饮用水管网生物膜中弧菌数量影响因素研究

史亚,施晓峰,张永静,柳景青,程东庆*

(1.浙江中医药大学医学技术学院,杭州310053;2.浙江大学建筑工程学院,杭州310058)

饮用水管网生物膜中弧菌数量影响因素研究

史亚1,施晓峰1,张永静1,柳景青2,程东庆1*

(1.浙江中医药大学医学技术学院,杭州310053;2.浙江大学建筑工程学院,杭州310058)

摘要研究饮用水管网中管材、管龄、管径等因素对生物膜弧菌数量的影响.采集中国东部某市管网生物膜样本12份,用硫代硫酸盐-柠檬酸盐-胆盐-蔗糖琼脂培养基分离培养弧菌,并用弧菌科细菌生化鉴定系统鉴定并计数.结果表明,弧菌检出率为50%(6/12).在管龄相近的不同管材管壁生物膜中,弧菌数量为球墨铸铁管(212± 39.40 CFU/cm2)＞镀锌钢管(4.85±1.03 CFU/cm2)＞不锈钢复合管(0.66±0.21 CFU/cm2),并且球墨铸铁管中的弧菌数量与其他管材间差异有统计学意义(P＜0.05),而在塑料管中未检出弧菌;而不同管龄的球墨铸铁管以及镀锌钢管生物膜中弧菌数量变化不明显;在不同管径(公称直径为200、150和300 mm)的球墨铸铁管生物膜中,弧菌数量分别为2.65×103±204、212±39.40和44.20±6.88 CFU/cm2.综上表明,管材对生物膜弧菌数量有较大的影响,其中,以球墨铸铁管中的弧菌数量最多;因此,饮用水管段的铺设应考虑铸铁管的潜在弧菌风险.

关键词饮用水管网;弧菌;管材;管龄;管径

随着现代工业的发展,目前居民小区、公共建筑等终端用户的饮用水以管网系统供水为主.饮用水供水系统作为出厂水到达用户的最后一个环节,它是城市供水水质安全保障的最后屏障.研究表明,饮用水管网中有大量细菌以生物膜的形式存在[1].管网中的细菌可诱发管壁腐蚀和结垢,降低管网的输水能力,引起水质恶化,更为严重的是病原微生物也易在生物膜中滋生,其随管网水传播更会增加饮用水的微生物风险[23].

在供水系统生物膜中存在诸如硝化螺菌属、浮霉状菌属、酸杆菌属、鞘氨醇单胞菌、假单胞菌属等细菌[4].弧菌广泛分布于自然界,以水体中最多.在已发现的76种弧菌中,至少有12种对人类有致病作用,常引起人体严重腹泻和脱水[5].目前,关于饮用水管网中弧菌检测以及其数量影响因素等研究鲜有报道.本研究以中国东部某市实际饮用水管网中的生物膜为试验对象,分析管材、管龄、管径等因素对生物膜中弧菌数量的影响.

1　材料与方法

1.1采样点及采样方法

2013年4月—10月,在中国东部某市12个饮用水管网采样点进行采样,管道具体参数见表1.截取所需管段,用无菌试管刷对内壁反复刷洗,再用生理盐水进行冲洗.将采集的生物膜样品置于无菌玻璃瓶中,并放入低温采样箱中迅速转移至实验室低温保藏,用于后续试验.

1.2样品预处理

在样品瓶中加入无菌玻璃珠后置于摇床振荡15 min,制成菌悬液,使用60目的无菌筛网对样品进行过滤,并用无菌生理盐水冲洗;离心后弃上清,将沉淀物定容至一定体积,用于弧菌的培养鉴定.

1.3弧菌的分离培养及鉴定

将预处理后的样品逐级稀释,取1 m L接种于硫代硫酸盐-柠檬酸盐-胆盐-蔗糖(thiosulfate-citrate-bile salts-sucrose,TCBS)琼脂培养基上,于37℃恒温培养24～48 h,根据菌落形态特征进行分类并计数.挑取典型菌落进行分离纯化,革兰氏染色,氧化酶试验和葡萄糖氧化-发酵试验(O/F试验).其中,氧化酶阳性、葡萄糖发酵型的菌为疑似弧菌,采用弧菌科细菌生化鉴定系统(GYZ-9V)进一步鉴定.

