Different chemicals used in this study, including acetic acid (CH₃COOH), ethanol (C₂H₅OH), hexane (C₆H₁₄), chloroform (CHCl₃), ethyl acetate (C₄H₈O₂), and toluene (C₇H₈), were purchased from Thermo Fisher Scientific, Waltham, USA. Crystal violet, propidium iodide, ethidium bromide, glutaraldehyde, phosphate buffer saline (PBS) were purchased from Sigma-Aldrich, St. Louis, USA. Methanol (CH₃OH) and hydrochloric acid (HCl), purchased from Sigma-Aldrich, St. Louis, USA, and Milli-Q H₂O were mixed together with the intended ratio for extraction procedure. Luria–Bertani (LB) broth and Mueller–Hinton (MH) broth (Sigma-Aldrich, St. Louis, USA) were used to culture bacteria in liquid media. LB and MH agar (Sigma-Aldrich, St. Louis, USA) were used to culture bacteria on solid media. Tryptone soy agar (Oxoid, Basingstoke, Hampshire, United Kingdom) was used to determine twitching motility of bacteria. Peptone, tryptone, and NaCl were purchased from Sigma-Aldrich, St. Louis, USA, while yeast extract was purchased from Difco, USA, and used to prepare the culture media to validate the bacterial motility.

Environmental isolate K. pneumoniae KPM9 and clinical isolate K. pneumoniae KPi1627 strains were obtained from the State Collection of Pathogenic Microorganisms and Cell Cultures (SCPM, Obolensk, Russia). K. pneumoniae ATCC BAA-2473 laboratory strain was purchased from ATCC (American Type Culture Collection, United States). All tested bacteria strains were identified according to Lev et al. (2018). K. pneumoniae KPi1627 strain was isolated from a clinical sample (trachea) at Moscow Infectious Hospital No. 1 in 2014, while K. pneumoniae KPM9 strain was isolated from the environment (fresh-water) in the Krasnodar Region of Russia in 2011 (Lev et al., 2018) and collected in the Burdenko Neurosurgery Institution.

The identification and detection of bacterial strains were confirmed using a Vitek-2 Compact instrument with a VITEK^® 2 Gram-negative (GN) ID card (SKU number 21341; BioMérieux, Paris, France) and a MALDI-TOF Biotyper (Bruker Daltonics, Bremen, Germany) instrument, which is capable of distinguishing among Klebsiella oxytoca, K. pneumoniae subsp. ozaenae, K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. rhinoscleromatis, and K. variicola. After that, identified K. pneumoniae strains were stored in 15% glycerol and kept at −80°C. A single colony from each strain was inoculated in 10 ml of the LB broth and incubated overnight at 37°C by shaking at 210 rpm/min. The overnight culture was adjusted to half of the McFarland standard (1 × 10⁸ CFU/ml) to be used in biofilms assays under static conditions.

Acidic water–methanol extract (AWME3) was isolated from live Black Soldier Fly (H. illucens) larvae, 15 days old, brownish color, wheat fed, and provided by the NordTechSad, LLC company (Arkhangelsk, Russia). The H. illucens larvae fat was extracted according to Mohamed et al. (2022). Briefly, 3 g of larvae fat was subjected to sequential extraction using water (Milli Q quality), methanol (99.9%, HPLC grade), and hydrochloric acid (37%) with a ratio of 90:9:1, v/v/v. AWME3 was selected for our experiments in this study because of its highest activity among other extracts against Aeromonas sp (Mohamed et al., 2021).

The impact of AWME3 on the alteration of the membrane permeability of all K. pneumoniae strains was determined using crystal violet (CV) uptake assay, in which stain is passed through the cell membrane (Hobby et al., 2019). After incubation in LB medium at 37°C overnight, K. pneumoniae strains were harvested and washed with PBS, pH 7.4, three times. The pellets were resuspended in PBS and mixed with AWME3 at a concentration from ¼ to 2 MIC for 4 h. Bacterial cells were incubated with 1% CV in the dark for 15 min. After centrifugation, the absorbance of the supernatant was determined by measuring the OD₅₇₀ nm using CLARIOstar^® Plus multimodal plate reader (BMG Labtech, Ortenberg, Germany). The absorbance of CV was considered as 100%. The crystal violet uptake was calculated using the following formula: % of uptake = (OD of the sample)/(OD of the crystal violet solution) × 100.

