Bacteria In Aquatic Environments Research Articles

English

Arsenic (As) contamination of surface and ground waters in Uttar Pradesh (UP) is geological in nature, even though some degree of As contamination may be due to anthropogenic activities. Little is known about the arsenite oxidizing bacteria in aquatic environments of UP. In this study we isolated the As resistant bacteria from both surface and ground waters of UP. Seven potential strains were isolated by culture method using chemically defined medium (CDM). Two strains (AOB-GP1 and AOB-MT1) were Gram negative and other five were found to be Gram positive. The presence of arsenite oxidizing bacteria in groundwater is limited if compared to surface waters. 16S rDNA revealed highest homology of 99 - 88%. The strains were similar with Acinetobacter sp. PGSI1, Rummeliibacillus stabekisii, Cronobacter sakazakii, Bacillus tequilensis, Brevibacillus agri strain, Bacillus thuringiensis and Virgibacillus marismortui. The 16S rDNA gene sequence analysis for the As-resistant strains in surface and ground waters points to prevalence of γ-proteobacteria and Firmicutes, solely isolated γ-Proteobacteria (Acinetobacter sp. and Cronobactersakazakii sp.) from ground water samples. Based on morphological, cultural, biochemical and physiological characteristics the arsenite oxidizing bacteria in this study were grouped into three orders viz,. pseudomonadales, enterobacteriales and bacillales. Key words: Arsenite oxidizing bacteria, Pseudomonadales, Firmicuites, surface water, ground water.

Molecular epidemiology of Vibrio cholerae associated with flood in Brahamputra River valley, Assam, India

Cholera is often caused when drinking water is contaminated through environmental sources. In recent years, the drastic cholera epidemics in Odisha (2007) and Haiti (2010) were associated with natural disasters (flood and Earthquake). Almost every year the state of Assam India witnesses flood in Brahamputra River valley during reversal of wind system (monsoon).This is often followed by outbreak of diarrheal diseases including cholera. Beside the incidence of cholera outbreaks, there is lack of experimental evidence for prevalence of the bacterium in aquatic environment and its association with cholera during/after flood in the state. A molecular surveillance during 2012–14 was carried out to study prevalence, strain differentiation, and clonality of Vibrio cholerae in inland aquatic reservoirs flooded by Brahamputra River in Assam. Water samples were collected, filtered, enriched in alkaline peptone water followed by selective culturing on thiosulfate bile salt sucrose agar. Environmental isolates were identified as V. cholerae, based on biochemical assays followed by sero-grouping and detailed molecular characterization.The incidence of the presence of the bacterium in potable water sources was higher after flood. Except one O1 isolate, all of the strains were broadly grouped under non-O1/non-O139 whereas some of them did have cholera toxin (CT). Surprisingly, we have noticed Haitian ctxB in two non-O1/non-O139 strains. MLST analyses based on pyrH, recA and rpoA genes revealed clonality in the environmental strains. The isolates showed varying degree of antimicrobial resistance including tetracycline and ciprofloxacin. The strains harbored the genetic elements SXT constins and integrons responsible for multidrug resistance. Genetic characterization is useful as phenotypic characters alone have proven to be unsatisfactory for strain discrimination. An assurance to safe drinking water, sanitation and monitoring of the aquatic reservoirs is of utmost importance for combating the impending epidemic threat in the flood affected areas. Further, the management of flood through multi-prong approaches and sustainable utilization of environmental resources would be effective in disease management.

Ecology of aerobic anoxygenic phototrophs in aquatic environments.

Recognition of the environmental role of photoheterotrophic bacteria has been one of the main themes of aquatic microbiology over the last 15 years. Aside from cyanobacteria and proteorhodopsin-containing bacteria, aerobic anoxygenic phototrophic (AAP) bacteria are the third most numerous group of phototrophic prokaryotes in the ocean. This functional group represents a diverse assembly of species which taxonomically belong to various subgroups of Alpha-, Beta- and Gammaproteobacteria. AAP bacteria are facultative photoheterotrophs which use bacteriochlorophyll-containing reaction centers to harvest light energy. The light-derived energy increases their bacterial growth efficiency, which provides a competitive advantage over heterotrophic species. Thanks to their enzymatic machinery AAP bacteria are active, rapidly growing organisms which contribute significantly to the recycling of organic matter. This chapter summarizes the current knowledge of the ecology of AAP bacteria in aquatic environments, implying their specific role in the microbial loop.

