rabin 2015

FutureMedicinalChemistry Review

part of

Agents that inhibit bacterial biofilm formation

Nira Rabin1, Yue Zheng1, Clement Opoku-Temeng1, Yixuan Du1, Eric Bonsu2 & Herman O Sintim*,11Department of Chemistry &

Biochemistry, University of Maryland,

College Park, MD 20742, USA 2Department of Natural Sciences, Bowie

State University, 14000 Jericho Park

Road, Bowie, MD 20715, USA

*Author for correspondence:

Tel.: +1 301 405 0633

Fax: +1 301 314 9121

[email protected]

647Future Med. Chem. (2015) 7(5), 647671 ISSN 1756-891910.4155/FMC.15.7 2015 N Rabin et al.

Future Med. Chem.

Review7

5

2015

In the biofilm form, bacteria are more resistant to various antimicrobial treatments. Bacteria in a biofilm can also survive harsh conditions and withstand the hosts immune system. Therefore, there is a need for new treatment options to treat biofilm-associated infections. Currently, research is focused on the development of antibiofilm agents that are nontoxic, as it is believed that such molecules will not lead to future drug resistance. In this review, we discuss recent discoveries of antibiofilm agents and different approaches to inhibit/disperse biofilms. These new antibiofilm agents, which contain moieties such as imidazole, phenols, indole, triazole, sulfide, furanone, bromopyrrole, peptides, etc. have the potential to disperse bacterial biofilms in vivo and could positively impact human medicine in the future.

The ability of bacteria to form biofilm has drawn considerable interest from researchers over the past decade [1]. Biofilms are usually made up of diverse microorganisms, which are attached to a surface. These microor-ganisms are usually embedded in polymeric matrix. Bacteria foul medical devices and implants, such as catheters, components of cardiac pacemakers, artificial heart valves and joints. These implant-associated biofilms can be treated with antibiotics but in certain situations replacement surgeries, which of course come with risks, would be needed [2,3].

A potential antibiofilm drug that can either facilitate the dispersion of preformed biofilms or inhibit the formation of new biofilms in vivo is needed. So far, a plethora of potential antibiofilm agents with unique structures, mainly inspired by natural prod-ucts, have been developed and shown great promise in dispersing existing biofilms or preventing bacteria from forming biofilms in vitro. In contrast to conventional antibi-otics, the majority of the recently developed antibiofilm molecules do not directly affect bacterial survival and thus the expectation is that resistance to these molecules will not readily occur. In the coming years it is

hoped that some of these lead compounds would be translated into antibiofilm drugs. A complementary approach to prevent bio-film formation on medical is to passivate the surfaces of the implants with molecules that discourage biofilm formation. In this review, we summarize the current state-of-the-art compounds that are capable of inhibiting biofilm formation of a spectrum of clinically relevant Gram-positive and Gram-negative bacteria.

Biofilm inhibition by small moleculesNature-inspired synthetic moleculesNature continues to inspire the discovery of novel compounds with interesting struc-tures and biological activity. These naturally derived compounds have served as scaffolds for the development of a plethora of synthetic therapeutic agents. Currently, many groups are focusing their research on the discov-ery of novel compounds capable of inhibit-ing biofilms [4,5]. To date, many antibiofilm compounds have been identified from diverse natural sources, for example, brominated furanones [6], garlic [7], ursine triterpenes [8], corosolic acid and asiatic acid [9], ginseng [10] and 3-indolylacetonitrile [11].

For reprint orders, please contact [email protected]

Figure 1. Natural compounds with 2-aminoimidazole moiety.

HN

N

H2N

H2NNH

N NH

O

Br

HN O

Br

Br

NH

NH

HN

HN

NH

Br

Br

O

NNH2

Bromoageliferin

Oroidin

648 Future Med. Chem. (2015) 7(5) future science group

Review Rabin, Zheng, Opoku-Temeng, Du, Bonsu, Sintim

Imidazole derivativesThe 2-aminoimidazole functionality is essentially a guanidine mimetic and is found in numerous marine natural products. Prototypical examples in this class include natural products such as bromoageleferin and oroidin (Figure 1), which were isolated from the sponge Agelas conifer.

The Melander group has developed an array of novel molecular scaffolds that both inhibit and disperse bac-terial biofilms [1216]. The 2-aminoimidazole heterocy-cle has proven to be crucial for the observed biological activity of these compounds.

Reyes et al. have synthesized a library of 2-amino-imidazole triazoles (2-AITs) and checked their activity on Acinetobacter baumannii (ATCC 19606) and Meth-icillin-resistant Staphylococcus aureus (MRSA) [17]. Compounds 1(a-e) (Figure 2) were found to inhibit biofilm formation upwards of 94% at the initial con-centration of 100 M. However, compounds 1(b-d) were also found to have potent antibacterial activities. Compounds 1(a-d) also possessed the highest biofilm dispersal activity at 200 M, with compound 1c as the most effective at dispersing preformed A. baumannii biofilms with an EC

50 of 44.70 M. In addition these

compounds were tested against MRSA (ATCC BAA-44) in biofilm inhibition assays. Compounds 1a, 1c

and 1d showed IC50 values of 9.86, 8.55 and 4.50 M, respectively and were nonmicrobicidal.

Melander et al. synthesized a series of analogs (2b-l, Figure 2) of compound 2a, a potent antibiofilm com-pound [18], and tested them against three MRSA strains: BAA-1770, BAA-1765 and 43 300 (200 M) [19]. Ana-logs with alkyl chain substituents of less than four car-bons (2b-d) possessed a similar inhibition activity to compound 2a. Analogs substituted with alkyl chains more than four carbons or benzyl substituent exhib-ited an increased ability to inhibit biofilm formation in comparison to compound 2a, especially analog 2j. The 2-amino substituted library was also screened against MRSA strain for oxacillin resistance suppres-sion activity. Substitution with short aliphatic chains (2c and 2d) led to an increase in synergetic activity with oxacillin against MRSA.

Recently, Melander and colleagues discovered another series of 1,4-disubstituted-2-aminoimidazoles (3a-l, Figure 2) and explored their antibiofilm activity against multidrug resistant bacteria [20]. These com-pounds were found to be active biofilm inhibitors of MRSA with the lead compound 3h exhibiting single digit IC

50 value (IC

50 = 4.14 2.03 M).

Melander et al. also found a class of molecules based on a menthyl carbamate scaffold that possesses potent nonmicrobicidal biofilm inhibition activity against vari-ous staphylococcal strains [21]. A series of blended 2-ami-noimidazole (2-AI) head group together with menthyl carbamate moiety were synthesized (Figure 3) and tested for antibiofilm activity. The antibiofilm activity and their ability to disperse mature preformed biofilms were tested against MRSA, Staphylococcus aureus (ATCC# 29213), Pseudomonas aeruginosa (PA14) and A. bau-mannii (ATCC# 19606) [22]. Compounds 47 inhib-ited biofilm formation in P. aeruginosa (PA14) with IC

50 of 18, 18, 58.7 and 40.3 M, respectively, while 8

inhibited biofilm in A. baumannii with IC50

of 16.7 M.Frei et al. showed that 2-aminobenzimidazoles

(2-ABIs) are biofilm inhibitors in P. aeruginosa [23]. Based on the three known inhibitors: bromoagelif-erin (Figure 1), 3-indolylacetonitrile and resveratrol (Figure 4), they proposed a scaffold that combined all of the three compounds. They designed and synthe-sized a small set of stilbenes containing 2-aminoimid-azole or indole moieties (Figure 4). Compounds 9 and 10 (100 M) were able to inhibit P. aeruginosa biofilm growth at 24 h by 56 and 48%, respectively.

In order to check which feature in those molecules is responsible for their antibiofilm activity, they syn-thesized each part separately and found that molecule 11 (Figure 4) exhibited greater activity than the lead compound 9 and almost completely inhibited biofilm formation at 24 h (94% inhibition, IC

50= 47 M).

Key term

IC50: Quantifies the ability of a compound to inhibit a specific biological or biochemical function. For this review, it refers to the concentration of a compound that inhibits biofilm formation by 50%.

Figure 2. 2-aminoimidazole based biofilm inhibitors.

