Anticandidal Efficacy of Oral Probiont Limosilactobacillus fermentum Against Dental Caries Pathogens in Children, Tamil Nadu, India

Abstract

Dental caries remains a prevalent concern among children globally and is associated with Candida sp. Some researchers have suggested probiotic supplements as a possible solution for reducing dental caries. A study conducted in Tamil Nadu focused on collecting 80 dental caries samples from both males and females, obtained from two different locations. The samples underwent processing using the spread plate technique, followed by anticandidal activity assessments. Through ITS sequence analysis, candida strains were identified, including C. albicans (DDGRPO1, DDGRPO2). The study specifically investigated the ability of the probiotic bacterial strain Lb. fermentum cell-free filtrate to inhibit C. albicans. The research revealed that Lb. fermentum probiotics effectively inhibited the growth of C. albicans DDGRP01, displaying strong antifungal activity against Candida sp. (98%). While these results are promising, it is worth mentioning the increasing interest in exploring innovative alternatives to probiotic-based treatments. This avenue of research offers potential for a more comprehensive approach to addressing this issue. Notably, Lb. fermentum, derived from the human oral cavity, emerges as a significant postbiotic candidate for dental prophylaxis, indicating a hopeful direction for future studies.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-023-01175-5.

Introduction

Dental caries remains a critical global health concern, constituting 34.1% of health issues worldwide in 2015, as reported by the Global Burden of Disease (GBD) [1]. Dental caries is recognized as the most widespread oral health problem among children worldwide. According to reports from the World Health Organization, dental caries affects a significant percentage, ranging from 60 to 90%, of children in both developing countries [2, 3]. Dental caries and periodontal diseases are prevalent infectious conditions worldwide. Beyond causing oral health issues, oral microorganisms have been linked to various systemic diseases outside of the mouth [4]. The oral cavity functions as a crucial, self-contained ecosystem within the human body [5]. Hence, accurate identification of Candida sp. holds significant importance in diagnostic laboratories. Such identification carries prognostic and therapeutic implications, enabling timely and precise antifungal treatments [6]. Insufficient oral health has profound repercussions for children, affecting their nutrition, growth, and overall development. Unaddressed oral diseases in childhood lead to pain, the emergence of dentofacial anomalies, and other significant health complications. Scientific research continues to advance in identifying optimal approaches for diagnosing, treating, and preventing dental caries. Consumption of sugary foods in developing countries, coupled with inadequate oral hygiene practise, are contributing factors to the increasing levels of dental caries [7].

Probiotics are beneficial microorganisms often referred to as "good" bacteria. These probiotic strains play a crucial role in promoting healthy digestion and strengthening the immune system, providing a diverse array of health benefits to the host. Traditionally, probiotics have primarily been associated with enhancing gut health, with the most common applications focused on treating or preventing gastrointestinal infections and diseases. Probiotic strains such as Lactobacillus and Bifidobacterium have become increasingly popular due to their numerous antibacterial and antifungal properties. These beneficial bacteria, known as probiotics, provide a host of benefits, including safeguarding against pathogen invasion and promoting improved nutritional health [8]. Probiotics have emerged as a potential treatment and preventive measure against Candida infections. Different species of Lactobacillus have shown significant potential in combating fungal infections caused by C. albicans. Lactobacillus species include Lb. plantarum, Lb. fermentum, Lb. reuteri, Lb. paracasei [9].

In recent years, a significant change has been observed, with studies increasingly suggesting the use of probiotics for promoting oral health. This evolving research suggests that probiotics could potentially have a positive influence on maintaining and enhancing oral health.

Maintaining oral hygiene is essential for overall health, aligning with the goal of "Health for All by 2025." Dental health constitutes a significant aspect of this objective. Dental caries is a crucial health concern prevalent in both developed and developing countries, particularly among children worldwide. The government of Tamil Nadu is actively working on enhancing oral health initiatives. To ensure the evidence-based success of these population-focused efforts in improving Tamil Nadu's oral health, there is an urgent need for probiotic-based treatments.

The objective of this study was to isolate and identify Candida sp. strains from the oral cavities of dentistry patients. Furthermore, the research sought to evaluate the anticandidal activity of these Candida strains on Lactobacillus sp. using probiotic isolates.

Materials and Methods

Ethical Statement

We obtained ethical approval from the Bharathidasan University Institutional Ethical Committee (Ref. No. BDU/IEC/2020/03 issued on 24.06.2020) in Tiruchirappalli, Tamil Nadu, India.

