IN VIVO STUDY EVALUATES EFFECTIVENESS OF ADDING CIPROFLOXACIN TO CALCIUM HYDROXIDE VERSUS SAUSSUREA COSTUS TO CALCIUM HYDROXIDE MEDICATION AGAINST BACTERIAL BIOFILM IN ROOT CANAL TREATMENT

Amal Abdu Al Kareem AL Muhalel1image, Hassan Abdulwahab Al-Shamahy1,2image, 

1Departement of Basic Sciences, Faculty of Dentistry, Sana’a University, Republic of Yemen.

 2Medical Microbiology and Clinical Immunology Department, Faculty of Medicine and Health Sciences, Sana’a University.

 

Abstract

Background and objectives: Elimination of root canal bacteria is a perquisite for a successful root canal treatment. This research aims to determine the effectiveness of adding ciprofloxacin versus Saussurea costus extract to calcium hydroxide medicament against bacterial biofilm in root canal treatment in vivo study. 

Materials and methods: In vivo study on 70 single-rooted human teeth at Sana'a University dental clinics used Saussurea costus extract preparation, including washing, drying, crushing, mixing, centrifugation, drying, and homogenization.The intracanal drug concentration was standardized by mixing 1 g of calcium hydroxide powder with 1 ml of ciprofloxacin or 1 ml of Saussurea costus extract in both groups. Three bacteriological samples were taken before root canal treatment, before Medicaments were placed, and 7 days after, and CFU and bacterial isolation were performed using standard methods. 

Results: An intragroup analysis showed that all drug groups had a significant decrease in the number of intracanal bacterial cells from S1 to S2 and from S2 to S3 (p<0.0001). When comparing quantitative S1 or S2 data, there was no significant difference between the groups; however, at Sample 3, the Saussurea costus extract calcium hydroxide (34.05±21.8) had considerably higher bacterial counts than the calcium hydroxide + Ciprofloxacin group (11.9±29 CFU). The most isolated bacteria in S1 and S2 were Staphylococcus aureus (58.6%, 51.4%), followed by Klebsiella species (27.1%, 18.6%), Escherichia coli (25.7%, 17.1%), Enterococcus faecalis (22.9%, 15.7%) and Pseudomonas aeruginosa (21.4%, 12.9%). 

Conclusions: The addition of Ciprofloxacin to calcium hydroxide and Saussurea costus- calcium hydroxide provided further antibacterial effectiveness when used as an intracanal medicament in vivo during root canal treatment.

Keywords: Bacteria, biofilm, calcium hydroxide, ciprofloxacin, root canal treatment, Saussurea costus.

 

INTRODUCTION

 

Since apical periodontitis is mostly caused by bacterial infection, minimizing or getting rid of bacteria is essential to maximizing treatment results1,2. The bacterial burden in the root canal system has been demonstrated to be significantly decreased by mechanical instrumentation combined with chemical disinfection3. Nonetheless, chemomechanical instru-mentation is not sufficient to eradicate all root canal bacteria, according to multiple studies4-7. Intracanal medicine has been suggested as a means of achieving this goal, as it facilitates a notable increase in bacterial clearance during chemo-mechanical preparation8,9. Due to its antibacterial and biologic properties, calcium hydroxide is the most often utilized intracanal medication10,11. Nevertheless, calcium hydroxide has a limited antibacterial impact, especially in situations of persistent infections12.

In their evaluation of the effectiveness of calcium hydroxide nanoparticles in eliminating Enterococcus faecalis in human root dentine, Louwakul et al.13, found that calcium hydroxide was not able to destroy the bacteria. Comparably, Javidi et al.14, assessed calcium hydroxide's antibacterial efficiency in terms of killing E. faecalis in root canals, both with and without a silver nanoparticle suspension. They found that calcium hydroxide was less effective than calcium hydroxide mixed with other antibacterial agents. Nonetheless, prior research assessing the antibacterial efficacy of calcium hydroxide as an intracanal medication has yielded inconsistent results. While some research1,4 observed further bacterial clearance following intracanal treatment with calcium hydroxide, other studies15,16 reported an increase in the percentage of positive cultures and bacterial counts. It has been previously established that the presence of bacteria at the time of root filling has a substantial impact on the root canal treatment's success16-18.

