**Antimicrobial Effect of Probiotics against Common Pathogens**

### Sabina Fijan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63141

#### **Abstract**

The antimicrobial or antagonistic activity of probiotics is an important property that includes the production of antimicrobial compounds, competitive exclusion of pathogens, enhancement of the intestinal barrier function and others. There are many methods to ascertain probiotic properties, including various *in vitro* and *in vivo* methods. The *in vivo* methods include various modifications of the spot‐on lawn assay, agar well diffusion assay (AWDA), co‐culturing methods, usage of cell lines and others. In many cases *in vitro* antagonist activity is observed, but in real settings it is not observed. The *in vivo* methods mainly used are animal models; however, their use is being restricted according to the European legislation OJ L136. The justification of animal models is also questionable as the results of studies on animals do not predict the same results for humans. The use of replacement alternative methods, for example incorporating human cells and tissues, avoids such confounding variables. Most important studies are double‐ blinded randomized clinical trials; however, these studies are difficult to perform as it is not easy to achieve uniform conditions. There is a clear need for more elaborate assays that would better represent the complex interactions between the probiotics and the final host. This complex situation is a challenge for scientists.

**Keywords:** antimicrobial effect, *in vitro* methods, *in vivo* methods, pathogens, probiot‐ ics

### **1. Introduction**

Throughout the history of microbiology, most human studies have been focused on the disease‐ causing organisms found on or in people; whilst fewer studies have examined the benefits of the resident bacteria. However, we are surrounded by beneficial microorganisms that live in or

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

on the human body. The intestinal microbiota is very well adapted, exceptionally stable and very specific for each individual. In normal conditions of stable functioning of the digestive system, neutral and beneficial microorganisms dominate. It is estimated that there are 100 trillion microorganisms in the intestine of a human adult and this is 10 times larger than the number of cells in the human body [1, 2]. However, the balance of the intestinal microbiota is negatively influenced by modern lifestyle, leading to increased numbers of pathogenic microorganisms that disrupt microbial balance and cause a reverse from beneficial to harmful functioning. In such cases, the external support with probiotics is very welcome and supported by several scientific studies [3].

According to the Food and Agriculture Organisation of the United Nations (FAO) and the World Health Organisation (WHO), probiotics are defined as live microorganisms, which when administered in adequate amounts confer a health benefit on the host [4, 5]. The most common probiotic bacteria are certain strains from the genera *Lactobacillus* (i.e., *L. rhamnosus, L. acidophilus, L. plantarum, L. casei, L. delbrueckii subsp. Bulgaricus,* etc.) and *Bifidobacterium* (i.e., *B. infantis, B. animalis* subsp*. lactis, B. longum,* etc.). Other probiotic bacteria include *Pediococcus acidilactici, Lactococcus lactis* subsp*. lactis, Leuconostoc mesenteroides, Bacillus subtilis, Enterococcus faecium, Streptococcus thermophilus*, *Escherichia coli* Nissle 1917, etc. Certain yeasts such as *Saccharomyces boulardii* are also probiotics [6, 7].

Probiotics together with other beneficial microbes are commensals of the gut and differ from pathogenic bacteria in the terms of their actions on immune cells in the gut as they do not stimulate the proliferation of mononuclear cells or trigger an inflammatory action [8]. Regard‐ less of whether the probiotics are used for human or animal consumption, there are several characteristics that a probiotic must achieve. Some of the important characteristics of probiotics include the following: a probiotic must be generally required as safe (GRAS); a probiotic should exhibit bile and acid tolerance in order to survive the path from the oral cavity to the small intestine where it lives, multiplies and excretes beneficial nutrients and molecules; a probiotic should have the ability to adhere to mucus and/or epithelial cells, and/or other surfaces; a probiotic should be susceptible to antibiotics; a probiotic should exhibit antimicrobial activity against pathogens [3, 5, 9, 10]. Although it is accepted that probiotics must be of human origin [4, 5], many authors have found that some strains that are not normally isolated from human have shown to be effective [11, 12], which negates this requirement. As noted above, one of the important attributes of probiotics is their antimicrobial effect against pathogens by maintaining the homeostasis of the intestinal flora. Assessing the antimicrobial effect of various probiotics against pathogenic microorganisms is the guiding concept of this chapter. This chapter reviews the principles and results from various authors of different methods for determining the antimicrobial or antagonistic effect of probiotics against potential pathogens.

### **2. Antimicrobial or antagonistic properties of probiotics**

In literature both the terms "antimicrobial" and "antagonistic" are found to determine the ability of one species to inhibit the growth of another species. According to the online Ency‐ clopaedia Britannica, "antagonism" refers to an "association between organisms in which one benefits at the expense of the other,." However, this encyclopaedia does not include the adverb "antimicrobial." On the other hand, it contains "antimicrobial agent" that refers to "a large variety of chemical compounds that are used to destroy microorganisms or to prevent their development.." The online Merriam Webster dictionary defines "antimicrobial" as "destroy‐ ing or inhibiting the growth of microorganisms and especially pathogenic microorganisms" and "antagonistic" as "showing dislike or opposition: showing antagonism."

This antimicrobial/antagonistic ability is especially important for probiotics as one of the functional beneficial requirements of probiotics is a broad antimicrobial spectrum as well as antagonism against pathogenic bacteria with strong antimicrobial activity. The antagonistic activity of one microorganism against another can be caused by competitive exclusion, immune modulation, stimulation of host defence systems, production of organic acids or hydrogen peroxide that lower pH, production of antimicrobials such as bacteriocins, antioxi‐ dants, production of signalling molecules that trigger changes in gene expression [13–15]. Antimicrobial substances produced by beneficial microorganisms are known to include lactic acid, acetic acid, formic acid, phenyllactic acid, benzoic acid as well as other organic acids, short chain fatty acids, hydrogen peroxide, carbon dioxide, acetaldehyde, acetoin, diacetyl, bacteriocins and bacteriocins‐like inhibitory substances and others [10, 16, 17]. The most common bacteriocins include lacticin, lactocin, pediocin, pisciolin, enterocin, reuterin, plantaricin, enterolysin and nisin [18, 19].

### **3. Methods**

on the human body. The intestinal microbiota is very well adapted, exceptionally stable and very specific for each individual. In normal conditions of stable functioning of the digestive system, neutral and beneficial microorganisms dominate. It is estimated that there are 100 trillion microorganisms in the intestine of a human adult and this is 10 times larger than the number of cells in the human body [1, 2]. However, the balance of the intestinal microbiota is negatively influenced by modern lifestyle, leading to increased numbers of pathogenic microorganisms that disrupt microbial balance and cause a reverse from beneficial to harmful functioning. In such cases, the external support with probiotics is very welcome and supported by several

According to the Food and Agriculture Organisation of the United Nations (FAO) and the World Health Organisation (WHO), probiotics are defined as live microorganisms, which when administered in adequate amounts confer a health benefit on the host [4, 5]. The most common probiotic bacteria are certain strains from the genera *Lactobacillus* (i.e., *L. rhamnosus, L. acidophilus, L. plantarum, L. casei, L. delbrueckii subsp. Bulgaricus,* etc.) and *Bifidobacterium* (i.e., *B. infantis, B. animalis* subsp*. lactis, B. longum,* etc.). Other probiotic bacteria include *Pediococcus acidilactici, Lactococcus lactis* subsp*. lactis, Leuconostoc mesenteroides, Bacillus subtilis, Enterococcus faecium, Streptococcus thermophilus*, *Escherichia coli* Nissle 1917, etc. Certain yeasts such as

Probiotics together with other beneficial microbes are commensals of the gut and differ from pathogenic bacteria in the terms of their actions on immune cells in the gut as they do not stimulate the proliferation of mononuclear cells or trigger an inflammatory action [8]. Regard‐ less of whether the probiotics are used for human or animal consumption, there are several characteristics that a probiotic must achieve. Some of the important characteristics of probiotics include the following: a probiotic must be generally required as safe (GRAS); a probiotic should exhibit bile and acid tolerance in order to survive the path from the oral cavity to the small intestine where it lives, multiplies and excretes beneficial nutrients and molecules; a probiotic should have the ability to adhere to mucus and/or epithelial cells, and/or other surfaces; a probiotic should be susceptible to antibiotics; a probiotic should exhibit antimicrobial activity against pathogens [3, 5, 9, 10]. Although it is accepted that probiotics must be of human origin [4, 5], many authors have found that some strains that are not normally isolated from human have shown to be effective [11, 12], which negates this requirement. As noted above, one of the important attributes of probiotics is their antimicrobial effect against pathogens by maintaining the homeostasis of the intestinal flora. Assessing the antimicrobial effect of various probiotics against pathogenic microorganisms is the guiding concept of this chapter. This chapter reviews the principles and results from various authors of different methods for determining the antimicrobial or antagonistic effect of probiotics against potential pathogens.

**2. Antimicrobial or antagonistic properties of probiotics**

In literature both the terms "antimicrobial" and "antagonistic" are found to determine the ability of one species to inhibit the growth of another species. According to the online Ency‐ clopaedia Britannica, "antagonism" refers to an "association between organisms in which one

scientific studies [3].

192 Probiotics and Prebiotics in Human Nutrition and Health

*Saccharomyces boulardii* are also probiotics [6, 7].

#### **3.1. Methods of literature research**

A literature overview of three databases was conducted using the two following keywords: "probiotic" and "antimicrobial" between the years 1980 and 2016. The search yielded 2882, 1017, and 6200 publications in PubMed, Web of Science, and Science Direct databases, respectively (**Figure 1**).

**Figure 1.** Results of the publications in PubMed, Web of Science and Science Direct databases using keywords: "probi‐ otic" and "antimicrobial" between the years 1980 and 2015.

All three databases showed great increase in the number of publications in the past 10 years. The highest results were obtained via Science Direct due to the fact that this database includes various journals and books from the area of food and dairy sciences, the area of probiotics for animals as well as the area of human probiotics. PubMed on the other hand contains only research of probiotics for humans. Also, the programs for keyword searching for each chosen database seem to differ among each other thus yielding very different numbers of publications in journals, chapters and conference proceedings. The research on the methods for determining the antimicrobial effect of strain‐specific probiotics was conducted by adding an additional keyword aside "probiotic." This keyword described the various investigated methods for determining the antimicrobial/antagonistic activity of probiotics (i.e., spot‐on lawn, agar spot, agar well diffusion, paper disc, co‐culturing, *in vivo*). Of course, it was not possible to screen every single article therefore only a selection of the most recent or relevant were used.

#### **3.2.** *In vitro* **methods for determining the antimicrobial/antagonistic effect of probiotics against other microorganisms**

#### *3.2.1. Spot‐on lawn antimicrobial assay/agar spot antimicrobial assay*

The spot‐on lawn antimicrobial assay has been described by several authors. Several modifi‐ cations of the method have been made. Also various other expressions are used such as agar spot assay, critical dilution assay and deferred antagonism assay [10, 16, 20–22].

One of the simplest published principles of the spot‐on lawn antimicrobial assay (**Figure 2a**) consists of the following steps: different nutrients, selective or differential media, are prepared and various chosen indicator microorganisms or pathogens at different initial concentrations are either inoculated in a confluent manner after hardening of agar or are mixed with the agar in liquid state and poured into the plate. Different dilutions of aliquots of the investigated probiotic or cell‐free supernatant with bacteriocins are then spotted onto the media already inoculated with chosen indicator microorganisms [20, 23, 24].

After incubation, the antimicrobial activity is expressed either as inhibition zone or as arbitrary units (AU/mL). The zone of inhibition is noted either as the diameter or the area of the inhibition zone. The critical dilution is noted as the last dilution that produces a zone of inhibition larger than 6 mm. Arbitrary units are defined as the reciprocal of the highest dilution at which the growth of the indicator microorganism or pathogen is inhibited and are calculated as (1000/*a*) x *D* in AU/mL, where *a* is the aliquot of cell‐free supernatant added to well in μL and *D* is the dilution factor [22, 24].

A modification is the agar spot antimicrobial assay (**Figure 2b**) and consists of the following steps: MRS agar or other specified agar is prepared and the probiotic bacteria or test cultures (few μL) are spotted on. These agars are then incubated to develop spots. Next, the indicator bacteria (pathogenic species, spoilage species and other probiotic species) are mixed into specific soft agar (0.7%) and poured over a plate. The plates are then incubated aerobically or anaerobically and the inhibition zones are read. A clear zone of more than 1 mm around the spot is considered as positive [25]. A third modification is the spot‐on lawn antimicrobial assay with wells (**Figure 2c**), which consists of the following steps: chosen nutrients, selective or differential media, are prepared. Wells (6 mL, 7 mm or other dimensions) are bored in each plate and the bottom of the wells is sealed with agar. Aliquots of active cultures at different dilutions are pipetted into the wells. The plates are left at room temperature to allow migration and settling of the test cultures. The samples are then incubated for 3 h at 37°C and the plates are then overlaid with agar seeded with indicator pathogenic microorganisms (or other indicator organisms) and incubated at suitable incubation conditions. After incubation, the antimicrobial activity is expressed either as inhibition zone or as arbitrary units (AU/mL) [16].

**Figure 2.** Schemes of different versions of spot‐on lawn/agar spot assays: (a) simple spot‐on lawn assay, (b) agar spot assay and (c) spot‐on lawn assay with wells.

The fourth modification is the cross streak assay [26] where each probiotic strain is streaked in three parallel lines onto agar using a 1‐mL loop. Once these lines have dried, test pathogenic strains are streaked perpendicular to these initial strains in the same fashion, giving three possible zones of inhibition for each combination of strains. It was assumed that when there is inhibition, it is caused by the tester probiotic strain hindering the growth of the second‐ streaked (indicator) strain.

#### *3.2.2. Agar well diffusion assay/paper disc assay*

All three databases showed great increase in the number of publications in the past 10 years. The highest results were obtained via Science Direct due to the fact that this database includes various journals and books from the area of food and dairy sciences, the area of probiotics for animals as well as the area of human probiotics. PubMed on the other hand contains only research of probiotics for humans. Also, the programs for keyword searching for each chosen database seem to differ among each other thus yielding very different numbers of publications in journals, chapters and conference proceedings. The research on the methods for determining the antimicrobial effect of strain‐specific probiotics was conducted by adding an additional keyword aside "probiotic." This keyword described the various investigated methods for determining the antimicrobial/antagonistic activity of probiotics (i.e., spot‐on lawn, agar spot, agar well diffusion, paper disc, co‐culturing, *in vivo*). Of course, it was not possible to screen every single article therefore only a selection of the most recent or relevant were used.

