trans-Cinnamaldehyde as a Novel Candidate to Overcome Bacterial Resistance: An Overview of In Vitro Studies

The increasing of drug-resistant bacteria and the scanty availability of novel effective antibacterial agents represent alarming problems of the modern society, which stimulated researchers to investigate novel strategies to replace or assist synthetic antibiotics. A great deal of attention has been devoted over the years to essential oils that contain mixtures of volatile compounds and have been traditionally exploited as antimicrobial remedies. Among the essential oil phytochemicals, remarkable antimicrobial and antibiotic-potentiating activities have been highlighted for cinnamaldehyde, an α,β-unsaturated aldehyde, particularly abundant in the essential oils of Cinnamomum spp., and widely used as a food additive in industrial products. In line with this evidence, in the present study, an overview of the available literature has been carried out in order to define the bacterial sensitizing profile of cinnamaldehyde. In vitro studies displayed the ability of the substance to resensitize microbial strains to drugs and increase the efficacy of different antibiotics, especially cefotaxime, ciprofloxacin, and gentamicin; however, in vivo, and clinical trials are lacking. Based on the collected findings, cinnamaldehyde appears to be of interest as an adjuvant agent to overcome superbug infections and antibiotic resistance; however, future more in-dept studies and clinical investigations should be encouraged to clarify its efficacy and the mechanisms involved.


Introduction
The discovery of antibiotics is considered one of the most important achievements in the history of medicine since their use has significantly reduced morbidity and mortality associated with bacterial infections [1]. However, their inappropriate use and abuse have led to the emergence of antibiotic resistance at an alarming rate, which has resulted in drug treatment failure and the development of recurrent infections [2]. This phenomenon has been favored by incorrect prescriptions and a lack of adherence to therapies [3,4]. Approximately 700,000 people die every year due to infections caused by multidrugresistant bacteria (MDR), and this number is expected to exceed 10 million deaths by 2050 [2].
An irresponsible use of antimicrobial agents has also been highlighted in veterinary and agricultural fields. In fact, large volumes of antibiotics, often unnecessary, are administered to food-producing animals, endangering human health due to the possible presence of drug residues in food and the selection of resistant bacteria [3].
Resistant bacteria, also known as superbugs, have limited treatment options, thus representing a serious threat to public health, and increasing the risk of death, especially in critically ill patients, immunocompromised subjects, and in the hospital setting [3,5]. The most severe chronic infections are frequently caused by six pathogenic bacteria, known by their acronym ESKAPE, which means Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteria sp. [6]. aldehydes, phenols, alcohols, and heterocycles) [12].
Essential oils are known to possess a broad spectrum of bioa microbial, anti-inflammatory, antioxidant, genoprotective, and a The antimicrobial properties of essential oils have been known si sent the most exploited up until now. They may act as both bacte agents, being able to inhibit bacterial growth, thus blocking the ability, and to kill bacterial cells [22][23][24]. Usually, these effects are lipophilicity of the essential oil constituents, especially monoterpe bacterial wall and alter the cell permeability [11,24]. Moreover, th mation of different fatty acids, polysaccharides, and phospholipid gration of the bacterial cell wall [11,24]. These events can be reflec tial changes, disruption of transporters, and intracellular content ally lead to cell lysis and death [11]. The complex composition of e for hypothesizing the involvement of additional antimicrobial me inhibition of bacterial enzymes and the interference with systems duction and the synthesis of structural components [24].
Among the essential oil compounds, a great interest has bee dehyde, also known as cinnamic aldehyde or 3-phenyl-2-propen saturated aldehyde, belonging to the class of phenylpropanoids food additive in industrial products, such as drinks, candies, ice cr condiments [25], and it is rated safe (GRAS) by the United States istration (FDA) and by the Flavor and Extract Manufacturer's Asso FDA and the Council of Europe have recommended a daily intak Cinnamaldehyde occurs naturally as a trans stereoisom nylprop-2-enal or trans-cinnamaldehyde, which is especially abun from Cinnamomum spp. (Fam. Lauraceae), where it contribute [26,27]. However, minor amounts (≤ 0.9%) of (2Z)-cinnamaldehyd Cinnamomum spp. from Madagascar have been reported as well [ The bark of Cinnamomum cassia Nees (or Chinese cinnamon) J. Presl (or true cinnamon), which achieve about 85% and Cinnamaldehyde occurs naturally as a trans stereoisomer, namely (2E)-3-phenylprop-2-enal or trans-cinnamaldehyde, which is especially abundant in the essential oils from Cinnamomum spp. (Fam. Lauraceae), where it contributes to the typical aroma [26,27]. However, minor amounts (≤0.9%) of (2Z)-cinnamaldehyde in the essential oils of Cinnamomum spp. from Madagascar have been reported as well [28].
