Molecular Detection and Antibiotic Susceptibility of Bacillus cereus Isolated from Periodontitis Patients in Misan City
Mohammed A. Abd Ali*
| Aqeel A. A. Al-Waeli
© 2025 The authors. This article is published by IIETA and is licensed under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).
OPEN ACCESS
This research aims to detect and isolate Bacillus cereus, a facultative anaerobic Gram-positive bacillus, from the oral cavities of patients aged 18 to 23 years with dental diseases such as gingivitis or periodontitis. Furthermore, this research addresses the gap in understanding the role of Bis-(2-ethylhexyl) phthalate in periodontal therapy and explores the pathogenic pathways of B. cereus in dental biofilms. Additionally, the study investigates the minimum inhibitory concentration (MIC) of B. cereus strains isolated from dental patients in Al-Amarah City. Bis-(2-ethylhexyl) phthalate was identified as an antibacterial agent derived from Penicillium digitatum and was evaluated for its effectiveness against B. cereus. The isolates were identified using phenotypic and biochemical tests, as well as polymerase chain reaction (PCR). All isolates showed resistance to Optochin, Doxycycline, Flucloxacillin, Tetracycline, Nalidixic Acid, and Ciprofloxacin. Besides, the six B. cereus strains that were tested showed a fair level of sensitivity to Ciprofloxacin (CIP.5 µg) with an inhibition zone of 21 mm per disk. Based on FT-IR analysis followed by GC-MS, the active compound was identified to be Bis-(2-ethylhexyl) phthalate. As well, the current findings show that the antibiotic Bis-(2-ethylhexyl) phthalate as a secondary metabolite is effective against B. cereus. Finally, the study showed the possibility of treating infections caused by Gram-positive pathogenic bacteria and Gram-negative pathogens like Escherichia coli using these bacteria that have been isolated from the mouths of patients with dental illness.
MDR Bacillus cereus, dental diseases infection, antibiotic resistance, antimicrobial compound, Bis-(2-ethylhexyl) phthalate
Periodontal disease is a chronic multifactorial inflammatory illness associated with dysbiotic plaque biofilm, characterized by the degradation of the tooth-supporting tissues [1]. Periodontal diseases, both marginal and apical, arise from virulence factors produced by the dental plaque microbiota. These microorganisms release proteolytic enzymes and toxic byproducts that damage the periodontium's cellular and structural integrity. Notably, bacterial proteases degrade critical matrix components, including collagen, elastin, fibronectin, fibrin, and other elements of epithelial and connective tissue [1]. Dental biofilms develop as communities of interacting bacteria that are physically and functionally organized. These interactions, which can also be antagonistic or synergistic, result in the formation of a biofilm that shields the tooth surfaces [2, 3]. B. cereus is a microbe that causes opportunistic infections in humans and is primarily associated with periodontal diseases. The B. cereus group consists of six species: B. weihenstephanensis, B. cereus, B. thuringiensis, B. mycoides, and B. pseudomycoides [4]. Bacillus cereus is widespread in various habitats and can be easily isolated from foods such as meat, grains, and dairy products. They are large gram-positive bacilli forming spores, usually arranged in pairs or chains. They can move by peritrichous flagella; cells of B. cereus are 1-1.2 µm in width and 3-5 µm in length. It is a facultative aerobe with the optimum growth between 35-40℃ [5].
The organism is frequently implicated in invasive B. cereus infections, including pneumonia and bacteremia [6]. Hospital environments have been linked to reports of eye infections [7] and soft tissue infections [8]. B. cereus is a significant contributor to morbidity and mortality. As soon as B. cereus is detected, appropriate empirical therapy should begin [9]. Furthermore, the evolution of antibiotic resistance in B. cereus raises concerns since resistant strains of the bacteria can aid in the enrichment and emergence of further strains resistant to antibiotics.
Penicillium is a diverse genus of ascomycetous fungi found in various terrestrial environments, including soil [10, 11]. Certain species play a significant role in producing bioactive compounds, including antifungal, antibacterial, and immunosuppressant agents [12]. These fungi have been widely studied for their potential in pharmaceutical applications, yet antifungal drug development remains limited compared to antibiotics [13]. Given the increasing need for novel antimicrobial solutions, investigating Penicillium-derived compounds may offer valuable insights relevant to this study’s focus on bioactive agents [14-16].
