Recent Trends and Methods in Antimicrobial Drug Discovery from Plant Sources

  

    
Extraction method
    
Commonly used solvents
    
Solvent volume (mL)
    
Time period
    
Temperature
    
References
  
  

    
Soxhlet extraction
    
Methanol, ethanol or water and alcohol mixture
    
150-200
    
3-18 hrs
    
Depends    upon the solvent used
    
[58,87,88]
  
  

    
Sonification
    
Methanol, ethanol or water and alcohol mixture
    
50-100
    
1 hr
    
Heat    can be used
    
[58,87]
  
  

    
Maceration
    
Methanol, ethanol or water and alcohol mixture
    
Can be varied based on sample size
    
At least 3 days
    
Room    temperature
    
[89-92]
  
  

    
Percolation
    
Methanol, ethanol or water and alcohol mixture
    
Can be varied based on sample size
    
Upto 24 hrs
    
Room temperature
    
[92]
  

Table 2:  Experimental conditions for various methods of extraction for plant material.

Some modern extraction techniques have evolved recently which present certain advantages like comparatively reduced organic sample consumption and sample degradation, fewer steps, improved extraction efficiency, extraction kinetics and ease of automation. It makes these modern techniques much favorable for the extraction of plant materials. Few examples of these modern extraction techniques are; solid-phase micro-extraction, pressurized-liquid extraction, supercritical-fluid extraction, solid-phase extraction, microwaveassisted extraction and surfactant-mediated extraction [58]. These modern techniques are proving to be more efficient than the conventional methods [93].

Soxhlet extraction is also a widely used technique using organic solvents for the extraction purpose [94]. In this case, fresh solvent is continuously introduced to the dried plant material, which improves the overall extraction efficiency. However, this method cannot be used for thermolabile compounds as prolong heating and exposure of plant material to the boiling solvent may lead to degradation of compounds [95].

Serial exhaustive extraction is another method commonly used. It involves serial extraction with an array of solvents of increasing polarity. A range of solvents from non-polar (hexane) to a more polar (methanol) can be used to ensure that compounds with different polarity range could be extracted successfully [96]. This is the ideal method when the aim is to screen the plant for a variety of compounds. There are some methods which are suitable for the extraction of a particular group of compounds. For instance, steam distillation, volatile solvent extraction or Supercritical Fluid Extraction (SFE) are the methods of choice when it comes to the extraction of essential oils [97].

Variations exist in extraction methods based on length of the extraction period, solvent used, and particle size, solvent to sample ratio, temperature and pH. In a study, extractions performed using fine particles of diameter 10 μm gave higher extraction efficiency in 5 min as compared to 24 hrs extraction performed in shaking machine using less finely ground plant material [98]. In one experiment, sequential extraction performed at room temperature using various solvents was compared with extraction performed in a water bath with acidified distilled water (pH 2.0 with HCl), which was neutralized with NaOH before extraction with diethyl ether. It was found that the latter method was more efficient due to the easy extraction promoted by the acidified aqueous environment [84]. Similarly, solvent to sample ratio also participate in affecting the quantity and quality of obtained constituents. Studies reported that solvent to sample ratios (v/w) of 10 mL: 1g has been used and found as ideal [96].

Antimicrobial Agents Screening Techniques

A large number of assessment techniques are currently in use for the investigation of plant products as a source of new antimicrobial compounds. Since the principle is not the same for all these methods, results obtained will also be influenced to a great deal by method selected, the test microorganisms and the degree of solubility of each test compound [99,100]. The test systems should ideally be simple, inexpensive, rapid, and reproducible and should maximize high sample throughput in order to deal with a large number of extracts and fractions. The complexity of bioassays must be defined according to the laboratory facilities and skill of available personnel [101,102]. The most common antimicrobial screening techniques employed presently include; bioautographic, diffusion and dilution methods. The first two are the qualitative techniques as these methods only perform a qualitative analysis of the samples and give an idea of the presence or absence of antimicrobial substances. Dilution methods, on the other hand are considered quantitative assays [103].

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing is an important technique used in clinical microbiology laboratory. The performance of Antimicrobial Susceptibility Test (AST) is imperative to confirm susceptibility to chosen antimicrobial agents or to detect resistant strains [104]. In ethnopharmacological research, it is used to determine the efficacy of potential antimicrobials from biological extracts against various microorganisms. Plants with prospective antimicrobial compounds are screened using AST methods but their most common uses are; determining the efficacy of an antimicrobial in treating infections by finding its Minimum Inhibitory Concentration (MIC), epidemiological studies of susceptibility and in comparisons of new and existing antimicrobial agents [105].