Cinnamoyl moiety is a characteristic scaffold of cinnamaldehyde and its derivatives: it is considered as a Michael acceptor due to the presence of a α,α-unsaturated carbonyl pharmacophore, which can react with different electrophilic structures (e.g., enzymes, receptors), leading to several pharmacological effects [28]. Indeed, the substance has been found endowed with remarkable bioactivities in preclinical models (Figure 2), including antioxidant, anti-inflammatory, antimutagenic, antiproliferative, and neuroprotective ones [51,52]; moreover, its chemopreventive power has been reported [53]. Semisynthetic derivatives (e.g., α-hexylcinnamaldehyde) of cinnamaldehyde have also been studied to exploit the pharmacological properties of the lead compound and achieve improvements in its chemical stability [54][55][56]. Among the bioactivities of cinnamaldehyde, a remarkable broad spectru bacterial and antifungal properties has been highlighted: the substance was esp fective against Escherichia coli, Staphylococcus aureus, Salmonella spp., and Ba strains, acting through bactericidal mechanisms [28].
The antimicrobial capacity seems to arise from the ability of the substance with the bacterial wall and disrupt its integrity; indeed, the aldehydic group ca absorbed by the hydrophilic group of the bacterial surfaces, then it can pass th cell wall and start a process of inhibition and sterilization by destroying the p ride structure, leading to leakage of ions, proteins, and nucleic acids [25,28]. O anisms, such as the inhibition of biofilm formation and ATP production, alon interference with the quorum sensing systems, have been reported too [28]. A tention has also been devoted in the years to the antibiotic-potentiating proper namaldehyde, especially in superbug strains. In line with this evidence, in t study, an overview of the available literature has been carried out in order to bacterial sensitizing profile of cinnamaldehyde and to highlight a future inte natural substance as a novel strategy to overcome antibiotic resistance.

Methodology
The existing literature in PubMed and Scopus databases was searched in 2022 to select journal articles over a 20-year period (2002-today) focused on the rial combination of cinnamaldehyde and antibiotics in resistant bacteria; com with antifungal agents have also been considered. English was chosen as the language. The keywords "trans-cinnamaldehyde", "cinnamaldehyde" "antibio Among the bioactivities of cinnamaldehyde, a remarkable broad spectrum of antibacterial and antifungal properties has been highlighted: the substance was especially effective against Escherichia coli, Staphylococcus aureus, Salmonella spp., and Bacillus spp. strains, acting through bactericidal mechanisms [28].
The antimicrobial capacity seems to arise from the ability of the substance to interact with the bacterial wall and disrupt its integrity; indeed, the aldehydic group can be easily absorbed by the hydrophilic group of the bacterial surfaces, then it can pass through the cell wall and start a process of inhibition and sterilization by destroying the polysaccharide structure, leading to leakage of ions, proteins, and nucleic acids [25,28]. Other mechanisms, such as the inhibition of biofilm formation and ATP production, along with the interference with the quorum sensing systems, have been reported too [28]. A special attention has also been devoted in the years to the antibiotic-potentiating properties of cinnamaldehyde, especially in superbug strains. In line with this evidence, in the present study, an overview of the available literature has been carried out in order to define the bacterial sensitizing profile of cinnamaldehyde and to highlight a future interest in this natural substance as a novel strategy to overcome antibiotic resistance.

Methodology
The existing literature in PubMed and Scopus databases was searched in November 2022 to select journal articles over a 20-year period (2002-today) focused on the antibacterial combination of cinnamaldehyde and antibiotics in resistant bacteria; combinations with antifungal agents have also been considered. English was chosen as the preferred language. The keywords "trans-cinnamaldehyde", "cinnamaldehyde" "antibiotic", "synergism", and "combination", and their combinations through the Boolean logical operator "AND" have been used. As a research strategy, the PRISMA methodology was applied to select eligible papers for the study [57]. Notably, the studies focused on herbal extracts or essential oils containing cinnamaldehyde, but not on the pure compound, along with studies in which the purity of the substance was low (<90%) or not specified, and studies assessing other substances, diverse bioactivities, or lacking data, were excluded.

