Microbial Biofilms: Role in Human Infectious Diseases

Microbial biofilms: Role in Human Infectious Diseases focuses on new and emerging concepts in microbial biofilm research, such as the mechanisms of biofilm formation, biofilm-induced pathogenesis, biofilm detection/and diagnosis, gene exchange within biofilms, strategies to control microbial biofilms and the burden of biofilm associated infections. In addition, it highlights the various anti-biofilm strategies such as surface coating, signal quenching, novel compounds that can be translated to curb biofilm-associated infections and the escalation of antimicrobial resistance determinants.

Microbial biofilms can be a serious problem in medical settings as they are associated with significant mortality and morbidity. Infection related to biofilms increases recovery time and the cost of disease management. Biofilms are resistant to known antibiotics and human defense mechanism. In addition, due to close proximity of microbes within biofilms, increase genetic transformation has been detected results in increases frequency of antibiotic gene spread. With the advancement in science and technology, novel strategies have been proposed to combat the impact of biofilms on human health.

• Fulfills the knowledge gap in biofilm study
• Focuses on new and emerging concepts in microbial biofilm research
• Highlights the various anti-biofilm strategies
• Provides concise, thorough and up-to-date information about the important role of microbial biofilms in human diseases

• Language: English
• Publisher: Academic Press
• Release date: Apr 10, 2024
• ISBN: 9780443192531

Book preview

1

Application of nanoparticles to combat dental biofilms

Chipo Chapusha¹, Jennifer Bain² and Amol V. Janorkar¹,    ¹Department of Biomedical Materials Science, School of Dentistry, University of Mississippi Medical Center, Jackson, MS, United States,    ²Department of Periodontics and Preventive Science, School of Dentistry, University of Mississippi Medical Center, Jackson, MS, United States

Abstract

Dental biofilms are highly organized communities of microbiota that adhere to soft or hard surfaces in the oral cavity and are a result of harmful changes in composition and relative abundance of oral microbiomes. This chapter details the process of dental biofilm formation along with the current methods of treatment and their shortfalls. We have focused on the use of nanoparticles as a fast-emerging approach for the treatment of dental biofilms. Nanoparticles can be loaded with therapeutic agents and their surface properties can be tailored to make them more effective for antimicrobial treatments. Starting with the examples of nanoparticles used in the field of nanomedicine, we have described the various types of nanoparticles, recent efforts in their use in the dental arena, as well as their future applications. Finally, we have discussed periodontal disease, an outcome of uncontrolled dental biofilm growth, as a serious health concern and the use of nanoparticles for the potential treatment of periodontal disease.

Keywords

Biofilms; nanoparticles; dental materials; periodontal disease; drug delivery

1.1 Dental biofilms

1.1.1 Background

The oral cavity is home to about 500–700 species of microbes that work to maintain its health; only 50% of these microbiomes are cultivable. Harmful changes in composition and relative abundance of oral microbiomes compared to healthy states can occur; a phenomenon known as oral dysbiosis. Oral dysbiosis is mainly caused by poor oral hygiene but factors such as smoking, diet, genetics, and weak host immunity can increase the risk for oral dysbiosis. Oral dysbiosis leads to the aggregation of harmful microorganisms and the formation of dental biofilms [1].

Dental biofilms are highly organized communities of microbiota that adhere to soft or hard surfaces in the oral cavity such as the teeth, tongue, and periodontal pockets (Fig. 1–1). Dental biofilms are exposed to various stressors such as salivary flow, and changing temperature and pH levels from substances ingested in the oral cavity environment; however, dental biofilms adapt to this environment. Dental biofilms cause infections in the oral cavity, such as caries, tonsillitis, and periodontal diseases [1], and may lead to systemic health issues such as cardiovascular disease and diabetes [2]. The cost of treatment of biofilms, in general, is estimated at USD 94 billion annually [3]. Biofilms are a medical concern because of their ability to build resistance to a host’s immune system and antimicrobial treatments such as antibiotics.

Figure 1–1 Surfaces in the oral cavity where bacteria can attach and form dental biofilms. These include the tongue, tooth surfaces, periodontal pockets, dental implants, and orthodontic appliances. Figure made using Biorender.com.

