宮永 一彦

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller-Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline's activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.

To breed resistance to an assortment of infectious phages, continuous cultures of Escherichia coli JM109 grown in a chemostat were exposed to phage mixtures prepared from sewage influent. Four sequential chemostat-grown cultures were each infected with a different phage mixture. At the end of a chemostat run, one phage-resistant colony was isolated and used to inoculate the subsequent culture. This process was repeated, and increased phage resistance of the input bacterial strain resulted from the successive challenges with different phage cocktails. Multiple mutations apparently accumulated progressively. A mutant isolated at the end of the four runs, designated D198, showed resistance to 38 of 40 phages that infect the parent strain, JM109. D198 produced less outer membrane protein C (OmpC) than JM109. However, restoration of the OmpC protein by plasmid-mediated complementation did not completely restore the susceptibility of D198 to the 38 phages. Therefore, alterations beyond the level of OmpC protein production contribute to the phage resistance of D198. PCR-based genetic analysis revealed that D198 has a genome that is 209 kbp (about 200 genes) smaller than JM109. The deletion includes the chromosomal section from ompC to wbbL that encodes the rhamnosyl transferase involved in lipopolysaccharide biosynthesis. Strains D198 and JM109 were comparable in their growth characteristics and their abilities to express a recombinant protein.

Heat-treatment has been used for the reduction of excess sludge as a simple process. To clarify the relationship between reduction efficiency and biological response of sludge matrix during heat-treatment, microbial population and hydrolytic enzyme (protease) activity of a municipal activated sludge were studied. Culture-dependent analysis showed the rapid increase in the population of thermophilic bacteria at the early stage of heat-treatment and the emergence of protease-secreting bacteria. Culture-independent analysis by denaturing gradient gel electrophoresis (DGGE) revealed that the Bacilli, which include most of thermophiles, became the dominant class in the community by the treatment. The protease activity in supernatant of the sludge increased instantly after I h heat-treatment, which was considered to be released from microbial cells by lysis. The protease activity succession was correlated with the microbial succession and also with the change in MLSS and TOC concentrations during heat-treatment, suggesting that the protease activity plays an important role in the lysis-cryptic growth induced by heat-treatment. (c) 2007 Elsevier B.V. All rights reserved.

Escherichia coli is used as an indicator microorganism in public health. The conventional way to detect E. coli requires several days to produce a result, because it requires incubation of cells. Therefore a rapid and sensitive detection method is needed. T4e(-)/GFP phage, characterized by suppression of lysozyme and fusion of GFP (green fluorescent protein) to its SOC (small outer capsid) protein, was constructed, and it was shown to be able to detect E. coli K12 sensitively within several hours. However, because the host range of T4 phage to E. coli present in sewage water and sea water is narrow, this phage cannot be used to detect E. coli in environmental water. Two phages named IP008 and IP052, which have a broad host range to E. coli present in sewage influent, were screened from sewage influent. Mixture of these two phages produced clear plaques on 50% of E. coli screened from sewage influent. To use these phages as a tool for detection of E. coli, gfp was inserted into gene e, which encodes a lytic enzyme, and thus lytic-activity-suppressed phages were constructed (IP008e-/GFP and IP052e-/GFP). However, the fluorescent intensity of E. coli cells infected with IP008e-/GFP and IP052e-/GFP was not enough for visualization of the cell. Therefore, in addition to the insertion of gfp into gene e, fusion of GFP to SOC of IP008e-/GFP and IP052e-/GFP was conducted to produce IP008e-/ 2xGFP and IP052e-/2xGFP. E. coli cells infected with IP008e-/2xGFP and IP052e-/2xGFP showed much stronger fluorescence intensity than E coli cells infected by IP008e-/GFP and IP052e-/GFP. It is anticipated that, using these GFP-labeled phages, a broad range of E. coli present in sewage influent water can be detected rapidly.

