Exploring the Antimicrobial Properties of 99 Natural Flavour and Fragrance Raw Materials against Pathogenic Bacteria: A Comparative Study with Antibiotics

Currently, one of the most serious global problems is the increasing incidence of infectious diseases. This is closely related to the increase in antibiotic use, which has resulted in the development of multidrug resistance in microorganisms. Another problem is the numerous microbiological contaminations of cosmetic products, which can lead to dangerous bacterial infections in humans. Natural fragrance raw materials exhibit a wide spectrum of biological properties, including antimicrobial properties. Despite their prevalence and availability on the commercial market, there is little research into their effects on multidrug-resistant microorganisms. This study examines the inhibitory effect of natural substances on Gram-positive and Gram-negative bacteria. For this purpose, screening and appropriate assays were carried out to determine the minimum inhibitory concentration (MIC) value of individual substances, using the alamarBlueTM reagent. The lowest MIC values were observed for Staphylococcus aureus (black seed (Nigella sativa) expressed oil, MIC = 25 µg/mL), Kocuria rhizophila (fir balsam absolute, MIC = 12.5 µg/mL), and Pseudomonas putida (cubeb oil and fir balsam absolute, MIC = 12.5 µg/mL). The most resistant Gram-negative species was Enterobacter gergoviae, while Staphylococcus epidermidis was the most resistant Gram-positive species.


