**Modulation of Biofilm Growth by Sub‐Inhibitory Amounts of Antibacterial Substances**

Stoyanka R. Stoitsova, Tsvetelina S. Paunova‐Krasteva and Dayana B. Borisova

Additional information is available at the end of the chapter

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

#### **Abstract**

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 strain‐specific mechanisms of biofilm formation. extent or other the bacterial growth in liquid media, these same amounts of drugs, naturalantimicrobial

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

### **1. Introduction**

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

© 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.

necessitates the development of key measures. Among these, the identification of critical points of control, the development of surveillance measures, and the prevention of environmental contamination are in focus [1].

The idea on the effects of antibiotic sub‐MICs on biofilms is getting more and more compli‐ cated with the accumulation of experimental data. This puts forward the question of method‐ ology. The conventional approaches to antibiotic sensitivity do not apply to biofilm‐grown bacteria [9]. Due to the potentially very high intrinsic biofilm resistance, the focus has mainly been put on their prevention [16]. Probably for this reason, most results have been obtained while applying the drug during the sessile growth, with only a few studies testing the agent's effects on pre‐formed biofilms [17–19]. The routinely applied methodology is to test biofilm biomass on 96‐well plates by the crystal violet assay, with only a few other studies that explore cell viability as well, for example, the viable cell counts [20] or live‐dead staining for fluores‐

Modulation of Biofilm Growth by Sub‐Inhibitory Amounts of Antibacterial Substances

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

443

We have summarized the available experimental data on the action of sub‐MICs of antibiot‐ ics on biofilms in **Table 1**–**5**. We could find no strict pattern with regard to the effects of the separate groups of antibiotics. All groups were shown to influence some biofilms positively, and others, negatively. An important observation is the bacterial species and strain specifici‐ ty of the response to the sub‐MICs. Thus, sub‐MICs of ampicillin increased biofilm growth of *Staphylococcus saprophyticus* [6], reduced it in *Escherichia coli* K‐12 [21], and had no effect on *E. coli* UTI8 and *Mycobacterium avium* [6, 22] (**Table 1**). Sub‐MICs of ciprofloxacin promoted biofilms of *S. saprophyticus* [6] and *E. coli* UTI8 [6], but reduction was registered in *Streptococ‐ cus suis*, *Salmonella enterica* serovar Typhimurium clinical strains, *Stenotrophomonas maltophil‐ ia*, *Streptococcus pyogenes*, *Staphylococcus epidermidis*, and *Proteus mirabilis* [17, 18, 20, 23–25] (**Table 2**). Diverse effects have been illustrated for sub‐MICs of erythromycin on biofilms of *S. suis*, *Corynebacterium diphtheriae*, and *S. epidermidis* [25–27] (**Table 3**); for gentamycin on *S. enterica* serovar Typhimurium, *S. saprophyticus*, *E. coli*, *Haemophilus influenzae*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* (**Table 4**); and for tetracyclin on *E. coli*, *Staphylococcus*

*lugdunensis*, *M. avium*, *P. aeruginosa*, and *S. epidermidis* [9, 15, 21, 22, 28] (**Table 5**).

8 μg/ml *S. epidermidis* M187

Nafcillin 0.0625–0.5 MIC

> 0.0625–0.5 MIC

*S. haemolyticus* M176

infections, etc.)

infections, etc.)

**Antibiotics Amount Bacteria/strains Effect on biofilm Ref. Penicillins** Dicloxacillin 1/2 MIC *S. epidermidis* strains 9142, IE186, and M187 32–60% BF inhibition [29]

**Penicillin** 1/16–1/2 MIC*S. suis* NJ‐3 Dose‐dependent BF suppression [25]

**Cephalosporins** Cefazolin 1/2 MIC *S. epidermidis* strains 9142, IE186, and M187 32–55% BF inhibition [29]

Methicillin 1/3–1/8 MIC *S. aureus* Newman Denser BF formed by the strain and its small‐colony

*S. lugdunensis*—15 isolates from BF‐related infections (endocarditis, prosthetic joint

*S. lugdunensis*—15 isolates from BF‐related infections (endocarditis, prosthetic joint

1/8 MIC *C. diphtheriae* subsp. mitis strains No effect [26]

1/3–1/8 MIC *S. aureus* Newman No effect on BF [30]

0.5 MIC *S. epidermidis* strains SE5 and RP62A BF decrease [31]

decrease in 7%

variants

BF biomass reduction; decreased synthesis of the EPS,

Increase in 93% of the tested strains, no effect in 7% [9]

Increase in 13% of the tested strains, no effect in 80%, and

[16]

[30]

[9]

poly‐*N*‐acetyl‐glucosamine

cence microscopy.

In the aquatic and terrestrial environments, the contaminated sites (wastewater systems, pharmaceutical factories effluents, animal husbandry facilities, etc.) are characterized by the presence of subtherapeutic concentrations of antibiotics [1–3]. Thus, bacteria present in the environment are often subjected to drug amounts lower than the minimal inhibitory concen‐ trations (MICs) [4]. Antimicrobial sub‐MICs are encountered in the human body as well, during treatment, which can occur irregularly at intervals at the site of infection [5] or in cases of low‐dose antibiotic prophylaxis [6]. When microorganisms grow in the presence of sub‐MICs, the antibiotics can potentially alter the physicochemical characteristics of micro‐ bial cells, their functions, and the expression of some virulence genes [7]. While sub‐MICs generally do not interfere with bacterial growth dynamics, the microorganisms are subjected to stress. As a way to counter stress, microbes would often form biofilms both in external environments and on indwelling medical devices [3, 8, 9].

It is noteworthy that, when MICs or sub‐MICs are considered, this concerns values obtained with bacteria grown in liquid media, that is, as plankton. Such will be the use of the term also in the present review. In biofilms, the inhibitory doses exceed 10 to even 1000 times these of plankton [1, 9, 10]. Interestingly, when plankton and bacteria dispersed from bio‐ film have been examined, they were shown to have similar antibiotic susceptibility [11]. Hence, increased resistance is likely associated with characteristics that are a consequence of the structure of the sessile microbial communities. They themselves represent heterologous microenvironments in which gradients of physical and chemical parameters exist [3]. The advantages of these structured bacterial communities comprise limited antibiotic diffusion, enhanced transmission of resistance genes, expression of efflux pumps, drug adsorption by extracellular matrix, as well as the presence of metabolically inactive persister cells [12].

Provided the growing concern about the wide spread and the role of environments contain‐ ing subinhibitory amounts of antibacterials, the present review will focus on the interplay of sub‐MICs with biofilm growth and/or detachment. In a previous review, the antibiotic‐in‐ duced biofilm formation has been discussed [13]. However, the sub‐MIC of antibiotics, but also other antibacterials (e.g. antibacterial peptides, natural and synthetic substances, etc.), dependent on the combination drug‐bacterial strain or species, may have diverse effects on biofilm, from suppression through no effect to promotion. This determined the aim of the present review: to summarize current data and concepts about the modulation of biofilm growth by sub‐MICs of antibacterial substances.

### **2. Sub‐MIC of antibiotics and biofilms**

While it was initially believed that antibiotics in nature have the role for fighting against competitors, and that therefore also sub‐MICs would reduce virulence, recent evidence re‐ veals a more complicated picture, showing the capacity of some antibiotics at low dose to act as chemical signals to modulate metabolic processes [14] or regulate gene (including vir‐ ulence gene) expression [15].

