Subinhibitory concentrations of the cationic ...

Microbiology (2009), 155, 2826–2837

DOI 10.1099/mic.0.025643-0

Subinhibitory concentrations of the cationic antimicrobial peptide colistin induce the pseudomonas quinolone signal in Pseudomonas aeruginosa Joanne Cummins, F. Jerry Reen, Christine Baysse3, Marlies J. Mooij and Fergal O’Gara Correspondence

BIOMERIT Research Centre, Department of Microbiology, University College Cork, Ireland

Fergal O’Gara [email protected]

Received 31 October 2008 Revised

21 May 2009

Accepted 23 May 2009

Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection in cystic fibrosis (CF) lungs. The effects of subinhibitory concentrations of colistin on gene expression in P. aeruginosa were investigated by transcriptome and functional genomic approaches. Analysis revealed altered expression of 30 genes representing a variety of pathways associated with virulence and bacterial colonization in chronic infection. These included response to osmotic stress, motility, and biofilm formation, as well as genes associated with LPS modification and quorum sensing (QS). Most striking was the upregulation of Pseudomonas quinolone signal (PQS) biosynthesis genes, including pqsH, pqsB and pqsE, and the phenazine biosynthesis operon. Induction of this central component of the QS network following exposure to subinhibitory concentrations of colistin may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF lung.

INTRODUCTION Pseudomonas aeruginosa is an opportunistic pathogen, and is associated with both chronic and acute infections, including sepsis and wound and pulmonary infections (Bodey et al., 1983; Govan & Deretic, 1996). It is a major cause of pulmonary damage, especially in patients suffering from cystic fibrosis (CF), and its emergence as a nosocomial pathogen is a growing concern. Infections with P. aeruginosa are particularly difficult to cure through antimicrobial therapy because of the bacterium’s intrinsically impermeable outer membrane and active efflux of toxic agents from the cytoplasm (Hancock, 1997a; Nikaido, 1989). A major challenge has arisen regarding the treatment of infections caused by P. aeruginosa and other multi-drug 3Present address: Equipe DUALS UMR6026, Universite´ de Rennes, Campus de Beaulieu, F-35043, Rennes, France Abbreviations: AHQ, 2-alkyl-4-quinolone; CF, cystic fibrosis; MDR, multidrug resistant; PQS, Pseudomonas quinolone signal; qRT-PCR, quantitative real-time RT-PCR; QS, quorum sensing; RMA, robust multiarray average. The microarray data discussed in this study have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE13252. Three supplementary tables, listing primers used in this study, genes showing significantly altered expression in the colistin-treated sample, and motility in the presence of colistin, are available with the online version of this paper.

2826

resistant (MDR) Gram-negative bacteria (Bonomo & Szabo, 2006; Rahal, 2006). This has led to renewed interest in an ‘old class of antibiotics’ known as the polymyxins, first discovered in the 1950s in the soil bacterium Bacillus polymyxa (Ainsworth et al., 1947). Despite their effectiveness, intravenous formulations of colistin (polymyxin E) and polymyxin B were gradually abandoned in most parts of the world in the early 1980s because of the reported high incidence of nephrotoxicity (Beringer, 2001; Brown et al., 1970). Recently, a number of studies have confirmed the safety of colistin and provided increased support for treatment of acute pulmonary infections caused by P. aeruginosa as well as infections caused by other multiresistant Gram-negative bacteria (Garnacho-Montero et al., 2003; Hanberger et al., 2001; Markou et al., 2003). Despite the fact that polymyxins have been used clinically for nearly 60 years, there is a dearth of information regarding their pharmacokinetics and pharmacodynamics. This creates an underlying risk of sublethal treatment and the potential development of resistance. Indeed, the first reports of colistin-tolerant clinical isolates have already been published (Denton et al., 2002). P. aeruginosa has developed ‘adaptive tolerance’ to colistin, mediated through the PhoPQ and PmrAB two-component systems that control aminoarabinose modification of the LPS (McPhee et al., 2003). Pseudomonas strains isolated from patients treated with colistin have aminoarabinose-modi-

Downloaded from www.microbiologyresearch.org by 025643 G 2009 SGM IP: 178.18.19.221 On: Sun, 18 Oct 2015 22:02:08

Printed in Great Britain

Subinhibitory concentrations of colistin induce PQS

fied lipid A, providing evidence that P. aeruginosa can adapt to the presence of colistin in the airways of the CF lung (Denton et al., 2002; Frederiksen et al., 1999). While microbes have been subjected to antimicrobial agents throughout evolution, the clinical use of antibiotics is a relatively new phenomenon. The antibiotic concentrations that bacteria have encountered in nature are generally lower than the concentrations used to treat bacterial infections. Recent studies have shown that non-lethal concentrations can act as stimuli, in a dose-dependent manner, to elicit specific bacterial responses (Brazas & Hancock, 2005; Davies et al., 2006; Linares et al., 2006; Oh et al., 2000; Skindersoe et al., 2008; Tomasinsig et al., 2004). This modulation of transcription can lead to subtle changes in cell physiology with specific consequences for the collective behaviour of the bacterial population (Davies et al., 2006). The focus of this study was to investigate the effects of subinhibitory concentrations of colistin, one of the last reserve agents available for MDR bacteria, on P. aeruginosa.

METHODS Bacterial strains, plasmids and growth conditions. Strains and

plasmids used in this study are listed in Table 1. All cultures of P. aeruginosa PAO1 were routinely grown in Mueller–Hinton (MH) or Luria–Bertani (LB) media at 37 uC with shaking at 180 r.p.m. Escherichia coli strains were routinely grown in LB at 37 uC. Where appropriate, antibiotics were added to growth media at the following concentrations: 50 mg kanamycin ml21, 200 mg carbenicillin ml21, 60 mg tetracycline ml21 and 50 mg gentamicin ml21 for P. aeruginosa; and 40 mg X-Gal ml21 25 mg gentamicin ml21 and 12.5 mg tetracycline ml21 for E. coli. Colistin (Sigma-Aldrich) was prepared in 0.01 % (v/v) acetic acid and 0.2 % (w/v) BSA.

