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In vitro antibacterial efficacy of Acacia farnesiana seed extracts specifically directed against B. cepacia, B. multivorans, B. cenocepacia of Burkholderia cepacia complex
* Corresponding author: Mr. Sunil Kumar Mugle, M.Sc., Department of School of Sciences, Mahaveer Academy of Technology and Science University, Raipur, Chhattisgarh, India. sunilmugle@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Mugle SK, Sohal JK. In vitro antibacterial efficacy of Acacia farnesiana seed extracts specifically directed against B. cepacia, B. multivorans, and B. cenocepacia of Burkholderia cepacia complex. Ann Natl Acad Med Sci (India). doi: 10.25259/ANAMS_62_2024
Abstract
Objectives
The emergence of antibiotic-resistant pathogens and the constraints of existing pharmaceuticals have intensified the quest for alternative therapeutic strategies. Phytochemically-rich plant and seed extracts present a promising avenue for the development of novel antimicrobial agents.
Material and Methods
Acacia farnesiana was selected for this study due to its traditional medicinal applications in the treatment of many diseases. The seed extract of Acacia farnesiana was investigated to identify its antimicrobial properties. The dried seeds of Acacia farnesiana were sequentially extracted using methanol, ethanol, petroleum ether, and distilled water.
Results
The highest extraction yield, 18.5%, has been found with the methanol extract. Culture plates were inoculated for sensitivity testing, and the minimum inhibitory concentration (MIC) has been established. Three strains of the Burkholderia cepacia complex (BCC), Burkholderia cenocepacia, Burkholderia cepacia, and Burkholderia multivorans, have been utilized to evaluate the extracts’ antimicrobial activity. The Acacia farnesiana seed extracts included MICs that range from 0.50- 0.80 mg/mL. The zones of inhibition (ZOI) showed that the methanolic extracts exhibited antimicrobial evaluation results, thereby inhibiting the growth of all three bacterial strains. Specifically, Burkholderia cepacia exhibited a ZOI of 9.0±0.82 mm at 0.60 mg/mL, Burkholderia cenocepacia showed a ZOI of 15.4±0.94 mm at 0.80 mg/mL, and Burkholderia multivorans presented a ZOI of 8.7±0.47 mm at 0.50 mg/mL.
Conclusion
The outcomes give promising baseline data for the potential application of these crude extracts in drug development programs within the pharmaceutical industry. The extracts exhibit substantial potential as alternative or adjunctive therapies for pulmonary infections. Their efficacy, attributable to a diverse array of phytochemicals, addresses a critical need in the context of escalating antibiotic resistance. However, challenges such as standardization, safety, and clinical validation persist. Further research, including rigorously controlled clinical trials, is essential to establish these extracts as viable therapeutic options.
Keywords
Antimicrobial activity
Burkholderia cepacia complex
Medicinal plants
Minimum inhibitory concentration
Zones of inhibition
INTRODUCTION
With a rich history rooted in folk medicine, the use of plants along with their products has evolved over time, finding integration into both traditional and allopathic medicinal practices.1 This long-standing association with health is attributed to the existence of diverse secondary metabolites within plants, including saponins, glycosides, steroids, flavonoids, alkaloids, tannins, and terpenes. These compounds present potential for combating disease-causing pathogens, warranting further exploration for their therapeutic applications.2-4
The discovery of numerous synthetic and natural medications has led to amazing advancements in the field of medicine because of advances in science and technology.5-7 Antibiotics, a revolutionary discovery of the 20th century, have been instrumental in combating serious bacterial infections. However, their effectiveness is limited, treating only a third of known infectious diseases.8-11 Resistant pathogens, a result of widespread antibiotic overuse as well as misuse, are the cause of this limitation.12-14 Antibiotic resistance poses a growing threat, demanding novel therapeutic solutions. One promising approach involves exploring plants as a source of antibiotic resistance inhibitors.15-17 Plants, known for producing diverse compounds for self-defense against pathogens, offer the potential for targeting mechanisms distinct from those of conventional antibiotics, potentially overcoming drug resistance.18,19 This aligns with the long-standing tradition of using medicinal plants in various cultures for centuries to treat various ailments.20-22 To develop novel drugs, researchers have recently focused on safer phyto medicines as well as biologically active compounds that have been isolated from plant species and utilized in herbal medicines that have an acceptable therapeutic index.23-25 Historically, Acacia farnesiana has been used as a stringent, stomachic, and antiviral.26-28 Numerous academic studies have documented anti-inflammatory, analgesic, anti-cancer, and anti-malarial properties.29-31
Burkholderia is a genus of bacteria posing specific concerns for pharmaceutical products. Due to its potential presence in various drug forms, including inhalation solutions and aqueous preparations for oromucosal, oral, nasal, and cutaneous administration, screening for this bacterium is crucial.32
Several reasons necessitate screening for Burkholderia. First, belonging to the Gram-negative group, Burkholderia can produce endotoxins. These endotoxins can trigger pyrogenic effects, causing fever in patients. Additionally, Burkholderia acts as an opportunistic pathogen, commonly causing pneumonia in individuals with compromised immune systems or pre-existing lung conditions. Lung infections by Burkholderia are particularly dangerous, often leading to a rapid decline in lung function and, potentially, death. Studies by Lyczak33 suggest that mortality rates could be nearly five times greater in Cystic Fibrosis (CF) patients infected with Burkholderia compared to non-infected CF individuals.
