Original article / research

Year : 2024 | Month : July | Volume : 18 | Issue : 7 | Page : DC01 - DC04

Full Version

Detection of gbpA and gbpB in Streptococcus mutans Isolated from Patients with Oral Potentially Malignant Disorders: A Pilot Study

Published: July 1, 2024 | DOI: https://doi.org/10.7860/JCDR/2024/69002.19582

I Shabnam Tamanna, AS Smiline Girija, J Vijayashree Priyadharsini

1. Undergraduate Student, Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, Tamil Nadu, India. 2. Professor and Head, Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, Tamil Nadu, India. 3. Associate Professor, Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, Tamil Nadu, India.

Correspondence Address :
Dr. AS Smiline Girija,
Professor and Head, Department of Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, PH Road, Chennai-600077, Tamil Nadu, India.
E-mail: smilinejames25@gmail.com

Abstract

Introduction: Glucan-binding proteins (gbps) in Streptococcus mutans (S. mutans) are considered vital virulence factors contributing to plaque formation and caries progression. These proteins also aid in maintaining biofilm formation on the tooth surface and further colonisation of S. mutans.

Aim: To phenotypically characterise S. mutans from clinical samples of patients with Oral Potentially Malignant Disorder (OPMD), healthy individuals with and without caries, and to assess the frequency of the gbpA and gbpB genes among the groups.

Materials and Methods: This pilot study was conducted for a period of two months from May to June 2023 in the Department of Microbiology at Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India. Saliva samples (N=60) were collected from 20 patients in each of the three different groups: Group 1 included OPMD cases, Group 2 comprised healthy individuals with caries, and Group 3 consisted of healthy individuals without caries (control). Demographic data including age, gender, geographical location, and any previous history of clinical illness were recorded. The samples were promptly transferred to the microbiology lab and cultured on sterile Mutans Sanguis (MS) agar, followed by incubation at 37°C for 48 hours. S. mutans were phenotypically characterised, and the frequency of the gbpA and gbpB genes was assessed using Polymerase Chain Reaction (PCR).

Results: The prevalence of S. mutans among the study population was found to be 9 (45%) in Group 1, 8 (40%) in Group 2, and 3 (15%) in Group 3. The study findings revealed the presence of gbps in S. mutans isolated from OPMD cases, patients with caries, and non-cariogenic healthy patients, with the frequency of gbpA as 8 (88%), 7 (87.5%), and 1 (33.3%), and gbpB as 9 (100%), 5 (62.5%), and 1 (33.3%), respectively.

Conclusion: The frequency of gbpA and gbpB from the clinical strains of S. mutans associated with caries and OPMD cases was observed in the present study. Periodic surveillance of such virulent determinants would aid in a theragnostic approach to alleviate the complications caused by S. mutans in OPMD cases.

Keywords

Caries, Pathogenesis, Oral health, Virulence

Introduction

Cancer is considered a major global health and economic burden in developing countries (1). According to the International Agency for Research on Cancer (IARC), India’s cancer incidence is expected to rise by over 1.7 million cases by 2035, along with an increase in the cancer-related death rate to 2 million cases (2). Premalignant diseases are typically located on the floor of the mouth, tongue, gingivae, and buccal mucosa (3). As stated by the World Health Organisation (WHO), Candida leukoplakia, lichen planus, oral submucous fibrosis, verrucous hyperplasia, keratoacanthoma, and erythroplakia are some of the Oral Potentially or Premalignant Malignant Disorders that often act as precursors to invasive squamous cell carcinoma of the oral cavity (4). The most common risk factors in the development of OPMDs are tobacco chewing, smoking, and long-term alcohol consumption (5).

One widely researched area is the potential link between the altered oral microbiota of oral lesions, molecular alterations, and the development of cancer (6). The oral cavity of healthy individuals is home to over 700 different types of bacteria, mostly commensals that are crucial for maintaining oral homeostasis by reducing inflammation and producing proinflammatory cytokines (7),(8). In association with OPMD lesions, the most commonly reported organisms include Fusobacterium spp., Veillonella spp., Actinomyces spp., Clostridium spp., Haemophilus spp., Enterobacteriaceae spp., Prevotella spp., Porphyromonas gingivalis, Capnocytophaga gingivalis, and Streptococci spp. Among these, the most prevalent gram-positive, facultative anaerobic coccus in the human oral cavity is S. mutans. Despite being a commensal of the oral cavity (9), S. mutans is a major bacterial organism responsible for the pathogenesis of dental caries, periodontal diseases, and other oral diseases. The production of extracellular polysaccharides, development of biofilm, protein alterations, and acid generation due to the role of virulent genes and proteins like Gtfs, Gbps, and Ftfs attribute to S. mutans pathogenesis, leading to caries (10). Due to its significant presence in oral diseases, the presence of S. mutans was suspected in oral premalignant lesions.

