**Adverse Influences of Antimicrobial Strategy against Mature Oral Biofilm**

Shoji Takenaka, Masataka Oda, Hisanori Domon, Rika Wakamatsu, Tatsuya Ohsumi, Yutaka Terao and Yuichiro Noiri

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63564

#### **Abstract**

Antimicrobial measures, such as topical antiseptics and local drug delivery, have proven effective as complements to mechanical control. However, recent investigations have reported some adverse influences of antimicrobial strategy.

One possible negative reaction is that residual structure may serve as a scaffold for redevelopment of biofilm. It is reported that no or little biofilm structure was removed when oral biofilms were treated with chemical compounds and that the secondary adhesion was promoted in the presence of residual structure.

Second, residual structure may also act as pathogens. It is well known that various microbial components in the biofilm can play a role in disease pathogenesis, even if the microorganisms in the biofilm are completely killed.

Third, low-dose antibiotics may promote bacterial biofilm formation. The short-time exposure of chemical agents will cause gradient of concentration inside biofilm. In this case, the cells in deeper area may be exposed to subminimal inhibitory concentrations (sub-MICs) of antimicrobial agents. Recent studies have demonstrated that a variety of antibiotics or antimicrobial agents at sub-MIC levels can induce biofilm formation *in vitro*, interfering with bacterial biofilm virulence expression. residual structure may also act as It is well known that low-dose antibiotics may promote bacterial biofilm The antibiotics or antimicrobial agents at sub-MIC levels can induce biofilm formation This chapter reviews studies demonstrating adverse influences of antimicrobial oral antimicrobial residual stress

This chapterreviews studies demonstrating adverse influences of antimicrobial strategy against mature oral biofilm.

**Keywords:** oral biofilm, antimicrobial agent, residual structure, sub-MIC, stress re‐ sponse

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

Mechanical approach by procedures such as self-performed oral hygiene, scaling and root planning (SRP), or periodontal surgery is fundamental in the control of mature oral biofilms [1]. Chemical approaches such as topical antiseptics, local drug delivery, and systemic antibiotics are used with the expectation of producing an adjunctive effect [2‒5]. In fact, it has been demonstrated that adjunctive antimicrobials improve clinical parameters, including plaque index, gingival inflammation, and probing pocket depth [3, 5‒7]. It has also been reported that antiplaque biocides do not cause the microbial resistance and alterations of microbial flora [8].

that bind to the EPS strands and partially cross-link them to the matrix. Diffusion limitation arises readily in these polymer strands because the fluid flow is reduced and the diffusion distance is increased in the biofilm mode of growth [17]. On the other hand, prolonged antimicrobial stress causes the biofilms facilitating the spread of antibiotic resistance by promoting horizontal gene transfer [18]. The existence of tolerant or dormant cells is critical

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427

**Figure 2.** Mechanisms of biofilm tolerance. Antimicrobial penetration is retarded in the presence of EPS (yellow). The some microorganisms in the biofilm change activity in response to antimicrobial stress (green). The microenvironment in deeper area is altered to resist eradicating (pink). Persister cells are present in higher concentration in biofilm (vio‐ let). This image was modified from CBE Image Library by the Center for Biofilm Engineering at Montana State Univer‐

This chapter is focusing to the studies demonstrating adverse influences of antimicrobial

Recent investigations have demonstrated that chemical disinfection for oral biofilm may leave intact biofilm structures. We performed a direct time-lapse microscopic observation through‐ out continuous exposure of commercial mouthrinses to an oral biofilm model [10]. Conse‐ quently, no removal of biomass was observed in control, ethanol (EtOH), 0.12% chlorhexidine gluconate (CHG), and Biotene, which contains lysozyme, lactoferrin, lactoperoxidase, glucose

factor in chronic infection [19, 20] (**Figure 2**).

sity.

strategy against mature oral biofilm.

**2.1. Residual structure**

**2. Adverse influences of antimicrobial strategy**

However, recentinvestigations have demonstrated that antimicrobial compounds do not work as intended [9‒12. Especially in short-time exposure, the antimicrobials failed to penetrate into deeper area inside biofilm. Wakamatsu et al. have reported the penetration kinetics of mouthrinses into *in vitro Streptococcus mutans* biofilms by direct time-lapse microscopic analysis. The antimicrobial penetration was critically restricted within 30 s of exposure; the average penetration velocity was ranging from 4.2 to 30.1 μm/min [13]. This phenomenon can be explained by retarded penetration due to degradation and/or modification by the biofilm matrix. Extracellular polymeric substance (EPS) produced by microorganisms make up the intercellular space of microbial aggregates and form the structure and architecture of the biofilm matrix that reduces antimicrobial penetration [14,15]. Representative four models of how these polymer strands might interact are shown in **Figure 1** [16]. Panel A is the alginate paradigm. Calcium forms a complex with negatively charged polymer strands. Panel B shows tight adhesion of a negatively charged polymer and a positively charged polymer. Panel C indicates an insoluble polymer. Polymer complex formation is probably driven by hydrogen bonding or hydrophobic interactions. Panel D indicates that bacteria have surface receptors

