International Journal of Antimicrobial Agents 40 (2012) 177–181

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International Journal of Antimicrobial Agents
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Nutrient dispersion enhances conventional antibiotic activity against Pseudomonas aeruginosa biofilms
Stacy Sommerfeld Ross a , Jennifer Fiegel a,b,∗
a b

University of Iowa, College of Pharmacy, Department of Pharmaceutical Sciences and Experimental Therapeutics, Iowa City, IA 52242, USA University of Iowa, College of Engineering, Department of Chemical and Biochemical Engineering, Iowa City, IA 52242, USA

a r t i c l e

i n f o

a b s t r a c t
Bacterial biofilms cause significant infections in the medical field. Antibiotics commonly used to treat these infections often do not achieve complete bacterial eradication. New approaches to eliminate biofilms have focused on dispersion compounds to entice the bacteria to actively escape or disperse from the biofilm, where the bacteria may become more susceptible to antibiotics. The aim of this study was to demonstrate that combining antibiotics with nutrient dispersion compounds can synergistically decrease the viability of Pseudomonas aeruginosa biofilms. The effects of various co-treatments were studied on mature biofilms through qualitative and quantitative confocal microscopy. Combined treatment of P. aeruginosa biofilms with antibiotic and dispersion compounds resulted in a significant reduction in the live bacterial population compared with the untreated control in all cases, with four combinations displaying synergistic action (citrate with amikacin disulphate, colistin methanesulphonate or erythromycin, and succinic acid with colistin methanesulphonate). © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Article history: Received 17 January 2012 Accepted 20 April 2012 Keywords: Biofilm Synergy Antibiotic Dispersion Pseudomonas aeruginosa

1. Introduction Pseudomonas aeruginosa bacteria play a significant role in tracheal, urinary and wound infections. Pseudomonas aeruginosa can develop into biofilms where the bacteria secrete an external matrix of polysaccharides, extracellular DNA and proteins for added protection. Conversion of bacteria from a non-mucoid phenotype to a mucoid phenotype provides a defence against antibiotics and the human immune system [1]. Therefore, mucoid P. aeruginosa biofilms are not easily eradicated with antibiotics, including multiple antibiotics [2]. Because of the difficulty in treating P. aeruginosa with antibiotics alone, researchers have begun investigating methods to increase the effectiveness of antibiotics with dispersion-cueing agents. Dispersion results when bacteria actively escape the biofilm, where they become more susceptible to antibiotics [3–5]. Nutrient dispersion compounds, chelators, salt solutions, quorum-sensing inhibitors and altered pH have been shown to induce changes to the physicochemical environment surrounding the bacteria, leading to bacterial dispersal [4,6,7]. Nutrient dispersion compounds, such as the conjugate bases of simple acids, provide a nutrient-rich environment around the biofilm [7]. Moulton and Montie showed

nutrient compounds, such as organic acids, attract motile bacteria [8]. Sauer et al. observed that the bacteria in biofilms treated with dispersion compounds have increased expression of a flagella gene and decreased expression of a pili attachment gene [7]. Since released motile bacteria can easily be 100 times more susceptible to antibiotics, co-treatment with dispersion compounds and antimicrobials may enhance biofilm eradication. To date, nutrient dispersion compounds have not been investigated for synergistic biofilm reduction with antibiotics. However, previous studies have reported improved biofilm killing when combining antibiotics with other compounds that cue dispersion [3,4]. In a study by Rogers et al., synergistic eradication of P. aeruginosa biofilms was found with a 2-aminoimidazole-derived dispersion compound and tobramycin [3]. In another study, Brackman et al. showed increased bacterial killing with tobramycin and quorumsensing inhibitors [4]. The objective of this study was to identify combinations of antibiotics and nutrient dispersion compounds that synergistically decrease P. aeruginosa biofilm viability using qualitative and quantitative confocal microscopy studies.

2. Materials and methods 2.1. Materials

∗ Corresponding author. Present address: The University of Iowa, 115 S. Grand Ave., S215 PHAR, Iowa City, IA 52242, USA. Tel.: +1 319 335 8830; fax: +1 319 335 9349. E-mail address: (J. Fiegel).

