Antibacterial evaluation of (E)-3,7-dimethylocta-2,6-dien-1-yl acetate against Klebsiella pneumoniae strains: an in silico and in vitro study

Avaliação antibacteriana do acetato de (E)-3,7-dimetilocta-2,6-dien-1-ila contra cepas de Klebsiella pneumoniae: um estudo in silico e in vitro

Abstract

Pressure ulcers (PUs) carry a high risk of infection by multidrug-resistant pathogens, including Klebsiella pneumoniae. This study evaluated the antibacterial activity of (E)-3,7-dimethylocta-2,6-dien-1-yl acetate (TSM-16), a synthetic derivative of geraniol, against various strains of K. pneumoniae. In silico analyses were performed using PASS online®, Molinspiration®, AutoDock, PyMol, and Discovery Studio, in addition to in vitro broth microdilution tests to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), and disk diffusion assays to evaluate the combination of TSM-16 with conventional antibacterials. The in silico results indicated that TSM-16 complies with Lipinski’s Rule and has good bioavailability. In in vitro tests, the compound exhibited moderate to strong antibacterial activity (MIC 500-1000 µg/mL), a bacteriostatic profile (MBC >1000 µg/mL), and a predominance of synergistic effects when combined with synthetic antibacterials. These findings suggest that TSM-16 has potential as a safe antibacterial agent, serving as a basis for the development of new drugs against multidrug-resistant strains of K. pneumoniae.

Keywords:
pharmacology; physiotherapy; microbiology

Resumo

As lesões por pressão (LPP) apresentam alto risco de infecção por patógenos multirresistentes, incluindo Klebsiella pneumoniae. Este estudo avaliou a atividade antibacteriana do acetato de (E)-3,7-dimetilocta-2,6-dien-1-ila (TSM-16), derivado sintético do geraniol, contra diversas cepas de K. pneumoniae. Foram realizadas análises in silico utilizando PASS online®, Molinspiration®, AutoDock, PyMol e Discovery Studio, além de testes in vitro de microdiluição em caldo para determinar concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) e ensaios de disco-difusão para avaliação da associação do TSM-16 com antibacterianos convencionais. Os resultados in silico indicaram que o TSM-16 cumpre a Regra de Lipinski e possui boa biodisponibilidade. Nos testes in vitro, o composto apresentou atividade antibacteriana moderada a forte (CIM 500-1000 µg/mL), perfil bacteriostático (CBM >1000 µg/mL) e predominância de efeitos sinérgicos quando associado a antibacterianos sintéticos. Esses achados sugerem que o TSM-16 possui potencial como agente antibacteriano seguro, servindo como base para o desenvolvimento de novos fármacos contra cepas multirresistentes de K. pneumoniae.

Palavras-chave:
farmacologia; fisioterapia; microbiologia

1. Introduction

The human skin consists of three layers—the epidermis, dermis, and hypodermis—and acts as a protective barrier against the external environment (Khavkin and Ellis, 2011). In this context, skin lesions may arise due to prolonged hospitalizations, immobility, mechanical ventilation, or prolonged prone positioning; these are known as pressure injuries (PI), resulting from deformation, inflammatory processes, and ischemia that degrade tissues and cells (Zajac et al., 2024).

Thus, early intervention is essential to prevent colonization by multidrug-resistant bacteria, such as K. pneumoniae, E. coli, P. aeruginosa, and S. aureus, which form biofilms and hinder healing; K. pneumoniae, more specifically, also produces extended-spectrum beta-lactamases (ESBLs), reinforcing the need for new antibacterial molecules, such as terpenes (Seifi et al., 2016).

Terpenes, found in essential oils, act individually, additively, or synergistically, and can serve as prototypes for synthetic compounds with therapeutic potential (Lira et al., 2020). Geraniol (C10H18O), an aliphatic monoterpene present in various flowers, exhibits antifungal (Tsai et al., 2017), antibacterial (Taha and Eldahshan, 2017), and anti-biofilm (Lira et al., 2020) activity. Its synthetic derivative, (E)-3,7-dimethylocta-2,6-dien-1-yl acetate (TSM-16), although easily obtained chemically (Takabe et al., 1997), still lacks specific studies. Therefore, the present study aimed to evaluate the antibacterial activity of TSM-16 against strains of Klebsiella pneumoniae.

2. Materials and Methods

2.1. Research location

Laboratory tests of antibacterial activity were conducted at the Biochemistry Laboratory of the Academic Unit of Biological Sciences, at the Center for Rural Health and Technology of the Federal University of Campina Grande, under the coordination of Prof. Dr. Abrahão Alves de Oliveira Filho.