Influence of wave action on the partitioning and transport of unattached and floc-associated bacteria in fresh water.

The dynamic interaction of bacteria within bed sediment and suspended sediment (i.e., floc) in a wave-dominated beach environment was assessed using a laboratory wave flume. The influence of shear stress (wave energy) on bacterial concentrations and on the partitioning and transport of unattached and floc-associated bacteria was investigated. The study showed that increasing wave energy (0.60 and 5.35 N/s) resulted in a 0.5 to 1.5 log increase in unattached cells of the test bacterium Pseudomonas sp. strain CTO7::gfp-2 in the water column. There was a positive correlation between the bacterial concentrations in water and the total suspended solids, with the latter increasing from values of near 0 to up to 200 mg/L over the same wave energy increase. The median equivalent spherical diameter of flocs in suspension also increased by an order of magnitude in all experimental trials. Under both low (0.60 N/s) and high (5.35 N/s) energy regime, bacteria were shown to preferentially associate with flocs upon cessation of wave activity. The results suggest that collecting water samples during periods of low wave action for the purpose of monitoring the microbiological quality of water may underestimate bacterial concentrations partly because of an inability to account for the effect of shear stress on the erosion and mobilization of bacteria from bed sediment to the water column. This highlights the need to develop a more comprehensive beach analysis strategy that not only addresses presently uncharacterized shores and sediments but also recognizes the importance of eroded flocs as a vector for the transport of bacteria in aquatic environments.

Theoretical analysis of the cost of antagonistic activity for aquatic bacteria in oligotrophic environments.

Many strains of bacteria produce antagonistic substances that restrain the growth of others, and potentially give them a competitive advantage. These substances are commonly released to the surrounding environment, involving metabolic costs in terms of energy and nutrients. The rate at which these molecules need to be produced to maintain a certain amount of them close to the producing cell before they are diluted into the environment has not been explored so far. To understand the potential cost of production of antagonistic substances in water environments, we used two different theoretical approaches. Using a probabilistic model, we determined the rate at which a cell needs to produce individual molecules in order to keep on average a single molecule in its vicinity at all times. For this minimum protection, a cell would need to invest 3.92 × 10−22 kg s−1 of organic matter, which is 9 orders of magnitude lower than the estimated expense for growth. Next, we used a continuous model, based on Fick's laws, to explore the production rate needed to sustain minimum inhibitory concentrations around a cell, which would provide much more protection from competitors. In this scenario, cells would need to invest 1.20 × 10−11 kg s−1, which is 2 orders of magnitude higher than the estimated expense for growth, and thus not sustainable. We hypothesize that the production of antimicrobial compounds by bacteria in aquatic environments lies between these two extremes.

Occurrence of Multiple Antibiotic Resistant Bacteria in Aquatic Environments in Central Minnesota

The purpose of this study was to investigate the possibility that antibiotic resistant bacteria could be isolated and identified in aquatic ecosystems in the lakes on the campus of Saint John’s University and the nearby Sauk and Watab Rivers. A total of 125 isolates were collected. Seventy-nine percent of the isolates were gram negative rods. Twenty-six isolates that were resistant to seven or more antibiotics were selected for further investigation. The 26 isolates were all gram negative and members of seven different genera with Flavobacterium and Acinetobacter being the most common. Resistance coefficients were calculated based on optical density values relative to cells grown without antibiotics. Multi-drug resistant, gram negative bacteria were shown to be common in aquatic environments in central Minnesota.

Inflammatory responses and potencies of various lipopolysaccharides from bacteria and cyanobacteria in aquatic environments and water supply systems.

