This article was downloaded by: [University of Saskatchewan Library] On: 13 September 2012, At: 03:05 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Biofouling: The Journal of Bioadhesion and Biofilm Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gbif20 N-acyl homoserine lactone mediated interspecies interactions between A. baumannii and P. aeruginosa Nidhi Bhargava a , Prince Sharma b & Neena Capalash a a Department of Biotechnology, Panjab University, Chandigarh, 160014, India b Department of Microbiology, Panjab University, Chandigarh, 160014, India Version of record first published: 06 Aug 2012. To cite this article: Nidhi Bhargava, Prince Sharma & Neena Capalash (2012): N-acyl homoserine lactone mediated interspecies interactions between A. baumannii and P. aeruginosa , Biofouling: The Journal of Bioadhesion and Biofilm Research, 28:8, 813-822 To link to this article: http://dx.doi.org/10.1080/08927014.2012.714372 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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This article was downloaded by: [University of Saskatchewan Library]On: 13 September 2012, At: 03:05Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
Biofouling: The Journal of Bioadhesion and BiofilmResearchPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/gbif20
N-acyl homoserine lactone mediated interspeciesinteractions between A. baumannii and P. aeruginosaNidhi Bhargava a , Prince Sharma b & Neena Capalash aa Department of Biotechnology, Panjab University, Chandigarh, 160014, Indiab Department of Microbiology, Panjab University, Chandigarh, 160014, India
Version of record first published: 06 Aug 2012.
To cite this article: Nidhi Bhargava, Prince Sharma & Neena Capalash (2012): N-acyl homoserine lactone mediatedinterspecies interactions between A. baumannii and P. aeruginosa , Biofouling: The Journal of Bioadhesion and BiofilmResearch, 28:8, 813-822
To link to this article: http://dx.doi.org/10.1080/08927014.2012.714372
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
N-acyl homoserine lactone mediated interspecies interactions between A. baumannii andP. aeruginosa
Nidhi Bhargavaa, Prince Sharmab and Neena Capalasha*
aDepartment of Biotechnology, Panjab University, Chandigarh 160014, India; bDepartment of Microbiology, Panjab University,Chandigarh 160014, India
(Received 22 February 2012; final version received 16 July 2012)
Acinetobacter baumannii and Pseudomonas aeruginosa are pathogens capable of colonizing the same infection sitesand employing N-acyl homoserine lactone (AHL) based quorum-sensing systems to co-ordinate biofilm formation.Hence, the effect of P. aeruginosa AHLs on biofilm formation by A. baumannii and vice versa were investigated usingthe biofilm impaired quorum sensing mutants, A. baumannii M2 (abaI::Km) and P. aeruginosa PAO-JP2.Complementing the mutants with heterologous, extracted and pure AHLs increased biofilm mass significantly. Thesurface area coverage and biovolume also increased significantly as observed by confocal scanning laser microscopywhich corroborated scanning electron microscope analysis. Autoinducer synthase gene promoters of A. baumannii,PabaI-lacZ, and P. aeruginosa, PlasI-lacZ, were induced (p 5 0.05) by heterologous AHLs. Growth of A. baumanniiwas not inhibited by pyocyanin of P. aeruginosa which may allow their co-existence and interaction in the clinicalsetting, thereby affecting the severity of combined infections and therapeutic measures to control them.
Acinetobacter baumannii, Enterococcus faecium,Staphylococcus aureus, Klebsiella pneumoniae, Pseudo-monas aeruginosa and Enterobacter pneumoniaegrouped as ESKAPE, are the top six organismsresponsible for nosocomial infections (Sandiumengeet al. 2011). A. baumannii, is one of the most difficultpathogens to control and treat (Fishbain and Peleg2010) as it causes bacteremia, nosocomial pneumonia,urinary tract infections, secondary meningitis andsuper-infections in burn patients with varying fre-quency of occurrence (Gaynes and Edwards 2005;Adams et al. 2011). Its ability to form a biofilm helps itto exist in the hospital environment and successfullyescape the effect of antibacterial agents (Tomaras et al.2003; Nucleo et al. 2009).
