Halogen-furan-2(5 H )-one derivatives decrease biofilm production in Pseudomonas aeruginosa .

: Clinical evidence has shown that bacterial infections are more difficult to eradicate when forming a biofilm aggregate than when are produced by bacteria in planktonic form. Therefore, compounds that inhibit biofilm formation could be used against severe infections. It has been reported that bromo 2-(5H) furanones inhibited biofilm formation by their anti-quorum sensing properties. To determine if the 2-(5H) furanone moiety is essential to induce inhibition of biofilm formation, we evaluated ten halogen 2-(5H) furanones derivates previously synthesized. Besides evaluating the inhibition of biofilm formation, we assessed pyocyanin production, swarming motility, and transcription of essential QS genes: rsaL , rhlA , pqsA and phz1 genes. Our results showed that although three bromo-furan-2(5H)-one-type derivatives (A1-A3) and two bromo-4-(phenylamino)-furan-2(5H)-one-type compounds (B2 and B6) inhibited the biofilm formation in both P. aeruginosa PA14 (reference) and PA64 (drug-resistant) strains only the furanones A1-A3 were efficient to inhibit QSS.


Introduction
Multiple reports in the specialized literature indicate that bacterial multidrug resistance (MDR) is an emerging problem of universal dimensions' public health. To find new therapies against these resistance microorganisms, the World Health Organization (WHO) announced a list of priority microbes that is urgent to prevent due to high pathogenicity. One of these is Pseudomonas aeruginosa [1]. P. aeruginosa is the most common Gram-negative pathogen causing nosocomial pneumonia. For example, in Mexico, from 2007 to 2013, forty-six nosocomial infections produced by P. aeruginosa were detected at a pediatric hospital with a significant mortality rate (17.39%) [2]. depend on population density. Nowadays, three well-organized and hierarchy established quorum sensing pathways have been reported in P. aeruginosa, two LuxI/R-type systems (LasI/R and RhlI/R) and pseudomonas quinolone signal (PQS). It is generally accepted that LasI/R is at the top of the hierarchy in the QSS cascade except in conditions of limiting phosphate concentrations [3]. QSS is characterized by the conjugation of gene regulation and the synthesis of diffusible chemical signals called autoinducers (AIs). P. aeruginosa synthesized acyl-homoserine lactone (AHL) derivatives as AIs. When the cell density rises, reaching a certain concentration threshold, AI concentration increases, allowing the interaction between AHLs and the LuxR-type receptor. This complex binds to the corresponding promoter region and turn on the expression of several genes encoding diverse virulence factors. Thus, interrupting P. aeruginosa QSS could decrease bacterial virulence. It could become a new therapy to fight MDR bacteria. Furthermore, because this therapy does not exert a direct selection pressure, therefore, it may have a low probability of developing resistance [4].
It has been published that those infections caused by P. aeruginosa in its single, freefloating planktonic form can be eradicated with adequate antibiotic treatment. However, the infection is complicated to eradicate when this bacterium forms part of an aggregate known as biofilm [5]. Secreted proteins, polysaccharides, nucleic acids, and cellular debris are fundamental parts of bacterial biofilms. This aggregate could be attached to abiotic or biotic surfaces. The immersion of the bacteria in the biofilm gives it a better resistance against the antibiotics and the defenses of the infected host's immune system. The relationship between QSS and biofilm formation arises because lasI, which encodes for the synthetase responsible for 3O-C12-HSL production, is critical for mature biofilm formation in P. aeruginosa PAO1 [6].
Several compounds have been found which disrupt the quorum-sensing system. Historically, the first inhibitors of QSS have been synthesized using the 2-furanone structural template, the basic unit of acylated homoserine lactones (AHLs). It has been reported that AHLs controls both the virulence genes of bacteria and the comportment of host cells. For example, 3-oxo-C12 HSL, besides its QS regulation, is also responsible for the immunomodulation of the host immune system [7]. Among the anti-QSS furanones, C30 (5Z-4bromo-5-(bromomethylidene) furan-2-one) is one of the more relevant [8,9]. C30 possess a compelling profile against several virulence factors in such a way that it has become the usually reference compound in evaluating new anti-QSS compounds [8]. Besides, in vivo studies, C30 diminishes the bacterial population in pulmonary infection in mice [9].
On the other hand, recently, there has been an interest in evaluating anticancer drugs as antibacterial compounds [10]. So, we decided to evaluate two series of cytotoxic halogen-furan-2[5H]-one derivative [11] as an inhibitor of Pseudomonas aeruginosa QSS. The structural diversity of the different derivatives, together with the results of their biological activities, will allow the finding of critical structural components for the inhibition of virulence factors.
2.1 Growth of PA14 and INP64 P. aeruginosa strains in the presence of furanones Our results showed that, in PA14 bacteria growth, is slightly diminished by A3, although it was not statically significant. In the PA64 strain, the furanones did not inhibit the growth of the bacteria (data not shown). Furanones A1-A3 were evaluated at 100 µM, while B1-B7 furanones were assessed at 1 mM.

