The mild early inflammation of the gums that occurs as a result of the accumulation of bacteria, and plaque in the mouth and lead to infection, associated with bleeding and swelling of the gums, pain, and may it progresses to periodontitis when left untreated [1]. Eikenella corrodens, a unique species of the Eikenella genus, is a HACEK group of microorganisms [2]. In 1958, by Eiken E. corrodens were first isolated, and called “Bacteroides corrodens”[3]. It is a small, gram-negative bacillus, facultative anaerobic, fastidious, non-motile; slowly growing on chocolate or blood agar at 37 ^◦C. E. corrodens can cause abscesses in the brain, liver and thigh [4]. Dental plaque is a complicated biofilm that consists of bacteria surrounded by a protective matrix. This matrix is made up of extracellular polysaccharide and glycoproteins that create a safe space for bacteria within the dental biofilm [5]. E. corrodens possesses the luxS gene in its DNA and releases AI-2 into its culture medium. LuxS may assist in the last phase of the growth, maturation, and release of biofilms. The detachment of biofilms is crucial for the spread of various pathogens, and the presence of AI-2 in E. corrodens could enhance both the maturation of biofilms and the transmission of periodontal disease [6]. Chlorhexidine showed a stronger ability to prevent the formation of biofilms and inhibit demineralization when compared to 0.1% chlorhexidine mouthwash. Therefore, using chlorhexidine on patients could be a simple and effective way to prevent diseases related to biofilms [7] Various ways in which adaptations occur were explored, such as alterations in the cell membrane that make it harder for CHX to enter the cell and the activation of multidrug efflux pumps. These adaptations may be triggered by the mutagenic properties of certain substances. It is still unclear if CHX itself has mutagenic effects, CHX was pointed out to show increased mutagenicity [8]. Research using different biofilm models has indicated that there is a trend of improved biofilm removal as the concentration of NaOCl increases. However, contradictory results have been found in studies where lower concentrations of NaOCl were used [9].

The aim of this research is highlighted on one of the most important and virulent new locally isolated periodontal pathogens using the molecular technique, study its ability to produce biofilm and effect of both effect of Chlorhexidine (CHX) and SodiumHypochlorite (NaOCl) on Biofilm formation.

Sample Collection: Twenty-five samples were collected from patients with chronic periodontitis depending on clinically examination by a specialist dentist at Dental Specialized Center / Al-Noor and Mosul General Hospital. Absorbent paper points (size #30) were inserted to the base of pockets with a pocket depth (PD) of ≥ 4 mm for one minute. The absorbent paper points were transferred directly into tubes containing a modified transport medium. The patients (14 male and 11 female with average age of 19-65 years) proved that they had no systemic disease, no smokers and not taking antibiotics 3 months before sampling [10].

Estimation Biofilms Formation:

By using Modified Congo Red Agar medium:

Bacteria have ability to produce slime layer was studied by modifying Congo red agar medium by adding the Congo Red (CR) dye (0.8 g/l of distilled water and sucrose 36 g to the Modified Todd hewitt agar medium (MTHA), this modification was performed for the first time. MTHA medium and Congo Red dye were sterilized separately by autoclave, while sucrose was sterilized by filtration as a concentrated aqueous solution. After MTHA medium cooling to (45-50) °C, sugar and CR dye were added.The samples were inoculated on CR-MTHA medium and incubation at 37 C for 72 hours, then left for 48 hours at room temperature.

By using Microtiter plate: 

1. The fresh bacterial culture of E. corrodens and Streptococcus mitis were taken to get a suspension of Alternative Thioglycollate Broth medium to be ready (antibiotics free) matched to McFarland 0.5.

2. Micro-titter plate of 96- flat bottom well, bacterial broth (200 µl) was poured to the wells as a “mono-microbial population.” 

3. The plates were closed and incubated at microaerophilic condition for 24-30 hrs.

4. The wells contents were poured out then, wells rinsed many times with distilled water to remove the non-adherent cells.

5. With 0.1% crystal violet (100 µl) for 15 minutes, staining the adherent biofilm and washing it many times with distilled water.

6. To each well, 200 microliters of Ethanol at a concentration of 95% for 15 minutes was added.

7. The optical density was read by  ELISA reader, at a wavelength of 630 nm to determine the efficiency of the isolates in producing biofilm by comparing the readings to the following equation according to Al-Azzawi, (2018) [11] with modification.

8. The isolate is non-adherent (Non biofilm-forming) according to the equation (OD ≤ ODc).

9. The isolate is a weak adherent (weakly biofilm formation) according to the equation (ODc<OD≤2×ODc).

10. The isolate is considered to be of moderate biofilm formation (moderately adherent) according to the equation (2×ODc<OD≤4×ODc).

The isolate was considered to be highly adherent (strong biofilm-forming) according to the equation (OD>4×ODc).