Dental biofilm formation in the oral cavity occurs in the following stages (Fig. 1–2).

Figure 1–2 Stages of biofilm formation. Figure made using biorender.com.

1.1.1.1 Acquired pellicle formation

Saliva contains macromolecules that adhere to soft and hard surfaces in the oral cavity, creating a thin protein-containing film called salivary or acquired pellicle. Acquired pellicle acts as a lubricant and protects surfaces from acidic substances which lead to the demineralization of teeth [4].

1.1.1.2 Initial attachment to a surface

Acquired pellicle is composed of peptides, glycoproteins, and other proteins which adhere to surfaces in the oral cavity. Glycoproteins, specifically the proline-rich glycoproteins, are also receptors for bacterial attachment [4]. Primary colonizers such as Streptococcus spp., in the planktonic state, attach to the glycoproteins of acquired pellicle on a tooth surface. This initial attachment is driven by van der Waals or electrostatic forces. At this stage, bacteria can be eliminated. However, with time, the primary colonizers form an extracellular polymeric substance using the carbohydrates ingested in the oral cavity. This extracellular polymeric substance contains exopolysaccharides, water, and proteins that protect the budding biofilm. Extracellular polymeric substance protects the biofilm by providing structural and functional integrity, hence strengthening their attachment to pellicle. From here, the bacteria grow laterally, creating a monolayer over the surface.

1.1.1.3 Biofilm maturation

When the initial attachment is complete, primary colonizers expose their receptors for secondary colonizers such as gram-negative Fusobacterium nucleatum. As the biofilm matures, the composition changes to include tertiary colonizers such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis [5]. This causes the environment to become more anaerobic which supports the growth of these pathogenic bacteria. The oral cavity is rich in moisture and nutrients, and when coupled with poor oral hygiene, such a microenvironment supports the growth of dental biofilms. Although bacteria in the biofilm are at different stages of growth, they collectively work together to support and sustain the integrity of the biofilm. As bacteria species mature, eliminating the bacteria using antibiotics becomes more challenging because there are various species of microbiota at play, and they are shielded by extracellular polymeric substance.

1.1.1.4 Dispersal of bacteria

Once the biofilm matures, it ruptures and disperses to colonize other sites. This is done actively or passively. Active dispersal is initiated by bacteria and passive dispersal is initiated by external factors, such as fluid shear forces or humans intervening. Biofilm disperses using three main mechanisms: erosion, sloughing, and seeding. Biofilm dispersal occurs mainly due to changes in the amount of nutrients at a particular site and/or host defense systems which limit biofilm development [5,6].

1.1.2 Current treatment methods

Dental biofilms attach to different surfaces in the oral cavity. When dental biofilms attach to the subgingival and supragingival surface of the tooth, it is called plaque. Plaque is a soft and sticky bacterial biofilm. When left untreated, plaque mineralizes to form calculus also known as tartar. Calculus is a hard and porous deposit that forms around teeth and the gingiva line. Calculus, due to its porosity and rough surface, easily absorbs harmful substances which may irritate healthy tissue [7]. Studies have shown that starchy granules, bacteria in plaque, and even toxic metals such as lead from foods ingested may lodge in calculus due to its porous nature [8]. The following methods (Table 1–1) are currently being used to control or remove plaque and calculus.

Table 1–1

1.1.2.1 Physical or mechanical removal

Brushing: Brushing is used with toothpaste to remove plaque on the surface of teeth and gums. However, brushing does not remove all the bacteria and food substances lodged between teeth and will not remove calculus once it is formed.

Flossing: Flossing removes plaque and food substances lodged between teeth. This improves the removal of bacteria in places that are beyond reach for brushing but cannot be used as a stand-alone method of treatment. Flossing also will not remove calculus once formed.

Scaling and root planing: According to the glossary of periodontal terms, scaling is the use of instruments to remove plaque, calculus, and stains from the crown and root surfaces of the teeth. Scaling is done with a combination of hand instrumentation and ultrasonic handpieces. Root planing is a treatment procedure designed to remove cementum or surface dentin that is rough, impregnated with calculus, or contaminated with toxins or microorganisms on the surface of the roots. Root planing promotes the reattachment of periodontal fibers to teeth after scaling is done.