Dental Caries Sample Collection

Oral swabs were collected from school students residing around two districts in Tamil Nadu: Thanjavur and Tiruchirappalli. During the sample collection, students were instructed to rinse their mouths with tap water. Around 80 dental swabs were obtained from both male and female patients, all of whom were dealing with dental caries. These collected swab samples were carefully stored in a container and preserved at 4 °C in a refrigerator.

Isolation of Candida from Dental Caries

The dental swab samples collected were serially diluted in 1 ml of saline solution (Fig. 1). The Sabouraud dextrose agar (SDA) medium [10 g peptone, 40 g dextrose, and 20 g agar per litre] was prepared for the isolation of Candida. After sterilization, 100 µl of the diluted samples was evenly spread onto the SDA plates. Inoculated plates were incubated for 48 h at 28 °C [10].

Fig. 1.

Overview of the study, enrolment and swab collection, storage of collected swab, molecular identification and antifungal activity against probiotic bacteria

Morphological Characterization of Candida sp.

Microscopic Observation

Gram staining was performed using a Gram stain kit (Hi Media K001-1KT) to find the gram-positive Candida isolates.

Germ Tube Test

Candida sp. was suspended in 0.5 ml of human serum in a microfuge tube and incubated at 37 °C for 4 h, with meticulous care to prevent prolonged incubation. Using a loop, a small amount of the inoculum was gently transferred onto a clean, grease-free slide. A cover slip was delicately placed over it, ensuring no air bubbles were trapped between the slides. The prepared slide was examined under stereo zoom microscope at 40 × magnification to observe the presence of germ tubes.

Carbohydrate Fermentation Test

Candida sp. was prepared in SDB, ensuring that the density did not exceed the McFarland No. 1 standard. Subsequently, 0.1 ml of Candida cell suspension was added to each fermentation broth tube. These tubes contained 1% peptone, 2% sugars (including dextrose, maltose, lactose, galactose, sucrose, and xylose), and a bromothymol blue indicator. The prepared tubes were incubated for 10–14 days at 30 °C. During this incubation period, the tubes were regularly observed for both acid and gas production.

Hi Chrom Candida Differential Agar

Chrom agar is a specific chromogenic medium used for the primary identification of Candida species. Various Candida colonies produce distinct colours, facilitating the direct identification of the species. Candida cultures were allowed to incubate at 37 °C for a period of 24 to 48 h. The identification of species was established by observing the type and coloration of the colonies formed on the plate [11].

Molecular Characterization of Candida sp.

DNA Isolation

Genomic DNA was isolated using the phenol chloroform method, with slight modifications according to the protocols described by Vijayakumar et al., (2012).

Polymerase Chain Reaction

PCR amplification of the ITS region was performed using universal fungal primers ITS1 (5′ TCC GTA GGT GAA CCT GCG G 3′) and ITS4 (5′ TCC TCC GCT TGA TAT GC 3′). The PCR reaction involved initial denaturation at 94 °C for 40 s, followed by denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. These steps were repeated for a total of 35 cycles [12].

Restriction Fragment Length Polymorphism Analysis

The PCR products were treated with the Msp I restriction enzyme (Synergy Pvt. Ltd., Chennai). The reaction mix was prepared, including 10× enzyme buffer (2 μl), Msp I enzyme (5U), PCR product (10 μl), and nuclease-free water to reach a total volume of 20 μl. The resulting mixture was incubated at 37 °C for 1 h [13].

Antifungal Susceptibility Testing

Candida was cultured on SDA plates incubated for 24 h at 37 °C. After culture had grown, a single loop of colonies was inoculated into 10 ml of sterile 0.85% saline solution. The suspension was 11 × 10⁶ to 5 × 10⁶ cells/mL, which is equivalent to the 0.5 McFarland standard. Mueller–Hinton Agar (MHA) was then enriched by methylene blue at a concentration of 0.5 g/mL and 2 g of glucose. After sterilization, the mixture was poured into Petri dishes. Using a cotton swab soaked with the inoculum suspension, the MHA plates were evenly swabbed. The plates were allowed to air dry for 5–15 min before placing the antifungal discs onto the agar. The antifungal discs used were Fluconazole (25 µg), Amphotericin-B (20 µg), Ketoconazole (10 µg), Itraconazole (50 µg), and Voriconazole (1 µg) (Hi Media, India). [14].

