Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host,” which was defined by the Food and Drug Organization of the United Nations (FAO) and World Health Organization (WHO) [1, 2, 3]. Probiotics should have some fundamental characteristics, such as human origin, nonpathogenic in nature, resistance to destruction by technical processing, acid and bile tolerances, adequate adherence and colonization on epithelial surfaces, antagonistic activity against pathogens, regulation of immune response, and influence human metabolic activities [4, 5, 6, 7].
Bacteria belonging to the genera Lactobacillusand Bifidobacteriumare the most frequently used probiotics. Besides, Enterococcus, Streptococcus, Saccharomyces, and Bacillusare also commonly used probiotics (representative species are listed in Table 1). The administration of probiotics has been confirmed as an alternative biological approach to combat bacterial and fungal pathogens in the oral cavity, GI tract, and urogenital system [4, 5, 7, 8, 9, 10, 11, 12, 13, 14]. It has been reported that probiotics could reduce Candida, which cause fungal infections in different organ systems of the human body and prevent bacterial infectious diseases [9, 10, 15]. Probiotics were capable of preventing cancers , modulating blood pressure [17, 18], and repressing cholesterol levels . Recently, species of Akkermansia muciniphila, Eubacterium hallii, and Faecalibacterium prausnitziiare identified as new potential probiotics because of their great benefits to the microbial metabolic networks and human health, especially the effects on correcting the imbalance of gut microbiota composition [7, 20, 21, 22]. A combination of probiotics with traditional treatment has been thought to be a potential approach for treatment of certain diseases.
|Lactobacillus||Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus paracasei, Lactobacillus sporogenes, Lactobacillus lactis, Lactobacillus helveticus, and Lactobacillus fermentium|
|Lactococcus||Lactococcus lactis, Lactococcus lactissubsp. lactis, Lactococcus lactissubsp. diacetylactis, and Lactococcus Lactissubsp. cremoris|
|Bifidobacterium||Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium bifidus, and Bifidobacterium lactis|
|Enterococcus||Enterococcus faecalisand Enterococcus faecium|
|Saccharomyces||Saccharomyces cerevisiaeand Saccharomyces boulardii|
|Bacillus||Bacillus coagulansand Bacillus subtilis|
|Others||Akkermansia muciniphila, Eubacterium hallii, and Faecalibacterium prausnitzii|
Representative microbe commonly considered as probiotics.
It is noteworthy that health benefits of probiotic bacteria are strain specific, which cannot be generalized to other strains, not even the same species, although some properties may be common for different strains because of the similarities in the metabolism of ecological functionality [5, 6]. Thus, the selection of certain probiotics for therapeutic purposes should be targeted for specific pathogens. Probiotics effects are dose specific [5, 6]. It has been suggested that a daily intake of 106–109 colony-forming units (CFUs) of probiotic microorganisms is the minimum effective dose for therapeutic purposes [5, 6, 8].
A number of probiotics are currently commercially available, and they have been categorized into single-strain or multi-strain/multispecies products [7, 23, 24]. Multi-strain/multispecies probiotics exhibited better effects than single-strain probiotics. The multispecies probiotic consortium VSL#3 (Streptococcus thermophilus, Eubacterium faecium, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus delbrueckiisubsp. bulgaricus) was proven more effective than single-strain probiotics for the treatment of ulcerative colitis . The multispecies probiotic consortium, Ecologic AAD (Bifidobacterium bifidumW23, Bifidobacterium lactisW18, Bifidobacterium longumW51, Enterococcus faeciumW54, Lactobacillus acidophilusW37 and W55, Lactobacillus paracaseiW72, Lactobacillus plantarumW62, Lactobacillus rhamnosusW71, and Lactobacillus salivariusW24), combined with amoxicillin, could reduce diarrhea-like bowel movements, while the single strain could not . Thus, the combination-specific probiotic effects from diverse strains can lead to synergistic effects.
