Next Article in Journal
Development of a Nomogram to Predict the Outcome for Patients with Soft Tissue Sarcoma
Previous Article in Journal
Retrospective Investigation and Genetic Variation Analysis of Chicken Infectious Anemia in Shandong Province, 2020–2022
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Veterinary Sciences
Volume 10
Issue 4
10.3390/vetsci10040265
vetsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Antimicrobial Activity of Sempervivum tectorum L. Extract on Pathogenic Bacteria Isolated from Otitis Externa of Dogs

by
Diana Maria Dégi
1,†,
Kálmán Imre
2,
Viorel Herman
3,
János Dégi
3,*,†,
Romeo Teodor Cristina
4,
Adela Marcu
5,
Florica Morariu
5,* and
Florin Muselin
1
1
Department of Toxicology and Toxicoses, Plant Biology and Medicinal Plants, University of Life Science “King Mihai I” from Timișoara, 300645 Timisoara, Romania
2
Department of Public Health, University of Life Science “King Michael I” from Timișoara, 300645 Timisoara, Romania
3
Department of Infectious Diseases and Preventive Medicine, University of Life Science “King Mihai I” from Timișoara, 300645 Timisoara, Romania
4
Department of Pharmacology and Veterinary Pharmacy, University of Life Science “King Mihai I” from Timișoara, 300645 Timisoara, Romania
5
Department of Animal Production Engineering, Faculty of Bioengineering of Animal Recourses, University of Life Science “King Mihai I” from Timișoara, 300645 Timisoara, Romania
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Vet. Sci. 2023, 10(4), 265; https://doi.org/10.3390/vetsci10040265
Submission received: 13 March 2023 / Revised: 27 March 2023 / Accepted: 28 March 2023 / Published: 29 March 2023
(This article belongs to the Section Veterinary Physiology, Pharmacology, and Toxicology)
Review Reports Versions Notes

Abstract

:

Simple Summary

Two distinct but strongly related aims directed our work. First, we aimed to evaluate the phytotherapeutic potential of Sempervivum tectorum L., a traditionally well-known but chemically less characterized medicinal plant, through quantitative analysis of its biologically active substances. Modern methods of phytoanalytics (microwave extraction, lyophilization, and UV spectrophotometry) were applied to investigate the polyphenol and proanthocyanidin contents of Sempervivum tectorum L., which have significant biological and pharmacological activities, including antimicrobial activity. Our second aim was to determine the antimicrobial activity of Sempervivum tectorum L. extract against pathogenic bacteria isolated from ear swabs taken from dogs with with chronic and recurrent otitis externa and standard ATCC strains (respectively, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853). Sempervivum tectorum L. represents a particular model plant selected to represent the wide structural and therapeutical variety of phenolics, from simple phenolic acids to macrocyclic polyphenols.

Abstract

The present study investigated the antimicrobial activity, total phenolic content, and proanthocyanidin concentration of ethanolic extracts from fresh leaves of Sempervivum tectorum L. The extracts were phytochemically analyzed and evaluated for antimicrobial activity. The broth microdilution method was used to assess antimicrobial activity against pathogenic bacteria isolated from ear swabs taken from dogs with otitis externa. Many compounds were present in the ethanolic aqueous extracts, which exhibited a broad spectrum of antimicrobial activity. They showed strong antibacterial activity against standard clinical Gram-positive strains such as S. aureus and Gram-negative strains such as P. aeruginosa. In our study, the obtained quantity of total phenolic compounds in the ethanol:water extract of leaves was 126.17 mg GAE/g. The proanthocyanidin concentration in the tested Sempervivum tectorum L. extracts was 15.39 mg PAC/g material. The high contents of total phenolics and proanthocyanidin indicated that these compounds contribute to antimicrobial activity. The antimicrobial activity of the tested S. tectorum L. extracts ranged from 1.47 to 63.75 µg/mL, starting with 1.47 µg/mL and 1.75 µg/mL against S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 strains, respectively. Likewise, S. tectorum L. ethanol extract demonstrated a bacteriostatic effect against S. aureus clinical isolate with a median MIC of 23.25 µg/mL and MBC of 37.23 µg/mL; and bactericidal against S. aureus ATCC 25923 with the median MIC of 20.33 µg/mL and MBC of 37.29 µg/mL. In the Gram-negative P. aeruginosa clinical and standard strains, the expressed MIC and MBC values were 24.234 and 20.53 µg/mL for MIC, and 37.30 and 37.02 µg/mL for MBC, respectively.

1. Introduction

Plants have long been essential sources of therapeutic agents for both humans and animals [1,2,3]. The resurgence of interest in the pharmacological activities of plants, in recent decades has fueled the development of standardized methods of preparation and extraction [4].
Traditional veterinary care using plants is called ethnoveterinary medicine, and it is practiced widely across Europe, including in Romania [3,4,5,6,7,8,9].
Herbal remedies have traditionally been administered in the context of a holistic approach to health maintenance and well-being that integrates enhancement of various body processes with symptom relief [10]. Sempervivum tectorum L. is an evergreen plant from the Crassulaceae family [11,12,13]. S. tectorum L. is widely recognized in traditional veterinary medicine, and its juice is still employed as a cooling agent and astringent to treat wounds, burns, insect bites, and the treatment of ear infections in dogs [13,14,15,16].
S. tectorum L., known as the common houseleek, is a thorny perennial herb with a dense basal rosette of fleshy obovate to oblong, sharp-tipped, bluish-green leaves [11,14,15]. The flowers are arranged in terminal cymes and are a dull red color. Houseleek is native to central and southern Europe and grows on weathered rocks, roofs, and scree. Traditionally, the leaves are used fresh rather than dried.Houseleek contains tannins, bitter compounds, sugars, flavonoids, and mucilage [17]. The plant has been linked to antinociceptive, liver-protective, and membrane-stabilizing effects, which have been partly attributed to the antioxidant activity of its phenolic compounds [18,19]. It has numerous external applications: the pressed juice of the leaves can be applied to herpetic skin eruptions, suppurating wounds, inflamed insect bites, and external ear inflammation in dogs [15,17]. Romania’s flora consists of approximately 4000 species, 800 of which have therapeutic characteristics validated by scientific investigations in over fifty percent of cases [7,8,20]. Extraction by conventional method relies on heating the solvent using the open microwave-assisted extraction (MAE) method, which involves contact with the sample matrix and disrupts cellulosic cell walls in plants, allowing easy acquisition of active components [21,22,23,24]. Otitis externa (OE) in dogs, a common veterinary complaint, can be caused by primary, secondary, predisposing, or perpetuating factors [25,26]. Chronic or recurrent otitis externa in dogs is more difficult to treat, although acute and uncomplicated otitis externa in dogs can frequently be successfully managed [27]. Antimicrobial, antimycotic, and anti-inflammatory medications are used as pharmacological support, but treatment failure and antimicrobial resistance are coercing the development of alternative approaches based on phytotherapeutic agents [28]. The most common bacteria isolated in canine OE cultures are Staphylococcus pseudintermedius, P. aeruginosa, S. aureus, Enterococcus spp., Corynebacterium spp., Streptococcus spp., and Escherichia coli [27,28,29,30,31]. Colonization by opportunistic bacteria, yeast (such as Malassezia), and antimicrobial resistance could complicate the pathology and can pose a therapeutic challenge for veterinarians. Plant bioactive compounds are commonly used in veterinary medicine, and some have been found to have antibacterial effect against pathogenic bacteria isolated from clinical cases of canine otitis externa [32,33]. Given the increase in multi-antimicrobial-resistant bacteria, which is likely owing to selective pressure resulting from excessive/inappropriate antimicrobial use, this is a fascinating concept [34]. Unlike antibiotics, bioactive substances work at the point of application and are less prone to produce resistance when used often [32]. An increasing antimicrobial resistance in the Staphylococcus spp. and P. aeruginosa, isolated from dogs’ ear clinical samples, has been reported [27,35]. Typically, both bacterial species are known as common dog colonizers but are also considered opportunistic pathogens that can cause various diseases [36]. Methicillin-resistant S. aureus (MRSA) and multi-drug-resistantant P. aeruginosa in healthcare settings have become a global concern for the health of dogs and humans [27,37]. They are typically linked with a multidrug-resistant phenotype, limiting veterinarians’ therapeutic options [38].
This study aimed to evaluated the antibacterial activity of extracts of S. tectorum L. samples against isolated bacteria and in standard strains (S. aureus and P. aeruginosa) via chemical analysis using UV spectrophotometry. The antibacterial activity, total phenolic content, and concentration of flavonoids of ethanolic extracts of fresh Sempervivum tectorum L. leaves were studied.