Inflammatory substances derived from indigenous bacteria in aquatic environments or water systems are of great concern. Lipopolysaccharides (LPSs), one of the major inflammatory substances in water, are usually identified using Limurus amoebocyte lysate (LAL) assay on the basis of their endotoxic activity, but endotoxin levels do not accurately represent their inflammatory potency in humans. In this investigation, the cellular endotoxin contents of pure-cultured bacteria/cyanobacteria, which are frequently detected in water sources and distribution systems, and of indigenous bacteria in a river and in biologically activated carbon (BAC) effluent, were investigated. The indigenous bacteria showed the highest endotoxin contents exceeding 10(-3)EU/cell. The LPSs were then purified from those samples, and their inflammatory potencies were examined using a human monocytic cell line. The LPSs from Acinetobacter lwoffii culture, the river water, and the BAC effluent sample revealed a unique cytokine secretion pattern; they induced both IL-8 and TNF-α more strongly than the other tested bacterial LPSs. These results suggest that natural bacterial/cyanobacterial flora in aquatic environments and water distribution systems have the potential to induce relatively strong inflammatory responses in humans; therefore, further accumulation of data on water quality from the perspective of not just endotoxins but inflammatory potency is needed.

Characterization of the microbial community in a lotic environment to assess the effect of pollution on nitrifying and potentially pathogenic bacteria.

This study aimed to investigate microbes involved in the nitrogen cycle and potentially pathogenic bacteria from urban and rural sites of the São Pedro stream. Water samples were collected from two sites. A seasonal survey of bacterial abundance was conducted. The dissolved nutrient content was analysed. PCR and FISH analysis were performed to identify and quantify microbes involved in the nitrogen cycle and potentially pathogenic bacteria. The seasonal survey revealed that the bacterial abundance was similar along the year on the rural area but varied on the urban site. Higher concentration of dissolved nutrients in the urban area indicated a eutrophic system. Considering the nitrifying microbes, the genus Nitrobacter was found, especially in the urban area, and may act as the principal bacteria in converting nitrite into nitrate at this site. The molecular markers napA, amoA, and nfrA were more accumulated at the urban site, justifying the higher content of nutrients metabolised by these enzymes. Finally, high intensity of amplicons from Enterococcus, Streptococcus, Bacteroides/Prevotella/Porphyromonas, Salmonella, S. aureus, P. aeruginosa and the diarrheagenic lineages of E. coli were observed at the urban site. These results indicate a change in the structure of the microbial community imposed by anthrophic actions. The incidence of pathogenic bacteria in aquatic environments is of particular importance to public health, emphasising the need for sewage treatment to minimise the environmental impacts associated with urbanisation.

Reduction of clarithromycin and sulfamethoxazole-resistant Enterococcus by pilot-scale solar-driven Fenton oxidation

The presence of pathogenic antibiotic-resistant bacteria in aquatic environments has become a health threat in the last few years. Their presence has increased due to the presence of antibiotics in wastewater effluents, which are not efficiently removed by conventional wastewater treatments. As a result there is a need to study the possible ways of removal of the mixtures of antibiotics present in wastewater effluents and the antibiotic-resistant bacteria, which may also spread the antibiotic resistance genes to other bacterial populations. In this study the degradation of a mixture of antibiotics i.e. sulfamethoxazole and clarithromycin, the disinfection of total enterococci and the removal of those resistant to: a) sulfamethoxazole, b) clarithromycin and c) to both antibiotics have been examined, along with the toxicity of the whole effluent mixture after treatment to the luminescent aquatic bacterium Vibrio fischeri. Solar Fenton treatment (natural solar driven oxidation) using Fenton reagent doses of 50mgL−1 of hydrogen peroxide and 5mgL−1 of Fe3+ in a pilot-scale compound parabolic collector plant was used to examine the disinfection and antibiotic resistance removal efficiency in different aqueous matrices, namely distilled water, simulated and real wastewater effluents. There was a faster complete removal of enterococci and of antibiotics in all aqueous matrices by applying solar Fenton when compared to photolytic treatment of the matrices. Sulfamethoxazole was more efficiently degraded than clarithromycin in all three aqueous matrices (95% removal of sulfamethoxazole and 70% removal of clarithromycin in real wastewater). The antibiotic resistance of enterococci towards both antibiotics exhibited a 5-log reduction with solar Fenton in real wastewater effluent. Also after solar Fenton treatment, there were 10 times more antibiotic-resistant enterococci in the presence of sulfamethoxazole than in the presence of clarithromycin. Finally, the toxicity of the treated wastewater to V. fischeri remained very low throughout the treatment time.

