The Antibacterial and Antibiofilm Properties of Different Strains of Lactobacillus spp. Isolated from Traditional Kefir Dough on Oral Pathogens with Real Time PCR

doi:10.21203/rs.3.rs-1654174/v1

Oral illnesses such as dental caries and gingivitis are frequent. In these kinds of infection, the bacteria are thought to use biofilm formation as a pathogenic mechanism. Today, due to the side effects of chemical medications and resistance to antibiotics, the use of probiotics is of great importance as a suitable alternative way for infection treatment. Probiotics have shown favourable properties in terms of maintaining oral health. Kefir, which is made up of complex microbiota mainly Lactobacillus spp., has been suggested as a potential reservoir for probiotic. The present research aimed to isolate and identify lactic acid bacteria from traditional kefir dough as a probiotic that suppresses S. mutans and P. gingivalis growth, biofilm formation, and gene expression. In this study, Lactobacillus spp. was tested for antibacterial (well agar diffusion technique) and antibiofilm (crystal violet assay) properties against Streptococcus mutans (ATCC 35668) and Porphyromonas gingivalis (ATCC 33277). Through the use of real-time polymerase chain reaction, we explored whether Lactobacillus plantarum and Lactobacillus rhamnosus inhibited expression of Streptococcus mutans genes involved in biofilm formation and stress survival. Additionally, Lactobacillus plantarum and Lactobacillus rhamnosus dramatically inhibited the expression of gtfB and brpA.

Antibacterial

Kefir dough

Lactobacillus strains

Oral pathogens

Real time PCR

gene expression

Tooth caries and periodontal disorders are common oral infectious illnesses linked to a dysbiosis of the bacteria that live in dental plaque biofilms and can affect adults and children's health [1]. Streptococcus mutans is a gram-positive anaerobic coccus, one of the most common main colonizing bacteria causing demineralization of the tooth structure by producing acids and resists acidic conditions. Moreover, Porphyromonas gingivalis is a non-motile anaerobic gram-negative bacterium that causes periodontal diseases [2]. The incidence of these illnesses is associated with a number of variable factors. Bacterial biofilms, on the other hand, are now widely regarded as the primary pathogenic mechanism. Bacterial biofilms are collections of microorganisms encased in an extracellular polymeric viscosity produced by themselves [3]. In fact, several disorders are caused through the interactions between common oral microbiota, host susceptibility, and environmental factors including nutrition, smoking, and oral hygiene. Biofilms are a physical barrier to the host's immunological response and act as a reservoir for prolonged infections [4]. A particular enzyme called glucosyltransferase is required for the formation of these biofilms. The GTF gene encodes this enzyme, which belongs to the GH70 enzymatic family of glycosides hydrolases [5]. The brpA gene codes a predicted surface-associated protein that appears to be involved in biofilm formation, autolysis, and cell division [6]. Probiotics are believed to limit the proliferation of cariogenic bacteria and the creation of biofilms in order to provide anti-caries effects.

Probiotics inhibit growth and biofilm formation of cariogenic bacteria by exerting anti-caries effects. They are "live microorganisms that, when administered in sufficient amounts, confer a health benefit on the host and have shown favorable properties in maintaining oral health," according to the WHO/FAO [7].

Fermented dairy and non-dairy products, such as meat, fruits, cheese, and fermented milks, such as kefir, can potentially provide novel bacterial strains with possible probiotic properties [8]. Kefir is manufactured by fermenting milk with various bacteria, and is one of the most common probiotic sources. Kefir consists of over than 50 different species of LAB, yeasts, and acetic acid bacteria, as well as their metabolites including lactic acid, exopolysaccharides, and peptides, which have a variety of health benefits. The word kefir comes from the Turkish word keyif, which means "pleasant feeling" for the feelings experienced after drinking it [9]. Antimicrobial, antihypertensive, anti-inflammatory, anti-cariogenic, anti-allergic, and antioxidant properties were found in kefir [10]. Probiotic strains commonly belong to the Lactobacillus and Bifidobacterium. Lactobacillus is divided into three groups: namely obligatory homofermentative, facultative homofermentative, and obligate heterofermentative Lactobacillus (7). Some Lactobacillus species were introduced as potential probiotics in caries prevention owing to their inhibitory action against cariogenic S. mutans and P. gingivalis (2) The current of this study was conducted to determine the antibacterial effect of Lactobacillus plantarum and Lactobacillus rhamnosus Supernatant on S. mutans and P. gingivalis, furthermore, inhibition of biofilm formation and the effect of these LAB on gtfB and brpA, which are involved in biofilm formation were investigated.

