- Email: [email protected]
N-butanoyl-l -homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit
ARTICLE IN PRESS Microbiological Research 160 (2005) 399—403
www.elsevier.de/micres
N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit ˘anb, Gu ¨lgu ¨n Bos-gelmez-Tınaza,, Seyhan Ulusoya, Buket Arıdog b b ˘lu , Selc-uk Kaya Fu ¨sun Erog a
Department of Biology, Suleyman Demirel University, 32260 Isparta, Turkey Faculty of Medicine, Department of Clinical Microbiology, Suleyman Demirel University, 32260 Ispart, Turkey
b
Accepted 9 March 2005
KEYWORDS N-Acyl-homoserine lactone; Gram-negative bacteria; Quorum-sensing; Pseudomonas aeruginosa
Summary Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low. & 2005 Elsevier GmbH. All rights reserved.
Introduction Many host-associated bacteria use an intercellular signalling mechanism to control expression of specific genes in response to population density.
This type of gene regulation is termed quorum sensing (Fuqua et al., 1994). A wide spectrum of important processes is reported to be regulated by quorum sensing including bioluminescence, antibiotic production, biofilm formation, swarming,
Corresponding author.
E-mail address: [email protected] (G. Bos-gelmez-Tınaz). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2005.03.005
ARTICLE IN PRESS G. Bos- gelmez-Tınaz et al.
400 motility and virulence. Several Gram-negative bacteria utilise small diffusible N-acyl-homoserine lactone (AHL) molecules for quorum sensing (Pesci et al., 1997; Fuqua et al., 2001; Whitehead et al., 2001). These small signal molecules diffuse from bacterial cells and accumulate in the medium as a function of cell growth. At a threshold population density, the accumulated AHLs interact with cellular receptors on the cell surface and trigger the expression of target genes (Gray, 1997; Whitehead et al., 2001). Pseudomonas aeruginosa is perhaps the bestunderstood Gram-negative bacterium in terms of AHL mediated quorum sensing. P. aeruginosa is an opportunistic human pathogen that preferentially infects patients with cancer or AIDS, people with cystic fibrosis or blood, skin, eye and genitourinary tract infections, or patients immunocompromised by surgery, cytotoxic drugs or burn wounds (Passador and Iglewski, 1995; Williams et al., 1996). It can also colonise implanted devices, catheters, heartvalves or dental implants (de Kievit and Iglewski, 2000; Dunn and Wunderink, 1995). One of the reasons for P. aeruginosa being a successful opportunistic pathogen is the production of many secreted virulence factors (Passador and Iglewski, 1995). These virulence factors include elastase, alkaline protease, hemolysin, cyanide, pyocyanin and exotoxin A (Nicas and Iglewski, 1985; van Delden and Iglewski, 1998). The genes for these exoproducts are often controlled by quorum sensing systems. In P. aeruginosa, two quorum sensing system were identified, i.e. LasR/I and RhlR/I. The Las system comprises LasI, which is responsible for the synthesis of N-(3oxododecanoyl)-L-homoserine lactone (OdDHL), and the transcriptional activator LasR (Pesci et al., 1997; Whitehead et al., 2001). This system was shown to regulate the production of elastase, exotoxin A and alkaline protease. The Rhl system, relying on Nbutanoyl-L-homoserine lactone (BHL), was shown to be involved in the regulation of alkaline protease, elastase, cyanide and pyocyanin production (Pearson et al., 1995; Winson et al., 1995). Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae are also important human pathogens causing severe infections in hospitalised patients. However, there are limited reports regarding AHL quorum sensing signal molecules in these organisms. In this paper, we describe the isolation of 80 strains of Gram-negative bacteria from the intensive care unit of a university hospital and investigate the ability of these isolates to produce signal molecules. To our knowledge, this is the first report of lack of BHL production in clinical isolates of P. aeruginosa.