A technique to distinguish viable and dead cells has long been considered necessary in various fields such as sterilization, toxicity assessment, sanitary evaluation and so on. A bacterial staining method using fluorescent dye is a popular tool, although the weakness of fluorescence intensity and its fading over time constitute notable drawbacks. In the process of esterase-active bacteria staining with carboxyfluorescein diacetate (CFDA), we have reported glutaraldehyde (GTA) affected the discriminative recognition of bacteria due to prevention of fluorescence leakage from the cell. In this study, CFDA was applied to four pure bacterial strains (two Gram-negative strains and two Gram-positive strains) during the exponential growth phase and to activated sludge as an indicator of microbial viability. GTA concentration was also optimized and the effect of GTA addition was compared to the conventional method using ethylenediamine tetraacetic acid (EDTA) and the control without pretreatment. At higher concentrations of GTA, microbial viability decreased because of GTA toxicity. In the case of all conditions where CFDA staining was carried out in the assay of microbial viability, the highest viability was achieved by using of 1 g/L GTA. © 2007 Elsevier B.V. All rights reserved.

A technique to distinguish viable and dead cells has long been considered necessary in various fields such as sterilization, toxicity assessment, sanitary evaluation and so on. A bacterial staining method using fluorescent dye is a popular tool, although the weakness of fluorescence intensity and its fading over time constitute notable drawbacks. In the process of esterase-active bacteria staining with carboxyfluorescein diacetate (CFDA), we have reported glutaraldehyde (GTA) affected the discriminative recognition of bacteria due to prevention of fluorescence leakage from the cell. In this study, CFDA was applied to four pure bacterial strains (two Gram-negative strains and two Gram-positive strains) during the exponential growth phase and to activated sludge as an indicator of microbial viability. GTA concentration was also optimized and the effect of GTA addition was compared to the conventional method using ethylenediamine tetraacetic acid (EDTA) and the control without pretreatment. At higher concentrations of GTA, microbial viability decreased because of GTA toxicity. In the case of all conditions where CFDA staining was carried out in the assay of microbial viability, the highest viability was achieved by using of 1 g/L GTA. (C) 2007 Elsevier B.V. All rights reserved.

Biofilm formation in cooling water systems causes many problems such as increase of the frictional resistance in tubes and decrease in the heat exchange capacity of heat exchanger. Monitoring of biofilm formation in the system is necessary to avoid biofilm related problems and optimize biocide addition. However, detection of biofilm formation in the whole system is difficult. To solve this problem, a novel and simple method for monitoring biofilm was developed in this study. Biofilm consists of living microorganisms, and nonliving organic and inorganic substances. Microorganisms in the biofilm consume substrate in the circulating cooling water. Therefore substrate consumption rate reflects biofilm activity in the system. Experiments were scaled up stepwisely; i.e. beaker-scale, bench-scale, and operating plant experiments. The beaker-scale experiment was conducted to select suitable substrate. Among the eight substrates examined, lactic acid was the most bio-consumable one by the biofilm formed on carbon steel coupon. The lactic acid consumption rate (R-lac) was estimated at 273 mg/(m(2)h). The R-lac value was compared before and after the hydrogen peroxide wash of the bench-scale plant. The R-lac before the wash was 36.3 mg/(m(2) h). On the other hand, the R-lac after the wash was negligible. An experimental study was next conducted in the operating cooling water plant. A rapid decrease of lactic acid concentration in the circulating water of a relatively unclean system was shown. On the other hand., R-lac in a clean system was low. This simple method provided us information on biologically active biofilm formation in the system. (c) 2007 Elsevier B.V. All rights reserved.