O

Br

F O

NH

N

RHN N N

N HN

5

O4

2

H2NHN

N

N

N N

N5

O4

R 3

1a: R =

3b: R =

3c: R =

3d: R =

3j: R =

3k: R =

3l: R =

3a: R = 3e: R =

3f: R =

3g: R =

3h: R =

3i: R =

2a: R = H

2b: R = Me

2c: R =

2d: R =

2e: R =

2f: R =

2g: R =

2h: R =

CF3

2j: R =

2k: R =

2l: R = 2i: R =

1b: R = 1d: R =

1e: R =

1c: R =

NH

N

H2N N N

N

R

5

1

www.future-science.com 649future science group

Agents that inhibit bacterial biofilm formation Review

Then, the effect of different substitutions on the ring was investigated. Halide or methyl substitution on 2-ABI aryl ring exhibited greater activity. The most potent biofilm inhibitor identified was compound 12 (IC

50= 4.0 M). It is one of the most active P. aeru-

ginosa biofilm inhibitors known to date. Moreover, it was found that the 2-ABIs, 10 and 11, were capable of strongly dispersing P. aeruginosa biofilms. The mechanism of these 2-ABIs is still not fully understood.

Another modification by the Melander group in the 2-aminobenzimidazole (2-ABI) class of compounds (compounds 13 (R1-R19), Figure 4) involved the preparation of N-1-substituted-2-aminobenzimidazole as zinc-dependent S. aureus biofilm inhibitors [24]. The indole derivative (13, R15) was the most potent of these 2-ABIs with IC

50 values of 3.7 and 4.4 M

for 43300 and BAA-44, respectively. However, none of these compounds were effective dispersal agents.

Figure 3. 2-aminoimidazole and menthyl carbamate library.

HN

N

O

O

H2NN

NH

O

O

HNHCl

HCl

H2N

HCl

N

NH

O

OHN

H2N

HN

N

N

N

NH

O

O

N

H2N

HCl

NH

N NH

NN

O

ON

NH2HCl

NH

NN

NN

N

N O

O

Menthyl carbamate

4 5

67

8



Sambanthamoorthy et al. investigated the activi-ties of two structurally related benzimidazole com-pounds, 14 and 15 (Figure 4), which possess antibio-film activity in Vibrio cholerae [25]. Both compounds inhibited biofilm formation in V. cholerae at concen-tration of 10 M. Another six human pathogens were examined for the effect of compound 14: P. aerugi-nosa (CF-145), Klebsiella pneumoniae, Erwinia amy-lovora, Shigella boydii, MRSA strain USA300 and S. aureus strain Newman [25]. Compound 14 was used at a concentration of 100 M for the Gram-negative bacteria and 25 M for S. aureus. Compound 14 inhibited biofilm formation in each of these bacteria in a static biofilm assay, while it did not have an effect of their growth. Biofilm formation by P. aeruginosa strain PA01 on the surface of a medical silicone cath-eter, under flow conditions, was inhibited by 100 M of compound 14.

Indole derivativesIndole, which is generated by the degradation of tryptophan by tryptophanase [26], is an intercellu-lar signal molecule that can affect multiple aspects of some bacterial species [27]. Particularly, it has the potential to inhibit biofilm formation in several clini-

cal relevant bacterial strains. Escherichia coli O157:H7 biofilm formation and bacterial motility was inhib-ited by indole at a concentration of 500 M [28,29]. Gene expression in indole-treated biofilms was inves-tigated and it was found that indole reduced expres-sion of cold shock regulator genes (cspGH) by 2.5- to fourfold. Phosphate-related genes of E. coli O157:H7 were also affected by indole [28].

There are many oxygenases in bacteria and indoles are found to be readily converted to hydroxylated indoles by these oxygenases. These hydroxylated products include 5-hydroxyindole and 7-hydroxyin-dole (see Supplementary Figure 1) [30]. As indole can affect bacterial biofilm formation, the antibiofilm activities of oxidized indole derivatives were investi-gated. Indole was found to decrease E. coli O157:H7 biofilm formation by sixfold, while 7-hydroxyindole decreased biofilm formation by tenfold [31]. On poly-styrene, biofilm formation of E. coli O157:H7 and E. coli K12 could be diminished both by 7-hydroxy-indole and 5-hydroxyindole [31]. Specifically, 1000 M concentration of 7-hydroxyindole caused 27-fold reduction on E. coli O157:H7 biofilm formation while 1000 M 5-dydroxyindole caused 11-fold reduc-tion. For E. coli K12, bacterial biofilm was inhibited

Figure 4. Benzimidazoles analogs.

R1 = H; R2 = -CH(CH3)2; R3 = -(CH2)3CH3; R4 = -(CH2)5CH3; R5 = -(CH2)7CH3; R6 = -cyclopentane; R7 = -Bn; R8 = -Bn(4-OMe); R9 = -CH2CH2Ph; R10 = -CH2CH2Ph(o-OMe);R11 = -CH2CH2Ph(m-OMe); R12 = -CH2CH2Ph(p-OMe); R13 = -CH2CH2-napthalene; R14 = -CH2CH2-(2-pyridine); R15 = -CH2CH2-indole: R16 = -CH2CH2-benzimidazole;R17 = -CH2CH2CH2Ph; R18 = -CH2CH2CH2CH2Ph; R19 = -CH2CH2CH2CH2-indole.

Resveratrol 3-Indolylacetonitrile

9 1110 12

13

1514

HO

OH

OH

NH

N

NH

NNH2

NH2

NHN

NH

NH2

N

NH

NH2

N

NH

N

N

R

H2NO

HCl

N

HN

S

MeO

HN

MeO N

SCl

Cl



eightfold by 1000 M 7-hydroxyindole and sixfold by 1000 M 5-hydroxyindole.

The plant pathogen Rhodococcus sp. BFI 332 pro-duces significant amounts of indole-3-acetaldehyde and indole-3-acetic acid (see Supplementary Figure 1). Indole-3-acetaldehyde was found to be better than indole at reducing E. coli O157:H7 biofilm formation. E. coli O157:H7 biofilm formation was diminished by tenfold using 100 g/ml indole-3-acetaldehyde and only by fourfold using 100 g/ml indole [32].

The plant secondary metabolites, 3-indolylacetoni-trile (IAN) and indole-3-carboxyaldehyde (I3CA) (see Supplementary Figure 1), were also proven to reduce E. coli O157:H7 biofilm formation [11]. Indole, IAN and I3CA were compared under the same conditions for their ability to inhibit E. coli O157:H7 biofilm formation. IAN and I3CA were more effective than indole. E. coli O157:H7 biofilm formation was dimin-ished by threefold in the presence of 100 g/ml indole (see Supplementary Figure 2). Meanwhile, 100 g/ml

IAN and I3CA suppressed E. coli O157:H7 biofilm formation by 24-fold and 11-fold, respectively [11]. In addition, biofilm formation by P. aeruginosa was also inhibited by IAN (2.3-fold) and I3CA (1.9-fold). On the other hand, biofilm formation by P. aeruginosa was induced by indole and 7-hydroxyindole.

As some compounds with pyrroloindoline-triazole-amide scaffold have been identified to affect bacterial biofilm formation in E. coli, A. baumannii, S. aureus and MRSA [33], indoletriazole-amide analogs were investigated (Figure 5) [34]. Compounds 16(a-c) decreased 50% biofilm growth, relative to control, of S. aureus at the concentrations of 44.4, 173.6 and 174.8 M, respectively [34]. Also, compound 17 inhibited 12% biofilm formation of E. coli at con-centration of 150 M, about 50% biofilm growth of A. baumannii and MRSA at concentrations of 325.5 and 151.4 M, respectively [34]. Moreover, these ana-logs inhibited biofilm formation in a nontoxic way. They did not affect planktonic bacterial growth.

Figure 5. Indoletriazole-amide analogs.

O

N

NN

NH

NH

n

NH

NH

O

N

NN

Br16a: n = 416b: n = 516c: n = 6 17

Figure 6. Plant-derived biofilm inhibitors.

