Experimental rationale for the parameters of a rapid method for oxidase activity determination

Experimental rationale is provided for the parameters of a rapid (1-2-min) test to concurrently determine the oxidase activity of all bacteria grown on the membrane filter after water filtration. Oxidase reagents that are the aqueous solutions of tetramethyl-p-phenylenediamine dihydrochloride and demethyl-p-phenylenediamine dihydrochloride have been first ascertained to exert no effect on the viability and enzymatic activity of bacteria after one-hour contact. An algorithm has been improved for the rapid oxidase activity test: the allowable time for bacteria to contact oxidase reagents and procedures for minimizing the effect on bacterial biochemical activity following the contact. An accelerated method based on lactose medium with tergitol 7 and Endo agar has been devised to determine coliform bacteria, by applying the rapid oxidase test: the time of a final response is 18-24 hours. The method has been included into GOST 52426-2005.     

Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system

The accuracy of a traditional method (lactose utilization with acid and gas production) for the detection of coliform bacteria and E. coli was tested in comparison with method ISO 9308-1 (based on acid formation from lactose) and the Colilert-18 system (detection of beta-galactosidase). A total of 345 isolates were identified after isolation from water samples using API 20E strips. The Colilert-18 led to the highest number of positive findings (95% of the isolates were assigned to coliforms), whereas the ISO-9308-1 method resulted only in 29% coliform findings. With the traditional method only 15% were rated positive. Most of the isolates were identified by the API 20E system as Enterobacter spp. (species of the Enterobacter cloacae complex), Serratia spp., Citrobacter spp.and Klebsiella spp.; but species identification remained vague in several cases. A more detailed identification of 126 pure cultures by using 16S rRNA gene sequence analysis and analysis of the hsp60 gene resulted in the identification of Enterobacter nimipressuralis, E. amnigenus, E. asburiae, E. hormaechei, and Serratia fonticola as predominat coliforms. These species are beta-galactosidase positive, but show acid formation from lactose often after a prolonged incubation time. They are often not of fecal origin and may interfere with the ability to accurately detect coliforms of fecal origin.     

一株与伤寒沙门菌呈血清学交叉反应的哈夫尼亚菌

目的:了解哈夫尼亚菌的生物学特性。方法:检测哈夫尼亚菌的一般特征、生化特性和血清学特性。结果:本次研究中分离出的细菌为革兰阴性小杆菌,氧化酶阴性,硝酸盐还原阳性,能够发酵葡萄糖产酸产气,不分解乳糖、蔗糖等,不产生H2S,与沙门菌多价血清以及O9,Hd,Vi因子血清发生凝集反应,在生理盐水中不发生自凝。结论:本次研究中分离出的细菌为血清学特征与伤寒沙门菌类似的哈夫尼亚菌。     

Monitoring of bacterial contamination of water reservoirs in the Rostov region

The paper estimates the epidemic value of indicators for microbial contamination of waters from their main supply sources (waters from the Tsimlyansk Reservoir and the Nizhni Don River and drinking waters from the towns of Azov and Tsimlyansk) in the Rostov Region. The Tsimlyansk Reservoir water met the SanPiN 2.1.5.980-00 requirements for the normalizable indices of total coliform bacteria (TCB) and thermotolerant coliform bacteria (TtCB) in 54% of the samples; glucose-positive coliform bacteria (GPCB) and Salmonella were isolated in 100 and 60%, respectively. The Azov drinking water that met the SanPiN 2.1.4.1074-01 requirements for TCB and TtCB) was found to contain GPCB, Klebsiella, and Pseudomonas aeruginosa in 5.7, 4.8, and 3% of the samples, respectively Direct detection of pathogenic and potentially pathogenic microorganisms and the integral GPCB index are of the greatest prognostic value in assessing a microbial risk for waterborne enteric infections.     

