The Surface Structure, Chemical Molecules, and Thickness of Aggregatibacter actinomycetemcomitans biofilms by Different Inducers Agent

 

Indah Listiana Kriswandini¹*, Tantiana¹, Aulya Ariffany Mahardhika², Aqsa Sjuhada Oki¹

¹Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

²Undergraduate Student Faculty of Dental Medicine, Universitas Airlangga, Surabaya- Indonesia.

 

ABSTRACT:

Aggregatibacter actinomycetemcomitans caused oral infection in recent years. It requires advancements in health technology, such as infection and sickness detection kits. This study purposed to provide data to aid in the detection kits. Aggregatibacter actinomycetemcomitans was prepared to be biofilm cultured sample. Then the samples were induced by glucose, lactose, soy protein, and iron. Scanning Electron Microscopy Energy Dispersive X-Ray, Confocal Laser Scanning Microscopy, and Olympus FluoView ver 4.2a were used to analyze the ultrastructural dimension of biofilms. The surface structure of biofilm induced by iron differed from that of biofilms induced by other inducers. O(78.65±10.32) and Cl(1.28±0.21) were the most abundant chemical compounds induced by glucose; S(14.67±4.56) and Fe(37.46±59.62) were induced by lactose; C(25.41±16.36) and N(13.49±9.37) were induced by soy protein; and P(58.53±17.80) was induced by iron. Iron induced the highest amount of polysaccharides (605.641±71.66). Lactose (857.877±70.86) induced the greatest number of bacterium cells. Iron (11.000±1000)nm caused the thickest layer. The biofilm of Aggregatibacter actinomycetemcomitans induced by glucose, lactose, soy protein, and iron are all different.

 

 

 

INTRODUCTION: 

Dental and problems had been increasing for the latest years. Based on Basic Health Research, Indonesia confirmed that the dental and problems in 2007 were 23.2% and increasing in 2013 reached 25.9%. The second-highest rank of dental and problems was periodontal disease which reached 96.58% caused by Aggregatibacter actinomycetemcomitans^1-5.

 

The reasons above provoked the researcher to develop health research which was an infection disease detection kit. The kit is meant to detect infection earlier with biofilm as an indicator because 80% of infections are caused by biofilm^6-9. Biofilm could form after being induced by daily food such as glucose, lactose, soy protein, and iron.

 

Glucose and lactose were contained by monosaccharide, increasing the biomass that affects Extracellular Polymer Substances (EPS) to form a biofilm^10-13. Soy protein is contained with protein that directly forms the biofilm by making fibril on the surface^14-15. Iron, either Fe²⁺ or Fe³⁺, stimulated microcolony to form biofilm early and acted directly to EPS^16-18.

 

The size of Aggregatibacter actinomycetemcomitans bacteria, which was 0.4-0.5 x 1.0-1.5 µm, needed microscopy to examine biofilm in ultrastructural dimension. Scanning Electron Microscopy-Energy Dispersive X-ray (SEM-EDX) could be used to examine the surface structure and analyze the chemical molecules contained in biofilm^19-21. Confocal Laser Scanning Microscopy (CLSM) could be used to examine the 3D structure and thickness of biofilm because of its sensitivity, resolution, and ability to penetrate the biofilm^22,23.

 

 

In this work, the observation of Aggregatibacter actinomycetemcomitans biofilm properties with different inducers is concerned. The purpose of the research was to examine the difference in surface structure, chemical molecules, and thickness of Aggregatibacter actinomycetemcomitans' s biofilm induced with several inducers such as glucose, lactose, soy protein, and iron. The research data can be used to support the kit for detecting infectious diseases using biofilm as the indicator.

 

MATERIALS AND METHODS:

The research design was analytical descriptive research with ethical clearance number 249/HRECC. FODM/V/2019. The samples were one for each inducer and after the sample preparation was conducted, the sample was examined with the microscope in five different regions for SEM-EDX and three different regions for CLSM. The study was carried out in the Laboratory of Microbiology in Faculty of Dental Medicine and Electron Microscopy in Faculty of Medicine, Universitas Airlangga; Scanning Electron Microscopy Laboratory in Mechanical Engineering, Institut Teknologi Sepuluh Nopember; and Central Laboratory of Life Science, Universitas Brawijaya. The procedures that were prepared biofilm culture, SEM sample, and CLSM sample.