**3.2.** *In vitro* **methods for determining the antimicrobial/antagonistic effect of probiotics**

The spot‐on lawn antimicrobial assay has been described by several authors. Several modifi‐ cations of the method have been made. Also various other expressions are used such as agar

One of the simplest published principles of the spot‐on lawn antimicrobial assay (**Figure 2a**) consists of the following steps: different nutrients, selective or differential media, are prepared and various chosen indicator microorganisms or pathogens at different initial concentrations are either inoculated in a confluent manner after hardening of agar or are mixed with the agar in liquid state and poured into the plate. Different dilutions of aliquots of the investigated probiotic or cell‐free supernatant with bacteriocins are then spotted onto the media already

After incubation, the antimicrobial activity is expressed either as inhibition zone or as arbitrary units (AU/mL). The zone of inhibition is noted either as the diameter or the area of the inhibition zone. The critical dilution is noted as the last dilution that produces a zone of inhibition larger than 6 mm. Arbitrary units are defined as the reciprocal of the highest dilution at which the growth of the indicator microorganism or pathogen is inhibited and are calculated as (1000/*a*) x *D* in AU/mL, where *a* is the aliquot of cell‐free supernatant added to well in μL

A modification is the agar spot antimicrobial assay (**Figure 2b**) and consists of the following steps: MRS agar or other specified agar is prepared and the probiotic bacteria or test cultures (few μL) are spotted on. These agars are then incubated to develop spots. Next, the indicator bacteria (pathogenic species, spoilage species and other probiotic species) are mixed into specific soft agar (0.7%) and poured over a plate. The plates are then incubated aerobically or anaerobically and the inhibition zones are read. A clear zone of more than 1 mm around the spot is considered as positive [25]. A third modification is the spot‐on lawn antimicrobial assay with wells (**Figure 2c**), which consists of the following steps: chosen nutrients, selective or

spot assay, critical dilution assay and deferred antagonism assay [10, 16, 20–22].

*3.2.1. Spot‐on lawn antimicrobial assay/agar spot antimicrobial assay*

inoculated with chosen indicator microorganisms [20, 23, 24].

**against other microorganisms**

194 Probiotics and Prebiotics in Human Nutrition and Health

and *D* is the dilution factor [22, 24].

The agar well diffusion assay (AWDA) (**Figure 3a**) is used for determining the antagonistic effects of cell‐free supernatants. The general principle of agar well diffusion assay consists of the following steps: different nutrients, selective or differential media, are prepared. The plates are inoculated with the chosen indicator microorganism. The 6‐mm or 7‐mm wells are bored in each plate. Aliquots of different dilutions of cell‐free supernatants are pipetted into the wells. After incubation, the antimicrobial activity is expressed either as inhibition zone or as arbitrary units (AU/mL) [20, 22]. The paper disc assay (**Figure 3b**) is a modification where instead of making wells, discs measuring 6 mm are absorbed with aliquots of cell‐free supernatant and placed on the agar inoculated with indicator strains. After incubation, the inhibition zone is evaluated based on the clear zone around the paper disc [23].

**Figure 3.** Scheme of the agar well diffusion assay and paper disc assay: (a) agar well diffusion assay and (b) paper disc assay.

#### *3.2.3. Co‐culturing assays for determining the antimicrobial activity*

Determining the antimicrobial activity of probiotics against common pathogens is also possible with the co‐culturing assay. This method includes the following steps: preparation of incuba‐ tion media (i.e., nutrient broth, reconstituted skim milk, sterilized milk, yogurt, whey, etc.). Aliquots of pathogenic and probiotics microorganisms are inoculated into the incubation media. The samples are mixed well and incubated. After incubation, the population of pathogenic bacteria are counted on appropriate agars. Values are usually expressed as log cfu/ mL [14–16, 27, 28].

The microtitre plate assay is a version of the co‐culturing assay that includes the following steps: cell‐free supernatant of active probiotic or other investigated microorganism is prepared and divided into several parts that undergo different conditions (i.e., NaOH added to neu‐ tralize pH, left acidic, heated, etc.). Pathogenic microorganisms are cultured and added to appropriate broth. The microtitre plate is used to prepare mixes of probiotics/cell‐free super‐ natants and pathogenic microorganisms and incubated at suitable incubation conditions. Before and after incubation, the optical density at 620 nm is measured and the suppressive activity is calculated as a percentage of inhibition of pathogen growth [15].

Another important type of co‐culturing assay is using cell lines. As several important mecha‐ nisms underlying the beneficial effects of probiotics include the effects of probiotic properties on specific tissues, particularly on the intestine, the evaluation of probiotic effects on human intestinal cell lines *in vitro* is meaningful as these cells mimic the systemic environment of an organism and are used as a biological matrix alternative to *in vivo* tests. In fact, *in vitro* evidence is particularly important considering that the EU directives tend to discourage *in vivo* experiments on animals [29]. Several different cell lines have been used, such as HT‐29 cell line from human colon [29], IPEC‐J2 porcine neonatal jejunal cell line [30], Vero African green monkey kidney epithelial cell line [31], Caco‐2 colon adenocarcinoma cell line [32], HIEC‐6 normal epithelial small intestine cell line [33], BALB/c3T3 murine embryonal fibroblast cell line [29] and many others. Cells are routinely grown in Dubelco's modified Eagles' medium (DMEM), McCoy's 5a medium or other medium and seeded in well plates or microtitre plates that are incubated at 37°C for 24 h in 1 mL medium in 5% CO2. Probiotic and pathogenic microorganisms are then added and the cell viability is determined after incubation.

the following steps: different nutrients, selective or differential media, are prepared. The plates are inoculated with the chosen indicator microorganism. The 6‐mm or 7‐mm wells are bored in each plate. Aliquots of different dilutions of cell‐free supernatants are pipetted into the wells. After incubation, the antimicrobial activity is expressed either as inhibition zone or as arbitrary units (AU/mL) [20, 22]. The paper disc assay (**Figure 3b**) is a modification where instead of making wells, discs measuring 6 mm are absorbed with aliquots of cell‐free supernatant and placed on the agar inoculated with indicator strains. After incubation, the inhibition zone is

**Figure 3.** Scheme of the agar well diffusion assay and paper disc assay: (a) agar well diffusion assay and (b) paper disc

Determining the antimicrobial activity of probiotics against common pathogens is also possible with the co‐culturing assay. This method includes the following steps: preparation of incuba‐ tion media (i.e., nutrient broth, reconstituted skim milk, sterilized milk, yogurt, whey, etc.). Aliquots of pathogenic and probiotics microorganisms are inoculated into the incubation media. The samples are mixed well and incubated. After incubation, the population of pathogenic bacteria are counted on appropriate agars. Values are usually expressed as log cfu/

The microtitre plate assay is a version of the co‐culturing assay that includes the following steps: cell‐free supernatant of active probiotic or other investigated microorganism is prepared and divided into several parts that undergo different conditions (i.e., NaOH added to neu‐ tralize pH, left acidic, heated, etc.). Pathogenic microorganisms are cultured and added to appropriate broth. The microtitre plate is used to prepare mixes of probiotics/cell‐free super‐ natants and pathogenic microorganisms and incubated at suitable incubation conditions. Before and after incubation, the optical density at 620 nm is measured and the suppressive

Another important type of co‐culturing assay is using cell lines. As several important mecha‐ nisms underlying the beneficial effects of probiotics include the effects of probiotic properties on specific tissues, particularly on the intestine, the evaluation of probiotic effects on human

activity is calculated as a percentage of inhibition of pathogen growth [15].

evaluated based on the clear zone around the paper disc [23].

196 Probiotics and Prebiotics in Human Nutrition and Health

*3.2.3. Co‐culturing assays for determining the antimicrobial activity*

assay.

mL [14–16, 27, 28].

#### **3.3.** *In vivo* **methods for determining the antimicrobial/antagonistic effect of probiotics against other microorganisms**

For *in vivo* testing, randomized double blind, placebo‐controlled human trials should be undertaken to establish the efficacy of the probiotic product. The consultation recognized that there is a need for human studies in which adequate numbers of subjects are enrolled to achieve statistical significance. In order to ascertain that a given probiotic can prevent or treat a specific pathogen infection, a clinical study must be designed to verify exposure to the said pathogen (preventive study) or that the infecting microorganism is that specific pathogen (treatment study). If the goal is to apply probiotics in general to prevent or treat a number of infectious gastroenteritis or urogenital conditions, the study design must define the clinical presentation, symptoms and signs of infection, and include appropriate controls [4].

However, many scientists have reverted to *in vivo* animal studies. The animal models do not necessarily provide scientifically appropriate and relevant results for human, due to obvious species‐specific differences in anatomy, biochemistry, physiology, pharmacokinetics and toxic responses. Especially, in medicine and pharmacy, the "safety testing" on animals led to thousands of deaths worldwide due to the evidence that animal tests are not only worthless, but they are also dangerously unpredictable. The use of replacement alternative methods, especially incorporating human cells and tissues, avoids such confounding variables [32].

The European legislation OJ L136 of 08.06.2000 includes the 3Rs regulation that results in important reduction of studies on animal models and consists of the following. *Reduction*: using alternative methods for obtaining comparable levels of information from the use of fewer animals in scientific procedure or for obtaining more information from the same number of animals. *Refinement*: using alternatives methods that alleviate or minimise potential pain, suffering and distress, and which enhance animal wellbeing. *Replacement*: using alternative methods that permit a given purpose to be achieved without conducting experiments or other scientific procedures on animals [32].

The *in vivo* animal model antimicrobial study is described as follows: briefly, all animal models include at least two groups under controlled settings. One group receives chosen probiotic and pathogen (treated infected group) and the other receives only the pathogen (untreated infected group). The observed difference includes the exanimation of faeces as well as the examination of different cells after scarifying the animals (spleens, lymph nodes, blood, liver, colon, cecum, etc.). Animals used in these studies include mice, rats, chicks, rabbits, pigs, Fish and even worms [30, 34–37]. Work is done under accordance with the guidelines of the European convention for the protection of vertebrate animals used for experimental and other scientific purposes (Directive 86/609/EEC).

### **4. Recent results of** *in vitro* **antimicrobial/antagonistic assays for various probiotic strains**

The following section contains results of the antimicrobial/antagonistic assays for various probiotic strains or strains with probiotic‐like properties against various potential pathogens, spoilage microorganisms, or other probiotic microorganisms. The results reported using different assays (spot‐on lawn/agar spot, agar well diffusion/paper disc, co‐culturing, micro‐ titre plate and cell line assays) are published by various authors stated in the text and some of the individual procedures are briefly explained.

#### **4.1. Recent results of selected spot‐on lawn/agar spot antimicrobial assays**

The antimicrobial activity of *Lactobacillus plantarum* EM against seven potential pathogens using the spot‐on‐lawn assay was conducted by Choi and Chang [10]. The following nutrient and selective media were used: Luria‐Bertani agar for *E. coli* O157:H7 ATCC 43895, *P. aerugi‐ nosa* ATCC 27853, *S. enterica* serovar Typhi ATCC 19430, nutrient agar supplemented with 2% NaCl for *V. parahaemolyticus* ATCC 17802 and tryptic soy agar for *B. cereus* KCTC 3624, *M. luteus* ATCC 15307 and *S. aureus* ATCC 29123. All potential pathogenic bacteria had an initial inoculum of 6 log cfu/mL. An aliquot of 10 μL of *L. plantarum* EM was spotted onto each plate. The plates were incubated aerobically at 37°C. After incubation the arbitrary units (AU/mL) were determined. *L. plantarum* EM exhibited strong antimicrobial activity against the seven chosen potential pathogenic bacteria. The strongest activity was noted against *V. parahaemo‐ lyticus*, ATCC 17802 (25600 units) and the weakest activity was noted for *S. aureus*, ATCC 29123 (200 units). As one of the most important requirements of probiotics is a broad antimicrobial spectrum, the authors found that *L. plantarum* EM fulfilled the beneficial requirements of probiotics [10].