Bis-(2-ethylhexyl) phthalate, previously isolated from P. digitatum, has been identified as an antimicrobial compound; however, its specific role in regenerative therapy, especially in the aspect of periodontal therapy has not been investigated. Its mechanism of action and effectiveness against periodontal pathogens, especially Bacillus cereus, a potential causative agent of dental infections, are poorly studied. Therefore, this study was designed to address these knowledge gaps by investigating the contribution of B. cereus to periodontal disease pathogenesis, its interaction with dental biofilms, and its Minimum Inhibitory Concentration (MIC) in B. cereus strains isolated from dental patients in Al-Amarah City. Additionally, this study unveils novel insights on the antimicrobial effect of 2-(2-ethylhexoxy)-1-methoxy-2-propyl-2,4-hexadiene (another bioactive compound isolated from P. digitatum) against B. cereus and other periodontal pathogens. The present findings broaden our understanding of the potential use of compounds derived from P. digitatum as therapeutics for periodontal disease by elucidating these mechanisms.
2.1 Clinical diagnoses of the patients
The patient was taking non-steroidal anti-inflammatory drugs; however, no antibiotics were administered during the previous dental procedure. On examination, facial asymmetry and palpable pterygomandibular swelling were observed.
2.2 Collection of gingivalis samples
Twenty-two swabs of the bacterial isolate were collected from 12 male and 10 female patients attending a clinic at the Special Dental Center in Misan City, Iraq; the swabs were taken from pocked lesions of gingivalis following diagnosis by certified dentists (gingivitis, periodontitis). The collected samples were immediately sent to the Microbiology laboratory in the Department of Biology, College of Science, University of Misan. The specimens are collected from acute gingivitis and patients aged from 2-70 years, had samples taken. From January 1, 2023, until April 30, 2023. Twenty samples were collected from a total of twenty-two oral cavities (gingivitis, periodontitis).
2.3 Isolation and identification of bacterial isolated strains Bacillus cereus
One strain selected (B2) from seven strains (B1, B2, and B7) belonging to Bacillus cereus bacterial isolated strains, were identified by 16S r DNA sequenced, extracted and DNA amplified using PCR, sequenced, and aligned with other identified strains in Gene Bank database by using BLAST tool an online to determine the similarity score.
2.4 Preparation of media
The culture media was prepared according to the instructions of the manufacturer.
2.4.1 Blood agar
About 40 g of blood agar was dissolved in 1 liter of distilled water (DW) and heated to the boiling point to ensure complete dissolution of the powder; the medium was sterilized for 15 min at 121℃, then cooled to 50℃ before 5% sterile blood was aseptically added. The mixture was mixed thoroughly and poured into sterile agar plates.
2.4.2 Chocolate agar
About 40 g of blood agar was dissolved in 1 liter of distilled water (DW) and brought to boiling point to ensure complete dissolving of the powder; the media was sterilized for 15 min at 121℃ and cooled to 50℃ before aseptically adding 5% sterile blood to the media. The medium was placed in a water bath at 75-80℃ with gentle swirling until it turned dark brown. The medium was aseptically poured into sterile Petri dishes after being cooled to approximately 50℃.
2.4.3 MacConkey agar
About 51.5 g of the powder (i.e., MacConkey agar) was added to 1 liter of DW and boiled to ensure it completely dissolved; the mixture was sterilized for 15 min at 121℃ and cooled to 50℃ before being aseptically poured into Petri dishes.
2.4.4 Nutrient agar
About 28 g of the powder (i.e., Nutrient agar) was added to 1 liter of DW and boiled to ensure it completely dissolved; the medium was sterilized for 15 min at 121℃, cooled to 50℃, and then poured into aliquots in sterile Petri dishes.
