The community structure of methanogenic archaea is relatively stable,i.e.,it is sustained at a high abundance with minimal changes in composition,in paddy field soils irrespective of submergence and drainage.In contra...The community structure of methanogenic archaea is relatively stable,i.e.,it is sustained at a high abundance with minimal changes in composition,in paddy field soils irrespective of submergence and drainage.In contrast,the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils.This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments.In the pot experiment,a soil sample obtained from an upland field was incubated under submerged conditions for 275 d.Soil samples periodically collected were subjected to culture-dependent most probable number(MPN)enumeration,polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)analysis of archaeal 16 S r RNA gene,and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene(mcr A)of methanogenic archaea.The abundance of methanogenic archaea increased from 102 to 103 cells g-1 dry soil and 104 to 107 copies of mcr A gene g-1 dry soil after submergence.Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis,members from Methanocellales,Methanosarcinaceae,and Methanosaetaceae proliferated in the soils,and the community structure was relatively stable once established.In the field experiment,the number of viable methanogenic archaea in a rice paddy field converted from meadow(reclaimed paddy field)was monitored by MPN enumeration over five annual cycles of field operations.Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer’s paddy field was dressed onto the meadow(dressed paddy field)and an upland crop field converted from the meadow(reclaimed upland field).The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field.Then,the number gradually increased over five years and finally reached 103–104 展开更多
Spreading of antibiotic resistant bacteria into environment is becoming a major public health problem, implicating affair of the indirect transmission of antibiotic resistant bacteria to human through drinking water, ...Spreading of antibiotic resistant bacteria into environment is becoming a major public health problem, implicating affair of the indirect transmission of antibiotic resistant bacteria to human through drinking water, or vegetables, or daily products. Until now, the risk of nosocomial infection of antibiotic resistant bacteria has mainly been evaluated using clinical isolates by phenotypic method. To evaluate a risk of community-acquired infection of antibiotic resistant bacteria, a new method has been developed based on PCR-RFLP without isolation. By comparing restriction fragment lengths of the 16S rDNA gene from bacterial mixture grown under antibiotic treatment to those simulated from the DNA sequence, bacterial taxonomies were elucidated using the method of Okuda and Watanabe [1] [2]. In this study, taxonomies of polymyxin B resistant bacteria group in field soils, paddy field with organic manure and upland field without organic manure were estimated without isolation. In the both field soils, the major bacteria grown under the antibiotic were B. cereus group, which had natural resistance to this antibiotic. In field applied with organic manure, Prevotella spp., and the other Cytophagales, which were suggested to be of feces origin and to acquire resistance to the antibiotic, were detected. When numbers of each bacterial group were roughly estimated by the most probable number method, B. cereus group was enumerated to be 3.30 × 106 MPN/g dry soil in paddy field soil and 1.32 × 106 MPN/g dry soil in upland filed. Prevotella spp. and the other Cytophagales in paddy field were enumerated to be 1.31 × 106 MPN, and 1.07 × 106 MPN·g-1 dry soil.展开更多
In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial group...In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L<sup>-1</sup><sup></sup>) were included in row cattle feces (1.78 × 10<sup>4</sup> MPN g<sup>-1</sup>) and cattle feces manure (>3.84 × 10<sup>4</sup> MPN g<sup>-1</sup>). Compost originated from leftover food (>44.8 × 10<sup>4</sup> MPN g<sup>-1</sup>) and shochu lee (>320 × 10<sup>4</sup> MPN g<sup>-1</sup>) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L<sup>-1</sup><sup></sup>), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 10<sup>4</sup> MPN g<sup>-1</sup>), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L<sup>-1 </sup>of<sup> </sup>ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 10<sup>4</sup> MPN g<sup>-1</sup>), followed by cattle feces manure (4.19 × 10<sup>4</sup> MPN g<sup>-1</sup>), and shochu lee (0.36 × 10<sup>4</sup> MPN g<sup>-1</sup>), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.展开更多
Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection metho...Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g<sup>-1</sup>) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as 展开更多
Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and ...Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 10<sup>6</sup> MPN g<sup>-1</sup> dry soil), followed by γ-Proteobacteria (1.32 × 10<sup>6</sup> MPN), δ-Proteobacteria (0.006 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.006 × 10<sup>6</sup> MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 10<sup>6</sup> MPN), followed by α-Proteobacteria (26.0 × 10<sup>6</sup> MPN), β-Proteobacteria (17.1 × 10<sup>6</sup> MPN), δ-Proteobacteria (11.2 × 10<sup>6</sup> MPN), the other Firmicutes (1.71 × 10<sup>6</sup> MPN), γ-Proteobacteria (0.5 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.05 × 10<sup>6</sup> MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 10<sup>6</sup> MPN), followed by Actinobacteria (2.14 × 10<sup>6</sup> MPN), Bacillus sp. (2.14 × 10<sup>6</sup> MPN), and γ-Proteobacteria (0.35 × 10<sup>6</sup> MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candi展开更多