Material and methods Bacterial strains and growth media The clinical strains of P. aeruginosa, Acinetobacter baumannii, E. coli and K. pneumoniae were obtained from patients in the intensive care unit. These bacterial species were isolated from infected wounds, blood, urine or body fluid. All strains were grown in or on trypticase soy broth solidified with 1.2% agar when required. Detection of AHLs was achieved using either the Chromobacterium violaceum strain CV026 (McClean et al., 1997) or the Agrobacterium tumefaciens strain NT1 bioassay (Shaw et al., 1997; Cha et al., 1998; Ravn et al., 2001). C. violaceum CV026 (a mini-Tn5 mutant) was used as indicator strain for the detection of AHL with Nacyl side chains of four to eight carbons. C. violaceum CV026 responds to AHLs by inducing the synthesis of the purple pigment violacein (McClean et al., 1997). Agrobacterium tumefaciens strain NT1, carrying the plasmid pZLR4, was used as additional indicator. Agrobacterium tumefaciens NT1 produces a blue colour in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) in response to AHLs with N-acyl chain lengths from 6 to 12 carbon atoms (Shaw et al., 1997; Cha et al., 1998; Ravn et al., 2001). These strains were grown in or on LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl) solidified with 1.2% agar when required and supplemented with appropriate antibiotics, Agrobacterium tumefaciens NT1 with 20 mg/ml gentamycin, and C. violaceum with 20 mg/ml kanamycin.
Cross-feeding assay for AHL detection N-acyl-homoserine lactone production was examined by streaking the strains to be tested in parallel with a lane of the monitor strains CV026 and NT1.
Diffusion assay Five millilitre of molten LB agar (0.3%, w/v) were inoculated with 50 ml of a culture of C. violaceum grown overnight in LB. The agar-culture solution was immediately poured on the surface of prewarmed LB agar plates (100 mm 100 mm). Up to 100 ml supernatants to be tested were pipetted into wells, punched in the solidified agar with a sterile cork borer. The plates were incubated overnight at 30 1C and examined for violacein production.
ARTICLE IN PRESS N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa
Results The use of two indicator strains, C. violaceum CV026 and Agrobacterium tumefaciens NT1, allowed for screening a range of AHL from all the test strains (Table 1). Strong positive reactions were observed for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii tested in the Agrobacterium tumefaciens NT1 system (Fig. 1B). Negative reactions were recorded for isolates of K. pneumoniae and E. coli. Examples of cross-feeding assays with P. aeruginosa, Acinetobacter baumannii, K. pneumoniae and E. coli are shown in Fig. 1B. Incubation of P. aeruginosa and Acinetobacter baumannii in the presence of the indicator strain, Agrobacterium tumefaciens NT1 produces a blue colour in the bioassay medium due to expression of the lacZ reporter gene. K. pneumoniae and E. coli were AHL negative. In experiments with C. violaceum CV026 for detecting short-chained AHLs Acinetobacter baumannii, K. pneumoniae and E. coli did not activate violacein production in the C. violaceum CV026 system (Fig. 1A). While the well-characterised P. aeruginosa strain PA01 activated C. violaceum CV026, our clinical Table 1. AHL production of Gram-negative bacteria isolated from the intensive care unit of the hospital Species
No. of strains
Monitor system CV026
Pseudomomas aeruginosa Acinetobacter baumannii Klebsiella pnemonia Escherichia coli
50 10 10 10
NT1 + +
401
isolates of P. aeruginosa failed to activate violacein production in C. violaceum CV026 (Fig. 1A). P. aeruginosa strain PA01 was used as positive control for induction of C. violaceum CV026. Negative controls were the indicator strains themselves. Results of well-diffusion assays using these indicator strains were in agreement with observations in the cross-feeding test (data not shown).