Emodin Phloretin

7-EpiclusianoneIsolimonic acid

Casbane diterpene

Proanthocyanidin A2-phosphatidylcholine

19a : R = iPr19b : R = sec-Bu19c : R = Ph

Hyperforin and its hydrogenated analog

Chelerythrine

Carvacrol18a: R = H18b: R = CH3

O

O OH

OH

OHOH

OH

O

HO

HO HO

O

O

O

OHO

O

O

O

HOOC

OO

HOH

H

O

H

HOH

OH

OHHO

O

R

OH

OHHO

OO

R

-O O

OO

-O O

OO

OH

OH

OH

OH

OH

OH

HO

HO

HO

O

O

O

N

O

O

+



Plants-derived compoundsEmodin (Figure 6) is a naturally occurring anthra-quinone found in the roots and barks of numerous plants, molds and lichens. Ding et al. showed that emodin significantly inhibits biofilm formation at 20 M in P. aeruginosa and Stenotrophomonas malto-philia [35]. Cells that were incubated with emodin were detached and dispersed from the surface. It is likely that emodin penetrated into the biofilm and interfered with the quorum sensing (QS) system in P. aeruginosa.

Flavonoids have been shown to exhibit antibiofilm activity. Phloretin (Figure 6), a flavonoid found in apples was observed to control E. coli O157:H7 biofilm formation by inhibiting the production of fimbriae, which is required for biofilm formation [36]. Lee et al. showed that 50 g/ml phloretin achieved a 98% decrease in E. coli O157:H7 biofilm without affect-ing the growth of planktonic cells. Most importantly phloretin also exhibited no inhibitory activity against the commensal E. coli K-12 biofilm [36]. This is partic-ularly advantageous since a potential antibiofilm agent

Figure 6. Plant-derived biofilm inhibitors (cont.).

Ginkgoneolic acidC13:0

Ginkgolic acid C15:1(R)-norbgugaine

Tannic acid

Ellagic acidxylopyranoside

Ellagic acidmannopyranoside

Ellagic acid

OH

OH

OH

HO

O

O

O

O

OH

OH

OH

OH

HO

HO

O

O

O

O

O

O

OH

OH

OH

OH

OH

HO

HO

O

O

O

O

O

O

OH

OHOH

OHOH

OH

OH

OH

OH

OH

OH

OH

HO

HO

HOHO

HO

HO

HO

HO

HO

HOHO

HO

O

O

O

OO

O

O O

O

O

O

O

O

O

O

O

OH

OO

O

OO

OH

HO

O

NH

(CH2)11CH3H

OH O

CH2(CH2)4CH3



should selectively inhibit pathogenic strains without wiping out the commensal flora [37,38].

Extracts of ginger were shown to reduce biofilm formation in P. aeruginosa strain PA14 by 3956% without affecting the growth of the cells [39]. Further analysis revealed that the extracts caused a reduction in the extracellular polymeric substance, an increase in swarming motility and reduced levels of cellular c-di-GMP. In that same study, ginger extract showed broad spectrum antibiofilm activity in the Gram posi-tive bacteria S. aureus and Bacillus megaterium and in the Gram negative bacteria E. coli.

The therapeutic effects of the extracts of Hypericum perforatum (also known as St. Johns Wort) are well known. Hyperforin is a major constituent of H. per-foratum and because it is unstable, it was probably believed that it did not contribute to the therapeutic

effects of H. perforatum. Contrary to this notion, Schiavone et al. showed that a stable form of hyper-forin, hydrogenated hyperforin (Figure 6), inhibited the growth and biofilm formation of S. aureus ATCC 29213, MRSA, Enterococcus faecalis ATCC 29212 and S. aureus Ig5 [40]. Treating S. aureus ATCC 29213 and S. aureus Ig5 with 150 and 37.5 g/ml of the hydrogenated hyperforin, respectively, caused over 50% biofilm reduction. Also, at a concentration of

Key terms

Quorum sensing: Response to a signal in a population-dependent manner. In bacteria, this is used to coordinate gene expression.

C-di-GMP: Cyclic dinucleotide containing two guanine nucleobases with two 2-5-phosphodiester linkages and which regulates biofilm-related genes in many bacteria.



37.5 g/ml, hydrogenated hyperforin reduced the biofilms of S. aureus ATCC 43300 and Enterococcus faecalis ATCC 29212 by 47 and 45%, respectively.

Sarkisian et al. have also tested other secondary metabolites from Hypericum spp. for biofilm inhibi-tion [41]. They found that five of these compounds (18a-b and 19a-c, Figure 6) inhibited biofilm forma-tion by Staphylococcus epidermidis and S. aureus. The minimum biofilm inhibitory concentrations of these five compounds were lower than 8 g/ml.

7-Epiclusianone (Figure 6) is a natural compound purified from Rheedia brasiliensis. Murata et al. reported that 250 g/ml 7-epiclusianone with or with-out 125 ppm fluoride (F) inhibited Streptococcus mutans biofilm development [42]. 7-Epiclusianone could also reduce the formation of dental caries in vivo.

Isolimonic acid (Figure 6), a triterpenoid second-ary metabolite from citrus species has previously been shown to interfere with QS in Vibrio harveyi and also exhibit a dose-dependent inhibition of biofilm forma-tion with an IC

50 value of 94.18 M [43]. Isolimonic

acid inhibited E. coli O157:H7 biofilm formation at IC

50 of 19.7 M. It also demonstrated an ability to

modulate the type III secretion system and adhesion of E. coli O157:H7 [44].

Chelerythrine (Figure 6) was isolated from the plant Chelidonium majus and it showed inhibitory effects on biofilm formation of S. aureus ATCC 6538P and S. epidermidis ATCC 35984 in a dose-dependent man-ner with IC

50 values of 15.2 M and 8.6 M respec-

tively [45]. Another plant derived compound, proan-thocyanidin A2-phosphatidylcholine (Figure 6) was isolated from the medicinal plant Krameria lappacea and was also shown to inhibit biofilm formation in the Staphylococcus strains with EC

50 values of 6.9 and

7.6 M, respectively. Both molecules exhibited no inhibitory activity on the growth of the Staphylococcus strains, as there was no significant difference between growth curves in the presence and absence of sub-minimum inhibitory concentrations (MIC) of the compounds. They also lack the ability to disrupt mature biofilm.

Different adherent S. aureus biofilm forming cells were observed to form scattered clumps when treated with fractionated root extracts of Rubus ulmi-folius Schott (Rosaceae-Elmleaf blackberry), a wild shrub native to the Mediterranean. A limited num-ber of published studies have explored the antibac-terial properties of R. ulmifolius [46,47]. Quave et al.

observed an inhibition of S. aureus biofilm formation by R. ulmifolius extracts with concentrations in the range of 50200 g/ml [48]. The extract had no effect on mature biofilms, but when dosed together with selected antibiotics its efficacy against mature biofilms was significantly enhanced. LC-UV/MS/MS analysis of the extract revealed that the antibiofilm activity of the extract was probably due to its constituent ellagic acid and its derivatives (ellagic acid mannopyranoside and ellagic acid xylopyranoside; Figure 6).

The polyphenolic compound tannic acid (Figure 6) also inhibits S. aureus biofilm formation without inhib-iting cell growth (20 M) [49]. Tannic acid is found in teas and other plant-derived foods. Black tea, a source of tannic acid, also causes inhibition of biofilm forma-tion in S. aureus. When cells were grown in the pres-ence of tannic acid, an increased level of the protein IsaA was found. IsaA is a putative lytic transglyco-sylase that has previously been implicated in cleaving peptidoglycan.

Ginkgolic acid C15:1 was found among several plant-derived compounds to inhibit biofilm formation of E. coli O157:H7 at 5 g/ml without affecting the growth of the cells (see Supplementary Figure 3) [50]. The inhibition was observed to be as a result of the sup-pression of curli genes and consequently the produc-tion of fimbriae. When tested against the commensal E. coli K-12 strain, ginkgolic acid C15:1 had no inhibi-tory effect but interestingly it boosted biofilm forma-tion. At 5 g/ml, ginkgolic acid C15:1 inhibited bio-film formation by MRSA and other S. aureus strains but had no inhibitory effect on cell growth.