Among the most frequently used probiotics, the genera Lactobacillus, Bifidobacterium, Lactococcus, and Saccharomyceshave been included in the category of “generally regarded as safe” (GRAS) [4, 6]; however, other probiotic organisms such as Enterococcus, Bacillus, and Streptococcusare not generally regarded as safe. Since probiotics have been applied in food production, disease treatment, and others, it is important to undergo safety evaluation of probiotics before human consumption.
In this chapter, we briefly review the mechanisms of action of probiotics, the safety concern of probiotics, and their potentials for prevention and treatment of diseases. Here, we discuss the application of probiotics in the fungal Candida-infected and invasion candidiasis.
2. Probiotics mechanism of action
Probiotics mechanism of action is with important differences among different species and strain, examples are listed in Table 2.
|Mechanism of action||Probiotics||Study outcomes||References|
|Maintenance flora healthy by reduction the growth and colonization of pathogens||L. rhamnosusGG, L. caseiShirota, L. reuteriSD2112 and L. brevisCD2||L. rhamnosusGG showed the strongest inhibitory activity in fructose and glucose medium against C. albicans, followed by L. caseiShirota, L. reuteriSD2112 and L. brevisCD2|||
|L. plantarum,commercial preparation LactoLevure®||Increased survival of mice infected by multidrug resistant P. aeruginosaand E. coli|||
|B. breve, L. casei(randomized controlled trial, RCT)||Levels of beneficial organic acids significantly increased in the gut, and the incidences of infectious (pneumonia and bacteremia) complications were significantly lower in the probiotic group|||
|Synbiotic (Lactobacillus, Bifidobacterium, and galactooligosaccharides) for 8 weeks (RCT)||Acetic acid concentration significantly increased (100 times), pH value decreased, Gram-negative rod (1/10) in the gut decreased, and P. aeruginosadecreased in the probiotic group|||
|Multi-strain synbiotic for 7 days (RCT)||Synbiotic group had lower pathogenic bacteria (43% versus 75%) and multiple organisms (39% versus 75%) in nasogastric aspirates than controls|||
|B. lactisBb12 for 7–21 days (RCT)||Probiotic group had great higher counts of Bifidobacterium(P = 0.001) and lower counts of Enterobacteriaceae(P = 0.015) and Clostridiumspp. (P = 0.014) than in placebo group|||
|L. caseisubsp. rhamnosusfor 6 weeks (RCT)||Colonization of Candidain gut was reduced in probiotic group (P = 0.01)|||
|Enhancement of mucosal barrier integrity||L. plantarum299v for 8 days (RCT)||Bacterial translocation in mesenteric lymph nodes and liver was reduced to 0 and 12%, respectively|||
|Microencapsulated Bifidobacteria||Bacterial translocation to mesenteric lymph nodes was reduced by encapsulated Bifidobacteria(P < 0.05)|||
|VSL#3 (RCT)||Decreased incidence of bacterial translocation in VSL#3 group than in water group (8% versus 50%; P = 0.03)|||
|Immune modulation||VSL#3 (Lactobacillus, Bifidobacterium, and S. thermophilus) for 7 days (RCT)||Reduced acute physiology and chronic health evaluation II score; reduced sequential organ failure assessment, IL-6, procalcitonin, and protein|||
|L. plantarum299v (RCT)||Late attenuating effect (after 15 days), serum IL-6 levels reduced|||
Mechanism of action of probiotics.
2.1. Maintenance flora healthy by reduction the growth and colonization of pathogens
The ability of probiotics to establish in the gastrointestinal (GI) tract, maintain flora healthy, and reduce the growth of pathogens and colonization is enhanced by their ability to eliminate competitors. Probiotic strains release different antimicrobial molecules such as organic acids, hydrogen peroxide (Н2О2), and antimicrobial peptide bacteriocins into the intestinal environment to limit the growth of bacterial and fungal pathogens [6, 39, 40, 41, 42, 43].
