Antimicrobial resistance poses a major threat to human and animal health worldwide [2, 3]. Many treatments are currently under exploration, but cell-based therapeutics show a promising, sustainable antimicrobial alternative [17].

For the longest time, adipose tissue was considered an inert physical barrier playing a role in metabolism and heat insulation [18], and when the therapeutic effects were investigated, most studies focused on adipose-derived stem cells and the stromal vascular fraction rather than the adipocyte fraction [8, 19,20,21].

The most common method of adipose tissue processing depends on using enzymatic digestion (collagenase and trypsin). However, enzymatic isolation techniques are quite costly and time-consuming; this led to the consideration of mechanical tissue dispersion methods using shear force, centrifugation, or pressure [22,23,24]. The work describes the use of the Adipolyzer (under review at the Egyptian Patent Office – application number EG/P/2024/228), a mechanical adipocyte isolation system that performs in a reproducible, timely, and cost-efficient manner. The isolated adipocyte quantity was estimated by cell count and OD measurement at 600 nm, given that OD600 has long been used as a method of cell count estimation [25,26,27]. The present study showed a correspondence between both techniques, enabling the use of OD600 instead of the more time-consuming procedure of cell counting; this should facilitate future MIMACs' count estimation.

Subcutaneous adipose tissue was selected to be used in this study for its high content of connective tissue, saturated and unsaturated fatty acids, as well as its semisolid state at room temperature being quickly processed and getting high yield, compared to perirenal adipose tissue that has low connective tissue, high unsaturated fatty acids, solid in room temperature being hardly processed with low yield [28].

Evidence of direct immunological functions for adipocytes has been slight and mostly limited to specialized adipose tissue proximal to lymph nodes known as perinodal adipose tissue (PAT). PAT was thought to provide a readily available supply of fatty acids to activate lymphocytes, preventing them from competing for blood nutrients and thus improving immune responses [29]. Interestingly, subcutaneous inoculation of bacteria resulted in infection of PAT, and these adipocytes were able to clear intracellular bacteria by functional refocusing towards an immune response by upregulating genes associated with host defense and downregulating those related to fat metabolism [30].

Previous work has demonstrated that a crucial host defense mechanism against skin infections is a localized rise in subcutaneous adipocytes, size and number, after a S. aureus subdermal infection in mice; this finding demonstrates that adipocytes create an antimicrobial peptide cathelicidin, which has been shown to suppress the development of bacteria, activate neutrophils, and have pro-inflammatory properties [10, 31]. Additionally, adipocytes can exert immunological effects by sharing common antigen receptors with macrophages and by secretion of pro-inflammatory cytokines and chemokines such as TNF and IL-6 upon stimulation, as well as the production of adipokines and AMPs [11, 12, 29, 32].

The current study focused on the antibacterial role of adipocytes and Adipolysate on one of the most common opportunistic pathogens and commensal bacteria, S. aureus. It can cause potentially fatal diseases and a variety of illnesses, such as skin infections, pneumonia, and sepsis. Moreover, S. aureus was the most common cause of contagious mastitis in the bovine dairy herd; according to the survey study conducted on 20 randomly selected Egyptian dairies, contagious mastitis was reported on 45.2% of the study dairies, specifically S. aureus (35.7%) [33].

The study findings demonstrated that MIMACs could inhibit the growth of MRSA in vitro, with a Minimum Lethal volume of 75 µl and 100 µl for bacterial concentration of 1010 and 1012 cfu/ml. The extract Adipolysate showed a high antibacterial effect on MRSA, which was close to the results of the antibiotics used, vancomycin and erythromycin. These findings support the potential use of MIMACs and their Adipolysate as a safe, effective, and sustainable antimicrobial alternative.

Arguably, this is the first documented interaction between S. aureus and adipocytes by bright-field and transmission electron microscopy; the photomicrographs display the membrane attachment, disruption, and subsequent invasion of S. aureus into MIMACs, which supported the results of the in vitro antibacterial activity. S. aureus can infect adipocytes and remain viable within these cells; in fact, S. aureus infection of adipocytes results in decreased release of adiponectin and resistin, while secretion of IL-6, visfatin, and monocyte chemoattractant protein-1 (MCP-1) is enhanced.

There has been speculation that adipose tissue and the adipocytes within them could serve as hosts for a persistent S. aureus infection because adipocyte viability is unaffected by infection [10]. The presented results implied adipocytes' engulfment and bactericidal effects after contact with the bacterial cells that parallel previous findings, whichdemonstrated the wide range of antibacterial activities of adipose-derived mesenchymal stromal cells (ASCs) against both gram-positive and gram-negative bacteria (L. casei, S. aureus, and E. coli, En. Faecalis). After six hours of contact, ASCs have a bactericide-like effect through oxygenated free radical release, phagocytosis, and antibacterial molecule production. Following ASCs contact with S. aureus cells, abnormalities in S. aureus division resulted in pseudo-multicellular structures. These findings show that contact is crucial for the antibacterial actions of ASCs, which reduce bacterial growth and membrane permeabilization [20].

