Agnieszka Stącel-Stępniewska1, Ilona Rowińska2, *Paweł Kowalczyk3
Curasept pastes and gels as new killers against pathogenic strains from green and purple complexes present in the oral cavity
Pasty i żele Curasept jako nowi zabójcy szczepów patogennych z kompleksów zielonych i niebieskich obecnych w jamie ustnej
1Indent, Wroclaw, Poland
2The Medical and Social Center for Vocational and Continuing Education in Torun, Poland
3Department of Animal Nutrition, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland
Streszczenie
Wstęp. Bakterie łącząc się w określone grupy względem zużywania składników pokarmowych oraz odpowiedniego pH w jamie ustnej, tworzą specyficzne kompleksy, dzięki którym poszczególne osobniki mogą bardzo skutecznie wykorzystywać składniki odżywcze i dobrze chronić się przed układem odpornościowym gospodarza. Szczególnie na bytowanie siedliskowe poszczególnych patogennych szczepów bakterii narażone są kieszenie dziąsłowe, zwłaszcza jeśli są patologicznie pogłębione. Stają się one wówczas niszami, w których osadzają się resztki organiczne i mikroorganizmy bakteryjne. Socransky podzielił mikroorganizmy żyjące w biofilmie jamy ustnej na kilka grup, kierując się relacjami między gatunkami mikroorganizmów, a także przypisując im odpowiednie kolory. Kompleksy wyróżniono w następujący sposób: czerwone – obejmują bakterie najbardziej patogenne dla tkanek przyzębia; pomarańczowe – gatunki bakterii, które są bezpośrednio związane z mikroflorą należącą do kompleksu czerwonego; żółte – bakterie związane z płytkimi kieszonkami przyzębia; fioletowe – tworzą bakterie, które są umiarkowanie patogenne, jak mikroorganizmy z kompleksów czerwonego i żółtego; niebieskie – rozwijają się u osób z uporczywymi zmianami okołowierzchołkowymi, obejmują one bakterie z rodziny promieniowców; zielone – mają istotny wpływ na rozwój próchnicy.
Cel pracy. Celem badania było sprawdzenie właściwości antybakteryjnych past i żeli firmy Indent – lidera w profilaktyce stomatologicznej i dystrybutora takich firm, jak: Curasept, Tello, Frezyderm, które pomagają przyspieszyć regenerację kieszonek dziąsłowych oraz ich gojenie, a także zapobiegać przewlekłym stanom zapalnym w jamie ustnej w postaci długich infekcji lub wrzodziejących aft, chroniąc przed próchnicą oraz uporczywymi zmianami okołowierzchołkowymi tkanek przyzębia.
Materiał i metody. Referencyjne szczepy bakterii probiotycznych jako kontrola Bifidobacterium lactis HNO 19, Kluyveromyces marxianus fragilis B0399, Lactobactilius reuterii Prodentis, Lactobacillus salivarius SGL03 oraz patogenne szczepy kompleksów zielonego i fioletowego. Zielony kompleks bakteryjny występuje w kieszonkach powyżej 6 mm a należą do niego nastepujace bakterie: Eikinella corrodens (ATCC 23834), Capnocytophaga gingivalis (ATCC 33624), Agregatibacter typ a (ATCC 29522), Agregatibacter typ b (ATCC 43718) oraz kompleksu fioletowego: Vellionella parvula (ATCC 17745), Actinomyces odontolyticus (ATCC 17929) zostały dostarczone przez (LGC Standards U.K.) i były używane zgodnie z zaleceniami normy ISO 11133:2014. Szczepów tych użyto do przetestowania aktywności przeciwbakteryjnej z analizowanymi związkami przy użyciu minimalnego stężenia hamującego (MIC) i minimalnego stężenia bakteriobójczego (MBC).
Wyniki. Przebadane pięć związków wykazało superselektywność i aktywność przeciwdrobnoustrojową we wszystkich analizowanych szczepach bakteryjnych kompleksów zielonego i fioletowego – miały profil podobny do uzyskanego przy użyciu obecnie stosowanych antybiotyków, takich jak cyprofloksacyna, bleomycyna i kloksacylina, obserwowanych w testach MIC i MBC.
Wnioski. Należy również zauważyć, że koszt uzyskanych związków jest niski, co może stanowić atrakcyjną alternatywę dla obecnie stosowanych środków przeciwdrobnoustrojowych. Pozwoli to znacznie obniżyć koszty powszechnie stosowanych antybiotyków oraz przyczyni się do właściwości prozdrowotnych całego organizmu.
Summary
Introduction. Bacteria, by combining into specific groups according to the consumption of nutrients and the appropriate pH in the oral cavity, create specific complexes, thanks to which individual individuals can very effectively use nutrients and protect themselves well against the host’s immune system. Gingival pockets are particularly exposed to the habitat existence of individual pathogenic strains of bacteria, especially if they are pathologically deepened. They then become niches in which organic debris and bacterial microorganisms settle. Socransky divided the microorganisms living in the oral biofilm into several groups, guided by the relationships between the species of microorganisms, and also assigning them appropriate colors. The complexes were distinguished in this way: red – include bacteria most pathogenic to periodontal tissues; orange – include bacterial species that are directly related to the microflora belonging to the red complex; yellow – include bacteria associated with shallow periodontal pockets; purple – formed by bacteria that are moderately pathogenic like microorganisms from the red and yellow complexes; blue – develops in people with persistent periapical changes; these include bacteria from the Actinomycetes family; green – has a significant impact on the development of caries.
Aim. The aim of the study was to test the antibacterial properties of pastes and gels from Indent – a leader in dental prophylaxis and distributor of companies such as Curasept, Tello, Frezyderm, which help accelerate the regeneration of gingival pockets and their healing, as well as prevent chronic inflammation in the oral cavity in the form of long infections or ulcerative aphthae. Protecting against caries and persistent periapical changes of periodontal tissues.
