Introduction
Gingivitis considers a broad term that denotes gingivae inflammation . This condition represents widespread across the globe. The majority of gingivitis cases are associated with plaque, although several forms that are not related to plaque are also acknowledged. Plaque-related gingivitis generally arises when inadequate hygiene leads to the buildup of dental calculus and plaque . According to Neville (2019), it is characterized by inflammation of the gingivae as a response to a mature dental plaque biofilm. In individuals who are susceptible, ongoing gingivitis can progress to chronic periodontitis (Kistler et al., 2013).
Moreover, the clinical manifestation of plaque-related gingivitis can be influenced by various systemic factors, including a) Sex steroid hormones (e.g., puberty, menstrual cycle, pregnancy, oral contraceptives), b) Hyperglycemia, c) Leukemia, d) Smoking, e) Malnutrition, and f) Medications (e.g., phenytoin, calcium channel blockers, cyclosporine) as noted by Murakami(2018). Additionally, local agents may exacerbate gingivitis, encompassing trauma, tooth fractures, dental crowding, dental prostheses/appliances, defective dental restorations, and mouth breathing. Research also indicates that the host response is a significant factor in the etiopathogenesis of gingivitis (Neville et al., 2019).
Gingivitis has been observed the spread of this condition was higher in men than in females, as research indicates that females generally adhere to superior oral care practices, thereby enhancing their oral hygiene. This condition is frequently observed in both children and adults. According to Kashetty M., et al. (2018), younger children exhibit lower levels of plaque compared to adults and seem to be less sensitive to equivalent amounts of plaque. This phenomenon can be attributed to variations in the composition bacteria of plaque as well as changes in the inflammation response. By the age of 4 or 5 years, gingivitis affects approximately half of the population, with its incidence continuing to rise with age. The prevalence of gingivitis reaches nearly 100% during the puberty, although it experiences a slight decline post-puberty, remaining stable into the adulthood. Some children may infected with severe gingivitis during puberty. The gingivitis associated with puberty is linked to elevated levels of steroid hormones, as noted by Stenberg et al. (2019). An increase in gingivitis was noted during the 2nd and 3rd trimesters of pregnancy compared to the 1st trimester and control groups. However, plaque scores did not reveal any significant differences between non-pregnant and pregnant women. Specific bacterial analyses indicated a higher proportion of P. intermedia in pregnant women during both the second and third trimesters compared to those in the first trimester and non-pregnant women (Emmatty et al., 2013). Gingival inflammation can manifest as either acute ,or chronic and may be localized , or generalized. In certain instances, the inflammation restricted to the gingival margin or the interdental papillae . Clinical manifestations of gingivitis include swelling, erythema,and bleeding , a consistency ranging from boggy to firm, and stippling loss, blunting of the papillae interdental (Neville et al., 2019).
The initial stages of gingivitis in a healthy individual are characterized by a relatively straightforward bacterial community, primarily consisting of Gram-positive cocci and rods. As the condition of gingivitis progresses, these communities become more intricate, exhibiting an increased presence of Gram-negative rods, filaments, spirilla, , fusiforms, spirochete)Kistler, 2013)
Treatment of gingivitis consists of instituting a daily homecare regimen by the owner – ideally, toothbrushing, removing dental deposits (plaque and calculus) Gorrel,2013)
The mechanical elimination of plaque can be enhanced through the application of various chemicals agents, including mouth rinse that contain chlorhexidine and essential oil, as well as dentifrices formulated with triclosan and )2.0%) Gantrez copolymer. In context, research has investigated the incorporation of these chemopreventive agents into standard oral hygiene practices, demonstrating a statistically significant positive effect of these products on the management of plaque and gingivitis (Alawad, 2018).
This study aimed to isolate and diagnose of gingivitis-causing bacteria. Determining the efficiency of mouthwash solutions in their ability to eliminate the bacteria that cause inflammation.
Material and Methods
Specimens collection
It was collected 30 samples from the specialized dental center in Ramadi as well as the clinics in Ramadi, and swabs were taken from the inflamed gums, and the samples were placed in a tube containing 3 ml of nutrient broth medium. After collecting the samples, the samples cultivated at 37 ° C / 24 hours, The research was conducted in the laboratories of the Department of Medical Laboratory Techniques in Almaaref university college
Cultivation of samples
We make sure that the samples are not exposed to contamination after making sure we grow the samples on Petri dishes containing nutrient agar medium using a swap, and then we put the Petri dish at 37 ° C / 24 hours
Samples were taken and cultivated on Petri dishes containing MacConkey agar medium and Blood agar medium to distinguish between negative and positive bacteria for their identification.