National Committee of Laboratory Safety and Standards (NCLSS) guidelines. In brief, PAO1 was grown at 37 uC overnight in MH broth. The overnight culture was then diluted down to OD600 0.05 together with the appropriate final concentration of colistin and grown up in polypropylene 96-well plates. Colistin was added to the media at concentrations ranging from 0.15 to 2.4 mg ml21. Plates were incubated at 37 uC overnight in a growth curve machine (Bioscreen C). OD600 readings were taken every hour. Microarray sample and preparation. Three independent cultures of the P. aeruginosa strain PAO1 were exposed to 0.15 mg colistin ml21. This concentration was chosen as it has been clearly shown to have no effect on the growth of P. aeruginosa under the experimental conditions described (Fig. 1a). The untreated and treated samples were grown from OD600 0.05 to 0.8, and subsequently total RNA was extracted using the Ambion RiboPure Bacteria kit according to the manufacturer’s instructions. The RNA was treated with RQ1 RNasefree DNase (Promega) at 37 uC for 1 h and purified using the RNeasy Midi RNA Isolation kit (Qiagen). The RNA was precipitated with three volumes of ethanol and 1/10 volume of 3 M sodium acetate buffer (pH 5.2), and the RNA pellet was resuspended in sterile distilled H2O to a final volume of 20 ml. cDNA was synthesized according to the Affymetrix Expression Analysis Protocol guide. Transcripts corresponding to Bacillus subtilis genes dap, thr, phe, lys and trp were spiked into the cDNA synthesis reaction mixtures as controls to monitor cDNA synthesis, labelling, hybridization and staining efficiency. The cDNA was treated with 2 mg DNase-free RNase (Roche) at 37 uC for 1 h, purified using the QIAquick PCR Purification kit (Qiagen) and eluted in sterile distilled H2O. The cDNA was fragmented using RQ1 RNase-free DNase for 1 h at 37 uC, and biotin-labelled using the BioArray Terminal Labelling kit with biotin–ddUTP (Enzo Life Sciences). The cDNA was snap-frozen on dry ice and hybridized at MRC Geneservice (Cambridge, UK) on Affymetrix GeneChip P. aeruginosa Genome arrays. A Student’s paired t test was carried out on the robust multiarray average (RMA)normalized data generated from the microarray analysis using GeneDirector and Gene Spring software to give a list of genes with altered expression greater than 1.5 and a P value ¡0.05. Semiquantitative RT-PCR. Microarray data were validated by

Determination of MIC. The MIC of P. aeruginosa for colistin was

determined using the broth microdilution technique, according to the

semiquantitative RT-PCR on RNA isolated from three independent experiments. Specific RT-PCR primers were designed for each gene

Table 1. Bacterial strains and plasmids used in this study Abbreviations: Apr, ampicillin resistance; ATCC, American Type Culture Collection; bla, carbenicillin resistance; Kmr, kanamycin resistance; Tcr, tetracycline resistance. Strain or plasmid Strains P. aeruginosa PAO1 pMP220 3552 pLP0996 pqsR2 L. rhamnosus GG E. coli DH5a Plasmids pMP220 pCR2.1 TOPO

Description

Reference or source

Wild-type PAO1 pmrH–lacZ transcriptional fusion; Tcr pqsA–lacZ transcriptional fusion in pLP170, bla pqsR mutant in PAO1 Intestinal tract isolate

B. W. Holloway* This study McGrath et al. (2004) Xiao et al. (2006) ATCC Invitrogen

Promoterless lacZ vector, IncP; Tcr TOPO TA cloning vector; Apr, Kmr

Spaink et al. (1987) Invitrogen

*Monash University, Clayton, Victoria, Australia. http://mic.sgmjournals.org

Downloaded from www.microbiologyresearch.org by IP: 178.18.19.221 On: Sun, 18 Oct 2015 22:02:08

2827

J. Cummins and others

Fig. 1. (a) Growth curve of PAO1 in Mueller– Hinton broth over 24 h. P. aeruginosa PAO1 was exposed to increasing concentrations of colistin (0.15–2.4 mg ml”1) to determine the MIC. Growth was analysed using Bioscreen C and was measured spectrophotometrically every hour at 600 nm. (b) Quantitative RTPCR analysis of genes expressed in PAO1 exposed to 0.15 mg colistin ml”1 compared with untreated PAO1. The values are the mean and SD of five independent replicates. (c) Semiquantitative RT-PCR analysis of genes expressed in PAO1 exposed to 0.15 and 0.3 mg colistin ml”1 compared with untreated PAO1. The values are the mean and SD of three independent replicates.

based on the PAO1 genome sequence, GenBank accession number NC002516 (Supplementary Table S1). RT-PCRs were performed using 50 ng template cDNA ml21, PCR Master Mix (Promega), and 0.5 mM each primer for 20–25 cycles. The constitutively expressed housekeeping gene proC (Savli et al., 2003) was used as a reference to ensure harmonization of samples in all replicates. 2828

Promoter fusion analysis. The transcriptional fusion of the region upstream of PA3552 (including the transcriptional start site) was generated in pMP220, a plasmid containing a promoterless lacZ gene (Spaink et al., 1987). Specific primers were designed based on the PAO1 genome sequence (Supplementary Table S1). The PCR product was amplified using High Fidelity Taq polymerase (Promega) and


Microbiology 155

Subinhibitory concentrations of colistin induce PQS subsequently cloned into pCR2.1 TOPO according to the manufacturer’s recommendations (Invitrogen). The PCR product was subcloned as an Asp718 and XbaI fragment into KpnI–XbaI sites of pMP220, transformed into DH5a competent E. coli cells, and subsequently electroporated into P. aeruginosa strain PAO1. The untreated and treated (0.3 mg colistin ml21) samples were grown from OD600 0.05 to 0.4 in 40 ml MH. b-Galactosidase activity was measured as described by Miller (1972). All assays were carried out in triplicate and the mean and SE were calculated. Pseudomonas quinolone signal (PQS) extraction and analysis. Cultures of P. aeruginosa were grown in the presence of 0.3 mg colistin

ml21 in MH broth for 20 h. PQS was extracted and analysed by TLC using the protocol described by Fletcher et al. (2007). Motility analysis. Swimming, swarming and twitching ability was analysed on LB 0.3 % (w/v) agar, Eiken 0.5 % (w/v) agar (Eiken Chemical Tokyo) and LB 1 % (w/v) agar plates, respectively. Colistin was added at the following concentrations: 0.15, 0.3 and 0.6 mg ml21. The plates were inoculated with bacteria grown overnight on LB agar using a sterile toothpick. The plates were assessed qualitatively by examining the turbid zone formed by bacterial cells migrating from the inoculation point. Unlike PA14, PAO1 does not typically form large tendrils on swarm agar plates, and this allows measurement of the diameter of the swarm front. The twitching plates were analysed by staining with 0.1 % (w/v) crystal violet. Effect of supernatant from P. aeruginosa on the growth of Lactobacillus spp. The virulence assay to detect the antimicrobial

effects of supernatant from P. aeruginosa on Lactobacillus spp. was adapted from that of Zaborina et al. (2007). In brief, P. aeruginosa PAO1 was grown overnight in MH broth. This was diluted down to OD600 0.05 and colistin was subsequently added at a final concentration of 0.3 and 0.6 mg colistin ml21. After 24 h growth, the cells were pelleted by centrifugation and the supernatant was filtered through a 0.20 mm pore-size filter and maintained on ice. The Lactobacillus strains were grown overnight in DeMan, Rogosa and Sharpe (MRS) broth (Oxoid) and diluted down to OD600 0.01. Cultures (100 ml) were placed in 100-well plates, followed by 100 ml supernatant from P. aeruginosa. Control samples contained 100 ml MH and 0.6 mg colistin ml21. Plates with Lactobacillus spp. were incubated at 37 uC in the BioScreen C plate reader, and growth was monitored by measuring OD600.