The health-related microbiological quality of bottled drinking water sold in Dar es Salaam, Tanzania

The consumption of bottled and plastic-bagged drinking water in Tanzania has increased largely because of the deteriorating quality of tap water. It is uncertain whether these water products are safe for drinking. In this study, the microbiological quality of bottled and plastic-bagged drinking water sold in Dar es Salaam, Tanzania, was investigated. One hundred and thirty samples representing 13 brands of bottled water collected from shops, supermarkets and street vendors were analysed for total coliform and faecal coliform organisms as well as heterotrophic bacteria. These were compared with 61 samples of tap water. Heterotrophic bacteria were detected in 92% of the bottled water samples analysed. Total and faecal coliform bacteria were present in 4.6% and 3.6%, respectively, of samples analysed with a tendency for higher contamination rates in plastic-bagged drinking water. Microbiological quality of tap water was found to be worse compared with bottled water, with 49.2% and 26.2% of sampling points showing the presence of total coliform and faecal coliform organisms, respectively. The results suggest caution and vigilance to avert outbreaks of waterborne diseases from these types of drinking water.     

The health-related microbiological quality of bottled drinking water sold in Dar es Salaam, Tanzania

The consumption of bottled and plastic-bagged drinking water in Tanzania has increased largely because of the deteriorating quality of tap water. It is uncertain whether these water products are safe for drinking. In this study, the microbiological quality of bottled and plastic-bagged drinking water sold in Dar es Salaam, Tanzania, was investigated. One hundred and thirty samples representing 13 brands of bottled water collected from shops, supermarkets and street vendors were analysed for total coliform and faecal coliform organisms as well as heterotrophic bacteria. These were compared with 61 samples of tap water. Heterotrophic bacteria were detected in 92% of the bottled water samples analysed. Total and faecal coliform bacteria were present in 4.6% and 3.6%, respectively, of samples analysed with a tendency for higher contamination rates in plastic-bagged drinking water. Microbiological quality of tap water was found to be worse compared with bottled water, with 49.2% and 26.2% of sampling points showing the presence of total coliform and faecal coliform organisms, respectively. The results suggest caution and vigilance to avert outbreaks of waterborne diseases from these types of drinking water.     

The presence-absence coliform test for monitoring drinking water quality.

The concern for improved monitoring of the sanitary quality of drinking water has prompted interest in alternative methods for the detection of total coliform bacteria. A simplified qualitative presence-absence test has been proposed as an alternate procedure for detecting coliform bacteria in potable water. In this paper data from four comparative studies were analyzed to compare the recovery of total coliform bacteria from drinking water using the presence-absence test, the multiple fermentation tube procedure, and the membrane filter technique. The four studies were of water samples taken from four different geographic areas of the United States: Hawaii, New England (Vermont and New Hampshire), Oregon, and Pennsylvania. Analysis of the results of these studies were compared, based upon the number of positive samples detected by each method. Combined recoveries showed the presence-absence test detected significantly higher numbers of samples with coliforms than either the fermentation tube or membrane filter methods, P less than 0.01. The fermentation tube procedure detected significantly more positive samples than the membrane filter technique, P less than 0.01. Based upon the analysis of the combined data base, it is clear that the presence-absence test is as sensitive as the current coliform methods for the examination of potable water. The presence-absence test offers a viable alternative to water utility companies that elect to use the frequency-of-occurrence approach for compliance monitoring.     

Improving the standard and methodological basis for bacteriological monitoring the quality of drinking water

The paper presents materials to provide a scientific rationale for the microbiological parameters enhancing the efficiency of drinking water quality control from the point of view of reliability in the provision of epidemic water consumption safety (coliform bacteria being identified by the glucose index; E. coli). Based on the criterion assessment of the significance of microbial water contamination in relation to morbidity rates, the authors have been developed bacteriological standards--the absence of microorganisms in a 300-ml water sample. Evidence is provided for the epidemically significant level--more than 2 CFU of coliform bacteria in 100 ml. A rapid determination procedure has been substantiated in the experimental studies of the effects of oxidase reagents on the viability and biochemical properties of E. coli bacteria. In search for current technologies for membrane filtration, experimental rationale has been made for the possibility of using track membranes for sanitary and bacteriological water analyses.     

