*3.1.1 Antagonism on agar plate methods*

## *3.1.1.1 Simple spot-on lawn assay*

To screen postbiotics or probiotic microorganism using this method, the indicator pathogen is first inoculated, then probiotic microorganisms are spotted at specific points on solid media [37]. **Modification** to the method entails spotting probiotic microorganisms as parallel lines [33]. Its strength includes (a) media can be modified [33, 34], (b) it has an option of different incubation conditions, i.e., probiotic microorganism incubation conditions are first optimized, followed by optimizations for indicator pathogen.

### *3.1.1.2 Spot on agar assay*

Probiotic microorganisms are first spotted on agar media and then incubated [39]. An indicator pathogen is added, and soft agar at around 45–50°C is poured to

#### **Figure 3.**

*Abridgement of methods involved in screening probiotic microorganisms both* In vitro *and* In vivo*. (a)* In vitro *preliminary screening experiments on agar. (b)* In vitro *confirmatory experiments. (c)* In vivo *animal confirmatory experiment. (d)* In vivo *clinical confirmatory experiment.*

*Probiotics and Postbiotics from Food to Health: Antimicrobial Experimental Confirmation DOI: http://dx.doi.org/10.5772/intechopen.99675*

the previously prepared plate spotted with a probiotic microorganism [25, 35]. The advantage of the Agar spot test is that two different media can be used, one for spotting and the other as overlaid soft agar. Indicator and probiotic microorganisms can be grown at different times, meaning incubation conditions can be adjusted for each microorganism. The disadvantage is the high temperature of soft agar, i.e., between 45°C and 50°C, killing heat-labile indicator microorganisms. The strict aerobes may not grow well due to the pour plate method.

#### *3.1.1.3 Spot on lawn assay with wells also referred to as Agar well diffusion assay or as conventional whole plate method*

The wells are dug and indicator pathogen inoculated. Then postbiotic/Probiotic is dispensed [37, 40]. Unlike the simple spot-on lawn assay method, the probiotic microorganism can be allowed to grow first before introducing the indicator microorganisms or vice versa.

#### *3.1.1.4 Paper disc assay*

The postbiotic/probiotic is dispensed on the paper discs and placed on the inoculated media. The inoculation of both indicator pathogen and probiotic microorganism is simultaneous. The disadvantage of this method is that the results are not reproducible [41]. This is mainly attributed to the production of non-diffusible antimicrobials.

#### *3.1.1.5 Cross streak on agar assay*

Entails streaking the probiotic microorganism as parallel lines on media. A perpendicular line of indicator pathogen is then streaked. Growth inhibition is determined at the interception point [40].

#### *3.1.1.6 The radial streak on agar assay*

The probiotic microorganism is inoculated as a circle in the middle of the agar plate. The indicator pathogen is then streaked as radial lines from the edge of the petri dish to the center, and growth inhibition is examined [42]. Another method closely related to this method is cutting the media with the probiotic microorganisms and placing it on top of the indicator pathogen inoculated plate.

#### *3.1.2 Liquid coculture method*

The probiotic and indicator pathogens are both introduced to optimized broth culture media, then incubated. Samples are intermittently collected, and viability (cfu/ml) of indicator pathogen is established. It is used to determine if the probiotic effect is static or cidal [13, 24]. It may also be used to reveal the mechanism by which the probiotic bacteria exert their antimicrobial activity [35]. Microtitre assay is used to screen minimum inhibitory concentration (MIC) of postbiotics using microdilution method, macro serial dilution, or conventional kill time assay [35, 43]. Liquid coculture assay is recommended as a confirmatory test (**Figures 2** and **3**).

#### *3.1.3 Summary of antagonism assay*

**Antagonism assay** on agar plates has the advantage of being fast and straightforward. The disadvantage is that it does not directly interact with the probiotic

microorganism or postbiotics and indicator pathogen. Consequently, the probiotic microorganism should produce sufficient antimicrobial agent that should have the potential to diffuse through solid media in terms of size and spatial centrifugation [44]. Accordingly, it is not prudent to use these methods solely to ascertain antimicrobial activity of probiotic microorganisms. Hence, it is recommended to combine antagonism on agar plates and liquid coculture to establish the antimicrobial activity of probiotic microorganisms and postbiotics (**Figures 2** and **3**).