Isolation and identification

Isfahan University of Medical Sciences' microbiology lab received five samples of traditional kefir dough from the local sources. Lyophilized Standard strains S. mutans (ATCC 35668) and P. gingivalis (ATCC 33277) were prepared from the Iranian industrial microbial collection.

Examination of morphological characteristics of LAB

LAB strains were identified morphologically using the catalase test, gram staining, temperature growth at 15°C and 45°C and pH levels using the procedures in Bergey's book. The physical properties of each colony, as well as the cell appearance characteristics were examined via gram staining. To perform the catalase test and confirm the lactobacillus genus, colonies identical to Lactobacillus with bacterial features of rod-shape, gram positive and without spores were sampled. Each strain was cultivated several times in MRS medium to ensure the purity of MRS culture bacterial [11].

Fermentation of carbohydrates of LAB

The bacteria were cultivated on MRS in order so that fermentation and acid generation tests could be performed. The grown isolate was inoculated in a- tube containing fermentation liquid medium (1% of the desired sugar and phenol red reagent). The tubes were incubated at 37 ° C under 5% carbon dioxide for 72 hours. Sugar consumption and acid production were attributed to the red color change of the culture media to yellow and the formation of bubbles in the tubes [12].

Molecular identification of LAB

To begin with, Murray and Thomson procedures were used to extract the isolate's DNA, with minor modifications. This procedure involved centrifuging 10 mL of bacterial solution (from a 24-hour culture) for 10 minutes at 13,000 rpm. Sediments were transferred into the micro tube. A milliliter of TBE lubricating buffer was added to them after half an hour. Subsequently, at a temperature of 60°C (on a hot plate), chloroform isoamyl alcohol (1:24) was added and well mixed. The materials were again centrifuged at 13,000 rpm for 5 minutes. The supernatant was transferred to new vials and the same volume of pure isopropanol was added. After completely mixing the contents of each vial, they were placed on ice for 10 minutes. The materials were centrifuged at 13,000 rpm for 10 minutes to precipitate, following which 500 µl of 70% ethanol was added. The samples were centrifuged for 5 minutes at 5,000 rpm, with the supernatant removed gently. The DNA vials were then left to dry for an hour at the room temperature. After the sediment dried, each microtube was filled with 100 liters of sterile deionized distilled water. A 1% agarose gel was used to validate the quality of the extracted DNA through electrophoresis[13]. For checking the specificity of the selected primers in genus and species, they were blasted at NCBI. Afterwards the primers were purchased from Gene Technologies.

Table 1

Optimized primer

Reference	Product Size	Sequence	Target Gene	Bacteria
[14]	245	F:CTCAAAACTAAACAAAGTTTC R:CTTGTACACACCGCCCGTCA		Lacto genus
12))	176	F:CGAGACAGCAATTCCTGCACTCG CCTCAGAAACAGTCCGGTTGA:R	apbE2	Lactobacillus plantarum
12))	124	F:ATTTAACCGCAAGTGGCAGC AAATTGTGTGAACCGGCGTA:R	aes	Lactobacillus rhamnosus

Probiotic Lactobacilli preparation:

Lactic acid bacteria (LAB) were cultured on MRS broth and incubated at 37° C for 18-24 hours under anaerobic conditions. The bacterial growth was then centrifuged for 15 minutes at 13000 rpm/min. The supernatant was collected and filtered through a Millipore 0.22 m filter after being adjusted to a pH of 7.5 with NaOH[14].

Assay for sensitivity to lactobacillus

Sensitivity of pathogenic bacteria to different strains of lactobacillus was determined via the minimum inhibitory concentration (MIC) assay. The assay was done using the method described by Andrews [16]. According to this method, stock solutions containing 20 mg/ml of 10 different selected Lactobacillus were prepared. For preparation of the test inoculums the 48 h active cultures of pathogenic bacteria were adjusted to 0.5 McFarland standards (10⁷ to 10⁸ cfu/ml of bacteria) by adding several dilution of each probiotic powder in each well and microtiter plate incubated for 48 h at 37°C[15, 16].