The MIBC assay was conducted via the microdilution assay described by Cepas et al. (2019) with some modifications. For all K. pneumoniae strains, 100 µl of twofold dilutions of AWME3 in LB broth media with 1-, 0.5-, 0.25-, 0.125-, 0.063-, 0.032-, 0.016-, and 0.08-mg/ml concentrations in LB broth was inoculated with 100 µl of bacterial suspension 1 × 10⁶ CFU/ml and incubated for 24 h at 37°C without shaking. A negative control (culture medium without inoculum) and a positive control (culture medium with inoculum) were included in each 96-well plate. All plates were covered with adhesive film to avoid evaporation. After incubation, the unattached cells were carefully removed and washed twice with PBS and dried at 60°C for 20 min. Attached biofilms were stained with 125 µl of 1% (v/v) of CV and incubated for 10 min at room temperature. Afterward, the CV was completely removed, washed with PBS, and dried at 65°C for 60 min. The plates were rinsed with d.H₂O and dried, followed by the addition of 125 µl of 30% acetic acid to dissolve the biofilm-bound dye. Optical density was measured at 570 nm (OD₅₇₀) using a CLARIOstar^® Plus multimodal plate reader (BMG Labtech, Ortenberg, Germany). The MBIC was defined as the lowest concentration of AWME3, which resulted in a threefold decrease in OD₅₇₀, in comparison with the positive growth-control value (only bacteria). Additionally, MIBC of AWME3 and positive control (Dox) was determined in 96-well plates and identified as the lowest concentration, which inhibits biofilm growth of K. pneumoniae strains.

The minimum eradication biofilm concentration (MEBC) was determined according to Balkrishna et al. (2021) with minor changes. The MEBC was determined based on the MIBC test, where different concentrations of AWME3, in the range 0.08–1.0 mg/ml, were inoculated with fixed 1 × 10⁶-CFU/ml concentration of each K. pneumoniae strain in sterile LB broth and incubated at 37°C for 24 h. Subsequently, an aliquot of 30 μl of each of the MIBC, 2 MIBC, and 4 MIBC was scraped and spread on sterile MH agar plates then incubated for 48 h at 37°C. The lowest concentration of AWME3, which prevented bacterial growth, was identified as the MEBC. Likewise, doxycycline was used as a positive control, and MEBC was determined by the same manner.

The biofilm disruption assay was performed in a 96-well polystyrene plate (TPP, Trasadingen Switzerland) following the procedure (Chmielewska et al., 2020; Wijesinghe et al., 2021) with minor modifications. The bacteria were grown in microtiter plates for 72 h at 37°C to form mature biofilms in the wells. Then, media was discarded gently, and wells were washed using PBS buffer to remove loosely adhered cells. Freshly prepared LB broth was added to each well, and then AWME3 was added to give final concentrations of 0.25, 0.500, 1.0, and 2.0 mg/ml. The plate was incubated for the next 24 h under static conditions at 37°C. The wells of the plates were washed by sterile PBS to remove the planktonic cells followed by staining with 0.1% CV solution in water for 30 min. The stain was removed and gently washed with d.H₂O, dried at 60°C for 60 min, and the remaining biofilm-bound dye was dissolved using 30% acetic acid. The OD570 was recorded using a CLARIOstar microplate reader, and the percent of biofilm disruption was calculated with respect to the control group. The MEBC of AWME3 and doxycycline was determined by counting formed colonies (CFU). Briefly, adhered treated biofilms were completely scraped and serially diluted in PBS. Of prepared dilutions, 30 µl was spread on MH agar medium separately, then incubated at 37°C for 48 h; MEBC identified as the lowest concentration of AWME3 or doxycycline, which was able to eliminate the bacterial biofilm growth.

Biofilm architecture, in the absence and presence of AWME3 antimicrobial, was evaluated using the fluorescence microscopy protocol of Sateriale et al. (2020) and described in the Supplementary Methods in light microscopy examination with some changes. After fixing wells with 90% ethanol for 15 min and completely drying at 30°C, the biofilms were stained with 1 mM propidium iodide (PI) for 15 min at room temperature. The excess dye was washed with d.H₂O. Finally, biofilms were observed with a fluorescent microscope (Life technologies, Bothell, WA, USA) equipped with a digital camera. Digital images were acquired using a ×4 objective at PI excitation/emission wavelength of 543/617 nm. All obtained images were analyzed using Fiji Image J software (National Institutes of Health, Bethesda, USA) to obtain the mean fluorescence intensities from digital fluorescent images of biofilms.