Dynamics of heterotrophic bacteria in temperate coastal waters: similar net growth but different controls in low and high nucleic acid cells

Heterotrophic bacteria in aquatic environments are almost universally distributed into 2 distinct flow cytometric clusters based on their relative nucleic acid content: low (LNA) and high (HNA). The dynamics and possible regulation processes of both subgroups in southern Bay of Biscay coastal waters were examined by comparing weekly measurements over 2 yr at a shallow environment (L’Arbeyal beach) with monthly samples at 3 continental shelf stations off Xixon (Spain). Similar seasonal variations of temperature and chlorophyll a characterized inshore and offshore waters. Total bacterial abundances were also similar in shallow and shelf waters (from 0.22 to 3.72 × 106 cells ml−1), with the most remarkable difference being the higher abundance and larger size of HNA bacteria at L’Arbeyal, probably related to greater substrate availability. There, apparent net growth rates estimated from coherent periods (6 to 26 wk) of sustained increase or decrease (−0.02 to 0.04 d−1) were similar for LNA and HNA cells, although related to different factors. LNA bacteria net growth rates were strongly positively correlated with temperature (r = 0.95, p < 0.01, n = 6) and negatively with chlorophyll a (r = −0.90, p = 0.01, n = 6), supporting the hypothesis that they are independent from phytoplankton, which was recently suggested for this and other coastal sites using different approaches. We also show an opposite relationship between cell size and apparent net growth rates of LNA and HNA bacteria, providing further evidence of fundamentally different ecological roles.

Comparison of inflammatory responses in human cells caused by lipopolysaccharides from Escherichia coli and from indigenous bacteria in aquatic environment

The endotoxic activities of lipopolysaccharides (LPSs) in water samples are usually determined using a Limulus amoebocyte lysate (LAL) assay, but it is known that the determined activities do not always represent their inflammatory potency in humans. In this investigation, the inflammatory responses in three different human cells stimulated with Escherichia coli LPS, keratinocyte, CD14+ monocyte, and THP-1, were compared using cytokine secretion as biomarkers to develop novel in vitro assay systems for detecting changes in inflammatory potencies of endotoxins in aquatic environment. Only THP-1 with 6-h stimulation showed dose-dependent responses in the range of normal endotoxin levels in aquatic environment. Then, the inflammatory potency of environmental LPS, which was purified from river water, was tested using THP-1. The levels and patterns of cytokine secretion after the environmental LPS stimulation were completely different from E. coli LPS. Interleukin 8 (IL-8) secretions after the environmental LPS stimulation were approximately 10-fold higher than those after E. coli LPS stimulation. The environmental LPS also induced much higher levels of TNF-α secretions in THP-1. These results suggest that a diversity of LPS structures in aquatic environment could contribute to stronger and different inflammatory responses. This investigation indicated that the proposed THP-1 assay system could be useful for detecting the changes in inflammatory potencies caused by aquatic bacteria.

Possible alkylation of inorganic Hg(II) by photochemical processes in the environment

The methylation of inorganic Hg by anaerobic bacteria in aquatic environments is considered to be the major pathway for methylmercury (MeHg) production. However, recent research has suggested that abiotic or chemical methylation by humic substances and other low-molecular-weight organic compounds in natural environments is also possible. Here, the aqueous photo-transformation of Hg2+ to organomercurials was investigated in the presence of ketones, aldehydes and low molecular weight organic acids under UV irradiation. MeHg and/or ethylmercury (EtHg) were identified as the main organomercurial products by multiple analytical techniques, including chromatography-atomic spectrometry and molecular mass spectrometry and further confirmed by stable isotope tracer experiments. The yield of organomercurials was markedly influenced by pH, NaCl concentration, alkylation donor concentration and the presence of chelating ligands in the aqueous solution. Electron paramagnetic resonance spectrometry demonstrated that the radical reaction was not the predominating alkylation pathway, although methyl radicals were detected in the photo-alkylation procedure. A mechanism based on intra-molecular alkyl transfer in the Hg2+-low-molecular-weight organic compound complex is proposed. The present work helps us better understand of MeHg and EtHg photo-generation in natural environments.