In another research [24], screening for bacteriocins using the spot‐on lawn method was used. One hundred and fifty lactic acid bacteria were isolated from samples of traditional fermented Vietnamese pork. The isolate named *L. plantarum* KL‐1 was found to produce bacteriocins, effective against various Gram‐positive and Gram‐negative bacteria. The test was conducted by preparing two layers of soft agar (0.8% agar). The first layers were poured into the plate; then the top layer, which included 5 mL of soft agar together with 10 μL (about 107 cfu/mL) of freshly grown test bacterial strains, was added. The Gram‐positive strains included *S. aureus* TISTR 118, *E. faecalis* JCM 5803, *E. faecalis* TISTR 888, *L. lactis* subsp. *cremoris* TISTR1344, *L. mesenteroides* JCM 6124, *L. mesenteroides* TISTR 942, *L. sakei* subsp. *sakei* JCM 1157, *L. sakei* TISTR 890, *L. plantarum* ATCC 14917, *L. inoccua* ATCC 33090, *Streptococcus* sp. TISTR 1030, *B. coagu‐* *lans* JCM 2257, *B. coagulans* TISTR 1447 and *B. campeatris* NBRC 11547. The Gram‐negative test strains included *P. fluorescens* TISTR 358, *P. fluorescens* JCM 5963 and A. *hydrophila* TISTR 1321. Bacteriocin activity was tested by spotting 10 μL of previously prepared cell‐free supernatant (in different dilutions) of the isolate *L. plantarum* KL‐1. The inhibition zone was observed after overnight incubation at the proper temperature for each indicator microorganisms and the spectrum was expressed at arbitrary units (AU) [22]. The results show that the antimicrobial activity of the bacteriocin was strain specific. The bacteriocin was most effective against both strains of *L. sakei*. The bacteriocin was less effective against certain Gram‐positive bacteria (both strains of *E. faecalis*, *L. plantarum* and both strains of *L. mesenteroides* subsp. *mesenteroides*). However, it was not effective against *S. aureus*, both strains of *B. coagulans* and any of the chosen Gram‐negative bacteria.

of different cells after scarifying the animals (spleens, lymph nodes, blood, liver, colon, cecum, etc.). Animals used in these studies include mice, rats, chicks, rabbits, pigs, Fish and even worms [30, 34–37]. Work is done under accordance with the guidelines of the European convention for the protection of vertebrate animals used for experimental and other scientific

**4. Recent results of** *in vitro* **antimicrobial/antagonistic assays for various**

The following section contains results of the antimicrobial/antagonistic assays for various probiotic strains or strains with probiotic‐like properties against various potential pathogens, spoilage microorganisms, or other probiotic microorganisms. The results reported using different assays (spot‐on lawn/agar spot, agar well diffusion/paper disc, co‐culturing, micro‐ titre plate and cell line assays) are published by various authors stated in the text and some of

The antimicrobial activity of *Lactobacillus plantarum* EM against seven potential pathogens using the spot‐on‐lawn assay was conducted by Choi and Chang [10]. The following nutrient and selective media were used: Luria‐Bertani agar for *E. coli* O157:H7 ATCC 43895, *P. aerugi‐ nosa* ATCC 27853, *S. enterica* serovar Typhi ATCC 19430, nutrient agar supplemented with 2% NaCl for *V. parahaemolyticus* ATCC 17802 and tryptic soy agar for *B. cereus* KCTC 3624, *M. luteus* ATCC 15307 and *S. aureus* ATCC 29123. All potential pathogenic bacteria had an initial inoculum of 6 log cfu/mL. An aliquot of 10 μL of *L. plantarum* EM was spotted onto each plate. The plates were incubated aerobically at 37°C. After incubation the arbitrary units (AU/mL) were determined. *L. plantarum* EM exhibited strong antimicrobial activity against the seven chosen potential pathogenic bacteria. The strongest activity was noted against *V. parahaemo‐ lyticus*, ATCC 17802 (25600 units) and the weakest activity was noted for *S. aureus*, ATCC 29123 (200 units). As one of the most important requirements of probiotics is a broad antimicrobial spectrum, the authors found that *L. plantarum* EM fulfilled the beneficial requirements of

In another research [24], screening for bacteriocins using the spot‐on lawn method was used. One hundred and fifty lactic acid bacteria were isolated from samples of traditional fermented Vietnamese pork. The isolate named *L. plantarum* KL‐1 was found to produce bacteriocins, effective against various Gram‐positive and Gram‐negative bacteria. The test was conducted by preparing two layers of soft agar (0.8% agar). The first layers were poured into the plate;

freshly grown test bacterial strains, was added. The Gram‐positive strains included *S. aureus* TISTR 118, *E. faecalis* JCM 5803, *E. faecalis* TISTR 888, *L. lactis* subsp. *cremoris* TISTR1344, *L. mesenteroides* JCM 6124, *L. mesenteroides* TISTR 942, *L. sakei* subsp. *sakei* JCM 1157, *L. sakei* TISTR 890, *L. plantarum* ATCC 14917, *L. inoccua* ATCC 33090, *Streptococcus* sp. TISTR 1030, *B. coagu‐*

cfu/mL) of

then the top layer, which included 5 mL of soft agar together with 10 μL (about 107

**4.1. Recent results of selected spot‐on lawn/agar spot antimicrobial assays**

purposes (Directive 86/609/EEC).

198 Probiotics and Prebiotics in Human Nutrition and Health

the individual procedures are briefly explained.

**probiotic strains**

probiotics [10].

Tharmaraj and Shah [16] used the spot‐on‐lawn technique with wells to test the inhibition of chosen pathogenic bacteria (*E. coli*, *S. typhimurium*, *S. aureus* and *B. cereus*) and spoilage bacteria (*B. stearothermophilus* and *P. aeruginosa*) by one strain of *L. casei* (Shirota YLC); two strains of *L. paracasei* subsp. *paracasei* (LCS1, LC01), *L. acidophilus* (LA5, LAC1) and *B. animalis* (BB12, BLC1); three strains of *P. freudenreichii* subsp. *shermanii* (P, PS1, PB10360) and four strains of *L. rhamnosus* (LC705, LBA, LGG, LR1524). MRS agar was used for lactobacilli (*L. acidophilus*, *L. casei*, *L. paracasei* subsp. *paracasei* and *L. rhamnosus*), MRS agar + L‐cysteine (0.05%) for *B. animalis*, NaLa agar for *P. freudenreichii* subsp. *shermanii* and nutrient agar was used for pathogenic bacteria. Suitable agar (25 mL) was poured into the plates and wells were cut with a sterile metal borer. The bottom of the wells was sealed with 0.8% agar. Active culture (50 μL) of producing probiotics was then filled into the wells and left at room temperature for 2 h, followed by incubating at 37°C for 3 h. The remaining depth of the well was sealed with 1% agar. Finally, the spotted plates were overlaid with 10 mL of 0.8% agar seeded with about 10<sup>7</sup> cfu/mL of pathogenic bacteria. The plates were incubated anaerobically for 72 h at 37°C for all chosen pathogenic and spoilage bacteria. Both chosen spoilage bacteria were additionally incubated aerobically for 24 h at 37°C. On average, among all the probiotic and spoilage bacterial interactions, the spore formers were inhibited by the probiotic microorganisms to a greater extent (average zone of inhibition, 19 mm) than the non‐spore formers (average zone of inhibition, 14 mm). Also, the Gram‐positive bacteria (average zone of inhibition, 18 mm) were inhibited more than the Gram‐negative bacteria (average zone of inhibition, 14 mm). Strains of *P. freudenreichii* subsp. *shermanii* did not show notable inhibitory effect.

Soomro et al. [23] also used the spot‐on lawn method to determine antimicrobial activity of various *Lactobacillus* species. They found that *L. acidophilus* J1 showed an inhibitory effect against *E. coli*. The plates were prepared by inoculating 100 μL of indicator strain *E. coli* grown in broth with 3.5 mL soft MRS agar and were overlaid over MRS agar. The plates were incubated for 2 h at 37°C after which 30 μL of cell‐free supernatant of *L. acidophilus* J1 was spotted onto the overlaid surface. The pH of the cell‐free supernatant was adjusted to 5.5 to eliminate the effect of organic acids and hydrogen peroxide. The plates were incubated at 37°C for 18 h and were subsequently examined for inhibition zones. It was found that the inhibitory effect against *E. coli* was due to the production of a bacteriocin.


Where probiotics are divided as follows: lb: lactobacilli; bb: Bifidobacterium; oth: other; and pathogens are divided as follows: G+: Gram positive; G−: Gram negative; fng: fungi.

**Table 1.** A selection of assays published since 2013 of successful antimicrobial activity of chosen probiotics using the spot‐on lawn/agar spot assay on chosen pathogenic microorganisms.

Assays published since 2013 of antimicrobial activity of chosen probiotics using the spot‐on lawn/agar spot assay on chosen pathogenic microorganisms are noted in **Table 1**.

**Probiotic strains or strains with probiotic potential with efficient antimicrobial**

200 Probiotics and Prebiotics in Human Nutrition and Health

**Lb:***L. acidophilus* La‐5, **Bb:** *B. longum* ATCC

**Lb:***L. acidophilus* JN188382, *L. fermentum* JN188383, *L. fermentum* JN188384, *L. buchneri* JN188385, *L. buchneri* JN188386, *L. buchneri* JN188387, *L. casei* JN188388, *L. casei* JN188389,

**Oth:***E. faecium* CV1, LPP29, *W. cibaria* P71, *L.*

**Oth:***B. subtilis* DCU, *B. pumilus* BP, *B. cereus*

**Lb:***L. plantarum* CK06, CK19, B01, B07, K09, K10, K21, LM11, ZS07, ZS11 and ZS15

follows: G+: Gram positive; G−: Gram negative; fng: fungi.

spot‐on lawn/agar spot assay on chosen pathogenic microorganisms.

*lactis* subsp. *cremoris* SMF110, *Lc. mesenteroides* subsp. *cremoris* SMM69, *P.*

*pentosaceus* SMM73, TPP3

**Lb:***L. casei* C1; *L. plantarum* C4 **G+:***L. monocytogenes*

**Indicator pathogenic microorganisms Reference**

[35]

[38]

[42]

[43]

[44]

[45]

[47]

[48]

**G‐:***S.* Typhimurium CECT 4157, *Y. enterocolitica* IP383

*parahaemolyticus* DMST 5665, *S. dysenteriae* DMST 15111

**G+:***S. aureus, L. monocytogenes;***G‐:***E. coli* O157:H7 [41]

**G+:***E. faecium* ATCC 51558, *S. epidermidis* ATCC *12228*, *P. acnes ATCC 6919, L. monocytogenes, S. aureus* S244; **G‐:** *E. coli ATCC 29181, K. pneumoniae* K36, *E. cloacae, S. sonnei ATCC 25931, H. pylori ATCC 43579, V. parahaemolyticus,* **fng:** *C. albicans* ATCC

**G‐:***T. maritimum* NCIMB2154*,* LL01.8.3.8, *V. splendidus*

*19119;***G‐:***E. coli ATCC 25921, S. enterica, P. aeruginosa*

**G+:***S. aureus SSV25, S. epidermidis SSV30, S. lentus CCM 3472, E. faecalis* V583, *L. monocytogenes* CCM *4699,* **G‐:** *A. calcoaceticus* CCM *4503; S. paucimobilis* CCM *3293; S. enterica*

**G‐:***V. parahaemolyticus* [46]

**Lb:***Lactobacillus* MSMC64‐1 **G+:** MRSA DMST 20651, 20654, **G‐:** *S.* Typhi DMST 5784, *V.*

44831

**Lb:***L. plantarum* P6 **G+:***S. aureus ATCC 25923, B. cereus, L. ivanovii ATCC*

**Oth:***E. faecalis* AP‐216, *E. faecalis* AP 45 **G+:***C. perfringens* KCTC 3269, KCTC 5100, *L. monocytogenes*

**Lb:** LAB 18, LAB 48 **G‐:***S. enterica* serovar Enteritidis phage type 13A, *E. coli* O157:H7, *C. jejuni*

KCTC 3569, 3586, 3710

*subsp. enterica* TA100 CCM 3812

**Lb:***L. fermentum* PXN 44, *L. plantarum* PXN 47 **G+:***E. faecalis* NCTC 00775; **G‐:** *E. coli* NCTC 9001 [49] **Lb:***L. paraplantarum* FT259 **G+:***L. monocytogenes* IAL 633, *L. innocua* ATCC 3309 [50]

Where probiotics are divided as follows: lb: lactobacilli; bb: Bifidobacterium; oth: other; and pathogens are divided as

**Table 1.** A selection of assays published since 2013 of successful antimicrobial activity of chosen probiotics using the

CECT528, DMC‐1

**Oth:***S.cerevisiae* JCM7255 **G+:***S. agalactiae* [39] **Oth:***B. pumilus* B16, *B. mojavensis* J7 **G‐:***V. parahaemolyticus* [40]

**activity** *in vitro*

15707

HL7

*L. casei* JN188390

As noted in **Table 1**, the most common investigated probiotic strains or strains with probiot‐ ic potential were from the genus *Lactobacillus* (*L. plantarum, L. acidophilus, L casei* and *L. fermentum*). Several studies included probiotic strains of the genus *Bacillus*. The most com‐ mon pathogens included in the assays were *S. aureus*, *L. monocytogenes*, *E. coli*, *Vibrio* spp. and *Salmonella* spp. One study examined the antimycotic properties of probiotics against various strains of the genus *Aspergillus*. Different strains of *E. faecium* were on the one hand used as probiotics and on the other hand used as potential pathogen, thus proving the dualistic nature of this species.

#### **4.2. Recent results of selected agar well diffusion assays/paper disc methods**

The agar well diffusion assay was conducted in the research by Ali et al. [51], where 14 isolates with probiotic potential were screened for antimicrobial activity against *E. coli* and *S. aureus*. The probiotic isolates were identified as two *Lactobacillus* spp. (S2a3 and S4b1), eleven *Bifidobacterium* spp. (FCb1, Kb2, LZa7, LZb8, RC1b8, RC2b4, RC4a3, RC4b2, SCa4, SCb2 and Y2a5) and one *Streptococcus* spp. (RC2b3). A volume of 100 μL of cell‐free supernatant of isolates with probiotic potential was filled in 7‐mm wells cut in nutrient agar previously inoculated with *E. coli* or *S. aureus*. The diameter of inhibition zone was measured after 48 h of incubation. The supernatant obtained from all 14 isolates exhibited varying degrees of inhibitory activity against *S. aureus* and *E. coli*. The isolates LZb8, S4b1 and RC4a3 exhibited the superior antibacterial activity with inhibition zones ranged 8.3–8.4 mm. The least activity was recorded for the isolates SCa4 and RC4b2 (inhibition zone ranged 2.3–2.5 mm), whereas the isolates Kb2, LZa7, RC2b4, RC2b3, SCb2 and Y2a5 (inhibition zone ranged 3.5–4.8 mm) were moderately active against *S. aureus*. It is worth mentioning that the inhibitory activity of the tested isolates supernatants was slightly less against *E. coli* as compared to that obtained against *S. aureus*, indicating that *E. coli* could be less sensitive.

In the previously mentioned research by Soomro et al. [23], the paper disc method was also used. Sterile filter discs measuring 6‐mm diameter with an absorbed aliquot of 20 μL of cell‐ free supernatant of *L. acidophilus* J1 were placed on MRS and nutrient agar plates containing the target strain *E. coli*. After incubation at 37°C, the inhibitory activity was evaluated. It was found that the paper disc assay yielded an inhibition zone of 10 mm and was more appropri‐ ate compared to the spot‐on lawn assay.