Treated and untreated biofilms of K. pneumoniae ATCC BAA-2473 strain were examined using a scanning electron microscope (SEM) according to Ceruso et al. (2020) with minor modifications. Briefly, the K. pneumoniae cells were cultured and grown on 1-cm² cover glass (Thermo Fisher Scientific, Waltham, USA) in a six-well plate (TPP, Switzerland) for 6 h as mentioned above. Next, 0.0-, 0.125-, 0.250-, and 0.500-mg/mL concentrations of AWME3 were added to the formed biofilm in a six-well microtiter plate, then incubated for 24 h at 37°C without shaking. Further, the planktonic cells were removed by washing with PBS, pH7.4, three times. All adhered biofilms on the surface of the glass coverslips were fixed with 2.5% glutaraldehyde, pH 7.2, overnight at 4°C, washed three times in the rinsing buffer PBS at 4°C for 15 min, and then dehydrated by ethanol solutions in following concentrations: 30%, 50%, 70%, 80%, 90%, and 95%. All dehydrated samples were visualized under SEM (TESCAN, Kohoutovice, Czech Republic).

Statistical analysis was conducted, and graphs were generated using the software GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, United States). All experiments were performed in triplicate validating the statistical significance by one-way ANOVA test with Dunnett’s multiple comparison test and two-way ANOVA test with Dunnett’s, Tukey’s, and Sidak’s corrections, and the statistical significance level was p < 0.05.

K. pneumoniae KPi1627 and K. pneumoniae KPM9 strains demonstrated multidrug-resistant phenotypes to more than three different classes of antibiotics (Magiorakos et al., 2012), while K. pneumoniae ATCC BAA-2473 was classified as extensive drug resistant (XDR) (Mohamed et al., 2022). In addition, K. pneumoniae KPi1627 and K. pneumoniae KPM9 displayed high hypermucoviscosity after string assay, while K. pneumoniae ATCC BAA-2473 was negative to the same test.

To enhance the diagnosis, treatment, and prevention of infections, it is crucial to differentiate between acute infections caused by the independent growth of individual microorganisms (the so-called “planktonic” growth) and biofilm infections, which involve clusters of microbial cells (Mirzaei et al., 2024). The minimum inhibitory concentration (MIC) is the lowest concentration of antibiotics that stops visible bacterial growth. The minimum bactericidal concentration (MBC) is the lowest concentration needed to kill the bacteria. Diagnostic laboratories use MICs primarily to confirm the presence of resistance. MIC and MBC are determined based on planktonic cells, while the minimum biofilm eradication concentration (MEBC) indicates the lowest antibiotic concentration needed to eliminate the biofilm.

In a previous study, we determined MIC and MBC of AWME3 extract against planktonic bacterial cells of three K. pneumoniae isolates, including KPi1627, KPM9, and KP ATCC BAA-2473 (Mohamed et al., 2022). The MIC and MBC were recorded as 250 µg/ml under planktonic growth conditions in these experiments. In the present study, we identified the minimum inhibition biofilm concentration (MIBC) and MEBC for the same strains as 500 µg/ml, when they were exposed to static conditions after being adherent to polystyrene microtiter plates with the exceptional resilience ( Table 1 ). Doxycycline (Dox) used as the positive control displayed remarkable MIC values of 6.25, 3.12, and 12.5 µg/ml against K. pneumoniae KPi1627, K. pneumoniae KPM9, and K. pneumoniae ATCC BAA-2473, respectively. At the same time, Dox exhibited MEBC values exceeding 50 µg/ml for all three strains.

These results demonstrate a significant reduction of sensitivity to AWME3 treatment by all hvKp-tested strains when they were grown in biofilm mode compared to that of planktonic cell growth. Moreover, the AWME3 extract demonstrated bactericidal activity, comparable to the standard antibiotic (Dox) against all biofilms formed by K. pneumoniae strains ( Table 1 ).

Biofilms are constructed as bacterial colonies formed in multiple layers on biotic or abiotic material that facilitates bacterial survival and persistence against harmful conditions, as well as contributes to virulence during infection. We performed biofilm formation assay using LB broth medium in a 96-well microtiter plate. The biofilms grown for 24 h were assessed by staining with crystal violet (CV). Crystal violet, an aniline dye, represents the initial stain employed in the process of Gram staining. When cells are exposed to a 95% ethanol or acetone solution, they create a vivid and eye-catching purple color after interacting with the crystal violet pigment. The intensity of CV staining in K. pneumoniae, Gram-negative bacteria, reveals the extent of the thin peptidoglycan layer covered by lipopolysaccharides and lipoproteins, proteins, and DNA, which form a biofilm on the plastic surface.