 

The biofilm cultures were started with culturing Aggregatibacter actinomycetemcomitans ATCC 29522 in Brain Heart Infusion Broth incubated for 24 hours in the anaerobic jar until it reached Mc Farland standard 5. Then the samples were induced by 5% glucose, 5% lactose, 5% soy protein, and 2% iron. The samples were incubated in the anaerobic jar for 24 hours before they became fixated with 2% glutaraldehyde and proceeded to freeze-drying and critical point drying. After the drying process, samples were stamped into the holder and coated with pure gold for the conductor. The samples then became examined with Scanning Electron Microscopy HITACHI FLEXSEM 1000.

 

The biofilm cultures were started with culturing Aggregatibacter actinomycetemcomitans ATCC 29522 in a 24-well microplate with Brain Heart Infusion Broth incubated for 24 hours’ anaerobic jar until it reached Mc Farland standard 5. Then the samples were induced by 5% glucose, 5% lactose, 5% soy protein, and 2% iron. The samples were incubated in the anaerobic jar for 24 hours before they became fixated with 4% paraformaldehyde and washed with saline solution. After it became fixated, the samples were stained with Propidium Iodide for 15 minutes in the darkroom^24,25. The samples were then rewashed with saline solution and stained with ConA-FITC for 15 minutes in the darkroom and then proceed to wash with saline solution again and examined with CLSM Olympus Type FV1000.

 

The qualitative and quantitative analysis of biofilm used SEM EDX APEX for surface structure and chemical molecules also Olympus FluoView ver 4.2a. The qualitative data were being analyzed with the description of surface structure.

 

 

RESULT:

 

Figure 1: (A) Aggregatibacter actinomycetemcomitans biofilm induced by glucose, (B) Biofilm induced by lactose (C) Biofilm induced by soy protein, and (D) Biofilm induced by iron.

 

The surface structure of Aggregatibacter actinomycetemcomitans biofilms induced by glucose, lactose, soy protein, and iron was revealed by SEM-EDX imaging (Figure 1). Aggregatibacter actinomycetemcomitans biofilm induced by glucose showed the island-like biofilm with low-density microcolony that performed with cavities in biofilm. The lactose and soy protein grew widespread with the high density of microcolony without a cavity in lactose biofilm; meanwhile, it showed cavities in soy protein biofilm. The most different biofilm was biofilm induced by iron which showed bacterias coated with amorf caused by Fe³⁺.

 

The image was then confirmed with an EDX analysis image (Figure 2). The white-black area meant the conductor could not coat the biofilm equally, which meant there were cavities in the biofilm that showed in glucose and soy protein biofilm ^25,26. The homogenous color in lactose biofilm means there were no cavities in the biofilm. The white-black area in soy protein biofilm explained that there were cavities. The amorf form could be seen in the biofilm induced by iron.

 

Table 1: Chemical molecules of Aggregatibacter actinomycetemcomitans biofilm that induced by glucose, lactose, soy Protein, and Iron.

Molecules	Glucose	Lactose	Soy Protein	Iron
C	15.92 ± 5.11	21.06 ± 2.31	25.41 ± 16.36	10.35 ± 4.06
N	12.65 ± 2.36	13.14 ± 1.50	13.49 ± 9.37	9.83 ± 4.25
O	78.65± 10.32	36.60 ± 3.79	31.46 ± 23.39	44.46 ± 17.78
P	33.16 ± 4.86	47.06± 14.19	44.40 ± 34.29	58.53 ± 17.80
S	9.86 ± 0.39	14.67 ± 4.56	13.20 ± 5.07	14.02 ± 4.28
Fe	2.06 ± 0.50	37.46± 59.62	3.52 ± 5.19	18.24 ± 7.65
Cl	1.28 ± 0.21	1.24 ± 0.57	0.97 ± 0.11	1.10 ± 0.32

 

 

Figure 2: (A) Glucose showed the black-white area, (B) Lactose showed homogenous area, (C) Soy protein showed black-white are, (D) Iron showed bacteria coated with amorf.