Some fatty acids have antibacterial properties by directly inhibiting or killing bacteria, while others have an impact on virulence factors or stop bacteria from adhering to surfaces; these effects depend on their structures, morphologies, carbon chain length, and the quantity, locations, and orientation of double bonds [13, 14]. Many organisms use fatty acid methyl ester's antibacterial qualities to defend against bacteria. Its primary action target is the bacterial cell membrane; it obstructs the synthesis of cellular energy, inhibits the functioning of enzymes, and ultimately causes the direct lysis of bacterial cells.

Additionally, fatty acids inhibit biofilm formation and lower hyphae or fimbriae production [34, 35]. Thus, the fatty acid methyl ester is a potentially effective antimicrobial drug due to its safety and efficacy [36]. The fatty acid profile of bovine Adipolysate represented Methyl tetradecanoate, Methyl myristoleate, Pentadecanoic acid, Hexadecanoic acid, Palmitoleic acid, Heptadecanoic acid, Methyl stearate, Octadecenoic acid, Octadecadienoic acid and Docasadienoic acid as the main components. Several studies demonstrated the antimicrobial activity of different fatty acids, such as tetra-decanoic acid and pentadecanoic acid, which were reported to cause significant inhibition and disruption of E. coli, En. Faecalis, and S. enterica serovar Typhimurium [37,38,39]. Tetradecanoic acid (myristic acid) alleviates a number of C. albicans virulence pathways, including oxidative stress mitigation, sphingolipid metabolism, and ergosterol production [40].

Additionally, the presented findings were analogous to the results of other conducted studies that analyzed the chemical compounds of Imperata cylindrica and Scenedesmus intermedius using GC/MS analysis. Certain substances examined, including hexadecanoic acid methyl ester, were discovered to possess antibacterial influence against gram-positive and gram-negative bacteria such as S. aureus, P. aeruginosa, B. subtilis, and K. [41, 42].

Myristoleic acid inhibited the formation of S. aureus biofilm, as it influences the expression of various genes relevant to biofilm formation, including virulence-related genes, lipase, and hyaluronate lyase [43]. 9,12-octadecadienoic acid was among the fatty acid esters with antimicrobial activity against S. aureus, S. epidermidis, B. subtilis, E. coli, and Ps. Aeruginosa [44], while another work showed 9,12-octadecadienoic acid and hexadecanoic acid possess significant inhibitory activity against S. aureus and B. subtilis [45]. The antibacterial effect against gram-positive and gram-negative bacteria was attributed to several fatty acids, including 9-octadecenoic acid, methyl ester, and heptadecanoic acid [46].

Furthermore, several fatty acids exhibited diverse biological properties; tridecanoic acid (TDA) has anthelminthic, anti-inflammatory, and anti-cancer effects [47]. Pentadecanoic acid shows promise in augmenting the effectiveness of endocrine therapy for the treatment of breast cancer cells [48]. Monounsaturated hexadecenoic fatty acids have been given more attention in recent years as potential health indicators because of their essential roles in physiology and pathology. According to some descriptions, palmitoleic acid (an isomer of hexadecenoic acid) is a lipokine that can control a variety of metabolic processes, including lipogenic activity in white adipocytes, β cell proliferation, muscle insulin sensitivity, and endoplasmic reticulum stress prevention. Palmitoleic acid has been linked to several advantageous effects in rodent models and cell lines [49].

Antimicrobial resistance results in S. aureus treatment failure, this raises an urgent need to limit traditional treatment with antibiotics and to explore new alternatives of combat [50]. This study aimed to provide such an alternative, but it had some limitations including a small sample size, which may affect the generalizability of the results and variability in antimicrobial properties. Antimicrobial effects were assessed in vitro, potentially overlooking the complexities of in vivo conditions, and the mechanisms behind the observed activity were not thoroughly investigated. While comparisons were made with certain antibiotics, a broader range of antimicrobials and multidrug-resistant pathogens should be included in future research. The need to evaluate the cytotoxicity of MIMACs and Adipolysate remains unaddressed, along with the implications of storage conditions on their efficacy over time.

The research offers novel insights into the potential of bovine adipose tissue derivatives, specifically mechanically isolated mature adipose cells (MIMACs) and their lysate (Adipolysate), as sustainable and effective antimicrobial agents against methicillin-resistant Staphylococcus aureus (MRSA). The study demonstrates significant antimicrobial activity, with complete bacterial inhibition observed at specific volumes, suggesting that these adipose-derived products could serve as alternatives to traditional antibiotics, thus addressing the critical issue of antimicrobial resistance. Additionally, the characterization of the fatty acid profile through GC/MS spectrometry highlights specific components with known antimicrobial properties, paving the way for further exploration into their mechanisms of action and potential applications in medical and agricultural fields.