Journal de Mycologie Médicale (2014) 24, 34—43
Available online at
ScienceDirect www.sciencedirect.com
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Antimicrobial activity of Mentha piperita and Saturenja hortensis in a murine model of cutaneous protothecosis ´ antimicrobienne de Mentha piperita et Saturenja hortensis Activite ` le murin de protothe ´ cose cutane ´e dans un mode ˘s¸ b, C. Ca ˘toi a, N. Fit¸ b C. Bouari a, P. Bolfa a,*, G. Borza a, G. Nada a Pathology Department, University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary – na – s¸tur Street, 400372, Cluj-Napoca, Romania Medicine, 3-5, Ma b Microbiology Department, University of Agricultural Sciences and Veterinary Medicine, Faculty of – na – s¸tur Street, 400372, Cluj-Napoca, Romania Veterinary Medicine, 3-5, Ma
Received 29 January 2013; received in revised form 29 October 2013; accepted 21 November 2013 Available online 31 December 2013
KEYWORDS Protothecosis; Animal model; Therapy; Antimicrobial; Natural products
Summary Background. — To date there is no defined pharmacologic treatment protocol available against cutaneous protothecosis, which is difficult to combat using conventional drugs. Objectives. — Our experiment aimed to comparatively investigate the effect of two essential oils (Mentha piperita and Saturenja hortensis) against cutaneous protothecosis experimentally induced by Prototheca zopfii in mice. Materials and methods. — Immunosuppressed BALB/c female mice, were divided into six experimental groups, infected with P. zopfii, and then treated for 21 days against the infection. The effectiveness of the different treatments was assessed clinically and histologically by quantifying the degree of inflammation (immunohistochemical quantification of macrophages, T lymphocytes and neutrophils) and fibrosis. Results. — Skin lesions in experimental protothecosis from non-treated mice were more severe as compared to the four groups of treated animals. Both M. piperita and S. hortensis have proved to be efficient in vivo in the treatment of cutaneous protothecosis by reducing the clinical signs and significantly reducing the degree of inflammation (P < 0.05 for the number of macrophages, T lymphocytes and neutrophils) and fibrosis as compared to untreated animals. Conclusion. — Interestingly, our study shows that M. piperita and S. hortensis could represent a potential source of natural antimicrobial products in the treatment of cutaneous protothecosis. # 2013 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (P. Bolfa). 1156-5233/$ — see front matter # 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.mycmed.2013.11.005
Natural products in the treatment of protothecosis
MOTS CLÉS Protothécose ; Modèle animal ; Thérapie ; Antimicrobien ; Produit naturel
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Re ´sume ´ Origine. — Actuellement, il n’existe pas de protocole défini de traitement contre la protothécose cutanée, qui est difficile à combattre en utilisant des médicaments conventionnels. Objectif. — Notre travail a étudié comparativement l’effet de deux huiles essentielles (Mentha piperita et Saturenja hortensis) contre la protothécose cutanée induite expérimentalement par Prototheca zopfii chez la souris. Mate´riels et me´thodes. — Des souris immunodéprimées BALB/c femelles ont été divisées en six groupes expérimentaux, infectées par la P. zopfii, puis traitées pendant 21 jours contre l’infection. L’efficacité des différents traitements a été évaluée cliniquement et histologiquement en quantifiant le degré d’inflammation (quantification immunohistochimique des macrophages, des lymphocytes T et des neutrophiles) et de la fibrose. Re ´sultats. — Les lésions cutanées dans la protothécose expérimentale de la souris non traitées étaient plus sévères par rapport aux quatre groupes d’animaux traités. Les deux M. piperita et S. hortensis ont prouvé leur efficacité in vivo dans le traitement de la protothécose cutanée en diminuant les signes cliniques et en réduisant considérablement le degré d’inflammation ( p < 0,05 pour le nombre de macrophages, des lymphocytes T et des neutrophiles) et de la fibrose par rapport aux animaux non traités. Conclusion. — En conclusion, notre étude montre que M. piperita et S. hortensis pourrait représenter une source potentielle de produits naturels antimicrobiens dans le traitement de protothécose cutanée. # 2013 Elsevier Masson SAS. Tous droits réservés.
Introduction The genus Prototheca comprises ubiquitous, unicellular, achlorophyllos microalgae that reproduce asexually by formation of variable numbers of sporangiospores within a sporangium [25,30]. Currently four species namely Prototheca zopfii, Prototheca wickerhamii, Prototheca blaschkeae, and Prototheca cutis (recently isolated from inflamed human skin) have been involved as having pathogenic potential in both humans and animals, especially when predisposing factors occur or when the host immunologic defense are impaired [30,40,42,43]. The organisms have been isolated from a variety of environmental sources, with a particular affinity for wet areas with high organic matter content such as: soil, plants, sludge, lakes, rivers or marine water, feces of domestic or wild animals, barn floors and meat products [2,9,25,26,31]. In humans, protothecosis presents three clinical forms: localized cutaneous (the most common), articular (olecranon bursitis) and disseminated or systemic infections involving many internal organs [16,26,38]. Cutaneous lesions are usually described as papules, nodules, erythematous plaque, verrucous lesions, ulcerated and herpetiform lesions, with crusting and purulent discharge [10,16,22,26,27]. The incidence of infection depends on predisposing factors and it is believed that cutaneous protothecosis occur following an unknown skin injury through contact with contaminated sources or by traumatic inoculation with the algae [26]. In veterinary medicine the most prevalent form of protothecosis is bovine mastitis [24,32,41] but it is also reported as localized or disseminated infections in goats (cutaneous) [7,28], dogs (cutaneous, enteric, ocular and disseminated) and cats (cutaneous) [19]. Essential oils are a complex mixture of many different bioactive compounds, lately increasingly studied by researchers because of their therapeutic potential, similar to that
of some classical antimicrobial agents [12]. The main therapeutic activities of these natural products are reported to be anti-inflammatory, decongestant, immunomodulant, antimicrobial, antimycotic, antiviral and antioxidant [12,35]. Mentha piperita essential oil, rich in phenolic compounds such as: flavonoids and tannins, known for its many applications and therapeutic properties is frequently used in dermatology to calm pruritus and to attenuate skin irritation and inflammation [15,36]. Also in vitro the essential oil of Saturenja hortensis, which contains as active compounds carvacrol, thymol, gamma-terpinene and p-cymene, possesses a wide fungicidal effect, inhibiting the growth of different phytopathogenic fungi such as: Alternaria mali, Botrytis cinerea and Candida albicans [1,23]. Even though protothecosis represents a sporadic and completely curable disease, it should not be ignored [20]. A standard treatment protocol for cutaneous protothecosis has not been well established and it may vary from case to case. The most common pharmaceutical agents used are antifungal drugs such as: ketoconazole, itraconazole, fluconazole, nystatin and conventional amphotericin B [6,10,14,17,26]. Although there are few studies that mention the inhibitory effect of some essential oils such as: Maleleuca alternifolia (tea tree), Citrus bergamia (bergamot), Camelia sinensis (green tea), Cupressus sempervirens (cypress tree) and Pistacia lentiscus (mastic tree) upon Prototheca in vitro growth [44,45], no data is available concerning the in vivo therapeutic potential of natural products in protothecosis. Moreover, as reviewed by Milanov and Suvajdy¨iã [33], the treatment sensitivity of Prototheca spp. in vitro is not correlated with its efficacy in vivo. Thus, this study aimed to comparatively investigate the effect of some essential oils and antifungal drugs on an experimental murine model of cutaneous protothecosis.
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C. Bouari et al.
Materials and methods
Collection of skin samples
Inoculum preparation
At 24 hours after the last treatment, mice were sacrificed under anesthesia and skin biopsies were aseptically collected for microbiological and histopathological analysis. The skin samples were grossly examined for lesions. Each tissue samples was divided into two parts, one Petri dish for bacterioscopical and bacteriological exams, and the other to be used for histopathological evaluation.
P. zopfii isolate from cow mastitic milk sample kept in the Prototheca collection of the Microbiology Department within the Faculty of Veterinary Medicine, Cluj-Napoca was used. The organism was identified following the usual methods based on their cultural, microscopic and biochemical features (carbohydrates assimilation and growth at 27—37 8C). Gene sequence analysis using specific PCR showed that our isolate belonged to genotype 2, according to previous classification [3], which is involved in the etiopathogenesis of bovine mastitis. The isolate was grown under aerophilic condition, on nutrient agar supplemented with 2—3 drops of 10% glucose, at 37 8C, for 48—72 h. Then an inoculum with an optical density of 106 c.f.u./mL was prepared in sterile physiologic saline.