The intensity of biofilm staining was accurately measured by meticulously dissolving the dye in a 30% acetic acid and water mixture. Our results of the CV dye biofilm staining demonstrate that KPi1627, KPM9, and KP ATCC BAA-2473 strains form strong biofilms ( Figure 1 ). Significant inhibition of biofilm formation (p < 0.0001) by all K. pneumoniae strains was observed at concentrations MIBC (0.5 mg/ml) and 2 MIBC (1.0 mg/ml). KPi1627 and KPM9 biofilms were significantly reduced (p = 0.001) at 0.5 MIBC (0.25 mg/mL) of AWME3 ( Figures 1A, B ). Of note, the KP ATCC B AA-2473 strain biofilm was significantly reduced (p = 0.007) after exposition to 0.125 MIBC (0.0625 mg/ml) of AWME3 ( Figure 1C ).

The tested opportunistic MDR K. pneumoniae pathogens frequently form mixed biofilms that can lead to nosocomial infections in healthy individuals. We wanted to test how well AWME3 works against resistant strains and if it can remove the strong biofilms created by these strains. Mixed biofilm established by equal volumes (1:1:1) of the three different strains was the strongest one (p = 0.003) compared to biofilms formed by each K. pneumoniae strains alone ( Supplementary Figure S1 ). On the other hand, KP ATCC BAA-2437 was the lowest (p = 0.0005) in biofilm formation capacity, compared to mixed biofilm ( Supplementary Figure S1 ). The results depicted in Supplementary Figure S2 provide compelling evidence of the dose-related eradication of mixed biofilms formed by three strains of K. pneumoniae. Notably, when exposed to a concentration of 2 MIBC (1,000 µg/ml) of AWME3, the mixed biofilms consisting of KPi1627, KPM9, and KP ATCC BAA-2473 were completely eradicated (p < 0.0001) (see Supplementary Figure S2 ). Concentrations of 0.25 MIBC (125 µg/ml), 0.5 MIBC (250 µg/ml), MIBC (500 µg/ml), and 2 MIC (1,000 µg/ml) exhibit a remarkable (p < 0.0001) inhibition of the mixed biofilms resulting in 76.3%, 80.01%, 88.2%, and 98.56% reductions, respectively. In addition, our AWME3 extract effectively reduces mixed biofilms to 28.3% and 33.97% at low concentrations of 1/16 MIBC (62.5 µg/ml) and 1/32 MIBC (31.25 µg/ml), respectively, against mixed hvKp strains ( Supplementary Figure S2 ) with significant reductions (p = 0.010, p = 0.038).

Our results have conclusively demonstrated that the AWME3 extract derived from HI larvae fat possesses remarkable antimicrobial properties against both single and mixed biofilms formed by various MDR strains of K. pneumoniae, such as KPi1627, KPM9, and KP ATCC BAA-2473.

Considering the dynamic growth of biofilms and their greater tolerance to antibiotics, we explored in our study the biofilm growth during 72 h first, and then subjected them to antibiotic challenge. We used light, fluorescence, and scanning electron microscopy as direct microscopic methods to gather evident information about the effect of AWME3 on treated biofilms.

Through the light microscopy technique, we investigated the effect of AWME3 at 0.5 MIBC (0.25 mg/ml), MIBC (0.5 mg/ml), and 2 MIBC (1 mg/ml) on mature biofilm formation on glass coverslips using the CV assay. We found that the control group of bacteria without treatments revealed a remarkable sight. A dense and intricately woven mat of biofilms emerged resembling a heavy knit fabric. These biofilms displayed multiple layers, clearly intact, and adorned the uneven surfaces. Additionally, intriguing clusters of cells gave rise to darkened areas adding to the captivating spectacle. The EPS creates a vast, intertwined network that serves as a matrix for connected threads, effectively shielding the biofilm from a variety of hazardous conditions ( Supplementary Figure S3A ). AWME3 exposures to a sub-MIC (0.25 mg/mL) led to a remarkable decrease in cell count, formation of fragile mats, degradation of clusters, and a noticeable absence of cell aggregation ( Supplementary Figure S3B ). The highest concentration of AWME3 at 2 MIBC (1.0 mg/ml) successfully prevented the formation of mature biofilm in K. pneumoniae KP ATCC BAA-2473 leading to the complete absence of cell clusters or aggregates. Furthermore, numerous vacant spaces were observed, and the bacterial cells appeared minimal ( Supplementary Figure S3D ). Notably, there was no bacterial growth after culturing the 30 µl of scraped biofilm on MH agar plates incubated for 24 h, compared to standard antibiotic (Dox), which could not inhibit the mature biofilms at 4 mg/ml (data not shown).