One of the major components of ginkgolic acid, ginkgoneolic acid (C

13:0) has been demonstrated to

possess antibacterial activities [51]. He et al. observed that at 2 g/ml, ginkgoneolic acid inhibited the adher-ence of S. mutans, a dental pathogen, on saliva-treated hydroxyapatite (S-HA) beads [52]. Ginkgoneolic acid at 32 g/ml caused 50% or more inhibition of S. mutans biofilm formation. It also caused a change of the bio-film morphology. The study by He et al. therefore demonstrates that ginkgoneolic acid has potential to be used to treat dental caries.

Carvacrol (Figure 6), a component of oregano essen-tial oil, reduces bacterial motility and virulence at sub-lethal concentrations [53]. Nostro et al. reported that vapor forms of oregano oil, carvacrol and thymol were effective at inhibiting biofilm formation as well as erad-icating the preformed biofilms of S. aureus and S. epi-dermidis [5456]. Carvacrol reduces biofilm formation by Chromobacterium violaceum (0.10.3 mM), Sal-monella typhimurium (0.751.25 mM) and S. aureus (0.51 mM) [57]. At these concentrations there was no effect on bacterial growth. Preformed biofilms were

Key term

Minimum inhibitory concentration: Refers to the lowest concentration of a compound to inhibit bacterial growth after overnight incubation.

Figure 7. Resveratrol and its oligomers.

O

OH

OH

OH

OH

HO

OH

OH

HO

O

O

OHOH

OH

OH

OH

HO

HO

HO

HO

O

Trans-resveratrol

-viniferin Vitisin B



not affected by the addition of carvacrol. Carvacrol affects genes coding for QS and inhibits the produc-tion of acylhomoserine lactones (AHLs), chitinase and violacein in C. violaceum. Thyme oil, oregano oil and carvacrol also reduced the amount of biofilm produced at 0.012% by the S. typhimurium strains [55].

Bgugaine is a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare [58]. Recently Majik et al. described the synthesis of the N-demethylated form of the natural pyrrolidine alkaloid and explored its QS inhibition in P. aeruginosa [59]. P. aeruginosa showed a decrease in biofilm density, with increasing concentra-tion of (R)-norbgugaine (04 mM). Eighty three percent inhibition was observed at a concentration of 1.8 mM and above. P. aeruginosa growth was not affected by norbgugaine. Besides biofilm inhibition, norbgugaine was also found to inhibit motility, pyocyanin, LasA protease and rhamnolipid productions.

Another natural product, casbane diterpene (Figure 6), has been identified to have antibiofilm activ-ity [60]. Casbane diterpene is an extract of the plant Croton nepetaefolius and was screened against both Gram-positive and Gram-negative bacteria. Casbane diterpene was able to inhibit the biofilm formation of both Gram-positive and Gram-negative bacteria. 125 and 250 g/ml casbane diterpene significantly reduced biofilm formations by S. aureus and S. epider-midis CECT 4183, respectively. 15.6 and 250 g/ml of casbane diterpene reduced biofilm formation of K. pneumoniae ATCC 11296 (45%) and P. aeruginosa ATCC 10145 (80%), respectively. Also, biofilm forma-tions of P. aeruginosa CGCT 111, E. coli K12 strains and Pseudomonas fluorescens were reduced by high concentrations of casbane diterpene.

A screen of close to 500 plant extracts revealed that, trans-resveratrol (Figure 7), a major component of the extracts of Carex dimorpholepsis, demonstrated

antibiofilm formation activity against E. coli O157:H7 [61]. Interestingly, no inhibitory effect was observed against commensal E. coli strains. Resveratrol is found in red grapes, peanuts and some woody plants and has been reported to have other antibacterial and antioxidant properties in humans [62,63].

Resveratrol oligomers are also found to have nutri-tional benefits [64,65]. -Viniferin, a dimer of resveratrol (Figure 7), is found in grape vines and carex plants and has been reported to have fungicidal and antioxidant activities [66]. Biofilm inhibition experiments showed that trans-resveratrol and -viniferin inhibited the bio-film formation of two P. aeruginosa strains, PAO1 and PA14, in a dose-dependent fashion [67]. Trans-resvera-trol and -viniferin (50 g/ml) inhibited biofilm for-mation by P. aeruginosa PAO1 and P. aeruginosa PA14 by 92 and 82%, respectively. Also -viniferin inhib-ited E. coli O157:H7 biofilm formation at 10 g/ml by 98%, without affecting planktonic growth.

Lee et al. [68] tested trimers and tetramers of resvera-trol for biofilm inhibition; they found vitisin B (Figure 7) to inhibit biofilm formation of E. coli O157:H7 and P. aeruginosa PA14. In E. coli, 5 g/ml vitisin B inhib-ited biofilm formation by more than 90%. In addition, at concentrations lower than 50 g/ml, visitin B did not inhibit E. coli O157:H7 growth. The mechanism of the inhibition of E. coli O157:H7 biofilm formation was found to involve inhibition of fimbriae production.

Garlic-derived natural products have been reported to inhibit QS systems in Pseudomonas and Vibrio spe-cies [7,69]. Bioassays showed that the main compounds in the garlic extracts responsible for QS inhibition are ajoene, sulfides, polysulfides and vinyl dithiins [7,70]. Ajoene (Figure 8), a component extracted from garlic, has been identified to be able to decrease the produc-tion of QS signal molecules of P. aeruginosa and can synergize biofilm inhibition by tobramycin [70]. Ajoene

Figure 8. Sulfur derivatives with antibiofilm activities.

S

SS

O

CO2H

NH2

S SS

O

Diphenyl disulde S-phenyl-L-cystein sulfoxide

Ajoene



(80 g/ml) inhibited the production of N-butyryl-l-homoserine lactone in P. aeruginosa by almost three-fold. Tobramycin (as high as 340 g/ml) was ineffec-tive at killing bacteria in a P. aeruginosa biofilm but in the presence of ajoene, up to 90% of biofilm-forming P. aeruginosa were killed by 10 g/ml of tobramycin.

Extracts from the Amazonian medicinal plant, Petiveria alliacea L. (Phytolaccaceae), contain sulfur derivatives such as S-phenyl-l-cystein sulfoxide and diphenyl disulfide [71]. The effects of these compounds on P. aeruginosa biofilms were investigated and it was found that at 1 mM concentration, both S-phenyl-l-cystein sulfoxide and diphenyl disulfide (Figure 8) did not inhibit planktonic cell growth but lowered cell viability within a biofilm by 41 and 45%, respectively (determined by the MTT assay) [71].

Marine-derived compoundsPeach et al. reported the discovery of a new struc-tural class of biofilm inhibitors by applying an image-based, high-throughput-screening platform to a marine natural products library [72]. Auromomycin (see Supplementary Figure 4) was found to have bio-film inhibition activity against V. cholerae with IC

50 of

60.1 M. Moreover, addition of subinhibitory concen-trations of antibiotics (tetracycline, ceftazidime and ciprofloxacin) enhanced the biofilm inhibitory activity of auromomycin [72].

Natural halogenated furanones, isolated from the marine red algae Delisea pulchra, have also shown anti-biofilm properties. Both natural and synthetic bromi-nated furanones have been identified as the effective QS inhibitors (QSIs) in both Gram-positive and Gram-negative bacteria [6,7376]. Some synthetic furanones show an ability to penetrate P. aeruginosa biofilm and cause changes in biofilm maturation (compound 18,

Figure 9) [6]. Both nitrogen and sulfur analogs of bro-minated furanones have also been synthesized and tested (see Figure 9) [77,78].

Compound 19 (R1/R2/R3= H) had an inhibi-tory effect on biofilm formation (IC

50= 20 M) with

minimum effect on planktonic cell growth. How-ever, the use of halogenated furanones is limited due to their toxicity in mammalian cells. Analogs 2022, (Figure 9) on the other hand, reduced biofilm forma-tion and thickness in E. coli and P. aeruginosa (see Supplementary Figure 5) and inhibited elastase B pro-duction by P. aeruginosa at nonmicrobicidal concentra-tions and are less toxic to human neuroblastoma SK-N-SH cells than natural bromofuranones [79]. However, these analogs are not as active as the more toxic haloge-nated furanones and require concentrations that are far greater than 100 M to achieve significant antibiofilm effects.