Material and methods. Reference strains of probiotic bacteria as a control Bifidobacterium lactis HNO 19, Kluyveromyces marxianus fragilis B0399, Lactobactilius reuterii Prodentis, Lactobacillus salivarius SGL03 and pathogenic strains of green and purple complexes. Green Complex bacteria in pockets above 6 mm: Eikinella corrodens (ATCC 23834), Capnocytophaga gingivalis (ATCC 33624), Agregatibacter type a (ATCC 29522), Agregatibacter type b (ATCC 43718) and purple complex: Vellionella parvula (ATCC 17745), Actinomyces odontolyticus (ATCC 17929) were provided by (LGC Standards U.K.) and were used according to the recommendations of ISO 11133:2014. These strains were used to test the antibacterial activity with the analyzed compounds using the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).
Results. The five compounds tested showed superselectivity and antimicrobial activity, in all analyzed bacterial strains of both green and purple complexes which had a profile similar to that obtained using currently used antibiotics, such as ciprofloxacin, bleomycin and cloxacillin, observed in MIC and MBC tests.
Conclusions. It should also be noted that the cost of the obtained compounds is low, which may be an attractive alternative to currently used antimicrobials. This will allow for a significant reduction in the costs of commonly used antibiotics and will contribute to the health-promoting properties of the entire organism.
Introduction
The microbial colonization depends on the availability of nutrients and the breaking of the natural limit of non-specific immunity. The first to inhabit the oral cavity are the so-called pioneer species transmitted by the macroorganism and obtained from the environment. They colonize specific ecological niches and, over time, multiply, creating ecological communities. The environment in which they live changes as a result of their metabolic activity, which facilitates the penetration of other bacterial species into them, and thus begins ecological succession, which leads to a large and significantly diversified environment of sessile microflora, or to pathological changes (1-17).
The colonization of the tooth surface by microorganisms is possible due to adhesion or adherence processes. Adhesins enable the connection of the lipophilic surface of bacterial cells with the hydrophobic surface of epithelial cells. Adhesins are proteins that bind to carbohydrates using structures called fimbriae (fibrils of various lengths found on the cell surface). Fimbriae have been detected on streptococci: S. anginosus, S. salivarius, S. oralis, S. mutans, on Actinomycetes – A. viscosus, A. naeslundi and in Gram-negative rods – Prevotella, P. intermedia, P. oralis, P. bucceae, P. melaniogenica and the species Porphyromonas gingivalis (18-20).
Other adhesive substances are substances that stick together or transport substrates, e.g. polypeptides that bind salivary glycoproteins to bacterial cells or calcium ions. Extracellular polypeptides that react with streptococcal proteins behave similarly. The cell walls of streptococci contain lipoteichoic acids that can bind to the acquired salivary sheath (a thin layer that forms from salivary glycoproteins on teeth just a few minutes after brushing and adheres to them). An important role in colonization processes is played by cytoplasmic membrane polyproteins that act as a „transporter” of components, e.g. sugar or peptides associated with the acquired salivary sheath or the surfaces of other bacterial cells. So-called ligands take part in the adhesion processes – negatively charged salivary glycoproteins that can occur on epithelial cells of the mucous membrane, hard tissues of the tooth above and below the gums and on prosthetic restorations. These compounds are positively charged (Ca+2), so they bind to negatively charged bacterial adhesins of streptococci – S. mutans, S. oralis, Actinomyces rods, Gram-negative bacteria, i.e. P. intermedia, F. nucelatum, P. gingivitis, E. corrdens (1-13, 18-20).
Another important adhesion mechanism is bacterial aggregation and colonization process, which involves the attachment of microorganisms to each other and their adherence to various surfaces. The main substrates in the oral cavity are: the acquired membrane on the hard surfaces of enamel and root cement (bones) and on the oral mucosa. The membrane covering the surfaces is about 1 micron thick and is characteristic of each surface. The membranes on the surfaces of hard tissues are not identical; on the epithelium they are referred to as the mucosal membrane (5, 13, 20-33).
In the oral cavity, bacteria group into specific groups, also known as complexes, thanks to which they use nutrients better and defend themselves more effectively against the defense mechanisms of the macroorganism (32, 33). The concept of „bacterial complex” was introduced by Socransky (34). The division criterion is the relationship between bacteria. Socransky divided pathogens in the biofilm into 6 groups, to which he assigned an appropriate color – blue, yellow, green, purple, orange and red. The last three complexes (purple, orange and red) dominate in the subgingival space (34-48). In our studies, we focused on bacteria of the green and purple complexes (fig. 1).
Fig. 1. Bacterial complexes in the oral cavity according to Socransky (34)
The green group consists of Eikenella corrodens is a Gram-negative bacteria found in the upper respiratory tract and the digestive tract of humans. It constitutes the physiological bacterial flora of the periodontium. It was identified in 1958 by M. Eiken and named Bacteroides corrodens (48). It is a pleomorphic cell (often takes the form of a rod), Gram-negative. It is a relative anaerobe, it grows better in an atmosphere enriched with 5-10% CO2. It has high growth requirements – it grows on blood agar and chocolate agar (48). It grows in the form of light yellow colonies, often with a depression in the center. It does not produce catalase, it produces cytochrome oxidase (49). It belongs to the HACEK group (40) (slow-growing Gram-negative bacteria constituting the physiological flora), which can cause endocarditis in children. This group also includes: Haemophilus sp., Actinobacillus actinomycetemcomitan, Cardiobacterium hominis, Kingella kingae (50). Eikenella corrodens is resistant to: macrolides, aminoglycosides, lincosamides, metronidazole and intermediately susceptible to quinolones (51).
The genus Capnocytophaga is a group of spindle-shaped, Gram-negative, rod-shaped organisms with gliding motility. These organisms are part of the normal oral flora and were first described as Bacteroides ochraceus and CDC group DF-1. In patients without evidence of immune defects, cellulitis or wound infection occurs. Infections caused by Capnocytophaga can be treated with antibiotics, but complications of the disease can occur rapidly and can be potentially fatal. Aggregatibacter actinomycetemcomitans is a bacterium commonly associated with aggressive periodontitis, most severe in the central incisors and first molars. As in previous classifications, the occurrence of this organism correlated with juvenile periodontitis, and to a lesser extent with rapidly progressive periodontitis. It is a Gram-negative, nonmotile, facultatively anaerobic rod, as the name suggests, associated with actinomycetes, belonging to the family Pasteurellaceae. Based on the structure of membrane polysaccharides, six basic serotypes of this pathogen have been distinguished, of which three occur most frequently in the oral cavity, marked with the letters a, b and c (51). According to a study by Perry et al. (51) serotypes a and b are detected in almost equal proportions in healthy individuals and those with chronic periodontitis (then referred to as adult periodontitis), serotype b occurs almost twice as often in aggressive periodontitis (then referred to as juvenile periodontitis), serotype c appears less frequently, in equal proportions, and is primarily responsible for infections outside the oral cavity. The most common such infections are brain abscesses, osteitis and endocarditis (51).