Lactic acid and acetic acid are the main metabolites formed by lactic acid bacteria (LAB). Both lactic acid and acetic acid could result in acidity environment and thus inhibit the growth of various microorganisms. Acetic acid has a broader spectrum of antimicrobial activity when compared to lactic acid. Moreover, it is known that a synergistic effect exists between the two acids: mixtures of acetic and lactic acids suppress the growth of the pathogenic enteric bacterium Salmonella typhimurium.
LAB can also produce Н2О2, the antimicrobial activity of which is linked to the strong oxidizing effect. Hydrogen peroxide showed a bactericidal effect on most pathogens when in combination with lactoperoxidase-thiocyanate milk system . L. johnsoniiNCC933 and L. gasseriKS120.1 killed enteric uropathogenic and vaginosis-associated pathogens due to the production of lactic acid and hydrogen peroxide .
Bacteriocins are ribosomally synthesized antimicrobial peptides, which have broad spectrum of inhibitory effect against Gram-positive and Gram-negative bacteria, viruses, and fungi [47, 48, 49, 50]. L. plantarum2.9, a bacteriocinogenic strain, inhibited a set of foodborne pathogens including B. cereus, E. coliO157:H7, and S. enterica. Bacteriocin-producing strains identified in our lab, e.g., L. plantarumZJ316, L. plantarumLZ95, L. plantarumZJ008, and L. plantarumZJ005, showed antimicrobial activity against various pathogens in vitrosuch as S. aureus, E. coli, S. enterica, L. monocytogenes, and C. albicans[42, 52, 53, 54].
2.2. Enhancement of mucosal barrier integrity
Probiotics have been shown to improve barrier function and the mechanisms of barrier function including alteration of tight junction protein expression and/or localization, induction of mucus secretion, increased production of cytoprotective molecules such as heat-shock proteins, inhibition of apoptosis of epithelial cells, and promoting cell survival [29, 55, 56]. They compete with pathogens and prevent their invasion through the epithelium by the ability of adherence to the intestinal epithelium and mucus. L. plantarumhas been shown to enhance mucosal barrier by adhering to the mucosal membrane and reducing Gram-negative bacteria . Probiotics also compete for limiting resources, thus suppressing the growth of bacterial and fungal pathogens. The probiotic E. coliNissle 1917 is able to effectively take up multiple limited environmental irons and simultaneously competitively inhibit the growth of other intestinal microbes and pathogens .
Furthermore, butyrate, a short-chain fatty acid (SCFA), could reduce bacterial translocation, improve the organization of tight junctions, modulate intestinal motility in addition to being an energy source for colonocytes, and maintain the integrity of the intestinal epithelium [29, 30, 31, 58, 59, 60]. E. halliiis an important anaerobic butyrate producer resident in our gut, which influences the intestinal metabolic balance and enhances the host-gut microbiota homeostasis . Thus, the administration of probiotics with butyrate-producing bacteria, in particular, could be an effective way to achieve health benefits.
2.3. Immune modulation
Probiotics are reported to enhance phagocytic activity of granulocytes and cytokine excretion in lymphocytes, increase immunoglobulin-secreting cells, and attenuate inflammasome activation. They are able to affect cells involved in immune responses, including epithelial cells, dendritic cells (DCs), T cells, regulatory T (Treg) cells, monocytes/macrophages, immunoglobulin A (IgA)-producing B cells, and natural killer cells [62, 63].
Probiotic bacteria have an effect on intestinal DCs, which have the ability to recognize and respond to different bacteria by linking the innate immune system to the adaptive immune response and to develop T- and B-cell responses. Badia et al. found that the immunomodulatory role of S. boulardiiin the DCs prior to infection was related to the upregulation of tumor necrosis factor alpha (TNFα) and C─C chemokine receptor type 7 mRNAs, which might make the DCs more effective in antagonizing bacteria [64, 65]. Smith et al. reported that S. boulardiistimulated the production of cytokines TNFα, IL-1, IL-12, IL-6, and IL-10 in DCs and also induced high levels of costimulatory molecules CD80 and CD86, thus modulated the immune system and led to an efficient clearing of enteropathogenic bacteria from the blood stream coupled with a faster cytokine response [65, 66].
