S. maltophilia is an emerging nosocomial pathogen with intrinsic antibiotic resistance that primarily affects immunocompromised patients. S. maltophilia usually causes respiratory tract infections with a mortality rate of 20–60% [1]. These bacteria cause respiratory and urinary tract infections by colonizing the surfaces of medical devices and treatment equipment such as urinary catheters, endoscopes, and ventilators [2]. S. maltophilia has many structural components that contribute to its virulence and pathogenicity. Pili/flagella/fimbrial/adhesins enable colonization on living and non-living surfaces; outer membrane lipopolysaccharide causes antibiotic resistance and biofilm formation as well as cell death; diffusible signaling factor causes motility, extracellular enzyme production, lipopolysaccharide synthesis, microcolony formation, and tolerance to antibiotics and heavy metal ions [3, 4]. S. maltophilia has the ability to form biofilms on abiotic surfaces and host (biotic) tissues. This is an important virulence factor for bacteria that plays an important role in nosocomial and multi-bacterial infections, significantly reducing the therapeutic efficacy of important antibiotics, including aminoglycosides, fluoroquinolones, and tetracyclines [5].

Trimethoprim/sulfamethoxazole (TMP-SMX) is the first-choice therapeutic option for treating infections due to its in vitro efficacy and favorable clinical outcomes. Meanwhile, alternative antibiotics such as levofloxacin, tigecycline, ceftazidime, colistin, and ticarcillin-clavulanic acid are also used in cases of resistance to TMP-SMX [6]. Due to the recent increase in antimicrobial resistance in S. maltophilia and because of the biofilm component that contributes to this resistance, there is a need to develop new antimicrobial and antibiofilm agents against S. maltophilia.

Studies have shown that T. serpyllum (wild thyme) plant extracts are effective against both Gram-positive and Gram-negative bacteria, including Escherichia coli, Staphylococcus aureus, and Bacillus subtilis [7, 8]. M. piperita (peppermint), R. officinalis (rosemary), T. cordata (T. linden), S. officinalis (sage), and T. spicata (spiked thyme) plants have also been reported to promote antimicrobial effects [9, 10, 11, 12]. Although some studies have examined the antimicrobial effects of these plant species, no comprehensive research has evaluated the activity of these extracts against multidrug-resistant S. maltophilia, except for T. serpyllum. Unlike previous studies, this study compares six plant extracts and further integrates a molecular docking analysis to explore potential mechanisms of action, thereby offering a more mechanistic and comparative insight into the antimicrobial and antibiofilm potential of these extracts.

In silico molecular docking is used to describe the reactions of molecules and predict the potential associated macroscopic properties. Data obtained from experiments can provide novel information; however, these data do not reveal the mechanism of action in the biological system. Therefore, these mechanisms should be further studied in computer modeling analyses using the structures of biological systems [13].

This study aimed to investigate the antimicrobial and antibiofilm activities of T. serpyllum, M. piperita, R. officinalis, T. cordata, S. officinalis, and T. spicata extracts against S. maltophilia strains and to investigate the possible interactions of the compounds in the most active extracts with the targets in terms of antibacterial activity through the molecular docking method.

S. maltophilia is naturally resistant to many antibiotics, including beta-lactams, aminoglycosides, macrolides, tetracyclines, and carbapenems. This poses serious challenges in the treatment of S. maltophilia infections. Currently, the most effective antibiotic is TMP-SMX; however, resistance to TMP-SMX has increased over recent years [24, 25]. Indeed, a systematic review reported that the resistance rate of S. maltophilia to TMP/SMX was 9.2% worldwide between 2000 and 2022 [26]. These data highlight that reliance on TMP-SMX alone may not be sustainable in the long term, and alternative therapeutic strategies are urgently required.

Moreover, in a study by Pompilio et al. [16], which analyzed 109 S. maltophilia isolates from various clinical samples, the authors noted that the majority of the assessed strains (91.7%) were capable of producing biofilms. Additionally, Bilgin et al. [27] determined that a significant portion (87.2%) of the 78 evaluated S. maltophilia isolates from pulmonary and extrapulmonary samples formed biofilms, and Sun et al. [28] reported that 42 (82%) of 51 hospital-acquired S. maltophilia isolates also formed biofilms.