Biofilm formed on carbon steel by various species of bacterial cells causes serious problems such as corrosion of steel, choking of flow in the pipe; deterioration of the heat-transfer efficiency, and so on. Cathodic protection is known to be a reliable method for protecting carbon steel from corrosion. However, the initial attachment of bacteria to the surface and the effects of cathodic protection on bacterial viability in the biofilm have not been clarified. In this study, cathodic protection was applied to an artificial. biofilm containing Pseudomonas aeruginosa (PAOI), a bio-film constituent, on carbon steel. The aims of this study were to, evaluate the inhibition effect of cathodic protection on biofilm formation and to reveal the inhibition mechanisms. The viability of PAO1 in artificial biofilm of 5 mm thickness on cathodically protected steel decreased to 1% of the initial cell concentration. Analysis of pH distribution in the artificial biofilm by pH microelectrode revealed that pH in proximity to carbon steel increased to approximately 11 G after cathodic protection for 5 h. Moreover, 99% of region in the artificial biofilm was under the pH conditions of over nine. A simulation of pH profile was shown to correspond to L experimental values. These results indicate cells in the artificial biofilm were killed or damaged by cathodic protection due to pH increase.

Municipal sewage influent was screened for the presence of the virulence genes encoding Shiga-like toxins SLT-I and SLT-II (slt-I and slt-II) and intimin (eaeA) and those involved in biosynthesis of O157 (rfbE) and H7 (fliC) antigens by multiplex PCR to simultaneously identify the enterohemorrhagic Escherichia coli (EHEC) O157:H7 and its virulence factors in a single reaction. The screening was carried out monthly from October 2004 to September 2005. Direct PCR analysis using total DNA from sewage concentrate showed the presence of at least one virulence gene in 100% samples (n = 12). Sixty six percent of these samples were also positive for rfbE (O157) gene and fliC (H7) gene. The PCR amplification of these genes was possible when the concentration was above 20 cells ml(-1). From the multiplex PCR of the isolates following plating on Cefixime-Tellurite Sorbitol MacConkey (CT-SMAC) agar to detect non-sorbitol fermenting (NSF) colonies (n = 600), one E. coli strain carrying slt-II gene and two strains of E coli O157:H7 carrying slt-I were detected. The results show that municipal sewage represents a potential reservoir of EHEC. CT-SMAC agar was proved to have limited E. coli O157:H7 selectivity and only 0.005% (3/600) sensitivity for sewage samples due to the high frequency (43%) of NSF strains in sewage. The enrichment of sewage sample in modified E. coli broth (mEC) increased the sensitivity of PCR resulting in the clearer amplification of five genes. Amplification of target cell type in mEC broth implied that EHEC were present in sewage in a culturable and hence potentially infectious state. However, pre-enrichment did not affect the selectivity of CT-SMAC because frequency of NSF colonies remained the same as that obtained without enrichment. The study, therefore, underscores the need for more sensitive screening techniques that can be routinely employed for the regular monitoring of sewage influent. (c) 2006 Elsevier B.V. All rights reserved.

Municipal sewage influent was screened for the presence of the virulence genes encoding Shiga-like toxins SLT-I and SLT-II (slt-I and slt-II) and intimin (eaeA) and those involved in biosynthesis of O157 (rfbE) and H7 (fliC) antigens by multiplex PCR to simultaneously identify the enterohemorrhagic Escherichia coli (EHEC) O157:H7 and its virulence factors in a single reaction. The screening was carried out monthly from October 2004 to September 2005. Direct PCR analysis using total DNA from sewage concentrate showed the presence of at least one virulence gene in 100% samples (n = 12). Sixty six percent of these samples were also positive for rfbE (O157) gene and fliC (H7) gene. The PCR amplification of these genes was possible when the concentration was above 20 cells ml-1. From the multiplex PCR of the isolates following plating on Cefixime-Tellurite Sorbitol MacConkey (CT-SMAC) agar to detect non-sorbitol fermenting (NSF) colonies (n = 600), one E. coli strain carrying slt-II gene and two strains of E. coli O157:H7 carrying slt-I were detected. The results show that municipal sewage represents a potential reservoir of EHEC. CT-SMAC agar was proved to have limited E. coli O157:H7 selectivity and only 0.005% (3/600) sensitivity for sewage samples due to the high frequency (43%) of NSF strains in sewage. The enrichment of sewage sample in modified E. coli broth (mEC) increased the sensitivity of PCR resulting in the clearer amplification of five genes. Amplification of target cell type in mEC broth implied that EHEC were present in sewage in a culturable and hence potentially infectious state. However, pre-enrichment did not affect the selectivity of CT-SMAC because frequency of NSF colonies remained the same as that obtained without enrichment. The study, therefore, underscores the need for more sensitive screening techniques that can be routinely employed for the regular monitoring of sewage influent. © 2006 Elsevier B.V. All rights reserved.

