Germination, Outgrowth, and Vegetative-Growth Kinetics of Dry-Heat-Treated Individual Spores of Bacillus Species

Abstract

DNA damage kills dry-heated spores of , but dry-heat-treatment effects on spore germination and outgrowth have not been studied. This is important, since if dry-heat-killed spores germinate and undergo outgrowth, toxic proteins could be synthesized. Here, Raman spectroscopy and differential interference contrast microscopy were used to study germination and outgrowth of individual dry-heat-treated and spores. The major findings in this work were as follows: (i) spores dry-heat-treated at 140°C for 20 min lost nearly all viability but retained their Ca-dipicolinic acid (CaDPA) depot; (ii) in most cases, dry-heat treatment increased the average times and variability of all major germination events in spore germination with nutrient germinants or CaDPA, and in one nutrient germination event with spores; (iii) spore germination with dodecylamine, which activates the spore CaDPA release channel, was unaffected by dry-heat treatment; (iv) these results indicate that dry-heat treatment likely damages spore proteins important in nutrient germinant recognition and cortex peptidoglycan hydrolysis, but not CaDPA release itself; and (v) analysis of single spores incubated on nutrient-rich agar showed that while dry-heat-treated spores that are dead can complete germination, they cannot proceed into outgrowth and thus not to vegetative growth. The results of this study provide new information on the effects of dry heat on bacterial spores and indicate that dry-heat sterilization regimens should produce spores that cannot outgrow and thus cannot synthesize potentially dangerous proteins. Much research has shown that high-temperature dry heat is a promising means for the inactivation of spores on medical devices and spacecraft decontamination. Dry heat is known to kill spores by DNA damage. However, knowledge about the effects of dry-heat treatment on spore germination and outgrowth is limited, especially at the single spore level. In the current work, Raman spectroscopy and differential interference contrast microscopy were used to analyze CaDPA levels in and kinetics of nutrient- and non-nutrient germination of multiple individual dry-heat-treated and spores that were largely dead. The outgrowth and subsequent cell division of these germinated but dead dry-heat-treated spores were also examined. The knowledge obtained in this study will help understand the effects of dry heat on spores both on Earth and in space, and indicates that dry heat can be safely used for sterilization purposes.

Single-cell analysis reveals individual spore responses to simulated space vacuum

“…As seen previously with dry heat- or wet heat-killed spores, 20 , 35 , 36 after high vacuum exposure non-viable spores also retained CaDPA and did not release CaDPA upon suspension in water. These results indicate that high vacuum treatment does minimal damage to spores’ IM permeability barrier that retains CaDPA.…”

“…Finally, it is notable that there was no detectable outgrowth of high vacuum-killed spores, just as seen recently with dry heat-killed B. subtilis spores. 35 , 36 This may be significant from an applied perspective, since if high vacuum-killed spores don’t outgrow, they will thus likely not synthesize any proteins. This could be especially important with spores from pathogenic bacteria, such as B. anthracis and Clostrdium botulium .…”

“…Spores of B. subtilis strains were prepared at 37 °C on double strength Schaeffer’s (2×SG) medium agar plates and B. cereus spores were also prepared on plates, all as described previously. 35 – 37 All purified spores were stored at 4 °C in water protected from light and were >98% free from growing or sporulating cells, germinated spores and cell debris as was observed by phase-contrast microscopy. In some experiments, B. subtilis sleB spoVF spores that make stable DPA-less spores were sporulated on plates with 100 μg/ml DPA.…”

“… 31 PS832 Wild type Laboratory 168 strain PS533 Wild type Ref. 35 PS578 sspA sspB α − β − PS283 sspA Lacking the α/β-type SASP SspA PS338 sspB Lacking the α/β-type SASP SspB PS355 sspA sspB α − β − PS483 sspE Lacking the γ-type SASP SspE, γ − PS482 sspA sspB α − β − γ − PS1899 dacB Increased spore core water content PS2211 dacB sspA sspB Increased spore core water content, α − β − FB122 sleB spoVF No spore CaDPA FB122 (+DPA) sleB spoVF Some spore CaDPA (sporulation with DPA) PS3664 sleB spoVF sspA sspB No spore CaDPA and α − β − PS3664 (+DPA) sleB spoVF sspA sspB Some spore CaDPA (sporulation with DPA), α − β − PS3722 ligD Ku Non-homologous DNA end joining defect PS3751 ligD Ku sspA sspB Non-homologous end DNA joining defect, α − β − PERM454 exoA nfo Apurinic/apyrimidinic endonucleases defect PERM450 exoA nfo sspA sspB Apurinic/apyrimidinic endonucleases defect, α − β − PS2496 mrgA DNA-binding stress protein defect PS2507 mrgA sspA sspB DNA-binding stress protein, α − β − PS2558 katX Lacks spore catalase ...…”

Effects of the microbicide ceragenin CSA‐13 on and properties ofBacillus subtilisspores prepared on two very different media

“…Inner membrane GR‐dependent germination of one batch each of multiple individual spores prepared on 2xSG or LB plates was also carried out and monitored by differential interference contrast (DIC) microscopy essentially as described previously (Wang et al ; He et al ). Briefly, heat‐activated spores (~1μl of ~10 8 spores per ml −1 in water) were spread on the surface of a microscope coverslip that was dried in a vacuum desiccator for 5–10 min, and the coverslip was mounted on and sealed to a microscope sample holder held at 37°C.…”