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Kiyoshi Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric Microbial Viability assay based on the reduction of water-soluble tetrazolium WST-1
European Journal of Clinical Microbiology & Infectious Diseases, 2015Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Munetaka Ishiyama, Masanobu Shiga, Takashi Ikegami, Kiyoshi MatsumotoAbstract:Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric Microbial Viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7–14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3–4 days using the WST-1 colorimetric method and those obtained after 10–14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.
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A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric Microbial Viability assay.
Letters in applied microbiology, 2014Co-Authors: Tadayuki Tsukatani, H. Suenaga, M. Shiga, Kiyoshi MatsumotoAbstract:UNLABELLED A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric Microbial Viability assay based on the reduction in a tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) with 2-methyl-1,4-napthoquinone as the electron mediator was developed. The proposed method was useful to measure the proliferation of 18 kinds of moulds and seven kinds of yeasts, including representative pathogens such as Aspergillus spp., Candida spp. and Cryptococcus spp. Linear relationships between the absorbance and viable fungal cell density were obtained for all fungi, suggesting that the absorbance change reflected the fungal proliferation. In addition, the minimum inhibitory concentrations (MICs) against a variety of different pathogenic moulds and yeasts for amphotericin B, itraconazole and 5-flucytosine were determined by susceptibility testing using the proposed method and compared with those obtained using the conventional broth microdilution method. There was an excellent agreement between the results obtained using the WST-8 colorimetric method and those obtained using the conventional Clinical and Laboratory Standard Institute method. The WST-8 colorimetric assay is a useful method for rapid determination of accurate MICs for a variety of different fungi. SIGNIFICANCE AND IMPACT OF THE STUDY A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric Microbial Viability assay based on reduction in a tetrazolium salt (WST-8) was developed. The WST-8 colorimetric method was useful to measure the proliferation of a variety of different fungi. In the antifungal susceptibility testing, there was a good agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The proposed method was superior to conventional methods in terms of its rapidity towards a variety of different fungi.
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Determination of water-soluble vitamins using a colorimetric Microbial Viability assay based on the reduction of water-soluble tetrazolium salts.
Food Chemistry, 2011Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Munetaka Ishiyama, Kiyoshi MatsumotoAbstract:Abstract A method for the determination of water-soluble vitamins using a colorimetric Microbial Viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H -tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B 6 , biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B 6 in various foodstuffs. There was good agreement between vitamin B 6 concentrations determined after 24 h using the WST-8 colorimetric method and those obtained after 48 h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate.
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distinction of gram positive and negative bacteria using a colorimetric Microbial Viability assay based on the reduction of water soluble tetrazolium salts with a selection medium
Journal of General and Applied Microbiology, 2011Co-Authors: Tadayuki Tsukatani, Katsuya Noguchi, Hikaru Suenaga, Tomoko Higuchi, Masanobu Shiga, Kiyoshi MatsumotoAbstract:Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric Microbial Viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric Viability assay was carried out in a selection medium containing 0.5μg/ml crystal violet, 5.0 μg/ml daptomycin, and 5.0μg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.
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colorimetric Microbial Viability assay based on reduction of water soluble tetrazolium salts for antiMicrobial susceptibility testing and screening of antiMicrobial substances
Analytical Biochemistry, 2009Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Tomoko Higuchi, Munetaka Ishiyama, Tetsuyuki Akao, Kiyoshi MatsumotoAbstract:The applicability of a colorimetric Microbial Viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antiMicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antiMicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.
Tadayuki Tsukatani - One of the best experts on this subject based on the ideXlab platform.
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Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric Microbial Viability assay based on the reduction of water-soluble tetrazolium WST-1
European Journal of Clinical Microbiology & Infectious Diseases, 2015Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Munetaka Ishiyama, Masanobu Shiga, Takashi Ikegami, Kiyoshi MatsumotoAbstract:Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric Microbial Viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7–14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3–4 days using the WST-1 colorimetric method and those obtained after 10–14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.
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A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric Microbial Viability assay.
Letters in applied microbiology, 2014Co-Authors: Tadayuki Tsukatani, H. Suenaga, M. Shiga, Kiyoshi MatsumotoAbstract:UNLABELLED A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric Microbial Viability assay based on the reduction in a tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) with 2-methyl-1,4-napthoquinone as the electron mediator was developed. The proposed method was useful to measure the proliferation of 18 kinds of moulds and seven kinds of yeasts, including representative pathogens such as Aspergillus spp., Candida spp. and Cryptococcus spp. Linear relationships between the absorbance and viable fungal cell density were obtained for all fungi, suggesting that the absorbance change reflected the fungal proliferation. In addition, the minimum inhibitory concentrations (MICs) against a variety of different pathogenic moulds and yeasts for amphotericin B, itraconazole and 5-flucytosine were determined by susceptibility testing using the proposed method and compared with those obtained using the conventional broth microdilution method. There was an excellent agreement between the results obtained using the WST-8 colorimetric method and those obtained using the conventional Clinical and Laboratory Standard Institute method. The WST-8 colorimetric assay is a useful method for rapid determination of accurate MICs for a variety of different fungi. SIGNIFICANCE AND IMPACT OF THE STUDY A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric Microbial Viability assay based on reduction in a tetrazolium salt (WST-8) was developed. The WST-8 colorimetric method was useful to measure the proliferation of a variety of different fungi. In the antifungal susceptibility testing, there was a good agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The proposed method was superior to conventional methods in terms of its rapidity towards a variety of different fungi.