Veillonella parvula is a strictly anaerobic, Gram-negative bacterium, resembling a granuloma (52). It is a natural part of the oral flora, but as a result of changes in pH in the oral cavity it can participate in periodontitis and tooth decay, as well as systemic infections, which include meningitis, osteomyelitis (53). Along with Gardanella it can participate in bacterial vaginosis in women. It can also be associated with arterial hypertension together with Campylobacter rectus and Prevotella melaninogenica (54). Veillonella parvula feeds on lactates provided by Streptococcus species, present in the oral cavity (55). Streptococcus mutans and Veillonella parvula can form multi-species biofilms, which lead to reduced susceptibility to antibacterial treatment in the oral cavity, which can lead to tooth decay and periodontitis (56). Bacteria of the genus Veillonella are sensitive to metronidazole and penicillin (57, 58). Active antibiotics against Veillonella parvula are cephalosporin, clindamycin and chloramphenicol (57).
Actinomyces odontolyticus was first described in the 20th century (59). In humans, these bacteria occur mainly on the mucous membranes of the oral cavity, gastrointestinal tract and female reproductive organs. After tissue disruption, bacteremia, sepsis and endocarditis often develop (60). Actinomyces odontolyticus can induce individual cases of infection leading to meningeal or brain abscesses (61-64).
Aim
The aim of the study was to test the antibacterial properties of pastes and gels from Indent – a leader in dental prophylaxis and distributor of companies such as Curasept, Tello, Frezyderm, which help accelerate the regeneration of gingival pockets and their healing, as well as prevent chronic inflammation in the oral cavity in the form of long infections or ulcerative aphthae. Protecting against caries and persistent periapical changes of periodontal tissues.
Material and methods
Microorganisms and media
Reference strains of probiotic bacteria as a control Bifidobacterium lactis HNO 19, Kluyveromyces marxianus fragilis B0399, Lactobactilius reuterii Prodentis, Lactobacillus salivarius SGL03 and pathogenic strains of green and purple complexes. Green Complex bacteria in pockets above 6 mm: Eikinella corrodens (ATCC 23834), Capnocytophaga gingivalis (ATCC 33624), Agregatibacter type a (ATCC 29522), Agregagibacter type b (ATCC 43718) and purple complex: Vellionella parvula (ATCC 17745), Actinomyces odontolyticus (ATCC 17929) were provided by (LGC Standards U.K.) and were used according to the recommendations of ISO 11133:2014. These strains were used to test the antibacterial activity with the analyzed compounds using the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as described in (21, 47).
MIC and MBC tests
The MIC was estimated by a microtiter plate method using sterile 48 or 96-well plates (47). First, precursor and TIL solutions were prepared sterile deionized water at 20 mg mL-1. Fifty microliters of the solutions was placed in the first row of the plate. Next, 25 μL of sterile TSB medium was added to the other wells, and serial dilutions were performed. Then, 200 μL of inoculated TSB medium containing resazurin (0.02 mgmL-1) as an indicator was added to all the wells. TSB medium was inoculated with 106 colony-forming units (CFU) mL-1 (approximately 0.5 McFarland units) of the bacterial strains. The plates were incubated at 30°C for 24 h. Color changes from blue to pink or yellowish with turbidity were taken as positive, and the lowest concentration at which there was no visible color change was the MIC. The MBC was estimated based on the measurement of the dehydrogenases activity in the cultures after a 24-h incubation without the ILs. Four millilitres of a dense culture (approximately 109 CFU mL-1) that was incubated for 24 h in TSB medium at 25°C was added to identical test tubes. Next, the tested compounds were added to the test tubes until the mixture reached final concentrations of 10-250 mg mL-1. Then, the cultures containing the TILs were incubated for 1 h at 30°C. Next, 0.1 g of CaCO3 and 0.1 mL of a 3% triphenyltetrazolium chloride (TTC) solution were added to the test tubes. Then, the test tubes were sealed with parafilm and incubated for 1 h 30°C in darkness.
MTT-assay
The cytotoxic effects of the tested peptidomimetics 1-16 on BALB/c3T3 mouse fibroblast cells, after 24 h of incubation at a concentration of 1 mmol/mL, were determined by MTT assay. The MTT test is based on the ability of the enzyme mitochondrial dehydrogenase to convert an orange, water-soluble tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into insoluble forma-zan, which is a dark blue product of the above reaction. After dissolving formazan crystals in DMSO or isopropanol, a coloured solution is formed, the intensity of which is measured spectrophotometrically in the wavelength range of 492-570 nm. The amount of coloured reduced MTT is proportional to the oxidative activity of the cell’s mitochondria, and under strictly defined experimental conditions to the number of metabolically active (living) cells in the population. The MTT test can also be used to determine cell viability in populations of cells that no longer divide but are metaboli-cally active. The MTT test is currently the most commonly used to assess cytotoxic activity and is recommended as a reference by international standards-setting organisations (65).
Statistical analysis
Fig. 2. Minimum inhibitory concentration (MIC) of the coumarin derivatives in model bacterial strains. The x-axis compounds 1-5 used sequentially. The y-axis shows the MIC value in μg/mL-1
All experimental data from at least three different trials (n = 3) are given as means ± standard error of the mean (SEM, manufacturer, Saint Louis, MO, USA). To compare pairs of means, the Tukey post hoc test was used, indicating statistical significance with * p < 0.05 ,** p < 0.1, and *** p < 0.01 (10).
