Preliminary studies on the inhibition potential of Indian domestic curd against coliforms, an emerging periodontal pathogen.

BACKGROUND: Coliforms colonize in dental plaques via oral route and may lead to systemic complications. Escherichia coli and its lipopolysaccharide-induced periodontitis is an emerging threat. Clinical management necessitates antibiotic regimens with risk of resistance and upsetting the gut. There is urgent need for better, sustainable, and economical alternative. AIM: To investigate the inhibition of coliforms, a potential periodontopathogen directly by Indian domestic curd (IDC) "in situ".
MATERIALS AND METHODS: Coliforms from natural habitat (Municipal sewage in Agartala, Tripura), a source of infection through food and water, were used as target organism. Domestically prepared curd without any fortification is used to explore its true inhibition potential. Assays of agar well diffusion performed with IDC (ultraviolet sterilized and pH adjusted 6.5) against isolated pure cultures of coliforms. The study protocol nullified effect of organic acids, volatile compounds, bacteriophages, and peroxides in IDC. Peptide nature of inhibitory ingredient was studied by Sodium Dodecyle Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), urea treatment. Computational phylogenetics revealed structural features of inhibitory ingredient. Statistical comparisons were done by analysis of variance. Second-order polynomial regression was done to evaluate the effect of IDC dilution on coliform inhibition. Mann-Whitney U-test performed to analyze different sample treatments.
RESULTS: Agar well diffusion (sealed bottom wells) shows inhibition of catalase-negative coliforms (confirmed by Gram staining and triple sugar iron agar assay) in pure culture (MacConkey agar). Activity diminished in urea, potentiated in ethylene diamine tetra acetic acid, remains unchanged by heat treatment (121°C, 15 min). SDS-PAGE revealed three distinct peptides (>10-15KDa). Hence, thermostable inhibitory peptides attached to target cell lead to observed activity (titer up to 1204.82 AU/ml with minimum 8 mm inhibition).
CONCLUSIONS: IDC adequately inhibits sewage coliforms and may prevent dental plaques coliform colonization and its associated risks.

INTRODUCTION

Escherichia coli colonization in dental plaques and oral cavities is becoming more widespread than ever.[123] Dental plaque bacteria not only influence the oral cavity locally but may also lead to cardiovascular,[4] pulmonary,[5] and renal complications.[6] Complexity of coliform infection can also be acknowledged from coexistence of high-frequency E. coli infection and other systemic conditions in patients of subgingival dental plaques.[7] It was observed that Porphyromonas gingivalis and E. coli lipopolysaccharides (LPSs) exhibit systemically different but similar local induction of inflammatory markers in dental mucosa and that E. coli is no less a threat to dental health.[8] Infants and elderly are more susceptible to E. coli contamination in dental plaques through oral route and successive spread of infection leading to life-threatening conditions.[91011] The studies showed that E. coli LPS stimulates unique set of immune and inflammatory responses in periodontitis.[121314] As a hallmark Gram-negative periodontal bacteria predominantly stimulate T-lymphocyte receptor 2 during immune response in periodontitis.[15] Hence, the role of E. coli LPS amplifies in the context of polymicrobial diseases such as in case of periodontal disease.

Therefore, substances that effectively minimize role of E. coli and its virulence factor LPS in periodontal diseases demands more emphasis. It is presumable that coliforms eventually gain entry to the oral cavity from their natural habitat such as sewage and contaminated food and water and becomes colonized. Conventional antibiotics are effective but in the long run increases the risk of drug-resistant super bugs. Hence, a novel approach for the inhibition of coliforms particularly via oral route to tackle dental colonization is the need of the day.

In the recent years, there has been a renewed interest on novel antimicrobial therapies for dental plaque-related diseases such as use of probiotics.[16] However, most study focuses on purified and highly concentrated preparations of lactic acid bacteria (LAB) in various forms.[1718] Commercial preparations of probiotics such as Wakamate D® tablets (Wakamoto Pharmaceutical Co., Tokyo, Japan, contain 6.7 × 108 colony forming units [CFUs]/tablet of Lactobacillus salivarius WB21 and xylitol) and Lactobacilli reuteri prodentis lozenges have been developed and are in use for periodontal deceases. It is easily perceptible that the commercial preparations are costly and may not serve to the general population. Moreover, studies that use concentrated and purified preparations of LAB indicate efficacy of LAB in pure and concentrated form only.