S. mutans produces a class of proteins called gbps that support dental biofilm maintenance and cell attachment to teeth, promoting the production of plaque and dental caries. There are four types of gbp genes-gbpA, gbpB, gbpC, and gbpD- that S. mutans produces and are responsible for biofilm formation. The glucosyltransferases (gtfs) genes encode glucan-binding enzymatic proteins whose ability to bind glucan keeps them cell-associated even in the absence of a cell wall anchor (11). The gbps significantly contribute to the development of biofilms, sucrose-dependent adherence, and the preservation of a solid, symbiotic microbial community in the oral cavity (12). The production of gbps is often influenced by pH (12). The GbpA protein encoded by gbpA is known to facilitate cellular adhesion to the surface (13), and the GbpB protein encoded by gbpB plays a vital role in peptidoglycan formation, maintaining cell integrity (14). With this literature background, it is evident that gbp are important virulence factors of S. mutans in dental caries pathogenesis, yet a gap exists regarding their frequency in cases with OPMDs. This highlights the need for a novel study to understand the frequency of gbps among S. mutans strains from different groups of individuals and to compare the frequency with healthy individuals. This study aimed to phenotypically characterise S. mutans from clinical samples of patients with OPMD, healthy individuals with and without caries, and to assess the frequency of gbpA and gbpB in the clinical strains of S. mutans.

Material and Methods

This pilot study was conducted at Saveetha Dental College, Chennai, Tamil Nadu, India for a period of two months from May to June 2023. The investigation was designed as a pilot study, so power analysis was not performed, and the study was conducted with a minimum sample size of 20 in each group. Institutional human ethical clearance and consents were obtained prior to the collection of the samples (Ref: SRB/SDC/UG-2141/23/MICRO/125, IHEC/SDC/UG-2141/23/MICRO/275). Saliva samples were collected in sterile specimen containers and immediately transferred to the Microbiology laboratory for processing. The samples were streaked onto sterile Mitis Salivarius (MS) agar supplemented with sucrose and incubated at 37°C for 48 hours. After incubation, the colonies were phenotypically characterised by Gram staining and a negative catalase test (15).

Sampling and isolation of S. mutans: The study population included three groups of patients (N=60), with 20 individuals (n) in each group: OPMD patients (group 1), healthy individuals with caries (group 2), and healthy individuals without caries (group 3). As a comparative evaluation of the case-control groups, healthy individuals with and without caries were included.

Inclusion criteria: Patients with specific clinical manifestations of oral premalignant disorders such as leukoplakia, lichen planus, oral submucous fibrosis, verrucous hyperplasia, keratoacanthoma, and erythroplakia were included in group 1. Since S. mutans is a potent cariogenic pathogen, healthy patients with caries were included in group 2. As S. mutans is a normal oral commensal, a third group was included comprising healthy individuals without caries, acting as the control group.

Exclusion criteria: Patients with other manifestations of wounds or accidental lesions and patients under antibiotic therapy were excluded from the study.

Molecular detection of gbpA and gbpB: Following the manufacturer’s recommendations, genomic DNA was isolated from fresh cultures of S. mutans using a Qiagen extraction kit, and it was then stored at -20°C until future need. A 15 μL amplification reaction mixture was prepared using 5.6 μL of double-distilled water and 7.8 μL of 2× Master Mix from Takara, Japan. Specific primers for gbpA and gbpB were added (Table/Fig 1), and the PCR conditions were set for 36 cycles in a thermocycler (Eppendorf Mastercycler, Germany). The PCR amplicons were visualised on a 1% agarose gel electrophoresis and confirmed using a 100-bp DNA ladder (Table/Fig 2).