Pereira et al. evaluated the antibiofilm activity of brominated alkylidene lactams (see Figure 9, com-pounds 2328), on S. aureus, P. aeruginosa, S. epi-dermidis and S. mutans [80]. Among all of the com-pounds that were evaluated, the most active against S. epidermidis were (E)--alkylidene--lactam 23 with IC

50 = 12.2 g/ml (25.4 M) and the -hydroxy--

lactam 24 with IC50

= 13.3 g/ml (22.6 M). Against P. aeruginosa, the most active compounds were (Z)--alkylidene--lactam 25 and 26 with IC

50 values of 0.6

and 0.7 /ml (1.3 M), respectively. The most active compound against S. aureus biofilm was -hydroxy--lactam 27 and 44 g/ml (80.9 M) inhibited 53.1% of biofilm formation. Compounds 23, 25 and 28 (Figure 9) inhibited S. mutans biofilm formation, with 23 being the most active.

Marine sponges produce an array of secondary metabolites, which they use to ward off fish that prey on them. One of such classes of compounds is the bro-mopyrrole alkaloids, which have been shown to exhibit a range of biological activities, including antibiofilm activities. 4-Thiazolidinones derivatives of pyrroles, in addition to antibiofilm properties, also have anti-HIV-1 [81] and anti-inflammatory [82,83] activities. 4-Thiazolidinone bromopyrrole derivatives (com-pounds 29 and 30, Figure 10) are against S. aureus bio-films [84]. Interestingly, the MIC of 29 and 30 against S. aureus biofilm is 0.78 g/ml, which is three-times less than the MIC value for vancomycin (3.125 g/ml).

Streptomyces spp. has served as a source of a plethora of structurally unique molecules with various bio-logical activities [85]. In a screening of marine-derived microbial prefractions for P. aeruginosa biofilm inhibi-tors, a class of structurally unique molecules, skylla-mycins, were purified from cultures of Streptomyces spp [86]. The cyclic depsipeptide skyllamycins B and C

Key term

N-acyl homoserine lactones: Signaling molecules that are involved in quorum sensing. They are secreted by different bacteria and their structures differ by the length of the side chain and the substitution of a carbonyl at the third carbon.

Figure 9. Brominated furanone analogs.

OBrO

Br

OBr

O SBrO

R2

R3

R1

OO

Br

OO

Br

O

O

Br

N

Br

OF3C

OMe OMe

N

Br

OBr HO

Br

OMe

N

Br

O

Cl

OMe

Cl

N

Br

O

Br

Naturalbromofuranone

18

22

2120

2425

23

27 2826

19, R1 = H, alkyl R2 = H, Br R3 = H, Br

Cl

N

Br

O

OMe

HO

Br

OMe

N

Br

O

Cl

Figure 10. Bromopyrrole alkaloids.

SN

O O N Br

Br

O

NH

29 30

NHS

N

O O N Br

Br

NO2



(Figure 11) were found to inhibit biofilm formation with respective EC

50 values of 30 and 60 M. Skylla-

mycin B demonstrated the ability to clear mature bio-films of P. aeruginosa strain PAO1 wspF (designed to overexpress c-di-GMP and hence is expected to form strong biofilms) in a nontoxic manner.

(-)-Ageloxime D (Figure 11) is a diterpene alkaloid, which was isolated from the Indonesian marine sponge Agelas nakamurai. Hertiani et al. found that (-)-agelox-ime D inhibits biofilm formation in S. epidermidis, but does not affect bacterial growth [87].

Tello et al. have reported the antibiofilm activities of natural (obtained from Colombian Caribbean octo-coral Pseudoplexaura flagellosa and Eunicea knighti) and synthetic cembranoid compounds (Figure 12) [8890]. At 100 ppm, cembranoid epimers at C8 inhibited P. aeruginosa biofilm maturation [88]. In V. harveyi, compounds 31 and 33 (10 ppm) inhibited V. harveyi biofilm maturation by 75%. Compounds 35 and 36 (100 ppm) inhibited by V. harveyi biofilm maturation by 40 and 95%, respectively. Compounds 32, 33 and 34 (10 ppm) inhibited S. aureus biofilm formation by 70%. These cembranoid epimers did not inhibit bacterial growth at 100 ppm.

Compound 38 was found to be a QSI in C. viola-ceum. Against P. aeruginosa, compounds 3743 were found to inhibit biofilm formation with IC

50 values less

than 50 M. Compounds 38 and 39 were particularly effective against S. aureus biofilm formation with IC

50

values of 0.16 and 0.08 M, respectively. Amongst the

cembranoids that were tested, biofilm formation by V. harveyi was inhibited only by compounds 4143, with IC

50 values of 5.20, 30.80 and 9.91 M, respectively.

These compounds did not affect bacterial growth.

AHLs-based inhibitorsN-acyl homoserine lactones mediate QS in Gram-negative bacteria. There are several types depending on the length of the acyl side chain. A considerable amount of modified AHL derivatives have been developed and tested for quorum sensing inhibition and sometimes for biofilm inhibition (Figure 13) [8995].

Lactone based analogs of AHL are susceptible to hydrolysis but a few hydrolytically more stable AHL analogs have also been developed and have the poten-tial to be used in practical applications. For example, thiolactanone 51, which is hydrolytically more stable than the lactone analog, inhibits biofilm formation by

Figure 11. Skyllamycins and (-)-ageloxime D structures.

O

H

CO2HHN

HN

O

NH

O

OO

O

HN

OH

O

NH

O

OH

HN O

NH

O

NH

O

HO

OCH3NH

HN

HN

N

OHO

O

CO2HHN

HN

HN

NH

NH

NH

OH

OH

O

H

O

O

OO

O

OO

OO

NH

NH

O

HO

OCH3

HN

HN

HN

N

OHO

O

HO

HN

H

N+ N

NN

Skyllamycin B Skyllamycin C

(-)-Ageloxime D



P. aeruginosa [96]. N-decanoyl-l-homoserine benzyl ester (52, Figure 13), a nonlactone analog but which could also undergo ester hydrolysis nonetheless could repress virulence factors production, swarming and the production of rhamnolipids by P. aeruginosa without affecting growth [97]. Compound 52 affects virulence by repressing las and rhl systems. In a recent study by Weng et al., it was found that compound 52 (200 M) inhibited biofilm formation by PAO1 and reduced biofilm tolerance to tobramycin [98].

The QS system in the bacterium Burkholderia cenocepacia is controlled by two AHLs, N-octanoyl-l-homoserine lactone and N-hexanoyl-l-homoserine lac-tone. Brackman et al. reported the synthesis of a series of AHL analogs, triazolyldihydrofuranones (Com-pounds 5355, Figure 13), in which the amide func-tion was replaced by a triazole group and they evaluated their effect on QS and biofilm formation in B. cenocepa-cia and P. aeruginosa [99]. In P. aeruginosa PAO1, the most effective were compounds 53 and 54, whereas in B. cenocepacia LMG16656, 54 and 55 were active.

Porphyromonas gingivalis is a major pathogen of peri-odontal disease. The QS mechanism in this bacterium is still not clear, although AI-2 signaling has been

implicated. Planktonic P. gingivalis cells respond to N-acyl HSL molecules and AHL analogs (5658, Figure 13) have been shown to reduce P. gingivalis bio-film thickness [100]. Some of these analogs could also enhance the potency of antibiotics (cefuroxine, mino-cycline and ofloxacin) against P. gingivalis biofilms (see Supplementary Figure 6) [101].

Other small moleculesMetabolites isolated from the myxobacterium Sorangium cellulosum strain So ce960 have been demonstrated to have antibiotic properties. One of such metabolites is car-olacton (Figure 14A), a macrolide ketocarbonic acid [102]. The antibiofilm activity of carolacton against caries and endocarditis-associated bacterium S. mutans has been investigated. It was observed that 5 g/ml of carolacton reduced S. mutans biofilms by 35%, with very little effect on planktonic cells growth [103]. Hallside et al. reported the effect of carolacton and its analog 59 (Figure 14B) on biofilm morphology [104]. When carolacton (>500 nM) and compound 59 (>62.5 M) were incubated with S. mutans in the presence of biofilm-inducing media, dramatic changes in the integrity and morphology of the biofilm matrix were observed (Figure 14C).

Figure 12. Cembranoids library for biofilm inhibition. Data taken from [8890].

