The emergence of antibiotic resistance has prompted scientists to explore medicinal plants, not only to ascertain claims of efficacy and safety, but also to develop phytochemical based new drugs  as  alternatives to antibiotics. Plants of the genus Capsicum, colloquially referred to as chile peppers, have been widely grown for food and medicinal purposes for centuries. It is believed that   the   Aztec   and   Tarahumara  peoples  used  chile peppers as herbal remedies for coughs and bronchitis. Chile peppers have traditionally been used as antiseptics in African cultures, and in gastrointestinal and anti-inflammatory applications in India (Corson and Crews, 2007; Cichewicz and Thorpe, 1996). Capsaicin, the major bioactive molecule in Capsicum, has garnered medical attention due to its analgesic, anticancer, cardioprotective and antimicrobial properties (Omolo et al., 2014; Dorantes et al., 2008).

Additionally, capsaicin provides a unique characteristic and pungent taste to each variety of Capsicum. All plants of the Capsicum genus produce varied amount of capsaicin, however, only a few chile pepper varieties have been scientifically examined for their antimicrobial properties against bacterial and fungal pathogens. The antimicrobial activity of chile pepper extracts have been tested on a small selection of Capsicum annum, Capsicum baccatum, Capsicum frutescens, Capsicum pubescens, and Capsicum chinense cultivars, representing some of the milder members of the genus Capsicum (Cichewicz and Thorpe, 1996; Sanatombi and Sharma, 2008; Al Othman et al., 2011; Dorantes et al., 2000; Bacon et al., 2017). Only the Habanero variety of the most pungent Capsicum species, C. chinense, has been examined for bactericidal activity. Similarly, C. chinense varieties with extremely high capsaicin concentrations, such as the Trinidad Moruga Scorpion, Trinidad Douglah, Trinidad 7-Pot, Trinidad Scorpion, and Bhut Jolokia (“Ghost Pepper”) have never been tested for medicinal use (Omolo et al., 2014; Mills-Robertson et al., 2012).

The “hotness” of chile peppers has historically been expressed in Scoville Heat Units (SHUs), which was determined by a panel of human taste testers according to the Scoville organoleptic test. Once analyzed according to this method, chile peppers are sorted by their degree of pungency. There are five levels of pungency classification on the Scoville scale: non-pungent (0 – 700 SHU), mildly pungent (700 – 3,000 SHU), moderately pungent (3,000 – 25,000 SHU), highly pungent (25,000 – 80,000 SHU) and very highly pungent (>80,000 SHU) (Al Othman et al., 2011).

The organoleptic method is neither precise nor objective, and has led to the advancement of analytical methods to determine the capsaicin content of chile peppers. One such method is an antibody based capsaicin assay determines the amount of capsaicinoids present in peppers through comparisons to known standards in 96 micro-well plates and the use of a spectrophotometer.

Alternatively, Liquid Chromatography (LC) and High- Performance Liquid Chromatography (HPLC)  have  been used to determine capsaicin content in peppers (Perkins et al., 2002; Sanatombi and Sharma, 2008; Al Othman et al., 2011). These analytical methods are more time consuming and costly than antibody-based assays. The antibody based capsaicin assay was selected for this study as it is a high throughput, rapid method that has demonstrated reliability of capsaicin content determinations for whole fruits in numerous other studies (Perkins et al., 2002; Stewart et al., 2007; Rollyson et al., 2014; Kantar et al., 2016).

Monitoring cellular respiration is a common way to assess the viability of cell cultures. This practice uses biochemically active compounds that change color in the presence of electrons generated by cellular respiration. Resazurin is a deep blue, tetrazolium-based, non-toxic, oxidation-reduction dye that is reduced intracellularly to the pink compound, resorufin, by electron-generating enzymes involved in microbial respiration (Ahmed et al., 1994; O’Brien et al., 2000; Byth et al., 2001; WÄ™sierska-GÄ…dek et al., 2005; Fai and Grant, 2009).

The resazurin assay is non-destructive to the cell, and is useful for monitoring the viability of numerous microorganisms under environmental stressors (Byth et al., 2001; Twigg, 1945; Liu, 1989; Guerin et al., 2001; Mariscal et al., 2009). The assay is simple, sensitive, rapid, robust, reliable, relatively inexpensive, and serves to assess antibacterial properties of natural products such as plant compounds (Zhi-Jun et al., 1997; Gloeckner et al., 2001; Sarker et al., 2007; Vega-Avila and Pugsley, 2011; Rampersad, 2012).

