Bifidobacterium longum and Galactooligosaccharide Improve Skin Barrier Dysfunction and Atopic Dermatitis-like Skin.

PURPOSE: The beneficial effects of a combination therapy using Bifidobacterium longum and galactooligosaccharide (GOS) for the treatment of atopic dermatitis (AD) have not been elucidated. METHODS: Gene expressions of interleukin (IL)-4 and IL-13 from peripheral blood mononuclear cells and fecal abundance of B. longum from 12-month-old infants were evaluated. Human primary epidermal keratinocytes (HEKs) and hairless mice were treated with B. longum, GOS, B. longum-derived extracellular vesicles (BLEVs), dinitrochlorobenzene (DNCB), or a synbiotic mixture of B. longum and GOS. Expression of epidermal barrier proteins and cytokines as well as serum immunoglobulin E (IgE) levels were analyzed in HEKs and mice. Dermatitis scores, transepidermal water loss (TEWL), epidermal thickness, and fecal B. longum abundance were evaluated in mice. RESULTS: Fecal abundance of B. longum was negatively correlated with blood IL-13 expression in infants. B. longum or BLEVs increased expression of filaggrin (FLG) and loricrin (LOR) in HEKs. B. longum increased the efficacy of GOS to upregulate FLG and LOR expressions in HEKs. Oral administration of GOS increased fecal abundance of B. longum in mice. Oral administration of B. longum attenuated DNCB-induced skin inflammation, abnormal TEWL, AD-like skin, and deficiency of epidermal barrier proteins. Moreover, the combination of B. longum and GOS showed greater effects to improve DNCB-induced skin inflammation, abnormal TEWL, AD-like skin, serum IgE levels, IL-4 over-expression, and the deficiency of epidermal barrier proteins than the administration of B. longum alone. CONCLUSIONS: B. longum and GOS improve DNCB-induced skin barrier dysfunction and AD-like skin.

Genomic and ecological study of two distinctive freshwater bacteriophages infecting a Comamonadaceae bacterium.

Bacteriophages of freshwater environments have not been well studied despite their numerical dominance and ecological importance. Currently, very few phages have been isolated for many abundant freshwater bacterial groups, especially for the family Comamonadaceae that is found ubiquitously in freshwater habitats. In this study, we report two novel phages, P26059A and P26059B, that were isolated from Lake Soyang in South Korea, and lytically infected bacterial strain IMCC26059, a member of the family Comamonadaceae. Morphological observations revealed that phages P26059A and P26059B belonged to the family Siphoviridae and Podoviridae, respectively. Of 12 bacterial strains tested, the two phages infected strain IMCC26059 only, showing a very narrow host range. The genomes of the two phages were different in length and highly distinct from each other with little sequence similarity. A comparison of the phage genome sequences and freshwater viral metagenomes showed that the phage populations represented by P26059A and P26059B exist in the environment with different distribution patterns. Presence of the phages in Lake Soyang and Lake Michigan also indicated a consistent lytic infection of the Comamonadaceae bacterium, which might control the population size of this bacterial group. Taken together, although the two phages shared a host strain, they showed completely distinctive characteristics from each other in morphological, genomic, and ecological analyses. Considering the abundance of the family Comamonadaceae in freshwater habitats and the rarity of phage isolates infecting this family, the two phages and their genomes in this study would be valuable resources for freshwater virus research.

Genome characteristics and environmental distribution of the first phage that infects the LD28 clade, a freshwater methylotrophic bacterial group.