**Figure 1.** Conceptual models of matrix cohesion. (A) Alginate paradigm. Calcium is cross-linked between alginate. (B) Adhesion of a negatively charged polymer and positively charged polymer. (C) Hydrogen bonding or hydrophobic interaction. (D) Bacteria are partially cross-linked to the matrix. Reproduced from Takenaka et al. [16] with permission.

that bind to the EPS strands and partially cross-link them to the matrix. Diffusion limitation arises readily in these polymer strands because the fluid flow is reduced and the diffusion distance is increased in the biofilm mode of growth [17]. On the other hand, prolonged antimicrobial stress causes the biofilms facilitating the spread of antibiotic resistance by promoting horizontal gene transfer [18]. The existence of tolerant or dormant cells is critical factor in chronic infection [19, 20] (**Figure 2**). 

**Figure 2.** Mechanisms of biofilm tolerance. Antimicrobial penetration is retarded in the presence of EPS (yellow). The some microorganisms in the biofilm change activity in response to antimicrobial stress (green). The microenvironment in deeper area is altered to resist eradicating (pink). Persister cells are present in higher concentration in biofilm (vio‐ let). This image was modified from CBE Image Library by the Center for Biofilm Engineering at Montana State Univer‐ sity.

This chapter is focusing to the studies demonstrating adverse influences of antimicrobial strategy against mature oral biofilm.

### **2. Adverse influences of antimicrobial strategy**

#### **2.1. Residual structure**

**1. Introduction**

426 Microbial Biofilms - Importance and Applications

microbial flora [8].

Mechanical approach by procedures such as self-performed oral hygiene, scaling and root planning (SRP), or periodontal surgery is fundamental in the control of mature oral biofilms [1]. Chemical approaches such as topical antiseptics, local drug delivery, and systemic antibiotics are used with the expectation of producing an adjunctive effect [2‒5]. In fact, it has been demonstrated that adjunctive antimicrobials improve clinical parameters, including plaque index, gingival inflammation, and probing pocket depth [3, 5‒7]. It has also been reported that antiplaque biocides do not cause the microbial resistance and alterations of

However, recentinvestigations have demonstrated that antimicrobial compounds do not work as intended [9‒12. Especially in short-time exposure, the antimicrobials failed to penetrate into deeper area inside biofilm. Wakamatsu et al. have reported the penetration kinetics of mouthrinses into *in vitro Streptococcus mutans* biofilms by direct time-lapse microscopic analysis. The antimicrobial penetration was critically restricted within 30 s of exposure; the average penetration velocity was ranging from 4.2 to 30.1 μm/min [13]. This phenomenon can be explained by retarded penetration due to degradation and/or modification by the biofilm matrix. Extracellular polymeric substance (EPS) produced by microorganisms make up the intercellular space of microbial aggregates and form the structure and architecture of the biofilm matrix that reduces antimicrobial penetration [14,15]. Representative four models of how these polymer strands might interact are shown in **Figure 1** [16]. Panel A is the alginate paradigm. Calcium forms a complex with negatively charged polymer strands. Panel B shows tight adhesion of a negatively charged polymer and a positively charged polymer. Panel C indicates an insoluble polymer. Polymer complex formation is probably driven by hydrogen bonding or hydrophobic interactions. Panel D indicates that bacteria have surface receptors

**Figure 1.** Conceptual models of matrix cohesion. (A) Alginate paradigm. Calcium is cross-linked between alginate. (B) Adhesion of a negatively charged polymer and positively charged polymer. (C) Hydrogen bonding or hydrophobic interaction. (D) Bacteria are partially cross-linked to the matrix. Reproduced from Takenaka et al. [16] with permission.

Recent investigations have demonstrated that chemical disinfection for oral biofilm may leave intact biofilm structures. We performed a direct time-lapse microscopic observation through‐ out continuous exposure of commercial mouthrinses to an oral biofilm model [10]. Conse‐ quently, no removal of biomass was observed in control, ethanol (EtOH), 0.12% chlorhexidine gluconate (CHG), and Biotene, which contains lysozyme, lactoferrin, lactoperoxidase, glucose oxidase, and potassium thiocyanate, even after 20 min exposure. Treatments with CHG and EtOH resulted in only a slight contraction of the biofilm (**Figure 3**).

**Bacterium Experimental**

*Streptococcus mutans* Glass-based

*Porphyromonasgingivalis* Chambered

*Staphylococcus epidermidis*

structure.

pulsing of CHG [26].

inflammatory reactions.

