Probiotics as Antifungals in Mucosal Candidiasis

Abstract

Candida is an opportunistic pathogen that causes mucosal and deep systemic candidiasis. The emergence of drug resistance and the side effects of currently available antifungals have restricted their use as long-term prophylactic agents for candidal infections. Given this scenario, probiotics have been suggested as a useful alternative for the management of candidiasis. We analyzed the available data on the efficacy of probiotics in candidal colonization of host surfaces. A number of well-controlled studies indicate that probiotics, particularly lactobacilli, suppress Candida growth and biofilm development in vitro. A few clinical trials have also shown the beneficial effects of probiotics in reducing oral, vaginal, and enteric colonization by Candida; alleviation of clinical signs and symptoms; and, in some cases, reducing the incidence of invasive fungal infection in critically ill patients. Probiotics may serve in the future as a worthy ally in the battle against chronic mucosal candidal infections.

probiotics, Candida, candidiasis, Lactobacillus

The high prevalence of human immunodeficiency virus (HIV) infection and other immunocompromising conditions globally has resulted in resurgence of Candida infections. These infections may be present on mucosal surfaces, including the oral cavity, oropharynx, esophagus, and vagina, as well as systemically [1].

Healthy individuals may also be the target of Candida infections, as this fungus is a commensal organism in human mucosal surfaces, inhabiting one-half of the human populace as an opportunist pathogen of the gastrointestinal and urogenital tracts [2]. When adverse conditions supervene, particularly in debilitated individuals, Candida is capable of causing superficial as well as deep invasive candidiasis, including fungemias. These diseases are essentially caused by candidal biofilms attached to body surfaces, as opposed to the planktonic form of the yeast, which exists in the suspended phase. Candida albicans is the most common Candida species inhabiting the mucosal surfaces both in health and disease, whereas other Candida species such as C. tropicalis, C. guilliermondii, C. krusei, and C. glabrata are less frequently isolated.

A range of adverse factors predisposes an individual to local or systemic candidal infection. The critical factors that precipitate systemic infections include the very low-birth-weight neonates [3] and immunosuppression as in HIV disease, or radiation and cytotoxic therapy [4]. Perturbation of mucosal ecosystem or marked changes in the microbial ecosystems due to antibiotics or corticosteroids, hypoendocrine states (eg, hypothyroidism, Addison disease, and diabetes mellitus), blood disorders such as acute leukemia, xerostomia due to irradiation or Sjogren syndrome, and ill-fitting appliances are predisposing factors for localized candidal infections either in healthy or diseased states [4]. Thus, Candida is considered to be an opportunistic pathogen, causing “diseases of the diseased.”

The aim of this review was to explore critically the available in vitro and in vivo data on the efficacy of probiotic therapy in managing mucosal candidiasis. For this purpose, a critical review of the literature was conducted to select pertinent articles published in the English literature from 2000 to 2015. An electronic search was performed in Medline using the following terms: “probiotic or Lactobacillus” AND “Candida or candidiasis” to garner clinical evidence, and “probiotic or Lactobacillus” AND “Candida” for the in vitro studies. Only clinical trials assessing Candida infection in the oral cavity, urogenital tract, and gastrointestinal tract were included.

In the following sections, we provide an overview of Candida infections, a summary of probiotics, in vitro and in vivo evidence of the antifungal effects of probiotics and their possible mechanisms of action, and the safety and risks of probiotic therapy.

CANDIDA INFECTIONS

Oral Candidiasis

Oral candidiasis can manifest in a variety of clinical guises. The classic triad of oral candidiasis is the pseudomembranous, the erythematous (atrophic), and the hyperplastic variants [4].

In addition, there are a number of other Candida-associated lesions where the etiology is multifactorial. These diseases include Candida-associated denture stomatitis, angular cheilitis or angular stomatitis, median rhomboid glossitis, and the newly described linear gingival erythema, the microbial etiology of which is still poorly understood [4].

Extraoral and Systemic Candida Infections

Vulvovaginal Candida infection (VVC) is the second most common cause of vaginitis after bacterial vaginosis. Transmission of this yeast from the vagina to the mouths of newborns during birth is a major portal of oral infections in newborns, leading to the development of thrush [2].

Candida inhabits the gastrointestinal (GI) tract in almost all humans, and most of the infections involving Candida are endogenously acquired from the GI tract. Candida can translocate into the bloodstream through the intact gastrointestinal mucosa and spread to visceral organs, leading to systemic candidiasis, especially in critically ill patients [3]. Disruption of normal physiological barriers, such as gastric acidity and perturbations of the indigenous microflora of the colon, facilitate Candida overgrowth.

Within the GI tract, the most common site of infection is the esophagus. Candida may be associated with gastric ulcers as an opportunistic pathogen that delays ulcer healing and aggravates the disease [5].

Management of Candidiasis

For some decades, systemic antifungal agents have been successfully used to prevent mucosal as well as invasive fungal infections. However, due to the drug side effects (nausea, vomiting, and diarrhea), and potential emergence of resistant strains, antifungal prophylaxis has not been totally successful.

