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      Antibacterial and antibiofilm activity of acetone leaf extracts of nine under-investigated south African Eugenia and Syzygium (Myrtaceae) species and their selectivity indices

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          Abstract

          Background

          Antimicrobial resistance (AMR) remains an important global health issue but the gap between AMR and development of new antimicrobials is increasing. Plant extracts may have good activity per se or may be sources of effective antimicrobial compounds which can act against planktonic and/or biofilms of pathogens. We determined the antimicrobial efficacy and cytotoxicity of some under-investigated plants from the Myrtaceae family endemic to South Africa. The ability of the plant extracts to inhibit or destroy pre-formed bacterial biofilms was also determined.

          Methods

          Based on previous preliminary in vitro screening and on chemotaxonomy, nine species from the Myrtaceae family were selected. The antimicrobial activity of the crude acetone leaf extracts was determined against six common nosocomial pathogens, namely: Gram-positive bacteria ( Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus), Gram-negative bacteria ( Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhimurium) using a two-fold serial microdilution assay with p-iodonitrotetrazolium violet as growth indicator. The number of antimicrobial compounds present in extracts was determined by bioautography. Cytotoxicity of extracts was determined against Vero kidney cells using a colorimetric tetrazolium-based assay. The total antibacterial activity (TAA) in ml/g and selectivity index (LC 50/MIC) of the plant extracts were calculated. A modified crystal violet assay was used to determine the antibiofilm activity of the extracts.

          Results

          Syzygium legatii, Syzygium masukuense, and Syzygium species A had the best activities against Gram-negative and Gram-positive bacteria (MIC) values ranging from 0.04–0.08 mg/ml. Eugenia erythrophylla had the best MIC (0.02 mg/ml) against Bacillus cereus. Many extracts had relatively low cytotoxicity (LC 50 > 20 μg/ml) leading to reasonable selectivity indices. Three leaf extracts ( Syzygium masukuense, Syzygium species A, and Eugenia natalitia) were moderately cytotoxic (20 μg/ml < LC 50 < 100 μg/ml). The plant extracts had a good capacity to reduce biofilm formation and good to poor potential to destroy pre-formed biofilms.

          Conclusions

          The plant species examined in this study had varying degrees of antibacterial activity against bacterial planktonic and biofilm forms with some having good activity against both forms. Several of these selected species may be potential candidates for further investigation to isolate antimicrobial compounds and to determine the mechanism of activity.

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          Most cited references61

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          A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria.

          J Eloff (1998)
          Agar diffusion techniques are used widely to assay plant extracts for antimicrobial activity, but there are problems associated with this technique. A micro-dilution technique was developed using 96-well microplates and tetrazolium salts to indicate bacterial growth. p-Iodonitrotetrazolium violet [0.2 mg/ml] gave better results than tetrazolium red or thiazolyl blue. The method is quick, worked well with Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli and with non-aqueous extracts from many different plants. The method gave reproducible results; required only 10-25 microliters of extract to determine minimal inhibitory concentrations, distinguished between microcidal and microstatic effects, and provided a permanent record of the results. Using S. aureus, and a Combretum molle extract, the technique was 32 times more sensitive than agar diffusion techniques and was not sensitive to culture age of the test organism up to 24 hours. The S. aureus culture could be stored up to 10 days in a cold room with little effect on the assay results. This method was useful in screening plants for antimicrobial activity and for the bioassay-guided isolation of antimicrobial compounds from plants. MIC values determined for sulfisoxazole, norfloxacin, gentamicin, and nitrofuratoin were similar to values indicated in the literature but values obtained with trimethroprim and ampicillin were higher with some bacteria.
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            Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies.

            Bacteria have evolved the ability to form multicellular, surface-adherent communities called biofilms that allow survival in hostile environments. In clinical settings, bacteria are exposed to various sources of stress, including antibiotics, nutrient limitation, anaerobiosis, heat shock, etc., which in turn trigger adaptive responses in bacterial cells. The combination of this and other defense mechanisms results in the formation of highly (adaptively) resistant multicellular structures that are recalcitrant to host immune clearance mechanisms and very difficult to eradicate with the currently available antimicrobial agents, which are generally developed for the eradication of free-swimming (planktonic) bacteria. However, novel strategies that specifically target the biofilm mode of growth have been recently described, thus providing the basis for future anti-biofilm therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.
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              Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation.

              Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.
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                Author and article information

                Contributors
                adeyerimi@gmail.com
                aroabimbola@yahoo.co.uk
                folorunso.fasina@fao.org
                kobus.eloff@up.ac.za
                lyndy.mcgaw@up.ac.za
                Journal
                BMC Complement Altern Med
                BMC Complement Altern Med
                BMC Complementary and Alternative Medicine
                BioMed Central (London )
                1472-6882
                20 June 2019
                20 June 2019
                2019
                : 19
                : 141
                Affiliations
                [1 ]ISNI 0000 0001 2107 2298, GRID grid.49697.35, Phytomedicine Programme, Faculty of Veterinary Science, , University of Pretoria, ; Private Bag X04, Onderstepoort, 0110 South Africa
                [2 ]ISNI 0000 0001 2107 2298, GRID grid.49697.35, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, , University of Pretoria, ; Private Bag X04, Onderstepoort, 0110 South Africa
                [3 ]Emergency Centre for Transboundary Animal Diseases-Food and Agriculture Organization of the United Nations (ECTAD-FAO), House H. Sida, Ali Hassan Mwinyi Road, Ada Estate, Dar es Salaam, Tanzania
                Author information
                http://orcid.org/0000-0003-1494-9842
                Article
                2547
                10.1186/s12906-019-2547-z
                6587284
                31221162
                89d80fb8-11e7-4edb-b2ca-5a4d19c217a9
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 15 October 2018
                : 3 June 2019
                Funding
                Funded by: World Academy of Science
                Award ID: grant 99808
                Award Recipient :
                Funded by: Nacionalna zaklada za razvoj civilnog društva (HR)
                Award ID: grant 105993
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2019

                Complementary & Alternative medicine
                antibacterial activity,antibiofilm activity,cellular safety,nosocomial bacteria,myrtaceae,syzygium,eugenia

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