表1　供水管网各管段相关参数_Table 1 Parameters of the pipes in drinking water distribution system

1.4细菌总数测定

参照《生活饮用水卫生标准检验方法》(GBT 5750—2006),将预处理后的样品逐级稀释,取1 m L涂布于营养琼脂培养基上,每个梯度做3个平行,置于(36±1)℃培养箱内培养24 h后进行菌落计数.

1.5数据分析

应用SPSS 19.0软件的最小显著差异法对试验所得数据进行差异显著性检验.

2　结果与分析

2.1不同管材管网生物膜样品中弧菌数量测定结果

从图1可以看出:在管龄相近的不同管材管壁生物膜上,在球墨铸铁管、镀锌管和不锈钢复合管中检出弧菌,而在塑料管中未检出;在球墨铸铁管中弧菌数量明显多于其他管材(P＜0.05),达到212±39.40 CFU/cm2,其次为镀锌管,数量为4.85±1.03 CFU/ cm2,再次为不锈钢复合管,数量为0.66±0.21 CFU/ cm2;而细菌总数在不同管材生物膜中的分布为:镀锌钢管(1.67×106±2.43×105CFU/cm2)＞球墨铸铁管(2.12×104±5.63×103CFU/cm2)≈塑料管(1.77×104±1.65×103CFU/cm2)＞不锈钢复合管(7.17×102±1.35×102CFU/cm2).

柱状图上的不同小写字母表示在P＜0.05水平差异有统计学意义.Different lowercase letters above the columns represent statistically significant differences at the 0.05 probability level.图1 不同管材生物膜中细菌和弧菌数量Fig.1 Amounts of bacteria and Vibrio on different materials

2.2不同管龄管网生物膜样品中弧菌数量测定结果

选择球墨铸铁管和镀锌管进行弧菌数量与管龄关系分析,结果见图2和图3.管龄为5年的球墨铸铁管生物膜中弧菌数量为35.4±4.26 CFU/cm2,6年和9年的未检出弧菌,管龄为11年的生物膜中弧菌数量最多,为212±39.4 CFU/cm2;管龄为12年的镀锌钢管生物膜中弧菌数量为4.85±1.03 CFU/ cm2,而在其他管龄(13、17和23年)中均未检出弧菌.总之,管龄与管网生物膜中的弧菌数量未见明显规律.

图2　不同管龄球墨铸铁管生物膜中细菌和弧菌数量Fig.2 Amounts of bacteria and Vibrio on nodular cast iron pipes of different ages

图3　不同管龄镀锌管生物膜中细菌和弧菌数量Fig.3 Amounts of bacteria and Vibrio on galvanized pipes of different ages

管龄为5、6和9年的球墨铸铁管生物膜中细菌菌落总数在1 000～10 CFU/cm2之间,11年的数量最多,为2.12×104±5.63×103CFU/cm2;镀锌钢管中细菌总数在1×105CFU/cm2以上,除管龄17年的生物膜细菌总数为346±26.5 CFU/cm2外.

2.3不同管径管网生物膜样品中弧菌数量测定结果

由图4可见:球墨铸铁管3个不同管径生物膜中弧菌数量以200 mm的最多(2.65×103±204 CFU/cm2),显著多于管径150 mm(212±39.40 CFU/cm2)和300 mm(44.20±6.88 CFU/cm2) (P＜0.05).管径与管网生物膜中的弧菌数量也未见明显规律.

从图4还可以看出:细菌总数与弧菌数量的变化规律基本一致,即管径200 mm(8.28×105± 7.87×103CFU/cm2)＞150 mm(2.12×104±5.63× 103CFU/cm2)≈300 mm(1.22×104±1.59×103CFU/cm2).

