Prevalence, quantification and typing of adenoviruses detected in river and treated drinking water in South Africa

AIMS: Human adenoviruses (HAds), of which there are 51 serotypes, are associated with gastrointestinal, respiratory, urinary tract and eye infections. The importance of water in the transmission of HAds and the potential health risks constituted by HAds in these environments are widely recognized. Adenoviruses have not previously been quantified in river and treated drinking water samples. In this study, HAds in river water and treated drinking water sources in South Africa were detected, quantified and typed. METHODS AND RESULTS: Adenoviruses were recovered from the water samples using a glass wool adsorption-elution method followed by polyethylene glycol/NaCl precipitation for secondary concentration. The sensitivity and specificity of two nested PCR methods were compared for detection of HAds in the water samples. Over a 1-year period (June 2002 to July 2003), HAds were detected in 5.32% (10/188) of the treated drinking water and 22.22% (10/45) of river water samples using the conventional nested PCR method. The HAds detected in the water samples were quantified using a real-time PCR method. The original treated drinking water and river water samples had an estimate of less than one copy per litre of HAd DNA present. The hexon-PCR products used for typing HAds were directly sequenced or cloned into plasmids before sequencing. In treated drinking water samples, species D HAds predominated. In addition, adenovirus serotypes 2, 40 and 41 were each detected in three different treated drinking water samples. Most (70%) of the HAds detected in river water samples analysed were enteric HAds (serotypes 40 and 41). One HAd serotype 2 and two species D HAds were detected in the river water. CONCLUSIONS: Adenoviruses detected in river and treated drinking water samples were successfully quantified and typed. The detection of HAds in drinking water supplies treated and disinfected by internationally recommended methods, and which conform to quality limits for indicator bacteria, warrants an investigation of the risk of infection constituted by these viruses. The risk of infection may have implications for the management of drinking water quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is unique as it is the first report on the quantification and typing of HAds in treated drinking water and river water. This baseline data is necessary for the meaningful assessment of the potential risk of infection constituted by these viruses.     

Diversity and dynamics of bacterial species in a biofilm at the end of the Seoul water distribution system

To investigate changes in the bacterial species and hygienic safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyse the bacterial population in the biofilm of a semi-pilot galvanized iron pipe model. The presence of sequences from aerobic Sphingomonas sp., anaerobic Rhodobacter sp., and unculturable bacteria indicated that these organisms coexisted after 1 day of model operation, demonstrating the ease of biofilm formation on galvanized iron pipes in the end region of the water distribution system studied. Sequences similar to those of unculturable bacteria, E. coli, and anaerobic bacteria were detected during the course of succession on the biofilm. More complicated band patterns were observed after 70 days of operation. PCR-DGGE illustrated changes in the biofilm during succession as well as the possibilities of anaerobic conditions and faecal contamination of the drinking water system. PCR-DGGE and culture-dependent fatty acid methyl ester (FAME) analysis showed different patterns for the same samples (Lee & Kim 2003); however, PCR-DGGE showed less diversity than did FAME analysis. This study compared the culture-dependent FAME and culture-independent PCR-DGGE methods directly, and their use in promoting the hygienic safety of drinking water.     

Harnessing microbial activities for environmental cleanup

Human activities have released large amounts of toxic organic and inorganic chemicals into the environment. Toxic waste streams threaten dwindling drinking water supplies and impact terrestrial, estuarine and marine ecosystems. Cleanup is technically challenging and the costs based on traditional technologies are exceeding the economic capabilities of even the richest countries. Recent advances in our understanding of the microbiology contributing to contaminant transformation and detoxification has led to successful field demonstrations. Hence, harnessing the activity of naturally occurring bacteria, particularly the power of anaerobic reductive processes, is a promising approach to restore contaminated subsurface environments, protect drinking water reservoirs and to safeguard ecosystem health.     

Rapid detection of sulfide-producing bacteria from sulfate and thiosulfate

An original liquid medium and a field broth bottle method for the rapid detection of the most probable number of sulfide-producing bacteria (SPB) from sulfate and thiosulfate are described. The broth bottle method enables after inoculation with a sample (water, sediment) the growth of ubiquitous aerobic bacteria, causing oxygen depletion, required for the growth of the present various anaerobic and facultative anaerobic SPB. The medium regularly gives higher numbers of the SPB than the used control medium (Postgate's E) for detection of sulfate-reducing bacteria and the final results are obtained just 36 h after the medium inoculation. The method is simple and suitable for the estimation of the physiological group of SPB in fresh waters, saline waters, sediments and industrial waters.     

Inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> and coliform bacteria in traditional brass and earthernware water storage vessels

The detection and enumeration of indicator bacteria such as Escherichia coli is used to assess the extent of faecal contamination of drinking water. On the basis of this approach, the effectiveness of storing water contaminated with faecal indicator bacteria in brass or earthern vessels (mutkas) of the type used in rural India have been investigated. Suspensions of bacteria in sterile distilled water were maintained for up to 48 h in each vessel and enumerated by surface plate counts on nutrient agar (non-selective) and several selective coliform media at 37 °C either under standard aerobic conditions, or under conditions designed to neutralise reactive oxygen species (ROS), e.g. using an anaerobic cabinet to prepare plates of pre-reduced growth medium or by inclusion of sodium pyruvate in the growth medium, with incubation of aerobically-prepared plates in an anaerobic jar. The counts obtained for E. coli decreased on short-term storage in a brass mutka; counts for selective media were lower than for equivalent counts for non-selective medium, with ROS-neutralised conditions giving consistently higher counts than aerobic incubation. However, after 48 h, no bacteria were cultivable under any conditions. Similar results were obtained using water from environmental sources in the Panjab, and from rural households where brass and earthern mutkas are used for storage of drinking water, with enumeration on selective coliform media (presumptive total coliforms). In all cases results indicated that, while storage of water in a brass mutka can inactivate E. coli and coliforms over a 48 h period, standard aerobic plate counting using selective media may not be fully effective in enumerating sub-lethally damaged bacteria.     

A simple, rapid and sensitive presence/absence detection test for bacteriophage in drinking water

R. ARMON AND Y. KOTT. 1993. A rapid, simple and sensitive direct bacteriophage presence detection method for 500 ml drinking water samples has been developed. The method includes a glass device consisting of a jar containing the water sample and an immersible probe filled with solidified soft agar containing bacterial host cells. Host bacteria in logarithmic phase were added to the experimental volume and the probe was submerged. The entire device was incubated in a water bath at 36C.
Plaques of somatic bacteriophage infecting Escherichia coli strain CN₁₃, could be detected within 3 h. Male-specific bacteriophages infecting E. coli F+ amp were detected within 6 h. Bacteriophage infecting the anaerobe Bacteroides fragilis subsp. fragilis HSP40 were detected after 8 h. Application of this device and the associated technique, enabled a one-step detection of 1 pfu of E. coli or Bact. fragilis specific bacteriophage in 500 ml drinking water samples.     

Detection and characterization of filterable heterotrophic bacteria from rural groundwater supplies

AIMS: The chemical/physical environment of groundwater may contribute to the existence of a subpopulation of small-sized bacteria (filterable bacteria) that fails to be trapped on conventional 0.45 microm-pore-size membrane filters during routine bacteriological water quality analyses. Efforts were directed to determining an efficient recovery method for detection of such cells. METHODS AND RESULTS: Individual groundwater supplies in a rural setting were examined by a double membrane filtration procedure to determine the presence of heterotrophic plate count (HPC) bacteria capable of escaping detection on conventional pore size (0.45 microm) membrane filters but retained on 0.22 microm-pore-size filters. Since optimum cultural conditions for recovery of filterable bacteria are not well defined, initial efforts focused on evaluation of various media (R2A, m-HPC and NWRI) and incubation temperatures (15, 20, 28 and 35 degrees C) for specific recovery of filterable bacteria. Maximum recovery of small-sized HPC bacteria occurred on low-nutrient concentration R2A agar incubated for 7 d at 28 degrees C. Similarly, identical cultural conditions gave enhanced detection of the general HPC population on 0.45 microm-pore-size filters. A 17-month survey of 10 well water supplies conducted with the cultural conditions described above resulted in detection of filterable bacteria (ranging in density from 9 to 175 cfu ml-1) in six of the groundwater sources. The proportion of filterable bacteria in any single sample never exceeded 10% of the total HPC population. A majority of the colonies appearing on the 0.22 microm membrane filters was pigmented (50-90%), whereas the proportion of colonies demonstrating pigmentation on the larger porosity filters failed to exceed 50% for any of the samples (19-49%). CONCLUSION: A reliable recovery method was developed for the detection of filterable bacteria from groundwater. During a subsequent survey study using this procedure, filterable bacteria were detected in a majority of the groundwater supplies examined; however, the density of filterable bacteria in any single sample never exceeded 10% of the total HPC population. Identification of randomly selected isolates obtained on the 0.22 microm filters indicated that some of these filterable bacteria have been implicated as opportunistic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: We have determined the presence of small-sized HPC bacteria in ground water that may go undetected when using standard porosity membrane filters for water quality analyses. Further study is needed to assess the significance and possible health risk associated with presence of filterable bacteria in drinking water supplies from groundwater sources.     

Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes

Aims:  To develop a PCR-based tracking method for the detection of a subset of bacteria in drinking water distribution systems capable of degrading haloacetic acids (HAAs).
Methods and Results:  Published degenerate PCR primers were used to determine that 54% of tap water samples (7/13) were positive for a deh gene, indicating that drinking water distribution systems may harbour bacteria capable of HAA degradation. As the published primer sets were not sufficiently specific for quantitative PCR, new primers were designed to amplify deh II genes from selected indicator strains. The developed primer sets were effective in directly amplifying deh II genes from enriched consortia samples, and the DNA extracted from tap water provided that an additional nested PCR step for detection of the deh II gene was used.
Conclusions:  This study demonstrates that drinking water distribution systems harbour microbes capable of degrading HAAs. In addition, a quantitative PCR method was developed to detect and quantify deh II genes in drinking water systems.
Significance and Impact of the Study:  The development of a technique to rapidly screen for the presence of dehalogenase genes in drinking water distribution systems could help water utilities determine if HAA biodegradation is occurring in the distribution system.     

Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.     

Isolation of Aeromonas spp. from water by using anaerobic incubation.

A membrane filtration method incorporating a combination of anaerobic and aerobic incubation has been developed for the enumeration of Aeromonas spp. in drinking water. The use of anaerobic incubation improved the detection of Aeromonas spp. by reducing the growth of nonaeromonads. The confirmation rate of presumptive Aeromonas spp. identified on the initial isolation agar exceeded 92%.     

Isolation of Aeromonas spp. from water by using anaerobic incubation

A membrane filtration method incorporating a combination of anaerobic and aerobic incubation has been developed for the enumeration of Aeromonas spp. in drinking water. The use of anaerobic incubation improved the detection of Aeromonas spp. by reducing the growth of nonaeromonads. The confirmation rate of presumptive Aeromonas spp. identified on the initial isolation agar exceeded 92%.     

饮用水微生物的安全快速检测

【目的】为了更好地分析饮用水中的微生物含量。【方法】利用流式细胞术(Flowcytometry,FCM)、ATP测定方法检测瓶装无气饮用水中的微生物数量、可同化有机碳(Assimilable organic carbon,AOC)含量以及微生物活性,并将检测结果与传统的饮用水微生物检测技术相对照。【结果】FCM方法可快速区分水样中的活性细菌和非活性细菌,AOC含量反映了水样中微生物再生能力;而ATP检测方法也能比异养细菌平板计数法(Heterotrophic plate count,HPC)更好地反映瓶装无气饮用水中的实际微生物含量。【结论】FCM、ATP测定方法要明显优于依赖于培养的传统方法。     

A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA)

Methods to detect the presence of coliform bacteria in drinking water usually involve a series of complex cultivating steps that are time-consuming and subject to external influences. For this reason, the new 16S rRNA probe has been developed in this study as an alternative detector PCR-ELISA technique that does not involve the culture of bacteria and that is able to detect, identify, and quantify the representative coliform species present in water samples. Our results indicate that this technique is both rapid (detection time of 4 h) and accurate (1.4% error rate). The limit of detection (LOD) was 5 CFU/100 ml for total coliforms, which meets the standards set by most countries for drinking water. Our comparative study demonstrated that this PCR-ELISA method is superior to current conventional methods in terms of detection time, LOD, and accuracy.     

Development of a rapid and sensitive method combining a cellulose ester microfilter and a real-time quantitative PCR assay to detect Campylobacter jejuni and Campylobacter coli in 20 liters of drinking water or low-turbidity waters

Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.     

Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.     

