pseudomonas aeruginosa is one of the major causes of nosocomial diseases ; it can secrete a diversity of virulence factors and in parallel forms biofilms to ensure the infection success . the production of key virulence factors in p. aeruginosa and other important pathogenic bacteria is regulated by a cell - to - cell communication mechanism known as quorum sensing ( qs ) . this mechanism enables bacteria to detect their population density through the production , release , and perception of small diffusible molecules called autoinducers and to coordinate gene expression accordingly . in p. aeruginosa , two qs systems ( las and rhl ) drive the production ( by the synthetases lasi and rhli ) and the perception ( by the transcription factors lasr and rhlr ) of the acyl - homoserine lactones ( ahls ) n-(3-oxododecanoyl)-l - homoserinelactone ( 3-oxo - c12-hsl ) and n - butanoyl - l - homoserine lactone ( c4-hsl ) , respectively . once lasr interacts with 3-oxo - c12-hsl , it induces the las system ( by increasing lasi expression ) and triggers the production of lasb elastase , lasa protease , apr alkaline protease , and exotoxin a . rhlr interacts with c4-hsl , resulting in an enhancement of the production of rhamnolipids , pyocyanin , lasb elastase , hydrogen cyanide , and cytotoxic lectins [ 3 - 5 ] . in addition , biofilm formation and maturation is also regulated by las system   and indirectly by rhl system under nutritional condition . indeed , some studies demonstrated the role of rhamnolipids in biofilm architecture and maintenance [ 10 - 12 ] . the las and the rhl systems are organized in a hierarchical manner where the las system regulates the rhl system at the transcriptional and posttranscriptional levels . in addition , p. aeruginosa releases a third intercellular signal , 2-heptyl - hydroxy-4-quinolone ( designated the pseudomonas quinolone signal ) , which interacts with the ahl systems in an intricate way   and acts as a link between the las and rhl quorum - sensing systems . since fundamental virulence processes in many pathogenic bacteria are regulated by qs systems , an interesting strategy to overcome the emergence of antibiotic - resistant microorganisms is to interfere with this cell - to - cell communication mechanism in order to attenuate their virulence . thus , medicinal plants traditionally used to treat infectious diseases should be screened , not only for their antimicrobial properties , but also for their capacity to inhibit qs mechanisms in bacteria . in this study , we investigated the qs inhibitory ( qsi ) effects of extracts from a congolese medicinal plant , cordia gilletii de wild . the root barks extracts from this plant species are used for the treatment of malaria and diarrhea ( decoction ) , for wounds and skin diseases ( topical application ) , whereas leaves decoction is used against malaria . root barks and leaves of c. gilletii were collected in kisantu area the ( democratic republic of congo ) in january 2005 , and voucher specimen has been deposited under the number br - sp.627986 at the national botanical garden of meise , belgium . powders of the two - plant parts were exhaustively and successively extracted with solvents of increasing polarity ( n - hexane , dicholoromethane , ethyl acetate and methanol ) . the evaporation of solvents in buchi rotavapor yielded crude extracts , which were dissolved in dimethyl sulfoxide ( dmso ) at a final concentration of 10 mg / ml . p. aeruginosa pao1 wild - type and reporter strains were grown in liquid lb cultures ( 5 ml ) supplemented with 50 mm 3-(n - morpholino ) propanesulfonic acid ( mops ; ph 7.0 ) at 37c supplemented with carbenicillin ( 300 g / ml ) when appropriate as described previously . for the detection of anti - qs activity , we used reporter strains including six pao1-derived strains harboring qs - related promoter - lacz fusions ( lasb - lacz ; rhla - lacz ; rhli - lacz , rhlr - lacz ; lasi - lacz and lasr - lacz ) , and pao1-derived strains harboring qs - independent acea gene ( acea - lacz ) , described previously . inhibition of pyocyanin and elastase production in p. aeruginosa pao1 wild type was assessed according to previously described procedures . briefly , p. aeruginosa pao1 were grown overnight polystyrene tube containing 5 ml of lb - mops medium ( 37c and agitation at 175 r.p.m ) . the cells were washed twice in fresh lb - mops medium , and the pellets were suspended in lb - mops medium . then , 50 l portions of the cell suspension were added to 940 ml of lb - mops , spectrometrically evaluated at 600 nm ( in order to obtain a a600 ranging between 0.020 and 0.025 , corresponding to ~10 cfu / ml ) using a spectramax m2 device ( molecular devices , california , usa ) and supplemented with 10 l of dmso ( 1% [ vol / vol ] , final concentration ) or 10 l of plant extract dissolved in dmso ( 100 g / ml , final concentration ) . after 18 h of growth , samples were taken to assess the growth ( a600 ) . after centrifugation ( 16,000 g , 5 min ) , 900 l of supernatant were mixed with 500 l of chloroform in eppendorf tube . the organic phase was transferred in a new eppendorf tube and pyocyanin was extracted with 300 l of hcl 0.2 n and quantified spectrometrically at 380 nm . lasb elastase production was assessed through the measurement of elastase activity using elastin - congo red ( a495 nm ) . the statistical significance of each test ( n = 6 ) was evaluated by conducting student s t - tests using the graphpad prism software ( graphpad software inc . pao1 reporter strains were prepared as described for pyocyanin quantification ( see previous section ) . pao1 strains ( 50 l ) were grown in 940 l of lb medium at 37c under agitation ( 175 r.p.m ) , supplemented with 10 l of plant extract or naringenin ( 4 mm , final concentration ) or dmso ( 1% [ vol / vol ] , final concentration ) and incubated for 18 h. after incubation , the cell growth was assessed as previously and the absorbance of the medium after centrifugation of the bacteria ( 16,000 g , 5 min ) was used as a blank . the sample used for cell growth assessment was used to perform the b - galactosidase assay with o - nitrophenyl - b - d - galactopyranoside as previously described . quantification of biofilm formation by p. aeruginosa pao1 was assessed according to previously described procedures . pao1 cells were incubated statically for 24 h at 37c in 24-well polystyrene plates containing biofilm broth medium ( na2hpo4 1.25 g / l , feso4.7h2o 0.0005 g / l , glucose 0.05 g / l , ( nh4)2so4 0.1 g / l , mgso4.2h2o 0.2 g / l , kh2po4 0.5 g / l ) supplemented with plant extract ( 100 g / ml ) or dmso 1% or naringenin ( 4 mm ) . after 24 h of incubation , biofilm biomass was quantified via crystal violet staining . root barks and leaves of c. gilletii were collected in kisantu area the ( democratic republic of congo ) in january 2005 , and voucher specimen has been deposited under the number br - sp.627986 at the national botanical garden of meise , belgium . powders of the two - plant parts were exhaustively and successively extracted with solvents of increasing polarity ( n - hexane , dicholoromethane , ethyl acetate and methanol ) . the evaporation of solvents in buchi rotavapor yielded crude extracts , which were dissolved in dimethyl sulfoxide ( dmso ) at a final concentration of 10 mg / ml . aeruginosa pao1 wild - type and reporter strains were grown in liquid lb cultures ( 5 ml ) supplemented with 50 mm 3-(n - morpholino ) propanesulfonic acid ( mops ; ph 7.0 ) at 37c supplemented with carbenicillin ( 300 g / ml ) when appropriate as described previously . for the detection of anti - qs activity , we used reporter strains including six pao1-derived strains harboring qs - related promoter - lacz fusions ( lasb - lacz ; rhla - lacz ; rhli - lacz , rhlr - lacz ; lasi - lacz and lasr - lacz ) , and pao1-derived strains harboring qs - independent acea gene ( acea - lacz ) , described previously . inhibition of pyocyanin and elastase production in p. aeruginosa pao1 wild type was assessed according to previously described procedures . briefly , p. aeruginosa pao1 were grown overnight polystyrene tube containing 5 ml of lb - mops medium ( 37c and agitation at 175 r.p.m ) . the cells were washed twice in fresh lb - mops medium , and the pellets were suspended in lb - mops medium . then , 50 l portions of the cell suspension were added to 940 ml of lb - mops , spectrometrically evaluated at 600 nm ( in order to obtain a a600 ranging between 0.020 and 0.025 , corresponding to ~10 cfu / ml ) using a spectramax m2 device ( molecular devices , california , usa ) and supplemented with 10 l of dmso ( 1% [ vol / vol ] , final concentration ) or 10 l of plant extract dissolved in dmso ( 100 g / ml , final concentration ) . after 18 h of growth , samples were taken to assess the growth ( a600 ) . after centrifugation ( 16,000 g , 5 min ) , 900 l of supernatant were mixed with 500 l of chloroform in eppendorf tube . the organic phase was transferred in a new eppendorf tube and pyocyanin was extracted with 