In the present study, 15 of the 16 assessed clinical S. maltophilia strains (93%) were found to produce biofilms; furthermore, 11 were determined to have a strong biofilm production capacity, two a moderate capacity, and two a weak capacity. The results of our study are consistent with those of other studies, indicating that S. maltophilia strains exhibit a high capacity for biofilm production. This strong biofilm-forming ability may partly explain the persistence and multidrug resistance of S. maltophilia in clinical environments. Meanwhile, no significant difference was found in our study between the biofilm production capacities of TMP-SMX-resistant and susceptible strains (p $>$ 0.05), indicating that biofilm formation is a common adaptive trait independent of TMP-SMX resistance.

To our knowledge, no previous studies have investigated the antibacterial activity of T. serpyllum, R. officinalis, T. cordata, and S. officinalis extracts against S. maltophilia. However, our study demonstrates significant antibacterial activity by the T. serpyllum methanolic extract. Balkan et al. [29] reported that essential oils from T. serpyllum have shown inhibition zones $>$20 mm with 20 µL applications, while Galovičová et al. [30] reported a 15.67 mm inhibition zone using 10 µL of essential oil. In our study, the methanolic extract at a concentration of 1024 µg/mL produced a larger inhibition zone of 20.5 mm. These results not only demonstrate a dose–response relationship for the essential oil but also suggest that the methanolic extract may be more effective than essential oils. This enhanced activity may be attributed to the synergistic effects of polar compounds (such as phenolic acids and flavonoids) extracted by methanol, emphasizing the importance of the extraction technique in maximizing antimicrobial potential.

A study on methanol and water extracts from T. spicata showed the formation of inhibition zones of 23 mm and 16 mm, respectively [31]. In this study, the inhibition zone of the methanol extract from T. spicata was found to be 12.63 $\pm$ 2.55 mm. This difference may be due to the dose used. Joma et al. [31] used an extract concentration of 50 mg/mL, whereas our study employed an extract concentration of 1 mg/mL. The results show that the diameter of the inhibition zone increases with the dose of the antibiotic. In this study, a dose-dependent increase in the inhibition zone was also demonstrated in measurements performed with concentrations of 512 µg/mL and 1000 µg/mL (p 0.05). These results confirm a dose–response effect, but also suggest the need for standardized extract concentrations in antimicrobial studies to allow comparability across research.

While the minimum inhibitory concentration (MIC) for the ethanol extract from M. piperita against S. maltophilia is reported in the literature as $>$80 mg/mL [32], our study observed inhibition of 17.56 $\pm$ 2.67 mm at a concentration of 1 mg/mL, demonstrating that this extract may be effective even at low concentrations.

Although, to our knowledge, no previous antimicrobial studies have been conducted on T. cordata extracts against S. maltophilia, Ali et al. [11] tested alkaloid, flavonoid, and glycoside extracts on S. aureus and E. coli. Notably, while no antimicrobial activity was observed against E. coli in this study [11], the flavonoid extract showed activity against S. aureus, producing an 18 mm inhibition zone at a high concentration of 3 mg/mL. This low level of antimicrobial activity is consistent with the weak antibacterial effect of T. cordata extracts observed in our study. Furthermore, the significantly reduced impact of T. cordata extracts on resistant S. maltophilia strains in our findings (p 0.05) provides additional evidence of the limited antimicrobial potential of this extract. Therefore, T. cordata extracts may not represent a promising candidate for therapeutic development against S. maltophilia infections.

Zhang et al. [33] reported that a disc impregnated with 50% R. officinalis essential oil produced a 13.83 mm inhibition zone against S. maltophilia. In our study, the methanol extract (primarily containing polar compounds) demonstrated a larger inhibition zone (17.25 mm). This observation suggests that the bioactive compounds in the polar fraction of R. officinalis could exhibit stronger antimicrobial effects compared to the essential oil. Additionally, partial evaporation of volatile components from the essential oil during testing might have contributed to this difference. These findings could be interpreted to indicate that the polar extract of R. officinalis may potentially offer therapeutic advantages over the essential oil against resistant S. maltophilia strains. Moreover, this finding implies that polar extracts may be advantageous for developing stable formulations compared to volatile essential oils.

