Effects of Cold Atmospheric Pressure Plasma on Streptococcus mutans isolated from Dental Caries

A total of (40) oral cavity swabs were sampled from various sites of dental caries (pit, fissure and dental roots) from patients who referred to the Dental Clinic at the College of Dentistry, Aliraqia University, Baghdad, Iraq. Initially, the swabs were placed in test tubes supplied with Stuart transport medium and then distributed by streaking on semi-selective Mitis Salivaris agar medium (MSA). The findings indicated that the isolates are gram-positive cocci that are arranged in chains. The isolates were further morphologically and biochemically identified, using the VITEK instrument, revealing only 32.5 % (n= 13) of S. mutans. The nonthermal plasma (cold atmospheric pressure plasma; CAP) has many uses in the area of research and development of clinical application worldwide. According to the biofilm assay, all isolates showed the capability of producing biofilm, with differences in thickness of the formed layer, with 3 isolates (7.5%) forming a strong layer, 6 (15%) forming a moderate layer, and 4 (10%) forming a weak layer. The non-thermal plasma (cold atmospheric pressure plasma; CAP) has many uses in the area of research and development of clinical application worldwide.


Introduction
Cariogenic bacteria, mainly S. mutans, colonize dental tissues as the main sign of the start of higher susceptibility to develop dental carries [1].S. mutans is a gram-positive, facultative anaerobic bacterial species with a close relation to the family of streptococci that has an ubiquitous distribution in the oral, throat, and intestinal tissues.This species also forms the main cause for the development of plaques and cavities in the teeth [2,3].S. mutans is highly capable of producing amyloids, that are found in high frequency in natural biofilms, and therefore it is known of high efficiency in creating' biofilms [2].Also, due to its well-recognized ability to produce acids, this species, in the presence of fermentable carbohydrates like sucrose and fructose, performs colonization to the teeth surface and causes damaging structural impacts.Moreover, it is capable of sticking to other plaque-causing bacterial species, as well as to the salivary pellicle on the enamel, leading to the production of acidified metabolic products and accumulation of glycogen stores.These functions, in addition to turning carbohydrates in food into fructans, glucans, and extracellular polysaccharides (EPS), lead to the development of caries.Despite several acidogenic and aciduric species that are present in the plaques and associated with the formation of caries, this species is still considered as the major source of EPS production, which renders the process of controlling biofilms difficult [3].The well-known capability of S. mutans of producing biofilms renders this species more difficult to treat, as these structure act as tiny rims which protect the engulfed microbial populations [4].S. mutans forms biofilms via several mechanisms, among which is the expression a surface protein known as adhesin P1 [5][6][7].Biofilms are formed based on two stages; the first involves the interaction of bacterial surface proteins with the material produced by the host, or the bacteria, and subjected to adsorption on the dental surface, while in the second, biofilms are formed upon the assemblage of bacteria with one another, or with other microorganisms, to produce an extracellular polysaccharide matrix [8].Adhesion P1 becomes involved in the process of dental surface colonization in the absence of sucrose.It undergoes interactions with high-molecular-mass glycoproteins, released in the saliva, in various site [9,10].S. mutans can perform an initial adherence to surfaces covered by the saliva via sucrose-independent pathways, mainly via a pathway in which one type of pellicle proteins interacts with bacterial adhesins [11,12].This pellicle protein, termed the SloC protein, is expressed on the surface of S. mutans and considered as one of its powerful virulence factors [13].It is an adhesin of the lipoprotein receptor antigen I (LraI) family, which was first described in various bacterial species and assists bacteria to actively move and adhere to other surfaces, including extracellular matrix, salivary agglutinin, other plaque bacteria, as well as receptors on the surface of epithelial cells [14].Cold Atmospheric Plasma (CAP) is a form of ionized gas that possesses similar numbers of ions and electrons with positive and negative charges [15,16].In thermal terms, plasma is typically found in two forms, namely the equilibrium plasma, in which ions and electrons show thermodynamic equilibrium, and the non-equilibrium plasma, where electrons show significantly higher temperature as compared to ions and neutrals [17].For biomedical applications, several different types of CAP have been created, such as the Dielectric Barrier Discharge (DBD), Atmospheric Pressure Plasma Jet (APPJ), Plasma Needle, and Plasma Pencil.In general, energy is needed to created Plasma and, more specifically, the electrical discharge is needed to product non-thermal plasma [18].Production of CAP takes place at room temperature and atmospheric pressure, rendering it safe to be applied in animal, including human, bodies [19,20] Non -thermal CAP or plasma needle is used in this research.