The idea on the effects of antibiotic sub‐MICs on biofilms is getting more and more compli‐ cated with the accumulation of experimental data. This puts forward the question of method‐ ology. The conventional approaches to antibiotic sensitivity do not apply to biofilm‐grown bacteria [9]. Due to the potentially very high intrinsic biofilm resistance, the focus has mainly been put on their prevention [16]. Probably for this reason, most results have been obtained while applying the drug during the sessile growth, with only a few studies testing the agent's effects on pre‐formed biofilms [17–19]. The routinely applied methodology is to test biofilm biomass on 96‐well plates by the crystal violet assay, with only a few other studies that explore cell viability as well, for example, the viable cell counts [20] or live‐dead staining for fluores‐ cence microscopy.

necessitates the development of key measures. Among these, the identification of critical points of control, the development of surveillance measures, and the prevention of environmental

In the aquatic and terrestrial environments, the contaminated sites (wastewater systems, pharmaceutical factories effluents, animal husbandry facilities, etc.) are characterized by the presence of subtherapeutic concentrations of antibiotics [1–3]. Thus, bacteria present in the environment are often subjected to drug amounts lower than the minimal inhibitory concen‐ trations (MICs) [4]. Antimicrobial sub‐MICs are encountered in the human body as well, during treatment, which can occur irregularly at intervals at the site of infection [5] or in cases of low‐dose antibiotic prophylaxis [6]. When microorganisms grow in the presence of sub‐MICs, the antibiotics can potentially alter the physicochemical characteristics of micro‐ bial cells, their functions, and the expression of some virulence genes [7]. While sub‐MICs generally do not interfere with bacterial growth dynamics, the microorganisms are subjected to stress. As a way to counter stress, microbes would often form biofilms both in external

It is noteworthy that, when MICs or sub‐MICs are considered, this concerns values obtained with bacteria grown in liquid media, that is, as plankton. Such will be the use of the term also in the present review. In biofilms, the inhibitory doses exceed 10 to even 1000 times these of plankton [1, 9, 10]. Interestingly, when plankton and bacteria dispersed from bio‐ film have been examined, they were shown to have similar antibiotic susceptibility [11]. Hence, increased resistance is likely associated with characteristics that are a consequence of the structure of the sessile microbial communities. They themselves represent heterologous microenvironments in which gradients of physical and chemical parameters exist [3]. The advantages of these structured bacterial communities comprise limited antibiotic diffusion, enhanced transmission of resistance genes, expression of efflux pumps, drug adsorption by extracellular matrix, as well as the presence of metabolically inactive persister cells [12].

Provided the growing concern about the wide spread and the role of environments contain‐ ing subinhibitory amounts of antibacterials, the present review will focus on the interplay of sub‐MICs with biofilm growth and/or detachment. In a previous review, the antibiotic‐in‐ duced biofilm formation has been discussed [13]. However, the sub‐MIC of antibiotics, but also other antibacterials (e.g. antibacterial peptides, natural and synthetic substances, etc.), dependent on the combination drug‐bacterial strain or species, may have diverse effects on biofilm, from suppression through no effect to promotion. This determined the aim of the present review: to summarize current data and concepts about the modulation of biofilm

While it was initially believed that antibiotics in nature have the role for fighting against competitors, and that therefore also sub‐MICs would reduce virulence, recent evidence re‐ veals a more complicated picture, showing the capacity of some antibiotics at low dose to act as chemical signals to modulate metabolic processes [14] or regulate gene (including vir‐

contamination are in focus [1].

442 Microbial Biofilms - Importance and Applications

environments and on indwelling medical devices [3, 8, 9].

growth by sub‐MICs of antibacterial substances.

**2. Sub‐MIC of antibiotics and biofilms**

ulence gene) expression [15].

We have summarized the available experimental data on the action of sub‐MICs of antibiot‐ ics on biofilms in **Table 1**–**5**. We could find no strict pattern with regard to the effects of the separate groups of antibiotics. All groups were shown to influence some biofilms positively, and others, negatively. An important observation is the bacterial species and strain specifici‐ ty of the response to the sub‐MICs. Thus, sub‐MICs of ampicillin increased biofilm growth of *Staphylococcus saprophyticus* [6], reduced it in *Escherichia coli* K‐12 [21], and had no effect on *E. coli* UTI8 and *Mycobacterium avium* [6, 22] (**Table 1**). Sub‐MICs of ciprofloxacin promoted biofilms of *S. saprophyticus* [6] and *E. coli* UTI8 [6], but reduction was registered in *Streptococ‐ cus suis*, *Salmonella enterica* serovar Typhimurium clinical strains, *Stenotrophomonas maltophil‐ ia*, *Streptococcus pyogenes*, *Staphylococcus epidermidis*, and *Proteus mirabilis* [17, 18, 20, 23–25] (**Table 2**). Diverse effects have been illustrated for sub‐MICs of erythromycin on biofilms of *S. suis*, *Corynebacterium diphtheriae*, and *S. epidermidis* [25–27] (**Table 3**); for gentamycin on *S. enterica* serovar Typhimurium, *S. saprophyticus*, *E. coli*, *Haemophilus influenzae*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* (**Table 4**); and for tetracyclin on *E. coli*, *Staphylococcus lugdunensis*, *M. avium*, *P. aeruginosa*, and *S. epidermidis* [9, 15, 21, 22, 28] (**Table 5**).



**Antibiotics Amount Bacteria/strains Effect on biofilm Ref.**

Norfloxacin 1–10,000 mg/l *S. aureus* ATCC 25923 BF stimulation by 1 mg/l [8]

1/2–1/8 MIC *S*. *maltophilia*—5 clinical isolates strains

1/2–1/8 MIC *Staphylococcus epidermidis*—20 clinical isolates

strains

1/2–1/8 MIC *Staphylococcus epidermidis—*20 clinical isolates

1/2–1/8 MIC *S*. *maltophilia*—5 clinical isolates strains

0.0625– 0.5 MIC *S. lugdunensis*—15 isolates from

infections, etc.)

3362‐33; 3362‐34)

1/2–1/8 MIC *S*. *maltophilia*—5 clinical isolates strains

0.03–0.06 MIC *S. maltophilia* strains Sm 132 and Sm 144

2; 10; 50; 100 x MIC*S. aureus*—6 strains of coagulase negative

1 μg/ml *M. avium* strains (101, 104, 109, and

1/2–1/8MIC *S*. *maltophilia* —5 clinical isolates strains

A5)

strains

Grepafloxacin 1/2–1/8MIC *S*. *maltophilia*—5 clinical isolates

**Table 2.** Effects of sub‐MIC of fluoroquinolones on biofilms.

M89; M65; M100; M74)

biofilm‐related infections (endocarditis, prosthetic joint

Ofloxacin 1/2–1/8MIC *S*. *maltophilia*—5 clinical isolates

Levofloxacin 1/2–1/64 MIC *S. pyogenes* isolates (M56; st38;

Moxifloxacin 1 μg/l *M. avium* strains (104; 101; A5;

1/2–1/64 MIC *S. pyogenes* isolates (M56; st38;M89; M65; M100; M74)

1/2–1/64 MIC *S. pyogenes* isolates (M56; st38;M89; M65; M100; M74)

0.001–100 mg/l *P. aeruginosa* BF reduction [15]

1–10,000 mg/l *P. aeruginosa* NNRL‐B3509 BF stimulation by 1 mg/l [8]

1/16–1/2 MIC *S. suis* NJ‐3 Dose‐dependent biofilm suppression [25]

0.5 × MIC *S. epidermidis* strains (SE5; RP62A) No effect on BF formation [31]

counts

Modulation of Biofilm Growth by Sub‐Inhibitory Amounts of Antibacterial Substances

counts

pre‐formed BF

pre‐formed BF

cell counts

counts

counts

counts

Pefloxacin 1/2–1/8 MIC *S. epidermidis*—20 clinical isolates Reduces BF growth and pre‐formed BF [17]

**Antibiotics Amount Bacteria/strains Effect on biofilm Ref.** Erythromycin 1/16–1/2 MIC *S. suis* NJ‐3 Dose‐dependent BF suppression [25]

1/8 MIC *C. diphtheriae* subsp. mitis strains BF increase [26]

BF inhibition and reduction of viable cell

Dose‐dependent BF inhibition [23]

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

Suppression of BF growth and reduction of

BF inhibition and reduction of viable cell

Dose‐dependent BF inhibition [23]

Suppression of BF growth and reduction of

Biofilm inhibition and reduction of viable

Dose‐dependent BF inhibition [23]

No effect on BF [22]

Decrease in adhesion and BF formation [7]

No effect on BF [35]

BF inhibition [36]

BF inhibition and reduction of viable cell

BF inhibition and reduction of viable cell

BF inhibition and reduction of viable cell

Increase in 20% of the tested strains, in 47%

no effect, and in 40% decrease

[20]

445

[17]

[20]

[17]

[20]

[9]

[20]

[20]

[20]

Abbreviations: BF, biofilm; EPS, exopolysaccharide; MIC, minimal inhibitory concentration.