Identification of samples
Cultural characteristics
After growing the samples on the medium after cultivated for 24 hours / 37 ° C, the characteristics of the bacterial colonies were observed (color, shape, size).
Biochemical examination
Biochemical tests were conducted, which included: Citrate utilization, Iodole test, VP (Voges Proskauer) ,MR (Methyl red)
Device Vitek 2 compact
The apparatus was utilized to verify the identification of bacterial isolates following the execution of biochemical tests on them. The apparatus is composed of a cassette holder and Reagent Cards that feature 64 holes, with each hole symbolizing the base material or medium designated for testing. Additionally, it includes plastic tubes, a DensiChek device, and both input and output units.
Determine the efficiency of mouthwash solutions
Mueller Hinton agar was prepared in dishes and incubated at 37 ° C / 24 hours. The next day, after making sure that there was no contamination in the dishes, samples were cultivated on dishes.
Three types of mouthwash solutions were taken and each type was placed in a beaker. The beaker was numbered for each type of mouthwash, after that we took the filter paper and punched it with perforations. We took the perforated parts in a circular shape and put each group in each type of mouthwash after waiting for half an hour. We took these papers and put each type. mouthwash on one side of the plate on which the sample was grown and our number on the back of the plate is the number of the mouthwash solution. After completion, we put the dishes in the incubator at a temperature of 37 ° C for 24 hours, the next day we learned the extent of the effect of the mouthwash solutions.
Results and Discussion
Sample collection
In the figure (1) presented below, bacterial growth is observed on the nutrient broth medium. The presence of this bacterial growth suggests the occurrence of gingivitis, which can arise from various factors. Primarily, it is caused by the accumulation of plaque, with poor dental and oral hygiene being significant contributors to plaque formation and propagation. Gingivitis is an early and reversible condition. In this ailment, the gums become red, swollen, and prone to bleeding, which may occur during activities such as touching, brushing, or even spontaneously. Additionally, there are several risk factors associated with gingivitis, including inadequate care practices, tobacco use, advancing age, dry mouth, nutrition (deficiency of vit C ), improperly fitting dental restorations, or misaligned teeth that are challenging to clean. Other contributing factors include conditions that impair immunity. Hormonal fluctuation related to menstrual cycle,pregnancy , use contraceptive pills, as well as genetic predispositions and specific medical conditions like certain viral and fungal infections, also play a role(Singh et al.,2013).
Figure 1: Bacteria cultivated in nutrient broth.
Subculture of samples
After the samples were collected, the samples were activated on nutrient agar medium The figures (2) illustrate the activation of samples.
Figure 2: the samples activation on nutrient agar
Following the activation of the samples on nutrient agar medium, they were subsequently cultivated on MacConkey agar medium and Blood agar medium to distinguish between positive and negative samples (table 1). This process also facilitated diagnosis using the VITEK device, as the bacteria that are positive for the test absorb the crystal violet stain, resulting in a purple appearance when observed under an optical microscope. This phenomenon occurs because the thick peptidoglycan layer in the bacterial cell wall retains the stain even after it has been washed away during the decolorization phase of the test. Conversely, bacteria that are negative for the test fail to retain the violet stain post-decolorization; the alcohol used in this stage disrupts the outer membrane of gram-negative cells, rendering the cell wall more porous and incapable of holding onto the crystal violet stain. Their peptidoglycan layer is considerably thinner and is situated between an inner cell membrane and an outer bacterial membrane, which leads them to absorb the counterstain (safranin or fuchsine) and appear red or pink. The distinctions between the negative and positive bacteria are illustrated in figures (3) and (4) below.
Figure 3:Gram-positive bacteria because it grew on blood agar and did not grow on MaCconky agar.
Figure 4: Gram-negative bacteria on MacConkey agar.
