Bactericidal effect of citronellal and molecular docking with KPC-2 of Klebsiella pneumoniae and PBP2A of Staphylococcus aureus Abstract
Infectious diseases remain a leading cause of death worldwide. Due to selective pressure, resistant bacteria, especially Klebsiella pneumoniae and Staphylococcus aureus, are frequently selected and disseminated in various environments. This study aimed to evaluate the antibacterial activity of citronellal against K. pneumoniae and S. aureus, as well as its interactions with enzymes associated with resistance to carbapenems and methicillin, KPC-2 and PBP2a, respectively. Bioassays were conducted to determine the minimum inhibitory and bactericidal concentrations. Rigid molecular docking assays were performed with fixed amino acid residues of KPC-2 and PBP2a, while the ligand was flexible. Citronellal inhibited bacterial growth and showed bactericidal activity against K. pneumoniae at 128 µg/mL and S. aureus at 64 µg/mL. Additionally, citronellal interacted with the active sites of KPC-2 and PBP2a, with binding energies of −4.30 and −4.05 kcal/mol, respectively. In conclusion, citronellal demonstrated bactericidal effects against the tested strains. However, despite its affinity for the target enzymes, the binding activity was lower compared to avibactam and penicillin G in the docking simulations.
Key words
Citronellal; Molecular docking; K pneumoniae; S aureus
INTRODUCTION
The increasing incidence of nosocomial infections poses a significant public health challenge worldwide, particularly due to the involvement of multidrug-resistant (MDR) bacteria. These pathogens not only complicate treatment options but also contribute to higher morbidity, mortality, and healthcare costs. The etiology of nosocomial infections can be attributed to various multidrug-resistant pathogens, among which Klebsiella pneumoniae and Staphylococcus aureus are particularly noteworthy (Sousa et al. 2021). Both species are frequently associated with healthcare-associated infections and are listed by the World Health Organization as priority pathogens for which new antibiotics are urgently needed. Their ability to develop and spread resistance mechanisms has made them emblematic of the broader crisis in antimicrobial therapy.
K. pneumoniae is part of the intestinal microbiota, and a member of the Enterobacteriaceae family (Kollef et al. 2021). The diseases caused by this Gram-negative bacillus are commonly linked to the host immune status, and their severity can be attributed to the high rates of antimicrobial resistance, driven by indiscriminate use of antibiotics (Sousa et al. 2019). One of the resistance mechanisms of K. pneumoniae involves its ability to produce enzymes such as carbapenemases (KPC), which can inactivate carbapenem antibiotics, including meropenem, ertapenem, and imipenem (Kołpa et al. 2018), as well as other beta-lactams such as penicillins and cephalosporins (Sawa et al. 2020).
S. aureus, in turn, is primarily found on the human skin microbiota. This microorganism is also a major pathogen responsible for hospital-acquired infections and has become a growing public health concern due to its increasing antimicrobial resistance profile (Demessant-Flavigny et al. 2023). Specifically, methicillin-resistant S. aureus (MRSA) strains exhibit resistance to all beta-lactam antibiotics, except for fifth-generation cephalosporins. In addition, these strains produce penicillin-binding proteins (PBPs), such as PBP2a, which is responsible for the resistant phenotype (Ambade et al. 2023).
Consequently, the main challenges in the treatment of infectious diseases are related to the significant increase in multidrug-resistant pathogens, high mortality rates, therapeutic failure, and the financial burden of treatments. These alarming trends underscore the urgent need for alternative therapeutic approaches capable of overcoming existing resistance mechanisms. Therefore, the search for novel therapeutics and antimicrobial alternatives is essential, particularly those involving molecules derived from natural sources, such as plants and microorganisms (Genilloud 2019).
Most natural products are safe, which renders the study of their antimicrobial activity highly relevant. Plant secondary metabolites, such as essential oils (EOs), encompass substances containing various bioactive compounds, contributing to their functional versatility and positioning them as an interesting group for exploration (Lopez-Romero et al. 2015, Singh et al. 2016, Tahya et al. 2022). In this regard, EOs have become attractive alternatives owing to a wide range of biological activities, including antifungal, antibacterial, and antioxidant potential. These compounds are found in different parts of plants, such as stems, leaves, and fruits. Among leaf-derived EOs, citronella oil is prominent, with its major component, citronellal, a monoterpene, exhibiting favorable oral bioavailability and low toxicity (Jacob et al. 2017, Medeiros et al. 2017).