The commonly used antifungals are the polyenes (nystatin and amphotericin B) and azoles (fluconazole, itraconazole, voriconazole). Interestingly, the biofilm phase of Candida is much more resistant to all these antifungals compared with their planktonic counterparts [6]. The limited spectrum and toxicity of available antifungals and the gradual emergence of resistance to these drugs are a concern; thus, alternative therapies are urgently warranted.

PROBIOTICS

The use of probiotic bacteria against microbial infections has emerged as an alternative therapeutic technique for Candida infections in view of the limitations of the currently available antimicrobials.

Probiotics are defined as live microorganisms that, when administered or consumed in adequate quantities, confer health benefits on the host. Bacteria belonging to the genera Lactobacillus and Bifidobacterium and, to a lesser extent, Enterococcus, Streptococcus, and Saccharomyces have often been used as probiotics in food supplements for a considerable period of time [7].

A safe probiotic needs to be of human origin, devoid of intrinsic and transmissible antibiotic resistance genes. The functional requirements of a probiotic include acid and bile tolerances, adequate adherence and colonization on epithelial surfaces, immunostimulation, and antagonistic activity against pathogens [7].

Therapeutic Potential of Probiotics

In therapeutic terms, probiotics are known to reduce Candida infections in different organ systems of the human body, and are generally considered to be beneficial for overall health. For instance, probiotics can combat diarrhea (mainly in children), and relieve lactose intolerance and symptoms of inflammatory bowel diseases [7]. Additionally, probiotic bacteria have been investigated for their potential for preventing cancers such as colorectal cancer [8], regulating blood pressure [9], and suppressing cholesterol levels [10]. The combination of probiotics with traditional treatment options are thought to generate better outcomes and disease resolution in different loci, with only a marginal increase in the treatment cost [7, 11, 12].

Probiotics as an Antimicrobial

Organisms of the genus Lactobacillus have been traditionally used as probiotics for decades, and they are deemed worthy as an alternative biological approach to combat bacterial and fungal pathogens in the oral cavity, GI tract, and urogenital system [1, 3, 11, 13–21]. It is noteworthy that the antimicrobial effect of probiotic bacteria is strain-specific; hence, the selection of probiotics for therapeutic purposes should be targeted for specific pathogens and their beneficial effects cannot be generalized [14]. In addition, there are reports of putative antiviral effects of probiotics, mainly against respiratory viral pathogens, in people of all ages [22].

IN VITRO EVIDENCE OF THE ANTIFUNGAL EFFECTS OF PROBIOTICS

A number of in vitro studies have demonstrated the antifungal effect of polymicrobial combinations of probiotics against human C. albicans isolates from the oral cavity, GI tract, and genitourinary tract [13, 14, 23–31]. Table 1 illustrates the variety and the extent of bacterial strains used to evaluate the candidicidal activity of probiotic bacteria, beginning this millennium.

Table 1.