Mature biofilms were also quantified using the CV staining assay in 96-well microplates. The graph in Figure 2 clearly illustrates that at concentrations of 1,000 and 2,000 µg/ml of AWME3, the biofilms created by K. pneumoniae KPi1627, KPM9, and KP ATCC BAA-2473 strains were impressively and significantly disrupted (*p < 0.01–****p < 0.0001).

The mature biofilms formed by the K. pneumoniae ATCC BAA-2473 strain clearly exhibited the highest susceptibility to AWME3, with a biofilm reduction of 63.1% when treated with 0.25 mg/ml ( Figure 2C ).

The propidium iodide (PI) staining method is extensively utilized and endorsed in biofilm research giving valuable information regarding eDNA release and degradation that affects biofilm maturation. PI, which can only traverse compromised bacterial membranes, is regarded as an indicator of the integrity of the membrane. Staining based on intact membrane impermeable DNA-binding stains like PI is occasionally used even while specifically studying eDNA (Mann et al., 2009).

The AWME3 ability to disrupt biofilms formed by the tested K. pneumoniae strains was further investigated using fluorescence microscopy study of biofilms stained with propidium iodide (PI) ( Figure 3 ). The images were obtained using 543/617 nm excitation/emission filter. As shown in control images, tested bacterial strain formed very dense biofilms on glass coverslips. Dense clumps of cells in biofilms were visualized, and bacteria were seen to be heavily colonized and adherent in multiple layers. Exposure to varying concentrations of AWME3 resulted in a significant (p < 0.0001) decrease and scattered bacterial cell presence ( Figures 3B–E ). Upon treating bacterial biofilms with 0.5 MIBC (0.25 mg/ml), the presence of reduced mats, threads, and clumps was observed, with cells displaying dispersion and noticeable gaps between them ( Figure 3B ). AWME3 at a concentration of 0.5 mg/ml (MIBC) significantly decreased the number of cells and the overall bacterial cell count and intensity ( Figure 3C ). The highest impact of AWME3 was clearly observed when the concentration of MIBC was 2 MIBC (1.0 mg/ml) ( Figure 3D ). There was almost 10-fold decrease (p < 0.0001) in fluorescence intensity compared to that of the control group, as shown in Figure 3E . These results demonstrate that AWME3 effectively breaks down the pre-existing deposits of extracellular nucleic acids (eNAs) found in mature K. pneumoniae biofilms, and the level of disruption is proportional to the used AWME3 concentration.

SEM analysis was performed to visually observe the biofilm disruption after AWME3 treatment. Cells appear aggregated and accumulated in multiple layers in untreated biofilms established by K. pneumoniae KP ATCC BAA-2473 ( Figures 4A–C ). Furthermore, no morphological alterations were detected in untreated cells, where cells were smooth with intact cell wall and bacilli ( Figure 4C ).

AWME3 treatment caused a significant decrease in the number of adherent bacteria, compared to that of the control cells. The SEM analysis revealed rough surfaces, with wrinkled cell walls and visible pores in the bacterial cells treated with AWME3, as indicated by the blue arrows ( Figures 4D–F ). Lysed cells, cell wall debris, and ghost cells were obvious when biofilms were treated with 2 MIBC (1.0 mg/ml) of AWME3 (blue arrows and red cycle in Figure 4F ). The biofilm was reduced down to a monolayer of adherent cells, and even single cells were detected ( Figures 4D–F ). These findings suggest that AWME3 is a potent anti-biofilm agent, which can disrupt the mature biofilms.

Thus, three independent microscopy techniques all showed that AWME3 disrupts mature biofilms composed of peptidoglycan layers covered by lipopolysaccharides, lipoproteins, proteins, and eNAs, produced by various MDR hvKp strains on the plastic surface in a dose-related manner.

The hypermucoviscous (HMV) phenotype is one of the key virulence factors of K. pneumoniae. This phenotype is associated with serious infections such as liver abscesses, pneumonia, and bloodstream infections. The HMV phenotype is characterized by its ability to form a thick, sticky biofilm, which contributes to its pathogenicity (Kawai, 2006). Understanding this phenotype is important for developing effective treatment strategies against K. pneumoniae infections.

