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.

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.

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.

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.

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 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.

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.

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.

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. (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 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.

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.

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.

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.

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.

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.