Evaluation of antimicrobial effects of Rosemary and Withania somnifera methanol extract prepared by ultrasound waveform on Escherichia coli biofilm isolated from urinary tract infection

1 Department of Food Science and Technology, Zahedan University of Medical Sciences, Zahedan, Iran

2 Department of Chemistry, University of Sistan and Baluchestan, Zahedan, Iran

3 Department of Surgery, School of Medicine, Amir al momenin Hospital, Zabol University of Medical Sciences, Zabol, Iran

*Corresponding Author: Maryam Beigomi ([email protected])

A B S T R A C T Generally, bacteria co-exist setting up communities associated to solid superficies, this is to which we refer as biofilms that serve as a survival strategy. This type of formation cause serious sanitary problems for both humans and animals. Nowadays, chemical or natural compounds able to block this formation are looked for. In this project, we have evaluation of antimicrobial effects of Rosemary and Withania somnifera extract prepared by ultrasound waveform on Escherichia coli biofilm isolated from urinary tract infection. Rosemary and Withania somnifera extracts were obtained by using rotary devices. E. coli was collected from the patients being cured in Zabol hospitals. The least hindering and killing concentration of the samples were calculated by lowering their densityin sinks. The growth and biofilm formation of strains were determined by microtiterplate method. Data were analyzed statistically by determination of significant difference using analysis of variance (ANOVA) test. All tests were analyzed at the significance level P<0.05. The results revealed that the concentrations of 5 and 10 mg/mL are the most restrain in the biofilm formation of the isolated plates. Rosemary and Withania somnifera extracts have considerable antimicrobial and ant biofilm effect on the samples of the E. coli resistant to antibiotics.

Article info Received: 11 Feb 2021 Revised: 08 Apr 2021 Accepted: 25 May 2021          Keywords: Extract plant, Anti biofilm, ultrasound, Escherichia coli

1. Introduction

The use of herbal compounds for the treatment of infections is an old method in many parts of the world, especially developed countries. Paying attention to medicinal herbs with microbial properties can restore the common problems with the use of antibiotics. Rosemary is a plant that is a perennial lamacae that forms in the lush greenery of all seasons. The characteristics of this plant are a great smell of leaves, which is known as an aromatic herb. Due to its antimicrobial, anti-mutagenic and chemical precursor properties, it has been recognized as a valuable medicinal plant in the pharmaceutical and medical industries [1, 2].

Essential oils of this plant include boronol, limonene, camphon, camphor and other herbal compounds such as phenolic acids, including daily acids, caffeic acid and chlorogenic acid [3].

Withania somnifera is a plant from the Solanaceae family. It grows in Africa, the Mediterranean, and India, and is used as antibacterial, antioxidant, anti-inflammatory and liver diseases [4].

Conventional extraction methods are based on plant placement in a solvent that is used to increase the process speed of mixing or heating. Soxhlet method is a standard method used as the reference for evaluating other methods. This method is general, mainly used to extract low or moderate volatility compounds that are resistant to heat(5). The use of ultrasound in extraction is one of the new methods of extraction that the waves penetrating into the material at a frequency of 20 kHz, causing continuous expansion and contractions in the internal molecules resulting in the formation of cavities. These cavities, asymmetrically interconnected, cause the material to be extruded from the cells out of it [5].

Microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces( [6]. Biofilm-associated cells have the ability to adhere irreversibly on a wide variety of surfaces, including living tissues and indwelling medical devices as catheters, valves, prosthesis, and so forth.

2. Methods

2.1 Extract preparation and investigation of the antimicrobial effects of the plant extract

The wind cheese used in this study was gathered from Sistan and Baluchistan province (fig. 1). They were detected to be wind cheese by a researcher from the University of Zabol. The samples were cut and 10g of the dry plant powder was put into half-liter erlens containing 100ml of methanol. The contents of erlens were shaken 24 hours at room temperature b shaker device with speed of 130 rpm and were kept in ultrasonic carrier for 10 minutes and were filtered with Wattman paper. Separation of solution from extract was done by rotary device and vacuum pump. The obtained extract was weighed and solved in DMSO. The samples were used in fridge at 4 degrees of temperature to be used in antimicrobial tests.