Implications of coliform variability in the assessment of the sanitary quality of recreational waters

The most widely used indicator of the sanitary quality of recreational waters is the coliform group of bacteria. Present techniques of coliform enumeration are imprecise, and this fact is too often overlooked in routine water quality surveys as well as in research efforts seeking quantitative relationships between coliform density and the health effects of recreational waters. To illustrate this point, three years of data gathered by the New York City Department of Health as part of their routine beach water sampling programme were re-analysed, taking the limited precision of each coliform estimate into account. Re-analysis showed 56.6% of the data were not significantly different (P greater than 0.05) from the standard being used. This large percentage of the data was of little value in determining the acceptability of the waters being sampled relative to the standard being used and thus represented a substantial waste of time and expense. Of the remaining data, half indicated acceptable water quality and half indicated unacceptable water quality relative to the standard. These three years of data, therefore, gave little information on the acceptability of the water quality at this location with respect to the standard being used. The data further suggest significant differences in coliform density within sample dates. It is recommended that in future water quality surveys, or in studies of the health effects of recreational waters as related to coliform density, emphasis should shift from maximizing the number of sample dates to maximizing the number of replicate determinations made per sample date.     

A single medium for the rapid detection of Escherichia coli at 44��C

Lactose tryptone ricinoleate broth (0·3% lactose, 2% Oxoid tryptone, 0·1% Na ricinoleate; pH 7·1) was found to be as good as the currently recommended combination of lactose ricinoleate broth and tryptone water for the detection of Escherichia coli in the positive tubes of presumptive coliform counts.     

Implications of coliform variability in the assessment of the sanitary quality of recreational waters.

The most widely used indicator of the sanitary quality of recreational waters is the coliform group of bacteria. Present techniques of coliform enumeration are imprecise, and this fact is too often overlooked in routine water quality surveys as well as in research efforts seeking quantitative relationships between coliform density and the health effects of recreational waters. To illustrate this point, three years of data gathered by the New York City Department of Health as part of their routine beach water sampling programme were re-analysed, taking the limited precision of each coliform estimate into account. Re-analysis showed 56.6% of the data were not significantly different (P greater than 0.05) from the standard being used. This large percentage of the data was of little value in determining the acceptability of the waters being sampled relative to the standard being used and thus represented a substantial waste of time and expense. Of the remaining data, half indicated acceptable water quality and half indicated unacceptable water quality relative to the standard. These three years of data, therefore, gave little information on the acceptability of the water quality at this location with respect to the standard being used. The data further suggest significant differences in coliform density within sample dates. It is recommended that in future water quality surveys, or in studies of the health effects of recreational waters as related to coliform density, emphasis should shift from maximizing the number of sample dates to maximizing the number of replicate determinations made per sample date.     

Contamination of potable water distribution systems by multiantimicrobial-resistant enterohemorrhagic Escherichia coli

BACKGROUND: The contamination of processed or unprocessed drinking water by fecal coliform bacteria has been reported worldwide. Despite a high incidence of waterborne diseases, entero-hemorrhagic Escherichia coli (EHEC) is an underacknowledged pathogen of concern to public health in India. Although the presence of EHEC is recorded in surface water resources of India, drinking water sources are yet to be investigated. OBJECTIVES: The goal of this study was to analyze potable water samples for the presence of virulence determinants of EHEC and to determine the sensitivity of the virulence determinants to antimicrobials. METHODS: We enumerated coliform bacteria in potable water samples collected from six locations in Lucknow, a major city in northern India, using the most probable number method. E. coli (n = 81), randomly isolated by membrane-filtration technique from four sites, were identified by biochemical characterization. E. coli were not detected in samples from two other sites. We screened 15 randomly selected isolates from each site for virulence determinants of EHEC using polymerase chain reaction (PCR). The isolates positive for virulence determinants (n = 18) were screened for sensitivity to 15 antimicrobials by the disk diffusion method. RESULTS: Both stx1 and stx2 genes were present in 33.3% of isolates, whereas others possessed either stx1 (11.1%) or stx2 (55.6%). eaeA, hlyA, and chuA genes were present in 100, 23.3, and 16.7% of isolates, respectively. Resistance to multiple antimicrobials was observed in potential EHEC. CONCLUSIONS: The occurrence of multiantimicrobial-resistant EHEC in potable water is an important health concern because of the risk of waterborne outbreaks.     