Despite increasing interest in plant-derived antimicrobials, most research has focused on whole essential oils rather than isolated constituents. Understanding the activity of specific phytochemicals may enhance the development of targeted antimicrobial agents. Therefore, the present study aimed to analyze the activity of citronellal against K. pneumoniae and S. aureus, as well as explore, through molecular docking, the interactions of this compound with the microbial resistance enzymes KPC-2 and PBP2a.
MATERIALS AND METHODS
Chemicals
Citronellal (3,7-dimetiloct-6-enal, >95% purity) was purchased from Quinarí® (Ponta Grossa, Brazil). For all antibacterial assays, citronellal was dissolved in 3% dimethyl sulfoxide (DMSO) and 2% Tween® 80 to prepare a stock solution at 1,024 µg/mL stock solution. The stock solutions were stored at −20 °C and further diluted in the culture medium to obtain the final concentrations used in each assay (Hood et al. 2003, Nascimento et al. 2007).
Microorganisms
The selection of bacterial strains was based on their clinical significance and resistance characteristics. Clinical isolates were chosen due to their multidrug-resistant phenotypes, commonly associated with healthcare-associated infections, which pose therapeutic challenges and represent relevant models for the evaluation of novel antimicrobials. Standard ATCC strains were included to provide genotypic and phenotypic consistency, ensuring reproducibility and enabling comparison with results from the existing scientific literature. Clinical isolates of S. aureus (101, 103, and 105) and K. pneumoniae (101, 103, 104, and 105), along with S. aureus ATCC 25925, S. aureus ATCC 29213, and K. pneumoniae ATCC 13883, were included in the study. These strains are maintained within the microbial collection of the Laboratory of Research in Microbiology (LPM) at the university center (Centro Universitário de Patos, UniFIP), Patos City, Paraíba State, Brazil. The strains were cultivated in Brain Heart Infusion (BHI) broth at 37 °C for 24 h and stored at 4 °C. For the experiments, three colonies were transferred to Sabouraud Dextrose Broth (SDB) and incubated at 37 °C for 24 h. To prepare standard bacterial suspensions, cells were harvested by centrifugation (10,000 × g for 1 min) and resuspended in sterile 0.85% NaCl (saline) to achieve a turbidity equivalent to the 0.5 McFarland standard, using the DensiCHEK™ PLUS colorimeter (bioMérieux, Rio de Janeiro, Brazil). The standard bacterial suspensions, corresponding to 1.5 × 10⁸ colony-forming units (CFUs)/mL, were subsequently diluted in the culture medium to obtain the inoculum used in the assays (Hadacek & Greger 2000, CLSI 2012).
Minimum Inhibitory (MIC) and Minimum Bactericidal (MBC) Concentrations
The MIC values of citronellal were determined using the broth microdilution method in 96-well U-bottom polystyrene plates, following the guidelines outlined in the Clinical and Laboratory Standards Institute document (CLSI 2015). Stock solutions of citronellal were prepared and serially diluted in BHI broth to obtain final concentrations ranging from 2 to 1,024 µg/mL. Wells containing BHI with 3% DMSO and 2% Tween® 80, along with bacterial cells, served as the growth control. All experiments were performed in duplicate to ensure reproducibility and reliability of the results. The MIC values were defined as the lowest concentration of the compound that completely inhibited visible bacterial growth after 24 h of incubation at 37 °C, in comparison to the growth control.