**design**

dish

coverglass

**Incubation time**

Flow-cell 24h 0.14mM QAC

24h 0.12% CHG EO CPC IPMP

> 0.5mM Glutaraldehyde 14.9μM nisin

EtOH: ethanol; CHG: chlorhexidinegluconate; SLS: sodium lauryl sulfate; TRN: triclosan; CPC; cetylpyridinium

**Table 1.** A summary of representative experiments demonstrating that chemical approach failed to detach the biofilm

In contrast, there are some reports that the biofilm structure has been successfully degraded by repeated exposures of mouthrinse [23‒25]. Although it is likely that biofilm reduction may be enhanced by repeated pulse of a mouthrinse, this approach may not always be effective. Pratten and Wilson have reported that anaerobic counts in dental plaque biofilm returned to pretreatment levels with altered bacterial composition after 4 days, despite the continuous

Summarizing the above, these results suggest that chemical approach such as the mouth‐ rinse, especially without repeated use, may not be sufficient to eradicate oral biofilm struc‐ ture. Residual structure may cause adverse effects in oral environment, even if the

As the remaining biofilm matrix contains carbohydrates, proteins, polysaccharide, lipids, and nucleic acid [27], dead bacteria and biofilm components could work as antigens and induce

For example, *Actinobacillus actinomycetemcomitans, P. gingivalis, Tannerella forsythia*, and *Treponema denticola* have been implicated in the development of various forms of periodonti‐

chloride; IPMP: isopropyl methyl phenol; QAC: quaternary ammonium compound

microorganisms in the biofilm are completely killed.

*2.1.1. Antigen and host inflammatory reaction*

24h 0.05 to 0.2% CHG 5min Microscopic

**Antimicrobial agent**

**Exposure time**

Adverse Influences of Antimicrobial Strategy against Mature Oral Biofilm

5 min Microscopic

60 min Microscopic

observation (transmission image)

observation (transmission image)

observation (transmission image), Quantitative analysis of protein and carbohydrate composition

**Judgment Reference**

http://dx.doi.org/10.5772/63564

[13]

429

[21]

[22]

**Figure 3.** Transmission images of biofilm cluster before (A) and after (B) 0.12% chlorhexidine (CHG) treatment. The biofilm was exposed to CHG continuously inside glass capillary biofilm reactor for 20 min. Scale bar, 30 μm. Repro‐ duced from Takenaka et al. [10] with permission.

Davison et al. investigated the dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of *Staphylococ‐ cusepidermidis* using time-lapse confocal scanning laser microscopy [21]. Chlorine among these chemicals was the only antimicrobial agent that caused any biofilm removal. Yamaguchi et al. showed that treatment of *Porphyromonas gingivalis* biofilms with CHG for 5 min does not degrade their external structure, or reduce the volumes of protein and carbohydrate constit‐ uents [22]. A summary of representative experiments demonstrating that chemical approach failed to detach the biofilm structure is shown in **Table 1**.



EtOH: ethanol; CHG: chlorhexidinegluconate; SLS: sodium lauryl sulfate; TRN: triclosan; CPC; cetylpyridinium chloride; IPMP: isopropyl methyl phenol; QAC: quaternary ammonium compound

**Table 1.** A summary of representative experiments demonstrating that chemical approach failed to detach the biofilm structure.

In contrast, there are some reports that the biofilm structure has been successfully degraded by repeated exposures of mouthrinse [23‒25]. Although it is likely that biofilm reduction may be enhanced by repeated pulse of a mouthrinse, this approach may not always be effective. Pratten and Wilson have reported that anaerobic counts in dental plaque biofilm returned to pretreatment levels with altered bacterial composition after 4 days, despite the continuous pulsing of CHG [26].

Summarizing the above, these results suggest that chemical approach such as the mouth‐ rinse, especially without repeated use, may not be sufficient to eradicate oral biofilm struc‐ ture. Residual structure may cause adverse effects in oral environment, even if the microorganisms in the biofilm are completely killed.

#### *2.1.1. Antigen and host inflammatory reaction*

oxidase, and potassium thiocyanate, even after 20 min exposure. Treatments with CHG and

**Figure 3.** Transmission images of biofilm cluster before (A) and after (B) 0.12% chlorhexidine (CHG) treatment. The biofilm was exposed to CHG continuously inside glass capillary biofilm reactor for 20 min. Scale bar, 30 μm. Repro‐

Davison et al. investigated the dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of *Staphylococ‐ cusepidermidis* using time-lapse confocal scanning laser microscopy [21]. Chlorine among these chemicals was the only antimicrobial agent that caused any biofilm removal. Yamaguchi et al. showed that treatment of *Porphyromonas gingivalis* biofilms with CHG for 5 min does not degrade their external structure, or reduce the volumes of protein and carbohydrate constit‐ uents [22]. A summary of representative experiments demonstrating that chemical approach

> **Antimicrobial agent**

0.12% CHG Biotene

Biotene

0.1% SLS 0.03% TRN 0.12% CHG 0.05% CPC 0.005% nisin **Exposure time**

20 min Microscopic

60 min Microscopic

observation (transmission image)

observation (transmission image)

**Judgment Reference**

[10]

[12]

EtOH resulted in only a slight contraction of the biofilm (**Figure 3**).

duced from Takenaka et al. [10] with permission.