However, the approach in the present study is completely different and focuses on the assessment of inhibition by an easily available, economical, and safe alternative against a potential periodontopathic organism. In this work, evaluation of the inhibition potential of domestically prepared curd (Indian domestic curd [IDC]) is reported against sewage coliforms. Therefore, the study may have significant public health impact that may promote IDC as a protective mean against dental colonization with no risk of drug resistance.

MATERIALS AND METHODS

Study criteria

To replicate an in situ study design, coliforms were isolated from a natural reservoir that serves as potential source of contamination through civil supplies. Similarly, household preparation of IDC from Tripura that was meant to be consumed was collected. The active ingredient of IDC was examined in its raw form and by various treatments that are assessed by standard assays as described in the section.

Isolation of coliforms in pure culture from a natural reservoir

For identification and isolation of E. coli from sewage samples in pure culture, the sewage samples were collected from an area within urban Agartala Municipality in sterile plastic containers (50 mL) which were transported within 2 h of collection to the Microbiology Laboratory. MacConkey agar (HiMedia) plates were directly inoculated by streaking the sewage sample and incubated at 37°C for 24 h. Coliforms selectively grows on MacConkey agar (due to sodium taurocholate 5.0 g/L, Neutral Red 0.04 g/L) as bright pink colonies. Bright pink, round colonies were isolated and Gram-stained for preliminary identification of coliforms. MacConkey agar plates were further streaked by a bacterial suspension (pink colonies in MacConkey agar) in sterile water (1.0 McFarland or from 2 × 108 CFU/mL to 3 × 108 CFU/mL) and were incubated as earlier [Figure 1]. This was the pure culture of coliforms which were further confirmed by Gram staining [Figure 2] and culturing in triple sugar iron (TSI) agar (HiMedia) slants. TSI agar contains multiple carbohydrates (lactose, sucrose, and dextrose) with ferrous sulfate and sodium thiosulfate besides various protein source and yeast extract. Only coliforms can change the color of TSI agar slants from pink to yellow due to large-scale fermentation of carbohydrates and change in pH in the presence of phenol red [Figure 3]. Coliforms identified likewise were used as indicator organisms.

Figure 1

Coliform colonies in MacConkey agar (isolated from Municipal sewage)

Figure 2

Identification of coliforms by Gram staining: Gram-negative rods are seen

Figure 3

Identification of coliforms by biochemical test: Coliforms growing in triple sugar iron agar with yellow coloration

Identification of lactic acid bacteria in Indian domestic curd samples

IDC samples were routinely examined by Gram staining and catalase test directly. From each batch of IDC sample several smears were made such that a part of the inoculum was spread on to the slide and another part of the inoculum was kept intact in the same slide. The smear on one half is Gram stained (HiMedia/CDH) carefully and observed microscopically for Gram-positive bacilli and its high abundance. Catalase test was done on other half of the slides having smears with high abundance of Gram-positive bacilli by adding few drops of 3% hydrogen peroxide solution (on the IDC inoculum on other half of the slide). This ensured in situ identification bacteria responsible for curd formation [Figure 4]. Culturing of curd-forming bacteria (as LAB) in artificial medium (in MRS agar, etc.,) was not done in this study to simulate an in situ condition. Catalase-negative Gram-positive bacilli were identified as LAB.

Figure 4

Identification of lactic acid bacteria by catalase test: 1st Slide with high abundance of Gram-positive bacilli showed no bubble formation after adding H2O2, 2nd Slide with positive control of catalase test with aerobes shows bubble formation

Each batch of IDC samples were sterilized in a laminar airflow cabinet under ultraviolet (UV). For this, samples were poured in small amounts in Petri plates without lids and kept in laminar airflow with occasional stirring. This also ensured that organic volatile matters of curd are eliminated. Initial acidity of IDC samples was pH 5≤–6. The pH of IDC samples adjusted to 6.5 with 4N NaOH to exclude inhibitory effect of organic acids. This alkali-treated IDC sample was centrifuged (3000 rpm/5 min) and supernatant was collected carefully avoiding the fat. Supernatant was translucent, the pellet was semisolid, and pH of these fractions (supernatant and the pelleted IDC) was thus 6.5. Both the supernatant and the pelleted IDC were used for in situ inhibitory assay of IDC, followed by partial characterization of IDC supernatant (pH 6.5).