This research has been designed to examine the antimicrobial activity of specific plant seeds against Burkholderia, drawing on the established knowledge of plant-based medicines. Various techniques for extraction were applied. Against the backdrop of Burkholderia bacteria complex (BCC) bacteria (BCC strains like Burkholderia cepacia, Burkholderia cenocepacia, and Burkholderia multivorans), which are a feared contamination risk in water-based pharmaceutical products, the current study was designed to assess Acacia farnesiana antimicrobial activity.
MATERIAL AND METHODS
Collection of plant material
Mature seeds of A. farnesiana were collected for this research from the vicinity of Chhattisgarh Regional Science Centre, Raipur. Plant identification was conducted at the Plant Identification Cell, Department of Botany, Guru Ghasidas Vishwavidyalaya (a central university, Bilaspur). Various extraction solvents (methanol, ethanol, ether, and distilled water) were employed to determine the extraction yield percentage.
Culture and maintenance of microorganisms
Every experimental bacterium was obtained as a pure culture from the American Type Culture Collection (ATCC). On Burkholderia cepacia selective agar (BCSA), these pure bacterial cultures were grown. Before being utilized in experiments, each bacterial strain was kept alive by routinely subculturing it on the same medium and storing it between 2 and 8°C.
Preparation of plant extract
Seeds of Acacia farnesiana collected from the source plant underwent a cleaning process involving washing with tap water which has been followed by distilled water, and then ethanol, before drying overnight at 50°C and subsequently milling into coarse powder. Soxhlet extraction was performed on 100 g of powdered material using ethanol, methanol, ether, and distilled water (each for 12 hours). In a vacuum chamber, the extracts were allowed to evaporate at a low pressure to extract the solvents. Up to additional screening, all extracts were kept in sterile containers between 2 and 8°C.34
Yield percentage is calculated as follows: Y (%) = M2/M1*100
Where, Y: Yield, M1: Dry weight of seeds, and M2: Dry weight of extract.
The agar well diffusion approach and MIC have been utilized to assess the methanol extract antimicrobial activities.
Media preparation and its sterilization
Solid agar media in Petri plates were used for antimicrobial susceptibility testing in the agar well diffusion approach to encourage the growth of bacterial surface colonies. Mueller Hinton Agar (MHA) supplemented with 38.0 g/L (HIMedia Lot No. 0000569021) was utilized. A serial microdilution assay was utilized to ascertain the values of the minimum inhibitory concentration (MIC). To prepare the bacterial cultures, 2% Luria Broth (w/v) was inoculated. By autoclaving at a temperature of 121°C and 15 psi for the time of 15 minutes, all media were sterilized.
The antimicrobial activity was evaluated by utilizing the agar well diffusion approach. Using sterile spreaders, 8-hour-old broth cultures of the corresponding bacteria were added to MHA plates for inoculation. In the agar, wells were made using sterile corn borers. The plant extract has been prepared as a stock solution with a concentration of 1mg/mL. Using sterile microtips, 100 µL aliquots of various extract concentrations were added to the wells, which were then left to diffuse for two hours at room temperature.