图4　球墨铸铁管不同管径生物膜中细菌和弧菌数量Fig.4 Amounts of bacteria and Vibrio on nodular cast iron pipes of different nominal diameters

3　讨论

细菌在饮用水管网中的再繁殖方式包括在水体中悬浮生长和在管内壁附着生长2种.由于饮用水管网属于贫营养生长环境,细菌在管壁的附着生长更占优势,即形成生物膜[6].生物膜的存在增大了病原菌在管网中滋生的机会[2-3].弧菌是外环境水体的定殖菌群,出厂水若消毒不彻底,在输水过程中外源弧菌可进入管道,并且在适宜条件下大量增殖.研究发现,在夏秋季急性腹泻患者中24.31%由病原性弧菌感染所致[7].此次调查的12份生物膜样品中6份检出弧菌,检出率为50%,数量最多的达到2.65×103±204 CFU/cm2.多种因素(如水剪切力、流速等)皆会导致生物膜从管壁上脱落进入水体中[8].研究发现,摄入1×105CFU以上的副溶血弧菌可引起发病[9].假设100 cm2的生物膜脱落,则自来水局部弧菌含量很容易达到人感染所需的菌量,不慎摄入,即可对健康造成极大威胁.另有研究证实,海鱼弧菌浓度达到1×106CFU/m L,可引起伤口感染,并导致多脏器的组织细胞损伤[10].由此可见,管壁生物膜弧菌对居民健康存在潜在威胁.

供水管道长年运行,随着管龄增加,其中生物膜细菌数量也发生着变化.MARTINY等[4]研究饮用水供水管网生物膜形成过程中细菌数量及群落组成的变化发现,当管龄超过500 d(约1.3年)后生物膜的群落结构进入稳定期,超过709 d(约2年)后,生物膜中细菌总量进入稳定阶段,细菌数量达到最大值.我们选取的管道管龄均在5年以上,对不同管龄的镀锌管和球墨铸铁管中细菌总数和弧菌数量的研究结果显示,细菌总数和弧菌数量与管龄的关系未见明显规律.

供水管道管径对生物膜的形成亦存在一定影响.在水流流速一致的情况下,管径越小,生物膜中细菌数量越少[11].袁一星等[12]认为,管径会影响余氯衰减速率,管径愈大,余氯衰减速度越慢,从而使水体中保持较高的氯抑制细菌的生长,细菌数量越少.为了减少水流流速和余氯对生物膜的影响,本文所研究的管段水流流速基本为0.1 m/s,余氯质量浓度在0.18～0.48 mg/L范围内.本试验结果表明,管径对细菌总数和弧菌数量的影响均不明显.

管网材质与管壁表面特征对生物膜细菌有重要影响.金属管材在水环境下易发生腐蚀,增加了管道内壁的粗糙度、含水率和黏滞性,有利于有机物的吸附,为细菌吸附繁殖生成生物膜提供了物质基础.此外,多孔结构还可保护生物膜免受水流的冲刷[13].管材也可能通过对消毒剂的衰减进而影响细菌生长.付军等[14]研究发现,金属管材中的氯胺衰减速率最快,其生物膜中的细菌数量也明显多于其他管材.我们研究发现,镀锌管生物膜细菌数量最多,不锈钢复合管最少.这是由于镀锌管内壁比较粗糙,而不锈钢复合管内壁较为光滑,细菌附着生长的条件相对较差,营养物质也较难在管壁沉积,因此,细菌数量明显较低.

此外,不同管材的生物膜细菌群落组成有很大差异.LIU等[15]研究表明,塑料管生物膜细菌群落以酸硫杆状菌属(Acidithiobacillus)、水小杆菌属(Aquabacterium)和Limnobacter为主,铸铁管生物膜细菌群落以硫匣菌属(Thiocapsa)为主.REN等[16]研究发现,铸铁管中以生丝微菌属(Hyphomicrobium)和脱硫弧菌属(Desulfovibrio)为主,而在镀锌管、不锈钢管、塑料管中以鞘脂单胞菌属(Sphingomonas)和假单胞菌属(Pseudomonas)为主.本研究发现,弧菌数量在球墨铸铁管中最多,可能是球墨铸铁管释放的铁(Fe)离子有利于弧菌生长.刘连生等[17]研究Fe2+、Fe3+对弧菌生长的影响结果表明,添加0.1%的Fe2+使弧菌的生长量增加2倍,添加0.1%的Fe3+使弧菌生长量增加25%:说明Fe离子能促进弧菌的生长.关于球墨铸铁管有利于弧菌生长的机制有待于进一步研究.

4　结论

管龄和管径对生物膜中弧菌数量的影响无明显规律,而管材对生物膜弧菌数量有较大的影响.在球墨铸铁管中弧菌数量最多,其次为镀锌管,再次为不锈钢复合管,在塑料管中未检出弧菌.因此,饮用水管段的铺设应考虑铸铁管的潜在弧菌风险.

