)現(xiàn)只要增加光通量,波長(zhǎng)在400nm以上的可見光與紫外光一樣可以滅活細(xì)菌[18],當(dāng) DNA損傷率超過修復(fù)率,細(xì)胞即發(fā)生死亡[18-19].目前,可見光殺菌的研究光源多為單波長(zhǎng)可見光,相關(guān)研究發(fā)現(xiàn),可見光輻照是否具有殺菌效果與波長(zhǎng)有很大關(guān)系,如405、435、470、525、610、630、810和880nm波長(zhǎng)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(huán)丙沙星耐藥菌的滅活應(yīng)該是具有殺菌功能的不同單波段可見光共同作用的結(jié)果.

圖1 可見光輻照對(duì)環(huán)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(huán)丙沙星耐藥菌的滅活率隨光照強(qiáng)度增加而升高,光照強(qiáng)度越高,滅活效果越好.可見光輻照60min時(shí),100、300、500和 1000W 可見光輻照下環(huán)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(duì)環(huán)丙沙星耐藥菌的滅活效果相當(dāng),其滅活率分別是 0.34和 0.39log,而前者光照強(qiáng)度遠(yuǎn)高于后者光照強(qiáng)度,這可能是因?yàn)椴煌庠窗l(fā)射的可見光各波段光強(qiáng)有所差別.另外,同一光照強(qiáng)度下,環(huán)丙沙星耐藥菌的滅活率隨輻照時(shí)間的增加而升高.光照強(qiáng)度在時(shí)間上的累積即為輻照劑量,可見光對(duì)細(xì)菌的影響與輻照劑量密切相關(guān)[21-22].例如,470nm 可見光輻照劑量達(dá)到10J/cm2以上才能滅活金黃色葡萄球菌[21]; 405nm藍(lán)光和880nm紅外光同時(shí)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(huán)丙沙星耐藥菌滅活動(dòng)力學(xué)擬合結(jié)果Table 3 Kinetic of visible light irradiation on ciprofloxacin-resistant bacteria

將可見光輻照 0～60min的環(huán)丙沙星耐藥菌濃度變化進(jìn)行動(dòng)力學(xué)方程擬合,如表3所示,得出100、300和1000W輻照下環(huán)丙沙星耐藥菌的變化符合零級(jí)反應(yīng)動(dòng)力學(xué),反應(yīng)速率隨光照強(qiáng)度的增加而升高,在1000W輻照下反應(yīng)速率常數(shù)最大為 11345.24CFU/(mL·min),500W 輻照下環(huán)丙沙星耐藥菌的變化符合一級(jí)反應(yīng)動(dòng)力學(xué),光輻照反應(yīng)速率常數(shù)為0.01min-1.

2.4 可見光+UVA對(duì)環(huán)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(chǎn)生的可見光+UVA的光強(qiáng)分別是25.4、37.3、61.2和118.63mW/cm2,其中UVA光強(qiáng)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(shí),環(huán)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(dá)到0.30～5.63log.500W可見光+UVA對(duì)環(huán)丙沙星耐藥菌的滅活效果最好,反應(yīng)20min,即輻照劑量為414J/cm2時(shí),環(huán)丙沙星耐藥菌的滅活率達(dá)到5.63log,遠(yuǎn)遠(yuǎn)高于500W可見光輻照60min時(shí)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?yàn)榈凸β使療舭l(fā)射的 UVA較弱.而1000W輻照下,10min時(shí)可見光+UVA與可見光對(duì)環(huán)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(duì)環(huán)丙沙星耐藥菌的滅活率才迅速增加,60min時(shí)其滅活率達(dá)到1.60log,高于可見光輻照的滅活率 0.39log,這說明UVA在反應(yīng)前期未對(duì)環(huán)丙沙星耐藥菌產(chǎn)生明顯的滅活作用,而在后期開始發(fā)揮顯著的消毒效果,這可能是UVA氧化損傷積累的結(jié)果.UVA對(duì)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(huán)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(yīng)[27].

圖2 可見光+UVA(＞300nm)輻照對(duì)環(huán)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(huán)丙沙星耐藥菌濃度的變化進(jìn)行動(dòng)力學(xué)方程擬合,如表4所示,得出 100W輻照下環(huán)丙沙星耐藥菌的變化符合一級(jí)反應(yīng)動(dòng)力學(xué),光輻照反應(yīng)速率常數(shù)為0.01min-1,300W 輻照下環(huán)丙沙星耐藥菌的變化符合零級(jí)反應(yīng)動(dòng)力學(xué),光輻照反應(yīng)速率常數(shù)為2572.02CFU/(mL·min),其他2種輻照條件下環(huán)丙沙星耐藥菌的變化既不符合零級(jí)反應(yīng)動(dòng)力學(xué),也不符合一級(jí)反應(yīng)動(dòng)力學(xué).