2.2. Test substances

Geraniol and its synthetic derivative, (E)-3,7-dimethylocta-2,6-dien-1-yl acetate, were supplied by the Laboratory of Organic Synthesis and Medicinal Chemistry at the Federal University of Campina Grande, under the supervision of Prof. Dr. Juliano Carlo Rufino de Freitas. The antimicrobials used—chloramphenicol, ampicillin, gentamicin, ciprofloxacin, ceftriaxone, tetracycline, and penicillin—were purchased from Sigma-Aldrich® (São Paulo, SP).

2.3. Bacterial strains

Strains of Klebsiella pneumoniae (ATCC 13883, Kp101, Kp104, Kp105, and Kp110) were obtained from the Micology Laboratory’s Culture Collection at the Department of Pharmaceutical Sciences/CCS/UFPB. The strains were maintained on Muller-Hinton Agar (DIFCO Laboratories, USA/France), prepared according to the manufacturer’s instructions, and stored at 4 °C. (Bona et al., 2014).

2.4. In silico pharmacological activity

2.4.1. PASS Online®

To evaluate the pharmacological properties of (E)-3,7-dimethylocta-2,6-dien-1-yl acetate, the free software PASS Online® was used. The tool predicts the biological potential of organic molecules based on their chemical structure, estimating different biological activities. Activity potential is expressed by the Pa (probability of being active) and Pi (probability of being inactive) indices, available at NCSS (2026).

2.4.1.1. In silico analysis of absorption, distribution, metabolism, and excretion parameters

The theoretical oral bioavailability of (E)-3,7-dimethylocta-2,6-dien-1-yl acetate was evaluated using the free software Molinspiration Cheminformatics® (Molinspiration, 2026), applying Lipinski’s “Rule of Five,” which considers compounds with a molecular weight ≤500 Da, cLogP ≤5, ≤10 hydrogen acceptors, and ≤5 hydrogen donors as potentially well-absorbed (Lipinski et al., 2001).

2.4.1.2. Molecular docking

Rigid molecular docking simulations were performed with the bacterial proteins DNA gyrase (PDB ID: 5L3J, 2.83 Å) and topoisomerase IV (PDB ID: 5EIX, 3.35 Å), obtained from the Protein Data Bank and processed in PyMol 2.5.3 to remove water and crystallographic artifacts. The ligands geraniol, TSM-16, levofloxacin, and norfloxacin were built in Marvin Sketch 16.3.7, with energy minimization in Avogadro 1.2.0 (pH 7.4) and Mopac 2012 (PM6), followed by optimization with the AM1-BCC force field in Chimera 1.16, generating .mol2 files (Hanwell et al., 2012).

The proteins were prepared in AutoDockTools 1.5.4 (ADT) with the addition of polar hydrogens, Kollman charges, and blending of nonpolar hydrogens. The active site grids were defined as follows: DNA gyrase (-11.953; 20.487; 23.401 Å; 40×40×40 Å) and topoisomerase IV (138.155; -20.934; 60.138 Å; 40×40×40 Å), with a spacing of 0.375 Å. Docking was performed in AutoDock 4.2 with 100 runs of the Lamarckian genetic algorithm, yielding binding free energy (ΔG) and inhibition constant (Ki), with the conformations having the lowest ΔG selected. The interactions were analyzed in PyMol 2.5.3 and Discovery Studio 2021 (Bell and Zhang, 2019).

2.5. Determination of the Minimum Inhibitory Concentration (MIC)

The MIC of (E)-3,7-dimethylocta-2,6-dien-1-yl acetate was determined by microdilution in a 96-well U-bottom plate, according to CLSI (2012), in duplicate. The plates were incubated at 35-37 °C for 24-48 h. The reading was performed visually based on the absence or presence of bacterial growth, observing the formation of cell clusters (buds) and the color change of the solution from blue to pink. The MIC was defined as the lowest concentration capable of inhibiting visible growth of the microorganism (Palomino et al., 2002; Ostrosky et al., 2008; CLSI, 2012; Bona et al., 2014).