In Vitro Investigations on the Antifungal Effects of Probiotics

Reference	Probiotics	Pathogen	Method	Results	Comments
Strus et al, 2005 [27]	14 different strains: Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus	Candida albicans, Candida pseudotropicalis	Antagonism on agar plates	- All probiotics inhibited the growth of C. albicans to a certain degree - Most lactobacilli were able at least slightly inhibit the growth of C. pseudotropicalis	Anticandidal activity related to H2O2 production and alternative mechanism.
Thein et al, 2006 [28]	L. acidophilus, Actinomyces israelii, Prevotella nigrescens, Porphyromonas gingivalis, Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, and Streptococcus intermedius	C. albicans 2560 g	Biofilm assay (scanning electron microscopy)	- 48 h co-culture: All bacteria, except S. mutans and S. intermedius, reduced viable yeasts cells - C. albicans biofilm + P. aeruginosa: reduced hyphal growth compared with bacteria-free biofilm	Bacteria modulate C. albicans biofilm formation in mixed species co-cultures and affected the morphogenesis of the yeast.
Hasslöf et al, 2010 [14]	L. plantarum 299v, L. plantarum 931, L. rhamnosus GG ATCC 53103, L. rhamnosus LB21, and Lactobacillus paracasei	MS: - Reference strains: S. mutans NCTC 10449, S. mutans Ingbritt, and Streptococcus sobrinus OMZ176 - Clinical isolates: S. mutans P1:27 and S. mutans P2:29	Agar overlay interference tests. Four concentrations of probiotics were tested (109, 107, 105, and 103 CFU/mL)	MS: - 109 to 105 CFU/mL: all lactobacilli strains inhibited the growth of the MS strains completely (except L. acidophilus La5) - 103 CFU/mL: only L. plantarum 299v and L. plantarum 931 displayed a total growth inhibition for all MS. L. rhamnosus GG ATCC 53103 inhibited the growth slightly for 3 MS	L. acidophilus La5: weaker inhibition capacity in comparison with the other probiotic strains (P < .05). All the tested Lactobacillus strains reduced Candida growth, but the effect was generally weaker than for MS.
		C. albicans: - Reference strains: C. albicans ATCC 28366, C. albicans ATCC 10231 - Clinical isolates: C. albicans 1957, C. albicans 3339 and C. albicans GDM8		C. albicans: - 109 and 107 CFU/mL: all lactobacilli except L. acidophilus La5 and Lactobacillus reuteri PTA 5289 inhibited all Candida strains completely - 105 CFU/mL: L. rhamnosus strains, L. paracasei, and L. reuteri PTA 5289 displayed a slight inhibition. L. acidophilus La5 showed no inhibition. L. plantarum and L. reuteri ATCC 55730 executed a total inhibition - 103 CFU/mL: No inhibition was recorded except for the L plantarum strains
Murzyn et al, 2010 [26]	Saccharomyces boulardii	C. albicans SC5314	- Hyphae formation assay - Adhesion assay - Gene expression assay	- Active compounds of probiotic yeast reduced Candida virulence factors (hyphae formation, cell adhesion, and biofilm formation) - Yeast extract and capric acid reduced expression of HWP1, INO1, and CSH1 genes in C. albicans cells	Capric acid was the main compound affecting hyphae formation, Candida adhesion, and biofilm formation.
Ishijima et al, 2012 [23]	Streptococcus salivarius K12	C. albicans (clinical isolate)	- Germ tube formation and mycelial growth of C. albicans (adherence to plastic substratum) - Deferred streak assay	- S. salivarius reduced adherence of mycelial form to plastic substratum, increased the number of planktonic Candida cells in culture medium, but did not inhibit C. albicans strain - Probiotic bacteria preferentially bound to hyphae	S. salivarius K12 was not directly fungicidal, but appeared to inhibit Candida adhesion to the substratum.
Köhler et al, 2012 [25]	L. rhamnosus GR-1 and L. reuteri RC-14 Lactobacillus johnsonii PV016 and Staphylococcus aureus ATCC 25923 (controls)	C. albicans SC5314.	Antagonism on agar plates and in broth cultures	- 48 h: Lactobacillus GR-1 and RC-1 showed visible zones of fungal growth inhibition around them - L. johnsonii: very weak inhibition zone - S. aureus: no inhibition zones - C. albicans growth was suppressed at low pH by the Lactobacillus culture supernatants - Probiotics inhibited genes associated with biofilm formation	Lactic acid at low pH environment: major role in fungal growth inhibition. Glucose or other nutrient exhaustion was not a likely cause for fungal inhibition. H2O2 production may be an anti-Candida factor.
Coman et al, 2014 [13]	L. rhamnosus IMC 501	- Gram-positive, gram-negative bacteria - C. albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis	- Modified cross-streak method - Radial streak method - Agar well diffusion method	L. rhamnosus - Inhibitory activity against both gram-positive and gram-negative bacteria, and against 2 C. albicans strains (ATCC 10261 and ISS7)	L. paracasei IMC 502: higher activity toward all the pathogens, especially Candida strains; strong inhibition registered for SYNBIO.
	L. paracasei IMC 502			L. paracasei - Inhibitory effect on gram-positive and gram-negative bacteria, especially S. aureus and Proteus mirabilis. All Candida spp were inhibited except C. glabrata and C. tropicalis
	Combination of both (SYNBIO)			SYNBIO- Inhibitory activity against most of the bacteria and fungi strains, especially C. albicans and C. krusei
Verdenelli et al, 2014 [30]	L. paracasei subsp paracasei, L. plantarum, L. fermentum, L. rhamnosus IMC 501, and L. paracasei IMC 502	C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis (clinical isolates)	- Agar well diffusion assay and radial method - Coaggregation assay	- Well diffusion assay: no inhibition against Candida spp - Radial method: All lactobacilli had the capacity to inhibit Candida in different degrees	Inhibition and coaggregation ability vary according to the Lactobacillus strain and the pathogen involved
Kheradmand et al, 2014 [24]	L. plantarum (ATCC 8014) and L. johnsonii (clinical isolate) enriched or not with SeNPs	C. albicans (ATCC 14053)	- Conventional hole-plate diffusion method and time-kill assay using probiotic supernatant (grown with or without selenium dioxide)	Conventional hole-plate diffusion: - L. plantarum and L. johnsonii supernatant grown with selenium dioxide showed potent anti-Candida activity. - No antifungal effect was observed with supernatant without selenium Time-kill assay: - No viable C. albicans was present after 4 h incubation with culture supernatants grown with selenium dioxide - Viable C. albicans cells were present even after 24 h incubation with culture supernatants grown without selenium dioxide	Direct antifungal effect was observed when selenium-enriched Lactobacillus spp were co-cultured with C. albicans. The strong inhibition of C. albicans by supernatant of selenium-enriched Lactobacillus spp indicated the release of potent exometabolites.
			- Time-kill assay using probiotic cell suspension (grown with or without selenium dioxide)	After 0.5 h, Lactobacillus strains without SeNPs decreased the viability of C. albicans by approximately 10-fold. SeNP-enriched species decreased 1000-fold
Ujaoney et al, 2014 [29]	L. acidophilus, L. rhamnosus, L. salivarius, Bifidobacteriumbifidum, Streptococcus thermophilus, Bifidobacterium infantis, Lactobacillus GG, and Bacillus coagulans BC30	C. albicans 10341	Biofilm assay on denture strips using bacterial suspensions and probiotic supernatants (XTT quantification)	Probiotics’ supernatant provided a stronger and significant inhibitory effect on biofilm formation than their bacterial counterparts	Depletion of nutrients in the culture media by overgrowth of the probiotic bacteria may inhibit fungal growth.
Vilela et al, 2015 [31]	L. acidophilus ATCC 4356	C. albicans ATCC 18804	Biofilm assay and C. albicans filamentation assay using light microscope	- L. acidophilus culture filtrate reduced the growth of C. albicans cells by 45.1% - Less hyphae formation in the presence of L. acidophilus cells or culture filtrate	L. acidophilus produced substances with anti-Candida activity, presenting an indirect effect on Candida.
Chew et al, 2015 [32]	L. rhamnosus GR-1 and L. reuteri RC-14	C. glabrata ATCC 2001 and clinical isolates	- Spot overlay assay - Plate-based microtiter assay - Candida viability assay using confocal laser scanning microscopy - Aggregation assay - MATH assay	- Probiotic strains exhibit growth inhibitory activities (bacterial cells and supernatant) and candidicidal activity against C. glabrata - Both probiotic strains exhibited strong autoaggregation and coaggregation in the presence of Candida	Lactobacilli may prevent C. glabrata colonization through the formation of aggregates. Reduction of pH plays role on the antifungal effect of probiotic, but not H2O2. Other inhibitory mechanisms or pathways may be involved.