By using Tube Method (TM): 

This is a qualitative technique used to detect biofilms. A loopful of E. corrodens culture placed in 10 ml of alternative thioglycollate broth containing 1% glucose. At 37oC for 24 h, tubes were incubated. After the incubation period, the tubes were poured out and rinsed with a phosphate-buffered saline solution (with a pH of 7.3) and then allowed to dry. The tubes were subsequently stained using a solution of crystal violet (0.1%). Any extra stain was rinsed off with distilled water. The tubes were then dried while turned upside down. Scoring for the tube method was based on the outcomes of the control strains. Biofilm formation was deemed positive if a thick film was visibly present on both the wall and bottom of the tube [12].

The Effect ofChlorhexidine(CHX) and SodiumHypochlorite (NaOCl) on Biofilm:

The fresh bacterial culture of E. corrodens and S. mitis were taken to get a suspension of Alternative Thioglycollate Broth medium to be ready (antibiotics free) matched to McFarland 0.5.

1.  100 µl of each individual type of bacteria was placed into the wells of a 96-well plate to create a mono-microbial population. 

2. 100 µl of both  CHX 0.02% and NaOCl  % were      added separately.

3. Wells contained only bacterial inoculums with no antibacterial agent as a control.

4. The well plates were sealed and incubated at microaerophilic condition for 24 hrs.

5.  Afterward, the contents of the wells were poured out and the cells that were not sticking were rinsed multiple times with distilled water. 

6. The biofilm that stuck to the surface was treated with 100 µl of 0.1% crystal violet dye for 15 minutes, and then rinsed multiple times with distilled water.

7. Extraction of stained biofilm with (95 % Ethanol) and after 15 minutes, a microplate reader read the absorbance at 630nm.

E. corrodens and Streptococcus mitis were isolated from patients with periodontitis in Mosul city / Iraq. This bacterium was diagnosed by molecular methods; the sequence of 16S rRNA gene was determined for E. corrodens and Streptococcus mitis, then compared with that of (NCBI) National Center Biotechnology Information. Deposition the sequence of 16S rRNA of E. corrodens and Streptococcus mitis at GenBank, under the accession number OQ996282.1, then this strain has given MeAm as name (Table 1).

Table (1) The bacteria, strains and accession number

Detection of biofilm production performed by a simple qualitative method “Congo Red Agar (CRA) medium”. E. corrodens ability to form a biofilm was tested by the Congo red process, note that the culture medium (CR-MTHA-1) was used in this study has been modified, so that it can be used to grow bacteria and study their ability to form biofilm, considering that E. corrodens are Fastidious bacteria. After inoculation of (CR-MTHA-1) medium with E. corrodens isolate, the results were strongly positive, where there was a change in color to dry black, it is an indication that this bacterium has the capacity to produce slime layer. While the isolate that did not have the ability to form a slime layer appeared pink or red in colour (Figure 1).

Figure (1) Slime layer production on CR-MTHA-1 medium by E. corrodens

After the incubation was carried out under the appropriate conditions, the petri dishes were taken out of the incubator and left for three days, which led to an increase in the darkness of the black color (Figure 2).

Figure (2) Slime layer production on CR-MTHA medium by E. corrodens

MTP assay 

The susceptibility of bacterial isolates isolated from periodontitis to biofilm formation was performed by MTP method, where the results were showed that all isolates of E. corrodens and Streptococcus mitis were forming biofilm with different degrees, and after comparing results with the quality control and applying the equation mentioned in material and methods, all bacterial types have strong biofilm-forming (strong adherent). 

Also, the best suitable culture medium to be used in the study of biofilm was determined by making a comparison between Alternative Thioglycollate Broth with hemain and MTHB medium, Where the last one has a higher result when compared with quality control and the application of the equation and here E. corrodens was used only.

Figure (3) Biofilm formation by MTP assay

Tube method:

The experiment was performed in triplicate and repeated three times.The result of the inoculation of E. corrodens on the alternative thioglycollate broth with 1% glucose was showed a positive result in terms of the formation of biofilm on the inner walls in the form of a purple layer adhering to the inner surface of the tube (Figure 4).

Figure (4) Showing ability of E. corrodens to produce  biofilm by tube method.

The Effect ofChlorhexidine(CHX) and SodiumHypochlorite (NaOCl) on Biofilm:

 In this study, Chlorhexidine (CHX) and Sodium Hypochlorite (NaOCl) have been shown to have an effect on biofilm formation in E. corrodens and Streptococcus mitis.

In this study, both (CHX and NaOCl) gave a positive result and affected the biofilm, and the agents affected both types of bacteria (E. corrodens and Streptococcus mitis). The effect of the two agents on the bacteria of E. corrodens was stronger than for Streptococcus. For E. corrodens, the effect of CHX was stronger than NaOCl and clearly affected biofilm as shown in the final reading on 630 nm. The result, belonging to Streptococcusmitis, was revealed that the CHX also showed a higher and greater effect than the NaOCl on the biofilm of this bacteria (Table 2).