Analysed compounds – characteristics
All counds were used with manufacturer recommendation
1. CURASEPT DAYCARE HERBAL INVASION – protective toothpaste with CPC-HAP 75 ml
Curasept Daycare Protection Booster toothpaste contains essential oils with innovative cetylpyridinium chloride and hydroxyapatite complex (CPC-HAP), whose synergistic action prevents the multiplication of microorganisms in the oral cavity for up to 4 hours after use. Contains a herbal flavor. It is SLS-free. Contains 900 ppm of fluoride. Fights caries and gum disease, halitosis, disinfects and remineralizes:
Active ingredients of CURASEPT DAYCARE HERBAL INVASION toothpaste:
1) Cetylpyridinium chloride – Has an antiseptic effect. 2) CPC-HA Complex (Cetylpyridinium Chloride + Hydroxyapatite) – Hydroxyapatite creates a protective layer that remineralizes while gradually releasing cetylpyridine, providing extended protection for up to 4 hours. 3) Essential oils (menthol, eucalyptol, thymol, methyl salicylate) – Prevent bacterial aggregation and slow down the growth of biofilm in the oral cavity. Penetrate the dental plaque and have a long-lasting effect at the supragingival and subgingival levels. They also help prevent gingivitis, help with daily mechanical plaque control and fight bad breath. Basic composition: Aqua, Sorbitol, Hydrated Silica, Xylitol, Propylene Glycol, Peg-32, Cellulose Gum, Cocamidopropyl Betaine, Hydroxyapatite, Calcium Glycerophosphate, Sodium Fluoride, Cetylopiyridinium Chloride, Calcium Phantothenate, 1,2-hexanediol, Potassium Nitrate, Cetraria Islandica Extract, Thymol, Elettaria Card amomum Seed Oil, Eucalyptol, Menthol, Methyl Salicilate, Stevia Rebaudiana Leaf/stem Extract, Sodium Saccharin, Aroma, Propanediol, Xanthan Gum, Phenoxyethanol, Ethylhexylglycerin, P-anisic Acid, Sodium Benzoate, Chlorphenesin, Tetrasodium Glutamate Diacetate, C.l. 19140, C.l. 42090.
2. CURASEPT ADS 100 – 1% chlorhexidine gum gel 30 ml
The gum gel contains 1% chlorhexidine digluconate, which is one of the best ingredients on the market in effectively combating daily plaque. It also contains Purified water, Propylene Glycol, Hydroxy Ethyl Cellulose, PVP/VA copolymer, PEG-40 hydrogenated castor oil, Chlorhexidine digluconate, Sodium acetate, Aroma, Acetic acid, Sodium metabisulfite, Ascorbic acid.
Using the gel protects the gums and interdental spaces, and also prevents infection of the oral cavity by pathogens, which may be bacteria, fungi or viruses. In addition, the gel has a healing effect on ulcers in the oral cavity, treats periodontal disease, and also combats inflammation caused by irritation of the dental prosthesis. The ADS 100 gel in a 30 ml package is distinguished by its highly concentrated 1% chlorhexidine which acts radically against bacteria and dental plaque. It acts locally in the dental pocket for up to 12 hours after application. And also in the local treatment of gums during dental treatment, for owners of dentures, orthodontic appliances. It is also intended for patients with periodontal disease, having dental implants and also having a tendency to excessive accumulation of dental plaque. It does not change the taste of consumed food and drinks. The CURASEPT A.D.S. function slows down and inhibits the formation of bacterial plaque and prevents irritation of the gums and oral cavity.
3. CURASEPT PREVENT – toothpaste 75 ml
CURASEP PREVENT is a toothpaste designed for prophylaxis and maintaining the health of gums and peri-implant tissues. Moreover, it helps maintain the effect of dental therapy in patients with chronic problems or during dental procedures. The synergistic selection of ingredients means that the paste helps maintain the ideal oral microflora. Additionally, Curasept Prevent paste hinders the multiplication of the most aggressive microorganisms, restores the balance of bacterial flora and improves the health of gum and periodontal tissues, while counteracting halitosis. CURASEPT PREVENT toothpaste is intended especially for: diabetics, smokers, people suffering from metabolic diseases, with dry mouth, with weakened immunity, undergoing chemotherapy and radiotherapy, during orthodontic and implant treatment, patients (with complex prosthetic rehabilitation) treated for periodontal problems in the maintenance phase. Contains Sorbitol, Aqua, Hydrated Silica, Xylitol, PEG-32, Sodium Lauroyl Sarcosinate, Sodium Cocoyl Glutamate, VP-VA Copolymer, Cellulose Gum, Phenoxyethanol, Sodium Saccharin, Sodium Citrate, Sodium Benzoate, Hexetidine, Ozonized Olive Oil, Melaleuca Alternifolia Leaf Oil, Stevia Rebaudiana Leaf/Steam Extract, Colostrum, Aroma, C.l. 19140, C.i. 42090.
a) Ozonated Oil – Gradually releases ozone and creates a favorable micro-environment for maintaining the balance of oral microflora, as well as improving the health of tissues and mucous membrane.
2) Colostrum – Supports regeneration and strengthens gums and oral mucosa, thanks to the content of enzymes and proteins with a defensive effect that interact with lactoferrin and lysozyme.
3) PVP/VA – Thanks to its ability to adhere to teeth and mucous membrane, it creates a protective layer, hindering the adhesion and proliferation of bacterial plaque and delays the formation of biofilm.
4) Tea Tree Oil – Contains a natural set of substances with antioxidant action. Effectively prevents the formation of biofilm in the oral cavity.
5) Stevia – Natural sweetener with anti-caries effect. Widely used to inhibit the formation of biofilm in the oral cavity.
4. CURASEPT PREVENT – oral gel 30 ml
CURASEP PREVENT is an oral gel indicated for the protection, prevention and maintenance of implants and control of risky situations related to periodontal disease. Thanks to its ability to adhere to teeth and mucous membrane, it creates a protective coating that inhibits the adhesion and proliferation of bacterial plaque and the formation of biofilm. Additionally, it improves the health of gum tissue, even in the case of comorbidities and balances the oral microflora.
Active ingredients of the gel:
a) Ozonated Oil – Gradually releases ozone and creates a favorable micro-environment for maintaining the balance of oral microflora, as well as improves the health of tissues and mucous membrane.
b) Colostrum – Supports regeneration and strengthens gums and oral mucosa, thanks to the content of enzymes and proteins with a defensive effect that interact with lactoferrin and lysozyme.
c) PVP/VA – Thanks to its ability to adhere to teeth and mucous membrane, it creates a protective layer, hindering the adhesion and proliferation of bacterial plaque and delays the formation of biofilm.
d) Tea tree oil – Contains a natural set of substances with antioxidant action. Effectively prevents the formation of biofilm in the oral cavity.
e) Stevia – A natural sweetener with anti-caries action. Widely used to inhibit the formation of biofilm in the oral cavity.