Discussion Bacterial isolates obtained from the intensive care unit of Suleyman Demirel University Hospital, Isparta, Turkey, were assayed for the presence of quorum sensing signal molecules using the two indicator strains. Among four bacterial species, all strains of P. aeruginosa and Acinetobacter baumannii have been found to produce AHL molecules active in the Agrobacterium tumefaciens NT1 system. In agreement with published data, tester strains of E. coli and K. pneumonia were AHL negative (Zhu et al., 2001; Stickler et al., 1998). P. aeruginosa and Acinetobacter baumannii activated Agrobacterium tumefaciens NT1 indicator strain on plates. These findings agree with previous reports (Winson et al., 1995; Stickler et al., 1998; Gonzalez et al., 2001). Surprisingly, we found that all P. aeruginosa isolates were negative in the C. violaceum CV026 system. From published data, it is well-known that P. aeruginosa supernatants contain two major AHLs, BHL and OdDHL, in addition to at least two minor components, HHL and OHHL (Winson et al., 1995; Williams et al., 1996). The production of violacein in C. violaceum is stimulated by total AHLs from P. aeruginosa (Winson et al., 1995; Blosser and Gray, 2000; Zhu et al., 2001; Aendekerk et al., 2002; Hu et al., 2003).
Figure 1. Cross-feeding assays for detection of AHLs (A) violacein production by C. violaceum as an indication of shortchain AHLs. (1) P. aeruginosa, (2) Acinetobacter baumannii, (3) PA01, (4) E. coli, (5) K. pneumonia, and (6) PA01 (control). (B) blue colouration by Agrobacterium tumefaciens NT1 as an indication for long-chain AHLs. (1) P. aeruginosa, (2) Agrobacterium baumannii, and (3) PA01 (control).
ARTICLE IN PRESS 402 In our study, total AHL fractions from P. aeruginosa isolates failed to stimulate the production of violacein by C. violaceum. There are several possible explanations for this behaviour. It may be due to drastic imbalance in the BHL/OdDHL ratio in these isolates. If the proportion of long-chain molecules is higher in these strains, an inhibition of the indicator strain is likely to occur (McClean et al., 1997). Alternatively, it shows an inability to produce enough BHL to stimulate violacein production in C. violaceum due to a deficiency in the RhlI/ R system. In addition, linkage between the gacA gene and BHL production was previously demonstrated. The disruption of gacA in strain PAO1 resulted in reduced or delayed production of BHL (Reimmann et al., 1997). Further work is needed to elucidate the reasons for failure of BHL production and how these isolates differ in virulence determinants from other P. aeruginosa strains. Such information may offer an approach for controlling P. aeruginosa infections by designing novel strategies which interfere with AHL-based signalling systems. The disruption of signalling systems might provide an opportunity to prevent the bacteria from responding to the signal and consequently might prevent the expression of virulence factors.
Acknowledgements This work was funded by Suleyman Demirel University, Turkey. We thank Dr. Miguel Camara and Dr. Steve Diggle for providing C. violaceum strain CV026 and Dr. Lars Ravn Flodgaard for Agrobacterium tumefaciens strain NT1.
References Aendekerk, S., Ghysels, B., Cornelis, P., Baysse, C., 2002. Characterization of a new efflux pump, MexGHIOpmD, from Pseudomonas aeruginosa that confers resistance to vanadium. Microbiology 148, 2371–2381. Blosser, R.S., Gray, M.K., 2000. Extraction of violacein from Chromobacterium violeceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers. J. Microbiol. Methods 40, 47–55. Cha, C., Gao, P., Chen, Y.C., Shaw, P.D., Farrand, S.K., 1998. Production of acyl-homoserine lactone signals by Gram-negative plant-associated bacteria. Mol. Plant Microbe Interact. 11, 1119–1129. de Kievit, T.R., Iglewski, B.H., 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 68, 4839–4849.