Table 1: Bacteria on MacConkey and blood agar
| Samples number | MacConkey agar | Blood agar |
| 1 | – | + |
| 2 | – | + |
| 3 | – | + |
| 4 | – | + |
| 5 | + | + |
| 6 | – | + |
| 7 | + | + |
| 8 | + | + |
| 9 | + | + |
| 10 | + | + |
| 11 | + | + |
| 12 | + | + |
| 13 | – | + |
| 14 | – | + |
| 15 | + | + |
| 16 | – | + |
| 17 | – | + |
| 18 | – | + |
| 19 | + | + |
| 20 | + | + |
| 21 | – | + |
| 22 | + | + |
| 23 | – | + |
| 24 | – | + |
| 25 | – | + |
| 26 | + | + |
| 27 | – | + |
| 28 | – | + |
| 29 | – | + |
| 30 | – | + |
Biochemical test
Table 2: biochemical test of bacterial
| Number of sample |
Indole |
citrate |
Methyl red |
Voges proskauer |
Gram stain |
| 1 | – | – | + | + | + |
| 2 | + | – | – | + | + |
| 3 | + | – | + | + | + |
| 4 | – | + | – | + | + |
| 5 | + | – | + | – | – |
| 6 | + | + | – | – | + |
| 7 | – | + | – | + | – |
| 8 | – | + | – | – | – |
| 9 | + | + | – | – | – |
| 10 | – | + | + | + | – |
| 11 | – | + | – | – | – |
| 12 | + | – | + | – | – |
| 13 | + | – | – | + | + |
| 14 | – | + | – | + | + |
| 15 | – | + | – | – | – |
| 16 | + | – | – | + | + |
| 17 | – | – | + | + | + |
| 18 | + | + | – | – | + |
| 19 | + | – | + | – | – |
| 20 | – | + | – | + | – |
| 21 | – | – | + | + | + |
| 22 | – | + | – | + | – |
| 23 | + | + | – | – | + |
| 24 | – | + | – | + | + |
| 25 | – | – | + | + | + |
| 26 | – | + | – | + | – |
| 27 | + | – | – | + | + |
| 28 | + | + | – | – | + |
| 29 | – | – | + | + | + |
| 30 | + | – | + | + | + |
( + ) positive , ( – ) negative
The biochemical tests are presented in Table 2 above. The findings from the bacterial isolates indicated that some were positive while others were negative. The isolates that tested positive in the citrate test exhibited the capability of certain bacteria to use citrate as their only carbon source, utilizing the enzyme citrase or citrate-permease to aid in the transport of citrate into the cell. These organisms also transform ammonium dihydrogen phosphate into ammonia and ammonium hydroxide, leading to an alkaline environment within the medium. At a pH of 7.5 or above, bromothymol blue displays a royal blue color. Conversely, at neutral pH, bromothymol blue appears green, signifying a negative result due to the organism’s inability to use citrate as the sole carbon source. A blue coloration in the medium indicates that the bacteria are citrate positive, while a lack of color change signifies a negative citrate result, as demonstrated in Figure 5. The indole test produces a positive result due to the bacteria’s ability to convert tryptophan into indole. This conversion is facilitated by a series of various intracellular enzymes, collectively referred to as tryptophanase. On the other hand, a negative indole test occurs when bacteria fail to convert tryptophan into indole. A positive indole result is marked by the development of a pink to red color, commonly known as a ‘cherry-red ring,’ whereas a negative result shows no formation of a red ring, as illustrated in Figure 6. The methyl red test is positive when bacteria can utilize glucose and convert it into stable acids such as lactic acid, acetic acid, or formic acid as end products. A negative methyl red test indicates the bacteria’s inability to carry out this conversion. The positive result is indicated by a color change of methyl red from yellow to red, while a negative result shows no color change, as depicted in Figure 7. The Voges-Proskauer test is positive when acetylmethyl carbinol is generated from glucose fermentation. If present, acetylmethyl carbinol is converted to diacetyl in the presence of α-naphthal.
(1) (2)
Figure 5: (1) positive growth bacteria for citrate test , (2) negative growth bacteria for citrate test
(1) (2)
Figure 6: (1) positive growth bacteria for indole test , (2)negative growth bacteria for indole test(2)
Figure 7: (1) positive growth bacteria for methyl red test , (2) negative growth bacteria for methyl red test
(1) (2)
Figure 8: (1) positive growth bacteria for Voges Proskauer test (2) negative growth bacteria for Voges Proskauer test
Vitek 2 compact
After identification the isolates on biochemical tests, the Vitek device was used to identification bacterial isolates, in order to identification very accurately the isolates. The table(3) and reports of appendices below shows the isolates that were identification on a device Vitek 2
Table 3: Results of bacterial isolates
|
Isolate of bacteria |
Repetition |
|
Staphylococcus aureus |
5 |
|
Staphylococcus epidermidis |
4 |
|
Staphylococcus pseudintermedius |
2 |
|
Staphylococcus lentus |
3 |
|
Escherichia coli |
3 |
|
Enterococcus casseliflavus |
4 |
|
Klebsiella pneumonia |
4 |
|
Rhizobium radiobacter |
3 |
|
Sphingomonas paucimobilis |
1 |
|
Enterobacter hormaechei |
1 |
The effect of mouthwash
The table (4) presented below illustrates the impact of mouthwash solutions on the bacteria responsible for gingivitis. The findings indicate that certain bacteria exhibit resistance while others are susceptible to these mouthwash solutions. The resistance observed in some bacteria arises from their ability to develop resistance mechanisms, guided by the genetic instructions encoded in their DNA. Typically, resistance genes are located within plasmids, which are small segments of DNA that facilitate the transfer of genetic information between different microorganisms. Consequently, this allows some bacteria to share their DNA, leading to the development of resistance in other germs. Furthermore, it is important to note that bacteria do not recognize antibiotics and do not evolve from resistance mechanisms, as depicted in figure (9).