Restores the balance of bacterial flora and improves the health of gum tissues, also in the case of comorbidities. Ideal for patients with implants, smokers, diabetics, with dry mouth and patients with weakened immune resistance. Prevents halitosis.
5. CURASEPT ADS 350 – office set of gels for gums with chlorhexidine 0.5% – 5 ml
The gel for gums contains chlorhexidine digluconate in a concentration of 0.5%, which is one of the best ingredients on the market in the effective fight against daily plaque. Using the gel protects the gums and interdental spaces, and also prevents infection of the oral cavity by pathogens, which may be bacteria, fungi or viruses. Additionally, the gel has a healing effect on ulcers in the oral cavity, treats gingivitis, and also combats inflammation caused by irritation of the dental prosthesis. CURASEPT ADS 350 gel is intended for patients after complicated extraction, after maxillofacial surgery, with post-bite trauma and mucosal damage with ulcers and aphthous stomatitis, with mucosal infections.
Contains A.D.S.® System – ANTI DISCOLORATION SYSTEM
Composition of the gel: Aqua, Propylene Glycol, Xylitol, Hydroxyethyl Cellulose, Chlorhexidine Digluconate, Ascorbic Amide. Peg-40 Hydrogenated Castor Oil, Sodium Metabisulphite, Aroma, Methylparaben
Results
The effects of the 5 compounds investigated (illustrated in fig. 3-5) were evaluated within bacterial cells using a previously established methodology (21, 47). Following the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), the MIC values ranged between 0.5-1.5 μM, while the MBC values fell within the range of 1.5-2.5 (+/- 0.5) μM for analysed for four bacterials strains used as control and, Gram-negative strains and Staphylococcus, as depicted in figure 4. MIC serves as a reference for assessing the susceptibility or resistance of bacterial strains to the antibiotic applied in vitro. On the contrary, MBC represents the minimum concentration of an antibacterial agent required to eradicate bacteria, distinguishing it as bactericidal rather than bacteriostatic (34, 47). Basically, all compounds tested against Gram-positive and Gram-negative bacterial strains show good activity against them. And all MIC values are below 1.5 μg/mL-2 (fig. 2). Interestingly, the change in the different compounds had a vital impact on activity. However, the fluorine compounds and other active ingredients was playing a role in the activity against some bacteria, which we confirmed with the activity of compound no. 1 and 5. This means that the compounds had a good potency compared to the analysed antibiotics (fig. 6). Interestingly, all five compounds showing broad spectrum activity against both Gram-negative as well as Gram-positive bacteria. The steric hindrance created by the bulky fluorine atom and other active ingredients might prevent proper binding and interaction with the active site residues, thereby reducing the compound’s overall efficacy. The analyzed probiotic strains were resistant to the action of pastes and gels, while the pathogenic strains were destroyed by strong cytotoxic properties damaging both Gram-negative and Gram-positive pathogens. This indicates their selective action on red and yellow complex bacteria.
Fig. 3. Minimum bactericidal concentration (MBC) of coumarin derivatives in model bacterial strains. The x-axis features compounds 1-5 used sequentially. The y-axis shows the MBC value in μg/mL-1
Fig. 4. The ratio of MBC/MIC of the investigated agents 1-16 in selected bacterial strains. The x-axis compounds 1-5. The y-axis shows the MBC/MIC value in μM shows the MIC value in μM
Fig. 5. Examples of MIC in selected bacterial strains for studied antibiotics ciprofloxacin (cipro), bleomycin (bleo), and cloxacillin (clox). The x-axis features antibiotics used sequentially. The y-axis shows the MIC value in μM
Fig. 6. Measurement of cell viability (%) (y-axis) on human dental pulp stem cells (hDPSC) when exposed to analysed compounds 1 – during 24 hours incubation. The x-axis features compounds 1-5
Antimicrobial agents are often classified as either bacteriostatic or bactericidal. A medicine is deemed bactericidal if the MBC-MIC ratio is low (less than 1.5), and it is feasible to achieve drug concentrations that eradicate 99.9% of the exposed organisms. Should the proportion of antibiotic be classified as bacteriostatic; nevertheless, the MBC to MIC ratio is high and it may not be safe to take doses of the medication to kill 99.9% of bacteria. The exact distinction between the bacteriostatic and bactericidal properties of many substances depends on the concentration of the pathogen and the drug that is reached in the target tissue. In the case of all tested compounds 1-5, we are dealing with bactericidal agents (fig. 4).
Furthermore, as can be seen in figures 3 and 5, investigated compounds typically exhibited greater antibacterial activity than commonly used antibiotics. This is especially crucial because tested microorganism resistance to known antibiotics has evidently increased, such as cloxacillin (clox) or ciprofloxacin (cipro) (fig. 6). The rate at which microorganisms will become resistant to bleomycin (bleo) is unknown, but this will cause a major disruption in the antibiotic arsenal that is now used to treat hospital infections. Interestingly, our compounds exhibit activity comparable to that of bleomycin (bleo).
Model of analysed strains with tested compounds were statistical significance at p < 0.05 (tab. 1).