During the present study, IDC samples were collected from households in Tripura. IDC in these households were prepared from Jersy cow's milk in sterilized stainless steel utensils. Milk is generally sterilized by boiling and cooled in room temperature. The warm milk is inoculated with a starter Culture (small amount of curd sample from the previous day) followed by incubation overnight, approximately for <18 h at room temperature. Prolong incubation is avoided to control organic acidity as very sour flavor is undesirable in IDC. IDC samples are transported to the laboratory in sterile container and divided into two portions, for identification of LAB and for inhibitory assays.

Inhibition assay

Nutrient agar (SRL) plates were seeded with coliforms (1.0 McFarland suspension) using sterile cotton swab and dried. Approximately 6-mm wells were cut on nutrient agar plates carefully with sterile micropipette tip top. The wells were sealed at the bottom by adding a drop of molten nutrient agar and cooled. 10 μl of supernatant or pelleted IDC samples (which is already alkali-treated and UV-sterilized) were added in the wells and the plates were incubated at 37°C for 24 h. After incubation, plates were checked for inhibition zone around the sample wells. Gentamicin at various concentrations was used as standard antibiotic (disk diffusion in separate nutrient agar plates) to compare inhibitory potential of IDC against a common indicator organism [Figure 5]. Zone of inhibition was measured in mm. In this assay, IDC sample as supernatant as well as pellet was directly used for inhibitory assay. The concentrate of artificial broth culture supernatant (of cultured LAB) was not used in this study.

Figure 5

Inhibition by standard antibiotic discs (gentamicin)

To estimate the activity of the IDC supernatant, the crude IDC supernatant was diluted in sterile distilled water and inhibition potential was studied against the indicator organism until a dilution shows a minimum inhibition zone (at least 8 mm diameter of zone of inhibition including well diameter of 6 mm) which was expressed as titer of inhibitory ingredient in arbitrary units/ml (AU/ml). AU/ml was calculated by 1000/d × D/100, where D is dilution in percent and d is volume in dilution (modified from Parente et al., 1995).[19] Enumeration of inhibitory potency was done by determining unit of inhibitory activity (U) and activity in mm2/ml based on the mean of inhibition zone observed. The IDC supernatant was subjected to heat treatment at 121°C for 15 min and was assayed for antagonistic activity. Furthermore, the effect of ethylene diamine tetra acetic acid (EDTA) was assayed for inhibition potential of IDC supernatant. One gram EDTA was dissolved in 100 ml sterile distilled water and added to IDC supernatant in 1:1 ratio, 10 μl of this was tested in vitro. One unit (U) of inhibitory activity was defined as equal to 1 mm of the zone of inhibition.[2021]

Activity in mm2/ml was determined by “Lz − Ls/V", where “Lz” is clear zone area (mm2), “Ls” is well area (mm2) and “V” is volume of sample (ml).[22] Calculations were done from mean of inhibition zone (mm) observed.

Evaluation of peptide nature of inhibitory component in Indian domestic curd

Subsequently, the IDC supernatant was treated with urea (2% W/V) for 1 h at room temperature and the treated sample was tested for inhibitory activity against the indicator organism. If antagonistic potential of IDC supernatant is diminished after treatment with urea, it may indicate peptide nature of the inhibitory ingredient if all other sources of antagonistic activity are suitably nullified in IDC samples. Total protein in the IDC supernatant was estimated by Biuret method (Tulip). The crude IDC supernatant (not purified) was analyzed by SDS-PAGE (12.5%).