 

The chemical molecules analysis with SEM-EDX analysis showed quantitative differences in chemical molecules that consisted in biofilm formation (Table 1). The highest amount of chemical molecules of Aggregatibacter actinomycetemcomitans that induced by glucose were O (78.65 ± 10.32) and Cl (1.28 ± 0.21). The highest amount of chemical molecules of biofilm that induced by lactose were S (14.67 ± 4.56) and Fe (37.46 ± 59.62). The highest chemical molecules induced by soya protein were C (25.41 ± 16.36) and N (13.49 ± 9.37). The highest amount of chemical molecules induced by iron was P (58.53 ± 17.80). That also happened in polysaccharides, dead cells, and thickness of biofilm formation in CLSM examination (Table 2). It happened because of the different pathways of inducers to form a biofilm.

 

 

Figure 3: (A) Glucose-induced biofilm; (B) Lactose-induced biofilm; (C) Soy protein-induced biofilm; (D) Iron-induced biofilm.

 

Table 2. 3D structures of Aggregatibacter actinomycetemcomitans biofilm induced glucose, lactose, soy protein, and iron.

3D Structures	Glucose	Lactose	Soy Protein	Iron
Polysaccharide	497.225 ± 59.52	566.829 ± 20.10	256.92 ± 9.57	605.641 ± 71.66
Bacteria Cells	652.019± 184.01	857.877 ± 70.86	461.77 ± 226.68	843.280 ± 28.24

 

Table 3: The thickness of Aggregatibacter actinomycetemcomitans biofilm induced glucose, lactose, soy protein, and iron.

Properties	Glucose	Lactose	Soy Protein	Iron
Thickness	7.000 ± 0 nm	8.333 ± 1154 nm	6.000 ± 1000 nm	11.000 ± 1000 nm

 

The 3D structures with CLSM examination could not give any interpretation except the appearance of fluorescence which gave the interpretation of polysaccharides and bacteria cells. The polysaccharides showed the green fluorescence in the image, and the bacteria cells showed red fluorescence (Figure 3). Further analysis for the existence of polysaccharides and bacteria cells was examined in quantitative data (Table 2). The highest amount of polysaccharides was in Aggregatibacter actinomycetemcomitans that induced by Iron. Aggregatibacter actinomycetemcomitans which was stimulated by lactose, had the most bacteria cells. The thickest biofilm was in Aggregatibacter actinomycetemcomitans that induced by iron (Table 2 and Table 3).

 

DISCUSSION:

The surface structure of Aggregatibacter actinomycetemcomitans induced by glucose, lactose, soy protein, and iron was different. The most different biofilm was induced by iron because the iron pathway started with the extracellular membrane of biofilm, which absorbed Fe³⁺ in the bacterial stage and formed the conductive area used as electron transport to every part of EPS that differs from the biofilm induced by logam and non-logam^27-29.

 

The chemical molecules in the biofilm, C, O, and S, had a strong bond from the EPS component, which was polysaccharides- sulfate. The chemical molecules of Fe in biofilm had the function to stabilize the colonization of bacteria in a strong bonded glocoronic acid- iron³⁰. The chemical molecules of Cl in biofilm had an antibacterial activity that made the biofilm easily damaged³¹. The chemical molecules of S, P, and N, had the function to form a biofilm granule that coated the heterotroph, autotroph, and microalgae species to maintain growth and the biofilm mass²⁸.

 

The number of polysaccharides as the adhesive agent, cohesion, protective barrier, the integrity of structure stabilization had the function in protecting biofilm from antimicrobial penetration so that the higher amount of polysaccharides, the more substantial biofilm protect itself from damage^29,32-34. The thickness and density of bacteria cells were formed because of quorum sensing, which affects the biofilm virulence. The thicker and denser biofilm, the easier biofilm to protect itself from antimicrobial penetration^35-37.

