周 賀,王雙玲,徐夢瑤,王 禮,王永京,張 燦,張明露*(.北京工商大學(xué)環(huán)境科學(xué)與工程系,北京00048；.軍事醫(yī)學(xué)科學(xué)院疾病預(yù)防控制所,北京 0007)

管網(wǎng)多相界面下抗生素抗性菌的分布特征研究

周 賀1,王雙玲1,徐夢瑤1,王 禮1,王永京1,張 燦2,張明露1*(1.北京工商大學(xué)環(huán)境科學(xué)與工程系,北京100048；2.軍事醫(yī)學(xué)科學(xué)院疾病預(yù)防控制所,北京 100071)

以CDC生物膜反應(yīng)器模擬給水管網(wǎng)輸配系統(tǒng),探究管網(wǎng)水相、生物膜相和顆粒物相三相界面下6種典型抗生素抗性細(xì)菌的分布特征.反應(yīng)器穩(wěn)定運(yùn)行30d后,出水余氯從0.66mg/L下降到0.26mg/L,出水濁度和顆粒物濃度分別從進(jìn)水的0.08NTU和377counts/mL增高至0.86NTU和 4151counts/mL,出水水質(zhì)變差.進(jìn)水中僅有紅霉素和氨芐西林的抗性細(xì)菌的數(shù)量較高,分別為36和99CFU/100mL,出水中,鏈霉素和氨芐西林的抗性細(xì)菌數(shù)量最高,分別為432和155CFU/100mL,遠(yuǎn)遠(yuǎn)高于進(jìn)水中抗性細(xì)菌數(shù)量.生物膜相中異養(yǎng)菌總數(shù)和細(xì)胞總數(shù)達(dá)到了4089CFU/cm2和1.5×106cells/cm2,鏈霉素和紅霉素的抗性細(xì)菌數(shù)量較高,為3432和2508CFU/cm2,其抗性細(xì)菌比例分別達(dá)到了83.9%和61.4%.顆粒物相中,氯霉素和氨芐西林的抗性細(xì)菌比例較高,都在45%左右.生物膜和顆粒物都會(huì)給細(xì)菌提供一個(gè)安全穩(wěn)定的生長場所,使細(xì)菌能夠抵抗殘留消毒劑和部分抗生素的抑制作用,更易產(chǎn)生耐藥性,對人體健康的威脅也更大.

抗生素；多相界面；細(xì)菌數(shù)量；抗性細(xì)菌比例

由于抗生素在控制感染性疾病方面發(fā)揮了重要的作用,因此在醫(yī)療事業(yè)、集中化畜牧養(yǎng)殖業(yè)和工業(yè)生產(chǎn)中被大量使用.使用過程中殘留的抗生素會(huì)隨生活垃圾或工業(yè)廢水進(jìn)入到污水處理系統(tǒng)和水環(huán)境中,由于現(xiàn)行的污水處理工藝不能有效地去除抗生素,會(huì)使進(jìn)入到水環(huán)境中的抗生素含量增多,為微生物提供一定的選擇壓力[1-3],促進(jìn)抗性菌株的產(chǎn)生,其中還可能有一些病原微生物,對人體健康以及整個(gè)生態(tài)系統(tǒng)都構(gòu)成長期潛在危害,也對自來水廠的處理工藝提出了新的挑戰(zhàn)[4-7].

管網(wǎng)輸配系統(tǒng)是保障飲用水水質(zhì)安全的關(guān)鍵環(huán)節(jié)[8].進(jìn)入管網(wǎng)的出廠水中,不僅含有少量微生物,還包括顆粒物、有機(jī)物等營養(yǎng)物質(zhì).在管網(wǎng)輸送過程中,細(xì)菌會(huì)黏附在管網(wǎng)內(nèi)壁上富集生長形成穩(wěn)定的生物膜,生物膜不僅可以吸取水中營養(yǎng)物質(zhì),而且可以給細(xì)菌提供一個(gè)安全穩(wěn)定的生長場所,使細(xì)菌可以大量繁殖.傳統(tǒng)上將管網(wǎng)系統(tǒng)中的細(xì)菌分為兩部分,生物膜相(95%)和水相(5%)[9],對管網(wǎng)系統(tǒng)的相關(guān)研究也大多集中在水相和生物膜相,對顆粒物相的探究較少.研究表明,管網(wǎng)中的顆粒物會(huì)吸附一些營養(yǎng)物質(zhì)和細(xì)菌,包括某些病原微生物[10],而且細(xì)菌可能在水相、生物膜和顆粒物上進(jìn)行遷移轉(zhuǎn)化[11-12],因此實(shí)際研究中不應(yīng)被忽視[13-14].本研究選取美國疾病預(yù)防與控制中心 (CDC)研制的生物膜反應(yīng)器來模擬給水管網(wǎng)系統(tǒng),該反應(yīng)器具有剪切力均勻、出水穩(wěn)定等優(yōu)點(diǎn),能夠較好地模擬實(shí)際給水管網(wǎng)的水力條件[15-17].本文對管網(wǎng)水相、生物膜相和顆粒物相三相界面下抗性細(xì)菌分布特征進(jìn)行探究,旨在為保障管網(wǎng)水質(zhì)提供參考.