A. baumannii isolates obtained from multiple sitesof surgical patients were found to be associated withother pathogens as co-infecting organisms (Dent et al.2011). Comparative genomics analysis of A. baumanniireveals that it shares 65% orthologs with P. aeruginosawhich is a successful opportunistic pathogen (Gospo-darek et al. 2009). Both the organisms exhibit quorumsensing which involves acyl homoserine lactone(AHL) – mediated regulation of virulence geneexpression. In P. aeruginosa, quorum sensing regulates
the expression of many secreted virulence factors andbiofilm development (Uroz et al. 2009). In A.baumannii, biofilm development (Niu et al. 2008) andmotility (Clemmer et al. 2011) have been shown to beregulated by a quorum sensing system.
The two AHL-dependent quorum sensing systemsdeciphered in P. aeruginosa are lasI-R and rhlI-R.LasR transcriptional regulator protein responds to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12HSL) generated by lasI and RhlR transcriptionalregulator responds best to rhlI generated signal N-butanoyl-L-homoserine lactone (C4-HSL). The overallregulatory quorum sensing cascade is controlled byLasR-I system (Riedel et al. 2001). Besides AHLmolecules, quorum sensing in P. aeruginosa is alsomediated by Pseudomonas quinolone system (PQS)which participates in biofilm maturation (Diggle et al.2007). A. baumannii has only one such quorum sensingsystem known which involves AbaR transcriptionalactivator that forms a complex with abaI generatedsignal N-(3-hydroxydodecanoyl)-L-homoserine lac-tone (3-OH-C12 HSL) and regulates transcription ofvarious genes (Niu et al. 2008).
Both A. baumannii and P. aeruginosa are associatedwith upper respiratory tract colonization and cysticfibrosis (Riedel et al. 2001; Wagner and Iglewski 2008;
Rogers et al. 2010). Co-infection with both mayincrease the severity and management of disease.Interactions in mixed species biofilms result in in-creased resistance to antibiotics comparative to pureculture biofilms (Burmølle et al. 2006). In a case studyon multidrug resistant A. baumannii in surgicalpatients, co-infection with other organisms, usuallyS. aureus or P. aeruginosa, was identified. Co-infectionwith a non-A. baumannii organism did not significantlyaffect mortality (p ¼ 0.75). However the only organismthat approached significance (p ¼ 0.07) with respect tomortality was P. aeruginosa (Dent et al. 2011). AHLbased one way communication between P. aeruginosaand Burkholderia cepacia co-existing at the sameinfection site has been shown in a mouse model(Riedel et al. 2001). In the present study, the effect ofAHL based interaction between A. baumannii and P.aeruginosa on biofilm formation was studied usingquorum sensing mutants.
Materials and methods
Organisms and culture conditions
Strains used in this study are listed in Table 1. Forvirulence assays P. aeruginosa PAO1 and PAO-JP2were grown in peptone water (2% w/v peptone, pH7.0) as it resulted in better expression of extracellularvirulence factors. All other organisms were grown inLuria Bertani broth at 378C.
Cross streak inhibition assay
P. aeruginosa was spread on one half of a LB agarplate. A. baumannii was streaked perpendicular to itand incubated at 378C for 16 h (Tomlin et al. 2001).
Planktonic co-culture
A. baumannii and P. aeruginosa PAO1 were co-cultured in 1:1 ratio in LB at 378C, 160 rpm for24 h. Viable plate counting was done to determine thegrowth of individual species in co-culture on LB platewith amikacin (100 mg ml71) and Pseudomonas agarwith 0.03% cetrimide.
Effect of pyocyanin on growth of A. baumannii
Pyocyanin was extracted from 50 ml of cell freesupernatant (CFS) of P. aeruginosa PAO1, grown for16 h in LB at 378C with an equal volume of chloro-form (Tomlin et al. 2001). The extracted fraction waslyophilized and dissolved in 50 ml of DMSO. LB agarplates were overlaid with soft agar (0.75%, w/v)containing A. baumannii M2 and extracted pyocyanin(2–20 mg) was added into the wells made in overlaidagar. Plates were incubated at 378C for 16 h to checkgrowth inhibition (Baron and Rowe 1981). Tobramy-cin (10 mg) was used as positive control and DMSO asnegative control.