DifcoTM nutrient agar and nutrient broth were purchased from BD Diagnostic Systems (Sparks, MD); glycerol, amikacin disulphate, tobramycin sulphate, erythromycin, colistin methanesulphonate,

0924-8579/$ – see front matter © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

0 mg/mL. colistin methanesulphonate at 2. As a proof of concept.9 18. Biofilm treatments Biofilms were treated on Day 4 with an aqueous solution containing either antibiotic alone.8 13. Then. was added due to its additional anti-inflammatory properties. CA). 2.132 M dipotassium phosphate. † 22. † Significant compared with the antibiotic control and dispersion control (P < 0. OH). 2. Dubuque. morpholinepropanesulphonic (MOPS) free acid (10×) and dipotassium phosphate (0. was defined as a statistically significant reduction in percent live bacteria compared with all three controls (i. Sommerfeld Ross. 5 mL of 0. which led to the z-step size of 0. NJ).5 g/mL [13]. Confocal images were quantified using COMSTAT biomass calculations [9].7* . Results and discussion Prior to imaging via confocal microscopy.7 32.4 ± 10. which was determined from the reported mean sputum concentration achievable (1237 ␮g/g sputum) [12].2 13. VA) was cultured overnight in nutrient broth (37 ◦ C). aeruginosa laboratory strain BAA-47. Four of the antibiotics were tested at the maximum solubility concentration in water: amikacin disulphate at 1. Image quantification and statistical analysis Images were rendered using Volocity® (PerkinElmer.0 67.9 35. MO).178 S.8 89. 2.8%) (Table 1). colistin methanesulphonate and polymyxin B sulphate) since they are commonly used against P. b N = 3 for all samples. Images were obtained using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss. Roskilde.0 6. dispersion compound alone or a combination of antibiotic and dispersion compound. † 10.3. J. † 4.8 29. Then. each well was stained with 2 ␮L of a mixture containing 3.2.6 3. Dispersion compounds were tested at the same molar concentration of 10 mM. the amikacin disulphate controls and co-treatments where N = 6 and the polymyxin B antibiotic control where N = 5. Erythromycin.4. 3. the medium was replaced with fresh medium to remove free-swimming bacteria.5* . tobramycin. the pinhole was set to 1 Airy Unit with an optimal size of <1 ␮m. 3. (Mt Prospect. sodium citrate dihydrate was from RPI Corp. assuming a sputum density of 1. aeruginosa biofilms.3 2. aeruginosa strain BAA-47 (ATCC. Propidium iodide was excited with the 543-nm HeNe laser and the emission was collected with the long-pass 560-nm filter.8* 7.3 42. the untreated control.6* 0. Four antibiotic compounds were investigated (amikacin. Treatment with tobramycin at a higher concentration near the solubility limit (6. and ferrous sulphate heptahydrate was from Fisher Scientific (Fair Lawn. SYTO 9 was excited with the 488-nm argon laser and the emission was collected with a band pass 505–530-nm filter. After treatment.5 38. aeruginosa biofilms. Germany).3 mg/mL) did not enhance biofilm eradication (data not shown).34 mM SYTO® 9 (to stain all bacteria green) and 19.48 ␮m. Four of the five antibiotics tested (amikacin disulphate. Z-stack image sequences were obtained via a Plan-Neofluar 40×/1.97 mM propidium iodide (to stain bacteria with damaged membranes red).3 20. Fiegel / International Journal of Antimicrobial Agents 40 (2012) 177–181 Table 1 Percent live bacteria (mean ± standard deviation) quantified from confocal microscopy image stacksa . polymyxin B sulphate at 2.0064 g of magnesium sulphate and 0.8 mg/mL. the study focused solely on a mucoid P.0* 1. 