2.4.5 Potato Dextrose Agar (PDA)
About 200 g of peeled potatoes were boiled in 1 liter of distilled water, and the infusion was filtered to remove solid particles. To the potato infusion, 20 g of dextrose was added, and the mixture was autoclaved for 15-20 minutes at 121℃. The medium was then cooled to 50℃ before being aseptically poured into sterile Petri dishes.
2.5 Identification of Penicillium digitatum
Pure mold colonies were grown on temporary slides and analyzed under a microscope. Mycelia and spore structures of Penicillium digitatum were examined for their unique morphological characteristics. The morphological characteristics of the fungi captured in the photographs were identified using a combination of in-depth literature research and expert opinion.
Distinct morphological features Colonies grown at 32℃ on PDA are initially white but rapidly become black during conidial development.
2.6 Antimicrobial susceptibility test
The antibacterial activity of the fungal compound was determined using the disc diffusion assay technique against the Gram-positive (MDR Bacillus cereus, and Staphylococcus aureus) and Gram-negative (Escherichia coli) isolates. Each bacterial strain was suspended to a turbidity of 0.5 McFarland and plated on Muller-Hinton Agar. Discs containing concentrations of 100 μg, 200 μg and 400 μg of the fungal compound were placed on the agar plates, and incubated at 37℃ for 24h. The diameter of the inhibitory zones was measured.
2.7 Cultivation and extraction
A fungal strain and culture medium were obtained from citrus fruits infected with characteristic green mold symptoms. The P. digitatum isolates were then cultivated on potato dextrose agar (PDA) at 27℃ for seven days before being stored at 4℃. Mature spores were collected by rinsing the PDA surface with autoclaved distilled water. The hyphae were then removed by filtering the spore suspensions through a sterilized cotton ball placed in a funnel. The spore suspension of P. digitatum was quantified using a Countess-II FL automatic cell counter (Thermo Fisher Scientific, Waltham, MA) and adjusted to 1 × 10⁶ CFU/mL using autoclaved distilled H₂O.
2.7.1 UV-visible
UV-visible spectroscopy was employed in this study (λ_max = 277 nm, corresponding to the C=O carbonyl group). Additionally, the IR spectrum exhibited a strong absorption at 3446.7–3377.3 cm⁻¹ and 3130.4 cm⁻¹, corresponding to the strong N-H stretching vibration of amines. Absorptions at 3094.38–2950.48 cm⁻¹ correspond to aliphatic C-H stretching vibrations. A strong peak at 1718.58 cm⁻1 corresponds to the C=O stretching vibration of the carbonyl group. A peak at 1575.84 cm⁻¹ corresponds to the aromatic C=C stretching vibration. Absorptions at 1124.50–1136.07 cm⁻¹ indicate the presence of C-O stretching vibrations. Figure 1 shows the FTIR spectrum of the bioactive compound isolated from P. digitatum cultured on spoiled citrus fruits, and Table 1 presents the identified chemical compounds in the extract.
Figure 1. FTIR spectrum of the bioactive compound isolated from P. digitatum cultured on citrus spoilage fruits
Table 1. Some of the identified compounds in the P. digitatum cultured on spoiled citrus [17]
|
No. |
Compounds |
Molecular Weight |
Chemical Formula |
RT (min) |
Area (%) |
|
1. |
Bis-(2-ethylhexyl)-phthalate |
390.50 |
C6H42 |
34.460 |
5.750 |
|
2. |
Bis-(2-ethylhexyl)-Sebacate |
426.60 |
C26H50O4 |
37.740 |
1.300 |
|
3. |
5-Eicosene |
282.50 |
C20H42 |
20.470 |
4.100 |
|
4. |
1-Octadecene |
252.50 |
C18H36 |
24.130 |
5.670 |
|
5. |
Squalene |
422.80 |
C30H50 |
38.050 |
53.540 |
|
6. |
2-(phenyl)thio)-octanal |
216.30 |
C14H16O5 |
44.460 |
2.570 |
|
7. |
Cyclotetradecanetrimethyl |
280.50 |
C20H40 |
48.540 |
4.310 |
2.7.2 GC-MS analysis
The aqueous extract of P. digitatum was analyzed using GC-MS. A 1-µL aliquot of the extract was injected into the chromatographic column (30 m × 0.32 mm × 0.25 µm) via the Autosampler AS 1300. For the first analysis, the initial temperature was set to 60℃ and then gradually increased to 240℃. It was further increased to 290℃ and maintained for 2 minutes. Helium was used as the carrier gas at a flow rate of 1 mL/min. Mass spectra were obtained using electron ionization (EI) at 70 eV in full scan mode over an m/z range of 40–1000. The compounds were identified by comparing their mass spectra and retention times (RT) with those retrieved from the National Institute of Standards and Technology (NIST) library [15]. Figure 2 presents the GC-MS chromatogram of the isolated bis(2-ethylhexyl) phthalate from P. digitatum. Figure 3 displays the determined MIC of the crude penicillin extract isolated from P. digitatum cultured on spoiled citrus against MDR B. cereus.