OHO

OH

OHO

OH

O

O

O

O

O

O

O

O

O

OO

H

OAc

OH

OHOAc

OH

HO

HO

OH

OH

OH

OAc

HO

OH O

H

CHO

HO

OH

CHO

31 32 33 34 35

36 37 38 39

40 41 42 43 44



Peptides & biofilmsMicrobial amyloidsMany neurodegenerative diseases such as Alzheimers and Parkinsons are probably caused by the aggregation of proteins into amyloid fibers. Amyloid formation was originally viewed as a consequence of protein misfolding and aggregation. Ongoing studies show that functional amyloids have a ubiquitous role in living systems, espe-cially in bacteria. Proteins that are folded into amyloid state are found in many microorganisms and they have a role in cell-cell communication and biofilm forma-tion [105,106]. Once formed, amyloid fibers are resistant to disassembly by enzymatic and chemical digestion [107].

S. aureus biofilm matrix contains polysaccharides and DNA that interact with structural and enzymatic proteins. At certain conditions, S. aureus biofilms are composed of small peptides, amyloid-like fibers that are called phenol soluble modulins (PSMs) [108]. PSMs con-trol biofilm integrity. Mutants incapable of producing PSMs formed biofilms that were degraded by enzymes and mechanical stress. PSMs can modulate biofilm dis-assembly using amyloid-like aggregation as a control point for their activity. It is known that PSMs are reg-ulated by the agr QS network [109,110]. Schwartz et al. have found that an agr deficient strain did not produce

fibers [108]. PSMs may be stored as inert fibrils in a sessile biofilm until conditions are suitable for their dissociation and promoting biofilm disassembly.

These microbial amyloid fibers are structurally simi-lar to the pathogenic variants found in humans; there-fore, they are good targets in screening for molecules with antibiofilm and antiamyloids activities.

Bacillus subtilis produces a spore-coat protein called TasA. TasA forms amyloid structures in these bacterias biofilms. AA-861 (Figure 15) is a benzo-quinone derivative with anti-inflammatory activity. Parthenolide (Figure 15) is a sesquiterpene lactone with anti-inflammation and anticancer activities [111]. Romero et al. showed that AA-861 and parthenolide inhibit biofilm in B. subtilis by interfering with the formation of amyloid-like fibers [112]. Parthenolide was also shown to interfere with pre-established biofilms.

Key terms

Biofilm matrix: Network of macromolecules that form a protective scaffold around bacteria in a biofilm.

Amyloids: Misfolded polypeptides that aggregate to form a cross- structure. They are associated with diseases such as Alzheimer and diabetes. They are also found in the matrices of some bacterial biofilms.

Figure 13. Synthetic N-acyl homoserine lactone analogs.

ON

O

HN

O

OSC

8

HN

OO

O

Br

NH

SR

O O

On

NH

BrO

OSO

HN

O O

8HN

O

8

NH

O OH

O

O

NNN

O

O

NNN

O

O11

NNN

O

O7

N

N

O

OH

NH

OHN

NH

HOO O

45

O

HN

O

O

HN46 47

51

504948

52

53 5455

5857

56

O



These compounds also inhibit biofilm formation by Bacillus cereus and E. coli, which have amyloid proteins as major matrix components.

Cationic peptidesAntimicrobial peptides in plants and animals are usu-ally cationic molecules (contain an excess of lysine and arginine residues) and are composed of 1250 amino acids residues [113]. Cationic peptides are able to pass through the cell membrane, bind to the DNA and induce gene expression. They play an important role in the innate defenses of all species of life [114].

The human cathelicidin peptide LL-37 is able to block biofilm formation by P. aeruginosa [115]. LL-37 contains 37 residues and is a -helical peptide that is produced at mucosal surfaces by epithelial cells (Table 1). The attachment of P. aeruginosa cells to the surface was significantly reduced in the presence of LL-37.

Looking for shorter peptides for biofilm inhibi-tion, de la Fuente-Nez et al. found four peptides

with antibiofilm activity (Table 1). One of them, a 9-amino acid peptide 1037, has a weak antimicrobial activity MIC (304 g/ml) but demonstrated signifi-cant decrease in biofilm mass (78%) at half the MIC value [114]. When they compared the sequences of the peptides they found a conserved part (RIRVR). In B. cenocepacia and Listeria monocytogenes, treatment with 5 g/ml 1037 resulted in a substantial reduction in biofilm growth in both organisms [114].

LL-37 also has antimicrobial activity toward group A streptococcus (GAS) and it can cause cell lysis by disturbing cell membranes [116]. hCAP18, which LL-37 is originated from, is widespread in human cells and tissues, including neutrophils, monocytes, NK cells and mast cells. Also, LL-37 can attract neutrophils and CD4 T cells to infection sites.

LL-37 is also active against S. epidermidis [117]. In this bacterium, exopolysaccharide intercellular adhe-sion mediates immune evasion. LL-37 has higher anti-bacterial activities toward ica-mutant strain, which lacks exopolysaccharide intercellular adhesion, than the wild type strain. Interaction between S. epidermi-dis and LL37 was also investigated by Hell et al. [118] They showed that at low concentration (1 mg/l), LL37 inhibits bacterial attachment and biofilm formation

Key term

Planktonic bacteria: Bacteria living in a free state without being attached to a surface or physically connected to other bacterial cells and can freely drift.

Figure 14. Biofilm inhibition by carolacton. (A) Carolacton structure. (B) Analog 59 structure. (C) Confocal microscopy imaging of Streptococcus mutans UA159 biofilm cells treated with 250 M: (i) DMSO, (ii) carolacton and (iii) 59. Reproduced with permission from [104] American Chemical Society (2014).

O OMe

OH

OO

OH

OH

O

OMe

OH

OO

OO

O

Carolacton

59

A

B

C i. ii. iii.

Figure 15. Molecules that interfere with the formation of amyloid-like fibers.

O

O

OH

O

O

O

Parthenolide

AA-861



significantly. LL37 up to 16 mg/l did not inhibit bacterial growth.

d-amino acidsCells in B. subtilis biofilm are held together by exo-polysaccharides and amyloid-like fibers. The dispersal of biofilm involves the release of planktonic cells from the exopolysaccharides and protein components of the matrix. The d-amino acids, d-Tyr, d-Leu, d-Trp and d-Met, which are incorporated into the peptidogly-can, can trigger the release of the TasA fibers [119,120]. d-amino acids, such as d-tyrosine, d-methionine, d-tryptophan or d-leucine, are also known to inhibit biofilm formation in B. subtilis, P. aeruginosa and S. aureus [119]. The biofilm inhibitory effects of d-amino acids could be reversed by their cognate l-amino acids.

B. subtilis biofilm has been documented to be depen-dent on the availability of the polyamine spermidine. Kolodkin-Gal et al. found that a shorter structural analog, norspermidine, is another biofilm-disassembly factor, present in conditioned medium from aging B. subtilis biofilms and it directly interacts with the exo-polysaccharide [121]. Exogenous norspermidine (25 M) added to the growth medium prior to inoculation, fully inhibited biofilm formation without inhibiting plank-tonic growth. d-amino acids and norspermidine acted together in breaking down existing, mature pellicles. Norspermidine was also found to inhibit biofilm forma-tion by S. aureus and E. coli. These findings have how-ever been refuted by Hobley et al., who observed that exogenous norspermidine inhibited B. subtilis plank-tonic growth and pellicle development in an exopolysac-charide-independent manner and was not required in the formation of robust biofilms [122]. They also found that norspermidine is not synthesized by B. subtilis and is not naturally present in biofilms formed by B. subtilis. They reconfirmed the requirement of spermidine for the formation of robust B. subtilis biofilms.

Polysaccharides & biofilmsKingella kingae is a Gram negative bacterium that can cause infections such as endocarditis, osteomyelitis and arthritis. Its ability to cause infections is mainly due to its ability to form biofilms. Bendaoud et al. showed that Kingella kingae produces extracellular galactan, a poly-saccharide that inhibits its own biofilm formation and also the biofilm formation by other bacteria such as, Aggregatibacter actinomycetemcomitans, K. pneumoniae, S. aureus, S. epidermidis and Candida albicans [123]. Galactan consists of a linear polymer of galactofura-nose residues. The biological role of galactan is still unknown. One hypothesis is that it regulates the bio-film architecture. Another possibility is that it mediates the release of cells in the dispersal stage.