Aggregatibacter actinomycetemcomitans induced by glucose, lactose, soy protein, and iron showed the chemical components such as carbon, nitrogen, oxygen, sulfur, phosphate, ferric, and chloride were different because they can be associated with an amino acid that involving biofilm formation³⁸. The specific differences of chemical molecules could not be assessed accurately because of the lack of SEM-EDX measurements so that it needs further research to examine chemical compounds. Chemical compounds would accurately give the information of the way chemical components were being in a biofilm.

 

The lack of SEM-EDX was the measurement that limited chemical molecules could not explain how samples contained with kind of chemical molecules. Meanwhile, the lack of CLSM was the limited magnification, which could not support the more explicit images if the samples size was smaller than a nanometer²⁵. The finding of the different chemical molecules in this research proved differences in the biofilm induced with glucose, lactose, soy protein, and iron. It would be better if the new research conducts another examination of chemical compounds that will explain the dominancy of chemical molecules in each Aggregatibacter actinomycetemcomitans biofilm induced by glucose lactose, soy protein, and iron.

 

The differences in surface structures, chemical molecules, and thickness were caused by the different pathways of inducers to form a biofilm. The kind of inducers would create the specific appearance that can be understood after being examined with SEM-EDX, which will appear as the density of bacteria, the specific form in iron-induced biofilm, and the composition of polysaccharides, dead cells, and thickness that were being examined with CLSM. The lack of information for the research of chemical molecules was limiting the researcher to explain why chemical molecules exist in each biofilm so that it needs further examination about chemical compounds to explain clearly about chemical molecules.

 

 

CONCLUSION:

Aggregatibacter actinomycetemcomitans biofilm induced by glucose, lactose, soy protein, and iron had differences in surface structure, chemical molecules, and thickness of biofilm that will support the infection disease detection kit research. Different paths of inducers to build a biofilm induced variances in surface structure, chemical molecules, and thickness.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

REFERENCES:

1.     DEPKES R. Riset Kesehatan Dasar. 2013

2.     Ridwan RD, Sidarningsih, Wijayanti U. The Anti-Bacterial Activity of Gingival Mucoadhesive Patch from Thymus vulgaris Essential Oil towards Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. Research Journal of Pharmacy and Technology 2021; 14: 645–649. doi.org/10.5958/0974-360X.2021.00115.3

3.     Wijaksana I. Infectobesity dan Periodontitis: Hubungan Dua Arah Obesitas dan Penyakit Periodontal. Odonto : Dental Journal 2016; 3: 67–73. doi.org/10.30659/odj.3.1.67-73

4.     Nazir MA. Prevalence of periodontal disease, its association with systemic diseases and prevention. Int J Health Sci (Qassim) 2017; 1: 72–80.

5.     Nugraha AP, Sibero MT, Nugraha AP, et al. Anti-Periodontopathogenic Ability of Mangrove Leaves (Aegiceras corniculatum) Ethanol Extract: In silico and in vitro study. European Journal of Dentistry 2022. doi.org/10.1055/s-0041-1741374

6.     Ni’mah M, Kriswandini IL, Baktir A. Antigenic Protein Profile of Streptococcus mutans Biofilm for Developing of Dental Caries and Periodontal Disease Risk Biomarker. IOP Conf Ser: Earth Environ Sci 2019; 217: 012050. doi.org/10.1088/1755-1315/217/ 1/012050

7.     R V, P DG. Characterization and Biofilm Detection among Clinically Important Candida Species. Research Journal of Pharmacy and Technology 2016; 9: 1375–1378. doi.org/10.5958/ 0974-360X.2016.00263.8

8.     Da Cunda P, Iribarnegaray V, Papa-Ezdra R, et al. Characterization of the Different Stages of Biofilm Formation and Antibiotic Susceptibility in a Clinical Acinetobacter baumannii Strain. Microb Drug Resist 2020; 26: 569–575. doi.org/ 10.1089/mdr.2019.0145