RESULTS

An expected feature of the microarray data was the upregulation of genes known to be involved in tolerance to polymyxins (Supplementary Table S2). The pmrHFIJKLM-ugd (PA3552–PA3559) operon has been shown to be induced in response to subinhibitory concentrations of colistin (McPhee et al., 2003), and a sixfold induction of a pmrH–lacZ fusion was observed 3 h post-inoculation (Fig. 2a). Quantitative real-time RT-PCR (qRT-PCR) was carried out on selected targets and alteration of these transcripts in response to 0.15 mg colistin ml21 was confirmed (Fig. 1b). Results obtained from the qRT-PCR were in agreement with the findings from the microarray data, further indicating that it was a biologically relevant dataset. To determine whether an increase in colistin concentration would increase the magnitude of the transcriptional response, we carried out semiquantitative RT-PCR on cells treated with 0.3 mg colistin ml21 (Fig. 1c). Although this approach is less sensitive than qRT-PCR, there was a clear increase in expression of target genes at 0.3 mg colistin ml21 (Fig. 1c), and this concentration was chosen for analysis of colistinresponsive genes identified in the microarray analysis. Colistin exposure triggers induction of 4-hydroxy2-heptylquinoline (HHQ) and PQS

Transcriptome analysis of P. aeruginosa PAO1 exposed to a subinhibitory concentration of colistin There is growing evidence that subinhibitory concentrations of antibiotics can modulate gene expression in bacteria, leading to adaptive responses. To understand the implications of P. aeruginosa exposure to subinhibitory concentrations of colistin, global changes in the pattern of gene expression were assessed by microarray analysis. The experimental approach for the microarray study was designed to encapsulate the direct and indirect effects of exposure to subinhibitory concentrations of colistin, some of which might be expected to be subtle. A Student’s paired t test was performed on the RMA-normalized data, using a 1.5-fold cut-off to ensure detection of these subtle effects, http://mic.sgmjournals.org

which could then be further validated. Approximately 0.5 % (30 out of the 5500 probe sets present on the array) of genes showed significantly altered expression in the colistin-treated sample. Of these, 13 were upregulated and 17 were downregulated (Table 2 and Supplementary Table S2). Data analysis revealed upregulation of genes involved in quorum sensing (QS), LPS modification and biofilm formation, while genes involved in motility and osmotolerance were downregulated (Table 2 and Supplementary Table S2). The low fold-change is indicative of the subtle but biologically significant effect that exposure to subinhibitory concentrations of colistin has on P. aeruginosa cellular physiology. The unexpected prevalence of PQSassociated genes led us to investigate the hypothesis that subinhibitory concentrations of colistin induce the PQS signalling molecule and its regulon in P. aeruginosa.

An unexpected yet striking feature of the transcriptome analysis was the upregulation of genes involved in synthesis of 2-alkyl-4-quinolones (AHQs), including HHQ and PQS (Table 2 and Supplementary Table S2). PQS is one of three QS molecules found in P. aeruginosa along with C4-HSL and C12-HSL, and its expression is usually induced during the onset of the stationary phase of growth. Genes involved in synthesis of AHQs (pqsB) and conversion of HHQ to PQS (pqsH) were upregulated in response to subinhibitory concentrations of colistin (Supplementary Table S2). Furthermore, pqsE, which plays a role in mediating the effects of the PQS signal molecule (Bredenbruch et al., 2006), was also upregulated in response to colistin (Table 2). Upregulation of pqsB and pqsE was confirmed by RTPCR (Fig. 1b, c). As the pqsABCDE operon is known to be


2829


Table 2. Microarray analysis of P. aeruginosa PAO1 exposed to a subinhibitory concentration of colistin Shown are genes with a twofold or greater altered expression (P ¡0.05) in the presence of 0.15 mg colistin ml21. Positive values represent genes that are upregulated and negative values represent genes that are downregulated in the presence of colistin. Gene number Upregulated genes PA1000 PA1667 PA1904 PA2233 PA3334 PA3478 PA4139 Downregulated genes PA0715 PA0720 PA1513 PA2821 PA5372 PA5525

Gene name pqsE phzF* pslC rhlB

betA

Description

Fold change

Quinolone signal response Hypothetical protein Phenazine biosynthesis protein PhzF Probable glycosyltransferase Probable acyl carrier protein Rhamnosyltransferase chain B Hypothetical protein

+2.1 +2.3 +3.6 +2.0 +2.4 +2.3 +2.6

Hypothetical protein Bacteriophage Pf1 Hypothetical protein Probable glutathione S-transferase Choline dehydrogenase Hypothetical protein

22.3 22.2 22.0 22.0 22.3 22.4

*The Affymetrix P. aeruginosa microarray contains probe sets for only one copy of each phzA–G gene.

controlled by the pqsA promoter, a PpqsA–lacZ fusion was used to monitor gene expression. Exposure to subinhibitory concentrations of colistin led to fivefold upregulation of the pqsABCDE operon after 3 h (Fig. 2b). Expression of the pqsABCDE operon remained high relative to untreated cells until entry to stationary phase, at which point the operon was induced in the absence of colistin (Fig. 2b). Increasing the concentration of colistin led to a further increase in the level of pqsABCDE induction (data not shown), indicating that the response is dose-dependent. Induction of PQS biosynthetic gene expression in P. aeruginosa cells in response to subinhibitory concentrations of colistin might be expected to lead to increased production of PQS. Therefore, PQS was extracted from the supernatant of P. aeruginosa cultures that had been treated with subinhibitory concentrations of colistin. Samples were analysed by TLC and increased production of PQS was observed in the colistin-treated samples (Fig. 3). Although there was no observable change in pqsR expression in response to colistin, induction of the PQS biosynthetic genes by colistin requires a functionally intact PqsR (data not shown). PqsR (also called MvfR) is a LysRtype regulator that binds directly to the pqsA and phnA promoters and positively influences expression of pqsABCDE and phnAB (Cao et al., 2001; Wade et al., 2005; Xiao et al., 2006). Expression of pqsR is positively influenced by LasR and negatively influenced by RhlR, creating a regulatory loop between the three QS circuits (Wade et al., 2005). Promoter fusion analysis revealed that the colistin-mediated PQS gene expression was not a direct result of upregulation of the LasIR and RhlIR QS systems, 2830