**Table 1.** Effects of sub‐MIC of β‐lactam antibiotics on biofilms.



**Table 2.** Effects of sub‐MIC of fluoroquinolones on biofilms.

**Antibiotics Amount Bacteria/strains Effect on biofilm Ref.** Cefalotin 1/3–1/8 MIC *S. aureus* Newman Three‐ to fourfold denser BF formed by the strain and its

Cefoperazone 1/3–1/8 MIC *S. aureus* Newman No effect on BF [30]

*E. coli* UTI89 BF stimulation at MIC, no effect of

**Carbapenems** Imipenem 2–4 μg/ml *P. aeruginosa* PA01 BF induction, changes in BF morphology, upregulation of

Ceftazidime 1/2–1/8 MIC 5 clinical isolates strains *S*. *maltophilia* BF inhibition and reduction of viable cell counts [20]

2; 8; 32 mg/l 6 clinical isolates of *P. aeruginosa* Synergistic effect with polymorphonuclears against

**Antibiotics Amount Bacteria/strains Effect on biofilm Ref.** Ciprofloxacin 1/16–1/2 MIC *S. suis* NJ‐3 Dose‐dependent BF suppression [25]

*Typhimurium* clinical isolates 75

EPS

0.1–1.1 μg/ml*S. saprophyticus* 15305 Sub‐MIC (0.3–0.7 μg/ml) stimulate BF formation [6]

sub‐MIC

Cefotaxime 1/2–1/16 MIC*Salmonella enterica* serovar Typhimurium

3362‐34)

**Table 1.** Effects of sub‐MIC of β‐lactam antibiotics on biofilms.

1/2–1/16MIC *Salmonella enterica* serovar

strains

1/2–1/64 MIC *S. pyogenes* isolates (M56; st38;M89; M65; M100; M74)

1/2–1/8 MIC *Staphylococcus epidermidis*—20 clinical isolates

Ampicillin 0.005–500 μg/ml

> 1/2–1/1024 MIC

444 Microbial Biofilms - Importance and Applications

0.03 and 0.125 μg/ml

0.125–1.0 MIC

clinical isolates 75 strains

10 μg/l *M. avium* strains 104; 101, A5; 3362‐33 and

Abbreviations: BF, biofilm; EPS, exopolysaccharide; MIC, minimal inhibitory concentration.

small‐colony variants

*E. coli* MG1655 wt and MG1655 (pBR322) BF reduction [21]

73 isolates *A. baumannii* BF stimulation [33]

5 strains *P. mirabilis* BF inhibition [24]

developed 48 h BF

synthesis

MIC

counts

the BF bacteria

0.1–1.1 μg/ml *S. saprophyticus* 15305 BF stimulation by sub‐MIC (0.4–0.9 μg/ml) [6] 1/2–1/1024 MIC *E. coli* UTI89 Statistically significant BF increase and

1/2–1/8MIC 5 clinical isolates *S*. *maltophilia* BF inhibition and reduction of viable cell

0.5 × MIC *E. coli* strains (8; 9; 10; 31 ;1583) Reduction of BF formation and survival of

0.125–1.0 MIC 5 strains *P. mirabilis* BF inhibition [24]

h BF

2; 8; 32 mg/l MIC *P. aeruginosa*—6 clinical isolates Synergistic effect with

At 1/2 MIC—significantly increased production of BF and

No effect on BF [22]

Inhibition of BF formation and EPS

upregulation of BF‐associated genes at 1/4

Dose‐dependent BF inhibition [23]

Reduces BF growth and pre‐formed BF [17]

polymorphonuclears against developed 48‐

*ampC* and genes for alginate biosynthesis

[30]

[5]

[6]

[32]

[34]

[5]

[6]

[20]

[18]

[32]



**Antibiotics Amount Bacteria/strains Effect on biofilm Ref.**

**Antibiotics Amount Bacteria/ strains Effect on biofilm Ref.**

1/2 MIC *S. epidermidis* strains 9142, IE186 and M187 8–24% BF inhibition [29]

Modulation of Biofilm Growth by Sub‐Inhibitory Amounts of Antibacterial Substances

0.5× MIC *S. epidermidis* strains SE5 and RP62A Decrease in BF [31]

0.5–2 μg/ml *M. avium* strains (104; 101; A5; 3362‐33; 3362‐34)BF increase [22] 0.01–100 mg/l *P. aeruginosa* BF reduction [15]

1/2–1/8 MIC 5 clinical isolates strains *S*. *maltophilia* BF inhibition and reduction of viable cell

(pBR322)

93%

counts

0.005–500 μg/ml *E. coli* MG1655 wt and MG1655 (pBR322) Significant BF increase in the presence of

BF increase [40]

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

Enhancement of *ica*ADBC operon expression and EPS synthesis

Increase in 20% of the tested strains; in 47% no effect and in 33% decrease

Increase in 27% of the tested strains; in 47% no effect and in 27% decrease

Increase 7% of tested strains; decrease

Enhancement of *ica*ADBC operon expression and EPS synthesis

Increase in 20% of the tested strains, in 40% no effect, and in 40% decrease

In 80% no effect, in 20% decrease [9]

[28]

447

[9]

[9]

[21]

[9]

[28]

[9]

[20]

Kanamycin 10–110 μg/ml *P. aeruginosa* PAK and isogenic

**Table 4.** Effects of sub‐MIC of aminoglycosides on biofilms.

Streptogramins Quinupristin‐dalfopristin

Glycopeptides Vancomycin

Tetracyclins Tetracyclin

DHFR inhibitors Trimethroprim‐ sulfamethoxazole

Oxazolidonones Linezolid

and oxazolidonones on biofilms.

action of sub‐MICs [9].

mutants

0.5 μg/ml *S. epidermidis* strains 567 and *S. epidermidis* 561

infections, etc.)

infections, etc.)

infections, etc.)

0.5 μg/ml *S. epidermidis* strains 567 and *S. epidermidis* 561

infections, etc.)

infections, etc.)

0.0625–0.5 MIC *S. lugdunensis*—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint

0.0625–0.5 MIC *S. lugdunensis*—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint

0.0625–0.5 MIC *S. lugdunensis*—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint

0.0625–0.5 MIC *S. lugdunensis*—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint

0.0625–0.5 MIC *S. lugdunensis*—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint

**Table 5.** Effects of sub‐MIC of streptogramins, glycopeptides, tetracyclins, dihydrofolate reductase (DHFR) inhibitors

Antibiotics with identical mechanisms of antibacterial action, for example, gentamicin and erythromycin, may have different effects on biofilm [19, 29]. In addition, the sub‐MICs of a given antibiotic may have diverse effects on different strains of one species of microorgan‐ ism. For example, such is the case with the effects of cefazolin and levofloxacin on 15 isolates of *S. lugdunensis* [9] (**Table 1**), the effects of erythromycin on 69 clinical isolates of *S. epidermi‐ dis* [27] (**Table 3**), of vancomycin on 3 strains of *S. epidermidis* and 15 isolates of *S. lugdunensis* [9, 30] (**Table 5**), and of trimethoprim‐sulfamethoxazole on 15 isolates of *S. lugdunensis* [9] (**Table 5**). Obviously, individual strains use different response mechanisms to oppose the

Sub‐MICs of antibiotics have the potential to affect the structure of individual bacterial cells. Changes of morphology have been registered in several studies. For instance, sub‐MIC of

#### **Table 3.** Effects of sub‐MIC of macrolides on biofilms.



**Table 4.** Effects of sub‐MIC of aminoglycosides on biofilms.

**Antibiotics Amount Bacteria/strains Effect on biofilm Ref.**

Azithromycin 1/2–1/16 MIC *S. suis* NJ‐3 BF inhibition by 1/4 and 1/2 MIC [25]

Clarithromycin 1 μg/ml *M. avium* strains (101, 104, 109, and A5) BF inhibition [36] MIC b/n 50–550 mg/ml*P. aeruginosa* Sub‐MIC result in altered structure and

**Antibiotics Amount Bacteria/strains Effect on biofilm Ref.**

*Typhimurium* clinical isolates 75

0.1–1.1 μg/ml *S. saprophyticus* 15305 Statistically significant BF

1/2–1/1024 MIC *E. coli* UTI89 Statistically significant BF

*S. aureus* strains (RN6390 ATCC

3362‐33; 3362‐34; 8G12; 5G4; 6H9)

8 μg/ml *H. influenzae* NTHi2019 No effect on BF [19]