Biofilm inhibition.
Our findings showed that in P. aeruginosa PA14 strain, A1 at 50 and 100 µM doses decreased biofilm formation. However, it was not statistically significant. Unexpectedly, furanone A2 became more active at 50 µM dose than at 100 µM dose, achieving inhibition of approximately 50% in biofilm formation. A3 reduces the biofilm by 50% at 100 µM dose. Concerning the derivatives of the bromo-4-(phenylamino)-furan-2 (5H)-one type, the furanones B2 and B6 showed biofilm inhibition from the 250 µM dose. It should be noted that the inhibitory activities of type B furanone are shown at higher doses than those shown for type A (Figure 3). On the other hand, our results indicated that in P. aeruginosa INP64, drug-resistant strain, A1 and A3 inhibit biofilm formation from 10 µM dose, while furanone A2 did it from 50 µM (Figure 4a). In contrast, B2 and B6 inhibit biofilm formation from 500 µM ( Figure 4b). Again, note the difference between the small doses of active furanones of type A and those of type B active furanones.

Pyocyanin production.
The A1-A3 furanones inhibited the pyocyanin production on the P. aeruginosa PA14 strain. In general, these furanones showed a decrease from 35% at 10 µM to 50% at 100 µM doses. On the other hand, group B furanones did not inhibit the pyocyanin generation (data not shown) (Fig. 5). In the INP64 strain, the furanones A1-A3 decreased the pyocyanin production from 10 to 100 M doses (Fig. 6). In this strain, the furanones B type did not reduce the pyocyanin production (data not shown).

Rhamnolipids production
The results showed that in the PA14 strain, A1, A2 and A3 at 100 µM, while B2 and B6 at 1 mM decrease rhamnolipids production. However, in the INP64 strain, there have not been results because in cultures of 24 h rhamnolipids have not been detected. Figure 6. Effect of different furanones on rhamnolipids production of P. aeruginosa PA14 strain. Bacteria were incubated in liquid LB medium at 37°C in the presence of furanones for 18 h. Later, the rhamnolipids production was quantified with orcinol. NT: not treatment, D: dimethyl sulfoxide, C30: furanone C30. * P≤0.05, ** P≤0.005, *** P≤0.001.
Our results indicate that A1, A2, A3, B2 and B6 inhibited the biofilm formation in both P. aeruginosa PA14 (reference) and INP64 (drug-resistant) strains. However, A1-A3 inhibited biofilm production, in both P. aeruginosa strains, with one order of magnitude less of concentration than B2 and B6, furanones. For example, A3, in the PA 14 reference strain, decreases biofilm formation 50% at 100 mM, while B2 inhibits approximately 60% at a dose of 1000 M. The results showed that in the PA14 strain, A1, A2, A3 at 100 µM dose decrease rhamnolipids production. At the same time, B2 and B6 decreased it at 1 mM doses.
One point to considerer is that furanones of B series did not inhibit pyocyanin production and swarming motility in both P. aeruginosa strains evaluated. These results suggest that furanones of type B inhibit biofilm formation and rhamnolipids production in the strains of P. aeruginosa by a different mechanism than QSS, which could explain the high doses used compared to those of A1-A3. This proposal is supported by the fact that the furanones of the B series had no activity on the transcription of the rsal, rhlA, pqsA and phz genes, all involved in QSS.
A feature that attracts attention is that the A2 and A3 did not reduce the transcription of rsaL as A1 did. The rasL gene is located between the lasR and lasI genes. Overexpression of rsaL in P. aeruginosa PA01 resulted in a 20-fold reduction of 3-O-C12-HSL production [12]. Conversely, 3-O-C12-HSL production is dramatically enhanced in a rsaL mutant. Unexpectedly, the rsaL is among the genes most strongly activated by the LasR-3-OC12-HSL complex [13]. Therefore, the rsaL gene could be considered a repressor of QSS. It has been reported that due to their vicinity RsaL and LasR binding sites, these two proteins compete for attaching to the rsaL-lasI bidirectional promoter. Consequently, LasR would triggers lasI transcription by outcompeting the transcriptional repressor RsaL at high cell densities when the 3OC12-HSL levels reach a certain threshold. The fact that rsaL expression itself is also dependent upon LasR adds further complexity to this regulatory mechanism.
In our case, the administration of A1-A3 inhibited biofilm formation and reduced pyocyanin production and swarming both virulence factors governed by QSS. These results indicated that the inhibition of rsaL gene transcription by A1 did not affect the expression of virulence factors.
Considering that biofilm formation is a multifactorial development involving processes, such as producing exopolysaccharides that QS does not regulate, furanones B1and B6 may affect biofilm production without affecting the QS response [14]. In the case of the rhamnolipids production, the rhlA promoter expression is dependent on RhlR, and if the expression of this promoter is not decreased, but the production of rhamnolipids does a decrease in the presence of furanones B1-B7, it may be that because these compounds affected the manufacture of the rhamnolipids by other mechanism [15] Even though all the synthesized derivatives share a base system of 2-(5H) furanone core and halogen atoms, the reactivity that gives them the size and conformation of their structures makes only A1-A3 show anti-QSS.