An isothiazolone biocide, 5-chloro-2-methyl-4-isothiazolin-3-one (CMI), was degraded in the presence of iron. According to the Fe-dependent degradation of CMI, stoichiometric production of chloride was observed. Copper and stainless steel did not enhance the physico-chemical degradation of CMI, whilst phosphate inhibited the Fe-dependent degradation. Neither aerobic nor anaerobic conditions influenced the Fe-dependent CMI degradation. Furthermore, FeO(OH)-powder and Fe3O4-powder did not lead to the physico-chemical degradation of CMI. Rapid disappearance of CMI was observed in an operating cooling water plant. CMI added to the cooling tower declined from 1.4 mg l(-1) to < 0.1 mg l(-1) in 2 d. This finding is important in optimising the use of CMI and combating resistance if encountered.

To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4°C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4°C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28°C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28°C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28°C. © Springer-Verlag 2006.

To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4 degrees C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4 degrees C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28 degrees C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28 degrees C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28 degrees C.

To estimate the self-purification capacity of sewer pipe, six different types of concrete blocks were installed in a domestic sewer pipe for nine months. The concrete blocks used were plain, grain, porous, and wet concrete (no-hole, or perforated with holes of d = 1 or 12 mm). After a 79-day exposure to sewage, a heterogeneous biofilm formed on the surface of each block. The self-purification capacities of the blocks were estimated by measuring the decrease in substrate concentration in artificial sewage. The analyzed substrates were dissolved oxygen (DO), total organic carbon (TOC), NH4+, and NO3-. Wet concrete with holes (d = 12 mm) showed the highest substrate consumption rates: DO = 460, TOC = 480, NH4-N = 87, and nitrogen as NO3-N = 170, in mg substrate/(m(2)h). These results indicate that sewers have a considerable potential for removing organic material and nutrients, and modification of sewer surfaces may increase these activities. (c) 2006 Elsevier B.V. All rights reserved.

A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e(-)/GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation of E. coli O157:H7 cells with PP01e(-)/GFP did not lead to cell lysis, while the propagation of PP01e(-)/GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e(-)/GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count.

For a precise estimation of sanitary condition, Escherichia coli detection system using E. coli-specific bacteriophage T4 wits constructed. To facilitate E coli detection, T4e(-) phage which did not produce the lysozyme was constructed and green fluorescent protein (GFP) was displayed oil T4e- small outer capside (SOC) protein. This T4e(-)/GFP call detect E. coli K12 without cell lysis. In this Study, we applied T4e-/GFP to the detection of E. coli in the sewage influent. Chromocult((R)) coliforin (CC) agar plates are generally used for simultaneous detection of total coliforms and E. coli. We investigated the number of coliforms and E. coli in the municipal sewage influent by using CC agar plates for 1 year. There were 10(5)-10(6) CFU/ml of total coliforms and 104 105 CFU/ml of E. coli throughout the year. More than 20 strains of E. coli selected from CC agar were infected by T4e-/GFP. The ratio of clear plaque forming 1-7. coli was different every month and very low (annual average 8%). Most of the E. coli showed turbid plaque or no plaque. E. coli formed the turbid plaque showed no-fluorescence, fluorescence only on cell Surface due to phage adsorption, or heterogenous fluorescence among the cells, while E. coli formed clear plaque Could be detected because of T4e-/GFP amplification in the cell. (c) 2005 Elsevier B.V. All rights reserved.