Abbreviations: CFU, colony-forming units; H₂O₂, hydrogen peroxide; MATH, microbial adhesion to hydrocarbons; MS, mutans streptococci; SeNPs, selenium dioxide nanoparticles; XTT, tetrazolium salt.

The probiotic bacteria that have been investigated against Candida species to date include Streptococcus salivarius K12 [23], Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 [25], and also clinical isolates of Lactobacillus [27, 30].

Antimicrobial activity of lactobacilli is generally well known. Studies using antagonism in agar diffusion assays have demonstrated that Lactobacillus species inhibit the growth of both gram-positive and gram-negative pathogens (eg, Streptococcus mutans and Escherichia coli, respectively) [13, 14], in addition to Candida species [13, 14, 25, 27, 30, 32]. Candida albicans was found to be more susceptible to the antifungal effect of Lactobacillus than C. tropicalis [27]. Moreover, probiotic bacteria and their supernatant also exhibited growth inhibitory activities against C. glabrata [32]. The production of hydrogen peroxide by the probiotics that antagonize candidal growth was a notable phenomenon observed in a number of these studies [27, 30].

Hyphae formation and adhesion assays were used to evaluate the effect of Saccharomyces boulardii [26] and S. salivarius [23] on C. albicans. Saccharomyces boulardii appears to secrete an active compound that inhibits filamentation of C. albicans and its mycelial development, a crucial virulence attribute of this fungal pathogen. Streptococcus salivarius K12 was not directly fungicidal, but appeared to inhibit Candida adhesion to the substratum and increase the planktonic cells in culture medium [23].

The effect of probiotics may be time dependent. Using a time-kill assay, some investigators have attempted to reinforce the probiotic effect of bacteria by supplementing the medium with chemical adjuvants, such as selenium. The latter is an essential micromineral that regulates metabolism and is known to reinforce immunity. Selenium nanoparticle-enriched Lactobacillus plantarum and Lactobacillus johnsonii cells and supernatant have shown higher antifungal activity against C. albicans than controls devoid of the nanoparticles [24]. These data, yet to be confirmed, exemplify how probiotics could be synergized and deserve further study.

Experiments on the effect of probiotics on Candida biofilms, as opposed to their suspended planktonic phase, provide another fascinating glimpse of how probiotics behave [28, 29, 31]. It has been shown that a number of bacteria can interfere with the biofilm growth by reducing hyphal development [28, 31], a result akin to that described above [26]. Ujaoney et al [29] reported that the probiotic cell-free supernatant had a strong and significant inhibitory effect on biofilm development on denture acrylic strips than the bacteria per se, indicating that the inhibitory agent is an exometabolite secreted into the medium.

Chew et al [32], using confocal laser scanning microscopy, also demonstrated the candidicidal effect of planktonic lactobacilli and their supernatant against C. glabrata, another common fungal pathogen.

As summarized in Table 1, there is now a convincing body of in vitro data to indicate the antifungal effect of probiotics against Candida species. The challenge now is to clarify the mechanisms involved and harness these in further translational work. Investigations of the molecular mechanisms underlying the probiotic effect using gene expression and related technology are likely to yield interesting data in this regard.

IN VIVO EVIDENCE OF THE ANTIFUNGAL EFFECTS OF PROBIOTICS

As opposed to the in vitro studies reported above, a number of in vivo studies have also been performed over the past decade or so to substantiate the antifungal activity of probiotics in humans (Table 2). The oral cavity, GI tract, and urogenital tract have been the major loci of investigation, as these sites are susceptible to Candida infections.