Composting is a biological aerobic decomposition process consisted from different phases. Although the Japanese Standards for manure recommended that it took at least 6 months to complete the maturing phase, there was...Composting is a biological aerobic decomposition process consisted from different phases. Although the Japanese Standards for manure recommended that it took at least 6 months to complete the maturing phase, there was no reliable ground. In order to find out shortening method of the maturing phase, the microorganisms concerned with a progress of the maturing was determined by using the most probable number method (MPN) and PCR-RFLP of the 16S rDNA, which was found effective to provide numbers and taxonomy of polymyxin B resistant bacterial groups in the former paper [1]. Compared to the numbers after thermophilic phase, those of Actinobacteria, δ-proteobacteria, and the other gram negative bacteria increased to 50 times, 20 times, and 105 times, respectively, after maturing phase, while those of Bacillus spp., and α and β-proteobacteria decreased to 1/10, and 1/105 after maturing phase. Numbers of the other Fumicutes, and γ-proteobacteria remained in the same revel. Actinobacteria, δ-proteobacteria, and the other gram negative bacteria might be concerned with a progress of the maturing phase, because these bacterial groups were detected and enumerated due to their proliferation ability. Although number of Acitinobacteria might be underestimated because of a PCR bias, the method was found effective for the purpose to monitor bacteria actively proliferated in culture medium.展开更多
The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for indus...The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g<sup>-1</sup>, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g<sup>-1</sup>. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g<sup>-1</sup>. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g<sup>-1</sup>) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g<sup>-1</sup>), and the other Firmicutes (3.7 × 106 MPN g<sup>-1</sup>). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g<sup>-1</sup>), Burkhokderia sp. (1.5 × 106 MPN g<sup>-1</sup>), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g<sup>-1</sup>). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.展开更多
基金a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows (No. 19 6611)JSPS KAKENHI (No. 24780318).
文摘The community structure of methanogenic archaea is relatively stable,i.e.,it is sustained at a high abundance with minimal changes in composition,in paddy field soils irrespective of submergence and drainage.In contrast,the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils.This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments.In the pot experiment,a soil sample obtained from an upland field was incubated under submerged conditions for 275 d.Soil samples periodically collected were subjected to culture-dependent most probable number(MPN)enumeration,polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)analysis of archaeal 16 S r RNA gene,and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene(mcr A)of methanogenic archaea.The abundance of methanogenic archaea increased from 102 to 103 cells g-1 dry soil and 104 to 107 copies of mcr A gene g-1 dry soil after submergence.Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis,members from Methanocellales,Methanosarcinaceae,and Methanosaetaceae proliferated in the soils,and the community structure was relatively stable once established.In the field experiment,the number of viable methanogenic archaea in a rice paddy field converted from meadow(reclaimed paddy field)was monitored by MPN enumeration over five annual cycles of field operations.Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer’s paddy field was dressed onto the meadow(dressed paddy field)and an upland crop field converted from the meadow(reclaimed upland field).The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field.Then,the number gradually increased over five years and finally reached 103–104
文摘Spreading of antibiotic resistant bacteria into environment is becoming a major public health problem, implicating affair of the indirect transmission of antibiotic resistant bacteria to human through drinking water, or vegetables, or daily products. Until now, the risk of nosocomial infection of antibiotic resistant bacteria has mainly been evaluated using clinical isolates by phenotypic method. To evaluate a risk of community-acquired infection of antibiotic resistant bacteria, a new method has been developed based on PCR-RFLP without isolation. By comparing restriction fragment lengths of the 16S rDNA gene from bacterial mixture grown under antibiotic treatment to those simulated from the DNA sequence, bacterial taxonomies were elucidated using the method of Okuda and Watanabe [1] [2]. In this study, taxonomies of polymyxin B resistant bacteria group in field soils, paddy field with organic manure and upland field without organic manure were estimated without isolation. In the both field soils, the major bacteria grown under the antibiotic were B. cereus group, which had natural resistance to this antibiotic. In field applied with organic manure, Prevotella spp., and the other Cytophagales, which were suggested to be of feces origin and to acquire resistance to the antibiotic, were detected. When numbers of each bacterial group were roughly estimated by the most probable number method, B. cereus group was enumerated to be 3.30 × 106 MPN/g dry soil in paddy field soil and 1.32 × 106 MPN/g dry soil in upland filed. Prevotella spp. and the other Cytophagales in paddy field were enumerated to be 1.31 × 106 MPN, and 1.07 × 106 MPN·g-1 dry soil.
