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Phytochemical‑driven antibiofilm effects of Ruellia tuberosa L. against Escherichia coli and Klebsiella pneumoniae

  • Authors:
    • Anuranjini Charackal
    • Arun Baskarapillai
    • Geetha Royapuram Veeraragavan
  • View Affiliations / Copyright

    Affiliations: Department of Medical Microbiology, AKG Co‑Operative Institute of Health Sciences (AKG CIHS), Mavilayi, Kannur, Kerala 670622, India, Department of Biotechnology and Microbiology, Janakiammal Campus, Kannur University, Kannur, Kerala 670002, India, Department of Microbiology, Centre for Infectious Diseases, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University (Deemed to be University), Chennai, Tamil Nadu 600077, India
    Copyright: © Charackal et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY 4.0].
  • Article Number: 42
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    Published online on: March 27, 2026
       https://doi.org/10.3892/wasj.2026.457
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Abstract

Multidrug‑resistant Gram‑negative pathogens, including Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) represent major clinical concerns due to their potent biofilm‑forming ability and associated virulence traits that contribute to persistent infections and antibiotic failure. In response to the growing limitations of conventional antibiotics against biofilm‑associated infections, the present study evaluated the antibiofilm potential of the ethanolic leaf extract of Ruellia tuberosa L. (R. tuberosa L.) using in vitro assays and gas chromatography‑mass spectrometry‑guided molecular docking analysis. The extract demonstrated antibacterial activity at the minimum inhibitory concentration (MIC) level and effectively inhibited biofilm formation at sub‑MIC concentrations, achieving 55% inhibition in E. coli at 10 mg/ml and 67.81% inhibition in K. pneumoniae at 20 mg/ml, without significantly affecting planktonic growth. Phytochemical profiling revealed a diverse compound composition, with diethyl phthalate identified as the major constituent. Molecular docking demonstrated stable interactions of this compound with key biofilm‑ and virulence‑associated targets in both pathogens, suggesting interference with bacterial adhesion, biofilm development, and persistence. Overall, these findings highlight R. tuberosa L. as a promising natural source of antibiofilm agents capable of attenuating biofilm formation in multidrug‑resistan Gram‑negative pathogens. These plant‑derived compound may serve as adjunct strategies for managing biofilm‑associated infections caused by E. coli and K. pneumoniae, warranting further in vivo and molecular validation.
View Figures

Figure 1

Preparation of Ruellia
tuberosa L. ethanolic leaf extract. (A) Shade-dried powdered
leaves. (B) Maceration in ethanol (4 days). (C) Filtration of the
extract.

Figure 2

Identification of bacterial isolates
on MacConkey agar. (A) E. coli exhibiting lactose-fermenting
pink colonies. (B) K.pneumoniae exhibiting large mucoid
lactose-fermenting colonies.

Figure 3

Agar well diffusion assay showing
antimicrobial activity of Ruellia tuberosa L. extract
against (A) E. coli and (B) K. pneumoniae, with
inhibition zones of 14 and 13 mm, respectively, along with the
negative control.

Figure 4

Inhibition of biofilm formation by
R. tuberosa L. extract. (A) Representative images of
CV-stained biofilms of E. coli and K. pneumoniae at
different concentrations, including control and sterility control
Biofilm inhibition in (B) E. coli and (C) K.
pneumoniae was quantified using the CV staining assay
(OD520), with bacterial growth measured at
OD600. The R. tuberosa L. extract significantly
reduced biofilm formation by 55% in E. coli at 10 mg/ml and
by 67.81% in K. pneumoniae at 20 mg/ml compared to the
control. Data are presented as the mean ± SD. *P<0.05
and **P<0.01. R. tuberosa L, Ruellia
tuberosa L.; E. coli, Escherichia coli; K. pneumoniae,
Klebsiella pneumoniae.