Fig. 1. Characteristic of Rosemary (A) and Withania somnifera (B)

 

2.2 The antimicrobial effect of the extract on E. coli

Sensitivity of the bacteria samples with multiple resistances to the wind cheese extract against E. coli was analyzed by dilution method in broths. To this end, seven broths of microtitre plates were injected 100 ml of MHB. The first broth was added 100ml of the diluted extract. Then, 100ml of the first broth was transferred to the second one and the same was done to the last broth. 100ml of the last broth was removed and 100ml of the microbial suspension with 107 units per ml was added to all broths. The mixture was kept 24 hours at temperature of 37 degrees. The first broth inhibiting the growth of bacteria after being positioned in the incubator was considered as MIC and for more precision; 10 ml of the light broths was transferred to Moller environment. After 24 hours, the first concentration removing 99.9% of the bacteria was regarded as MBC.

2.3 Biofilm formation assay in presence of the biocides:

After performing the procedure described above, the microplate was covered and incubated aerobically for 24 h at suitable temperature. At first, the OD (Optical Density) was measured (600 nm) by using an automated ELISA counter, then, the content of each well of the microplate was aspirated and each well was washed three times with 250 μL of sterile physiological saline. The remaining attached bacteria were fixed with 200 μL of 99% methanol per well and after 15 min all of the wells were emptied and left to dry. Then, each well was stained for 5 minute with 0.2 mL of 2% crystal violet. Excess stain was rinsed off by washing the plate slowly with distilled water. After the plate was air dried, the dye bound to the adherent cells was re-solubilized with 160 mL of 33% (v/v) glacial acetic acid per well. The OD of each well was measured at 492 nm by using an automated ELISA counter.

2.4 Antibiotic activity

Four pure strains of Escherichia coli were determined by Kirby-Bauer antibiogram and their susceptibility to antibiotics was evaluated. Antibiotics used in this study included gentamicin, amoxiclav, azithromycin, and amikacin (manufactured by Antibody Medicine). After 24 hours of incubation at 37 ° C, the diameter of the inhibitory halos was measured and the sensitivity and resistance of the strains were determined and the results were compared with the NCCLS standard table[7].

2.4 Statistical analyses:

The growth was compared at each experiment using analysis of variance (ANOVA) repeated measures (GraphPad 8). The level of statistical significance was set at p< 0.01.

3. Results

The results of this study showed that the highest resistance to ceftazidime, gentamicin, amoxiclav and azithromycin were 75%, 50%, 25% and 25%, respectively. While the highest sensitivity to antibiotics azithromycin, gentamicin and amoxiclav was equal to 50% (Table 1).

The results of this study showed that the lowest inhibitory concentration of cheese extract was 2.5 mg/ml, while the highest inhibitory concentration was 5 mg/ml, the highest lethal concentration was 10 mg. mg/ml, in which two strains were completely eliminated at this concentration, and the lowest lethal concentration was equal to 2.5 mg/ml, in which one strain was eliminated in this concentration.

The lowest and highest inhibitory concentrations of rosemary extract were 5 and 10 mg/ml, in which 3 and 1 strains were inhibited, while the highest lethal concentration was 20 mg/ml, which was one. The strain is eliminated at this concentration (Table 2).

The results of this study showed that by increasing the concentration of the extract, the amount of biofilm formation decreases. As the concentration of the Withania somnifera extract increased, biofilm formation decreased more than rosemary. The results revealed that the concentrations of 5 and 10 mg/mL are the most restrain in the biofilm formation of the isolated plates (fig. 3 and 4).