Biofilm development and quantification
One ml of LB, containing glycerol (1% v/v), wasinoculated with 50 ml of overnight pure culture (26108
cfu ml71) in polystyrene micro-centrifuge tubes.Biofilm was statically developed for 5 days at 378C.The tubes were washed with phosphate buffered saline(PBS, 10 mM) thrice to remove the loosely adheredcells. The remaining bacterial cells on the walls andbottom of the tubes were stained with crystal violet(0.05%, w/v) for 30 min and the excess stain waswashed off with PBS. The tubes were air dried, and thedye bound to the adherent cells was re-solubilized with1 ml of 33% (v/v) glacial acetic acid. The absorbancewas measured at 570 nm which was indicative of thebiofilm mass (Figure S1) (Zeng et al. 2008). [Supple-mentary material is available via a multimedia link onthe online article webpage.] A. baumannii M2 and P.aeruginosa PAO1, biofilms were also developed in LBmedium (26) conditioned with an equal amount offilter sterilized autologous or heterologous CFS. Heattreated CFS (658C for 15 min) was added as control.
Biofilms of the mutant strains were developed inLB medium as explained above after supplementingwith 20 mM (0.2 ml from a stock of 100 mM) ofeither N-butanoyl-L-homoserine lactone (C4 HSL),N-hexanoyl-L-homoserine lactone (C6 HSL), N-octa-noyl-L-homoserine lactone (C8 HSL), N-(3-oxodode-canoyl)-L-homoserine lactone (3-oxo-C12 HSL), N-(3-hydroxydodecanoyl)-L-homoserine lactone (3-OH-
Table 1. A. baumannii and P. aeruginosa strains.
Strain Characteristics Reference
A. baumannii M2 Clinical isolate Niu et al. (2008)A. baumannii M2 (abaI::Km) abaI mutant of M2 strain; KanR
A. baumannii M2(PabaI-lacZ) lac Z expressed under abaI promoterP. aeruginosa PAO1 Wild type Iglewski et al. (1997)P. aeruginosa PAO-JP2 lasI rhlI mutant of PAO1; HgR TcR Rumbaugh et al. (1999)E. coli DH5 a (pSC11 þ pJN105L) (i) pSC11 encoding PlasI-lacZ reporter;
ApR and (ii) pJN105L encoding lasRexpression; GmR
Chugani et al. (2001)Lee et al. (2006)
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C12 HSL), 2-heptyl-3-hydroxy-4-quinolone (PQS) orAHLs extracted from CFS of A. buamannii M2 and P.aeruginosa PAO1 with acidified ethyl acetate (0.01% v/v glacial acetic acid) and dissolved in dimethylforma-mide (DMF) (Shaw et al. 1997). C4, C6, C8, 3-oxo-C12 (Cayman Chemicals) and 3-OH-C12 HSLs(Sigma) were dissolved in DMF and PQS (Sigma) indimethyl sulfoxide (DMSO).
Induction of autoinducer synthase gene promoter byexogenous AHLs
A. baumannii M2 (PabaI-lacZ) and E. coli DH5a(pSC11 þ pJN105L) reporter strains were grown inLB for 16 h at 378C in the presence of ethyl acetateextracted AHLs from CFS of A. baumannii M2 and P.aeruginosa PAO1. b-Galactosidase activity was deter-mined according to Miller (1972) using ortho-nitro-phenyl-b-D-galactopyranoside (Sigma) as thesubstrate. The ethyl acetate extract of LB was usedas a control.