Agar well diffusion method
Quality control experiments were conducted with inocula lacking seed extract.
After that, the plates were incubated for 18-24 hours at 35 to 37°C. A digital Vernier caliper (Make: Aerospace) was used to measure the diameter of the inhibition zones (mm), and the activity index was computed based on that measurement. The experiment was conducted three times, maintaining triplicates. Three fixed directions were used to take readings for each replicate, and the average values were observed.
Minimum inhibitory concentration
The MIC refers to the lowest concentration of a substance that prevents observable bacterial growth on culture plates following incubation. Typically determined through methods such as tube or agar dilution techniques, these assays involve serially diluting the test substance in bacterial growth media, inoculating with test organisms, and subsequently assessing growth visually to identify the lowest concentration that inhibits visible growth. This procedure is a standardized assay for evaluating antimicrobial agents.
MIC values play a crucial role in research and development, in pharmaceutical and diagnostic laboratories, by confirming microbial resistance to antimicrobial agents and monitoring the effectiveness of new antimicrobial compounds. MIC serves as a fundamental measure for evaluating the efficacy of antimicrobial agents against specific organisms in laboratory settings. This information not only informs dosing strategies for patients but also guides antibiotic selection, thereby aiding in the prevention of microbial resistance to specific treatments.
Test for antimicrobial activity
The antibacterial activity of the tested compounds against BCC bacteria has been analyzed using the microdilution method in the antibacterial assay. Bacterial suspensions were adjusted to specific concentrations using sterile Buffered Sodium Chloride-Peptone Solution (BSCP, HiMedia, Lot No. 0000481841): “1.1 × 107 CFU/ml for Burkholderia cepacia, 1.4 × 107 CFU/mL” for Burkholderia cenocepacia, and 1.3 X 107 CFU/mL for Burkholderia multivorans. Before being used, the inoculum was prepared and kept at 4°C. Levofloxacin (5.0 µg/disc, Lot No. LE5-2215, Microexpress) is used as a standard antimicrobial drug [Table 1].
| S. No. | Name | Type | ATCC No. | Source |
|---|---|---|---|---|
| 1 | Burkholderia cepacia | Gram negative, oxidase positive | ATCC 25416 | Microbiologics |
| 2 | Burkholderia multivorans | Gram negative, oxidase positive | ATCC BAA-247 | Microbiologics |
| 3 | Burkholderia cenocepacia | Gram negative, oxidase positive | ATCC BAA-245 | Microbiologics |
BCC: Burkholderia cepacia complex, ATCC: American type culture collection, BAA: Biosafety level A.
The inoculum was diluted and then cultured on solid media to verify that there was no contamination and to ensure that the inoculum was viable. The experiment was conducted twice independently (in duplicate) and repeated a total of three times.
Determination of MIC
In 96-well microtiter plates, the MICs were ascertained by a serial dilution method. Various seed extracts were initially prepared at 1 mg/mL concentration, followed by serial dilutions with Soybean-Casein Digest Medium (SCDM, HiMedia, Lot No. 0000534275) containing the respective bacterial inocula. After that, the microplates were incubated at 35°C for 72 hours. The lowest concentrations at which binocular microscopy revealed no discernible growth were known as MICs.
RESULTS
In the current examination, the inhibitory effects of Acacia farnesiana seed extract were assessed against three strains of the BCC bacteria. The microdilution method and the agar well diffusion method have been utilized to assess the antimicrobial activity, and the results are summarized in Tables 2 and 3. The antimicrobial efficacy was quantitatively assessed through two approaches: measuring the activity index [Figure 1], the zone of inhibition [Figure 2], and determining the minimum inhibitory concentration (MIC).
| S. No. | Microorganisms | Seed Extract | Standard ZOI (in mm) | |
|---|---|---|---|---|
| ZOI (in mm) | AI | |||
| 1 | B. cepacia | 9.0±0.82 | 0.355 | 25.34±1.25 |
| 2 | B. multivorans | 8.7±0.47 | 0.338 | 25.67±1.25 |
| 3 | B. cenocepacia | 15.4±0.94 | 0.624 | 24.67±1.25 |
ZOI=Zone of Inhibition “(in mm) includes the diameter of disc(6mm):
AI-Activity Index=ZOI of test sample/ZOI of standard.
