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Alexandria Loconte
The rapid rise of antimicrobial resistance is a worldwide problem. This has necessitated the need to search for new antimicrobial agents. Mushrooms are rich sources of potential antimicrobial agents. This study investigated the antimicrobial properties of methanol extracts of Trametes gibbosa, Trametes elegans, Schizophyllum commune, and Volvariella volvacea. Agar well diffusion, broth microdilution, and time-kill kinetic assays were used to determine the antimicrobial activity of the extracts against selected test organisms. Preliminary mycochemical screening revealed the presence of tannins, flavonoids, triterpenoids, anthraquinones, and alkaloids in the extracts. Methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea showed mean zone of growth inhibition of 10.00 0.0 to 21.50 0.84, 10.00 0.0 to 22.00 1.10, 9.00 0.63 to 21.83 1.17, and 12.00 0.0 to 21.17 1.00 mm, respectively. The minimum inhibitory concentration of methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea ranged from 4.0 to 20, 6.0 to 30.0, 8.0 to 10.0, and 6.0 to 20.0 mg/mL, respectively. Time-kill kinetics studies showed that the extracts possess bacteriostatic action. Methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea exhibited antimicrobial activity and may contain bioactive compounds which may serve as potential antibacterial and antifungal agents.
Time-kill kinetics assays help understand interactions that exist between microbial strains and antimicrobial agents. The assay shows a concentration or time-dependent test effect of antimicrobial agents on strains of microorganisms. It determines antimicrobial agents as bacteriostatic/fungistatic or bactericidal/fungicidal [7]. Bacteria develop resistance to drugs in different ways including formation of biofilms, active efflux of drugs, drug inactivation caused by enzyme secretion, and drug target site alteration [5]. Biofilm formation reduces the penetrating abilities, and as such bacteria producing biofilms are not affected by the mode of action of antibiotics [8]. Biofilms refer to cells of microbes embedded in self-produced matrix of extrapolymeric substances attached irreversibly to a surface. Biofilms constitute 65% of microbial infections, and bacteria living in them develop resistance to antimicrobial agents a thousand times than those existing in free-living forms (planktonic forms) [9]. Potential agents to be considered in antimicrobial resistant drug development should therefore demonstrate strong biofilm reduction or inhibition activity [9].
Time-kill kinetic studies indicate that methanol extracts of T. gibbosa, T. elegans, S. commune, and V. volvacea exhibited bacteriostatic actions. There are few reports on the time-kill kinetic studies of mushrooms, and several reports of the natural product extracts have been reported [64, 66]. However, the findings in this study are in contrast with the study of Tinrat [67] who determined the time-kill kinetic activity of the mushrooms Pleurotus sajor-caju, Hypsizygus tessellatus, Lentinus edodes, Flammulina velutipes, and Pleurotus eryngii and found them to exhibit bactericidal activity. There is the need to isolate and characterize the bioactive compounds in the various extracts responsible for the antimicrobial activity.
In this study, a novel standardised in vitro time-kill curve assay was developed. The assay was validated using five World Health Organization N. gonorrhoeae reference strains and a range of ciprofloxacin concentrations below and above the MIC. Then the activity of nine antimicrobials with different target mechanisms was examined against a highly antimicrobial susceptible clinical strain isolated in 1964. The experimental time-kill curves were analysed and quantified with a previously established pharmacodynamic model. First, the bacterial growth rates at each antimicrobial concentration were estimated with linear regression. Second, we fitted the model to the growth rates, resulting in four parameters that describe the pharmacodynamic properties of each antimicrobial. A gradual decrease of bactericidal effects from ciprofloxacin to spectinomycin and gentamicin was found. The beta-lactams ceftriaxone, cefixime and benzylpenicillin showed bactericidal and time-dependent properties. Chloramphenicol and tetracycline were purely bacteriostatic as they fully inhibited the growth but did not kill the bacteria. We also tested ciprofloxacin resistant strains and found higher pharmacodynamic MICs (zMIC) in the resistant strains and attenuated bactericidal effects at concentrations above the zMIC.
N. gonorrhoeae time-kill curve experiments analysed with a pharmacodynamic model have potential for in vitro evaluation of new and existing antimicrobials. The pharmacodynamic parameters based on a wide range of concentrations below and above the MIC provide information that could support improving future dosing strategies to treat gonorrhoea.