which were unchanged in the presence of subinhibitory concentrations of colistin (data not shown). Physiological consequences of colistin-induced AHQs To date, P. aeruginosa and Burkholderia spp. are the only organisms in which AHQs have been identified (Dubern & Diggle, 2008), and the antimicrobial properties of these compounds towards Gram-positive bacteria have been reported (Hays et al., 1945; Wells et al., 1952). However, the role of PQS in this is uncertain as a DpqsE mutant had the same killing effect as PA14 wild-type (De´ziel et al., 2004). As induction of AHQs may increase antimicrobial activity against Gram-positive bacteria, supernatants from colistin-treated cultures were tested for inhibition of Lactobacillus rhamnosus GG growth. While colistin alone did not affect growth, P. aeruginosa culture supernatants had a slight inhibitory effect on the growth of L. rhamnosus GG. Addition of a subinhibitory concentration of colistin to P. aeruginosa cultures resulted in supernatant that had a marked inhibitory effect on the growth of L. rhamnosus GG (Fig. 4a). Although there was no significant increase in growth inhibition in the presence of culture supernatants treated with 0.3 mg colistin ml21, a pronounced inhibitory effect was observed upon addition of supernatant from cells treated with 0.6 mg colistin ml21. Increased inhibition was not observed upon addition of supernatant from colistin-treated pqsR (Fig. 4b) or pqsA (data not shown) mutant cells. This would suggest that the increased antimicrobial activity is a direct result of increased AHQ


Microbiology 155


Fig. 3. PQS extracted after 20 h of growth and analysed by TLC. Lanes: a, untreated PAO1; b, treated with 0.3 mg colistin ml”1; c, anthranilate control. Compound B is an unidentified organic molecule co-purified with PQS.

Fig. 2. (a) Effect of 0.3 mg colistin ml”1 on pmrH–lacZ expression in P. aeruginosa PAO1; white bar, untreated; black bar, treated. (b) Effect of 0.3 mg colistin ml”1 on pqsA–lacZ expression in P. aeruginosa PAO1; white bars, untreated; black bars, treated. Values are the mean and SE of triplicate measurements.

production in response to subinhibitory concentrations of colistin. As PQS is maximally produced at the onset of the stationary phase of growth, it may act as a secondary regulatory signal for a subset of QS-controlled genes (De´ziel et al., 2004). Therefore, induction of PQS in the early exponential phase might be expected to affect the cellular physiology of P. aeruginosa. Indeed, an interesting feature of PQS is that, unlike the acyl homoserine lactone (AHL) signal molecules, addition of exogenous PQS can overcome density-dependent gene expression in P. aeruginosa (Diggle et al., 2003). Recent work by Haüssler and http://mic.sgmjournals.org

colleagues has indicated that PQS predisposes P. aeruginosa to exogenous stress (Haüssler & Becker, 2008). To study the effects of colistin-induced PQS on P. aeruginosa fitness, cells were challenged with both subinhibitory concentrations of colistin and purified PQS. An increased lag-time relative to addition of either colistin or PQS alone was observed (Fig. 5a). Under the same conditions, no effect was observed in the pqsR mutant (Fig. 5b). Increasing the concentration of exogenous PQS in the presence of colistin led to an increased lag-time in the pqsR mutant. However, growth was also inhibited in the presence of PQS alone at these higher concentrations (data not shown), suggesting that a threshold had been reached. It would therefore appear that colistin-induced PQS, once accumulated above a threshold level, has a negative effect on the cellular physiology of P. aeruginosa. As well as playing a role in signalling and virulence, PQS is also a critical component in the formation of biofilms in P. aeruginosa. Expressed in cells that form the stalk of maturing biofilms, PQS contributes to the formation of the mushroom structure and secretion of extracellular DNA (Yang et al., 2007). Transcriptome analysis revealed regulation of several genes involved in biofilm formation and maturation in response to subinhibitory concentrations of colistin (Fig. 1c, Table 2 and Supplementary Table S2). Phentoypic assays revealed that both swarming (44 % inhibition) and swimming motility (31 % inhibition) were affected; however, no effect on twitching motility was observed (Supplementary Table S3). To examine the possibility that colistin exposure influences biofilm formation in P. aeruginosa, crystal violet assays were carried out on static cultures in the presence of subinhibitory concentrations of colistin. No difference in attachment or


2831


Fig. 4. Exposure of PAO1 to subinhibitory concentrations of colistin increases the inhibiting effect of its supernatant (SN) on the growth of probiotic organisms. (a) Addition of SN extracted from PAO1 cells treated with 0.6 mg colistin ml”1 resulted in marked growth inhibition of L. rhamnosus GG. (b) Addition of SN extracted from pqsR mutant cells treated with 0.6 mg colistin ml”1 did not cause this growth-inhibiting effect. This experiment was carried out three times with independent cultures and the outcome of one of the representative experiments is shown.

pellicle formation was observed. Flow-cell analysis also revealed no difference in biomass or ability to form mushroom structures in the presence of the antimicrobial (data not shown). This may be a reflection of the distinct environments and expression profiles that exist in planktonic cultures and mature biofilms (Waite et al., 2005, 2006; Whiteley et al., 2001). It must also be considered that other genes, which are not responsive to subinhibitory concentrations of colistin under the conditions tested, are required for induction of the biofilm mode of growth.

the phenazine biosynthesis operon (Table 2). qRT-PCR confirmed increased expression of phzF and this was found to coincide with an increase in pyocyanin production, particularly evident in the earlier stages of growth (Fig. 6). Induction of the phzA–G operon by colistin provides evidence that exposure to subinhibitory concentrations influences P. aeruginosa pathogenicity, a finding that has implications for the use of this antibiotic as a monotherapeutic option.