For MBC determination, 10µL aliquots from wells showing no visible growth were homogenized, spread onto BHI agar plates, and incubated at 37 °C for 24 h. The MBC values were defined as the lowest concentration that resulted in a 99.9% reduction in colony-forming unit (CFU) counts compared to the untreated control. The antibacterial effect of citronellal was classified according to the MBC/MIC ratio as follows: bactericidal, MBC/MIC = 1–4; bacteriostatic, MBC/MIC > 4 (Ostrosky et al. 2008, CLSI 2012). Positive control antibiotics were not included in this study due to the experimental design being focused on assessing the intrinsic antimicrobial potential of citronellal in isolation. This approach aimed to eliminate interference from known standards in the interpretation of the compound’s efficacy, particularly in preliminary screenings involving plant-derived molecules. Nevertheless, internal validation was ensured through the use of ATCC reference strains and rigorous adherence to standardized protocols.
Molecular docking
Rigid molecular docking simulations were performed with the respective target proteins: KPC-2 from K. pneumoniae (PDB ID: 4ZBE) at 1.80 Å and PBP2a from S. aureus (PDB ID: 1MWT) at 2.45 Å. The protein structures were retrieved from the Protein Data Bank (PDB, https://www.rcsb.org/?ref=nav_home) and loaded into PyMol 2.5.3 for the removal of water molecules and co-crystallized ligands (avibactam and penicillin G, respectively). The structure of the ligand citronellal (CIT) was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/), and its energy minimization and molecular optimization were performed at pH 7.4 using Avogadro 1.2.0 and Mopac2012 at the PM6 level, applying the MMFF94 force field (Halgren 1999, Hanwell et al. 2012).
AutoDock 4.2 was selected for the docking simulations due to its flexibility, widespread use in academic research, and ability to provide detailed control over docking parameters. Although more recent tools offer enhanced scoring functions, AutoDock remains a robust and validated platform, particularly for ligand–enzyme interaction studies involving natural compounds.
Subsequently, the proteins were loaded into AutoDock Tools (Morris et al. 2009) for the addition of hydrogen atoms and Kollman charges, as well as the merging of nonpolar hydrogens. The docking simulations were then conducted by identifying the active sites of the targets using the following grid centers: KPC-2 (9.280; 1.468; 4.539 Å), dimensions (32 × 32 × 32 Å), and spacing of 0.300 Å; PBP2a (27.987; 28.938; 87.527 Å), dimensions (36 × 36 × 36 Å), and spacing of 0.300 Å.
Following the localization of the active sites, the docking process was carried out in AutoDock 4.2 using 100 runs of the Lamarckian genetic algorithm with default AutoDock Tools parameters. Binding Free Energy (ΔG) and Inhibition Constant (Ki) values were generated, and the conformations with the lowest ΔG values were selected. Finally, the results were analyzed using PyMol 2.5.3 and Discovery Studio 2021 to determine the binding regions of the target with the ligand molecule, the types of molecular interactions involved, and the active sites of the amino acid residues participating in the binding.
Method validation was performed through molecular redocking, which consists of reproducing the position and orientation of the ligand as observed in the crystallographic structure. Subsequently, the ligand conformation displaying the lowest Root Mean Square Deviation (RMSD) value was selected, ensuring that atomic distance deviations between atoms were ≤ 2.0 Å (Bell & Zhang 2019).
RESULTS AND DISCUSSION
Citronellal exhibits bactericidal activity Against K. pneumoniae and S. aureus isolates
In the present study, the antibacterial activity of citronellal was evaluated by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for each tested strain. To classify the nature of the antimicrobial effect, we used the MBC/MIC ratio, a well-established parameter for distinguishing between bactericidal and bacteriostatic activities: values ≤ 4 are indicative of a bactericidal effect, whereas values > 4 denote a bacteriostatic action (Ostrosky et al. 2008, CLSI 2012).
Based on this criterion, citronellal demonstrated a bactericidal effect against all tested strains of S. aureus and K. pneumoniae, including both clinical isolates and ATCC reference strains (Table I and II). MIC values for S. aureus ranged from 64 to 256 µg/mL, while for K. pneumoniae, values ranged from 128 to 512 µg/mL. MBC values were consistent with a bactericidal classification. Notably, citronellal showed greater potency against S. aureus clinical isolates than against K. pneumoniae, although for ATCC strains the MIC values were equivalent.