Cutaneous protothecosis and murine model The investigation was carried out in accordance with all Institutional Ethical and Health guidelines. Cutaneous protothecosis was studied in animal model of BALB/c mice 10 weeks old female, specified pathogen free (SPF), with an approximate body mass of 20 grams, purchased from Cantacuzino Institute, Bucharest (CIB). Animals were allowed an acclimatization period of one week. Three consecutive days previous administration of Prototheca suspension, the mice were immunosuppressed by intraperitoneal injection of 400 micrograms/mouse/day dexamethasone. Then, the animals were randomly assigned into six groups of 12 mice each: group 1: control, sterile physiologic saline; groups 2—6: topical administration of P. zopfii (0.5 mL of 1 106 c.f.u./mL, according to Dai et al. protocol [11] to shaved, anaesthetized mice, in the dorsal area, 3 consecutive days, following skin abrasions (last day of administration was considered day 0). Starting day 7 animals from groups 3 to 6 received daily, for 21 days, the following treatments: group 3: topical 2% M. piperita essential oil (Carl Roth, 9318.1) in olive oil; group 4: topical 2% S. hortensis essential oil (Carl Roth, 3763.1) in olive oil; group 5: intraperitoneal nystatin (Sigma, N1638, 10,000 U/ mL) — 5 mg/kg of body weight; group 6: intraperitoneal amphotericin B (Sigma, A2942, 25 mg/mL) — 0.5 mg/kg of body weight. Throughout the whole experimental period, animals were daily monitored, maintained in different cages and fed ad libitum with mouse food pellets (CIB).
Essential oils and antimicrobial agents Before evaluating the in vivo effect of natural essential oils and antimicrobial agents, their antimycotic activity on Prototheca isolates was determined in vitro by broth microdilution assay following the CLSI (formerly NCCLS — the National Committee for Clinical Laboratory Standards) guidelines based on documents M27-A2 for yeasts [34]. M. piperita and S. hortensis were selected because previous tests carried out in vitro have shown an inhibitory effect on P. zopfii isolates used in the present study [4,5].
Culture and staining of Prototheca zopfii The samples from skin lesions were cultured on Sabouraud’s dextrose agar (Difco) plates, containing 0.05 mg/mL chloramphenicol — in order to inhibit bacterial growth. Streaked plates were incubated under aerophilic condition, at 37 8C, for 48—72 hours and monitored daily. Identification of Prototheca spp. was based on their macroscopical features and confirmed microscopically by cell typical morphology. From the growing colonies, wet microscopic smears were stained using Lügol and lactophenol blue (Roth, 3097.1) and examined using light microscopy.
Tissue preparation for histopathology and immunohistochemistry Dorsal skin samples with associated dermis were taken from the middle of the back and fixed in 10% phosphate-buffered formalin for 18—24 h at 4 8C, dehydrated in ascending concentrations (80, 95, and 100%) of ethanol, cleared in xylene, and embedded in Histowax (Histo-Lab. Ltd., Gothenburg, Sweden). Serial sections of skin containing epidermis, 5-mm thick, were cut deparaffinized, rehydrated with water, and used for all hematoxylin and eosin (HE), periodic acidSchiff (PAS — Roth, HP01.1) staining or immunohistochemical (IHC) analyses. These sections were used for the quantification of inflammation (macrophages, T lymphocytes and neutrophils). For IHC, tissue sections were deparaffinized, rehydrated and treated for 30 min with peroxidase block (Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) to block endogenous peroxidase. To quantify the inflammatory response associated with experimental protothecosis, we incubated the sections with the following primary antibodies: CD8 antibody for T lymphocytes YTS169.4 (Abcam, 22378, 1:100), macrophage antibody SPM281 (Abcam, 75693, ready-to-use) and neutrophil antibody NIMP-R14 (Abcam, 2557, 1:50). For every antibody, negative controls were treated only with PBS under identical conditions. Pretreatment of sections by heating in sodium citrate buffer, 10 mM, pH 6.0 (using a pressure cooker), was performed to enhance immunostaining. All primary antibodies were diluted in Novocastra Antibody Diluent (RE7133-CE). Incubations with primary antibodies was done overnight at 4 8C. After washing the sections thoroughly with PBS, we used Novostain Universal Detection Kit reagents (Novocastra Laboratories Ltd., Benton Lane, Newcastle upon Tyne, UK). Thus, we incubated with biotinylated universal secondary antibody for 10 min, followed by incubation with ready-to-use streptavidin—peroxidase conjugate for 10 min. The immunostaining reaction product was developed using diaminobenzidine
Natural products in the treatment of protothecosis tetrahydrochloride (DAB, Novocastra) for 10 min and counterstained lightly with Gill 2 hematoxylin, followed by dehydration in ethanol, and mounting in an anhydrous mounting medium (Neo-Mount, Merck, Darmstadt, Germany). Microscopic acquisition and image analyses were done using an Olympus B 41microscope. Pictures were taken using a DP 25 camera (Olympus) under 400 magnification (for IHC) and processed using Cell B software (Olympus). All samples were coded and evaluated by two independent counters (B.P. and B.G.). For the quantification of the inflammatory response, macrophages, neutrophils and CD8 positive cells were averaged per HPF (HPF = high power field, 400-fold magnification), separately for every slide, then the mean S.E. values were obtained from the evaluation of five to 10 randomly chosen high power fields per slide.
Statistical analysis Mean comparisons were carried out using ANOVA, followed by a t-test. A confidence level of 95% (P < 0.05) was considered significant. ‘R’ software was used in all statistical analyses [37].
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Results Clinical evaluation of experimental cutaneous protothecosis At 7 days following Prototheca administration, all animals were clinically evaluated for skin lesions. No lesions were observed in animals from group 1 (control), whereas different degrees of cutaneous lesions were seen in the other five groups. A minimum of 7 mice par group (10 in group 2, eight in group 3, eight in group 4, seven in group 5 and nine in group 6) were considered to be clinically infected, according to dorsal skin lesions. These animals were selected for further treatment with the different natural and conventional agents for 21 consecutive days. All mice survived throughout the experimental period and animals in groups 2—6 presented different degrees of cutaneous lesions, which were further evaluated in the pathological and microbiological studies. The most severe clinical lesions were observed in the untreated control mice, whereas the clinical degree of severity was reduced in all four groups of treated animals (reduction in size of skin lesions). The number infected
Table 1 Clinical results following cutaneous experimental protothecosis in BALB/c mouse. ´ sultats cliniques suivants de la protothe ´ cose expe ´ rimental cutane ´ e dans la souris BALB/c. Les re
[(Figure_1)TD$IG]
Group
Number of animals per group
With lesions
Improved
Cured
1 2 3 4 5 6
12 12 12 12 12 12
— 10a (100%) 8a (100%) 8a (100%) 7a (100%) 9a (100%)
— 0 (0%) 6 (75%) 4 (50%) 6 (85.71%) 7 (77.77%)
— 0 (0%) 2 (25%) 4 (50%) 1 (14.29%) 2 (22.23%)
(negative control) (Prototheca control) (Mentha piperita) (Saturenja hortensis) (nystatin) (amphotericin B) a
All animals with lesions were microbiologically positive, and those with no lesions were negative.