Samples collection and cultivation
Collection of samples (n= 40) was performed by swabbing the oral cavities of patients having various forms of dental caries (pit, fissure and dental roots) who attended to the Dental Clinic at the College of Dentistry, Aliraqia University, Baghdad, Iraq.The sterile disposable cotton swabs were placed within test tubes that contain Stuart transport medium, followed by streaking on semi-selective Mitis Salivaris agar medium (MSA) and storage for 4 months (January-April 2021).

Synthetic media
• Stuart transport medium (Stuart, 1954) For the preparation of this medium, an amount of 14.1 g was added to 1L of distilled water (DW), mixed well, dissolved by heating with frequent agitations, dispensed in screw-capped tubes, sterilized by autoclaving, and stored in 2-8 °C.
•Mitis salivarius bacitracin agar (MSBA) [21] MSBA was prepared by dissolving Mitis salivaris agar (90 g/L) and sucrose (150 g/L) in DW (950 ml).After adjusting pH to 7.2, the volume was brought up to 1000 ml with DW, followed by sterilization in autoclave.The preparation was then cooled to 45°C and bacitracin solution (1 ml; 00 I.U/ L) was added.After pouring into sterile plates, MSBA was utilized to isolate and identify S. mutans.
•Carbohydrates fermentation medium [22] For the preparation of this medium, brain-heart infusion broth (3.7 g) and phenol red (0.1g) were dissolved in distilled water (100 ml).After adjusting pH to 7.0, sterilization was performed in autoclave.The medium was then cooled to room temperature and kept in sterilized screw-capped tubes (10 ml), into each 1 ml of filter-sterilized carbohydrates solution (10%; mannitol, sorbitol and raffinose) was added in aseptic conditions.
•Blood agar [23] For the preparation of this medium, blood base agar (40 g) was dissolved in DW (900 ml), then pH was adjusted to 7 and the overall volume was brought up to 950 ml using DW.Following sterilization with autoclave, the medium was cooled to room temperature, followed by the addition of sterile defibrinated blood (50 ml) in aseptic conditions, gentle mixing, pouring into sterile Petri dishes, and storage at 4°C.

Biochemical tests
Catalase test [24] A wooden stick was utilized to deposit a growth cluster of pure cultures of individual bacterial isolates onto glass slides, followed by treatment with hydrogen peroxide solution (3%; 2 drops).Positive result (i.e., catalase release) was confirmed by the appearance of gas bubbles.[25] Following the streaking of individual bacterial isolates on blood agar, anaerobic incubation (37°C; 48 h) was performed for the detection of hemolysin produced and the identification of hemolysis type.[26] (Yoo et al., 2005) This test was achieved for determining the capability of bacterial isolates of utilizing mannitol, sorbitol, and raffinose as the only source of carbon and energy.Individual bacterial isolates were introduced into separate test tubes containing brain heart infusion broth with one of the carbon sources mentioned above (3%).Anaerobic incubation (24 h; 37°C) was then performed.Colour change from red to yellow was used to indicate positive result.

Identification of bacteria by VITEK 2 system
VITEK 2 is a fully automated instrument that is used to investigate microorganisms depending on their growth.The three versions of the VITEK 2, namely VITEK 2 small, VITEK 2, and VITEK 2 XL, are characterized by different capacities as well as various degrees of automation.However, these three versions of VITEK 2 support similar colorimetric reagent cards, which are automatically incubated and analysed.
These reagent cards have 64 wells capable of holding one test substrate.The entire set of substrates are used to measure various metabolic processes, e.g.acidification, alkalinization, enzyme hydrolysis, and growth in the presence of inhibitors.On each side of the reagent card, there is an optically transparent coating which is used for the maintenance of a sealed vessel, which allows the correct level of oxygen transfer while avoiding any contact with the organism-substrate interphase.Attached to every card is an inoculation transfer tube, as clarified below, while bar codes provide information related to product type, lot number, expiry date, and a unique identification which can be utilized to link the sample either prior or subsequent to the loading of the card onto the system.
At the present time, four types of reagent cards are commonly utilized to identify various classes of microorganism, which are (table 1): GN: fermenter and non-fermenter Gram-negative bacteria.GP: Non-spore-forming bacilli and gram-positive cocci.YST: yeasts and yeast-like organisms.BCL: Bacillus spore-forming Gram-positive.Sterile cotton swabs were used to transfer sufficient amounts of pure bacterial colony growths into transparent polystyrene tubes (12 x 75 mm) containing sterile normal saline solution (1 ml, pH 4.5-pH 7.0).Turbidity was measured and modified using a turbidity meter.