Microscopy of biofilms
The optical distribution and architecture of biofilmsformed by A. baumannii M2 (abaI::Km) and P.aeruginosa PAO-JP2 in response to exogenous ex-tracted AHLs (autologous and heterologous) werestudied by scanning electron microscopy (SEM). Fiveday old static biofilms were developed on the surface ofa glass cover slip immersed in medium supplementedwith extracted autologous and heterologous AHLs in a35 mm Petri dish and incubated at 378C. The cover-slips were washed thrice with 10 mM phosphatebuffered saline (PBS, pH 7.0) and placed in 2.5%(v/v) glutaraldehyde for 2 h. The samples were thendehydrated through a series of 10 min ethanol rinses(30, 50, 70 and 95%) followed by six washes of 10 mineach in absolute ethanol (Fratesi et al. 2004). Thecoverslips were then cut into 0.5 cm60.5 cm squares,affixed to SEM stubs, coated with gold and viewed at avoltage of 20 KV (Jeol JSM 6100).
Confocal laser scanning microscopy (CLSM) of 5-day old biofilms was carried out with Carl Zeiss LSM510 microscope. The coverslip was washed thrice with10 mM PBS (pH 7.0) to remove the weakly adheredcells and biofilm was stained with FITC (10 mg ml71 inDMF) for 2 h in dark followed by three washings with10 mM PBS (pH 7.0) (Jefferson et al. 2005). Imageswere recorded at 488 nm (argon lazer) excitation and530 nm (long pass filter set) emission and examinedwith 636 oil immersion objective lens. Three imagesfrom three independent biofilms were analyzed usingCOMSTAT software (Heydorn et al. 2000) to quantifythe bio-volume which provided an estimate of the
biomass in the biofilm. Digital image analysis ofoptical thin sections viewed by CLSM was performedwith open-source software packages viz. Zeiss LSMImage Browser Version 4.2.0.121 and ImageJ 1.44p.
Effect of autologous and heterologous AHLs onvirulence factors of P. aeruginosa
P. aeruginosa PAO-JP2 was grown in 2.0 ml ofpeptone water at 378C for 24 h in the presence ofAHLs extracted with ethyl acetate from CFS of a2.0 ml culture of A. baumannii M2 or P. aeruginosaPAO1 grown under similar conditions to provide anAHL concentration equivalent to that produced by thewild type strain used for comparison. Pyocyanin wasextracted from CFS with chloroform in the ratio of3:2, re-extracted with an equal volume of 0.2 N HCland quantified colorimetrically by taking the ratioAb490/OD600 and expressed as relative pyocyaninproduction (Essar et al. 1990). Protease activity wasmeasured as described by Kessler et al. (1982). TheCFS was incubated with azocasein (2% in 50 mMphosphate buffer, pH 7.0) in a 1:1 ratio for 1 h at 378C.The reaction was stopped with 500 ml of 10%trichloroacetic acid and centrifuged at 60006g for5 min. The absorbance (Ab520) of the supernatant wasmeasured and relative protease activity was calculatedas Ab520/OD600 (Riedel et al. 2001).
Statistical analysis
Data were expressed as means + standard deviation(SD), and all experiments were repeated at least threetimes in triplicate. The Students t-test was used todetermine the significance, and p � 0.05 was consid-ered significant.
Results
A cross streak assay showed that P. aeruginosa PAO1and A. baumannii M2 could coexist without anyinhibitory effect on each other (Figure 1a) which wasfurther supported by recovery of A. baumannii and P.aeruginosa from planktonic co-culture. Both thecultures followed a similar growth pattern over 24 hof co-culturing (Figure 1b). It was also observed thatA. baumannii M2 was not inhibited by antibacterialactivity of pyocyanin produced by P. aeruginosa PAO1when checked by well diffusion assay (Figure S2)[Supplementary material is available via a multimedialink on the online article webpage.].
A. baumannii M2 formed biofilm, albeit 2.0 fold(p 5 0.01) less than P. aeruginosa PAO1. Mutantstrains of A. baumannii M2 (abaI::Km) and P.aeruginosa PAO-JP2 were impaired in their biofilm
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formation and formed 2.4 (p 5 0.01) and 2 (p 5 0.01)fold less biofilm compared with their wild types(Figure 2).