0.001–100 μg/l *P. aeruginosa* BF reduction [15]

0.125 μg/ml *H. influenzae* NTHi2019 Decreased BF formation, reduction of

8 μg/m *M. avium* strains (101, 104, 109, and A5) BF inhibition [36] sub‐MICs *P. aeruginosa*—35 clinical isolates Dose‐dependent BF reduction [38]

2.5–10 mg/ml *S. aureus* strains (B1 487; B1 493; B1 412; B 391; B1 468; B1 483; B1 379; B1 472)

**Table 3.** Effects of sub‐MIC of macrolides on biofilms.

446 Microbial Biofilms - Importance and Applications

From sub‐MIC up to

100× MIC

Gentamycin 1/2–1/16 *Salmonella enterica* serovar

strains

25923)

0.1–1.5 μg/ml MIC *P. aeruginosa* PAK and isogenic mutants

mutants

0.3; 0.5; 1.0 μg/ml *Xylella fastidiosa* ATCC 700964 and 3 isogenic mutants

Amikacin 0.5× MIC *E. coli* strains (8; 9; 10; 31; 1583) Reduction of BF formation

2; 8; 32 mg/l *P. aeruginosa*—6 clinical isolates Synergistic effect with

Streptomycin 0.5–2 μg/ml *M. avium* strains (104; 101; A5;

Tobramycin 0.05–2 μg/ml *P. aeruginosa* PAK and isogenic

0.25 MIC *S. epidermidis*—96 clinical isolates BF inhibition in 4 strains; BF enhancement in

20, other strains—unaffected

established BF

architecture of BF

synthesis

Inhibition of BF formation and EPS

BF increase [11]

BF increase [40]

BF increase [40]

BF reduction [41]

increase by 0.6—0.7 μg/ml

increase by 1/32 MIC

BF increase, induction of BF‐associated genes

and survival of the BF‐bacteria

polymorphonuclears for developed 48‐h BF

BF reduction [37]

[27]

[19]

[39]

[25]

[6]

[6]

[22]

[18]

[32]


**Table 5.** Effects of sub‐MIC of streptogramins, glycopeptides, tetracyclins, dihydrofolate reductase (DHFR) inhibitors and oxazolidonones on biofilms.

Antibiotics with identical mechanisms of antibacterial action, for example, gentamicin and erythromycin, may have different effects on biofilm [19, 29]. In addition, the sub‐MICs of a given antibiotic may have diverse effects on different strains of one species of microorgan‐ ism. For example, such is the case with the effects of cefazolin and levofloxacin on 15 isolates of *S. lugdunensis* [9] (**Table 1**), the effects of erythromycin on 69 clinical isolates of *S. epidermi‐ dis* [27] (**Table 3**), of vancomycin on 3 strains of *S. epidermidis* and 15 isolates of *S. lugdunensis* [9, 30] (**Table 5**), and of trimethoprim‐sulfamethoxazole on 15 isolates of *S. lugdunensis* [9] (**Table 5**). Obviously, individual strains use different response mechanisms to oppose the action of sub‐MICs [9].

Sub‐MICs of antibiotics have the potential to affect the structure of individual bacterial cells. Changes of morphology have been registered in several studies. For instance, sub‐MIC of penicillin induced filamentation of cells of *C. diphtheriae*, while erythromycin reduced cell size of this microorganism [26]. The sub‐MIC of the drug combination piperacillin/tazobactam induced the occurrence of filamentous forms in *P. aeruginosa*, while subinhibitory amounts of imipenem resulted in the formation of roundish forms (coccobacilli) of this bacterium [43]. Sub‐MICs of ciprofloxacin caused the occurrence of filamentous cells when applied to *E. coli* isolated from urinary tract infections. This was accompanied by alterations in the morpholo‐ gy of the outer membrane cardiolipin domains of the strain [44]. The cell‐surface physico‐ chemical characteristics of the bacteria would also be affected. Several studies focus on cell‐ surface hydrophobicity. For example, sub‐MICs of penicillin and streptomycin which enhanced biofilm formation in *C. diphtheriae* also rendered the cell surface more hydropho‐ bic [26]. The combination piperacillin/tazobactam applied as sub‐MIC that suppressed biofilm growth also reduced cell‐surface hydrophobicity of *P. aeruginosa* [45]. While these examples appear to show a likely positive correlation between cell‐surface hydrophobicity (which is also related with cell adhesion) and the effects on biofilm growth, this may not be the rule throughout. Thus, sub‐MICs of moxifloxacin that reduced biofilm growth had no influence on the cell‐surface hydrophobicity of *S. maltophilia* [7]. Changes in zeta potential [8], flagellum‐ mediated swimming [45] and type IV fimbria‐related twitching motility [39, 45] have been registered as well. When examined, the overall morphology of the biofilm, its thickness, substratum coverage, and roughness would change as well [16, 39].

reducing the production of C4‐homoserine lactone [53]. Azithromycin also antagonized quorum sensing in *P. aeruginosa* [4]. Cephalotin and cephalotaxime suppressed the *agr*

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449

Sub‐MICs of antibiotics can interact with bacterial regulation mechanisms and gene expres‐ sion. Transcriptomic studies indicated that the expression of approximately 5% of bacterial promoters may be affected [13]. Genes related with antibiotic resistance should be men‐ tioned in the first place. There was a correlation between the transcription of the *ermC* gene and the biofilm formation of erythromycin‐treated *S. epidermidis* [27]. In *P. aeruginosa*, among the 34 genes influenced by imipenem, the most strongly induced gene was *ampC* coding for chromosomal β‐lactamase [54]. Genes related with the synthesis of EPS or bacterial capsules may be affected, like the genes from the *ica*ADBC operon in *S. epidermidis* [28, 49]. Other genes related with adhesion and biofilm development that should be mentioned are *comD*, *gtfC*, *luxS*, *gtfB*, and *atlA* of *Streptococcus mutans* upregulated by sub‐MIC of triclosan [55] and *guaB2* and *gtf* in *M. avium*, upregulated under the action of sub‐MICs of streptomycin and tetracycline [22]. The biofilm growth and detachment have been related with the intracellular levels of the second messenger cyclic‐di‐GMP [40]. The *eal* gene participating in the pathways for its synthesis was upregulated by sub‐MIC of tobramycin [41]. Definitely, the effects of sub‐MICs on gene expression are not confined to these related to biofilm, many other genes can be

The capacity of released metabolites of bacterial species and strains to modulate biofilm growth of other bacteria is continuously in focus because of the potential for the isolation of novel biofilm modulating substances. As an initial screening step, the action of cell‐free superna‐ tants (CFSs) is tested. The activities may vary from stimulation [56, 57] to suppression [58, 59]. Noteworthy, the effects of CFSs on bacterial growth in liquid media do not predict the effects on sessile growth. Thus, subinhibitory amounts of 10-2 diluted CFSs from two bacteriocino‐ genic strains of *Lactobacillus plantarum* slowed down the growth of laboratory and uropatho‐ genic strains of *E. coli*, but stimulated significantly the biofilm development [59]. The active substances in CFSs may be proteins/peptides, carbohydrates, low molecular weight metabo‐ lites, etc. [see comments by 57] and for some of them the nature, structure, and mode of action have been explored. However, we shall restrict our review to molecules which have inhibito‐ ry activity on bacterial growth, and for which there is data on the effects of subinhibitory

Bacteriocins are proteins/peptides produced by prokaryotes which are active against other bacterial species or strains. For example, colicins are produced by some strains of *E. coli*. One of them, colicin M, is a phosphatase that hydrolyses the peptidoglycan lipid II intermediate, thus interfering with peptidoglycan synthesis and causing cell lysis. In subinhibitory amounts, it upregulated in *P. aeruginosa* PAO1 the *ydeH* gene related with the synthesis of cyclic‐di‐GMP, as well as several biofilm‐related genes, *ycfJ*, *rprA*, *omrA*, and *omrB.* However, no biofilm

quorum‐sensing system in *S. aureus* [54].

influenced as well [40].

amounts on biofilms.