Results

S. mutans formed smooth, raised, adhesive colonies with a characteristic frosted glass appearance on the MS agar. Gram staining showed gram-positive cocci in short chains. The negative catalase test excluded the possibility of Staphylococci spp. being present, further confirming the presence of Streptococci spp. (Table/Fig 3).

Frequency of S. mutans and detection of gbpA and gbpB: (Table/Fig 4) displays the frequency of S. mutans, gbpA and gbpB.

Discussion

The OPMD are a group of lesions and conditions that may precede the development of Oral Squamous Cell Carcinoma (OSCC). They usually form due to prolonged mechanical and chemical irritation of the oral mucosa in association with various risk factors. Such abnormal OPMD lesions have an altered microbiome compared to the healthy oral mucosa, contributing to the progression of the lesion to tumorous tissues. S. mutans is known to be attributed to this progression with its potent virulence factors. Three key characteristics of S. mutans, such as the production of large amounts of glucan extracellular polymers from sucrose, acidogenicity, and the ability to thrive in harsh environments, make it a potent pathogen in the oral cavity (16). However, the role of S. mutans and its mechanisms in OPMD conditions are still unclear.

Alterations in the oral microbiome transform Streptococci into a major potential biomarker, as documented in a study where oral swabs from OSCC lesions showed differences in the Streptococcus spp. count between the case and the control (17). In this context, in the present investigation, it was found that the frequency of S. mutans in the saliva of patients with OPMDs was the highest (45%), slightly higher than in healthy individuals with caries (40%), and lowest (15%) in the group with healthy individuals. The property of bacterial adherence to mucosal lesions and surfaces suggests that S. mutans adheres to OPMD lesions and plays an important role in colonisation and the spread of infection in such cases (18),(19).

In recent years, there could be a drastic change in the prevalence and frequencies, may be due to the host and environmental factors. Being a polymicrobial conglomeration, the carious scrapings in the present study would have shown the presence of S. mutans in only eight cases and not in the other 12 cases and this is a unique finding if this study.

Similar findings were found in a study conducted by Salman HA et al., (20).

The increased prevalence of S. mutans in OPMD patients could indicate that the ability to form biofilms and adhere to the lesions is potent among these clinical strains. Among various virulence determinants, gbps are a group of virulent proteins responsible for their ability to bind glucan and assist S. mutans colonies in colonising the oral mucosa even in the absence of cell wall membrane proteins (11). gbpA and gbpB proteins encoded by the respective genes are known for their roles in cellular adhesion and peptidoglycan formation, respectively, underlying caries pathogenesis. Glucans are glucose homopolymers that make up glycans and play a vital role in pathogenesis by their ability to attach to the oral mucosal epithelium (21). The high frequency of gbpA and gbpB in this study may be linked to the S. mutans’ ability to adhere more strongly and form robust biofilms. When compared to its frequency among individuals with caries, its role may be correlated with increased colonisation and progression of the lesions in OPMD cases. Similar observations were documented in an earlier study where higher synthesis of glycans and lactic acid was observed from the clinical strains of S. mutans derived from prosthesis patients and patients with cancer (22).

Another observation made in this study is the low percentage (62.5%) of gbpB in healthy individuals with caries, while it was a striking 100% in OPMD patients. This contrasts with an earlier study on the gpbB genes, which showed nearly 85% in comparison with the gpbA gene, with a lesser prevalence of 80% in the subjects studied (23). This difference could be due to the unclear function of gbpB in the pathogenesis of caries and the fact that caries pathogenesis is mainly due to the property of adherence of S. mutans to the tooth structure, which is facilitated by GbpA and GbpC proteins (24). The frequency of gbpA and gbpB was lower in the control group, supporting less virulence among the commensal traits in the oral cavity. Looking towards future prospects, the information provided in this study could be beneficial in developing novel drugs that target these specific genes gbpA and gbpB, leading to reduced complications in cancerous lesions and other orodental disease manifestations. The frequency of various other virulent genes should also be assessed using a larger sample size. This is crucial because the clinical significance of gpbA and gpbB is closely associated with bacterial adherence to the tooth surface and is critical in the formation of the plaque matrix. Additionally, with the prevalence of S. mutans and drug-resistant traits being high in dental settings (25),(26), it is crucial to regularly monitor their occurrence in all dental clinics. Predicting putative vaccine candidates for priority pathogens is common through computational approaches (27),(28), and gbps may be considered as a novel target in designing an anti-caries vaccine.