1 材料與方法

CDC生物膜反應(yīng)器[18],示意圖見圖1,由體積為1L的玻璃容器組成,出水量達(dá)350mL,停留時(shí)間約為6h;8個(gè)獨(dú)立的、可拆卸的聚丙烯桿支撐著聚氯乙烯材料的頂蓋,頂蓋上有一個(gè)進(jìn)水口、一個(gè)取樣口和一個(gè)氣體交換口;每個(gè)桿上可以固定 3個(gè)可拆卸的掛片(直徑約 1.27cm,厚度約0.3cm),細(xì)菌在掛片表面附著生長形成生物膜;容器底部固定一個(gè)帶轉(zhuǎn)子的隔板,反應(yīng)器運(yùn)行過程中與磁力攪拌器相連,轉(zhuǎn)速控制在200～300r/min,給掛片表面提供均勻的剪切力,模擬實(shí)際管網(wǎng)中的水力條件.本實(shí)驗(yàn)選擇的掛片材質(zhì)為聚乙烯(PE),進(jìn)水經(jīng)蠕動(dòng)泵輸送到 CDC反應(yīng)器中,對反應(yīng)器出水進(jìn)行收集.反應(yīng)器穩(wěn)定運(yùn)行30d后,對樣品進(jìn)行收集,對各指標(biāo)進(jìn)行檢測.

圖1 CDC生物膜反應(yīng)器示意Fig.1 Schematic diagram of CDC biofilm reactor

1.1 水質(zhì)基礎(chǔ)指標(biāo)

收集反應(yīng)器進(jìn)出水,對水樣的余氯、濁度、總有機(jī)碳(TOC)以及顆粒物濃度和粒徑進(jìn)行測量.用鉻酸酸化過的玻璃瓶收集出水水樣,水樣經(jīng)0.45μm的一次性聚醚砜濾膜的濾器過濾后,放入總有機(jī)碳分析儀(Aurora 1030W)進(jìn)行測定;使用 GR-1500A臺(tái)式激光顆粒計(jì)數(shù)儀分析顆粒物濃度及粒徑分布;余氯測量使用HACH 5870000便攜式余氯儀;濁度測量使用HACH 1900C便攜式濁度儀.

1.2 三相界面樣品的收集

用無菌玻璃瓶分別收集1L進(jìn)水和出水;取3片掛片放入無菌50mL離心管內(nèi),加5mL無菌水,震蕩1min后放于超聲儀內(nèi),40kHz下超聲5min,再次震蕩超聲,重復(fù)兩次,即得到生物膜相菌懸液;收集50L反應(yīng)器出水進(jìn)行過濾,采用Swinnex濾器(Millipore SX0004700),直徑47mm,孔徑1.2μm的玻璃纖維素濾膜(Millipore APFC04700).將過濾完畢的濾膜放入50mL離心管內(nèi),加10mL無菌水,震蕩5min后,40kHz下超聲20min,得到顆粒物相菌懸液.

1.3 異養(yǎng)菌平板計(jì)數(shù)(HPC)

取1mL進(jìn)出水與滅菌后未凝固的R2A瓊脂培養(yǎng)基(AOBOX)在平板上均勻混合;對于生物膜和顆粒物相樣品,分別吸取100μL菌懸液均勻涂布于 R2A瓊脂培養(yǎng)基平板上.將平板倒置放于22℃恒溫培養(yǎng)箱內(nèi)培養(yǎng)7d,每個(gè)樣品5個(gè)平行.

