The field of probiotics is up-and-coming, especially in management of microbial pathogens. Probiotics confer nutritional benefits, reduce inflammation and infection. Probiotics have also shown to be helpful in the management of microbial pathogens, which include bacteria, fungi, and viruses. To ernes this potential maximumly, there is a need for an elaborate screening system for new isolates. This entails; rigorous screening methods and thorough confirmatory systems. There is need also to come up with standard methods used to evaluate the probiotics mechanism of action both in vivo and in vitro. In summary, there is a need for a standard screening process for probiotic microorganisms that is reproducible. The aim is to ensure that, the candidate microbial cultures are not written off without proper investigations. This will also fasten the screening process and save time and resources wasted in pre-screening experiments.
- screening methods
- confirmatory methods
- animal model
Fermentation is one of the oldest technologies used for food preservation. It involves converting carbohydrates to alcohol, carbon dioxide, and organic acids using microorganisms under anaerobic conditions. The fermentation process improves food by developing diverse flavors, aromas, and textures in food substrates. Also, it enriches food substrates with protein, essential amino acids, essential fatty acids, and vitamins. The primary mechanism of the preservation of foods is the production of acid, which lowers the pH to a level at which most of the spoilage-causing microorganisms cannot grow, hence prolonging the shelf life of such foods . At present, various fermented foods are produced worldwide at household and industrial levels, in both small-scale and large commercial enterprises. Associated with fermentation are beneficial microorganisms known as probiotics. The vast majority of the probiotics are lactic acid microorganisms  to produce fermented dairy products.
Among the beneficial effects of probiotics include improved intestinal health, enhancement of the immune response, reduction of serum cholesterol, and cancer prevention [3, 4, 5]. There is also substantial evidence to support probiotic use in treating acute diarrhoeal diseases, prevention of antibiotic-associated diarrhea, and improvement of lactose metabolism . The range of food products containing probiotic strains is vast and still growing. And so is the list of beneficial effects. More so, with an increasing desire for quality life, preference for minimal use of chemicals, and the rising cost of healthcare. Natural products like probiotics is a promising alternative. Related to probiotics are prebiotics. Biogenics involves the use of beneficial bioactive substances produced by probiotic bacteria whose activities are independent of the viability of probiotic bacteria.
This book chapter focuses on the use of probiotics in the management of microbial pathogens, emphasizing the need to have a reproducible standard screening process both
1.1 Mechanism of action of probiotics
WHO/FAO defines probiotics as
The probiotics have myriad of mechanisms in which it protects against infection. These include; (1) they lower pH, (2) pathogen antagonism by producing antimicrobial compounds for example, bacteriocins and or other metabolic products, (3) competitive exclusion with the pathogen for binding sites and receptors sites, (4) competition for substrates that is, nutrients and growth factors, therefore, limiting resources, (5) stimulate immunomodulatory cells, (6) production of enzymes example, enzymes that neutralize toxins produced by pathogens (7) improve the barrier function of the intestinal mucosa, (8) modulate inflammatory responses, (9) aggregate with pathogens, (10) produce hydrogen peroxide (H2O2) a strong oxidizing agent that damage nucleic acids and proteins, (11) produce organic acids like lactic acid, acetic acid among others (12) produce CO2 thus creating anaerobic microenvironment (Figure 1) [3, 4, 13, 14, 15, 16].
The probiotics are generally regarded as safe . The few results obtained when probiotics are administered together with conventional drugs clinically are promising and include synergy with the drug, half dose of conventional drug needed, and faster healing [18, 19, 20, 21]. Further research is needed in this area.
1.2 Probiotics as antimicrobial agents
Besides the health-improving benefits, the antimicrobial activity of probiotics has been well documented, with promising results against microbial pathogens. Probiotics have been deemed as the following most crucial immune defense systems according to WHO . This is due to increasing antibiotic resistance to commonly prescribed antibiotics [22, 23]. There is a need, therefore, to come up with reproducible screening protocols for
2. Areas in the used technology that need harmonization
To obtain reproducible and conclusive results of probiotic antimicrobial activity, standardization of protocols is essential. This section reviews the essential areas that will inform on the choice of indicator pathogen, probiotic microorganism, inoculum size, incubation time and conditions, and technique of production of postbiotics (also referred to as cell-free supernatant (CFS)/ Biogenic/spent media) used in previous research and the need for harmonization.
2.1 The selection of experimental indicator pathogen
The choice has relied on the target disease that the probiotic is thought to treat. Thus, for vulvovaginal candidiasis
2.2 The choice of probiotic microorganism and postbiotics
The WHO/FAO has listed the criteria for evaluation of probiotic microorganism, which include; the ability of the microorganism to adhere to epithelial cells, bile salts, resist stomach acid and enzymes, persistence within the system, produce antimicrobial compounds, antibiotic resistance profile inability to confer resistance or genome stability and ability to stabilize the normal microbiota among others [7, 15, 30]. Probiotic antimicrobial activity is strain-specific; therefore, the species level and strain of the selected probiotic should be identified.