Limitation(s)

The limitation of the study was that, being relatively new, the available data for comparison ranges from minimal to none. Although the potential for result distortion exists due to the small sample size, statistical analysis could not be conducted. The limited sample size could also hinder the ability to predict results on a larger scale. While S. mutans is a significant microorganism, this study only assessed the frequency of gbp types A and B in S. mutans present in OPMDs and did not explore the association of other types of virulence factors.

Conclusion

The prevalence of S. mutans was comparatively higher in OPMD patients than in healthy individuals with and without caries. The findings of the study document the frequency of gbpA and gbpB among the clinical strains of S. mutans in association with OPMD cases and caries. gbpA and gbpB being found to be frequent among the test strains, the findings of the study may be used to design novel drugs targeting these genes both for the diagnosis and treatment of oral lesions in OPMD cases.

Authors contribution: IST contributed for the literature search, data collection analysis and manuscript drafting. ASSG contributed for the conceptualisation, design, data verification, manuscript review. JVP contributed for the final verification and review of the manuscript.

Reference


1.	Kanavos P. The rising burden of cancer in the developing world. Ann Oncol. 2006;17(S8):15-23. [CrossRef] [PubMed]
2.	Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends- An update. Cancer Epidemiol Biomarkers Prev. 2016;25(1):16-27. [CrossRef] [PubMed]
3.	Schwimmer E. Die idopathisches Schleimhaut-plaques der Mundhohle (leukoplakia buccalis). Arch Derm Syphilol. 1877;9:511-70.
4.	Van der Waal I. Oral leukoplakia, the ongoing discussion on definition and terminology. Med Oral Patol Oral Cir Bucal. 2015;20(6):e685-92. Doi: 10.4317/medoral.21007. PMID: 26449439; PMCID: PMC4670248. [CrossRef] [PubMed]
5.	Sabashvili M, Gigineishvili E, Jikia M, Chitaladze T. Role of tobacco in the development of oral leukoplakia and oral cancer. Dentistry. 2018;8(6):1000495. [CrossRef]
6.	Brook I, Gober AE. Effect of smoking cessation on the microbial flora. Arch Otolaryngol Head Neck Surg. 2007;133(2):135-38. [CrossRef] [PubMed]
7.	Hezel MP, Weitzberg E. The oral microbiome and nitric oxide homoeostasis. Oral Dis. 2015;21(1):07-16. [CrossRef] [PubMed]
8.	Bik EM, Long CD, Armitage GC, Loomer P, Emerson J, Mongodin EF, et al. Bacterial diversity in the oral cavity of 10 healthy individuals. ISME J. 2010;4(8):962-74. [CrossRef] [PubMed]
9.	Girija ASS, Ganesh PS. Functional biomes beyond the bacteriome in the oral ecosystem. Jpn Dent Sci Rev. 2022;58(19):217-26. [CrossRef] [PubMed]
10.	Shui Y, Jiang Q, Lyu X, Wang L, Ma Q, Gong T, et al. Inhibitory effects of sodium new houttuyfonate on growth and biofilm formation of Streptococcus mutans. Microb Pathog. 2021;157:104957. [CrossRef] [PubMed]
11.	Banas JA, Vickerman MM. Glucan-binding proteins of the oral streptococci. Crit Rev Oral Biol Med. 2003;14(2):89-99. [CrossRef] [PubMed]
12.	Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases: Role in extracellular matrix formation of cariogenic biofilms. Caries Res. 2011;45(1):69-86. [CrossRef] [PubMed]
13.	Matsumi Y, Fujita K, Takashima Y, Yanagida K, Morikawa Y, Matsumoto-Nakano M. Contribution of glucan-binding protein A to firm and stable biofilm formation by Streptococcus mutans. Mol Oral Microbiol. 2015;30(3):217-26. [CrossRef] [PubMed]