300 l of hcl 0.2 n and quantified spectrometrically at 380 nm . lasb elastase production was assessed through the measurement of elastase activity using elastin - congo red ( a495 nm ) . the statistical significance of each test ( n = 6 ) was evaluated by conducting student s t - tests using the graphpad prism software ( graphpad software inc . , ca , usa ) , and a p  0.01 was considered significant . pao1 reporter strains were prepared as described for pyocyanin quantification ( see previous section ) . pao1 strains ( 50 l ) were grown in 940 l of lb medium at 37c under agitation ( 175 r.p.m ) , supplemented with 10 l of plant extract or naringenin ( 4 mm , final concentration ) or dmso ( 1% [ vol / vol ] , final concentration ) and incubated for 18 h. after incubation , the cell growth was assessed as previously and the absorbance of the medium after centrifugation of the bacteria ( 16,000 g , 5 min ) was used as a blank . the sample used for cell growth assessment was used to perform the b - galactosidase assay with o - nitrophenyl - b - d - galactopyranoside as previously described . quantification of biofilm formation by p. aeruginosa pao1 was assessed according to previously described procedures . pao1 cells were incubated statically for 24 h at 37c in 24-well polystyrene plates containing biofilm broth medium ( na2hpo4 1.25 g / l , glucose 0.05 g / l , ( nh4)2so4 0.1 g / l , mgso4.2h2o 0.2 g / l , kh2po4 0.5 g / l ) supplemented with plant extract ( 100 g / ml ) or dmso 1% or naringenin ( 4 mm ) . after 24 h of incubation , biofilm biomass was quantified via crystal violet staining . c. gilletii root barks and leaves extracts were investigated for their effect on pyocyanin production . as shown in figure 1a , all tested extracts decreased drastically pyocyanin production with no significant effect on p. aeruginosa pao1 growth when compared to the dmso control . besides , all extracts ( except root barks methanol and leaves dichloromethane extracts ) decrease significantly elastase production in pao1 although less spectacular compared to pyocyanin reduction [ figure 1b ] . in addition , no elastase - like activities ( which could interfere with the tests ) were observed when the extracts were used in bacteria - free control tests ( data not shown ) . effect of cordia gilletii root barks and leaves extracts on pyocyanin and elastase production in pseudomonas aeruginosa pao1 . ( a ) effect of c. gilletii root barks and leaves extracts on pyocyanin production in p. aeruginosa pao1 . ( b ) effect of c. gilletii root barks and leaves extracts on elastase production in p. aeruginosa pao1 ( hexane : n - hexane , dcm : dichloromethane , etoac : ethyl acetate , meoh : methanol ) . all experiments were performed in six replicates in the case of the decrease of pyocyanin and elastase production was due to the interference of c. gilletii extracts with qs mechanisms , we assessed the impact of c. gilletii extracts on qs - regulated genes lasb and rhla genes ( coding for lasb elastase and rhamnolipid , respectively ) expression . therefore , the effect of c. gilletii extracts on lasb and rhla genes expression was monitored by using two pao1 reporter strains harboring qs - related ( lasb and rhla ) promoter - lacz fusions . pao1 reporter strain harboring qs - independent acea gene ( coding for isocitrate lyase ) was used to verify that the drop in b - galactosidase activity was indeed associated with a reduction in qs - related gene expression rather to a general effect on transcription / translation mechanisms . naringenin , a flavanone , which is known to affect qs signaling in p. aeruginosa pao1 without affecting bacterial growth , was used as a positive control . as shown in figure 2 , the results highlight that rbdcm extract and leaves methanol extract , at final concentration of 100 g / ml reduce qs - regulated lasb and rhla genes expression without affecting pao1 cells growth . indeed , colony - forming unit of p. aeruginosa pao1 wild - type and reporter strains grown in the presence of extracts for 18 h were similar to those of dmso - treated cells ( data not shown ) . more interesting , rbdcm extract does not affect the expression of the control gene acea [ figure 2c ] , contrarily to the leaves methanol extract . however , effects of leaves methanol extract on qs - independent acea gene and qs - regulated ( lasb and rhla ) genes may be the results of two or more different active compounds . indeed , some compounds could do affect specifically the expression of qs - related