Bacteriophages infecting major groups of freshwater heterotrophic bacteria have been rarely isolated, hampering analyses of freshwater viromes. Here, we report the isolation and genomic characterization of P19250A, the first phage that infects the LD28 clade, an abundant freshwater methylotrophic bacterial group. P19250A was isolated from Lake Soyang, an oligotrophic reservoir, using an LD28 strain as a host. Morphological and genomic analyses revealed that P19250A is a lytic siphovirus with a ∼38.6-kb genome. To analyze the distribution of P19250A genome within its habitat, six seasonal viral metagenome (virome) samples were prepared from Lake Soyang. Through binning analysis of freshwater viromes, P19250A was shown to be the most highly assigned freshwater phage that infects heterotrophic bacteria (up to 8.21%) in five viromes. Furthermore, when freshwater virome data collected worldwide were analyzed, P19250A genome also showed high abundance, especially in Lough Neagh, UK, where P19250A genome was recorded as the most abundant bacteriophage. From metagenome analysis, the proportion of P19250A-assigned reads showed seasonal fluctuation following the abundance of the LD28 clade in Lake Soyang. These results showed that P19250A would be an essential resource for analyses of freshwater viromes, and also suggest that phages of other abundant freshwater bacteria need to be isolated for better understanding of freshwater viruses.

Niche specialization of bacteria in permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica.

Perennially ice-covered lakes in the McMurdo Dry Valleys, Antarctica, are chemically stratified with depth and have distinct biological gradients. Despite long-term research on these unique environments, data on the structure of the microbial communities in the water columns of these lakes are scarce. Here, we examined bacterial diversity in five ice-covered Antarctic lakes by 16S rRNA gene-based pyrosequencing. Distinct communities were present in each lake, reflecting the unique biogeochemical characteristics of these environments. Further, certain bacterial lineages were confined exclusively to specific depths within each lake. For example, candidate division WM88 occurred solely at a depth of 15 m in Lake Fryxell, whereas unknown lineages of Chlorobi were found only at a depth of 18 m in Lake Miers, and two distinct classes of Firmicutes inhabited East and West Lobe Bonney at depths of 30 m. Redundancy analysis revealed that community variation of bacterioplankton could be explained by the distinct conditions of each lake and depth; in particular, assemblages from layers beneath the chemocline had biogeochemical associations that differed from those in the upper layers. These patterns of community composition may represent bacterial adaptations to the extreme and unique biogeochemical gradients of ice-covered lakes in the McMurdo Dry Valleys.

Chicken Egg Yolk Antibodies (IgY) for Prophylaxis and Treatment of Rotavirus Diarrhea in Human and Animal Neonates: A Concise Review.

The rotavirus-induced diarrhea of human and animal neonates is a major public health concern worldwide. Until recently, no effective therapy is available to specifically inactivate the rotavirion particles within the gut. Passive immunotherapy by oral administration of chicken egg yolk antibody (IgY) has emerged of late as a fresh alternative strategy to control infectious diseases of the alimentary tract and has been applied in the treatment of diarrhea due to rotavirus infection. The purpose of this concise review is to evaluate evidence on the properties and performance of anti-rotavirus immunoglobulin Y (IgY) for prevention and treatment of rotavirus diarrhea in human and animal neonates. A survey of relevant anti-rotavirus IgY basic studies and clinical trials among neonatal animals (since 1994-2015) and humans (since 1982-2015) have been reviewed and briefly summarized. Our analysis of a number of rotavirus investigations involving animal and human clinical trials revealed that anti-rotavirus IgY significantly reduced the severity of clinical manifestation of diarrhea among IgY-treated subjects relative to a corresponding control or placebo group. The accumulated information as a whole depicts oral IgY to be a safe and efficacious option for treatment of rotavirus diarrhea in neonates. There is however a clear need for more randomized, placebo controlled and double-blind trials with bigger sample size to further solidify and confirm claims of efficacy and safety in controlling diarrhea caused by rotavirus infection especially among human infants with health issues such as low birth weights or compromised immunity in whom it is most needed.

Complete genome sequence of bacteriophage P26218 infecting Rhodoferax sp. strain IMCC26218.