2. Materials and Methods

2.1. Study Area

Sempervivum tectorum L. plant materials were collected from spontaneous flora in the Socolari area (GPS Coordinates: 44°56’39,57” N, 21°43’45,00” E; Figures S1–S4), in the mountain region of western Romania, during spring and summer 2019.
S. tectorum L. samples and herbarium specimens were deposited at the Department of Vegetal Biology and Medicinal Plants, where sample authentication was also accomplished (voucher no. 112). The plants were identified according to the determination key given by Flora Europaea (Sempervivum tectorum LINN.; Sp. Plant. 464/1753), based on information provided by the Euro+Med PlantBase [39].

2.2. Chemicals and Plant Material

Ethanol, sodium hydrogen carbonate, triphenyl tetrazolium chloride (TTC), and Folin–Ciocalteu phenol reagent were acquired through from Sigma Aldrich (Merck KGaA, Darmstadt, Germany). Agar used for microbiological exams was obtained from Becton Dickinson (Becton Dickinson GmbH, Heidelberg, Germany). All other solvents and chemicals met analytical standards. The leaves were removed from the rosette and roots, washed, and filtered through grade 4 Whatman® qualitative filter paper (Merck KGaA, Darmstadt, Germany) before lyophilization (Figure S5). The samples were lyophilized to obtain the crude mass of juice for further experiments.

Lyophilization Method

The material was lyophilized using the Leybold Heraeus Lyovac GT2 (LH Leybold, Labexchange–Die Laborgerätebörse GmbH, Burladingen, Germany). The lyophilized S. tectorum L. leaves were then finely powdered in a blender as the pressure was slowly brought back up to room temperature and pressure. When a heat-dependent drying procedure could potentially denature thermosensitive components present in a material, lyophilization is the preferred method of drying [40,41].

2.3. Conventional Extraction Method

2.3.1. Sample Preparation for Conventional Extraction Method

Microwave-Assisted Extraction (MAE)

Each lyophilized sample (5 g) was placed in a round-bottom flask, followed by 50 mL of solvent (aqueous ethanol, 50:50 v:v). MAE was performed for 5 min at 50 W and 35 °C on a CEM Star 2 Plus Open Vessel Microwave Digestion System (CEM Corporation, Matthews, NC, USA), and the extract was centrifuged at 4000 rpm for 10 min and filtered through grade 4 Whatman® filter paper (two more measures of solvent were used to extract the press residue (total solvent volume: 150 mL)). Before further analysis, at 40 °C, the mixed extract was evaporated until dry and then redissolved in 30% solvent [17,42,43]. The S. tectorum L. leaves were ground to powder in a blender and subjected to MAE. After a specified length of selected conventional extraction, the filtrates were filtered and collected. A rotary evaporator (Julabo-Pura 4 water bath) was used to remove the solvent from the filtrate (Julabo UK Ltd., Stamford, UK). The extracts were stored at −20 °C until further examination.

2.3.2. Analytical Methods

Total Phenolic Content

Spectrometric quantification of polyphenols and proanthocyanins was carried out in accordance with the European Pharmacopeia directive.The total phenolic content (TPC) of extracts was determined by UV spectrophotometry (Varian Cary 50 UV-VIS, Agilent Technologies, Santa Clara, CA, USA) using the Folin–Ciocalteu (FC) method described by Anokwuru et al. [44]. Gallic acid (GA) equivalent (mg GA/g raw material) was used to express TPC. Total polyphenols were measured using a wavelength of 241 nm and compared to ethanol as a blank.To determine total phenolics using the FC technique [45], 800 mL of sodium carbonate (7.5 percent w/v) was added to 20 mL of each sample (range from 0.5 to 20 mg/mL), properly mixed, and allowed to stand for 2 min. Then, 1 mL of FC reagent was added while the mixture was vortexed. After leaving the samples in the dark for 30 min at room temperature, the absorbance was measured spectrophotometrically at 765 nm.

Proanthocyanin Content

According to Mannino et al. [46], we used UV spectrophotometry to determine the proanthocyanin (PAC) contents in extracts (Varian Cary 50 UV-Vis; Agilent Technologies, Santa Clara, CA, USA) based on acid hydrolysis and color formation.