Figure 5

Growth kinetics of E. coli and
K. pneumoniae in the presence of R. tuberosa L.
extract. Growth curves of (A) E. coli and (B) K.
pneumoniae were monitored at sub-MIC concentrations of R.
tuberosa L. extract (10 and 20 mg/ml, respectively). Data are
presented as the mean ± SD. R. tuberosa L, Ruellia
tuberosa L.; E. coli, Escherichia coli; K. pneumoniae,
Klebsiella pneumoniae.

Figure 6

Gas chromatography-mass spectrometry
chromatogram of Ruellia tuberosa L. Spectral peaks were
analyzed using the WILEY7 library, and the identified compounds are
summarized in Table III. Peak
10, detected at a retention time of 14.98 min, exhibited the
highest relative abundance.

Figure 7

Molecular interaction analysis of DEP
with E. coli target proteins. (A and C) 3D binding poses of
DEP within the active sites of 8ENQ and 5DFK proteins,
respectively, shown as electrostatic surface representations; (B
and D) 2D interaction maps illustrating key stabilizing
interactions. In the 2D diagrams, dark pink dashed lines indicate
π-π stacking interactions, light pink dashed lines represent
π-alkyl interactions, and pink lines denote alkyl interactions. DEP
binding to 8ENQ is characterized by π-π stacking with TRP A:106 and
alkyl/π-alkyl interactions with LEU A:59 and TRP A:106.
Interactions with 5DFK involve π-alkyl contacts with PRO A:41, ILE
A:120, and TYR A:169.

Figure 8

Molecular docking and interaction
analysis of K. pneumoniae proteins. (A and C) 3D binding
conformations of DEP within the active sites of 8FFK and 9HW9,
shown as surface representations; (B and D) 2D interaction diagrams
highlighting key stabilizing interactions. In the 2D maps, green
dashed lines represent conventional hydrogen bonds, light green
dashed lines indicate carbon-hydrogen bonds, pink lines denote
alkyl interactions, light pink dashed lines indicate π-alkyl
interactions, and dark pink dashed lines represent π-π stacking
interactions. DEP binding to 8FFK is characterized by π-π stacking
with PHE A:616 and alkyl/π-alkyl interactions involving LEU A:572,
PHE A:665, ILE A:625, and MET A:574. By contrast, DEP interaction
with 9HW9 involves three conventional hydrogen bonds with GLN
A:107, SER A:154, and GLN A:104, a carbon-hydrogen bond with ASN
A:105, and additional alkyl/π-alkyl interactions with LEU A:84 and
TYR A:94.
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Copy and paste a formatted citation
Spandidos Publications style
Charackal A, Baskarapillai A and Royapuram Veeraragavan G: Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>. World Acad Sci J 8: 42, 2026.
APA
Charackal, A., Baskarapillai, A., & Royapuram Veeraragavan, G. (2026). Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>. World Academy of Sciences Journal, 8, 42. https://doi.org/10.3892/wasj.2026.457
MLA
Charackal, A., Baskarapillai, A., Royapuram Veeraragavan, G."Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>". World Academy of Sciences Journal 8.3 (2026): 42.
Chicago
Charackal, A., Baskarapillai, A., Royapuram Veeraragavan, G."Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>". World Academy of Sciences Journal 8, no. 3 (2026): 42. https://doi.org/10.3892/wasj.2026.457
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Spandidos Publications style
Charackal A, Baskarapillai A and Royapuram Veeraragavan G: Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>. World Acad Sci J 8: 42, 2026.
APA
Charackal, A., Baskarapillai, A., & Royapuram Veeraragavan, G. (2026). Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>. World Academy of Sciences Journal, 8, 42. https://doi.org/10.3892/wasj.2026.457
MLA
Charackal, A., Baskarapillai, A., Royapuram Veeraragavan, G."Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>". World Academy of Sciences Journal 8.3 (2026): 42.
Chicago
Charackal, A., Baskarapillai, A., Royapuram Veeraragavan, G."Phytochemical‑driven antibiofilm effects of <em>Ruellia tuberosa </em>L. against <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em>". World Academy of Sciences Journal 8, no. 3 (2026): 42. https://doi.org/10.3892/wasj.2026.457
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