Abstract
Purpose: To evaluate the antimicrobial and antibiofilm activity of A. lakoocha extract against oral pathogens by an in vitro method.Methods: The dried powder of the aqueous extract of A. lakoocha was purchased from a Thai traditional drug store. Representative strains of oral pathogens (Streptococcus mutans ATCC 25175, Streptococcus sobrinus ATCC 33478, Enterococcus faecalis ATCC 19433, Lactobacillus fermentum ATCC 14931, Lactobacillus salivarius ATCC 11741, Aggregatibacter actinomycetemcomitans ATCC 33384, Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC 25611, Prevotella nigrescens ATCC 25261, Fusobacterium nucleatum ATCC 25586 and Tanerella forsythia ATCC 43037) were tested for minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using a microdilution technique, as well as by a time kill assay. Antibiofilm activity was investigated by a 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay.Results: All tested strains were susceptible to A. lakoocha extract with variable degrees of antimicrobial inhibition. The extract was effective against both Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis) and Gram-positive bacteria (Streptococcus mutans, Streptococcus sobrinus), with MIC ranging from 0.10 – 0.39 mg/ml and MBC from 0.10 – 3.12 mg/ml. Killing activity depended on time and concentrations of the extract. The extract acted as a potent antibiofilm agent with dual actions, preventing biofilm formation and also eradicating the existing biofilm.Conclusion: A. lakoocha extract possesses compounds with good antimicrobial properties that may be used for oral infectious diseases caused by certain oral pathogens associated with dental caries and/or periodontal diseases. For the application, A. lakoocha extract may be incorporated in mouthwash or toothpaste.Keywords: Artocarpus lakoocha, Antimicrobial, Biofilm, Dental caries, Periodontal diseases, Time-kill assay
Highlights
Antimicrobial properties have been derived from a wide range of plant extracts
The concentrations of A. lakoocha extract required to inhibit of the ≥ 50 % biofilm formation (MBIC50) of S. mutans and A. actinomycetemcomitans were 0.39 ± 0.11 and 0.10 ± 0.09 mg/ml, and for ≥ 90 % inhibition of biofilm growth (MBIC90) were 3.12 ± 0.02 and 0.39 ± 0.10 mg/ml, respectively (Figure 2A)
Bacterial cells of S. mutans and A. actinomycetemcomitans in control groups showed a regular, smooth surface as shown in Figure 3A and C, respectively. It revealed that the bacterial cells after treatment with A. lakoocha extract lost their original shape showing a distorted, irregular cell wall structure, which was clearly observed in A. actinomycetemcomitans (Figure 3D)
Summary
Antimicrobial properties have been derived from a wide range of plant extracts. Natural products are sources of chemical compounds that can be used as antimicrobial agents. Growing cultures (106 cfu/ml) of each representative strain, Gram positive (S. mutans ATCC 25175) and Gram negative bacteria (A. actinomycetemcomitans ATCC 33384), were added to appropriate medium and exposed to 1×, 2× and 4× the MIC of A. lakoocha extract. 200 μl (106 cfu/ml) of each representative strain, S. mutans ATCC 25175 and A. actinomycetemcomitans ATCC 33384, was inoculated into each well of the flatbottom 96-well microtiter plate and incubated for 24 h in appropriate conditions at 37 °C. At the end-point of the treatment of the biofilms with A. lakoocha extract, the adherent bacteria were washed three times with sterile phosphate buffered saline. A scanning electron microscopy (SEM) was performed to examine the morphology changes of each representative strain in S. mutans ATCC 25175 and A. actinomycetemcomitans ATCC 33384 after treatment with 10 % DMSO or 0.1 mg/ml A. lakoocha extract as the control and treated sample, respectively. Data were expressed as mean and standard deviation (S.D.) by computational analysis from triplicate independent experiments
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