Introduction
Multiple drug resistance (MDR) is defined as the resistance of a microorganism to at least one antimicrobial drug in three or more categories [1].Currently, it poses a significant obstacle to the treatment of bacterial and fungal infections in patients due to the limited possibility of selecting an effective and selective antibiotic therapy [2].The presence of resistant microorganisms in the hospital environment is a very serious problem that makes it difficult to perform surgeries, among other things.The main cause of resistance is the excessive and inappropriate use of antibiotics, as well as their widespread use in industries such as agriculture, food, and veterinary medicine in rapidly developing countries [3].Therefore, it is essential to find natural substances that can inhibit the growth of bacteria.It is important to note that bacteria of the same species are not always resistant or sensitive to a given antimicrobial compound in the same way.Resistance and susceptibility are determined by the minimum inhibitory concentration (MIC) of the biocidal compound that inhibits the growth of the microorganism.
Microorganisms have developed specific mechanisms to survive in the presence of toxic compounds due to their adaptation to various environmental conditions.Bacteria use mechanisms that can be classified into four categories: absorption of limitation of substances, modification of the target site, inactivation, and active pumping out of the cell interior [4].The main mechanisms of resistance in bacteria is shown in Figure 1.Red squares indicate substances toxic to bacteria.
Resistance and susceptibility are determined by the minimum inhibitory concentration (MIC) of the biocidal compound that inhibits the growth of the microorganism.
Microorganisms have developed specific mechanisms to survive in the presence of toxic compounds due to their adaptation to various environmental conditions.Bacteria use mechanisms that can be classified into four categories: absorption of limitation of substances, modification of the target site, inactivation, and active pumping out of the cell interior [4].The main mechanisms of resistance in bacteria is shown in Figure 1.Red squares indicate substances toxic to bacteria.So far, antibiotics and synthetic chemicals with antibacterial activity have been the most effective and widely used tools against pathogens.However, due to increasing multidrug resistance, other solutions should be sought.Taking into account the growing global market of natural fragrance raw materials, essential oils, absolutes, balsams, and concretes show great potential in this area.
Essential oils (EOs) are secondary metabolites that have a characteristic scent.Their secretion aims to protect against parasites and predators, limit the growth of competing plants, and prevent sprouting in the winter.In addition, EOs play an important ecological role in ecosystems, where they act as attractants and repellents.They are soluble in alcohols and ethers, but insoluble in water [5].Their lipophilic nature allows them to penetrate the cell wall and the cytoplasmic membrane of bacteria, causing their integrity and structure.The presence of various chemical compounds in EOs can reduce the membrane potential, interfere with the proton pump activity, coagulate the cytoplasm, and degrade structures such as lipids or proteins [6].Consequently, these activities lead to the leakage of cellular organelles into the environment and the lysis of the bacterial cell [6].EOs exhibit a wide spectrum of biological properties, including antimicrobial, antiviral, antifungal, antiparasitic, antioxidant, and insecticidal [7].The properties of the natural material depend on the main bioactive components.Numerous studies have confirmed the antimicrobial activity of natural fragrance raw materials [2,5,[7][8][9][10][11][12].Due to their antimicrobial properties, they can be used in the fight against pathogens in the cosmetics, pharmaceutical, and food industries.
The purpose of this study was to determine the inhibitory effect of selected natural fragrances, which were essential oils, balsams, concretes, and absolutes, on selected species of microorganisms that are considered pathogens capable of developing antibiotic resistance and contributing to the development of diseases.These bacterial strains were So far, antibiotics and synthetic chemicals with antibacterial activity have been the most effective and widely used tools against pathogens.However, due to increasing multidrug resistance, other solutions should be sought.Taking into account the growing global market of natural fragrance raw materials, essential oils, absolutes, balsams, and concretes show great potential in this area.
Essential oils (EOs) are secondary metabolites that have a characteristic scent.Their secretion aims to protect against parasites and predators, limit the growth of competing plants, and prevent sprouting in the winter.In addition, EOs play an important ecological role in ecosystems, where they act as attractants and repellents.They are soluble in alcohols and ethers, but insoluble in water [5].Their lipophilic nature allows them to penetrate the cell wall and the cytoplasmic membrane of bacteria, causing their integrity and structure.The presence of various chemical compounds in EOs can reduce the membrane potential, interfere with the proton pump activity, coagulate the cytoplasm, and degrade structures such as lipids or proteins [6].Consequently, these activities lead to the leakage of cellular organelles into the environment and the lysis of the bacterial cell [6].EOs exhibit a wide spectrum of biological properties, including antimicrobial, antiviral, antifungal, antiparasitic, antioxidant, and insecticidal [7].The properties of the natural material depend on the main bioactive components.Numerous studies have confirmed the antimicrobial activity of natural fragrance raw materials [2,5,[7][8][9][10][11][12].Due to their antimicrobial properties, they can be used in the fight against pathogens in the cosmetics, pharmaceutical, and food industries.
The purpose of this study was to determine the inhibitory effect of selected natural fragrances, which were essential oils, balsams, concretes, and absolutes, on selected species of microorganisms that are considered pathogens capable of developing antibiotic resistance and contributing to the development of diseases.These bacterial strains were selected due to their significant pathogenic capacity and high risk of product contamination in various areas.The results of this study may help find natural alternatives to antiseptics and antibiotics that will be equally effective against antibiotic-resistant microorganisms.In this study, we used Gram-negative bacteria from the genera Pluralibacter, Klebsiella, Pseudomonas, and Burkholderia, and Gram-positive bacteria from the genera Staphylococcus, Kocuria, and Cutibacterium.These bacteria are natural members of the skin microbiota and are not a threat in the case of healthy skin tissue.However, many studies have shown that these species are closely related to skin diseases, including atopic dermatitis or acne [13][14][15].Numerous other parts of the body can be colonised by pathogens, including the axillae, groin, and gastrointestinal tract.Colonisation provides a reservoir from which bacteria can be introduced into the bloodstream when the host defence is disrupted, whether by shaving, aspiration, or surgery [16].In the case of S. aureus, its presence can cause, for example, pneumonia, respiratory tract infections, endocarditis, osteomyelitis, conjunctivitis, and other diseases [17].Furthermore, the presence of bacteria in damaged skin tissue leads to the development of wounds, bacterial infections, and difficulties in healing [12].One of the most extreme developments in skin and soft tissue infections is necrotising fasciitis and necrotising soft tissue infections caused by the Streptococcus A group and methicillinresistant S. aureus (MRSA) [18].In our work, we focus on the use of essential oils and other fragrances due to their availability, simplicity of use, and biological properties.Due to their characteristic fragrances, they are mainly used in aromatherapy, where they have a relaxing function, improving emotional and physical health by penetrating subcutaneous tissues [19].Therefore, we focused on the use of natural fragrances as growth inhibitors of selected bacterial species.

