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Immunology and Microbiology » "Microbial Biofilms - Importance and Applications", book edited by Dharumadurai Dhanasekaran and Nooruddin Thajuddin, ISBN 978-953-51-2436-8, Print ISBN 978-953-51-2435-1, Published: July 13, 2016 under CC BY 3.0 license. © The Author(s).

Chapter 19

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

By Stoyanka R. Stoitsova, Tsvetelina S. Paunova‐Krasteva and Dayana B. Borisova
DOI: 10.5772/62939

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

Stoyanka R. Stoitsova, Tsvetelina S. Paunova‐Krasteva and Dayana B. Borisova
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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 extent or other the bacterial growth in liquid media, these same amounts of drugs, natural substances, etc., may have diverse effects on bacterial biofilms, ranging from suppression 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 modulation, differences in experimental protocols should be taken into account, as well as the strain‐specific mechanisms of biofilm formation.

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 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].

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 [13]. Thus, bacteria present in the environment are often subjected to drug amounts lower than the minimal inhibitory concentrations (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 microbial 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 biofilm 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 containing 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‐induced 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 reveals 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 virulence gene) expression [15].

The idea on the effects of antibiotic sub‐MICs on biofilms is getting more and more complicated with the accumulation of experimental data. This puts forward the question of methodology. 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 [1719]. 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 fluorescence microscopy.

We have summarized the available experimental data on the action of sub‐MICs of antibiotics on biofilms in Table 15. 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 specificity 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 Streptococcus suis, Salmonella enterica serovar Typhimurium clinical strains, Stenotrophomonas maltophilia, Streptococcus pyogenes, Staphylococcus epidermidis, and Proteus mirabilis [17, 18, 20, 2325] (Table 2). Diverse effects have been illustrated for sub‐MICs of erythromycin on biofilms of S. suis, Corynebacterium diphtheriae, and S. epidermidis [2527] (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).

AntibioticsAmountBacteria/strainsEffect on biofilmRef.
Dicloxacillin1/2 MICS. epidermidis strains 9142, IE186, and M18732–60% BF inhibition[29]
8 μg/mlS. epidermidis M187
S. haemolyticus M176
BF biomass reduction; decreased synthesis of the EPS, poly‐N‐acetyl‐glucosamine[16]
Penicillin1/16–1/2 MICS. suis NJ‐3Dose‐dependent BF suppression[25]
1/8 MICC. diphtheriae subsp. mitis strainsNo effect[26]
Methicillin1/3–1/8 MICS. aureus NewmanDenser BF formed by the strain and its small‐colony variants[30]
S. lugdunensis—15 isolates from BF‐related infections (endocarditis, prosthetic joint infections, etc.)Increase in 93% of the tested strains, no effect in 7%[9]
1/3–1/8 MICS. aureus NewmanNo effect on BF[30]
Cefazolin1/2 MICS. epidermidis strains 9142, IE186, and M18732–55% BF inhibition[29]
S. lugdunensis—15 isolates from BF‐related infections (endocarditis, prosthetic joint infections, etc.)Increase in 13% of the tested strains, no effect in 80%, and decrease in 7%[9]
0.5 MICS. epidermidis strains SE5 and RP62ABF decrease[31]
Cefalotin1/3–1/8 MICS. aureus NewmanThree‐ to fourfold denser BF formed by the strain and its small‐colony variants[30]
Cefoperazone 1/3–1/8 MICS. aureus NewmanNo effect on BF[30]
Cefotaxime1/2–1/16 MICSalmonella enterica serovar Typhimurium clinical isolates 75 strainsAt 1/2 MIC—significantly increased production of BF and EPS[5]
E. coli MG1655 wt and MG1655 (pBR322)BF reduction[21]
0.1–1.1 μg/mlS. saprophyticus 15305Sub‐MIC (0.3–0.7 μg/ml) stimulate BF formation[6]
E. coli UTI89BF stimulation at MIC, no effect of
10 μg/lM. avium strains 104; 101, A5; 3362‐33 and 3362‐34)No effect on BF[22]
Imipenem2–4 μg/mlP. aeruginosa PA01BF induction, changes in BF morphology, upregulation of ampC and genes for alginate biosynthesis[32]
0.03 and
0.125 μg/ml
73 isolates A. baumanniiBF stimulation[33]
Ceftazidime1/2–1/8 MIC5 clinical isolates strains S. maltophiliaBF inhibition and reduction of viable cell counts[20]
5 strains P. mirabilisBF inhibition[24]
2; 8; 32 mg/l6 clinical isolates of P. aeruginosaSynergistic effect with polymorphonuclears against developed 48 h BF[34]

