Exogenous Pyocyanin Alters Pseudomonas aeruginosa Susceptibility to Ciprofloxacin

This in vitro cell-based study identified the contributing role of pyocyanin in th e resistance of Pseudomonas aeruginosa to ciprofloxacin. Problem statement: P. aeruginosa is the major pathogen in the Cystic Fibrosis (CF) lung with pyoc yanin being a critical component of its virulence. Prevalence is high and, once acquired, chronic infe ction is difficult to eliminate. Ciprofloxacin rema ins a crucial oral agent effective against P. aeruginosa, but resistance is increasingly reported. Approach: Here we examined the extent to which exogenously ad ded pyocyanin affected P. aeruginosa susceptibility to ciprofloxacin and the contributio n f altered efflux activity and biofilm production with the aim of ultimately increasing sensitivity. Results: Metabolic conversion of resazurin to resorufin was used as an index of bacterial cell gr owth while fluorescent measurement of acriflavine efflux and crystal violet staining was used as mark ers of efflux activity and biofilm production, respectively. Pyocyanin (100 μM) added exogenously decreased susceptibility of tw P. aeruginosa strains, PAO1 and ATCC 27853 to ciprofloxacin at 12 5 and 500 μg L, respectively. Exogenously added pyocyanin decreased efflux activity in both s trains while biofilm production was significantly increased. Conclusion: We conclude that increased biofilm production may c ontribute to the observed decreased susceptibility of P. aeruginosa to ciprofloxacin. Ciprofloxacin is a crucial orally effectively agent against P. aeruginosa with resistance being increasingly reported. This i n tial study highlights a potential mechanism that may underlie this resistan ce which may be of clinical interest. Further studi es using additional antibiotics and the pyocyanin prec u sor 1-hydroxyphenazine are required.


INTRODUCTION
Cystic Fibrosis (CF) is one of the most frequently occurring fatal genetic diseases seen among the Caucasian population (Lau et al., 2004;Grant et al., 2009). Infection with Pseudomonas aeruginosa is responsible for the premature death of over 80% of CF patients (Lau et al., 2004) and a large proportion of the morbidity associated with the disease (Rosenfeld et al., 2003). Although current antibiotic therapeutic strategies have decreased the morbidity and mortality associated with CF, P. aeruginosa permanently colonises these patients' lungs, with eradication almost impossible (Lau et al., 2004). Ciprofloxacin is a crucial orally effectively agent against P. aeruginosa although resistance is increasingly reported (Hodson et al., 1987;Masaadeh and Jaran, 2009). P. aeruginosa populations in sputum samples taken from CF patients are mixed with respect to both genotype and phenotype, even when isolates represent the same clone (Fothergill et al., 2007). The spread of a drug-resistant strain of P. aeruginosa [named the Liverpool Epidemic Strain (LES)] in CF patients has been demonstrated by Cheng et al. (1996) whilst Fothergill et al. (2007) further showed that some of these isolates express an unusual phenotype characterised by the early and over-expression of QSregulated virulence genes, including those encoding the secretion of the virulence-factor pyocyanin, referred to as the Over-Production Phenotype (OP) (Fothergill et al., 2007). Pyocyanin has been shown to have a number of toxic effects on the respiratory system, with initial research focussed on the pro-oxidant properties of pyocyanin which results in morphological changes culminating in cellular damage (Dietrich et al., 2006). Dietrich et al. (2006) recently highlighted the signalling events associated with pyocyanin in P. aeruginosa strains PAO-1 and PA14. Pyocyanin was shown to act as a physiological signal for the upregulation of certain Quorum Sensing (QS) controlled genes during stationary phase, including mexGHI-opmD genes involved in efflux, redox process and iron acquisition (Dietrich et al., 2006). Fothergill et al. (2007) also found interesting correlations between the OP phenotype and resistance or susceptibility patterns to various antipseudomonal antibiotics (Fothergill et al., 2007). The author suggests that pyocyanin itself may play a direct role in the observed link between antimicrobial susceptibility and the OP phenotype (Fothergill et al., 2007).
This study was conducted to examine the effect of exogenously added pyocyanin in P. aeruginosa PAO1 and ATCC 27853 susceptibility to the quinolone antibiotic, ciprofloxacin. Furthermore we have investigated the contribution exogenously added pyocyanin has towards altered efflux activity and biofilm production.

MATERIALS AND METHODS
Pyocyanin was purchased from Sapphire Bioscience (Redfern, NSW, Australia). All other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Preparation of cultures:
Wild-type Pseudomonas aeruginosa strains PAO-1 and ATCC 27853 were stored at -70°C in glycerol and subcultured onto Luria-Bertani (LB) agar (Oxoid, Basingstoke, UK) slopes before testing. Overnight cultures of P. aeruginosa were prepared in Luria-Bertani (LB) broth and incubated for 18 h at 37°C on a shaker at 200 rpm.