2.4. Antimicrobial Activity Testing

Extracts were evaluated against a panel of bacteria, which included Gram-positive S. aureus clinical isolate and ATCC 25923, and Gram-negative P. aeruginosa clinical isolate and ATCC 27853, which were collected and cultured from ear swabs of dogs with chronic and recurrent otitis externa (Figure S6). The clinical strains are multi-drug- and methicillin-resistant (Tables S4 and S5). Clinical Laboratory Standards Institute (CLSI) [47,48] standards were followed for culture, identification, and susceptibility testing. After investigating the morphological and biochemical characteristics using standard laboratory methods reported and recommended by Bergey’s Manual of Systematic Bacteriology, the purified bacterial cultures were identified and confirmed (Figures S7 and S8). Clinical breakpoints of gentamicin (clinical breakpoint range: 0.5–8 g/mL, respectively 0.5–16 g/mL) and enrofloxacin (clinical breakpoint range: 0.5–4 g/mL, respectively 0.25–16 g/mL) for P. aeruginosa and S. aureus were used, taking into account the body site and host [49,50].
The bacterial isolates used were clinical isolates from the microbiological collection of the Microbiology Laboratory of the Faculty of Veterinary Medicine, Department of Infectious Diseases and Preventive Medicine, King Michael I University of Life Sciences, Timișoara. Antibacterial activity was assessed utilizing the CLSI M07-A9 broth microdilution method [51]. The minimum inhibitory concentration (MIC) was determined using serial dilution in 96-well microdilution plates (capacity: 200 μL). Mueller–Hinton agar for bacteria (Becton Dickinson GmbH, Heidelberg, Germany) was used to culture the test species at 37 °C. The final bacterial inoculum density was 5 × 105 CFU/mL [52].
To achieve a concentration of 1 mg/mL of lyophilized extract, stock solutions of Sempervivum extract were prepared in 70% ethanol (20 mg lyophilized extract dissolved in 250 mL ethanol to obtain an 80 g/mL concentration). The extracts were then serially diluted in concentrations ranging from 0.5 to 62 g/mL (0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, and 64.0 g/mL). The ethanol concentration in each well was never higher than 5%. The inoculum was added to each well, and the plates were cultured for 24 h at 37 °C. Positive controls included enrofloxacin (ENR, 5 µg) and gentamycin (GN, 10 µg) (both from Bio-Rad, Marnes-la-Coquette, France), while the solvent acted as a negative control. One inoculated well was included to allow for control of the broth’s suitability for organism growth. To ensure sterility of the medium, one non-inoculated well free of antimicrobial agents was also included (sterility control). The growth of bacteria was determined by adding 20 μL of 0.5% triphenyl tetrazolium chloride (TTC) aqueous solution (growth control). The minimal inhibitory concentration (MIC) was established as the lowest concentration of extract that inhibited observable growth (a red-colored pellet on the bottom of the well after the addition of TTC). To measure the minimal bactericidal concentration (MBC), broth from wells without apparent growth was inoculated at 37 °C for 24 h with Mueller–Hinton agar (Becton Dickinson GmbH, Heidelberg, Germany).

2.5. Statistics

The data represent the standard deviation of the mean of three replicates (SD). Using SPSS version 20.0, the results were subjected to multiway analysis of variance, and mean comparisons were performed using Tukey’s multiple range test (SPSS Inc., Chicago, IL, USA). Mean differences were considered significant at p < 0.05.

3. Results

Lyophilized S. tectorum L. from the mountain region was used. Freeze-drying was used since it is highly efficient and does not require high temperatures.

3.1. Total Phenolic Composition

Polyphenolic compounds are aromatic hydroxylated compounds that are present in a wide variety of plant species. Flavonoids, anthocyanidins, and resveratrol are the most well-known polyphenols. The total phenolic content was determined primarily based on the extracts’ antimicrobial activity. Polyphenols from S. tectorum L. can potentially contribute to research as components of treatments for well-known diseases such as otitis externa [16]. Table 1 shows the total phenolic content (TPC) of the S. tectorum L. extracts obtained using microwave-assisted extraction in this study.
The lyophilized S. tectorum L. samples had the highest TPC (126.17 mg GA/g material). Our results show that using the microwave-assisted extraction method and a 1:1 ethanol:water mixture was the best way to extract the highest TPC. This is ideal, because these solvents are preferable in plant compound extract applications. Microwave-assisted extraction with ethanol as a solvent yielded the highest total phenol content (126.17 mg GA/g material). The TPC can be influenced by the climatic conditions under which a plant is grown. This could be why the amounts of certain active substances in plants can vary so greatly. Another reason for the large differences reported in total phenolic contents could be the preparation process. Certain active compounds disintegrate when samples are dried at high temperatures, but most compounds retain their original activity when lyophilized because the process does not use high temperatures. We can confirm that the lyophilization process is the best option for sample preparation. Fresh biomass has the advantage of requiring minimum processing and resulting in minimal degradation of the target bioactive components. The issue is that it can include more than 70% water, diluting the extraction solvent. When preparing raw materials, water is also an issue because it impedes shredding and grinding. Even if the herb is in bulk, grinding it to the desired size prior to extraction is easier with dry biomass. Furthermore, the solvent/herb ratio is much easier to control, providing greater reproducibility in experimental work. As a result, using a microwave technique for plant extraction has benefits such as enhanced mass transfer, improved solvent penetration, less reliance on the solvent utilized, extraction at lower temperatures, faster extraction rates, and higher product yields [53,54].

3.2. Proanthocyanidin (PAC) Content

Proanthocyanidins (PACs) are oligomeric or polymeric polyphenols that are found in many plants. They mainly comprise catechin, epicatechin, and gallic acid ester oligomers. They have antibacterial properties, just like polyphenols [55]. However, their biological abilities are primarily determined by their structures [56].
The PAC content of the S. tectorum L. extracts is shown in Table 1. The highest PAC content (15.39 mg PAC/g material) was obtained in lyophilized S. tectorum L. material using microwave-assisted extraction and a 50:50 ethanol:water mixture.
The analysis revealed extremely high PAC content in lyophilized S. tectorum L. extracts; because the active components did not disintegrate, the lyophilization process was ideal for preparing the studied materials.

3.3. Antimicrobial Activity

The microdilution method was used to evaluate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the most effective S. tectorum L. extract. Table 2 and Table 3 show the concentration effect of this extract (Figures S9–S11). The MIC and MBC of this S. tectorum L. extract indicated high activity. The ethanolic leaf extract demonstrated the highest activity, with determined inhibitory activity against all tested strains.
The inhibitory effects of the S. tectorum L. extract became observable at 1.47 µg/mL against S. aureus ATCC 25923 and 1.75 µg/mL against P. aeruginosa ATCC 27853.
Antibacterial activity was determined according to the MBC/MIC ratio (Table 2). If the ratio was less than 4, the effect was considered bactericidal; if it was greater than 4, it was considered bacteriostatic. The S. tectorum L. ethanol extracts were bacteriostatic against the S. aureus clinical isolate strain, with a median MIC of 23.25 µg/mL and MBC of 37.23 µg/mL, and were bactericidal against S. aureus ATCC 25923, with a median MIC of 20.33 µg/mL and median MBC of 37.29 µg/mL. The median MIC and MBC of the Gram-negative P. aeruginosa clinical and standard strains were 24.234 and 20.53 µg/mL and 37.30 and 37.02 µg/mL, respectively.
The MBC was confirmed by the absence of bacterial growth in the inhibition zone corresponding to the tested strain’s lowest MIC. With an MBC of 15.75 µg/mL, S. tectorum L. extract demonstrated potential bactericidal activity against the tested pathogenic bacteria (S. aureus and P. aeruginosa) (Table 3). The MIC and MBC results of the most effective plant extract suggest that S. tectorum L. can be used to control and prevent the growth of bacteria implicated in the pathogenesis of otitis externa in dogs. The bacterial strains were chosen for their importance in the pathogenesis of canine otitis externa. P. aeruginosa is one of the most common pathogenic bacteria involved for otitis externa in dogs. Toxins and other metabolites produced by S. aureus cause skin lesions, a risk factor for otitis externa in dogs. S. tectorum L. extract inhibited the growth of all tested bacterial strains.
Because the Kruskal–Wallis p-value was less than the significance level (c2 = 12.222, p < 0.001), we concluded that there were significant differences in total phenolic and proanthocyanidin contents in extracts obtained using the microwave-assisted extraction method: S. tectorum L. extract had a median MIC of 22.086 (23.25, 20.33, 24.234, 20.53).