Standards: Levofloxacin (5.0 mcg/disc); (Values are mean of triplicate readings (mean ± S.D)”).
| S. no. | Microorganism | MIC (mg/ml) | Growth control | Negative control |
|---|---|---|---|---|
| 1 | B. cepacia | 0.60 | G | NG |
| 2 | B. multivorans | 0.50 | G | NG |
| 3 | B. cenocepacia | 0.80 | G | NG |
G: Growth, NG: No growth, MIC: Minimum inhibitory concentration
- Activity Index (AI) against various microorganisms.
- (a, b, c) Antimicrobial activity in zones of inhibition at respective concentrations against the mentioned Burkholderia cepacia complex annals organisms.
Methanol exhibited the highest extraction yield at 18.5%, followed by ethanol at 17.3%, ether at 14.0%, and distilled water at 15.5%.
Measurement of antimicrobial activity using agar well diffusion method
The zone of inhibition (ZOI) of the experimental plant seeds against different BCC bacteria was used to assess their antimicrobial efficacy. The ZOI results were then compared with the standard antibiotic activity, Levofloxacin (5.0 µg/disc, Lot No. LE5-2215, Microexpress). The findings indicated that the seed extract exhibited potent antimicrobial activity against all studied microorganisms.
The largest ZOI was observed for B. cenocepacia, measuring 15.4±0.94 mm [Figure 3]. Similarly, B. cepacia showed a ZOI of 9.0±0.82 mm [Figure 4], and B. multivorans exhibited a ZOI of 8.7±0.47 mm [Table 2, Figure 5].
-
B. cenocepacia.
-
B.cepacia.
-
B. multivorans
Determination of MIC values
The minimum concentration of an antimicrobial agent that stops a microorganism from growing visibly is known as the MIC. Acknowledging microbial resistance to an antimicrobial agent and tracking the effectiveness of novel antimicrobial compounds both depend on MIC determination.
The seed extract of Acacia farnesiana exhibited MIC values of 0.50 mg/mL against B. multivorans, 0.60 mg/mL against B. cepacia, and 0.80 mg/mL against B. cenocepacia [Table 3].
DISCUSSION
The exploration of natural sources such as plants for new antimicrobials has intensified in response to the escalating threat of antibiotic resistance and the potential of these compounds to offer safer and more efficacious alternatives to synthetic drugs.35,36 Phytochemical, bioactive compounds found in plants, present a promising avenue for developing medicines that are less toxic and more potent in combating an array of human pathogens, including fungi, bacteria, and viruses. Numerous studies have investigated the antimicrobial qualities of plant extracts and searched for new antimicrobial agents. As a result, dietary supplements, nutraceuticals, and pharmaceuticals are increasingly incorporating medicinal plants.
The current study examined the antimicrobial activity of Acacia farnesiana seed extract against particular BCC bacteria, which are known to pose a serious risk of contamination for pharmaceutical products containing water.37-39 In addition to assessment through the agar well diffusion method, the susceptibility of the seed extract has been analyzed by utilizing the serial microdilution approach for determining the MIC.
Our initial investigation demonstrated that the seed extract of Acacia farnesiana exhibited activity against BCC bacteria, including Burkholderia cepacia, Burkholderia cenocepacia, and Burkholderia multivorans. This approach of evaluating seed extract for its antimicrobial potential has also been undertaken by numerous researchers across various plant species such as Adhatoda zeylanica, Trianthema decandra L., Argemone mexicana L., Tinospora cordifolia, and Cassia fistula. The study also indicated varying antimicrobial activities of the seed extract against different strains of BCC at different concentrations. Murugesan40,41 demonstrated significant antimicrobial activity of the petroleum ether extract from Memecylon umbellatum Burm. f. Additionally, aqueous extracts from leaves of Pterospermum acerifolium have been reported to exhibit notable antimicrobial activity against several Gram-positive and Gram-negative human pathogenic bacteria. The antimicrobial evaluation, employing both agar well diffusion approach as well as MIC determination, has been widely employed in scientific research, and the observed antimicrobial activity may be clinically significant, as it shows a considerable ZOI comparable to that of the standard antimicrobial drug used.42-44
CONCLUSION
The current investigation reveals that Acacia farnesiana harbors promising antimicrobial constituents that could be valuable for pharmaceutical development aimed at combating diverse diseases. The seed extract of Acacia farnesiana demonstrates substantial inhibitory activity against the tested BCC bacteria. These findings validate traditional medicinal knowledge regarding the plant’s therapeutic properties, offering potential avenues for the innovation of novel antimicrobial therapeutics.