G. Bos- gelmez-Tınaz et al. Dunn, M., Wunderink, R.G., 1995. Ventilator-associated pneumonia caused by Pseudomonas infection. Clin. Chest Med. 16, 95–109. Fuqua, C., Parsek, M.R., Greenberg, E.P., 2001. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 35, 439–468. Fuqua, W.C., Winans, S.C., Greenberg, E.P., 1994. Quorum sensing in bacteria—the luxR–luxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176, 269–275. Gonzalez, R.H., Nusblat, A., Nudel, B.C., 2001. Detection and characterization of quorum sensing signal molecules in Acinetobacter strains. Microbiol. Res. 155, 271–277. Gray, K.M., 1997. Intercellular communication and group behaviour in bacteria. Trends Microbiol. 5, 184–188. Hu, J.Y., Fan, Y., Lin, Y., Zhang, H., Ong, S.L., Dong, N., Xu, J., Ng, W.J., Zhang, L., 2003. Microbial diversity and prevalence of virulent pathogens in biofilms developed in a water reclamation system. Res. Microbiol. 154, 623–629. McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft, B.W., Stewart, G.S.A.B., Williams, P., 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production for the detection of N-acylhomoserine lactones. Microbiology 143, 3703–3711. Nicas, T.I., Iglewski, B.H., 1985. The contribution of exoproducts to virulence of Pseudomonas aeruginosa. Can. J. Microbiol. 31, 387–392. Passador, L., Iglewski, B.H., 1995. Quorum sensing and virulence gene regulation in Pseudomonas aeruginosa. In: Roth, J.A., Bolin, C.A., Brodgen, K.A., Minion, F.C., Wannemuehler, M.J. (Eds.), Virulence Mechanisms of Bacterial Pathogens, second ed. ASM Press, Washington, DC, pp. 65–78. Pearson, J.P., Passador, L., Iglewski, B.H., Greenberg, E.P., 1995. A second N-acyl-L-homoserine lactone signal produced by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92, 1490–1494. Pesci, E.C., Pearson, J.P., Seed, P.C., Iglewski, B.H., 1997. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179, 3127–3132. Ravn, L., Christensen, A.B., Molin, S., Givskov, M., Gram, L., 2001. Methods for acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics. J. Microbiol. Methods 44, 239–251. Reimmann, C., Beyeler, M., Latifi, A., Winteler, H., Foglino, M., Lazdunski, A., Hass, D., 1997. The global activator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Mol. Microbiol. 24, 309–319. Shaw, P.D., Ping, G., Daly, S.L., 1997. Detecting and characterizing N-acyl-homoserine lactone signal
ARTICLE IN PRESS N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94, 6036–6041. Stickler, D.J., Morris, N.S., McLean, R.J.C., Fuqua, C., 1998. Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Appl. Environ. Microbiol. 64, 3486–3490. van Delden, C., Iglewski, B.H., 1998. Cell-to-cell signalling and Pseudomonas aeruginosa infections. Emerging Infect. Dis. 4, 551–560. Whitehead, N.A., Barnard, A.M.L., Slater, H., Simpson, N.J.L., Salmond, G.P.C., 2001. Quorum-sensing in Gramnegative bacteria. FEMS Microbiol. Rev. 25, 365–404. Williams, P., Stewart, G.S.A.B., Camara, M., 1996. Signal transduction through quorum sensing in Pseudomonas aeruginosa. In: Nakazawa, T., Furukawa, K., Haas, D.,
403
Silver, S. (Eds.), Pseudomonas: Molecular Biology and Biotechnology. ASM Press, Washington, DC, pp. 195–206. Winson, M.K., Camara, M., Latifi, A., Foglino, M., Chhabra, S.R., Daykin, M., Bally, M., Chapon, V., Salmond, G.P.C., Bycroft, B.W., Lazdunski, A., Stewart, G.S.A.B., Williams, P., 1995. Multiple N-acyl-Lhomoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92, 9427–9431. Zhu, H., Thuruthyil, S.J., Willcox, M.D.P., 2001. Production of acyl homoserine lactones by gram-negative bacteria isolated from contact lens wearers. Clin. Exp. Ophtalmol. 29, 150–152.