428 Microbial Biofilms - Importance and Applications

**Bacterium Experimental**

Multispecies (*Streptococcus oralis*, *Streptococcus gordonii*, *Actinomycesnaeslundii*)

Multispecies (*Streptococcus oralis*, *Streptococcus gordonii*, *Actinomycesnaeslundii*)

failed to detach the biofilm structure is shown in **Table 1**.

**Incubation time**

Flow-cell 20h 11.6% EtOH

Flow-cell 20h 40% EtOH

**design**

As the remaining biofilm matrix contains carbohydrates, proteins, polysaccharide, lipids, and nucleic acid [27], dead bacteria and biofilm components could work as antigens and induce inflammatory reactions.

For example, *Actinobacillus actinomycetemcomitans, P. gingivalis, Tannerella forsythia*, and *Treponema denticola* have been implicated in the development of various forms of periodonti‐

tis. An extensive review of the literature revealed that lipopolysaccharide or outer mem‐ brane lipids, polysaccharide, fimbriae and outer membrane, and secreted proteins are antigens of all four bacteria that may play a role in disease pathogenesis [28].

*gingivalis* adherent with the residual biofilm developed in saliva-coated well following a CHG treatment for 5 min using a confocal laser microscopy [22]. The volume of *P. gingivalis* adhering to the residual structure was greater than that in saliva-coated wells. This result indicates that the residual biofilm could serve as a scaffold for the secondary biofilm formation. Outer membrane vesicles produced by *P. gingivalis* promote autoaggregation and coaggregation of another bacterial species [41, 42]. In addition, they also enhance the attachment to and invasion

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431

Our research group has demonstrated that residual structure of *S. mutans* biofilm following complete disinfection favors secondary bacterial adhesion and biofilm redevelopment [40]. At first, *S. mutans* biofilm generated on a resin-composite disc in a rotating disc reactor was disinfected completely with 70% isopropyl alcohol, and returned to the reactor. The same bacterial strains in the logarithmic phase were then flowed into the reactorfor 4 h. The amount of secondary adhered cells on the remaining structure was compared with that on a disc without structure using confocal laser scanning microscopic (CLSM) analysis and quantita‐ tive analysis. Three-dimensional reconstruction revealed that viable bacteria appear to get caught to upstream edges of disinfected biofilm structure (**Figure 4**). The cryosectioned sample demonstrated stratified patterns of viable cells beside the structure. Mean viable count adhered on the structure was significantly higher than that on plane surface. This result showed that

**Figure 4.** Three-dimensional reconstructed images of 4-h secondary biofilm (green) on disinfected 72-h biofilm struc‐ ture (red). Fresh planktonic *S. mutans* cells flowed into the completely disinfected 72-h biofilm structure for 4 h. Viable bacteria were stained green by calcein fluorescence and appeared to get caught in upstream edges of disinfected bio‐

of epithelial cells by *T. forsythia* [43].

film structure.

In addition, even if the microorganisms in the biofilm are completely eradicated, various microbial components in the biofilm could play a role in disease pathogenesis. Augustin et al. reported that injection of dead components of *Enterococcus faecalis* into rats following mechan‐ ical aortic damage by a catheter produced endocarditic vegetation enriched with polymor‐ phonuclear cells [29]. Bacterial components have also been attracted considerable attention as an adjuvant. It has been reported that injection of structural components of the outer surface membrane led a variety of immunopotentiative actions following the activation of phago‐ cytes and leukocytes [30‒32].

### *2.1.2. Calculus formation*

The remaining dental biofilm structure will absorb calcium and phosphate from saliva for the formation of supragingival calculus and from crevicular fluid for the formation of subgingi‐ val calculus. Calculus formation begins with the deposition of kinetically favored precursor phases of calcium phosphate, octacalcium phosphate, and dicalcium phosphate dihydrate, which are gradually hydrolyzed and transformed into less soluble hydroxyapatite and whitlockite mineral phases [33].

The calculus surface may not in itself induce inflammation in the adjacent periodontal tissue [34, 35]. Jepsen et al. stated that periodontal healing occurs even in the presence of calculus as long as the bacteria is removed or disinfected [34]. For example, it has been reported that autoclaved calculus does not cause pronounced inflammation or abscess formation in connective tissues [36]. Listgarten et al. have demonstrated that a normal epithelial attach‐ ment can be formed on its structure when microorganisms on calculus surface were com‐ pletely disinfected with CHG [37]. Johnson et al. investigatedthe clinical outcomes oftreatment with locally delivered controlled-release doxycycline (DH) or SRP in adult periodontitis patients. Treatment with either DH or SRP resulted in significant statistical and clinical improvements in clinical attachment levels, pocket depth, and bleeding on probing. These clinical outcomes were equivalent regardless of the extent of subgingival calculus present at baseline, suggesting that positive clinical change depend on altering the subgingival biofilm rather than the removal of calculus [38].