Results
A total of 276 studies focused on the ability of cinnamaldehyde to potentiate the effect of antimicrobial drugs when used in combination ( Figure 3). Among them, 24 records were removed as publications other than journal articles, while 129 were replicates in searched databases.  The screened 123 papers were further selected; out of which 25 studies focused on essential oils or herbal extracts containing cinnamaldehyde, and the other 14 on other substances, and were excluded. Moreover, out of 83 eligible papers, 4 reports were not included since they focused on other bioactivities; similarly, 61 records evaluating the antimicrobial properties of cinnamaldehyde alone, but not in combination with antimicrobial drugs, were removed too. Furthermore, 8 studies were not included for lacking data and another one since purity was not specified. At the end of the literature analysis, a total of 10 studies were considered eligible since they met the inclusion criteria.
Based on the selected studies, cinnamaldehyde has been found to be able to potentiate the antimicrobial properties of different drugs, although with specific potency and efficacy with respect to the drug and bacterial (or fungal) strain [58][59][60][61][62][63][64][65][66][67]. Usually, it produces synergistic or additive effects and allows for a significant reduction in the MIC The screened 123 papers were further selected; out of which 25 studies focused on essential oils or herbal extracts containing cinnamaldehyde, and the other 14 on other substances, and were excluded. Moreover, out of 83 eligible papers, 4 reports were not included since they focused on other bioactivities; similarly, 61 records evaluating the antimicrobial properties of cinnamaldehyde alone, but not in combination with antimicrobial drugs, were removed too. Furthermore, 8 studies were not included for lacking data and another one since purity was not specified. At the end of the literature analysis, a total of 10 studies were considered eligible since they met the inclusion criteria.
Based on the selected studies, cinnamaldehyde has been found to be able to potentiate the antimicrobial properties of different drugs, although with specific potency and efficacy with respect to the drug and bacterial (or fungal) strain [58][59][60][61][62][63][64][65][66][67]. Usually, it produces synergistic or additive effects and allows for a significant reduction in the MIC (minimal inhibitory concentration) value of the combined drug, thus suggesting promising bacterial sensitizing properties. It is noteworthy that some of the susceptible bacteria [60,[62][63][64][65] belonged to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter sp.), known to be responsible for resistant infections.
To quantify the type of interaction (synergism or antagonism) between cinnamaldehyde and antibiotic drugs, the fractional inhibitory concentration index (FICI), which represents the sum of FIC concentrations of each component in the mixture, has been conserved [68].  [68]. The FICI value is the sum of FIC A and FIC B and reveals the degree of drug interaction: a lower than 0.5 FICI value indicates a synergistic interaction, values between 0.5 and 1 denote additive effects, while FICI values higher than 1 and 4 correspond to null and antagonistic effects, respectively [68].
The ability of cinnamaldehyde to affect drug efficacy in different bacterial strains (Gram-positive and Gram-negative) and fungi has been described and detailed in Table 2. For each microorganism, the strain, antibiotic drug, MIC value (expressed as µg/mL) of the antibiotic drug alone and in combination with cinnamaldehyde, the cinnamaldehyde concentration in combination (expressed as µg/mL), and the FICI value have been displayed.

Potentiating Effects of Cinnamaldehyde in Gram-Positive Bacteria
The potentiating effects of cinnamaldehyde were evaluated in different Gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus and its methicillin-resistant strains, namely MRSA (methicillin-resistant S. aureus), and Streptococcus pyogenes ( Table 2).
L. monocytogenes is a ubiquitous bacterium, implicated within the past decade in several outbreaks of foodborne disease [69]. It causes invasive syndromes, and case fatalities can be around 30% in specific high-risk population groups, such as the elderly, immunocompromised individuals, fetuses, and newborns [70]. Moreover, it may acquire antibiotic resistance genes from the plasmids and conjugative transposons of other organisms [71]. Only a few studies have evaluated the ability of cinnamaldehyde to synergize antibiotics in L. monocytogenes. Alves et al. [58] highlighted that the substance produced synergistic effects with nisin (0.50 FICI), a bacteriocin produced by Lactococcus lactis strains, reducing the MIC value by 4 folds.