www.elsevier.de/micres
N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit ˘anb, Gu ¨lgu ¨n Bos-gelmez-Tınaza,, Seyhan Ulusoya, Buket Arıdog b b ˘lu , Selc-uk Kaya Fu ¨sun Erog a
Department of Biology, Suleyman Demirel University, 32260 Isparta, Turkey Faculty of Medicine, Department of Clinical Microbiology, Suleyman Demirel University, 32260 Ispart, Turkey
b
Accepted 9 March 2005
KEYWORDS N-Acyl-homoserine lactone; Gram-negative bacteria; Quorum-sensing; Pseudomonas aeruginosa
Summary Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low. & 2005 Elsevier GmbH. All rights reserved.
Introduction Many host-associated bacteria use an intercellular signalling mechanism to control expression of specific genes in response to population density.
This type of gene regulation is termed quorum sensing (Fuqua et al., 1994). A wide spectrum of important processes is reported to be regulated by quorum sensing including bioluminescence, antibiotic production, biofilm formation, swarming,
Corresponding author.
E-mail address: [email protected] (G. Bos-gelmez-Tınaz). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2005.03.005
ARTICLE IN PRESS G. Bos- gelmez-Tınaz et al.
400 motility and virulence. Several Gram-negative bacteria utilise small diffusible N-acyl-homoserine lactone (AHL) molecules for quorum sensing (Pesci et al., 1997; Fuqua et al., 2001; Whitehead et al., 2001). These small signal molecules diffuse from bacterial cells and accumulate in the medium as a function of cell growth. At a threshold population density, the accumulated AHLs interact with cellular receptors on the cell surface and trigger the expression of target genes (Gray, 1997; Whitehead et al., 2001). Pseudomonas aeruginosa is perhaps the bestunderstood Gram-negative bacterium in terms of AHL mediated quorum sensing. P. aeruginosa is an opportunistic human pathogen that preferentially infects patients with cancer or AIDS, people with cystic fibrosis or blood, skin, eye and genitourinary tract infections, or patients immunocompromised by surgery, cytotoxic drugs or burn wounds (Passador and Iglewski, 1995; Williams et al., 1996). It can also colonise implanted devices, catheters, heartvalves or dental implants (de Kievit and Iglewski, 2000; Dunn and Wunderink, 1995). One of the reasons for P. aeruginosa being a successful opportunistic pathogen is the production of many secreted virulence factors (Passador and Iglewski, 1995). These virulence factors include elastase, alkaline protease, hemolysin, cyanide, pyocyanin and exotoxin A (Nicas and Iglewski, 1985; van Delden and Iglewski, 1998). The genes for these exoproducts are often controlled by quorum sensing systems. In P. aeruginosa, two quorum sensing system were identified, i.e. LasR/I and RhlR/I. The Las system comprises LasI, which is responsible for the synthesis of N-(3oxododecanoyl)-L-homoserine lactone (OdDHL), and the transcriptional activator LasR (Pesci et al., 1997; Whitehead et al., 2001). This system was shown to regulate the production of elastase, exotoxin A and alkaline protease. The Rhl system, relying on Nbutanoyl-L-homoserine lactone (BHL), was shown to be involved in the regulation of alkaline protease, elastase, cyanide and pyocyanin production (Pearson et al., 1995; Winson et al., 1995). Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae are also important human pathogens causing severe infections in hospitalised patients. However, there are limited reports regarding AHL quorum sensing signal molecules in these organisms. In this paper, we describe the isolation of 80 strains of Gram-negative bacteria from the intensive care unit of a university hospital and investigate the ability of these isolates to produce signal molecules. To our knowledge, this is the first report of lack of BHL production in clinical isolates of P. aeruginosa.