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Determination of water-soluble vitamins using a colorimetric Microbial Viability assay based on the reduction of water-soluble tetrazolium salts.
Food Chemistry, 2011Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Munetaka Ishiyama, Kiyoshi MatsumotoAbstract:Abstract A method for the determination of water-soluble vitamins using a colorimetric Microbial Viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H -tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B 6 , biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B 6 in various foodstuffs. There was good agreement between vitamin B 6 concentrations determined after 24 h using the WST-8 colorimetric method and those obtained after 48 h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate.
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distinction of gram positive and negative bacteria using a colorimetric Microbial Viability assay based on the reduction of water soluble tetrazolium salts with a selection medium
Journal of General and Applied Microbiology, 2011Co-Authors: Tadayuki Tsukatani, Katsuya Noguchi, Hikaru Suenaga, Tomoko Higuchi, Masanobu Shiga, Kiyoshi MatsumotoAbstract:Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric Microbial Viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric Viability assay was carried out in a selection medium containing 0.5μg/ml crystal violet, 5.0 μg/ml daptomycin, and 5.0μg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.
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colorimetric Microbial Viability assay based on reduction of water soluble tetrazolium salts for antiMicrobial susceptibility testing and screening of antiMicrobial substances
Analytical Biochemistry, 2009Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Tomoko Higuchi, Munetaka Ishiyama, Tetsuyuki Akao, Kiyoshi MatsumotoAbstract:The applicability of a colorimetric Microbial Viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antiMicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antiMicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.
J. Magarian Blander - One of the best experts on this subject based on the ideXlab platform.
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Sensing Microbial Viability through Bacterial RNA Augments T Follicular Helper Cell and Antibody Responses
Immunity, 2018Co-Authors: Gaetan Barbet, Leif E. Sander, Matthew Geswell, Irina Leonardi, Andrea Cerutti, Iliyan D. Iliev, J. Magarian BlanderAbstract:Summary Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial Viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2– monocytes instructed Tfh differentiation through interleukin-1β (IL-1β), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-β and IL-1β dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of Microbial Viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.
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Vita-PAMPs: Signatures of Microbial Viability
Advances in experimental medicine and biology, 2013Co-Authors: Diego Mourão-sá, Soumit Roy, J. Magarian BlanderAbstract:Can the innate immune system detect and respond to Microbial Viability? Using bacteria as a model, we found that indeed the very essence of Microbial infectivity, Viability itself, can be detected, and notably, in the absence of the activity of virulence factors. The Microbial molecule that serves as the signature of Viability is bacterial messenger RNA (mRNA), common to all bacteria, and without which bacteria cannot survive. Prokaryotic mRNAs also differ from eukaryotic mRNAs in several ways, and as such, these features all fulfill the criteria, and more, for a pathogen-associated molecular pattern (PAMP) as originally proposed by Charles Janeway. Because these mRNAs are lost from dead bacteria, they belong to a special class of PAMPs, which we call vita-PAMPs. Here we discuss the possible receptors and pathways involved in the detection of bacterial mRNAs, and thus Microbial Viability. We also consider examples of vita-PAMPs other than bacterial mRNA.
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Beyond pattern recognition: five immune checkpoints for scaling the Microbial threat.
Nature reviews. Immunology, 2012Co-Authors: J. Magarian Blander, Leif E. SanderAbstract:Pattern recognition by the innate immune system enables the detection of microorganisms, but how the level of Microbial threat is evaluated - a process that is crucial for eliciting measured antiMicrobial responses with minimal inflammatory tissue damage - is less well understood. New evidence has shown that features of Microbial Viability can be detected by the immune system and thereby induce robust responses that are not warranted for dead microorganisms. Here, we propose five immune checkpoints that, as defined here, collectively determine the gravity of Microbial encounters.
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Detection of prokaryotic mRNA signifies Microbial Viability and promotes immunity
Nature, 2011Co-Authors: Leif E. Sander, Michael J. Davis, Mark V. Boekschoten, Derk Amsen, Christopher C. Dascher, Bernard Ryffel, Joel A. Swanson, Michael Müller, J. Magarian BlanderAbstract:Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense Microbial Viability through detection of a special class of Viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by Viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of Microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.
Leif E. Sander - One of the best experts on this subject based on the ideXlab platform.
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Dead or alive: how the immune system detects Microbial Viability.