Tab. 1. Statistical analysis of all analyzed compounds by MIC, MBC, and MBC/MIC; < 0.05*, < 0.01**, < 0.001***
| No. of Samples | 1, 2 | 3 | 4-5 | Type of test |
| Bifidobacterium lactis HNO 19 | * | ** | ** | MIC |
| Kluyveromyces marxianus fragilis B0399 | * | ** | ** | MIC |
| Lactobactilius reuterii Prodentis | * | ** | ** | MIC |
| Lactobacillus salivarius SGL03 | * | ** | ** | MIC |
| Eikinella corrodens (ATCC 23834) | * | * | * | MBC |
| Capnocytophaga gingivalis (ATCC 33624) | * | * | * | MBC |
| Agregatibacter type a (ATCC 29522) | * | * | * | MBC |
| Agregagibacter type b (ATCC 43718) | * | * | * | MBC |
| Vellionella parvula (ATCC 17745) | * | * | * | MIC |
| Actinomyces odontolyticus (ATCC 17929) | * | – | | MIC |
The human dental pulp stem cells (hDPSC) (as good model for cell observed and obtained in oral cavity) were treated with tested compounds with concentrations ranging from 1 to 3.5 μg/mL and were incubated for 24 hours. All tested compounds were not cytotoxic in analysed cells at the lowest concentrations tested of 1 μg/mL, the viability percentages remains above 99.50%. However, gradual reduction in viability was caused by tested 5 agents at 2 μg/mL ranging the cell viability percentages from 87.00% for compound 5 and 66.50% respectively. The obtained results were used to calculate the half-maximal inhibitory concentration (IC50) after 24 hours of incubation with selected most active antimicrobial used oral cavity compounds 1-5. The IC50 value for hDPSC after 24 h of incubation ranging from 3.22 μg/mL for compound 1 up to 3.26 μg/mL for all compounds respectively.
Discussion
Cytotoxic studies of tested compounds
The effects of the 5 compounds investigated (illustrated in fig. 3-5) were evaluated within bacterial cells using a previously established methodology (21, 47). Following the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), the MIC values ranged between 0.25-4.5 μM, while the MBC values fell within the range of 1-8 (+/- 0.5) μM for bacterial strains of green and purple complexes; Eikinella corrodens (ATCC 23834), Capnocytophaga gingivalis (ATCC 33624), Agregatibacter type a (ATCC 29522), Agregagibacter type b (ATCC 43718) and purple complex: Vellionella parvula (ATCC 17745), Actinomyces odontolyticus (ATCC 17929) Bifidobacterium lactis HNO 19, Kluyveromyces marxianus fragilis B0399, Lactobactilius reuterii Prodentis, Lactobacillus salivarius SGL03.
MIC serves as a reference for assessing the susceptibility or resistance of bacterial strains to the antibiotic applied in vitro. On the contrary, MBC represents the minimum concentration of an antibacterial agent required to eradicate bacteria, distinguishing it as bactericidal rather than bacteriostatic (47). Basically, all our compounds tested against Gram-positive and Gram-negative bacterial strains show good activity against them. And all MIC values are below 5 μg/mL-1 (fig. 3). Interestingly, the change in the different toothpaste and gels had a vital impact on activity. The oral hygiene products containing active ingredients in their composition was playing a role in the activity against some bacteria, which we confirmed with the activity of compound no. 1 and 5. This means that the active comonents had a good potency compared to antibiotics. Interestingly, all compounds showing broad spectrum activity against both Gram-negative as well as Gram-positive bacteria but the highest the compound no 3 and 4. Compounds 1 to 5 have greater efficacy against: Eikinella corrodens (ATCC 23834), Capnocytophaga gingivalis (ATCC 33624), Agregatibacter type a (ATCC 29522), Agregagibacter type b (ATCC 43718) and purple complex: Vellionella parvula (ATCC 17745), Actinomyces odontolyticus (ATCC 17929) strains. Compounds No. 3 and 4 demonstrated a broad range and good action against all bacteria, as revealed confi-dentially with respect to the fluorine group. Furthermore, compounds with a para-fluoro group in had more activity overall. The obtained results were a good and very simple and cheap training test which allowed us to estimate to what extent the types of specific diets have an influence on the formation of specific bacterial biofilms on the induction of inflammatory conditions of periodontal disease with specific bacterial biofilms. The methods described in the manuscript significantly shorten the time of detection of disease entities induced by persistent bacterial biofilms belonging to different classes. They also allow the assessment of the actual inflammation of the periodontal tissues. By specifying the type of identifier, the doctor can directly estimate what type of bacteria he is dealing with and what treatment should be administered. The obtained results suggest that the substances contained in the analysed components may also interact with red complexes of bacteria showing cariogenic activity present in the human oral cavity (11, 12). Currently, antibiotic resistance among pathogenic bacteria is becoming more and more common, leading to super-resistance. The lack of colonization capabilities by such pathogens is the result of the lack of access to adhesins occupied by the microflora of the inhabited niche. Moreover, the sessile microflora can produce certain antagonistic compounds that interfere with the colonization of the niche by exogenous bacterial strains, e.g. hydrogen peroxide produced by S. mitis, salivaricin produced by S. salivarius streptococci (which inhibits the development of pyogenic streptococci S. pyogenes) or enocin (bacteriocin) produced by S. mutans, C. martuchotii and A. actinomycetemcomitans (which inhibits the growth of related organisms) of yellow and red complexes (36).
Conclusions
The observed results are especially important because of increasing resistance of bacteria to various drugs and antibiotics. All selected compounds showed super-selectivity in all analyzed bacterial strains and exhibited the highest cytotoxic activity, comparable or better than the commonly used antibiotics: ciprofloxacin, bleomycin, and cloxacillin.The bacterial flora of the oral cavity contributes to the development or aggravation of oxidative stress. As mentioned above, bacteria inhabiting the crowns of teeth breathe oxygen (Eh +200 mV) and produce carbon dioxide, which results in a decrease in the oxidative reduction potential to about +76 mV. As the bacterial plaque grows, this potential can decrease to as much as -140 mV. In the gingival crevices and pockets – in the subgingival dental plaque, the oxidative reduction potential fluctuates in the range of Eh +72 mV to -300 mV, which leads to the creation of an anaerobic environment. People with high levels of oxidative stress suffer from periodontal diseases twice as often and vice versa – periodontal diseases closely correlate with an increase in the level of oxidative stress parameters (22-26). The analyzed probiotic strains were resistant to the action of pastes and gels, while the pathogenic strains were destroyed by strong cytotoxic properties damaging both Gram-negative and Gram-positive pathogens. This indicates their selective action on red and yellow complex bacteria. The analyzed ingredients of pastes and gels act selectively on bacterial pathogens without affecting human dental pulp stem cells (hDPSC), thanks to which the human pulp is protected by the compounds used, which is an invaluable value in maintaining healthy enamel and teeth during daily use in oral hygiene.
Piśmiennictwo
1. Pietruska M, Dolińska E, Skurska-Czelej A: Zapobieganie chorobom przyzębia i ich leczenie. Wyd. I. Wydawnictwo Czelej, Lublin 2018.