Material and methods Bacterial strains and growth media The clinical strains of P. aeruginosa, Acinetobacter baumannii, E. coli and K. pneumoniae were obtained from patients in the intensive care unit. These bacterial species were isolated from infected wounds, blood, urine or body fluid. All strains were grown in or on trypticase soy broth solidified with 1.2% agar when required. Detection of AHLs was achieved using either the Chromobacterium violaceum strain CV026 (McClean et al., 1997) or the Agrobacterium tumefaciens strain NT1 bioassay (Shaw et al., 1997; Cha et al., 1998; Ravn et al., 2001). C. violaceum CV026 (a mini-Tn5 mutant) was used as indicator strain for the detection of AHL with Nacyl side chains of four to eight carbons. C. violaceum CV026 responds to AHLs by inducing the synthesis of the purple pigment violacein (McClean et al., 1997). Agrobacterium tumefaciens strain NT1, carrying the plasmid pZLR4, was used as additional indicator. Agrobacterium tumefaciens NT1 produces a blue colour in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) in response to AHLs with N-acyl chain lengths from 6 to 12 carbon atoms (Shaw et al., 1997; Cha et al., 1998; Ravn et al., 2001). These strains were grown in or on LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl) solidified with 1.2% agar when required and supplemented with appropriate antibiotics, Agrobacterium tumefaciens NT1 with 20 mg/ml gentamycin, and C. violaceum with 20 mg/ml kanamycin.
Cross-feeding assay for AHL detection N-acyl-homoserine lactone production was examined by streaking the strains to be tested in parallel with a lane of the monitor strains CV026 and NT1.
Diffusion assay Five millilitre of molten LB agar (0.3%, w/v) were inoculated with 50 ml of a culture of C. violaceum grown overnight in LB. The agar-culture solution was immediately poured on the surface of prewarmed LB agar plates (100 mm 100 mm). Up to 100 ml supernatants to be tested were pipetted into wells, punched in the solidified agar with a sterile cork borer. The plates were incubated overnight at 30 1C and examined for violacein production.
ARTICLE IN PRESS N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa
Results The use of two indicator strains, C. violaceum CV026 and Agrobacterium tumefaciens NT1, allowed for screening a range of AHL from all the test strains (Table 1). Strong positive reactions were observed for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii tested in the Agrobacterium tumefaciens NT1 system (Fig. 1B). Negative reactions were recorded for isolates of K. pneumoniae and E. coli. Examples of cross-feeding assays with P. aeruginosa, Acinetobacter baumannii, K. pneumoniae and E. coli are shown in Fig. 1B. Incubation of P. aeruginosa and Acinetobacter baumannii in the presence of the indicator strain, Agrobacterium tumefaciens NT1 produces a blue colour in the bioassay medium due to expression of the lacZ reporter gene. K. pneumoniae and E. coli were AHL negative. In experiments with C. violaceum CV026 for detecting short-chained AHLs Acinetobacter baumannii, K. pneumoniae and E. coli did not activate violacein production in the C. violaceum CV026 system (Fig. 1A). While the well-characterised P. aeruginosa strain PA01 activated C. violaceum CV026, our clinical Table 1. AHL production of Gram-negative bacteria isolated from the intensive care unit of the hospital Species
No. of strains
Monitor system CV026
Pseudomomas aeruginosa Acinetobacter baumannii Klebsiella pnemonia Escherichia coli
50 10 10 10
NT1 + +
401
isolates of P. aeruginosa failed to activate violacein production in C. violaceum CV026 (Fig. 1A). P. aeruginosa strain PA01 was used as positive control for induction of C. violaceum CV026. Negative controls were the indicator strains themselves. Results of well-diffusion assays using these indicator strains were in agreement with observations in the cross-feeding test (data not shown).