In our study, we observed an inhibition zone of 16.81 mm against S. maltophilia using a total extract of S. officinalis. Interestingly, this finding is comparable to results reported by Kačániová et al. [34], who demonstrated a 16.47 $\pm$ 0.58 mm inhibition zone using S. officinalis essential oils. Although essential oils generally contain a higher concentration of volatile bioactive compounds, the similar levels of antibacterial activity observed suggest that well-prepared total extracts—despite having a different composition—may achieve comparable efficacy. This supports our earlier observations that the synergistic interaction of both polar and nonpolar compounds in total extracts can significantly contribute to antimicrobial activity. Therefore, total extracts may offer not only a broader spectrum of bioactive components but also advantages in terms of stability and formulation potential. This indicates that both the volatile and non-volatile fractions of S. officinalis contribute toward the observed antimicrobial properties, potentially offering formulation flexibility for therapeutic use.

Pietruczuk-Padzik et al. [35] reported that among all the tested plant extracts against Staphylococcus species, T. serpyllum and Taraxacum officinale leaf extracts exhibited the most promising antibacterial activity, both against planktonic cultures and biofilm forms. Kačániová et al. [34] found that T. serpyllum essential oils inhibited Pseudomonas aeruginosa biofilm at a concentration of 0.236 mg/mL, while Čabarkapa et al. [36] reported that this essential oil prevented biofilm formation by Salmonella enteritidis strains. In our study, the T. serpyllum extract demonstrated biofilm inhibition activity in 66.7% of the S. maltophilia strains. These findings support the potential of T. serpyllum to suppress biofilm formation in various bacterial species.

Wijesundara and Rupasinghe [37] reported that the essential oils from S. officinalis significantly inhibited biofilm formation of Streptococcus pyogenes strains at 0.25 mg/mL and were able to eradicate existing biofilms at 0.5 mg/mL. Ünlü et al. [38] also reported that this essential oil inhibited biofilm formation in 41.2% of the tested methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) isolates, with varying degrees of inhibition. In the present study, S. officinalis extract exhibited biofilm inhibition activity in 40% of S. maltophilia strains. These findings indicate that S. officinalis can exert biofilm-suppressing effects on various bacterial species.

Ben Abdallah et al. [39] reported the potent antibiofilm activity of R. officinalis (rosemary) essential oils against MRSA, and Jardak et al. [40] demonstrated the effectiveness of the same essential oils against Staphylococcus epidermidis biofilms. Galovičová et al. [41] also reported that these essential oils inhibited S. maltophilia biofilms. In our study, the methanolic extract of R. officinalis showed lower inhibition (33.3%) against S. maltophilia biofilms. This difference in efficacy may be due to differences in extract type and concentration ratio.

Su et al. [42] reported that the minimum biofilm eradication concentration was above 1 mg/mL for all the studied strains. Similarly, Río-Chacón et al. [43] reported that the minimum biofilm eradication concentration value was above 5 mg/mL for the majority of strains. In our study, a TMP-SMX concentration of 4 mg/mL inhibited the formation of mature biofilm structures in all tested strains. Our results, when evaluated in conjunction with the literature, indicate that high doses of TMP-SMX are necessary for inhibiting S. maltophilia biofilms. However, systemic application of this concentration in humans is not possible. Therefore, the obtained data may simply guide the evaluation of alternative treatment strategies, such as topical applications, implant coatings, or the development of new drug delivery systems.

According to our results, the T. serpyllum extract exhibited the highest antibacterial and antibiofilm activity with statistical significance (p 0.05). This suggests that T. serpyllum may be a potential antimicrobial agent against S. maltophilia. While M. piperita, R. officinalis, and S. officinalis extracts showed similar levels of antibacterial activity (p $>$ 0.05), T. cordata and T. spicata extracts demonstrated lower activity (p 0.05). The fact that T. serpyllum was the most effective extract in terms of both antibacterial and antibiofilm activities suggests a possible relationship between these effects. Therefore, these findings indicate that T. serpyllum has the potential to be evaluated as a natural antimicrobial and antibiofilm agent.

In the analyses conducted by Sonmezdag et al. [44] using the gas chromatography–mass spectrometry–olfactometry (GC–MS–olfactometry) and liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) techniques, 24 volatile compounds were identified in the aroma profile of T. serpyllum, most of which were classified as terpenes. Phenolic compound analysis revealed 18 different phenolics, with luteolin-7-O-glucoside, luteolin, and rosmarinic acid being the most prominent.