Medicinal plant extracts have been utilized extensively around the world for therapeutic purposes, including the treatment of numerous microbial illnesses19. For a variety of reasons, including their ease of availability, low cost, and lack of side effects, plants are used as a natural source of medications and are seen as an alternative to modern treatment, particularly in developing nations20,21.  These traditional medicinal herbs can be investigated for their antimicrobial therapeutic potential in order to find and develop new antimicrobial medicines. Saussurea costus, sometimes known as S. costus, is one of the more significant medicinal herbs that have been utilized from ancient times. In addition to being used in traditional Chinese and Indian medicine, S. costus is well-known in Islamic medicine22. Some active biochemical components of S. costus, which have various physiological effects on the body, are what give it its medicinal usefulness. Flavonoids, alkaloids, sesquiterpenes, phenolic compounds, tannins, sugars, and glycosides are the primary biochemically active substances23. Many studies have documented the therapeutic benefits of S. costus root, including its ability to heal wounds, stimulate the immune system, be anti-inflammatory, choleretic, hepatoprotective, antiulcerogenic, larvicidal, gastro-protective, cytotoxic, cardiotonic, and anticancer properties24.

Ciprofloxacin, a second generation fluoroquinolone, has been used successfully as an intracanal drug for root canal treatment. It is effective against enteric bacteria, including E. coli and Enterococcus species with strong tubular penetration25-29.The purpose of this study is to compare the effectiveness of S. costus extract versus ciprofloxacin in addition to calcium hydroxide medication against bacterial biofilm in root canal treatment in an in vivo setting.

 

MATERIALS AND METHODS 

 

S. costus extract preparation

Botanist details of  the plant’s roots: The kingdom is Plantae, the Clade is Tracheophytes, the order is Asteraceae, the genus is Dolomiaea, and the species is D. costus. The plant's roots was brought from China by a documented S. costus dealer, as commercial records for this plant date back to the year 2024 AD, Junuary first. The roots of the plant were thoroughly dried after being thoroughly cleaned three times with tap water. The root was first reduced to tiny bits and then to a fine powder using an electronic grinder. After combining 25 g of powdered dried roots with 250 ml of solvent (water) and letting it sit for 24 hours, the solutions were filtered through gauze, centrifuged for 15 minutes at 3000 rpm, and then allowed to dry for 48 hours at 45 °C in a hot air oven. A sterile container is used to store the dried extract. The crude was then ready for use after the dried plant combination was steeped in 70% ethanol and homogenized using magnetic stirrers.

The preparation of the calcium hydroxide mixture:

In order to create a uniform combination (ratio of 1:1) with 1 ml of either Ciprofloxacin in group I or S. costus extract in group II, the concentration of intracanal medications was standardized across all groups using 1 g of calcium hydroxide powder. 

Ethical Consideration: The Medical Ethics and Research Committee of Sana'a University's Faculty of Dentistry granted consent for this study, which took place between January 2024 and May 2024 (approval number: 77; dated January 1, 2024). The review committee's set of ethical guidelines was regularly adhered to.

Patient selection

Patients in need of endodontic treatment who attended Sana'a University clinics were chosen. Every patient gave their informed consent to take part. Following that, a thorough medical and dental history was taken, and each patient underwent a clinical and radiographic examination. 

Inclusion criteria:

· The age of the patients was within a range of 19 to 65 years.

· All selected teeth are single root teeth.

· The patient is clinical and X-ray diagnostic with necrotic pulp and asymptomatic apical periodontitis around 2-4 mm.

· All selected teeth for making the root canal have enough crown structure for adequate isolation with a rubber dam.

Exclusion criteria:

· Patients who received antibiotic treatment during the last 3 months.