In this study, the resazurin assay was used as an indicator of cell viability after exposure to chile pepper extracts, to test 29 previously untested chile pepper varieties for their antimicrobial properties. The 29 chile pepper extracts were tested against several pathogenic organisms: the Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, Gram-negative bacteria Escherichia coli O157:H7 and Salmonella Typhimurium, and the pathogenic yeast Candida albicans.

Presently, there has been no report where the capsaicin contents of the fruits of several Capsicum cultivars have been correlated with antimicrobial activity against bacterial and fungal pathogens. Many of the Capsicum varieties regarded as the “hottest in the world” have never been examined for antimicrobial properties.

Therefore, the unique focus of the current study was to cultivate 29 varieties of Capsicum, including the hottest known varieties, together in a controlled environment and study the correlation of capsaicin contents with antimicrobial potential against bacterial and fungal pathogens.  We  identified  a  Capsicum  variety  with low capsaicin content and showed bactericidal activity, very similar to the variety with highest capsaicin content. These results of the current study provide new direction to explore derivatives of capsaicin or additional bioactive molecules with applications in the treatment of topical and digestive human diseases caused by bacterial and fungal pathogens.

The resazurin dye powder was obtained from Difco Inc. (Corpus Christi, TX). The Capsaicin Plate Kit (Cat. # 20-0027) was purchased from Beacon Analytical Systems Inc. (Saco, ME). The capsaicin powder (99.1% pure) was purchased from Chem-Impex Int’l Inc. (Wood Dale, IL) and stored at 4°C. SSB14B stainless steel beads (0.9-2.0 mm diameter) were purchased from Thomas Scientific (Swedesboro, NJ), for tissue homogenization using a Gold Bullet Blender (Next Advance, Inc., Averill Park, NY). BBLTM tryptic soy agar (TSA), BectoTM tryptic soy broth (TSB), yeast extract peptone dextrose (YPD) media and BBLTM brain heart infusion (BHI) broth were purchased from Becton, Dickinson and Company (Sparks, MD).

Cultivation of pepper plants

Seeds of 29 pepper varieties were purchased from various seed companies across North America (Table 1) to explore their phenotypic diversity. Seed samples were procured from different geographic regions, and represent two different Capsicum species (C. chinense and C. annuum) (Table 1). Seed were sown 0.6 cm deep in Master Garden Premium potting mix (Premier Tech Horticulture, Ltd., Ontario, Canada) in a 32-cell tray (volume = 150 cm³). Trays were covered with a clear plastic lid (approx. 10 cm above media) and were placed in a greenhouse maintained at 23°C air temperature. When seeds germinated and cotyledons unfolded, the lid was removed and plants were grown in the same environment for an additional 9-14 weeks under natural daylight plus 25 μmol×m^-2×s^-1 supplemental irradiance (0800-0200 HR; Sunblaze T5 fluorescent lights, Sunlight Supply, Inc., WA, USA; +1.62 mol m^-2 d^-1). Plants fertilized with Pure Blend Pro Grow (3-2-4), Pure Blend Pro Bloom (2-3-5), and Cal-Mag Plus (all from Botanicare, AZ) for a ten-week period following the manufacturer’s recommendations. All the chile pepper plants were transplanted in the University of Minnesota agricultural fields in June of 2015, developed by applying Sustane 4-6-4 Organic Plant Fertilizer (Sustane Natural Fertilizer, Inc., MN)  fertilizers as recommended by Bosland et al (Paul et al., 2012). Fruits were harvested in September 2014 (Figure 1). All the ripened pepper fruits were harvested from three plants of each chile pepper cultivar variety and stored at -20°C.

Microbiological methods

L. monocytogenes Scott A, Salmonella Typhimurium LT2, and Escherichia coli O157:H7 EDL933 bacterial cultures were obtained from the Food Safety Microbiology Laboratory (Department of Food Science and Nutrition), while the S. aureus 6538 and C. albicans ATCC 10231 cultures were obtained from the Hegeman Laboratory (Department of Horticultural Science), at the University of Minnesota – Twin Cities (St. Paul, MN).

L. monocytogenes was streak plated on BHI agar media, while the other bacterial strains were plated on TSA agar medium.

Candida albicans was grown in YPD agar medium. The plates were incubated overnight at 37°C for bacterial strains and at 30°C for C. albicans, and subsequently stored at 4°C. The test cultures were prepared by inoculating 5 ml of respective broth media and incubating overnight at 37°C for bacterial strains and at 30°C for C. albicans with shaking at 150 rpm.