250 ␮L of media (50 mL of 10× MOPS free acid.3* 8.0 mg/mL.9* . These concentrations were at or above the minimum biofilm eradication concentrations reported for biofilms grown for 4–24 h [5.05). Waltham.4* 2. colistin methanesulphonate and polymyxin B sulphate) resulted . 2. Denmark). except the untreated control where N = 21.5 mg/mL. † 27.1. Eugene.7 27. † 9. Controls: untreated biofilms and treatment with single antibiotic or dispersion compounds Untreated P. 1A) of primarily live bacteria (78.9 50.5.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3. Live/dead staining and confocal imaging a Three antibiotic compounds were synergistic with dispersion compounds in reducing the percent live bacteria.e.3) was added to Lab-TekTM 8-well chambered coverglass wells (Thermo Fisher Scientific.3 oil objective. treatment with antibiotic alone and treatment with nutrient dispersion compound alone).05).2 mg/mL. The sum of the live biomass and sum of the dead biomass from three images were used to calculate the percent live and percent dead biomass.00114 g of ferrous sulphate heptahydrate in 500 mL of purified water at pH 7. tobramycin sulphate. 108 CFU/mL was added to each well. Water was added to the untreated controls such that all wells contained 400 ␮L of fluid. the chambered coverglass plates were incubated for 24 h on an orbital shaker. Bacterial strain and culture conditions The mucoid P. Images were segmented with a threshold intensity value of 45 to uniformly separate bacterial fluorescence from background noise.3* polymyxin B sulphate and magnesium sulphate were from SigmaAldrich (St Louis. OR) was used to stain the cells remaining in the biofilm. Purified water was obtained from a NANOpure Infinity® Ultrapure Water System (Barnstead International. The plate was sealed with Parafilm and was incubated at 37 ◦ C and 80% relative humidity on an orbital shaker table (190 rpm) for 4 days.11]. Jena.132 M) were from Teknova (Hollister. other antibiotics provided no additional benefit or increased bacteria viability when combined with dispersion compounds.05) between the percent live of different treatment groups were determined using Minitab 15 via analysis of variance (ANOVA) with a post hoc Dunnett’s test to isolate treatment groups that were statistically significant compared with controls.9* . This batch device was chosen to provide low shear stress on the biofilm.4 ± 10. 0.3 10. To compare images.1* . * Significant compared with the untreated control (P < 0. Sampleb Untreated controls Dispersion compound controls Citrate Succinic acid Amikacin disulphate with citrate with succinic acid Tobramycin sulphate with citrate with succinic acid Colistin methanesulphonate with citrate with succinic acid Polymyxin B sulphate with citrate with succinic acid Erythromycin with citrate with succinic acid Live bacteria remaining (%) 78.4 8. Briefly. succinic acid was from MP Biomedicals LLC (Solon.2* 4. and erythromycin at 2. aeruginosa biofilms developed a flat lawn (Fig. Synergy for this study The focus of this study was to identify combinations of antibiotic and nutrient dispersion compounds that synergistically reduce the viability of P. 100 ␮L of bacterial suspension at ca. MA).8 91. A LIVE/DEAD BacLightTM Bacterial Viability Kit (Invitrogen.3* 4.1* 2. Manassas. Significant differences (P < 0. a broad-spectrum antibiotic. IL). Tobramycin sulphate was tested at a concentration of 1.10.2 5. IA).