These results are in agreement with previous studies that reported the antimicrobial activity of citronellal against S. aureus. Sartoratto et al. (2004), Thomas et al. (2012), and Mogana et al. (2020) also found a bactericidal effect of citronellal against this Gram-positive species. Hussain et al. (2011), using a modified resazurin microtitration assay, reported bactericidal concentrations of 590.9 µg/mL and 490 µg/mL for S. aureus NCTC-1803 and NCTC-6571, respectively. Additionally, the essential oil of Melissa officinalis, whose main component is citronellal, exhibited enhanced antibacterial activity with lower MIC values, suggesting a possible synergistic effect (Williamson 2001). Simic et al. (2008) evaluated the essential oil of Cymbopogon winterianus, which contains citronellal and citronellol, and found MIC values of 2 µg/mL and 6 µg/mL for S. aureus and E. coli, respectively. Guimarães et al. (2019) also observed MIC values of 250 µg/mL for citronellal against these species, aligning with our results.
Conversely, Pontes et al. (2018) reported no antimicrobial activity of citronellal (MIC > 2,048 µg/mL) against ESBL-producing K. pneumoniae strains. Similarly, Lima et al. (2020) and Bezerra et al. (2022) observed an absence of activity against ESBL-producing E. coli and K. pneumoniae. These discrepancies may be attributed to differences in bacterial resistance profiles, since our study evaluated non-ESBL-producing strains. In addition, variations in the chemical composition of essential oils, the purity of isolated compounds, the methodology employed (e.g., broth microdilution vs. agar diffusion), and the bacterial strains used can all influence antimicrobial outcomes. Cimanga et al. (2002) demonstrated that essential oil from Eucalyptus citriodora, composed of 73% citronellal, showed greater activity against E. coli than S. aureus. However, unlike our study, that work assessed only the complete essential oil, not the isolated compound. These conflicting findings highlight the relevance of standardizing methodologies and distinguishing between the effects of isolated constituents and their combinations in essential oils.
Citronellal exhibits affinity for the proteins under study
To evaluate the potential binding interactions of citronellal with bacterial enzymes, molecular docking studies were conducted using the KPC-2 and PBP2a proteins. The docking protocol was validated through redocking of the co-crystallized ligands, avibactam and penicillin G, into their respective targets. This step yielded RMSD values below 2.0 Å and demonstrated structural overlap with the original crystallographic conformations (Figures 1 and 2), confirming the reliability of the applied methodology.
Docking validation via redocking. (a) Best overlay of redocked avibactam (yellow) compared to the co-crystal structure from Protein Data Bank (PDB, green). (b) Two-dimensional interactions between avibactam and the amino acid residues within the active site of the KPC-2 protein (PDB ID: 4ZBE).
Docking validation via redocking. (b) Best overlay of redocked penicillin G (yellow) compared to the co-crystal structure from Protein Data Bank (PDB, green). (b) Two-dimensional interactions between penicillin G and the amino acid residues within the active site of the PBP2a protein (PDB ID: 1MWT).
Docking simulations revealed that citronellal exhibited moderate affinity for both KPC-2 and PBP2a when compared to avibactam and penicillin G, respectively (Table III). This difference in binding affinity is likely due to variations in molecular size, polarity, and the capacity to form multiple and stable interactions within the active sites. Citronellal, being a small, neutral, and hydrophobic molecule with a terminal carbonyl group, formed mainly weak van der Waals interactions, particularly with KPC-2, where it showed slightly greater affinity (Figure 3).
Table II Bactericidal effect of citronellal against S. aureus strains.| Strains | Citronellal | |||
|---|---|---|---|---|
| *MIC | *MBC | MBC/MIC | Effect | |
| S. aureus 101 | 32 | 64 | 2 | Bactericidal |
| S. aureus 103 | 64 | 64 | 1 | Bactericidal |
| S. aureus 105 | 64 | 64 | 1 | Bactericidal |
| S. aureus ATCC 25925 | 64 | 64 | 1 | Bactericidal |
| S. aureus ATCC 29213 | 64 | 64 | 1 | Bactericidal |
Hydrogen and hydrophobic bond interactions between citronellal and the active site of the KPC-2 enzyme (PDB ID: 4ZBE). (a) Two-dimensional (2D) representation of citronellal interactions with amino acid residues in the active site. (b) Three-dimensional (3D) view showing binding distances of citronellal within the KPC-2 active site. (c) Surface representation highlighting hydrogen bond interactions. (d) Surface representation illustrating hydrophobic interactions.