S. aureus is a Gram-positive opportunistic pathogen that is responsible for many nosocomial and community-acquired infections. The attachment to medical implants and host tissue, and the establishment of a mature biofilm, all play an important role in the persistence of chronic infections [72,73]. Clinical use of methicillin led to the development of methicillin-resistant S. aureus (MRSA) strains [74], which increased the need for new therapeutic strategies to sensitize these strains to the antibiotic treatment.
Cinnamaldehyde has been assessed against S. aureus in association with conventional antibiotics and other antibacterial substances, such as nisin. In particular, two studies have highlighted the ability of cinnamaldehyde to significantly synergize nisin with 0.26 to 0.50 FICI values [58,59]. Remarkable synergistic effects were reported in combination with ampicillin, piperacillin, and bacitracin (0.24-0.37 FICI), with antibiotic MIC values reduced by about 8 folds [60]. The substance was also found to greatly synergize amikacin, amoxicillin, and gentamicin (0.19-0.50 FICI) in MRSA strains [61]; moreover, it lowered by about 2-fold the MIC value of ampicillin and ceftazidime (1.00 FICI), although without exhibiting synergistic effects [61]. Both additive and synergistic interactions were recorded in combination with cefoxitin, oxacillin, and vancomycin [61].     At last, possible potentiating effects of cinnamaldehyde were evaluated in Streptococcus pyogenes, which is an exclusive human Gram-positive bacterial pathogen, characterized by high virulence and mortality risk [75]. Palaniappan et al. [60] highlighted synergistic effects of cinnamaldehyde with nitrofurantoin (0.13 FICI value) in Streptococcus pyogenes, while an additive effect was observed in combination with ampicillin (1.00 FICI).

Potentiating Effects of Cinnamaldehyde in Gram-Negative Bacteria
The substance was assessed in combination with different antibiotics in many Gramnegative strains, including Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, and Salmonella typhimurium ( Table 2). E. coli and Klebsiella spp., belonging to the Enterobacteriaceae, are usually part of the intestinal flora but can also contribute to a wide range of both community-and hospital-acquired infections [76]. Klebsiella spp. are also responsible for opportunistic nosocomial infections, with a high incidence of resistant strains [77,78]. β-Lactam antibiotics are usually administered to treat their infections, although the resistance to these drugs causes serious pharmacological and medical issues [76]. E. coli belongs to the resident flora in the lower intestinal tract of warm-blooded animals, such as humans, but can also be found as an environmental contaminant as a consequence of the release of feces or wastewater effluent [77].
As for Pseudomonas aeruginosa, a common Gram-negative environmental organism that can cause severe infections in humans owing to its natural resistance to antibiotics and the ability to form biofilms [79], Topa et al. [64] demonstrated that cinnamaldehyde produced synergistic effects with colistin (0.50 FICI) and additive effects with carbenicillin, tobramycin, and erythromycin (0.75 FICI). Recently, Chada et al. [65] highlighted a synergist interaction of cinnamaldehyde with gentamicin in P. aeruginosa (0.375 FICI), with a 4-fold lowering of the antibiotic MIC. Moreover, the substance exhibited a quorum quenching (QQ) potential, being able to attenuate the quorum sensing (QS) circuits, particularly by downregulating QS genes and abrogating the biosynthesis of key factors involved in bacterial virulence and biofilm formation [65]. The antivirulence properties of cinnamaldehyde in combination with gentamicin were also confirmed in a Caenorhabditis elegans model infected with a P. aeruginosa infection [65]. These findings highlight an interest in cinnamaldehyde as a possible anti-quorum sensing agent to be exploited in combination with antibiotics in the battle against P. aeruginosa and deserve further in vivo studies for confirmation.
At last, the possible synergistic potential of cinnamaldehyde has been evaluated in S. typhimurium in combination with different antibiotics [60]. This bacterium primarily affects the intestinal lumen and often causes diarrhea in infants and young children, leading to food poisoning. Furthermore, the development of drug resistance by S. typhimurium strains led to serious complications in clinical patients [80]. Palaniappan et al. [60] showed remarkable synergistic effects of cinnamaldehyde in combination with ampicillin, tetracycline, erythromycin, bacitracin, and novobiocin (0.24-0.37 FICI) in S. typhimurium, reducing the MIC values of all the tested antibiotics by about 8 folds.

