Nutrient Dispersion Enhances Conventional Antibiotic Activity Against

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International Journal of Antimicrobial Agents 40 (2012) 177181

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InternationalJournal ofAntimicrobial Agents

journal homepage: http: / /www.elsevier .com/ locate / i jant imicag

Short communication

Nutrient dispersion enhances conventional antibiotic activity against

Pseudomonas aeruginosa biofilms

Stacy Sommerfeld Ross a,Jennifer Fiegel a,b,

a University of Iowa, College of Pharmacy,Department of Pharmaceutical Sciences andExperimentalTherapeutics, Iowa City, IA 52242, USAb University of Iowa, College of Engineering, Department of Chemical andBiochemical Engineering, Iowa City, IA 52242,USA

a r t i c l e i n f o

Article history:

Received 17 January 2012Accepted 20 April 2012

Keywords:

Biofilm

Synergy

Antibiotic

Dispersion

Pseudomonasaeruginosa

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 ofthis study

was to demonstrate that combining antibiotics with nutrient dispersion compounds can synergistically

decrease the viability ofPseudomonasaeruginosabiofilms. The effects ofvarious co-treatments were stud-

ied on mature biofilms through qualitative and quantitative confocal microscopy. Combined treatment

ofP. 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 combi-

nations 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.

1. Introduction

Pseudomonas aeruginosa bacteria play a significant role in tra-

cheal, urinary and wound infections. Pseudomonas aeruginosa can

develop into biofilms where thebacteriasecrete an external matrix

of polysaccharides, extracellular DNA and proteins for added pro-

tection. 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 mul-

tiple antibiotics [2].

Because of thedifficultyin treatingP. aeruginosa with antibiotics

alone, researchers have begun investigating methods to increase

the effectiveness of antibiotics with dispersion-cueing agents. Dis-

persion results when bacteria actively escape the biofilm, where

they become more susceptible to antibiotics [35]. Nutrient dis-persion compounds, chelators, salt solutions, quorum-sensing

inhibitors and altered pH have been shown to induce changes to

the physicochemical environment surrounding the bacteria, lead-

ing 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

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:[email protected] (J. Fiegel).

nutrient compounds, such as organic acids, attract motile bacte-

ria [8]. Sauer et al. observed that the bacteria in biofilms treatedwith dispersion compounds have increased expression of a flag-

ella gene and decreased expression of a pili attachment gene [7].

Since released motilebacteriacan easilybe 100 times more suscep-

tible to antibiotics, co-treatment with dispersion compounds and

antimicrobials may enhance biofilm eradication.

To date, nutrient dispersion compounds have not been investi-

gated for synergistic biofilm reduction with antibiotics. However,

previous studies have reportedimprovedbiofilmkilling when com-

bining antibiotics with other compounds that cue dispersion [3,4].

In a study by Rogers et al., synergistic eradication ofP. 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 quorum-

sensing inhibitors [4]. The objective of this study was to identifycombinations 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

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.

http://dx.doi.org/10.1016/j.ijantimicag.2012.04.015


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178 S. Sommerfeld Ross, J. Fiegel / International Journal of Antimicrobial Agents40 (2012) 177181

polymyxin B sulphate and magnesium sulphate were from Sigma-

Aldrich (St Louis, MO); sodium citrate dihydrate was from RPI

Corp. (Mt Prospect, IL); succinic acid was from MP Biomedicals

LLC (Solon, OH); morpholinepropanesulphonic (MOPS) free acid

(10) and dipotassium phosphate (0.132M) were from Teknova

(Hollister, CA); and ferrous sulphate heptahydrate was from Fisher

Scientific (Fair Lawn, NJ). Purified water was obtained from a

NANOpure Infinity Ultrapure Water System (Barnstead Interna-

tional, Dubuque, IA).