4. Discussion

The antimicrobial activity of the tested S. tectorum L. extract ranged from 1.47 to 63.75 µg/mL, depending on the concentration of the extract and the tested microorganism. The tested concentrations showed a wide range of antimicrobial activity, which could be classified as high or moderate. S. tectorum L. extract was bacteriostatic at very high concentrations, or there was no bactericidal activity at most of the tested concentrations.
The results indicate that S. tectorum L. extract has a primarily inhibitory and bactericidal effect on microorganisms. The antimicrobial activity of dried leaf extracts was higher than that of fresh leaf extracts, indicating a higher concentration of antimicrobial compounds in the leaves. In addition, when antimicrobial activity against the tested bacterial species was observed, Gram-positive bacteria (S. aureus) were more susceptible. This is due to differences in the cell envelope composition of Gram-positive and Gram-negative bacteria, which affect the permeability and susceptibility of these organisms to different compounds [57,58,59].
Based on these results, we can conclude that ethanol extracted the compounds with the highest antimicrobial activity found in the plant’s leaves. This was expected, given that the leaves are described as the active plant parts in the ethnopharmacological usage of this plant. Furthermore, the obtained results can be attributed to phenolic and proanthocyanin compounds, which are well known for their antimicrobial activity [16,55,60,61]. Because of the slight discrepancy in results between antimicrobial activity and phenol composition (significant concentration in the roots but low antimicrobial activity), we can assume that other compounds, such as flavonoids (found in low concentrations in the leaf extract), play an important role in the antimicrobial effect of the tested extracts. In 2015, Rovanin et al. [62] used the Folin–Ciocalteu assay to determine TPC in extracts of S. tectorum (solvent extraction, solvent ethanol:water = 7:3) and other plants. The TPC of S. tectorum extract in the latter study was 16.00 mg GA/g extract. We found a much higher TPC of 139.42 mg GA/g when using ultrasound extraction. In the Rovanin et al. study, the leaves of S. tectorum L. were air-dried at 25–28 °C, which is most likely the reason for this difference. In our study, the leaves were lyophilized, allowing for a higher TPC to be preserved. In addition to TPC, the same authors reported PAC content, with the results expressed as mg of catechin equivalent (CE)/g extract [47]. They obtained a PAC value of 0.9 mg CE/g in an extract using a 7:3 ethanol:water mixture. Abram et al. [61] identified two major PAC compounds (4-dibenzyl-(-)-epigallocatechin and 4-dibenzyl-(-)-epigallocatechin-3-gallate) in S. tectorum extracts. Knez Marevci et al. [13] were the first to test and confirm the PAC content of dried S. tectorum extracts. They used conventional extraction methods and solvents to compare two lyophilized S. tectorum extracts, and obtained the following results using the same solvent mixture for all three methods: Soxhlet extraction yielded 13.40 mg PAC/g extract, cold solvent extraction yielded 15.89 mg PAC/g extract, and ultrasound extraction yielded 15.42 mg PAC/g extract. Several factors contribute to the increased interest in herbal medications in veterinary medicine. Among them, there is a widespread belief among the general public that medicinal plants are both practical and safer than synthetic compounds. When taken correctly, herbal medications often have few adverse effects, are well tolerated by most pets, and can be helpful in cases where conventional medications are not tolerated, or side effects are intolerable. Another primary reason is cost, as they are less expensive than traditional treatments [63,64,65]. Furthermore, they are regarded as a more sustainable approach. In this regard, phytotherapeutic remedies are also viable for treating otitis externa in dogs while avoiding synthetic drugs [66]. Plant-based medicine is becoming more popular in dogs due to its effectiveness and appropriate benefit–risk balance [32]. It may also aid in the treatment of subclinical or chronic diseases in the absence of conventional treatment. The widespread use of phytotherapy would also justify a discussion about the source of veterinarians’ knowledge in this field and increased academic training provided by veterinary faculties [66,67]. Otitis externa is one of the most common painful conditions in dogs. This a highly prevalent multifactorial skin disease that can be challenging to treat and accounts for up to 20% of pet counseling cases [68]. Bacterial reinfection becomes common if the root cause is not treated, needing prolonged antibiotic therapy [28,68]. The prolonged therapy increases the risk of antibiotic-resistant bacteria spreading from animals to humans and promotes their emergence. Resistance to antibiotics has emerged as one of the most serious public health concerns. This is how treatment failure appears to be coercing alternative approaches based on phytotherapeutic agents [32,69,70,71]. This study emphasizes the significance of antimicrobial resistance and the necessity for antibiotic alternatives, such as plant extract. As indicated in the results of our study, the S. aureus clinical isolate was resistant to the most frequently used or first-line antibiotic prescribed for otitis externa infection (gentamicin). Thus topical and oral antibiotics (gentamicin and enrofloxacin) are frequently used in canine bacterial ear infections, and resistance is a significant concern [72]. An increasing antimicrobial resistance in the Staphylococcus spp. and P. aeruginosa, isolated from dog ear clinical samples, has been reported [27,35].
Furthermore, this study opens the opportunity to initiate new investigations addressing some limitations of the current findings, especially regarding in vivo evaluation of the Sempervivum tectorum L. extract in the clinical cases of otitis externa in dogs and in vitro testing of other bacterial strains isolated from this disease.

5. Conclusions

Otitis externa in dogs is frequently complicated by the proliferation of numerous pathogenic bacterial strains. The treatment of this disorder in companion animals is primarily based on the use of synthetic antimicrobials. Increased resistance to potentially effective antimicrobials is among the adverse effects of these antimicrobials in veterinary medicine. Our findings indicate that S. tectorum L. has a high potential for use in pharmacy and phytotherapy. According to this information, parts of this plant are precious natural sources of antimicrobial substances.
Further research on this plant species should focus on a detailed qualitative analysis of its parts and in vivo studies of its medically active components to create a high-value natural pharmaceutical product.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vetsci10040265/s1, Figure S1. Sempervivum tectorum L. in the natural habitat (old fortress, Socolari/Ilidia—Ciclova Română, Caraș Severin County); Figure S2. Sempervivum tectorum L. in the natural habitat; Figure S3. Sempervivum tectorum L. (Sempervivum folium and Sempervivum flower); Figure S4. Sempervivum tectorum L.—preparation for processing in the laboratory; Figure S5. Sempervivum tectorum L., fresh leaf and lyophilized; Figure S6. Erythematous ceruminous otitis externa in dog; Figure S7. Pseudomonas aeruginosa (clinical strain); Figure S8. Staphylococcus aureus (clinical strain); Figure S9. Microtiter plate with varying concentrations of Sempervivum tectorum L. extract and tested bacterial strains; Figure S10. Microplate assay for determination of MIC /MBC of Sempervivum tectorum L. extract against clinical and standard bacterial strains (P. aeruginosa and S. aureus); Figure S11. Determination of MIC and MBC, in a 96-well microtiter plate of ethanolic:water extracts of Sempervivum tectorum L. against S. aureus and P. aeruginosa (clinical and standard) strains; Table S1. Total phenol and proanthocyanidin concentration of S. tectorum L. extract; Table S2. The median minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations activity of S. tectorum L. extracts (µg/mL); Table S3. The minimum inhibitory concentrations of the most effective plant extract against S. aureus and P. aeruginosa (clinical and standard) strains; Table S4. Phenotypic Antimicrobial Resistance (AMR) profiles of Staphylococcus aureus clinical strain; Table S5. Phenotypic Antimicrobial Resistance (AMR) profiles of Pseudomonas aeruginosa clinical strain.