1.1.2.2 Chemical removal

Oral rinse: Therapeutic oral rinses containing antimicrobial agents such as chlorhexidine and cetylpyridinium chloride [10], or essential oils are used to control or eliminate plaque. Oral antimicrobial rinses prevent the adhesion of bacteria to surfaces in the mouth, thus preventing biofilm formation [11]. However, oral rinses alone cannot remove biofilms once they form. Oral rinses need to be used as an adjunct treatment to the mechanical removal of biofilms such as brushing and scaling.

Antibiotics: Broad-spectrum antibiotics are used to accompany mechanical treatments such as scaling and root planing. Most antibiotics are administered systemically; however, local placement of antibiotics is beneficial as discussed later.

The current treatment methods mentioned above still face some challenges. Reinfection, tooth sensitivity, and gingival recession are common problems for procedures such as scaling and root planing. Antibiotics administered systemically may lead to adverse systemic effects and/or resistant strains. Some orally administered drugs tend to discolor teeth. For example, tetracyclines used to treat dental biofilms have been known to cause yellow staining of teeth in children 8 years and younger, with the severity of discoloration depending on dosage and length of tetracycline use [12]. Chlorhexidine may also cause brown or yellowish stains on teeth.

1.1.3 Newer antibacterial treatment methods

Essential oils: Essential oils have been used for protection against pests, aromatherapy, the treatment of inflammation, and antimicrobial action in mouthwashes. Most essential oils are known to change the bacterial cell wall. However, further research is required to understand the safety margins and the factors that may affect the antimicrobial potential of essential oils [13].

Bacteriophages: Bacteriophages are self-proliferating viruses that target specific bacteria but are nontoxic to human tissues [14]. This approach can be advantageous because bacteriophages can penetrate the extracellular polymeric matrix of biofilms. Nevertheless, this is still a challenge because biofilms are a community of bacteria and it is difficult to ensure that bacteriophages target specific bacteria in the biofilms [15].

Vaccines: Another proposed approach is the production of vaccines against bacteria. Vaccines work to help build immunity against disease-causing organisms by introducing weaker antigens of the disease to the body or by teaching the body how to create the antigens (mRNA vaccines). A clear understanding of biofilm formation and specific host immunity is needed for the successful development of such vaccines.

Nanoparticles: Using nanoparticles is a fast-emerging approach that is showing great promise. Nanoparticles can be loaded with therapeutic agents and their surface properties can be tailored to make them more effective for antimicrobial treatments. Nanoparticles, in addition, provide a more targeted treatment approach [16].

While many of these new/emerging therapeutics have potential, in this chapter, we will focus on the application of nanoparticles in combating dental biofilms.

1.2 Nanoparticles

1.2.1 Background

The National Nanotechnology Initiative defines nanotechnology as the manipulation of matter with at least one dimension sized from 1 to 100 nm. Consequently, nanoparticles are materials within the size range of 1–100 nm. Due to their size, nanoparticles have small volumes and large surface areas. This increases their surface area-to-volume ratio, making nanoparticles exhibit unique properties compared to bulk materials. Having a large surface area-to-volume ratio means most of the material is exposed to the surrounding environment. As a result, nanoparticles have shown to be more effective in their use as catalysts compared to bulk materials. Gold and Silver nanoparticles have shown adequate catalytic activity in the removal of organic dyes in water treatment systems [17]. Biological and organic molecules in living organisms, such as DNA and oxygen, are within the nanoscale size range. Nanoparticles can interact with biological systems at a molecular level compared to bulk materials. It is easier for nanoparticles to pass through cellular barriers, a property that is utilized in cancer treatment.

A material’s behavior and properties are sensitive to changes in size and shape at the nanoscale. This is why bulk materials will have different electrical, magnetic, thermal, and optical properties from the same material at the nanoscale. Ted Sargent, the author of The Dance of Molecules, says matter is tunable at the nanoscale [18]. Due to these benefits, nanoparticles are showing great promise for their use in the field of medicine. Nanoparticles are being considered for the diagnosis, treatment, and prevention of diseases, a field known as nanomedicine. Nanomedicine integrates multiple disciplines such as biology, chemistry, material science, and engineering.