2.3 Inoculum size
The actual number of viable indicator pathogens in the inoculum size directly influences the outcome. Too little may lead to false-positive results, while too heavy inoculum may give a false negative result [29, 31]. A foundation for the inoculum size can be suggested by CLSI . Researchers have used different inoculum size, incubation temperature, and time for both probiotic and indicator pathogen in
2.4 The experimental conditions and incubation time
Lactic acid bacteria and Bifidobacteria are fastidious; subsequently, the media chosen should have a specific nutrient requirement, for example, growth factors. MRS, which is an appropriate media for the growth of probiotic microorganisms, is widely used. MRS is both a selective and an enriched media for the growth and isolation of only lactic acid bacteria and other bacteria. Therefore, if this medium cannot support the indicator pathogen, for example, dermatophytes (J. ), probiotic growth factors can be incorporated in any media of choice such as potato dextrose agar (PDA), sabouraud dextrose agar (SDA) and nutrient agar (NA) to favor the growth of both the indicator pathogen and the probiotic microorganism. Proper choice of media and specific modifications is key to a successful experiment [33, 34]. Therefore, media supplemented with growth factors should be screened for the ability to grow both probiotic microorganisms and indicator pathogen. We propose that the specific incubation conditions such as time and oxygen requirements for both probiotic microorganisms and indicator pathogen be optimized before the experiment and confirmed by the growth curve of individual microorganisms (Figure 2). Furthermore, fresh media should always be prepared and used for reproductive results, especially in the case of disc diffusion results.
2.5 The technique of production of postbiotics
The postbiotics is also referred to as cell-free supernatant (CFS) or biogenics or spent media. The preparation of postbiotics is varied and attests to the need to harmonize the methods. The process entails the following steps; the probiotic microorganism is inoculated in broth media and incubated in an incushaker . Cells are then removed by centrifuging to obtain the CFS (; J. [33, 35, 37]). The supernatant obtained can then be screened for antimicrobial activity , or the supernatant is further filter-sterilized [35, 36]. The CFS is then used to screen for the microbial activity or concentrated to obtain concentrated CFS (cCFS)   or freeze-dried .
The advantage of using postbiotics is that the properties of the active component can be deduced. To ascertain if the active ingredient is proteinaceous, heat treatment and enzymes are used. If the activity is reduced or is lost compared to non-treated postbiotic, it infers that the antimicrobial agent is proteinaceous [33, 38]. To ascertain if the antimicrobial activity is pH-related, the postbiotic is neutralized and buffered .
3. Technology for investigation and confirmation of the antimicrobial activity of probiotics
3.1 Experiments for investigation of antimicrobial potential of probiotics
Included in this section are the
3.1.1 Antagonism on agar plate methods
184.108.40.206 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 .
220.127.116.11 Spot on agar assay
Probiotic microorganisms are first spotted on agar media and then incubated . An indicator pathogen is added, and soft agar at around 45–50°C is poured to 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.
18.104.22.168 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.
22.214.171.124 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 . This is mainly attributed to the production of non-diffusible antimicrobials.
126.96.36.199 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 .
188.8.131.52 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 . 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 . 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
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 .
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 . The virulent factors in
184.108.40.206 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, 47, 48]. The methods used include microarray analysis, RT-PCR techniques, and western blot .
220.127.116.11 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
18.104.22.168 Antibiofilm Assay
Biofilm produced by pathogens serves as a physical barrier and increases virulence. Antibiofilm assay includes (a)
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 .
22.214.171.124 Electron microscopy
126.96.36.199 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].
188.8.131.52 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
184.108.40.206 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 . Very high ROS concentrations induce necrosis . 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, 63, 64, 65, 66, 67];
3.3 Experiments that confirm the antimicrobial activity of probiotics
In vivoexperiments 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;
|Disease||Animal||Route of infection||Target organ||Reference|
|Dermatomycosis||Guinea pig||Skin abrasion||Skin localized infection|||
|Dermatomycosis||Guinea pig||Intravenous||cutaneous disseminated infection|||
|Bacterial and fungal systemic infection||Skin||media|||
|Bacterial and fungal systemic infection||Pregnant mice||Intravascular||placenta|||
|Bacterial and fungal systemic infection||Injection||systemic||[20, 85]|
|Bacterial and fungal systemic infection||Zebra fish (||Microinjection||disseminated infection|||
|Chronic vaginitis||Rats; oophectomised and kept permanently in pseudoestrous-weekly injection of estrogen||Intravaginal with blastospores||Vaginal swabs||[47, 75]|
|Localized oral candidosis (thrush)||Rats and several avian species||Peroral challenge with blastospores; favored by carbohydrate rich diet, antibiotic treatment and use of germ free or specific pathogen free animals||Mouth swabs||[75, 84]|
3.3.2 Clinical trials
Clinical trials are conducted after promising
The probiotic dosage administered; only 42% of the clinical studies reported dosage correctly. It is recommended that the probiotic dosage is reported in colony-forming unit (CFU). Some clinical studies reported the number of drops, grams, or not indicated at all .
The description of how the probiotic was prepared was incomplete in many studies .
Viability, which is the overall health of cells. It is crucial to check the viability of probiotics before administration and after a given duration since storage, transportation, and handling condition could kill some microorganisms.
It is essential to describe probiotic microorganisms not only to the species level but also to strain. This is because the diversity of probiotic microorganisms is enormous. Further, the probiotic activity is species and strain-specific [88, 89, 90]. This is incomplete in the majority of the clinical studies done. Only 49% of the studies conducted complete strain identification.
Route, frequency, and duration  of probiotic administration should always be reported. Many studies omit this vital aspect.
Sample size affects the power of the study to draw a conclusion and the precision of estimates. Therefore, the sample size should be big enough to reduce bias, especially when some patients discontinue the study.
It is important to note that, these details including probiotic dosage used in clinical studies, should be extrapolated from
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 . Probiotics clinical studies on the management of oral pathogens[9, 21, 93, 94, 95], urogenital infections [20, 96, 97, 98, 99] and gastrointestinal systems  had promising results thus, supporting some probiotics as potential antimicrobial agents .
In conclusion, clinical studies are essential. Successful clinical studies require thorough
4. Summary and future prospects of probiotics as antimicrobial agents
The probiotics are offering a ray of hope to solve dwindling antibiotic efficacy. Further, the number of immunocompromised persons, number of microbial infections and drug resistance, and probiotics could come in handy to solve these problems. Therefore, there is a need for detailed conclusive research on