A comparison between minerals-modified glutamate medium and lauryl tryptose lactose broth for the enumeration of Escherichia coli and coliform organisms in water by the multiple tube method.

In a multi-laboratory trial, minerals-modified glutamate medium (MMGM) was compared with lauryl tryptose lactose broth (LTLB) in the multiple tube method for the enumeration of coliform organisms, including Escherichia coli, in water. Samples of raw and chlorinated waters yielded a total of 2313 positive tube-reactions with MMGM and 2174 with LTLB. These were interpreted either as E. coli; other coliform organisms; or as false positive reactions. The results at first reading (18 or 24 h) and at 48 h have been analysed statistically in terms of (i) most probable numbers of coliform organisms; (ii) positive reactions and their interpretation; and (iii) whether or not the sample yielded any E. coli or other coliform organisms. All three analyses indicated the same trends. For the detection of E. coli in raw waters LTLB was better than MMGM at 18-24 h, but MMGM was better at 48 h with waters containing small numbers of coliform organisms; for raw waters with greater numbers of organisms, both media performed equally well. Analysis of a subset of samples read at both 18 and 24 h indicated that the superiority of LTLB over MMGM with raw waters disappeared by 24 h. For chlorinated waters, LTLB yielded more positive gas reactions at 18-24 h, but fewer of these were E. coli than with MMGM; at 48 h MMGM was clearly better than LTLB for total coliform organisms including E. coli--especially if the numbers were small. MMGM therefore remains the medium of choice for the detection of E. coli as an indicator of faecal contamination of chlorinated drinking water supplies. It is also better for the detection of small numbers of E. coli in other waters.     

饮用水常规指示菌与介水传染病相关性研究

目的研究GB 5749—2006《生活饮用水卫生标准》中常规监测指示菌与介水传染病之间的相关性。方法从公共卫生科学数据中心获取2014年全国介水传染病发病数据和饮用水常规指示菌监测数据;采用主成分分析对全国22个省市常规指示菌合格率作排名和分级;对常规指示菌与介水传染病及常规指示菌间的相关性采用Spearman偏相关分析。结果在22个省市饮用水常规指示菌合格率分级中,黑龙江省、江苏省、北京市和河北省等级较高。枯水期间除总大肠菌群水平与副伤寒发病率呈正相关(P0.05)外,其他指示菌与疾病之间均无显著相关性;4种常规指示菌间均两两呈正相关(P0.05)。丰水期间除总大肠菌群水平与伤寒、副伤寒发病率呈正相关(P0.05)外,其他指示菌与疾病之间无显著相关性;除大肠埃希菌与菌落总数无显著相关性外,其他常规指示菌间两两呈正相关。结论 GB 5749—2006中的常规指示菌与介水传染病相关性并不显著,建议进一步进行常规指示菌与介水传染病相关性的定量研究及探寻新型指示菌。     