Discussion Bacterial isolates obtained from the intensive care unit of Suleyman Demirel University Hospital, Isparta, Turkey, were assayed for the presence of quorum sensing signal molecules using the two indicator strains. Among four bacterial species, all strains of P. aeruginosa and Acinetobacter baumannii have been found to produce AHL molecules active in the Agrobacterium tumefaciens NT1 system. In agreement with published data, tester strains of E. coli and K. pneumonia were AHL negative (Zhu et al., 2001; Stickler et al., 1998). P. aeruginosa and Acinetobacter baumannii activated Agrobacterium tumefaciens NT1 indicator strain on plates. These findings agree with previous reports (Winson et al., 1995; Stickler et al., 1998; Gonzalez et al., 2001). Surprisingly, we found that all P. aeruginosa isolates were negative in the C. violaceum CV026 system. From published data, it is well-known that P. aeruginosa supernatants contain two major AHLs, BHL and OdDHL, in addition to at least two minor components, HHL and OHHL (Winson et al., 1995; Williams et al., 1996). The production of violacein in C. violaceum is stimulated by total AHLs from P. aeruginosa (Winson et al., 1995; Blosser and Gray, 2000; Zhu et al., 2001; Aendekerk et al., 2002; Hu et al., 2003).
Figure 1. Cross-feeding assays for detection of AHLs (A) violacein production by C. violaceum as an indication of shortchain AHLs. (1) P. aeruginosa, (2) Acinetobacter baumannii, (3) PA01, (4) E. coli, (5) K. pneumonia, and (6) PA01 (control). (B) blue colouration by Agrobacterium tumefaciens NT1 as an indication for long-chain AHLs. (1) P. aeruginosa, (2) Agrobacterium baumannii, and (3) PA01 (control).
ARTICLE IN PRESS 402 In our study, total AHL fractions from P. aeruginosa isolates failed to stimulate the production of violacein by C. violaceum. There are several possible explanations for this behaviour. It may be due to drastic imbalance in the BHL/OdDHL ratio in these isolates. If the proportion of long-chain molecules is higher in these strains, an inhibition of the indicator strain is likely to occur (McClean et al., 1997). Alternatively, it shows an inability to produce enough BHL to stimulate violacein production in C. violaceum due to a deficiency in the RhlI/ R system. In addition, linkage between the gacA gene and BHL production was previously demonstrated. The disruption of gacA in strain PAO1 resulted in reduced or delayed production of BHL (Reimmann et al., 1997). Further work is needed to elucidate the reasons for failure of BHL production and how these isolates differ in virulence determinants from other P. aeruginosa strains. Such information may offer an approach for controlling P. aeruginosa infections by designing novel strategies which interfere with AHL-based signalling systems. The disruption of signalling systems might provide an opportunity to prevent the bacteria from responding to the signal and consequently might prevent the expression of virulence factors.
Acknowledgements This work was funded by Suleyman Demirel University, Turkey. We thank Dr. Miguel Camara and Dr. Steve Diggle for providing C. violaceum strain CV026 and Dr. Lars Ravn Flodgaard for Agrobacterium tumefaciens strain NT1.
References Aendekerk, S., Ghysels, B., Cornelis, P., Baysse, C., 2002. Characterization of a new efflux pump, MexGHIOpmD, from Pseudomonas aeruginosa that confers resistance to vanadium. Microbiology 148, 2371–2381. Blosser, R.S., Gray, M.K., 2000. Extraction of violacein from Chromobacterium violeceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers. J. Microbiol. Methods 40, 47–55. Cha, C., Gao, P., Chen, Y.C., Shaw, P.D., Farrand, S.K., 1998. Production of acyl-homoserine lactone signals by Gram-negative plant-associated bacteria. Mol. Plant Microbe Interact. 11, 1119–1129. de Kievit, T.R., Iglewski, B.H., 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 68, 4839–4849.