However, calculus is known to be a plaque retention factor as well as a reservoir for toxic bacterial products and antigens. Histological section of a human tooth root showed that calculus is covered with viable bacterial plaque [34]. Nichols et al. reported that the dihydro‐ ceramide lipids produced by *P. gingivalis* were found in subgingival calculus [39]. Hence, the presence of calculus will be a secondary etiological factor.

#### *2.1.3. Scaffold for secondary bacterial adhesion*

Recent investigations revealed that residual structure would promote a secondary bacterial adhesion and biofilm redevelopment [22, 40]. Yamaguchi et al. compared the volume of *P.*

*gingivalis* adherent with the residual biofilm developed in saliva-coated well following a CHG treatment for 5 min using a confocal laser microscopy [22]. The volume of *P. gingivalis* adhering to the residual structure was greater than that in saliva-coated wells. This result indicates that the residual biofilm could serve as a scaffold for the secondary biofilm formation. Outer membrane vesicles produced by *P. gingivalis* promote autoaggregation and coaggregation of another bacterial species [41, 42]. In addition, they also enhance the attachment to and invasion of epithelial cells by *T. forsythia* [43].

tis. An extensive review of the literature revealed that lipopolysaccharide or outer mem‐ brane lipids, polysaccharide, fimbriae and outer membrane, and secreted proteins are antigens

In addition, even if the microorganisms in the biofilm are completely eradicated, various microbial components in the biofilm could play a role in disease pathogenesis. Augustin et al. reported that injection of dead components of *Enterococcus faecalis* into rats following mechan‐ ical aortic damage by a catheter produced endocarditic vegetation enriched with polymor‐ phonuclear cells [29]. Bacterial components have also been attracted considerable attention as an adjuvant. It has been reported that injection of structural components of the outer surface membrane led a variety of immunopotentiative actions following the activation of phago‐

The remaining dental biofilm structure will absorb calcium and phosphate from saliva for the formation of supragingival calculus and from crevicular fluid for the formation of subgingi‐ val calculus. Calculus formation begins with the deposition of kinetically favored precursor phases of calcium phosphate, octacalcium phosphate, and dicalcium phosphate dihydrate, which are gradually hydrolyzed and transformed into less soluble hydroxyapatite and

The calculus surface may not in itself induce inflammation in the adjacent periodontal tissue [34, 35]. Jepsen et al. stated that periodontal healing occurs even in the presence of calculus as long as the bacteria is removed or disinfected [34]. For example, it has been reported that autoclaved calculus does not cause pronounced inflammation or abscess formation in connective tissues [36]. Listgarten et al. have demonstrated that a normal epithelial attach‐ ment can be formed on its structure when microorganisms on calculus surface were com‐ pletely disinfected with CHG [37]. Johnson et al. investigatedthe clinical outcomes oftreatment with locally delivered controlled-release doxycycline (DH) or SRP in adult periodontitis patients. Treatment with either DH or SRP resulted in significant statistical and clinical improvements in clinical attachment levels, pocket depth, and bleeding on probing. These clinical outcomes were equivalent regardless of the extent of subgingival calculus present at baseline, suggesting that positive clinical change depend on altering the subgingival biofilm

However, calculus is known to be a plaque retention factor as well as a reservoir for toxic bacterial products and antigens. Histological section of a human tooth root showed that calculus is covered with viable bacterial plaque [34]. Nichols et al. reported that the dihydro‐ ceramide lipids produced by *P. gingivalis* were found in subgingival calculus [39]. Hence, the

the

Recent investigations revealed that residual structure would promote a secondary bacterial adhesion and biofilm redevelopment [22, 40]. Yamaguchi et al. compared the volume of *P.*

of all four bacteria that may play a role in disease pathogenesis [28].

cytes and leukocytes [30‒32].

430 Microbial Biofilms - Importance and Applications

whitlockite mineral phases [33].

rather than the removal of calculus [38].

*2.1.3. Scaffold for secondary bacterial adhesion*

presence of calculus will be a secondary etiological factor.

*2.1.2. Calculus formation*

Our research group has demonstrated that residual structure of *S. mutans* biofilm following complete disinfection favors secondary bacterial adhesion and biofilm redevelopment [40]. At first, *S. mutans* biofilm generated on a resin-composite disc in a rotating disc reactor was disinfected completely with 70% isopropyl alcohol, and returned to the reactor. The same bacterial strains in the logarithmic phase were then flowed into the reactorfor 4 h. The amount of secondary adhered cells on the remaining structure was compared with that on a disc without structure using confocal laser scanning microscopic (CLSM) analysis and quantita‐ tive analysis. Three-dimensional reconstruction revealed that viable bacteria appear to get caught to upstream edges of disinfected biofilm structure (**Figure 4**). The cryosectioned sample demonstrated stratified patterns of viable cells beside the structure. Mean viable count adhered on the structure was significantly higher than that on plane surface. This result showed that

**Figure 4.** Three-dimensional reconstructed images of 4-h secondary biofilm (green) on disinfected 72-h biofilm struc‐ ture (red). Fresh planktonic *S. mutans* cells flowed into the completely disinfected 72-h biofilm structure for 4 h. Viable bacteria were stained green by calcein fluorescence and appeared to get caught in upstream edges of disinfected bio‐ film structure.

the residual structure following antimicrobial disinfection promoted bacterial secondary adhesion and biofilm formation.

