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. 2024 Aug 15:14:1406429.
doi: 10.3389/fcimb.2024.1406429. eCollection 2024.

Effect of anti-biofilm peptide CRAMP-34 on the biofilms of Acinetobacter lwoffii derived from dairy cows

Lin Liu #  1   2 , Hui Li #  1   2   3 , Chengjun Ma  1   2 , Jingjing Liu  1   2 , Yang Zhang  2   4 , Dengfeng Xu  2   4 , Jing Xiong  2   4 , Yuzhang He  1   3 , Hongzao Yang  1   2   5 , Hongwei Chen  1   2   3   5
Affiliations

Effect of anti-biofilm peptide CRAMP-34 on the biofilms of Acinetobacter lwoffii derived from dairy cows

Lin Liu et al. Front Cell Infect Microbiol. .

Abstract

Dairy mastitis is one of the most common diseases in dairy farming, and the formation of pathogenic bacteria biofilms may be an important reason why traditional antibiotic therapy fails to resolve some cases of dairy mastitis. We isolated and identified three strains of A. lwoffii were with strong biofilm forming ability from dairy cow mastitis samples from Chongqing dairy farms in China. In order to investigate the effect of novel anti-biofilm peptide CRAMP-34 on A.lwoffii biofilms, the anti-biofilm effect was evaluated by crystal violet staining, biofilms viable bacteria counting and confocal laser scanning microscopy (CLSM). In addition, transcriptome sequencing analysis, qRT-PCR and phenotypic verification were used to explore the mechanism of its action. The results showed that CRAMP-34 had a dose-dependent eradicating effect on A. lwoffii biofilms. Transcriptome sequencing analysis showed that 36 differentially expressed genes (11 up-regulated and 25 down-regulated) were detected after the intervention with the sub-inhibitory concentration of CRAMP-34. These differentially expressed genes may be related to enzyme synthesis, fimbriae, iron uptake system, capsular polysaccharide and other virulence factors through the functional analysis of differential genes. The results of subsequent bacterial motility and adhesion tests showed that the motility of A.lwoffii were enhanced after the intervention of CRAMP-34, but there was no significant change in adhesion. It was speculated that CRAMP-34 may promote the dispersion of biofilm bacteria by enhancing the motility of biofilm bacteria, thereby achieving the effect of eradicating biofilms. Therefore, these results, along with our other previous findings, suggest that CRAMP-34 holds promise as a new biofilm eradicator and deserves further research and development.

Keywords: Acinetobacter lwoffii; anti-biofilm peptide; biofilms; dairy mastitis; transcriptomic.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Analysis of antimicrobial resistance of A. lwoffii biofilm bacteria. 0, 1/4 MIC, 1/2 MIC, 1 MIC, 2 MIC, 4 MIC, 8 MIC (MIC, minimal inhibitory concentration), respectively, Ampicillin, cefquinime, tetracycline, amikacin, ciprofloxacin, kanamycin, and gentamicin at 16 MIC, 32 MIC, 64 MIC, 128 MIC, and 256 MIC were used to measure OD600nm in A. lwoffii biofilms, and OD600nm were used to quantify the biofilms using CV-0.04%. The trial was replicated three times independently. Student’s t-tests were used to calculate the statistical significance. P < 0.05 was considered statistically significant.
Figure 2
Figure 2
Screening results of CRAMP-34 with optimal action time. 0 μg/mL, 31.25 μg/mL, 62.5 μg/mL, 125 μg/were used at 1 h, 2 h, 3 h, 4 h, and 5 h, respectively. mL and 250 μg/mL of CRAMP-34 were treated with A. lwoffii biofilm and quantified using 0.04% crystal violet stainin (CV-0.04%). The trial was replicated 3 times independently. Student’s t-tests were used to calculate the statistical significance. P < 0.05 was considered statistically significant.
Figure 3
Figure 3
CRAMP-3 inhibits the biofilm activity of A. lwoffii. (A) Crystal violet staining (CV) was used to determine the biofilm of A. lwoffii treated with different concentrations (1/2 MIC-8MIC) of CRAMP-34, ciprofloxacin and LL-37. (B) Plate colony count (CC) was used to determine the number of viable biofilms of A. lwoffii treated with different concentrations (1/2 MIC-8 MIC) of CRAMP-34, ciprofloxacin, and LL-37. The test was repeated 3 times independently. Student’s t-tests were used to calculate the statistical significance. P < 0.05 was considered statistically significant.
Figure 4
Figure 4
CLSM imaging of A. lwoffii before and after CRAMP-34 treatment. (A, B) 20× confocal laser scanning microscopy (CLSM) in three-dimensional image (3D) and orthogonal plots of the control group. (C, D) 20×CLSM 3D and orthogonal plots of CRAMP-34 treatment group at 2 MIC concentrations. (E, F) 3D and orthogonal plots of CRAMP-34 treatment group at 1/2 MIC concentrations of 20× CLSM. (G) Number of A. lwoffii biofilms treated with CRAMP-34 at 2 MIC and 1/2 MIC concentrations. (H) A. lwoffii biofilm volume after CRAMP-34 treatment with 2 MIC and 1/2 MIC concentrations. (I) Basal area of A. lwoffii biofilm after CRAMP-34 treatment with 2 MIC and 1/2 MIC concentrations. (J) Fluorescence intensity of A. lwoffii biofilm after treatment with cramp-34 at 2 mic and 1/2 mic concentrations. (K) Area ratio of A. lwoffii bioenvelope after CRAMP-34 treatment with 2 MIC and 1/2 MIC. The images were taken from three random shooting fields in three independent replicate experiments, and the fluorescence intensity, biofilm volume, and biofilm substrate area were analyzed and processed by the open-source software Biofilm Q. Statistical analysis was performed using Prism 8.0 software. One-way ANOVA and LSD multiple test were used to calculate the statistical significance.
Figure 5
Figure 5
Differential genes in A. lwoffii biofilm before and after CRAMP-34 treatment. (A) Volcanic diagram of differential genes between A. lwoffii biofilm control group and plankton group. (B) Differential genes in A lwoffii biofilm before and after 1/2 MIC CRAMP-34 treatment. The red dots indicate genes whose expression is upregulated, the green dots indicate genes whose expression is downregulated, and the blue dots indicate genes whose expression has not changed significantly. (C, D) Venn diagram of association analysis of differentially expressed genes between groups.
Figure 6
Figure 6
Validation of qRT-PCR and related phenotypic. (A) Validation of differentially expressed genes by qRT-PCR to demonstrate the reliability of transcriptome sequencing. Experiments were repeated at least 3 times independently. (B) Changes in the number of upper layer bacteria in culture system after the intervention of CRAMP-34 (1/4 MIC-4 MIC) on biofilm. (C) The change of movement diameter of biofilm bacteria after treatment with CRAMP-34. (D) Number of adhesion of A lwoffii on 96-well cell culture plates during the same period of time before and after CRAMP-34 treatment. The trial was replicated three times independently. Student’s t-tests were used to calculate the statistical significance. P < 0.05 was considered statistically significant.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by Chongqing Technical Innovation and Application Development Special General Project (CSTB2023TIAD-LDX0006), National Center of Technology Innovation for Pigs (NCTIP-XD/B12, NCTIP-XD/C17), The Project of Shandong Province on the Transformation of Scientific and Technological Achievements (2022LYXZ030), Science and Technology Plan Project of Yunnan Provincial Department of Science and Technology (202403AC100013) and Chongqing Natural Science Foundation (CSTB2024NSCQ-MSX0373).

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