Figure 1 Microbiological exam from skin lesions following cutaneous experimental protothecosis. (a) macroscopic appearance of Prototheca zopfii on Sabouraud’s dextrose agar supplemented with 0.05 mg/mL chloramphenicol, after 48 hours of incubation in aerobic condition at 37 8C. The colonies are creamy, whitish, with irregular edges, granular surface and typical mulberry aspect; (b) microscopic appearance of P. zopfii — wet preparation in lactophenol cotton blue solution showing sporangia in different phases of the life cycle: sporangia (arrows), sporangia-containing sporangiospores (arrowheads), some of which are being released (star) (1,000). ´ sions cutane ´ es apre ` s une protothe ´ cose expe ´ rimentale cutane ´ e. (a) aspect macroscopique de L’examen microbiologique des le ´ nicol (0,05 mg/mL), apre ` s 48 heures d’incubation en ae ´ robiose `a Prototheca zopfii sur un agar de type Sabouraud avec du chloramphe ` me, blancha ˆ tre, aux rebords irre ´ guliers et ayant une surface granuleuse ainsi qu’un aspect 37 8C. Les colonies sont de couleur cre ´ nol bleue ˆ re ; (b) aspect microscopique de P. zopfii — frottis humide dans une solution de lactophe typique ressemblant `a une mu ´ rentes phases du cycle de vie : sporanges (fle ` ches), sporanges avec des sporangiospores (pointes montrant des sporanges dans les diffe ` ches), certains sont libe ´ re ´ s (e ´ toile) ( 1000). des fle
38 animals, respectively the number and percentage of improved or cured skin lesions are summarized in Table 1.
Microbiological exams After aerobic incubation, at 37 8C, for 72 hours, on Sabouraud’s dextrose agar plates, visible colonies with typical
[(Figure_2)TD$IG]
C. Bouari et al. features for Prototheca were developed. The colonies’ dimensions ranged from 3 to 4 mm in diameter. They had generally irregular edges, granular or sometimes smooth surface, pale-gray or whitish color, creamy consistency, characteristic mulberry aspect and typically yeast smell (Fig. 1a). Usually they can easily be confused with Candida or Cryptococcus colonies. These data are similar with
Figure 2 Cutaneous experimental protothecosis in BALB/c mouse. (a) control group: no lesions (HE 100); (b) Prototheca control group: pandermal, severe, diffuse granulomatous inflammation (HE 40); (c) Mentha piperita treated group: focal granulomatous reaction, with extensive fibrosis surrounding it (PAS 40); (d) Saturenja hortensis treated group: subcutaneous focal granulomatous reaction, with necrotic center and surrounding fibrous tissue (PAS 100); (e) nystatine treated group: PAS positive Prototheca cells within the center of a small focal granuloma (PAS 400); (f) amphotericin B treated group: subcutaneous large focal granuloma surrounded by fibrous tissue and a layer of predominantly mononuclear cells (HE 100). ´ cose expe ´ rimentale cutane ´ e chez la souris BALB/c. (a) groupe de contro ˆ le : pas de le ´ sions (HE 100) ; (b) groupe infecte ´ Protothe ´ ve ` re affectant toutes les couches cutane ´ es (HE 40) ; (c) Mentha avec Prototheca : une inflammation granulomateuse diffuse et se ´ : re ´ action granulomateuse focale, avec une fibrose extensive qui l’entoure (PAS 40) ; (d) Saturenja hortensis piperita groupe traite ´ : re ´ action cutane ´ e granulomateuse focale, avec un centre ne ´ crotique et des tissus fibreux (PAS 100) ; (e) nystatine groupe traite ´ : cellules de Prototheca PAS positives situe ´ es au centre d’un petit granulome focal (PAS 400) (f) l’amphote ´ ricine B groupe traite ´ : important granulome sous-cutane ´ focal entoure ´ par un tissu fibreux et une couche de cellules principalement groupe traite ´ aires (HE 100). mononucle
Natural products in the treatment of protothecosis
[(Figure_3)TD$IG]
previous reports [29,39]. As to be seen in Table 1, all animals that developed dorsal skin lesions were positive microbiologically for protothecosis. Since differentiation from other yeasts is not possible based exclusively on their cultural aspects, microscopy is necessary to confirm Prototheca spp. Prototheca is easily directly stained by lactophenol blue or Lügol solution, but heat fixation may induce morphological impairment. Microscopic appearance of Prototheca cells was observed by
39 examining wet smears using light microscopy, when structures described as ‘‘morula’’ are visible (Fig. 1b). Prototheca spp. are spherical or oval in shape, 7—30 mm in diameter, and they reproduce asexually. During cell maturation the cytoplasm undergoes a process of cleavage to form 6—20 endospores of different sizes, range from 12 to 22 mm. These endospores increase in size, and sporangia (mother cell) wall break under pressure from the enlarging endospores (daughter cells) and every 5 to 6 hours they are passively
Figure 3 Cutaneous experimental protothecosis in BALB/c mouse, from group 2. (a) Prototheca untreated group: presence of foamy macrophages in the center of granuloma (arrowheads), and important number of neutrophils (HE 400); (b) Prothotheca control group: foamy macrophages (arrowhead), some with intracytoplasmatic stages (arrows) of Prototheca (HE 1000). Mentha piperita treated group: different life cycle phases of Prototheca zopfii within the lesions; (c) sporangia; (d) dividing sporangia; (e) formation of sporangiospores inside the mother cell; (f) release of sporangiospores (PAS 1,000). ´ cose expe ´ rimentale cutane ´ e chez la souris BALB/c, dans le groupe 2. (a) Prototheca groupe non traite ´ : pre ´ sence de Protothe ` ches), ainsi qu’un nombre important de neutrophiles (HE 400) ; macrophages spumeux dans le centre du granulome (pointes des fle ´ avec Prototheca : macrophages spumeux (te ˆ te de fle ` che), certains avec des stades intracytoplasmiques (fle ` ches) (b) groupe infecte ´ : diffe ´ rentes phases du cycle de vie de Prototheca zopfii dans les le ´ sions ; (c) de Prototheca (HE 1000). Mentha piperita groupe traite ´ rieur de la cellule me ` re ; (f) libe ´ ration de les sporanges ; (d) la division des sporanges ; (e) formation de sporangiospores `a l’inte sporangiospores (PAS 1000).
40 released outside (Fig. 1b). These findings are similar with previous reports [21,26].
Histopathological and immunohistochemical study Dorsal skin samples were collected from areas with lesions at the end of the 21 days of topical treatment and processed for the microscopic study. The pathological exam aimed to describe the lesions, check for the presence of Prototheca in the lesions and quantify the inflammation. From each skin sample of every animal, 3 different HE stained serial slides were examined. No microscopic lesions were observed in any
[(Figure_4)TD$IG]
C. Bouari et al. animal from group 1 (Fig. 2a). In all the groups with topical administration of P. zopfii, they were present within the lesions. As the organism is known to be haematoxilinophyllic [33], they were visualized using periodic acid-Schiff (PAS) staining technique, where different evolution stages from its life cycle were visible — sporangium, cell division, formation and release of sporangiospores (Fig. 3c—f). The majority of the lesions were localized between the superficial dermis and the subcutaneous connective tissue. In all groups the inflammation was of granulomatous type, with formation of focal granulomas in treated animals (groups 3, 4, 5 and 6), whereas in untreated animals (group 2) the inflammation was more severe, with a predominantly diffuse pattern (Fig. 2b).
Figure 4 Immunohistochemical staining from cutaneous protothecosis skin biopsies. (a) CD8 antibody for T lymphocytes in Prototheca untreated group; (b) amphotericin B treated group; (c) macrophage antibody in amphotericin B treated group; (d) Saturenja hortensis treated group; (e) neutrophil antibody in Prototheca untreated group; (f) amphotericin B treated group. ´ es. (a) anticorps CD8 et des lymphocytes T dans le groupe non traite ´ La coloration immunohistochimique des biopsies cutane ´ avec de l’amphote ´ ricine B ; (c) anticorps des macrophages dans le groupe traite ´ avec de de Prototheca ; (b) groupe traite ´ ricine B ; (d) groupe traite ´ avec de la Saturenja hortensis ; (e) anticorps des neutrophiles dans le groupe non traite ´ l’amphote ´ avec de l’amphote ´ ricine B. de Prototheca ; (f) groupe traite
Natural products in the treatment of protothecosis [(Figure_5)TD$IG]
41
Figure 5 Immunohistochemical quantification of macrophages (a), lymphocytes (b) and neutrophils (c) from dorsal skin biopsies of experimental protothecosis. Statistical significant differences between groups are marked with an asterisk (P < 0.05). ´ es Quantification immunohistochimique des macrophages (a), les lymphocytes (b) et des neutrophiles (c) `a partir de biopsies cutane ´ cose expe ´ rimentale. Les diffe ´ rences significatives statistiques entre les groupes sont marque ´ s d’une aste ´ risque de protothe ( p < 0,05).