· Patients had or have a systemic disease.

· Pregnant females.

· The relevant tooth shows periodontal pockets deeper than 4 mm during clinical examination.

· Apical radiolucency is smaller than 2 mm or bigger than 4 mm in preapical digital x-ray.

· Non-restorable teeth.

· No previous endodontic treatment.

· Incomplete root formation or root resorption, perforation, or calcification.

Root canal treatment and microbiological sampling procedures:

The study's patients' teeth were sized and polished prior to receiving treatment30. The patients were instructed to rinse their mouths with chlorhexidine digluconate (a chemical manufactured by the CHX DEXA firm). Before beginning the endodontic process, each tooth was topically anesthetized with 20% benzocaine gel (Prime-Dent). This was followed by a local anesthetic injection with 0.9 ml of a solution comprising 2% Lidocaine HCL and 1:100000 epinephrine (Lignospan standard). Aseptic approach and a rubber dam were used to clean the operative field, which included the tooth, during the endodontic process. The area was then disinfected with 30% hydrogen peroxide (H2O2) and 2.5% sodium hypochlorite. Sterile spherical burs were used to remove the decayed coronal structures (Mani, Inc.). The crown surface and the surrounding structures (the clamp and dam) were cleaned for thirty seconds, as previously mentioned. After that, the impact of the NaOCl was neutralized using 5% sodium thiosulfate. Sterile high-speed round and fissure bursts were used to create the access cavity in an aseptic environment. A tapered low-speed stone (Mani, Inc.) was used to smooth the access cavity in an aseptic setting. As previously mentioned, the disinfection treatment was carried out once more following the completion of the access cavity preparation. As stated by Karataş et al.31, sterile Teflon tape and cotton pellets were employed to seal the pulp chamber's floor and roof in order to stop disinfectants from penetrating the coronal and radicular pulp spaces.  Following the inactivation of the NaOCl effect using sodium thiosulfate, sterility control samples (SR) were collected from the coronal surface of the tooth, rubber dam, and clamp using a sterile swab with an ejectable tip (Sample 1). The swab was inserted into 1 milliliter of Brain Heart Infusion Broth (BHI Broth) and immediately inoculated into Brain Heart Infusion agar for the purpose of isolating and counting bacteria. It was then cultured for 48 hours at 37°C, after which the colonies were counted and the bacteria were identified. Next, using Fanta (AF) nickel titanium rotary files (Fanta) and an E-connect endo motor (Eighteeth Medical) in rotation mode with a setting of 325 rpm and 1.8 N cm torque as advised by the manufacturer, the root canal system was prepared till AF4 35/04. Hand files #40–60 (Micromega) were used for additional shaping in accordance with the architecture of each canal until clean, white dentine chips were clearly visible at the apical 3 mm of the master file. After every file use, 2 ml of sterile saline was irrigated using side-vented needles of size #30 G, positioned 1 mm below the WL. following the instrumentation of the canal up to the master apical file (MAF). Using an Irrisafe ultrasonic tip, irrigation was carried out using sterile saline solution with the assistance of passive ultrasonic irrigation (PUI). To activate the irrigant in each canal, the Varios 370 (woodpecker device) ultrasonic device's tip was inserted 1 mm from the working length at medium power. A 2–3 mm apical–coronal movement was used to produce the activation (3 cycles×20 s= 1 min). For a total volume of 3 ml, 1 ml of the irrigating solution was added to the root canal device in each cycle. In order to get the second microbiological sample (Sample 2), the canal content was collected, dried using three sterile absorbent paper points that matched the MAF size, and aspirated. The tested intracanal medications were then inserted into the appropriate root canals, stopping about 1 mm short of the WL. To administer the intracanal medications, Dentsply Maillefer's lentulo- spiral was utilized. inside groups I, II, and III of the canals. Using a Dentsply Maillefer plugger, the medications were packed up to the level of the opening. Following that, sterile Teflon tape was used to cover the canal orifices. The second allocator sealed the access cavity using sterile cotton and glass ionomer fillin (Medifil, Promedica Dental Material GmbH).

