Water quality indicators: bacteria,coliphages, enteric viruses

Water quality through the presence of pathogenic enteric microorganisms may affect human health. Coliform bacteria, Escherichia coli and coliphages are normally used as indicators of water quality. However, the presence of above-mentioned indicators do not always suggest the presence of human enteric viruses. It is important to study human enteric viruses in water. Human enteric viruses can tolerate fluctuating environmental conditions and survive in the environment for long periods of time becoming causal agents of diarrhoeal diseases. Therefore, the potential of human pathogenic viruses as significant indicators of water quality is emerging. Human Adenoviruses and other viruses have been proposed as suitable indices for the effective identification of such organisms of human origin contaminating water systems. This article reports on the recent developments in the management of water quality specifically focusing on human enteric viruses as indicators.     

指标菌和病毒对卤素消毒剂耐力的比较

本文报告了水中埃希氏大肠杆菌(270017),2株DNA大肠杆菌噬菌体T_2和T_7,2株RNA大肠杆菌噬菌体f_2和Qβ,9株肠道病毒即脊髓灰质炎病毒1、2、3型,柯萨奇病毒A_9、B_3、B_5和埃柯病毒1、2、12型对卤素消毒剂的耐力比较试验。结果2株RNA噬菌体f_2和Qβ对氯和碘的耐力比DNA T_2、T_7和大肠杆菌强。当用69-1饮水消毒剂灭活9株肠道病毒比较试验时,柯萨奇病毒B_3、B_5和埃柯病毒1型的耐力比其它几株病毒强。当把噬菌体f_2和Qβ与埃柯1型、柯萨奇B_3和B_5对69-1消毒剂的耐力比较,结果表明噬菌体f_2和Qβ与埃柯病毒1型的耐力相似,但它们均比柯萨奇病毒B_3和B_5强。     

Chemical and microbiological parameters as possible indicators for human enteric viruses in surface water

There are still conflicting results on the suitability of chemical and microbiological parameters as indicators for the viral contamination of surface waters. In this study, conducted over 20 months, the abundance of human adenovirus, human polyomavirus, enterovirus, group A rotavirus and norovirus was determined in Ruhr and Rhine rivers, Germany. Additionally, prevalence of different possible indicators such as somatic coliphages, E. coli, intestinal enterococci, and total coliforms was also considered. Moreover, the chemical parameter TCPP (tris-(2-chloro-, 1-methyl-ethyl)-phosphate), characterized by environmental stability and human origin, was included. Furthermore, chemical parameters (fluoride, chloride, nitrate, nitrite, bromide, phosphate, and sulfate) which may influence the stability and subsequently the detection rates of viruses in aquatic environment were measured. Quantitative Real-Time (RT-)PCR and double agar layer test were used for the quantification of human enteric viruses and somatic coliphages, respectively. The analyses for E. coli, total coliforms, and intestinal enterococci were done with respect to the standard reference method. The chemical parameters were measured by liquid chromatography of ions and by gas chromatography-flame photometer detector (GC-FPD), respectively. We demonstrated that human adenovirus had the highest detection rate (96.3%), followed by somatic coliphages (73.5%), human polyomavirus (68.6%), and rotavirus (63.5%). However, norovirus GII and enterovirus were found in only 25.7 and 17.8%, respectively. The concentration of the viral genome ranged between 16 and 1.1 × 10⁶ gen. equ./l (genome equivalents/l) whereas the concentrations for TCPP ranged between 0.01 and 0.9 μg/l. The results of the Pearson correlation showed no association between TCPP and any other microbiological parameter. None of the other tested chemical parameters correlated negatively, and therefore they do not influence the stability of enteric viruses. We conclude that neither TCPP nor any other chemical or microbiological parameter can be used as a reliable indicator for the presence of enteric viruses in river water.     