Screening Assays
The first part of the study was devoted to screening assays for each microbial species to identify raw materials that showed an inhibitory effect on bacterial growth.The assays were carried out with the use of alamarBlue TM reagent.The number of natural fragrances that showed an inhibitory effect (at a concentration of 200 µg/mL) on the growth of the tested bacterial species is as follows: out of 99 natural fragrance raw materials, 47 showed an inhibitory effect on the growth of Pseudomonas putida, 36 on Cutibacterium acnes and Pseudomonas fluorescens, 35 on Staphylococcus aureus, 32 on Kocuria rhizophila, 26 on Burkholderia cepacia, 22 on Staphylococcus epidermidis, 15 on Klebsiella pneumoniae, and 11 on Pluralibacter gergoviae (Figure 2).
selected due to their significant pathogenic capacity and high risk of product contamination in various areas.The results of this study may help find natural alternatives to antiseptics and antibiotics that will be equally effective against antibiotic-resistant microorganisms.In this study, we used Gram-negative bacteria from the genera Pluralibacter, Klebsiella, Pseudomonas, and Burkholderia, and Gram-positive bacteria from the genera Staphylococcus, Kocuria, and Cutibacterium.These bacteria are natural members of the skin microbiota and are not a threat in the case of healthy skin tissue.However, many studies have shown that these species are closely related to skin diseases, including atopic dermatitis or acne [13][14][15].Numerous other parts of the body can be colonised by pathogens, including the axillae, groin, and gastrointestinal tract.Colonisation provides a reservoir from which bacteria can be introduced into the bloodstream when the host defence is disrupted, whether by shaving, aspiration, or surgery [16].In the case of S. aureus, its presence can cause, for example, pneumonia, respiratory tract infections, endocarditis, osteomyelitis, conjunctivitis, and other diseases [17].Furthermore, the presence of bacteria in damaged skin tissue leads to the development of wounds, bacterial infections, and difficulties in healing [12].One of the most extreme developments in skin and soft tissue infections is necrotising fasciitis and necrotising soft tissue infections caused by the Streptococcus A group and methicillin-resistant S. aureus (MRSA) [18].In our work, we focus on the use of essential oils and other fragrances due to their availability, simplicity of use, and biological properties.Due to their characteristic fragrances, they are mainly used in aromatherapy, where they have a relaxing function, improving emotional and physical health by penetrating subcutaneous tissues [19].Therefore, we focused on the use of natural fragrances as growth inhibitors of selected bacterial species.

Screening Assays
The first part of the study was devoted to screening assays for each microbial species to identify raw materials that showed an inhibitory effect on bacterial growth.The assays were carried out with the use of alamarBlue TM reagent.The number of natural fragrances that showed an inhibitory effect (at a concentration of 200 µg/mL) on the growth of the tested bacterial species is as follows: out of 99 natural fragrance raw materials, 47 showed an inhibitory effect on the growth of Pseudomonas putida, 36
In particular, S. epidermidis was found to be the most resistant to the action of natural fragrances, as evidenced by its inhibition by only 22 fragrance materials, while C. acnes was the least resistant, with inhibition by 36 fragrance materials.
Regarding the number of fragrance materials that inhibit the tested pathogens, P. gergoviae is the most resistant species, as only 11 fragrance raw materials inhibited its growth, which is the smallest number among all the tested Gram-negative species.On the other hand, P. putida is the least resistant bacteria species, as it was inhibited by 47 fragrance raw materials.
All MIC values obtained for each of the bacterial strains are presented in Table S1.The most effective raw materials for each type of Gram-negative bacteria were essential oils of C. sativum and C. intratropica, J. communis CO 2 extract, and A. balsamea absolute, which had the lowest MIC values.

Discussion
The antimicrobial activity of essential oils has been confirmed in many studies [5,[7][8][9][10][11].In this study, we evaluated the antibacterial activity of ninety-nine fragrance raw materials against Gram-positive and Gram-negative pathogens.The essential oils of C. intratropica and C. sativum, N. sativa expressed oil, A. balsamea absolute, and J. communis CO 2 extract showed the lowest MIC values (12.5-50 µg/mL) for all evaluated species.Previous studies have shown that natural fragrance materials are more active against Gram-positive strains than Gram-negative strains [20][21][22].This is due to the structure of the cell wall and the natural resistance of Gram-negative bacteria caused by the presence of a double layer of phospholipids and LPS [23].However, in our study, we did not observe this dependence: the MIC values were the same or very similar for both Gram-positive and Gram-negative bacteria.For example, the MIC values for J. communis CO 2 extract for S. epidermidis and P. gergoviae were 50 µg/mL, and for A. balsamea absolute for K. rhizophila and P. putida, the MIC values were 12.5 µg/mL.
Most of the studies conducted so far have focused on the Gram-positive microorganisms of the S. aureus species, and there is still limited research into the effects of natural fragrance materials on other Gram-negative bacteria besides the E. coli species [21,[24][25][26][27]. Furthermore, there is a paucity of the literature investigating the effects of natural fragrance materials on bacterial species belonging to the genera Burkholderia, Pseudomonas, Klebsiella, Cutibacterium, and Kocuria.In existing studies, the MIC values are often given as the zone of inhibition of growth or concentration % (v/v) [21,24,28,29].To compare the results obtained with those of other authors, it is crucial to use the same method, culture conditions, specific bacterial strains, and tested fragrance compounds.Therefore, it is challenging to compare the obtained MIC values with other data.
On the basis of the MIC values obtained, several natural fragrance raw materials that exhibited the lowest MIC values were selected for the tested bacterial strains.For the selected raw materials, the main components that occur in their composition are presented in Table 2.Each of the selected natural raw materials listed in Table 2 has a different composition.Therefore, their antibacterial activity differs from that of the microorganisms tested.The composition and properties of essential oils and extracts are influenced by the time of harvesting the plant, the conditions under which it was grown, light interception, the part of the plant from which the raw fragrance material was extracted, or the manner and conditions under which the extraction process was carried out [40].The structures of the main components of natural fragrance raw materials showing the lowest MIC values are shown in Figure 3. the part of the plant from which the raw fragrance material was extracted, or the manner and conditions under which the extraction process was carried out [40].The structures of the main components of natural fragrance raw materials showing the lowest MIC values are shown in Figure 3.In the case of N. sativa expressed oil, for which the lowest MIC value was shown for S. aureus (MIC = 25 µg/mL), the main compounds found in this raw material are cuminaldehyde 1 and β-caryophyllene 2 [30].Li et al. showed that cuminaldehyde inhibits the growth of S. aureus (ATCC 6538) with an MIC result of 800 µg/mL, confirming its antibacterial properties [41].Chew Li Moo confirmed the biocidal properties for βcaryophyllene for Bacillus cereus (ATCC 14579), but not for S. aureus [42].However, these results make it possible to conclude that N. sativa expressed oil containing mainly cuminaldehyde and β-caryophyllene in the volatile fraction has antibacterial properties.
The essential oil of C. intratropica, which showed the highest antimicrobial activity for P. putida and B. cepacia (MIC = 25 µg/mL), contains mainly guaiol 3, bulnesol 4, dihydrocolumellarin 5, and γ-eudesmol 6 [31,32].Petard showed that the essential oil of Bulnesia sarmienti, which consists mainly of bulnesol and guaiol, exhibits an inhibitory effect on the growth of Gram-positive bacteria [43].This allows us to conclude that, In the case of N. sativa expressed oil, for which the lowest MIC value was shown for S. aureus (MIC = 25 µg/mL), the main compounds found in this raw material are cuminaldehyde 1 and β-caryophyllene 2 [30].Li et al. showed that cuminaldehyde inhibits the growth of S. aureus (ATCC 6538) with an MIC result of 800 µg/mL, confirming its antibacterial properties [41].Chew Li Moo confirmed the biocidal properties for β-caryophyllene for Bacillus cereus (ATCC 14579), but not for S. aureus [42].However, these results make it possible to conclude that N. sativa expressed oil containing mainly cuminaldehyde and β-caryophyllene in the volatile fraction has antibacterial properties.
The essential oil of C. intratropica, which showed the highest antimicrobial activity for P. putida and B. cepacia (MIC = 25 µg/mL), contains mainly guaiol 3, bulnesol 4, dihydrocolumellarin 5, and γ-eudesmol 6 [31,32].Petard showed that the essential oil of Bulnesia Plants 2023, 12, 3777 10 of 17 sarmienti, which consists mainly of bulnesol and guaiol, exhibits an inhibitory effect on the growth of Gram-positive bacteria [43].This allows us to conclude that, despite the different natural raw materials tested, the high percentage of bulnesol and guaiol influences the biocidal activity of the fragrance raw material.
In the case of the J. communis CO 2 extract, the lowest MIC values were shown for K. rhizophila, S. epidermidis, and K. aerogenes (MIC = 25 µg/mL).The main components of the volatile fraction of this raw material are (-)-α-pinene 7 and D-limonene 8 [33,34].Dhar et al. found no antibacterial activity exhibited by (-)-α-pinene against E. coli and S. aureus [44].Silva et al. also found no antibacterial activity for (-)-α-pinene against S. aureus [45].In the case of limonene, Han et al. confirmed its antibacterial activity against S. aureus [46].It can be suspected that limonene may contribute to the antimicrobial action, together with the non-volatile components of the J. communis CO 2 extract.
C. officinalis balsam showed the most prominent inhibitory effect against K. rhizophila (MIC = 25 µg/mL).The main volatile constituents of the raw material are β-caryophyllene (for which its antibacterial activity has already been described above) and α-copaene 19 [35,39].However, consistent with our results are the results of Martins et al. who found that the essential oil of the inner bark of Kielmetera coriacea, of which α-copaene (14.9%) is one of the main components, showed positive antimicrobial activity against Prevotella nigrescens (ATCC 33563) (MIC = 25 µg/mL), but not against the microorganisms we studied [47].
Our results showed that A. archangelica essential oil is the most effective against C. acnes with an MIC = 25 µg/mL.This raw material mainly contains compounds such as α-pinene, β-phellandrene 20, 3-carene, and limonene 21 in its composition [31].Juliano et al. showed that Santolina insularis essential oil containing β-phellandrene (18.87%) inhibited the growth of C. acnes (ATCC 6919) with an MIC value of 1 mg/mL [48].This is a higher result compared to that obtained in this study, but allows us to conclude that A. archangelica essential oil has antibacterial properties.The differences in the values presented may be due to the presence of other chemical compounds in this essential oil.
Lastly, P. cubeba essential oil showed the highest inhibitory effect against C. acnes (MIC = 25 µg/mL) and P. putida (MIC = 12.5 µg/mL).The main constituents of this raw material are γ-cadinene 22, β-cubebene 23, and α-copaene [39].The antibacterial effect of the raw materials, mainly γ-cadinene, in their composition has been confirmed against the species of C. acnes [49].Furthermore, the growth inhibitory activity of the P. putida species was assessed using Piper porphyrophyllum essential oil containing α-copaene (13.2%) in its composition [50].These results suggest that the described compounds significantly affect antimicrobial activity.The possible inconsistencies observed between our results and those reported by other investigators could be explained by differences in the experimental setup.
From the MIC values obtained in this study, all antibiotics showed antibacterial activity against different bacterial strains, but at different levels (the antibiotic results are presented in Table 3).All bacteria tested were more or less sensitive to the three antibiotics.Differences may be due to evolved resistance mechanisms, the cell structure of the species, or the chemical structure of the antibiotic [50].The MIC values obtained for antibiotics are lower than those for raw materials of natural fragrance.One way to achieve better results for essential oils is to use them in combination (synergism) [23,51,52].This approach can reduce the required concentration of essential oils and improve their antimicrobial effectiveness.The combination of selected components that exhibit synergism will then reduce the concentration needed to achieve the same inhibitory effect against bacteria, compared to the use of individual components.The selection of suitable components depends on the MIC values they exhibit, their chemical structure, the percentage of their chemical composition, and, when given a fragrance raw material for combination, the effect on the bacterial cell and the main chemical compounds in the formulation.An example mechanism is to increase the permeability of the cytoplasmic membrane by one component while allowing the other component to be transported into the bacterial cell.To obtain satisfactory synergistic results, lower concentrations of components should be chosen whenever possible compared to their individual inhibitory concentrations.To test the effect of synergism, the checkerboard method can be used by placing medium, appropriately diluted fragrance raw materials and inoculum in a 96-well plate.As microorganisms are constantly developing resistance to conventional antibiotics, it is essential to explore alternative ways to combat them.Pathogenic bacterial species employed in this study have already developed resistance mechanisms against specific types of antibiotics [53][54][55][56][57][58][59][60][61][62].However, most raw materials were able to inhibit their growth, indicating that the mechanisms responsible for antibiotic resistance may not guarantee resistance to natural fragrances.Although the tested raw materials may have a weaker effect on microorganism activity and growth compared to antibiotics, they exhibit great potential in the fight against pathogenic microorganisms and the treatment of bacterial infections caused by them.The results of the research obtained can contribute to the design of appropriate tools to combat resistant pathogens in the future in the pharmaceutical, cosmetic, or household industries.

Materials
The applied pathogenic bacteria strains, including

Natural Fragrance Materials
Natural fragrance raw materials with an initial concentration of 10 mg/mL in DMSO were used.DMSO at low concentrations does not have a significant effect on the growth in short-term experiments [63].In this study, the 8% DMSO concentration used was not toxic to bacterial strains.

Minimal Inhibitory Concentration (MIC)
The objective of the tests was to determine the minimum inhibitory concentration (MIC) of the natural fragrance materials against the microorganisms tested.Cell density was obtained in a way similar to that of the screening test.Natural fragrance materials dissolved in DMSO were diluted (two times) in 96-well plates in the concentration range of 800-3.125 µg/mL for all bacteria.The appropriate medium was added to the 96-well plates in volumes: col.1: 164 µL, col.2-10: 90 µL, col.11: 100 µL, and col.12: 110 µL.The natural material was added to the col. 1 in volume of 16 µL, and two-fold serial dilutions were prepared horizontally on the plate.Excess dilutions (90 µL) were discarded from the plate.The subsequent part of the MIC assay was carried out in the same way as for the screening tests (the procedure is described in Section 4.2.3).

Analysis of the Screening and MIC Assays
The basis for the change in the analysis of the assays was the colour of the alamarBlue TM reagent from pink to blue.The lack of colour change after the addition of the dye indicated inhibition of bacterial growth, as described by Rampersad [65].The MIC value was determined as the last well showing a noticeable blue colour, expressed in µg/mL.Screening and MIC assays were performed and analysed according to CLSI (Clinical and Laboratory Standards Institute) with slight modifications [66,67].

Conclusions
The growth inhibitory and microbial activity of the 99 fragrance materials was determined for the first time against specific bacterial species, namely S. aureus (ATCC 6538), S. epidermidis (ATCC 12228), K. rhizophila (ATCC 9341), K. aerogenes (ATCC 13048), K. pneumoniae (ATCC 13883), P. gergoviae (ATCC 33028), B. cepacia (ATCC 25416), P. putida (ATCC 49128), P. fluorescens (ATCC 13525), and C. acnes (ATCC 11827).Screening tests revealed that S. epidermidis was the most resistant Gram-positive bacterium, inhibited by 22 fragrance materials, whereas P. gergoviae was the most resistant Gram-negative bacterium, inhibited by 11 materials.The appropriate tests demonstrated that the lowest MIC values were obtained for S. aureus (Nigella sativa expressed oil, Cynara scolymus absolute, MIC = 25 µg/mL), K. rhizophila (Abies balsamea absolute, MIC = 12.5 µg/mL), and P. putida (Piper cubeba EO, Abies balsamea absolute, MIC = 12.5 µg/mL).On the basis of the results obtained, it indicates the antibacterial potential of natural fragrance raw materials.We can conclude that the most active aroma materials against the pathogens tested offer an alternative to chemical preservatives and antibiotics.Thus, they can be used as ingredients in various formulations of the cosmetic, chemical, medical, or food industries.In addition to providing unique characteristics such as fragrance and colour, they will exhibit antimicrobial activity to prevent product contamination.

Figure 1 .
Figure 1.The main mechanisms of resistance in bacteria.Own elaboration.

Figure 1 .
Figure 1.The main mechanisms of resistance in bacteria.Own elaboration.

Figure 2 .
Figure 2. Summary of the amount of inhibitory and non-inhibitory materials against all tested bacterial species.

Figure 2 .
Figure 2. Summary of the amount of inhibitory and non-inhibitory materials against all tested bacterial species.

Figure 3 .
Figure 3. Structures of the main components of natural fragrance raw materials showing the lowest MIC values.

Figure 3 .
Figure 3. Structures of the main components of natural fragrance raw materials showing the lowest MIC values.

Table 1 .
Minimal inhibitory concentration (MIC) values for the most active fragrance raw materials against all strains evaluated.

Table 2 .
Chemical composition of selected natural fragrance raw materials showing the lowest MIC values.

Table 3 .
Results for antibiotics obtained for tested bacterial strains.