Current opinion in immunology, 2018Co-Authors: Matteo Ugolini, Leif E. SanderAbstract:Immune detection of Microbial Viability is increasingly recognized as a potent driver of innate and adaptive immune responses. Here we describe recent mechanistic insights into the process of how the immune system discriminates between viable and non-viable Microbial matter. Accumulating evidence suggests a key role for Microbial RNA as a widely conserved Viability associated PAMP (vita-PAMP) and a molecular signal of increased infectious threat. Toll-like receptor 8 (TLR8) has recently emerged as a critical sensor for viable bacteria, ssRNA viruses, and archaea in human antigen presenting cells (APC). We discuss the role of Microbial RNA, and other potential vita-PAMPs in antiMicrobial immunity and vaccine responses.
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Sensing Microbial Viability through Bacterial RNA Augments T Follicular Helper Cell and Antibody Responses
Immunity, 2018Co-Authors: Gaetan Barbet, Leif E. Sander, Matthew Geswell, Irina Leonardi, Andrea Cerutti, Iliyan D. Iliev, J. Magarian BlanderAbstract:Summary Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial Viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2– monocytes instructed Tfh differentiation through interleukin-1β (IL-1β), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-β and IL-1β dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of Microbial Viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.
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Beyond pattern recognition: five immune checkpoints for scaling the Microbial threat.
Nature reviews. Immunology, 2012Co-Authors: J. Magarian Blander, Leif E. SanderAbstract:Pattern recognition by the innate immune system enables the detection of microorganisms, but how the level of Microbial threat is evaluated - a process that is crucial for eliciting measured antiMicrobial responses with minimal inflammatory tissue damage - is less well understood. New evidence has shown that features of Microbial Viability can be detected by the immune system and thereby induce robust responses that are not warranted for dead microorganisms. Here, we propose five immune checkpoints that, as defined here, collectively determine the gravity of Microbial encounters.
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erratum detection of prokaryotic mrna signifies Microbial Viability and promotes immunity
Nature, 2011Co-Authors: Leif E. Sander, Mark V. Boekschoten, Derk Amsen, Christopher C. Dascher, Bernard Ryffel, Joel A. Swanson, Michael Müller, Michael Davis, Magarian J BlanderAbstract:Nature 474, 385–389 (2011). In Fig. 1d of this Letter, the labels HKEC and EC were swapped in the print version. The lane labelled HKEC should be labelled EC and the lane labelled EC should be labelled HKEC. The error has been corrected online in the HTML and PDF versions.
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Detection of prokaryotic mRNA signifies Microbial Viability and promotes immunity
Nature, 2011Co-Authors: Leif E. Sander, Michael J. Davis, Mark V. Boekschoten, Derk Amsen, Christopher C. Dascher, Bernard Ryffel, Joel A. Swanson, Michael Müller, J. Magarian BlanderAbstract:Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense Microbial Viability through detection of a special class of Viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by Viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of Microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.
Hikaru Suenaga - One of the best experts on this subject based on the ideXlab platform.
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Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric Microbial Viability assay based on the reduction of water-soluble tetrazolium WST-1
European Journal of Clinical Microbiology & Infectious Diseases, 2015Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Munetaka Ishiyama, Masanobu Shiga, Takashi Ikegami, Kiyoshi MatsumotoAbstract:Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric Microbial Viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7–14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3–4 days using the WST-1 colorimetric method and those obtained after 10–14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.
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Determination of water-soluble vitamins using a colorimetric Microbial Viability assay based on the reduction of water-soluble tetrazolium salts.
Food Chemistry, 2011Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Munetaka Ishiyama, Kiyoshi MatsumotoAbstract:Abstract A method for the determination of water-soluble vitamins using a colorimetric Microbial Viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H -tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B 6 , biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B 6 in various foodstuffs. There was good agreement between vitamin B 6 concentrations determined after 24 h using the WST-8 colorimetric method and those obtained after 48 h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate.
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distinction of gram positive and negative bacteria using a colorimetric Microbial Viability assay based on the reduction of water soluble tetrazolium salts with a selection medium
Journal of General and Applied Microbiology, 2011Co-Authors: Tadayuki Tsukatani, Katsuya Noguchi, Hikaru Suenaga, Tomoko Higuchi, Masanobu Shiga, Kiyoshi MatsumotoAbstract:Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric Microbial Viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric Viability assay was carried out in a selection medium containing 0.5μg/ml crystal violet, 5.0 μg/ml daptomycin, and 5.0μg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.
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colorimetric Microbial Viability assay based on reduction of water soluble tetrazolium salts for antiMicrobial susceptibility testing and screening of antiMicrobial substances
Analytical Biochemistry, 2009Co-Authors: Tadayuki Tsukatani, Takatoshi Ezoe, Hikaru Suenaga, Tomoko Higuchi, Munetaka Ishiyama, Tetsuyuki Akao, Kiyoshi MatsumotoAbstract:The applicability of a colorimetric Microbial Viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antiMicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antiMicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.