Preparation of chile pepper extracts

The 29 chile pepper varieties included 24 very highly pungent varieties (samples 1 - 24) and five non-pungent varieties (samples 25 - 29) (Figure 1). For each variety, one pepper fruit was randomly selected from three different plants of each variety and treated independently, providing a total of 87 chile pepper extracts. The samples were prepared by crude methanol extraction as previously described (Beacon Analytical Systems, 2011), and this method was selected as methanol extractions of chile peppers have been shown to exhibit the greatest antimicrobial activity (Bacon et al., 2017). Whole pepper fruits were briefly washed under running tap water and dried with paper towels. 5 g of pepper fruits were cut into small pieces of approximately 5 mm and transferred to a 50 ml conical tube containing eight stainless steel beads, and homogenized in a Bullet Blender tissue homogenizer for 12 min to make a paste. The resultant pepper paste was mixed with 12.5 ml of 100% methanol and the slurry was further homogenized for 12 min. The methanolic fraction without the cell debris was centrifuged for 23 min at 4,000 rpm. The resultant pepper extracts were filter-sterilized through a 0.2 µm Acrodisc filter (Pall Corporation, NY). The methanolic pepper extracts were diluted 1:2 using sterile deionized water and stored at 20°C until used for the assays. The capsaicin contents of all the 87 extracts were determined using the Capsaicin Plate Kit, as per the manufacturer’s instructions (Beacon Analytical Systems, 2011).

For the resazurin based cell viability assays, 0.02% (w/v) resazurin dye was prepared in deionized water, filter-sterilized through a 0.2 µm Acrodisc syringe filter and stored at 4°C until use (Sittampalam et al., 2014). For each test, 50, 25, 12.5, and 6.25 µl of 1:2 diluted pepper extract were added into each well of a sterile 96 well microtiter plate and diluted with BHI for L. monocytogenes, or TSB for all other test microbes to obtain 1:4, 1:8, 1:16 and 1:32 dilutions respectively. The resulting volume in each test well was 100 µl.

The microbial inoculum was prepared from an overnight culture by adjusting the OD₆₀₀ to 0.08 or 0.13, representing approximately 5 × 10⁶ or 5 × 10⁷ CFU/ml yeast and bacteria cells respectively. 100 µl of the bacterial or yeast cell inoculum was pipetted into each test well. As a positive control, an equal amount of methanol was mixed with BHI broth for L. monocytogenes and TSB for all other microbes and was pipetted into the positive control well and diluted with sterile broth media to a volume of 100 µl. 100 µl of inoculum was then added for a total volume of 200 µl. 100 µl of the appropriate broth was pipetted into the growth control well, along with 100 µl of inoculum. An equivalent amount of the methanol in the samples used was pipetted into the negative control well. The methanol was diluted with the appropriate broth to a total of 200 µl.  20 µl of the 0.02% resazurin dye was added into each well. The plates were incubated for 2 h at 37°C, photographed, and the results were recorded based on change in color from deep blue to pink or purple. The changes in color were observed and scored as 1 (no change in resazurin color), 0.5 (changed to indigo/purple) and 0.0 (changed to pink). Averages < 0.5 are reported as 0. Averages <1 but ≥ 0.5 are reported as 0.5. Averages ≥ 1 are reported as 1.

Statistical analysis

A  student’s  t-test  analysis  was  used  to  determine  the statistical significance of the difference between samples, and between samples and standards.

The Beacon Capsaicin Plate Kit assay is a polyclonal antibody assay specific for capsaicin determination of chile pepper fruits with reactivity to a limited number of closely related compounds (e.g. other capsaicinoids). Capsaicin is the most abundant capsaicinoid in chile peppers. The other capsaicinoids include dihydro-capsaicin, nordihydrocapsaicin, homodihydrocapsaicin and  homocapsaicin.  It  has  been established that milder varieties have capsaicinoid concentrations ranging from 0.003- 0.010% (w/w). Slightly hot varieties have capsaicinoids ranging from 0.01- 0.30% (w/w), while the hot varieties have a concentration range of 0.3- 1.0% (w/w) (Barbero et al., 2006). Based on this classification, the samples used herein from the C. chinense species were determined to be highly pungent, or very highly pungent. The samples from the C. annuum species were all non-pungent.

The amount of capsaicinoids in a chile pepper pod is dependent on the genetic makeup of the plant, the environment in which the plant is grown, the age of the fruit, and the position of the fruit on the plant. The heat level can vary considerably between plants of the same variety grown in a single field at the same time (Al Othman et al., 2011). When single plant heat levels of genetically identical chile pepper  plants  were  compared with the field average, it was found that individual plants could possess heat levels as much as 78% higher than the field average, indicating that plant to plant variation of an individual variety contributes to the chile pepper heat levels (Bacon et al., 2017). Similar variations were observed in the data obtained from the capsaicin assay in this study (Figure 2).