Statistical analysis

The study was statistically analyzed using Origin Statistical Software. The results were expressed as the mean ± standard error of the mean of the parameters obtained. Statistical comparisons were done by one-way analysis of variance. Second-order polynomial regression analysis was done to evaluate the effect of dilution of the IDC sample on coliform inhibition. For different types of treatments of IDC samples such as pelleted IDC, crude IDC supernatant (unpurified/un-concentrated), heat-treated (121°C for 15 min), and EDTA-treated (1:1 of 1%W/V EDTA) IDC samples, the results are represented with Mann–Whitney U-test. MWW test examines the differences between two independent samples or samples that are treated differently.

For statistical analysis of the peptide sequence of Bacteriocin of LAB, “Lactobacillus bacteriocin” was queried in Uniprot database and in Research Collaboratory for Structural Bioinformatics - Protein Data Bank (RCSB-PDB) (for structural data). Thirty-six sequences of “Lactobacillus bacteriocin” of various organisms were retrieved as FASTA for assessment of diversity among bacteriocins of various LAB. Proteins sequences which are related but not bacteriocin such as bacteriocin biosynthesis proteins and bacteriocin transport/processing ATP-binding proteins were excluded from analysis. Selected sequences were aligned by ClustalW and substitution model was identified by maximum likelihood (ML) fits considering lowest Bayesian information criterion (BIC) scores. Gamma distribution was estimated to evaluate nonuniformity of evolutionary rates among informative sites in the aligned sequences. Amino acid composition pattern of sequenced bacteriocins were analyzed on the basis of physico-chemistry of constituent amino acids. ML method was used for phylogeny reconstruction using WAG model and Bootstrap tested (500 replicates). The ML heuristic method was nearest neighbor interchange. MEGA 6 was used for the analysis.[23]

RESULTS

Two varieties of LAB have been identified in IDC sample batches that were examined in the study, Gram-positive bacilli in cluster or in pairs and Gram-positive bacilli in chains. Both varieties were catalase negative with no bubble formation after adding 3% hydrogen peroxide solution [Figure 6].

Figure 6

Gram-positive bacilli in clusters/pairs or in chains, isolated from Indian domestic curd

Both IDC supernatant and the pelleted IDC showed inhibitory activity against municipal sewage coliforms [Figures 7 and 8]. Inhibitory activity of IDC supernatant is 27.271 U and 58,381.03 mm2/ml, while for pelleted IDC, it is 20.94444 U and 31,609.56 mm2/ml [Table 1]. However, in some batches, pelleted IDC had superior activity. Titer of the inhibitory ingredient in IDC supernatant can reach up to 1204.82 AU/ml showing 8.8 ± 1.0328 mm diameter of zone of inhibition (including well diameter) in 1.0 McFarland coliform load [Table 2].

Figure 7

Zone of inhibition of Indian domestic curd supernatant (low to high dilution)

Figure 8

Zone of inhibition of Indian domestic curd pellet and Indian domestic curd supernatant

Table 1

Bioactivity calculated from zone of inhibition in agar well diffusion assay against coliforms as indicator organisms

Table 2

Titer of inhibitory ingredient in Indian Domestic Curd supernatant showing at least 8 mm diameter of inhibition zone (including well diameter)

Prolong urea treatment completely denatured inhibitory ingredient in IDC supernatant. Total protein concentration in crude IDC supernatant (not purified and not concentrated) was <1000 mg/dl in all IDC batches. This indicates that the inhibitory ingredient in IDC supernatant is peptide in nature and present in low concentration. SDS-PAGE of this crude IDC supernatant showed the presence of 3 protein bands of molecular weight >10–15 KDa [Figure 9]. For regular SDS-PAGE protein, sample concentration should be somewhere around 2000 mg–2500 mg/100 ml (visualization with Coomasie Brilliant Blue) in general, but since the IDC supernatant was not purified to several folds and used in its raw form, thin bands and impurities were present in the gels.