2.6. Determination of the Minimum Bactericidal Concentration (MBC)

After reading the results, 10 μL of inoculum from three dilutions starting from the MIC was transferred to Mueller-Hinton broth (100 μL/well) in a sterile microdilution plate to determine the MBC. After incubation at 35±2 °C for 24 hours, 20 μL of resazurin was added. The assays were incubated at 35±2 °C for an additional 24 hours to confirm the concentration capable of inhibiting the total growth of the bacterial species, as evidenced by no change in the color of the indicator dye (Ncube et al., 2008; Guerra et al., 2012)

2.6.1. Study of the product’s combination with antibacterial agents

The study of the combination of (E)-3,7-dimethylocta-2,6-dien-1-yl with antibacterials (ampicillin, gentamicin, ciprofloxacin, ceftriaxone, tetracycline, and penicillin) was performed by disk diffusion on solid medium, using filter paper disks (Bauer et al., 1966). A 20 µL aliquot of the compound’s MIC was added to the discs containing the antibacterials at their respective concentrations, and then applied to sterile Petri dishes (140 × 15 mm) containing Mueller-Hinton agar previously inoculated with approximately 1 mL of the bacterial suspensions using a sterile swab.

The plates were incubated at 35 °C for 24-48 h and subsequently analyzed (Ostrosky et al., 2008). The effect of the combination of the synthetic derivative TSM-16 and the antibacterials was classified as synergistic, antagonistic, or indifferent, according to the methodology of Cleeland and Squires (1991).

3. Results and Discussion

Computational models allow for more accurate simulation of human systems, thereby increasing efficiency and regulatory credibility in drug testing. Thus, this study utilized in silico tools (Molinspiration®) to characterize molecular properties. Table 1 presents the pharmacokinetic and pharmacodynamic properties of the synthetic compound (E)-3,7-dimethylocta-2,6-dien-1-yl acetate (TSM-16).

Table 1
Molecular properties of the synthetic compound TSM-16.

Substância	MiLogP	MM	nALH	nDLH	nviolations	TPSA	nrotb
TSM-16	3.91	196.29	2	0	0	26.30	6

• Caption: miLogP = octanol/water partition coefficient; nviolations = number of violations; MM = molecular mass; nALH = number of hydrogen-bonding acceptor groups; nDLH = number of hydrogen-bonding donor groups; TPSA = topological polar surface area; nrotb = number of rotatable bands. Source: Author’s data (2025).

The synthetic compound met Lipinski’s Rule of Five, exhibiting <5 hydrogen-bonding donors and <10 hydrogen-bonding acceptors, MM < 500 Da, and miLogP < 5, indicating adequate lipophilicity and no violations. Furthermore, it exhibited nrotb ≤ 10 and TPSA < 140 Å, suggesting good permeability, absorption, and potential for oral bioavailability (Lipinski et al., 2001).

The potential biological activities of TSM-16 were also analyzed in this study, calculated using the Molinspiration® tool, which presents the results as scores (Table 2).

Table 2
Prediction of biological activity - synthetic compound TSM-16.

Substance	GPCR ligand	Ion channel modulator	Kinase inhibitor	Nuclear receptor ligand	Protease inhibitor	Enzyme inhibitor
TSM-16	-0.50	0.04	-1.11	-0.12	-0.80	0.21

• Caption: GPCR = G protein-coupled receptor. Source: Author’s data (2025).

The bioactivity data estimated by Molinspiration® for the TSM-16 compound indicate a promising profile, considering that molecules with a higher probability of bioactivity have scores close to 0.0 (Mohan et al., 2017). The results highlight ion channel modulation (0.04), good potential for enzyme inhibition (0.21), and interaction with nuclear receptors (-0.12). For GPCR ligands and protease inhibitors, the potential was moderate, while kinase inhibitor activity showed low biological relevance. In in silico analyses using PASS Online®, which estimates the probability of biological activity (PA), antibacterial, antifungal, and antimycobacterial activities were predicted. Antibacterial activity stood out, as the probability of being active (PA = 0.437) was higher than that of being inactive (PI = 0.023), as shown in Table 3.

Table 3
Therapeutic activities of TSM-16, as determined by PASS Online®, with the probability of being active and the probability of being inactive.

Therapeutic activity	PA	PI
Antibacterial	0.437	0.023
Antifungal	0.598	0.019
Antimycobacterial	0.546	0.014

• Caption: PA = Probability of being active; PI = Probability of being inactive. Source: Author’s data (2025).

These results corroborate the literature, which states that geraniol may be a biologically active compound with potential for treating infections caused by bacteria, including Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii (Lira et al., 2020; Kwiatkowski et al., 2022) and thus further justify the present study.

3.1. Molecular docking

In molecular docking with DNA gyrase and topoisomerase IV (Table 4), TSM-16 outperformed geraniol (GER) in affinity, with lower binding energies (∆G) and inhibition constants (Ki). For DNA gyrase, TSM-16 stood out with -4.58 kcal/mol (Ki = 437.01 µM). However, although TSM-16 exhibits higher affinity than GER for both enzymes, the antibacterials levofloxacin (LEV) and norfloxacin (NOR) maintain superior binding parameters.