Skin sections from group 2 revealed a pandermal, granulomatous, inflammatory infiltrate composed of lymphocytes, histiocytes, macrophages — epithelioid type, neutrophils, and rare eosinophils. Rare multinucleated giant cells and plasma cells were also present along with foamy macrophages, some containing different stages of Prototheca (Fig. 3a, b) as well as hyperplastic lymphoid tissue (only 2 animals). P. zopfii was observed on HE and PAS-stained sections and was found as clusters, as well as solitary structures, within granulomas and macrophages, as well as in necrotic areas. Within the treated groups (Fig. 2c—f), the degree of inflammation, evaluated by the number and type of inflammatory cells, was more severe in the animals treated with S. hortensis (group 4), followed by the ones treated with M. piperita (group 3) and by the animals treated with nystatine and amphotericin B (groups 5 and 6). The thickness of the fibrous tissue surrounding the granulomas, also varied, being more developed in animals treated with M. piperita, followed by the ones treated with amphotericin B, S. hortensis and nystatine. In order to quantify the inflammatory response associated with experimental cutaneous protothecosis, we determined separately, from every mouse, the number of T lymphocytes, macrophages and neutrophils (Fig. 4a—f). Thus, as anticipated by the histopathological exam, the number of macrophages, T lymphocytes and neutrophils was significantly increased in all experimentally infected groups, as compared to the negative controls (P < 0.05, Fig. 5a—c). On the other hand, the mean number of macrophages, T lymphocytes and neutrophils were significantly decreased (P < 0.05) in all treated animals (groups 3—6) as compared to the Prototheca control animals (group 2). This means that the different form of treatment attenuated the infection. There was a similar trend regarding the number of macrophages and neutrophils between all groups (Fig. 4a, c). Hence, the mean number of macrophages and neutrophils was significantly decreased (P < 0.05) in animals treated with M. piperita, nystatine and amphotericin B as compared with the ones topically treated with S. hortensis. Moreover, the mean number of neutrophils in animals treated with M. piperita was
significantly increased (P < 0.05) as compared with the mouse treated with both conventional antifungal drugs (Fig. 5c). Regarding the quantification of T lymphocytes, their mean number was significantly increased in the animals treated with amphotericin B as compared with the other 3 treated groups (Fig. 5b).
Discussion The most frequent form of infection with Prototheca spp. in humans is represented by cutaneous protothecosis [26]. The use of natural products in the treatment of different diseases in both human and veterinary medicines is representing an important approach as an alternative for conventional drugs. So far, there were several studies which showed that some essential oils have inhibitory effects in vitro upon Prototheca growth. Also, in other two of our previous in vitro studies [4,5] we have shown that S. hortensis and M. piperita essential oils are inhibiting the growth/survival of P. wickerhamii and P. zopfii, both involved in human and animal pathologies. In another study, Pramila et al. [36] showed that a methanolic leaf extract of M. piperita proved antifungal activity in vitro against another fungi: C. albicans and Candida glabrata. A similar study [8] demonstrated that M. piperita L. in different forms (hydroalcoholic extract, infusion and essential oil) presented significant in vitro antimicrobial activity against Candida spp. Similar to the study by Horiuchi and Masuzawa [18] in a mouse model of skin protothecosis induced by intradermal and/or subcutaneous inoculation we found that the main lesion induced by P. zopfii, at 28 days following inoculation, was represented by the formation of epitheloid cell granulomas. The lesion was more severe in mouse skin from group 2 (Prototheca only). Based on histopathological observations, we found that the degree of inflammation was reduced in all four treated groups, being less severe in the animals that received the conventional nystatine or amphotericin B treatment. It is a well-known fact that here are important differences between the in vitro and the in vivo sensitivity of Prototheca spp. [33]. To our knowledge, the present study is the first one
42 to focus on the in vivo therapeutic potential of M. piperita and S. hortensis essential oils, on an animal model of experimental cutaneous protothecosis. According to previous works [13] nystatine and amphotericin B have proved their in vivo therapeutic potential against human cutaneous protothecosis. In our study we confirmed that both agents have proved their in vivo efficiency against experimental protothecosis in mice. This was proved by clinical and histopathological results. In fact, both conventional antifungal agents that we employed in the current experiment have proved to be more efficient than the two essential oils used to topically control the infection. It seems like, of the four different treatments that we tested, nystatine was the most efficient in reducing the degree of lesions as quantified by the number and nature of inflammatory cells and the degree of fibrosis. On the other hand, both essential oils (M. piperita and S. hortensis) have proven in vivo efficiency in significantly reducing the degree of the protothecal induced skin lesions, as compared to the lesions from the infected, untreated group. Interestingly, our results show that M. piperita had almost similar efficiency, as the two classic antifungal drugs that we used, in reducing the degree of skin experimental protothecosis. Thus, a natural therapeutic approach using peppermint essential oil could prove itself useful in the treatment of cutaneous protothecosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements This work was supported by the Executive Unit for Financing Higher Education, Research, Development and Innovation, Romania, Human Resources Program, Postdoctoral Research Project, No. 175/2010 and by project No. 1356/2013 financed by University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania.
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C. Bouari et al. [7] Camboim KA, Garino FJ, Dantas AFM, et al. Protothecosis by P. wickerhamii in goats. Mycoses 2010;54:196—200. [8] Carreto CFP, Almeida RBA, Furlar MR, et al. Antimicrobial activity of Mentha piperita L against Candida spp. Braz Dent Sci 2010;13:4—9. [9] Casal M, Linares MJ, Solís F, et al. Appearance of colonies of Prototheca on CHROMagar Candida medium. Mycopathologia 1997;137:79—82. [10] Chao SC, Hsu M, Lee JY. Cutaneous protothecosis: report of five cases. Br J Dermatol 2002;146:688—93. [11] Dai T, Bil de Arce VJ, Tegos GP, et al. Blue dye and red light: a dynamic combination for prophylaxis and treatment of cutaneous Candida albicans infections in mice. Antimicrob Agents Chemother 2011;55:5710—7. [12] Edris EA. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review. Phytother Res 2007;21:308—23. [13] Galán F, Martos PG, Palomo MJ, et al. Onychoprotothecosis due to Prototheca wickerhamii. Mycopathologia 1997;137:75—7. [14] Gao J, Zhang HQ, He JZ, et al. Characterization of Prototheca zopfii associated with outbreak of bovine clinical mastitis in herd of Beijing, China. Mycopathologia 2012;173:275—81. [15] Herro E, Jacob SE. Mentha piperita (peppermint). Dermatitis 2010;21:327—9. [16] Hightower KD, Messina JL. Cutaneous protothecosis: a case report and review of the literature. Cutis 2007;80:129—31. [17] Hillesheim PB, Bahrami S. Cutaneous protothecosis. Arch Pathol Lab Med 2011;135:941—4. [18] Horiuchi Y, Masuzawa M. Epithelioid cell granulomas experimentally induced by Prototheca in the skin of mice: a light microscopic study. Hiroshima J Med Sci 1995;44:21—7. [19] Hosaka S, Hosaka M. A case report of canine protothecosis. J Vet Med Sci 2004;66:593—7. [20] Jagielski T, Lagneau PE. Protothecosis. A pseudofungal infection. J Mycol Med 2007;17:261—70. [21] Joshi K, Gavin J, Wheeler E. The ultrastructure of Prototheca wickerhamii. Mycopathologia 1975;56:9—13. [22] Kantrow SM, Boyd AS. Protothecosis. Dermatol Clin 2003;21: 249—55. [23] Krstev TM, Radnovic ´ D, Kitic ´ D, et al. Chemical composition and antimicrobial activity of Saturenja hortensis L. essential oil. Cent Eur J Biol 2009;4:411—6. [24] Lagneau PE. First isolation of Prototheca zopfii in bovine mastitis in Belgium. J Mycol Med 1996;6:145—8. [25] Lassa H, Jagielski T, Malinowski E. Effect of different heat treatments and disinfectants on the survival of Prototheca zopfii. Mycopathologia 2011;171:177—82. [26] Lass-Flörl C, Mayr A. Human protothecosis. Clin Microbiol Rev 2007;20:230—42. [27] Leimann BC, Monteiro PC, Lazera M, et al. Protothecosis. Med Mycol 2004;42:95—106. [28] Macedo JT, Riet-Correa F, Dantas AF, et al. Cutaneous and nasal protothecosis in a goat. Vet Pathol 2008;45:352—4. [29] Malinowski E, Lassa H, Kłossowska A. Isolation of Prototheca zopfii from inflamed secretion of udders. Bull Vet Inst Pulawy 2002;46:295—9. [30] Marques S, Silva E, Carvalheira J, Kraft C, et al. Bovine mastitis associated with Prototheca blaschkeae. J Clin Microbiol 2008; 46:1941—5. [31] Marques S, Silva E, Carvalheira J, et al. In vitro susceptibility of Prototheca to pH and salt concentration. Mycopathologia 2010;169:297—302. [32] Melville PA, Watanable ET, Benites NR, et al. Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization. Mycopathologia 1999;146:79—82. [33] Milanov DS, Suvajdy ¨iã LD. Characteristics and importance of the genus Prototheca in human and veterinary medicine. Prog Nat Sci 2006;110:15—27.