As a biochemical approach to decrease the amount of biological sludge, the sludge solubilization by repeatedly changing the microbial environments from aerobic to anaerobic was studied. The sludge reduction amount in batch operation was related to the degradation of intracellular materials released through sludge solubilization. The shorter the operation cycle time was, the more effective the sludge amount reduction was. On the other hand, under the cycle time of 3-24 h, longer anaerobic period compared to aerobic period was effective in removing nitrogenous compounds. This was partly ascribed to the excretion of enzyme such as protease and lipase. (c) 2005 Elsevier B.V. All rights reserved.

As a biochemical approach to decrease the amount of biological sludge, the sludge solubilization by repeatedly changing the microbial environments from aerobic to anaerobic was studied. The sludge reduction amount in batch operation was related to the degradation of intracellular materials released through sludge solubilization. The shorter the operation cycle time was, the more effective the sludge amount reduction was. On the other hand, under the cycle time of 3-24 h, longer anaerobic period compared to aerobic period was effective in removing nitrogenous compounds. This was partly ascribed to the excretion of enzyme such as protease and lipase. (c) 2005 Elsevier B.V. All rights reserved.

We designed a bacteriophage T2 system to display proteins fused at the N-terminus of the head protein small outer capsid (SOC) of a T2 phage. To facilitate selection of chimeric phage, a T2 phage encoding the beta-galactosidase gene (beta gal) upstream of the soc gene was constructed. The phage, named T2 Gal, produces blue plaques on agar plates containing XGal. Subsequently, a plasmid encoding the target protein upstream of soc was constructed and used to transform E. coli B-E cells. Transformed cells were infected with T2 beta Gal and homologous recombination between phage DNA and the plasmid resulted in a chimeric phage that produced transparent plaques due to the excision of the beta gal gene. Chitosanase of Bacillus sp. strain K17 (ChoK), consisting of 453 amino acids, was used as a model target protein. Recombinant T2 phage that produced ChoK was named T2ChoK. T2ChoK was produced from T2 beta Gal at a recombination frequency of about 0.1%. On the other hand, the value for T2 beta Gal produced from wild-type T2 was 0.001%. This new system enables us to select recombinant phage very quickly and accurately. The number of molecules of ChoK was calculated at 14.7 per single phage. Latent period and burst size were estimated for the chimeric phages.

A rapid and simple selection method of high-yield cells has been desired to establish highly productive cell lines for useful secondary metabolites. For this purpose, a new attempt was made to partition cultured plant cells in a poly(ethylene glycol)-dextran aqueous two-phase system (ATPS). The applicability of the ATPS in partitioning cultured strawberry cells (designated FAW) was investigated. The result of single-step partitioning in the ATPS supplemented with 0.4 mmol/kg lithium sulfate showed that FAW cells cultivated for 7 d under light-irradiation were separated into two cell populations with significantly different anthocyanin content. Additionally, the analysis technique of microscopic cell images showed that cells accumulating a high level of anthocyanin were partitioned completely into the bottom phase in a partitioning experiment of FAW cells cultivated for 10 d under light-irradiation in the ATPS supplemented with 1.8 mmol/kg potassium phosphate buffer. These results indicated that cell partitioning in ATPS increased the intracellular anthocyanin content and that the cultured strawberry cell population was heterogeneous in terms of cell surface properties. This is the first report of partitioning based on the heterogeneity of the cell surface properties correlated with the intracellular secondary metabolism in cultured plant cells. Our results also suggested that the ATPS was appropriate as a large-scale method for selecting useful cell lines among the cultured plant cells.

