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

Abstract

DNA damage kills dry-heated spores of Bacillus subtilis, 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 B. subtilis and Bacillus megaterium 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 Ca²⁺-dipicolinic acid (CaDPA) depot; (ii) in most cases, dry-heat treatment increased the average times and variability of all major germination events in B. subtilis spore germination with nutrient germinants or CaDPA, and in one nutrient germination event with B. megaterium spores; (iii) B. subtilis 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.IMPORTANCE 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 Bacillus subtilis 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 B. subtilis and Bacillus megaterium 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.

Keywords: Bacillus; dry-heat treatment; germination; outgrowth; spores; vegetative growth.

FIG 1

Effects of dry-heat treatment on B. subtilis spores. (A) Percentages of spores that survived and retained CaDPA as a function of treatment time at 140°C were determined as described in Materials and Methods. (B) Raman spectra of single spores of B. subtilis that retained CaDPA after various dry-heat treatment times. (C) Raman spectra of single spores of B. subtilis in the protein amide band regions. Band intensities are given in arbitrary units (AU).

FIG 2

l-Valine germination of B. subtilis spores after dry-heat treatment. (A) The percentage of the germination as a function of incubation time of untreated and dry-heat-treated spores with l-valine was determined as described in Materials and Methods. Approximately 300 spores were monitored to obtain the data shown. (B, C, D) Kinetics of the l-valine germination of individual untreated (B) and dry-heat-treated (C and D) B. subtilis spores. Spores were dry-heat treated and germinated with l-valine, and the germination of 10 individual spores was followed, all as described in Materials and Methods. Image intensities are given in arbitrary units (AU).

FIG 3

AGFK germination of B. subtilis spores after dry-heat treatment. (A) Spores with and without various dry-heat treatments at 140°C were germinated with AGFK, and the germination of 400 individual spores was followed as described in Materials and Methods. (B, C, D) AGFK germination of 10 individual spores with and without dry-heat treatment at 140°C for (B) 0, (C) 30, or (D) 60 min was monitored as described in Materials and Methods. Image intensities are given in arbitrary units (AU).

FIG 4

CaDPA germination of B. subtilis spores with and without dry-heat treatment. (A) CaDPA germination of spores given various treatments at 140°C was followed, and 400 individual spores were examined as described in Materials and Methods. (B, C, D) CaDPA germination of 10 single spores after dry-heat treatment at 140°C for (B) 0, (C) 30, or (D) 60 min, respectively. Germination was followed as described in Materials and Methods. Image intensities are given in arbitrary units (AU).

FIG 5

Dodecylamine germination of B. subtilis spores with and without dry-heat treatment. (A) Germination after various dry-heat treatment times with dodecylamine, with >300 spores examined for each condition as described in Materials and Methods. (B, C, D) Dodecylamine germination of 10 single spores after dry-heat treatment at 140°C for (B) 0, (C) 30, or (D) 60 min, respectively. Germination was followed as described in Materials and Methods. Image intensities are given in arbitrary units (AU).

FIG 6

Glucose germination of B. megaterium spores with and without dry-heat treatment. (A) Glucose germination of spores dry-heat treated at 140°C for various times. More than 500 spores were examined for each treatment as described in Materials and Methods. (B, C, D) Glucose germination of 10 single spores after dry-heat treatment at 140°C for (B) 0, (C) 30, or (D) 60 min, respectively, was followed as described in Materials and Materials and Methods. Image intensities are given in arbitrary units (AU).

FIG 7

Germination, outgrowth, and growth of individual B. subtilis spores with and without dry-heat treatment. Time-lapse images of single B. subtilis spores given various treatments at 140°C that were then incubated in LB medium agar at 37°C were recorded as described in Materials and Methods. (A) Time-lapse images of single spores without dry-heat treatment. (B, C) Time-lapse images of single spores after dry-heat treatments at 140°C for 7 min and 30 min, respectively. The two arrows in A and B indicate two individual spores that germinated, outgrew, and began vegetative growth. Note that in C, spores that germinated did not go through outgrowth, even after 15 h. Bar, 5 μm.