**3. Antibiofilm bacterial metabolites**

The extracellular biofilm matrix is an important component of these structured microbial consortia. It has both structural and protective functions. In the interplay with the antibiotics, its barrier role against drug penetration should be underlined [46]. For the time being, available publications show a strict correlation between the effects of sub‐MICs on the biofilm and on the extracellular matrix components. More data are available on the extracellular polysac‐ charide (EPS). In cases of biofilm biomass reduction (e.g. by gentamicin and ciprofloxacin on *S*. Typhimurium, by fluoroquinolones on *P. aeruginosa*, and by dicloxacillin on *S. epidermidis*) this was accompanied by reduced release of EPS [5, 16, 47]. In cases of biofilm biomass increase (by sub‐MIC of erythromycin on *S. epidermidis*, of cefotaxime on *S. enterica* serovar Typhimurium, and of azithromycin on representatives of several bacterial genera), this coincided with EPS increase [5, 48, 49]. While less studied, such correlation might also characterize another component of the extracellular matrix, the extracellular DNA. It was registered with increased amounts in *S. epidermidis* biofilms treated with sub‐MICs of vancomycin [50, 51].

Sub‐MICs of antibiotics can interact with the bacterial‐host interactions. Together with their capacity to affect phenotypes, they can influence bacterial sensitivity to oxidative stress [45], suppress host proinflammatory responses [6], and cooperate with host polymorphonuclear leucocytes to destroy biofilms [34].

There is evidence that in nature, antibiotics at non‐inhibitory concentrations can have the role of signalling molecules that can interfere with quorum sensing [4, 42]. It was shown that sub‐ MICs of antibiotics influence quorum‐sensing‐related phenotypes of *Chromobacterium violaceum*, like the production of the pigment violacein, of acyl‐homoserine lactones, and of chitinase [52]. Sub‐MICs of tobramycin inhibited the Rhl/R system of *P. aeruginosa* thus reducing the production of C4‐homoserine lactone [53]. Azithromycin also antagonized quorum sensing in *P. aeruginosa* [4]. Cephalotin and cephalotaxime suppressed the *agr* quorum‐sensing system in *S. aureus* [54].

Sub‐MICs of antibiotics can interact with bacterial regulation mechanisms and gene expres‐ sion. Transcriptomic studies indicated that the expression of approximately 5% of bacterial promoters may be affected [13]. Genes related with antibiotic resistance should be men‐ tioned in the first place. There was a correlation between the transcription of the *ermC* gene and the biofilm formation of erythromycin‐treated *S. epidermidis* [27]. In *P. aeruginosa*, among the 34 genes influenced by imipenem, the most strongly induced gene was *ampC* coding for chromosomal β‐lactamase [54]. Genes related with the synthesis of EPS or bacterial capsules may be affected, like the genes from the *ica*ADBC operon in *S. epidermidis* [28, 49]. Other genes related with adhesion and biofilm development that should be mentioned are *comD*, *gtfC*, *luxS*, *gtfB*, and *atlA* of *Streptococcus mutans* upregulated by sub‐MIC of triclosan [55] and *guaB2* and *gtf* in *M. avium*, upregulated under the action of sub‐MICs of streptomycin and tetracycline [22]. The biofilm growth and detachment have been related with the intracellular levels of the second messenger cyclic‐di‐GMP [40]. The *eal* gene participating in the pathways for its synthesis was upregulated by sub‐MIC of tobramycin [41]. Definitely, the effects of sub‐MICs on gene expression are not confined to these related to biofilm, many other genes can be influenced as well [40]. activities of component

### **3. Antibiofilm bacterial metabolites**

penicillin induced filamentation of cells of *C. diphtheriae*, while erythromycin reduced cell size of this microorganism [26]. The sub‐MIC of the drug combination piperacillin/tazobactam induced the occurrence of filamentous forms in *P. aeruginosa*, while subinhibitory amounts of imipenem resulted in the formation of roundish forms (coccobacilli) of this bacterium [43]. Sub‐MICs of ciprofloxacin caused the occurrence of filamentous cells when applied to *E. coli* isolated from urinary tract infections. This was accompanied by alterations in the morpholo‐ gy of the outer membrane cardiolipin domains of the strain [44]. The cell‐surface physico‐ chemical characteristics of the bacteria would also be affected. Several studies focus on cell‐ surface hydrophobicity. For example, sub‐MICs of penicillin and streptomycin which enhanced biofilm formation in *C. diphtheriae* also rendered the cell surface more hydropho‐ bic [26]. The combination piperacillin/tazobactam applied as sub‐MIC that suppressed biofilm growth also reduced cell‐surface hydrophobicity of *P. aeruginosa* [45]. While these examples appear to show a likely positive correlation between cell‐surface hydrophobicity (which is also related with cell adhesion) and the effects on biofilm growth, this may not be the rule throughout. Thus, sub‐MICs of moxifloxacin that reduced biofilm growth had no influence on the cell‐surface hydrophobicity of *S. maltophilia* [7]. Changes in zeta potential [8], flagellum‐ mediated swimming [45] and type IV fimbria‐related twitching motility [39, 45] have been registered as well. When examined, the overall morphology of the biofilm, its thickness,

The extracellular biofilm matrix is an important component of these structured microbial consortia. It has both structural and protective functions. In the interplay with the antibiotics, its barrier role against drug penetration should be underlined [46]. For the time being, available publications show a strict correlation between the effects of sub‐MICs on the biofilm and on the extracellular matrix components. More data are available on the extracellular polysac‐ charide (EPS). In cases of biofilm biomass reduction (e.g. by gentamicin and ciprofloxacin on *S*. Typhimurium, by fluoroquinolones on *P. aeruginosa*, and by dicloxacillin on *S. epidermidis*) this was accompanied by reduced release of EPS [5, 16, 47]. In cases of biofilm biomass increase (by sub‐MIC of erythromycin on *S. epidermidis*, of cefotaxime on *S. enterica* serovar Typhimurium, and of azithromycin on representatives of several bacterial genera), this coincided with EPS increase [5, 48, 49]. While less studied, such correlation might also characterize another component of the extracellular matrix, the extracellular DNA. It was registered with increased amounts in *S. epidermidis* biofilms treated with sub‐MICs of

Sub‐MICs of antibiotics can interact with the bacterial‐host interactions. Together with their capacity to affect phenotypes, they can influence bacterial sensitivity to oxidative stress [45], suppress host proinflammatory responses [6], and cooperate with host polymorphonuclear

There is evidence that in nature, antibiotics at non‐inhibitory concentrations can have the role of signalling molecules that can interfere with quorum sensing [4, 42]. It was shown that sub‐ MICs of antibiotics influence quorum‐sensing‐related phenotypes of *Chromobacterium violaceum*, like the production of the pigment violacein, of acyl‐homoserine lactones, and of chitinase [52]. Sub‐MICs of tobramycin inhibited the Rhl/R system of *P. aeruginosa* thus

*epidermidis* the

substratum coverage, and roughness would change as well [16, 39].

vancomycin [50, 51].

leucocytes to destroy biofilms [34].

448 Microbial Biofilms - Importance and Applications

The capacity of released metabolites of bacterial species and strains to modulate biofilm growth of other bacteria is continuously in focus because of the potential for the isolation of novel biofilm modulating substances. As an initial screening step, the action of cell‐free superna‐ tants (CFSs) is tested. The activities may vary from stimulation [56, 57] to suppression [58, 59]. Noteworthy, the effects of CFSs on bacterial growth in liquid media do not predict the effects on sessile growth. Thus, subinhibitory amounts of 10-2 diluted CFSs from two bacteriocino‐ genic strains of *Lactobacillus plantarum* slowed down the growth of laboratory and uropatho‐ genic strains of *E. coli*, but stimulated significantly the biofilm development [59]. The active substances in CFSs may be proteins/peptides, carbohydrates, low molecular weight metabo‐ lites, etc. [see comments by 57] and for some of them the nature, structure, and mode of action have been explored. However, we shall restrict our review to molecules which have inhibito‐ ry activity on bacterial growth, and for which there is data on the effects of subinhibitory amounts on biofilms.