Assay of biofilm production
Biofilm production was assessed and quantified using 96-well microtiter plates as earlier clarified by [27].Overnight bacterial cultures were diluted to an OD660 of 0.01 in 10 ml LB medium with 1% glucose.A volume of 200μl from the dilution was added to the wells of plates.Each 3 wells contained an equal volume of inoculum.The plates were covered with a lid and incubated overnight under 37°C.After that, the unattached bacteria were removed with PBS with shaking and removal of excess liquid by rinsing on paper towels twice.In each well, 200 μl of crystal-violated stain (CV) (0.1%) was been added.The plate was then left to settle for 10-15 min to manage un-inoculated wells.The excess stain was eliminated by rinsing twice.PBS was then applied to the wells to remove the remaining stain.Then 50 μl of ethanol was added.Finally, plates were read at absorption of 600 nm in ELISA reader.Biofilm was divided into three parts according to the mean absorption results, as follows [28].
•Weak biofilm layer: When the absorbance values were equal to or higher than cut off values for control.
•Moderated biofilm layer: When the absorbance values were equal to or higher than twice cut off values for control.
•Strong biofilm layer: When the absorbance values were equal to or higher than four time cut off values for control.Cold atmospheric pressure plasma Three testing approaches were employed in the present work to estimate the antimicrobial efficacy of the CAP against 48-hour biofilms of two-gram positive and two-gram negative bacteria.These included the counting of the colony forming units (CFU), as a tool to quantitively assess the antimicrobeal activity TMREES-2023 Journal of Physics: Conference Series 2754 (2024) 012010 of CAP, live/dead fluorescence labelling, as a tool to qualitatively characterize this activity, and scanning electron microscopy (SEM), as a tool for analyzing the morphological changes of the bacterial species under study.The present work employed the plasma jet (Figure 1), while a set operating distance with variable exposure periods was adopted.In addition, the bacterial species under study was subjected to exposure on agar plates in order to provide an estimation of the size of the cold plasma exposure zone.Exposure times of 1, 2, 5, and 10 min with a distance of 15 mm were applied.The results revealed an increase in the number of dead bacteria as the exposure time increase.

Figure (1):
The plasma jet kINPen used in the current study.

Isolation of S. mutans
Following their collection from the mouth cavity, bacterial samples were kept in test tubes containing Stuart transport media, in addition to sterile disposable cotton swabs, before their streaking on a semiselective Mitis Salivaris agar medium (MSA).The latter medium was selected due to its dual activity of stimulating the growth of streptococci while hindering that of the majority of other bacteria [29].Restreaking of the surviving bacteria on selective MSBA medium resulted in the inhibition of most bacterial species, except for S. mutans and S. sorbinu [30].The identification of S. mutans becomes easier due to the formation of glucan, in addition to the fact that the colonies appear characteristically as a result of adding sucrose to this medium [26].

Characterization of S. mutans isolates
Following the cultivation of S. mutans-like bacterial isolates on MSBA, identification was achieved depending on the results of their morphological, cultural, and biochemical examinations.Morphological and cultural features the initial identification of S. mutans was achieved by employing their physical and cultural features, following their cultivation on MSBA.The results revealed gram-positive isolates that have a shape of spheres and exist in chains, which agrees with the results of [31].
Biochemical analysis [26] The results obtained from the biochemical analysis of bacterial isolates that were presumed to belong to the genus Streptococcus revealed that these isolates were Gram positive, catalase-negative, and capable of fermenting mannitol, sorbitol, and raffinose sugars.These isolates couldn't make hemolysin that displayed gamma.The 10 bacterial isolates were further tested by the VITEK-II system to establish that they were S. mutans.The result indicated in table (3).which are highly frequent in natural biofilms, this species, which is closely linked to the streptococci group that inhabits the mouth, throat, and gut, is well fitted to create biofilms [2].
A study by Li et al.Our findings unmistakably demonstrated that S. mutans log-phase cell density influenced acid adaption, as S. mutans cultured at high cell density adapted to the signal pH more quickly than cells at lower density [5].
In recent years, non-thermal plasma (cold atmospheric pressure plasma CAP) has been utilized in the various areas of research and development of clinical applications worldwide, including blood coagulation, hand/skin sterilization, wound healing, cell apoptosis and tissue removal.This work was performed on 13 groups of microplates, including three groups that were exposed to the cold atomspheric plasma for different durations (5, 10, and 15 sec) at a fixed exposure distance of 3 mm.The results show a decreasing in the number of viable bacterial cells for S. mutans, the main responsible bacterial species for the development of dental caries [32].

Conclusions
Dental caries is one of the most common illnesses infecting the teeth.The results showed that the direct interaction between the streptococcal proteins and the non-thermal atmospheric pressure plasma (CAP) reduced the state of cytosolic proteins in these facultative anaerobes.