The biofilm mass of A. baumannii M2 (abaI::Km)increased significantly by 8.4 (p 5 0.001) and 7.4 fold(p ¼ 0.001) when grown in medium conditioned withCFS from A. baumannii M2 and P. aeruginosa PAO1,respectively, but no effect on the biofilm was observed(Figure 3) when grown in medium conditionedwith either CFS of P. aeruginosa PAO-JP2 or heatinactivated CFS (autologous and heterologous), in-dicating that the component(s) responsible for restor-ing the impaired biofilm of the mutant was present inthe CFS of the wild type strains only and was heatsensitive. This also showed that these results were notdue to an increase in the nutrients that might bepresent in the CFS.
To check if the increase in the biofilm mass of themutant was in response to AHLs present in thesupernatant, biofilms were formed in the presence of
AHLs extracted from CFS of overnight grown culturesof P. aeruginosa PAO1 and A. baumannii M2.Complementation of A. baumannii M2 (abaI::Km)with CFS extracted AHLs of P. aeruginosa PAO1showed a 1.8 fold (p � 0.05) increase in biofilmformation while PAO-JP2 biofilm increased by 1.5fold (p 5 0.05) in the presence of AHLs extractedfrom A. baumannii M2 CFS (Figure 4a).
A. baumannii M2 (abaI::Km) grown in the presenceof pure AHLs ie C8, 3-oxo-C12 and 3-OH-C12 HSL(20 mM) showed a significant increase in biofilmformation by 1.4 (p 5 0.05), 1.6 fold (p 5 0.05) and2.0 fold (p ¼ 0.01), respectively (Figure 4b). Combina-tions of C6 þ C8 HSLs (10 mM each) and C4 þ 3-oxo-C12 HSLs (10 mM each) also showed significant
Figure 1. (a) Cross-streak inhibition assay showing growthof A. baumannii M2 (three parallel streaks on right) alongwith P. aeruginosa PAO1 (left). (b) Planktonic co-culture.Data points represent mean CFU ml71 of P. aeruginosa andA. baumannii at different time intervals with a final count at24 h.
Figure 2. Biofilm formation on polystyrene by wild type A.baumannii M2 and P. aeruginosa PAO1 and their quorumsensing mutants, M2 (abaI::Km) and PAO-JP2. Bars ¼ themeans of three sets; * ¼ significant differences (*p-value � 0.01, t test) between the means.
Figure 3. Biofilm formation on polystyrene by mutant A.baumannii M2 (abaI::Km) in conditioned medium. CFS M2,CFS PAO1 and CFS PAO-JP2 are cell free supernatants ofA. baumannii M2, P. aeruginosa PAO1 and P. aeruginosaPAO-JP2, h.t.CFS is heat treated CFS. Bars ¼ the means ofthree experiments; * ¼ significant differences (*p-value �0.001, t test) between the means of biofilm formed inconditioned and unconditioned medium.
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increases of 2.5 (p ¼ 0.01) and 2.8 fold (p 5 0.01)respectively. Long chain AHLs (�C8) were found tobe more effective in restoration of biofilm formation byA. baumannii M2 (abaI::Km) while addition of shortchain AHLs (C4 and C6) and PQS quorum sensingsignal molecules in the growth medium did notincrease the biofilm formed by A. baumannii M2(abaI::Km) (data not shown).
Addition of C4 and 3-oxo-C12 HSL (autologousAHLs) increased JP2 biofilm by 1.2 fold (p 5 0.05)and 1.6 fold (p 5 0.05), respectively, whereas 3-OH-C12 HSL (heterologous AHL) resulted in a 1.3 fold(p 5 0.05) increase while a combination of C4 and 3-oxo-C12 HSL (10 mM each) increased biofilm forma-tion by 1.9 fold (p 5 0.01) (Figure 4c).
The effect of heterologous AHLs on the autoindu-cer synthase gene promoter of A. baumannii M2 and P.aeruginosa PAO1 was also studied. The expression ofPabaI-lacZ in the presence of AHLs extracted from A.baumannii M2 and P. aeruginosa PAO1 CFS, increasedby 1.6 (p 5 0.05) and 3.3 (p � 0.01) fold, respectively,over the control. PlasI-lacZ expression also increased by1.7 fold (p � 0.001) with both heterologous andautologous extracted AHLs (Figure 5).