Keeping in view the recent encroachments in antimicrobial drug discovery from plant sources, the development of more sensitive bioassay techniques has become inevitable. In vitro tests for screening of plant extracts should be standardized and in order to get comparable results to currently used antibiotics, further studies should be conducted for MIC determination of natural products [106]. The recent standard AST methods approved by various organizations like National Committee for Clinical Laboratory Science (NCCLS), now known as Clinical and Laboratory Standards Institute (CLSI), European Committee for Antimicrobial Susceptibility Testing (EUCAST) and British Society for Antimicrobial Chemotherapy (BSAC) are available and they serve as guidelines for antimicrobial susceptibility testing of a wide range of drugs. However, these might not be exactly applicable to plant extracts and certain modifications have to be made [107].

For convenience AST methods can be broadly classified into diffusion and dilution methods. Examples of the former include; agar disc diffusion, agar well diffusion and bioautography, while agar dilution, broth microdilution and broth macrodilution techniques are categorized under the dilution methods [108].

Diffusion methods

The earliest methods for detecting the antibacterial effects of extracts involved the use ditches cut into agar plates which were streaked with bacteria. Test solution was poured into the ditch and was allowed to diffuse out and inhibit bacterial growth. As an alternative source of diffusion, some researchers used paper strips impregnated with antibiotics [109]. The agar diffusion principles were further extended to develop the disc diffusion test [110]. Agar wells carrying the antibiotic solution were soon replaced by paper discs impregnated with antibiotics.

Due to its simplicity and ease of performance, diffusion tests were widely adopted by many investigators, but the lack of standardization resulted in unreliable and non-reproducible results. The first article describing a standardized technique called the ‘Paper Disc Method’ (PDM) for sensitivity testing of bacteria was published by Ericsson in 1954 [111]. Different types of diffusion tests are used nowadays and they are a vital part of current AST methodologies.

Agar disc diffusion assay: Disc diffusion refers to the diffusion of an antimicrobial agent of a specified concentration from discs, tablets or strips, into the solid culture medium that has been seeded with the selected inoculum isolated in a pure culture. The principle of disc diffusion testing was developed in 1940 and has been used in microbiology laboratories for over 70 years [112]. Bauer, Kirby, Sherris, and Turck diligently tested different variables used in the test, such as media, temperature and depth of agar. Their landmark paper was published in 1966, describing the test that is used today [113,114]. The agar disc diffusion susceptibility method (commonly known as Kirby-Bauer test) is simple, practical and has been well standardized [104].

AST of pure substances can be efficiently performed with agar disc diffusion technique but when used for mixtures containing constituents exhibiting different diffusion rates, it may give unreliable results [115]. The test is suitable for lead identification but is not considered much productive for the quantification of antimicrobial response. Furthermore, the bacteriostatic and bactericidal effects cannot be distinguished using this method [84,107,116].

The test is performed by applying a bacterial inoculum of approximately 1–2 ×10⁸ CFU/mL to the surface of a large (150 mm diameter) Mueller-Hinton agar plate [104].The medium of choice is generally Mueller-Hinton although Nutrient agar [117] and Tryptone Soya agar [80] have also been used by various researchers. Once the agar plate is completely dry, the antibiotic discs are applied either manually or with a dispensing apparatus. In general, no more than 12 discs should be placed on a 150-mm agar plate or 5 discs on a 90-mm plate [118]. Different researchers have reported the drying time of impregnated paper disc and it varies from 2 hrs to overnight under a laminar flow cabinet [83]. The plates are incubated in an inverted position (agar side up) under conditions appropriate for the test microorganism [118]. The incubation time for bacterial strains is 24 h at 37°C and for fungi it is 48 hrs at 25°C. The zones of growth inhibition are then measured to the nearest millimeter around each of the antibiotic discs. The diameter of the zone is related to the susceptibility of the test microorganism and to the diffusion rate of the drug through the agar medium. The results are interpreted using the criteria published by the Clinical and Laboratory Standards Institute (CLSI) [119]. Disc diffusion test provides us with “qualitative” results and MIC calculation is not possible. It gives results in the shape of categories of susceptibility and organisms are categorized as susceptible, intermediate or resistant to the tested antimicrobial compound. However, some zone reader systems available commercially claim to give an approximate MIC with certain organisms and antimicrobials by the comparison of zone diameters with the standard curves stored in an algorithm [120.121]. The design of the disc diffusion method for fungal strains is similar to that for bacteria using the same medium (Mueller–Hinton agar) supplemented with glucose and methylene blue dye both of which are easily available [118].

Agar well diffusion: It shares the same principle as the disc diffusion assay. A number of researchers have used this technique to test the antimicrobial action of different compounds extracted from plant sources. The plates inoculated with the test organism (approximately 1–2×10⁸ CFU/mL) have 6-8 mm wells cut into the surface of the agar using a sterile cork borer. A distance of at least 30 mm is kept between adjacent wells and walls of the petri plate. The wells are filled with a predetermined quantity of antimicrobial samples, the plates are then incubated at 35-37°C for 24-48 hours depending upon the test organism. The diameters of any clear zones around sample containing wells are measured using calipers [122- 126]. It has also proven to be an inexpensive, less complicated and reliable method of antifungal drug susceptibility testing for Candida sp. and it has given comparable results to the disc diffusion test [127].