To investigate the therapeutical use of phage mixture for controlling gastrointestinal Escherichia coli O157:H7 cells, in vitro and in vivo experiments were conducted. Three phages, SP15, SP21, and SP22 were selected from 26 phage stock screened from feces of stock animals and sewage influent. Addition of single or binary phage to the E. coli cell batch-culture reduced the turbidity of the culture. However, reascend of the turbidity due to the appearance of phage resistance cell was observed. On the other hand, addition of three phage mixture (SP15-21-22) did not produce reascend of culture turbidity under aerobic condition. Under anaerobic condition, slight reascend of culture turbidity was observed after SP15-21-22 addition. Chemostat continuous culture was operated under anaerobic condition to optimize the titer of phage cocktail and frequency of the addition for controlling E. coli cells. Five-log decrease of E. coli cell concentration after addition of phage cocktail of 10(9) Plaque forming unit (PFU)/ml was observed. However, reascend of cell concentration was observed after 1 d incubation. Repeated addition of phage cocktail was effective to reduce the cell concentration. Suspension of phage cocktail in the buffer containing 0.25% CaCO(3) neutralized 9 times much more buffer of pH 2. Based on this in vitro experiment, phage cocktail (SP15-21-22) suspended in the buffer containing 0.25% CaCO(3) was orally administrated to the mice in which E. coli O157: H7 cells was administrated in 2-d advance. E. coli and phage concentration in the feces was monitored for 9 d after phage addition. High titer of phage was detected in the feces when the phage cocktail administrated daily. E. coli O157: H7 concentration in the feces has been reduced according to the time period. However, difference of E. coli concentration in the feces of mice administrates with phage and in the control mice without phage addition became slight after 9-d test period. High titer of the phage settled down in the gastrointestinal tracts and reduced the concentration of E. coli cell. Repeated oral administration of SP15-21-22 was effective for rapid evacuation of E. coli O157:H7 from the feces and gastrointestinal tract of mice.

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (k(a)) of T2ppD1 (0.17 x 10(-9) (ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9) (ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage. (C) 2004 Elsevier B.V. All rights reserved.

As a biochemical approach to reduce the amount of excess sludge from wastewater treatment process, sludge solubilization induced by changing the sludge environment by alternating aerobic and anaerobic operations has been proposed. The sludge solubilization leads to the release of carbonaceous and nitrogenous compounds, which is to be reduced time by time due to the energy consumption by the repeated microbial assimilation. Batch experiments were carried out under various operating conditions alternating aerobic and anaerobic period to characterize the behavior of nitrogenous compounds in the sludge solubilization. Nitrogenous compounds among intracellular matters released by the lysis were successively transformed biochemically, finally having been removed completely under an optimal condition. However, under the conditions out of the optimum, the released nitrogen such as ammonia and nitrate is accumulated in the system. Ammonia accumulation was found to be due to the higher protease activity associated with the protein hydrolysis originated from cell lysis. (C) 2004 Elsevier B.V. All rights reserved.

Eucommia ulmoides was cultivated in suspended culture and formed aggregates with broad size spectrum. According to the morphological features, density and size, the aggregates were classified into daughter aggregate (DA), meristemoid-like aggregate (MA) and old aggregate (OA). DA can grow into MA, which proliferates DAs again at some vigorously growing points on their surface. Coexistence of DAs and MAs implies better conditions for callus growth. It was revealed that mechanical disintegration of callus into smaller size in the subculture has a negative effect on callus growth due to the damage of active cells located at the aggregate surface and/or the characteristics of original big aggregate which belongs to OA before fragmentation. The growth rates of spontaneous DA and MA were about two times higher than that of the disintegrated callus. As an approach for scaling-up process, spontaneously proliferated aggregates can provide the rapidly growing callus of E. ulmoides culture compared with the mechanically disintegrated callus. (C) 2004 Elsevier B.V. All rights reserved.

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour. (C) 2004 Elsevier B.V. All rights reserved.

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance. © Springer-Verlag 2003.

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.

Staining of esterase-active bacteria with carboxyfluorescein diacetate ( CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.