After seven days, patients were summoned back for another appointment. Leak detection was performed on the interim restoration during the second visit. No single case of teeth with leaky or damaged restorations was included in the study. Every tooth was isolated using a rubber dam, and the surgical field was cleaned as previously indicated. After removing the temporary restoration, the pulp chamber was thoroughly cleansed and saline flushed once more. With careful strokes, the intracanal medication was extracted from the canals using Headstorm hand files. Using syringes with 30-gauge needles, a circumferential filing was performed with sterile saline (10 ml) until the irrigant exited the canal devoid of any leftover medication32. The third postmedication sample (Sample 3) was obtained following the removal of the intracanal medication using three sterile paper points, and it was inculcated into one milliliter of peptone water as previously mentioned. Using the cold lateral condensation technique, gutta percha cones (Meta Biomed) and AH Plus sealer (Dentsply/Maillefer) were used to obturate the canals after they had been dried with sterile paper points. A composite restoration was placed on the tooth right away, and a follow-up radiograph was obtained to assess the level of obscuration. 

 

RESULTS 

 

Table 1 shows the sex and age distribution of patients included in the study at the Sana’a University dental clinics in Sana’a city. The male patients counted 40%, and the female patients were 60%. The mean age of our patients was 31.9 years, with SD equal to 11.3 years, and ages ranged from 14 to 58 years. Most of our patients were in the age group of 25-34 years (47.1%), followed by the group of 15-24 years (20%), while other age groups were less frequent.Table 2 shows the type of mixed materials used to reduce endodontic microorganisms for patients included in the root canal treatment study. The study evaluated S. costus alone, calcium hydroxide-ciprofloxacin, and calcium hydroxide- S. costus in reducing endodontic microorganisms in 70 Yemeni patients. In 26 patients, S. costus was applied, calcium hydroxide-ciprofloxacin was applied to 22 patients, and calcium hydroxide was applied to 22 patients. Table 3 shows the types of bacterial isolates before performing root canal drilling from the infected tooth and surrounding tissues (sample 1).

The most isolated bacteria was Staphylococcus aureus (S. aureus) (58.6%), followed by Klebsiella species (27.1%), Escherichia coli  (E. coli) (25.7%), Enterococcus faecalis (E. faecalis) (22.9%), and Pseudomonas aeruginosa (P. aeruginosa) (21.4%). Mixed growth was observed in 23 samples (32.9%). Table 4 shows the types of bacterial isolates after performing root canal drilling before irrigation with antibacterial agents (sample 2). The most isolated bacteria was S. aureus (51.4%), followed by Klebsiella species (18.6%), E. coli (17.1%), E.  faecalis (15.7%), and P. aeruginosa (12.9%). Mixed growth was observed in 13 samples (18.6%). 

Table 5 shows the types of bacterial isolates after performing root canal and after irrigation with antibacterial agents (sample 3). The most isolated bacteria was S. aureus (11.4%), followed by Klebsiella species (7.1%), E. coli (11.4%), E. faecalis (4.3%), and P. aeruginosa (8.6%). Mixed growth was not observed, and 40% showed complete sterilization for aerobic bacteria.Table 6 shows the comparison between the frequency of bacterial isolates from the root canal after applying antibacterial mixtures to the canal. For E. coli, there was a non-significant difference equal to 5.7% reduction after applying the antibacterial mixture (p=0.33). Considering Klebsiella species, there was a significant difference equal to 11.5% reduction after applying the antibacterial mixture (95% CI = 0.23-22.8 and p=0.04). Considering P. aeruginosa, there was a non-significant difference equal to 4.3% reduction after applying the antibacterial mixture (95% CI = 6.4- 15.2 and p=0.41). Considering S. aureus, there was a significant difference equal to a 40% reduction after applying the antibacterial mixture with a 95% CI of 25-552 and p<0.0001.