Author Contributions

Conceptualization, D.M.D., K.I. and F.M. (Florin Muselin); methodology, D.M.D. and F.M. (Florin Muselin); software, J.D.; validation, R.T.C., V.H. and F.M. (Florin Muselin); formal analysis, J.D.; investigation, D.M.D., J.D., A.M., F.M. (Florica Morariu) and V.H.; resources, R.T.C. and F.M. (Florica Morariu); data curation, J.D.; writing—original draft preparation, D.M.D., K.I. and F.M. (Florin Muselin); writing—review and editing, R.T.C.; visualization, V.H.; supervision, F.M. (Florin Muselin); project administration, F.M. (Florin Muselin); funding acquisition, J.D. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the project “Increasing the impact of excellence research on the capacity for innovation and technology transfer within USAMVB Timișoara,” code 6PFE, submitted to the competition Program 1: the development of the national system of research and development, subprogram 1.2: Institutional performance, institutional development projects—development projects of excellence in RDI.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We were able to carry out this research with the support of the Interdisciplinary Research Platform of King Michael I University of Life Sciences, Timisoara, where the microbiological and phytopharmaceutical analysis was conducted.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Dzobo, K. The Role of Natural Products as Sources of Therapeutic Agents for Innovative Drug Discovery. In Comprehensive Pharmacology; Elsevier: Amsterdam, The Netherlands, 2021; pp. 408–422. [Google Scholar] [CrossRef]
  2. Mathur, S.; Hoskins, C. Drug development: Lessons from nature. Biomed. Rep. 2017, 6, 612–614. [Google Scholar] [CrossRef] [Green Version]
  3. Pulak, K.M. (Ed.) Evidence—Based Validation of Herbal Medicine; Elsevier: Amsterdam, The Netherlands, 2015; ISBN 978-012-800874-4. [Google Scholar]
  4. Najmi, A.; Javed, S.A.; Al Bratty, M.; Alhazmi, H.A. Modern Approaches in the Discovery and Development of Plant-Based Natural Products and Their Analogues as Potential Therapeutic Agents. Molecules 2022, 27, 349. [Google Scholar] [CrossRef]
  5. Mattalia, G.; Belichenko, O.; Kalle, R.; Kolosova, V.; Kuznetsova, N.; Prakofjewa, J.; Stryamets, N.; Pieroni, A.; Volpato, G.; Sõukand, R. Multifarious Trajectories in Plant-Based Ethnoveterinary Knowledge in Northern and Southern Eastern Europe. Front. Vet. Sci. 2021, 8, 710019. [Google Scholar] [CrossRef] [PubMed]
  6. Schlittenlacher, T.; Knubben-Schweizer, G.; Cero, M.D.; Vogl, C.R.; Maeschli, A.; Hamburger, M.; Walkenhorst, M. What can we learn from past and recent Bavarian knowledge for the future development of European veterinary herbal medicine? An ethnoveterinary study. J. Ethnopharmacol. 2022, 288, 114933. [Google Scholar] [CrossRef] [PubMed]
  7. Bartha, S.G.; Quave, C.L.; Balogh, L.; Papp, N. Ethnoveterinary practices of Covasna County, Transylvania, Romania. J. Ethnobiol. Ethnomedicine 2015, 11, 35. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Cășaru, C.; Bulgaru, A.; Daneș, D. Medicinal plants used in traditional veterinary medicine to treat ruminants in the curvature Subcarpathians area, Romania. Scientific Works. Series C. Vet. Med. 2020, LXVI, 93–103. [Google Scholar]
  9. Gilca, M.; Tiplica, G.S.; Salavastru, C.M. Traditional and ethnobotanical dermatology practices in Romania and other Eastern European countries. Clin. Dermatol. 2018, 36, 338–352. [Google Scholar] [CrossRef]
  10. Pieroni, A.; Giusti, M.E.; Münz, H.; Lenzarini, C.; Turković, G.; Turković, A. Ethnobotanical knowledge of the Istro-Romanians of Zejane in Croatia. Fitoterapia 2003, 74, 710–719. [Google Scholar] [CrossRef] [PubMed]
  11. Florin, M.; Alexandra, T.; Gabriela, S.L.; Romeo, C.T.; Corina, G.; Ioan, M.; Eugenia, D. Protective Effects of Aqueous Extract of Sempervivum tectorum L. (Crassulaceae) on Aluminium-Induced Oxidative Stress in Rat Blood. Trop. J. Pharm. Res. 2014, 13, 179–184. [Google Scholar] [CrossRef] [Green Version]
  12. Byalt, V.V. The adventive species of Crassulaceae. Russ. J. Biol. Invasions 2021, 2, 155–157. [Google Scholar] [CrossRef]
  13. Marevci, M.K.; Bjelić, S.; Dariš, B.; Knez, Ž.; Leitgeb, M. The Influence of Extracts from Common Houseleek (Sempervivum tectorum) on the Metabolic Activity of Human Melanoma Cells WM-266-4. Processes 2021, 9, 1549. [Google Scholar] [CrossRef]
  14. Gentscheva, G.; Karadjova, I.; Minkova, S.; Nikolova, K.; Andonova, V.; Petkova, N.; Milkova-Tomova, I. Optical Properties and Antioxidant Activity of Water-Ethanolic Extracts from Sempervivum tectorum L. from Bulgaria. Horticulturae 2021, 7, 520. [Google Scholar] [CrossRef]
  15. Kovac-Besovic, E.E.; Duric, K.; Suljevic, L. Investigation of Flavonoids Presence in Houseleek, Sempervivum tectorum L. Sci. Pharm. 2009, 77, 256. [Google Scholar] [CrossRef]
  16. Stojković, D.; Barros, L.; Petrović, J.; Glamoclija, J.; Santos-Buelga, C.; Ferreira, I.C.; Soković, M. Ethnopharmacological uses of Sempervivum tectorum L. in southern Serbia: Scientific confirmation for the use against otitis linked bacteria. J. Ethnopharmacol. 2015, 176, 297–304. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Cattaneo, F.; De Marino, S.; Parisi, M.; Festa, C.; Castaldo, M.; Finamore, C.; Duraturo, F.; Zollo, C.; Ammendola, R.; Zollo, F.; et al. Wound healing activity and phytochemical screening of purified fractions of Sempervivum tectorum L. leaves on HCT 116. Phytochem. Anal. 2019, 30, 524–534. [Google Scholar] [CrossRef]