2.2. Bacterial strain and culture conditions

The mucoid P. aeruginosa strain BAA-47 (ATCC, Manassas, VA)

was cultured overnight in nutrient broth (37 C). Then, 250L of

media (50 mLof 10 MOPS free acid, 5 m L of 0.132 M dipotas-

sium phosphate, 0.0064g of magnesium sulphate and 0.00114g

of ferrous sulphate heptahydrate in 500 mLof purified water at pH

7.3) was added to Lab-TekTM 8-well chambered coverglass wells

(Thermo Fisher Scientific, Roskilde, Denmark). Then, 100L ofbac-

terial suspension at ca. 108 CFU/mL was added to each well. The

plate was sealed with Parafilm and was incubated at 37C and 80%

relative humidity on an orbital shaker table (190rpm) for 4 days.

This batch device was chosen to provide low shear stress on the

biofilm.

2.3. Biofilm treatments

Biofilms were treated on Day 4 with an aqueous solution con-

taining either antibiotic alone, dispersion compound alone or a

combination of antibiotic and dispersion compound. Water was

addedto theuntreatedcontrolssuchthatall wellscontained 400L

of fluid. After treatment, the chambered coverglass plates were

incubated for 24h on an orbital shaker.

2.4. Live/dead staining and confocal imaging

Prior to imaging via confocal microscopy, the medium was

replaced with fresh medium to remove free-swimming bacteria.A LIVE/DEADBacLightTM Bacterial Viability Kit (Invitrogen, Eugene,

OR)was used tostainthe cells remainingin thebiofilm.Briefly,each

well was stained with 2L of a mixture containing 3.34 mM SYTO

9 (to stain all bacteria green) and 19.97mM propidium iodide (to

stain bacteria with damaged membranes red).

Imageswere obtained using a Zeiss LSM510 confocal laser scan-

ning microscope (Carl Zeiss, Jena, Germany). SYTO 9 was excited

with the 488-nm argon laser and the emission was collected with

a band pass 505530-nm filter. Propidium iodide was excited with

the 543-nm HeNe laser and the emission was collected with the

long-pass 560-nm filter. Z-stack image sequences were obtained

via a Plan-Neofluar 40/1.3 oil objective. To compare images, the

pinhole was setto 1 Airy Unit with an optimal size of


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Fig. 1. Nutrient dispersion compounds sodium citrate and succinic acid developed dense lawns ofPseudomonas aeruginosa biofilm compared with the untreated control

after 24h. Confocal images ofP. aeruginosa biofilms (A) untreated or (B and C) treated with aqueous solutions containing sodium citrate (B) and succinic acid (C). Bacteria

were stained with SYTO 9 andpropidium iodide: green= live bacteria; red= dead bacteria. Top: aerial view, scale bar= 45m. Bottom: side view, each square= 23.1m on

each side. (For interpretation of thereferences to colourin this figure legend, thereader is referred to theweb version of the article.)

in significantly lower percent live bacteria than the untreatedcontrols, whilst erythromycin exhibited no significant effect on

viability (Fig. 2, left-hand column).

The two aminoglycosides investigated (amikacindisulphate and

tobramycin sulphate) significantly reduced the viable biovolume.

Aminoglycosidesare effective against metabolicallyactive bacteria,

suggesting that the biofilms in these studies contained metaboli-

cally active bacteria. Treatment with amikacin disulphate resulted

in a sparse lawn of dispersed dead cells (Fig. 2A) with 50.410.8%

live bacteria. Treatment with tobramycin sulphate resulted in

biofilms with lower viability (13.92.0% live), visually observed

as a lawn of live bacteria with clumps of dead bacteria (Fig. 2D).

Amikacin disulphate was less effective than tobramycin sulphate,

which is consistent with other literature reports [5,10].

Biofilms treated with the macrolide erythromycin displayedminimal death (67.08.2% live), but a significant morphology

change observed as a clumped architecture (Fig. 2G). Macrolides

require higher concentrations than aminoglycosides to eradi-

cate bacteria within mature biofilms [10,11]. These results are

consistent with this since erythromycin was not effective at a con-

centration double that of amikacin disulphate.