Assays published since 2013 of antimicrobial activity of chosen probiotics using the agar well diffusion assay or the paper disc assay on chosen pathogenic microorganisms are noted in **Table 2**. The results show that the agar well diffusion assay or the paper disc assay is the most common method used for determining the antimicrobial or antagonistic effect. The most common investigated probiotic strains or strains with probiotic potential were again from the genus *Lactobacillus*. Some assays included bifidobacteria and bacteria from the ge‐ nus *Pediococcus* and *Lactococcus*. The most common pathogens included in the assays were

again *S. aureus*, *L. monocytogenes*, *E coli*, *Vibrio* spp., *Aeromonas* spp. *Salmonella* spp. and *Pseu‐ domonas aeruginosa*. One study even assessed the antagonistic activity of probiotics against herpes simplex virus types 1 and 2 and one study investigated the antimicrobial activity of probiotics against the protozoa *Giardia lamblia*. However, using cell‐free supernatants does not mimic real conditions. Therefore, further assays are necessary.



again *S. aureus*, *L. monocytogenes*, *E coli*, *Vibrio* spp., *Aeromonas* spp. *Salmonella* spp. and *Pseu‐ domonas aeruginosa*. One study even assessed the antagonistic activity of probiotics against herpes simplex virus types 1 and 2 and one study investigated the antimicrobial activity of probiotics against the protozoa *Giardia lamblia*. However, using cell‐free supernatants does

**Indicator pathogenic microorganisms Reference**

[29]

[34]

[35]

[38]

[52]

[55]

[56]

[57]

**G+:***S. aureus* ATCC 6538, *E. faecalis* ATCC 29212, **G‐:** *E. coli* ATCC 25922, *P. aeruginosa* ATCC 9027, **fng:** C. *albicans* ATCC

**G+:***S. aureus* ATCC 29213, **G‐:** *E. coli* K88, 25922 and 1569, *S. Enteritidis* ATCC 13076, *S*. Typhimurium ATCC 14082

**G+:***L. monocytogenes,***G‐:***E. coli* C17, *S. enterica* ser Typhimurium

**G+:***S. aureus, L. monocytogenes,***G‐:***E. coli* O157:H7 [41]

**G‐:***P. aeruginosa* PTCC 1430 [53]

*parahaemolyticus* DMST 5665, *S. dysenteriae* DMST 15111

KCTC 191712, *L. monocytogenes* KACC1076420, *B. cereus* KACC11240 **G‐:** *S.* Typhi KCTC2514, *S. choleraesuis*

**G+:***S. aureus* ATCC 6538, *L. monocytogenes* DSM 12464, *E.*

**fng:***A. niger* PTCC 5012, *A. flavus* PTCC 5004, *A. parasiticus*

KCTC293215, *S. gallinarum* KCTC293126, *S. boydii* KACC10792 14, *Y. enterocolitica* KACC1532020, *E. coli* O138KCTC261511,

ATCC 43888, *S. enterica* ser *Enteritidis* CIP 81.3

O1KCTC2441, *P. aeruginosa* KCCM 1180218,

*faecalis*, **G‐:** *E. coli* ATCC 25922

PTCC 5286, *P. chrysogenum* PTCC 5035

CECT4156, *Y. enterocolitica* IP383

**Lb:***Lactobacillus* MSMC64‐1 **G+:** MRSA DMST 20651, 20654, **G‐:** *S.* Typhi DMST 5784, *V.*

**Lb:***L. plantarum* WCFS1, *L. plantarum* NA7 **G+:***L. monocytogenes* CIP 81.3 ILSI NA 39, **G‐:** *E. coli* O157:H7

**Oth:***B. amyloliqufaciens* **G+:***C. difficile* [54] **Oth:***P. pentosaceus* KID7 **G+:***S. aureus* KCCM1133515, MRSA CCM40510, *S. epidermidis*

not mimic real conditions. Therefore, further assays are necessary.

10231

**Probiotic strains or strains with probiotic potential with efficient antimicrobial**

202 Probiotics and Prebiotics in Human Nutrition and Health

**Lb:***L. acidophilus* PBS066, *L. fermentum*

*L. plantarum* PBS067, *L. rhamnosus* PBS070, *L. reuteri* PBS072, **Bb:** *B. animalis* subsp. *lactis* PBS075, *B. longum* subsp. *longum* PBS108

**Lb:***L. salivarius* JM41, JK21V, JM31, JS2A, JM14, JK22, JM2A1 and JM32, *L. plantarum PZ01* **Oth:** *P. acidilactici* JM241 and JH231, *P.*

*pentosaceus* JS233, *E. faecium JS11*

C1, *L. plantarum* C4, *L. acidophilus*

**Lb:***L. acidophilus* La‐5, **Bb:** *B. longum*

**Lb:***L. casei* PTCC 1608, *L. rhamnosus* PTCC

**Lb:***L. rhamnosus* FM13, FM14, FM22, FS2, FS10, PS2, PS11, SF6, SP13, *L. paracasei* CM1,

**Lb:** L. *casei* LC‐01, *L. acidophilus* LA‐5, *L.*

CM2, MF5, PM8

*paracasei*

**activity** *in vitro*

PBS073,

**Lb:***L. casei*

ATCC15707

1637

Where probiotics are divided as follows: lb: lactobacilli; bb: Bifidobacterium; oth: other; and pathogens are divided as follows: G+: Gram positive; G−: Gram negative; fng: fungi; prs: parasite.

**Table 2.** A selection of assays published since 2013 of successful antimicrobial activity of chosen probiotics using the agar well diffusion assay or the paper disc assay on chosen pathogenic microorganisms.

#### **4.3. Recent results of selected co‐culturing assays**

The influence of the potential pathogenic bacteria *P. aeruginosa* ATCC 27853 against various combinations of probiotic supplements and the kefir microbiota was investigated using the co‐culturing method [6]. One multispecies, one oligospecies and one monospecies probiotic supplement as well as kefir microbiota from kefir grains originally from the Caucasian mountains were used. The multispecies supplement contained *L.* acidophilus (NIZO 3678; NIZO 3887), *L. paracasei* NIZO 3672, *L. plantarum* NIZO 3684, *L. rhamnosus* NIZO 3689, *L. salivarius* NIZO 3675, *B. bifidum* NIZO 3804, *B. lactis* NIZO 3680 and *E. faecium* NIZO 3886. The oligospecies supplement contained *L. acidophilus* LA‐5*, B. infantis* BB‐12 and *E. faecium.* The monospecies supplement contained *L. reuteri* DSM 17938. Co‐culturing was conducted by adding 1 mL of probiotic samples and 1 mL of overnight *P. aeruginosa* suspension in 40 mL previously sterilized bovine milk. Samples were incubated for 4 days at 25°C with agitation (250/min). After incubation, serial 10‐fold dilutions were prepared for all samples and the *P. aeruginosa* populations were enumerated on cetrimide agar with added glycerol. It was found that the *P. aeruginosa* population in milk without probiotics reached an average of 9.2 log/mL, whilst the *P. aeruginosa* population in milk reached 5.2, 8.3, 8.3 and 5.0 for the samples with the multispecies, oligospecies and monospecies probiotic supplement as well as the kefir microbiota respectively. This research thus found that the decrease of the potential pathogen *P. aeruginosa* was dependent on the type of probiotic microbiota and that both multispecies microbiota (multispecies probiotic supplement kefir microbiota) with a much more diverse population than the other two samples (oligospecies probiotic supplement and the monospe‐ cies supplement) exhibited an efficient synergistic effect [7]. Both samples also exhibited a higher increase in the total population of anaerobic microorganisms after fermentation with *P. aeruginosa* thus indicating that a more successful quorum‐sensing regulatory network was established and yielded antagonistic effects against the potential pathogen *P. aeruginosa*.

Tharmaraj and Shah [16], as already mentioned in the previous section, also investigated the inhibition effect of various probiotics against chosen pathogenic and spoilage bacteria with the co‐culturing method. Briefly, 9 mL of reconstituted skim milk was inoculated with 1 mL of overnight culture of probiotic bacteria and 0.1 mL of pathogenic or spoilage bacteria. The medium was mixed well and incubated at 37°C for 24 h. / were counted on nutrient agar and the log population calculated (**Table 6**). All four pathogenic bacteria were inhibited by all probiotic strains tested to varying degrees. On average, the probiotic bacteria reduced the population of pathogenic bacteria by 2.8 log units. *B. cereus* was inhibited to a greater degree by all probiotic bacteria and strains than other pathogenic bacteria. On average, the inhibito‐ ry effect of all probiotic bacteria and strains was the weakest against *E. coli*. *S. aureus* was inhibited to a greater degree by *B. animalis* and *L. rhamnosus* than the other probiotic bacteria.

Ratsep et al. [15] published their research of a microtitre plate assay on the antimicrobial effect of *L. plantarum* (five strains: N11, N27, N33, N44 and E) supernatant against various *C. difficile* strains (six clinical isolates from Norwegian patients, six clinical isolates from Estonian patients and two reference strains: VPI 10463 and M13042). Overnight *C. difficile* cultures were added to BHI broth with a density according to McFarland 3.0. Various reaction mixes were pre‐ pared (natural (acidic), neutral (pH 6.0) and neutral, heated for 20 min at 100°C) and incubat‐

ed under anaerobic conditions for 48 h at 37°C. The optical density at 620 mm was measured at the beginning of incubation and after 48 h and the suppressive activity of *L. plantarum* strains calculated as a percentage of inhibition. The highest inhibitions of *C. difficile* growth were in the samples of heated neutralized supernatants and the lowest in cases of the neutralized samples. There was statistically higher inhibition in heated neutralized supernatants versus neutralized supernatants of N11 and E56 lactobacilli strains. When comparing antagonistic activity of *L. plantarum* strains, there was a relevant difference only between N11 and N33 strains in the samples of heated neutralized supernatants. The neutralization of supernatant did not reduce its inhibitory effect. Thus, lowering the pH of the environment is not the main mechanism in inhibition of *C. difficile* by lactobacilli. Also heating of supernatant did not reduce its activity; thus, some thermostable compounds may be involved in the inhibition. The above‐ mentioned authors [15] also performed the co‐culturing assay with the *L. plantarum* strains and *C. difficile* strains by inoculating 50 mL of brain heart infusion broth with 50 μL of lactobacilli suspension and 50 μL of *C. difficile* suspension and incubating under anaerobic conditions for 48 h at 37°C. After incubation, serial 10‐fold dilutions were prepared and the *C. difficile* populations were enumerated on fastidious anaerobe agar. It was found that the five *L. plantarum* strains (N11, N27, N33, N44 and E) were able to inhibit the growth of the 14 *C. difficile* strains (six clinical isolates from Norwegian patients and Estonian patients and two refer‐ ence strains: VPI 10463, M13042) in the co‐culture incubation as the average log cfu/mL after 48 h was 3.0, whereas the average log cfu/mL of the *C. difficile* strains alone was 7.0.

**4.3. Recent results of selected co‐culturing assays**

204 Probiotics and Prebiotics in Human Nutrition and Health

The influence of the potential pathogenic bacteria *P. aeruginosa* ATCC 27853 against various combinations of probiotic supplements and the kefir microbiota was investigated using the co‐culturing method [6]. One multispecies, one oligospecies and one monospecies probiotic supplement as well as kefir microbiota from kefir grains originally from the Caucasian mountains were used. The multispecies supplement contained *L.* acidophilus (NIZO 3678; NIZO 3887), *L. paracasei* NIZO 3672, *L. plantarum* NIZO 3684, *L. rhamnosus* NIZO 3689, *L. salivarius* NIZO 3675, *B. bifidum* NIZO 3804, *B. lactis* NIZO 3680 and *E. faecium* NIZO 3886. The oligospecies supplement contained *L. acidophilus* LA‐5*, B. infantis* BB‐12 and *E. faecium.* The monospecies supplement contained *L. reuteri* DSM 17938. Co‐culturing was conducted by adding 1 mL of probiotic samples and 1 mL of overnight *P. aeruginosa* suspension in 40 mL previously sterilized bovine milk. Samples were incubated for 4 days at 25°C with agitation (250/min). After incubation, serial 10‐fold dilutions were prepared for all samples and the *P. aeruginosa* populations were enumerated on cetrimide agar with added glycerol. It was found that the *P. aeruginosa* population in milk without probiotics reached an average of 9.2 log/mL, whilst the *P. aeruginosa* population in milk reached 5.2, 8.3, 8.3 and 5.0 for the samples with the multispecies, oligospecies and monospecies probiotic supplement as well as the kefir microbiota respectively. This research thus found that the decrease of the potential pathogen *P. aeruginosa* was dependent on the type of probiotic microbiota and that both multispecies microbiota (multispecies probiotic supplement kefir microbiota) with a much more diverse population than the other two samples (oligospecies probiotic supplement and the monospe‐ cies supplement) exhibited an efficient synergistic effect [7]. Both samples also exhibited a higher increase in the total population of anaerobic microorganisms after fermentation with *P. aeruginosa* thus indicating that a more successful quorum‐sensing regulatory network was established and yielded antagonistic effects against the potential pathogen *P. aeruginosa*.

Tharmaraj and Shah [16], as already mentioned in the previous section, also investigated the inhibition effect of various probiotics against chosen pathogenic and spoilage bacteria with the co‐culturing method. Briefly, 9 mL of reconstituted skim milk was inoculated with 1 mL of overnight culture of probiotic bacteria and 0.1 mL of pathogenic or spoilage bacteria. The medium was mixed well and incubated at 37°C for 24 h. / were counted on nutrient agar and the log population calculated (**Table 6**). All four pathogenic bacteria were inhibited by all probiotic strains tested to varying degrees. On average, the probiotic bacteria reduced the population of pathogenic bacteria by 2.8 log units. *B. cereus* was inhibited to a greater degree by all probiotic bacteria and strains than other pathogenic bacteria. On average, the inhibito‐ ry effect of all probiotic bacteria and strains was the weakest against *E. coli*. *S. aureus* was inhibited to a greater degree by *B. animalis* and *L. rhamnosus* than the other probiotic bacteria.