Table 1.

Effects of sub‐MIC of β‐lactam antibiotics on biofilms.

[i] - Abbreviations: BF, biofilm; EPS, exopolysaccharide; MIC, minimal inhibitory concentration.

AntibioticsAmountBacteria/strainsEffect on biofilmRef.
Ciprofloxacin1/16–1/2 MICS. suis NJ‐3Dose‐dependent BF suppression[25]
1/2–1/16MICSalmonella enterica serovar Typhimurium clinical isolates 75 strainsInhibition of BF formation and EPS synthesis[5]
0.1–1.1 μg/mlS. saprophyticus 15305BF stimulation by sub‐MIC (0.4–0.9 μg/ml)[6]
1/2–1/1024 MICE. coli UTI89Statistically significant BF increase and upregulation of BF‐associated genes at 1/4 MIC[6]
1/2–1/8MIC5 clinical isolates S. maltophiliaBF inhibition and reduction of viable cell counts[20]
1/2–1/64 MICS. pyogenes isolates (M56; st38;M89; M65; M100; M74)Dose‐dependent BF inhibition[23]
0.5 × MICE. coli strains (8; 9; 10; 31 ;1583)Reduction of BF formation and survival of the BF bacteria[18]
1/2–1/8 MICStaphylococcus epidermidis—20 clinical isolatesReduces BF growth and pre‐formed BF[17]
0.125–1.0 MIC5 strains P. mirabilisBF inhibition[24]
2; 8; 32 mg/l MICP. aeruginosa—6 clinical isolatesSynergistic effect with polymorphonuclears against developed 48‐h BF[32]
0.001–100 mg/lP. aeruginosaBF reduction[15]
Norfloxacin1–10,000 mg/lS. aureus ATCC 25923BF stimulation by 1 mg/l[8]
1–10,000 mg/lP. aeruginosa NNRL‐B3509BF stimulation by 1 mg/l[8]
1/2–1/8 MICS. maltophilia—5 clinical isolates strainsBF inhibition and reduction of viable cell counts[20]
1/2–1/64 MICS. pyogenes isolates (M56; st38;M89; M65; M100; M74)Dose‐dependent BF inhibition[23]
1/2–1/8 MICStaphylococcus epidermidis—20 clinical isolatesSuppression of BF growth and reduction of pre‐formed BF[17]
1/16–1/2 MICS. suis NJ‐3Dose‐dependent biofilm suppression[25]
Ofloxacin1/2–1/8MICS. maltophilia—5 clinical isolates strainsBF inhibition and reduction of viable cell counts[20]
1/2–1/64 MICS. pyogenes isolates (M56; st38;M89; M65; M100; M74)Dose‐dependent BF inhibition[23]
0.5 × MICS. epidermidis strains (SE5; RP62A)No effect on BF formation[31]
1/2–1/8 MICStaphylococcus epidermidis—20 clinical isolatesSuppression of BF growth and reduction of pre‐formed BF[17]
1/2–1/8 MICS. maltophilia—5 clinical isolates strainsBiofilm inhibition and reduction of viable cell counts[20]
Levofloxacin1/2–1/64 MICS. pyogenes isolates (M56; st38;
M89; M65; M100; M74)
Dose‐dependent BF inhibition[23]
0.0625– 0.5 MICS. lugdunensis—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint infections, etc.)Increase in 20% of the tested strains, in 47% no effect, and in 40% decrease[9]
Moxifloxacin1 μg/lM. avium strains (104; 101; A5;
3362‐33; 3362‐34)
No effect on BF[22]
1/2–1/8 MICS. maltophilia—5 clinical isolates strainsBF inhibition and reduction of viable cell counts[20]
0.03–0.06 MICS. maltophilia strains Sm 132 and Sm 144Decrease in adhesion and BF formation[7]
2; 10; 50; 100 x MICS. aureus—6 strains of coagulase negativeNo effect on BF[35]
1 μg/mlM. avium strains (101, 104, 109, and A5)BF inhibition[36]
1/2–1/8MICS. maltophilia —5 clinical isolates strainsBF inhibition and reduction of viable cell counts[20]
Grepafloxacin 1/2–1/8MICS. maltophilia—5 clinical isolates strainsBF inhibition and reduction of viable cell counts[20]
Pefloxacin1/2–1/8 MICS. epidermidis—20 clinical isolatesReduces BF growth and pre‐formed BF[17]