#### *3.1.4 Cell culture and tissues*

To closely mimic human infection, human cell cultures are infected with indicator pathogen, then treated with probiotic cultures or postbiotics [41].

## **3.2 Experiments for the discovery of antimicrobial mechanisms of probiotics**

The methods used to ascertain probiotic microorganism mechanisms of antimicrobial activity include; the ability to inhibit virulence factors and cell death.

## *3.2.1 Ability to inhibit virulence factors*

The virulence factors of pathogenic microorganisms vary from one microorganism to the other. For example, the virulence factor in bacteria includes adhesion, immunoevasion and immunosuppression, exo-enzymes, and exotoxin, among others [45]. The virulent factors in *Candida* include secretion of hydrolases, yeast to hypha transition, contact sensing, thigmotropism, biofilm formation, phenotypic switching, and range of fitness attributes [27, 37]. The following methods can be used to examine the ability of the probiotic microorganism to inhibit the virulence factors;

## *3.2.1.1 Gene expression levels*

The expression levels of specific genes controlling one or more of these virulence factors can be ascertained when checking for probiotic activity [14, 25, 35, 36, 46–48]. The methods used include microarray analysis, RT-PCR techniques, and western blot [49].

#### *3.2.1.2 Aggregation and coaggregation assay*

Aggregation assay using spectrophotometric autoaggregation and coaggregation is used to ascertain the antimicrobial activity of probiotics [26, 38, 50]. The morphological transition of *C. albicans* that is, germ tube formation contributes to adherence and invasion to the host tissue and increases virulence [51, 52]. Lactobacilli build aggregates and co-aggregates with Candida cells, and this process neutralizes germ tube growth [53]. In addition, the coaggregation protects access of pathogens to a cell receptor and, as a result, inhibit pathogen adhesion which is a prerequisite step for colonization and the subsequent development of disease [26, 44, 50, 54].

#### *3.2.1.3 Antibiofilm Assay*

Biofilm produced by pathogens serves as a physical barrier and increases virulence. Antibiofilm assay includes (a) **static systems** like microtiter plate, Molony biofilm, Calgary biofilm device, biofilm ring test (b) **open systems** such as Kadouri *Probiotics and Postbiotics from Food to Health: Antimicrobial Experimental Confirmation DOI: http://dx.doi.org/10.5772/intechopen.99675*

system, flow cell, perfused (membrane) biofilm fermenter, microfermentors, Modified Robbins Device, sorbarod devices (SBF), drip flow reactor, constant depth film fermenter, microfluidic biochips, rotating disc reactor, BioFlux device, annular reactors, CDC biofilm reactors (c) **microcosm** example airway epithelial cell model, reconstituted human epithelia (RHE), endothelial cells under flow model, Zürich oral biofilm-model, microfluidic coculture model, Zürich burn biofilm-model, multiple Sorbarod devices (MSD) (d) **ex-vivo which include;** candidiasis in the vaginal mucosa, RWV bioreactor, cardiac valve *ex vivo* model, root canal biofilms [55]. Viable colonies can also be used. While fluorescent labeling of biofilm coupled with mathematical labeling is used [41].

#### *3.2.1.4 Exo-enzymes*

The indicator microorganisms are treated with probiotics or CFS. The indicator microorganism is then examined for the ability to produce exo-enzymes on agar plate assays. The agar plate contains a suitable substrate specific to each enzyme activity [56].

#### *3.2.1.5 Electron microscopy*

Scanning electron microscopy and Transmission electron microscope are used to examine cell integrity which includes morphological adherence, distortion, biofilm, or apoptosis [27, 50, 57].

