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  • Antibacterial and Antibiofilm Activity of Lactobacillus-Derived Cell-Free Supernatants Against Multidrug-Resistant Pseudomonas aeruginosa: Synergistic Effects With Antibiotics and Suppression of Biofilm-Associated Genes

  • Sheyda Sharabiani,1 Mohammadamir yousefi,2 Shirin Mohammadipour,3 Hamed Abdollahi,4 Seyed Amirhossein Ghasemi,5 Leila Asadpour,6,*
    1. Department of Biology, Rasht Branch, Islamic Azad University, Rasht, Iran.
    2. Department of Surgical Technology, School of Paramedical Sciences, Semnan University of Medical Sciences, Semnan, Iran
    3. Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
    4. Department of Parasitology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Khuzestan, Iran
    5. Department of Biology, Rasht Branch, Islamic Azad University, Rasht, Iran.
    6. Department of Biology, Rasht Branch, Islamic Azad University, Rasht, Iran.


  • Introduction: Multidrug-resistant (MDR) Pseudomonas aeruginosa is a major cause of difficult-to-treat infections, particularly because of its ability to form biofilms and express virulence-associated factors. The development of alternative or adjunctive antimicrobial strategies is therefore increasingly important. This study evaluated the antibacterial, antibiofilm, and antibiotic-synergistic effects of cell-free supernatants (CFSs) derived from probiotic bacteria against MDR P. aeruginosa, with additional assessment of their effects on the biofilm-associated genes algD and pslA.
  • Methods: Among the isolates screened for antimicrobial susceptibility, four MDR P. aeruginosa isolates with biofilm-producing capacity were selected for further investigation. CFSs from Lactobacillus casei, L. acidophilus, L. paracasei, L. plantarum, and Bacillus pumilus were prepared individually and in combination. Antibacterial activity was evaluated using disk diffusion and minimum inhibitory concentration (MIC) assays. Biofilm inhibition was assessed using the crystal violet microtiter plate assay. The synergistic activity of selected CFSs with antibiotics was determined using the fractional inhibitory concentration index (FICI). The effects of sub-MIC concentrations of L. casei and L. acidophilus CFSs, individually and in combination, on the expression of algD and pslA were evaluated by real-time PCR.
  • Results: All tested CFSs exhibited inhibitory activity against the bacterial isolates, with effective activity observed at CFS dilutions of 1:4 to 1:8. The combined CFS formulation demonstrated the strongest antibiofilm activity, producing more than 65% inhibition of biofilm formation. In the antibiotic combination assays, L. acidophilus CFS showed synergistic interactions with amikacin and ciprofloxacin, with FICI values of 0.5, while L. casei CFS also demonstrated synergistic activity with several tested antibiotics, including amikacin (FICI = 0.75) and ciprofloxacin (FICI = 0.5). At the molecular level, treatment with L. casei and L. acidophilus CFSs individually reduced the expression of algD and pslA by more than 45%, whereas their combination produced a greater than 60% reduction in expression compared with the untreated control (p < 0.05).
  • Conclusion: Probiotic-derived CFSs demonstrated substantial antibacterial and antibiofilm activity against MDR P. aeruginosa. Their ability to enhance the activity of selected antibiotics and markedly suppress the expression of biofilm-associated genes suggests that CFS-based formulations may represent a promising adjunctive strategy for addressing antimicrobial resistance and targeting virulence-related mechanisms in MDR P. aeruginosa.
  • Keywords: Pseudomonas aeruginosa; multidrug resistance; cell-free supernatant; biofilm; antibiotic synergy

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