Galactose is a monosaccharide that is utilized by many organisms. Galactose metabolism plays a role in the biofilm formation in B. subtilis [124]. The sugar-nucleotide UDP-galactose is toxic for planktonic cells. However, it is essential for exopolysaccharide biosyn-thesis during biofilm formation. The metabolism of galactose is generally catalyzed by the enzymes GalK, GalT and GalE, each of which catalyzes one of three steps in converting galactose to UDP-glucose in the Leloir pathway (see Supplementary Figure 7) [125]. A deficiency of any one of these enzymes has been dem-onstrated to have deleterious effects on planktonic bacteria growth in the presence of galactose [124]. In



the third step, GalE catalyzes the reversible conversion of UDP-galactose to UDP-glucose. Chai et al. stud-ied a B. subtilis galE deletion mutant and observed that addition of galactose caused planktonic cell lysis due to the accumulation of UDP-galactose [124]. In that study, it was observed that biofilm-forming B. subtilis galE on the other hand survived in the presence of galactose by producing exopolysaccharide (EPS). Analysis of the EPS showed a large amount of galactose and so it was proposed that UDP-galactose was channeled into EPS production.

The antimicrobial properties of sugar esters have been previously demonstrated. One of such stud-ies used enzymatically synthesized lauroyl glucose to probe its antimicrobial properties against selected bac-terial and fungal test organisms [126]. It was observed that lauroyl glucose after 48 h of incubation with P. aeruginosa and Pseudomonas aureofaciens resulted in 51 and 57% disruption of preformed biofilms, respec-tively. Against the fungal test organisms, lauroyl glu-cose dislodged 45% of C. albicans and 65% of Candida lipolytica biofilms.

In another study, the tropical marine bacteria, Serra-tia marcescens was isolated from the hard marine coral Symphyllia sp [127]. The bacteria were found to produce a biosurfactant. A glycolipid made up of palmitic acid and glucose was subsequently purified from S. marc-escens and was determined to possess antimicrobial, antiadhesive and antibiofilm activities. It was observed that 50 g/ml of the purified glycolipid surfactant dis-lodged 70.6% of preformed P. aeruginosa PAO1 bio-films whilst at 100 g/ml, 90.5% of the P. aeruginosa PAO1 biofilms were disrupted.

Fatty acids & biofilmsFree fatty acids (FFAs) derive from fatty acids that escape from lipids with the aid of enzymes [128]. FFAs were observed to have antibacterial functions against both Gram-negative bacteria, such as Chlamydia tra-chomatis [129], Neisseria gonorrhoeae [130] and Gram-positive bacteria [131], such as Bacillus larvae [132]. FFAs exist in human skin [133] and they were found to be functional in controlling skin infection against

bacteria [134]. The relationship between FFAs and bac-terial biofilm formation has been studied extensively. FFAs have been proven to have the ability of inhibit-ing biofilm formation [135,136]. Oleic acid (Figure 16), which was identified as one of the primary unsatu-rated fatty acids, is able to repress biofilm formation of S. aureus by blocking bacterial adhesion [135]. Biofilm production of eight strains of S. aureus was reduced dramatically when oleic acid was added during the initial adhesion step. Another fatty acid, cis-2-dece-noic acid (Figure 16), which is produced by P. aeru-ginosa is able to induce the dispersion of established biofilms and inhibit biofilm development in P. aerugi-nosa, E. coli, K. pneumoniae, Proteus mirabilis, Strep-tococcus pyogenes, B. subtilis, S. aureus and the yeast C. albicans [136]. Cis-2-decenoic acid at concentrations greater than or equal to 125 g/ml inhibited biofilm formation in microtiter plates, but at concentrations at or above 500 g/ml it also inhibited bacterial growth. Jenings et al. showed that the combination with antibiotics resulted in decreased biofilm forma-tion, especially the combination with the antibiotic linezolid resulted in biofilm inhibition at concentra-tions 216-times lower than for either antimicrobial alone [137]. The mechanism of fatty acids antibacte-rial activity remains unclear. FFAs are able to affect some fundamental processes of bacteria by affecting the cell membrane, especially the process of energy production [128].

The combination of AI-2-based antibiofilm molecules with traditional antibioticsBiofilm-associated infections are often very difficult to treat with conventional antibiotics. Little is known about the relationship between the antibiofilm effect of QSI and the susceptibility of biofilms to antibiotics in the presence of these molecules. AI-2 is an autoin-ducer found in both Gram-negative and Gram-posi-tive bacteria. AI-2 signaling plays an important role in biofilm formation. AI-2 analogs, isobutyl-DPD and isopropyl-DPD, were found to be QSIs in E. coli, S. typhimurium and V. harveyi [138,139], and phenyl-DPD inhibits QS-related pyocyanin production in P.

Table 1. Peptides with antibiofilm activity.

Peptide Amino acid sequence MIC (g/ml) Biofilm inhibition at 1/2 MIC (%)

LL-37 LLGDFFRKSKIGKEFKRIVQRIKDFLRNLVPRTES 31 57

1026 VQWRIRVRIKK 5 54

1029 KQFRIRVRV 10 40

1036 VQFRIRVRIVIRK 10 43

1037 KRFRIRVRV 304 78

MIC: Minimum inhibitory concentrations.

Figure 16. Fatty acids that repress biofilm formation.

OH

O

HO O

Oleic acid

Cis-2-decanoic acid



aeruginosa [140]. AI-2 analogs, isobutyl-DPD and phe-nyl-DPD were also tested for their ability to alter bio-film formation, maturation and removal among E. coli and P. aeruginosa [141]. Biofilms treated with AI-2 ana-logs were thinner and less ordered. When AI-2 analogs were added together with the antibiotic gentamicin, pre-existing E. coli biofilm could be dispersed (see Supplementary Figure 8).

Nitric oxide & biofilm dispersalStrategies to induce biofilm dispersal are of interest because in most settings, therapeutic interventions would be applied after the biofilm has already formed. It has been shown that the important biological mes-senger, nitric oxide (NO) is a signal for biofilm disper-sal, inducing the transition from the biofilm mode of growth to the free swimming planktonic state [142,143]. The delivery of exogenous NO to biofilms was achieved via NO donors (Figure 17). At low concentra-tions of NO donors, Barraud et al. observed a decrease in biofilm biomass and an increase in planktonic bio-mass [142]. NO donors also enhanced the efficacy of antibacterial compounds, such as tobramycin, in the dispersal of P. aeruginosa biofilms.

Proteins harboring heme nitric oxide/oxygen bind-ing (HNOX) domains are known to play a role in NO sensing and modulation of intracellular c-di-GMP levels in bacteria, such as Shewanella woodyi and She-wanella oneidensis [144]. The HNOX protein forms a regulatory complex with a DGC/PDE (diguanylate cyclase/phosphodiesterase) protein. NO binding to HNOX inhibits cyclic-di-GMP production and some-times increases PDE activity, lowering cyclic-di-GMP concentrations, which causes biofilm dispersal [145]. It is known that NO induces an increase in PDE activity and as a result induces the dispersion of P. aeruginosa biofilms [146]. This is followed by a decrease in intra-cellular c-di-GMP levels [142,143]. In P. aeruginosa, the genome encodes two proteins containing an MHYT domain. MHYT domain acts as a sensor for diatomic gases such as O

2, CO or NO.

In order to identify proteins controlling c-di-GMP turnover in response to NO in P. aeruginosa, Li et al. [146] searched the Pseudomonas genome data-base for proteins containing c-di-GMP modulating activity, in particular the largely unexplored MHYT domain predicted to possess putative gas sensor func-tion. The P. aeruginosa genome encodes two proteins containing MHYT domains: the membrane-anchored proteins MucR (PA1727) and NbdA (NO-induced biofilm dispersion locus A; PA3311). NbdA is responsi-ble for the NO induced dispersion response. They con-cluded that MucR is an active PDE under planktonic growth conditions but act as a DGC in biofilms.