genes and others the expression of qs - independent acea gene and/or the transcription machinery without affecting pao1 cells growth . effect of cordia gilletii extracts on quorum sensing ( qs)-regulated genes ( a : lasb ; b : rhla ) expressions , and qs - independent acea gene ( c ) in pseudomonas aeruginosa pao1 . gene expression was measured as the b - galactosidase activity of the lacz gene fusions expressed in miller units . root barks and leaves extracts were tested at 100 g / ml ( hexane : n - hexane , dcm : dichloromethane , etoac : ethyl acetate , meoh : methanol ) . dimethyl sulfoxide ( dmso ) ( 1% [ vol / vol ] , final concentration ) was used as solvent control and naringenin ( nar : 4 mm , final concentration ) as qs inhibitory control . * significance at p < 0.05 . all experiments were performed in six replicates since qs - regulated ( lasb and rhla ) genes expression is impaired by rbdcm extract of c. gillettii , we were interested in its effect on qs systems ( lasri and rhlri ) in p. aeruginosa pao1 . therefore , the effect of root barks extract was further characterized by evaluating the expression of the ahl synthetase genes lasi and rhli and the qs regulator genes lasr and rhlr . the results highlight that the rbdcm affects both qs systems ( lasri and rhlri ) [ figure 3 ] . indeed , rbdcm inhibits significantly the expression of ahl synthetase genes lasi ( 24%  5% of inhibition ) and rhli ( 52%  5% of inhibition ) , and of the qs regulator genes lasr ( 25%  3% of inhibition ) and rhlr ( 23%  4% of inhibition ) . effect of root barks dichloromethane ( rbdcm ) extract in pseudomonas aeruginosa quorum sensing ( qs ) regulator genes ( a : lasi ; b : lasr ; c : rhli ; d : rhlr ) . dimethyl sulfoxide ( 1% [ vol / vol ] , final concentration ) was used as solvent control and naringenin ( nar : 4 mm , final concentration ) as qs inhibitory control . * significance at p < 0.05 . all experiments were performed in six replicates since biofilm formation is partially controlled by qs mechanisms , the effect of rbdcm extract on p. aeruginosa pao1 biofilm formation was assessed after 24 h. noticeably , there were a significant decrease ( 21%  5% of inhibition ) in biofilm formation when strain pao1 was grown in the presence of rbdcm extract ( 100 g / ml ) compared with that of the negative control ( dmso ) [ figure 4 ] . effect of root barks dichloromethane ( rbdcm ) extract in biofilm formation by pseudomonas aeruginosa pao1 . after 24 h of static incubation , biofilm biomass was quantified by using crystal violet staining . dimethyl sulfoxide ( 1% [ vol / vol ] , fi nal concentration ) was used as solvent control and naringenin ( nar : 4 mm , final concentration ) as quorum sensing inhibitory control . * c. gilletii root barks and leaves extracts were investigated for their effect on pyocyanin production . as shown in figure 1a , all tested extracts decreased drastically pyocyanin production with no significant effect on p. aeruginosa pao1 growth when compared to the dmso control . besides , all extracts ( except root barks methanol and leaves dichloromethane extracts ) decrease significantly elastase production in pao1 although less spectacular compared to pyocyanin reduction [ figure 1b ] . in addition , no elastase - like activities ( which could interfere with the tests ) were observed when the extracts were used in bacteria - free control tests ( data not shown ) . effect of cordia gilletii root barks and leaves extracts on pyocyanin and elastase production in pseudomonas aeruginosa pao1 . ( a ) effect of c. gilletii root barks and leaves extracts on pyocyanin production in p. aeruginosa pao1 . ( b ) effect of c. gilletii root barks and leaves extracts on elastase production in p. aeruginosa pao1 ( hexane : n - hexane , dcm : dichloromethane , etoac : ethyl acetate , meoh : methanol ) . in the case of the decrease of pyocyanin and elastase production was due to the interference of c. gilletii extracts with qs mechanisms , we assessed the impact of c. gilletii extracts on qs - regulated genes lasb and rhla genes ( coding for lasb elastase and rhamnolipid , respectively ) expression . therefore , the effect of c. gilletii extracts on lasb and rhla genes expression was monitored by using two pao1 reporter strains harboring qs - related ( lasb and rhla ) promoter - lacz fusions . pao1 reporter strain harboring qs - independent acea gene ( coding for isocitrate lyase ) was used to verify that the drop in b - galactosidase activity was indeed associated with a reduction