This phenomenon may have clinical relevance because bacteria are exposed to sub-MIC of antibiotics at the beginning and end of a dosing regimen [63]. In addition, antimicrobials are retarded to diffuse within the biofilm matrix [14, 15]. In such cases, the bacteria in deeper areas

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433

As for oral biofilm, there are a few studies reported that sub-MICs of antimicrobial agents upregulate the genes related to EPS production and induce biofilm formation. Dong et al. evaluated the expression of genes related to *S. mutans* biofilm formation following treatment with 1/2 MIC of CHG, tea polyphenols, and sodium fluoride (NaF) [64]. The results showed that expression of *gtfB, gtfC, luxS, comD*, and *comE* was significantly upregulated after treatment with each antimicrobial agent in planktonic cells. Similarly, *gtfB, luxS, comD*, and *comE* were also upregulated in biofilm. Morphological observation using a FE-SEM and CLSM revealed that the biofilms of *S. mutans* treated with sub-MICs of NaF or CHG became denser, containing more EPS and fewer water channels. However, tea polyphenols appear to not promote *S. mutans* biofilm formation, as evidenced by SEM and CLSM images. Little EPS was produced on the surface of teeth after *S. mutans* was treated with a sub-MIC of tea polyphe‐ nols, although the expressions of gtfB and gtfC genes were upregulated. The inconsistency of these results can be explained by that sub-MICs of tea polyphenols may prevent from bacterial adhesion to the surface of teeth in the presence of fluid shear force. Because the gene analy‐ sis was performed using a 24-well plate under a static condition, whereas the biofilm forma‐ tion for morphological analysis was prepared under a controlled flow. It has been reported that tea polyphenols could decrease the adherence of *S. mutans* to glass surface [65, 66].

Bedran et al. investigated the effect of triclosan at sub-MICs on *S. mutans* biofilm formation, adherence to oral epithelial cells and expression of several genes involved in adherence and biofilm formation [67]. The authors reported that biofilm formation increased six-fold in the presence of 1/4 MIC of triclosan. Growth of *S. mutans* in the presence of triclosan at sub-MICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. Furthermore, the expression of *comD, gtfC*, and *LuxS* was significantly upregulated in the presence of 1/2

Even in limited works with regard to oral biofilms, it is likely that short-time exposure of antimicrobial agents in oral cavity sometimes cause adverse influences because the survived microorganisms after exposure to the agents will alter gene expressions in a positive and

Although chemical agents provide some benefits in terms of controlling oral biofilms, they have the limitation of leaving biofilm structures that may induce adverse reactions such as biofilm regrowth. Furthermore, sub-MICs of certain antimicrobial agents might induce biofilm formation and upregulate pathogenic genes. Future strategies for the control of oral biofilms

and 1/4 MIC, although the expression of *atlA* and *gtfB* was less pronounced.

may therefore shift to the degradation and/or detachment of biofilm matrix.

are exposed to antimicrobials at sub-MICs.

negative way.

**3. Conclusion**

The mechanism of *S. mutans* adhesion on the residual structure can be explained by cell-cell aggregation and glucan-dependent aggregation. The cell surface protein antigen c (PAc) of *S. mutans* is known to correlate with cellular hydrophobicity, sucrose-independent adhesion to tooth surface and self-aggregation between cells [44, 45]. The glucan-dependent aggregation is mediated by glucosyltransferase enzymes and glucan-binding proteins [46]. Glucan-binding protein C, which is a cell-wall anchoring protein and a cell surface glucan receptor, plays an important role in sucrose-dependent adhesion by binding to soluble glucan synthesized by glucosyltranseferase D [47, 48].

Thus, since a numerous and diverse range of microorganisms reside in our intraoral environ‐ ment, the residual biofilm will contribute to biofilm redevelopment.

### **2.2. Antimicrobials-induced biofilm formation**

Numerous studies have shown that subminimum inhibitory concentrations (sub-MICs) of various antibiotics and chemicals can inhibit biofilm formation. A representative example is the macrolide antibiotics. Although *Pseudomonas aeruginosa* that contributes to progress respiratory infection is resistant to azithromycin, low-dose azithromycin has been shown to inhibit protein synthesis [49] and improve clinical symptom [50, 51]. Sub-MIC concentra‐ tions of azithromycin have also been shown to inhibit quorum sensing and alginate produc‐ tion [52, 53].