9.     Hollman B, Perkins M, Walsh D. Biofilms and Their Role in Pathogenesis. Centre of Biomolecular Sciences, University of Nottingham 2020

10.   Zhang Q, Ni Y, Kokot S. Competitive Interactions between Glucose and Lactose with BSA: which Sugar is Better for Children? Analyst 2016; 141: 2218–2227.doi.org/10.1039/ C5AN02420J

11.   I NZ, Prakasam G. Oral Biofilms. Research Journal of Pharmacy and Technology 2016; 9: 1812–1814. doi.org/10.5958/0974-360X.2016.00368.1

12.   Pratiwi SUT, Hamzah H. Inhibition and Degradation Activity of (Sapindus rarak seeds) Ethanol Extract Against Polymicrobial Biofilm. Research Journal of Pharmacy and Technology 2020; 13: 5425–5430. doi.org/10.5958/0974-360X.2020.00947.6

13.   Abdelghafar A, Yousef N, Askoura M. Combating Staphylococcus aureus biofilm with Antibiofilm Agents as an Efficient Strategy to Control Bacterial Infection. Research Journal of Pharmacy and Technology 2020; 13:5601–5606. doi.org/ 10.5958/0974-360X.2020.00977.4

14.   Nishinari K, Fang Y, Guo S, Phillips GO. Soy Proteins: A Review on Composition, Aggregation and Emulsification. Food Hydrocolloids 2014; 39: 301–318. doi.org/10.1016/ j.foodhyd.2014.01.013

15.   Speziale P, Pietrocola G, Foster TJ, Geoghegan JA. Protein-Based Biofilm Matrices in Staphylococci. Frontiers in Cellular and Infection Microbiology 2014; 4: 171. doi.org/10.3389/ fcimb.2014.00171

16.   Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nature Reviews Microbiology 2016; 14: 563–575. doi.org/10.1038/ nrmicro.2016.94

17.   Oh E, Andrews KJ, Jeon B. Enhanced Biofilm Formation by Ferrous and Ferric Iron Through Oxidative Stress in Campylobacter jejuni. Frontiers in Microbiology 2018; 9: 1204. doi.org/10.3389/fmicb.2018.01204

18.   Ramadhani NF, Nugraha AP, Putra Gofur NR, Hakiki D, Ridwan RD. Elevation of C-Reactive Protein in Chronic Periodontitis Patient As Cardiovascular Disease Risk Factor. Biochemical and Cellular Archives 2020; 20: 2875–2878. doi.org/10.35124/ bca.2020.20.S1.2875

19.   Vyas N, Sammons RL, Addison O, Dehghani H, Walmsley AD. A Quantitative Method To Measure Biofilm Removal Efficiency from Complex Biomaterial Surfaces Using SEM and Image Analysis. Scientific Reports 2016; 6: 32694. doi.org/10.1038/ srep32694

20.   Desmond P, Best JP, Morgenroth E, Derlon N. Linking Composition of Extracellular Polymeric Substances (EPS) to the Physical Structure and Hydraulic Resistance of Membrane Biofilms. Water Research 2018; 132: 211–221. doi.org/10.1016/ j.watres.2017.12.058

21.   Ramadhani NF, Nugraha AP, Gofur NRP, Permatasari RI, Ridwan RD. Increased Levels of Malondialdehyde and Cathepsin C by Aggregatibacter actinomycetemcomitans in Saliva as Aggressive Periodontitis Biomarkers. Biochemical and Cellular Archives 2020; 20: 2895–2901.