  18. Alberti, Á.; Béni, S.; Lackó, E.; Riba, P.; Al-Khrasani, M.; Kéry, Á. Characterization of phenolic compounds and antinociceptive activity of Sempervivum tectorum L. leaf juice. J. Pharm. Biomed. Anal. 2012, 70, 143–150. [Google Scholar] [CrossRef] [PubMed]
  19. Alberti, Á.; Riethmüller, E.; Béni, S.; Kéry, Á. Evaluation of Radical Scavenging Activity of Sempervivum tectorum and Corylus avellana Extracts with Different Phenolic Composition. Nat. Prod. Commun. 2016, 11, 469–474. [Google Scholar] [CrossRef] [Green Version]
  20. Segneanu, A.-E.; Cepan, C.; Grozescu, I.; Cziple, F.; Olariu, S.; Ratiu, S.; Lazar, V.; Murariu, S.M.; Velciov, S.M.; Marti, T.D. Therapeutic Use of Some Romanian Medicinal Plants. In Pharmacognosy-Medicinal Plants; IntechOpen: London, UK, 2019. [Google Scholar] [CrossRef] [Green Version]
  21. Chan, C.-H.; Yusoff, R.; Ngoh, G.-C.; Kung, F.W.-L. Microwave-assisted extractions of active ingredients from plants. J. Chromatogr. A 2011, 1218, 6213–6225. [Google Scholar] [CrossRef]
  22. Javad, S.; Mubarik, M.; Aftab, A.; Tariq, A. Microwave Assisted Extraction of Phenolics from Trachyspermum ammi (L.) Sprague. J. Bioresour. Manag. 2016, 3, 4. [Google Scholar] [CrossRef]
  23. Akhtar, I.; Javad, S.; Yousaf, Z.; Iqbal, S.; Jabeen, K. Review: Microwave assisted extraction of phytochemicals an efficient and modern approach for botanicals and pharmaceuticals. Pak. J. Pharm. Sci. 2019, 32, 223–230. [Google Scholar] [PubMed]
  24. Kontogeorgis, G.M.; Holster, A.; Kottaki, N.; Tsochantaris, E.; Topsøe, F.; Poulsen, J.; Bache, M.; Liang, X.; Blom, N.S.; Kronholm, J. Water structure, properties and some applications—A review. Chem. Thermodyn. Therm. Anal. 2022, 6, 100053. [Google Scholar] [CrossRef]
  25. Leonard, C.; Thiry, D.; Taminiau, B.; Daube, G.; Fontaine, J. External Ear Canal Evaluation in Dogs with Chronic Suppurative Otitis Externa: Comparison of Direct Cytology, Bacterial Culture and 16S Amplicon Profiling. Vet. Sci. 2022, 9, 366. [Google Scholar] [CrossRef]
  26. Dégi, J.; Imre, K.; Cătană, N.; Morar, A.; Sala, C.; Herman, V. Frequency of isolation and antibiotic resistance of staphylococcal flora from external otitis of dogs. Vet. Rec. 2013, 173, 42. [Google Scholar] [CrossRef]
  27. Dégi, J.; Moțco, O.-A.; Dégi, D.; Suici, T.; Mareș, M.; Imre, K.; Cristina, R. Antibiotic Susceptibility Profile of Pseudomonas aeruginosa Canine Isolates from a Multicentric Study in Romania. Antibiotics 2021, 10, 846. [Google Scholar] [CrossRef]
  28. Bourély, C.; Cazeau, G.; Jarrige, N.; Leblond, A.; Madec, J.; Haenni, M.; Gay, E. Antimicrobial resistance patterns of bacteria isolated from dogs with otitis. Epidemiol. Infect. 2019, 147, e121. [Google Scholar] [CrossRef] [Green Version]
  29. Moog, F.; Mivielle, J.; Brun, J.; Dumitrache, M.O.; Amalric, N.; Lecru, L.-A.; Pressanti, C.; Kondratjeva, J.; Combarros, D.; Fantini, O.; et al. Clinical and Microbiological Performances and Effects on Lipid and Cytokine Production of a Ceruminolytic Ear Cleaner in Canine Erythemato-Ceruminous Otitis Externa. Vet. Sci. 2022, 9, 185. [Google Scholar] [CrossRef]
  30. Harvey, R. A Review of Recent Developments in Veterinary Otology. Vet. Sci. 2022, 9, 161. [Google Scholar] [CrossRef]
  31. O’Neill, D.G.; Volk, A.V.; Soares, T.; Church, D.B.; Brodbelt, D.C.; Pegram, C. Frequency and predisposing factors for canine otitis externa in the UK—A primary veterinary care epidemiological view. Canine Med. Genet. 2021, 8, 7. [Google Scholar] [CrossRef] [PubMed]
  32. Tresch, M.; Mevissen, M.; Ayrle, H.; Melzig, M.; Roosje, P.; Walkenhorst, M. Medicinal plants as therapeutic options for topical treatment in canine dermatology? A systematic review. BMC Vet. Res. 2019, 15, 174. [Google Scholar] [CrossRef]
  33. Wynn, S.G.; Fougère, B.J. Chapter 20—Veterinary Herbal Medicine: A Systems-Based Approach. In Veterinary Herbal Medicine; Mosby: Maryland Heights, MO, USA, 2007; pp. 291–409. [Google Scholar]
  34. Beco, L.; Guaguere, E.; Méndez, C.L.; Noli, C.; Nuttall, T.; Vroom, M. Suggested guidelines for using systemic antimicrobials in bacterial skin infections: Part 2—Antimicrobial choice, treatment regimens and compliance. Vet. Rec. 2013, 172, 156–160. [Google Scholar] [CrossRef] [Green Version]
  35. Bertelloni, F.; Cagnoli, G.; Ebani, V. Virulence and Antimicrobial Resistance in Canine Staphylococcus spp. Isolates. Microorganisms 2021, 9, 515. [Google Scholar] [CrossRef] [PubMed]
  36. Monecke, S.; Gavier-Widén, D.; Hotzel, H.; Peters, M.; Guenther, S.; Lazaris, A.; Loncaric, I.; Müller, E.; Reissig, A.; Ruppelt-Lorz, A.; et al. Diversity of Staphylococcus aureus Isolates in European Wildlife. PLoS ONE 2016, 11, e0168433. [Google Scholar] [CrossRef] [Green Version]
  37. Belhout, C.; Elgroud, R.; Butaye, P. Methicillin-Resistant Staphylococcus aureus (MRSA) and Other Methicillin-Resistant Staphylococci and Mammaliicoccus (MRNaS) Associated with Animals and Food Products in Arab Countries: A Review. Vet. Sci. 2022, 9, 317. [Google Scholar] [CrossRef] [PubMed]
  38. Ruiz-Ripa, L.; Simón, C.; Ceballos, S.; Ortega, C.; Zarazaga, M.; Torres, C.; Gómez-Sanz, E.S. pseudintermedius and S. aureus lineages with transmission ability circulate as causative agents of infections in pets for years. BMC Vet. Res. 2021, 17, 42. [Google Scholar] [CrossRef]
  39. The Euro+Med PlantBase. Available online: https://www.emplantbase.org/information.html (accessed on 24 March 2022).
  40. Costantino, H.R.; Pikal, M.J. Lyophilization of Biopharmaceuticals; AAPS Press: Arlington, VA, USA, 2004. [Google Scholar]