Distribution of antibiotic-resistant bacteria in aquatic environments in Miyagi prefecture

Distribution of antibiotic-resistant bacteria in aquatic environments in Miyagi prefecture

Antibiotic contamination and occurrence of antibiotic-resistant bacteria in aquatic environments of northern Vietnam

The ubiquitous application and release of antibiotics to the environment can result in bacterial antibiotic resistance, which in turn can be a serious risk to humans and other animals. Southeast Asian countries commonly apply an integrated recycling farm system called VAC (Vegetable, Aquaculture and Caged animal). In the VAC environment, antibiotics are released from animal and human origins, which would cause antibiotic-resistant bacteria (ARB). This study evaluated occurrence of ARB in the VAC environment in northern Vietnam, with quantitative analysis of antibiotic pollution. We found that sulfonamides were commonly detected at all sites. In dry season, while sulfamethazine was a major contaminant in pig farm pond (475–6662 ng/l) and less common in city canal and aquaculture sites, sulfamethoxazole was a major one in city canal (612–4330 ng/l). Erythromycin (154–2246 ng/l) and clarithromycin (2.8–778 ng/ml) were the common macrolides in city canal, but very low concentrations in pig farm pond and aquaculture sites. High frequencies of sulfamethoxazole-resistant bacteria (2.14–94.44%) were found whereas the occurrence rates of erythromycin-resistant bacteria were lower (< 0.01–38.8%). A positive correlation was found between sulfamethoxazole concentration and occurrence of sulfamethoxazole-resistant bacteria in dry season. The sulfamethoxazole-resistant isolates were found to belong to 25 genera. Acinetobacter and Aeromonas were the major genera. Twenty three of 25 genera contained sul genes. This study showed specific contamination patterns in city and VAC environments and concluded that ARB occurred not only within contaminated sites but also those less contaminated. Various species can obtain resistance in VAC environment, which would be reservoir of drug resistance genes. Occurrence of ARB is suggested to relate with rainfall condition and horizontal gene transfer in diverse microbial community.

High frequency of phage-infected bacterial cells in a rice field soil in Japan

Phages cause significant mortality of bacteria in aquatic environments and thus play an important role in biogeochemical nutrient cycles through biomass turnover. This study evaluated the frequency of phage infection to bacterial cells and resultant potential for mortality and lysis of bacteria in three soil layers in a Japanese rice field. The frequency of visibly infected bacterial cells (FVIC) was from 8.9% to 12.1% on average in these soil layers. This was significantly greater than the reported FVIC values in aquatic environments, and the estimated fraction of bacterial mortality from phage lysis reached far over 100% in every soil layer. The phage infection to bacterial cells tended to be more frequent for short-rod cells than for long-rod cells. The medians of phage-like particles in a bacterial cell (burst size) ranged from 12.5 to 16 particles cell−1 with the low and high quartiles of 7 and 27, respectively.

Fluoroquinolone (FQ) Contamination Does Not Correlate with Occurrence of FQ-Resistant Bacteria in Aquatic Environments of Vietnam and Thailand

Fluoroquinolone antibiotics (FQs) have been used worldwide for chemotherapy, animal husbandry, and aquaculture, and the occurrence of FQ-resistant (FQs(r)) bacteria in natural environments has been reported. Plasmid-mediated transferable quinolone resistance (PMQR) genes are suspected to originate from the chromosomes of water-dwelling bacteria. However, the occurrence of and the potential reservoir of FQs(r) bacteria and PMQR genes in aquatic environments have not been elucidated. In this study, we detected FQs(r) bacteria and PMQR genes in aquatic environments in Thailand and Vietnam, and measured FQ contamination. Levels of contamination were greater Thailand (avg. 5130, max 46100 ng L(-1)) than in Vietnam (avg. 235, max 1130 ng L(-1)); however, the occurrence of FQs(r) bacteria was higher in Vietnam (~15%) than in Thailand (~7.0%), suggesting that contamination by FQs is not directly linked to the development of FQs(r) bacteria. Diverse taxonomic groups of FQs(r)-bacteria were identified, and one of the PMQR genes, qnrB, was detected from bacteria of environmental origin, not enteric bacteria. This suggests that the environmental bacteria are a potential reservoir of antibiotic resistance determinants even at un-contaminated sites.