Ratsep et al. [15] published their research of a microtitre plate assay on the antimicrobial effect of *L. plantarum* (five strains: N11, N27, N33, N44 and E) supernatant against various *C. difficile* strains (six clinical isolates from Norwegian patients, six clinical isolates from Estonian patients and two reference strains: VPI 10463 and M13042). Overnight *C. difficile* cultures were added to BHI broth with a density according to McFarland 3.0. Various reaction mixes were pre‐ pared (natural (acidic), neutral (pH 6.0) and neutral, heated for 20 min at 100°C) and incubat‐

Another important co‐culturing method is the use of cell lines as noted in the research by Abdel et al. [31] of 12 lactobacilli isolates interfering with the adherence and invasion of *S.* Typhi 66 using kidney epithelial cell line *Vero* (ATCC CCl‐81). The same authors also investigated this interference with the co‐culturing assay in MRS broth. It was found that nine lactobacilli isolates inhibited the growth of *S.* Typhi in the co‐culturing assay. Nine lactobacilli isolates were also successful in achieving a >50% inhibition of adherence of *S.* Typhi isolate (SS6) to Vero cells.

Assays published since 2013 of antimicrobial activity of chosen probiotics using various co‐ culturing assays on chosen pathogenic microorganisms are noted in **Table 3**. The results were similar to the results of spot‐on lawn and agar well diffuse assays.



Where probiotics are divided as follows: lb: lactobacilli; bb: Bifidobacterium; and pathogens are divided as follows: G+: Gram positive; G−: Gram negative; fng: fungi; prs: parasite.

**Table 3.** A selection of assays published since 2013 of successful antimicrobial activity of chosen probiotics using the co‐culturing assay on chosen pathogenic microorganisms.

### **5. Recent results of** *in vivo* **antimicrobial/antagonistic assays for various probiotic strains**

#### **5.1. Recent results of determining the** *in vivo* **antimicrobial assays using animal models**

In the research by Mazaya et al. [36], both *in vitro* and *in vivo* studies were conducted. The significant *in vitro* antimicrobial activity (no method specified) of two lactobacillus strains isolated from Egyptian dairy products (*L. plantarum* LA5 and *L. paracasei* LA7) was found against several potential pathogens: *S. aureus* ATCC 25923, *B. subtilis* ATCC 23857, *M. luteus* ATCC 21882, *P. aeruginosa* ATCC 27853 and *S.* Typhi. *In vivo* assays were also conducted on 5‐week‐old male mice, divided into six groups (10 mice/group). Animals within different treatment groups were treated daily for 8 days as follows: Group 1, untreated control; Group 2, animals challenged with single inoculation *S.* Typhi (200 μL aliquot of 1X 108/P.O); Group 3, animals treated orally with *L. plantarum* (LA5) (200 μL aliquot of 1X 108/P.O) for 7 days; Group 4, animals treated orally with *L. paracasei* (LA7) (200 μL aliquot of 1X 108/P.O) for 7 days; Group 5, animals challenged with single inoculation *S.* Typhi, then treated with LA5 for next 7 days; Group 6, animals challenged with single inoculation *S.* Typhi and treated with LA 7 for next 7 days. Administration of LA5 or LA7 counteracted the pathogenic effect resulting from Salmonella infection. The lactobacilli succeeded to get rid of salmonellosis based on its phagocytic and immunostimulant activity against typhoid antigen.

Lazarenko et al. [82] conducted an *in vitro* and *in vivo* assay to determine anti‐staphylococcal actions of certain probiotic cultures (*L. casei* IMV B‐7280, *L. acidophilus* IMV B‐7279; *B. longum* VK1 and *B. bifidum* VK2). *In vitro* assay using perpendicular strokes yielded antagonistic activity against all three strains of *S. aureus* (209‐P, 43, 8325‐4). The *in vivo* study with *S. aureus* 8325‐4 on mice showed that the combination of probiotics (*L. casei* IMV B‐7280, *B. longum* VK1 and *B. bifidum* VK2) was most successful as after day 9 no colonies of *S. aureus* 8325‐4 were found in the vagina of the mice.

In the study by Bujalance et al. [35], a lack of correlation between *in vitro* and *in vivo* meth‐ ods of the antimicrobial activity of probiotic lactobacilli against enteropathogenic bacteria was determined. In this study, the *in vitro* assay using the agar spot test showed that 20 strains of probiotic lactobacilli successfully inhibited *Y. enterocolitica*, *S. enterica* ser Typhimurium and *L. monocytogenes*. However, in the *in vivo* study using mouse models the selected strains (*L. casei* C1 and *L. plantarum* C4) lacked protective effects against *S.* Typhimurium. Similar conclu‐ sions of finding no antagonism *in vivo* are noted in the study by Bratz et al. [83]. Another study [79] found that the tea Yerba mate exhibited antagonistic activity *in vitro* but as a feed additive did not reduce S. Enteritidis colonization *in vivo* in broiler chickens. These studies prove that successful *in vitro* assays do not necessarily mean that the chosen microorganism with probiotic properties will be successful in real conditions. On the other hand, *in vivo* animal studies do not automatically prove antagonism in humans or other species. Therefore, the justification of using vertebrate animal models is questionable [32]. Gupta et al. [84] used *Drosophila melanogaster* commonly known as the "fruit fly," instead of a vertebrate animal model. It is a eukaryotic organism and is considered an alternative in the drug discovery process, mainly because the key physiological processes are well conserved from fly to humans. Moreover, a short life cycle, distinct developmental stages, easy cultivation, numer‐ ous offspring and a strong cytogenetic/genetic background make *Drosophila* a model organ‐ ism to study many biological processes including toxicity testing. Zhou et al. [30] used porcine neonatal jejunal epithelial cell lines (IPEC‐J2) for *in vitro* assay and worms (*Caenorhabditis elegans*) for *in vivo* testing of lactobacilli isolates against enterotoxigenic *E. coli*

**Probiotic strains or strains with probiotic potential with efficient antimicrobial activity** *in vitro*

206 Probiotics and Prebiotics in Human Nutrition and Health

**Lb:***L. paracasei* CNCM I‐4034, *L. rhamnosus* CNCM

Gram positive; G−: Gram negative; fng: fungi; prs: parasite.

co‐culturing assay on chosen pathogenic microorganisms.

I‐4036, **Bb:** *B. breve* CNCM I‐4035

**probiotic strains**

found in the vagina of the mice.

**Indicator pathogenic microorganisms Reference**

[80]

**G‐:***E. coli* ETEC CECT 501, *S.* Typhimurium CECT 443 *S.* Typhi CECT 725, *S. sonnei* CECT 457

**Lb:***L. reuteri***Oth:***B. subtilis* MA139 **G‐:***E. coli* K88 [77] **Lb:***L. plantarum* C014 **G‐:***A. hydrophila* TISTR 1321 [78] **Lb:***L. acidophilus*, **Oth:***Pediococcus* **G‐:***S.* Enteritidis 13A [79]

**Lb:***L. fermentum* 907, **Bb:** *B. longum* 1011 **G‐:***E. coli* O157:H7, *E. coli* O86 [81]

Where probiotics are divided as follows: lb: lactobacilli; bb: Bifidobacterium; and pathogens are divided as follows: G+:

**Table 3.** A selection of assays published since 2013 of successful antimicrobial activity of chosen probiotics using the

**5. Recent results of** *in vivo* **antimicrobial/antagonistic assays for various**

**5.1. Recent results of determining the** *in vivo* **antimicrobial assays using animal models**

on its phagocytic and immunostimulant activity against typhoid antigen.

In the research by Mazaya et al. [36], both *in vitro* and *in vivo* studies were conducted. The significant *in vitro* antimicrobial activity (no method specified) of two lactobacillus strains isolated from Egyptian dairy products (*L. plantarum* LA5 and *L. paracasei* LA7) was found against several potential pathogens: *S. aureus* ATCC 25923, *B. subtilis* ATCC 23857, *M. luteus* ATCC 21882, *P. aeruginosa* ATCC 27853 and *S.* Typhi. *In vivo* assays were also conducted on 5‐week‐old male mice, divided into six groups (10 mice/group). Animals within different treatment groups were treated daily for 8 days as follows: Group 1, untreated control; Group 2, animals challenged with single inoculation *S.* Typhi (200 μL aliquot of 1X 108/P.O); Group 3, animals treated orally with *L. plantarum* (LA5) (200 μL aliquot of 1X 108/P.O) for 7 days; Group 4, animals treated orally with *L. paracasei* (LA7) (200 μL aliquot of 1X 108/P.O) for 7 days; Group 5, animals challenged with single inoculation *S.* Typhi, then treated with LA5 for next 7 days; Group 6, animals challenged with single inoculation *S.* Typhi and treated with LA 7 for next 7 days. Administration of LA5 or LA7 counteracted the pathogenic effect resulting from Salmonella infection. The lactobacilli succeeded to get rid of salmonellosis based

Lazarenko et al. [82] conducted an *in vitro* and *in vivo* assay to determine anti‐staphylococcal actions of certain probiotic cultures (*L. casei* IMV B‐7280, *L. acidophilus* IMV B‐7279; *B. longum* VK1 and *B. bifidum* VK2). *In vitro* assay using perpendicular strokes yielded antagonistic activity against all three strains of *S. aureus* (209‐P, 43, 8325‐4). The *in vivo* study with *S. aureus* 8325‐4 on mice showed that the combination of probiotics (*L. casei* IMV B‐7280, *B. longum* VK1 and *B. bifidum* VK2) was most successful as after day 9 no colonies of *S. aureus* 8325‐4 were


Where probiotics are divided as follows: lb: lactobacilli; bb: Bifidobacterium; oth: other; and pathogens are divided as follows: G+: Gram positive; G−: Gram negative; fng: fungi; prs: parasite; ins: insect.

**Table 4.** A selection of assays published since 2013 of successful antimicrobial activity of chosen probiotics using the *in vivo* animal models on chosen pathogenic microorganisms.

The most recent *in vivo* antagonistic assays using animal models are noted in **Table 4**. These results confirm the strain specific antagonistic activity of chosen probiotics.

#### **5.2. Recent results of determining the** *in vivo* **antimicrobial assays using clinical trials**

Most important research on the antagonistic effect of probiotics are clinical trials, however only a few well conducted clinical studies have been reported. Most clinical studies include the comparison of antibiotic therapy with adjuvant probiotic therapy. In the study by Dore et al. [87], in this prospective, single centre, open label pilot study, patients scheduled for upper endoscopy for any reason and found to be positive for *H. pylori* infection were invited to enter. The intervention consisted of *L. reuteri* (DSM 17938, Reuflor, BioGaia AB, Sweden) 108 cfu/ tablet plus pantoprazole (proton pump inhibitor) 20 mg twice a day. A 76% decrease in urease activity was observed. The absence of a control group with pantoprazole without *L. reuteri* however prevents any definite conclusion.

In the study by Pendharkar et al. [88] the clinical outcome for women conventionally treated for bacterial vaginosis and yeast infection with probiotics bacilli was investigated. This study is an example of the antibiotic therapy with adjuvant probiotic therapy. In the clinical trial, women were recruited in three groups as follows: women with bacterial vaginosis receiving clindamycin and metronidazole treatment together with a prolonged administration of EcoVag® (containing *L. rhamnosus* DSM 14870 and *L. gasseri* DSM 14869) for 10 consecutive days after each antibiotic treatment followed by weekly administration of capsules for next four months, women with recurrent vulvovaginal candidiasis receiving extended flucona‐ zole and EcoVag® treatment, and women receiving extended fluconazole treatments only. The 6‐ and 12‐month cure rates for bacterial vaginosis were 67 %. The 6‐ and 12‐month cure rates for vulvovaginal candidiasis were 100 and 89% in women receiving fluconazole and EcoVag®, and 100 and 70% in women receiving fluconazole only. The study suggests that the treatment with antibiotics or anti‐fungal medication in combination with EcoVag® capsules provide long‐term cure against bacterial vaginosis and R‐VVC.

Some of the most recent *in vivo* antagonistic clinical trials are noted in **Table 5**. These results confirm that adjuvant therapy with antibiotics and chosen probiotics enhances the antagonis‐ tic activity.



Where probiotics are divided as follows: lb: lactobacilli; bb: Bifidobacterium; oth: other; pathogens are divided as follows: bc: bacteria; G+: Gram positive; G−: Gram negative; fng: fungi; and clinical trials are divided as follows: DBRCT: double‐ blinded randomized clinical trial; CT: clinical trial; AT: additional therapy; Dis: disease; AAD: antibiotic associated diarrhoea.

**Table 5.** A selection of published since 2013 of antimicrobial activity of chosen probiotics using *in vivo* clinical trials on chosen pathogenic microorganisms or treatment of diseases.

### **6. Discussion and conclusions**

The most recent *in vivo* antagonistic assays using animal models are noted in **Table 4**. These

Most important research on the antagonistic effect of probiotics are clinical trials, however only a few well conducted clinical studies have been reported. Most clinical studies include the comparison of antibiotic therapy with adjuvant probiotic therapy. In the study by Dore et al. [87], in this prospective, single centre, open label pilot study, patients scheduled for upper endoscopy for any reason and found to be positive for *H. pylori* infection were invited to enter. The intervention consisted of *L. reuteri* (DSM 17938, Reuflor, BioGaia AB, Sweden) 108

tablet plus pantoprazole (proton pump inhibitor) 20 mg twice a day. A 76% decrease in urease activity was observed. The absence of a control group with pantoprazole without *L. reuteri*

In the study by Pendharkar et al. [88] the clinical outcome for women conventionally treated for bacterial vaginosis and yeast infection with probiotics bacilli was investigated. This study is an example of the antibiotic therapy with adjuvant probiotic therapy. In the clinical trial, women were recruited in three groups as follows: women with bacterial vaginosis receiving clindamycin and metronidazole treatment together with a prolonged administration of EcoVag® (containing *L. rhamnosus* DSM 14870 and *L. gasseri* DSM 14869) for 10 consecutive days after each antibiotic treatment followed by weekly administration of capsules for next four months, women with recurrent vulvovaginal candidiasis receiving extended flucona‐ zole and EcoVag® treatment, and women receiving extended fluconazole treatments only. The 6‐ and 12‐month cure rates for bacterial vaginosis were 67 %. The 6‐ and 12‐month cure rates for vulvovaginal candidiasis were 100 and 89% in women receiving fluconazole and EcoVag®, and 100 and 70% in women receiving fluconazole only. The study suggests that the treatment with antibiotics or anti‐fungal medication in combination with EcoVag® capsules

Some of the most recent *in vivo* antagonistic clinical trials are noted in **Table 5**. These results confirm that adjuvant therapy with antibiotics and chosen probiotics enhances the antagonis‐

**Basic therapy Type of**

pantoprazole

**G+:***H. pylori* Standard triple *H. pylori*

Antibiotics: Clindamycin, metronidazole, fluconazole

eradication therapy with

**trial**

CT [87]

CT [88]

DBRCT [89]

**Reference**

cfu/

**5.2. Recent results of determining the** *in vivo* **antimicrobial assays using clinical trials**

results confirm the strain specific antagonistic activity of chosen probiotics.

however prevents any definite conclusion.