The well agar diffusion method for antimicrobial screening:

Using well diffusion technique proposed by Cadirci and Citak, the antibacterial activity of Lactobacillus spp. on and Porphyromonas gingivalis was tested. P. gingivalis was cultured on blood agar enriched with vitamin K1 and hemin (at a concentration equivalent to McFarland 1 standard) and S. mutans suspension (at a concentration comparable to McFarland 0.5 standard) was cultured on blood agar complete with 5% defibrinated blood sheep medium. A number of the wells were drilled in the culture media. The wells were filled with100 µl of SCS. For Porphyromonas gingivalis and Streptococcus mutans, inhibition zones were determined in millimeters after 72 hours of anaerobic incubation at 37°C and 48 hours of aerobic incubation at 37°C (14)[15].

The effect of Lactobacillus species on formation of biofilm:

For biofilm assays Streptococcus mutans was grown in BHI containing 2% sucrose, Porphyromonas gingivalis was grown in BHI containing vitamin K and hemin while Lactobacillus species were grown in deMan Rogosa and Sharpe (MRS) broth without sucrose. All the microorganisms were incubated at 37°C in an anaerobic jar for 48h. To evaluate the effect of Lactobacillus spp. on formation of the biofilm of Streptococcus mutans, the suspensions of each isolate were prepared as stated above. The suspensions were adjusted with their respected broth to 0.5 McFarland turbidity standards and several dilutions. Formation of S. mutans and P. gingivalis biofilm was assayed in the presence or absence of Lactobacillus strains in a 96-well polystyrene culture plate. Lactobacillus strains and pathogenic bacteria were mixed at an equal ratio (1:1). Blank wells contained culture medium instead of probiotic strains. The plates were incubated at 37° C for 48 hrs. Quantitation of biofilms was performed using crystal violet based microtiter plate assay [17].

Gas chromatography-mass spectrometry:

The selected strains were inoculated in MRS broth and incubated at 30°C for 4 days. Subsequently the samples were centrifuged at 4000 rpm for 20 minutes. Equal amounts of ethyl acetate were added to the broth and incubated in a rotary shaker for 1 h. The downer layer of the broth was separated. The samples were analyzed with a Hewlett Packard 6850 Gas chromatograph, 5973 mass selective detector, and 7683B series injector (Agilent Technologies, Palo Alto, CA, USA) with helium as the carrier gas at a flow of 1.0 mL/min. One microliter of each sample was injected with 1 min of split flow delay and resolved on a 30 m × 0.25 mm × 0.25 µm DB5MS column (Agilent Technologies, Palo Alto, CA, USA). Inlet, interface, and ion source temperatures were 300 ◦C. Oven starting and final temperatures were within the rate of 5 ◦C/min for 36 min and then for 2 min at a constant temperature. Metabolite annotation was achieved by mass spectra comparison with analytical standards, in house library and the NIST14 database (National Institute of Standards and Technology, Gaithersburg, MD, USA)To prepare dried probiotic powder the upper layer of the broth was incubated, and allowed to dry [18].

Streptococcus gene expression:

We used reverse transcription real-time PCR to evaluated the mRNA levels of S. mutans genes encoding virulence proteins related to carbohydrate metabolism (gtfB) and biofilm formation (brpA) to examine the anti-biofilm formation mechanism, as described previously. For growth curve analysis, the reagent Pars Tous kit was used to extract total RNA and cDNA. A Nanodrop ND-2000 spectrophotometer utilized to assess RNA concentration (ThermoFisher Scientific). The cDNA was amplified with SYBR Premix Taq and the primer sets are given in Table 2 on an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). For gtfB and brpA, the following real-time PCR conditions were used: 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds, 52°C for gtfB, 55° for brpA, and 60°C for 1 minute. The temperature was reduced at a rate of 0.1°C/s from 95°C to 60°C for melting curve analysis, while the fluorescence signal intensity was continuously measured. After adjusting to the 16SrRNA level, variations in mRNA expression levels were determined using ABI 7500 v.2.2 software (Applied Biosystems). The results are reported as a fold change compared to the control group, and fold changes were taken into account.