表4 可見光+UVA輻照0～60min環(huán)丙沙星耐藥菌滅活動(dòng)力學(xué)擬合結(jié)果Table 4 Kinetic of visible light with UVA irradiation on ciprofloxacin-resistant bacteria

2.5 光復(fù)活和暗修復(fù)

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

光輻照后環(huán)丙沙星耐藥菌持續(xù)消減可能與殘余消毒效果有關(guān).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(fù)情況,發(fā)現(xiàn)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(shí)間越長(zhǎng),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(chǎn)生的穩(wěn)定氧化劑如 H2O2有關(guān)[28],H2O2可在水中持續(xù)存在數(shù)小時(shí),起到抑制細(xì)菌再生的作用[29].

圖3 光輻照后環(huán)丙沙星耐藥菌的光復(fù)活(a)和暗修復(fù)(b)效果(“+”表示環(huán)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(huán)丙沙星耐藥菌耐藥性的去除

圖4 可見光+UVA對(duì)環(huán)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(xì)菌在抗生素存在條件下的生存和生長(zhǎng)能力[30].根據(jù)美國(guó)臨床和實(shí)驗(yàn)室標(biāo)準(zhǔn)協(xié)會(huì)制定的抗菌藥物敏感性試驗(yàn)執(zhí)行標(biāo)準(zhǔn),腸桿科細(xì)菌對(duì)環(huán)丙沙星耐受性的判定標(biāo)準(zhǔn)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(jié)果表明,1000W可見光+UVA輻照的環(huán)丙沙星耐藥菌抑菌圈直徑總體呈下降趨勢(shì),輻照 60min,抑菌圈直徑由11.0mm下降到8.0mm,即環(huán)丙沙星耐藥菌耐藥性增強(qiáng),如圖4所示,其他光輻照條件均不影響環(huán)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(guān)太陽光輻照對(duì)多重耐藥菌耐藥性影響的研究結(jié)果有所不同, Rizzo等[1]研究發(fā)現(xiàn)太陽光輻照 180min,多重耐藥性大腸桿菌對(duì)環(huán)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(huán)丙沙星耐藥菌在光復(fù)活和暗修復(fù)后,抑菌圈直徑不變.除 48h光復(fù)活,500W可見光輻照后的環(huán)丙沙星耐藥菌在復(fù)活后抑菌圈直徑由11.0mm增加到21.0～22.0mm,即環(huán)丙沙星耐藥菌的耐藥性有所降低.1000W可見光輻照后的環(huán)丙沙星耐藥菌只在暗修復(fù) 48h時(shí),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(huán)丙沙星耐藥菌在光復(fù)活和暗修復(fù)后抑菌圈直徑,分別從11.0mm減小到10.0mm和9.3mm,即環(huán)丙沙星耐藥菌耐藥性增強(qiáng),這說明環(huán)丙沙星耐藥菌對(duì)UVA具有一定的耐受性.Huang等發(fā)現(xiàn)紫外(UV254)消毒可使四環(huán)素耐藥菌的半抑制濃度(IC50)降低40%,即四環(huán)素耐藥菌對(duì) UV254無耐受性[31].可見抗生素耐藥菌對(duì)不同波長(zhǎng)光的耐受性不同.

圖5 光復(fù)活(a)和暗修復(fù)(b)后環(huán)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(jié)論

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

3.2 可見光和可見光+UVA對(duì)環(huán)丙沙星耐藥菌的滅活率基本隨光照強(qiáng)度和輻照時(shí)間的增加而升高.可見光中,1000W 輻照下環(huán)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(dá)到0.39log;可見光+UVA中, 500W 輻照下環(huán)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(dá)到5.63log.

3.3 不同光輻照條件對(duì)環(huán)丙沙星耐藥菌的光復(fù)活和暗修復(fù)影響不同.500W 可見光+UVA 輻照的環(huán)丙沙星耐藥菌完全滅活后無復(fù)活,未完全滅活的環(huán)丙沙星耐藥菌復(fù)活率達(dá)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(huán)丙沙星耐藥菌的耐藥性;光復(fù)活和暗修復(fù)后,100、300W可見光和可見光+UVA輻照后的環(huán)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(fā)生改變.

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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?yàn)樗廴究刂评碚撆c技術(shù).

2016-11-28

北京市屬高等學(xué)校高層次人才引進(jìn)與培養(yǎng)計(jì)劃項(xiàng)目(CIT&TCD201304032)

* 責(zé)任作者, 副教授, sunyx@th.btbu.edu.cn