Figure 9

Sodium Dodecyle Sulphate Polyacrylamide Gel Electrophoresis of crude (unpurified/unconcentrated) Indian domestic curd supernatant. Thin protein bands and impurities can be observed

Modifications of the study protocol were done to nullify antagonistic effect of IDC due to organic acids, LAB bacteriophages, and volatile compounds and may be of peroxides as follows: using crude IDC as supernatant (not concentrate) and not LAB culture broth, alkali treatment, sealing of sample wells in nutrient agar plates, UV sterilization of IDC samples in laminar airflow with stirring and using facultative anaerobe as indicator organism (coliforms). Heat treatment preserved inhibitory activity of supernatant as well as the pellet, but pellet showed somewhat decreased potential which may be due to heavy coagulum formation and subsequent entrapment of inhibitory potential in the coagulum [Figure 10]. Activity of IDC supernatant after heat treatment (121°C for 15 min) is 25.92U or 52739.94 mm2/ml with zone of inhibition 25.92 ± 3.98873 mm against coliforms (1.0 McFarland) [Table 3]. The IDC supernatant showed the potentiation of inhibitory activity to some extent when treated with EDTA, showing much clearer zone of inhibition [Figure 11]. Mean zone of inhibition was 26.94444 ± 4.18121 mm and activity was 26.94444U or 56991.22 mm2/ml [Table 3].

Figure 10

Heat treatment preserves antagonistic activity. Coagulum formation in pellet minimizes antagonistic potential

Table 3

Effect of various treatment on inhibitory activity of Indian Domestic Curd supernatant in agar well diffusion assay

Figure 11

Incubation with ethylene diamine tetraacetic acid potentiates antagonistic activity of Indian domestic curd supernatant. Increasing amount of Indian domestic curd supernatant and fixed amount of ethylene diamine tetraacetic acid

These observations [Figure 12] indicate that the inhibitory potential of IDC supernatant and pellet is due to production of various thermostable inhibitory peptides (and not due to LAB bacteriophages or organic acids, peroxides, and volatile compounds which may be produced during LAB fermentation in IDC because their effect has been nullified in the study design).

Figure 12

Inhibitory activity of Indian domestic curd samples in various treatment conditions (urea-treated Indian domestic curd lacks any activity). SD - Standard Deviation, IDC-Indian Domestic Curd, W/V - Weight / Volume, P Value - Probability Value, EDTA- ethylene diamine tetra acetic acid, Both P - Values are Significant (>95.5% Confidence Interval)

Whelan-Goldman WAG model (+G) describes the substitution pattern (of sequenced bacteriocin peptides of LAB origin used in this study) the best in the aligned sequences with BIC score (Bayesian Information Criterion) of 1532.919 and AICc value (Akaike Information Criterion, corrected) of 1298.088. The estimated value of the shape parameter for the discrete gamma distribution is 12.2073. There are 10 positions of evolutionary information in the final dataset of 36 amino acid sequences. Phylogenetic analysis of 36 sequenced bacteriocins of LAB comprises 10 different species. All sequences were from low molecular weight bacteriocins (1.4–14.073 kDa) of various organisms [Figure 13]. The analysis revealed several aspects of bacteriocin diversity; a single species may synthesize several types of bacteriocins with variable molecular weight and primary sequence, for example Lactobacillus plantarum produces glycocin with molecular weight as high as 7 kDa and another peptide of molecular weight as low as 2.9 kDa. Moreover, bacteriocins of this LAB have variable molecular weight in spite of being clustered together in the phylogenetic tree (PDB ID: 2RLW, 2JUI, 1YTR). They may be subunits of a large macromolecular structure. Furthermore, bacteriocins of a single LAB species may have significant sequence variability that they may be placed at distant sites in a phylogenetic tree as observed in Lactobacillus acidophilus. Amino acid frequencies of aligned sequence reveal (Single letter amino acid code) higher proportions of nonpolar amino acids [Figure 14].

Figure 13

Diversity among sequenced bacteriocins of lactic acid bacteria origin (molecular weight ranges from 1.4 kDa to 14.073 kDa)

Figure 14

Composition of amino acids in lactic acid bacteria bacteriocins

DISCUSSION

Recent studies on E. coli and its virulence factor LPS-induced periodontitis have revealed a complex pathophysiology of its manifestation. E. coli not only populate in oral mucosa in high numbers but also suppress growth of Actinomyces oris and Streptococcus mutants.[24] Moreover, drug-resistant strains of E. coli can colonize in high percentage among patients with fixed dental appliances and poor oral hygiene.[25] In this context, a study aimed at inhibition of E. coli through orally consumed IDC becomes thoroughly relevant. Upon infection, E. coli initiates unique immune response in the periodontal space. For example, E. coli LPS stimulates periodontal ligament cells (PDL cells) interleukin-6 production through a glucocorticoid-sensitive mechanism involving nitric oxide formation, probably via endothelial nitric oxide synthase in human PDL cell.[13]