#### *3.2.1.6 Germ tube and hyphal growth inhibition*

The pelleted spores of dermatophytes and dimorphic pathogenic fungus are allowed to develop germ tubes and hyphae. Probiotic or CFS is then added and incubated. Growth is determined by examining germ tubes and hyphae [36, 58].

#### *3.2.1.7 Spore germination inhibition assay*

The pelleted mycelia and probiotic or CFS are added to media and incubated. Samples are withdrawn and microscopically examined. Percentage spore germination is calculated by the following formula [33, 36, 58]:

% spore germination = [Numbers of germinated spores **/**Numbers of total spores] × 100

#### *3.2.1.8 Fluorescent metabolic dyes and Confocal laser scanning microscopy*

The indicator microorganisms are treated with probiotic cultures or CFS then stained with fluorescent dyes according to the manufacturer's instructions. The live or dead cells are counted, and their metabolic activity is ascertained [26, 27]. Live/ dead cells can also be confirmed by viable counts (cfu/ml).

#### *3.2.2 Ability to induce cell death*

A sequence of unique morphological changes outlines apoptosis. These include; visible cell shrinkage, extensive plasma membrane blebbing, chromatin condensation, nuclear fragmentation, formation of apoptotic bodies, which later undergoes decomposition within the phagosome and finally terminates with complete recycling of the components [59, 60]. Accumulation of reactive oxygen species (ROS) decreased membrane potential, biochemical and cytological

responses well known in programmed cell death (PCD), for instance, apoptosis [60]. Very high ROS concentrations induce necrosis [61]. These changes can be used to determine cell integrity. Of the biochemical and cytological methods used to check pathogen cell integrity after treatment with probiotics include but are not limited to; nuclear fragmentation using DAPI/Tunnel [62–67]; *in situ* ligation assay [65]; DNA laddering [65, 66]; externalization of Annexin V/PI by cell membrane [62, 64, 67–70]; mitochondrial and cytosolic calcium [66, 67, 69, 71]; depolarization of the mitochondria using mitochondria membrane potential detection kits for instance, JC fluorescent probes [62, 63, 66–71]; reactive oxygen species (ROS) accumulation [66, 67, 69–71]; detecting cytochrome c in cytoplasm using western blotting or color metric kits [63, 66–69, 71, 72]; cytosol / mitochondria intracellular glutathione [67, 69]lipid peroxidation [67, 69]; potassium release [67] and metacaspace activation detection using kits like CaspACE FITC-VAD-FMK *in situ* Marker [63, 67–69]. The antimicrobial activity of a probiotic microorganism can be assessed using a combination of a number of these methods, which can corroborate the integrity of the indicator pathogen. Careful choice of positive (example, antimicrobial drug) and negative (untreated) controls are important for interpreting the results.

### **3.3 Experiments that confirm the antimicrobial activity of probiotics** *in vivo*

The *in vitro* studies offer required information about antimicrobial agents on susceptibility responses [73], exposure times, and optimal concentrations [74]. However, these studies have their limitations, for instance, the bulk of antimicrobial agents that are active *in vitro* lack significant antimicrobial activity *in vivo,* and vice versa sometimes occurs [73]. The strength of animal models in determining antimicrobial efficacy is that the study can be ascertained at specific body sites, for example, skin, thigh, lung, peritoneum, meninges, and endocardia [74]. Furthermore, antimicrobial agents are altered by host factors such as metabolism and the immune system in an animal model [74]. Consequently, animal models bridge the gap between in vitro and clinical trials [73] and are indispensable for authentication of probiotic antimicrobial activity. In brief, *in vivo* animal models and clinical studies are an absolute requirement to provide proof of beneficial activities of probiotic antimicrobial activity. To achieve this, appropriate infectious models for the two groups are critical. One infected with indicator pathogen and treated with probiotic cultures, and the other group infected with indicator pathogen only (negative control).