Considering E. faecalis, there was significant difference equal to 11.4% reduction after applying the antibacterial mixture  with 95% CI equal to 1.2–22 and p=0.025. Considering mixed growth there was significant difference equal to 18.6% reduction after applying the antibacterial mixture  with 95% CI equal to 9.5–29.3 and p=0.0002. Considering no bacteria growth there was significant difference equal to 57.1% reduction after applying the antibacterial mixture  with 95% CI equal to 44.3–68 and p<0.0001. Considering Total bacteria growth there was significant difference equal to 32.2% reduction after applying the antibacterial mixture  with 95% CI equal to 8.8–36.4 and p=0.0016.

Table 7 shows the comparison of the effect of antibacterial agents in inhibiting bacterial growth and biofilm formation after root canal procedure and after irrigation with antibacterial agents (sample 3). For E. coli, the lowest number occurred in mixtures B (Calcium Hydroxide-Ciprofloxacine) and C (1, 4.5%), and with mixture B, a higher negative growth occurred (86%) than other mixtures (A, D). Table 8 shows the comparison of the effect of the 3 tested antibacterial agents in inhibiting bacterial growth and biofilm formation after root canal procedure and after irrigation with antibacterial agents (sample 3). Considering the total absent growth, the highest rate (86.4%) occurred for mixture A (Calcium Hydroxide-Ciprofloxacine), while for mixture B (Calcium Hydroxide- S. costus), the inhibition growth was only 18.2%, with a positive difference growth inhibition equal to 68% and a 95% CI equal to 39.7-82.1 with p=0.0001).

Calcium Hydroxide-Ciprofloxacine showed good effect by killing completely P. aeruginosa and E. faecalis, while 4.5% growth rates occurred for E. coli, Klebsiella species, and S. aureus. Calcium Hydroxide- S. Costus mixture showed lower effect in killing P. aeruginosa with a survival rate equal to 27.3%, and for E. coli it was 13.6%, while for E. faecalis and Klebsiella species the survival rate was 4.5% for the mixture and B. Table 9 shows the mean, median, mode, and SD of the number of bacterial colonies and reductions after using mixers to sterilize root canals. The mean±SD for S. costus was 598.3±50.04 CFU/ml before application and decreased to 11.7±27.7 CFU/ml after application. The mean±SD of calcium hydroxide-Ciprofloxacine was 72.5±26.8 CFU/ml before application and then decreased to 11.9±29 CFU/ml after application. Mean±SD for calcium hydroxide- S. costus was 290.3±121.4 CFU/ml before application and then decreased to 34.05±21.8 CFU/ml after application. 

 

DISCUSSION 

 

The current study assessed the antibacterial activity of calcium hydroxide in conjunction with ciprofloxacin or S. costus extract on the bacterial biofilm found in patient teeth root canals. The colony count in the groups receiving S. costus extract and calcium hydroxide + ciprofloxacin was inhibited, according to the results. The major study question's alternative hypothesis was verified by colony counting, which demonstrated that the in-vivo antibacterial effects of CH paste on bacterial biofilm were enhanced when calcium hydroxide was combined with either ciprofloxacin or S. costus. Bacteria can enter the pulp tissue when a carious lesion spreads to the pulp chamber and the hard tissue barrier is broken. Bacteria spread and colonize the entire root canal system in apical periodontitis. Consequently, bacteria within the root canal system need to be removed in order to facilitate the healing of apical periodontitis and cleaning of the root canal system.