The architecture of the biofilm formed by mutantstrains of A. baumannii M2 (abaI::Km) and P.aeruginosa PAO-JP2 in response to autologous andheterologous AHLs was studied by SEM. The mutantstrains of A. baumannii M2 (abaI::Km) attached to thesubstratum and appeared as single or scattered groupsof cells (Figure 6.1b) while A. baumannii M2 showedlarger area coverage and more cell to cell contact(Figure 6.1a). Supplementation of autologous AHLsto A. baumannii M2(abaI::Km) resulted in the
formation of a mat like structure with increased cellto cell contact (Figure 6.1c). In response to hetero-logous AHLs A. baumannii M2 (abaI::Km) formedbiofilm with increased surface area coverage havingthree dimensionally stacked cells which is a character-istic of biofilms in the later stages of development(Figure 6.1d).
P. aeruginosa PAO-JP2 also formed an impairedbiofilm (Figure 6.2b) as compared to its wild type(Figure 6.2a). Supplementation of the medium withautologous and heterologous AHLs resulted in biofilmthat was not as well structured as its wild type but
Figure 5. Response of lasI and abaI synthase genepromoters to autologous and heterologous AHL extractedfrom A. baumannii M2 and P. aeruginosa PAO1. R1 is M2(PabaI-lacZ) and R2 is E. coli (pJN105L þ pSC11). {Relativeexpression 1 ¼ 0.93 MU for R1 and 64.10 MU for R2.Bars ¼ the means of three readings; * ¼ significantdifferences (*p-value � 0.05; **p-value � 0.01; ***p-value � 0.001, t test) between the means of b-galactosidaseactivity of cultures grown in the presence and absence ofAHLs.
Figure 4. Biofilm on polystyrene in LB supplemented with extracted and pure AHLs. (a) A. baumannii M2 (abaI::Km) and P.aeruginosa PAO-JP2 grown in the presence of AHLs extracted from CFS of heterologous wild type organisms. Pure AHLsindividually (20 mM) and in combination (10 mM each) were added to (b) A. baumannii (abaI::Km), and (c) P. aeruginosa PAO-JP2. Bars ¼ the means for three experiments. * ¼ significant differences (*p-value � 0.05; **p-value � 0.01, t test) between themeans of biofilm developed in the presence and absence of AHLs. Ethyl acetate extracted LB and DMF alone were used ascontrols.
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showed enhanced aggregation and cell to cell contact,with greater surface area coverage of the substratumthan the mutant strain (Figure 6.2c, d).
CLSM scans corroborated the SEM observationsand clearly showed the conversion of scantily growingpatches of FITC stained aggregated biofilm cells of themutant strains to a full grown mat covering a largerarea in response to the heterologous AHLs (Figure 7a–d). A significant increase of 3.7 and 2.6 fold (p 5 0.05)in the surface area coverage (%) and biovolume(mm3 mm72), respectively, of A. baumannii M2 (abaI::Km) was observed when it was supplemented withAHLs extracted from P. aeruginosa PAO1. Also,increases of 7.1 (p 5 0.001) and 1.9 (p 5 0.05) foldin the surface area coverage and biovolume, respec-tively, were observed in P. aeruginosa PAO-JP2 biofilmin the presence of AHLs from A. baumannii M2. Thebiofilm formed by P. aeruginosa PAO-JP2 had a 6 fold(p 5 0.001) greater roughness co-efficient compared toA. baumannii M2 (abaI::Km). Supplementation ofheterologous AHLs to A. baumannii M2 (abaI::Km)and P. aeruginosa PAO-JP2 had no effect on theroughness coefficient, indicating that supplementationdid not affect the biofilm heterogeneity. Signalintensity plots along the z-axis and x-axis suggested
the formation of an uniformly spread biofilm bymutants supplemented with heterologous AHLs.