Authors’ contributions
JKS: Supervision, project administration, methodology. SKM: Conceptualization, data curation, writing – review & editing, resources
Ethical approval
Intitutional Review Borard approval is not required as ATCC cultures only used.
Declaration of patient consent
Patient’s consent not required as there are no patients in this study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
References
- Human Vaginal Pathogen Inhibition Studies on Aqueous, Methanolic and Saponins Extracts of Stem Barks of Ziziphus mauritiana. Int J Pharm Sci Res. 2011;2:659-63.
- [Google Scholar]
- Antibacterial activity and phytochemical screening of ethanolic extracts obtained from selected sudanese medicinal plants. Curr Res J of Bio Sci. 2010;2:143-46.
- [Google Scholar]
- Antimicrobial activity and phytochemical screening of an ornamental foliage plant, pothos aurea (Linden ex Andre) An Int J of Chem. 2010;1:63-71.
- [Google Scholar]
- Chemical constituents and antimicrobial activity of salix subserrata. Rec Nat Prod. 2011;5:133-37.
- [Google Scholar]
- Antimicrobial and antioxidant efficacy of some medicinal plants against food borne pathogens. Adv in Bio Res. 2010;4:122-25.
- [Google Scholar]
- Antibacterial activity of ethanolic extracts of some arid zone plants. Int J of Pharm Tech Res. 2011;3:283-86.
- [Google Scholar]
- Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction. Lancet. 2001;357:1325-8.
- [CrossRef] [PubMed] [Google Scholar]
- An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries. Microb Drug Resist. 2004;10:169-76.
- [CrossRef] [PubMed] [Google Scholar]
- Screening and isolation of antibiotic resistance inhibitors from herb material resistant inhibition of 21 Korean plants. Nat Prod Sci. 1995;1:50-4.
- [Google Scholar]
- Antimicrobial potential and phytochemical screening of andrographis affinis nees an endemic medicinal plant from India. Int J of Pharma and Pharmaceutical Sci. 2011;3:157-59.
- [Google Scholar]
- Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multiple drug resistant human pathogens. J Ethanopharma. 2001;74:113-23.
- [Google Scholar]
- Antibacterial activity of the plant used in Indian herbal medicine. Int J of green pharma. 2010;10:22-8.
- [Google Scholar]
- A convenient microdilution method screening natural products against bacteria and fungi. Pharm Biol. 2001;39:221-25.
- [Google Scholar]
- Indian medicinal plants, a compendium of 500 species. Hyderabad, India: : Orient Longman Ltd; 1995. p. :10-12.
- Investigating the anti-viral and anti-bacterial activities of jordanian medicinal plants: A narrative review. RJPT 2022:127-36.
- [CrossRef] [Google Scholar]
- Evaluation of hydroalcoholic extract of acacia farnesiana inn roots for analgesic and anti- inflammatory activity. Int J of Phytomed. 2010;2:341-44.
- [Google Scholar]
- Manual of clinical microbiology (6th Ed). Washington DC: ASM Press; 1995. p. :15-8.
- Alaboratory manual of pharmaceutical microbiology. Idu, Abuja, Nigeria 1996:69-105.
- [Google Scholar]
- Screening of crude extracts of six medicinal plants usedinSouth–West Nigerianun orthodox medicine for antimethicillin resistant staphylococcus aureus activity. BMC Comp Alt Med. 2005;5:1-7.
- [Google Scholar]
- Antimicrobial activity of piper ribesoides root extract against staphylococcus aureus. J App Biol Sci. 2007;1:87-90.