22.   Rai V, Dey N. The Basics of Confocal Microscopy. 2011. doi.org/ 10.5772/16214

23.   Mongkolrob R, Taweechaisupapong S, Tungpradabkul S. Correlation Between Biofilm Production, Antibiotic Susceptibility and Exopolysaccharide Composition in Burkholderia pseudomallei bpsI, ppk, and rpoS Mutant Strains. Microbiol Immunol 2015; 59: 653–663. doi.org/10.1111/1348-0421.12331

24.   Loterie D, Farahi S, Papadopoulos I, Goy A, Psaltis D, Moser C. Digital Confocal Microscopy Through A Multimode Fiber. Optics Express 2015; 23: 23845–23858. doi.org/10.1364/OE.23.023845

25.   Goldstein J, Newbury DE, Joy DC, et al. Scanning Electron Microscopy and X-Ray Microanalysis: Third Edition. 2003. doi.org/10.1007/978-1-4615-0215-9

26.   Avula A, Galor A, Blackwelder P, et al. Application of Scanning Electron Microscopy With Energy-Dispersive X-Ray Spectroscopy for Analyzing Ocular Surface Particles on Schirmer Strips. Cornea 2017; 36: 752–756. doi.org/10.1097/ ICO.0000000000001173

27.   Abbas HA, Serry FM, EL-Masry EM. Biofilms: The Microbial Castle of Resistance. Research Journal of Pharmacy and Technology 2013; 6: 01–03.

28.   Stauch-White K, Srinivasan VN, Kuo-Dahab WC, Park C, Butler CS. The Role of Inorganic Nitrogen in Successful Formation of Granular Biofilms for Wastewater Treatment That Support Cyanobacteria And Bacteria. AMB Express 2017; 7. doi.org/ 10.1186/s13568-017-0444-8

29.   Fulaz S, Vitale S, Quinn L, Casey E. Nanoparticle-Biofilm Interactions: The Role of the EPS Matrix. Trends Microbiol 2019; 27: 915–926. doi.org/10.1016/j.tim.2019.07.004

30.   Li X, Pei H, Hu W, et al. The fate of Microcystis aeruginosa Cells during The Ferric Chloride Coagulation and Flocs Storage Processes. Environ Technol 2015; 36: 920–928. doi.org/10.1080/ 09593330.2014.966768

31.   Lu L, Hu W, Tian Z, et al. Developing Natural Products as Potential Anti-Biofilm Agents. Chinese Medicine 2019; 14: 1–17. doi.org/10.1186/s13020-019-0232-2

32.   Tandra Das T, Gopinath P. Biofilm Formation Among Enterococci Species. Research Journal of Pharmacy and Technology 2016; 9: 1877–1879.

33.   Chaturvedi R, Chandra P, Mittal V. Biofilm Formation by Acinetobacter Spp. in Association with Antibiotic Resistance in Clinical Samples Obtained from Tertiary Care Hospital. Research Journal of Pharmacy and Technology 2019; 12: 3737–3742. doi.org/10.5958/0974-360X.2019.00620.6

34.   Utami DT, Pratiwi SUT, Haniastuti T, Hertiani T. Degradation of Oral Biofilms by Zerumbone from Zingiber zerumbet (L.). Research Journal of Pharmacy and Technology 2020; 13: 3559–3564. doi.org/10.5958/0974-360X.2020.00629.0

35.   Rajkumari J, Borkotoky S, Murali A, Suchiang K, Mohanty SK, Busi S. Attenuation of Quorum Sensing Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa by Pentacyclic Triterpenes, Betulin and Betulinic Acid. Microbial Pathogenesis 2018; 118: 48–60. Doi.Org/10.1016/ J.Micpath.2018.03.012

36.   Henry AJ, R SA, R D. Evaluation of Disinfectant Action on Biofilm Bacteria. Research Journal of Pharmacy and Technology 2018; 11: 910–912. doi.org/10.5958/0974-360X.2018.00168.3

37.   Abdul-Lateef LA, Hassan E, TahaAbd-AllaBactash E. Effect of Cranberry on Biofilm Formation by P. mirabilis Isolated from Patients Suffering from Urinary Tract Infections. Research Journal of Pharmacy and Technology 2018; 11: 1097–1100. doi.org/ 10.5958/0974-360X.2018.00206.8

38.   Ampornaramveth RS, Akeatichod N, Lertnukkhid J, Songsang N. Application of D-Amino Acids as Biofilm Dispersing Agent in Dental Unit Waterlines. International Journal of Dentistry 2018; 2018: e9413925. doi.org/10.1155/2018/9413925

 

 

Accepted on 05.08.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):799-803.