Table 2.

Effects of sub‐MIC of fluoroquinolones on biofilms.

AntibioticsAmountBacteria/strainsEffect on biofilmRef.
Erythromycin1/16–1/2 MICS. suis NJ‐3Dose‐dependent BF suppression[25]
1/8 MICC. diphtheriae subsp. mitis strainsBF increase[26]
0.25 MICS. epidermidis—96 clinical isolatesBF inhibition in 4 strains; BF enhancement in 20, other strains—unaffected[27]
Azithromycin1/2–1/16 MICS. suis NJ‐3BF inhibition by 1/4 and 1/2 MIC[25]
2.5–10 mg/mlS. aureus strains (B1 487; B1 493; B1 412; B 391; B1 468; B1 483; B1 379; B1 472)BF reduction[37]
0.125 μg/mlH. influenzae NTHi2019Decreased BF formation, reduction of established BF[19]
8 μg/mM. avium strains (101, 104, 109, and A5)BF inhibition[36]
sub‐MICsP. aeruginosa—35 clinical isolatesDose‐dependent BF reduction[38]
Clarithromycin 1 μg/mlM. avium strains (101, 104, 109, and A5)BF inhibition[36]
MIC b/n 50–550 mg/mlP. aeruginosaSub‐MIC result in altered structure and architecture of BF[39]

Table 3.

Effects of sub‐MIC of macrolides on biofilms.

AntibioticsAmountBacteria/strainsEffect on biofilmRef.
Gentamycin1/2–1/16Salmonella enterica serovar Typhimurium clinical isolates 75 strainsInhibition of BF formation and EPS synthesis[25]
0.1–1.1 μg/mlS. saprophyticus 15305Statistically significant BF
increase by 0.6—0.7 μg/ml
1/2–1/1024 MICE. coli UTI89Statistically significant BF
increase by 1/32 MIC
8 μg/mlH. influenzae NTHi2019No effect on BF[19]
From sub‐MIC up to 100× MICS. aureus strains (RN6390 ATCC 25923)BF increase[11]
0.1–1.5 μg/ml MICP. aeruginosa PAK and isogenic mutantsBF increase[40]
Streptomycin 0.5–2 μg/mlM. avium strains (104; 101; A5; 3362‐33; 3362‐34; 8G12; 5G4; 6H9)BF increase, induction of
BF‐associated genes
Tobramycin0.05–2 μg/mlP. aeruginosa PAK and isogenic mutantsBF increase[40]
0.3; 0.5; 1.0 μg/mlXylella fastidiosa ATCC 700964 and 3 isogenic mutantsBF reduction[41]
0.001–100 μg/lP. aeruginosaBF reduction[15]
Amikacin0.5× MICE. coli strains (8; 9; 10; 31; 1583)Reduction of BF formation
and survival of the BF‐bacteria
2; 8; 32 mg/lP. aeruginosa—6 clinical isolatesSynergistic effect with polymorphonuclears for
developed 48‐h BF
Kanamycin10–110 μg/mlP. aeruginosa PAK and isogenic mutantsBF increase[40]

Table 4.