Bacteriocins are proteins/peptides produced by prokaryotes which are active against other bacterial species or strains. For example, colicins are produced by some strains of *E. coli*. One of them, colicin M, is a phosphatase that hydrolyses the peptidoglycan lipid II intermediate, thus interfering with peptidoglycan synthesis and causing cell lysis. In subinhibitory amounts, it upregulated in *P. aeruginosa* PAO1 the *ydeH* gene related with the synthesis of cyclic‐di‐GMP, as well as several biofilm‐related genes, *ycfJ*, *rprA*, *omrA*, and *omrB.* However, no biofilm

stimulation was confirmed by the crystal violet assay [60]. RIP is an RNAIII‐inhibiting heptapeptide originally isolated from CFS of *Staphylococcus xylosus.* It inhibits staphylococcal pathogenesis. In sub‐MICs, it suppressed biofilm formation by interfering with the quorum‐ sensing mechanisms [61]. Nisin is a polycyclic antibacterial peptide produced by *Lactococcus lactis*. At growth inhibitory concentrations, it suppressed sessile growth of *S. aureus*; howev‐ er, sub‐MICs had no effect on biofilm [62].

Invertebrate antibacterial responses are also explored. Thus, thanatin is an insect antimicro‐ bial peptide on the basis of which a shorter synthetic derivative, R‐thanatin, was synthe‐ sized. When applied in sub‐MIC amounts to *S. epidermidis* (including methicillin‐resistant staphylococcus epidermidis, MRSE), *Staphylococcus haemolyticus*, and *Staphylococcus hominis*, it inhibited biofilm formation. Parallelly, MRSE underwent serious morphological alterations

Modulation of Biofilm Growth by Sub‐Inhibitory Amounts of Antibacterial Substances

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

451

The application of plants for treatment of illness dates back to the very early moments of mankind history, and has laid the basis of modern phytotherapy. The studies on the antibac‐ terial activities of medicinal plant products have a long tradition, more recently expanded to biofilm research. Studies include tests on essential oils and plant extracts, partially purified enriched fractions, as well as isolated pure substances. Also, plant products may be used as a

Among the plant products, essential oils are most popular for their wide use in ethnomedi‐ cine. Some of them that have antibacterial action proved successful against bacterial biofilms as well. Among the essential oils that at subinhibitory amounts could suppress sessile growth are, for example, these from *Satureja hortensis* L. (active against *Prevotella nigrescens* biofilm) [71], from *Thymus vulgaris* (active against *P. aeruginosa* and *E. coli* biofilms) [72], and from *Mentha piperita* (active against biofilms of *P. aeruginosa* and *A. hydrophylla*) [73]. In addition,

Methanol and aqueous branch extracts of five *Juniperus* sp. were examined for their activi‐ ties against two *S. aureus* strains. The extracts had minimal activity on planktonic growth of *S. aureus* ATCC 3538P but suppressed biofilm formation, while the other strain, *S. aureus* 810, was not affected in either mode of growth [74]. The extract from *Leonurus cardiaca* L. sup‐ pressed the adherence of *S. aureus* to both abiotic surfaces and surfaces covered with fibrino‐ gen, fibronectin, or collagen [75]. Sub‐MICs (1/2 to 1/32 MIC) of extracts from *Boesenbergia pandurata* (Roxb.) Schltr. and *Eleutherine americana* Merr. significantly prevented biofilm formation. Together with this, the extract from *E. americana* also suppressed quorum sensing

In a study on 14 fractions from plant extracts, the total extract and the phenyl propanoid‐ containing fraction from *Rhodiola rosea*, and the total extract and the sesquiterpene lactone‐ containing fraction (Am2) from *Arnica montana*, were shown to have no antibacterial effects. However, they suppressed the biofilm growth in *E. coli* urinary tract infection isolates. These same extracts had the opposite effect—biofilm stimulation, on a multidrug‐resistant *E. coli* strain isolated from asymptomatic bacteriuria [77]. Noteworthy, the sesquiterpene lactone‐ containing fraction Am2 also suppressed the quorum‐sensing‐controlled bioluminescence in

Carvacol is an antimicrobial monotherpenic phenol with antibacterial potential that is present in many essential oils. In subinhibitory doses, it suppressed sessile growth of a number of

*Vibrio harveyi* bioreporter strains (ATCC1116 and ATCC1117) [78, 79].

like swelling and abnormal divisions [70].

**5. Subinhibitory amounts of plant substances**

peppermint oil suppressed EPS production [73].

in *C. violaceum* test system [76].

basis for chemical modifications aiming improved antibiofilm activity.

Mupirocin is an antibacterial substance of the monoxycarbolic acid class that was originally isolated from *Pseudomonas fluorescens*. At sub‐MIC, it can reduce both biofilm formation and glycocalyx production by *P. aeruginosa* [63]. Phenyl lactic acid is a metabolite of *Lactobacillus* probiotic strains. At subinhibitory amounts, it attenuated the virulence and pathogenicity of *P. aeruginosa* and *S. aureus*, including biofilm formation, by interacting with quorum sensing [64]. The antifungal and antibacterial molecule, 2,4‐diacetylphloroglucinol, was isolated from the CFS of *Pseudomonas protegens*. At subinhibitory amounts, it reduced pellicle and biofilm formation, and sporulation of *B. subtilis* [65].

### **4. Antimicrobial peptides and biofilm modulation**

The host‐bacterial interactions are also explored with the aim of identifying of novel mole‐ cules that would help overcoming the bacterial resistance mechanisms and combating infections. One important group of substances is that of the antibacterial peptides, an impor‐ tant part of the innate immune system.

Colistin is a cationic antimicrobial peptide which is gaining importance in the fight against *P. aeruginosa* cystic fibrosis infections. Subinhibitory concentrations altered the expression of 30 genes of the bacterium. Genes related with quorum sensing, lipopolysaccharides (LPSs) modifications, quinolone biosynthesis, and biofilm formation were upregulated while genes involved with motility and osmotolerance were downregulated. However, biofilm biomass remained unaffected [66].

The major human host defence peptide LL‐37 is found in mucosal surfaces, the granules of phagocytes, as well as in bodily fluids. At very low concentrations, far below those that kill or inhibit the growth of *P. aeruginosa*, LL‐37 prevented the *in vitro* biofilm formation [67]. It interfered with biofilm growth in at least three ways: by reduction of initial attachment, promotion of twitching motility, and downregulation of key components of the Las and Rhl quorum‐sensing systems [67].

The synthetic antimicrobial peptide 1018, derived from the bovine neutrophil defence peptide bactenecin, has recently been identified as biofilm inhibitory compound. While not reflect‐ ing on bacterial growth, it could prevent the biofilm growth of *P. aeruginosa*, *E. coli*, *Acineto‐ bacter baumannii*, *Klebsiella pneumoniae*, *S. enterica*, *Burkholderia cenocepacia*, and methicillin‐ resistant *S. aureus*. This co‐related with degradation of ppGpp [68]. In addition, peptide 1018 acted in synergism with conventional antibiotics, like ceftazidime, ciprofloxacin, imipenem, or tobramycin, to both prevent development and disperse existing biofilms [69].

Invertebrate antibacterial responses are also explored. Thus, thanatin is an insect antimicro‐ bial peptide on the basis of which a shorter synthetic derivative, R‐thanatin, was synthe‐ sized. When applied in sub‐MIC amounts to *S. epidermidis* (including methicillin‐resistant staphylococcus epidermidis, MRSE), *Staphylococcus haemolyticus*, and *Staphylococcus hominis*, it inhibited biofilm formation. Parallelly, MRSE underwent serious morphological alterations like swelling and abnormal divisions [70].

