Lack of efficient killing of purified dormant spores of Bacillales and Clostridiales species by glycerol monolaurate in a non‐aqueous gel

Abstract

Significance and Impact of the Study: Given potential deleterious effects of spores of Bacillales and Clostridiales, there is an ongoing interest in new ways of spore killing. A recent paper (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of many species. We now find that (i) the Bacillus subtilis spores prepared as in the previous report were impure and (ii) GMLg gave no killing of purified spores of Bacillales and Clostridiales species in ≤5 h under the published conditions. Thus, GMLg is not an effective sporicide, though may prevent spore germination or kill germinated spores. AbstractInactivation of Bacillales and Clostridiales spores is of interest, since some cause food spoilage and human diseases. A recent publication (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of Bacillus subtilis, Bacillus cereus and Clostridioides difficile, and Bacillus anthracis spores to a lesser extent. We now show that (i) the B. subtilis spores prepared as in the prior work were impure; (ii) if spore viability was measured by diluting spores 1/10 in GMLg, serially diluting incubations 10-fold and spotting aliquots on recovery plates, there was no colony formation from the 1/10 to 1/1000 dilutions due to GMLg carryover, although thorough ethanol washes of incubated spores eliminated this problem and (iii) GMLg did not kill highly purified spores of B. subtilis, B. cereus, Bacillus megaterium and C. difficile in 3-20 h in the conditions used in the recent publication. GMLg also gave no killing of crude B. subtilis spores prepared as in the recent publication in 5 h but gave~1Á5 log killing at 24 h. Thus, GMLg does not appear to be an effective sporicide, although the gel likely inhibits spore germination and could kill spores somewhat upon long incubations.

What’s new and notable in bacterial spore killing!

“…A common problem in spore killing research is the use of spore preparations that are not especially pure, with large amounts of debris remaining from lysed mother cells or cells which did not sporulate (Green et al 2020 ; Schlievert et al 2018 ). This debris can include nucleic acids, proteins and cell wall fragments, and this can interfere with spore killing by radiation, heat and chemicals, such that reproducibility of killing regimens between labs can be poor and the design of regimens for spore killing potentially more difficult than it ought to be.…”

“…However, the situation with chemical agents can be more complicated, as the agent can adsorb to dormant spores and then kill the spores when they are exposed to germinants and nutrients to allow measurements of spore killing. Indeed, there have been agents suggested to kill dormant spores, but actually do not, as when the agent was either neutralized or well removed from the treated dormant spores, they were fully alive; one example being illustrated in Green et al ( 2020 ). Thus, in analysis of any chemical treatment of spores, it is essential that the chemical be either neutralized or removed prior to spores’ conversion to the much less resistant germinated spores.…”