Inhibitory activity of clove oil and cinnamic acid on Klebsiella pneumonia biofilm

Main Article Content

Aseel Qassim Hussein
Safaa A.L. Al-Meani

Keywords

Klebsiella pneumonia, MTT, FBLC, FIC, CFU

Abstract

New approaches must be developed to treat illnesses brought on by drug-resistant bacteria because of the rise in antibiotic-resistant bacteria and the insufficiency of new antibiotics on the market. Combining current antibiotics with phytochemicals (PCs) may be a method to boost antibiotic efficacy. Lab studies have linked essential oils (EOs) and their constituents to a set of phytochemicals
that have these effects. As an illustration, we chose cinnamic acid (CAc) and clove oil (COs) as phytochemicals that can work in concert with antibiotics (cefepime) to combat an isolate of Klebsiella pneumonia. In the present study, initial evaluation of the biofilm forming abilities, haem agglutination, yeast agglutination & string test of 84 isolates of K. pneumoniae were performed. The strongest
biofilm forming & positive virulence factors isolates was selected for further experimentation. The COs and CAc's impact on early cell attachment was dosage dependent, according to the crystal violet (CV) assay (p less than 0.05). The inhibition of biofilm formation was highest at 4 MIC of clove oil then 0.25 MIC of CAc. When the CAc & COs were tested against a preformed biofilm, the percentage inhibition was increased with increasing with incubation time. As determined by the CV assay.
The methyl-thiazolyl blue tetrazolium bromide (MTT) assay results showed that the COs considerably decreased the metabolic activity of K. pneumonia biofilms at 4 MIC (P˂ 0.05). The addition of COS and CAC to prevent initial cell attachment lowered both the biomass of the cells (as shown by the CV assay) and their metabolic activity, resulting in 95.03% inhibitor of COs and 93.5% inhibitor of CAc.
The K. pneumonia was grown on Foley balloon latex catheter (FBLC) and then calculated sessile cells. The number of cells shows statistically significant differences between adherent cells and free cells (P˂0.05), and the median of growth for sessile cells treated with CAc & COs were reduced to almost half compared to untreated bacterial biofilm.
By using sonicating water bath (SWB), Foley catheter was put into SWB to dislodge the sessile cells in the solution to made suspension of bacteria, with measuring colony-forming unit (CFU), the suitable time was 6 min among (6, 10 & 15) min.
Synergistic interaction by combination between cefepime and phytochemicals (Cos & CAc), in cefipime-resistant strains of K. pneumonia, synergistic effects measured as metabolic activity decrease and restoration of cefipime sensitivity. AMN3 “Human murine mammary adenocarcinoma” cell attachment phenotypes phenotypes appeared by K. pneumonia strains. The results showed the dissolution of the biofilm adhesions when treated with phytochemicals compared to the control.
Finally, based on K. pneumoniae's reported genome, genes (magA, Aerobactin, mrkJ, mrkA, AcrAB, blaKPC, blaCTX-M and entB) were found. To further confirm the inhibitory effects of the cinnamic acid and clove oil on the K. pneumoniae, which is strong biofilm-producing and their biofilmassociated genes (mrkA and mrkJ) were determined by quantitative RT- PCR. This study showed that
the down-regulation of type III fimbria (mrkA) biosynthesis gene and Phosphodiesterase (mrkJ ) responsible for biofilm development after treatment with cinnamic acid and clove oil.

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