Probiotics also influence intestinal epithelial cells through interaction with Toll-like receptors (TLRs) and downregulate the expression of NF-κB and proinflammatory cytokines [67, 68]. This effect is supported by the following studies: the supernatant of probiotic Faecalibacterium prausnitziiinhibited the NF-κB pathway in vitroand in vivoand showed protective effects in different models such as dinitrobenzene sulfate (DNBS)-induced colitis model and dextran sodium sulfate (DSS)-induced colitis ; the probiotic strain L. rhamnosusGG prevented cytokine-induced apoptosis in intestinal epithelial cells ; and L. rhamnosusGR-1 reduced the adhesion of E. coliby promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation .
3. Probiotic as antifungals for prevention and treatment of candidiasis
Candidais an opportunistic pathogen, causing mucosal infections including infections in the oral cavity, oropharynx, esophagus, and vagina, and potentially life-threatening systemic candidiasis. Candida albicansis the most common fungal pathogen in humans responsible for causing superficial as well as deep invasive candidiasis, which are essentially caused by Candidabiofilms attached to body surfaces. Other Candidaspecies such as Candida tropicalis, Candida guilliermondii, Candida krusei, and Candida glabrataare less frequently isolated in healthy and diseased humans [72, 73, 74]. Probiotics are known to reduce Candidainfection in different organs and are generally considered to be beneficial for overall health. They appear to assist the host combat the pathogen by suppressing filamentation formation and reducing biofilm development, the mechanism of which may be related to expression of genes associated with biofilm formation and filamentation in Candidaspecies. In vitroand in vivostudies have demonstrated the role of probiotics in the prevention of Candidacolonization and invasive candidiasis [38, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86].
3.1. In vitroevidences: probiotics in prevention/treatment of Candidainfections
Several in vitrostudies have addressed the antifungal effects of probiotics against Candidaisolated from the human oral cavity, GI tract, and genitourinary tract [77, 78, 79, 80, 81, 86, 87]. The probiotics that have been investigated against Candidaspecies include Lactobacillus(e.g., L. rhamnosus, L. plantarum, L. fermentum, L. acidophilus, L. paracasei, L. johnsonii, and L. salivarius), Bifidobacterium(e.g., B. bifidumand B. infantis), Saccharomyces(e.g., S. boulardii), and Streptococcus(e.g., S. thermophilus). Table 3 shows candidacidal activity of probiotic strains in different studies. C. albicansappears to be more susceptible to the antifungal effect of Lactobacillusthan C. pseudotropicalis, and the probiotics exhibited growth inhibitory activities against C. glabrata, C. krusei, and C. parapsilosis[79, 87].
|Probiotics||Target pathogen||Study outcome||References|
L. fermentum, L. rhamnosus, L. plantarum, and L. acidophilus
|C. albicansand C. pseudotropicalis||All probiotics inhibited the growth of C. albicansby H2O2 production and alternative mechanism|||
|S. boulardii||C. albicansSC5314||S. boulardiiinhibited the affecting hyphae formation, Candidaadhesion, and biofilm formation by capric acid production|||
|L. paracaseiIMC 502||C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis||High activity toward Candidastrains except C. glabrataand C. tropicalis|||
|L. plantarumATCC 8014 and L. johnsoniienriched or not with SeNPs||C. albicansATCC 14053||Strong inhibition of C. albicansby supernatant of selenium-enriched Lactobacillusspp.|||
|L. acidophilus, L. rhamnosus, L. salivarius, B. bifidum, S. thermophiles, and B. infantis||C. albicans10341||Significant inhibitory effect on biofilm formation and reduce viability of Candida|||
|L. rhamnosusGR-1 and L. reuteriRC-14||C. albicansSC5314||Visible inhibition zones of fungal C. albicansby probiotic treatment; low pH environment caused by lactic acid and the H2O2 production may be anti-Candidafactors|||