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SHI Ya1,SHI Xiaofeng1,ZHANG Yongjing1,LIU Jingqing2,CHENG Dongqing1*
(1.College of Medical Technology,Zhejiang Chinese Medical University,Hangzhou 310053,China;2.College of Civil Engineering and Architecture,Zhejiang University,Hangzhou 310058,China)

Summary With the development of modern industries,drinking water in residential area and public building were supplied by water distribution systems.The drinking water distribution system is the final step from water plants to the users,and therefore is essential to assure drinking water safety for customers.Previous studies confirmed that many bacteria in the drinking water distribution systems existed as biofilm,which could induce corrosion and scaling of the pipe walls,reduce the water quality and water-carrying capacity of the pipelines. Moreover,as the pathogens growing in the biofilms transferred in the pipelines,the risks caused by these microorganisms would increase.Vibrio species is an estuarine bacterium widely distributed in the natural aquatic environment around the world.Among the discovered 76 kinds of Vibrio species,at least 12 kinds have pathogenic effect on human,and often result in severe diarrhea and dehydration.We found the Vibrio species in our previoussurvey of pathogenic bacteria in biofilm,indicating that the residents were at potential risk from Vibrio species. Therefore,the effect of pipe materials,pipe ages,and pipe diameters on amount of Vibrio species in biofilm of drinking water distribution system was necessary to investigate.

In this study,12 biofilm samples were collected from the drinking water distribution system in east China. Vibrio was isolated using thiosulfate-citrate-bile salts-sucrose(TCBS)agar medium and was identified according to their biochemical reaction characteristics using biochemical identification kit for Vibrionaceae GYZ-9V.The amounts of total bacteria were determined by a spread-plating method according to the Ministry of Health Standard Examination Methods for Drinking Water(2006).Samples and their dilutions were spread on Petri dishes with nutrient agar medium.Analysis of variance(ANOVA)by a least significant difference test was used to compare Vibrio counts from different pipe materials,ages and diameters.

The results showed that Vibrio species could be detected in nodular cast iron,galvanized steel and stainless steel clad pipes,but not in plastic pipe.The Vibrio species amount on the biofilm attached to the nodular cast iron pipe(212±39.40 CFU/cm2)was significantly higher than those attached to the galvanized steel pipe(4.85±1.03 CFU/cm2)and stainless steel clad pipe(0.66±0.21 CFU/cm2).However,the rank of total bacteria amount was galvanized steel pipe＞nodular cast iron pipe≈plastic pipe＞stainless steel clad pipe.Pipe age had little influence on Vibrio amounts from galvanized steel and nodular cast iron pipes.Nodular cast iron pipe with a nominal diameter of 200 mm had the highest amount of Vibrio species(2.65×103±204 CFU/cm2),followed by 150 mm(212±39.40 CFU/cm2)and 300 mm(44.20±6.88 CFU/cm2),and the tendency of total bacteria amount was similar with Vibrio species.

In conclusion,contamination from Vibrio species with varying levels is observed in nodular cast iron, galvanized steel and stainless steel clad pipes,but not in plastic pipe.The different pipe materials,especially nodular cart iron pipe,have strong influence on Vibrio species amount in biofilm from drinking water distribution system;therefore,the potential risks of Vibrio species from nodular cast iron pipe should be taken into consideration while laying the pipelines.

Key wordsdrinking water distribution system;Vibrio;pipe material;pipe age;pipe diameter

Effects of drinking water distribution system on Vibrio amounts in biofilm.Journal of Zhejiang University(Agric.&Life Sci.),2016,42(6):665- 670

DOI:10.3785/j.issn.1008-9209.2016.01.271

中图分类号R 123

文献标志码A

基金项目:水体污染控制与治理国家科技重大专项(2012ZX07403-003).

*通信作者(Corresponding author):程东庆(http://orcid.org/0000-0002-2025-6420),E-mail:B2006069@163.com

第一作者联系方式:史亚(http://orcid.org/0000-0001-8433-1584),E-mail:shiya711@126.com

收稿日期(Received):2016 01 27;接受日期(Accepted):2016 04 12;网络出版日期(Published online):2016 11 19 URL:http://www.zjujournals.com/agr/CN/article/download ArticleFile.do?attach Type=PDF&id=10426