2.7.3 Antimicrobial susceptibility test
The antibacterial activity of Bis-(2-ethylhexyl) phthalate isolated compound from Penicillium digitatum was decisive by disc diffusion methods against the Gram-positive MDR B. cereus and Staphylococcus aureus and Gram-negative (Escherichia coli) [16]. Each bacterial isolate was suspended to a turbidity of 0.5 McFarland and plated on Muller-Hinton Agar. Discs containing concentrations of 100, 200, and 400 μg of the antibacterial activity of Bis-(2-ethylhexyl) phthalate compound were placed on the agar plates and incubated at 37℃ for 24 h. The zone of inhibition was detected.
Figure 2. GC-MS chromatogram of the isolated and identified Bis-(2-ethylhexyl) phthalate from P. digitatum cultured on spoiled citrus [17]
Figure 3. MIC of crude Penicillin compound extracted from P. digitatum cultured on spoiled citrus against MDR B. cereus (Arrow pointed to MIC ≥ 16 mg/mL)
2.7.4 MIC
The MIC of Bis-(2-ethylhexyl) phthalate was detected using the dilution assay [18].
2.8 Identification of bacterial isolates
The culture features of the bacteria were used to identify them, and a Gram stain was used to confirm the identification. Gram-positive and Gram-negative ID kits from Biomerieux, France, were used in conjunction with an automated microbiological system called Vitek2 for the identification process.
2.8.1 Bacterial morphological characterization
The gram-stained bacteria were examined under a microscope and categorized into different shapes (cocci, bacilli, irregular single, pairs, chains, or clusters) using the gram staining concept.
2.8.2 Detection of B. cereus by PCR
All PCR reactions were carried out in 50 µL reaction volume. The reaction mixture was prepared in a PCR tube. The primer used were:
Forward: 5-AGAGTTTGATCCTGGCTCAG-3
Backward: 5-GGTTACCTTGTTACGACTT-3
The PCR reaction included a step at 95℃ as an initial denaturation for 5 min, followed by 35 cycles of denaturation at 95℃ for 30 sec; primer annealing was done at 55℃ for 30 sec and extension at 75℃ for 30 sec; then, the final extension was done at 75℃ for 5 min.
2.8.3 Phylogenetic tree analysis
The 16S rDNA gene sequences were used to carry out BLAST with the database of the NCBI gene bank and were provisionally identified.
Six Bacillus spp. (27% of the total) were identified in this study. This result is in line with previous research showing that Bacillus spp. comprise 20% of clinical bacterial isolates from dental diseases [19]. Additionally, the current results agree with previous research, showing that various bacterial species are prevalent in dental plaque [20]. Based on morphological and biochemical properties, six isolates were identified as Gram-positive, motile, rod- or short rod-shaped bacteria, and all tested positive for IMViC. The results showed that none of the six isolates produced indole, but the spore-forming isolates were positive for nitrate reduction. The ability to hydrolyze starch was positive, while the ability to hydrolyze gelatin was negative. Table 2 and Figures 4 and 5 showed that all six isolates tested negative for oxidase and positive for catalase. Bacillus spp. are Gram-positive, rod-shaped, endospore-forming bacteria that were initially identified in the nineteenth century by Cohn and Koch. Gram-positive isolates C1–C6 were used in the present investigation. B. subtilis and short-rod-shaped B. cereus were also isolated and identified from oral samples [21].