Time-kill curves that monitor bacterial growth and death over a wide range of antimicrobial concentrations have been frequently used to evaluate the effect of antimicrobials over time. These data can be analysed using mathematical models and are often the first step in PK/PD modelling. Regoes et al. [7] analysed time-kill curves from E. coli exposed to different classes of antimicrobials using a pharmacodynamic model that is characterised by four parameters: the maximal bacterial growth rate in the absence of antimicrobial (ψ max), the minimal bacterial growth rate at high concentrations of antimicrobial (ψ min), the Hill coefficient (к), and the pharmacodynamic MIC (zMIC) (Fig. 1). This model, which is closely related to E max models [5], has also been applied to study the effects of antibiotics alone and in combinations against other pathogens, such as Staphylococcus aureus [8] and Mycobacterium marinum [9].
Pharmacodynamic model with four parameters. The bacterial growth rates (ψ) in response to each antimicrobial concentration are estimated from time-kill data with linear regression. The maximal bacterial growth rate ψ max, the minimal bacterial growth rate at high concentrations of antimicrobial ψ min, the pharmacodynamic MIC (zMIC) and the Hill coefficient к are shown and define the shape of the curve
In this study, a standardised in vitro time-kill curve assay for N. gonorrhoeae was developed using Graver-Wade (GW) medium. GW medium is a chemically defined, nutritious, liquid medium that supports growth of a wide range of N. gonorrhoeae auxotypes and clinical isolates starting from very low inocula [13]. The novel time-kill curve assay was validated on five World Health Organization N. gonorrhoeae reference strains with fluoroquinolone resistance determinants. A highly susceptible clinical N. gonorrhoeae isolate (DG666, isolated in 1964) was subsequently studied in detail and time-kill curve experiments performed for nine antimicrobials that have been, or currently are, used to treat gonorrhoea. In a second step we analysed the time-kill data using a pharmacodynamic model [7] for a comparative analysis of the pharmacodynamic properties of different antimicrobials.
Prior to growth curve experiments, strains were subcultured once on chocolate agar PolyViteX (Biomerieux). A 0.5 McFarland inoculum was prepared and diluted to 100 CFU/ml (1:106) in GW Medium (35 C). A volume of 100 μl diluted bacteria per well was transferred to Sarstedt round-bottom 96 well plates. The plates were tightly sealed with adhesive polyester foil (Sarstedt) and bacteria were grown shaking at 100 rpm at 35 C in a humid 5 % CO2-enriched atmosphere. Bacterial growth was monitored over a time-course of 60 h (0, 2, 4, 6, 8, 10, 12, 20, 22, 24, 26, 28, 30, 32, 34, 40, 44, 48, 60 h). For every sampled time point, the content of one well was removed and viable counts determined [16]. Growth curves were analysed by plotting the log CFU/ml against the time and fitting a Gompertz growth model [17] to the data as implemented in the package cellGrowth [18] for the R software environment for statistical computing [19]. Only lag, log and stationary phases were included in the analysis and the decline phase excluded.
The bacterial growth rates (ψ) were determined from changes in the density of viable bacteria (CFU/ml) during the first 6 h of the time-kill experiments. The bacterial populations were assumed to grow or die at a constant rate, resulting in an exponential increase or decrease in bacterial density:
The growth rate was estimated as the coefficient of a linear regression from the logarithm of the colony counts. Maximum likelihood estimation was used to account for the censored data (values below the limit of detection of 100 CFU/ml). For a given antimicrobial, the geometric mean of all measurements at zero hours was used as the first data point. From the growth rate, the bacterial doubling time can be calculated as follows:
Time-kill curves for ciprofloxacin and six different Neisseria gonorrhoeae strains. Time-kill curves for WHO G (a), WHO K (b), WHO L (c), WHO M (d), WHO N (e) and DG666 (f) are shown. Twelve doubling dilutions are plotted, the highest concentration (black line) corresponds to 16 MIC as measured with Etest and growth in absence of antimicrobial is drawn in red. The antimicrobial was added at timepoint 0 and monitored until 6 h. The limit of detection in the assay was 100 CFU/ml
Time-kill curves for eight additional antimicrobials were also made (spectinomycin, gentamicin, azithromycin, benzylpenicillin, ceftriaxone, cefixime, chloramphenicol and tetracycline) using the highly antimicrobial susceptible DG666 strain (Fig. 3). Similar to the effect of ciprofloxacin (Fig. 2f), gentamicin and spectinomycin exhibited rapid killing during the first 2 h of the assay for concentrations above MIC. Cefixime and ceftriaxone showed little effect from zero to 3 h but the growth rate then decreased rapidly. For benzylpenicillin and azithromycin, at concentrations above MIC, the killing started after 1 h and decreased rapidly at later time points. The time-kill curves for tetracycline and chloramphenicol looked similar with almost no killing of bacteria within the assay time of 4 h. Chloramphenicol showed a weak bactericidal effect at the highest antimicrobial concentration (Fig. 3).
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