Potentiating Effects of Cinnamaldehyde in Fungi
Cinnamaldehyde has also been assayed as a possible strategy to counteract fungi infections, and some studies highlighted its ability to potentiate the effects of some antifungal drugs (Table 2): particularly, it partly synergized azole drugs in Aspergillus fumigatus, Trichophyton rubrum, and Malassezia pachydermatis fungi, being especially effective in combination with fluconazole (<0.2 FICI) [66,67].

Discussion
The increasing prevalence of drug-resistant bacteria and the lack of effective antibiotics have highly alarmed the scientific community, leading researchers to investigate natural substances as novel strategies to both directly affect bacterial infections and synergize synthetic antibiotics. Among natural compounds, cinnamaldehyde attracted special attention owing to its antibacterial properties and the ability to resensitize microbial strains to drugs [60], thus suggesting a possible interest in the battle against antibiotic resistance.
In this study, we selected ten in vitro studies, which are not available in vivo or in clinical trials, using the following criteria: >90% purity of cinnamaldehyde and combination of this substance with antimicrobial agents to counteract resistant bacteria. The purity of cinnamaldehyde is a key issue, since the presence of impurities in minor compounds can affect the activity of the tested substance, leading to unreliable results.
Based on the selected studies, the most efficient synergism was found when cinnamaldehyde (0.03-0.05 µg/mL) was assessed in combination with cefotaxime or ciprofloxacin in 33 clinical isolates of Klebsiella sp; in fact, the MIC values were lowered by 1024 and 256 folds, respectively [62]. Similar results were obtained in 28 clinical isolates of Escherichia coli, where cinnamaldehyde (0.11-0.22 µg/mL) lowered the MIC value of cefotaxime by 512 folds, and that of ciprofloxacin by 64 folds [62]. Interesting synergistic effects of cinnamaldehyde were also highlighted in combination with tetracycline in Escherichia coli, where a MIC reduction of 4-to 8-fold was registered; similar potentiating effects were produced in combination with erythromycin, novobiocin, ampicillin, and piperacillin [62,63].
Cinnamaldehyde also produced synergistic effects in combination with colistin and gentamicin in Pseudomonas aeruginosa, reducing the MIC values by 4-and 16-fold, respectively [64,65], and in MRSA strains in combination with amikacin (16-fold reduction of the antibiotic MIC), gentamicin, and vancomycin, followed by oxacillin and amoxicillin; the substance was found effective at concentrations from 31.25 to 62.5 µg/mL, corresponding to 1/8 and 1/4 of the MIC value [61]. Similarly, a notable antibacterial activity of the combination of cinnamaldehyde and nisin (i.e., 25 to 125 µg/mL cinnamaldehyde and 1/8 of the antibiotic MIC) was reported in S. aureus [58,59]. The substance (16.25-41.3 µg/mL) also potentiated the antibiotic effects of ampicillin, tetracycline, erythromycin, bacitracin, and novobiocin in Salmonella typhimurium and those of nisin (62.5 µg/mL) in Listeria monocytogenes ATCC 15313, reducing the MIC value by 4-to 8-fold [58,60].
In this respect, Tetard et al. [81] showed that cinnamaldehyde (>256 µg/mL) triggers an upregulation of the efflux pumps of the resistance-nodulation-cell division (RND) family in P. aeruginosa, especially of the multidrug efflux system MexAB-OprM, which can lead to increased drug extrusion and lowered antibiotic efficacy. This effect was found to be transient and persistent until the compound is degraded into cinnamic alcohol, which lacks the ability to induce the efflux pumps [81]. Moreover, the authors highlighted that the resistance induced by cinnamaldehyde in P. aeruginosa was modest and gained after several days of exposure at concentrations higher than 900 µg/mL [82]. Furthermore, the mutation mechanisms and the clinical impact remain to be clarified. It is important to outline that the concentrations of cinnamaldehyde inducing bacterial sensitization in P. aeruginosa [64] were at least 3-to 120-fold lower than those responsible for the resistance, suggesting that opposite effects can occur depending on the concentrations of the substance; more in-depth studies could clarify this issue.
In regard to the mechanisms accounting for the bacterial sensitizing properties of cinnamaldehyde, the substance has been shown to affect multiple targets, including the bacterial wall, biofilm, quorum sensing system, cell metabolism, and factors involved in cell survival (Figure 4), which in turn can contribute to the potentiation of the antibiotic efficacy and the overcoming of resistance.