Somatic coliphages and bacterial indicators of bathing water quality in the beaches of Gipuzkoa, Spain

Monitoring the quality of the bathing waters of Gipuzkoa (the Basque Country, Spain) makes it possible to assess the suitability of its 15 beaches for bathing throughout each season. In 1998, the parameters E. coli, somatic coliphages (SOMCPH) and F-specific RNA bacteriophages (FRNAPH) were incorporated into the bathing water quality monitoring system. This enabled the study of the link between bacterial and viral indicators as well as the analysis of the ratios between both types of indicators in waters with different levels of pollution. Although bacterial indicators (total coliforms (TC) and faecal coliforms (FC)) and enterococci showed a strong correlation between them, the correlations between the viral indicators and between the viral and bacterial indicators were weaker, though significant in all cases. The ratio between SOMCPH and E. coli indicates that at low levels of bacterial pollution (E coli <100 MPN/100 ml) SOMCPH outnumber E coli. In contrast, at higher levels of pollution (E coli >100 MPN/100 ml), SOMCPH numbers are lower than those of E-coli. The data reveal the presence of viral indicators in waters classified as suitable for bathing by the European Directive and alert us to their suitability.     

Active microbial decontamination of tilapia fish

Fish grown in contaminated ponds can be exposed to a variety of human pathogenic microorganisms which may accumulate in its tissues. This study was conducted in order to examine the efficiency of changing the water, in the holding tanks, to reduce the level of microorganisms in various fish tissues. Fish contamination was accomplished by seeding 10⁹, 10¹¹ 10¹⁰cfu or pfu ml^‐1 of E.coli, MS2 coliphage and poliovirus 1, respectively, into 1001 of water. These microorganisms were tested in the skin, muscle, liver, spleen and the digestive tract (DT). A single inoculation of the test microorganisms was followed by 3–24 water changes (tank volumes) and compared with self‐purification (control) experiments without changing the water. The highest levels of microorganisms were found in the DT, 6 h after inoculation for E.coli and 12 h after inoculation for MS2 and poliovirus 1. Lower levels of the microorganisms were found in the spleen, liver and on the skin and none were detected in the muscle. In the decontamination experiments, no microorganisms were detected in the DT 5–6 days after inoculation, as compared to 11–21 days in the control experiments. The results of this study indicate that faster decontamination of fish is achieved by repeated changing of the water in the holding tanks and this may lower the risk of disease transmission by fish handling or consumption.     

Association of fish and environmental strains of Aeromonas hydrophila in the causation of ulcerative disease syndrome in fish

Ulcerative disease syndrome in fish was first noticed in 1972 in Australia. It has affected several countries in south‐east Asia and recently north‐east Asia and eastern part of India (including west Bengal). No definite cause has yet been known as the etiologic agent of this syndrome. However, this paper presents evidence that in India it appears to be caused by Aeromonas hydrophila. The isolated strains of A.hydrophila from affected fishes are enterotoxigenic, cytotoxic and haemolytic which might contribute to the pathogenesis of the disease process.     

Development and efficacy of novobiocin and rifampicin-resistant Aeromonas hydrophila as novel vaccines in channel catfish and Nile tilapia

Three attenuated Aeromonas hydrophila vaccines were developed from the virulent 2009 West Alabama isolates through selection for resistance to both novobiocin and rifampicin. When channel catfish (Ictalurus punctatus) were IP injected with 4 × 105 colony-forming unit (CFU) of the mutants, no fish died. However, when the same age and size matched channel catfish were IP injected with similar amount of their virulent parents, 80-100% fish died. Similarly, when Nile tilapia (Oreochromis niloticus) were IP injected with 2 × 108 CFU of the mutants, no fish died. However, when Nile tilapia were IP injected with similar amount of the mutants, all fish died. Vaccination of channel catfish with the mutants at dose of 4 × 105 CFU/fish offered 86-100% protection against their virulent parents at 14 days post vaccination (dpv). Vaccination of Nile tilapia with the mutants at dose of 2 × 108 CFU/fish offered 100% protection against their virulent parents at 14, 28, and 56 dpv. Agglutination assay results suggested that protection elicited by the mutants was partially due to antibody-mediated immunity. Taken together, our results suggest that the three attenuated vaccines might be used to protect channel catfish and Nile tilapia against the highly virulent 2009 West Alabama isolates of A. hydrophila.     