Bacteriophage P26218 is a virus that thrives in freshwater and infects Rhodoferax sp. strain IMCC26218, both of which were isolated from Soyang Lake, Korea. The bacterial host, IMCC26218, belongs to the genus Rhodoferax and is closely related to R. saidenbachensis, with 98.7 % 16S rRNA gene sequence similarity. Bacteriophage P26218 has an icosahedral head structure with a diameter of ~52 nm and short tail of ~9 nm, which is a typical morphology of the Podoviridae family. Its complete dsDNA genome was 36,315 bp with 56.7 % G + C content. This is the first genome sequence reported for a lytic phage of the genus Rhodoferax.

High abundance and role of antifungal bacteria in compost-treated soils in a wildfire area.

Compost has been widely used in order to promote vegetation growth in post-harvested and burned soils. The effects on soil microorganisms were scarcely known, so we performed the microbial analyses in a wildfire area of the Taebaek Mountains, Korea, during field surveys from May to September 2007. Using culture-dependent and -independent methods, we found that compost used in burned soils influenced a greater impact on soil fungi than bacteria. Compost-treated soils contained higher levels of antifungal strains in the genera Bacillus and Burkholderia than non-treated soils. When the antifungal activity of Burkholderia sp. strain O1a_RA002, which had been isolated from a compost-treated soil, was tested for the growth inhibition of bacteria and fungi isolated from burned soils, the membrane-filtered culture supernatant inhibited 19/37 fungal strains including soil fungi, Eupenicillium spp. and Devriesia americana; plant pathogens, Polyschema larviformis and Massaria platani; an animal pathogen, Mortierella verticillata; and an unidentified Ascomycota. However, this organism only inhibited 11/151 bacterial strains tested. These patterns were compatible with the culture-independent DGGE results, suggesting that the compost used in burned soils had a greater impact on soil fungi than bacteria through the promotion of the growth of antifungal bacteria. Our findings indicate that compost used in burned soils is effective in restoring soil conditions to a state closer to those of nearby unburned forest soils at the early stage of secondary succession.

Evaluation of the sensitivity and specificity of primer pairs and the efficiency of RNA extraction procedures to improve noroviral detection from oysters by nested reverse transcription-polymerase chain reaction.

Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoV-polluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoV-seeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp® Viral RNA Mini kit with a QIAshredder™ Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.

Single-strand conformation polymorphism analysis for the study of adenoviral diversity in urban rivers.

The diversity of human adenoviruses (AdVs) in river waters was studied by single-strand conformational polymorphism (SSCP) analysis. Water samples were collected between 2002 and 2003 from 4 rivers in the Gyeonggi Province, South Korea. Forty-six (79.3%) of the 58 samples were positive for AdVs as determined on PCR amplification. Nine different SSCP profiles (profiles A to I) were detected in all the AdVs-positive samples by SSCP analysis, and most of the AdVs-positive samples (38 of 46 samples; 82.6%) showed the SSCP profile D. Nine different sequences were obtained in the SSCP profiles; sequence alignments and phylogenetic analysis identified 5 different sequences that were closely related to the human AdV type 41, and the 4 different sequences that were closely related to human AdV types 3, 4, 12, and 40. Two AdVs genomic variants were detected (types 3 and 41 in A, types 12 and 41 in B, and 2 genomic variants of type 41 in C) in SSCP profiles A, B, and C, respectively. SSCP analysis could be a useful technique for the identification of genetic variants of AdVs and for studying AdVs diversity in urban rivers.

Enteric viruses in raw vegetables and groundwater used for irrigation in South Korea.