DISCUSSION Exposure to subinhibitory concentrations of colistin results in increased pyocyanin production in P. aeruginosa PQS regulates the production of several virulence factors in P. aeruginosa, including elastase, rhamnolipids and phenazines (De´ziel et al., 2004). Enhanced production of phenazines in response to subinhibitory concentrations of other antibiotics has already been reported (Mitova et al., 2008; Shen et al., 2008). Exposure to subinhibitory concentrations of colistin resulted in upregulation of 2832

Colistin is considered one of the last reserve agents for treating Gram-negative MDR bacterial infections. To develop an effective response to the emergence of colistin resistance, it is imperative that targets of the antibiotic are identified and characterized. While the mechanism of polymyxin-mediated killing has been studied by several groups, it is becoming increasingly clear that suboptimal doses of antibiotics modulate markedly different changes in bacterial gene expression (Brazas & Hancock, 2005; Gerber et al., 2008; Labro et al., 1992; Skindersoe et al., 2008). The


Microbiology 155


Fig. 5. (a) Growth kinetics of P. aeruginosa PAO1 in the presence of colistin (0.6 mg ml”1) and PQS (1 ml ml”1). In the presence of both colistin and PQS there is an inhibiting effect on growth. (b) Growth kinetics of P. aeruginosa pqsR mutant cells under the same conditions. There was no effect on growth when PQS was present in the media while there was a slight effect on growth in the presence of colistin. This experiment was repeated three times with independent cultures and the outcome of one representative experiment is shown.

Fig. 6. Quantification of pyocyanin. White bars, untreated; black bars, treated with 0.3 mg colistin ml”1. Results are the mean and SE of triplicate measurements. http://mic.sgmjournals.org


2833


lack of definitive data regarding the pharmacokinetics and pharmacodynamics of colistin necessitates elucidating the subinhibitory colistin signature as a matter of priority. Antibiotics are increasingly being thought of as hormetic compounds; in other words, they have contrasting effects at low and high concentrations. Exposure to subinhibitory concentrations is not necessarily detrimental to susceptible bacteria and may in some cases result in phenotypes that are advantageous (Linares et al., 2006). Biofilm formation, cytotoxicity, motility, expression of virulence determinants and QS may be induced or repressed by bacteria in response to subinhibitory concentrations of different classes of antimicrobials (Brazas & Hancock, 2005; Linares et al., 2006; Skindersoe et al., 2008). The transcriptional changes induced tend to be both speciesand antibiotic-specific. For instance, while tobramycin and colistin may inhibit biofilm formation in Klebsiella pneumoniae, tobramycin increases biofilm formation in P. aeruginosa and E. coli (Hoffman et al., 2005; Hostacka & Ciznar, 2008; Linares et al., 2006). Tetracycline increases the cytotoxicity of P. aeruginosa, while ciprofloxacin induces the R2/F2 pyocins in P. aeruginosa, causing susceptibility to fluoroquinolones (Brazas & Hancock, 2005; Linares et al., 2006). Plate assays reveal increased expression of lasIR in E. coli in the presence of both colistin and polymyxin B, while expression is unchanged in liquid cultures (Goh et al., 2002). In contrast, several recent studies have reported downregulation of the rhlIR and lasIR N-acylhomoserine lactone signalling systems in P. aeruginosa in response to sublethal concentrations of LL37, azithromycin, ceftazidime and ciprofloxacin (Nalca et al., 2006; Overhage et al., 2008; Skindersoe et al., 2008). Therefore, the emerging model is one where subinhibitory concentrations of antibiotics elicit a niche-specific response in bacteria, which may contribute to adaptation within a natural ecosystem. Revealing the induction of the PQS virulence regulon in response to subinhibitory concentrations of colistin is an important contribution to this emerging field. The transcriptional response to subinhibitory concentrations of antibiotics can often involve genes that appear to be unrelated to the previously defined targets of the antibiotic. Polymyxins are known to displace the calcium and magnesium bridges that stabilize the LPS (Evans et al., 1999; Hancock, 1997b; Hermsen et al., 2003; Tam et al., 2005). Both pmrHFIJKLM and PQS are induced in the exponential growth phase when P. aeruginosa is grown in magnesium-limiting media (Guina et al., 2003). Therefore, it is possible that the displacement of Mg2+ following colistin binding is responsible, at least in part, for the observed transcriptional changes. Furthermore, ~60 % of PQS appears to be cell wall-associated (Diggle et al., 2007; Le´pine et al., 2003), and this may also be a critical factor in the response. A recent study has shown that inactivation of bptS (PA1396), a sensor kinase, results in upregulation of PA4773, a predicted polyamine biosynthesis gene, and increased tolerance to colistin and polymyxin B (Ryan et al., 2834

2008). Understanding how subinhibitory concentrations of antimicrobials elicit changes in bacterial gene expression will require a comprehensive analysis of the nature of the interaction and identification of all sensory and regulatory components involved. To address the question of how exposure to colistin may influence P. aeruginosa within a natural ecosystem, the consequences of PQS induction were assessed. Subinhibitory concentrations of colistin result in a PqsRdependent increase in antimicrobial activity against L. rhamnosus GG. A recent report by Zaborina and colleagues has shown similar results using a subinhibitory concentration of the K-opoid dynorphin (Zaborina et al., 2007). Accumulation of PQS also has a significant effect on the cellular physiology of P. aeruginosa. Treatment of P. aeruginosa with both PQS and colistin resulted in a PqsR-dependent enhanced inhibitory effect on growth relative to either PQS or colistin alone. Haüssler and colleagues propose that PQS mediates an endogenous stress that predisposes P. aeruginosa to subsequent external stresses (Haüssler & Becker, 2008). They suggest that PQS moderates the P. aeruginosa population through its dual function as both pro- and anti-oxidant. The increased susceptibility may also be due to the interaction of PQS with the LPS layer of the cell, whereby it maintains the LPS in a well-ordered state (Mashburn-Warren et al., 2008). Alterations in membrane fluidity are known to contribute to protection against antimicrobial peptides (CAMPs), with fluid membranes being more resistant (Peschel, 2002). The contrasting roles of PQS highlight the need for a systems biology approach in studying the effect of subinhibitory concentrations of antibiotics on mixed microbial communities. While an increase in PQS may enable P. aeruginosa to adapt in a multi-species ecosystem, it also has important implications for the use of colistin as a monotherapeutic strategy. PQS controls a battery of genes necessary for virulence and biofilm formation (Diggle et al., 2007). Its production results in upregulation of virulence factors such as phenazines and rhamnolipid, which are important for pathogenesis in several virulence models (Gallagher & Manoil, 2001; Mahajan-Miklos et al., 1999; Mavrodi et al., 2001; Pesci et al., 1999; Rahme et al., 2000). PQS is also known to promote biofilm formation and DNA release in P. aeruginosa, when iron is limiting (Yang et al., 2007). While our genetic and phenotypic data would suggest that subinhibitory concentrations of colistin influence biofilm formation in P. aeruginosa, no significant difference was observed in biomass using flow cells (data not shown). It is important to note that gene expression varies in P. aeruginosa planktonic cultures relative to the biofilm mode of growth (Waite et al., 2005, 2006; Whiteley et al., 2001). Furthermore, culture conditions may influence the patterns of gene expression in response to subinhibitory concentrations of colistin. Exposure to different nutrients and media constituents affects cellular physiology and may influence the response of P. aeruginosa to colistin. Indeed,