The cyclic polypeptides colistin methanesulphonate and

polymyxin B sulphate were equally effective in reducing the bac-

terial burden. Colistin methanesulphonate treatment decreased

the total bacteria within the wells (Fig. 2J) and significantly

reduced the percent live remaining after treatment (29.32.6%

live). Polymyxin B sulphate reduced the live bacteria population

to 18.79.3%, resulting in a thick lawn of dead bacteria (Fig. 2M).

Cyclic polypeptides are effective at eradicating biofilm bacteriawith low metabolic activity [14]. 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.

This study focused on nutrient dispersioncompounds since bac-

teria released out of the biofilm would be more susceptible to

antibiotics [3,4]. In the current study, sodium citrate and succinic

acid were tested at a concentration of 102 M. Sauer et al. have

observed that citrate and succinate (the carboxylate anion of suc-

cinic acid) successfully dispersed 4-day-old P. aeruginosa biofilms

within 100 min following addition of the nutrient compound in

a flow cell system [7]. Dispersion was not observed within the

wells of the Lab-Tek chambered coverglass device in the current

study. We did, however, observe an increase in biomass owing to

the addition of nutrient dispersion compounds compared with theuntreatedcontrols (Fig.1B andC), which wasnot observed in previ-

ous flow cell studies. This was expected as the addition of nutrient

dispersion compounds provided a carbon source for the bacteria to

consume.

3.2. Co-treatment with amikacin disulphate and dispersion

compounds

Amikacin disulphate was synergistic with citrate, providing a

reduction in total bacteria (Fig. 2B) and decrease in live bacteria

to 8.37.5% after treatment (Table 1). Since amikacin is effective

against metabolically active bacteria, the addition of citrate to the

treatment enhanced the activity of amikacin. Combination treat-

ment with succinic acid resulted in more biomass (Fig. 2C) and a

decrease in percent live bacteria (42.222.1%) compared with the

untreated control and dispersion compound control, butno signifi-

cant differencecompared with the antibiotic control (50.410.8%)

(Table 1).

3.3. Co-treatment with tobramycin sulphate and dispersion

compounds

Co-treatment ofP.aeruginosabiofilmswith tobramycin sulphate

and citrate (Fig. 2E) resulted in a lawn of bacteria with a signifi-

cant increase in bacteria viability (35.31.9% live) compared with

the antibiotic control. Co-treatment with succinic acid (Fig. 2F)

resulted in an increase in bacteria viability (20.8

4.4% live) thatwas not significant compared with the antibiotic control (Table 1).

The percent live for both treatments was significantly reduced

compared with the untreated control. Increased metabolic activity

from the addition of dispersion compounds may promote bacte-

ria to release inactivating enzymes that decrease the effectiveness

of tobramycin sulphate. Miller et al. have shown that aminoglyco-

sides respond differently to resistance mechanisms developed by

the bacteria [15]. They observed that tobramycin resistance was

increased in the presence of 9 of 11 modifying enzymes, whilst

amikacin resistance was increased in the presence of only 3 of

the 11 modifying enzymes. In the current studies, combination

treatments with tobramycin were not synergistic at eradicating P.

aeruginosa biofilms, whilst combination treatments with amikacin

were minimally effective.


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Fig. 2. Treatment ofPseudomonas aeruginosa biofilms with antibiotic alone or withcombination treatments of an antibioticand dispersion compoundhad varying effects on

biofilm growth and eradication. Confocal images ofP. aeruginosabiofilms treated with (A) amikacin disulphate, (B) amikacin disulphate and citrate, (C) amikacin disulphate

and succinic acid,(D) tobramycin sulphate, (E)tobramycin sulphateand citrate, (F) tobramycin sulphateand succinic acid,(G) erythromycin, (H) erythromycin andcitrate,(I)

erythromycin and succinic acid,(J) colistin methanesulphonate, (K) colistin methanesulphonate and citrate, (L)colistin methanesulphonate and succinic acid,(M) polymyxin

B sulphate, (N) polymyxin B sulphate and citrate and (O) polymyxin B sulphate and succinic acid. Bacteria were stained with SYTO 9 and propidium iodide: green= live

bacteria; red= dead bacteria. Top: aerialview, scale bar =45m. Bottom: side view, each square =23.1m on each side. (For interpretation of thereferencesto colour in this

figure legend, thereader is referred to the web version of thearticle.)