Raw vegetables irrigated with groundwater that may contain enteric viruses can be associated with food-borne viral disease outbreaks. In this study, we performed reverse transcription-PCR (RT-PCR) and cell culture-PCR to monitor the occurrence of enteric viruses in groundwater samples and in raw vegetables that were cultivated using that groundwater in South Korea. Samples were collected 10 times from three farms located in Gyeonggi Province, South Korea. RT-PCR and cell culture-PCR were performed to detect adenoviruses (AdVs), enteroviruses (EVs), noroviruses (NoVs), and rotaviruses, followed by sequence analyses of the detected strains. Of the 29 groundwater samples and the 30 vegetable samples, five (17%) and three (10%) were positive for enteric viruses, respectively. AdVs were the most frequently detected viruses in four groundwater and three vegetable samples. EVs and NoVs were detected in only one groundwater sample and one spinach sample, respectively. The occurrence of enteric viruses in groundwater and vegetable samples was not correlated with the water temperature and the levels of indicator bacteria, respectively. Phylogenetic analysis indicated that most of the detected AdVs were temporally distributed, irrespective of sample type. Our results indicate that raw vegetables may be contaminated with a broad range of enteric viruses, which may originate from virus-infected farmers and virus-contaminated irrigation water, and these vegetables may act as a potential vector of food-borne viral transmission.

Concentration method for the detection of enteric viruses from large volumes of foods.

Enteric viruses are the major cause of outbreaks of foodborne viral disease worldwide, and vegetables and fruits are considered significant vectors of virus transmission. In this study, we compared viral elution concentration methods in strawberry and lettuce and tested the secondary concentration step for concentrating viruses from large volumes of lettuce samples. Among the tested procedures, the combination of a 0.05 M glycine plus 100 mM Tris elution buffer (pH 9.5) and a polyethylene glycol precipitation concentration was most efficient for the detection of norovirus genogroup II from strawberries (50% of samples) and lettuce (2.9% of samples). The secondary concentration step using ultrafiltration devices could be applied to large lettuce samples without any decrease in detection limit and efficiency, and other cultivable enteric viruses including enteroviruses, adenoviruses, and rotaviruses were recovered from lettuce at efficiencies of 11.4, 9.05, and 11.3%, respectively. This method could be useful for detecting enteric viruses in fresh foods.

The genetic diversity of human noroviruses detected in river water in Korea.

We studied the genetic diversity of human noroviruses in river waters by RT-nested PCR and phylogenetic analysis. During 2002-2003, water samples were collected from four rivers in Gyeonggi Province, South Korea. Among the 58 samples, 32 (55.2%) and 26 (44.8%) showed positive results with noroviruses belonging to genogroups I (GI) and II (GII), respectively. The phylogenetic analysis grouped 8 and 7 genotypes in GI and GII, respectively. The major types were GI/1, GI/13, and GII/15, and GI/1 and GI/3 were temporarily distributed. Most GI- and GII-grouped strains were closely related to the reference strains from neighboring countries, China and Japan, and GII/4-related strains had similar sequences to strains recognized as worldwide epidemic outbreaks. The strains circulating between countries are of particular concern to the outbreaks of noroviral diseases in Korea and must be periodically monitored in the natural environments.

Degradation of various alkyl ethers by alkyl ether-degrading Actinobacteria isolated from activated sludge of a mixed wastewater treatment.

Various substrate specificity groups of alkyl ether (AE)-degrading Actinobacteria coexisted in activated sewage sludge of a mixed wastewater treatment. There were substrate niche overlaps including diethyl ether between linear AE- and cyclic AE-degrading strains and phenetole between monoalkoxybenzene- and linear AE-degrading strains. Representatives of each group showed different substrate specificities and degradation pathways for the preferred substrates. Determining the rates of initial reactions and the initial metabolite(s) from whole cell biotransformation helped us to get information about the degradation pathways. Rhodococcus sp. strain DEE5311 and Rhodococcus rhodochrous strain 117 both were able to degrade anisole and phenetole through aromatic 2-monooxygenation to form 2-alkoxyphenols. In contrast, diethyl ether-oxidizing strain DEE5311 capable of degrading a broad range of linear AE, dibenzyl ether and monoalkoxybenzenes initially transformed anisole and phenetole to phenol via direct O-dealkylation. Compared to this, cyclic AE-degrading Rhodococcus sp. strain THF100 preferred tetrahydrofuran (265 ± 35 nmol min(-1)mg(-1) protein) to diethyl ether (<30), but it cannot oxidize bulkier AE than diethyl ether. Otherwise, 1,4-diethoxybenzene-degrading Rhodococcus sp. strain DEOB100 and Gordonia sp. strain DEOB200 transformed 1,3-/1,4-dialkoxybenzenes to 3-/4-alkoxyphenols by similar manners in the order of rates (nmol min(-1) mg(-1) protein): 1,4-diethoxybenzene (11.1 vs. 3.9)>1,4-dimethoxybenzene (1.6 vs. 2.6)>1,3-dimethoxybenzene (0.6 vs. 0.6). This study suggests that the AE-degrading Actinobacteria can orchestrate various substrate specificity responses to the degradation of various categories of AE pollutants in activated sludge communities.