1.2.2 Applications in medicine

There are various types of nanoparticles, but they fall under three main categories: organic, inorganic, and hybrid nanoparticles. Each category is based on the composition of the materials used to make the nanoparticles. Below are a few examples of such nanoparticles used in the field of nanomedicine.

1.2.3 Organic nanoparticles

1.2.3.1 Liposomes and lipid nanoparticles

Liposomes are structures containing a vesicle and phospholipid bilayer (Fig. 1–3A). Vesicles are liquid-containing sacs surrounded by a lipid bilayer. The lipid bilayer is made of amphiphilic molecules containing a hydrophilic head and a hydrophobic tail. Liposomes are being used in clinical applications. For example, liposomes are used as vaccine adjuvant delivery systems (VADs). VADs are used to enhance the body’s reaction to antigens found in vaccines thus improving the action of a vaccine [19]. Epaxal is a hepatitis A vaccine that uses liposomes [20]. Liposomes are also used in drug delivery due to their biocompatibility and biodegradability. Vesicles can be loaded with therapeutic agents, and depending on the arrangement of the lipid bilayer, they can carry either hydrophilic or hydrophobic materials [21]. Drugs such as doxorubicin, which is used in chemotherapy, amphotericin B in antifungal treatments, and Depodur for pain management use liposomes to deliver the active-drug ingredients [20].

Figure 1–3 Structures of (A) liposome (i) without drugs loaded and (ii) drug loaded and (B) lipid nanoparticle (i) without drugs loaded and (ii) drug loaded. Figures made using Biorender.com.

Lipid nanoparticles are like liposomes; however, they contain a single layer of phospholipids surrounding a vesicle (Fig. 1–3B). They are used to carry nucleic acids and small molecules [22]. Drugs such as Onpattro use lipid nanoparticles to deliver active-drug ingredients for the treatment of polyneuropathy due to hereditary transthyretin-mediated amyloidosis, hATTR amyloidosis [23]. Lipid nanoparticles are also being used to deliver COVID mRNA vaccines [24].

1.2.3.2 Polymeric nanoparticles

Polymeric nanoparticles are made from synthetic or natural polymers. They are within the size range of 1–1000 nm. Therapeutic agents can be loaded in polymeric nanoparticles or adsorbed on the surface [25]. Polymeric nanoparticles are being used in drug-delivery applications because they provide stability for drugs, show controlled drug release, and can be biodegraded. Examples are materials such as polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) that have been used in the delivery of vaccines, proteins, and antiinflammatory drugs [26].

1.2.3.3 Dendritic nanoparticles

These are compact materials with multiple functional groups attached to a central core. Dendritic nanoparticles have low polydispersity indices due to their uniformity in size. They are being used as contrasting agents in medical imaging. Dendritic branches can be made of contrasting materials such as iron oxide for use in optical imaging and magnetic resonance imaging (MRI) [27]. They are also being studied for use in drug delivery for cardiovascular therapy and cancer therapy [28]. However, the cytotoxicity of cationic dendrimers may increase with concentration and size.

1.2.4 Inorganic nanoparticles

These are nanoparticles made using metallic materials such as gold, silver, copper, or iron oxide.

1.2.4.1 Gold nanoparticles

At the macroscale, gold has a yellow color. However, nanogold particles interact differently with light due to changes in size and shape. For example, gold nanoparticles in the size range of 10–20 nm have a red color, and gold nanoparticles of about 80 nm have an orange color. This property was used in medieval times to stain glass windows. Currently, this property allows gold nanoparticles to be used as a contrasting agent in X-ray-computed microtomography [29].

1.2.4.2 Silver nanoparticles

Silver nanoparticles have been used to stain glass yellow [29]. They are currently being used in the antimicrobial treatment of surfaces. It is proposed that because of silver’s nanoparticle size, it can easily penetrate bacteria’s cell walls to inhibit protein synthesis and DNA replication [30]. However, more research is needed to define silver’s antimicrobial mechanisms.

1.2.4.3 Iron oxide nanoparticles

Iron oxide nanoparticles are being used as contrast agents for MRI and as a drug to treat iron deficiency anemia in patients with chronic kidney disease [31]. Iron oxide nanoparticles have been investigated for targeted drug delivery in chemotherapy [32].