14.	Mattos-Graner RO, Jin S, King WF, Chen T, Smith DJ, Duncan MJ. Cloning of the Streptococcus mutans gene encoding glucan binding protein B and analysis of genetic diversity and protein production in clinical isolates. Infect Immun. 2001;69(11):6931-41. [CrossRef] [PubMed]
15.	Ge Y, Caufield PW, Fisch GS, Lia Y. Streptococcus mutans and Streptococcus sanguinis colonization correlated with caries experience in children. Caries Res. 2008;42(6):444-48. [CrossRef] [PubMed]
16.	Lemos JA, Palmer SR, Zeng L, Wen ZT, Kajfasz JK, Freires IA, et al. The biology of Streptococcus mutans. Microbiol Spectr. 2019;7(1):10-128. [CrossRef] [PubMed]
17.	Su SC, Chang LC, Huang HD, Peng CY, Chuang CY, Chen YT, et al. Oral microbial dysbiosis and its performance in predicting oral cancer. Carcinogenesis. 2021;42(1):127-35. [CrossRef] [PubMed]
18.	Beachey EH. Bacterial adherence: Adhesin-receptor interactions mediating the attachment of bacteria to mucosal surface. J Infect Dis. 1981;143(3):325-45. [CrossRef] [PubMed]
19.	Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: From the natural environment to infectious diseases. Nat Rev Microbiol. 2004;2(2):95-108. [CrossRef] [PubMed]
20.	Salman HA, Senthilkumar R, Imran K, Selvam KP. Isolation and typing of Streptococcus mutans and Streptococcus sobrinus from caries-active subjects. Contemp Clin Dent. 2017;8(4):587-93. Doi: 10.4103/ccd.ccd_610_17. PMID: 29326510; PMCID: PMC5754980. [CrossRef] [PubMed]
21.	Pinho SS, Reis CA. Glycosylation in cancer: Mechanisms and clinical implications. Nat Rev Cancer. 2015;15(9):540-55. [CrossRef] [PubMed]
22.	Alanazi SA, Alduaiji KT, Shetty B, Alrashedi HA, Acharya BG, Vellappally S, et al. Pathogenic features of Streptococcus mutans isolated from dental prosthesis patients and diagnosed cancer patients with dental prosthesis. Microb Pathog. 2018;116:356-61. [CrossRef] [PubMed]
23.	Hassan AT, Al-Alaq FT. Correlation between cell surface adhesins genes and biofilm formation in Streptococcus mutans bacteria isolated from dental caries. HIV Nursing. 2023;23(1):753-58.
24.	Matsumura M, Izumi T, Matsumoto M, Tsuji M, Fujiwara T, Ooshima T. The role of glucan-binding proteins in the cariogenicity of Streptococcus mutans. Microbiol Immunol. 2003;47(3):213-15. [CrossRef] [PubMed]
25.	Malini M, Thomas TK, Bhargava D, Girija S. Microbiology of the white coat in a dental operatory. Indian J Dent Res. 2012;23(6):841. [CrossRef] [PubMed]
26.	Smiline GAS, Pandi SK, Hariprasad P, Raguraman R. A preliminary study on the screening of emerging drug resistance among the caries pathogens isolated from carious dentine. Indian J Dent Res. 2012;23(1):26-30. [CrossRef] [PubMed]
27.	Smiline Girija AS. Delineating the immuno-dominant antigen vaccine peptides against gacs sensor kinase in: An investigational approach. Front Microbiol. 2020;11:2078. [CrossRef] [PubMed]
28.	Sankar S. In silico design of a multi-epitope Chimera from Aedes aegypti salivary proteins OBP 22 and OBP 10: A promising candidate vaccine. J Vector Borne Dis. 2022;59(4):327-36. Doi: 10.4103/0972-9062.353271. PMID: 36751764. [CrossRef] [PubMed]

DOI and Others

DOI: 10.7860/JCDR/2024/69002.19582

Date of Submission: Dec 09, 2023
Date of Peer Review: Feb 15, 2024
Date of Acceptance: May 15, 2024
Date of Publishing: Jul 01, 2024

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Dec 11, 2023
• Manual Googling: Feb 20, 2024
• iThenticate Software: May 14, 2024 (10%)

ETYMOLOGY: Author Origin

EMENDATIONS: 7

Tables and Figures

[Table/Fig-1]

[Table/Fig-2]

[Table/Fig-3]

[Table/Fig-4]