1.4 細(xì)菌總數(shù)計(jì)數(shù)(TCC)

取1.2中采集的水樣、生物膜和顆粒物樣品,分別取 100μL于 96孔板中,加 1μL SYBR GREEN熒光染料(1×),25℃下避光孵化20min.孵化完畢后,使用多維高清流式細(xì)胞儀(BD LSRFortessa)進(jìn)行樣品分析,波長 488nm,以無菌水做空白參比,每個(gè)樣品5個(gè)平行.

1.5 抗性細(xì)菌比例測定

選取氯霉素、紅霉素、四環(huán)素、鏈霉素、氨芐西林和卡那霉素為實(shí)驗(yàn)所用抗生素(抗生素均為 Dr.Ehrenstorfer標(biāo)準(zhǔn)品,純度 97%以上).將滅菌后的 R2A培養(yǎng)基冷卻至不燙手時(shí),稱取抗生素純品加到培養(yǎng)基內(nèi)混勻,制成含抗生素的平板培養(yǎng)基,同時(shí)以未添加抗生素的培養(yǎng)基為對照.將生物膜相和顆粒物相菌懸液涂布在含抗生素的培養(yǎng)基上,22℃下恒溫培養(yǎng) 7d.對于進(jìn)出水樣品,使用聚醚砜材質(zhì)的濾膜(GPWP04700)將 100mL水樣濃縮過濾,將濾膜貼在培養(yǎng)基上,22℃下恒溫培養(yǎng) 7d.細(xì)菌的存活率即為抗性細(xì)菌比例[19].

存活率(%)=(A/A0)×100%式中：A為添加抗生素平板上長出的菌落數(shù);A0為未添加抗生素平板上長出的菌落數(shù).

2 結(jié)果與討論

2.1 水質(zhì)基礎(chǔ)指標(biāo)

反應(yīng)器穩(wěn)定運(yùn)行 30d后,余氯由初始的0.66mg/L降低到0.26mg/L,出水的TOC略有增加.在實(shí)際管網(wǎng)中,水廠出水經(jīng)管道運(yùn)送到用戶過程中,部分無機(jī)鹽會(huì)轉(zhuǎn)變成能被微生物利用的有機(jī)營養(yǎng)物質(zhì)[20],會(huì)使水中的有機(jī)物含量增多,促進(jìn)細(xì)菌生長,從而使余氯消耗增加.

反應(yīng)器進(jìn)出水顆粒物的粒徑分布見圖 2.反應(yīng)器出水的濁度和顆粒物濃度較進(jìn)水有明顯升高,濁度由0.08NTU增高至0.86NTU,顆粒物濃度由 377counts/mL增高至 4151counts/mL.由圖 2可知,進(jìn)水和反應(yīng)器出水都以小顆粒物為主,1～7μm的小顆粒占90%左右.在反應(yīng)器運(yùn)行過程中,隔板轉(zhuǎn)動(dòng)會(huì)使部分沉積物懸浮,同時(shí)顆粒物也會(huì)富集增長,掛片最外層成熟的生物膜會(huì)脫落,導(dǎo)致一些細(xì)菌及其代謝產(chǎn)物也會(huì)脫落到水中,使出水的濁度和顆粒物濃度升高[10].

圖2 反應(yīng)器進(jìn)出水的顆粒物粒徑分布Fig.2 Distribution of particle size in influent and effluent of reactor

2.2 HPC和TCC計(jì)數(shù)

進(jìn)水的HPC和TCC分別為9CFU/mL和2.7×103cells/mL,出水的HPC和TCC增長明顯,分別為60CFU/mL和1.8×104cells/mL;生物膜相的 HPC和 TCC分別為 4089CFU/cm2和 1.5× 106cells/cm2,單位面積上的細(xì)菌量非常高;顆粒物相的細(xì)菌量較低,表明只有少部分細(xì)菌會(huì)粘附在顆粒物上,但由于顆粒物濃度較高,能夠吸附更多細(xì)菌,使顆粒物上粘附的細(xì)菌總數(shù)增加.