Tabela 4
Energias livres de ligação (ΔG) e constante inibitória (Ki) dos ligantes GER, TSM-16, LEV e NOR frente as enzimas DNA girase (PBD ID: 5L3J) e Topoisomerase IV (PBD ID: 5EIX).

Enzyme	Binding sites	ΔG (kcal/mol)	Ki (µM)
DNA gyrase	GER	- 4.53	474.53
	TSM-16	- 4.58	437.01
	LEV	- 4.46	542.34
	NOR	- 4.40	594.93
Topoisomerase IV	GER	- 4.63	401.32
	TSM-16	- 5.02	207.60
	LEV	- 6.49	17.62
	NOR	- 6.06	36.86

• Source: Author’s data (2025).

The interactions between GER and TSM-16 with the active site of DNA gyrase (Figures 11B) are predominantly hydrophobic, involving alkyl-alkyl and π-alkyl bonds, as well as van der Waals forces, which contribute to the stabilization of the ligands within the enzyme. In the case of GER, a hydrogen bond between the Thr165 residue and the geraniol OH group, with a distance of 2.02 Å, further enhances its stability at the active site. In comparison, levofloxacin also interacts via polar and nonpolar bonds, but exhibits slightly lower affinity, possibly due to lower molecular complementarity with the active site of DNA gyrase.

Figure 1
Molecular docking of the TSM-16 and LEV ligands with the active site of the DNA gyrase enzyme (PBD ID: 5L3J). Main types of interactions between TSM-16 and the enzyme’s active site in 2D. 3D distribution and distances of the chemical bonds between the TSM-16 ligand and the amino acids of the enzyme’s active site, and a 3D surface model showing the regions of the ligand with higher or lower degrees of hydrophobicity. Source: Author’s own work (2025).

TSM-16 interacts with the active site of topoisomerase IV (Figure 2A) by forming two hydrogen bonds with guanine 1 (G1), with distances of 1.83 Å and 2.08 Å, as well as a carbon-hydrogen interaction between the oxygen of G1 and carbon 7 (C7) of TSM-16 of 2.91 Å, two π-sigma bonds with adenine and cytosine, and hydrophobic interactions, such as van der Waals forces. The oxygen atoms of TSM-16 play a key role as hydrogen donors. In comparison, levofloxacin (LEV) exhibits greater affinity for the active site of topoisomerase IV (Figure 2B), due to lower ∆G and Ki values, better molecular complementarity, and multiple hydrogen bonds with residues Ser1080, Arg1119, and the adenine 5 and guanine 1 nucleotides, in addition to other interactions that confer greater stability to the compound in a predominantly polar active site.

Figure 2
Molecular docking of the TSM-16 and LEV ligands with the active site of the topoisomerase IV enzyme (PBD ID: 5EIX). Main types of interactions between TSM-16 and the enzyme’s active site in 2D. 3D distribution and distances of the chemical bonds between the TSM-16 ligand and the amino acids of the enzyme’s active site, and a 3D surface model of the region of the enzyme’s active site occupied by the ligand and the hydrogen donor and acceptor sites. Source: Author’s own work (2025).

Based on the in silico results, TSM-16 exhibits a favorable pharmacokinetic profile, justifying in vitro testing. In the present study, TSM-16 exhibited MICs between 500 and 1000 µg/mL against Klebsiella pneumoniae (Table 5), indicating moderate to strong activity (Sartoratto et al., 2004), superior to natural geraniol, which exhibited an MIC >1500 µg/mL. Monoterpenes exert antimicrobial activity primarily by disrupting the cell membrane (Singh et al., 2021), and, considering the hydrophobic nature of TSM-16, it is plausible that it interacts with lipid bilayers, altering the biophysical properties of membranes and inhibiting microorganisms (Belin et al., 2019). These in vitro findings are consistent with the in silico results, which indicated a possible enzymatic inhibitory effect. The MIC of TSM-16 was >1000 µg/mL against all analyzed strains (Table 6).

Table 5
Minimum Bactericidal Concentration (MBC) of TSM-16 against Klebsiella pneumoniae strains.

Cepas bactérias		TSM-16
Kp101	MIC	500
	MBC	> 1000
Kp104	MIC	1000
	MBC	> 1000
Kp105	MIC	1000
	MBC	> 1000
Kp110	MIC	500
	MBC	> 1000
ATCC	MIC	500
	MBC	> 1000

• Source: Author’s data (2025).

Table 6
Study of the combination of the synthetic compound TSM-16 with conventional antimicrobials against strains of K. pneumoniae.