Figure 4. Colony of B. cereus on blood agar (a) after 3 days of incubation at 37℃, (b) after 7 days of incubation at 37℃
Figure 5. B. cereus under microscope (100x)
Table 2. Biochemical characterization of B. cereus
|
Number of Isolates |
Gram Stain |
Catalase |
Oxidase |
Indol |
Motility |
Spore |
|
1 |
+ rods |
+ |
- |
- |
+ |
+ |
Figure 6. Agarose gel electrophoresis showing 16S rDNA amplicon of B. cereus at lanes 1, 2, and 3, where M refers to a 2 kb ladder
Six strains were amplified by polymerase chain reaction (PCR), and the identification was obtained by a comparative study of 16S rDNA gene sequences. When resolved on agarose, a single distinct PCR amplicon band of 1500 pb was seen in Figure 6. The results of identifying the B. cereus using NCBI Blast nucleotide sequences were shown in Figure 7 for detection and Table 3 for identification. In a study by Anitha et al. [19], the results for the identification of the B. cereus using NCBI Blast nucleotide sequence and comparison with reference strains in the same study are displayed in Table 3.
The activity testing on Mueller Hinton agar was conducted using six different antibiotic discs (Oxoid) that included the following antibiotics: Optochin (Op.5 µg), Doxycycline (DO.10 µg), Flucloxacillin (FOX.5 µg), Ciprofloxacin (CIP.5 µg), Tetracycline (TE.30 µg), and Nalidixic acid (NA. 5 µg). Based on the results of the zone of inhibition measurements, the susceptibility of the B. cereus bacterial isolates to the antibiotics was classified as sensitive, intermediate, or resistant according to the criteria set out by EUCAST. Ciprofloxacin (CIP.5 µg) was the sole antibiotic that proved effective against all six strains of B. cereus strains tested in this investigation while Optochin (OP.5 µg), Doxycycline (DO.10 µg), Flucloxacillin (FOX.5 µg), Tetracycline (TE.30 µg), and Nalidixic acid (NA.5 µg) all proved ineffective. Ciprofloxacin (CIP.5 µg) was found to have a 21 mm inhibition zone against all six strains of B. cereus (Figure 9 and Tables 4 and 5). All six bacterial strains of B. cereus exhibited high sensitivity to the antibiotic. One class of antibiotics known as fluoroquinolones includes ciprofloxacin (CIP.5 µg). There are other documented cases of invitation as well by Horii et al. [22]. It has been demonstrated by Citron and Appleman [23]. B. cereus usually shows resistance to penicillin and other β-lactam antibiotics.
Figure 7. Data of the identification of the B. cereus using NCBI Blast nucleotide sequence
Table 3. Data of the identification of the B. cereus using NCBI Blast nucleotide sequence and comparison with the reference strains
|
Description |
Score |
E Value |
Accession |
|
Buxilinx screen |
N/A |
N/A |
N/A |
|
Bacillinx screen, strain CoRIT, 155 ribosomal RNA gene, partial sequence |
4.4 |
0.407 |
MR341231 |
|
Uncoloured bacterium |
N/A |
N/A |
N/A |
|
Uncoloured bacterium clone 200_312, 155 ribosomal RNA gene, partial sequence |
4.4 |
0.409 |
K203284 |
|
Uncoloured bacterium clone DolO2, 45654, 153 ribosomal RNA gene, partial sequence |
4.4 |
0.007 |
J2031931 |
|
Uncoloured bacterium clone RNA/MP3N-155 ribosomal RNA gene, partial sequence |
4.4 |
0.009 |
K0033931 |
|
Uncoloured bacterium and full 15x DNA gene, clone D14815627 |
4.4 |
0.007 |
DR99329 |
|
Uncoloured bacterium clone OTU_161, 155 ribosomal RNA gene, partial sequence |
4.6 |
0.007 |
K004384 |
|
Uncoloured bacterium clone TTP-821, 155 ribosomal RNA gene, partial sequence |
4.5 |
0.007 |
K203286 |
|
Uncoloured bacterium clone 2_14, 155 ribosomal RNA gene, partial sequence |
4.5 |