Within-group analysis in the present investigation showed a statistically significant decrease in the quantity of intra-canal bacterial cells from S1 to S2 and from S2 to S3 across all tested treatment groups (p< 0.0001). Intracanal medicine and chemo-mechanical methods both markedly decreased the amount of comparable bacterial cells from infected root canals. This result is consistent with other research that found that intra-canal medicine and chemo-mechanical treatments significantly decreased the number of germs in the treated area33,34. On the other hand, a number of earlier investigations5,35 noted a rise in bacteria following intracanal treatment. The increase in bacterial counts following intra-canal treatment in earlier investigations may have been caused by bacterial leakage through transient repair or contamination. Another possibility is that bacteria were present in areas of the chemo-mechanical preparation that were not treated, such as the isthmus, ramifications, or dentinal tubules, and that this led to the regrowth of leftover bacteria after intra-canal therapy. The current study's findings indicate that there was no discernible variation in the groups' S1 or S2 bacterial counts. The calcium hydroxide+Ciprofloxacin group (mean=11.7, range from 0-81CFU/ml) had considerably lower bacterial counts in S3 compared to the pure S. costus (11.7, 0-141 CFU) and calcium hydroxide + S. costus (mean=34.05, range from 0-84) groups. Furthermore, compared to the pure S. costus and calcium hydroxide+ S. costus groups, the percentage of the decrease in bacterial counts from S1 to S3 and from S2 to S3 was larger in the calcium hydroxide+Ciprofloxacin group.

Notable antibacterial action is shown by the second-generation fluoroquinolone ciprofloxacin, which has good pharmacokinetic qualities and little adverse effects36. Quinolones have been found to have antibacterial action and to be well tolerated (low toxicity, low allergenicity, and high intrinsic solubility) making them appropriate for the prophylaxis of infectious problems37. With a frequency of 0.4% to 2%38, immediate hypersensitivity reactions to quinolones are uncommon and can cause urticaria, itching, erythema, skin rash, and shock. Furthermore, ciprofloxacin, which has a sensitivity rate ranging from 90% to 100%, is among the most effective antibiotics in Yemen39-41.

This study also revealed that the use of pure S. costus in intracanal medication, calcium hydroxide + Ciprofloxacin, and calcium hydroxide + S. costus in calcium hydroxide + S. costus resulted in 17 of 26, 19 of 22, and 4 of 22 root canals being negative for bacteria, respectively (Table 7). On the other hand, no cases of chemo-mechanical preparation were found to be negative for root canal bacteria, indicating that while chemo-mechanical preparation is effective in significantly lowering the amount of intracanal bacteria, it cannot completely eradicate all bacteria from root canals. Therefore, intracanal medication provides additional antibacterial effect in the form of bacteria-free root canals, which is consistent with the findings of earlier studies5,33.

The intracanal application period in this trial was seven days. Regarding the intracanal administration period of calcium hydroxide or other combinations, there is inconsistent evidence available. In the past, Sj€ogren et al.42,  revealed that all it took to achieve bacteria-free root canals was a seven days administration of a calcium hydroxide medication. Similar to this, Shuping et al.8, study showed that 92.5% of root canals with negative cultures were treated after applying calcium hydroxide for one week. On the other hand, data addressing the timing of calcium hydroxide intracanal application indicates that the pH in the outer root dentine rises 1-6 days before peaking, at least 1-3 weeks later43. In a similar vein, Estrela et al.44, observed that microbial inactivation required a 60-day application of calcium hydroxide. Nonetheless, it has been noted that applying calcium hydroxide intracanally for at least 30 days reduces the resistance to root fracture45. For this reason, calcium hydroxide was applied for seven days in the current investigation. 

Following that, sterile Teflon tape was used to cover the canal orifices in the current investigation. To stop bacteria from entering the root canal, the second allocator sealed the access cavity using sterile cotton and glass ionomer fillin. Cavit is a substance made of zinc oxide and ethylene that could leak tiny amounts. According to Weston et al.46, Cavit only stops bacterial leaks when it is applied with a minimum thickness of 4 mm. In the current investigation, access cavities were sealed with Cavit-G and canal orifices were sealed with a sterile Teflon pellet. Nevertheless, not every teeth's Cavit thickness was standardized or measured. The significant number of negative instances found in S3 for bacteria could indicate that the Cavit shielded root canals against bacterial leaks. It has been shown in the past that Cavit, when used to treat root canals with calcium hydroxide, offers sufficient protection against bacterial leakage for a month47. Finally, despite a significant decrease in colony counts across all experimental groups, no intra-canal medication was able to completely remove all germs from each patient's root canal. This fact highlights the value of mechanical instruments that can be used in conjunction with various chemical antibacterial treatments.

