Total Direct Counts Research Articles

INVASIVE INSECT SPECIES CYDALIMA PERSPECTALIS (WALKER, 1859): GLOBAL DISTRIBUTION, INVASION IN UKRAINE AND DAMAGE IN CHERNIVTSI REGION

Invasions of species into ecosystems cause their transformation and, in certain cases, their degradation. The study was carried out to determine the extent of the invasion of Cydalima perspectalis (Walker, 1859). For this purpose, the following aspects were analyzed: distribution of the species in different countries of the worldі, history of invasion of the pest in Ukraine, degree of damage to plantations of different box (Buxus L.) species and colony density in certain areas. The chronology of the distribution of C. perspectalis was studied using literature sources and international databases: Global Biodiversity Information Facility (GBIF), Delivering Alien Invasive Species Inventories for Europe (DAISIE), European and Mediterranean Plant Protection Organization (EPPO). The information on the history of the species' invasion in Ukraine and in particular in the Chernivtsi region was found in the literature, using the database of the National Biodiversity Information Network (UkrBIN), through questionnaires and personal observations using the route method (counting the number of C. perspectalis on a certain route). The degree of damage to Buxus sp. shrubs was determined visually using a scale. The density of C. perspectalis in each area was assessed by total counts and direct counts in situ with a parallel assessment of habitat size. It was found that the expansion of C. perspectalis beyond its native range began in 2006 from southwestern Germany. In 15 years, the species has occupied the territories of about 40 mainland and island countries of the North American and European continents. Since 2014, after the invasion of the Zakarpattia region, C. perspectalis has begun to spread throughout the Ukrainian territories. Currently, the secondary range of the species is represented by 14 regions. The presence of C. perspectalis has been detected in 16 settlements of Chernivtsi region, where the degree of damage to box shrubs is mostly severe. The density of caterpillars on most bushes is up to 100 individuals/m2, which indicates the high damage of the species. Key words: Cydalima perspectalis (Walker, 1859), invasive species, damage, expansion.

Groundwater flow system and microbial dynamics of groundwater in a headwater catchment

Besides surface water systems, groundwater also provides a habitat for prokaryotes. The prokaryotes inhabiting groundwater are affected by the environment that they grow in. Owing to the environmental specificity, some prokaryotic community constituents might thus provide information on local subsurface growth conditions as well as advective transport processes in case of rapid dislocation. In this study, we investigated the spatiotemporal distribution of prokaryotic communities in groundwater and spring water to examine the relationship between the groundwater flow system and prokaryotic dynamics. We sampled and monitored the water in a mountainous forested catchment in Kawamata Town, Fukushima Prefecture, Japan. Seven piezometers and a spring located across a ridge, hillslope, and valley were monitored. Prokaryotic information, such as density estimated by total direct counting, community constituents and diversity using the next-generation sequencing methods, physicochemical parameters, general water quality, and groundwater mean residence time using sulfur hexafluoride (SF6) were analyzed. Our results showed that the mean residence time of groundwater and spring water ranged from less than 1 year to 30 years. The prokaryotic density ranged from 5.37 × 103cells/mL to 2.01 × 106cells/mL (n = 60). As the mean residence time increased, the density of prokaryotes in the shallow subsurface also increased, whereas that of the prokaryotes in the deep subsurface decreased. The contribution of deep groundwater to the examined groundwater is inferred using the groundwater residence time and prokaryotic information, including density and specific constituents. The stable water isotopes did not suggest the deep groundwater contribution to the groundwater at the ridge in the high-rainfall period; however, the SiO2 concentration indicated that. Elucidating the prokaryotic information along with existing hydrological tracers may enable a better understanding of groundwater flow processes.

The Effect of Yeast and Roughage Concentrate Ratio on Ruminal pH and Protozoal Population in Thai Native Beef Cattle.