Table 1. Antibiotic pattern of Escherichia coli strains (%)

Resistance level	Ceftazidime	Amoxiclav	Amikacin	Gentamicin	Azithromycin
S	0	50	25	50	50
I	25	25	75	0	25
R	75	25	0	50	25

S= sensitive; I= Intermediate; R= resistant

 

Table 2. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) (mg/ml)

Withania coagulans		Rosemary		Bacterial Strains
MIC	MBC	MIC	MBC
5	10	5	10	1
5	5	5	10	2
2.5	2.5	5	5	3
5	10	10	20	4

 

Fig. 2. The effects of different concentrations of extract plant of Withania somnifera on the biofilm formation of bacterial and control

 

Fig. 2. The effects of different concentrations of extract plant of Rosemary on the biofilm formation of bacterial and control

 

4. Discussion

Natural products derived from medicinal plants are abundant source of biologically active compounds. Many plant compounds have been used for development of new antimicrobial agents [8, 9]. However, few plant extract have been investigated for their antibiofilm activity.

The study of W,AL Shahwany, The result showed that the isolates behaved differently in their sensitivity to the different extracts added to their growth medium. Among the different plant phenolic extracts tested, Z. officinale showed antibiofilm efficacy against k. pneumoniae and S. aureus followed by T. vulgaris and C. zeylanicum extracts exhibited [10].

The study of Nikolic, The antibacterial and anti-biofilm activity of ethanolic extract from the rhizome of Zingiber officinale were evaluated. The values were in the range from 0.0024 to > 20 mg/ml. The most sensitive bacteria were Gram-positive bacteria: Staphylococcus aureus ATCC 25923. Anti-biofilm activity was tested by crystal violet assay. Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis and E. coli ATCC 25922 were used as the test organisms. Ethanolic extract showed the best result on Proteus mirabilis biofilm where biofilm inhibitory concentration (BIC50) was 19 mg/ml [11].

Yahya et al. (2013) found out that the ethanolic extract of Z. officinale inhibited P. aeruginosa biofilm formation under both aerobic and anaerobic environments [12].

Lycium chilense and Schinus fasciculatus were the most effective antimicrobial plant extracts (15.62µg/ml and 62.50µg/ml for Staphylococcus sp. Mcr1 and Bacillus sp. Mcn4, respectively). The highest (66%) anti-biofilm activity against Bacillus sp. Mcn4 was observed with T. absinthioides and L. divaricate extracts. The highest (68%) anti-biofilm activity against Staphylococcus sp. Mcr1 was observed with L. chilense extract. T. minuta, T. absinthioides, and L. divaricata showed percentages of anti-biofilm activity of between 55% and 62%. The anti-adherence effects of T. minuta and L. chilense observed in Bacillus sp. Mcn4 reflected a difference of only 22% and 10%, respectively, between anti-adherence and biofilm inhibition. Thus, the inhibition of biofilm could be related to cell adherence. In Staphylococcus sp. Mcr1, all plant extracts produced low anti-adherence percentages [13].

The study of Geethashri, demonstrate the efficacy of aqueous extract of Azadirachta indica, Mangifera indica, Piper betel and Pepper nigrum for antibiofilm activity against E. faecalis and S. aureus. The aqueous extracts were obtained by cold percolation method. The antibiofilm activity of plants extract was evaluated at 30, 15 and 7.5 mg/ml concentration. The percentage yield of extract was maximum in P. nigrum. The aqueous extract of A. indica significantly suppressed E. faecalis and S. aureus biofilm at 7.5 mg/ml at p<0.01 and p<0.001 significance level. P. betel significantly (p<0.001) disintegrated the E. faecalis biofilm at 30 mg/ml and S. aureus at 15 mg/ml (p<0.01). P. nigrum disintegrated E. faecalis and S. aureus biofilm significantly (p<0.05 and p<0.001) at 30 and 15 mg/ml respectively. M. indica significantly (p<0.05) suppressed S. aureus biofilm at 30 mg/ml [14].

The study of Varposhti, biofilm formation of P. aeruginosa strain 214 was determined in presence of three plant extracts, Cyclamen coum, Dianthus orieltalis and Origanum majorana, and Zataria multiflora Bio essential oil. The C. coam extract and Z. multiflora Bio essential oil inhibited biofilm formation completely at concentrations<0.062 mg/ml and 4 7l/ml, respectively. The D. orientalis and O. majorana extracts did not inhibit biofilm formation at the used concentrations (0.003 – 8 mg/ml) [6].

