Analysis of the germination kinetics of individual Bacillus subtilis spores treated with hydrogen peroxide or sodium hypochlorite

Abstract

Significance and Impact of the Study: This work shows that with Bacillus subtilis spore populations in which approximately 95% of individual spores were killed by several oxidizing agents, >95% of the spores in these populations germinated with nutrients, albeit slowly. This is important, as assay of an early germination event, release of dipicolinic acid, has been suggested as a rapid assay for spore viability and would give false-positive readings for the level of the killing of oxidizing agent-treated spore populations. Analysis of the germination kinetics of multiple individual untreated or oxidizing agenttreated spores also provides new information on proteins damaged by oxidizing agent treatment, and at least some of which are in spores' inner membrane. AbstractMore than 95% of individuals in populations of Bacillus subtilis spores killed approximately 95% by hydrogen peroxide or hypochlorite germinated with a nutrient, although the germination of the treated spores was slower than that of untreated spores. The slow germination of individual oxidizing agent-treated spores was due to: (i) 3-to 5-fold longer lag times (T lag ) between germinant addition and initiation of fast release of spores' large dipicolinic acid (DPA) depot (ii) 2-to 10-fold longer times (DT release ) for rapid DPA release, once this process had been initiated; and (iii) 3-to 7-fold longer times needed for lysis of spores' peptidoglycan cortex. These results indicate that effects of oxidizing agent treatment on subsequent spore germination are on: (i) nutrient germinant receptors in spores' inner membrane (ii) components of the DPA release process, possibly SpoVA proteins also in spores' inner membrane, or the cortex-lytic enzyme CwlJ; and (iii) the cortex-lytic enzyme SleB, also largely in spores' inner membrane. This study further indicates that rapid assays of spore viability based on measurement of DPA release in spore germination can give false-positive readings.

Microbial Growth under Supercritical CO 2

“…S8A in the supplemental material). Second, it has been shown that certain stresses, such as pH and oxidative stresses, significantly extend the lag time for germination of Bacillus spores (55,56). We have observed extended lag phases for growth of MIT0214 under 1 atm CO 2 compared to 1 atm N 2 (see Fig.…”

“…First, germination frequencies under CO 2 headspaces are known to be significantly lower than those under aerobic or anaerobic nitrogen headspaces, with only 10 to 30% of B. cereus spores germinating under 1 atm CO 2 (55). Growth dynamics for isolate B. cereus MIT0214 (see Fig.…”

Killing the spores of Bacillus species by molecular iodine

“…However, DPA release from the I 2 ‐treated spores exposed to nutrient germinants is slower than that of untreated spores, as seen with B. subtilis spores treated with other oxidizing agents (Setlow et al . ). This slowed DPA release in germination conditions by I 2 ‐treated spores seems likely due in part due to inactivation of spores’ IM germinant receptors (GRs) that recognize the nutrient germinants (Setlow ), since the rate of germination with AGFK via the GerB and GerK GRs was decreased much more than with l ‐valine via the GerA GR, as reported for spores treated with another oxidizing agent (Setlow et al .…”

Evaluation of the Efficacy of a Hydrogen Peroxide Disinfectant

“…The results obtained in this study, for each microorganism, show that the inhibitory effect of the disinfectant is directly influenced by the exposure time; for this case, it is recommended to use the highlevel hydrogen peroxide-based disinfectant, STERIS, at a concentration of 2%, with a contact time of 5 min, as established by the producer. Several studies [10,[13][14][15], in which the microorganisms Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus were used as a control, showed that hydrogen peroxide is 100% effective against these microorganisms at a concentration of 3% with a time of 5 min contact for vegetative bacteria and more than 2 h for the sporulated microorganism. The same effect was achieved in the present study, compared with all the strains tested, with the same time but at a lower concentration of the product at 2%.…”