BACTÉRIAS E BIOINDICADORES DE QUALIDADE DE ÁGUAS DE ECOSSISTEMAS ORIZÍCOLAS DA REGIÃO SUL DO BRASIL

The presence of heterotrophic bacteria in aquatic environments, such as those in the agricultural systems of irrigated rice fields, is determined by biotic and abiotic processes, with the focus on physical-chemical factors and highly complex interactions with other organisms. In this study, we address aspects related to these bacteria, such as: bacterial diversity in rice growing areas, total coliforms and thermotolerant as environmental indicators. Are also mentioned topics related to natural and artificial wetlands, the viability of enteric bacteria, the environmental impacts, reuse and monitoring of water in rice fields ecosystems.

Functional bacteria as potential indicators of water quality in Three Gorges Reservoir, China

The distributions of culturable bacteria and functional bacteria associated with nitrogen (N) or phosphorus (P) in the backwater areas of the Three Gorges Reservoir (TGR) were investigated. Results from seven locations in the TGR indicated that the abundance of total bacteria was high, with 8.12 x 10(6), 2.70 x 10(7), and 6.73 x 10(10) colony-forming units per milliliter or per gram dry weight in surface water, bottom water, and sediments, respectively. Aquatic environments with higher nutrient loadings possessed higher bacteria densities and lower bacteria community diversities. Eight kinds of functional bacteria ratios, including surface water to bottom water and ratios of water to sediments, were calculated, in which four kinds of functional bacteria, namely, nitrogen-fixing bacteria, ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, and organophosphate-solubilizing bacteria, displayed obvious differences in different locations. Based on the functional bacteria ratios of water to sediments, it was found that the obtained result of location grouping from cluster analysis was similar to that based on the community-level physiological profiles studies. The above results showed that the ratios of functional bacteria could distinguish the aquatic environments with different trophic conditions in the TGR. This demonstrated that the distribution ratios of functional bacteria in aquatic environments could work as potential bioindicators to reflect the trophic condition of the water.

Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp.

The occurrence and spread of multi-drug resistant bacteria is a pressing public health problem. The emergence of bacterial resistance to antibiotics is common in areas where antibiotics are heavily used, and antibiotic-resistant bacteria also increasingly occur in aquatic environments. The purpose of the present study was to evaluate the impact of the wastewater treatment process on the prevalence of antibiotic resistance in Acinetobacter spp. in the wastewater and its receiving water. During two different events (high-temperature, high-flow, 31 °C; and low-temperature, low-flow, 8 °C), 366 strains of Acinetobacter spp. were isolated from five different sites, three in a wastewater treatment plant (raw influent, second effluent, and final effluent) and two in the receiving body (upstream and downstream of the treated wastewater discharge point). The antibiotic susceptibility phenotypes were determined by the disc-diffusion method for 8 antibiotics, amoxicillin/clavulanic acid (AMC), chloramphenicol (CHL), ciprofloxacin (CIP), colistin (CL), gentamicin (GM), rifampin (RA), sulfisoxazole (SU), and trimethoprim (TMP). The prevalence of antibiotic resistance in Acinetobacter isolates to AMC, CHL, RA, and multi-drug (three antibiotics or more) significantly increased ( p < 0.01) from the raw influent samples (AMC, 8.7%; CHL, 25.2%; RA, 63.1%; multi-drug, 33.0%) to the final effluent samples (AMC, 37.9%; CHL, 69.0%; RA, 84.5%; multi-drug, 72.4%), and was significantly higher ( p < 0.05) in the downstream samples (AMC, 25.8%; CHL, 48.4%; RA, 85.5%; multi-drug, 56.5%) than in the upstream samples (AMC, 9.5%; CHL, 27.0%; RA, 65.1%; multi-drug, 28.6%). These results suggest that wastewater treatment process contributes to the selective increase of antibiotic resistant bacteria and the occurrence of multi-drug resistant bacteria in aquatic environments.

Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.