in qs - related gene expression rather to a general effect on transcription / translation mechanisms . naringenin , a flavanone , which is known to affect qs signaling in p. aeruginosa pao1 without affecting bacterial growth , was used as a positive control . as shown in figure 2 , the results highlight that rbdcm extract and leaves methanol extract , at final concentration of 100 g / ml reduce qs - regulated lasb and rhla genes expression without affecting pao1 cells growth . indeed , colony - forming unit of p. aeruginosa pao1 wild - type and reporter strains grown in the presence of extracts for 18 h were similar to those of dmso - treated cells ( data not shown ) . more interesting , rbdcm extract does not affect the expression of the control gene acea [ figure 2c ] , contrarily to the leaves methanol extract . however , effects of leaves methanol extract on qs - independent acea gene and qs - regulated ( lasb and rhla ) genes may be the results of two or more different active compounds . indeed , some compounds could do affect specifically the expression of qs - related genes and others the expression of qs - independent acea gene and/or the transcription machinery without affecting pao1 cells growth . effect of cordia gilletii extracts on quorum sensing ( qs)-regulated genes ( a : lasb ; b : rhla ) expressions , and qs - independent acea gene ( c ) in pseudomonas aeruginosa pao1 . gene expression was measured as the b - galactosidase activity of the lacz gene fusions expressed in miller units . root barks and leaves extracts were tested at 100 g / ml ( hexane : n - hexane , dcm : dichloromethane , etoac : ethyl acetate , meoh : methanol ) . dimethyl sulfoxide ( dmso ) ( 1% [ vol / vol ] , final concentration ) was used as solvent control and naringenin ( nar : 4 mm , final concentration ) as qs inhibitory control . * since qs - regulated ( lasb and rhla ) genes expression is impaired by rbdcm extract of c. gillettii , we were interested in its effect on qs systems ( lasri and rhlri ) in p. aeruginosa pao1 . therefore , the effect of root barks extract was further characterized by evaluating the expression of the ahl synthetase genes lasi and rhli and the qs regulator genes lasr and rhlr . the results highlight that the rbdcm affects both qs systems ( lasri and rhlri ) [ figure 3 ] . indeed , rbdcm inhibits significantly the expression of ahl synthetase genes lasi ( 24%  5% of inhibition ) and rhli ( 52%  5% of inhibition ) , and of the qs regulator genes lasr ( 25%  3% of inhibition ) and rhlr ( 23%  4% of inhibition ) . effect of root barks dichloromethane ( rbdcm ) extract in pseudomonas aeruginosa quorum sensing ( qs ) regulator genes ( a : lasi ; b : lasr ; c : rhli ; d : rhlr ) . dimethyl sulfoxide ( 1% [ vol / vol ] , final concentration ) was used as solvent control and naringenin ( nar : 4 mm , final concentration ) as qs inhibitory control . * significance at p < 0.05 . since biofilm formation is partially controlled by qs mechanisms , the effect of rbdcm extract on p. aeruginosa pao1 biofilm formation was assessed after 24 h. noticeably , there were a significant decrease ( 21%  5% of inhibition ) in biofilm formation when strain pao1 was grown in the presence of rbdcm extract ( 100 g / ml ) compared with that of the negative control ( dmso ) [ figure 4 ] . effect of root barks dichloromethane ( rbdcm ) extract in biofilm formation by pseudomonas aeruginosa pao1 . after 24 h of static incubation , biofilm biomass was quantified by using crystal violet staining . rbdcm extract was tested at 100 g / ml . dimethyl sulfoxide ( 1% [ vol / vol ] , fi nal concentration ) was used as solvent control and naringenin ( nar : 4 mm , final concentration ) as quorum sensing inhibitory control . * signifi cance at p < 0.05 . few studies have already been reported the anti - qs effects of plants traditionally used in the treatment of infectious diseases [ 23 - 26 ] . c. gilletii belongs to the family of boraginaceae and it is used in congolese traditional medicine . previously we have shown direct and indirect antimicrobial activities against pathogenic microorganisms . to the best of our knowledge , none of the members of this plant family has been screened so far for inhibitory effects on qs , except for an interfering effect in the vibrio fischerii bioluminescence . in the present investigation , we have shown that p. aeruginosa pao1 growth is not affected by any of the tested c. gilletii root barks or leaves extracts ( at 100 g / ml final concentration ) . besides , by