|L. acidophilusATCC 4356||C. albicansATCC 18804||Reduce growth of C. albicanscells by 45.1%|||
|L. caseisubsp. rhamnosus||Candidaspp.||80 preterm neonates with a very low birth weight: probiotic reduced incidence and intensity of enteric colonization by Candidaspp. (RCT)|||
|L. rhamnosus GG, L. rhamnosus LC705, P. freudenreichiisubsp. shermaniiJS||Candidaspp.||276 elderly people: probiotic intervention reduced the risk of high yeast counts by 75% and the prevalence of hyposalivation (RCT)|||
|L. rhamnosusGR-1 and L. reuteriRC-14||Candidaspp.||55 women: probiotics significant reduced vaginal discharge, itching, and/or burning vaginal feeling, dyspareunia, and/or dysuria, and reduced the presence of Candidaspp. (RCT)|||
|L. acidophilus, L. rhamnosus, B. longum, B. bifidum, S. boulardii, and S. thermophilus||Candidaspp.||150 children (aged 3 month to 12 year) on broad-spectrum antibiotics for at least 48 h: probiotic therapy avoided a significant increase in the number of patients colonized by Candidaspp., significantly reduced the presence of Candidain the urine (RCT)|||
|L. bulgaricus, B. longum, and S. thermophilus||Candidaspp.||65 patients with Candida-associated stomatitis: detection rate of Candidaspp. was reduced in the probiotic group; significant relief of clinical signs and symptoms after probiotic administration (RCT)|||
|L. acidophilus, B. lactis, B. longum, and B. bifidum||Candidaspp.||112 preterm neonates (gestational age < 37 wk and birth weight < 2500 g): probiotics may reduce enteral fungal colonization and invasive fungal sepsis in low-birth-weight neonates (RCT)|||
|L. reuteriDSM 17938 and L. reuteriATCC PTA 5289||Candidaspp.||215 elderly people (aged 60–102 y): significant reduction of Candidacells in saliva and plaque (RCT)|||
Probiotics in prevention/treatment of Candidainfections.
However, the mechanisms involved in antifungal activity of probiotics against Candidaremain unclarified. Strus et al. found that Lactobacillusstrains could inhibit the growth of C. albicansto a certain degree and their anticandidal activity related to H2O2 production . Murzyn et al. reported that S. boulardiiwas able to secrete active compounds, mainly capric acid, reduced the expression of hwp1, ino1, and csh1genes that encode virulence factors in C. albicansSC5314 cells, and inhibited filamentation of C. albicansand its mycelial development . Therefore, it is likely that the antimicrobial molecules, organic acids, and Н2О2 produced by probiotic are major factors to limit growth of fungal pathogen Candida.This idea was supported by the research of Köhler et al. They demonstrated that low pH environment caused by lactic acid and the H2O2 production of L. rhamnosusGR-1 and L. reuteriRC-14 strains played important role in their inhibition activity to C. albicansSC5314. Moreover, L. rhamnosusGR-1 and L. reuteriRC-14 inhibited genes associated with C. albicansbiofilm formation . This result, together with the findings in Murzyn et al. study, shed light on a novel approach for uncovering the molecular mechanisms of the probiotic effect by using gene expression and related technology.
3.2. In vivoevidences: probiotics in prevention/treatment of Candidainfections
In vivostudies, especially RCTs, have also been performed to substantiate the antifungal activity of probiotics in humans. These studies mostly focus on the sites of oral cavity, GI tract, and urogenital tract, which are susceptible to Candidainfections (Table 3).
The elderly are a group particularly susceptible to oral candidiasis, because of frequent usage of dentures, hyposalivation, and their weakened immune status. Researches by Hatakka et al. and Kraft-Bodi et al. have shown that the daily consumption of food with L. reuteriDSM17938, L. reuteriATCC PTA 5289, and L. rhamnosusGG ATCC 53103 significantly reduced the high yeast counts in saliva and biofilms in the elderly [76, 85]. The removal of biofilms by the use of probiotics that reduce the oral burden of Candidacould play a major role in preventing oral candidiasis in denture wearers.