Assessment of bacterial cell growth:
In order to detect subtle differences in P. aeruginosa PAO1 and ATCC 27853 sensitivity to ciprofloxacin methods were adapted from the method described by Sarker et al. (2007) using resazurin in a 96-well microtitre plate format. Briefly, the inoculum was adjusted to a final density of 5×10 5 CFU mL −1 in a 96-well plate and incubated with 100 µM pyocyanin. Following the addition of 0.5% resazurin ciprofloxacin (31-1000 µg L −1 ) was added and plates were incubated at 35°C, ambient air. After 20 h resorufin fluorescence was measured (Excitation 535 nm; Emission 590 nm) using a Fluoroskan Ascent microplate fluorometer. Appropriate cell free controls were included to test for potential interaction between resazurin and pyocyanin.
Efflux activity assay: The method for assessing efflux activity was adopted from the protocol of Poole and Srikumar (2001). Briefly, cultures were adjusted to 1×10 8 CFU mL −1 and incubated with 20 µM carbonylcyanide-M-Chlorophenylhydrazone (CCCP) and acriflavine. Phenylalanine-arginine b-naphthylamide (PabN) (40 µM) was included as positive controls for the inhibition of efflux. Change in extracellular acriflavin fluorescence, indicative of efflux pump activity was measured using a Fluoroskan Ascent microplate fluorometer (Excitation 450 nm; Emission 500 nm).
Biofilm assay: The colorimetric assay for biofilm quantification with Crystal Violet (CV) staining was adapted from a previously described method (Jackson et al., 2002) and performed under semi-static conditions. Briefly, 1cm lengths of silicone (1.58 mm ID) tubes were used as carriers in the study (Sigma Aldrich, St Louis, MO) and prepared by washing with alcohol-acetone (1:1) and soaking in 70% ethanol for 15min. Following three rinses with sterile distilled water the carriers were transferred to 6 well plates and incubated with adjusted cell cultures (10 6 CFU mL −1 ) in the presence of pyocyanin (100 µM) for 36h at 37°C. Following incubation the carriers were washed with both water and saline, fixed with methanol (99%) and stained with 0.05% CV in water. Adherent CV was then extracted with 3 mL of 33% acetic acid and absorbance of the extract was read at 550 nm using a Titretek Multiskan MKII microtiter plate reader (Labsystems). Experiments were repeated in triplicate and appropriate controls included.
Data analysis: Statistical analysis was performed using GraphPad Instat 3 (GraphPad Software, San Diego, CA, USA). Significance was determined by one-way Analysis Of Variance (ANOVA) with Dunnett's multiple comparison test or Student's t-test. Experiments were performed a minimum of three times and the results are presented as the means ± standard deviations. Significance levels were defined as *p<0.05, **p<0.01, ***p<0.001.

RESULTS
Effects of pyocyanin on P. aeruginosa susceptibility to Ciprofloxacin: Ciprofloxacin has been demonstrated to have good activity against both PAO-1 and ATCC 27853 with MIC values of 125 and 500 µg L −1 respectively. In this study the exogenous addition of PCN (100 µM) did not affect susceptibility of either strain to ciprofloxacin determined visually (not shown). However using metabolic conversion of resazurin to resorufin as a more sensitive measure of bacterial growth we show that PCN (100 µM) decreased susceptibility of both PAO-1 and ATCC27853 to ciprofloxacin at 125 µg L −1 (p<0.001) and 500 µg L −1 (p<0.01) respectively compared to control cultures (Fig. 1). PCN did not affect the growth of either strain by itself (not shown). aeruginosa to ciprofloxacin. P. aeruginosa PAO1 and P. aeruginosa ATCC 27853 were incubated with ciprofloxacin (31-1000 µg L −1 ) in the absence or presence of PCN (100 µM) for 20 h. Cell growth was determined using reassuring reduction to Resorufin by metabolically active bacteria. Data are normalized relative to untreated controls and represent the mean ±SD of three independent experiments. Comparisons: * treated Vs. untreated Effects of 100 µ µ µ µM pyocyanin on bacterial efflux activity: Acriflavine is a fluorescent molecule that is exported by a number of multi-drug resistance efflux systems including MexAB-OprM. Export of the compound, via the efflux mechanism, results in increased fluorescence that can then be measured fluorometrically. Here we show that exogenously added PCN (100 µM) significantly inhibits acriflavine efflux in both Pseudomonas strains relative to untreated controls, p<0.001 (Fig. 2). This inhibition of acriflavine efflux was largely unaffected in cultures grown continuously in media containing 100 µM PCN (Fig. 2). In both strains the observed inhibition of acriflavine efflux by PCN was less than the positive control PabN (40 µM). aeruginosa. P. aeruginosa PAO1 and P. aeruginosa ATCC 27853 were loaded with acriflavine (20µM) and then treated with PCN (100 µM). PabN (40 µM) was included as the positive control for efflux inhibition. Data are normalised relative to untreated controls and represent the mean ±SD of three independent experiments. Comparisons: * treated Vs. untreated Effects of 100 µ µ µ µM pyocyanin on biofilm production: Crystal violet staining is widely used to determine biofilm production under static conditions. Here we show that the exogenous addition of PCN (100 µM) produced a 1.6±0.1 and 3.4±0.1 fold increase in crystal violet absorbance in PAO-1 and ATCC27853 respectively relative to untreated control. Chlorhexidine (0.2%) was included as the negative control and completely inhibited biofilm production (data not shown).