In recent years, probiotics has been harnessed to overcome periodontal pathogens as an adjunct therapy.[26] There are encouraging results of randomized placebo-controlled clinical trials and follow-up studies as well.[172728293031] However, these studies used a probiotic preparation as concentrate in highly modified form such as LAB tablets or lozenges. Further, these studies have various organisms rather E. coli as a target. In the previous studies, several LAB have been isolated from fermented food-like curd which are cultured in artificial broth medium and the several folds concentrated cell-free supernatant found to have inhibitory activity mostly against various Gram-positive organisms due to production of bioactive peptides such as bacteriocin.[323334]

In the present study, the E. coli strain is isolated from municipal sewage which generally has high occurrence of coliforms including antibiotic-resistant strains that may contaminate public supplies.[353637] Inhibition of E. coli has been carried out by IDC which have significant nutritional value and is very popular throughout India.[383940] While investigation of the inhibitory component of IDC during this study, it was found that dilute and pelleted IDC both can inhibit coliforms and heat treatment did not altered the inhibition potential. It is also observed that EDTA enhances the inhibition potential of IDC and urea treatment diminishes its activity. EDTA destabilizes the cell wall of target pathogen and potentiates the action of the inhibitory peptide in IDC. This inhibitory peptide undergoes degradation in the presence of urea since urea is a chaotropic agent [Figure 15]. The presence of multiple protein in the molecular weight range >10–15 KDa may be justified by the fact that LAB can synthesize multiple inhibitory peptides in culture. Therefore, there is clear indication of peptide nature of the inhibitory ingredient in IDC and the inhibitory action remains stable even after heat treatment and serial dilution [Figure 16]. The mode of action of inhibitory component is through surface attachment to the target cells which is feature of bioactive peptides of LAB origin.[41] Although there are other factors in curd that are inhibitory in nature (such as the acidity of curd, peroxides, bacteriophages, and volatile components ) but, in this study, all these effects were nullified (by neutralization of IDC using alkali, stirring of samples in UV, and sealed bottom agar wells in diffusion assays). Furthermore, Coliforms may remain unaffected to the peroxide and organic acids of LAB origin.[42]

Figure 15

Loss of inhibitory potential of Indian domestic curd supernatant after treatment with urea

Figure 16

Detection of bacteriocin titer by dilution

Therefore it appears that IDC, a popular traditional fermented food in which LAB Bacteriocin occurs may be standardized as probiotic food (against E. coli a potential periodontal pathogen) that demonstrates significant inhibitory potential as per ICMR guidelines.[43] To establish the beneficial aspects of LAB against dental plaque coliforms a future study may be designed by isolating coliforms from patients suffering of dental plaques. In a synthetic dental model ex-situ the inhibition potential of IDC again st plaque formation may be studied.

In a rapidly changing etiology of periodontal infections, emerging pathogens such as E. coli need to be tackled on a priority basis. The present study demonstrated adequate inhibition against E. coli in vitro by IDC even in dilution [Figure 17]. Therefore, this work can be a good starting point toward a clinical trial with IDC that would include E. coli isolated from periodontitis patients.

Figure 17

Effect of dilution on Indian domestic curd inhibitory potential

CONCLUSIONS

Within the limitation of the present study, it can be concluded that IDC has inhibitory activity against coliforms. This inhibitory activity does not require any special treatment or fortification of IDC for its effectiveness against E. coli, a potential and emerging periodontal pathogen. This study clearly indicates that IDC in regular diet may be beneficial for oral health and hygiene.

Limitations

Neither IDC sample preparation and collection were standardized, nor the IDC samples were prepared in the laboratory. However, this was done to reflect an in situ” format of the study which focuses on natural inhibition property of domestically prepared curd against natural coliforms which can be a potential periodontal pathogen. In the study, coliforms were not isolated from patients. A future study in this laboratory is intended against coliforms isolated from dental plaques of periodontitis patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.