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. 2023 May 15;23(1):131.
doi: 10.1186/s12866-023-02886-x.

In vitro and in vivo antibiofilm activity of the synthetic antimicrobial peptide WLBU2 against multiple drug resistant Pseudomonas aeruginosa strains

Sara Masihzadeh  1   2 , Mansour Amin  1   2 , Zahra Farshadzadeh  3   4
Affiliations

In vitro and in vivo antibiofilm activity of the synthetic antimicrobial peptide WLBU2 against multiple drug resistant Pseudomonas aeruginosa strains

Sara Masihzadeh et al. BMC Microbiol. .

Abstract

Background: The global crisis of antibiotic resistance increases the demand for the novel promising alternative drugs such as antimicrobial peptides (AMPs). Here, the antibiofilm activity of the WLBU2 peptide against Pseudomonas aeruginosa (P. aeruginosa) isolates was investigated in this study.

Methods: Two clinical MDR and carbapenem resistant P. aeruginosa (CRPA) isolates, and standard P. aeruginosa ATCC 27,853 were investigated. The MIC and MBC of WLBU2 were determined. The MBIC was determined to evaluate inhibitory activity of WLBU2 on biofilm formation and MBEC to dispersal activity on preformed biofilm. The relative expression levels of biofilm-associated genes including rhlI, rhlR, lasI and lasR were analyzed using RT-qPCR. In vivo evaluation of inhibitory effect of WLBU2 on biofilm formation was performed in the murine models of P. aeruginosa biofilm-associated subcutaneous catheter infection.

Results: MIC and MBC of WLBU2 for both MDR and ATCC 27,853 P. aeruginosa strains were 8 and 16 µg/mL, respectively, while both the MIC and MBC against the CR strain were 4 µg/mL. MBIC was estimated to be 64 µg/ml for all strains. MBEC against MDR and ATCC 27,853- P. aeruginosa strains was 128 µg/ml and against CRPA was 64 µg/ml. The bacterial adhesion to a static abiotic solid surface (the surface in the polypropylene microtiter wells) was significantly inhibited at 1/4× MIC in all P. aeruginosa strains and at 1/8× MIC in CRPA strain (P < 0.05). Following treatment with WLBU2 at 1/8× MIC, significant inhibition in biofilm formation was observed in all isolates (P < 0.05). Results of the colorimetric assay showed that WLBU2 at 4× MIC was able to disperse 69.7% and 81.3% of pre-formed biofilms on abiotic surface produced by MDR and standard (ATCC 27,853) P. aeruginosa, respectively (P < 0.03), while a 92.2% reduction in the CRPA biofilm was observed after treatment with 4× MIC WLBU2 (P < 0.03). The expression levels of all genes in isolates treated with 1/2 MIC of WLBU2 were down-regulated by more than four-fold compared to the untreated isolates (P < 0.05). WLBU2 significantly inhibited biofilm formation in murine catheter-associated CRPA infection model at 1/4×MIC, 1/2×MIC, and 1×MIC by 33%, 52%, and 67%, respectively.

Conclusion: Considering relatively strong inhibitory and eradication potency of WLBU2 on the P. aeruginosa biofilms in in vitro and in vivo conditions, the peptide can be considered as a promising candidate for designing an antibiofilm drug.

Keywords: Antimicrobial peptides; Biofilm; Pseudomonas aeruginosa; WLBU2.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Time-Kill Kinetics of WLBU2 and Colistin against P. aeruginosa. 1 A: concentration-dependent killing of P. aeruginosa ATCC27853 by WLBU2 and colistin; 1B: concentration-dependent killing of CRPA by WLBU2 and colistin. There was no significant difference in the killing activity between WLBU2 and colistin (P > 0.05). All data represent mean ± standard error of the mean (SEM) of 3 independent experiments
Fig. 2
Fig. 2
The effect of WLBU2 in different concentrations on bacterial attachment inhibition (2a) and biofilm formation inhibition (2b). Control is untreated strain and represent 100% biofilm formation. Data are mean ± SD. Statistical analyses were carried out by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test. Experiments with P < 0.05 were considered significant. P < 0.05 (∗), P < 0.01 (∗∗), P < 0.001 (∗∗∗), P < 0.0001 (∗∗∗∗), P > 0.05 non-significant compared to untreated positive controls
Fig. 3
Fig. 3
The effect of WLBU2 in different concentrations on dispersion of biofilm structure. MDR: Multidrug Resistant; CRPA: Carbapenem Resistant P. aeruginosa; SS: Standard Strain (ATCC 27,853)
Fig. 4
Fig. 4
The expression levels of genes involved in P. aeruginosa biofilm formation influenced by WLBU2
Fig. 5
Fig. 5
In vivo examination of CRPA biofilm biomass formed on the surface of catheters after treatment by different concentration of WLBU2; control (5a), treated biofilm at 1/4 MIC (b), 1/2MIC (c) and 1 MIC (d)
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