Prodigiosin is a red bacterial pigment that exhibits antimicrobial activity against a wide range of microorganisms. Bacillus oleronius is a bacterium that is associated with rosacea, a long-term skin condition that affects the face and typically results in pimples, redness, and swelling of the skin. We explored the effect of prodigiosin on the growth of B. oleronius. Prodigiosin completely inhibited the growth of B. oleronius in a broth microdilution assay at 0.75 (0.289) μg/mL, lower than previously reported minimum inhibitory concentration values for other species of Bacillus. We also observed a rapid decline in the optical density of mid-log phase B. oleronius cultures immediately upon treatment with prodigiosin, suggesting that prodigiosin exerts bactericidal activity on actively dividing cells.

Rosacea is a facial skin disorder that causes a wide range of symptoms from persistent redness of the skin to eye irritation. It is a chronic inflammatory disease with a typical onset at age 30 or older^[1][2]. Rosacea is estimated to affect 5.46% of the global adult population^[3]. In the United States, an estimated 82% of rosacea cases remain untreated, resulting in long-term skin conditions that are sometimes characterized by cycles of remission and relapse^[4].

While the exact cause of rosacea is unknown, a growing body of literature suggests that the condition may be caused or exacerbated by the presence of a bacterium called Bacillus oleronius^[5]. Compared to the general population, rosacea patients are more likely to have circulating antibodies against B. oleronius, suggesting previous exposure to and immune response against the bacterium^[6][7][8]. Additionally, B. oleronius proteins have the ability to activate peripheral blood mononuclear cells and neutrophils, causing the release of pro-inflammatory cytokines^[9][10]. More generally, some antibiotics of the tetracycline and macrolide groups are effective in the treatment of some types of rosacea, suggesting that a bacterial agent may be involved in the pathogenesis of the disease^[11][12][13]. However, these antibiotics also have an anti-inflammatory response that likely alleviates rosacea symptoms^[12][13]. The potential association between B. oleronius and rosacea is further complicated by the bacterium’s commensal relationship with Demodex folliculorum, a parasitic mite that colonizes human skin and is also associated with rosacea^{[9][14][15][16]}. However, the relative contributions of D. folliculorum and B. oleronius to the pathology of rosacea have not yet been determined.

Prodigiosin is a red pigment that is produced by bacteria from a number of genera: Serratia, Hahella, Streptomyces, Saccharopolyspora, Zooshikella, Vibrio, and others in the class Gammaproteobacteria^[17][18]. In isolation, the pigment exhibits antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria^{[19][20][21][22]}. It is also been demonstrated to possess antifungal and antiprotozoal activity^[23][24] as well as the ability to prevent biofouling by marine barnacles^[25]. While many potential medical and technological applications for prodigiosin and related pigments have been identified, the biological role of prodigiosin for the bacterial species producing the pigment has not been determined^[26][27]. Prodigiosin production did not impact the virulence of Serratia marcescens in artificial infections of the model organism silkworm, Bombyx mori^[28].

Multiple species of bacteria from the genus Bacillus have exhibited sensitivity to prodigiosin^[19][25][22]. More specifically, prodigiosin treatment of Bacillus subtilis during the exponential phase caused immediate bacterial death and a corresponding decrease in the optical density of the culture^[22]. In a series of follow-up experiments by Danevčič et al. the mechanism of death was further defined. Using an ONPG/β-galactosidase substrate internalization assay, the researchers demonstrated that prodigiosin treatment resulted in cytoplasmic membrane leakage^[22]. Furthermore, a modified comet assay revealed the release of nucleic acid diffusion from the cells, a hallmark of cellular lysis^[22]. Herein we build on the work of Danevčič et al. and demonstrate the bactericidal effect of prodigiosin on B. oleronius.

We first became interested in exploring the prodigiosin sensitivity of B. oleronius after learning of the sensitivity of several Bacillus species to prodigiosin and related compounds^{[19][29][25][22]}. As a growing body of scientific evidence has implicated B. oleronius in the pathology of rosacea, we believed defining the sensitivity of B. oleronius to prodigiosin was scientifically warranted.

Prodigiosin and Topical Antibiotic Minimum Inhibitory Concentrations

In the microdilution assay, the average MIC of prodigiosin against B. oleronius was 0.75 (0.289) μg/mL and ranged between 0.5 μg/mL and 1 μg/mL in 4 independent experiments. This MIC is lower than the MICs that have been reported for other species in the genus Bacillus, which range between 5.2 μg/mL and 6.9 μg/mL in one study^[22] and 100 μg/mL in another study^[25]. As we utilized the same experimental methodology as Danevčič et al. for this experiment, our results are directly comparable^[30][22].

As a means of comparison, we also determined the MICs of common topical antibiotics against B. oleronius. The prodigiosin MIC was similar to the MICs of bacitracin, 0.5 (0) μg/mL and clindamycin, 1 (0) μg/mL, but it was greater than the MIC of neomycin, 32 (0) ng/mL.

