D-cycloserine or similar physiochemical compounds may be uniquely suited for use inBacillus anthracisspore decontamination strategies

Abstract

Aims: As observed in the aftermath of the anthrax attacks of 2001, decontamination and remediation of a site contaminated by the accidental or intentional release of Bacillus anthracis spores is difficult, costly and potentially damaging to the environment. The identification of novel strategies that neutralize the threat of spores while minimizing environmental damage remains a high priority. We investigated the efficacy of D-cycloserine (DCS), an antibiotic and inhibitor of the spore-associated enzyme (alanine racemase) responsible for converting L-alanine to D-alanine, as a spore germination enhancer and antimicrobial agent. Methods and Results: We characterized the impact of DCS exposure on both germinating spores and vegetative cells of fully virulent B. anthracis by evaluating spore germination kinetics, determining the minimum inhibitory concentrations (MICs) required to affect growth of the bacteria and performing macrophage viability assays. DCS enhanced germination induced by L-alanine and also efficiently killed the newly germinated spores. Furthermore, DCS proved nontoxic to macrophages at concentrations that provided protection from the killing effects of spores. Similar tests were conducted with Bacillus thuringiensis (subspecies kurstaki and Al Hakam) to determine its potential as a possible surrogate for B. anthracis field trials. Bacillus thuringiensis spores responded in a similar manner to B. anthracis spores when exposed to DCS. Conclusions: These results further support that DCS augments the germination response of spores in the presence of L-alanine but also reveal that DCS is bactericidal towards germinating spores. Significance and Impact of the Study: DCS (or similar compounds) may be uniquely suited for use as part of decontamination strategies by augmenting the induction of spore germination and then rendering the germinated spores nonviable.

Germination of Spores of Bacillus Species: What We Know and Do Not Know

“…While the inhibition or stimulation of spore germination in applied settings is somewhat off the focus of this review, these topics have attracted renewed interest in recent years because of the need for effective decontamination regimens for spores of organisms such as C. difficile and B. anthracis but without the concerns unique to the food industry. Thus, there are some recent reports of promising results in using a germination step prior to spore decontamination for promoting inactivation of B. anthracis and C. difficile spores (80)(81)(82), as well as enzymatic spore coat removal by lytic enzymes such as lysozyme to allow spore killing (83). Several compounds have also been identified that may be effective in inhibiting the germination or outgrowth of spores of organisms such as B. anthracis and C. difficile (84)(85)(86)(87)(88), and perhaps compounds analogous to these could be useful in applied settings.…”

Comparison of sampling methods to recover germinatedBacillus anthracisandBacillus thuringiensisendospores from surface coupons

“…When compared to more nutritionally complex media, previous studies in our lab showed that a germination‐induction solution consisting of 10 mmol l −1 l ‐alanine and 10 mmol l −1 inosine (AI) was sufficient for effective spore germination (Omotade et al . ). In those studies, germination‐induction solutions for B. anthracis and B. thuringiensis spores were prepared in WFI and PBS respectively.…”

“…; Omotade et al . ). All quantification was performed using serial dilution and plating of samples on sheep's blood agar (SBA) plates (Remel‐Thermo Scientific, Lenexa, KS), which were incubated at 37°C.…”

The impact of inducing germination ofBacillus anthracisandBacillus thuringiensisspores on potential secondary decontamination strategies

“…; Omotade et al . ). Accordingly, we characterized the effects of several germination media of various nutritional complexities to identify advantages or disadvantages associated with each medium.…”

“…, ; Omotade et al . ) and has been shown to be generally nonhazardous to humans through extensive testing as a widespread commercial insecticide (Green et al . ; Bernstein et al .…”