2. Harsman P (red. wyd. pol. U. Kaczmarek): Master Dentistry Stomatologia zachowawcza, stomatologia dziecięca, ortodoncja, periodontologia, protetyka. Wyd. I. Elsevier Urban & Partner, Wrocław 2010.
3. Coulthard P, Horner K, Sloan P, Theaker E: Choroby błony śluzowej jamy ustnej, radiologia, chirurgia stomatologiczna. Elsevier Urban & Partner, Wrocław 2008.
4. Górska R (red.): Diagnostyka i leczenie chorób błony śluzowej jamy ustnej. Med Tur Press International, Otwock 2011.
5. Jańczuk Z (red.): Praktyczna periodontologia kliniczna. Wydawnictwo Kwintesencja, Warszawa 2004.
6. Jańczuk Z (red.): Profilaktyka profesjonalna w stomatologii. Wydawnictwo Lekarskie PZWL, Warszawa 2001.
7. Felton A, Chapman A, Felton S (red. nauk. tłum. U. Kaczmarek): Zdrowie jamy ustnej. Wydawnictwo Lekarskie PZWL, Warszawa 2009.
8. Jańczuk Z (red.): Podręcznik dla asystentek i higienistek stomatologicznych. Wydawnictwo PZWL, Warszawa 2011.
9. Mielczarek A, Kowalik R, Najman M (red.) Podręcznik dla asystentek i higienistek stomatologicznych. Wydawnictwo PZWL, Warszawa 2018.
10. Krajewski W: Podstawy profilaktyki stomatologicznej. Med Tour Press International, Otwock 2019.
11. Lewis MAO, Jordan RCK: Medycyna jamy ustnej. Wydawnictwo Lekarskie PZWL, Warszawa 2013.
12. Borakowska-Siennicka M: Wpływ diety na stan tkanek przyzębia – na podstawie piśmiennictwa. Nowa Stomatol 2012; 17(3): 130-133.
13. Górska R: Choroby przyzębia. Klasyfikacja 2017. Wydawnictwo PZWL, Warszawa 2018.
14. Konopka T, Gerber H: Stan kliniczny przyzębia pacjentów badanych w kierunku stresu oksydacyjnego związanego z zapaleniem przyzębia. Dent Med Probl 2008; 45(1): 42-49.
15. Rutkowski R, Pancewicz SA, Rutkowski K, Rutkowska J: Znaczenie reaktywnych form tlenu i azotu w patomechanizmie procesu zapalnego. Pol Merkuriusz Lek 2007; 23(134): 131-136.
16. Kamecka-Białowarczuk EA, Dąbrowska E: Równowaga oksydoredukcyjna w środowisku jamy ustnej. e-Dentico 2009; 2: 58-68.
17. Kamecka-Białowarczuk EA, Dąbrowska E: Równowaga oksydoredukcyjna w środowisku jamy ustnej. e-Dentico 2008; 2: 42-51.
18. Łuszczewski A, Matyska-Piekarska E, Trefler J et al.: Reaktywne formy tlenu – znaczenie w fizjologii i stanach patologii organizmu. Reumatologia 2007; 45(5): 284-289.
19. Olędzki R, Kędziora-Kornatowska K: Mechanizmy antyoksydacyjne w organizmie człowieka. Valetudinaria – Post Med Klin Wojsk 2006; 11(1): 15-20.
20. Król K, Grocholewicz K: Wybrane białka śliny jako biomarkery miejscowych i ogólnych procesów chorobowych. Przegląd piśmiennictwa. Zakład Stomatologii Ogólnej Pomorskiej Akademii w Szczecinie 2007; 53(1): 78-82.
21. Kucia M, Wietrak E, Szymczak M, Kowalczyk P: Preliminary in vitro study on effect of Lactobacillus salivarius and other natural components against anaerobic periodontal bacteria. Molecules 2020; 25: 4519.
22. Caton JG, Armitage G, Berglundh T et al.: New classification scheme for periodontal and peri-implant diseases and conditions – introduction and key changes from the 1999 classification. J Clin Periodontol 2018; 45(suppl. S20): S1-S8.
23. Chen Y, Yang YC, Zhu BL et al.: Association between periodontal disease, tooth loss and liver diseases risk. J Clin Periodontol 2020; 47(9): 1053-1063.
24. Carrizales-Sepúlveda EF, Ordaz-Farías A, Vera-Pineda R, Flores-Ramírez R: Periodontal Disease, Systemic Inflammation and the Risk of Cardiovascular Disease. Heart Lung Circ 2018; 27(11): 1327-1334.
25. Sroussi HY, Epstein JB, Bensadoun RJ et al.: Common oral complications of head and neck cancer radiation therapy: mucositis, infections, saliva change, fibrosis, sensory dysfunctions, dental caries, periodontal disease, and osteoradionecrosis. Cancer Med 2017; 6(12): 2918-2931.
26. Güll FD, Deppe H, Kesting M, Schwarzer C: Periodontal disease-like bone loss after adjuvant radiotherapy in the head and neck region: A case report and review of the literature. Quintessence Int 2017; 48(6): 451-457.
27. Rowińska I, Szyperska-Ślaska A, Zariczny P et al.: Impact of the Diet on the Formation of Oxidative Stress and Inflammation Induced by Bacterial Biofilm in the Oral Cavity. Materials 2021; 14: 1372.
28. Rowińska I, Szyperska-Ślaska A, Zariczny P et al.: The Influence of Diet on Oxidative Stress and Inflammation Induced by Bacterial Biofilms in the Human Oral Cavity. Materials 2021; 14: 1444.
29. Smith AJ, Hall V, Thakker B, Gemmell CG: Antimicrobial susceptibility testing of Actinomyces species with 12 antimicrobial agents. J Antimicrob Chemother 2005; 56: 407-409.
30. Hansen JM, Fjeldsoe-Nielsen H, Sulim A et al.: Actinomyces species: A Danish survey on human infections and microbial characteristics. Open Microbiol J 2009; 3: 113.
31. Brook I: Actinomycosis: diagnosis and management. South Med J 2008; 101: 1019-1023.
32. Hall V: Actinomyces gathering evidence of human colonization and infection. Anaerobe 2008; 14: 1-7.
33. Binda C, Lopetuso LR, Rizzatti G et al.: Actinobacteria: A relevant minority for the maintenance of gut homeostasis. Dig Liver Dis 2018; 50(5): 421-428.