  41. Vilas, C.; Alonso, A.A.; Balsa-Canto, E.; López-Quiroga, E.; Trelea, I.C. Model-Based Real Time Operation of the Freeze-Drying Process. Processes 2020, 8, 325. [Google Scholar] [CrossRef] [Green Version]
  42. Albero, B.; Tadeo, J.L.; Pérez, R.A. Ultrasound-assisted extraction of organic contaminants. TrAC Trends Anal. Chem. 2019, 118, 739–750. [Google Scholar] [CrossRef]
  43. Brglez Mojzer, E.; Knez Hrnčič, M.; Škerget, M.; Knez, Ž.; Bren, U. Polyphenols: Extraction Methods, Antioxidative Action, Bioavailability and Anticarcinogenic Effects. Molecules 2016, 21, 901. [Google Scholar] [CrossRef]
  44. Anokwuru, C.; Sigidi, M.; Zininga, T.; Tshisikhawe, M.; Shonhai, A.; Ramaite, I.; Traoré, A.; Potgieter, N. Phenolic contents, antioxidant activity and spectroscopic characteristics of pterocarpus angolensis DC. Stem bark fractions. Indian J. Tradit. Knowl. 2017, 16, 400–406. [Google Scholar]
  45. Blainski, A.; Lopes, G.C.; De Mello, J.C.P. Application and Analysis of the Folin Ciocalteu Method for the Determination of the Total Phenolic Content from Limonium brasiliense L. Molecules 2013, 18, 6852–6865. [Google Scholar] [CrossRef] [Green Version]
  46. Mannino, G.; Maffei, M.E. Metabolomics-Based Profiling, Antioxidant Power, and Uropathogenic Bacterial Anti-Adhesion Activity of SP4TM, a Formulation with a High Content of Type-A Proanthocyanidins. Antioxidants 2022, 11, 1234. [Google Scholar] [CrossRef]
  47. Clinical Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. In CLSI Standard VET01S, 5th ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2020. [Google Scholar]
  48. Clinical Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, 30th ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2020; CLSI Supplement M100. [Google Scholar]
  49. KuKanich, K.S.; Bagladi-Swanson, M.; KuKanich, B. Pseudomonas aeruginosa susceptibility, antibiogram and clinical interpretation, and antimicrobial prescribing behaviors for dogs with otitis in the Midwestern United States. J. Vet. Pharmacol. Ther. 2022, 45, 440–449. [Google Scholar] [CrossRef]
  50. Azzariti, S.; Bond, R.; Loeffler, A.; Zendri, F.; Timofte, D.; Chang, Y.-M.; Pelligand, L. Investigation of In Vitro Susceptibility and Resistance Mechanisms in Skin Pathogens: Perspectives for Fluoroquinolone Therapy in Canine Pyoderma. Antibiotics 2022, 11, 1204. [Google Scholar] [CrossRef] [PubMed]
  51. Clinical Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—11th Edition; CLSI document M07-A; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
  52. Smith, K.P.; Kirby, J.E. The Inoculum Effect in the Era of Multidrug Resistance: Minor Differences in Inoculum Have Dramatic Effect on MIC Determination. Antimicrob. Agents Chemother. 2018, 62, e00433-18. [Google Scholar] [CrossRef] [Green Version]
  53. Garcia-Vaquero, M.; Ummat, V.; Tiwari, B.; Rajauria, G. Exploring Ultrasound, Microwave and Ultrasound–Microwave Assisted Extraction Technologies to Increase the Extraction of Bioactive Compounds and Antioxidants from Brown Macroalgae. Mar. Drugs 2020, 18, 172. [Google Scholar] [CrossRef] [Green Version]
  54. Arya, P.; Kumar, P. Comparison of ultrasound and microwave assisted extraction of diosgenin from Trigonella foenum graceum seed. Ultrason. Sonochem. 2021, 74, 105572. [Google Scholar] [CrossRef]
  55. Nawrot-Hadzik, I.; Matkowski, A.; Hadzik, J.; Dobrowolska-Czopor, B.; Olchowy, C.; Dominiak, M.; Kubasiewicz-Ross, P. Proanthocyanidins and Flavan-3-Ols in the Prevention and Treatment of Periodontitis—Antibacterial Effects. Nutrients 2021, 13, 165. [Google Scholar] [CrossRef]
  56. Ou-Yang, C.; Chai, W.; Xu, X.; Song, S.; Wei, Q.; Huang, Q.; Zou, Z. Inhibitory potential of proanthocyanidins from the fruit pulp of Clausena lansium (Lour.) Skeels against α-glucosidase and non-enzymatic glycation: Activity and mechanism. Process Biochem. 2020, 91, 364–373. [Google Scholar] [CrossRef]
  57. Barreto-Santamaría, A.; Arévalo-Pinzón, G.; Patarroyo, M.A.; Patarroyo, M.E. How to Combat Gram-Negative Bacteria Using Antimicrobial Peptides: A Challenge or an Unattainable Goal? Antibiotics 2021, 10, 1499. [Google Scholar] [CrossRef]
  58. Segovia, R.; Solé, J.; Marqués, A.M.; Cajal, Y.; Rabanal, F. Unveiling the Membrane and Cell Wall Action of Antimicrobial Cyclic Lipopeptides: Modulation of the Spectrum of Activity. Pharmaceutics 2021, 13, 2180. [Google Scholar] [CrossRef]
  59. Epand, R.M.; Walker, C.; Epand, R.F.; Magarvey, N.A. Molecular mechanisms of membrane targeting antibiotics. Biochim. Biophys. Acta 2016, 1858, 980–987. [Google Scholar] [CrossRef] [PubMed]
  60. Dai, J.; Mumper, R.J. Plant Phenolics: Extraction, Analysis and Their Antioxidant and Anticancer Properties. Molecules 2010, 15, 7313–7352. [Google Scholar] [CrossRef]
  61. Abram, V.; Donko, M. Tentative Identification of Polyphenols in Sempervivum tectorum and Assessment of the Antimicrobial Activity of Sempervivum L. J. Agric. Food Chem. 1999, 47, 485–489. [Google Scholar] [CrossRef] [PubMed]
  62. Rovčanin, B.R.; Ćebović, T.; Stešević, D.; Kekić, D.; Ristić, M. Antibacterial effect of Herniaria hirsuta, Prunus avium, Rubia tinctorum and Sempervivum tectorum plant extracts on multiple antibiotic resistant Escherichia coli. Biosci. J. 2015, 31, 1852–1861. [Google Scholar] [CrossRef] [Green Version]
  63. Karimi, A.; Majlesi, M.; Rafieian-Kopaei, M. Herbal versus synthetic drugs; beliefs and facts. J. Nephropharmacology 2015, 4, 27–30. [Google Scholar]
  64. Romero, B.; Susperregui, J.; Sahagún, A.M.; Diez, M.J.; Fernández, N.; García, J.J.; López, C.; Sierra, M.; Díez, R. Use of medicinal plants by veterinary practitioners in Spain: A cross-sectional survey. Front. Vet. Sci. 2022, 9, 1060738. [Google Scholar] [CrossRef]