Microbiology 155


while this study provides clear evidence that exposure to subinhibitory concentrations of colistin induces PQS production in P. aeruginosa, the extent to which all metabolic enzymes for PQS synthesis are influenced remains to be elucidated. Subinhibitory concentrations of tetracycline (1/16 MIC) have recently been found to positively influence expression of phzA1, phzA2, rhlA, lasB, exoS, exoY and rhlR in P. aeruginosa, whereas rifampicin (1/4 MIC) reduces expression of rhlA, lasB and phzA (Liang et al., 2008). A novel regulator of the PQS regulon has been identified in a YebC family protein, PA0964, which was found to regulate expression of the PQS regulon in response to subinhibitory concentrations of tetracycline. Surprisingly, in that study, polymyxin did not induce expression of phzA1, phzA2 or rhlA, although neither the concentration of antibiotic used in these assays nor the specific polymyxin used was stated. As we have shown, induction of the PQS biosynthetic operon is dose-dependent, with increased activation as the concentration approaches sublethal levels. It is probable, therefore, that the concentration used in the study described by Liang and colleagues was simply too low to elicit activation of this system. Microarray analysis has also revealed that exposure to azithromycin (1/64 MIC) increases pqsA expression in P. aeruginosa (Nalca et al., 2006; Skindersoe et al., 2008), suggesting that this critical component of the QS network is responsive to a wide class of compounds, perhaps reflecting an ability to command a rapid adaptive response in diverse ecological niches.

Research Board (RP/2004/145; RP/2006/271; RP/2007/290), the Environmental Protection Agency (EPA 2006-PhD-S-21) and the Higher Education Authority of Ireland (PRTLI3).

REFERENCES Ainsworth, G. C., Brown, A. M. & Brownlee, G. (1947). Aerosporin.

An antibiotic produced by Bacillus aerosporus. Nature 160, 263. Beringer, P. (2001). The clinical use of colistin in patients with cystic

fibrosis. Curr Opin Pulm Med 7, 434–440. Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. (1983). Infections

caused by Pseudomonas aeruginosa. Rev Infect Dis 5, 279–313. Bonomo, R. A. & Szabo, D. (2006). Mechanisms of multidrug

resistance in Acinetobacter species and Pseudomonas aeruginosa. Clin Infect Dis 43 (suppl 2), S49–S56. Brazas, M. D. & Hancock, R. E. (2005). Ciprofloxacin induction of a

susceptibility determinant in Pseudomonas aeruginosa. Antimicrob Agents Chemother 49, 3222–3227. Bredenbruch, F., Geffers, R., Nimtz, M., Buer, J. & Haüssler, S. (2006). The Pseudomonas aeruginosa quinolone signal (PQS) has an

iron-chelating activity. Environ Microbiol 8, 1318–1329. Brown, J. M., Dorman, D. C. & Roy, L. P. (1970). Acute renal failure

due to overdosage of colistin. Med J Aust 2, 923–924. Cao, H., Krishnan, G., Goumnerov, B., Tsongalis, J., Tompkins, R. & Rahme, L. G. (2001). A quorum sensing-associated virulence gene of

Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism. Proc Natl Acad Sci U S A 98, 14613–14618. Davies, J., Spiegelman, G. B. & Yim, G. (2006). The world of

subinhibitory antibiotic concentrations. Curr Opin Microbiol 9, 445– 453.

Conclusion

Denton, M., Kerr, K., Mooney, L., Keer, V., Rajgopal, A., Brownlee, K., Arundel, P. & Conway, S. (2002). Transmission of colistin-resistant

The results presented in this paper offer an important contribution to the field of subinhibitory antibiotics and have clear implications for the treatment of P. aeruginosa infections with colistin. Concerns have already been raised about colistin monotherapy, not least with the emergence of heteroresistance among Gram-negative pathogens (Hawley et al., 2008; Li et al., 2006). In the light of the role played by PQS in P. aeruginosa infection and its induction by subinhibitory concentrations of colistin, combinatorial strategies may have to be considered. An integrated approach is required to fully understand the effect of subinhibitory concentrations of colistin on P. aeruginosa in mixed microbial communities, including the CF lung. This knowledge is crucial for the successful development and application of colistin therapies in the future.

Pseudomonas aeruginosa between patients attending a pediatric cystic fibrosis center. Pediatr Pulmonol 34, 257–261.

hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2heptylquinoline in cell-to-cell communication. Proc Natl Acad Sci U S A 101, 1339–1344. Diggle, S. P., Winzer, K., Chhabra, S. R., Worrall, K. E., Camara, M. & Williams, P. (2003). The Pseudomonas aeruginosa quinolone signal

molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR. Mol Microbiol 50, 29–43. Diggle, S. P., Matthijs, S., Wright, V. J., Fletcher, M. P., Chhabra, S. R., Lamont, I. L., Kong, X., Hider, R. C., Cornelis, P. & other authors (2007). The Pseudomonas aeruginosa 4-quinolone signal molecules

HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment. Chem Biol 14, 87–96. Dubern, J. F. & Diggle, S. P. (2008). Quorum sensing by 2-alkyl-4-

ACKNOWLEDGEMENTS We acknowledge David Clarke and Claire Adams for useful advice and discussions. This research was supported in part by grants awarded by the European Commission (MTKD-CT-2006-042062; O36314), the Science Foundation of Ireland (SFI 02/IN.1/B1261; 04/ BR/B0597), the Department of Agriculture and Food (DAF RSF 06 321; DAF RSF 06 377), the Irish Research Council for Science, Engineering and Technology (IRCSET) (05/EDIV/FP107), the Health http://mic.sgmjournals.org

De´ziel, E., Le´pine, F., Milot, S., He, J., Mindrinos, M. N., Tompkins, R. G. & Rahme, L. G. (2004). Analysis of Pseudomonas aeruginosa 4-

quinolones in Pseudomonas aeruginosa and other bacterial species. Mol Biosyst 4, 882–888. Evans, M. E., Feola, D. J. & Rapp, R. P. (1999). Polymyxin B sulfate

and colistin: old antibiotics for emerging multiresistant Gramnegative bacteria. Ann Pharmacother 33, 960–967. Fletcher, M. P., Diggle, S. P., Camara, M. & Williams, P. (2007).