Natural products in the treatment of protothecosis [34] National Committee for Clinical Laboratory Standards. Reference method for broth dilution testing of yeasts. Approved standard M27-A2. 2nd ed, Wayne, PA: NCCLS; 2002. [35] Pisseri F, Bertolia A, Pistelli L. Essential oils in medicine: principles of therapy. Parassitologia 2008;50:89—91. [36] Pramila DM, Xavier R, Marimuthu K, et al. Phytochemical analysis and antimicrobial potential of methanolic leaf extract of peppermint (Mentha piperita: Lamiaceae). J Med Plant Res 2012;6:331—5. [37] R Development Core Team. R Foundation for statistical computing, Vienna, Austria;http://www.R-project.org. [38] Ramírez-Romero R, Rodríguez-Tovar LE, Nevárez-Garza AM, et al. Chlorella infection in a sheep in Mexico and minireview of published reports from humans and domestic animals. Mycopathologia 2010;169:461—6. [39] Ra ˘puntean S, Ra ˘puntean G, Fit¸ N, et al. Morphological and cultural characterization of some strains of unicellular algae of the genus Prototheca sampled from mastitic cow milk. Not Bot Hort Agrobot Cluj 2009;37:31—40. [40] Roesler U, Moller A, Hensel A, et al. Diversity within the current algal species Prototheca zopfii: a proposal for two Prototheca
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[41]
[42]
[43] [44]
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zopfii genotypes and descriptions of a novel species, Prototheca blaschkeae sp. nov. Int J Syst Evol Microbiol 2006;56: 1419—25. Roesler U, Hensel A. Longitudinal analysis of Prototheca zopfii-specific immune responses: correlation with disease progression and carriage in dairy cows. J Clin Microbiol 2003;41: 1181—6. Satoh K, Ooe K, Nagayama H, et al. Prototheca cutis sp. nov., a newly discovered pathogen of protothecosis isolated from inflamed human skin. Int J Syst Evol Microbiol 2010;60: 1236—40. Thompson G, Silva E, Marques S, et al. Algaemia in a dairy cow by Prototheca blaschkeae. Med Micol 2009;47:1—5. Tortorano AM, Prigitano A, Dho G, et al. In vitro activity of conventional antifungal drugs and natural essences against the yeast-like alga Prototheca. J Antimicrob Chemother 2008;61: 1312—4. Turchetti B, Pinelli P, Buzzini P, et al. In vitro antimycotic activity of some plant extracts towards yeast and yeast-like strains. Phytother Res 2005;19:44—9.
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Antimicrobial activity of Mentha piperita and Saturenja hortensis in a murine model of cutaneous protothecosis ´ antimicrobienne de Mentha piperita et Saturenja hortensis Activite ` le murin de protothe ´ cose cutane ´e dans un mode ˘s¸ b, C. Ca ˘toi a, N. Fit¸ b C. Bouari a, P. Bolfa a,*, G. Borza a, G. Nada a Pathology Department, University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary – na – s¸tur Street, 400372, Cluj-Napoca, Romania Medicine, 3-5, Ma b Microbiology Department, University of Agricultural Sciences and Veterinary Medicine, Faculty of – na – s¸tur Street, 400372, Cluj-Napoca, Romania Veterinary Medicine, 3-5, Ma
Received 29 January 2013; received in revised form 29 October 2013; accepted 21 November 2013 Available online 31 December 2013
KEYWORDS Protothecosis; Animal model; Therapy; Antimicrobial; Natural products
Summary Background. — To date there is no defined pharmacologic treatment protocol available against cutaneous protothecosis, which is difficult to combat using conventional drugs. Objectives. — Our experiment aimed to comparatively investigate the effect of two essential oils (Mentha piperita and Saturenja hortensis) against cutaneous protothecosis experimentally induced by Prototheca zopfii in mice. Materials and methods. — Immunosuppressed BALB/c female mice, were divided into six experimental groups, infected with P. zopfii, and then treated for 21 days against the infection. The effectiveness of the different treatments was assessed clinically and histologically by quantifying the degree of inflammation (immunohistochemical quantification of macrophages, T lymphocytes and neutrophils) and fibrosis. Results. — Skin lesions in experimental protothecosis from non-treated mice were more severe as compared to the four groups of treated animals. Both M. piperita and S. hortensis have proved to be efficient in vivo in the treatment of cutaneous protothecosis by reducing the clinical signs and significantly reducing the degree of inflammation (P < 0.05 for the number of macrophages, T lymphocytes and neutrophils) and fibrosis as compared to untreated animals. Conclusion. — Interestingly, our study shows that M. piperita and S. hortensis could represent a potential source of natural antimicrobial products in the treatment of cutaneous protothecosis. # 2013 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (P. Bolfa). 1156-5233/$ — see front matter # 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.mycmed.2013.11.005
Natural products in the treatment of protothecosis
MOTS CLÉS Protothécose ; Modèle animal ; Thérapie ; Antimicrobien ; Produit naturel
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Re ´sume ´ Origine. — Actuellement, il n’existe pas de protocole défini de traitement contre la protothécose cutanée, qui est difficile à combattre en utilisant des médicaments conventionnels. Objectif. — Notre travail a étudié comparativement l’effet de deux huiles essentielles (Mentha piperita et Saturenja hortensis) contre la protothécose cutanée induite expérimentalement par Prototheca zopfii chez la souris. Mate´riels et me´thodes. — Des souris immunodéprimées BALB/c femelles ont été divisées en six groupes expérimentaux, infectées par la P. zopfii, puis traitées pendant 21 jours contre l’infection. L’efficacité des différents traitements a été évaluée cliniquement et histologiquement en quantifiant le degré d’inflammation (quantification immunohistochimique des macrophages, des lymphocytes T et des neutrophiles) et de la fibrose. Re ´sultats. — Les lésions cutanées dans la protothécose expérimentale de la souris non traitées étaient plus sévères par rapport aux quatre groupes d’animaux traités. Les deux M. piperita et S. hortensis ont prouvé leur efficacité in vivo dans le traitement de la protothécose cutanée en diminuant les signes cliniques et en réduisant considérablement le degré d’inflammation ( p < 0,05 pour le nombre de macrophages, des lymphocytes T et des neutrophiles) et de la fibrose par rapport aux animaux non traités. Conclusion. — En conclusion, notre étude montre que M. piperita et S. hortensis pourrait représenter une source potentielle de produits naturels antimicrobiens dans le traitement de protothécose cutanée. # 2013 Elsevier Masson SAS. Tous droits réservés.
