Chemical Characterization and Antibacterial Activity of Essential Oil of Medicinal Plants from Eastern Serbia.

The objective of this study was to evaluate wild growing Satureja kitaibelii, Thymus serpyllum, Origanum vulgare, Achillea millefolium and Achillea clypeolata with respect to their essential oil (EO) content, composition and antimicrobial activity. The five species were collected at Mt. Rtanj and the village of Sesalac, Eastern Serbia. The main EO constituents of Lamiaceae plants were p-cymene (24.4%), geraniol (63.4%) and germacrene D (21.5%) in Satureja kitaibelii, Thymus serpyllum and Origanum vulgare ssp. vulgare, respectively. A. millefolium EO had multiple constituents with major ones being camphor (9.8%), caryophyllene oxide (6.5%), terpinen-4-ol (6.3%) and 1,8-cineole (5.6%), while the main EO constituents of A. clypeolata were 1,8-cineole (45.1%) and camphor (18.2%). Antimicrobial testing of the EO showed that Staphylococcus aureus (Gram-positive) was more sensitive to all of the tested EOs than Escherichia coli (Gram-negative). S. kitaibelii EO showed the highest antimicrobial activity against both tested bacterial strains. This is the first study to characterize the EO composition and antimicrobial activity of these five medicinal species from Eastern Serbia in comparison with comprehensive literature data. The results can be utilized by the perfumery, cosmetics, food and pharmaceutical industries, but also for healing purposes in self-medication.

Anti-biofilm activities of essential oils rich in carvacrol and thymol against Salmonella Enteritidis.

The aim of the present study was to determine the bioactive compounds in four essential oils (EO's) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains were previously characterized depending on the expression of the extracellular matrix components cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry and rough). This study revealed that the EO's and EOC's (carvacrol and thymol) investigated showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the efficacy of EO's and EOC's in the inhibition of biofilm formation between the strains with different morphotype (rdar and bdar) did not show a statistically significant difference. Results related to the effectiveness of EO's and EOC's (the essential oil components, carvacrol and thymol) on eradication of preformed 48 h old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time.

Antibacterial and antibiofilm effect of essential oils on staphylococci isolated from cheese - application of the oil mixture in a cheese model.

The aim of the research was to examine the antimicrobial and antibiofilm effects of angelica, immortelle, laurel, hyssop, and sage plant dust essential oils (EOs) against isolated strains of Staphylococcus spp. from cheeses, in vitro and in the model of white cheese. MALDI-TOF MS analysis confirmed two Staphylococcus aureus strains and two coagulase-negative, identified as S. saprophyticus and S. warneri. All isolates produce biofilm, where the strains of S. aureus showed slightly better adherence. The main component of angelica EO was ß-phellandrene (48.19 %), while α-pinene (20.33 %) were dominant in immortelle EO, in hyssop EO cis-pinocamphone (37.25 %), in laurel EO 1,8-cineole (43.15 %) and in sage EO epirosmanol (26.25 %). The sage EO exhibited the strongest antistaphylococcal activity against all isolates. Synergism was also detected in combination of sage with hyssop or laurel EO. Better antibiofilm activity was confirmed for sage EO compared to hyssop EO. The mixture of sage/laurel EOs reduced the total number of staphylococci in the cheese after 4 days. Results indicate that in vitro applied EOs showed significant antistaphylococcal and antibiofilm activity, while the oil mixture reduced the initial total number of staphylococci.

Antibacterial and Antifungal Potential of Helichrysum italicum (Roth) G. Don Essential Oil.

Helichrysum italicum (Roth) G. Don is a typical Mediterranean plant, with limited distribution on the islands of Sardinia, Corsica, and the Iberian Peninsula, as well as the islands of the Adriatic Sea and the Balkan Peninsula. In these regions, H. italicum is mainly collected from spontaneous nature, while in recent years, there has been a pronounced cultivation trend due to increased demand and market requirements for constant quality of raw materials. Bearing in mind that biological activity is linked with chemical composition, this review aimed to collect data from different scientific databases (Scopus, PubMed, Web of Science, and Google Scholar) on the antimicrobial activity of essential oil and its chemical composition. A total of 20 papers investigating the antibacterial, antibiofilm, and antifungal activities of H. italicum essential oil were found. Furthermore, in these samples, several compounds occurred as dominant: neryl acetate, α-pinene, and γ-curcumene. These compounds are known for their antimicrobial properties, which likely contribute to the essential oil's efficacy against various microbial strains.