实时荧光PCR检测肠致泻性大肠杆菌在腹泻人群中的分布

目的调查致泻性大肠杆菌(DEC)在福建省腹泻人群中的分布情况,为做好防治工作提供本底资料。方法对监测医院腹泻患者的粪便样本提取增菌液的DNA,再用实时荧光PCR(RT-PCR)技术进行相关病原菌检测,以了解5种病原菌在腹泻人群中的分布情况。结果检测206份样本,DEC总检出率为18.9%,各相关病原菌检出率:肠致病性大肠杆菌(EPEC)为8.3%,均为不典型肠致病性大肠杆菌;肠产毒性大肠杆菌(ETEC)为1.5%,均为产耐热肠毒素-SL阴性、不耐热型肠毒素-LT阳性;肠侵袭性大肠杆菌(EIEC)为1.9%,肠黏附性大肠杆菌(EAEC)为7.3%,肠出血性大肠杆菌(EHEC)未检出。结论 DEC相关病原菌以EPEC和EAEC为主,其次为EIEC和ETEC,未检出EHEC,与20世纪80年代相比,病原谱发生了变化。     

大肠埃希菌、克雷伯菌产超广谱β-内酰胺酶的检测及耐药性分析

目的研究本院3年中大肠埃希菌、克雷伯菌ESBLs的检出率及对常用抗生素的耐药情况。方法对山西医科大学第二医院2001～2003年分离的837株大肠埃希菌、216株克雷伯菌用标准纸片扩散确证法检测其ESBLs产生率；采用K-B法进行药敏检测。结果大肠埃希菌产ESBLs分离率为2001、2002、2003分别为12．27％、15．90％、30，85％；克雷伯菌产ESBLs分离率为2001、2002、2003分别为23．88％、28．81％、30．00％；产ESBLs株对头孢菌素类、氨基糖苷类、氟喹喏酮类耐药率均高于非产ESBLs菌株；未发现对亚胺培南耐药的菌株。结论近年来大肠埃希菌、克雷伯菌产ESBLs菌株逐年升高，应引起足够的重视，采取有效措施控制产ESBLs菌的感染。     

A comparison of ten USEPA approved total coliform/E. coli tests

Since 2002, the United States Environmental Protection Agency (USEPA) has approved ten enzyme-based total coliform and E. coli detection tests for examination of drinking water. These tests include: Colilert^®, Colilert-18^®, Colisure^®, m-Coli Blue 24^®, Readycult^® Coliforms 100, Chromocult^®, Coliscan^®, E*Colite^®, Colitag™ and MI Agar. The utility of the enzyme based test systems is based on both the ability of the test to detect the target organisms at low levels and the ability of the test system to suppress the growth of non-target organisms that might result in false positive results. Differences in the ability of some of these methods to detect total coliform and E. coli, as well as suppress Aeromonas spp., a common cause of “false positive” results, have been observed. As a result, this study was undertaken to elucidate the strengths and weaknesses of each method. Water samples were collected from three geographically and chemically diverse groundwaters in Wisconsin. One-hundred milliliter aliquots were individually spiked with both low concentrations (one to ten organisms) and high concentrations (fifty to one-hundred) of each of five different total coliform organisms (Serratia, Citrobacter, Enterobacter, E. coli, & Klebsiella). These spiked samples were used to test the capability of ten enzyme-based test systems to both detect and enumerate the spiked organisms. In addition, 100 ml samples were independently spiked with two different strains of Aeromonas spp. at six different levels, to assess the ability of each enzyme-based test to suppress Aeromonas spp. Analysis of the data indicated that wide variability exists among USEPA approved tests to detect and quantify total coliforms, as well as suppress Aeromonas spp.     