Introduction The genus Prototheca comprises ubiquitous, unicellular, achlorophyllos microalgae that reproduce asexually by formation of variable numbers of sporangiospores within a sporangium [25,30]. Currently four species namely Prototheca zopfii, Prototheca wickerhamii, Prototheca blaschkeae, and Prototheca cutis (recently isolated from inflamed human skin) have been involved as having pathogenic potential in both humans and animals, especially when predisposing factors occur or when the host immunologic defense are impaired [30,40,42,43]. The organisms have been isolated from a variety of environmental sources, with a particular affinity for wet areas with high organic matter content such as: soil, plants, sludge, lakes, rivers or marine water, feces of domestic or wild animals, barn floors and meat products [2,9,25,26,31]. In humans, protothecosis presents three clinical forms: localized cutaneous (the most common), articular (olecranon bursitis) and disseminated or systemic infections involving many internal organs [16,26,38]. Cutaneous lesions are usually described as papules, nodules, erythematous plaque, verrucous lesions, ulcerated and herpetiform lesions, with crusting and purulent discharge [10,16,22,26,27]. The incidence of infection depends on predisposing factors and it is believed that cutaneous protothecosis occur following an unknown skin injury through contact with contaminated sources or by traumatic inoculation with the algae [26]. In veterinary medicine the most prevalent form of protothecosis is bovine mastitis [24,32,41] but it is also reported as localized or disseminated infections in goats (cutaneous) [7,28], dogs (cutaneous, enteric, ocular and disseminated) and cats (cutaneous) [19]. Essential oils are a complex mixture of many different bioactive compounds, lately increasingly studied by researchers because of their therapeutic potential, similar to that
of some classical antimicrobial agents [12]. The main therapeutic activities of these natural products are reported to be anti-inflammatory, decongestant, immunomodulant, antimicrobial, antimycotic, antiviral and antioxidant [12,35]. Mentha piperita essential oil, rich in phenolic compounds such as: flavonoids and tannins, known for its many applications and therapeutic properties is frequently used in dermatology to calm pruritus and to attenuate skin irritation and inflammation [15,36]. Also in vitro the essential oil of Saturenja hortensis, which contains as active compounds carvacrol, thymol, gamma-terpinene and p-cymene, possesses a wide fungicidal effect, inhibiting the growth of different phytopathogenic fungi such as: Alternaria mali, Botrytis cinerea and Candida albicans [1,23]. Even though protothecosis represents a sporadic and completely curable disease, it should not be ignored [20]. A standard treatment protocol for cutaneous protothecosis has not been well established and it may vary from case to case. The most common pharmaceutical agents used are antifungal drugs such as: ketoconazole, itraconazole, fluconazole, nystatin and conventional amphotericin B [6,10,14,17,26]. Although there are few studies that mention the inhibitory effect of some essential oils such as: Maleleuca alternifolia (tea tree), Citrus bergamia (bergamot), Camelia sinensis (green tea), Cupressus sempervirens (cypress tree) and Pistacia lentiscus (mastic tree) upon Prototheca in vitro growth [44,45], no data is available concerning the in vivo therapeutic potential of natural products in protothecosis. Moreover, as reviewed by Milanov and Suvajdy¨iã [33], the treatment sensitivity of Prototheca spp. in vitro is not correlated with its efficacy in vivo. Thus, this study aimed to comparatively investigate the effect of some essential oils and antifungal drugs on an experimental murine model of cutaneous protothecosis.
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C. Bouari et al.
Materials and methods
Collection of skin samples
Inoculum preparation
At 24 hours after the last treatment, mice were sacrificed under anesthesia and skin biopsies were aseptically collected for microbiological and histopathological analysis. The skin samples were grossly examined for lesions. Each tissue samples was divided into two parts, one Petri dish for bacterioscopical and bacteriological exams, and the other to be used for histopathological evaluation.
P. zopfii isolate from cow mastitic milk sample kept in the Prototheca collection of the Microbiology Department within the Faculty of Veterinary Medicine, Cluj-Napoca was used. The organism was identified following the usual methods based on their cultural, microscopic and biochemical features (carbohydrates assimilation and growth at 27—37 8C). Gene sequence analysis using specific PCR showed that our isolate belonged to genotype 2, according to previous classification [3], which is involved in the etiopathogenesis of bovine mastitis. The isolate was grown under aerophilic condition, on nutrient agar supplemented with 2—3 drops of 10% glucose, at 37 8C, for 48—72 h. Then an inoculum with an optical density of 106 c.f.u./mL was prepared in sterile physiologic saline.
Cutaneous protothecosis and murine model The investigation was carried out in accordance with all Institutional Ethical and Health guidelines. Cutaneous protothecosis was studied in animal model of BALB/c mice 10 weeks old female, specified pathogen free (SPF), with an approximate body mass of 20 grams, purchased from Cantacuzino Institute, Bucharest (CIB). Animals were allowed an acclimatization period of one week. Three consecutive days previous administration of Prototheca suspension, the mice were immunosuppressed by intraperitoneal injection of 400 micrograms/mouse/day dexamethasone. Then, the animals were randomly assigned into six groups of 12 mice each: group 1: control, sterile physiologic saline; groups 2—6: topical administration of P. zopfii (0.5 mL of 1 106 c.f.u./mL, according to Dai et al. protocol [11] to shaved, anaesthetized mice, in the dorsal area, 3 consecutive days, following skin abrasions (last day of administration was considered day 0). Starting day 7 animals from groups 3 to 6 received daily, for 21 days, the following treatments: group 3: topical 2% M. piperita essential oil (Carl Roth, 9318.1) in olive oil; group 4: topical 2% S. hortensis essential oil (Carl Roth, 3763.1) in olive oil; group 5: intraperitoneal nystatin (Sigma, N1638, 10,000 U/ mL) — 5 mg/kg of body weight; group 6: intraperitoneal amphotericin B (Sigma, A2942, 25 mg/mL) — 0.5 mg/kg of body weight. Throughout the whole experimental period, animals were daily monitored, maintained in different cages and fed ad libitum with mouse food pellets (CIB).
Essential oils and antimicrobial agents Before evaluating the in vivo effect of natural essential oils and antimicrobial agents, their antimycotic activity on Prototheca isolates was determined in vitro by broth microdilution assay following the CLSI (formerly NCCLS — the National Committee for Clinical Laboratory Standards) guidelines based on documents M27-A2 for yeasts [34]. M. piperita and S. hortensis were selected because previous tests carried out in vitro have shown an inhibitory effect on P. zopfii isolates used in the present study [4,5].
Culture and staining of Prototheca zopfii The samples from skin lesions were cultured on Sabouraud’s dextrose agar (Difco) plates, containing 0.05 mg/mL chloramphenicol — in order to inhibit bacterial growth. Streaked plates were incubated under aerophilic condition, at 37 8C, for 48—72 hours and monitored daily. Identification of Prototheca spp. was based on their macroscopical features and confirmed microscopically by cell typical morphology. From the growing colonies, wet microscopic smears were stained using Lügol and lactophenol blue (Roth, 3097.1) and examined using light microscopy.
Tissue preparation for histopathology and immunohistochemistry Dorsal skin samples with associated dermis were taken from the middle of the back and fixed in 10% phosphate-buffered formalin for 18—24 h at 4 8C, dehydrated in ascending concentrations (80, 95, and 100%) of ethanol, cleared in xylene, and embedded in Histowax (Histo-Lab. Ltd., Gothenburg, Sweden). Serial sections of skin containing epidermis, 5-mm thick, were cut deparaffinized, rehydrated with water, and used for all hematoxylin and eosin (HE), periodic acidSchiff (PAS — Roth, HP01.1) staining or immunohistochemical (IHC) analyses. These sections were used for the quantification of inflammation (macrophages, T lymphocytes and neutrophils). For IHC, tissue sections were deparaffinized, rehydrated and treated for 30 min with peroxidase block (Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) to block endogenous peroxidase. To quantify the inflammatory response associated with experimental protothecosis, we incubated the sections with the following primary antibodies: CD8 antibody for T lymphocytes YTS169.4 (Abcam, 22378, 1:100), macrophage antibody SPM281 (Abcam, 75693, ready-to-use) and neutrophil antibody NIMP-R14 (Abcam, 2557, 1:50). For every antibody, negative controls were treated only with PBS under identical conditions. Pretreatment of sections by heating in sodium citrate buffer, 10 mM, pH 6.0 (using a pressure cooker), was performed to enhance immunostaining. All primary antibodies were diluted in Novocastra Antibody Diluent (RE7133-CE). Incubations with primary antibodies was done overnight at 4 8C. After washing the sections thoroughly with PBS, we used Novostain Universal Detection Kit reagents (Novocastra Laboratories Ltd., Benton Lane, Newcastle upon Tyne, UK). Thus, we incubated with biotinylated universal secondary antibody for 10 min, followed by incubation with ready-to-use streptavidin—peroxidase conjugate for 10 min. The immunostaining reaction product was developed using diaminobenzidine
Natural products in the treatment of protothecosis tetrahydrochloride (DAB, Novocastra) for 10 min and counterstained lightly with Gill 2 hematoxylin, followed by dehydration in ethanol, and mounting in an anhydrous mounting medium (Neo-Mount, Merck, Darmstadt, Germany). Microscopic acquisition and image analyses were done using an Olympus B 41microscope. Pictures were taken using a DP 25 camera (Olympus) under 400 magnification (for IHC) and processed using Cell B software (Olympus). All samples were coded and evaluated by two independent counters (B.P. and B.G.). For the quantification of the inflammatory response, macrophages, neutrophils and CD8 positive cells were averaged per HPF (HPF = high power field, 400-fold magnification), separately for every slide, then the mean S.E. values were obtained from the evaluation of five to 10 randomly chosen high power fields per slide.