Table (2): The effect of CHX and NaOCl on biofilm formation

Bacterial Growth of E. corrodens on Conventional Media:

Once the E. corrodens inoculum was used to make a suspension in its appropriate growth medium (which did not contain any antibiotics) equivalent to McFarland 0.5, culturing E. corrodens on conventional media including:

1.  Nutrient agar

2.  macConkey agar

3. mueller hinton agar, and under appropriate incubation conditions.

The result of our study revealed that these media mentioned above did not support the growth of this type of bacteria.

The methods used to detect the production of mucous substance and the ability to form a biofilm are different, it mainly includes Congo red agar medium CRA and MTP method [13]. Samie and Nkgau, (2012) [14] study that the MTP method is the most sensitive and easy to detect biofilm formation among strains. The CRA method is relied to show the difference in  the morphological pattern of isolates which produce slim layer with highly virulence, and that detection of this pattern may help in distinguishing between strong and weak biofilm producing strains  that reflect the degree of   infecting and helping in  the determination  the primary treatment, and the different  production of multiple polysaccharide adhesion (PIA) causes this different degree in the production of biofilm , which indicates changing in the genetic regulation [13]. Bacteria growing in the biofilm show various differences morphologically from the original one growing on the culture, these differences involve movement change, high production of extracellular polysaccharides and high antibiotic resistance [15]. The method of MTP is very important in the study of the early stage of biofilm formation and classification of degree of infection into low, medium and high, as it is the production of the biofilm is an important virulence factor, as it support bacteria with protection for from phagocytosis, lead to the continuation of the infection [16].

Bacteria that form biofilms are often the cause of infections and were known to be extremely challenging to eliminate. They can resist antibiotics through mechanisms such as limiting the entry of antibiotics into the biofilm, slowing their growth, and activating genes that make them resistant. Close contact between microbial communities allows them not only to communicate, but also exchange genetic material. The rate of gene transfer between these communities was higher than when compared to individual microbes. This means that horizontal microbial gene transfer and the formation of biofilms are closely connected [17].

Karim et al., (2013) [6] study mentioned that LuxS could assist in the last phase of biofilm growth, maturation, and detachment. Given that the spread of many pathogens relies on detachment of biofilm, AI-2 in E. corrodens may not only help in maturation of biofilm but also in the transmission of periodontal disease. In E. corrodens, the process of forming biofilms is influenced by a signaling molecule known as autoinducer-2 (AI-2). When purified AI-2 was added, it increased the formation of biofilms in E. corrodens. Dental plaque is a diverse collection of bacteria that form a complex biofilm on the surface of the teeth. E. corrodens was one of the bacterial species found in dental plaque and was believed to contribute to its harmful effects due to a lectin on its cell surface. This bacterium has the ability to form a biofilm on a polystyrene surface. After 24 hours, a biofilm developed at the bottom of the wells in microtiter plates. As the biofilm grew, microcolonies could be seen. When anaerobic respiration was restricted because of a lack of nitrate, the biofilm only formed at the interface of the air and water [18].

Chlorhexidine (CHX) has been studied for its effect on biofilm formation. Several papers have shown that CHX can increase the ability of biofilm formation in Enterococcus faecalis, leading to the emergence of biocide-resistant microorganisms . In dentistry, CHX has been found to disrupt the matrix integrity of multispecies oral biofilms, resulting in a reduction in biofilm thickness and bacterial vitality [19]. Additionally, CHX mouth rinses have been shown to inhibit biofilm formation on dental enamel and induce biofilm disruption, making it a promising approach for biofilm management [20]. Furthermore, CHX has been found to have a limited effect on the biofilm matrix, primarily reducing bacterial cell volumes rather than dissolving glycoconjugates [21]. Overall, the studies suggest that CHX can have both positive and negative effects on biofilm formation, depending on the specific bacteria and conditions being studied. The study of Singh et al., (2022) [22] revealed that NaOCl, when used as a mouthwash twice a day, can be suggested as a component of home care for patients with chronic periodontitis disease. It is a cost-effective and readily accessible option. Enhancing the disinfection process during non-surgical periodontal treatment is crucial. The effectiveness of sodium hypochlorite and a 980 nm diode laser in this treatment was being evaluated [23]. The study of Sinha et al., (2022) [24] was revealed that both 0.05% sodium hypochlorite and 0.2% chlorhexidine were successful in decreasing aerosol contamination during ultrasonic scaling, but 0.05% sodium hypochlorite had a significantly greater impact than 0.2% chlorhexidine.

The addition of sodium hypochlorite did not have a positive impact on the clinical outcomes compared to using mechanical instrumentation alone over the 12-month period., the data that was currently available did not demonstrate any further clinical advantages when sodium hypochlorite is used alongside nonsurgical periodontal treatment [25]. 

Bottone et al, (2006) [26] study was showed that E. corrodens have no growth on majority selective media, such as eosin methylene blue (EMB) or MacConkey agars, and also the study was mentioned that E. corrodens seems to rely heavily on hemin for growth when first isolated, showing slow development on media like Mueller-Hinton or Brucella agar with 5% blood, or on chocolate agar.

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