Clinical Characteristics and Molecular Subtyping of Vibrio vulnificus Illnesses, Israel

During 1996–1997, a new Vibrio vulnificus biotype 3, which caused severe soft tissue infection after fishbone injury, emerged in Israel. We conducted a follow-up study from 1998 through 2005 to assess changing trends, outcomes, and molecular relatedness of the implicated strains. A total of 132 cases (71% confirmed and 29% suspected) of V. vulnificus biotype 3 infection were found. Most infections (95%) were related to percutaneous fish exposure, mainly tilapia (83%) or common carp (13%). Bacteremia, altered immune status, and history of ischemic heart disease were identified as independent risk factors for death, which reached a prevalence of 7.6%. Pulsed-field gel electrophoresis patterns of strains from 1998 through 2005 and from 1996 through 1997 showed a high degree of homogeneity and were distinct from those of V. vulnificus biotype 1. Infections caused by V. vulnificus biotype 3 continue affect the public’s health in Israel.     

大肠杆菌O157特异基因的PCR检测方法

目的：建立一种灵敏、特异的检测肠出血性大肠杆菌O157的PCR方法。方法：针对大肠杆菌O157特异性基因rfbO157设计引物，扩增-497bp的O157标识序列。结果：应用该PCR反应体系，对4株O157菌株和14株非O157菌株扩增结果表明，O157菌株均扩增出预期的497bp片段，14株非O157菌株则为阴性。纯菌液检测灵敏度为60cfu／PCR反应，模拟混合菌液检测灵敏度为400cfu／PCR反应。结论：该方法用于肠出血性大肠杆菌O157的检测鉴定简便、快捷，具有很高的特异性和灵敏度，为O157的临床辅助诊断提供了有力手段。     

我国腹泻病患者和家畜家禽粪便标本中携带小肠结肠炎耶尔森氏菌HPI毒力岛的大肠杆菌的调查

目的　了解携带小肠结肠炎耶尔森氏菌HPI毒力岛的大肠杆菌在我国腹泻病患者粪便标本、动物粪便标本及部分食品标本中的存在情况和所致腹泻病的临床特征。方法　使用菌落原位杂交、DNA打点杂交和PCR扩增等方法。结果　在各地送检的从腹泻病患者粪便标本中分离到的、用常规方法不能鉴定的大肠杆菌中 ,均检出带有小肠结肠炎耶尔森氏菌HPI毒力岛的大肠杆菌菌株 ,检出率为 2 7.0 5 % (436 / 16 12 )。从市售食品标本中分离的大肠杆菌中 ,携带小肠结肠炎耶尔森氏菌HPI毒力岛的大肠杆菌的检出率为 10 .2 3% (9/ 88)。从家畜家禽粪便中分离的大肠杆菌中 ,携带小肠结肠炎耶尔森氏菌HPI毒力岛的大肠杆菌检出率为 5 .71% (16 / 2 80 )。携带小肠结肠炎耶尔森氏菌HPI毒力岛的大肠杆菌所致腹泻的典型临床表现为食欲不振、腹痛、寒战乏力、正常体温或低热、每日腹泻多在 6次以上、多为粘液便。结论　携带小肠结肠炎耶尔森氏菌HPI毒力岛的大肠杆菌在我国分布广泛 ,检出率高于其他几类致泻性大肠杆菌 ,对我国人民的健康是一个潜在的威胁     

