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Publications (3)7.16 Total impact

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    ABSTRACT: To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens. Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl. perfringens with various germinants. When incubated in water at 90-100°C for 10-30 min, more than 90% of spores were inactivated but 50-80% retained their Ca(2+) -dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640-1680 cm(-1) (amide I) and 1230-1340 cm(-1) (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of T(lag) , ΔT(release) and ΔT(lys) , during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl. perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer T(lag) and ΔT(release) values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased T(lag) , ΔT(release) and ΔT(lys) values, as did wet heat-treated sleC spores germinating with dodecylamine. (i) Some proteins important in Cl. perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat. This study provides information on the germination of individual Cl. perfringens spores and improves the understanding of effects of wet heat treatment on spores.
    Journal of Applied Microbiology 07/2012; 113(4):824-36. · 2.39 Impact Factor
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    ABSTRACT: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Germination of individual wild-type Cl. perfringens spores was followed by monitoring Ca-dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T(lag)) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT(release)) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex-lytic enzyme (CLE) (sleC spores) exhibited the same features during GR-dependent germination, but with longer average T(lag) values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T(lag) of wild-type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild-type and sleC spores had identical average T(lag) and ΔT(release) values in dodecylamine germination that does not utilize GRs. Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE-deficient Bacillus spores, indicating that germination of these species' spores is not completely identical. This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.
    Journal of Applied Microbiology 08/2011; 111(5):1212-23. · 2.39 Impact Factor
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    ABSTRACT: Aims: To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens.Methods and Results: Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (T lag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔT release) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average T lag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The T lag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average T lag and ΔT release values in dodecylamine germination that does not utilize GRs.Conclusions: Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical.Significance and Impact of the Study: This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.
    Journal of Applied Microbiology 01/2011; 111(5). · 2.39 Impact Factor