### *3.3.1 In vivo experiments on animal models*

The infection route of dermatophytes is strictly dependent on the goal of the study, indicator fungus, and animal disease model of interest. Examples, to study geophilic and anthrophilic dermatophytes; *Microsporum gypseum* and *Trichophyton rubrum* that is difficult to establish infections in laboratory animals', zoophilic dermatophytes especially *Trichophyton metangrophytes* var. *mentagrophtes*, var. *quinckeanum* and var. *granulae*, *Trichophyton verrucosum,* and *Microsporum canis* are used instead. The most recommended animal model for dermatophytoses is hairless guinea pigs as the infection resembles infections in humans, and topical treatment is applicable. Mouse, rat, hamster, and dog are disadvantaged for dermatophytoses animal model since they defecate, lick, and bite itching or irritating lesions intensively [75].

#### *Probiotics and Postbiotics from Food to Health: Antimicrobial Experimental Confirmation DOI: http://dx.doi.org/10.5772/intechopen.99675*

*C. albicans* and *Candida tropicalis* have high virulence in systemically induced mice model [76–79]. Pregnant mice [75], zebrafish [80] and *Caenorhabditis elegans* [46] have also been utilized in disseminated systemic infection models. *Candida metapsilosis* is virulent in the vaginal mouse model [81]. Furthermore, oophectomised rats are used for chronic vaginitis [47, 75]. However, *C. parapsilosis, C. glabrata,* and *C. krusei* do not induce mice mortality [77]. Further, *C. albicans* [82]*, Ctropicalis, C. parapsilosis* complex (*C. parapsilosis, C. orthopsilosis,* and *C. metapsilosis*), are virulent in the invertebrate *Galleria monella* model [77]. Induced immunosuppressed mice in murine oral candidiasis model of choice. To cause the immunosuppressed condition, administration of prednisolone 100 mg per kg [83] or ketamine: xylazine 90-100 mg/kg and 10 mg/kg respectively [84] of body weight administered by injected subcutaneously 24 h before inoculation with Candida orally is given. Additionally, avian and rats species can be used as oral candidiasis models [75, 84]; a summary of these *in vivo* models is given in **Table 1**.


**Table 1.**

*Précis of* in vivo *animal models for dermatomycosis, candidiasis and bacterial infections.*

#### *3.3.2 Clinical trials*

Clinical trials are conducted after promising *in vitro* and *in vivo* animal model experiments. The randomized placebo-controlled clinical trial is the most recommended method [10]. The number of clinical researches conducted on probiotics is about 1000, with *Lactobacillus rhamnosus* GG and *B. animalis* sp. *Lactis* being the most studied [41, 86]. The majority of these studies are on gastrointestinal diseases and the digestive system [86]. However, currently, there is a shift to metabolic disorders, communicable and infection [86]. The primary concerns in these clinical studies that need to be addressed for harmonization of probiotic clinical research include:


It is important to note that, these details including probiotic dosage used in clinical studies, should be extrapolated from *in vitro* and *in vivo* models. Therefore, this emphasizes the importance of prior quality research.

Few clinical trials on confirmation of the antimicrobial effect of probiotics have been reported so far, yet they have been considered the final confirmative experiment. Probiotics are regarded as safe [13, 17]; thus, many researchers skip this critical step. This is the case in which many commercially marketed probiotics have pending clinical studies [92]. Probiotics clinical studies on the management of oral pathogens [9, 21, 93–95], urogenital infections [20, 96–99] and gastrointestinal systems [100] had promising results thus, supporting some probiotics as potential antimicrobial agents [10].

In conclusion, clinical studies are essential. Successful clinical studies require thorough *in vitro* and *in vivo* experiments, especially estimating the dosage,

*Probiotics and Postbiotics from Food to Health: Antimicrobial Experimental Confirmation DOI: http://dx.doi.org/10.5772/intechopen.99675*

duration, and frequency of probiotic administration. Areas that need urgent reporting and harmonization in clinical studies include probiotic viability, probiotic species and strain, dosage (CFU), duration, frequency of administration, and route of probiotics administration.