2.3 最高抗性界定濃度下各相界面的HPC數(shù)量

美國臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化協(xié)會(huì)(CLSI)規(guī)定了氯霉素、紅霉素、四環(huán)素、鏈霉素、氨芐西林和卡那霉素 6種抗生素的最高抗性界定濃度,在此濃度下細(xì)菌能夠長出則表明細(xì)菌對此種抗生素具有耐藥性.在 6種抗生素的最高抗性界定濃度下,不同相界面的HPC響應(yīng)情況見表1.由表1可得,四環(huán)素和卡那霉素對細(xì)菌的抑制作用非常明顯,在最高抗性界定濃度下,三相界面的抗性細(xì)菌數(shù)量都為 0,其余 4種抗生素的最高抗性界定濃度下,進(jìn)水和顆粒物相中抗性細(xì)菌的數(shù)量很少,而在出水中,對鏈霉素和氨芐西林產(chǎn)生耐藥性的HPC數(shù)量分別為434和155CFU/100mL;在生物膜相中,對鏈霉素和紅霉素產(chǎn)生耐藥性的HPC數(shù)量高達(dá)3432和2508CFU/cm2.結(jié)果表明生物膜上存在大量的抗性細(xì)菌,因此應(yīng)特別關(guān)注管網(wǎng)生物 膜對水質(zhì)及人體健康的影響.

表1 CLSI最高抗性界定濃度下的6種抗生素抗性細(xì)菌濃度Table 1 The total number of 6kinds of antibiotic resistant bacteria at the highest inhibitory concentration defined by CLSI

2.4 多相界面下抗性細(xì)菌的比例

在 6種抗生素的最高抗性界定濃度下,三相界面下的抗性細(xì)菌比例見圖3.由圖3可得,四環(huán)素和卡那霉素對細(xì)菌的抑制作用很強(qiáng),所有樣品中抗性細(xì)菌的比例均為 0%.四環(huán)素屬于廣譜抗生素,具有較強(qiáng)的殺菌作用,在較高濃度下,細(xì)菌很難存活;卡那霉素屬于氨基糖苷類抗生素,對多數(shù)腸桿菌科細(xì)菌具有良好抑制作用.在反應(yīng)器進(jìn)水中,抗性細(xì)菌比例都較小.氨芐西林抗性細(xì)菌的比例最高,為23.0%.在顆粒物相中,氯霉素和氨芐西林的抗性細(xì)菌比例較高,均在45%左右.在生物膜相中,鏈霉素的抗性細(xì)菌比例最高,達(dá)到了83.9%,紅霉素的抗性細(xì)菌比例也較高,為 61.4%,其余4種抗生素的抗性細(xì)菌比例均小于1%.氯霉素和鏈霉素也屬于廣譜抗生素,但毒性較低,細(xì)菌易產(chǎn)生耐藥性;氨芐西林和紅霉素屬于窄譜抗生素,主要抗菌譜為革蘭氏陽性菌,對水體中的革蘭氏陰性菌作用不大.在出水中,鏈霉素的抗性細(xì)菌比例與生物膜相樣品相似,為 84.3%,但氨芐西林的抗性細(xì)菌比例高于生物膜,為 30.06%,紅霉素和氯霉素的抗性細(xì)菌比例分別為15.9%和5.6%.

出水中抗性菌比例最高的為鏈霉素和氨芐西林,其中鏈霉素為生物膜相中抗性菌比例較高的抗生素,而氨芐西林為顆粒物相中抗性菌比例較高的抗生素,這可能是因?yàn)樯锬は嗪皖w粒物相的細(xì)菌會(huì)脫落到水中,同時(shí)也可能發(fā)生基因的水平遷移致使出水中的抗性菌比例增高[20-21].在生物膜相樣品中,對鏈霉素和紅霉素的抗性菌比例都大于60%,相比進(jìn)水,鏈霉素和紅霉素的抗性菌比例有了很大地升高,表明在生物膜中,細(xì)菌對鏈霉素和紅霉素更易產(chǎn)生耐藥性.研究表明,由于生物膜對細(xì)菌的保護(hù)作用,以附著形式生長的細(xì)菌對抗生素具有更強(qiáng)的耐受性,其抗性能達(dá)到懸浮態(tài)細(xì)菌的 1000～1500倍[22].此外,有文獻(xiàn)報(bào)道,經(jīng)管道運(yùn)輸后的用戶出水中抗性細(xì)菌比例高于水廠出水,說明在管網(wǎng)運(yùn)輸過程中,水中細(xì)菌的抗性增強(qiáng)[23].進(jìn)一步研究發(fā)現(xiàn),氯消毒產(chǎn)生的消毒副產(chǎn)物與細(xì)菌長時(shí)間接觸會(huì)誘導(dǎo)細(xì)菌發(fā)生基因突變,因此增加了細(xì)菌耐藥性的產(chǎn)生.基因突變會(huì)使細(xì)胞結(jié)構(gòu)發(fā)生改變或產(chǎn)生了抗生素降解酶從而提高其對抗生素的抗性[21,24-25].