Biosensor-based assays for PQS, HHQ and related 2-alkyl-4quinolone quorum sensing signal molecules. Nat Protoc 2, 1254–1262.


2835

J. Cummins and others Frederiksen, B., Koch, C. & Høiby, N. (1999). Changing epidemiology

of Pseudomonas aeruginosa infection in Danish cystic fibrosis patients (1974–1995). Pediatr Pulmonol 28, 159–166. Gallagher, L. A. & Manoil, C. (2001). Pseudomonas aeruginosa PAO1

kills Caenorhabditis elegans by cyanide poisoning. J Bacteriol 183, 6207–6214. Garnacho-Montero, J., Ortiz-Leyba, C., Jimeńez-Jimeńez, F. J., Barrero-Almodo´var, A. E., Garcıá-Garmendia, J. L., BernabeuWittel, I. M., Gallego-Lara, S. L. & Madrazo-Osuna, J. (2003).

Treatment of multidrug-resistant Acinetobacter baumannii ventilatorassociated pneumonia (VAP) with intravenous colistin: a comparison with imipenem-susceptible VAP. Clin Infect Dis 36, 1111–1118. Gerber, M., Walch, C., Lo¨ffler, B., Tischendorf, K., Reischl, U. & Ackermann, G. (2008). Effect of sub-MIC concentrations of

metronidazole, vancomycin, clindamycin and linezolid on toxin gene transcription and production in Clostridium difficile. J Med Microbiol 57, 776–783. Goh, E. B., Yim, G., Tsui, W., McClure, J., Surette, M. G. & Davies, J. (2002). Transcriptional modulation of bacterial gene expression by

subinhibitory concentrations of antibiotics. Proc Natl Acad Sci U S A 99, 17025–17030. Govan, J. R. & Deretic, V. (1996). Microbial pathogenesis in cystic

fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60, 539–574. Guina, T., Purvine, S. O., Yi, E. C., Eng, J., Goodlett, D. R., Aebersold, R. & Miller, S. I. (2003). Quantitative proteomic analysis indicates

increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways. Proc Natl Acad Sci U S A 100, 2771–2776. Hanberger, H., Diekema, D., Fluit, A., Jones, R., Struelens, M., Spencer, R. & Wolff, M. (2001). Surveillance of antibiotic resistance in

Li, J., Rayner, C. R., Nation, R. L., Owen, R. J., Spelman, D., Tan, K. E. & Liolios, L. (2006). Heteroresistance to colistin in multidrug-resistant

Acinetobacter baumannii. Antimicrob Agents Chemother 50, 2946– 2950. Liang, H., Li, L., Dong, Z., Surette, M. G. & Duan, K. (2008). The YebC

family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production. J Bacteriol 190, 6217–6227. Linares, J. F., Gustafsson, I., Baquero, F. & Martinez, J. L. (2006).

Antibiotics as intermicrobial signaling agents instead of weapons. Proc Natl Acad Sci U S A 103, 19484–19489. Mahajan-Miklos, S., Tan, M. W., Rahme, L. G. & Ausubel, F. M. (1999). Molecular mechanisms of bacterial virulence elucidated using

a Pseudomonas aeruginosa–Caenorhabditis elegans pathogenesis model. Cell 96, 47–56. Markou, N., Apostolakos, H., Koumoudiou, C., Athanasiou, M., Koutsoukou, A., Alamanos, I. & Gregorakos, L. (2003). Intravenous

colistin in the treatment of sepsis from multi-resistant Gram-negative bacilli in critically ill patients. Crit Care 7, R78–R83. Mashburn-Warren, L., Howe, J., Garidel, P., Richter, W., Steiniger, F., Roessle, M., Brandenburg, K. & Whiteley, M. (2008). Interaction of

quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation. Mol Microbiol 69, 491–502. Mavrodi, D. V., Bonsall, R. F., Delaney, S. M., Soule, M. J., Phillips, G. & Thomashow, L. S. (2001). Functional analysis of genes for

biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 183, 6454–6465. McGrath, S., Wade, D. S. & Pesci, E. C. (2004). Dueling quorum

European ICUs. J Hosp Infect 48, 161–176.

sensing systems in Pseudomonas aeruginosa control the production of the Pseudomonas quinolone signal (PQS). FEMS Microbiol Lett 230, 27–34.

Hancock, R. E. (1997a). The bacterial outer membrane as a drug

McPhee, J. B., Lewenza, S. & Hancock, R. E. (2003). Cationic

barrier. Trends Microbiol 5, 37–42.

antimicrobial peptides activate a two-component regulatory system, PmrA–PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50, 205–217.

Hancock, R. E. (1997b). Peptide antibiotics. Lancet 349, 418–422. Haüssler, S. & Becker, T. (2008). The pseudomonas quinolone signal

(PQS) balances life and death in Pseudomonas aeruginosa populations. PLoS Pathog 4, e1000166. Hawley, J. S., Murray, C. K. & Jorgensen, J. H. (2008). Colistin

heteroresistance in Acinetobacter and its association with previous colistin therapy. Antimicrob Agents Chemother 52, 351–352. Hays, E. E., Wells, I. C., Katzman, P. A., Cain, C. K., Jacobs, F. A., Thayer, S. A., Doisy, E. A., Gaby, W. L., Roberts, E. C. & other authors (1945). Antibiotic substances produced by Pseudomonas aeruginosa.

J Biol Chem 159, 725–750. Hermsen, E. D., Sullivan, C. J. & Rotschafer, J. C. (2003). Polymyxins:

pharmacology, pharmacokinetics, pharmacodynamics, and clinical applications. Infect Dis Clin North Am 17, 545–562. Hoffman, L. R., D’Argenio, D. A., MacCoss, M. J., Zhang, Z., Jones, R. A. & Miller, S. I. (2005). Aminoglycoside antibiotics induce bacterial

biofilm formation. Nature 436, 1171–1175. Hostacka, A. & Ciznar, I. (2008). Aminoglycosides and colistin inhibit

biofilm formation in Klebsiella pneumoniae. )..Epidemiol Mikrobiol Imunol 57, 101–105. Labro, M. T., el Benna, J. & Jemni, A. (1992 ). Alteration of bacteria

induced by subinhibitory concentrations of cefixime: consequences on bactericidal activity of human polynuclear neutrophils. Pathol Biol (Paris) 40, 427–432. Le´pine, F., De´ziel, E., Milot, S. & Rahme, L. G. (2003). A stable isotope

dilution assay for the quantification of the Pseudomonas quinolone signal in Pseudomonas aeruginosa cultures. Biochim Biophys Acta 1622, 36–41. 2836