The response of P. aeruginosa PAO-JP2 to hetero-logous A. baumannii M2 AHLs with respect to theexpression of virulence factors other than biofilmformation was also studied. The relative proteaseactivity and relative pyocyanin production by thequorum sensing mutant strain P. aeruginosa PAO-JP2were 4 fold (p 5 0.05) and 15 fold (p 5 0.01),respectively, less compared to its wild type P.aeruginosa PAO1. The PAO-JP2 culture showed a 2fold (p 5 0.05) increase in proteolytic activity (3-oxo-C12 HSL mediated virulence factor) while there was noeffect on pyocyanin production (C4 regulated virulencefactor) (Figure 8a). However, addition of autologousextracted AHLs from P. aeruginosa PAO1 led to a 13fold (p ¼ 0.01) increase in pyocyanin production and a3.7 fold (p 5 0.05) increase in proteolytic activity(Figure 8b).
Discussion
The present study shows that both A. baumannii and P.aeruginosa PAO1 can co-exist in vitro in co-culturewithout any antibacterial effect of the pyocyanin
Figure 6.1. SEM images of A. baumannii biofilms on glass coverslips. (a) M2; (b) abaI::Km; (c) abaI::Km biofilm developed inthe presence of AHLs extracted from A. baumannii M2; (d) abaI::Km biofilm developed in the presence of AHLs extracted fromP. aeruginosa PAO1. Magnification 30006.
Figure 6.2. SEM images of P. aeruginosa biofilms on glass coverslips. (a) PAO1; (b) PAO-JP2; (c) PAO-JP2 biofilm developedin the presence of AHLs extracted from P. aeruginosa PAO1; (d) PAO-JP2 biofilm developed in the presence of AHLs extractedfrom A. baumannii M2. Magnification 60006.
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produced by P. aeruginosa PAO1 on A. baumannii,contrary to what has been reported in the case of amixed biofilm formed by P. aeruginosa with B. cepacia(Tomlin et al. 2001). This finding is significant as itmay raise the possibility of mixed species biofilmformation by A. baumannii and P. aeruginosa whichcould be difficult to control as such mixed speciesbiofilms have been reported to show increasedresistance to antibiotics (Burmølle et al. 2006).
The role of quorum sensing in biofilm formation byA. baumannii and P. aeruginosa is well established.Hence pure culture biofilms of quorum sensingmutants of these organisms showed reduced biomassdue to their inability to form a well architecturedbiofilm like their wild types. The deficiency observed inbiofilm formed by the mutants was restored byexogenous addition of autologous and heterologousCFS from A. baumannii M2 and P. aeruginosa PAO1,while the CFS of P. aeruginosa PAO-JP2 did not haveany impact on the biofilm formed by A. baumannii M2(abaI::Km). The CFS of A. baumannii M2 and P.aeruginosa PAO1 left after extraction of AHLs was still
found to increase biofilm formation significantly in A.baumannii M2 (abaI::Km) (Figure S3) [Supplementarymaterial is available via a multimedia link on the onlinearticle webpage.]. This showed that in addition toAHLs, CFS may also contain extracellular factors thatstimulate biofilm formation. However, no substantialincrease in biofilm was seen with heat treated CFS ofthe wild types, which suggested the heat labile natureof the biofilm promoting components. Kristich et al.(2004) also showed a 4.5 fold increase in biofilmformation in E. faecalis JH2 when grown in condi-tioned medium with CFS from strain OG1RF and thebiofilm promoting activity was lost on heating theCFS. Lactonolysis of AHLs by heating have beenreported to disrupt its signalling potential (Yates et al.2002). Heat treatment was prefered as it also has theadvantage of inactivating AHLs irreversibly and also itdoes not affect the growth of the culture which is likelyin the case of alkali inactivation of AHLs.