Microbiological Quality of Raw Donkey Milk from Serbia and Its Antibacterial Properties at Pre-Cooling Temperature.

The aim of this study was to examine the microbiological quality of raw donkey milk of an indigenous Serbian breed as well as the changes in the microbial populations during storage at 4 °C. In addition, antibacterial activity of donkey milk against E. coli, L. monocytogenes and S. aureus at 15 °C as well as the content of the two main antibacterial proteins lysozyme and lactoferrin were investigated. Microbiological examination of 137 individual milk samples collected over a period of 21 months showed good microbiological quality since foodborne pathogens such as Salmonella spp. and L. monocytogenes were not detected in any of the analyzed samples, while the number of E. coli, Enterobacteriaceae, total coliform bacteria, sulfite-reducing Clostridia and aerobic sporogenic bacteria was below the limit of quantification (<1 cfu mL-1). During the six-days storage at 4 °C, total bacterial counts and the counts of lactic acid bacteria remained at the initial level while pathogenic bacteria were not detected. The strongest antibacterial activity of the tested milk was observed against E. coli, while S. aureus was the least sensitive to milk antibacterial compounds. Although further research is needed to fully elucidate the antibacterial mechanism and synergistic activity of different compounds in donkey milk, the high content lysozyme (2.63 ± 0.03 g L-1) and lactoferrin (15.48 mg L-1) observed in tested milk could contribute to its strong antibacterial activity and extension of the storage period during which it can be safely consumed.

Comparison of the Efficiency of Selected Disinfectants against Planktonic and Biofilm Populations of Escherichia coli and Staphylococcus aureus.

The aim of this study is to compare the efficacy of selected food disinfectants on planktonic populations of Staphylococcus aureus and Escherichia coli and on the same microorganisms (MOs) incorporated in a biofilm. Two disinfectants were used for treatment: peracetic acid-based disinfectant (P) and benzalkonium chloride-based disinfectant (D). Testing of their efficacy on the selected MO populations was performed using a quantitative suspension test. The standard colony counting procedure was used to determine their efficacy on bacterial suspensions in tryptone soy agar (TSA). The germicidal effect (GE) of the disinfectants was determined based on the decimal reduction ratio. For both MOs, 100% GE was achieved at the lowest concentration (0.1%) and after the shortest exposure time (5 min). Biofilm production was confirmed with a crystal violet test on microtitre plates. Both E. coli and S. aureus showed strong biofilm production at 25 °C with E. coli showing significantly higher adherence capacity. Both disinfectants show a significantly weaker GE on 48 h biofilms compared to the GE observed after application of the same concentrations on planktonic cells of the same MOs. Complete destruction of the viable cells of the biofilms was observed after 5 min of exposure to the highest concentration tested (2%) for both disinfectants and MOs tested. The anti-quorum sensing activity (anti-QS) of disinfectants P and D was determined via a qualitative disc diffusion method applied to the biosensor bacterial strain Chromobacterium violaceum CV026. The results obtained indicate that the disinfectants studied have no anti-QS effect. The inhibition zones around the disc therefore only represent their antimicrobial effect.

Environmental Bovine Mastitis Pathogens: Prevalence, Antimicrobial Susceptibility, and Sensitivity to Thymus vulgaris L., Thymus serpyllum L., and Origanum vulgare L. Essential Oils.