8 ± 4. aeruginosa biofilms (A) untreated or (B and C) treated with aqueous solutions containing sodium citrate (B) and succinic acid (C). 1. 2G).5% after treatment (Table 1).0 ± 8.9 ± 2.4 ± 10.2 ± 22. Combination treatment with succinic acid resulted in more biomass (Fig. 2F) resulted in an increase in bacteria viability (20.10]. 2E) resulted in a lawn of bacteria with a significant increase in bacteria viability (35. The cyclic polypeptides colistin methanesulphonate and polymyxin B sulphate were equally effective in reducing the bacterial burden. 3. Top: aerial view. Amikacin disulphate was less effective than tobramycin sulphate.11]. which was not observed in previous flow cell studies. This was expected as the addition of nutrient dispersion compounds provided a carbon source for the bacteria to consume. Bacteria were stained with SYTO® 9 and propidium iodide: green = live bacteria. 2C) and a decrease in percent live bacteria (42. Dispersion was not observed within the wells of the Lab-Tek chambered coverglass device in the current study. In the current studies.) in significantly lower percent live bacteria than the untreated controls. aeruginosa biofilms. In the current study. providing a reduction in total bacteria (Fig. Sommerfeld Ross. Co-treatment with tobramycin sulphate and dispersion compounds Co-treatment of P. The success of cyclic polypeptides and aminoglycosides in killing biofilm bacteria in these studies suggests that there were subpopulations of bacteria with varying metabolic activities. the addition of citrate to the treatment enhanced the activity of amikacin.3 ± 2. This study focused on nutrient dispersion compounds since bacteria released out of the biofilm would be more susceptible to antibiotics [3. sodium citrate and succinic acid were tested at a concentration of 10−2 M. Biofilms treated with the macrolide erythromycin displayed minimal death (67.8%) (Table 1). left-hand column). Colistin methanesulphonate treatment decreased the total bacteria within the wells (Fig.3 ± 7. have shown that aminoglycosides respond differently to resistance mechanisms developed by the bacteria [15]. Aminoglycosides are effective against metabolically active bacteria. Increased metabolic activity from the addition of dispersion compounds may promote bacteria to release inactivating enzymes that decrease the effectiveness of tobramycin sulphate.4% live) that was not significant compared with the antibiotic control (Table 1). visually observed as a lawn of live bacteria with clumps of dead bacteria (Fig. 1B and C). 2M). The percent live for both treatments was significantly reduced compared with the untreated control. They observed that tobramycin resistance was increased in the presence of 9 of 11 modifying enzymes. resulting in a thick lawn of dead bacteria (Fig.1%) compared with the untreated control and dispersion compound control. which is consistent with other literature reports [5. Fiegel / International Journal of Antimicrobial Agents 40 (2012) 177–181 179 Fig. each square = 23. J.2% live). Nutrient dispersion compounds sodium citrate and succinic acid developed dense lawns of Pseudomonas aeruginosa biofilm compared with the untreated control after 24 h. however.6% live).2. whilst combination treatments with amikacin were minimally effective. 2J) and significantly reduced the percent live remaining after treatment (29. 2D).8% live bacteria.3 ± 1.4 ± 10. the reader is referred to the web version of the article.0% live). Confocal images of P. (For interpretation of the references to colour in this figure legend.7 ± 9. but a significant morphology change observed as a clumped architecture (Fig. 3. red = dead bacteria.1 ␮m on each side. Bottom: side view.3.9% live) compared with the antibiotic control.4]. whilst amikacin resistance was increased in the presence of only 3 of the 11 modifying enzymes.3%. . 2. Treatment with amikacin disulphate resulted in a sparse lawn of dispersed dead cells (Fig. Macrolides require higher concentrations than aminoglycosides to eradicate bacteria within mature biofilms [10. but no significant difference compared with the antibiotic control (50. Co-treatment with amikacin disulphate and dispersion compounds Amikacin disulphate was synergistic with citrate. combination treatments with tobramycin were not synergistic at eradicating P. aeruginosa biofilms within 100 min following addition of the nutrient compound in a flow cell system [7]. 2B) and decrease in live bacteria to 8. The two aminoglycosides investigated (amikacin disulphate and tobramycin sulphate) significantly reduced the viable biovolume. Since amikacin is effective against metabolically active bacteria. Sauer et al. Polymyxin B sulphate reduced the live bacteria population to 18. have observed that citrate and succinate (the carboxylate anion of succinic acid) successfully dispersed 4-day-old P. suggesting that the biofilms in these studies contained metabolically active bacteria. Miller et al. observe an increase in biomass owing to the addition of nutrient dispersion compounds compared with the untreated controls (Fig. Cyclic polypeptides are effective at eradicating biofilm bacteria with low metabolic activity [14]. These results are consistent with this since erythromycin was not effective at a concentration double that of amikacin disulphate. 2A) with 50. aeruginosa biofilms with tobramycin sulphate and citrate (Fig. We did. scale bar = 45 ␮m.S. Co-treatment with succinic acid (Fig. Treatment with tobramycin sulphate resulted in biofilms with lower viability (13. whilst erythromycin exhibited no significant effect on viability (Fig.

(J) colistin methanesulphonate. (K) colistin methanesulphonate and citrate.) . Fiegel / International Journal of Antimicrobial Agents 40 (2012) 177–181 Fig. each square = 23. (C) amikacin disulphate and succinic acid. the reader is referred to the web version of the article. (E) tobramycin sulphate and citrate. 2. red = dead bacteria. Confocal images of P. (H) erythromycin and citrate.180 S. (B) amikacin disulphate and citrate. (For interpretation of the references to colour in this figure legend. Treatment of Pseudomonas aeruginosa biofilms with antibiotic alone or with combination treatments of an antibiotic and dispersion compound had varying effects on biofilm growth and eradication. aeruginosa biofilms treated with (A) amikacin disulphate. (D) tobramycin sulphate. Sommerfeld Ross. Top: aerial view. (I) erythromycin and succinic acid. (G) erythromycin. J. (M) polymyxin B sulphate. Bacteria were stained with SYTO® 9 and propidium iodide: green = live bacteria. scale bar = 45 ␮m. Bottom: side view.1 ␮m on each side. (N) polymyxin B sulphate and citrate and (O) polymyxin B sulphate and succinic acid. (L) colistin methanesulphonate and succinic acid. (F) tobramycin sulphate and succinic acid.

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