Bioautography: Bioautography is a method of separation and detection of plant antimicrobials by performing a suitable chromatographic process followed by a biological detection system. Even though sophisticated online high performance liquid chromatography coupled bioassays are available, bioautography offers process simplicity and rapidity for the chemical and biological evaluation of plant extracts [128]. Bioautography is a very useful and inexpensive method of evaluating antimicrobial potential of plant extracts both in their crude form and as purified phytochemicals. It can also be used effectively in the target directed isolation of active plant secondary metabolites on chromatogram [100]. It has simplified the complex process of isolation of antimicrobial phytoconstituents from crude plant extracts. A very little amount of sample is required to determine the bioactivity moreover it also determines the polarities of active compounds [129]. The three different bioautographic methods are agar diffusion or contact bioautography, direct TLC bioautographic detection and the immersion or agar overlay bioautography [130].

Contact bioautography: In contact bioautography, a TLC or paper chromatogram is developed first from which the antimicrobials diffuse to an inoculated agar plate. The chromatogram is inverted onto the inoculated agar layer for a defined period to allow diffusion, after which the chromatogram is removed and the agar layer is incubated. The zones of inhibition on the agar surface, conforming to the spots in chromatographic plates are the indicators of antimicrobial compounds. Growth incubation time varies between 16–24 hrs [131].

Direct TLC bioautography: In direct TLC bioautography, the developed TLC plate is sprayed with or dipped into a suitable seeding density of fungal or bacterial inoculum. A suspension of test bacteria or fungi is used for spraying or dipping purpose. The bioautogram is then incubated for 48 hrs at 25°C under humid condition. For visualization of microbial growth, tetrazolium salts are used. These salts are converted by the dehydrogenases of living microorganisms to intensely colored formazen [132]. The bioautogram after been sprayed with the salts are re-incubated at 25°C for 24 hrs [133] or at 37°C for 3-4 hrs [61,129]. Antimicrobial activity is indicated by the appearance of clear white zones against a purple background on the TLC plate.

Immersion/agar overlay bioautography: In Immersion or agar overlay bioautography, the chromatogram is completely covered with an agar medium containing the test microorganism. After solidification, incubation and staining which is usually done with tetrazolium dye, the inhibition zones can be easily visualized to locate the antimicrobial compounds. Agar overlay is a fusion of contact and direct bioautography [115].

Dilution methods

Dilution methods are most commonly used to determine the MIC of antimicrobial agents, including antibiotics and other substances that either kill (bactericidal activity) or inhibit the growth (bacteriostatic activity) of microorganisms [134]. Different types of dilution tests are broth microdilution, broth macrodilution and agar dilution methods [108]. The final volume of the test defines whether the method is termed macrodilution i.e. when using a total volume of 2 mL , or microdilution, if performed in microtiter plates using =500 μL per well. In both the agar and broth dilution approaches, the lowest concentration of the antimicrobial agent that prevents visible growth of a microorganism under known conditions is termed as MIC [134]. Dilution methods offer certain advantages over diffusion techniques which include; enhanced sensitivity for smaller extract volumes, quantitative analysis and ability to differentiate bacteriostatic and bactericidal effects of test sample [116]. It is an economical test which can be used for a number of microorganisms and it gives fairly reproducible results. Dilution methods are considered as reference methods for in vitro susceptibility testing and are also used to evaluate the performance of other AST techniques.

Broth macrodilution: Broth macrodilution or tube dilution method was one of the earliest AST methods [135]. It was among the first to be developed and still serves as a reference method. The assay is generally performed in test tubes, two fold dilutions of test antibiotic is prepared in a liquid growth medium of volume greater than 1mL [104]. Test tubes without antimicrobial test sample serving as controls are also included in the test. A standardized bacterial suspension of 1–5×10⁵ CFU/mL is used to inoculate the test tube containing the antibiotic. Following overnight incubation, the tubes are examined for changes in turbidity as an indicator of growth. The lowest concentration of antibiotic that prevented growth represents the MIC. The major disadvantage of this method is extensive manual handling of antibiotic solutions, thus increasing the possibility of errors in solution preparation [118].

Broth microdilution: The broth microdilution method, also known as microtiter/microwell plate method is an adaptation of the broth dilution method in which the assays are performed using small volumes of test antimicrobial and allows a number of bacteria to be tested quite rapidly. The mechanization of test by use of microtiter plastic plates and reduction in required test volumes of sample and reagents has made this assay significantly useful and popular. Standard plates having 96 wells are used and it allows approximately 12 antibiotics to be tested in a range of 8 twofold dilutions in a single tray [118].