Recent metabolomic studies using ultra-high-performance liquid chromatography–electrospray ionization–quadrupole-time-of-flight mass spectrometry (UHPLC–ESI–QTOF-MS) have elucidated the distinct yet overlapping phytochemical profiles of R. officinalis and S. officinalis [45, 46]. These analyses revealed that rosemary is particularly rich in bioactive diterpenes, with Soxhlet extraction yielding high concentrations of carnosol (22,000.67 $\pm$ 77.39 µg/g) and carnosic acid (2915.40 $\pm$ 33.23 µg/g), along with notable levels of luteolin-7-O-glucoside (209.95 $\pm$ 8.78 µg/g). In contrast, sage demonstrated higher accumulations of rosmarinic acid (17,678.7 $\pm$ 673.4 µg/g) and 12-methoxy carnosic acid (21,918.3 $\pm$ 715.4 µg/g), while still containing detectable quantities of luteolin derivatives. Previous studies have reported that carnosol, carnosic acid, and luteolin compounds, which are biocomponents of R. officinalis, T. serpyllmus, and S. officinalis species, exhibited strong antimicrobial and antibiofilm activities, and that the mechanism of action of these compounds occurred on the cell membrane [47, 48, 49]. In line with these findings, our study identified an active transport mechanism located in the cell membrane, which maintains membrane integrity through lipid transport, as a potential target for improving the understanding of the possible antimicrobial mechanism of action. In this context, a molecular docking analysis was performed to evaluate the interaction of the selected compounds through this mechanism.

The Mla ABC transport system, which is known to transport phospholipids across the periplasm in Gram-negative bacteria and plays a role in maintaining outer membrane homeostasis, is also found in S. maltophilia. The Mla system plays a role in basic resistance to antimicrobial and antibiofilm agents. The periplasmic substrate-binding protein MlaC is an important component in this system and has been shown to bind to phospholipids, thereby contributing to basic resistance to various antimicrobial agents [50]. In this study, the E. coli phospholipid-binding protein MlaC (PDB ID: 5UWA) was selected as a model receptor in the docking analysis. When we examined the molecular docking results, strong hydrogen bond interactions were observed with MET31 in the amino acid residues of the carnossic acid molecule and the MlaC receptor-binding motif (RBM), while van der Waals interactions were observed with the THR38, VAL135, ILE119, ASP32, LEU30, TYR67, ALA163, and VAL68 residues. In addition, a Pi-Sigma bond interaction was observed with LEU64, while alkyl and Pi–alkyl bond interactions were determined with ALA34, LEU109, LEU148, ALA35, PHE39, ILE137, TRP152, and PHE150. When the luteolin and MlaC docking interaction was examined, hydrogen bonds were observed for the TYR164, ASP165, and MET166 residues, and van der Waals interactions were observed for the ILE174, LEU101, LEU65, VAL60, PHE150, and MET173 residues. The MET166 residue also formed a second interaction: a Pi–sulfur bond. While the TYR105 residue showed a Pi–Pi interaction, the LEU65, ALA73, and VAL68 residues exhibited Pi–alkyl interactions.

In the carnosol–MlaC docking interaction, van der Waals interactions were observed for the MET166, ASP165, MET173, TYR164, GLN69, VAL70, PHE97, LEU65, VAL60, and TYR67 residues, and both hydrogen bonding and Pi–Pi–T interactions were observed for TYR105. Additionally, carnosol exhibited Pi–Sigma interactions for the VAL68 and ALA163 residues. Moreover, the ILE174, ALA73, LEU101, PHE150, and LEU64 residues showed alkyl and Pi–alkyl interactions.