### **5. Subinhibitory amounts of plant substances**

stimulation was confirmed by the crystal violet assay [60]. RIP is an RNAIII‐inhibiting heptapeptide originally isolated from CFS of *Staphylococcus xylosus.* It inhibits staphylococcal pathogenesis. In sub‐MICs, it suppressed biofilm formation by interfering with the quorum‐ sensing mechanisms [61]. Nisin is a polycyclic antibacterial peptide produced by *Lactococcus lactis*. At growth inhibitory concentrations, it suppressed sessile growth of *S. aureus*; howev‐

Mupirocin is an antibacterial substance of the monoxycarbolic acid class that was originally isolated from *Pseudomonas fluorescens*. At sub‐MIC, it can reduce both biofilm formation and glycocalyx production by *P. aeruginosa* [63]. Phenyl lactic acid is a metabolite of *Lactobacillus* probiotic strains. At subinhibitory amounts, it attenuated the virulence and pathogenicity of *P. aeruginosa* and *S. aureus*, including biofilm formation, by interacting with quorum sensing [64]. The antifungal and antibacterial molecule, 2,4‐diacetylphloroglucinol, was isolated from the CFS of *Pseudomonas protegens*. At subinhibitory amounts, it reduced pellicle and biofilm

The host‐bacterial interactions are also explored with the aim of identifying of novel mole‐ cules that would help overcoming the bacterial resistance mechanisms and combating infections. One important group of substances is that of the antibacterial peptides, an impor‐

Colistin is a cationic antimicrobial peptide which is gaining importance in the fight against *P. aeruginosa* cystic fibrosis infections. Subinhibitory concentrations altered the expression of 30 genes of the bacterium. Genes related with quorum sensing, lipopolysaccharides (LPSs) modifications, quinolone biosynthesis, and biofilm formation were upregulated while genes involved with motility and osmotolerance were downregulated. However, biofilm biomass

The major human host defence peptide LL‐37 is found in mucosal surfaces, the granules of phagocytes, as well as in bodily fluids. At very low concentrations, far below those that kill or inhibit the growth of *P. aeruginosa*, LL‐37 prevented the *in vitro* biofilm formation [67]. It interfered with biofilm growth in at least three ways: by reduction of initial attachment, promotion of twitching motility, and downregulation of key components of the Las and Rhl

The synthetic antimicrobial peptide 1018, derived from the bovine neutrophil defence peptide bactenecin, has recently been identified as biofilm inhibitory compound. While not reflect‐ ing on bacterial growth, it could prevent the biofilm growth of *P. aeruginosa*, *E. coli*, *Acineto‐ bacter baumannii*, *Klebsiella pneumoniae*, *S. enterica*, *Burkholderia cenocepacia*, and methicillin‐ resistant *S. aureus*. This co‐related with degradation of ppGpp [68]. In addition, peptide 1018 acted in synergism with conventional antibiotics, like ceftazidime, ciprofloxacin, imipenem,

or tobramycin, to both prevent development and disperse existing biofilms [69].

er, sub‐MICs had no effect on biofilm [62].

450 Microbial Biofilms - Importance and Applications

formation, and sporulation of *B. subtilis* [65].

tant part of the innate immune system.

remained unaffected [66].

quorum‐sensing systems [67].

**4. Antimicrobial peptides and biofilm modulation**

The application of plants for treatment of illness dates back to the very early moments of mankind history, and has laid the basis of modern phytotherapy. The studies on the antibac‐ terial activities of medicinal plant products have a long tradition, more recently expanded to biofilm research. Studies include tests on essential oils and plant extracts, partially purified enriched fractions, as well as isolated pure substances. Also, plant products may be used as a basis for chemical modifications aiming improved antibiofilm activity.

Among the plant products, essential oils are most popular for their wide use in ethnomedi‐ cine. Some of them that have antibacterial action proved successful against bacterial biofilms as well. Among the essential oils that at subinhibitory amounts could suppress sessile growth are, for example, these from *Satureja hortensis* L. (active against *Prevotella nigrescens* biofilm) [71], from *Thymus vulgaris* (active against *P. aeruginosa* and *E. coli* biofilms) [72], and from *Mentha piperita* (active against biofilms of *P. aeruginosa* and *A. hydrophylla*) [73]. In addition, peppermint oil suppressed EPS production [73].

Methanol and aqueous branch extracts of five *Juniperus* sp. were examined for their activi‐ ties against two *S. aureus* strains. The extracts had minimal activity on planktonic growth of *S. aureus* ATCC 3538P but suppressed biofilm formation, while the other strain, *S. aureus* 810, was not affected in either mode of growth [74]. The extract from *Leonurus cardiaca* L. sup‐ pressed the adherence of *S. aureus* to both abiotic surfaces and surfaces covered with fibrino‐ gen, fibronectin, or collagen [75]. Sub‐MICs (1/2 to 1/32 MIC) of extracts from *Boesenbergia pandurata* (Roxb.) Schltr. and *Eleutherine americana* Merr. significantly prevented biofilm formation. Together with this, the extract from *E. americana* also suppressed quorum sensing in *C. violaceum* test system [76].

In a study on 14 fractions from plant extracts, the total extract and the phenyl propanoid‐ containing fraction from *Rhodiola rosea*, and the total extract and the sesquiterpene lactone‐ containing fraction (Am2) from *Arnica montana*, were shown to have no antibacterial effects. However, they suppressed the biofilm growth in *E. coli* urinary tract infection isolates. These same extracts had the opposite effect—biofilm stimulation, on a multidrug‐resistant *E. coli* strain isolated from asymptomatic bacteriuria [77]. Noteworthy, the sesquiterpene lactone‐ containing fraction Am2 also suppressed the quorum‐sensing‐controlled bioluminescence in *Vibrio harveyi* bioreporter strains (ATCC1116 and ATCC1117) [78, 79].

Carvacol is an antimicrobial monotherpenic phenol with antibacterial potential that is present in many essential oils. In subinhibitory doses, it suppressed sessile growth of a number of Gram‐positive and Gram‐negative bacteria [80, 81]. Polyphenols from muscadine grapes with antioxidant and antibacterial activity, at 0.5 MIC, inhibited biofilm growth of *S. aureus* [82]. Similar was the effect of the essential oil components eugenol and citral on *S. aureus* and *Listeria monocytogenes* [83], epigallocatechin‐3‐gallate from green tea on *S. maltophilia* [84], ursolic acid, genistein, cranberry extract, *p*‐hydroxybenzoic acid, and resveratol on *S. aureus* [75, 85]. Menthol, together with biofilm suppression, was shown to inhibit both the *las*‐ and *pqs*‐related quorum sensing [73].

antibacterials [1]. As a major risk for human health, biofilm communities provide the bacte‐ ria with prerequisites for rapid resistance development [99]. Among the other more direct risks that biofilms may cause to human health, should be mentioned the possibility for enhanced colonization of indwelling medical devices in the presence of subinhibitory amounts of antibacterial substances, and the contamination of surfaces in medical or food‐processing environments. Depending on the aim of a given antibiofilm strategy, different effects may be in the focus. Disinfection of outer surfaces in hospitals and in food industry requires that the used agents have the capacity to detach established biofilms. On the opposite, if biofilms on indwelling devices are concerned, once established, their detachment is hazardous. It may be accompanied with dissemination of the bacteria to other sites in the human body, and there are risks of sepsis [69]. Therefore, the development of medical materials should be directed to biofilm prevention. However, when the effects of a given substance are estimated, the biology of the biofilm as a whole is better to be addressed, starting from the attachment and establish‐ ment of the sessile community, and going as far as its detachment. The methodologies used by the predominant amount of the present‐day studies search for the effects of sub‐MICs by applying the tested agents during biofilm growth. It can be recommended that in the future a more standardized methodology is applied which includes as well tests for dispersion of established biofilms and for microbial vitality. The present review showed several critical points in the effects of sub‐MICs of antibacterial substances as biofilm modulators. Among these, the strain‐ and species‐specific responses of the bacteria in their biofilm development, the expression of virulence factors and quorum sensing should necessarily be taken into

Modulation of Biofilm Growth by Sub‐Inhibitory Amounts of Antibacterial Substances

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453

, Tsvetelina S. Paunova‐Krasteva and Dayana B. Borisova

Department of General Microbiology, Institute of Microbiology, Bulgarian Academy of

framework. Nat. Rev. 2015;13:310–317. DOI: 10.1038/nrmicro3439

[1] Berendonk T, Manaia C, Merlin C, Fatta‐Kassinos D, Cytryn E, Walsh F, Bürgmann H, Sørum H, Norström M, Pons M, Kreuzinger N, Huovinen P, Stefani S, Schwartz T, Kisand V, Baquero F, Martinez J. Tackling antibiotic resistance: the environmental

[2] Bernier S, Surette M. Concentration‐dependent activity of antibiotics in natural environments. Front. Microbiol. 2013;4:1–14. DOI: 10.3389/fmicb.2013.00020

account when novel antibacterials are tested.