In contrast, the co-crystallized ligands displayed a higher number and diversity of interactions, reflecting their structural complementarity and stronger binding with the active sites of their respective enzymes (Figures 3 and 4). This highlights citronellal’s limited but notable capacity to interact with clinically relevant bacterial targets.
The relevance of KPC-2 and PBP2a stems from their roles in antimicrobial resistance. KPC-2 is a carbapenemase associated with resistance in K. pneumoniae and other Gram-negative pathogens. Its global dissemination, including in E. coli, Providencia stuartii, and Citrobacter freundii, poses a significant clinical threat, especially in Brazil where it has also been detected in Pseudomonas spp. (Jácome et al. 2012, Almeida et al. 2012, Tavares et al. 2015). Avibactam remains the only inhibitor currently available against KPC-producing strains (Papp-Wallace et al. 2010), underscoring the need for additional compounds with inhibitory potential.
On the other hand, PBP2a is a penicillin-binding protein encoded by methicillin-resistant S. aureus (MRSA) strains. Its low affinity for β-lactams enables continued peptidoglycan synthesis even in the presence of these antibiotics, contributing to resistance (Peacock & Paterson 2015, Das 2023). Although citronellal showed weaker binding to PBP2a (Figure 4), its interaction suggests potential for future structural modifications aimed at enhancing inhibitory efficacy.
Hydrogen and hydrophobic bond interactions between citronellal and the active site of the PBP2a enzyme (PDB ID: 1MWT). (a) Two-dimensional (2D) representation of citronellal interactions with amino acid residues in the active site. (b) Three-dimensional (3D) view showing binding distances of citronellal within the PBP2a active site. (c) Surface representation highlighting hydrogen bond interactions. (d) Surface representation illustrating hydrophobic interactions.
Furthermore, previous studies suggest that citronellal’s antibacterial action may also involve membrane disruption. Lopez-Romero et al. (2015) reported significant alterations in S. aureus membrane integrity, with increased permeability and potassium leakage after exposure to citronellal.
In summary, our findings confirm that citronellal exerts bactericidal effects against K. pneumoniae and S. aureus, and interacts moderately with KPC-2 and PBP2a. While its binding affinity is lower than established inhibitors, its structural simplicity and partial activity point to a promising scaffold for the development of new antibacterial agents targeting resistant enzymes.
Acknowledgements
The authors thank Centro Universitário UniFIP for the institutional support provided for the development of this research.
References
- ALMEIDA AC, VILELA MA, CAVALCANTI FL, MARTINS WM, MORAIS MAJ & MORAIS MM. 2012. First description of KPC-2-producing Pseudomonas putida in Brazil. Antimicrob Agents Chemother 56(4): 2205-2206.
- AMBADE SS, GUPTA VK, BHOLE RP, KHEDEKAR PB & CHIKHALE RV. 2023. A review on five and six-membered heterocyclic compounds targeting the penicillin-binding protein 2 (PBP2a) of methicillin-resistant Staphylococcus aureus (MRSA). Molecules 28(17): 7008.
- BELL EW & ZHANG Y. 2019. DockRMSD: an open-source tool for atom mapping and RMSD calculation of symmetric molecules through graph isomorphism. J Cheminform 11(1): 1-9.
- BEZERRA RV, PONTES MAN, ANDRADE-JÚNIOR FP, SANTOS BHC & LIMA IO. 2022. Avaliação da atividade antibacteriana do monoterpeno (R)-(+)-citronelal contra cepas de Pseudomonas aeruginosa Rev Colomb Cienc Quím Farm 51(2): 691-702.
- CIMANGA K ET AL. 2002. Correlation between chemical composition and antibacterial activity of essential oils of some aromatic medicinal plants growing in the Democratic Republic of Congo. J Ethnopharmacol 79(2): 213-220.