A significant (p 5 0.05) increase in the biofilmmass of A. baumannii M2 (abaI::km) and P. aeruginosaPAO-JP2 was obtained with heterologous AHLs,
Figure 7. CLSM images of biofilm of A. baumannii and P. aeruginosamutants on glass coverslips. a, e and i – abaI::Km; b, f andj – abaI::Km developed in the presence of AHLs extracted from P. aeruginosa PAO1 CFS; c, g and k – PAO-JP2; d, h and l –PAO-JP2 biofilm developed in the presence of AHLs extracted from A. baumanniiM2 CFS. Top panel images are 2D averages ofimage Z stacks while the middle panel images are iso-surface rendered 3D surface plots of the same data set and the lower panelshows gradient of fluorescence intensity quantified along the line indicated with white arrow, using ImageJ 1.44p software. Areasof exposed substratum are highlighted with black arrows. Each image represents a square surface area of 1466146 mm.
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highlighting the possibility of an AHL based interac-tion between A. baumannii and P. aeruginosa. Sincethere was no increase in the biofilm with syntheticshort chain AHLs (C4 and C6), indicating that AHLsdo not have any growth stimulating effect, the increasein biofilm mass and restoration of biofilm architecturewas due to quorum sensing mediated by AHLs ofspecific chain length.
The primary AHL signal coded by abaI in A.baumannii M2 has been identified as 3-OH-C12 HSL(Niu et al. 2008), whereas in P. aeruginosa C4 HSLis coded by rhlI and 3-oxo-C12 HSL by lasI (Riedelet al. 2001). The AHL molecules in the cell interactwith cognate receptor proteins and the entirecomplex then binds to the promoter and regulatesthe expression of quourm sensing target genes(Egland and Greenberg 2001). Since both thereceptor proteins AbaR (A. baumannii) and LasR(P. aeruginosa) shared a 31.4% identity and a 49%similarity (Bhargava et al. 2010), they could respondto heterologous AHLs irrespective of the modifica-tion at the C3 position. The X-ray crystal structureof LasR also revealed that the C-3 position of theacyl chain of the AHL molecule does not interactwith the conserved amino acid residues of the LuxRhomologs belonging to HTH family (Gambello et al.1993). LasR is known to exhibit broad rangespecificity (Savka et al. 2011) hence it responded to3-OH-C12 HSL effectively, the signal molecule of A.baumannii. Similar flexibility of AbaR to AHL withdifferent chain lengths and modifications at the C3position is reported in this study.
In addition to the effect of the AHL basedinterspecies interaction on biofilm formation, theexpression of other virulence factors of P. aeruginosawas also investigated. The production of virulencefactors of P. aeruginosa PAO-JP2 was found to bebetter in minimal medium than in rich medium, thuspeptone water was used for the study (data notshown). As A. baumannii produces only the long chainAHLs, complementation of P. aeruginosa PAO-JP2with A. baumannii AHLs increased proteolytic activityonly as it is controlled by long chain AHL, 3-oxo-C12HSL (Riedel et al. 2001).
P. aeruginosa has been reported to predisposeinfection by B. cepacia in patients with cystic fibrosisby producing extracellular factors that modify theepithelial cell surface in the lungs, thereby facilitatingattachment of B. cepacia. A purely unidirectionalinteraction between B. cepacia (producing C6 and C8HSL) and P. aeruginosa was reported where only theformer could perceive the AHLs of the latter (Riedelet al. 2001). The present study reveals quorum basedinteractions between A. baumannii and P. aeruginosawhich could also have consequences on manifestationof disease as these pathogens have overlapping sites ofinfection. Further studies are warranted on the impactof such communication in regard to biofilm formation,disease courses and therapeutic control during co-infection.
Acknowledgments
The authors are grateful to Dr PN Rather, Dr BH Iglewski,Dr KP Rumbaugh and Dr Greenberg for providing the
Figure 8. (a) Relative protease activity (%) and (b) relative pyocyanin production (%) in a 24 h old culture of P. aeruginosaPAO-JP2 grown in peptone water at 378C in the presence of autologous and heterologous AHLs. Bars ¼ the means of threereadings; * ¼ significant differences (*p-value � 0.05; **p-value � 0.01, t test) between the means.
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strains used in the study. This work was supported by UGC,New Delhi, India.
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