In the field of dentistry, it has also been reported that sub-MICs of antimicrobial agents or compounds can inhibit bacterial attachment [54, 56, 57], biofilm formation [54, 55, 57, 58], and downregulate virulence genes [54, 56, 59, 60]. Moon et al. reported N-acetyl cysteine (NAC) that is an antioxidant possessing anti-inflammatory activities, showed a significant decrease of *Prevotella intermedia* biofilm formation in the presence of sub-MIC [55]. NAC was demon‐ strated to present the expression of LPS-induced inflammatory mediators in phagocytic cells and gingival fibroblasts during the inflammatory process. Lee and Tan showed that treat‐ ment of *E. faecalis* with 1/2 sub-MIC of (–)-epigallocatechin-3-gallate (EGCG) significantly inhibited the expression of virulence genes related to collagen adhesion, cytolysins activator, gelatinase, and serine protease compared with the untreated control [60].

In contrast to the inhibitory effects of sub-MIC antimicrobials against biofilm formation, recent studies have shown that some antibiotics at sub-MIC can significantly induce biofilm formation in a variety of bacterial species such as *S. epidermidis, Staphylococcus aureus, Staphylococcus lugdunensis, Escherichia coli*, and *P. aeruginosa* [61]. Kaplan et al. demonstrated that sub-MIC of four different β-lactam antibiotics significantly induce biofilm formation in some strains of *S. aureus* [62]. The amount of biofilm induction was 10-fold in maximum and sub-MIC β-lactamantibiotics induce autolysin-dependent extracellular DNA release. However, the pattern of biofilm induction was strain and antibiotic dependent, indicating that biofilm formation by sub-MICs of antimicrobial agents do not always occur in all the strains of the same species.

This phenomenon may have clinical relevance because bacteria are exposed to sub-MIC of antibiotics at the beginning and end of a dosing regimen [63]. In addition, antimicrobials are retarded to diffuse within the biofilm matrix [14, 15]. In such cases, the bacteria in deeper areas are exposed to antimicrobials at sub-MICs.

As for oral biofilm, there are a few studies reported that sub-MICs of antimicrobial agents upregulate the genes related to EPS production and induce biofilm formation. Dong et al. evaluated the expression of genes related to *S. mutans* biofilm formation following treatment with 1/2 MIC of CHG, tea polyphenols, and sodium fluoride (NaF) [64]. The results showed that expression of *gtfB, gtfC, luxS, comD*, and *comE* was significantly upregulated after treatment with each antimicrobial agent in planktonic cells. Similarly, *gtfB, luxS, comD*, and *comE* were also upregulated in biofilm. Morphological observation using a FE-SEM and CLSM revealed that the biofilms of *S. mutans* treated with sub-MICs of NaF or CHG became denser, containing more EPS and fewer water channels. However, tea polyphenols appear to not promote *S. mutans* biofilm formation, as evidenced by SEM and CLSM images. Little EPS was produced on the surface of teeth after *S. mutans* was treated with a sub-MIC of tea polyphe‐ nols, although the expressions of gtfB and gtfC genes were upregulated. The inconsistency of these results can be explained by that sub-MICs of tea polyphenols may prevent from bacterial adhesion to the surface of teeth in the presence of fluid shear force. Because the gene analy‐ sis was performed using a 24-well plate under a static condition, whereas the biofilm forma‐ tion for morphological analysis was prepared under a controlled flow. It has been reported that tea polyphenols could decrease the adherence of *S. mutans* to glass surface [65, 66].

Bedran et al. investigated the effect of triclosan at sub-MICs on *S. mutans* biofilm formation, adherence to oral epithelial cells and expression of several genes involved in adherence and biofilm formation [67]. The authors reported that biofilm formation increased six-fold in the presence of 1/4 MIC of triclosan. Growth of *S. mutans* in the presence of triclosan at sub-MICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. Furthermore, the expression of *comD, gtfC*, and *LuxS* was significantly upregulated in the presence of 1/2 and 1/4 MIC, although the expression of *atlA* and *gtfB* was less pronounced. sub-MICsindicating

Even in limited works with regard to oral biofilms, it is likely that short-time exposure of antimicrobial agents in oral cavity sometimes cause adverse influences because the survived microorganisms after exposure to the agents will alter gene expressions in a positive and negative way.

### **3. Conclusion**

the residual structure following antimicrobial disinfection promoted bacterial secondary

The mechanism of *S. mutans* adhesion on the residual structure can be explained by cell-cell aggregation and glucan-dependent aggregation. The cell surface protein antigen c (PAc) of *S. mutans* is known to correlate with cellular hydrophobicity, sucrose-independent adhesion to tooth surface and self-aggregation between cells [44, 45]. The glucan-dependent aggregation is mediated by glucosyltransferase enzymes and glucan-binding proteins [46]. Glucan-binding protein C, which is a cell-wall anchoring protein and a cell surface glucan receptor, plays an important role in sucrose-dependent adhesion by binding to soluble glucan synthesized by

Thus, since a numerous and diverse range of microorganisms reside in our intraoral environ‐