文摘In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L<sup>-1</sup><sup></sup>) were included in row cattle feces (1.78 × 10<sup>4</sup> MPN g<sup>-1</sup>) and cattle feces manure (>3.84 × 10<sup>4</sup> MPN g<sup>-1</sup>). Compost originated from leftover food (>44.8 × 10<sup>4</sup> MPN g<sup>-1</sup>) and shochu lee (>320 × 10<sup>4</sup> MPN g<sup>-1</sup>) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L<sup>-1</sup><sup></sup>), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 10<sup>4</sup> MPN g<sup>-1</sup>), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L<sup>-1 </sup>of<sup> </sup>ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 10<sup>4</sup> MPN g<sup>-1</sup>), followed by cattle feces manure (4.19 × 10<sup>4</sup> MPN g<sup>-1</sup>), and shochu lee (0.36 × 10<sup>4</sup> MPN g<sup>-1</sup>), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.
文摘Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g<sup>-1</sup>) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as
文摘Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 10<sup>6</sup> MPN g<sup>-1</sup> dry soil), followed by γ-Proteobacteria (1.32 × 10<sup>6</sup> MPN), δ-Proteobacteria (0.006 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.006 × 10<sup>6</sup> MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 10<sup>6</sup> MPN), followed by α-Proteobacteria (26.0 × 10<sup>6</sup> MPN), β-Proteobacteria (17.1 × 10<sup>6</sup> MPN), δ-Proteobacteria (11.2 × 10<sup>6</sup> MPN), the other Firmicutes (1.71 × 10<sup>6</sup> MPN), γ-Proteobacteria (0.5 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.05 × 10<sup>6</sup> MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 10<sup>6</sup> MPN), followed by Actinobacteria (2.14 × 10<sup>6</sup> MPN), Bacillus sp. (2.14 × 10<sup>6</sup> MPN), and γ-Proteobacteria (0.35 × 10<sup>6</sup> MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candi
文摘Composting is a biological aerobic decomposition process consisted from different phases. Although the Japanese Standards for manure recommended that it took at least 6 months to complete the maturing phase, there was no reliable ground. In order to find out shortening method of the maturing phase, the microorganisms concerned with a progress of the maturing was determined by using the most probable number method (MPN) and PCR-RFLP of the 16S rDNA, which was found effective to provide numbers and taxonomy of polymyxin B resistant bacterial groups in the former paper [1]. Compared to the numbers after thermophilic phase, those of Actinobacteria, δ-proteobacteria, and the other gram negative bacteria increased to 50 times, 20 times, and 105 times, respectively, after maturing phase, while those of Bacillus spp., and α and β-proteobacteria decreased to 1/10, and 1/105 after maturing phase. Numbers of the other Fumicutes, and γ-proteobacteria remained in the same revel. Actinobacteria, δ-proteobacteria, and the other gram negative bacteria might be concerned with a progress of the maturing phase, because these bacterial groups were detected and enumerated due to their proliferation ability. Although number of Acitinobacteria might be underestimated because of a PCR bias, the method was found effective for the purpose to monitor bacteria actively proliferated in culture medium.
文摘The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g<sup>-1</sup>, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g<sup>-1</sup>. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g<sup>-1</sup>. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g<sup>-1</sup>) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g<sup>-1</sup>), and the other Firmicutes (3.7 × 106 MPN g<sup>-1</sup>). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g<sup>-1</sup>), Burkhokderia sp. (1.5 × 106 MPN g<sup>-1</sup>), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g<sup>-1</sup>). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.