水源水中致泻大肠埃希菌的检测与分析

目的　调查致泻大肠埃希菌在不同年限、不同季节、不同水源水中的分布规律以及菌型分布特点 ,为制定相应的防治对策提供参考依据。方法　在六安市区 3个主要饮用水源各设 5个采水点 ,每点分上、中、下游间隔10 0m各采集 1份水样 ,每月 1次 ,连续 5年检测致泻大肠埃希菌的分布 ,并对分离菌株进行生化学、血清学鉴定和药物敏感试验。结果　检测水源水 10 80份 ,分离到致泻大肠埃希菌 14 9株 ,阳性率为 13 80 % ,河水、塘水的检出率分别为 13 6 1%和 14 17% ,无显著性差异 (χ2 =0 0 6 2 ,P >0 0 5 )。 14 9株致泻大肠埃希菌分属 2 3个血清型 ;2 0 0 0年 5月份在河水中检出 1株H2 S肠道致病性大肠埃希菌O12 8∶K67。结论　检测结果揭示了水源水中致泻大肠埃希菌的分布规律及菌型分布特点 ;发现了产H2 SPEECO12 8∶K67。     

东莞口岸首次检出肠产毒性大肠埃希菌O6

目的：检测进口水产品中是否污染致泻性大肠埃希菌。方法：按照GB/T4789.6-2003致泻性大肠埃希菌检验进行。结果：从进口冻贝肉中检出肠产毒性大肠埃希菌O6,并对该菌的形态及生化等特性进行研究。结论：东莞口岸首次检出该菌,该批冻贝肉被彻底销毁,消除了疫病隐患,确保了卫生安全。     

太原地区携带OI一28毒力基因组岛致泻大肠埃希菌性小儿腹泻

目的探讨携带OI-28毒力基因组岛致泻大肠埃希菌在小儿腹泻中的病原学作用。方法在山西省儿童医院采集257例腹泻病患儿粪便,做肠道病原菌常规培养和致泻大肠埃希菌血清学分型鉴定,用PCR和DNA斑点杂交检测致泻大肠埃希菌EHEC OI-28毒力基因组岛中与RTX相关的5个毒力基因。结果257例腹泻病患儿检出病原菌206株(80.16%)。其中大肠埃希菌149株(57.98%),其他肠道致病菌57株(22.18%)。血清分型检出致泻大肠埃希菌EPEC 3株(2.01%)、ETEC 2株(1.34%)、EHEC 2株(1.34%),其余142株为"疑似致泻大肠埃希菌"(55.25%)。149株大肠埃希菌中OI-28 5个基因全阳性者21株(14.09%),1个基因阳性者8株(5.37%),2个基因阳性者2株(1.34%)。21例携带OI-28毒力基因组岛大肠埃希菌感染的腹泻患儿,以3岁以下小儿为主(80.95%)。结论携带OI-28毒力基因组岛大肠埃希菌是夏季小儿腹泻的重要病原菌之一。     

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hly A/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hly A/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.     

Occurrence of norovirus and other enteric viruses in untreated groundwaters of Korea

A total of 39 water samples from 23 different groundwater wells in Korea were collected and analyzed in order to monitor the occurrence of norovirus (NoV) and other indicator microbes as the first part of a national survey of groundwater. More than 500 L of untreated groundwater were filtered through 1MDS filters. Following elution and concentration by organic flocculation, PCR and sequence analysis were employed to detect and identify NoV, enterovirus, rotavirus, hepatitis A virus and adenovirus (Adv). Somatic and F-specific phages, heterotrophic bacteria, total coliforms and Escherichia coli were also analyzed to infer possible fecal contamination. NoVs were detected in 18% of the 39 samples. Five out of seven NoV-positive samples (71%) were identified as GI while the other two (29%) were GII. Enteroviruses and Advs were detected in two and three samples, respectively. Rotavirus and hepatitis A virus were not detected. Total coliforms, E. coli and coliphages were detected in 49, 15 and 13% of the samples, respectively, but did not appear to be suitable indicators of enteric virus contamination in groundwater. These results suggest that additional treatment may be needed for a significant number of groundwaters prior to use as drinking water.