Table2

Group-specific primer sets used for quantitative reverse transcription real-time PCR :

Reference	Sequence(5ˊ–3)	Primers	Target gene	Function
[19]	ACGAACTTTGCCGTTATTGTCA AGCAATGCAGCCAATCTACAA	For Rev	gtfB	Carbohydrate metabolism-promoting genes
[20]	CGTGAGGTCATCAGCAAGGTC CGCTGTACCCCAAAAGTTTAGG	For Rev	brpA	Regulatory protein-encoding genes
[21]	ATGTTGGGTTAAGTCCCG CTAGCGATTCCRRCTTCA	For Rev	16SrRNA	Housekeeping gene

Bacterial culture:

Streptococcus mutans and Porphyromonas gingivalis were taken from the Department of Iranian industrial microbial collection and validated using Gram staining, biochemical and molecular analysis with particular primers. Five Lactobacillus plantarum strains and five Lactobacillus rhamnosus strains were identified from traditional kefir dough in Different areas of Isfahan. According to the methods recommended by Bergey in manual of systematic bacteriology and Wood & Holzapfel in the genera of lactic acid bacteria, all isolates were studied for their morphological identification of strains using gram staining, catalase, oxidase and sugar fermentation test, grown at temperatures of 15° and 45 ° C and different pHs[11].The results are shown in Table 3. All the isolates are Gram-positive, and polymorphic, but often filamentous with short-chain. Oxidase, catalase, nitrate reduction and VP (Voges Proskauer) were negative. Based on the tests described five isolates were Lactobacillus plantarum and five were Lactobacillus rhamnosus. They all grew at NaCl %4 and %6.5, pH of 3.8 and 7.8. Lactobacillus plantarum grew at 15°C and 45°C, but the Lactobacillus rhamnosus grow at 15°.Elevation of Lactobacillus plantarum was convex and flat whereas that of Lactobacillus rhamnosus was flat. Colony color of Lactobacillus plantarum were creamy shine and creamy white but Lactobacillus rhamnosus were off white and white.

Fermentation of carbohydrates:

All the isolates of Lactobacillus plantarum were able to ferment Arabinose, Cellobiose, Mannitol, Gluconate, Raffinose, Ribose, Sorbitol, Sucrose and Xylose.

All the isolates of Lactobacillus rhamnosus were able to ferment Arabinose, Cellobiose, Mannitol, Gluconate, Ribose, Sorbitol and Sucrose but not Raffinose and Xylose.

Table 3

Fermentation of different carbohydrates

Carbohydrate

Bacteria

Xylose

Sucrose

Sorbitol

Ribose

Raffinose

Gluconate

Mannitol

Cellobiose

Arabinose

Lactobacillus plantarum

d

+

d

Lactobacillus rhamnosus

-

+

-

+

d

Molecular identification of Lactobacillus strains:

The results of the PCR assays were 200 bp for the genus of Lactobacillus,176 bp for L. plantarum and 124 bp for the L. rhamnosus (Fig1,2).

Antimicrobial susceptibility:

The MICs of 10 antimicrobials of Lactobacillus were determined for all the strains. The MIC for Streptococcus mutans was 5 mg/ml and for Porphyromonas gingivalis, it was 20 mg/ml.

Antimicrobial assay:

Lactobacilli were tested for their antibacterial properties against cariogenic (S. mutans) and periodontal pathogenic (P. gingivalis) bacteria (Table4). After 48 hours, all of the Lactobacilli strains tested displayed an antibacterial activity against S. mutans and P. gingivalis, according to the results of the disk diffusion method.

Table 4

Antibacterial efficacy of probiotic supernatant against oral infections

Number of isolate LAB

Lactobacillus isolated

L.p1^*

L.p2

L.p3

L.p4

L.p5

L.r1^*

L.r2

L.r3

L.r4

L.r5

Pathogenic bacteria

Streptococcus mutans

13mm

15mm

17mm

12mm

21mm

14mm

12mm

15mm

17mm

12mm

Porphyromonas gingivalis

13mm

15mm

19mm

15mm

19mm

15mm

14mm

17mm

20mm

16mm

*L.r= Lactobacillus rhamnosus * L.p= Lactobacillus plantarum

Overall, the mean diameter of the growth inhibition halo for Lactobacillus plantarum vs. Streptococcus mutans was 15.60 mm, which was 14 mm for Lactobacillus rhamnosus vs. Streptococcus mutans. Moreover, the mean diameter of the growth inhibition halo for Lactobacillus plantarum vs. Porphyromonas gingivalis was 15.60 mm and that for Lactobacillus rhamnosus vs. Porphyromonas gingivalis was 16.40.