A stock solution of the extract is obtained in the solvent already been used for extraction [136]. Other investigators have confirmed the use of DMSO as well [62,63,84,103]. Before transferring the predetermined volume of stock solution to the plate, an equal volume of sterile Mueller Hinton broth or water is usually used as diluents in the wells of the microtiter plate [105]. For the susceptibility testing of rapidly growing aerobic or facultative organisms, Mueller- Hinton broth with a pH between 7.2 and 7.4 at room temperature is often recommended as the medium of choice. The media may be supplemented with 2–5% lysed horse blood in case of fastidious organisms [137]. Similarly, EUCAST (2003) recommends certain supplemented Mueller Hinton broth for non-fastidious microorganisms. It should be noted that this assay procedure results the final concentration of the antimicrobial agents in the assay wells being two fold lower than in the aliquots added to the wells [118]. For effective analysis of MIC, 5-8 concentrations representing achievable concentrations for the antimicrobial are usually tested [138].

Inoculation is done with the standard inoculum size of 5×10⁵ CFU/mL [104], while some have reported an inoculum size of 1×10⁶ CFU/mL [80,83]. It is accomplished by a micropipette or mechanized dispenser that transfers 0.01 to 0.05 mL of standardized bacterial suspension into each well of the microdilution plate [104]. Some investigators have used a microbial culture with an optical density of 0.4 at 620 nm or a 12 hrs broth culture adjusted to a 0.5 McFarland turbidity standard [62]. EUCAST recommended incubation temperature of 35–37°C in air for 16–20 hrs in case of non-fastidious organisms [105].

After incubation, MICs are calculated using either a manual or automated viewing device for the examination of each well for growth. Growth is indicated by change in turbidity and the concentration of first well that appears clear is taken to be the MIC of the extract. The presence of growth is also determined by use of indicators like tetrazolium salts or resazurin dye [139] and also by spectrophotometric analysis [106,140]. The absorbance is usually measured at 620 nm when using spectrophotometric method for growth determination. The lowest sample concentration giving a zero absorbance reading [63] or the concentration with a sharp dip in absorbance value [106] is the MIC of the test compound.

Agar dilution: Agar dilution method is a well-established method for investigating the antimicrobial potential of extracts and is commonly used as a reference method for evaluation of new antimicrobial agents and susceptibility test protocols. It offers various advantages like the simultaneous testing of a number of biological isolates, ability to observe heterogeneous populations or mixed cultures, and the versatility and flexibility in sample selection and concentration range to be tested [118].

In agar dilution method, the antimicrobial agent after been filter sterilized (0.22μm) is incorporated in the molten agar medium with each plate containing a different concentration of the agent. Muller- Hinton agar [105] is used most commonly although the media used for testing is determined on the basis of the organisms and in some cases the antibiotic to be tested [105]. Some researchers have used nutrient agar [136,141]. Supplemented Muller-Hinton agar is used for non-fastidious bacteria [105,118].The pH of the agar must be between 7.2 and 7.4 at room temperature. The inoculum can be applied rapidly and simultaneously to the agar surfaces, EUCAST (2003) recommends an inoculum density of about 107 CFU/mL and using micropipette or replicator pins to transfer about 1μL (104 CFU/mL) of the inoculum. Most available replicators transfer 32–36 inoculums to each plate [137]. Streaking is done in radial patterns and the plates are then incubated at 37°C for 24-48 hrs. The MIC is read as the lowest antibiotic concentration that inhibits the growth of the organism completely [84,107]. A faint haze caused by the inoculum or a single colony should not be read as growth. The disadvantages of this method include the labor required to prepare the agar dilution plates and their relatively short shelf life. In addition, the manual evaluation and interpretation of susceptibility results can be time consuming [118].

Etest

It is an innovative commercial antimicrobial susceptibility test developed in Sweden, a gradient technique that combined the principles of both disc diffusion and agar dilution methods. It was first presented at the Inter-science Conference on Antimicrobial Agents and Chemotherapy (ICAAC) meeting in 1988. Etest was approved by U.S. FDA in 1991.

Plastic strips are impregnated with a gradient of antimicrobial concentrations in dry form. The upper surface of the Etest strip has a scale; it is calibrated across a corresponding MIC range. When placed on the surface of the agar plate that has been inoculated with the test organism, the antimicrobial on the test strip is immediately transferred to the plate in a continuous gradient manner. After optimum incubation period, the antimicrobial activity is seen as an ellipse of inhibited growth around the strip. MIC value is obtained from the point where the ellipse intersects the scale (Figure 1).

    

    

    Figure 1:  Etest inhibition ellipse showing the MIC at the intersection of the
growth of the organism and the calibrated strip [118].