Strains and compounds.
The PA14 strain were kindly provided by Dr. Ausubel and were used as reference strain and INP64 strain was provided by Dr. Rafael Coria Jimenez of National Institute of Pediatrics. The bacteria were kept in glycerol and frozen at -70°C. The furanones were previously synthesized as described [11] (Table 1). The furanones were dissolved in DMSO at a stock of 1 M and stored at -20°C.

Bacteria growth in the presence of furanones.
Overnight of bacteria was diluted in fresh LB medium at OD600= 0.05. The furanones were added, at beginning of culture, in different concentrations: 10, 50, 100, 250, 500 and 1000 µM. The cultures were incubated for 18 h at 37°C/200 rpm. At 18 h samples were taken to measure growth on a spectrophotometer at OD 600.
Overnight of bacteria was diluted in 5 ml of fresh LB medium at OD 600=0.05. The furanones were added at beginning of culture in different concentrations: 10, 50, 100, 250, 500 and 1000 µM. The cultures were incubated for 18 at 37°C/200 rpm. Later, pyocyanin was extracted and quantified as reported previously [16]. Briefly, one milliliter of culture was centrifuged, and supernatant was used for extraction. Pyocyanin was extracted with chloroform and HCl and quantified on a spectrophotometer OD520 using HCl 0.2 N as blank.

P. aeruginosa biofilm formation.
Overnight of bacteria was diluted in 2 ml of fresh LB medium at OD600 =0.05 in tubes and incubated for 18 h at 37°C/200rpm. Later, the biofilm formation was quantified as reported previously [17] with slightly modifications. Briefly, the culture was discarded, and the tube was washed 2 times with water to remove the remaining planktonic cells. Subsequently 2.5 ml of violet crystal dye at 0.1% was added and left for 15 min. The excess of dye was removed rinsing with distilled water. Later, 3 ml of ethanol 70% was added and left for 15 min. The violet crystal concentration was measured in a spectrophotometer OD570 using EtOH 70% as blank.

Swarming motility.
Overnight of bacteria was diluted in 1 ml of fresh LB medium at OD600 =0.05 and were inoculated on BM2 medium plates (K2HPO4 62 mM, MgSO4 2 mM, FeSO4 10 µM, glucose 0.4%, casamino acids 0.1% and agar 0.5%) [18]. Furanones A1, A2 y A3 were separately added at 100 µM and B2, B6, C1 and C7 at 1000 µM. The plates were incubated at 37°C during 18 h. Later, the plates were photographed in Gel Logic 200 Imaging System by Kodak. The measure of swarming area was performed in Molecular Imaging Software v. 4.0.3 by Kodak. 4.6 Rhamnolipids production. The PA14 strain was incubated in liquid LB medium. The rhamnolipids production was quantified as reported previously [19] with some modifications. Briefly, cultures were centrifuged to remove bacteria and supernatant was used to extract rhamnolipids. The extraction was performed with diethyl ether (1:1) twice, let dry at room temperature. Rhamnolipids were dissolved in 100 µl of distilled water and added 900 µl of orcinol 1.6% (dissolved in 60% H2SO4). A standard curve of rhamnose was used to quantify rhamnolipids (1µg of rhamnose equals to 2.5 µg of rhamnolipids).