圖3 三相界面下抗性細(xì)菌的比例Fig.3 The proportion of resistant bacteria in the multi-phase interfaces

3 結(jié)論

3.1 反應(yīng)器穩(wěn)定運(yùn)行30d后,出水的余氯下降明顯,濁度和顆粒物濃度增長明顯,同時(shí) TOC含量增高;出水的HPC和TCC較進(jìn)水有了明顯的升高,生物膜相的HPC和TCC數(shù)量較高;顆粒物相中的細(xì)菌數(shù)量較少,但出水的顆粒物濃度較高,其危害也不容忽視.

3.2 進(jìn)出水、生物膜和顆粒物樣品對四環(huán)素和卡那霉素均不具有耐藥性,對于鏈霉素、紅霉素、氯霉素和氨芐西林 4種抗生素,出水中抗性菌的數(shù)量和抗性菌比例均高于進(jìn)水,管網(wǎng)輸送過程對抗性菌的產(chǎn)生具有顯著影響.

3.3 出水中比例最高的是鏈霉素和氨芐西林抗性菌,鏈霉素抗性菌是生物膜相抗性比例最高的細(xì)菌,而氨芐西林抗性菌是顆粒物相抗性比例最高的細(xì)菌,提示出水中的抗性菌可能來自于生物膜和顆粒物.因此出廠水中應(yīng)嚴(yán)格控制顆粒物濃度,在實(shí)際管網(wǎng)建設(shè)中,應(yīng)選取不易形成生物膜的管材.

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Study on the distribution characteristics of antibiotic resistant bacteria (ARB) in the multi-phase interfaces of water pipe network.

ZHOU He1, WANG Shuang-ling1, XU Meng-yao1, WANG Li1, WANG Yong-jing1, ZHANG Can2, ZHANG Ming-lu1*(1.Department of Environmental Science and Engineering, Beijing Technology and Business University, Beijing 100048, China；2.Institute of Disease Control and Prevention,Academy of Military Medical Sciences,Beijing 100071, China). China Environmental Science, 2017,37(6)：2347～2351

A CDC biofilm reactor was used in the study to simulate the drinking water distribution system. The distribution characteristics of six typical antibiotic-resistant bacteria (ARB) in the three-phase interface of water, biofilm and particulate matter were evaluated. After 30days operation, the concentration of chlorine in the effluent decreased from 0.66mg/L to 0.26mg/L, the turbidity and the concentration of particulate matter increased from 0.08NTU and 377counts /mL to 0.86NTU and 4151counts/mL. The largest number of ARB in the effluent were streptomycin and ampicillin resistant bacteria (432CFU/100mL and 155CFU/100mL, respectively), which was much higher than the number of resistant bacteria in the inlet. The highest level of ARB in the inlet were erythromycin and ampicillin resistant bacteria, which were only 36CFU/100mL and 99CFU/100mL, respectively. In the biofilm phase, the total number of heterotrophic bacteria and total number of cells reached as high as 4089CFU/cm2and 1.5×106cells/cm2. Streptomycin and erythromycin resistance bacteria were 3432CFU/cm2and 2508CFU/cm2, the ratio of which to the total heterotrophic bacteria were 83.9% and 61.4%. The resistant bacteria ratio of chloramphenicol and ampicillin in the particulate phase were approximately 45%. Both biofilm and particulate matters can provide a safe and stable place for bacterial growth, and protect bacteria from the residual disinfectants and some antibiotics, which facilitates antibiotic resistant bacteria propagation and poses a greater threat to human health.

antibiotic；multi-phase；bacterial amount；ratio of resistant bacteria

X703,X172

A

1000-6923(2017)06-2347-05

周 賀(1991-),男,河北衡水人,碩士研究生,主要從事飲用水管網(wǎng)微生物研究.

2016-11-30

國家自然科學(xué)基金資助項(xiàng)目(51408010,51608011);北京工商大學(xué)兩科基金培育項(xiàng)目(LKJJ2016-17)

* 責(zé)任作者, 副教授, zhangminglu@th.btbu.edu.cn