208 Probiotics and Prebiotics in Human Nutrition and Health

tic activity.

DSM 14869

*bacterium* BB‐12

**Probiotic strains with efficient antimicrobial activity using** *in vivo* **clinical trials and other therapies**

**Lb:***L. rhamnosus* DSM 14870, *L. gasseri*

**Lb:***L. rhamnosus* GG (LGG), **Bb:** *B.*

provide long‐term cure against bacterial vaginosis and R‐VVC.

**Pathogenic microorganisms or treated disease**

**Lb:***L. reuteri* DSM 17938 **G+:***H. pylori* Proton pump inhibitor:

*Candida* spp.

**Bc:** not specified, **Fng**:

The antimicrobial ability of probiotics is a very important trait and includes the production of antimicrobial compounds, competitive exclusion of pathogens, enhancement of the intesti‐ nal barrier function and others. Usually, probiotic strains produce more than one antimicro‐ bial substance that may act synergistically, increasing the spectrum of targeted microorganisms. This property may be desirable as long as this antimicrobial spectrum is restricted to pathogenic microorganisms but it cannot be excluded that it will not affect the normal microbiota of the gut or other microbiotas as well [94]. The results show that probiot‐ ic properties are strain dependent and that strain identification is imperative [3].

Probiotic candidates have been accessed from very diverse habitats including faeces of breast‐ fed human infants [65, 69, 80, 85, 95], faeces of healthy adults [9, 15, 65, 70], faeces of elderly [81], faeces of children [25, 96], breast milk [42], human saliva [52], vaginal isolates of healthy women [66, 75], various fermented foods or beverages including raw or fermented milk [23, 35, 44], kefir [97], cheese [51, 56, 98], whey [99], yogurt [16, 41], dahi [100, 101], other dairy products [25, 36, 61], sourdough [102], sausages [17], fermented meat [24], kimchi [10, 62], maize [25, 59], fermented olives [103], Yerba mate [79], ragi [64], soy sauce [86], soil [104], as well as animal origin including rat faeces [71], geese [68], calves [105], pigs [45], fish [39, 60, 63, 78] and other seafood [40, 43, 46] and many others.

By far, the most commonly investigated probiotic were bacteria of the genus *Lactobacillus* (*L. plantarum, L. acidophilus, L. fermentum, L. casei, L. paracasei* and *L. reuteri*). The genus *Bifidobac‐ terium* and other probiotic microorganisms (*Lactococcus, Pediococcus, Enterococcus, Bacillus* and *Saccharomyces*) have been also been investigated, but to a somewhat lesser extent. Studies were also conducted on known probiotics from various tissue type collections. The most common pathogens used to test the antagonistic activity of probiotics were different strains of S. aureus, E. faecium, E. faecalis, L. monocytogenes, E. coli, various Salmonella, Vibrio and Yersinia spp., and P. aeruginosa.

The antimicrobial activity of probiotic microorganisms has a very wide area application including adjuvant therapy to antibiotic consumption or for correcting dysbiosis of the gastrointestinal tract microbiome due to diarrhoea [37, 38, 106], antagonistic activity in humans against urinary tract infections [26, 66, 75], eradicating *H. pylori* infections [87], nosocomial infections [15, 96], dental biofilm formation [72], lowering serum cholesterol [10, 55], treating fevers [31], as well as in the agro‐food industry for manufacturing fermented products [44, 62, 107], preventing food spoilage [16, 23, 41], as food additives for functional foods [50, 56, 57, 59, 67, 97], as prophylactic agents, adjuvants or alternatives to antibiotic therapies to antibiot‐ ic therapy in poultry [34, 47, 68], cattle [105, 108], pigs [45], fish [74, 78] and other livestock industry [104], just to name a few.

The process of determining antimicrobial properties of probiotic is complex and includes *in vitro* assays, *in vivo* models or substitute models, clinical studies, metagenomic analyses and mathematical modelling. Only after all these steps are completed, a probiotic candidate can be identified as such [109]. *In vitro* studies are the most represented. Although, they are a crucial step in selecting probiotic candidates, they are only the first step as efficient antimicrobial activity via *in vitro* studies does not necessarily mean that the antimicrobial activity is present in *in vivo* assays. Therefore, further research methods (double‐blinded randomized clinical trials) are necessary to prove the important antimicrobial trait of probiotic candidates. As noted in Section 5.2, there are only a few well‐conducted published clinical studies. Most clinical studies include the comparison of antibiotic therapy with adjuvant probiotic therapy which is an important aim of probiotic consumption.

There is a clear need for more elaborate assays that would better represent the complex interactions between the probiotics and the host microbiome to understand the consequen‐ ces of the *in situ* production of antimicrobials by the former [94]. Another important fact is that probiotics are often found to have higher antagonist activity as multispecies groups [6, 7, 26]. Quorum sensing among probiotics is also an important factor; however, quorum‐sensing studies among probiotics are sparse. It is well known that microorganisms coordinate collective behaviour in response to environmental challenges using sophisticated intercellu‐ lar communication networks and that they are not limited to communication within their own species but are capable of intercepting messages and coerce cohabitants into behavioural modifications [110], therefore probiotics are included. Although all these facts make re‐ search of the antimicrobial/antagonistic activity of probiotics even more complex, it also presents a great opportunity for future research.

### **Author details**

Sabina Fijan

well as animal origin including rat faeces [71], geese [68], calves [105], pigs [45], fish [39, 60,

By far, the most commonly investigated probiotic were bacteria of the genus *Lactobacillus* (*L. plantarum, L. acidophilus, L. fermentum, L. casei, L. paracasei* and *L. reuteri*). The genus *Bifidobac‐ terium* and other probiotic microorganisms (*Lactococcus, Pediococcus, Enterococcus, Bacillus* and *Saccharomyces*) have been also been investigated, but to a somewhat lesser extent. Studies were also conducted on known probiotics from various tissue type collections. The most common pathogens used to test the antagonistic activity of probiotics were different strains of S. aureus, E. faecium, E. faecalis, L. monocytogenes, E. coli, various Salmonella, Vibrio and Yersinia spp.,

The antimicrobial activity of probiotic microorganisms has a very wide area application including adjuvant therapy to antibiotic consumption or for correcting dysbiosis of the gastrointestinal tract microbiome due to diarrhoea [37, 38, 106], antagonistic activity in humans against urinary tract infections [26, 66, 75], eradicating *H. pylori* infections [87], nosocomial infections [15, 96], dental biofilm formation [72], lowering serum cholesterol [10, 55], treating fevers [31], as well as in the agro‐food industry for manufacturing fermented products [44, 62, 107], preventing food spoilage [16, 23, 41], as food additives for functional foods [50, 56, 57, 59, 67, 97], as prophylactic agents, adjuvants or alternatives to antibiotic therapies to antibiot‐ ic therapy in poultry [34, 47, 68], cattle [105, 108], pigs [45], fish [74, 78] and other livestock

The process of determining antimicrobial properties of probiotic is complex and includes *in vitro* assays, *in vivo* models or substitute models, clinical studies, metagenomic analyses and mathematical modelling. Only after all these steps are completed, a probiotic candidate can be identified as such [109]. *In vitro* studies are the most represented. Although, they are a crucial step in selecting probiotic candidates, they are only the first step as efficient antimicrobial activity via *in vitro* studies does not necessarily mean that the antimicrobial activity is present in *in vivo* assays. Therefore, further research methods (double‐blinded randomized clinical trials) are necessary to prove the important antimicrobial trait of probiotic candidates. As noted in Section 5.2, there are only a few well‐conducted published clinical studies. Most clinical studies include the comparison of antibiotic therapy with adjuvant probiotic therapy which is

There is a clear need for more elaborate assays that would better represent the complex interactions between the probiotics and the host microbiome to understand the consequen‐ ces of the *in situ* production of antimicrobials by the former [94]. Another important fact is that probiotics are often found to have higher antagonist activity as multispecies groups [6, 7, 26]. Quorum sensing among probiotics is also an important factor; however, quorum‐sensing studies among probiotics are sparse. It is well known that microorganisms coordinate collective behaviour in response to environmental challenges using sophisticated intercellu‐ lar communication networks and that they are not limited to communication within their own species but are capable of intercepting messages and coerce cohabitants into behavioural modifications [110], therefore probiotics are included. Although all these facts make re‐

63, 78] and other seafood [40, 43, 46] and many others.

210 Probiotics and Prebiotics in Human Nutrition and Health

and P. aeruginosa.

industry [104], just to name a few.

an important aim of probiotic consumption.

Address all correspondence to: sabina.fijan@um.si

Faculty of Health Sciences, University of Maribor, Maribor, Slovenia

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### **Chapter 11**

## **Probiotics in Childhood Celiac Disease**

Caterina Anania, Francesca Olivero, Eugenia Olivero and Lucia Pacifico

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63119

#### **Abstract**

Celiac disease (CD) is an autoimmune enteropathy induced by gluten ingestion in genetically susceptible individuals. Genetic predisposition plays an important role in the development of CD, but it is not sufficient by itself for the disease development. Although gluten proteins are the main environmental factor involved in CD pathogen‐ esis and ingestion of gluten is necessary to manifest the disease, recent studies have suggested that alteration of the microbiota could be involved and, in particular, the interplay between gut microbiota and the mucosal immune system. Dysbiosis, the alteration of the microbiota, has been associated with a variety of intestinal patholo‐ gies including Crohn disease and CD. Most observational studies in children and adults with CD have shown alterations in the intestinal microbiota composition compared to control subjects, which is only partially recovered after treatment with a gluten‐free diet (GFD). At this time, the only treatment for CD is lifelong adherence to a GFD, which involves the elimination of grains containing gluten, wheat, rye, and barley. However, it is difficult for many patients to follow a GFD. Abnormalities in the gut microbiome in CD patients have led to the use of probiotics as a promising alternative as a therapeutic or preventative approach.

**Keywords:** celiac disease, gluten free diet, intestinal microbiota, dysbiosis, probiotics

### **1. Introduction**

Celiac disease (CD) is an autoimmune enteropathy induced by gluten ingestion in genetically susceptible individuals [1]. The major genetic risk factor for CD is represented by HLA‐DQ genes. Ninety percent of affected individuals carry the HLA‐DQ2 haplotype, 5% the DQ8 haplotype, and the remaining 5% carry at least one of the two DQ2 alleles [1, 2]. Genetic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

predisposition plays an important role in the development of CD but it is not sufficient by itself for the disease development [3]. Approximately, 30% of the general population carry the HLA‐ DQ2/8 CD susceptibility genes, however, only 2–5% of these individuals will develop CD, suggesting that additional environmental factors contribute to disease development [4]. Although gluten proteins are the main environmental factor involved in CD pathogenesis and ingestion of gluten is necessary to manifest the disease, recent studies have suggested that potential factors such as birth delivery, breast‐feeding, infectious agents, and antibiotic intake could contribute to the development of CD [5–7]. The alteration of the microbiota could also be involved and, in particular, the interplay between gut microbiota and the mucosal immune system [8].

The microbiota, the set of microorganisms that colonize the human body, has a fundamental role for the host. It is important for both physiological and metabolic factors, ranging from the absorption of nutrients to the regulation and development of the immune system [9]. Dysbiosis, the alteration of the microbiota, has been associated with a variety of pathologies like Crohn disease and obesity [10, 11]. Most observational studies in children and adults with CD have shown alterations in the intestinal microbiota composition compared to control subjects, which is partially recovered after treatment with a gluten‐free diet (GFD) [12–14]. It has been demonstrated that levels of *Bifidobacteria* and *Lactobacilli* are reduced in CD patients [14, 15]. Specific alterations in the microbiota could contribute to the etiopathogenesis of CD by providing proteolytic activities that influence the generation of toxic and immunogenic peptides from gluten, and compromise the intestinal barrier function [16]. Probiotics are nonpathogenic live microorganisms, which, when orally administered in adequate amounts, alter the microflora of the host and have beneficial health effect [17].

At this time, the only treatment for CD is lifelong adherence to a GFD, which involves the elimination of grains containing gluten, wheat, rye, and barley. However, it is difficult for many patients to follow a GFD. Some probiotics have been found to digest or alter gluten polypeptides [18]. Abnormalities in the gut microbiome in CD patients have led to the use of probiotics as a promising alternative as a therapeutic or preventative approach.

Here we focus on the role of microbiota in the pathogenesis of CD and on the chances for probiotics to be involved in an alternative treatment strategy.

### **2. Microbiota composition in celiac children**

Several research papers have suggested that an important risk factor involved in the etiolo‐ gy of CD could be the gut microbiota. Multiple studies investigating the role of gut microbio‐ ta in CD have been performed on fecal samples and, later, on duodenal biopsies.