Numerous studies have shown that subminimum inhibitory concentrations (sub-MICs) of various antibiotics and chemicals can inhibit biofilm formation. A representative example is the macrolide antibiotics. Although *Pseudomonas aeruginosa* that contributes to progress respiratory infection is resistant to azithromycin, low-dose azithromycin has been shown to inhibit protein synthesis [49] and improve clinical symptom [50, 51]. Sub-MIC concentra‐ tions of azithromycin have also been shown to inhibit quorum sensing and alginate produc‐

In the field of dentistry, it has also been reported that sub-MICs of antimicrobial agents or compounds can inhibit bacterial attachment [54, 56, 57], biofilm formation [54, 55, 57, 58], and downregulate virulence genes [54, 56, 59, 60]. Moon et al. reported N-acetyl cysteine (NAC) that is an antioxidant possessing anti-inflammatory activities, showed a significant decrease of *Prevotella intermedia* biofilm formation in the presence of sub-MIC [55]. NAC was demon‐ strated to present the expression of LPS-induced inflammatory mediators in phagocytic cells and gingival fibroblasts during the inflammatory process. Lee and Tan showed that treat‐ ment of *E. faecalis* with 1/2 sub-MIC of (–)-epigallocatechin-3-gallate (EGCG) significantly inhibited the expression of virulence genes related to collagen adhesion, cytolysins activator,

In contrast to the inhibitory effects of sub-MIC antimicrobials against biofilm formation, recent studies have shown that some antibiotics at sub-MIC can significantly induce biofilm formation in a variety of bacterial species such as *S. epidermidis, Staphylococcus aureus, Staphylococcus lugdunensis, Escherichia coli*, and *P. aeruginosa* [61]. Kaplan et al. demonstrated that sub-MIC of four different β-lactam antibiotics significantly induce biofilm formation in some strains of *S. aureus* [62]. The amount of biofilm induction was 10-fold in maximum and sub-MIC β-lactamantibiotics induce autolysin-dependent extracellular DNA release. However, the pattern of biofilm induction was strain and antibiotic dependent, indicating that biofilm formation by sub-MICs of antimicrobial agents do not always occur in all the strains

ment, the residual biofilm will contribute to biofilm redevelopment.

gelatinase, and serine protease compared with the untreated control [60].

**2.2. Antimicrobials-induced biofilm formation**

adhesion and biofilm formation.

432 Microbial Biofilms - Importance and Applications

glucosyltranseferase D [47, 48].

tion [52, 53].

of the same species.

Although chemical agents provide some benefits in terms of controlling oral biofilms, they have the limitation of leaving biofilm structures that may induce adverse reactions such as biofilm regrowth. Furthermore, sub-MICs of certain antimicrobial agents might induce biofilm formation and upregulate pathogenic genes. Future strategies for the control of oral biofilms may therefore shift to the degradation and/or detachment of biofilm matrix.

### **Author details**

Shoji Takenaka1\*, Masataka Oda2 , Hisanori Domon2 , Rika Wakamatsu1 , Tatsuya Ohsumi1 , Yutaka Terao2 and Yuichiro Noiri1

[10] Takenaka S, Trivedi HM, Corbin A, Pitts B, Stewart PS. Direct visualization of spatial and temporal patterns of antimicrobial action within model oral biofilms. Appl Environ

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\*Address all correspondence to: takenaka@dent.niigata-u.ac.jp

1 Division of Cariology, Operative Dentistry and Endodontics, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

2 Division of Microbiology and Infectious Diseases, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

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**Chapter 19**

**Modulation of Biofilm Growth by Sub‐Inhibitory**

It is generally accepted that bacteria in biofilm are more resistant to antibacterials than their planktonic counterparts. For numerous antibiotics, it has been shown that minimal inhibitory concentrations (MICs) for bacteria grown in broth are much lower than the minimal biofilm inhibition concentrations. While sub‐inhibitory concentrations, that is, amounts of antibacterials below the MIC, do not either influence or suppress to some extentorotherthebacterialgrowthinliquidmedia,these same amountsofdrugs,natural substances, etc., may have diverse effects on bacterial biofilms, ranging from suppres‐ sion to stimulation of the sessile growth and varying with regard to the bacterial species and strains. This is a source of additional risks for both biofilm infection of host tissues and contamination indwelling devices. When considering the data for biofilm modula‐ tion, differences in experimental protocols should be taken into account, as well as the

**Keywords:** biofilm, sub‐MIC, antibiotics, bacteriocins, antimicrobial peptides, plant

While the development of antibiotics during the twentieth century resulted in remarkable advances in the fight against infectious microorganisms, it was unfortunately paralleled with the highly increasing risks for the development of antibiotic resistance. These risks are a consequence of the extensive use of antibacterial preparations in both human medicine and agriculture. Resistance has become a threat to human and animal health worldwide, and it

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Amounts of Antibacterial Substances**

Stoyanka R. Stoitsova,

http://dx.doi.org/10.5772/62939

Dayana B. Borisova

**Abstract**

metabolites

**1. Introduction**

Tsvetelina S. Paunova‐Krasteva and

Additional information is available at the end of the chapter

strain‐specific mechanisms of biofilm formation.