NO signaling is also found in Gram positive bac-teria such as B. subtilis, where it provides protec-tion against oxidative stress caused by H

2O

2. NO is

proposed to activate catalase, the H2O

2 degrading

enzyme [147]. In addition, NO suppresses the forma-tion of DNA damaging OH radicals from the oxi-dation of Fe2+ with H

2O

2 [147]. Biofilm formation in

B. subtilis is characterized by the formation of robust pellicles at the air-liquid interface and the formation of structurally complex spot colonies on agar surfaces. Schreiber et al. examined the effect of exogenously supplied NO on biofilm formation and dispersal in B. subtilis [148]. They found that NO affects biofilm dispersal of B. subtilis.

Antibiofilm surfacesAttachment of cells to surfaces is important for bio-film formation. This community of sedentary cells has become a problem in healthcare settings where biofilms are found to colonize and contaminate the surfaces of

Figure 17. NO donors.

ON

S

HN

OH

O

O

Fe

N

NN

N

O

NN C

C

C

CC

Na2+

2-

N

OO

CO2

Na

Na

+

+

+

--

HO NH

HN

OH

O

OS

NO

O

NH2

O

SNP

SNAP

GSNO

PROLI/NO

Figure 18. Ionic liquids.

NN CnH2n+1CnH2n+1

Cl-

NBr -+ +

1-alkyl-3-methyl imidazolium chloride 1-alkylquinolinium

bromide



medical implants. It has therefore become necessary to develop surfaces that can inhibit the formation and growth of biofilms. Efforts have generally been tar-geted toward the use of materials that are mostly used for medical implants.

In 2007, Chung et al. studied the effect of engi-neered microtopographies on the biofilm formation ability of S. aureus. They used engineered microto-pography Sharklet AFTM on poly (dimethyl siloxane) elastomer (PDMSe) with 2 m feature width and spacing and 3 m feature height [149]. By comparing the smooth PDMSe to the topographically modified surface, it was observed that S. aureus biofilms were inhibited on the Sharklet AFTM, recording only a 7% growth as compared with the 54% growth observed on the smooth PDMSe. The cultures were allowed to grow for 21 days, enough for S. aureus to contami-nate an implanted device in vivo. The disruption of S. aureus biofilms on this topographically modified surface did not involve the release of any bactericidal agents.

Monomeric trimethylsilane (TMS) was used to coat the surfaces of stainless steel and titanium alloy grade 5 and used to assess S. epidermidis biofilm forma-tion [150]. Ma et al. showed that TMS coated surfaces inhibited the formation of biofilms by preventing the attachment of cells. Biofilms of S. epidermidis were formed successfully on uncoated stainless steel and titanium alloy surfaces. It was also demonstrated that susceptibility of the biofilms to antibiotic treatment was significantly enhanced with TMS coating of the surfaces tested.

A different study used polystyrene coated with non-ionic surfactants containing poly (ethylene oxide), a brush coating, to study its effect on biofilm formation by S. epidermidis. The study observed that Pluronic F127 was the most active surfactant, as it achieved 90% inhibition of S. epidermidis biofilm [151]. Both studies however showed that the coated surfaces did not inhibit biofilms formed by P. aureginosa.

Ionic liquidsIonic liquids are a class of liquid salts with discrete anions and cations that can independently be modified. They are widely used in both chemistry and biology fields. Due to their flexibility, it is thought that ionic liquids could be designed to exhibit antimicrobial and antibiofilm properties [152,153]. A typical example of such class is the 1-alkylquinolinium bromide ionic liq-uids (Figure 18). These compounds have been shown to possess broad spectrum antimicrobial and antibiofilm activities. They significantly inhibited the planktonic cell growth and biofilm formation of a panel of Gram positive and Gram negative test organisms [152]. A pre-vious study also demonstrated the antibiofilm activity of a series of 1-alkyl-3-methylimidazolium chloride ionic liquids against a range of clinically relevant patho-gens [153]. Both studies observed a dependence of antib-iofilm potency on alkyl chain length, generally peaking at an alkyl chain length of 14.

ConclusionIn the last decade, there has been an explosion in the discovery of small molecules that modulate bacterial biofilm formation. The majority of the antibiofilm molecules that have been developed have either been natural product-inspired or modifications of signaling molecules that regulate biofilm formation. For example the modification of several quorum sensing molecules, which regulate biofilm formation, has resulted in modulators of biofilm formation. The biofilm inhibi-tory concentrations of the majority of the antibiofilm molecules discovered or developed to date are in the micromolar range, and hence further optimizations of these molecules to improve potency are required if any of these molecules are to become clinical candidates.

Future perspectiveOur current understanding of the molecular players of bacterial biofilm formation has increased [154] but there is still a great challenge in the development of antibiofilm drugs and to date, no antibiofilm drug has been registered and is in clinical use. This has made the treatment of biofilm-related infections very problematic. Research toward identifying antibio-film agents includes but not limited to the develop-ment of various technologies ranging from synthetic/extracted small molecules to modified surfaces. Approaches such as the modified surfaces, if success-ful, would be instrumental in combating medical and dental implant-associated biofilms. Nature continues to inspire researchers by providing unique chemical scaffolds such as the bromoageleferin and oroidin, which have been demonstrated to exhibit antibiofilm activities. Efforts could be targeted at fine-tuning such

Key term

Ionic liquids: Salts that are liquids under 100C and could even be a liquid at room temperature. The anion is poorly coordinated and thus no stable crystal is formed.



natural scaffolds to arrive at more potent antibiofilm drugs. Any potent antibiofilm agent that makes it to the drug development stage could be administered as a single drug compound to alleviate biofilm infections. Nonetheless antibiotics, which are otherwise ineffec-tive in the treatment of bacterial infections, could be combined with potent antibiofilm agents to augment the activities of the antibiotics and hence afford some leverage in the treatment of biofilm-related infections. Antibiofilm agents that can both disperse and kill bio-film bacteria could have some useful applicationse but remain rare [155,156,157].

Supplementary dataTo view the supplementary data that accompany this paper

please visit the journal website at: www.future-science.com/

doi/full/10.4155/FMC.15.7

Financial & competing interests disclosureThe authors are grateful to NSF (CBET 1264509 and CHEM

1307218) and Camille Dreyfus Foundation (Teacher-Scholar fel-

lowship to HOS) for funding. Y Zheng is supported by Kraybill

biochemistry fellowship. The authors have no other relevant

affiliations or financial involvement with any organization or

entity with a financial interest in or financial conflict with the

subject matter or materials discussed in the manuscript apart

from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

Open AccessThis work is licensed under the Creative Commons Attribu-

tion-NonCommercial 3.0 Unported License. To view a copy

of this license, visit http://creativecommons.org/licenses/by-

nc-nd/3.0/

Executive summary

Background Biofilms are defined as aggregated microorganism communities attached to surfaces and embedded in a self-

produced matrix. In the biofilm structure the cells are tolerant to harsh environmental conditions, resistant to antibiotics and

host immune systems.Biofilm inhibition by small molecules Natural compounds (isolated from plants, marine and bacteria) have served as biofilm inhibitors or as

scaffolds for the development of synthetic antibiofilm compounds.Peptides & biofilms Amyloids exist in many microorganisms and they are involved in cellcell communication and biofilm

formation. Cationic peptides are able to alter gene expression directly. D-amino acids also demonstrated the ability to inhibit biofilm formation.

Polysaccharides & biofilms Galactan can inhibit biofilm formation. Galactose metabolism plays a role in the biofilm formation in

B. subtilis. Sugar esters and a biosurfactant, produced by S. marcescens, possess antibiofilm properties.Fatty acids & biofilms Free fatty acids can affect bacterial cell membrane, especially the process of energy production. Some free

fatty acids, such as oleic acid and cis-2-decenoic acid, inhibit biofilm of some bacteria.The combination of antibiofilm molecules & traditional antibiotics The combination of QS molecule analogs (such as AI-2 analogs) together with the antibiotic gentamicin

resulted in reduction in biofilm formation.Nitric oxide & biofilm dispersal Nitric oxide induces the transition from the biofilm mode to planktonic state.Antibiofilm surfaces Various methods have been used to develop surfaces that can inhibit the formation and growth of biofilms.Ionic liquids Ionic liquids are designed to be biofilm inhibitors, such as 1-alkylquinolinium bromide ionic liquids.

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