The studies that have addressed the relation between fecal microbiota and CD in the pedia‐ tric population are summarized in **Table 1** [13, 19–24]. In the earliest report involving a total of 49 children, 26 celiac patients aged 12–48 months and 23 age‐matched controls, Collado et al. evaluated the composition of the fecal microbiota by both culture‐dependent and culture‐

independent methods using fluorescent in situ hybridization (FISH) [13]. They showed a high level of *Bacteroides*, *Clostridium*, and *Staphylococcus* in fecal samples from CD children com‐ pared to healthy subjects when analyzed by culture methods. The numbers of *Bacteroides‐ Prevotella*, *Clostridium histolyticum*, *Eubacterium rectale‐Clostridium coccoides*, *Atopobium*, and sulfate‐reducing bacterial groups were also significantly higher in fecal samples from CD children analyzed by FISH [13]. Subsequently, Sanz et al. [19], using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) in 10 CD children aged 15–45 months and 10 age‐matched healthy controls, demonstrated that the presence of species such as *Lactobacillus curvatus*, *Leuconostoc mesenteroides*, and *Leuconostoc carnosus* were charac‐ teristic of coeliac patients, while the *Lactobacillus casei* group was characteristic of healthy controls. Moreover, the authors found a reduction in *Bifidobacterium* population diversity in CD patients. Collado et al. [20], using real‐time PCR, evaluated duodenal and fecal microbio‐ ta in three groups of children: (1) untreated CD patients on a gluten‐containing diet (GCD); (2) treated CD patients who had been on a GFD for a minimum of two years; and (3) healthy controls. They found that feces and biopsies of CD patients had an increased presence of *Bifidobacterium, Bacteroides*, *and Clostridium leptum* groups with respect to the control group; *Escherichia coli and Staphylococcus* were otherwise predominant in CD subjects on GFD. GFD determined a complete normalization of gut microbiota [20]. De Palma et al. examined fecal microbiology and immunoglobulin‐associated features in active and non‐active stages of CD in children and in age‐matched controls [21]. They found that in CD patients there was an alteration in the type of fecal immunoglobulin‐coated bacteria along with a shift in the composition of the microbiota. In fact, they demonstrated a reduction of the percentages of the IgA‐coated bacteria in CD patients on a GFD and in those not following a GFD compared to the control group. They also found a reduction of the percentages of IgG‐ and IgM‐coated bacteria in treated CD patients with respect to untreated CD subjects and control group. Moreover, treated and untreated CD subjects showed a predominance of *Bacteroides‐Prevotella* as well as an impaired mucosal barrier, as suggested by the reduction of IgA‐coated bacteria with respect to the controls [21]. Sanchez et al., in an attempt to determine whether intestinal *Staphylococcus* spp. and their pathogenic features differed between CD patients and healthy controls, studied 40 CD children (20 active CD and 20 non‐active CD) and 20 healthy con‐ trols [22]. *Staphylococci* were isolated from feces and identified by PCR and DNA sequencing. CD was associated with alterations in species diversity and composition of the fecal *Staphylo‐ coccus* population. *Staphylococcus epidermidis* isolates carrying the *mecA* gene and both the *mecA* and *atIE* genes were more abundant in CD patients than in controls, most likely reflecting increased exposure of these subjects to opportunistic staphylococcal pathogens and antimi‐ crobials, which in turn affected the composition/features of their intestinal microbiota [22]. Di Cagno et al. in a study including seven CD patients on GFD, seven CD patients on a GCD, and seven healthy controls, utilizing DGGE analysis and gas chromatography‐mass spectrome‐ try‐solid‐phase microextraction analysis of fecal volatile organic compounds (VOCs), found that the fecal microbiota and VOCs of CD patients on GFD were more similar to those of healthy patients than to those of CD patients on GCD [23]. Consequently, the authors speculated that *Lactobacillus* and *Bifidobacterium* strains isolated from healthy children could be a potential probiotic treatment to restore the balance of intestinal microbiota in treated and untreated CD

predisposition plays an important role in the development of CD but it is not sufficient by itself for the disease development [3]. Approximately, 30% of the general population carry the HLA‐ DQ2/8 CD susceptibility genes, however, only 2–5% of these individuals will develop CD, suggesting that additional environmental factors contribute to disease development [4]. Although gluten proteins are the main environmental factor involved in CD pathogenesis and ingestion of gluten is necessary to manifest the disease, recent studies have suggested that potential factors such as birth delivery, breast‐feeding, infectious agents, and antibiotic intake could contribute to the development of CD [5–7]. The alteration of the microbiota could also be involved and, in particular, the interplay between gut microbiota and the mucosal immune

The microbiota, the set of microorganisms that colonize the human body, has a fundamental role for the host. It is important for both physiological and metabolic factors, ranging from the absorption of nutrients to the regulation and development of the immune system [9]. Dysbiosis, the alteration of the microbiota, has been associated with a variety of pathologies like Crohn disease and obesity [10, 11]. Most observational studies in children and adults with CD have shown alterations in the intestinal microbiota composition compared to control subjects, which is partially recovered after treatment with a gluten‐free diet (GFD) [12–14]. It has been demonstrated that levels of *Bifidobacteria* and *Lactobacilli* are reduced in CD patients [14, 15]. Specific alterations in the microbiota could contribute to the etiopathogenesis of CD by providing proteolytic activities that influence the generation of toxic and immunogenic peptides from gluten, and compromise the intestinal barrier function [16]. Probiotics are nonpathogenic live microorganisms, which, when orally administered in adequate amounts,

At this time, the only treatment for CD is lifelong adherence to a GFD, which involves the elimination of grains containing gluten, wheat, rye, and barley. However, it is difficult for many patients to follow a GFD. Some probiotics have been found to digest or alter gluten polypeptides [18]. Abnormalities in the gut microbiome in CD patients have led to the use of

Here we focus on the role of microbiota in the pathogenesis of CD and on the chances for

Several research papers have suggested that an important risk factor involved in the etiolo‐ gy of CD could be the gut microbiota. Multiple studies investigating the role of gut microbio‐

The studies that have addressed the relation between fecal microbiota and CD in the pedia‐ tric population are summarized in **Table 1** [13, 19–24]. In the earliest report involving a total of 49 children, 26 celiac patients aged 12–48 months and 23 age‐matched controls, Collado et al. evaluated the composition of the fecal microbiota by both culture‐dependent and culture‐

alter the microflora of the host and have beneficial health effect [17].

probiotics to be involved in an alternative treatment strategy.

**2. Microbiota composition in celiac children**

probiotics as a promising alternative as a therapeutic or preventative approach.

ta in CD have been performed on fecal samples and, later, on duodenal biopsies.

system [8].

224 Probiotics and Prebiotics in Human Nutrition and Health

patients [23]. Similar conclusions have been reached by Lorenzo Pisarello et al. [24] in a very recent work. They found lower counts of *Lactobacillus* in the feces of CD compared to con‐ trols. Furthermore, the authors selected from feces of controls 5 *Lactobacillus* strains because of their high resistance percentages to gastrointestinal tract conditions. *Lactobacillus rhamnosus* (LC4) showed the highest percentage of autoaggregation and *Lactobacillus paracasei* showed high hydrophobicity suggesting a potential use of these strains as probiotics in CD [24].



*CD* celiac disease, *FISH* fluorescent in situ hybridization, *DGGE* denaturing gradient gel electrophoresis, *PCR* polymerase chain reaction, *qPCR* quantitative polymerase chain reaction.

**Table 1.** Fecal microbiota in celiac disease.

patients [23]. Similar conclusions have been reached by Lorenzo Pisarello et al. [24] in a very recent work. They found lower counts of *Lactobacillus* in the feces of CD compared to con‐ trols. Furthermore, the authors selected from feces of controls 5 *Lactobacillus* strains because of their high resistance percentages to gastrointestinal tract conditions. *Lactobacillus rhamnosus* (LC4) showed the highest percentage of autoaggregation and *Lactobacillus paracasei* showed high hydrophobicity suggesting a potential use of these strains as probiotics in CD [24].

**Methods Main results**

Culture+qPCR +DGGE

Culture+ FISH In untreated CD:

↑ *Bacteroides* ↑ *Staphylococcus* ↑ *Clostridium*

↑ *Bacteroides‐Prevotella,* ↑ *Clostridium hystoliticum,* ↑ *Eubacterium rectale‐C. coccoides,* ↑ *Atopobium, Staphylococcus*

↓ *Bifidobacterium*

In untreated CD:

qPCR In untreated and treated CD: ↑ Bacterial count ↑ *E. coli*, ↑ B*acteroides,* ↑ *Clostridium leptum Staphylococcus* prevalence ↓ *Bifidobacterium* In treated CD: ↑ *Lactobacillus*

PCR+DGGE In treated and untreated CD:

Only in controls:

↓ Ratio of cultivable lactic acid bacteria and *Bifidobacterium* to *Bacteroides* and *Enterobacteria* In treated CD and in controls: *Lactobacillus brevis, Lactobacillus rossiae, Lactobacillus pentosus*

↑ *Leuconostoc carnosum,* ↑ *Leuconostoc mesenteroides,* ↑ *Lactobacillus curvatus* ↓ *Lactobacillus casei,* ↓ *Bifidobacterium adolescentis*

High diversity of fecal microbiota

**Year Country Patients population**

226 Probiotics and Prebiotics in Human Nutrition and Health

2007 Spain 26 untreated CD (mean

Sanz et al. [19] 2007 Spain 10 untreated CD (mean

**and sample size**

age, 26 months) 23 controls (mean age,

23.1 months)

age, 28 months)

age, 38.5 months) 18 treated CD (mean age,

30 controls (mean age,

7 treated CD (range, 6–12

7 (range, 6–12 years)

37.7 months)

33.5 months)

2009 Italy 7 untreated CD (range, 6– 12 years)

years)

controls

months)

2009 Spain 30 untreated CD (mean

10 controls (mean age, 24

**Author/ References** 

[13]

Collado et al.

Collado et al.

Di Cagno et al.

[23]

[20]

Duodenal microbial composition of pediatric CD patients was explored more extensively later on, with the main findings summarized in **Table 2** [20, 25–33]. Microbiota characterization from duodenal biopsy specimens was initially carried out on CD Spanish children by Nadal et al. [25] in 2007. The authors, in an attempt to identify the specific composition of the duodenal microbiota of celiac patients (with active and non‐active disease), evaluated 20 CD patients on GCD, 10 CD patients on GFD for 1–2 years, and 8 healthy controls. Bacteriological analyses of duodenal biopsy specimens, carried out by fluorescent in situ hybridization coupled with flow cytometry, showed that the proportions of total and Gram‐negative potentially pro‐inflam‐ matory bacteria were significantly higher in CD patients with active disease than in patients on GFD and controls. Although, the ratio of beneficial bacterial groups (*Lactobacillus‐*

*Bifidobacterium*) to potentially harmful *Bacteroides‐E. coli* was significantly reduced in CD patients on GFD, there was not a complete normalization of gut microbiota compared with controls [25]. Several subsequent Spanish studies confirmed these results [20, 26–28]. Particularly, these studies found that the *Bacteroides, E. coli, Bifidobacterium, Enterobacteriacae,* and *Staphylococcus* groups were significantly more abundant in GCD patients than in the controls with a greater diversity of these species [20, 26, 28], while, in contrast, members of the family Streptococcaceae were less abundant in CD patients [28]. Furthermore, the *Prevotella* genera were more frequent in healthy subjects than in celiac patients [27]. Ou et al. identi‐ fied Clostridium, Prevotella and Actinomyces as predominant bacteria in the proximal small intestine biopsies from a cohort of 45 CD children and 18 healthy controls born during the so‐ called "Swedish CD epidemic" (2004‐2007). This could explain the four‐fold increase in the incidence of CD in children less than two years of age observed between 2004 and 2007 [29]. Schippa et al. [30] analyzed the mucosa‐associated microbiota of CD children, before and after a GFD, and controls by temporal temperature gradient gel electrophoresis (TTGE). The most important findings of the study were: a demonstration of a presence of peculiar microbial TTGE profile and a significant higher biodiversity in CD pediatric patients' duodenal mucosa after 9 months of GFD compared to healthy controls. Di Cagno et al. [31], utilizing culture‐ dependent and culture‐independent methods and metabolomics analyses, investigated the differences in the microbiota and metabolome of 19 treated CD patients and 15 controls. They confirmed the lower levels of *Lactobacillus* and increased levels of *Bacteroides* in CD patients. Moreover, the authors showed that a GFD lasting at least two years did not completely restore the microbiota and metabolome in CD patients [31]. A recent Spanish study demonstrated that the intestinal microbiota of patients with duodenal Marsh 3c lesions showed similarity of 98% and differed from that of CD patients with other type of histologic lesion as Marsh 3a, Marsh 3b, and Marsh 2 [32]. This indicated that the composition of duodenal microbiota differed depending on the grade of intestinal damage.



*Bifidobacterium*) to potentially harmful *Bacteroides‐E. coli* was significantly reduced in CD patients on GFD, there was not a complete normalization of gut microbiota compared with controls [25]. Several subsequent Spanish studies confirmed these results [20, 26–28]. Particularly, these studies found that the *Bacteroides, E. coli, Bifidobacterium, Enterobacteriacae,* and *Staphylococcus* groups were significantly more abundant in GCD patients than in the controls with a greater diversity of these species [20, 26, 28], while, in contrast, members of the family Streptococcaceae were less abundant in CD patients [28]. Furthermore, the *Prevotella* genera were more frequent in healthy subjects than in celiac patients [27]. Ou et al. identi‐ fied Clostridium, Prevotella and Actinomyces as predominant bacteria in the proximal small intestine biopsies from a cohort of 45 CD children and 18 healthy controls born during the so‐ called "Swedish CD epidemic" (2004‐2007). This could explain the four‐fold increase in the incidence of CD in children less than two years of age observed between 2004 and 2007 [29]. Schippa et al. [30] analyzed the mucosa‐associated microbiota of CD children, before and after a GFD, and controls by temporal temperature gradient gel electrophoresis (TTGE). The most important findings of the study were: a demonstration of a presence of peculiar microbial TTGE profile and a significant higher biodiversity in CD pediatric patients' duodenal mucosa after 9 months of GFD compared to healthy controls. Di Cagno et al. [31], utilizing culture‐ dependent and culture‐independent methods and metabolomics analyses, investigated the differences in the microbiota and metabolome of 19 treated CD patients and 15 controls. They confirmed the lower levels of *Lactobacillus* and increased levels of *Bacteroides* in CD patients. Moreover, the authors showed that a GFD lasting at least two years did not completely restore the microbiota and metabolome in CD patients [31]. A recent Spanish study demonstrated that the intestinal microbiota of patients with duodenal Marsh 3c lesions showed similarity of 98% and differed from that of CD patients with other type of histologic lesion as Marsh 3a, Marsh 3b, and Marsh 2 [32]. This indicated that the composition of duodenal microbiota differed

depending on the grade of intestinal damage.