Reduced biofilm formation of supernatant of Lactobacillus against two oral pathogens:

Biofilm formation of Streptococcus mutans and Porphyromonas gingivalis was compared in the absence and presence of Lactobacillus species. Oral pathogens biofilm production was inhibited considerably by all the Lactobacillus species utilized in this experiment which were significantly (Fig3, 4). Different concentrations of both genera of Lactobacillus had a decreasing effect on oral pathogens, but from a concentration of 10^-5 down, a greater decreasing effect was observed.

Decrease in gtfB and brpA expression of S. mutans by L. plantarum and L. rhamnosus:

The expression of the genes involved in S. mutans pathogenicity was studied in order to assess the effect of L. rhamnosus and L. plantarum supernatants on S. mutans. The signal transduction system was regulated by the brpA genes, and the representative genes involved in glucan synthesis included gtfB. Both genes were suppressed by L. rhamnosus and L. plantarum.

Gas chromatography-mass spectrometry analysis:

Antimicrobial compounds detected in the culture supernatant of five L. plantarum fermentations in kefir dough (table 5).

Table 5

Isolated antimicrobial compounds of five Lactobacillus plantarum in kefir dough

Xylene	3 methyl 3 pyrazoline-5-one	2(5H) Furanone-3 methyl	Eicosane
Benzene 1.3 dimethyl	2,4 dimethylcyclopent-4-ENE-1,3	O-Cyanobenzoic acid	2-Pyrrolidione
Benzene 1.4 dimethyl	(3,4-Dimethoxybenzyl)-3,4-dihydro6,7dimethoxyisoquinolinium chloride	N-hexadecanioc acid	Heptacosane
9-octadecenamide	Tridecane	Nonadecane	Cyclotridecane
Tetradecane	Cycloheptasiloxane

Antimicrobial compounds detected in the culture supernatant of five Lactobacillus rhamnosus fermentations in kefir dough (Table6).

Table 6

Isolated antimicrobial compounds of five Lactobacillus rhamnosus in kefir dough

P-xylene	Cyclotrisiloxane-hexamethyl	Cycloheptasiloxane	Tetracosamethyl-cyclododecasiloxane
Benzene,1,3 dimethyl	Tetradecane	Hexadecane	Gamma terpinene
Gibberellic acid	Cycloheptasiloxane	Eicosane	Beta-Pinene

In general, several compounds were common in both species of Lactobacillus including Xylene, Benzene 1,3 dimethyl, Tetradecane, Cycloheptasiloxane, hexadecane and Eicosane.

Oral diseases are among the most prevalent diseases globally and have serious health threatening effects because one of the predisposing factors for systemic diseases, diagnosis and treatment of this disease is important. Two frequent human infectious illnesses are dental caries and periodontitis [22]. Today, due to the various side effects of chemical drugs and antibiotics, researchers have come up with new methods and utilization of beneficial microorganisms for treating and controlling diseases [23]. Probiotics have been studied for their importance in the treatment of a variety of illnesses. Kefir grains include lactic acid bacteria, acetic acid bacteria, yeasts, and potentially other microbes, and have been connected with health benefits for decades [24]Certain Lactobacillus strains have been demonstrated to have the capacity to disrupt oral ecology by suppressing pathogenic microbes such as S. mutans and P. gingivalis [10]. Due to the fact that there are no complete details on the separation of lactic acid bacteria from traditional kefir dough, studies have been performed to identify lactic acid bacteria from these kefir doughs. Lactic acid bacteria are found in a wide range of foods and are abundant in a wide range geographical conditions [25]. This variety in dairy products in the world is also very complex and can be an incentive to screen these products with the goal of achieving to suitable strains with special functional and technological characteristics[14] In 2002 Comeli al. studied the effect of bacterial strains of Lactobacillus lactis and Bacillus stearothermophilus in dairy products on oral health, They concluded that dairy strains were able to alter some of the characteristics of some oral bacteria, especially Streptococcus oralis colony count was significantly reduced in the presence of probiotics used [26].