- CLSI - CLINICAL AND LABORATORY STANDARDS INSTITUTE. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard – 9th ed. CLSI document M07-A9. Wayne: CLSI.
- CLSI - CLINICAL AND LABORATORY STANDARDS INSTITUTE. 2015. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard – 10th ed. CLSI document M07-A10. Wayne: CLSI.
- DAS S. 2023. The crisis of carbapenemase-mediated carbapenem resistance across the human-animal-environmental interface in India. Infect Dis Now 53: 104628.
- DEMESSANT-FLAVIGNY AL, CONNÉTABLE S, KEROB D, MOREAU M, AGUILAR L & WOLLENBERG A. 2023. Skin microbiome dysbiosis and the role of Staphylococcus aureus in atopic dermatitis in adults and children: a narrative review. J Eur Acad Dermatol Venereol 5(3): 3-17.
- GENILLOUD O. 2019. Natural products discovery and potential for new antibiotics. Curr Opin Microbiol 51: 81-87.
- GUIMARÃES AC ET AL. 2019. Antibacterial activity of terpenes and terpenoids present in essential oil. Molecules 24(13): 2471.
- HADACEK F & GREGER H. 2000. Testing of antifungal natural products: methodologies, comparability of results and assay choice. Phytochem Anal 11(3): 137-147.
- HALGREN TA. 1999. MMFF VII. Characterization of MMFF94, MMFF94s, and other widely available force fields for conformational energies and for intermolecular-interaction energies and geometries. J Comput Chem 20(7): 730-748.
- HANWELL MD, CURTIS DE, LONIE DC, VANDERMEERSCH T, ZUREK E & HUTCHISON GR. 2012. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J Cheminform 4: 17.
- HOOD JR, WILKINSON JM & CAVANAGH HMA. 2003. Evaluation of common antibacterial screening methods utilized in essential oil research. J Essent Oil Res 15: 428-433.
- HUSSAIN AI ET AL. 2011. Antibacterial activity of some Lamiaceae essential oils using resazurin as an indicator of cell growth. LWT Food Sci Technol 44(5): 1199-1206.
- JACOB RG, OLIVEIRA DH, DIAS IFC, SCHUMACHER RF & SAVEGNAGO L. 2017. Óleos essenciais como matéria-prima sustentável para o preparo de produtos com maior valor agregado. Rev Virtual Quim 9: 294-316.
- JÁCOME PRLA, ALVES LR, CABRAL AB, LOPES ACS & MACIEL MAV. 2012. First report of KPC-producing Pseudomonas aeruginosa in Brazil. Antimicrob Agents Chemother 56(9): 4990.
- KOLLEF MH, TORRES A, SHORR AF, MARTIN-LOECHES I & MICEK ST. 2021. Nosocomial infection. Crit Care Med 49(2): 169-187.
- KOŁPA M, WAŁASZEK M, GNIADEK A, WOLAK Z & DOBROŚ W. 2018. Incidence, microbiological profile and risk factors of healthcare-associated infections in intensive care units: a 10-year observation in a provincial hospital in southern Poland. Int J Environ Res Public Health 15(1): 112.
- LIMA DS, PONTES MAN, ANDRADE-JÚNIOR FP, SANTOS BHC, OLIVEIRA WA & LIMA IO. 2020. Atividade antibacteriana de citronelal e citronelol contra cepas de Escherichia coli produtoras de ESBL. Arch Health Invest 9: 238-241.
- LOPEZ-ROMERO JC, GONZÁLEZ-RÍOS H, BORGES A & SIMÕES M. 2015. Antibacterial effects and mode of action of selected essential oils components against Escherichia coli and Staphylococcus aureus Evid Based Complement Alternat Med 2015: 1-9. doi: 10.1155/2015/795435.
- MEDEIROS CIS, SILVA DF, OLIVEIRA FILHO AAO & LIMA EO. 2017. Pharmacological and toxicological analysis of two enantiomers derived from citronellal monoterpene: an in silico approach. REBES 7(1): 84-89.