Table 2.

Clinical Investigations on the Antifungal Effects of Probiotics in the Oral Cavity, Urogenital Tract, and Gastrointestinal Tract of Humans

Reference	Site of Action	Probiotic	Pathogen	Method	Results	Comments
Hatakka et al, 2007 [15]	Oral cavity	Lactobacillus rhamnosus GG (ATCC 53103), L. rhamnosus LC705, Propionibacterium freudenreichii subsp shermanii JS	Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis	- RCT: 276 elderly people - Probiotic therapy: daily consumption of 50 g of probiotic cheese or control cheese for 16 wk - Community periodontal index and mucosal lesions were recorded. Sampling for oral yeasts was undertaken	- Prevalence of yeast in saliva decreased in the probiotic group from 30% to 21% (32% reduction), and increased in the control group from 28% to 34% - Probiotic intervention reduced the risk of high yeast counts by 75%	Probiotic reduced the prevalence of hyposalivation; No adverse events were observed.
Mendonça et al, 2012 [39]	Oral cavity	Lactobacillus casei and Bifidobacterium breve	C. albicans, C. tropicalis, Candida guilliermondii, C. glabrata, Candida lipolytica, C. krusei, Candida kefyr, and Candida parapsilosis	- 42 women (aged ≥65 y) - Probiotic therapy: 3 times weekly for 30 d - Saliva sample collection for Candida cell quantification (CFU counting) and IgA analysis (ELISA)	- Reduction of Candida prevalence from 92.9% to 85.7% - Increase of anti-Candida IgA levels	C. albicans was the most frequently species isolated before and after probiotic consumption.
Sutula et al, 2012 [33]	Oral cavity	L. casei Shirota	Candida spp Streptococcus mutans and gram-negative anaerobic species	- 7 healthy complete denture wearers (aged ≥55 y) - Probiotic therapy: once daily, with the denture in position, for 28 d - Samples of saliva, tongue, and denture biofilm were collected	- No effect of probiotic on occurrence and viability of Candida - No significant change in the viability of Streptococcus mutans and gram-negative anaerobes	Small sample group (n = 7) completed the study protocol.
Sutula et al, 2013 [34]	Oral cavity	L. casei Shirota	- Candida spp - Gram-negative anaerobic species	- 21 healthy dentate subjects (aged 18–45 y) - Probiotic therapy: once daily for 28 d - Saliva and tongue-coating samples were collected; Morning breath samples were obtained using portable sulphide monitors	- Lactobacillus level in saliva was increased during probiotic consumption period - Candida and anaerobic species levels were unaffected by the therapy - Morning breath scores measured were not significantly affected	Confirmation of the temporary and intake-dependent presence of Lactobacillus.
Li et al, 2014 [11]	Oral cavity	Lactobacillus bulgaricus, Bifidobacterium longum, and Streptococcus thermophilus	Candida spp	- RCT: 65 patients with Candida-associated stomatitis - Probiotic therapy: antifungal alone (sodium bicarbonate solution + nystatin paste) or associated with probiotic, 3 times daily for 4 wk - Parameters of hyperemia, visual analogue scale scores, culture of saliva, and lingual dorsum swab were assessed	Detection rate of Candida spp was reduced in the probiotic group; Significant relief of clinical signs and symptoms after probiotic administration	No adverse events were observed.
Ishikawa et al, 2015 [16]	Oral cavity	L. rhamnosus HS111, Lactobacillus acidophillus HS101, and Bifidobacterium bifidum	Candida spp	- RCT: 55 denture wearers harboring Candida spp with no clinical symptoms of oral candidiasis - Probiotic therapy: once daily for 5 wk (probiotic or placebo) - Palatal swab sample for Candida cell quantification and identification	- Significant reduction of Candida infection after probiotic administration - C. albicans was the most prevalent species before and after the probiotic therapy	Reduction of Candida infection was independent of initial Candida level, colonizing species, or age of denture.
Kraft-Bodi et al, 2015 [17]	Oral cavity	Lactobacillus reuteri DSM 17938 and L. reuteri ATCC PTA 5289	Candida spp	- RCT: 215 elderly people (aged 60–102 y) - Probiotic therapy: twice daily for 12 wk (placebo or probiotic) - Prevalence and amount of Candida growth (saliva and plaque samples), oral hygiene, and gingival inflammation were assessed	- Significant reduction of Candida cells in saliva and plaque after probiotic administration - No differences in the levels of supragingival plaque or bleeding on probing were observed	“Strong taste” of the tablets and gastric upset were compliances reported in both control and experimental groups.
Pirotta et al, 2004 [38]	Urogenital tract	L. rhamnosus and B. longum (oral powder); L. rhamnosus, Lactobacillus delbrueckii, L. acidophilus, and Streptococcus thermophiles (vaginal pessary)	Candida spp	- RCT: 235 nonpregnant women (aged 18–50 y) with a short course of oral antibiotic administration - Probiotic therapy: powder administration twice daily and pessary once daily, for 6 d of antibiotic course and 4 d after - Vaginal swab was collected for Candida identification - Identification of symptomatic VVC	The use of oral or vaginal forms of probiotic bacteria could not prevent postantibiotic vulvovaginitis	10 d of probiotic therapy may be insufficient time for the occurrence of beneficial effects against Candida spp in the vagina.
Martinez et al, 2009 [12]	Urogenital tract	L. rhamnosus GR-1 and L. reuteri RC-14	Candida spp	- RCT: 55 women diagnosed with VVC - Probiotic therapy: single dose of fluconazole plus probiotic or placebo once daily for 4 wk - Vaginal swab was collected for Candida identification Clinical evaluation to detect signs and symptoms of VVC	- Probiotic significant reduced vaginal discharge, itching and/or burning vaginal feeling, dyspareunia, and/or dysuria - Probiotic reduced the presence of Candida spp	Mild adverse effects were reported, but could not be definitely associated with probiotic administration.