MIC Determination

The MIC of the Candida inoculum was confirmed using the broth microdilution technique. A 24-h Candida suspension was prepared. MIC testing was conducted on a 96-well plate, with each well loaded with 100 µl of Candida culture. SDA broth alone served as a negative control, while Candida cultured in SDA broth with antibiotics was used as a test sample. Serial dilutions were performed, resulting in final test concentrations of 100% or 3.13% v/v. The 96-well plates were incubated in a dark environment at 37 °C for 24 h. Following incubation, the OD at 580 nm. [15].

Anticandidal Activity of Probiotic Bacteria

Preparation of Probiotics

The overnight culture of Limosilactobacillus fermentum, Lactiplantibacillus plantarum, and Lactobacillus helviticus was inoculated into 100 μl of MRS broth incubated at 37 °C for 2 days. Broth culture was centrifuged at 9000 rpm for 20 min. After centrifugation, the cell-free filtrate was passed through a membrane filter.

Anticandidal Activity of Probiotics

The anti-candidal activity of cell-free filtrates (CFF) from Lb. fermentum, Lb. plantarum, and Lb. helviticus culture was carried out using agar well diffusion technique. MH agar plates were swabbed by 10⁵ CFU/mL on C. albicans (DDGRPO1 and DDGRPO2) strains. Wells were punctured on MHA plates, and 100 μl of CFF from Lb. fermentum, Lb. plantarum, and Lb. helviticus were loaded into the wells. Subsequently, the plates were incubated for 48 h at 37 °C. The zone of inhibition was observed. All experiments were conducted in duplicate [16].

Statistical Analysis

A significance level of p < 0.05 indicates that differences with p values below this threshold were considered statistically significant.

Results

Dental swabs were collected from individuals residing in Tiruchirappalli and Thanjavur Districts around Tamil Nadu, India (Fig. 2).

Fig. 2.

Dental caries sample collection from children A dental swab sample collection, B school students and C collected oral swab

Totally 80 samples were obtained from dental caries affected individuals. The causative agents of dental caries, specifically Candida species, were identified in the oral cavities of both male and female patients. Out of the 80 respondents, 40 were male and 40 were female, it includes a higher number of isolates in children (Fig. 3). This suggests that children are highly susceptible to dental caries.

Fig. 3.

Occurrence and distribution of Candida sp. from children in two districts

Isolation and Identification of Candida Species

Candida sp. observed as cream-coloured, smooth, and pasty with a convex shape (Fig. S1). Among the Candida sp. isolates identified from dental caries samples, 64.8% were children in Tiruchirappalli, while 35.2% were from Thanjavur. Gram staining was performed for all the isolates, revealing gram-positive unicellular or multicellular forms. Additionally, the presence of pseudohyphae and oval budding yeast cells was observed (Fig. S2). In germ tube method, the formation of Candida sp. was observed, specifically for C. albicans and other candida sp. did not produce germ tube (Fig. S3). Carbohydrate fermentation tests were performed on all the isolates using various sugars fermented in xylose, glucose, galactose, maltose, lactose and sucrose as outlined in Table 1.

Table 1.

Cultural, Microscopic morphology, and carbohydrate fermentation of Candida isolates

Species	Microscopic observation in SDA	Germ tube	Carbohydrate fermentation
			Xylose	Glucose	Galactose	Maltose	Lactose	Sucrose
C. albicans	Hyphae, Pseudohyphae, Gram Positive	Positive	Positive	Positive	Positive	Positive	Negative	Positive
C. tropicalis	Mycelium well developed	Negative	Positive	Positive	Positive	Positive	Negative	Positive
C. krusei	Branched mycelium	Negative	Positive	Positive	Negative	Negative	Negetive	Negative
C. glabrata	Pseudohyphae, Gram Positive	Negative	Positive	Positive	Negative	Negative	Negative	Negative

Candida species were phenotypically identified using CHROM agar (Fig. 4). Among the 44 isolates tested, the results showed that 29 isolates observed as light green colonies, 7 dull blue coloured colonies, 4 pink colonies, and 4 white colonies.

Fig. 4.

Candida sp. colony morphology on CHROM agar

Molecular Characterization

The 18S rRNA gene was amplified using the primers ITS1 and ITS4, resulting in an amplicon of approximately 514 bp (Fig. 5A). RFLP identification of C. albicans is 297 bp length illustrated in (Fig. 5A).