Antibiotics 2023, 12, x FOR PEER REVIEW 12 of 17 mutation mechanisms and the clinical impact remain to be clarified. It is important to outline that the concentrations of cinnamaldehyde inducing bacterial sensitization in P. aeruginosa [64] were at least 3-to 120-fold lower than those responsible for the resistance, suggesting that opposite effects can occur depending on the concentrations of the substance; more in-depth studies could clarify this issue. In regard to the mechanisms accounting for the bacterial sensitizing properties of cinnamaldehyde, the substance has been shown to affect multiple targets, including the bacterial wall, biofilm, quorum sensing system, cell metabolism, and factors involved in cell survival (Figure 4), which in turn can contribute to the potentiation of the antibiotic efficacy and the overcoming of resistance. As also reported for other essential oil compounds [21,22], Shi et al. [59] hypothesized that the synergistic effects of cinnamaldehyde in combination with nisin in S. aureus ATCC 29213 could arise from its ability to damage the bacterial wall and alter its permeability, thus affecting the antibiotic absorption and impairing the bacterial cell homeostasis, leading to autolysis and cell death. Indeed, an 54.5% membrane damage was induced by the combination of cinnamaldehyde and nisin with respect to the drug alone (28% damage) [59]. Similarly, Chadha et al. [65] hypothesized that cinnamaldehyde can cause membrane permeabilization and disruption along with oxidative damage, thus facilitating the penetration of the antibiotic gentamicin into cell and making the bacterial cell more susceptible to its antimicrobial activity.
Wang et al. [61] highlighted that the substance was able to destroy the bacterial wall and biofilm of MRSA and to downregulate the transcription and translation of the antibiotic resistance gene mecA; these effects could explain the synergism with non-beta-lactam antibiotics. Dhara et al. [62] also reported alterations in the cell surface morphology, shrinkage of the cell surface, and cytoplasm lowering in Gram-negative bacteria, i.e., E. coli and K. pneumoniae, after treatment with cinnamaldehyde, likely as a consequence of permeability and osmotic changes induced by the substance. Moreover, deep pores, disruption of the cytoplasmic membrane, and decomposition of inner organelles on cell surfaces were revealed after treatment with the combination of cinnamaldehyde and cefotaxime/ciprofloxacin [62].
Gram-negative bacteria carry an outer membrane characterized by an asymmetric hydrophobic bilayer composed of phospholipids and lipopolysaccharides (LPS), the latter playing a crucial role in the bacteria's protection [83]. Most antibiotics are absorbed As also reported for other essential oil compounds [21,22], Shi et al. [59] hypothesized that the synergistic effects of cinnamaldehyde in combination with nisin in S. aureus ATCC 29213 could arise from its ability to damage the bacterial wall and alter its permeability, thus affecting the antibiotic absorption and impairing the bacterial cell homeostasis, leading to autolysis and cell death. Indeed, an 54.5% membrane damage was induced by the combination of cinnamaldehyde and nisin with respect to the drug alone (28% damage) [59]. Similarly, Chadha et al. [65] hypothesized that cinnamaldehyde can cause membrane permeabilization and disruption along with oxidative damage, thus facilitating the penetration of the antibiotic gentamicin into cell and making the bacterial cell more susceptible to its antimicrobial activity.
Wang et al. [61] highlighted that the substance was able to destroy the bacterial wall and biofilm of MRSA and to downregulate the transcription and translation of the antibiotic resistance gene mecA; these effects could explain the synergism with non-betalactam antibiotics. Dhara et al. [62] also reported alterations in the cell surface morphology, shrinkage of the cell surface, and cytoplasm lowering in Gram-negative bacteria, i.e., E. coli and K. pneumoniae, after treatment with cinnamaldehyde, likely as a consequence of permeability and osmotic changes induced by the substance. Moreover, deep pores, disruption of the cytoplasmic membrane, and decomposition of inner organelles on cell surfaces were revealed after treatment with the combination of cinnamaldehyde and cefotaxime/ciprofloxacin [62].
Gram-negative bacteria carry an outer membrane characterized by an asymmetric hydrophobic bilayer composed of phospholipids and lipopolysaccharides (LPS), the latter playing a crucial role in the bacteria's protection [83]. Most antibiotics are absorbed through the outer membrane to reach their targets: hydrophobic drugs are able to pass the membrane by diffusion mechanisms, while hydrophilic ones, like β-lactams, exploit the bacterial porins to be transferred into cells [84]. Any alteration in the outer membrane of Gram-negative bacteria, including changes in the hydrophobic properties and porin mutations, can lower the antibiotic permeability, thus leading to bacterial resistance [84].