1.2.5 Hybrid nanoparticles

Hybrid nanoparticles are made from a combination of inorganic and organic materials (Fig. 1–4). This is done to improve properties such as stability, biocompatibility, and ability to target specific sites [33]. Chlorotoxin (CTX) is a biocompatible iron oxide nanoprobe that is coated with poly(ethylene glycol) (PEG). CTX can target glioma tumors via the surface-bound targeting peptide [29].

Figure 1–4 A metallic nanoparticle conjugated with organic polymer chains to improve the ability to target specific cells. Figure made using Biorender.com.

As seen, nanoparticles are being considered and used in medicine because of their many benefits. Some of their benefits are being utilized in cancer drug delivery to improve solubility of drugs, delivery of drugs to target sites, and increase circulation time in the body [34].

Their large surface area-to-volume ratios make lower concentrations of nanoparticles to be as effective as higher concentrations of bulk materials. Also, the sizes of most biological molecules are at the nanoscale making it easier for nanoparticles to penetrate cells and react with cell organelles.

1.3 Application of nanoparticles to treat dental biofilms

1.3.1 Surface coatings of dental materials

Microbiota’s attachment to surfaces in the oral cavity is paramount to successful dental biofilm formation. Microbiota attaches to surfaces such as teeth, dental implants, and restorative materials. Using nanoparticles as surface coatings on dental materials and oral surfaces can prevent the attachment of microbiota. Nanoparticles that have been used for such applications are metallic nanoparticles, for example, silver. This is because metallic nanoparticles, compared to antibiotics, have shown to be more effective in reducing the ability of microbes to build resistance against them. Antibiotics use a single antibacterial mechanism while metallic nanoparticles fight microbes using multiple antibacterial mechanisms, making it difficult for microbes to build resistance against them [35]. Antibacterial mechanisms of metal-based nanoparticles include destructive interactions with the bacteria cell wall and DNA, interfering with bacteria quorum sensing and production of reactive oxygen species [16]. For example, Gutta-percha is dental material used as a filling in root canal procedures. However, it shows insufficient antimicrobial activity. Mohan et al. were able to coat Gutta-percha with chitosan polymeric nanoparticles of the size 30–40 nm and silver nanoparticles of the size 20–30 nm. Coating Gutta-percha with silver and chitosan nanoparticles improved its antimicrobial activity against Enterococcus faecalis, a pathogen that causes root canal infections in the oral cavity. The authors concluded that the nanoparticles’ interaction with the bacteria led to the destruction of the bacteria membrane and the induction of oxidation stress [36]. Venugopal et al. showed that coating the surface of an orthodontic titanium implant with silver had an antibacterial effect against Streptococcus mutans, Streptococcus sanguinis, and A. actinomycetemcomitans. S. mutans and S. sanguinis are bacteria associated with dental caries, while A. actinomycetemcomitans is associated with periodontitis. The authors had three groups of orthodontic titanium implants: novel biopolymer-coated titanium implant with silver nanoparticles, titanium implant with silver directly coated on surface, and titanium implant without silver coating. Their novel biopolymer-coated titanium implant with silver nanoparticles on its surface showed the highest antibacterial activity followed by titanium implants that had silver nanoparticles coated directly on the surface. The biopolymer-coated titanium implant with silver nanoparticles showed zone of inhibitions ranging from 25 to 50 mm² against the tested bacteria. The authors attributed this enhanced performance to the biopolymer, hydroxyapatite/chitosan, retaining more nanoparticles (21.2 at.% silver) compared to the control (1.1 at.% silver) [37].

Size and shape of nanoparticles also plays a vital role in antimicrobial action. Pal et al. showed that truncated triangular silver nanoparticles had the highest antimicrobial action compared to spherical and rod-shaped nanoparticles because the reactivity of silver favored the high-atom-density facets. Truncated triangular silver nanoparticles have {1 1 1} top basal surfaces, and the spherical and rod-shaped silver nanoparticles have {1 0 0} with a few {1 1 1} surfaces [38]. Another study tested spherical, disk, and triangular-shaped silver nanoparticles against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. In Poland, E. coli and P. aeruginosa have been found in the oral cavities of immune-compromised individuals such as the older population [39]. All shapes showed the highest antibacterial action against E. coli compared to the other two bacteria, with nanospheres exhibiting the widest inhibition zone. The spherical nanoparticles’ superior antibacterial action against E. coli was attributed to a higher silver ion release as quantified using optical density measurements [40].