According to Table 3, Table 4, and Table 5, the most frequently identified aerobic bacteria from root canals in the current investigation were S. aureus, E. coli, Klebsiella species, E. faecalis, and P. aeruginosa. It is well recognized that the etiology of endodontic infections cannot be determined just by culturing and isolating aerobic bacteria. Therefore, molecular techniques have been widely employed to characterize bacterial communities in various contexts48, and their capacity to incorporate as-yet-uncultured species in analysis49 is one of their major advantages in this respect. Analyses of the endodontic microbiota using community profiling produced some intriguing results: (a). Different endodontic infections have been shown to consist of mixed communities50,51, including persistent/secondary infections linked to treated teeth52. (b). Underrepresented uncultivated bacteria may frequently be found in infected root canals53,54. (c). Depending on the clinical situation (chronic apical periodontitis, acute apical abscesses, treated teeth), bacterial communities may follow a particular pattern51,52. (d). Endodontic communities linked to the same clinical disease exhibit significant interindividual variation51, meaning that each individual has a distinct microbiota in terms of species richness and abundance. (e). This interindividual variation is further pronounced when analyzing individuals from different geographic locations. Nonetheless, endodontic infections are still largely caused by cultivated aerobic bacteria.

Since endodontic infections arise in previously sterile areas, their microbiota is abnormal. As a result, any species found in the canal has the potential to cause endodontic infections or at the very least contribute to the ecology of the microbial community. The aerobic bacteria that we recovered for current study also play a part in this process. For obvious ethical considerations, practically all studies to date that have attempted to identify the genotype or phenotype of endodontic bacteria have used a cross-sectional approach. It is only from these investigations that species prevalence and, consequently, species-disease correlation can be deduced. Causality can be reinforced by possible pathogenicity (in animal models or inferred from relationship with other human diseases), in addition to repeat detection. Numerous species have surfaced as candidates or suspected endodontic pathogens based on cross-sectional research; candidate or putative categories should be kept in place until a particular involvement in disease pathogenesis is demonstrated56.

Limitation of the study

Similar to earlier research, bacteriological samples were gathered for this investigation utilizing a similar methodology5,6. In this study, various tooth kinds (incisors and canines) with various root canal morphology were treated. The structure of the root canal may have an impact on bacteria that are sampled with paper points. Since adherent biofilms and inaccessible regions such dentinal tubules, accessory canals, and fins are not included in the microbiological root canal sample method utilizing paper points, it only provides information on the bacteriological conditions of the main root canal space12. Nonetheless, the present study's findings indicate that there was no statistically significant variation in the distribution of teeth among the groups. Furthermore, anaerobic culturing was not used to separate only anaerobic bacteria.

 

CONCLUSIONS 

 

The adding of Ciprofloxacin to calcium hydroxide and S. costus- calcium hydroxide given extra antibacterial effectiveness as used as an intracanal medicament in vivo during root canal treatment. Additional research is required to assess the antibacterial efficacy of calcium hydroxide + Ciprofloxacin and S. costus - calcium hydroxide.

 

ACKNOWLEDGEMENTS 

 

The authors would like to thank Yemen and the Faculty of Dentistry at Sana'a University for their kind cooperation.

 

CONFLICT OF INTEREST 

 

There are no conflicts of interest in regard to this project.

 

AUTHOR’S CONTRIBUTIONS 

 

Al Kareem AL Muhalel AA: Formal analysis, conceptualization, data organization, and clinical and laboratory examinations. Al-Shamahy HA: Methodology, formal analysis, visualization. Al-Maswary AA: supervision, clinical part. All authors reviewed the article and approved the final version.

 

DATA AVAILABILITY

 

The accompanying author can provide the empirical data that were utilized to support the study's conclusions upon request.

 

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