Vicariotto et al, 2012 [2]	Urogenital tract	Lactobacillus fermentum LF10 and L. acidophilus LA02 (arabinogalactan and fructooligosaccharides as prebiotics)	Candida spp	- Thirty female patients (aged 23–64 y) - Probiotic therapy: once daily for 7 d, then once every 3 d for additional 3 wk. In the following month, once weekly - Vaginal swabs were collected for yeast identification	- Probiotic significantly solved Candida yeast symptoms in 86.6% of patients after 28 d - At the end of the second month, recurrences were recorded in 11.5% of patients	Probiotic may establish and maintain a protective barrier effect against vaginal Candida.
Hu et al, 2013 [1]	Urogenital tract and oral cavity	Bifidobacterium and Lactobacillus (DanActive or YoPlus yogurt)	Candida spp	- 24 women (17 HIV infected, 7 HIV uninfected); - Probiotic therapy: 15 d consuming each yogurt. 30-day washout period between the 2 yogurt consumption periods - Oral and vaginal culture swabs were collected	- Less fungal colonization among women was observed after probiotic consumption - HIV-infected women had significantly lower vaginal fungal colonization after DanActive yogurt consumption	Reduced oral fungal colonization was observed in HIV-infected women consuming probiotic yogurts, but not statistically significant.
Kovachev et al, 2015 [35]	Urogenital tract	L. acidophilus, L. rhamnosus, L. delbrueckii subsp bulgaricus, and S. thermophiles	C. albicans	- 436 women (aged 17–50 y) with C. albicans vaginal infection - Probiotic therapy: antifungals alone (fluconazole and fenticonazole) or associated with vaginal probiotic agent - Clinical and microbiological tests were performed	- Probiotic reduced clinical complaints - Probiotic therapy improved the investigated parameters: vaginal fluorine, vaginal tissue changes, and pH	Local application of probiotics may improve the efficacy of conventional antifungals and prevent relapse.
Manzoni et al, 2006 [19]	Gastrointestinal tract	L. casei subsp rhamnosus	Candida spp	- RCT: 80 preterm neonates with a very low birth weight - Probiotic therapy: human milk alone or added with probiotic, for up to 6 wk - Samples from oropharyngeal, stool, gastric aspirate, and rectal specimens were collected to assess fungal colonization in the GI tract	- Human milk supplemented with probiotic reduced significantly the incidence of Candida enteric colonization - Probiotic reduced significantly the numbers of fungal isolates	Probiotic reduced incidence and intensity of enteric colonization by Candida spp; no adverse events were observed.
Romeo et al, 2011 [21]	Gastrointestinal tract	L. reuteri (ATCC 55730) and L. rhamnosus (ATCC 53103)	Candida spp	- 249 preterms neonates with a birth weight <2500 g and a gestational age <37 wk - Probiotic therapy: breast milk or formula milk alone, or supplemented with 1 probiotic, for up to 6 wk - Clinical evaluations were performed. Stool samples, oropharyngeal, and gastric aspirate specimens were collected for Candida detection. Blood cultures and Platelia Candida test were conducted for the diagnosis of invasive candidiasis	- Probiotic reduced significantly Candida stool colonization - L. reuteri group had a significant higher reduction in gastrointestinal symptoms than the L. rhamnosus and control groups - Probiotics reduced the incidence of abnormal neurological outcome	Probiotics may prevent gastrointestinal colonization by Candida, protect from late-onset sepsis, and reduce abnormal neurological outcomes in preterms.
Demirel et al, 2013 [3]	Gastrointestinal tract.	Saccharomyces boulardii	Candida spp	- 181 preterm neonates with a gestational age ≤32 wk and birth weight ≤1500 g - Probiotic therapy: nystatin suspension every 8 h, or breast milk or formula supplemented with probiotic once daily - Samples of blood, urine, and cerebrospinal fluid were collected to identify invasive fungal infection Skin, stool, or rectal cultures were obtained for Candida detection	- Candida colonization of the skin and stool were similar between probiotic and nystatin groups - Clinical sepsis and number of sepsis attacks were significantly lower in the probiotic group	Prophylactic S. boulardii and nystatin were equally effective in reducing candidal colonization and invasive fungal infection.
Kumar et al, 2013 [18]	Gastrointestinal tract	L. acidophillus, L. rhamnosus, B. longum, B. bifidum, S. boulardii, and Saccharomyces thermophilus	Candida spp	- RCT: 150 children (aged 3 mo–12 y) on broad-spectrum antibiotics for at least 48 h - Probiotic therapy: probiotic or placebo twice daily for 7 d - Rectal swab, samples of urine and blood were collected for Candida detection	- Probiotic therapy avoided a significant increase in the number of patients colonized by Candida spp - Probiotic significantly reduced the presence of Candida in the urine, but not in the blood	Probiotics may be an alternative strategy to reduce Candida infection in GI tract and urine in children receiving broad-spectrum antibiotics.
Roy et al, 2014 [20]	Gastrointestinal tract	L. acidophilus, Bifidobacterium lactis, B. longum, and B. bifidum	Candida spp	- RCT: 112 preterm neonates (gestational age <37 wk and birth weight <2500 g) - Probiotic therapy: breast milk supplemented with probiotic or placebo, twice daily for up to 6 wk - Clinical evaluations were performed. Stool samples and gastric aspirate specimens were collected for Candida detection. Blood cultures and Platelia Candida test were conducted for the diagnosis of invasive candidiasis	- Probiotic therapy reduced the duration of hospitalization and stool fungal colonization - Fungal infection was significant less in the probiotic group - Full feed establishment was earlier in probiotic group.	Probiotics may reduce enteral fungal colonization and reduce invasive fungal sepsis in low–birth-weight neonates.