Mastitis is considered to be one of the most important diseases of dairy cows in terms of health, production, and economy. Being the most common cause of antibiotic consumption in dairy cows, treatment of this disease is one of the biggest challenges in the veterinary profession as an increasing number of pathogens develop resistance to antibiotics used in the treatment. Therefore, new alternative approaches for limiting the use of antibiotics in livestock are required. For this reason, our study aimed to investigate prevalence of environmental mastitis associated bacterial strains, as well as the sensitivity of isolated strains to different antibiotics. Additionally, the therapeutic potential of three essential oils (EOs) was tested against bovine Serratia spp. and Proteus spp. mastitis pathogens, based on their chemical composition, as well as antibacterial potential. The study was carried out on 81 milk samples collected from dairy cows with mastitis. In order to determine prevalence of S. marcescens and P. mirabilis, microbiological isolation and identification were performed. Antimicrobial susceptibility testing was performed by disk diffusion method and the microdilution method was used to determine the antibacterial activity of selected EOs. In the oregano EO, a total of 23 compounds were detected, with carvacrol as a dominant component (78.94%). A total of 26 components were present in the EO of common thyme, where thymol was the most abundant compound (46.37%). Thymol also dominated (55.11%) the wild thyme EO. All tested EOs displayed antibacterial activity against all strains to different extents, while wild and common thyme EOs were the most effective. It could be concluded that the tested EOs represent promising therapeutic candidates for effective non-antibiotic treatment of mastitis.

Chemical Composition, Antimicrobial Activity, and Withdrawal Period of Essential Oil-Based Pharmaceutical Formulation in Bovine Mastitis Treatment.

Due to the emergence of antibiotic-resistant bacteria, the risk it represents to public health, and the possible consequences for animal health and welfare, there is an increasing focus on reducing antimicrobial usage (AMU) in animal husbandry. Therefore, a great interest in developing alternatives to AMU in livestock production is present worldwide. Recently, essential oils (EOs) have gained great attention as promising possibilities for the replacement of antibiotics. The current study aimed to test the potential of using a novel EO-based pharmaceutical formulation (Phyto-Bomat) in bovine mastitis treatment. The antibacterial activity was performed using the microdilution technique. Lactating dairy cows were treated with 15 mL of Phyto-Bomat in the inflamed quarter for 5 consecutive days in order to analyze blood and milk samples for thymol and carvacrol residues using gas chromatography and mass spectrometry (GC-MS). Antimicrobial activity expressed as the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) indicates that this formulation has the highest activity against Gram-positive strains. The dominant compounds in Phyto-Bomat were thymol and carvacrol, at 12.58 ± 1.23 mg/mL and 23.11 ± 2.31 mg/mL, respectively. The quantification of these two compounds in evaluated biological samples showed that 24 h after administration the concentration of thymol and carvacrol in milk samples was at the same level as before application. On the other hand, thymol and carvacrol were detectable in plasma samples even after 24 h post-treatment, with values ranging from 0.15-0.38 and 0.21-0.66 µg/mL, respectively. The tested formulation showed encouraging results of antibacterial activity against bovine mastitis pathogens, as well as the withdrawal period of dominant compounds, which implies that further testing regarding the bacteriological and clinical cure rates in clinical settings is needed.

Invasive Crayfish Faxonius limosus: Meat Safety, Nutritional Quality and Sensory Profile.

The aim of the present study was to evaluate the safety parameters, nutritional value and sensory profile of the meat from spiny-cheek crayfish (Faxonius limosus), captured from the Danube River in Serbia. To achieve this, we determined their microbiological safety, chemical composition, minerals and heavy metals, fatty acid and amino acid profile, as well as a sensory profile of the meat. The obtained results showed that the meat from crayfish was microbiologically safe. Crayfish meat has a high nutritional quality, high protein content (18.12%) and a total of 17 detected amino acids, of which essential amino acids constituted 6.96 g/100 g sample. Additionally, the crayfish meat was characterized by high levels of essential polyunsaturated fatty acids (PUFA), particularly n-3 PUFA, at an optimal ratio of n-3/n-6 and with low values of atherogenic and thrombogenic indices. Predominant macrominerals in the meat are K, followed by Na, Ca, P and Mg, whereas the content of microminerals was in the following order: Zn > Cu > Fe > Mn. The concentrations of accumulated toxic metals (Cd, Pb, As and Hg) did not exceed the maximum allowed levels. Sensory analysis confirmed that the meat from spiny-cheek crayfish has the potential to become a new food source of essential nutrients.

Natural Agents against Bovine Mastitis Pathogens.