Fig. 5.

A Electrophoresis of amplified PCR product on 1.5% agarose gel, B RFLP using 2% gel

Phylogenetic Analysis of Candida Species

A phylogenetic tree serves as a visual representation of evolutionary relationships between organisms or genes. Constructing a phylogenetic tree is a fundamental technique used to depict evolutionary connections among different species or groups. In this study, the GenBank database was used for similarity searches, and the matched sequences were compiled.

A phylogenetic tree of Candida species (ON358400 and ON358401) was constructed using the neighbor-joining technique based on 18S rRNA gene sequences, and taxon identifiers were color-coded. The accession codes of the 18S rRNA sequences are provided for the reference isolates used in the comparison. MEGA11 software was used to display the bootstrap values, which represent 100 repetitions at the nodes of the tree. The scale bar on the tree represents 1 unit of the number of base substitutions at each location, indicating the evolutionary distances between the sequences (Fig. 6).

Fig. 6.

Relative position of Candida species in relation to their NJ tree

Antifungal Susceptibility Testing

The susceptibility pattern of Candida species is illustrated through the observation of the zone of inhibition in (Fig. 7). Candida species including C. albicans, C. tropicalis, and C. glabrata exhibited resistance against ketoconazole, voriconazole, and fluconazole.

Fig. 7.

Antifungal susceptibility test candida sp using disc diffusion method. A C. albicans, Fluconazole, Amphotericin B, Voriconazole, Ketoconazole, and Itraconazole B C. tropicalis using fluconazole, amphotericin B, voriconazole, and ketoconazole. C C. glabrata, fluconazole, amphotericin B, voriconazole, and ketoconazole D C. krusei, using fluconazole, amphotericin B, voriconazole, and ketoconazole

The determination of MIC for Candida species against fluconazole, amphotericin B, and voriconazole is depicted. The concentrations of the fluconazole drugs tested C. albicans ranged from 0.04 mg/ml-1 (lowest) to 13 mg/ml-1 (highest) (Fig. S5).

Anticandidal Activity of Probiotic Bacteria

The probiotic bacteria Lb. fermentum, Lb. plantarum, and Lb. helviticus have exhibited their capability to inhibit dental pathogens, particularly Candida species. Notably, strains of C. albicans [DDGRPO1 and DDGRPO2]. Lb. fermentum, displayed zones of inhibition greater than 25 mm, indicating a strong inhibitory effect against C. albicans DDGRPO1 (Fig. 8).

Fig. 8.

The anticandidal activity of probiotics, specifically (1) Lb. fermentum GD5MG, (2) Lb. plantarum, and (3) Lb. helviticus, as well as (4) control. Probiotic strains were used as inhibitors against C. albicans strains DDGRP01 using the agar-well diffusion method

Discussion

Dental caries and the prevalence of fungi in the oral cavity pose increasing challenges for the species C. albicans in children. Among dentistry and oral care patients, C. albicans is the most commonly identified pathogen [17]. This study investigates the distribution of oral Candida species among children in Tiruchirappalli and Thanjavur, Tamil Nadu.

In this study, the initial identification medium was Hichrom Candida Differential Agar Media [18] to identify the Candida genus. The white, opaque background of Chromogenic Candida Agar (CCA) appears to provide satisfactory differentiation among colonies that have very similar colors (Table S1). Carbohydrate assimilation and fermentation tests are widely relied upon in clinical settings to accurately identify yeast species of clinical significance [19]. In this context, C. albicans demonstrates the ability to ferment xylose, glucose, galactose, maltose, sucrose, and all other sugars except lactose. Similarly, all candida species exhibit fermentation capabilities to xylose and glucose. Conversely, these species do not ferment galactose, maltose, lactose, or sucrose. While phenotypic methods for identifying Candida spp. are affordable and straightforward, they do have certain limitations. The most precise techniques for rapidly identifying Candida spp. involve the use of the germ tube test and PCR based on molecular biology methods. In the present study, all strains were initially identified through morphological characterization and biochemical tests, including the germ tube test. However, to ensure accuracy and confirm the Candida spp. identification, a cost-effective method employing 18SrRNA gene sequencing PCR was employed. Antifungal activity against candida sp. Show 90% susceptible to all the azole drugs [20]. In this study, the antifungal activity of Candida species was examined in dental caries patients from Tamil Nadu. The findings of the antifungal susceptibility test showed that 50% of the isolates exhibited resistance, with C. albicans showing resistance to all five antifungal drugs tested, including fluconazole, amphotericin B, voriconazole, ketoconazole, and itraconazole. C. glabrata displayed resistance to all five antifungal drugs as well. Additionally, C. krusei demonstrated resistance to 50% of the antifungal drugs, including fluconazole and ketoconazole (Fig. S4).