The study of Gaetti-Jardim, antimicrobial activity of plants extracts on microbial biofilms was determined in microplates. Psidium cattleianum and Myracrodruon urundeuva extracts demonstrated significant inhibitory activity on all bacterial strains tested; alcoholic and aqueous extracts showed similar results [15].

The study of Shafiei, The PEM compared with its respective constituent plants showed the lowest MIC towards S. sanguinis (3.81 mg/ml) and S. mutans (1.91 mg/ml) and exhibited a synergistic effect. The Psidium sp. (15.24 mg/ml) and, PEM and Psidium sp. (30.48mg/ml) showed the lowest MBC towards S. sanguinis and S. mutans respectively. The anti-adherence effect of the PEM and its respective constituent plants (except Psidium sp.) was different for the two bacteria in the single-species biofilm [16].

The study of Mohsenipour and Hassanshahian, was aimed to examine the effect of Thymus vulgaris (T. vulgaris) extracts on the planktonic form and biofilm structures of six pathogenic bacteria. According to disc diffusion test (MIC and MBC), the ability of Thymus vulgaris (T. vulgaris ) extracts for inhibition of bacteria in planktonic form was confirmed. In dealing with biofilm structures, the inhibitory effect of the extracts was directly correlated to their concentration. Except for the inhibition of biofilm formation, efficacy of each extract was independent from type of solvent [17].

The study of Chevalier, Saponin concentrations reached 0.7 and 0.95 mg ml”1 in S. virgaurea subsp. virgaurea and S. virgaurea subsp. Alpestris extracts, respectively. C. albicans was grown in liquid medium and cells were counted by microscopic examination after 0, 4 and 24 h of incubation. Solidago extracts did not inhibit the growth of C. albicans (four strains), E. coli, Pseudomonas aeruginosa, S. aureus, S. mutans, Streptococcus salivarius or Enterococcus faecalis. When inocula were incubated with Solidago extract for 4 and 24 h, we observed a decrease in Candida yeast–hyphal transition. Candida biofilms were then prepared in microtitre plates and treated with plant extracts at 0 h, to estimate biofilm formation, or at 18 h to estimate the effect of the saponin on pre-formed biofilms [18].

The study of Bhunu, the effect of the leaf extracts of P. curatellifolia on M. smegmatis growth and biofilm formation was investigated in order to determine the basis of its use in traditional medicinal use. The minimum inhibitory concentrations (MIC) of the extracts were found to be 6.2 μg/ml for the acetone extract, 12.5 μg/ml for both the ethanol and the total extract and 50 μg/ml for both the methanol and ethyl acetate extracts. The ethanol extract, dichloromethane extract and water extract were the only extracts that effectively inhibited biofilm formation in M. smegmatis. Combining the ethanol extract with kanamycin enhanced the effect of the ethanol extract in terms of inhibition of biofilm formation [19].

The study of Sasirekha, an attempt has been made to screen different plant extracts against microbial biofilms. Out of 33 isolates BF5 was found to be a high biofilm producer by crystal violet assay with high hydrophobicity. On studying the factors influencing biofilm production increase in glucose concentration, acidic and alkaline pH, high osmolarity and low iron concentration promoted BF 5 biofilm formation. Among the different plant extracts tested, turmeric showed 100% efficacy against BF5 followed by cloves (97%), Indian borage (91%) and Aloe vera (84%)[20].

The study of Teanpaisan, evaluate the antimicrobial and antibiofilm activity of A. lakoocha extract against oral pathogens by an in vitro method. 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 [21].