Miller, J. H. (1972). Experiments in Molecular Genetics Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory. Mitova, M. I., Lang, G., Wiese, J. & Imhoff, J. F. (2008). Subinhibitory

concentrations of antibiotics induce phenazine production in a marine Streptomyces sp. J Nat Prod 71, 824–827. Nalca, Y., Ja¨nsch, L., Bredenbruch, F., Geffers, R., Buer, J. & Haüssler, S. (2006). Quorum-sensing antagonistic activities of

azithromycin in Pseudomonas aeruginosa PAO1: a global approach. Antimicrob Agents Chemother 50, 1680–1688. Nikaido, H. (1989). Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrob Agents Chemother 33, 1831– 1836. Oh, J. T., Cajal, Y., Skowronska, E. M., Belkin, S., Chen, J., Van Dyk, T. K., Sasser, M. & Jain, M. K. (2000). Cationic peptide antimicrobials

induce selective transcription of micF and osmY in Escherichia coli. Biochim Biophys Acta 1463, 43–54. Overhage, J., Campisano, A., Bains, M., Torfs, E. C., Rehm, B. H. & Hancock, R. E. (2008). The human host defence peptide LL-37

prevents bacterial biofilm formation. Infect Immun 76, 4176–4182. Peschel, A. (2002). How do bacteria resist human antimicrobial

peptides? Trends Microbiol 10, 179–186. Pesci, E. C., Milbank, J. B., Pearson, J. P., McKnight, S., Kende, A. S., Greenberg, E. P. & Iglewski, B. H. (1999). Quinolone signaling in the

cell-to-cell communication system of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 96, 11229–11234.


Microbiology 155

Subinhibitory concentrations of colistin induce PQS Rahal, J. J. (2006). Novel antibiotic combinations against infections

with almost completely resistant Pseudomonas aeruginosa and Acinetobacter species. Clin Infect Dis 43 (suppl. 2), S95–S99. Rahme, L. G., Ausubel, F. M., Cao, H., Drenkard, E., Goumnerov, B. C., Lau, G. W., Mahajan-Miklos, S., Plotnikova, J., Tan, M. W. & other authors (2000). Plants and animals share functionally common

bacterial virulence factors. Proc Natl Acad Sci U S A 97, 8815–8821. Ryan, R. P., Fouhy, Y., Garcia, B. F., Watt, S. A., Niehaus, K., Yang, L., Tolker-Nielsen, T. & Dow, J. M. (2008). Interspecies signalling via the

Stenotrophomonas maltophilia diffusible signal factor influences biofilm formation and polymyxin tolerance in Pseudomonas aeruginosa. Mol Microbiol 68, 75–86. Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U. & Vahaboglu, H. (2003). Expression stability of six housekeeping genes:

a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 52, 403–408. Shen, L., Shi, Y., Zhang, D., Wei, J., Surette, M. G. & Duan, K. (2008).

Modulation of secreted virulence factor genes by subinhibitory concentrations of antibiotics in Pseudomonas aeruginosa. J Microbiol 46, 441–447. Skindersoe, M. E., Alhede, M., Phipps, R., Yang, L., Jensen, P. O., Rasmussen, T. B., Bjarnsholt, T., Tolker-Nielsen, T., Høiby, N. & Givskov, M. (2008). Effects of antibiotics on quorum sensing in

Pseudomonas aeruginosa. Antimicrob Agents Chemother 52, 3648–3663. Spaink, H. P., Okker, R. J. H., Wijffelman, C. A., Pees, E. & Lugtenberg, B. J. J. (1987). Promoters of nodulation region of the

Rhizobium leguminosarum Sym plasmid pRL1J1. Plant Mol Biol 9, 27–39. Tam, V. H., Schilling, A. N., Vo, G., Kabbara, S., Kwa, A. L., Wiederhold, N. P. & Lewis, R. E. (2005). Pharmacodynamics of

polymyxin B against Pseudomonas aeruginosa. Antimicrob Agents Chemother 49, 3624–3630.

to a proline-rich antimicrobial peptide. Antimicrob Agents Chemother 48, 3260–3267. Wade, D. S., Calfee, M. W., Rocha, E. R., Ling, E. A., Engstrom, E., Coleman, J. P. & Pesci, E. C. (2005). Regulation of Pseudomonas

quinolone signal synthesis in Pseudomonas aeruginosa. J Bacteriol 187, 4372–4380. Waite, R. D., Papakonstantinopoulou, A., Littler, E. & Curtis, M. A. (2005). Transcriptome analysis of Pseudomonas aeruginosa growth:

comparison of gene expression in planktonic cultures and developing and mature biofilms. J Bacteriol 187, 6571–6576. Waite, R. D., Paccanaro, A., Papakonstantinopoulou, A., Hurst, J. M., Saqi, M., Littler, E. & Curtis, M. A. (2006). Clustering of Pseudomonas

aeruginosa transcriptomes from planktonic cultures, developing and mature biofilms reveals distinct expression profiles. BMC Genomics 7, 162. Wells, I. C., Elliott, W. H., Thayer, S. A. & Doisy, E. A. (1952).

Ozonization of some antibiotic substances produced by Pseudomonas aeruginosa. J Biol Chem 196, 321–330. Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R., Teitzel, G. M., Lory, S. & Greenberg, E. P. (2001). Gene expression in

Pseudomonas aeruginosa biofilms. Nature 413, 860–864. Xiao, G., He, J. & Rahme, L. G. (2006). Mutation analysis of the

Pseudomonas aeruginosa mvfR and pqsABCDE gene promoters demonstrates complex quorum-sensing circuitry. Microbiology 152, 1679–1686. Yang, L., Barken, K. B., Skindersoe, M. E., Christensen, A. B., Givskov, M. & Tolker-Nielsen, T. (2007). Effects of iron on DNA

release and biofilm development by Pseudomonas aeruginosa. Microbiology 153, 1318–1328. Zaborina, O., Le´pine, F., Xiao, G., Valuckaite, V., Chen, Y., Li, T., Ciancio, M., Zaborin, A., Petrof, E. O. & other authors (2007).

Dynorphin activates quorum sensing quinolone signaling in Pseudomonas aeruginosa. PLoS Pathog 3, e35.

Tomasinsig, L., Scocchi, M., Mettulio, R. & Zanetti, M. (2004).

Genome-wide transcriptional profiling of the Escherichia coli response

http://mic.sgmjournals.org

Edited by: W. J Quax


2837