Molecular detection of human enteric viruses in urban rivers in Korea.

We performed RT-nested PCR to study the distribution of human enteric viruses in urban rivers in Korea. During 2002-2003, water samples were collected from four rivers in Gyeonggi Province, South Korea. Among 58 samples, 45 (77.6%), 32 (55.2%), 12 (20.7%), 2 (3.4%), 4 (6.9%), and 4 (6.9%) showed positive results with adenoviruses (AdVs), enteroviruses (EVs), reoviruses (ReVs), hepatitis A viruses (HAVs), rotaviruses (RoVs), and sapoviruses (SVs), respectively. According to the binary logistic regression model, the occurrence of each enteric virus, except ReVs and HAVs, was not statistically correlated with the water temperature and levels of fecal coliforms (P<0.05). AdVs were most often detected; only 4 samples (6.9%) were negative for AdVs while positive for other enteric viruses in the studied sites. Our results indicated that monitoring human enteric viruses is necessary to improve microbial quality, and that AdVs detection by PCR can be a useful index for the presence of other enteric viruses in aquatic environments.

Influence of pipe materials and VBNC cells on culturable bacteria in a chlorinated drinking water model system.

To elucidate the influence of pipe materials on the VBNC (viable but nonculturable) state and bacterial numbers in drinking water, biofilm and effluent from stainless steel, galvanized iron, and polyvinyl chloride pipe wafers were analyzed. Although no HPC (heterotrophic plate count) was detected in the chlorinated influent of the model system, a DVC (direct viable count) still existed in the range between 3- and 4-log cells/ml. Significantly high numbers of HPC and DVC were found both in biofilm and in the effluent of the model system. The pipe material, exposure time, and the season were all relevant to the concentrations of VBNC and HPC bacteria detected. These findings indicate the importance of determining the number of VBNC cells and the type of pipe materials to estimate the HPC concentration in water distribution systems and thus the need of determining a DVC in evaluating disinfection efficiency.

Physiological, numerical and molecular characterization of alkyl ether-utilizing rhodococci.

Twenty-seven Gram-positive strains were characterized physiologically and numerically and classified them into four groups according to their specific activities for utilization of linear alkyl ethers (AEs), cyclic AEs, monoalkoxybenzenes and 1,4-diethoxybenzene. The comparative analysis of the 16S ribosomal RNA gene and 16S-23S intergenic spacer region showed that they belonged to the genera Rhodococcus and Gordonia. Alkyl ether-utilizing rhodococci appeared to involve various and diverse cytochromes P450 of the families CYP116 (25 positive strains from 27), CYP153 (5/27), CYP249 (1/27) and a new family P450RR1 (27/27). The presence of P450RR1 was strongly related to the specific activity for utilization of 2-methoxyphenol and 2-ethoxyphenol. In addition, 26 of 27 strains contained multiple alkB genes coding for probable non-haem iron containing alkane monooxygenases and hydroxylases. Similar DNA fragments coding for a tetrahydrofuran monooxygenase A subunit (ThmA) were found in all cyclic AE-utilizing strains and nearly identical DNA fragments coding for likely orthologues of a propane monooxygenase A subunit (PrmA) in all linear AE-utilizing strains. The substrate availability in the degradation of aryl AEs, cyclic AEs and linear AEs agreed with the molecular probing of the respective genes encoding cytochrome P450RR1, ThmA and PrmA.