In the study of Fu et al., Which investigated the antimicrobial effects of Rosemary essential , it has been determined that the amount of non-growth of this essential oil on S. aureus is 18 mm and MIC is 0.125 [22]. Tsai et al. In a study on Streptococcus subbrinus, the effect of rosemary ethanoic extract was found to be 1.42 mg / ml [23]. In the study of Soltan Dallal et al., The antimicrobial effects of rosemary essential oil were evaluated by disk diffusion method and dilution in tubes on methicillin-resistant S. aureus. The diameter of the pathogen’s inhibition zone was about 20 mm. The minimum inhibitory concentration was 1.4 mg / ml and the lowest lethal concentration of growth was 2.8 [24].

The study of Moreno, The antibacterial efficacy of phytochemicals as 1,8-cineole, monoterpene constituent of Rosmarinus officinalis (rosemary) essential oils, and carnosic acid, main diterpene of rosemary leaves, was evaluated against nosocomial strains of MDR-Klebsiella pneumoniae and methicillin-resistant S. aureus (MRSA) grown in planktonic culture and biofilm. The fluorescent SYTOX green dye was used to assess the integrity of the bacterial plasma membranes. Results showed that 1,8-cineole decreased planktonic growth of MDR-K. pneumoniae cells, exhibited a clear permeabilizing effect on the bacterial plasma membrane, and disrupted biofilms developed by all MDR-K. pneumoniae strains tested. Carnosic acid inhibited MRSA strains, having up 8 antibiotic resistance, growing in a planktonic state [25].

The study of Elhariry, determine the bioactive compounds in two rosemary water extracts (RWE1 and RWE2) and to assess their antimicrobial, anti-adhesive and antibiofilm potentials against the food-related Bacillus and Pseudomonas species at concentrations; 4, 8, 12, 16 and 20 mg mL^−1. The anti-adhesive and antibiofilm doses were higher than MIC₉₀. RWE1 and RWE2 showed potential reduction in the bacterial cell adhesion to HEp-2 cells – 17.5–64.7 and 12.2–52.9%, respectively[26].

The Sandasi, The antibiofilm activity of extracts obtained from selected herbs, spices, beverages and commercially important medicinal plants was investigated on Listeria monocytogenes. The respiratory activity was assessed using the 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. The majority of extracts tested prevented cell adhesion to the polyvinyl chloride (PVC) surface. Seven of the 15 extracts reduced biofilm adhesion of both the clinical and the type strains by at least 50%. In contrast, inhibition of a preformed biofilm was more difficult to achieve, with only three extracts (Rosmarinus officinalis, Mentha piperita and Melaleuca alternifolia) inhibiting the growth of both strains by at least 50% [27].

The study of A. Abdulhasan, The ability to form biofilm formation was performed and antimicrobial activity for antibiotics and rosemary EO were investigated by minimal inhibitory concentration (MIC) in broth microdilution method. The results showed that all isolates had the ability to produce biofilm (100%). The MIC value of gentamicin, trimethoprim/sulfamethoxazole, ciprofloxacin and Rosemary EO against E. coli were 256, 2000, 500 and 104 μg/ml, respectively for strong biofilm producer isolates while the MIC values were 512, 2000, 1000 and 104 μg/ml, respectively for moderate biofilm producer isolates. Combination of gentamicin and ciprofloxacin with EO were reduced the MIC of antibiotics when used alone in both strong and moderate biofilm producer isolates [28].

5. Conclusion

This is the first report on evaluation of plants extracts for their anti-biofilm activity against oral pathogens. Our study results highlight the scientific evidence for the use of plants in oral care and treat the emergence of multidrug resistant microorganisms and potential side effects of allopathic health care products. Further this study result requires support by the evaluation of antimicrobial activity against drug resistant clinical isolates and cytotoxicity on human gingival fibroblast cells.

Conflict Of Interest

All authors disclose any financial and personal relationships with other people or organizations and the authors declare that there are not any potential conflicts of interest.

 

Acknowledgement

The authors thank Mrs. Hamideh Khajeh, Laboratory experts of institute of plant biotechnology in the University of Zabol for supplying required instruments.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publications

The author read and proved the final manuscript for publication.

Availability of data and material

All data generated during this study are included in this published article

Funding

This article was achieved based on the material and equipment of University of Zabol, that the authors thanks it

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