Capsaicinoid biosynthesis is unique to the genus Capsicum and results from the acylation of the aromatic compound, vanillylamine, with a branched-chain fatty acid. The presence of capsaicinoids is controlled by the Pun1 locus, which encodes a putative acyltransferase. In its homozygous recessive state, pun1/pun1, capsaicinoids are not produced by the pepper plant (Rollyson et al., 2014). The trace amounts of capsaicin in the “sweet” chile peppers were most likely because ancestral  chile  peppers  all  produced capsaicin, and the “sweet” chile varieties can revert to this ancestral trait.

The capsaicin data obtained in this study was used to determine whether there is a correlation between capsaicin content and bactericidal effects of the chile pepper extracts. While previous research suggests that capsaicin is the primary antimicrobial compound in chile peppers, data from this study implies that there might be other compounds or peptides with antimicrobial properties. Although Tobago Scotch Bonnet Red had very little capsaicin compared to the other samples of the C. chinense species, this cultivar showed some antimicrobial effects. This finding was unique because the other samples that had little capsaicin, but belonged to the C. annuum species, showed no antimicrobial properties. Since bactericidal activity of pure capsaicin was observed against all test organisms at >250 ppm (4,000 SHU), chile peppers lower than 4,000 SHU exhibiting partial or full bactericidal effect likely contain other antimicrobial compounds.

In this case, the Tobago Scotch Bonnet Red variety likely contains non-capsaicinoid antimicrobial compounds or peptides. Peptides isolated from the seeds of the habanero variety C. chinense have demonstrated antimicrobial effects against S. aureus, E. coli and other pathogenic organisms (Brito-Argaez et al., 2009). Such antimicrobial   peptides   may   be   responsible    for   the inhibitory effects observed in the Tobago Scotch Bonnet Red cultivar. Additional studies to isolate and determine antimicrobial properties of these chemicals are underway.

Figure 3 summarizes the average results from the resazurin assay at the various dilution levels of chile pepper extracts against all organisms. Viable cells with active metabolism can reduce resazurin dye into the resorufin product, resulting in a color change in the test wells from dark blue to purple or pink. The quantity of resorufin produced is proportional to the number of viable cells (Promega Corporation, 2016). Therefore, a purple color in a test well implies fewer viable and consequently, fewer metabolically active cells.

S. aureus was tested against four different dilution levels of the pepper samples (1:4, 1:8, 1:16, and 1:32). At the 1:4 dilutions, 16 of the samples high in capsaicin showed total bactericidal effects, while 7 varieties showed partially bactericidal effects. When the samples were diluted 1:8, five of the samples high in capsaicin were partially bactericidal, while the rest showed no observable effects. At the 1:16 dilution, only Bhut Jolokia affected cellular viability of S. aureus. No bactericidal effects were observed for any of the samples at the 1:32 dilution.

None of the samples showed total bactericidal effects against  Salmonella  Typhimurium  at  the  concentrations tested. Instead, a partial bactericidal effect was observed (at the 1:4 dilution), indicated by a resazurin change in color to purple for 23 samples. A similar effect was noted for E. coli O157:H7 with a partial bactericidal effect observed for the same 23 samples.

Against L. monocytogenes, 9 samples had a bactericidal effect on the cells at the 1:4 dilution, while 14 samples were partially bactericidal. At the 1:8 dilution, 5 samples, Bhut Jolokia, Trinidad Scorpion, Brown Trinidad Moruga Scorpion, Trinidad Scorpion Chocolate and Habanero Orange Blob, exhibited partial bactericidal effects.

C. albicans showed greatest susceptibility to the samples with 14 samples showing a partial bactericidal effect at the 1:16 dilution. 11 samples diluted as much as 1:32 were still partially bactericidal to C. albicans cells. At the 1:4 and 1:8 dilutions, the methanol concentrations were high enough to kill the C. albicans cells, so the data for these two tests were included for completion only (Figure 3). Based on the findings shown in Table 3, it was concluded that E. coli 0157:H7 and Salmonella Typhimurium were the least susceptible to the chile pepper extracts. This was expected, because both bacteria are contain an additional outer cell membrane and are generally more resistant to antimicrobials (Ibrahim et al., 2010).