拟杆菌作为粪便污染指示微生物的初探

目的 通过外环境中拟杆菌属菌量与大肠菌群存活菌量之间的相关性分析,探讨拟杆菌作为粪便污染指示微生物,并采用实时荧光定量PCR实现快速检测的可能性.方法 用大肠菌群最大或然数(most probable number,MPN)计数法和实时荧光定量PCR法分别定时检测模拟水样中大肠菌群的存活菌量及拟杆菌属菌量,探讨二者的污染检出时限和相关性.同时分别检测在3个时间点采集4个位点的12份河流水样,比较二者的检出情况.结果 MPN计数法检测大肠菌群需要72 h,而实时荧光定量PCR法检测拟杆菌不超过3 h;室内外模拟水样大肠菌群污染检出时限分别为＞40 d和9 d,而拟杆菌的污染检出时限分别为13 d和5 d;室外模拟水样拟杆菌属菌量(从第1天的8.3×106拷贝/ml下降到第5天的小于检出限104拷贝/ml)和大肠菌群存活菌量(从第1天的大于4.3×106MPN/100 ml下降到第5天的2.4×103 MPN/100 ml)之间呈正相关;12份河水水样两种方法检出均为阳性,符合率为100%,均说明二者对粪便污染的检测结果具有很好的一致性.结论 拟杆菌有可能作为粪便污染的指示微生物,并通过实时荧光定量PCR实现快速检测.

Abstract：

Objective To explore the possibility of Bacteriodes spp. as fecal contamination indicator bacteria with real-time quantitative PCR (RT-PCR) assay through analyzing the correlation between Bacteriodes spp. and coliform group in external environment. Methods Quantity of coliform group and Bacteriodes in water samples were detected by most-probable-number method (MPN) and RT-PCR,respectively, and their detection correlation was evaluated with linear correlation analysis. Both methods were also applied to detect the contaminated time limits and river water samples collected at four sampling sites in three different times. Results Seventy two hours were needed for the numeration of coliform group with MPN method,while RT-PCR could detect Bacteriodes within 3 hours. The contaminated time limit of indoor and outdoor water samples of coliform group was more than 40 days and 9 days, and Bacteriodes 13 days and 5 days,respectively. Also,the positive correlation between the quantity of Bacteroides and coliform group in outdoor water samples was obtained,the quantity of Bacteroides was from 8. 3 × 106 copise/ml to less than 104 copise/ml during the first day to the fifth day,while coliform group was 4. 3 × 106 MPN/100 ml to 2.4 × 103 MPN/100 ml. A 100% coincidence rate of the detection results with both methods was also observed. These results indicated that the detection results of both methods had perfect consistency. Conclusion Bacteriodes spp. can be potentially used as fecal contamination indicator bacteria with RT-PCR rapid detection.     

城镇污水综合处理卫生学指标评价

目的观察城镇污水处理厂污水处理前后微生物数值变化,分析对环境的影响,探讨排放标准.方法定点定时采集污水源水和经处理后的出厂水,按国家规定的方法检测细菌和病毒,同时收集理化指标检测数据.结果出厂水菌落总数高达720000cfu/L总大肠菌群和粪大肠菌群＞16000个/L;源水肠道病毒阳性率为58.33%出厂水肠道病毒阳性率为16.67%.结论理化指标处理效果较好,细菌、病毒处理效果较差,应加强对城镇污水综合处理厂出厂水有关标准的研究.     

哈夫尼亚菌生物学特性研究

目的了解哈夫尼亚菌的生物学特性。方法检测哈夫尼亚菌的一般特征、生化特性和血清学特性。结果本研究中的4株哈夫尼亚菌为革兰氏阴性小杆菌,氧化酶阴性,硝酸盐还原阳性,能够发酵葡萄糖产酸产气,不分解乳糖、蔗糖等,不产生H2S,各菌株能分别与沙门菌或志贺菌多价血清发生凝集反应,在生理盐水中不发生自凝。结论哈夫尼亚菌的生化特性复杂,主要特征稳定,但没有统一的系统反应模式;抗原结构也非常复杂,部分类似于沙门菌,部分类似于志贺菌,部分类似于大肠埃希菌等等。     