Physiological, biochemical, and genetic characterization of an alicyclic amine-degrading Mycobacterium sp. strain THO100 isolated from a morpholine-containing culture of activated sewage sludge.

Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282(T). The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1' were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450(mor) system remained unaltered. In addition, we found a spontaneous deletion of P450(mor) from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA.

Detection of human adenoviruses and enteroviruses in Korean oysters using cell culture, integrated cell culture-PCR, and direct PCR.

Oysters are known to be carriers of food-born diseases, but research on viruses in Korean oysters is scarce despite its importance for public health. We therefore tested oysters cultivated in Goheung, Seosan, Chungmu, and Tongyeong, for viral contamination using cell culture and integrated cell culture PCR (ICC-PCR) with Buffalo green monkey kidney (BGMK) and human lung epithelial (A549) cells. Additional screens via PCR, amplifying viral nucleic acids extracted from oysters supplemented our analysis. Our methods found 23.6%, 50.9%, and 89.1% of all oysters to be positive for adenoviruses when cell culture, ICC-PCR, and direct PCR, respectively, was used to conduct the screen. The same methodology identified enteroviruses in 5.45%, 30.9%, and 10.9% of all cases. Most of the detected enteroviruses (81.3%) were similar to poliovirus type 1; the remainder resembled coxsackievirus type A1. A homology search with the adenoviral sequences revealed similarities to adenovirus subgenera C (type 2, 5, and 6), D (type 44), and F (enteric type 40 and 41). Adenovirus-positive samples were more abundant in A549 cells (47.3%) than in BGMK cells (18.2%), while the reverse was true for enteroviruses (21.8% vs. 14.5%). Our data demonstrate that Korean oysters are heavily contaminated with enteric viruses, which is readily detectable via ICC-PCR using a combination of A549 and BGMK cells.

Use of cell culture-PCR assay based on combination of A549 and BGMK cell lines and molecular identification as a tool to monitor infectious adenoviruses and enteroviruses in river water.

Viral contamination in environmental samples can be underestimated because a single cell line might reproduce only some enteric viruses and some enteric viruses do not exhibit apparent cytopathic effects in cell culture. To overcome this problem, we evaluated a cell culture-PCR assay based on a combination of A549 and Buffalo green monkey kidney (BGMK) cell lines as a tool to monitor infectious adenoviruses and enteroviruses in river water. Water samples were collected 10 times at each of four rivers located in Gyeonggi Province, South Korea, and then cultured on group 1 cells (BGMK cells alone) and group 2 cells (BGMK and A549 cells). Reverse transcription and multiplex PCR were performed, followed by phylogenetic analysis of the amplicons. Thirty (75.0%) of the 40 samples were positive for viruses based on cell culture, and the frequency of positive samples grown on group 2 cells (65.0%) was higher than the frequency of positive samples grown on group 1 cells (50.0%). The number of samples positive for adenoviruses was higher with A549 cells (13 samples) than with BGMK cells (one sample); the numbers of samples positive for enteroviruses were similar with both types of cells. By using phylogenetic analysis, adenoviral amplicons were grouped into subgenera A, C, D, and F, and enteroviral amplicons were grouped into coxsackieviruses B3 and B4 and echoviruses 6, 7, and 30, indicating that A549 and BGMK cells were suitable for recovering a wide range of adenoviral and enteroviral types. The cell culture-PCR assay with a combination of A549 and BGMK cells and molecular identification could be a useful tool for monitoring infectious adenoviruses and enteroviruses in aquatic environments.

A novel continuous toxicity test system using a luminously modified freshwater bacterium.

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.