JKMU. Antimicrobial Activity of Combined Extracts of Trachyspermum, Thymus and Pistachio against Some Pathogenic Bacteria ARTICLE INFO.

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1 JKMU Journal of Kerman University of Medical Sciences, 2018; 25 (2): Antimicrobial Activity of Combined Extracts of Trachyspermum, Thymus and Pistachio against Some Pathogenic Bacteria Fatemeh Masoumipour, M.Sc. 1, Mehdi Hassanshahian, Ph.D. 2, Tayebeh Jafarinasab, 1 1- MSc of Microbiology,Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran 2- Associate Professor of Microbiology, Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran (Corresponding author;; mshahi@uk.ac.ir) Received: 3 November, 2017 Accepted: 8 June, 2018 ARTICLE INFO Original article Antibiotic resistant Antibacterial activity Biofilm Medicinal plants Background: Microbial biofilms are responsible for many human infections and increase of antibiotic resistant bacteria. Therefore, finding an efficient way to prevent infection and biofilm formation of bacteria is essential. Medicinal plants are among suitable candidates to inhibit biofilm formation of bacteria. The aim of this study was to evaluate the antimicrobial effects of Trachyspermum, Thymus and Pistachio (T.T.P) combined s (methanolic and ethanolic) against six pathogenic bacteria in planktonic and biofilm forms. Methods: The antibacterial activities of T.T.P combined s against planktonic form of pathogenic bacteria were evaluated by disc diffusion and macro-broth dilution methods. Also, antibiofilm activity of the combined s was evaluated by microtiter plate method. Results: The results of disc diffusion analysis (MBC and MIC) showed that both methanolic and ethanolic s of T.T.P combined s were efficient for inhibition of planktonic forms of bacteria, although ethanol was more effective compared to the methanolic. The T.T.P combined s had inhibitory effect against biofilm formation of pathogenic bacteria. Also, these s efficiently demolished biofilm formation and prevent metabolic activity of bacteria in biofilm structures. The concentration of each had a direct relationship with its inhibitory effect. The maximum inhibitory effects of T.T.P combined s on biofilm formation, demolish of biofilm structure and inhibition of the metabolic activity of bacteria in the biofilm were 95.3% for P. aeruginosa, 97.2% for P. aeruginosa and 81.16% for E. coli. Conclusion: The results of this research demonstrated that T.T.P combined s can be used as an alternative component with inhibitory effect against pathogenic bacteria in planktonic and biofilm forms. Introduction 153

2 Trachyspermum, Thymus and Pistachio against Pathogenic Bacteria Masoumipour, et al Materials & Methods Plant collection, identification and preparation 154

3 Journal of Kerman University of Medical Sciences 2018, Vol. 25, Issue 2 MIC and MBC determinations Bacteria and culture conditions Disc diffusion assay Inhibition of biofilm formation 155

4 Trachyspermum, Thymus and Pistachio against Pathogenic Bacteria Masoumipour, et al Statistical analysis Inhibition of constructed biofilm Results The inhibitory effects of T.T.P combined s against planktonic forms of bacteria Assessment of biofilm metabolic activity 156

5 % Biofilm formation inhibiti Journal of Kerman University of Medical Sciences 2018, Vol. 25, Issue 2 Table 1. Antibacterial activity, minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of T.T.P combined s Bacteria Disk Diffusion Zone of inhibition (mm) Ethanolic Methanolic (mg/ml) (mg/ml) against pathogenic bacteria. Methanolic (mg/ml) MIC Ethanolic (mg/ml) Methanolic (mg/ml) MBC Ethanolic (mg/ml) S. aureus 16± ± ± ± ±0.65 B. cereus 7±0.87 8± ± ± ±1.12 P. aeruginosa 15± ± ± ± ±1.02 A. baumannii 11± ± ± ± ±0.46 E. coli 21± ± ± ± ±1.04 K. pneumoniae 6±0.89 6± ± ± ±0.23 The inhibitory effects of T.T.P combined s against Biofilm formation S.aureus B.cereus A.baumannii P.aeruginosa E.coli K.pneumonia 6.25 mg/ml ethanol 12.5 mg/ml ethanol 25 mg/ml ethanol 6.25 mg/ml methanol 12.5 mg/ml methanol 25 mg/ml methanol Figure 1. The efficiency of T.T.P combined to inhibit biofilm formation of bacteria at different concentrations The inhibitory effects of T.T.P combined s for destruction of biofilm structures 157

6 OD 490 nm OD 490 nm % Biofilm Destruction Trachyspermum, Thymus and Pistachio against Pathogenic Bacteria Masoumipour, et al S.aureus B.cereus A.baumannii P.aeruginosa E.coli K.pneumonia 6.25 mg/ml ethanol 12.5 mg/ml ethanol 25 mg/ml ethanol 6.25 mg/ml methanol 12.5 mg/ml methanol 25 mg/ml methanol Figure 2. The efficiency of T.T.P combined in destruction of pathogenic bacteria biofilm at different concentrations The inhibitory effects of T.T.P combined s against biofilm metabolic activity A S.aureus B.cereus A.baumannii P.aeruginosa E.coli K.pneumonia control 6.25 ethanolic 12.5 ethanolic 25 ethanolic 2.5 B S.aureus B.cereus A.baumannii P.aeruginosa E.coli K.pneumonia control 6.25 methanolic 12.5 methanolic 25 methanolic Figure 3. The inhibitory effect of different concentrations of methanolic (mg ml -1 ) (A) and ethanolic (mg ml -1 ) (B) of T.P.P combined on dehydrogenase enzyme activity of bacterial biofilm 158

7 Journal of Kerman University of Medical Sciences 2018, Vol. 25, Issue 2 Discussion 159

8 Trachyspermum, Thymus and Pistachio against Pathogenic Bacteria Masoumipour, et al Acknowledgments The authors of this article appreciate the Vice-Chancellor for Research of the Shahid Bahonar University of Kerman for supporting this project. References 160

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10 Trachyspermum, Thymus and Pistachio against Pathogenic Bacteria Masoumipour, et al 162

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