Effects of sub‐MIC of aminoglycosides on biofilms.

AntibioticsAmountBacteria/ strainsEffect on biofilmRef.
0.5 μg/mlS. epidermidis strains 567 and
S. epidermidis 561
Enhancement of icaADBC operon expression and EPS synthesis[28]
0.0625–0.5 MICS. lugdunensis—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint infections, etc.)Increase in 20% of the tested strains; in 47% no effect and in 33% decrease[9]
1/2 MICS. epidermidis strains 9142, IE186 and M1878–24% BF inhibition[29]
0.0625–0.5 MICS. lugdunensis—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint infections, etc.)Increase in 27% of the tested strains; in 47% no effect and in 27% decrease[9]
0.5× MICS. epidermidis strains SE5 and RP62ADecrease in BF[31]
0.005–500 μg/ml E. coli MG1655 wt and MG1655 (pBR322)Significant BF increase in the presence of (pBR322)[21]
0.0625–0.5 MICS. lugdunensis—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint infections, etc.)Increase 7% of tested strains; decrease 93%[9]
0.5–2 μg/mlM. avium strains (104; 101; A5; 3362‐33; 3362‐34)BF increase[22]
0.01–100 mg/lP. aeruginosaBF reduction[15]
0.5 μg/mlS. epidermidis strains 567 and
S. epidermidis 561
Enhancement of icaADBC operon expression and EPS synthesis[28]
DHFR inhibitors
0.0625–0.5 MICS. lugdunensis—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint infections, etc.)Increase in 20% of the tested strains, in 40% no effect, and in 40% decrease[9]
1/2–1/8 MIC5 clinical isolates strains S. maltophiliaBF inhibition and reduction of viable cell counts[20]
0.0625–0.5 MICS. lugdunensis—15 isolates from biofilm‐related infections (endocarditis, prosthetic joint infections, etc.)In 80% no effect, in 20% decrease[9]

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 microorganism. 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. epidermidis [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 morphology of the outer membrane cardiolipin domains of the strain [44]. The cell‐surface physicochemical 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 hydrophobic [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].

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 polysaccharide (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 expression. Transcriptomic studies indicated that the expression of approximately 5% of bacterial promoters may be affected [13]. Genes related with antibiotic resistance should be mentioned 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 icaADBC 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].

3. Antibiofilm bacterial metabolites

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 supernatants (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 bacteriocinogenic strains of Lactobacillus plantarum slowed down the growth of laboratory and uropathogenic strains of E. coli, but stimulated significantly the biofilm development [59]. The active substances in CFSs may be proteins/peptides, carbohydrates, low molecular weight metabolites, 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 inhibitory 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; however, sub‐MICs had no effect on biofilm [62].

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 molecules that would help overcoming the bacterial resistance mechanisms and combating infections. One important group of substances is that of the antibacterial peptides, an important 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 reflecting on bacterial growth, it could prevent the biofilm growth of P. aeruginosa, E. coli, Acinetobacter 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 antimicrobial peptide on the basis of which a shorter synthetic derivative, R‐thanatin, was synthesized. 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

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 antibacterial 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 ethnomedicine. 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 activities 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. suppressed the adherence of S. aureus to both abiotic surfaces and surfaces covered with fibrinogen, 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].

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 of three newly synthesized chalcones 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 substances are considered as possible coating agents for indwelling 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 susceptibility [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 bacteria 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 establishment 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.


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