Impact of Prodigiosin During Bacillus oleronius Exponential Growth Phase

Despite the apparent difference in prodigiosin sensitivity, prodigiosin exerts a similar bactericidal effect on B. oleronius as it does on B. subtilis when cells are treated during the exponential growth phase^[22]. We treated B. oleronius cultures with 4 concentrations of prodigiosin that bracket the 0.75 μg/mL MIC or an equivalent concentration of 0.05% (v/v) ethanol (Fig. 1). Bacterial growth in ethanol control remained identical to the untreated control throughout the 405 min growth curve. All of the prodigiosin treatments resulted in a reduction in culture density within 30 min of addition as measured by OD650. Both the 0.25 μg/mL and 0.5 μg/mL prodigiosin conditions maintained a positive growth trajectory following treatment, although both exhibited a marked reduction in growth rate compared to the ethanol control. The 1 μg/mL and 2 μg/mL prodigiosin conditions had even more dramatic effects on the culture density. The 1 μg/mL prodigiosin treatment resulted in the OD650 remaining unchanged for the duration of the experiment, indicating a complete inhibition of bacterial growth. The 2 μg/mL prodigiosin treatment resulted in a steady decline in culture density, similar to the 5.9 μg/mL prodigiosin treatment of exponential phase B. subtilis by Danevčič et al.. While we did not explore the nature of this decline in OD650, we hypothesize that the 2 μg/mL prodigiosin condition caused the lysis of B. oleronius as previously demonstrated for B. subtilis. Our observation builds on the body of scientific literature that demonstrates the potential medically relevant applications of prodigiosin.

In summation, we demonstrate that the average MIC of prodigiosin against B. oleronius was 0.75 (0.289) μg/mL in a broth microdilution assay. As previously demonstrated for B. subtilis^[22], prodigiosin exerts a bactericidal effect against B. oleronius when the compound is applied during the exponential growth phase.

Prodigiosin is produced by a number of bacterial genera. The compound used in our study was isolated from Gammaproteobacterium strain MS-02-063^[31], while Danevčič et al. extracted prodigiosin from Vibrio ruber strain DSM 14379^[22]. As the prodigiosin from these distinct species of bacteria has the same chemical structure, the results of our experiments should be comparable. However, the difference in prodigiosin source should be considered as a potential source of variation between the experiments.

Additionally, our experiments were conducted with a single strain of B. oleronius. In order to explore the potential for prodigiosin to be utilized in a clinical context for the treatment of rosacea, future experiments should include additional strains of B. oleronius that have been isolated from human skin.

As the presence of B. oleronius is associated with rosacea, future studies could examine the effectiveness of topical prodigiosin application for treatment of the skin condition. If tested, prodigiosin should be explored in isolation and in conjunction with other rosacea treatments.

Bacterial Strain and Media

The bacterial strain used in this study was Bacillus oleronius strain ATCC 700005 (ATCC, Manassas, VA, USA). Bacteria were cultured in Muller Hinton II (MHII) broth at 37°C (Becton Dickson and Company, Sparks, MD, USA).

Broth Microdilution Assay

Minimum inhibitory concentrations (MIC) for bacitracin, CAS 1405-87-4 (Acros Organics, Fair Lawn, NJ, USA), clindamycin, CAS 58207-19-5 (Tokyo Chemical Industry CO., Tokyo, Japan), neomycin, CAS 1405-10-3 (Fisher BioReagents, Fair Lawn, NJ, USA), and prodigiosin, CAS 82-89-3 (AdipoGen, San Diego, CA, USA) were determined using the broth microdilution assay described by the Clinical and Laboratory Standards Institute^[30]. Briefly, a 1:2 dilution series of prodigiosin was prepared in a 96-well microtiter plate. Overnight cultures of B. oleronius were diluted in MHII broth and inoculated to a final concentration of 5×104 CFU/well. The total volume in each microtiter well was 100 μL. For the prodigiosin experiment, the concentration of ethanol was maintained at 0.8% (v/v) in all conditions. MICs were determined by visual inspection following 16–18 h of static incubation at 37°C. Using unaided eye observation, we determined the MIC to be the lowest concentration of a compound that completely inhibited bacterial growth. The MIC values reported here represent the average of 4 independent experiments for prodigiosin and clindamycin and the average of 5 independent experiments for bacitracin and neomycin. The MIC values are reported in the following format: average (SD).

Growth Curve with Prodigiosin Addition

An overnight culture of B. oleronius was diluted 1:100 into fresh MHII broth and divided into 7 mL cultures. The freshly diluted cultures were grown at 37°C with shaking at 200 rpm. Bacterial growth was monitored by measuring optical density at 650 nm (OD650) using a Spectronic 20D (Milton Roy, Dallas, TX, USA). For the first 180 min, OD650 was measured at 60 min intervals. After 195 min of growth, either prodigiosin, an equivalent volume of ethanol, or plain MHII broth, was aseptically added to the cultures. The tubes were then returned to the shaking incubator for 30 min prior to the next OD650 measurement. Subsequently, culture density at OD650 was measured at 30 min intervals for an additional 210 min. The concentration of ethanol was maintained at 0.05% (v/v) in prodigiosin and ethanol control conditions. The growth curves reported here represent the average of 3 independent experiments.

The authors declare no conflicts of interest.

Not Applicable.

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