Statistical analysis Mean comparisons were carried out using ANOVA, followed by a t-test. A confidence level of 95% (P < 0.05) was considered significant. ‘R’ software was used in all statistical analyses [37].
37
Results Clinical evaluation of experimental cutaneous protothecosis At 7 days following Prototheca administration, all animals were clinically evaluated for skin lesions. No lesions were observed in animals from group 1 (control), whereas different degrees of cutaneous lesions were seen in the other five groups. A minimum of 7 mice par group (10 in group 2, eight in group 3, eight in group 4, seven in group 5 and nine in group 6) were considered to be clinically infected, according to dorsal skin lesions. These animals were selected for further treatment with the different natural and conventional agents for 21 consecutive days. All mice survived throughout the experimental period and animals in groups 2—6 presented different degrees of cutaneous lesions, which were further evaluated in the pathological and microbiological studies. The most severe clinical lesions were observed in the untreated control mice, whereas the clinical degree of severity was reduced in all four groups of treated animals (reduction in size of skin lesions). The number infected
Table 1 Clinical results following cutaneous experimental protothecosis in BALB/c mouse. ´ sultats cliniques suivants de la protothe ´ cose expe ´ rimental cutane ´ e dans la souris BALB/c. Les re
[(Figure_1)TD$IG]
Group
Number of animals per group
With lesions
Improved
Cured
1 2 3 4 5 6
12 12 12 12 12 12
— 10a (100%) 8a (100%) 8a (100%) 7a (100%) 9a (100%)
— 0 (0%) 6 (75%) 4 (50%) 6 (85.71%) 7 (77.77%)
— 0 (0%) 2 (25%) 4 (50%) 1 (14.29%) 2 (22.23%)
(negative control) (Prototheca control) (Mentha piperita) (Saturenja hortensis) (nystatin) (amphotericin B) a
All animals with lesions were microbiologically positive, and those with no lesions were negative.
Figure 1 Microbiological exam from skin lesions following cutaneous experimental protothecosis. (a) macroscopic appearance of Prototheca zopfii on Sabouraud’s dextrose agar supplemented with 0.05 mg/mL chloramphenicol, after 48 hours of incubation in aerobic condition at 37 8C. The colonies are creamy, whitish, with irregular edges, granular surface and typical mulberry aspect; (b) microscopic appearance of P. zopfii — wet preparation in lactophenol cotton blue solution showing sporangia in different phases of the life cycle: sporangia (arrows), sporangia-containing sporangiospores (arrowheads), some of which are being released (star) (1,000). ´ sions cutane ´ es apre ` s une protothe ´ cose expe ´ rimentale cutane ´ e. (a) aspect macroscopique de L’examen microbiologique des le ´ nicol (0,05 mg/mL), apre ` s 48 heures d’incubation en ae ´ robiose `a Prototheca zopfii sur un agar de type Sabouraud avec du chloramphe ` me, blancha ˆ tre, aux rebords irre ´ guliers et ayant une surface granuleuse ainsi qu’un aspect 37 8C. Les colonies sont de couleur cre ´ nol bleue ˆ re ; (b) aspect microscopique de P. zopfii — frottis humide dans une solution de lactophe typique ressemblant `a une mu ´ rentes phases du cycle de vie : sporanges (fle ` ches), sporanges avec des sporangiospores (pointes montrant des sporanges dans les diffe ` ches), certains sont libe ´ re ´ s (e ´ toile) ( 1000). des fle
38 animals, respectively the number and percentage of improved or cured skin lesions are summarized in Table 1.
Microbiological exams After aerobic incubation, at 37 8C, for 72 hours, on Sabouraud’s dextrose agar plates, visible colonies with typical
[(Figure_2)TD$IG]
C. Bouari et al. features for Prototheca were developed. The colonies’ dimensions ranged from 3 to 4 mm in diameter. They had generally irregular edges, granular or sometimes smooth surface, pale-gray or whitish color, creamy consistency, characteristic mulberry aspect and typically yeast smell (Fig. 1a). Usually they can easily be confused with Candida or Cryptococcus colonies. These data are similar with
Figure 2 Cutaneous experimental protothecosis in BALB/c mouse. (a) control group: no lesions (HE 100); (b) Prototheca control group: pandermal, severe, diffuse granulomatous inflammation (HE 40); (c) Mentha piperita treated group: focal granulomatous reaction, with extensive fibrosis surrounding it (PAS 40); (d) Saturenja hortensis treated group: subcutaneous focal granulomatous reaction, with necrotic center and surrounding fibrous tissue (PAS 100); (e) nystatine treated group: PAS positive Prototheca cells within the center of a small focal granuloma (PAS 400); (f) amphotericin B treated group: subcutaneous large focal granuloma surrounded by fibrous tissue and a layer of predominantly mononuclear cells (HE 100). ´ cose expe ´ rimentale cutane ´ e chez la souris BALB/c. (a) groupe de contro ˆ le : pas de le ´ sions (HE 100) ; (b) groupe infecte ´ Protothe ´ ve ` re affectant toutes les couches cutane ´ es (HE 40) ; (c) Mentha avec Prototheca : une inflammation granulomateuse diffuse et se ´ : re ´ action granulomateuse focale, avec une fibrose extensive qui l’entoure (PAS 40) ; (d) Saturenja hortensis piperita groupe traite ´ : re ´ action cutane ´ e granulomateuse focale, avec un centre ne ´ crotique et des tissus fibreux (PAS 100) ; (e) nystatine groupe traite ´ : cellules de Prototheca PAS positives situe ´ es au centre d’un petit granulome focal (PAS 400) (f) l’amphote ´ ricine B groupe traite ´ : important granulome sous-cutane ´ focal entoure ´ par un tissu fibreux et une couche de cellules principalement groupe traite ´ aires (HE 100). mononucle
Natural products in the treatment of protothecosis
[(Figure_3)TD$IG]
previous reports [29,39]. As to be seen in Table 1, all animals that developed dorsal skin lesions were positive microbiologically for protothecosis. Since differentiation from other yeasts is not possible based exclusively on their cultural aspects, microscopy is necessary to confirm Prototheca spp. Prototheca is easily directly stained by lactophenol blue or Lügol solution, but heat fixation may induce morphological impairment. Microscopic appearance of Prototheca cells was observed by
39 examining wet smears using light microscopy, when structures described as ‘‘morula’’ are visible (Fig. 1b). Prototheca spp. are spherical or oval in shape, 7—30 mm in diameter, and they reproduce asexually. During cell maturation the cytoplasm undergoes a process of cleavage to form 6—20 endospores of different sizes, range from 12 to 22 mm. These endospores increase in size, and sporangia (mother cell) wall break under pressure from the enlarging endospores (daughter cells) and every 5 to 6 hours they are passively
Figure 3 Cutaneous experimental protothecosis in BALB/c mouse, from group 2. (a) Prototheca untreated group: presence of foamy macrophages in the center of granuloma (arrowheads), and important number of neutrophils (HE 400); (b) Prothotheca control group: foamy macrophages (arrowhead), some with intracytoplasmatic stages (arrows) of Prototheca (HE 1000). Mentha piperita treated group: different life cycle phases of Prototheca zopfii within the lesions; (c) sporangia; (d) dividing sporangia; (e) formation of sporangiospores inside the mother cell; (f) release of sporangiospores (PAS 1,000). ´ cose expe ´ rimentale cutane ´ e chez la souris BALB/c, dans le groupe 2. (a) Prototheca groupe non traite ´ : pre ´ sence de Protothe ` ches), ainsi qu’un nombre important de neutrophiles (HE 400) ; macrophages spumeux dans le centre du granulome (pointes des fle ´ avec Prototheca : macrophages spumeux (te ˆ te de fle ` che), certains avec des stades intracytoplasmiques (fle ` ches) (b) groupe infecte ´ : diffe ´ rentes phases du cycle de vie de Prototheca zopfii dans les le ´ sions ; (c) de Prototheca (HE 1000). Mentha piperita groupe traite ´ rieur de la cellule me ` re ; (f) libe ´ ration de les sporanges ; (d) la division des sporanges ; (e) formation de sporangiospores `a l’inte sporangiospores (PAS 1000).