using a reporter strain coupled to acea gene to evaluate the effect of the c. gilletii extracts on gene transcription machinery , we have discarded all leaves extracts as well as hexane , ethyl - acetate and methanol root barks extracts . accordingly , only dichloromethane root barks extract was found to specifically reduce in the same time qs - dependent virulence factors ( pyocyanin and elastase ) production , qs - regulated genes ( lasb and rhla ) expression as well as qs - regulatory genes , suggesting the occurrence of a tissue specific compound(s ) in c. gilletii that affect qs machinery in p. aeruginosa pao1 . however , we can not exclude that other tested extract contain qsi compounds , particularly for leaves methanol extract which inhibits transcription of the qs - regulated lasb and rhla genes and qs - independent acea gene . moreover , inhibition kinetic analysis should be led in order to detect time points in which the highest inhibition level of the qs phenotype and the qs genes could be recorded . biofilm formation in p. aeruginosa represents a protective mode of growth which may enhance bacterial survival under conditions of environmental stress . interestingly , rbdcm extract was found to specifically reduce biofilm formation by p. aeruginosa pao1 , which could be attributed to its qsi propriety . .   demonstrated that the qs dependence of biofilm formation is nutritionally conditional ( i.e. , qs systems are needed for biofilm formation in growth media with succinate as the sole carbon source but not glucose ) . accordingly , as we used glucose as the sole carbon source , we can not amputate biofilm reduction in the presence of rbdcm extract to the sole qs systems disruption . since root barks of c. gilletii are known to contain phenolic compounds , this class of molecules could represent one of the putative active compounds as some of them , catechin , naringenin   and perbergin   have already demonstrated anti - qs effect . however , qsi compounds from c. gilletii can be a new class of chemical structure compared with those flavonoids reported elsewhere and may show a different mechanism of inhibition . besides , at this stage we do not have sufficient data to speculate the quorum inhibitory mechanism and the transcriptional and/or post - transcriptional level of interference of root barks of c. gilletii . this study highlights anti - virulence propriety of c. gilletii , which could contribute to explain its efficacy in the traditional treatment of infectious diseases caused by p. aeruginosa . further investigations are needed in order to identify the chemical nature of compound(s ) responsible for these observed effects . isolated compounds will have a greater advantage for human use and search for such compounds may contribute to the prevention of bacterial diseases without the concern of antibiotic resistance . finally , we must point out that c. gilletii , although belonging to the family of boraginaceae , one of the most important botanical families of plants producing pyrrolizidine alkaloids ( pas ) , does not harbor these alkaloids in investigated root barks and leaves samples ( detection limit , 2 g of pas per gram of plant material ) , excluding thus toxicological risk due to pas and ensuring a probable safe use of this plant .
aim : the fight against infectious diseases and antimicrobial resistances needs the exploration of new active compounds with new proprieties like disrupting quorum sensing ( qs ) mechanisms , which is a cell - to - cell communication that regulates bacterial virulence factors . in this work , leaves and root barks extracts of a congolese medicinal plant , cordia gilletii , were investigated for their effect on the production of pseudomonas aeruginosa major virulence factors regulated by qs.materials and methods : the effect of c. gilletii extracts on virulence factors of p. aeruginosa pao1 was studied by the evaluation of the production of pyocyanine , elastase and biofilm ; and by the measurement of the expression of qs - related genes.results:the dichloromethane extract from root barks was found to quench the production of pyocyanin , a qs - dependent virulence factor in p. aeruginosa pao1 . 
 moreover , this extract specifically inhibits the expression of several qs - regulated genes ( i.e. lasb , rhla , lasi , lasr , rhli , and rhlr ) and reduces biofilm formation by pao1.conclusion:this study contributes to explain the efficacy of c. gilletii in the traditional treatment of infectious diseases caused by p. aeruginosa .