In this respect, the results obtained by Dhara et al. [62] strengthen the hypothesis that an impairment in the bacterial wall by cinnamaldehyde is a key mechanism of its bacterial sensitizing activity.
Some studies also highlighted that cinnamaldehyde significantly inhibited the biofilm formation and the expression of the biofilm regulatory gene hld in methicillin-resistant Staphylococcus aureus [61]. Moreover, the combined treatments of cinnamaldehyde with colistin and tobramycin potentiated the drug's ability to inhibit biofilm formation, leading to a complete inhibition of the process [64].
Biofilm is composed by a complex community of microbes that can adhere to a surface or form aggregates, enclosed in an extracellular polysaccharide matrix [85,86]. It enhances the bacterial resistance to hostile environmental conditions, allows the cellular exchange of plasmids encoding for antibiotic resistance, and impairs the activation of the immune system response, thus favoring the bacterial invasion [86]. It is also responsible for the development of persistent infections [87]. The biofilm inhibition by cinnamaldehyde can arise from different mechanisms, among which is a block of the quorum sensing system (QS), as recently highlighted by Chadha et al. [65] in combination with gentamicin in P. aeruginosa.
QS represents a cell-to-cell communication mechanism that occurs extensively in both Gram-positive and Gram-negative bacteria [88]. It consists of enzymes, receptors, and factors that regulate various bacterial functions, including biofilm production, sporulation, motility, and virulence [88,89]. The QS signal molecules are characterized by a low molecular weight and can be classified into different classes, including acyl homoserine lactones (AHLs), furanosyl borate diesters (AI2), cis-unsaturated fatty acids (DSF family signals), and peptides [89]. In P. aeruginosa, the QS system harbors two complete AHL circuits, namely LasI/LasR and RhlI/RhlR, with LasI/R being hierarchically positioned upstream of the RhlI/R circuit [88].
Chadha et al. [65] reported that cinnamaldehyde was able to affect the QS system in P. aeruginosa PAO1 by downregulating the QS and virulence genes (e.g., las, rhl, rhlAB, aprA, toxA, plcH) and abrogating the biosynthesis of AHL (acyl-homoserine lactones) molecules, involved in the QS processes. Similarly, Topa et al. [74] showed that cinnamaldehyde inhibited the expression of the LasB, RhlA, and PqsA QS systems in P. aeruginosa. Cinnamaldehyde exhibited a quorum quenching (QQ) potential, being able to affect the QS system at subinhibitory concentrations [65]. As also confirmed by molecular docking studies, the effect can be attributed to the ability of the substance to easily gain access to the active site of the QS receptors of P. aeruginosa, owing to its relatively small size; furthermore, being structurally similar to the AHL molecules, 3-oxo-C12-HSL and C4-HSL, it can strongly interact with the QS receptors, thus attenuating the QS circuits, inhibiting the biofilm formation, and lowering the bacterial virulence and motility [65]. Particularly, it has been hypothesized that cinnamaldehyde may abrogate the twitching motility in P. aeruginosa by inhibiting the mechanotactic functions of type IV pilus and the swimming and swarming motilities because of its anti-QS properties [65]. Furthermore, an inhibition of the EPS (extracellular polymeric substance) production by cinnamaldehyde, especially in relation to the alginate and rhamnolipid components, seems to directly modulate the pseudomonal biofilm formation and demonstrates the anti-fouling properties of the natural substance against P. aeruginosa [65].
Other mechanisms have also been proposed to explain the synergistic effects of cinnamaldehyde in combination with antibiotics. Particularly, Thirapanmethee et al. [90] showed that the substance blocked the polymerization, assembly, and bundling of the bacterial protein FtsZ in Acinetobacter baumanni, involved in the control of cell division [90,91]. Furthermore, some studies highlighted an ATP depletion by cinnamaldehyde [92,93], which could be reflected in an impairment of the bacterial function and survival.

Conclusions
Altogether, the collected evidence suggests a possible interest in cinnamaldehyde as an adjuvant strategy to synergize or support the effects of synthetic antibiotics against bacteria, especially against resistant strains and superbugs. However, as a small and heterogeneous group of in vitro studies, more in-depth mechanistic evidence and clinical investigations should be encouraged to clarify the promises and challenges of cinnamaldehyde in antibiotic resistance.