40 released outside (Fig. 1b). These findings are similar with previous reports [21,26].
Histopathological and immunohistochemical study Dorsal skin samples were collected from areas with lesions at the end of the 21 days of topical treatment and processed for the microscopic study. The pathological exam aimed to describe the lesions, check for the presence of Prototheca in the lesions and quantify the inflammation. From each skin sample of every animal, 3 different HE stained serial slides were examined. No microscopic lesions were observed in any
[(Figure_4)TD$IG]
C. Bouari et al. animal from group 1 (Fig. 2a). In all the groups with topical administration of P. zopfii, they were present within the lesions. As the organism is known to be haematoxilinophyllic [33], they were visualized using periodic acid-Schiff (PAS) staining technique, where different evolution stages from its life cycle were visible — sporangium, cell division, formation and release of sporangiospores (Fig. 3c—f). The majority of the lesions were localized between the superficial dermis and the subcutaneous connective tissue. In all groups the inflammation was of granulomatous type, with formation of focal granulomas in treated animals (groups 3, 4, 5 and 6), whereas in untreated animals (group 2) the inflammation was more severe, with a predominantly diffuse pattern (Fig. 2b).
Figure 4 Immunohistochemical staining from cutaneous protothecosis skin biopsies. (a) CD8 antibody for T lymphocytes in Prototheca untreated group; (b) amphotericin B treated group; (c) macrophage antibody in amphotericin B treated group; (d) Saturenja hortensis treated group; (e) neutrophil antibody in Prototheca untreated group; (f) amphotericin B treated group. ´ es. (a) anticorps CD8 et des lymphocytes T dans le groupe non traite ´ La coloration immunohistochimique des biopsies cutane ´ avec de l’amphote ´ ricine B ; (c) anticorps des macrophages dans le groupe traite ´ avec de de Prototheca ; (b) groupe traite ´ ricine B ; (d) groupe traite ´ avec de la Saturenja hortensis ; (e) anticorps des neutrophiles dans le groupe non traite ´ l’amphote ´ avec de l’amphote ´ ricine B. de Prototheca ; (f) groupe traite
Natural products in the treatment of protothecosis [(Figure_5)TD$IG]
41
Figure 5 Immunohistochemical quantification of macrophages (a), lymphocytes (b) and neutrophils (c) from dorsal skin biopsies of experimental protothecosis. Statistical significant differences between groups are marked with an asterisk (P < 0.05). ´ es Quantification immunohistochimique des macrophages (a), les lymphocytes (b) et des neutrophiles (c) `a partir de biopsies cutane ´ cose expe ´ rimentale. Les diffe ´ rences significatives statistiques entre les groupes sont marque ´ s d’une aste ´ risque de protothe ( p < 0,05).
Skin sections from group 2 revealed a pandermal, granulomatous, inflammatory infiltrate composed of lymphocytes, histiocytes, macrophages — epithelioid type, neutrophils, and rare eosinophils. Rare multinucleated giant cells and plasma cells were also present along with foamy macrophages, some containing different stages of Prototheca (Fig. 3a, b) as well as hyperplastic lymphoid tissue (only 2 animals). P. zopfii was observed on HE and PAS-stained sections and was found as clusters, as well as solitary structures, within granulomas and macrophages, as well as in necrotic areas. Within the treated groups (Fig. 2c—f), the degree of inflammation, evaluated by the number and type of inflammatory cells, was more severe in the animals treated with S. hortensis (group 4), followed by the ones treated with M. piperita (group 3) and by the animals treated with nystatine and amphotericin B (groups 5 and 6). The thickness of the fibrous tissue surrounding the granulomas, also varied, being more developed in animals treated with M. piperita, followed by the ones treated with amphotericin B, S. hortensis and nystatine. In order to quantify the inflammatory response associated with experimental cutaneous protothecosis, we determined separately, from every mouse, the number of T lymphocytes, macrophages and neutrophils (Fig. 4a—f). Thus, as anticipated by the histopathological exam, the number of macrophages, T lymphocytes and neutrophils was significantly increased in all experimentally infected groups, as compared to the negative controls (P < 0.05, Fig. 5a—c). On the other hand, the mean number of macrophages, T lymphocytes and neutrophils were significantly decreased (P < 0.05) in all treated animals (groups 3—6) as compared to the Prototheca control animals (group 2). This means that the different form of treatment attenuated the infection. There was a similar trend regarding the number of macrophages and neutrophils between all groups (Fig. 4a, c). Hence, the mean number of macrophages and neutrophils was significantly decreased (P < 0.05) in animals treated with M. piperita, nystatine and amphotericin B as compared with the ones topically treated with S. hortensis. Moreover, the mean number of neutrophils in animals treated with M. piperita was
significantly increased (P < 0.05) as compared with the mouse treated with both conventional antifungal drugs (Fig. 5c). Regarding the quantification of T lymphocytes, their mean number was significantly increased in the animals treated with amphotericin B as compared with the other 3 treated groups (Fig. 5b).
Discussion The most frequent form of infection with Prototheca spp. in humans is represented by cutaneous protothecosis [26]. The use of natural products in the treatment of different diseases in both human and veterinary medicines is representing an important approach as an alternative for conventional drugs. So far, there were several studies which showed that some essential oils have inhibitory effects in vitro upon Prototheca growth. Also, in other two of our previous in vitro studies [4,5] we have shown that S. hortensis and M. piperita essential oils are inhibiting the growth/survival of P. wickerhamii and P. zopfii, both involved in human and animal pathologies. In another study, Pramila et al. [36] showed that a methanolic leaf extract of M. piperita proved antifungal activity in vitro against another fungi: C. albicans and Candida glabrata. A similar study [8] demonstrated that M. piperita L. in different forms (hydroalcoholic extract, infusion and essential oil) presented significant in vitro antimicrobial activity against Candida spp. Similar to the study by Horiuchi and Masuzawa [18] in a mouse model of skin protothecosis induced by intradermal and/or subcutaneous inoculation we found that the main lesion induced by P. zopfii, at 28 days following inoculation, was represented by the formation of epitheloid cell granulomas. The lesion was more severe in mouse skin from group 2 (Prototheca only). Based on histopathological observations, we found that the degree of inflammation was reduced in all four treated groups, being less severe in the animals that received the conventional nystatine or amphotericin B treatment. It is a well-known fact that here are important differences between the in vitro and the in vivo sensitivity of Prototheca spp. [33]. To our knowledge, the present study is the first one
42 to focus on the in vivo therapeutic potential of M. piperita and S. hortensis essential oils, on an animal model of experimental cutaneous protothecosis. According to previous works [13] nystatine and amphotericin B have proved their in vivo therapeutic potential against human cutaneous protothecosis. In our study we confirmed that both agents have proved their in vivo efficiency against experimental protothecosis in mice. This was proved by clinical and histopathological results. In fact, both conventional antifungal agents that we employed in the current experiment have proved to be more efficient than the two essential oils used to topically control the infection. It seems like, of the four different treatments that we tested, nystatine was the most efficient in reducing the degree of lesions as quantified by the number and nature of inflammatory cells and the degree of fibrosis. On the other hand, both essential oils (M. piperita and S. hortensis) have proven in vivo efficiency in significantly reducing the degree of the protothecal induced skin lesions, as compared to the lesions from the infected, untreated group. Interestingly, our results show that M. piperita had almost similar efficiency, as the two classic antifungal drugs that we used, in reducing the degree of skin experimental protothecosis. Thus, a natural therapeutic approach using peppermint essential oil could prove itself useful in the treatment of cutaneous protothecosis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements This work was supported by the Executive Unit for Financing Higher Education, Research, Development and Innovation, Romania, Human Resources Program, Postdoctoral Research Project, No. 175/2010 and by project No. 1356/2013 financed by University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania.
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