The binding energy threshold is accepted as –6.0 kcal/mol. In our study, the results were accepted as significant because all ligand–receptor Gibbs free binding energies were more negative than –6.0 kcal/mol. When designing molecular docking analyses, the antimicrobial activity was predicted to occur via the Mla pathway. The Mla system consists of 6 proteins, MlaA, MlaB, MlaC, MlaD, MlaE, and MlaF, which transport phospholipids from the outer leaflet of the outer membrane to the inner membrane. MlaA is a lipoprotein located in the outer membrane and transfers phospholipids to MlaC, which is a periplasmic protein [51]. MlaC transfers phospholipids to the Mla–FEDB complex in the inner membrane, and Mla–FEDB then inserts phospholipids into the inner membrane [52]. In this study, the MlaC protein within the Mla system was specifically targeted owing to the central and unique role MlaC plays in the lipid transport function in the Mla system. MlaC is the sole periplasmic, soluble protein that transports phospholipids between MlaA in the outer membrane and the Mla–FEDB complex in the inner membrane. Since most other Mla components are membrane proteins, these components are structurally less accessible; in contrast, MlaC offers a directly inhibitory target [53]. Previous studies have demonstrated that the functional loss of mlaC increases bacterial susceptibility to several classes of antibiotics. Bernier et al. [54] showed that mlaC mutants in Burkholderia cepacia complex strains exhibited increased sensitivity to tetracyclines, chloramphenicol, macrolides, fluoroquinolones, and rifampin compared to wild-type strains. This highlights the functional relevance of MlaC as a potential target in antimicrobial resistance mechanisms. Moreover, inactivating the Mla system eliminates the lipid asymmetry of the outer membrane and increases the sensitivity to various antimicrobial agents in many Gram-negative bacteria [53]. When these results are evaluated in conjunction with in vitro antimicrobial activity studies, carnosic acid, carnosol, and luteolin compounds, which are abundantly found in T. serpyllum, R. officinalis, and S. officinalis extracts, are highlighted in terms of antimicrobial activity and may be potential active antibacterial substances.

This study has certain limitations, including the relatively small number of clinical isolates and the exclusive use of in vitro assays, which may not fully reflect the complexity of in vivo conditions. Moreover, variations in extract composition related to plant origin, harvest time, and extraction method could affect reproducibility. Despite these limitations, these findings provide preliminary evidence supporting the antibacterial and antibiofilm activity of selected plant extracts against S. maltophilia. Importantly, this work addresses a gap in the literature by, to our knowledge, being the first to evaluate the activity of T. serpyllum, R. officinalis, T. cordata, and S. officinalis extracts against S. maltophilia and by proposing the Mla system as a possible molecular target. Nonetheless, further investigations focusing on the standardization of extract preparation, the exploration of potential synergistic effects with existing antibiotics, and the experimental validation of molecular docking predictions would strengthen the clinical relevance of these results.

This study demonstrated that extracts of T. serpyllum, S. officinalis, and R. officinalis possess measurable antimicrobial and antibiofilm activities against multidrug-resistant S. maltophilia. The most active extract was from T. serpyllum, producing an inhibition zone of 20.5 $\pm$ 2.8 mm at a concentration of 1024 µg/mL and inhibiting biofilm formation in 66.7% of the tested strains. S. officinalis and R. officinalis were also effective, with inhibition zones of 16.8 $\pm$ 2.0 mm and 17.3 $\pm$ 2.4 mm, and observed biofilm inhibition in 40% and 33.3% of the evaluated strains, respectively. Notably, all three extracts retained activity against TMP-SMX-resistant isolates. Meanwhile, molecular docking analyses revealed strong binding of carnosol (–9.23 kcal/mol), carnosic acid (–8.51 kcal/mol), and luteolin (–7.62 kcal/mol) to the MlaC protein, suggesting a possible mechanism via the disruption of the Mla system. These findings provide the first evidence that these plant extracts and their key phytochemicals could serve as promising alternative therapeutic agents or potent adjuvants against challenging S. maltophilia infections.

The datasets used in this study are available from the corresponding author upon reasonable request.

YA and ZAT designed the research study. YA, HA, and IHK performed the research and conducted experiments. YA, HA, and IHK analyzed the data. BNE and FFS contributed to literature review, methodology development, and writing – review & editing. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Approval for this study was obtained from the Istanbul Health Sciences University Umraniye Training and Research Hospital Clinical Research Ethics Committee (approval number: 228473746, date: 02 November 2023). The study was carried out in accordance with the guidelines of the Declaration of Helsinki. Written consent was obtained from the patients or their families/legal guardians.

This research received no external funding.

The authors declare no conflict of interest.