- MOGANA R, ADHIKARI A, TZAR MN, RAMLIZA R & WIART C. 2020. Antibacterial activities of the extracts, fractions and isolated compounds from Canarium patentinervium Miq. against bacterial clinical isolates. BMC Complement Med Ther 20: 1-11.
- MORRIS GM, HUEY R, LINDSTROM W, SANNER MF, BELEW RK, GOODSELL DS & OLSON AJ. 2009. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30(16): 2785-2791.
- NASCIMENTO PFC ET AL. 2007. Atividade antimicrobiana dos óleos essenciais: uma abordagem multifatorial dos métodos. Rev Bras Farmacogn 17(1): 108-113.
- OSTROSKY EA, MIZUMOTO MK, LIMA MEL, KANEKO TM, NISHIKAWA SO & FREITAS BR. 2008. Métodos para avaliação da atividade antimicrobiana e determinação da concentração mínima inibitória (CMI) de plantas medicinais. Rev Bras Farmacogn 18(2): 301-307.
- PAPP-WALLACE KM, BETHEL CR, DISTLER AM, KASUBOSKI C, TARACILA M & BONOMO RA. 2010. Elucidating the role of Trp105 in the KPC-2 β-lactamase. Protein Sci 19(9): 1714-1727.
- PEACOCK SJ & PATERSON GK. 2015. Mechanisms of methicillin resistance in Staphylococcus aureus Annu Rev Biochem 84: 577-5601.
- PONTES MAN, GOMES SM, SANTOS BHC & LIMA IO. 2018. Efeito inibitório de monoterpenos frente a Klebsiella pneumoniae produtoras de ESBL. Rev Bras Cien Saúde 22(1): 51-56.
- SARTORATTO A, MACHADO ALM, DELARMELINA C, FIGUEIRA GM, DUARTE MCT & REHDER VLG. 2004. Composition and antimicrobial activity of essential oils from aromatic plants used in Brazil. Braz J Microbiol 35(4): 275-280.
- SAWA T, KOOGUCHI K & MORIYAMA K. 2020. Molecular diversity of extended-spectrum β-lactamases and carbapenemases, and antimicrobial resistance. J Intensive Care 8: 13.
- SIMIC A, RANCIC A, SOKOVIC MD, RISTIC M, GRUJIC-JOVANOVIC S, VUKOJEVIC J & MARIN PD. 2008. Essential oil composition of Cymbopogon winterianus and Carum carvi and their antimicrobial activities. Pharm Biol 46(6): 437-441.
- SINGH S, FATIMA Z & HAMEED S. 2016. Citronellal-induced disruption of membrane homeostasis in Candida albicans and attenuation of its virulence attributes. Rev Soc Bras Med Trop 49(4): 465-472.
- SOUSA ATHI ET AL. 2019. Perfil de resistência antimicrobiana de Klebsiella pneumoniae isoladas de animais domésticos e silvestres. Arq Bras Med Vet Zootec 71(2): 584-593.
- SOUSA SA, SOARES-SANTOS A, MORAIS MM, SILVA-JÚNIOR MF, NAKAZATO L & DUTRA V. 2021. Bacterial nosocomial infections: multidrug resistance as a trigger for the development of novel antimicrobials. Antibiotics 10(8): 942.
- TAHYA CY, KOLO SMD & KARNELASATRI K. 2022. Antimicrobial and antioxidant properties of Cymbopogon nardus L. (citronella grass) oil from Kefamenamu, Timor Tengah Utara Regency, Indonesia. AIP Conf Proc 2391: 050003.
- TAVARES CP ET AL. 2015. Molecular epidemiology of KPC-2-producing Enterobacteriaceae (non-Klebsiella pneumoniae) isolated from Brazil. Diagn Microbiol Infect Dis 82(4): 326-330.
- THOMAS BT, ADELEKE AJ, RAHEEM-ADEMOLA RR, KOLAWOLE R & MUSA OS. 2012. Efficiency of some disinfectants on bacterial wound pathogens. Life Sci J 9(4): 752-755.
- WILLIAMSON EM. 2001. Synergy and other interactions in phytomedicines. Phytomedicine 8(5): 401-409.