DISCUSSION
Among all current drug treatment options, ciprofloxacin remains a crucial orally effective antibiotic for treating P. aeruginosa infections (Hodson et al., 1987). Over-Production Phenotypes (OP) phenotypes were recently described by Fothergill et al. (2007) and shown to increase production of exoproducts including pyocyanin. These researchers highlighted interesting correlations between the OP phenotype and patterns of antibiotic resistance. The OP phenotype was shown to have a higher proportion of intermediate antimicrobial susceptibility to ciprofloxacin (Fothergill et al., 2007). Pyocyanin has previously been shown to behave as a signaling molecule influencing the regulation of a number of genes in P. aeruginosa (Dietrich et al., 2006).
Exogenous addition of 100 µM pyocyanin was shown in this study to have a modest inhibitory effect on the susceptibility of P. aeruginosa PAO-1 and ATCC27853 to ciprofloxacin when compared to untreated controls. Pyocyanin was shown to have no significant growth inhibitory effects of its own at 100 µM. Exogenous addition of pyocyanin produced a decrease in susceptibility of both strains tested at concentrations well in excess of the MIC that may be associated with an increase in the persister cell population (Moker et al., 2010). Persister cells constitute a small portion of a culture which is tolerant to killing by lethal doses of bactericidal antibiotics. The phenazine pyocyanin was recently shown to significantly increase the persister numbers in logarithmic P. aeruginosa PAO-1 or PA14 cultures (Moker et al., 2010). Although persister cells are believed to contribute to difficulties in treatment, the underlying mechanisms affecting persister formation are not well understood (Moker et al., 2010).
Acriflavine is a known substrate of the MexAB-OprM multidrug resistant pump (Poole and Srikumar, 2001). The MexAB-OprM system of P. aeruginosa has the broadest-spectrum of activity of the efflux pumps described to date and overproduction of this pump, in so called nalB mutants, has been associated with increased MICs for a number of unrelated antibiotics by 8-fold or more (Aeschlimann, 2003). Our results clearly show that exogenously added pyocyanin inhibits acriflavin efflux in both strains when compared to controls. This effect remained unaffected when overnight cultures were grown in the presence of 100µM pyocyanin. This would suggest that the decreased susceptibility to ciprofloxacin observed in both strains is unlikely to be related to alterations in efflux activity of MexAB-OprM. This statement is supported by Tohidpour et al (2009) who have demonstrated the association between PabN-inhibition of efflux activity and increased ciprofloxacin activity (Tohidpour et al., 2009). Furthermore our data also suggests that pyocyanin may be a competitive substrate for this system.
Studies have shown that the biofilm producing mucoid phenotype, P. aeruginosa can be up to 1,000 times more resistant to antibiotics than their planktonic counterparts (Drenkard, 2003). In our studies, exogenous addition of pyocyanin was shown to significantly increase crystal violet staining for biofilm production in both strains when compared to controls. This would suggest that the observed decrease in ciprofloxacin activity may in part be related to pyocyanin's influence on biofilm production. Interestingly, increased crystal violet stain was not observed for lower concentrations of exogenous pyocyanin (data not shown).
Sputum from CF patients colonized with P. aeruginosa has been shown to contain pyocyanin at concentrations up to 130 µM (Wilson et al., 1988). Our results would suggest that at these concentrations it is likely that pyocyanin may contribute to reduced P. aeruginosa susceptibility to ciprofloxacin and therefore may play a role in this organism's ability to evade eradication. However this study needs to be expanded to include other antibiotics including tobramycin and merepenam as well as the pyocyanin precursor 1hydroxyphenazine.

CONCLUSION
In conclusion, it appears from the results of our initial investigations that the presence of pyocyanin influences biofilm production by a currently undetermined mechanism and that this may contribute to the decreased effectiveness of ciprofloxacin against P. aeruginosa.