Table 4: resistant and sensitive bacteria for mouthwash solutions
|
Isolate of bacteria |
Mouthwash zac |
Mouthwash alpha zac |
Mouthwash Listerine |
| Staphylococcus aureus |
S (2m) |
R |
R |
| Staphylococcus epidermidis |
S (1m) |
R |
S (2m) |
| Staphylococcus pseudintermedius |
S (3m) |
S (1m) |
R |
| Staphylococcus lentus |
R |
R |
S (1m) |
| Escherichia coli |
S (1m) |
R |
S (2m) |
| Enterococcus casseliflavus |
S (2m) |
R |
S (1m) |
| Klebsiella pneumonia |
S (2m) |
R |
R |
| Rhizobium radiobacter |
R |
S (1m) |
R |
| Sphingomonas paucimobilis |
S (1m) |
R |
R |
| Enterobacter hormaechei |
R |
R |
S (1m) |
(R) Resistant , (S) Sensitive
Figure 9: The effect of mouthwash on bacteria, A zone will be around the sensitive disc, and not zone will not be around the disc, it is resistant
Mouthwashes that contain four essential oils related to phenol—thymol, eucalyptol, menthol, and methyl salicylate, with alcohol levels reaching as high as 26%—claim to effectively penetrate the plaque biofilm, thus eliminating the microorganisms responsible for gingivitis. These mouthwashes demonstrate a broad spectrum of antimicrobial properties, inhibit the aggregation of bacteria, slow bacterial reproduction, delay the maturation of plaque, and diminish both the mass of plaque and its pathogenicity (Fine et al., 2005).
Thymol, a compound found in essential oils, was identified by Lister, after whom the popular mouthwash—Listerine—was named. Listerine contains various essential oil components, including thymol, menthol, and eucalyptol. Mouthwashes and/or gargles that incorporate essential oils or their derivatives are highly effective in addressing dental issues (Sharma et al., 2010), as well as infections of the mouth or throat, and can help alleviate radiation-induced oral mucositis (Sharma et al., 2010; Buckingham, 2010).
Moreover, gargles enriched with essential oils can be quite advantageous in treating oropharyngeal candidiasis in individuals with AIDS, or for soothing a simple sore throat. For the latter, it is advisable to combine three drops of an essential oil, such as Palma Rosa (Cymbopogon citratus), with 15 mL of warm water to create a comforting gargle that also helps combat the infection. This procedure should be repeated every four hours throughout the day for three days (Jose et al., 2002).
Intrinsic resistance refers to the inherent characteristic of an organism, whereas acquired resistance arises from genetic alterations, which may occur through mutations or the uptake of genetic material, such as plasmids(Russell, . (1995
Resistance should also be distinguished from tolerance; resistance indicates the genetic capability of microorganisms to endure elevated levels of an antimicrobial agent measured by MIC, while tolerance means to the survive capacity a temporary exposure to high antimicrobial concentrations without altering the MIC, primarily due to a slowdown in metabolic activities. Conversely, persistence denotes the existence of a sub-population that can endure treatment by concentrations exceeding MIC of a specific antimicrobial, despite the individual being genetically identical(Brauner et al,(2016.
intrinsic resistance to Chlorhexidine gluconate is recognized in mycobacteria, and bacterial spores attributed to their outer cell membrane that create impermeable barriers against the penetration of Chlorhexidine gluconate molecules. Mechanisms of acquired genetic resistance to Chlorhexidine gluconate include multidrug efflux pumps and alterations in the cell membrane(Jaglic et al.,2012).
Conclusions
There are various types of bacteria that cause gingivitis, and they are not limited to specific strains. Zak’s mouthwash solution has proven effective in sterilizing and protecting the gums by eliminating the bacteria responsible for gingivitis, making it the most effective mouthwash solution used in the study.
Acknowledgment
Non.
Conflicts Interest
Non
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