Abbreviations: CFU, colony-forming units; ELISA, enzyme-linked immunosorbent assay; GI, gastrointestinal; HIV, human immunodeficiency virus; IgA, immunoglobulin A; RCT, randomized controlled trial; VVC, vulvovaginal candidiasis.

Oral Cavity

Despite the high prevalence of oral candidal infections in predisposed populations the world over, and the recalcitrance and chronicity of these diseases, there are only a few in vivo studies evaluating the effect of probiotics on suppressing oral candidiasis. These indicate that probiotics may be a useful adjunct in the battle against oral candidiasis, especially as a prophylactic agent in immunocompetent individuals.

The elderly are a group particularly susceptible to oral candidiasis even in health, due to the prosthesis (dentures) they frequently wear and hyposalivation. Their weakened immune status may favor the recurrence of candidiasis. Two research groups have shown that the daily consumption of lactobacilli-laced cheese [15] or lozenges [17] significantly reduces the high yeast counts in saliva and biofilms in the elderly. Because biofilms on oral prosthetic devices act as potent reservoirs of the yeast, the mechanical removal of biofilms associated with the regular use of probiotics that reduce the oral burden of Candida could play a major role in preventing oral candidiasis in denture wearers. Interestingly, one study reported increased salivary flow as a salutary accompaniment to probiotic administration [15].

As mentioned, full denture wearers suffer frequently from Candida-associated denture stomatitis [4], which lowers the quality of life. Ishikawa et al [16] have reported that a probiotic product, when regularly placed on the palatal surface of maxillary dentures, reduced oral candidal burden in healthy denture wearers. These preliminary data imply that multispecies probiotics, together with good denture hygiene, may help suppress recurrence of these chronic infections [11].

Commercial food products with probiotics are common worldwide. A widely available probiotic-laced drink containing Lactobacillus casei and Bifidobacterium breve was able to reduce the prevalence of oral Candida in healthy individuals [39]. A significant increase in anti-Candida immunoglobulin A levels was associated with probiotic consumption [39]. In contrast, the identical product did not significantly affect the oral candidal colonization in complete denture wearers [33] and in healthy dentate people [34], after 4 weeks of administration. The lower dose of probiotic intake and the small number of individuals included in the latter studies may explain these divergent observations.

Urogenital Tract

Chronic VVC is a widely prevalent disease and impacts the life quality of thousands of women the world over. Although standard antifungals are effective, there is no alternative approach for suppressing these recalcitrant infections. Several groups have therefore evaluated the efficacy of probiotics in the treatment and prophylaxis of VVC [1, 2, 12, 35].

Two studies conducted on healthy women have reported that the coadministration of probiotics with standard antifungal therapy (fluconazole) was more effective in reducing symptoms of VVC, including vaginal discharge, pruritus vulvae, vulvar and vaginal erythema, dyspareunia, and dysuria compared with a group treated with antifungals alone [12, 35]. Clinical improvement was also observed after local administration of a commercial slow-release probiotic product alone, without an antifungal agent, in healthy women with recurrent VVC [2]. Similarly, in a study conducted in immunocompromised women, who are highly susceptible to recurrent and complicated VVC infection, probiotic yogurt consumption led to a decreased frequency of infection [1].

In contrast, another well-controlled study reported that probiotic bacteria taken both orally and locally were unable to prevent postantibiotic VVC in immunocompetent individuals who took oral antibiotics [38]. Qualitative and quantitative differences in the probiotic strains, as well as the period of probiotic administration, are likely to be the reasons for the divergent results between the foregoing studies.

Gastrointestinal Tract

Candida species are common inhabitants of the GI tract of humans. Perturbation of the local microbiome, however, leads to dysbiosis within this ecosystem, leading to candidal overgrowth and possible invasive infections, especially in infants [21].

Hence, immunocompromised children, especially preterm neonates with low birth weight, have been the target population of a number of studies evaluating the efficacy of probiotics against candidal colonization of the GI tract [3, 19–21]. Within this population, most researchers have reported a significant reduction in the incidence and intensity of enteric candidal colonization with probiotic-laced human milk, administered either with or without concurrent antifungals [19–21]. Important secondary effects of the probiotics observed in these studies include reduction of sepsis episodes [3], early establishment of full feeding associated with reduction in the duration of hospitalization [20], and the decrease in the incidence of abnormal neurological outcomes associated with late-onset sepsis [21].

Broad-spectrum antibiotics are notorious for their ability to cause GI tract dysbiosis and candidiasis [18, 38]. In immunocompetent children who had received broad-spectrum antibiotics, probiotic therapy led to a reduction of gastrointestinal candidal colonization as well as candiduria—a surrogate marker of invasive fungal infection [18].

POSSIBLE MECHANISMS OF ACTION OF PROBIOTICS

Clearly, the major attribute of probiotics appears to be the restoration of a natural healthy microbiome in a given habitat, turning it from a catastrophic, disease-inducing, dysbiotic microbiota to a healthy, symbiotic, stable equilibrium. A number of hypotheses, most unproven as yet, has been proposed for the genesis of this well-balanced state from disease to health. Probiotics may compete for nutrients and receptors on the cell surfaces with the pathogenic microorganisms, thus preventing their adhesion and colonization on the mucosal surfaces [2, 29]. Co- and auto-aggregation of probiotics with the formation of a critical mass required for a healthy biofilm development may act as a protective lining against pathogenic infection [30]. Apart from the above, the production of biosurfactants that interfere with microbial adhesion and desorption [37], the release of exometabolites such as lactic, acetic, and capric acid, and the production of bacteriocins and hydrogen peroxide (H₂O₂) are other possible attributes postulated as mechanisms for probiotic activity [24–26]. Despite such in vitro data on the inhibitory effect of probiotic products on yeasts, the direct effect of probiotics on mucosal candidiasis is yet be shown in a laboratory environment mimicking the oral cavity, vagina, or GI tract.

The host response to probiotics is likely to play an important role in probiotic-mediated microbiome effects. The modulation of both innate and adaptive immune systems is probably associated with alteration of the cytokine profile and Candida recognition by epithelial and immune defense cells [28, 36, 40]. Evidence to imply probiotic interference with these host defense factors during candidal infestation is still needed.

With respect to candidal infection, probiotics were found to reduce filamentation and biofilm development in C. albicans, 2 key virulence attributes of this fungus [25, 28]. As the yeast form of Candida, as opposed to the hyphal form, is more susceptible to phagocytosis [40], probiotics appear to assist the host combat the pathogen more effectively by suppressing filamentation. Despite the evidence that probiotic bacteria may affect the expression of genes associated with biofilm formation and filamentation of Candida species [25], the mechanisms by which probiotics affect these yeasts attributes are still unclear.

Administration of probiotics in tandem with antifungal drugs synergizes clearance of Candida [11, 12, 35]. Apart from the obvious antifungal effect of the drug, the role of the probiotic under these conditions remains to be elucidated. The increased expression of stress-related genes and decreased expression of genes involved in drug resistance in Candida, promoted by the probiotics, would possibly increase the fungus's susceptibility to the antifungal agent administered [25].

SAFETY AND RISKS OF PROBIOTIC THERAPY

A range of bacteria has been utilized as probiotics in humans, depending on the pathological condition. None of the clinical studies mentioned above have reported adverse effects directly related to probiotics, suggesting their safety. Nevertheless, the safety, efficacy, and functionality of probiotic bacteria should be tested in healthy as well as in compromised individuals prior to their administration as therapeutic agents.

FUTURE PERSPECTIVES AND CONCLUSIONS

Clinical studies indicate that probiotics may reduce Candida colonization on human mucosal surfaces, relieve signs and symptoms of fungal infection, and enhance the antifungal effect of conventional therapy, implying that probiotics have the potential to sustain a healthy mucosal microbiota by acting both as prophylactic and adjunctive therapy against candidiasis. In vitro studies indicate that the antifungal effect of probiotics is likely to be due to their interference with Candida biofilm development and hyphal differentiation. However, it is premature to designate probiotics as an alternative to antifungals as yet, due to the paucity of available clinical trials. In particular, case-control clinical trials with adequate patient numbers are warranted, not only to ascertain the activity of the probiotic formulations, but also to evaluate their dosage, administration schedules, side effects, and biodynamics in humans. As with any formulations of live organisms, other concerns that need further investigation include the potential for selection of resistant strains, mutability, and tolerability on prolonged use, as well as pathogenic potential in immunocompromised patients. Given these caveats, probiotics may serve in the future as worthy allies in the battle against chronic mucosal fungal infections.

Note

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.