  65. Ahmad, A.S.; Sharma, R. Comparitive Analysis of Herbal and Allopathic Treatment systems. Eur. J. Mol. Clin. Med. 2020, 7, 2869–2876. [Google Scholar]
  66. Memon, M.; Shmalberg, J.; Adair, H.S., III; Allweiler, S.; Bryan, J.; Cantwell, S.; Carr, E.; Chrisman, C.; Egger, C.; Greene, S.; et al. Integrative veterinary medical education and consensus guidelines for an integrative veterinary medicine curriculum within veterinary colleges. Open Vet. J. 2016, 6, 44–56. [Google Scholar] [CrossRef] [Green Version]
  67. Memon, M.A.; Shmalberg, J.W.; Xie, H. Survey of Integrative Veterinary Medicine Training in AVMA-Accredited Veterinary Colleges. J. Vet. Med. Educ. 2021, 48, 289–294. [Google Scholar] [CrossRef] [PubMed]
  68. Kwon, J.; Yang, M.-H.; Ko, H.-J.; Kim, S.-G.; Park, C.; Park, S.-C. Antimicrobial Resistance and Virulence Factors of Proteus mirabilis Isolated from Dog with Chronic Otitis Externa. Pathogens 2022, 11, 1215. [Google Scholar] [CrossRef]
  69. Ruiz-Cano, D.; Sánchez-Carrasco, G.; El-Mihyaoui, A.; Arnao, M.B. Essential Oils and Melatonin as Functional Ingredients in Dogs. Animals 2022, 12, 2089. [Google Scholar] [CrossRef]
  70. Vercelli, C.; Pasquetti, M.; Giovannetti, G.; Visioni, S.; Re, G.; Giorgi, M.; Gambino, G.; Peano, A. In vitro and in vivo evaluation of a new phytotherapic blend to treat acute externa otitis in dogs. J. Vet. Pharmacol. Ther. 2021, 44, 910–918. [Google Scholar] [CrossRef] [PubMed]
  71. Sim, J.X.F.; Khazandi, M.; Pi, H.; Venter, H.; Trott, D.J.; Deo, P. Antimicrobial effects of cinnamon essential oil and cinnamaldehyde com-bined with EDTA against canine otitis externa pathogens. J. Appl. Microbiol. 2019, 127, 99–108. [Google Scholar] [CrossRef] [PubMed]
  72. Park, S.; Oh, T.; Bae, S. The stability and in vitro antibacterial efficacy of enrofloxacin and gentamicin solutions against Staphylococcus pseudintermedius over 28 days. Vet. Dermatol. 2023, 34, 28–32. [Google Scholar] [CrossRef] [PubMed]
Table
Table 1. Total phenol and proanthocyanidin contents of S. tectorum L. extract.
Table 1. Total phenol and proanthocyanidin contents of S. tectorum L. extract.
Type of ExtractProanthocyanidin Content
(mg PAC/g Extract)		Total Phenol Content
(mg GA/g Extract)
Ethanolic extract: 50% EtOH + 50% H2O (leaf)15.39 ± 0.667126.17 ± 0.334
All values are the average of three analyses ± SD.
Table
Table 2. Median minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of most effective S. tectorum L. extract (µg/mL).
Table 2. Median minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of most effective S. tectorum L. extract (µg/mL).
Bacterial StrainEthanolic Extract: 50% EtOH + 50% H2O (Leaf) (µg/mL)Enrofloxacin/5 µgGentamycin/10 µg
MICMBCMBC/MIC RatioMICMIC
S. aureus clinical isolate23.2537.231.600.512.5
S. aureus ATCC 2592320.3337.291.830.251.125
P. aeruginosa clinical isolate24.23437.301.530.56.25
P. aeruginosa ATCC 2785320.5337.021.800.1252.5
CLSI, 2020. P. aeruginosa breakpoints (µg/mL): enrofloxacin: ≤0.5 = susceptible, 1–2 = intermediate, ≥4 = resistant; gentamicin: ≤4 = susceptible, 4 = intermediate, ≥8 = resistant. S. aureus breakpoints (µg/mL): enrofloxacin: ≤0.5 = susceptible, 1–2 = intermediate, ≥4 = resistant; gentamicin: ≤4 = susceptible, 4 = intermediate, ≥8 = resistant.
Table
Table 3. Minimum inhibitory concentration of most effective S. tectorum extract against S. aureus and P. aeruginosa (clinical and standard) strains.
Table 3. Minimum inhibitory concentration of most effective S. tectorum extract against S. aureus and P. aeruginosa (clinical and standard) strains.
Plant ExtractConcentration of Extract (µg/mL)S. aureus Clinical IsolateS. aureus ATCC 25923P. aeruginosa Clinical IsolateP. aeruginosa ATCC 27853
MIC (µg/mL)MBC (µg/mL)MIC (µg/mL)MBC (µg/mL)MIC (µg/mL)MBC (µg/mL)MIC (µg/mL)MBC (µg/mL)
S. tectorum L. ethanolic extract 0.500000000
1.000000000
2.0 0 0 1.47 ± 0.301 00 0 1.75 ± 0.25 0
4.0 3.67 ± 0.381 0 3.53 ± 0.604 0 3.58 ± 0.378 0 3.66 ± 0.453 0
8.0 7.5 ± 0.661 07.11 ± 0.808 07.7 ± 0.396 07.63 ± 0.436 0
16.0 14.45 ± 0.5 15.93 ± 0.07615.7 ± 0.396 15.98 ± 0.02815.51 ± 0.475 15.96 ± 0.02815.53 ± 0.604 15.75 ± 0.433
32.0 30.05 ± 1.64 31.9 ± 0.132 30.83 ± 1.808 31.94 ± 0.06 30.95 ± 0.81 31.96 ± 0.02 30.86 ± 1.125 31.58 ± 0.381
64.0 63.67 ± 0.381 63.86 ± 0.13263.36 ± 0.58363.95 ± 0.02863.43 ± 0.419 64 ± 0.0063.75 ± 0.2563.73 ± 0.421
All values are the average of three analyses ± SD.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Dégi, D.M.; Imre, K.; Herman, V.; Dégi, J.; Cristina, R.T.; Marcu, A.; Morariu, F.; Muselin, F. Antimicrobial Activity of Sempervivum tectorum L. Extract on Pathogenic Bacteria Isolated from Otitis Externa of Dogs. Vet. Sci. 2023, 10, 265. https://doi.org/10.3390/vetsci10040265

AMA Style

Dégi DM, Imre K, Herman V, Dégi J, Cristina RT, Marcu A, Morariu F, Muselin F. Antimicrobial Activity of Sempervivum tectorum L. Extract on Pathogenic Bacteria Isolated from Otitis Externa of Dogs. Veterinary Sciences. 2023; 10(4):265. https://doi.org/10.3390/vetsci10040265

Chicago/Turabian Style

Dégi, Diana Maria, Kálmán Imre, Viorel Herman, János Dégi, Romeo Teodor Cristina, Adela Marcu, Florica Morariu, and Florin Muselin. 2023. "Antimicrobial Activity of Sempervivum tectorum L. Extract on Pathogenic Bacteria Isolated from Otitis Externa of Dogs" Veterinary Sciences 10, no. 4: 265. https://doi.org/10.3390/vetsci10040265

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop