## 2 Materials and Methods
### Microorganisms  
A comprehensive panel of microorganisms was selected to evaluate the antimicrobial efficacy of eugenol and isoeugenol, ensuring the inclusion of strains relevant to the cosmetic industry. The selected strains represent the key microbial contaminants commonly found in cosmetic products, encompassing both reference strains with well-documented susceptibility profiles and clinical strains with varying resistance mechanisms. Gram-Positive Bacteria:Gram-positive bacteria were chosen to assess the efficacy of eugenol and isoeugenol against common contaminants and potential pathogens in cosmetics. The following strains were used:  
Reference Strains:  
* _Staphylococcus aureus_ ATCC 29213--a model strain for testing antimicrobial agents, often implicated in skin infections and product contamination. * _Enterococcus faecalis_ ATCC 29212--a representative strain known for its resilience in various environments, including cosmetic formulations. Clinical Strains:
* _Staphylococcus epidermidis_ MRSE (Methicillin-Resistant _Staphylococcus epidermidis_)--commonly associated with biofilm formation and contamination of cosmetic products. * _Staphylococcus aureus_ MRSA (Methicillin-Resistant _Staphylococcus aureus_)--a significant pathogen in skin infections, highlighting the need for effective preservatives. * _Enterococcus faecalis_ VRE (Vancomycin-Resistant Enterococci) VanB and _Enterococcus ficeium_ VRE VanA/VanB--known for their resistance to antibiotics, posing challenges in cosmetic preservation. * Other clinically relevant strains included _Streptococcus pyogenes_, _Streptococcus pneumoniae_, _Streptococcus agalactique_, and _Listeria monocytogenes_, all of which are potential contaminants and pathogens in cosmetics. Gram-Negative Bacteria:  
The Gram-negative bacterial strains were selected based on their prevalence in cosmetic contamination and resistance to commonly used preservatives. Reference Strains:  
* _Escherichia coli_ ATCC 25922--a benchmark strain for assessing antimicrobial activity. * _Klebsiella pneumoniae_ ATCC 700603--a strain known for its environmental resilience. * _Pseudomonas aeruginosa_ ATCC 27853--a notorious biofilm-forming pathogen in aqueous cosmetic products. * _Haemophilus influenzae_ ATCC 49766--relevant in contamination due to its environmental adaptability. Clinical Strains:
* Multidrug-resistant strains such as _Klebsiella pneumoniae_ ESBL (Extended-Spectrum Beta-Lactamases) and NDM (New Delhi Metallo-beta-lactamase), _Escherichia coli_ ESBL and OXA-48, and _Enterobacter cloace_ ESBL were included due to their resistance mechanisms, which can compromise product safety. * Other strains such as _Neisseria gonorrhoeae_, _Pseudomonas aeruginosa_ VIM (Verona Integron-Mediated Metallo-beta-lactamase), _Acinetobacter baumannii_ MDR (Multi-Drug Resistant), and _Salmonella_ spp. were also tested for their relevance in product contamination. Yeast-Like Fungi:  
To assess antifungal properties, yeast-like fungi commonly encountered in cosmetic formulations were included. Reference Strains:  
* _Candida albicans_ ATCC 10231 and ATCC 24433--standard strains used in antifungal susceptibility testing. Clinical Strains:
* Clinically significant species such as _Candida paralysis_, _Candida glabrata_, and _Candida dubliniensis_ were included due to their prevalence in cosmetic contamination and resistance to antifungal agents. All the strains used in the study were obtained from the Department of Microbiology at the Multispecialty Provincial Hospital in Gorzow Wielkopolski. Reference strains were characterized by well-documented drug susceptibility profiles, while clinical strains were isolated from hospital patients, ensuring the relevance of the study to real-world contamination scenarios. The identification of bacterial and fungal strains was conducted using the mass spectrometry method on a MALDI-TOF device from Bruker.