Bovine mastitis is the most widespread and economically important disease worldwide. The present study aimed to determine bioactive compounds in two essential oils (EOs) from wild (Thymus serpyllum) and common thyme (Thymus vulgaris) and to assess the antioxidant potential as well as antibacterial efficacy of the EOs against mastitis-associated bacteria. The study also included antibiotic susceptibility tests. The strains were previously isolated from lactating animals with clinical and subclinical mastitis. The antioxidant potential of the commercial EOs of wild and common thyme was evaluated by five in vitro assays. The antibacterial activity was performed using the microdilution technique, while antibiotic susceptibility testing was performed by the Kirby-Bauer disc diffusion method. The dominant compound in wild thyme was thymol (45.22%), followed by p-cymene (23.83%) and γ-terpinene (3.12%), while in common thyme, it was thymol (54.17%), followed by γ-terpinene (22.18%) and p-cymene (16.66%). Among the fourteen mastitis-associated bacteria, strain IX Streptococcus spp. (ß-hemolytic) was the most sensitive to the tested EOs (minimum inhibitory concentration (MIC)/minimal bactericidal concentration (MBC) were 0.78/1.56 and 0.39/0.78 mg/mL for T. serpyllum (TS) and T. vulgaris (TV), respectively). Regarding Streptococcus spp. ß heamoliticus, MICs for TS ranged from 0.78 to 1.56 mg/mL, while for the same oil, MBCs ranged from 1.56 to 12.5 mg/mL. In the case of T. vulgaris, MICs ranged from 0.39 to 3.125 mg/ mL, while MBCs ranged from 3.125 to 6.25 mg/mL. TV is more active against E. coli, E. sakazakii, and Streptococcus spp., while it is less effective against Staphylococcus spp. than TS. The study revealed that the tested EOs possess remarkable antioxidative and antibacterial activities and could be used in the development of pharmaceutical formulation as an alternative to conventional mastitis therapy.

New Perspective of Origanum vulgare L. and Satureja montana L. Essential Oils as Bovine Mastitis Treatment Alternatives.

Mastitis represents a heavy burden for the dairy sector worldwide with high economic and animal welfare impact. Antibiotic treatment is an important component of mastitis control programs. However, emergence and transfer of antimicrobial-resistant (AMR) bacteria is becoming a growing concern. Therefore, the development of novel agents is required for prevention and treatment of mastitis. Hence, our aim was to assess the antibacterial properties of two essential oils (EOs) obtained from oregano (Origanum vulgare L., Lamiaceae) and mountain savory (Satureja montana L., Lamiaceae) against mastitis-associated bacteria in Serbia. The chemical composition and antioxidant potential of these EOs were also evaluated. The present study was conducted on strains derived from aseptic milk samples collected from Holstein-Friesian cows with clinical or subclinical mastitis, during the morning milking. Clinical mastitis was assessed by clinical examination, while subclinical mastitis was confirmed using somatic cell count in the milk samples. The microdilution method was used to determine the antibacterial activity, while antioxidant potential of the EOs was evaluated in several in vitro assays. The values of minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were used to quantitatively measure the antibacterial activity of each EO. MIC/MBC ranged from 0.78/6.25 and 0.39/0.78 mg/mL for oregano and mountain savory, respectively. A total of 25 compounds were identified in the oregano EO, while 47 were identified in winter savory EO, among which aromatic oxygenated monoterpenes were the most abundant compounds. The tested EOs have shown promising antimicrobial activity and could be considered as one of the treatment approaches in mastitis-affected cows.

Chemical Composition and Antibacterial Activity of Angelica archangelica Root Essential Oil.

Roots of wild growing Angelica archangelica L. from Mt. Ozren (Serbia) were subjected to hydrodistillation and GC-MS analysis. The roots contained 0.10% of essential oil with a-pinene (29.7%), 8-3-carene (14.2%), and a mixture of P-phellandrene and limonene (13.2%) as main compounds. The modified resazurin microtiter-plate assay was used to evaluate the antibacterial activity of the essential oil against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration (MIC) values were 14.2 µL/mL for S. aureus and 28.4 µL/mL for E. coli, while the minimum bactericidal concentrations (MBC) were 56.8 µL/mL and 113.6 µL/mL, respectively. According to the obtained results, the angelica root essential oil can be applied as a natural preservative in food and as a natural antibiotic for the treatment of several infectious diseases caused by these two bacteria.