For C. albicans, the MIC ranged from 0.160 to 0.160, and a notable number of samples exhibited higher MICs. Most of the antifungals tested in this study demonstrated effectiveness against C. albicans. Interestingly, C. albicans showed higher MICs for azoles compared to C. tropicalis, while C. glabrata exhibited higher MICs than C. albicans [21]. This discovery pinpointed three strains of Lactobacillus sp. for in-depth exploration as potential biotherapeutics for oral diseases. Specifically, Lb. fermentum has demonstrated effective antifungal properties against C. albicans.

Globally, dental caries continues to impact a significant number of children [3]. Its prevalence seems to be on the rise in economically disadvantaged nations, likely due to heightened sugar intake and limited access to fluoride resources. Moreover, certain communities in wealthier countries also bear a disproportionate burden on this issue. This highlights the significance of understanding the prevalence of Candida, particularly C. albicans, in preschool children and its correlation with their nutritional status. Notably, our study reveals a substantial presence of C. albicans among children in the Tiruchirappalli district. The anticandidal properties of Lactobacillus strongly supports their potential as a promising therapeutic alternative for preventing and treating candidiasis. Europe and North America have reported Candida carriage rates ranging from 30 to 40% in the population. In South Africa, the oral Candida infection rate was slightly higher at 53% [22]. Our research findings reveal a higher prevalence of dental caries among the population in Tiruchirappalli. This underscores the urgent necessity for implementing probiotic-based therapy, especially among children in the Tiruchirappalli district. Such interventions could significantly contribute to improving oral health and reducing the incidence of dental caries in this region.

However, there are challenges in fully understanding the mechanisms through which lactobacilli inhibit Candida growth [23, 24]. Our own research findings support this trend, specifically demonstrating that Lb. fermentum effectively inhibits the growth of C. albicans, indicating its potential as an agent in combating candidiasis.

Candida species are widely recognized as the predominant opportunistic yeast infections worldwide. Among the species within this genus, C. albicans remains the most prevalent, responsible for nearly 50–90% of candidiasis in humans. A notable characteristic of probiotic bacteria is their ability to generate various active compounds. These compounds contribute to the safety and efficacy of probiotic-derived agents. Probiotic strains, particularly lactic acid bacteria (LAB), have been extensively screened for their antifungal activity and yielding promising results.

Conclusions

In conclusion, this study underscores the increasing significance of probiotics, particularly strains like Lactobacillus, in promoting both gut and oral health. The research highlights the substantial potential of specific probiotic bacteria, including Lb. fermentum, Lb. plantarum, and Lb. helviticus, in inhibiting dental pathogens, especially Candida species. strong anticandidal activity against specific Candida strains, such as C. albicans DDGRPO1 and DDGRPO2, suggesting promising avenues for probiotics as a natural approach to combat dental infections.

The study’s findings not only contribute valuable insights to the field of probiotics and oral health but also emphasise the potential of leveraging natural microbial communities to enhance oral hygiene practices. Probiotics have shown effectiveness in targeting specific pathogens, opening doors for further research and practical applications in the prevention and treatment of oral infections. To fully capitalise on these promising results, future studies could explore additional probiotic strains and their specific mechanisms of action. Moving forward, it is crucial to conduct comprehensive clinical trials and long-term efficacy studies. These efforts will be instrumental in translating the current findings into practical and patient-centred dental care approaches, potentially reducing our dependence on conventional antimicrobial agents. Based on the results of the present research, it was observed that females exhibit a higher prevalence of Candida compared to males. This issue arises due to the excessive consumption of sugars, candy and cookies leading to alterations in the oral microbial ecology of the caries microbiota. Consequently, there is an imbalance between the demineralization and remineralization processes of the tooth. Ultimately, this research paves the way for a new era in oral health, where probiotics play a central role in promoting a healthy and balanced oral microbiome, offering innovative solutions for dental interventions and preventive strategies.

Supplementary Information

Below is the link to the electronic supplementary material.