Simple SummaryAs a result of the recent ban on antibiotics in feed, animal probiotics are becoming increasingly popular. Yeast is extensively used as both a probiotic and prebiotic in the gastrointestinal tracts of ruminants. The purpose of this study is to determine how adding yeast (Saccharomyces cerevisiae) to the diet and changing the roughage-to-concentrate ratio (R:C ratio) affects nutrient consumption, rumen fermentation, microbial protein synthesis, and protozoal population in Thai native beef cattle. The roughage source was urea–calcium-hydroxide-treated rice straw. The findings suggest that supplementing with a R:C ratio of 40:60 and a LY of 4 g/hd/d boosted nutrient digestibility, volatile fatty acid (VFA) production, propionic acid (C3) in particular, and microbial protein synthesis while lowering protozoal population.The objective of this research is to investigate the effect of yeast (Saccharomyces cerevisiae) adding and roughage-to-concentrate ratio (R:C ratio) on nutrients utilization, rumen fermentation efficiency, microbial protein synthesis, and protozoal population in Thai native beef cattle. Four Thai native beef cattle, weighing an average of 120 ± 10 kg live weight, were randomly assigned to four dietary treatments using a 2 × 2 factorial arrangement in a 4 × 4 Latin square design. Factor A was the level of roughage-to-concentrate ratio (R:C ratio) at 60:40 and 40:60; factor B was the levels of live yeast (LY) supplementation at 0 and 4 g/hd/d; urea–calcium-hydroxide-treated rice straw were used as a roughage source. Findings revealed that total intake and digestibility of dry matter (DM), organic matter (OM), and crude protein (CP) were increased (p < 0.05) by both factors, being greater for steers fed a R:C ratio of 40:60 supplemented with 4 g LY/hd/d. Ruminal ammonia nitrogen, total volatile fatty acid (VFA), and propionate (C3) were increased (p < 0.05) at the R:C ratio of 40:60 with LY supplementation at 4 g/hd/d, whereas rumen acetate (C2) and the C2 to C3 ratio were decreased (p < 0.05). With a high level of concentrate, LY addition increased total bacterial direct counts and fungal zoospores (p < 0.05), but decreased protozoal populations (p < 0.05). High-concentrate diet and LY supplementation increased nitrogen absorption and the efficiency of microbial nitrogen protein production. In conclusion, feeding beef cattle with 4 g/hd/d LY at a R:C ratio of 40:60 increased C3 and nutritional digestibility while lowering protozoal population.

Cronobacter sakazakii local isolates response to acid stress and their resuscitability

Cronobacter spp. has been reported to cause meningitis, necrotizing enterocolitis, and septicemia in a group of infants through the consumption of powder infant formula. These bacteria are reported to withstand various stress conditions such as heating, drying, low water activity, low pH, etc. A local isolate of Cronobacter sakazakii YRt2a was reportedly survived and entered Viable But Non-Culturable (VBNC) conditions during desiccation stress. This study aims to study the behavior of local isolates of Cronobacter spp. in response to acid stress and its resuscitability. C. sakazakii E2 and YRt2a were grown in TSB at pH 3.0±0.2 or 3.5±0.2. The number of culturable cells and viable cells were enumerated by the Total Plate Count and Direct Viable Count methods, respectively. Resuscitation was done by growing the stress or VBNC cells in TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer. The results showed that C. sakazakii E2 and YRt2a entered VBNC state after 60 mins of exposure to pH 3.0±0.2, while remained culturable after 120 minutes exposure to pH 3.5±0.2. TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer could resuscitate the stress or VBNC cells of C. sakazakii. Stress or VBNC state experienced by C. sakazakii in response to acid tends to be transient and can be resuscitated. C. sakazakii experiencing stress or VBNC may pose a risk for food safety.

Mechanism of increased microbial autofluorescence by heat treatment.

Autofluorescence of bacteria and fungi was prominently intensified by heat treatment at 200°C. This phenomenon was associated with advanced glycation end products formed in micro-organisms via the Maillard reaction. The fluorescence signal was strong enough to be utilized as an alternative probe for fluorescent dye in the total direct count method. This phenomenon could be incorporated in an automatic apparatus for microbial enumeration, as it does not require staining.

Evaluation of method bias for determining bacterial populations in bacterial community analyses

Various methods are used for analyzing a bacterial community. We recently developed a method for quantifying each bacterium constituting the microbiota by combining a next-generation sequencing (NGS) analysis with a quantitative polymerase chain reaction (NGS-qPCR) assay. Our NGS-qPCR method is useful for analyzing a comprehensive bacterial community because it is enables the easy calculation of the amounts of each bacterium constituting the microbiota. However, it has not been confirmed whether the estimated bacterial community obtained using this NGS-qPCR method corresponds to the results obtained using conventional methods. Accordingly, we prepared model bacterial community samples and analyzed them by several methods (NGS-qPCR, species-specific qPCR, flow cytometry, total direct counting by epifluorescent microscopy [TDC], and plate count). The total bacterial cell densities determined by the PCR-based methods were largely consistent with those determined by the TDC method. There was a difference between the amounts of each bacterium analyzed by NGS-qPCR and species-specific qPCR, although the same trend was shown by both species-specific qPCR and NGS-qPCR. Our findings also demonstrated that there is a strong positive correlation between the cell densities of a specific bacterial group in craft beer samples determined by group-specific qPCR and NGS-qPCR, and there were no significant differences among quantification methods (we tested two bacterial groups: lactic acid bacteria and acetic acid bacteria). Thus, the NGS-qPCR method is a practical method for analyzing a comprehensive bacterial community based on a bacterial cell density.

Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water

Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 104 in drinking water, was detected with the ATP assay, as well as 102 to 103 times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay.

Characterization of early microbial communities on volcanic deposits along a vegetation gradient on the island of Miyake, Japan.

The 2000 eruption of Mount Oyama on the island of Miyake (Miyake-jima) created a unique opportunity to study the early ecosystem development on newly exposed terrestrial substrates. In this study, bacterial and fungal communities on 9- and 11-year-old volcanic deposits at poorly to fully vegetation-recovered sites in Miyake-jima, Japan, were characterized by conventional culture-based methods and pyrosequencing of 16S rRNA and 18S rRNA genes. Despite the differences in the vegetation cover, the upper volcanic deposit layer samples displayed low among-site variation for chemical properties (pH, total organic carbon, and total nitrogen) and microbial population densities (total direct count and culturable count). Statistical analyses of pyrosequencing data revealed that the microbial communities of volcanic deposit samples were phylogenetically diverse, in spite of very low-carbon environmental conditions, and their diversity was comparable to that in the lower soil layer (buried soil) samples. Comparing with the microbial communities in buried soil, the volcanic deposit communities were characterized by the presence of Betaproteobacteria and Gammaproteobacteria as the main bacterial class, Deinococcus- Thermus as the minor bacterial phyla, and Ascomycota as the major fungal phyla. Multivariate analysis revealed that several bacterial families and fungal classes correlated positively or negatively with plant species.

Effect of dead cells on biofouling in the reverse osmosis process

Biofilm formation in membrane processes causes a flux decline, pressure drop increase, and other adverse effects. Understanding the mechanisms of fouling, control, and cleaning are important in order to resolve fouling problems. In this paper, the effect of cell viability on biofouling was studied via a feed water analysis and membrane autopsy. Pseudomonas aeruginosa PAO1 was used as the model bacteria. Biofouling tests were divided into two parts: live cells and dead cells (autoclaved cells). The feed water was periodically collected every 3 h to analyze the total direct counts (TDCs), heterotrophic plate counts (HPCs), and extracellular polymeric substances (EPS). A membrane autopsy was performed to characterize the fouled membrane through TDCs and EPS. When dead cells were inoculated into the feed water, low concentrations of TDCs and EPS were measured in both the feed water and fouled membrane. As a result, it was determined that initial flux decline by biofouling can be reduced if feedwater is disinfected before the reverse osmosis process.

Study on Fluorescent Staining of Bacteria for Total Direct Counts in Polycyclic Aromatic Hydrocarbons (PAHs) Contaminated Soil with 5-(4,6-DichloroTriazin-2-yl) Amino Fluorescein (DTAF)

Using DTAF fluorescent staining technique, the number of bacteria and the distributions of ΣPAHs in two different drilling vertical depths of a field contaminated by PAHs are counted and analyzed. The result showed that the distributions of the number of bacteria and the ΣPAHs in the vertical depth have obvious corresponding relations, and the higher the ΣPAHs is, the more the number of bacteria would be.

Structure and function of denitrifying bacterial assemblages in low-order Indiana streams

Anthropogenic activities increase rates of N input to the environment, and loss of this N is controlled by several factors, including denitrification. Streams are the initial receptors of terrestrial N, but the extent to which variability in stream denitrification rates are related to differences in microbial community structure are largely unexplored. In our study, the rate of denitrification and taxonomic and functional gene diversity and abundance were examined in 3 Indiana (USA) streams with differing amounts of watershed agriculture. Taxonomic and functional gene diversity were measured using terminal restriction length polymorphisms of the 16S ribosomal ribonucleic acid (rRNA) and nitrous oxide reductase (nosZ) genes, and abundance was examined using quantitative polymerase chain reaction (Q- PCR) and total direct cell counts. As expected, streams with highest amounts of watershed agriculture had highest NO3 2 concentrations and highest sediment organic matter (OM) content leading to higher denitrification rates. Overall, denitrification rates were controlled primarily by sediment OM content and secondarily by nosZ abundance and nosZ terminal restriction fragment (T-RF) number. However, total bacterial numbers were not related to peaks in denitrification rate. The 2 sites with the most substantial differences in watershed agriculture, NO3 2 concentrations, and sediment OM content also had the largest differences in both nosZ abundance and nosZ gene profiles. Overall, our results suggest that denitrification rates in agricultural streams are influenced by a combination of environmental variables (primarily benthic OM and NO3 2 concentrations) and microbial community composition.

Effects of energy level and Leucaena leucocephala leaf meal as a protein source on rumen fermentation efficiency and digestibility in swamp buffalo

Four Thai – rumen fistulated male swamp buffaloes (Bubalus bubalis), about 3 years old with 360±18kg liveweight, were randomly assigned according to a 2×2 factorial arrangement in a 4×4 Latin square design to receive four dietary treatments. The treatments were as follows: a cassava based supplement (CS) at 1g/kg BW and Leucaena leucocephala leaf meal (LLM) at 300g/d (T1); CS at 2g/kg BW with LLM at 300g/d (T2); CS at 1g/kg BW and heat treated LLM (HLLM) at 300g/d (T3); and CS at 2g/kg BW and HLLM at 300g/d. During the experiment, urea–calcium hydroxide treated rice straw was given on ad libitum basis. The results revealed an increase in roughage and total dry matter (DM) intake (P<0.05) by CS at 2g/kg BW (T2 and T4) as compared with CS at 1g/kg BW (T1 and T3). Digestion coefficients of DM, organic matter (OM), and crude protein (CP) were increased by CS at 2g/kg BW, while neutral detergent fiber (aNDF) and acid detergent fiber (ADF) were similar among treatments. However, there was no effect of neither energy level nor HLLM on ruminal pH and temperature (P>0.05). Concentration of ruminal ammonia nitrogen (NH3-N) was decreased by HLLM as compared with LLM (P<0.05), while blood urea–nitrogen was not altered. There was an increase (P<0.05) in total volatile fatty acid (TVFA), acetic acid (C2), propionic acid (C3), and butyric acid (C4) concentrations and the highest were found in CS at 2g/kg BW with HLLM (T4), while the lowest was in T1 and T3. However, no changes in C2–C3 ratio were found in this study. Total bacterial direct counts were found different (P<0.05), whereas fungal zoospores and protozoal populations were similar among treatments. Nevertheless, viable bacterial counts were found affected by both concentrate level and HLLM. The treatments with HLLM were lower than those in LLM and CS at 2g/kg BW were higher than those supplemented at CS at 1g/kg BW (P<0.05). In addition, efficiency of rumen microbial CP synthesis tended to be higher in treatment with higher level of energy and HLLM. Based on this study, it could be concluded that LLM could be used as a protein source, while the combination of HLLM and CS at 2g/kg BW could enhance the voluntary feed intake, nutrient digestibility and rumen fermentation in swamp buffalo fed on treated rice straw (urea–calcium hydroxide treatment).

Applicability of solid-phase cytometry and epifluorescence microscopy for rapid assessment of the microbiological quality of dialysis water

Currently, the gold standard to assess the microbiological quality of dialysis water is the determination of heterotrophic plate counts (HPC). The long waiting time of the HPC method and the fact that most bacteria are not culturable on agar plates provokes the search for rapid alternative methods for monitoring the microbiological quality of dialysis water. We tested the applicability of total viable counts (TVC) and total direct counts (TDC), determined via solid-phase cytometry and epifluorescence microscopy (EFM), in comparison to the standard HPC determination method in 113 samples from 13 dialysis water treatment units (59 drinking water and 54 dialysis water samples). Additionally, for a set of dialysis water samples (n = 22) endotoxin concentrations were also determined. TVC showed high correlation with HPC and results were of comparable magnitude for most investigated dialysis water samples [median: 3 cells/colony forming units (CFU) 100 mL(-1)]. However, in one dialysis water sample, HPC values (5800 CFU 100 mL(-1)) were >35-fold lower than TVC values (2.05 × 10(5) cells 100 mL(-1)) indicating severe limits of the HPC method to assess the microbiological quality of dialysis water. For drinking water, TVC (median: 4.8 × 10(4) cells 100 mL(-1)) was on average one order of magnitude higher than HPC (median: 2.5 × 10(3) cells 100 mL(-1)). TDCs (median dialysis water: 1.1 × 10(4) cells 100 mL(-1) and median drinking water: 4.9 × 10(6) cells 100 mL(-1)) were always several orders of magnitude higher than HPC or TVC. We propose that the TVC/solid-phase cytometry approach is a reliable and rapid alternative to the culture-dependent approach for assessment of the microbiological quality of dialysis water, especially when fast results are needed. TDC determined via EFM lacks sensitivity and reliability for assessing microbial concentrations in low-cell dialysis water samples since the limits of detection and quantification are high.

Total and Viable Legionella pneumophila Cells in Hot and Natural Waters as Measured by Immunofluorescence-Based Assays and Solid-Phase Cytometry

A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter(-1), and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 10(3) viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples.

Occurrence and diversity of epiphytic bacterial communities on two native plant species in a Michigan Creek

We examined the occurrence and diversity of bacteria of different phylogenetic groups in epiphytic assemblages on two native Michigan plant species mayapple (Podophyllum peltatum) and cow parsnip (Heracleum maximum) when the plant leaves were submerged in fresh water. Fresh leaves were incubated in triplicate for about a week within a creek in Hasting, Michigan, and in laboratory microcosm to develop mature epiphytic assemblages. We enumerated bacteria in these assemblages by nucleic acid staining (i.e., total direct counts using 4′,6-diamidino-2-phenyllindole) and fluorescent in situ hybridization (FISH), while community diversity was determined based on 16S rRNA gene sequencing. FISH indicated the dominance of members of the γ-Proteobacterial subclass (20%) on both plant species, while the 16S rRNA gene analysis revealed the predominance of the β-Proteobacteria (51%) on mayapple and the Fermicutes (26%) on cow parsnip, with the Bacteroidetes present equally within the epiphytic assemblages on both plants.

Soil Solarization in a Greenhouse for Controlling Fecal Contamination

Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. As a way of biosafety control, soil solarization in a closed greenhouse was examined for the efficiency on the inactivation of Escherichia coli which was inoculated into soil as a model microorganism for fecal pathogenic bacteria. Soil solarization greatly increased the soil temperature (depth of 15 cm), which reached up to 40°C within the first one week of solarization, thereafter fluctuated between 40 and 46°C in most of the solarization periods. The population of E. coli in the solarized soil decreased dramatically from 105 CFU g−1 dry soil to undetectable level (‹0.1 CFU g−1 dry soil) within one week as a result, whereas E. coli was detected even after four weeks in the non-solarized soil in an open greenhouse. Soil solarization, however, had little effect on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the rapid biosafety control of soil contaminated by human pathogens via immature compost or animal feces under greenhouse environments.

Threshold concentrations of biomass and iron for pressure drop increase in spiral-wound membrane elements

In a model feed channel for spiral-wound membranes the quantitative relationship of biomass and iron accumulation with pressure drop development was assessed. Biofouling was stimulated by the use of tap water enriched with acetate at a range of concentrations (1–1000 μg C l −1). Autopsies were performed to quantify biomass concentrations in the fouled feed channel at a range of Normalized Pressure Drop increase values (NPD i). Active biomass was determined with adenosinetriphosphate (ATP) and the concentration of bacterial cells with Total Direct Cell count (TDC). Carbohydrates (CH) were measured to include accumulated extracellular polymeric substances (EPS). The paired ATP and CH concentrations in the biofilm samples were significantly ( p < 0.001; R 2 = 0.62) correlated and both parameters were also significantly correlated with NPD i ( p < 0.001). TDC was not correlated with the pressure drop in this study. The threshold concentration for an NPD i of 100% was 3.7 ng ATP cm −2 and for CH 8.1 μg CH cm −2. Both parameters are recommended for diagnostic membrane autopsy studies. Iron concentrations of 100–400 mg m −2 accumulated in the biofilm by adsorption were not correlated with the observed NPD i, thus indicating a minor role of Fe particulates at these concentrations in fouling of spiral-wound membrane.

High-frequency phage-mediated gene transfer in freshwater environments determined at single-cell level

Lateral gene transfer by phages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency and range of phage-mediated gene transfer, it is important to understand the movement of DNA among microbes. Using an in situ DNA amplification technique (cycling primed in situ amplification-fluorescent in situ hybridization; CPRINS-FISH), we examined the propensity for phage-mediated gene transfer in freshwater environments at the single-cell level. Phage P1, T4 and isolated Escherichia coli phage EC10 were used as vectors. All E. coli phages mediated gene transfer from E. coli to both plaque-forming and non-plaque-forming Enterobacteriaceae strains at frequencies of 0.3-8 x 10(-3) per plaque-forming unit (PFU), whereas culture methods using selective agar media could not detect transductants in non-plaque-forming strains. The DNA transfer frequencies through phage EC10 ranged from undetectable to 9 x 10(-2) per PFU (undetectable to 2 x 10(-3) per total direct count) when natural bacterial communities were recipients. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viability in most cases. These results indicate that the exchange of DNA sequences among bacteria occurs frequently and in a wide range of bacteria, and may promote rapid evolution of the prokaryotic genome in freshwater environments.

Population Dynamics of Crenarchaeota and Euryarchaeota in the Mixing Front of River and Marine Waters

A transect from the Tomoe River Mouth through Shimizu Port to Suruga Bay, Japan, was examined between 2005 and 2009 to reveal the population dynamics of Crenarchaeota and Euryarchaeota in an estuary environment. Crenarchaeota tended to increase in abundance in waters deeper than 100 m compared with Euryarchaeota, and comprised 11% of total direct counts. Archaeal abundance was highest in the Tomoe River Mouth, with a strong negative correlation between surface euryarchaeal abundance and salinity (P<0.001). The diversity index for the phylotypic archaeal community in the mouth was three times higher than that at sites St1-1m and St1-10m in the estuary, and OTUs represented most of the OTU groups at the sites. Three of the seven total OTUs, which comprised 83.6% of the 140 sequenced clones in the estuary, were related to the OTUs in the mouth with similarities higher than 97%. A significant proportion of the archaeal community appears to be derived from the Tomoe River. The two dominant phylotypes of the archaeal community in Shimizu Port, belonging to MGI and MGII, occurred ubiquitously.

Unique distribution of deep groundwater bacteria constrained by geological setting

We collected groundwater samples at depths of up to 482 m from three boreholes drilled into sedimentary rock within two formations in Hokkaido, Japan. The prokaryotic community in each subsurface groundwater sample was analysed by microscopic counts and cloning-sequencing the 16S rRNA genes. On total direct counts, there were between 4.61 × 10(4) and 5.06 × 10(6) prokaryote cells ml(-1) in the samples, which is similar to the numbers observed at the marine subsurface. However, the vertical distribution of the prokaryotes did not show a simple decrease in abundance with increasing depth. A high abundance of cells with significant amounts of RNA was identified in the domain Bacteria using fluorescence in situ hybridization, with a high frequency of dividing cells at the transition zone between the two sedimentary rock formations. Cloning-sequencing analysis showed the predominance of γ-Proteobacteria at this transition zone at 281-312 m. The horizontal heterogeneity of the microbial distribution in the subsurface environment was also demonstrated by a relatively high density of members of the domain Archaea in borehole HDB-4, drilled only 1.5 km northeast of HDB-6 and in the same formation.