228 Probiotics and Prebiotics in Human Nutrition and Health

Nadal et al. [25] 2007 Spain 20 (untreated CD (mean

**Years Country Patients population**

**and sample size**

age, 5.1 years)

5.6 years)

years)

2009 Spain 8 untreated CD (mean age, 56.4 months)

65.2 months)

10 treated CD (mean age,

8 controls (mean age, 4.1

8 treated CD (mean age,

**Methods Main results**

qPCR In untreated CD:

In untreated CD: ↑ Total bacteria

↑Bacterial counts ↑ *Lactobacillus* prevalence ↓ *C. coccoides* prevalence ↑ *Staphylococcus*

↑ Gram‐negative bacteria ↑ *Bacteroides* and *E. coli,* which normalized after GFD In treated and untreated CD: ↓ The ratio of *Lactobacillus‐ Bifidobacterium* to *Bacteroides*

FISH+ flow cytometry

**Authors/ references** 

Collado et al.

[20]


*CD* celiac disease, *FISH* fluorescent in situ hybridization, *DGGE* denaturing gradient gel electrophoresis, *GFD* gluten‐free diet, *HIPchip* Human Intestinal Tract Chip, *IFN‐g* interferon‐gamma, *IL‐10* interleukin‐10, *IS‐pro* 16S‐23S interspacer, *PCR* polymerase chain reaction, *qPCR* quantitative polymerase chain reaction, *qRT‐PCR* quantitative reverse‐transcriptase‐ polymerase chain reaction, *TGGE* temporal temperature gradient gelelectrophoresis, *TLR2* toll‐like receptor 2, C-X-C chemokine receptor type 6.

**Table 2.** Duodenal‐associated microbiota in celiac disease.

In contrast, two recent studies reached different results. De Meij et al. [33], analyzing the total microbiome profile in small bowel biopsies of 21 untreated CD and 21 age‐matched controls, found that mucosa‐associated duodenal microbiome composition and diversity did not differ between children with untreated CD and controls. The same results were obtained by Cheng et al. using bacterial phylogenetic microarray to comprehensively profile the microbiota in duodenal biopsies of 10 CD and nine healthy children, suggesting that the duodenal mucosa‐ associated bacteria do not play an important role in the pathogenesis of CD [34].

In summary, although the majority of the studies available have confirmed the presence of intestinal dysbiosis in CD children characterized by low levels of *Lactobacilli* and *Bifidobacte‐ ria* and increase in Gram‐negative bacteria (*Bacteroides*), which were not completely normal‐ ized after GFD, some of them have failed to find a distinct signature that defines celiac microbiota. The available articles regarding the relationship between the gut microbiota and GFD, demonstrated that a GFD only allows a partial recovery of the gut microbiota in CD patients [30, 34, 35].

### **3. Pathogenetic role of intestinal dysbiosis in CD**

**Authors/ references** 

De Meij et al.

[32]

Giron Fernandez‐ Crehuet et al.

[34]

chemokine receptor type 6.

**Table 2.** Duodenal‐associated microbiota in celiac disease.

**Years Country Patients population**

230 Probiotics and Prebiotics in Human Nutrition and Health

Nistal et al. [27] 2012 8 untreated CD (mean age,

Cheng et al. [33] 2013 Finland 10 untreated CD (median

years)

3.75 years)

age, 6.8 years)

age 9.5 years)

8.1 years)

years)

2015 Spain 11untreated CD (median

years)

age, 5.0 years)

6 controls (median age, 8.8

*CD* celiac disease, *FISH* fluorescent in situ hybridization, *DGGE* denaturing gradient gel electrophoresis, *GFD* gluten‐free diet, *HIPchip* Human Intestinal Tract Chip, *IFN‐g* interferon‐gamma, *IL‐10* interleukin‐10, *IS‐pro* 16S‐23S interspacer, *PCR* polymerase chain reaction, *qPCR* quantitative polymerase chain reaction, *qRT‐PCR* quantitative reverse‐transcriptase‐ polymerase chain reaction, *TGGE* temporal temperature gradient gelelectrophoresis, *TLR2* toll‐like receptor 2, C-X-C

years)

2013 Netherland 21 untreated CD (median

**and sample size**

8 controls (mean age, 6.9

5 controls (mean age, 7.2

21 controls (median age,

9 controls (median age, 8.5

**Methods Main results**

16SrRNA gene sequencing

qRT‐PCR+ HIPchip microarray *Staphylococcus epidermidis, Staphylococcus pausteri*)

↓ *Streptococcus anginosus,* ↓ *Streptococcus mutans*

↓ *Streptococcus* and *Prevotella*

No significant differences in the abundance of bacterial phylum‐like groups between CD and controls The bacterial diversity was

comparable between CD and controls In treated and untreated CD:

diversity and abitability of *Lactobacillus* In untreated CD: ↓ *Streptococcus, Bacteroides, E. coli* In controls ↓ *Streptococcus, Bacteroides* ↑ *Bifidobacterium, Lactobacillus,*

↑ IL‐10, IFN‐g, C-X-C chemokine receptor type 6 expression

↓ *Firmicutes*

IS‐pro In treated and untreated CD: ↑*Streptococcus* ↑ *Lactobacillus* ↑ *Clostridium*

↑TLR2 expression

DGGE The intestinal microbiota of children with Marsh 3c lesion showed similarity of 98% and differs from other CD children with lesion as Marsh 3a, 3b and Marsh 2 In CD: ↓ Richness,

*Acinetobacter*

The intestinal microbiota composition and function play a fundamental role in the balance between the host's health and disease by different mechanisms: (1) regulation of epithelial cell proliferation and expression of tight junction proteins which act on intestinal permeability; (2) influence on mucin gene expression by goblet cells and their glycosylation pattern; 3) secretion of antimicrobial peptides (defensins, angiogenins, Reg3γ, etc.) by intestinal cells, which contribute to control gut bacterial adhesion. Certain components of the gut microbiota also affect the expression and activation of pattern recognition receptors (PRR), such as toll‐like receptors (TLRs), which are expressed by epithelial cells and innate immune cells. The mammalian TLR recognizes specific patterns of microbial components, called pathogen‐ associated molecular patterns (PAMPs). After the PRR‐PAMP interaction, activated innate immune cells start the adaptive immune response by presenting the antigen and by produc‐ ing cytokines, which leads to antigen‐specific, protective immune response. In inflammatory and autoimmune diseases this response causes damage to host's tissues [36]. The gut micro‐ biota impacts on adaptive immunity. Recently, specific commensal bacteria have been shown to influence T lymphocyte production (Th1, Th17) or anti‐inflammatory regulatory T cells (Tregs) [36].

To date, human microbiota and mucosal barrier function are the key players in etiology of many inflammatory and autoimmune diseases [37]. Changes in mechanisms regulating mucosal immunity and tolerance, can lead to impaired mucosal barrier function, increased penetration of microbial components from lumen into the mucosa and circulation, and consequently lead exaggeration of aberrant immune responses and inflammation.

The exact mechanisms through which the gut microbiota might influence CD onset or progression is unknown, but could include activation of innate immune system, modulation

of the epithelial barrier, or exacerbation of the gliadin‐specific immune response [38]. Moreover, the presence of microbiota can significantly influence the inflammatory effect of gluten. The microbiota may facilitate the access of gliadin peptides to the lamina propria and its interaction with infiltrated lymphocytes and antigen presenting cells (APCs) responsible for triggering the immune response via different mechanisms. In genetically predisposed individuals, gluten in association with microbial antigens can stimulate and modulate innate and adaptative immune response, sustaining a chronic mucosal inflammation, underlining this chronic disease [38].

### **4. Probiotics in the treatment of CD**

Probiotics are nonpathogenic live microorganisms, which when orally administered in adequate amounts, alter the microflora of the host and have beneficial health effects. Probiotics have shown to preserve the intestinal barrier promoting its integrity both in vitro and in vivo [39, 40] as well as regulating the response of the innate and adaptative immune system. The association of CD with intestinal dysbiosis and the evidence supporting a role for the microbiota and specific bacteria in maintaining gut barrier function and regulating the response of the innate and adaptive immune system, have supported the potential use of probiotics in CD treatment [41, 42]. Although the data regarding the use of probiotics for CD are encouraging, most of these data come from in vitro experimental models of CD [43, 44]. Studies regarding probiotics and CD in humans are very scarce [45–47]. Smecuol et al. evaluated the effect of the *Bifidobacterium* infantis natren life start (NLS) on gut permeability, the occurrence of symptoms, and presence of inflammatory cytokines in adult CD patients on GCD. Results have shown that probiotics did not modify intestinal permeability probably due to an insufficient dose or a short time of administration. However, probiotic administration improved gastrointestinal symptoms, alleviating and reducing constipation [47].

In children, the clinical trials performed on the effect of probiotics on CD are summarized in **Table 3**. In the earliest study Olivares et al. [45] evaluated the influence of *Bifidobacterium longum* CECT 7347 in addition to a GFD in children newly diagnosed with CD. They showed a decrease in peripheral CD3+ T lymphocytes and a trend in the reduction of tumor necrosis factor (TNF)‐α serum levels, and a reduction in the *Bacteroides fragilis* group(pro‐inflammato‐ ry bacteria) and in the content of IgA in stools. Klemenak et al. [46] evaluated the effect of a combination of the strains *Bifidobacterium breve* BR03 and *B. breve* B632, as compared to placebo. They reported that *B. breve* strains decreased the production of the pro‐inflammatory cyto‐ kine TNF‐α in children CD on a GFD.

At this time, the only treatment for CD is lifelong GFD, which involves the elimination of grains containing gluten, wheat, rye, and barley in addition to food products and additives derived from them [48]. To date, adherence to a diet is difficult for many patients. Studies have shown that dietary transgression in patients with CD is common and can occur anywhere from 32% to 55% [49]. Moreover, a GFD may be rich in high glycemic index foods which can increase

insulin resistance and, thus, the risk of obesity and cardiovascular disease. In the last decade, new therapies have been suggested to improve compliance to a GFD or to replace a GFD [50]. The use of probiotics appears to be able to reduce the damage caused by eating gluten‐ containing foods and may even accelerate mucosal healing after the initiation of GFD [50, 51]. A specific commercially available probiotic, VSL#3 (containing eight different bacteria), has been shown to reduce the toxicity of gluten when used in a fermentation process [52]. It is thought that the gut microbiota can be modified in its composition and function by probiotic administration. These may counteract or postpone the onset of CD, and it can be useful in patients on GFD, when the normal composition of the intestinal flora has not yet fully recovered.


*CD* celiac disease, DB double-blind, R randomized, PC placebo controlled.

**Table 3.** Clinical trials on the effect of probiotics for CD.

### **5. Conclusions**

of the epithelial barrier, or exacerbation of the gliadin‐specific immune response [38]. Moreover, the presence of microbiota can significantly influence the inflammatory effect of gluten. The microbiota may facilitate the access of gliadin peptides to the lamina propria and its interaction with infiltrated lymphocytes and antigen presenting cells (APCs) responsible for triggering the immune response via different mechanisms. In genetically predisposed individuals, gluten in association with microbial antigens can stimulate and modulate innate and adaptative immune response, sustaining a chronic mucosal inflammation, underlining

Probiotics are nonpathogenic live microorganisms, which when orally administered in adequate amounts, alter the microflora of the host and have beneficial health effects. Probiotics have shown to preserve the intestinal barrier promoting its integrity both in vitro and in vivo [39, 40] as well as regulating the response of the innate and adaptative immune system. The association of CD with intestinal dysbiosis and the evidence supporting a role for the microbiota and specific bacteria in maintaining gut barrier function and regulating the response of the innate and adaptive immune system, have supported the potential use of probiotics in CD treatment [41, 42]. Although the data regarding the use of probiotics for CD are encouraging, most of these data come from in vitro experimental models of CD [43, 44]. Studies regarding probiotics and CD in humans are very scarce [45–47]. Smecuol et al. evaluated the effect of the *Bifidobacterium* infantis natren life start (NLS) on gut permeability, the occurrence of symptoms, and presence of inflammatory cytokines in adult CD patients on GCD. Results have shown that probiotics did not modify intestinal permeability probably due to an insufficient dose or a short time of administration. However, probiotic administration

improved gastrointestinal symptoms, alleviating and reducing constipation [47].

In children, the clinical trials performed on the effect of probiotics on CD are summarized in **Table 3**. In the earliest study Olivares et al. [45] evaluated the influence of *Bifidobacterium longum* CECT 7347 in addition to a GFD in children newly diagnosed with CD. They showed a decrease in peripheral CD3+ T lymphocytes and a trend in the reduction of tumor necrosis factor (TNF)‐α serum levels, and a reduction in the *Bacteroides fragilis* group(pro‐inflammato‐ ry bacteria) and in the content of IgA in stools. Klemenak et al. [46] evaluated the effect of a combination of the strains *Bifidobacterium breve* BR03 and *B. breve* B632, as compared to placebo. They reported that *B. breve* strains decreased the production of the pro‐inflammatory cyto‐

At this time, the only treatment for CD is lifelong GFD, which involves the elimination of grains containing gluten, wheat, rye, and barley in addition to food products and additives derived from them [48]. To date, adherence to a diet is difficult for many patients. Studies have shown that dietary transgression in patients with CD is common and can occur anywhere from 32% to 55% [49]. Moreover, a GFD may be rich in high glycemic index foods which can increase

this chronic disease [38].

**4. Probiotics in the treatment of CD**

232 Probiotics and Prebiotics in Human Nutrition and Health

kine TNF‐α in children CD on a GFD.

An alternative treatment that can improve CD patients' quality of life may lie in probiotics. In particular, probiotics such as *Lactobacilli* and *Bifidobacterium* could be useful to reset altered gut microbiota, as well as reduce gliadin toxicity and immune activation. Their use as a primary prophylactic treatment for children at high risk of CD is also a potential consideration. However, their use in routine clinical practice is hindered by limited data from human studies. The role of specific probiotics and their mechanism of action need to be identified in a larger experimental population to confirm their effectiveness.

### **Author details**

Caterina Anania1 , Francesca Olivero1 , Eugenia Olivero2 and Lucia Pacifico1\*

\*Address all correspondence to: lucia.pacifico@uniroma1.it


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