We conducted an analysis of autoaggregation to test the hypothesis that it plays a role in biofilm formation. Specifically, we investigated how AWME3 affects the autoaggregative behavior of mucoid hypervirulent K. pneumoniae strains. The autoaggregation experiment was conducted at room temperature for 24 h. The results are presented in Supplementary Table S1 and Supplementary Figure S4A . The data indicate that 0.5 MIC of AWME3 (0.125 mg/ml) did not have an effect on the autoaggregation of KPi1627, KPM9, and KP ATCC BAA-2473 strains (p = 0.995, p = 0.971, p = 0.945, respectively) ( Supplementary Figure S4A ). Furthermore, the turbidity of the supernatant from the treated cells, measured after cell centrifugation, did not differ from that of the control group ( Supplementary Figure S4B ). Conversely, the same AWME3 concentration resulted in the formation of loose pellets, which differed from the dense pellets formed by all non-treated K. pneumoniae strains ( Supplementary Figure S4C ).

For better understanding of the impact of AWME3 on the virulence of hvKp strains, we conducted a straightforward string test considering the positive string as strings longer than 5 mm. The results of the KPi1627 and KPM9 strain tests indicated their virulent nature, except for the KP ATCC strain, which tested negative, suggesting a lack of virulence. Of note, the untreated KPi1627 strain showed the highest HMV-phenotype among all the tested bacteria, with a string 51.7 ± 3.5 mm in length. In comparison, the KPM9 and KP ATCC BAA-2473 strains had string lengths of 31 ± 3.63 and 3.81 ± 1 mm, respectively ( Supplementary Table S2 , Supplementary Figure S5 ). It is noteworthy that when all string-positive isolates were exposed to 0.5 MIC (0.125 mg/ml) of AWME3, they became negative in the string test highlighting the high efficacy of AWME3 in combating one of the key virulence factors of K. pneumonia strains with HMV phenotype.

Throughout history, K. pneumoniae has been recognized as a major cause of urinary tract infections (UTIs) further emphasizing its significance beyond its association with pneumonia (Paczosa and Mecsas, 2016). The expression of fimbriae is crucial for the successful colonization of the urinary epithelium by K. pneumoniae. These structures facilitate attachment to urothelial cells and play a pivotal role in promoting bacterial adhesion to abiotic surfaces, like urinary catheters (Stahlhut et al., 2012). Bacterial motility is a critical factor in the successful colonization of both living and non-living surfaces. Remarkably, the hyper fimbriae phenotype appears to grant the mutant strain a form of rudimentary mobility. While K. pneumoniae was previously considered to be non-motile, the discovery of rudimentary (limited) motility in this bacterium is well documented (Carabarin-Lima et al., 2016; Érika et al., 2021) representing another phenotype associated with virulence in K. pneumoniae infections.

Therefore, we conducted an assessment of the sub-MIC (0.125 mg/ml) impact of AWME3 on the three types of rudimentary motility (swimming, swarming, and twitching) of K. pneumoniae isolates. This experiment analysis showed that AWME3 extract has a significant effect on the basic (twitching) mobility of K. pneumoniae strains KPi1627, KPM9, and KP ATCC BAA-2473 ( Supplementary Table S 3 ). In particular, the AWME3 extract significantly reduced the swimming motility of KPM9 and KP ATCC BAA-2473 strains (p = 0.0025, p < 0.0001, respectively).

However, no notable effect on KPi1627 swimming motility was observed at 0.5 MIC (0.125 mg/ml) of AWME3. The swarming motility of MDR KPM9 and KP ATCC BAA-2473 strains was significantly (p = 0.007, p = 0.003) reduced after being exposed to 0.5 MIC (0.125 mg/mL) of AWME3. The sub-MIC (0.125 mg/ml) of AWME3 significantly (p < 0.0001) reduced the twitching motility zone diameters of all K. pneumoniae strains ( Supplementary Figure S6 , Figure 5C ). Furthermore, AWME3 at sub-MIC levels decreased the twitching motility of KPi1627, KPM9, and KP ATCC BAA-2473 bacterial strains by approximately 50% of the zone diameters. The treated bacterial strains exhibited twitching motility in the range of 4.23 ± 0.25 to 4.47 ± 0.25 mm, while the non-treated control groups showed significantly higher motility ranging from 8.5 ± 0.5 to 10.5 ± 0.5 mm ( Supplementary Table S 3 , Supplementary Figure S 6 ; Figure 5C ).