34. Socransky SS, Haffajee AD, Cugini MA et al.: Microbial complexes in sub-gingival plaque. J Clin Periodontol 1998; 25: 134-144.
35. Chaves BD, Brashears MM, Nightingale KK: Applications and safety considerations of Lactobacillus salivarius as a probiotic in animal and human health. J Appl Microbiol 2017; 123: 18-28.
36. Darveau RP, Hajishengallis G, Curtis MA: Porphyromonas gingivalis as a potential community activist for disease. J Dent Res 2012; 91: 816-820.
37. Jayaram P, Chatterjee A, Raghunathan V: Probiotics in the treatment of periodontal disease: A systematic review. J Indian Soc Periodontol 2016; 20: 488-495.
38. Kadowaki T, Baba A, Abe N et al.: Suppression of pathogenicity of Porphyromonas gingivalis by newly developed gingipain inhibitors. Mol Pharmacol 2004; 66: 1599-1606.
39. Haffajee AD, Socransky SS, Patel MR, Song X: Microbial complexes in suprag-ingival plaque. Oral Microbiol Immunol 2008; 23: 196-205.
40. Nędzi-Góra M, Kowalski J, Krajewski J, Górska R: Microbiological analysis of deep periodontal pockets in people with chronic periodontitis by PCR. Stomatol J 2007; 11: 717-725.
41. Paik S, Senty L, Das S et al.: Identification of Virulence Determinants for Endocarditis in Streptococcus sanguinis by Signature-Tagged Mutagenesis. Infect Immun 2005; 73: 6064-6074.
42. Servin AL: Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev 2004; 28: 405-440.
43. Sela MN: Role of Treponema denticola in periodontal diseases. Crit. Rev. Oral Biol. Med. 2001, 12, 399–413.
44. Cutler CW, Kalmar JR, Genco CA: Pathogenic strategies of the oral anaerobe Porhyromonas gingivalis. Trends Microbiol 1995; 3: 45-51.
45. Neville BA, O’Toole PW: Probiotic properties of Lactobacillus salivarius and closely related Lactobacillus species. Future Microbiol 2010; 5: 759-774.
46. Pidutti P, Federici F, Brandi J et al.: Purification and characterization of ribosomal proteins L27 and L30 having antimicrobial activity produced by the Lactobacillus salivarius SGL 03. J Appl Microbiol 2018; 124: 398-407.
47. Kowalczyk P, Koszelewski D, Brodzka A et al.: Evaluation of Antibacterial Activity against Nosocomial Pathogens of an Enzymatically Derived α-Aminophosphonates Possessing Coumarin Scaffold. Int J Mol Sci 2023; 24: 14886.
48. Eiken M: Studies on an anaerobic, rod-shaped, Gram-negative microorganism: Bacteroides corrodens N. sp. Acta Pathol Microbiol Scand 1958; 43: 404-416.
49. Nędzi-Góra M, Kowalski J, Górska R: The Immune Response in Periodontal Tissues. Arch Immunol Ther Exp 2017; 65: 421-429.
50. Norskov-Lauritsen N, Kilian M: Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilussegnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evol Microbiol 2006; 56: 2135-2146.
51. Perry MB, Maclean LM, Brisson JR, Wilson ME: Structures of the Antigenic O-Poly-saccharides of Lipopolysaccharides Produced by Actinobacillus actinomycetemcomitans Serotypes a, c, d and e. FEBS J 1996; 242: 682-688.
52. Potts TV, Zambon JJ, Genco RJ: Reassignment of Actinobacillus actinomycetemcomitans to the genus Haemophilus as Haemophilus actinomycetemcomitans. Int J Syst Bacteriol 1985; 35: 337-341.
53. Rahamat-Langendoen JC, van Vonderen MG, Engstrom LJ et al.: Brain abscess associated with Aggregatibacter actinomycetemcomitans: case report and review of literature. J Clin Periodontol 2011; 38: 702-706.
54. Matera G, Muto V, Vinci M et al.: Receptor recognition of and immune intracellular pathways for Veillonella parvula lipopolysaccharide. Clinical and Vaccine Immunology 2009; 16(12): 1804-1809.
55. Bongaerts GP, Schreurs BW, Lunel FV et al.: Was isolation of Veillonella from spinal osteomyelitis possible due to poor tissue perfusion? Medical Hypotheses 2004; 63(4): 659-661.
56. Pietropaoli D, Del Pinto R, Ferri C et al.: Definition of hypertension-associated oral pathogens in NHANES. Journal of Periodontology 2019; 90(8): 866-876.
57. Megrian D, Taib N, Witwinowski J et al.: One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide. Molecular Microbiology 2020; 113(3): 659-671.
58. Luppens SB, Kara D, Bandounas L et al.: Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm. Oral Microbiology and Immunology 2008; 23(3): 183-189.
59. Al-Otaibi FE, Al-Mohizea MM: Non-vertebral Veillonella species septicemia and osteomyelitis in a patient with diabetes: a case report and review of the literature. Journal of Medical Case Reports 2014; 8(1): 365.
60. Könönen E, Wade WG: Actinomyces and related organisms in human infections. Clin Microbiol Rev 2015; 28: 419-442.
61. Rueda MS, Hefter Y, Stone B et al.: A premature infant with neonatal Actinomyces odontolyticus sepsis. J Pediatric Infect Dis Soc 2021; 10: 533-535.
62. Huang Q, Hong Z, Hong Q: Cryptococcal meningoencephalitis with Actinomyces odontolyticus sepsis: a case report and literature review. BMC Infect Dis 2023; 23: 434.
63. Jain H, Singh G, Eranki A: Actinomyces odontolyticus causing meningitis and cervical abscess. Proc (Bayl Univ Med Cent) 2021; 34: 492-493.
64. Yesilbas O, Yozgat CY, Nizam OG et al.: Life-threatening multiple brain abscesses secondary to Actinomyces odontolyticus. Pediatr Int 2020; 62: 1307-1308.
65. Milovanovic V, Minic R, Vakic J et al.: MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation. Toxicon 2021; 192: 57-65.