The susceptibility of the microbes tested to the chile pepper samples brings into question the mechanism by which capsaicin affects fungal cells. Kurita et al. (2002) suggest that capsaicin enters the cells and functions as a toxin, possibly to the membrane structure, and/or as an osmotic stressor. Work to understand investigate the possible mechanism is currently underway in our lab. Since hospital acquired skin infections due to multidrug resistant Candida spp. are on the rise worldwide (Borman et al., 2016), many of the chile peppers studied in this work may hold promising treatment remedies.

As previously mentioned, the ancient Aztec and Tarahumara cultures used chile peppers to treat hundreds of human diseases, and some historians believe the ingestion of chile peppers promoted a healthier gut and could treat some diarrheal diseases, possibly due to multicellular eukaryotic parasites. There have been studies demonstrating anti-parasitic properties of chile peppers against the ectoparasite Ichthyophthirius multifiliis to control disease in the aquaculture industry (Ling et al., 2012), but studies with human parasites have not yet been conducted. Future studies in these areas are warranted, since chile peppers may represent a relatively inexpensive way to treat many human diseases.

Table 3 shows the total overall scores of each pepper type for each microorganism tested in this study. Bhut Jolokia Red had the highest score, meaning it had the greatest effect on microbial viability on the organisms tested. Figure 4 illustrates the linear correlation (r) between the antimicrobial scores and the capsaicin content of the C. chinense varieties. There was a moderate     positive     relationship    between   capsaicin concentration and antimicrobial scores (r = 0.426). Since there are over 30 classified chile pepper species, some may hold promise to treat human diseases, and studies in our lab are underway to investigate this further. Some question whether chile peppers are medically useful since they may irritate the patient. A recent study confirmed that human volunteers deemed capsaicinoid extract from the Bhut Jolokia chile pepper variety as an acceptable topical application for anti-arthritic and analgesic usage, thus overcoming an obstacle for future clinical applications of these important botanical sources (Sarwa et al., 2014, 2016). Since chile peppers are a widely accepted food item grown in every country in the world and known historically for broad-spectrum ethnopharmacological applications (Meghvansi et al., 2010), medical use of chile peppers, or isolated antimicrobial compounds, holds promise to treat many types of microbial infections and to numb pain caused by these associated diseases.

In this unique study, 29 cultivars of chile peppers were grown under controlled environmental conditions and correlated the capsaicin content with antimicrobial properties against fungal and Gram-negative and Gram-positive human pathogens. Prior to this study, the hottest chile pepper to be tested for antimicrobial properties was a Habanero pepper (~100,000 SHU). The presented study examines many unexplored chile pepper varieties that have recently garnered attention due to their very highly pungent flavor. Included in the examined peppers are the hottest chile pepper varieties in the world (>1,000,000 SHU). Of the 29 cultivars tested, three very

highly pungent cultivars (Trinidad 7 - Pot Brainstrain Red, Trinidad Moruga Scorpion, and HHP Moruga) with very high capsaicin content displayed bactericidal activity against S. aureus and C. albicans. Furthermore, the Bhut Jolokia (Ghost pepper) displayed bactericidal activity S. aureus, L. monocytogenes, and C. albicans even at 1:16 dilution of the extract, making it the most effective variety of C. chinense against the tested microbial panel. With the rise in multidrug resistant (MDR) “superbugs”, specifically methicillin-resistant and vancomycin-resistant St. aureus, capsaicin and its derivatives from chile peppers should be further explored as antibiotic alternatives in the treatment of bacterial and fungal pathogens.

The results obtained in thus study also highlight that capsaicin contents can vary widely between different cultivars of same Capsicum variety. Additionally, peppers with the highest capsaicin concentrations did not always correlate with the greatest antimicrobial activity, as the sixth most pungent variety, Bhut Jolokia Red, showed the greatest antimicrobial activity of all screen varieties.

Therefore, we propose the notion that different capsaicinoids have different antimicrobial properties. This opens the avenue for synthetic biology to improve the activity of capsaicin to treat multiple diseases.

The authors have not declared any conflict of interests.

Authors would like to thank Nina Le and Matthew Frederiksen for assistance in preparation of chile pepper homogenates. A special thanks to Dr. Joellen Feirtag and Dr. Adrian Hegeman for critique of this work and giving valuable feedback. We would like to thank Zachary Metz for writing assistance in manuscript preparation. We appreciate the technical expertise of Ms. Jayanti Suresh of the Hegeman Lab (UMN, Departments of Horticultural Science and Plant Biology) and Stephanie Nesbit of Beacon Analytical Systems, Inc. We thank Dr. Joe Delaney and Dr. “Pepper” Steve Marier who provided seeds for some of the chile pepper plants used in this study.