Microorganism	Association	PEN	AMP	TET	GEN	CRO	CIP
ATCC13883	ZOIATB	20mm	0mm	18mm	20mm	28mm	12mm
	ZOIATB +	20 mm	30 mm	20 mm	24 mm	30 mm	30 mm
		*	(↑)	(↑)	(↑)	(↑)	(↑)
Kp101	ZOIATB	6mm	0mm	8mm	20mm	26mm	24mm
	ZOIATB +	8 mm	6 mm	10 mm	24 mm	28 mm	6 mm
		(↑)	(↑)	(↑)	(↑)	(↑)	(↓)
Kp104	ZOIATB	6mm	10mm	10mm	16mm	12mm	28mm
	ZOIATB +	0 mm	0 mm	12 mm	22 mm	30 mm	10 mm
		(↓)	(↓)	(↑)	(↑)	(↑)	(↓)
Kp105	ZOIATB	6mm	12mm	28mm	20mm	24mm	36mm
	ZOIATB +	6 mm	12 mm	26 mm	26 mm	30 mm	36 mm
		*	*	(↓)	(↑)	(↑)	*
Kp110	ZOIATB	6mm	20mm	24mm	20mm	28mm	30mm
	ZOIATB +	8 mm	18 mm	30 mm	26 mm	30 mm	34 mm
		(↑)	(↓)	(↑)	(↑)	(↑)	(↑)

• ZOIATB: zone of inhibition in the presence of the antibiotic. ZOIATB +: inhibition zone in the presence of the antibiotic and TSM-16. Synergistic effect (↑); antagonistic effect (↓);
• ^*
  indifferent effect. Source: Author’s data (2025).

According to microbiological criteria, a compound is bactericidal when the MIC is up to four times the MIC, and predominantly bacteriostatic when it exceeds this limit (Pereira and Silva, 2022). In the present study, TSM-16 exhibited predominantly bacteriostatic activity against the evaluated strains, with a MIC >1000 µg/mL. These results differ from those observed by Kwiatkowski et al. (2022), who reported a bactericidal effect of geraniol against Klebsiella pneumoniae ATCC BAA-2473, but corroborate those of Lira et al. (2020), who recorded MBCs of geraniol ranging from 1386.8 to 5547.2 µg/mL against different bacteria. Furthermore, TSM-16 demonstrated efficacy when combined with conventional antibacterials, exhibiting synergy against some strains of K. pneumoniae (Table 6), reinforcing the strategy of combination therapies as a promising tool in combating bacterial infections (Bognár et al., 2024).

In tests with five strains and six antimicrobials, 30 combinations were evaluated, with a predominance of synergistic effects (70%), followed by antagonism (20%) and indifference (10%). This profile is consistent with monoterpenes and their derivatives, which are frequently associated with the inhibition of bacterial growth (Singh et al., 2021; Silva et al., 2022). Gentamicin and ceftriaxone showed synergy in 100% of the strains, tetracycline in 80%, and ciprofloxacin in 60%, while penicillin and ampicillin showed more heterogeneous results, possibly due to resistance mechanisms such as β-lactamases (Kwiatkowski et al., 2022; Papp-Wallace and Bhavsar, 2023).

Among the strains, ATCC 13883, Kp101, and Kp110 exhibited 83.3% synergy; Kp104, 50%; and Kp105 showed lower sensitivity (33.3% synergy and 50% indifferent), indicating an isolate-dependent effect (Li et al., 2024; Romo-Castillo et al., 2023). These results reinforce that terpenoid compounds can act as adjuvants, facilitating the entry of antimicrobials into bacterial cells (Zhang et al., 2024). Thus, the combination of TSM-16 with antibacterials shows promise against multidrug-resistant strains of K. pneumoniae, a priority critical pathogen designated by the WHO (2023), although further studies on mechanisms, cytotoxicity, stability, and in vivo efficacy are still needed.

4. Conclusion

Analysis of the results indicates that TSM-16 exhibits a high probability of interaction, a favorable pharmacokinetic profile, and significant antibacterial potential, with toxicity levels within acceptable ranges for scientific development. Molecular docking studies demonstrated efficient binding to the active site of DNA gyrase, while in vitro tests showed bacteriostatic activity and a predominant synergistic effect when combined with conventional antimicrobials against K. pneumoniae. Thus, further studies are essential to elucidate its toxicological profile, mechanisms of action, and in vivo efficacy, positioning TSM-16 as a promising option for new antimicrobial therapeutic strategies.

Data Availability Statement

All datasets supporting the results of this study have been published in the article itself. And available upon reasonable request from the corresponding author.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  24 July 2026
• Date of issue
  2026

History

• Received
  20 Mar 2026
• Accepted
  29 Apr 2026