Effect of oxygen on survival of faecal pollution indicators in drinking water

AIMS: The aim of this study was to determine the effect of oxygen on the survival of faecal pollution indicators including Escherichia coli in nondisinfected drinking water. METHODS AND RESULTS: Aerobic and anaerobic drinking water microcosms were inoculated with E. coli ATCC 25922 or raw sewage. Survival of E. coli was monitored by membrane filtration combined with cultivation on standard media, and by in situ hybridization with 16S rRNA-targeted fluorescent oligonucleotide probes. Anaerobic conditions significantly increased the survival of E. coli in drinking water compared with aerobic conditions. Escherichia coli ATCC 25922 showed a biphasic decrease in survival under aerobic conditions with an initial first-order decay rate of -0.11 day(-1) followed by a more rapid rate of -0.35 day(-1). In contrast, the first-order decay rate under anaerobic conditions was only -0.02 day(-1). After 35 days, <0.01% of the initial E. coli ATCC 25922 population remained detectable in aerobic microcosms compared with 48% in anaerobic microcosms. A poor survival was observed under aerobic conditions regardless of whether E. coli ATCC 25922 or sewage-derived E. coli was examined, and regardless of the detection method used (CFU or fluorescent in situ hybridization). Aerobic conditions in drinking water also appeared to decrease the survival of faecal enterococci, somatic coliphages and coliforms other than E. coli. CONCLUSIONS: The results indicate that oxygen is a major regulator of the survival of E. coli in nondisinfected drinking water. The results also suggest that faecal pollution indicators other than E. coli may persist longer in drinking water under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of oxygen should be considered when evaluating the survival potential of enteric pathogens in oligotrophic environments.     

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of blaNDM-1

New Delhi metallo-β-lactamase-1 gene (bla_NDM-1) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of β-lactam ring, and provides resistance against major classes of β-lactam antibiotics. Dissemination of the plasmid borne bla_NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla_NDM-1. Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6–25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla_NDM-1 gene.     

Review of Campylobacter spp. in drinking and environmental waters

Consumption of contaminated drinking water is a significant cause of Campylobacter infections. Drinking water contamination is known to result from septic seepage and wastewater intrusion into non-disinfected sources of groundwater and occasionally from cross-connection into drinking water distribution systems. Wastewater effluents, farm animals and wild birds are the primary sources contributing human-infectious Campylobacters in environmental waters, impacting on recreational activities and drinking water sources. Culturing of Campylobacter entails time-consuming steps that often provide qualitative or semi-quantitative results. Viable but non-culturable forms due to environmental stress are not detected, and thus may result in false-negative assessments of Campylobacter risks from drinking and environmental waters. Molecular methods, especially quantitative PCR applications, are therefore important to use in the detection of environmental Campylobacter spp. Processing large volumes of water may be required to reach the desired sensitivity for either culture or molecular detection methods. In the future, applications of novel molecular techniques such as isothermal amplification and high-throughput sequencing applications are awaited to develop and become more affordable and practical in environmental Campylobacter research. The new technologies may change the knowledge on the prevalence and pathogenicity of the different Campylobacter species in the water environment.     

羧酶体的组成、结构、功能和检测及其在脱氮细菌中的意义

羧酶体(Carboxysome)是一种具有CO2浓缩功能的"类细胞器",它存在于自养型脱氮细菌中,可增强细菌的自养生长能力。硝化细菌、厌氧氨氧化细菌和部分反硝化细菌都是重要的自养型脱氮细菌,探明其羧酶体的组成、结构和功能,将有助于揭示自养型脱氮菌的生长规律,进而强化生物脱氮过程。基于文献阅读和相关研究,本文对自养型细菌中羧酶体在组成、结构、功能和检测等方面的研究进展进行综述,以期为自养生物脱氮过程的深入理解和优化改进提供参考。     

Coliform species recovered from untreated surface water and drinking water by the membrane filter, standard, and modified most-probable-number techniques.

The species of total coliform bacteria isolated from drinking water and untreated surface water by the membrane filter (MF), the standard most-probable-number (S-MPN), and modified most-probable-number (M-MPN) techniques were compared. Each coliform detection technique selected for a different profile of coliform species from both types of water samples. The MF technique indicated that Citrobacter freundii was the most common coliform species in water samples. However, the fermentation tube techniques displayed selectivity towards the isolation of Escherichia coli and Klebsiella. The M-MPN technique selected for more C. freundii and Enterobacter spp. from untreated surface water samples and for more Enterobacter and Klebsiella spp. from drinking water samples than did the S-MPN technique. The lack of agreement between the number of coliforms detected in a water sample by the S-MPN, M-MPN, and MF techniques was a result of the selection for different coliform species by the various techniques.