In the study by Tahmourespour and et al. in 2011 the effect of a biosurfactant isolated from L. acidophilus on the formation of biofilm of S. mutans ATCC35668 and S. mutans isolated from dental plaque was investigated. The effect of the mentioned biosurfactant on the expression level of gtfA / B genes in these two strains was also shown. It was found that the derived biosurfactant was effective on the surface properties of biofilm formation, the ability to bind and express these genes[27]. In 2012 Zezhang T. Wen et al. studied the brpA gene, and concluded that brpA plays an important role in the pathophysiology of S.mutans and could be a potential target in modulating its virulence factors[28]. In 2014, Hoon Lee Sung et al. reported that Lactobacillus acidophilus can inhibit the effect of gtfB / gtfC / gtfD genes involved in tooth decay in their research on investigating the effect of probiotics on cariogenic biofilm model[29]. In 2018, DANA JEONG et al. isolated Lactobacillus kefiranofaciens from kefir and examined its inhibitory effect on eight genes in Streptococcus mutans,They concluded that Lactobacillus could reduce the expression of genes involved in tooth decay caused by Streptococcus mutans[8]. In 2020, the derivatives of Nonadecanoic acid, benzoic acid, hexadecanoic acid and pyrrol were separated from the Lactobacillus plantarum. The results of this research are in line with those reported here in [30]. We extracted LAB from kefir dough and tested its antibacterial properties against S. mutans and P. gingivalis biofilms. After treatment with probiotic supernatant, the biofilm generated by Streptococcus mutans and Porphyromonas gingivalis decreased significantly, while the inhibitory zone around colonies of Streptococcus mutans and Porphyromonas gingivalis increased. The impact of Lactobacillus plantarum and Lactobacillus rhamnosus on the expression of the gtfB and brpA genes in S. mutans was also investigated in this study. gtfB is a key virulence factor for Streptococcus mutans which is responsible for the synthesis of insoluble glucans, that form dental plaque, Furthermore, it might be a selective therapeutic target for cariogenic biofilm avoidance[19, 20]. brpA (lytR) encoding brpA regulatory protein has been described as an important regulator of biofilm formation. Furthermore this gene encodes a putative surface-associated protein that has a role in biofilm formation, autolysis, and cell division [6]. The obtained findings here in demonstrated the considerable antimicrobial and antibiofilm activity of probiotic bacteria L.plantarum and L.rhamnosus against S. mutans and P. gingivalis biofilms. The culture supernatant could also suppress the expression of virulence factors involved in polysaccharide synthesis and those that integrate external signals in a regulatory network composed of expression levels of gtfB and brpA, which are also involved in biofilm formation and play a key role in stress response regulation. Probiotic Lactobacilli can be used instead of antibiotics as a therapeutic alternative to inhibit growth and biofilms formation in Streptococcus mutans causing dental diseases, and Porphyromonas gingivalis causing gingivitis. Generally, Lactobacillus Plantarum has further inhibitory effects on both oral pathogens in comparison with Lactobacillus rhamnosus.

Finally, Lactobacillus plantarum and Lactobacillus rhamnosus reduce the expression of critical regulatory factors, which limits S. mutans and P. gingivalis biofilm development and stability.

Authors' contributions

KSE: Data curation; Formal analysis; Investigation; Methodology; Writing - original draft, TN: Funding acquisition; Formal analysis; Project administration; Supervision; Validation; Writing - review & editing. FP: Data curation, AM and MSD: Investigation.

Funding

This work was funded by Medical Sciences University of Isfahan (Grant number: 399075).

Statements and Declarations

Competing Interests:

The authors declare that they have no competing interests.

Ethics Approval:

The Ethics Committee of Isfahan University of Medical Sciences approved the ethical aspect of this study (IR. MUI.MED.REC.1399.260). This article does not contain any studies with human or animal subjects.

Consent to Participate:

Not applicable.

Consent for Publication:

Not applicable.

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No competing interests reported.

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The Antibacterial and Antibiofilm Properties of Different Strains of Lactobacillus spp. Isolated from Traditional Kefir Dough on Oral Pathogens with Real Time PCR

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Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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