0.007 |
R013137 |
|
Uncoloured bacterium clone 271424, 177 145 ribosomal RNA gene, partial sequence |
4.5 |
0.007 |
J000022 |
|
Uncoloured bacterium clone S19, 415 ribosomal RNA gene, partial sequence |
N/A |
N/A |
N/A |
According to a study by Khalifa et al. [24], a secondary metabolite of the antibacterial compound Bis-(2-ethylhexyl) phthalate showed antimicrobial activity against Gram-positive pathogenic bacteria B. cereus and Gram-negative pathogens isolates E. coli, suggesting its potential use in treating infections caused by these bacterial strains (Tables 4 and 5). Table 6 recorded ≥ 16 mg/ml as the MIC for the antimicrobial activity compound Bis-(2ethylhexy) phthalate against MDR B. cereus. The inhibition zones recorded for MDR B. cereus, S. aureus, and E. coli were 14 mm, 14.33 mm, and 12.82 mm, respectively. According to Tsoupras and Davi [25], the importance of fungi as a source of new molecules for drug discovery is clear; however, it is crucial to further investigate the compounds that have already been described in other to obtain more effective derivatives or analogs.
Figure 9. Effect of some antibiotics against B. cereus
Susceptibility testing: - OP = Optochin, DO= Doxycycline, NA= Nalidixic Acid, CIP= Ciprofloxacin, E= Erythromycin, FOX= Flucloxacillin
Table 4. Types of antibiotics under consideration in this study
|
No. |
Antibiotics |
Concentration (mcg) |
Antibiotics Inhibition Zone |
|
1 |
Optochin |
OP.5 |
R |
|
2 |
Doxycycline |
DO.10 |
R |
|
3 |
Flucloxacillin |
FOX. 5 |
R |
|
4 |
Ciprofloxacin |
Cip.5 |
S |
|
5 |
Tetracycline |
TE.30 |
R |
|
6 |
Nalidixic acid |
NA.5 |
R |
*R = Resistant, S = Sensitive
Table 5. The inhibition zone of some antibiotics and Bis-(2-ethylhexyl)-phthalate against MDR B. cereus isolated from some patients of dental diseases
|
No. |
Bacterial Isolate |
Inhibition Zone (mm) |
|
1 |
Bacillus cereus |
14.00 |
|
2 |
Staphylococcus aureus |
14.33 |
|
3 |
Escherichia coli |
12.82 |
Table 6. MIC for antimicrobial compound Bis-(2-ethylhexyl)-phthalate against MDR B. cereus isolated from some patients of dental diseases
|
Sample |
Dilution of Bis-(2-ethylhexyl) Phthalate Compound (mg/ml) |
||||||
|
Bacillus cereus |
≥ 64 |
≥ 32 |
≥ 16 |
≥ 8 |
≥ 4 |
≥ 2 |
≥ 1 |
|
- |
- |
- |
+ |
+ |
+ |
+ |
|
This study demonstrated that B. cereus accounts for 27% of the total bacterial isolates from patients with dental diseases, underscoring its role in dental infections. Early clinical resolution was closely associated with the use of appropriate empirical antibiotic therapy. Among the tested antibiotics, Ciprofloxacin (CIP.5 µg) emerged as the most effective agent for empirical treatment of B. cereus infections, although further clinical studies are required to substantiate these findings. Moreover, Bis-(2-ethylhexyl) phthalate, a microbial secondary metabolite from P. digitatum, exhibited significant activity against B. cereus. This compound holds promise as a potential therapeutic agent against infections caused by both Gram-positive pathogens like B. cereus and Gram-negative bacteria, such as E. coli. However, additional clinical and pharmacological studies are necessary to fully elucidate its therapeutic potential and to explore its application in treating dental and other systemic infections.
The authors wish to thank all staff of the Microbiology Unit, especially Mr. Qassem Al-Mousawy, the laboratory official, and all the laboratory workers.
This study was achieved by the research ethics committee at the University of Misan.
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