遵义市城区3种生吃蔬菜中肠道致病菌调查

目的 了解遵义市城区生吃蔬菜中沙门菌和志贺菌的污染状况,为该市生吃蔬菜卫生状况做出评价,以预防食源性疾病的暴发. 方法 样品采自遵义市较大的农贸市场中的个体菜农,按照卫生部有关规定,采取随机抽样的方法进行采集样品,对样本进行不同次数的日常清洗后,依据GB/T4789-2008,分别进行大肠菌群、沙门菌以及志贺菌分离培养、革兰染色、生化反应及因子血清学鉴定. 结果 检测葱、香菜以及生菜共135份,共检测出大肠菌群106株,总检出率78.52％,其95％置信区间为77.83％～85.45％,肠道致病菌47株,总检出率34.81％,其95％置信区间为26.78％～42.85％;其中两次清洗组大肠菌群检出率86.78％,肠道致病菌检出率53.33％,检出沙门菌15株,检出率33.33％,志贺菌9株,检出率20.00％;三次清洗组中大肠菌群检出率68.93％,肠道致病菌检出率37.78％,其中检出沙门菌11株,检出率24.44％,志贺菌6株,检出率13.34％;五次清洗组中大肠菌群检出率30.23％,肠道致病菌检出率13.33％,其中检出沙门菌5株,检出率11.11％,志贺菌1株,检出率2.22％.沙门菌在生吃蔬菜中污染率最高（22.96％）,而生菜带菌率较高;在不同清洗组中,清洗次数越多的组,其致病菌检出率越低（P＜0.005）. 结论 遵义市城区生吃蔬菜存在明显的食源性致病菌污染,建议有关部门加强对生食蔬菜安全监督和管理,制定相关标准,建立完善的食品安全监测体系.     

A microbiological study of bottled mineral water marketed in Ludhiana

The microbiological quality of bottled mineral water marketed in Ludhiana was examined, Twenty three brands were analyzed for presumptive coliform count by multiple tube tests, and E. coli count was confirmed by Eijkman test. Bacterial and fungal loads were tested by membrane filtration test. Out of 23 only one sample (4.4%) showed the presumptive coliform count to be 460 most probable number (MPN)l 1 00ml,and 1 was found to be positive when tested by Eijkman test for Ecoli. In the membrane filtration test three samples (13%) showed more than two types of bacteria. Different types of bacteria isolated included Bacillus sp (19/23). Pseudomonas spp (13123), Ecoli, Klebsiella sp and S.albus one each Fungi was isolated from five of twenty three. (22%) samples. Only one brand of mineral water was unfit for human consumption. The rest of the samples were contaminated with non pathogenic flora.     

遵义市城区3种生吃蔬菜中肠道致病菌调查

目的 了解遵义市城区生吃蔬菜中沙门菌和志贺菌的污染状况,为该市生吃蔬菜卫生状况做出评价,以预防食源性疾病的暴发. 方法 样品采自遵义市较大的农贸市场中的个体菜农,按照卫生部有关规定,采取随机抽样的方法进行采集样品,对样本进行不同次数的日常清洗后,依据GB/T4789-2008,分别进行大肠菌群、沙门菌以及志贺菌分离培养、革兰染色、生化反应及因子血清学鉴定. 结果 检测葱、香菜以及生菜共135份,共检测出大肠菌群106株,总检出率78.52％,其95％置信区间为77.83％～85.45％,肠道致病菌47株,总检出率34.81％,其95％置信区间为26.78％～42.85％;其中两次清洗组大肠菌群检出率86.78％,肠道致病菌检出率53.33％,检出沙门菌15株,检出率33.33％,志贺菌9株,检出率20.00％;三次清洗组中大肠菌群检出率68.93％,肠道致病菌检出率37.78％,其中检出沙门菌11株,检出率24.44％,志贺菌6株,检出率13.34％;五次清洗组中大肠菌群检出率30.23％,肠道致病菌检出率13.33％,其中检出沙门菌5株,检出率11.11％,志贺菌1株,检出率2.22％.沙门菌在生吃蔬菜中污染率最高（22.96％）,而生菜带菌率较高;在不同清洗组中,清洗次数越多的组,其致病菌检出率越低（P＜0.005）. 结论 遵义市城区生吃蔬菜存在明显的食源性致病菌污染,建议有关部门加强对生食蔬菜安全监督和管理,制定相关标准,建立完善的食品安全监测体系.