For the urogenital tract, chronic vulvovaginal candidiasis (VVC) is the most common candidiasis disease and impacts the life quality of thousands of women around the world. Researches on the effect of probiotics in the treatment and prophylaxis of VVC have been performed . Martinez et al., in an RCT involving 55 women, demonstrated that the administration of L. rhamnosusGR-1 and L. reuteriRC-14 significantly reduced the presence of Candidaand therefore reduced the vaginal discharge, itching, and/or burning vaginal feeling, dyspareunia, and/or dysuria .
For the GI tract, Candidaspecies are common inhabitants of GI tract. Dysbiosis of GI tract may lead to candidal overgrowth and possible invasive infections, especially in infants. Hence, immunocompromised children, especially preterm neonates with low birth weight, have been the target population of a large number of studies to evaluate the prevention or/and treatment potentials of probiotics to Candidainfections [28, 75, 83]. Manzoni et al., in an RCT involving 80 very low birth weight (VLBW) neonates, demonstrated that orally administered L. caseisubsp. rhamnosus significantly reduced incidence and intensity of enteric colonization by Candida. Another RCT, by Roy et al., found L. acidophilus, B. lactis, B. longum, and B. bifidumreduced enteral fungal colonization and invasive fungal sepsis in 112 preterm neonates (gestational age < 37 wk and birth weight < 2500 g) .
Together, both the laboratory studies and clinical studies showed that probiotics could prevent Candidacolonization by inhibiting adhesion, filamentation, and biofilm formation, and therefore supplementation of probiotics could be a potential approach for reducing Candidacolonization and invasive candidiasis.
4. Safety of probiotics
Although most commercially available probiotic strains are generally regarded as safe and none of the clinical studies mentioned above were reported to have adverse effects directly related to probiotics, there are some concerns regarding the safety of probiotics, including potential of bacteremia and/or endocarditis occurrence, toxicity to the gastrointestinal tract, and transfer of antibiotic resistance .
4.1. Potential of bacteremia and/or endocarditis occurrence
Lactic acid bacteria, including Bifidobacterium, have been reported to cause bacteremia as well as endocarditis [88, 89, 90, 91, 92]. Cannon et al. described that L. rhamnosuscaused liver abscess, lactobacillemia, and infective endocarditis in a few case studies, and also the occurrence of Lactobacillussepsis was directly linked with the ingestion of probiotic supplements, especially among immunocompromised patients and those with endocarditis . Kunz et al. found two premature infants with short gut syndrome developed Lactobacillus bacteremia while taking LactobacillusGG supplements. However, the risk of infection due to Lactobacilli is extremely rare. Statistic data from surveillance in Finland suggest that there was no increase in Lactobacillusbacteremia during 1990–2000, and Lactobacilli were isolated in 0.02% of all blood cultures .
4.2. Toxicity to the gastrointestinal tract
The role of probiotics on gastrointestinal physiology suggests a theoretical possibility that the production of metabolites might be undesirable and also might lead to malabsorption due to deconjugation of bile salts. These might increase the risk of colon cancer; however, there is no epidemiologic or clinical evidence to support this hypothesis [94, 95].
4.3. Transfer of antibiotic resistance
Another major safety concern of theoretical importance is genetic transfer of antibiotic resistance from probiotic strains to pathogenic cells in the gastrointestinal tract [96, 97]. Plasmids with antibiotic-resistance genes, including genes encoding resistance to tetracycline, erythromycin, chloramphenicol, and macrolide-lincosamide-streptogramin, have been found in L. plantarum, L. fermentum, L. acidophilus, and L. reuteristrains. L. plantarum5057 exhibited tetracycline resistance, and L. lactiswas with streptomycin, tetracycline, and chloramphenicol resistances [98, 99, 100]. Although the transfer of native Lactobacillusplasmids is quite rare, there are some cases, e.g., the antibiotic-resistance plasmids from Lactococcusspecies could transfer to Leuconostocspecies and Pediococcusspecies.
With respect to the potential risks of probiotics, it is important to conduct population-based surveillance for safety concern.