- [Google Scholar]
- Antidiabetic, antioxidant and antibacterial activities of leaf extracts of adhatoda zeylanica. Medic (Acanthaceae). J Pharm Sci Res 2009:67-73.
- [Google Scholar]
- Novel 4-thiazolidinone derivatives as potential antifungal and antibacterial drugs. Bioorg Med Chem. 2010;18:426-32.
- [CrossRef] [PubMed] [Google Scholar]
- Zulu medicinal plants with antibacterial activity. J Ethnopharmacol. 2000;69:241-6.
- [CrossRef] [PubMed] [Google Scholar]
- Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multiple drug resistant human pathogens. J Ethanopharma. 2001;74:113-23.
- [Google Scholar]
- Evalution of antimicrobial and antioxidant potentials of Trianthema decandra L. Asian J of Biotech. 2010;2:225-31.
- [Google Scholar]
- Antibacterial activity of Argemone mexicana L. against water brone microbes. Res J of Medicinal plant. 2011;5:621-26.
- [CrossRef] [Google Scholar]
- Antimicrobial activity of two Indian medicinal plants tinospora cordifolia (Family: Menispermaceae) and cassia fistula (Family: Caesalpinaceae) against human pathogenic bacteria. J of Pharma Res. 2011;4:167-70.
- [Google Scholar]
- A phytochemical screening and antimicrobial activity of the leaves of memecylon umbellatum Burm. F. J of App Pharma Sci. 2011;1:42-45.
- [Google Scholar]
- Antimicrobial activity and ethnomedicinal uses of some medicinal plants from similipal biosphere reserve, Orissa. Asian J of Plant Sciences. 2008;7:260-7.
- [CrossRef] [Google Scholar]
- Antibacterial activity of some Indian medicinal plants. J Nat Med. 2007;61:313-7.
- [CrossRef] [Google Scholar]
- Antimicrobial activity of Citrullus colocynthisin Gulf of Mannar. Int J of Curr Res. 2010;2:78-81.
- [Google Scholar]
- Burkholderia cepacia complex bacteria: A feared contamination risk in water-based pharmaceutical products. Clin Microbiol Rev. 2020;33:e00139-19.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]
- Lung infections associated with cystic fibrosis. Clin Microbiol Rev. 2002;15:194-222.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]
- Phytochemicals: Extraction methods, identification and detection of bioactive compounds from plant extracts. J Pharmacogn Phytochem. 2017;6:32-6.
- [Google Scholar]
- Antibacterial activity of the plant used in Indian herbal medicine. Int J of Green Pharma. 2010;10:22-8.
- [Google Scholar]
- In vitro antimicrobial potentialities of different Solvent extracts of ethnomedicinal plants against clinically isolated human pathogens. J Phytology. 2010;2:57-64.
- [Google Scholar]
- Antimicrobial activity of essential oils against multidrug-resistant clinical isolates of the Burkholderia cepacia complex. PLoS One. 2018;13:e0201835.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]
- Preparation and evaluation of antimicrobial herbal formulation of pterospermum acerifolium Willd. Int J Pharm Sci Res. 2017;8:4788-94.
- [Google Scholar]
- Evaluation of antimicrobial susceptibility testing methods for Burkholderia cepacia complex isolates from people with and without cystic fibrosis. J Clin Microbiol. 2025;63:e0148024.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]
- Umbellatum, B. Comparative in-vitro antibacterial and antifungal attributes of different solvent extracts from leaf, bark, root and inflorescence of memecylon.
- Phytochemical screening and Antimicrobial activity of the leaves of memecylon umbellatum burm. F. J applied Pharm science, (Issue) 2011:42-5.
- [Google Scholar]
- Antimicrobial activity of memecylon malabaricum leaves. Fitoterapia. 2004;75:409-11.
- [CrossRef] [PubMed] [Google Scholar]
- Evaluation of antimicrobial activity of different solvent extracts of medicinal plant: Melia azedarach L. Int J Curr Pharm Res. 2012;4:67-73.
- [Google Scholar]
- Evaluation of susceptibility testing methods for Burkholderia cepacia complex: A comparison of broth microdilution, agar dilution, gradient strip and EUCAST disc diffusion. Clin Microbiol Infect 2020:S1198-743X.
- [Google Scholar]