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S. Sommerfeld Ross, J. Fiegel / International Journal of Antimicrobial Agents40 (2012) 177181 181

3.4. Co-treatment with erythromycin and dispersion compounds

Erythromycin provided synergistic killing of biofilms with cit-

rate (6.55.1% live) (Table 1), resulting in biofilms with a clumped

architecture of mainly dead bacteria (Fig. 2H). Erythromycin

inhibits bacterial growth through inhibition of protein synthesis,

especiallyat high concentrations.Thus, erythromycin requires bac-

terial cells to be metabolically active to be effective. The promotion

of dispersion and bacterial metabolic activity with the addition of

citrate likely enhanced the synergistic activity of the compounds.

Co-treatment with succinic acid led to denser biofilm formation

(Fig. 2I) but no significant difference in percent live bacteria com-

pared with the antibiotic control (38.627.3% vs. 67.08.2%).

3.5. Co-treatment with colistin methanesulphonate and

dispersion compounds

Synergistic eradication ofP. aeruginosa biofilms occurred when

colistin methanesulphonate was combined with citrate or succinic

acid (Fig. 2K and L). Colistin methanesulphonate with citrate was

the best combination treatment found in this study, reducing the

percent live bacteria after treatment to 2.80.9%. Co-treatment

with succinic acid resulted in an increase in total bacteria present

but a decrease in percent live bacteria (13.910.7%). Cyclicpolypeptides are effective against all growth stages of planktonic

bacteria. The prodrug colistin methanesulphonate hydrolyses to

the active colistin form. This delayed antibiotic action likely led

to synergistic killing since dispersion was able to occur prior to the

drug hydrolysing into the active form.

3.6. Co-treatment with polymyxin B sulphate and dispersion

compounds

Co-treatment of polymyxin B with nutrient dispersion com-

pounds was not statistically different from the antibiotic control

(Table 1; Fig. 2N,O) and exhibited a trend towards antagonism.

Cyclicpolypeptides have also been shown to effectively kill interior

bacteria with low metabolic activity within the biofilm. However,the cells growing on the exterior of the biofilm can adapt to treat-

ment by reducing membrane permeability [14]. Thus, providing

nutrients at the same time as polymyxin B sulphate may have

encouraged cell adaptation, allowing them to grow and inhibit

antibiotic penetration.

3.7. Conclusions

This study showed that combined treatment ofP. aeruginosa

biofilms with antibiotic and nutrient dispersion compounds

resulted in a significant reduction in the live bacteria population

compared with the untreated control in all cases, with four combi-

nations displaying synergistic action. Further studies are needed to

identify the mechanisms by which these co-treatments operate.

Acknowledgments

The authors thank Tom Moninger and Jian Shao for helpful dis-

cussions andtechnical assistance, andDr Alexander Horwill for use

of the Volocity software.

Funding: This work was supportedby a Pharmaceutics Research

Starter Grant from the PhRMA Foundation (JF), a University of Iowa

Instituteof Clinical andTranslational Science Pilot Grant (NIH CTSA

grantno. UL1RR024979) (JF) and a PhRMAFoundation Pre-Doctoral

Fellowship in Pharmaceutics (SSR). Confocal microscopy wasmade

possible by theCentral Microscopy Facilityat theUniversityof Iowa

(Iowa City, IA) under the Office of the Vice President of Research.

Competing interests: None declared.

Ethical approval: Not required.

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