\*Address all correspondence to: stoitsova\_microbiobas@yahoo.com

**Author details**

Stoyanka R. Stoitsova\*

Sciences, Sofia, Bulgaria

**References**

Fenchone is a substance that is present in many essential oils. It had neither antibacterial nor antibiofilm effects on a panel of Gram‐positive and Gram‐negative strains. This molecule was used to synthesize its chemical derivatives. While the substitutions did not improve the antibacterial properties against *E. coli* ATCC 25922 and six *E. coli* K‐12 strains, some of the derivatives showed biofilm modulation potential [86]. Chalcones are a group of flavonoids with antibacterial potential, found in many plants. Synthetic chalcones are applied as well. The effects of sub‐MICs ofthree newly synthesizedchalcones on methicillin‐resistant *S. aureus* were examined. Both biofilm formation and adherence to human fibronectin were reduced, as well as the release of EPS [87].

### **6. Other compounds with biofilm‐modulating potential**

Other substances have also proved a good anti‐biofilm potential when applied in sub‐MICs. For example, sodium ascorbate, together with suppressing *P. aeruginosa* virulence factors (elastase, protease and haemolysin activities, pyocyanin production, and quorum sensing) also reduced biofilm formation [88]. Biofilm growth was inhibited by subinhibitory amounts of thiourea derivatives [89], thiazolinediones [90], and certain anthraquinones [91]. Organic complexes of metals are also elaborated as antibacterial and/or antibiofilm substances. Newly synthesized dimethylguanin‐copper complexes [92], the organo‐tellurium compound AS101 [93], and bismuth thiols [94] have shown anti‐biofilm activity at sub‐MICs. The latter substan‐ ces are considered as possible coating agents forindwelling devices. For prevention of medical devices from bacterial contamination, other substances may prove useful as coating material, like ovotransferrin, protamine sulfate, ethylenediaminetetraacetic acid (EDTA) [95], cerium nitrate, chitosan, hamamelitannin [96], polyvinyl pyrrilidine [97], etc.

As an opposite effect, the biocides used in food processing facilities, trisodium phosphate, sodium nitrite, and sodium hypochlorite, when applied in sub‐MICs, enhanced the capacity of *E. coli* to form biofilms. This was accompanied by a reduction of the antibiotic susceptibili‐ ty [98].

### **7. Some final considerations**

Presently, there is growing concern about the relationship between the rise of widespread antibiotic resistance and the role of environments containing subinhibitory amounts of

antibacterials [1]. As a major risk for human health, biofilm communities provide the bacte‐ ria with prerequisites for rapid resistance development [99]. Among the other more direct risks that biofilms may cause to human health, should be mentioned the possibility for enhanced colonization of indwelling medical devices in the presence of subinhibitory amounts of antibacterial substances, and the contamination of surfaces in medical or food‐processing environments. Depending on the aim of a given antibiofilm strategy, different effects may be in the focus. Disinfection of outer surfaces in hospitals and in food industry requires that the used agents have the capacity to detach established biofilms. On the opposite, if biofilms on indwelling devices are concerned, once established, their detachment is hazardous. It may be accompanied with dissemination of the bacteria to other sites in the human body, and there are risks of sepsis [69]. Therefore, the development of medical materials should be directed to biofilm prevention. However, when the effects of a given substance are estimated, the biology of the biofilm as a whole is better to be addressed, starting from the attachment and establish‐ ment of the sessile community, and going as far as its detachment. The methodologies used by the predominant amount of the present‐day studies search for the effects of sub‐MICs by applying the tested agents during biofilm growth. It can be recommended that in the future a more standardized methodology is applied which includes as well tests for dispersion of established biofilms and for microbial vitality. The present review showed several critical points in the effects of sub‐MICs of antibacterial substances as biofilm modulators. Among these, the strain‐ and species‐specific responses of the bacteria in their biofilm development, the expression of virulence factors and quorum sensing should necessarily be taken into account when novel antibacterials are tested.

### **Author details**

Gram‐positive and Gram‐negative bacteria [80, 81]. Polyphenols from muscadine grapes with antioxidant and antibacterial activity, at 0.5 MIC, inhibited biofilm growth of *S. aureus* [82]. Similar was the effect of the essential oil components eugenol and citral on *S. aureus* and *Listeria monocytogenes* [83], epigallocatechin‐3‐gallate from green tea on *S. maltophilia* [84], ursolic acid, genistein, cranberry extract, *p*‐hydroxybenzoic acid, and resveratol on *S. aureus* [75, 85]. Menthol, together with biofilm suppression, was shown to inhibit both the *las*‐ and *pqs*‐related

Fenchone is a substance that is present in many essential oils. It had neither antibacterial nor antibiofilm effects on a panel of Gram‐positive and Gram‐negative strains. This molecule was used to synthesize its chemical derivatives. While the substitutions did not improve the antibacterial properties against *E. coli* ATCC 25922 and six *E. coli* K‐12 strains, some of the derivatives showed biofilm modulation potential [86]. Chalcones are a group of flavonoids with antibacterial potential, found in many plants. Synthetic chalcones are applied as well. The effects of sub‐MICs ofthree newly synthesizedchalcones on methicillin‐resistant *S. aureus* were examined. Both biofilm formation and adherence to human fibronectin were reduced, as well

Other substances have also proved a good anti‐biofilm potential when applied in sub‐MICs. For example, sodium ascorbate, together with suppressing *P. aeruginosa* virulence factors (elastase, protease and haemolysin activities, pyocyanin production, and quorum sensing) also reduced biofilm formation [88]. Biofilm growth was inhibited by subinhibitory amounts of thiourea derivatives [89], thiazolinediones [90], and certain anthraquinones [91]. Organic complexes of metals are also elaborated as antibacterial and/or antibiofilm substances. Newly synthesized dimethylguanin‐copper complexes [92], the organo‐tellurium compound AS101 [93], and bismuth thiols [94] have shown anti‐biofilm activity at sub‐MICs. The latter substan‐ ces are considered as possible coating agents forindwelling devices. For prevention of medical devices from bacterial contamination, other substances may prove useful as coating material, like ovotransferrin, protamine sulfate, ethylenediaminetetraacetic acid (EDTA) [95], cerium

As an opposite effect, the biocides used in food processing facilities, trisodium phosphate, sodium nitrite, and sodium hypochlorite, when applied in sub‐MICs, enhanced the capacity of *E. coli* to form biofilms. This was accompanied by a reduction of the antibiotic susceptibili‐

Presently, there is growing concern about the relationship between the rise of widespread antibiotic resistance and the role of environments containing subinhibitory amounts of

**6. Other compounds with biofilm‐modulating potential**

nitrate, chitosan, hamamelitannin [96], polyvinyl pyrrilidine [97], etc.

quorum sensing [73].

452 Microbial Biofilms - Importance and Applications

as the release of EPS [87].

ty [98].

**7. Some final considerations**

Stoyanka R. Stoitsova\* , Tsvetelina S. Paunova‐Krasteva and Dayana B. Borisova

\*Address all correspondence to: stoitsova\_microbiobas@yahoo.com

Department of General Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

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**Chapter 20**

**Novel Models to Manage Biofilms on Microtextured**

Dental implants are used extensively to replace missing teeth. To enhance their integration with the bones of the jaws, the surfaces of titanium dental implants are modified to make them hydrophilic, high energy, and microtextured. These same features make biofilm development occur readily upon exposure to the saliva. The presence of mature biofilms on dental implant surfaces drives local inflammatory responses in the adjacent soft and hard tissues (peri-implantitis), which leads to pathological loss of bone and the forma‐ tion of a saucer shaped bone defects. This chapter examines the unique challenges posed by biofilms formed on highly complex dental implant surfaces, which are difficult to access for cleaning, and easily damaged by conventional cleaning approaches. We explore how biofilms can be removed from implant surfaces using a variety of novel methods, without causing surface damage or other undesirable modifications, and show how different laboratory and clinical models can be used to assess the performance of both

**Keywords:** biofilms, dental implants, surface characterization, debridement, lasers,

Dental implants have been used for over 40 years to replace missing teeth, with good clinical success when placed in most sites in the jaws [1]. They are inserted into specially prepared channels in the bone, and once fully integrated into the bone, can be restored with a ceramic crown to replace a single missing tooth (**Figure 1**). Typically, dental implants are fabricated from commercially pure titanium (Ti), or titanium alloys which include small amounts of vanadi‐ um and aluminum. Dental implants made from ceramic materials such as sapphire and

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,

**Dental Implant Surfaces**

Carol Tran and Laurence J. Walsh

http://dx.doi.org/10.5772/62803

**Abstract**

particle beams

**1. Introduction**

Additional information is available at the end of the chapter

conventional and novel methods of biofilm removal.