1.3.2 Incorporation into dental materials

Initially, nanoparticles were added to dental materials to improve mechanical properties, reduce shrinkage, and increase abrasion resistance [41]. Now, their antimicrobial properties are being explored and utilized. A common infectious disease caused by dental biofilms is dental caries. Treatment of dental caries, depending on the stage, will include teeth filling or teeth extraction which can be followed by the replacement of missing teeth with dental implants. The materials used to repair and replace damaged teeth are collectively known as dental restorative materials. Materials such as dental composites, dental cements, and resin-based materials are used for such applications. The challenge is the oral cavity houses several species of microorganisms, and preventing harmful bacterial accumulation on restorative materials is essential to maintaining the integrity of restorative materials and preventing secondary caries. Incorporating nanoparticles into restorative materials can help combat restorative material failure by making them less susceptible to bacterial accumulation. Gutiérrez et al. formulated experimental etch-and-rinse adhesives that incorporated copper nanoparticles in the size range of 63–154 nm. The experimental adhesive contained 0.0075, 0.015, 0.06, 0.1, 0.5, and 1.0 wt.% copper concentrations. All the experimental adhesives showed higher antimicrobial activity against S. mutans, bacteria known to cause caries, compared to the control that did not contain copper nanoparticles. However, they only recommended copper concentrations up to 0.5 wt.% as these did not have a negative effect on the physical properties of the experimental adhesive [42].

Chen et al. used direct contact test to evaluate the antibacterial activity of a reduced graphene–silver nanoparticle (R-GNs/Ag) modified glass ionomer cement against S. mutans. R-GNs/Ag at weight proportions (w/w): 0.05%, 0.10%, 0.50%, 1.00%, and 2.00% were added to commercially available glass ionomer cement. The original glass ionomer cement was set as a control. Results showed that the antibacterial effects of the glass ionomer cement against S. mutans increased as the concentration of graphene–silver nanoparticles increased. Even though glass ionomer cements are known to prevent the demineralization of teeth through the release of ions, secondary caries still lead to the failure of the restorative materials. Therefore their approach provides a possible solution to this problem [43].

1.3.3 Drug delivery

Drugs can be delivered orally, intravenously, via inhalation, or subcutaneously. Of all these methods of drug delivery, oral delivery of drugs is the most used. 84% of the best-selling pharmaceutical products are administered orally, mostly because oral drug delivery is noninvasive and shows the highest patient compliance [44]. However, the action of orally delivered drugs depends on wettability, solubility, and ability of the drug to penetrate the gastrointestinal tract. Orally delivered drugs interact with digestive enzymes which may degrade the drug before it reaches the intended site. Also, food substances in the digestive tract may interfere with the action of drugs [45]. Doxycycline, a drug used to treat bacterial infections, should not be taken with iron-rich foods, laxatives, and antacids because they reduce doxycycline’s ability to be absorbed across the digestive tract [46]. Such factors reduce therapeutic concentrations of drugs from reaching target sites.

To solve these problems, nanoparticles can be used as drug-delivery vehicles because drugs can be adsorbed on the surface or encapsulated in the nanoparticles. Encapsulation of drugs in nanoparticles provides stability by protecting drugs from digestive enzymes and interactions with food substances that may interfere with the drug’s action. Nanoparticles also provide local delivery of drugs, eliminating toxic systemic effects. Minocycline is a semisynthetic antibiotic belonging to the tetracycline family. It is an active ingredient in Arestin, an FDA-approved microparticle drug-delivery system. Arestin, PLGA microspheres, is in the size range of 20–60 μm and is used in combination with scaling and root planing to treat periodontitis in adults [47]. Another study showed that Minocycline loaded PEG–PLA nanoparticles with an average diameter of 100 nm significantly reduced the symptoms of periodontitis in dogs [48]. There is still room for the use of other nanoparticles, for example, dendrimers for drug delivery in the oral