G. Bos- gelmez-Tınaz et al. Dunn, M., Wunderink, R.G., 1995. Ventilator-associated pneumonia caused by Pseudomonas infection. Clin. Chest Med. 16, 95–109. Fuqua, C., Parsek, M.R., Greenberg, E.P., 2001. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 35, 439–468. Fuqua, W.C., Winans, S.C., Greenberg, E.P., 1994. Quorum sensing in bacteria—the luxR–luxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176, 269–275. Gonzalez, R.H., Nusblat, A., Nudel, B.C., 2001. Detection and characterization of quorum sensing signal molecules in Acinetobacter strains. Microbiol. Res. 155, 271–277. Gray, K.M., 1997. Intercellular communication and group behaviour in bacteria. Trends Microbiol. 5, 184–188. Hu, J.Y., Fan, Y., Lin, Y., Zhang, H., Ong, S.L., Dong, N., Xu, J., Ng, W.J., Zhang, L., 2003. Microbial diversity and prevalence of virulent pathogens in biofilms developed in a water reclamation system. Res. Microbiol. 154, 623–629. McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin, M., Lamb, J.H., Swift, S., Bycroft, B.W., Stewart, G.S.A.B., Williams, P., 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production for the detection of N-acylhomoserine lactones. Microbiology 143, 3703–3711. Nicas, T.I., Iglewski, B.H., 1985. The contribution of exoproducts to virulence of Pseudomonas aeruginosa. Can. J. Microbiol. 31, 387–392. Passador, L., Iglewski, B.H., 1995. Quorum sensing and virulence gene regulation in Pseudomonas aeruginosa. In: Roth, J.A., Bolin, C.A., Brodgen, K.A., Minion, F.C., Wannemuehler, M.J. (Eds.), Virulence Mechanisms of Bacterial Pathogens, second ed. ASM Press, Washington, DC, pp. 65–78. Pearson, J.P., Passador, L., Iglewski, B.H., Greenberg, E.P., 1995. A second N-acyl-L-homoserine lactone signal produced by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92, 1490–1494. Pesci, E.C., Pearson, J.P., Seed, P.C., Iglewski, B.H., 1997. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179, 3127–3132. Ravn, L., Christensen, A.B., Molin, S., Givskov, M., Gram, L., 2001. Methods for acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics. J. Microbiol. Methods 44, 239–251. Reimmann, C., Beyeler, M., Latifi, A., Winteler, H., Foglino, M., Lazdunski, A., Hass, D., 1997. The global activator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the autoinducer N-butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Mol. Microbiol. 24, 309–319. Shaw, P.D., Ping, G., Daly, S.L., 1997. Detecting and characterizing N-acyl-homoserine lactone signal
ARTICLE IN PRESS N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94, 6036–6041. Stickler, D.J., Morris, N.S., McLean, R.J.C., Fuqua, C., 1998. Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Appl. Environ. Microbiol. 64, 3486–3490. van Delden, C., Iglewski, B.H., 1998. Cell-to-cell signalling and Pseudomonas aeruginosa infections. Emerging Infect. Dis. 4, 551–560. Whitehead, N.A., Barnard, A.M.L., Slater, H., Simpson, N.J.L., Salmond, G.P.C., 2001. Quorum-sensing in Gramnegative bacteria. FEMS Microbiol. Rev. 25, 365–404. Williams, P., Stewart, G.S.A.B., Camara, M., 1996. Signal transduction through quorum sensing in Pseudomonas aeruginosa. In: Nakazawa, T., Furukawa, K., Haas, D.,
403
Silver, S. (Eds.), Pseudomonas: Molecular Biology and Biotechnology. ASM Press, Washington, DC, pp. 195–206. Winson, M.K., Camara, M., Latifi, A., Foglino, M., Chhabra, S.R., Daykin, M., Bally, M., Chapon, V., Salmond, G.P.C., Bycroft, B.W., Lazdunski, A., Stewart, G.S.A.B., Williams, P., 1995. Multiple N-acyl-Lhomoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92, 9427–9431. Zhu, H., Thuruthyil, S.J., Willcox, M.D.P., 2001. Production of acyl homoserine lactones by gram-negative bacteria isolated from contact lens wearers. Clin. Exp. Ophtalmol. 29, 150–152.