Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Accepted Manuscript Title: Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides Author: Sibel Dosle...

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Accepted Manuscript Title: Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides Author: Sibel Dosler Elif Karaaslan PII: DOI: Reference:

S0196-9781(14)00290-3 http://dx.doi.org/doi:10.1016/j.peptides.2014.09.021 PEP 69344

To appear in:

Peptides

Received date: Revised date: Accepted date:

7-7-2014 24-9-2014 24-9-2014

Please cite this article as: Dosler S, Karaaslan E, Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides, Peptides (2014), http://dx.doi.org/10.1016/j.peptides.2014.09.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.



Article title: Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics



and antimicrobial peptides.

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Authors: Sibel Dosler1*, Elif Karaaslan2

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Adresses: 1University of Istanbul, Faculty of Pharmacy, Department of Pharmaceutical



Microbiology, 34116, Beyazit-Istanbul, Turkiye.

University of Bezmi Alem, Faculty of Medicine, Department of Medical Microbiology,

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34116, Fatih-Istanbul, Turkiye.

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e-mail addresses: [email protected], [email protected]

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*Corresponding author: Sibel Dosler

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Address: Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical

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Microbiology, 34116 Beyazit, Istanbul, Turkiye.

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Tel / Fax: 90 212 440 02 57

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e-mail: [email protected]

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Abstract

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Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just

23 

because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm

24 

formation and production of multiple virulence factors. We investigated the in vitro activities

25 

of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin)

26 

and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1-7)-melittin A(2-9)

27 

amide, melittin, defensin and magainin-II) alone or in combination against biofilms of

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laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum

29 

inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum

30 

biofilm eradication concentrations (MBEC) were determined by microbroth dilution

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technique. The MBEC values of antibiotics and AMPs were 80->5120 and 640->640 mg/L,

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respectively. When combined with the LL-37 or CAMA at 1/10xMBEC, the MBEC values of

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antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and

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AMPs were able to inhibit the attachment of bacteria at the 1/10xMIC and biofilm formation

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at 1x or 1/10xMIC concentrations. Time killing curve studies showed 3-log10 killing against

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biofilms in 24-h with almost all studied antibiotics and AMPs. Synergism were seen in most

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of the studied combinations especially CAMA/LL-37+ciprofloxacin against at least one or

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two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic

39 

concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of

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biofilm formation by blocking the attachment of bacteria to surfaces might be alternative

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methods to fight with biofilm associated infections.

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Key words: Biofilm; inhibition; destruction; antimicrobial cationic peptides; P.

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aeruginosa; time kill curve; MBEC

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1. Introduction

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Pseudomonas aeruginosa is a major nosocomial pathogen that can cause a wide variety of acute and chronic infections, such as bacteraemia, pneumonia, intra-abdominal wound,

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burn and urinary tract infections. Patients with burn wounds, urinary tract infections,

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acquired-immune deficiency syndrome (AIDS), lung cancer, chronic obstructive pulmonary

50 

disease, bronchiectasis, or cystic fibrosis, or those with indwelling inert surfaces are

51 

especially vulnerable to such infections [15,27]. P. aeruginosa is a dreaded bacteria with high

52 

intrinsic and acquired antibiotic resistance rates; it also forms biofilms and produces multiple

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virulence factors. Biofilm-associated P. aeruginosa strains can cause several types of

54 

infection in patients with indwelling inert surfaces like medical devices for internal or external

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use. Because the bacteria in biofilms become more resistant to antibiotic treatment and the

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actions of the host immune system, the treatment of biofilm-associated infections is extremely

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difficult [16,20].

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A biofilm is a microbial community that is attached to abiotic surfaces and produces

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extracellular polysaccharides. It is characterized by the growth-dependent accumulation of

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multi-layered cell clusters that are in turn surrounded by an extracellular polysaccharide

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matrix. Microbial cells that grow in biofilms are physiologically distinct from planktonic cells

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of the same organism. When a cell switches to the biofilm mode of growth, it undergoes a

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phenotypic shift in behaviour, and large groups of genes are differentially regulated [6,10,13].

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Finally, the bacteria in biofilms become more resistant to antibiotics and the host’s immune

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defence mechanisms as they adapt to changing conditions together instead of as single cells.

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Antimicrobial cationic peptides (AMPs) have attracted attention as alternative or

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complementary antibiotics due to their prospective potency, rapid action, and broad spectrum

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of activities against Gram-negative and -positive bacteria, viruses, fungi, and parasites. AMPs

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are a major component of the innate immune systems of most living organisms, including

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insects, plants, microorganisms, and mammals. In addition to protect against environmental

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microorganisms, they exhibit multiple mechanisms of action and, consequently, a low

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potential to induce de novo resistance, which allows the limited use of other antibiotics

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[12,29]. AMPs seem to be a good candidate for treating biofilms, either alone or in combination

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with antibiotics [9,11,21,23]. In this study, we tried to determine the in vitro activities of

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multiple antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin, and

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colistin) and AMPs (LL-37, cecropin A [1-7]-melittin A [2-9] amide [CAMA], melittin,

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defensin, and magainin II) alone or in combination against biofilms of standard and clinical P.

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aeruginosa strains.

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2. Materials and methods

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2.1. Bacterial strains:

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P. aeruginosa laboratory strain ATCC 27853 (Rockville, MD, USA) and four clinical

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isolates obtained from specimens submitted to the routine Clinical Microbiology Laboratories

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of Istanbul University, Istanbul Faculty of Medicine were tested. Clinical strains were isolated

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from different patient with acute sepsis and the isolates were identified with API 20 NE

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(BioMerieux, France) diagnostic kits.

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2.2. Antimicrobial substances:

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Five AMPs (LL-37, CAMA, melittin, defensin, and magainin II) were obtained from

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Bachem AG (Bubendorf, Switzerland). Antibiotics (ceftazidime, tobramycin, ciprofloxacin,

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doripenem, piperacillin, and colistin) were kindly provided by their respective manufacturers.

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Except for doripenem, stock solutions from dry powders were prepared at a concentration of

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1280 mg/L for AMPs and 5120 mg/L for antibiotics according to the manufacturers’

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recommendation and stored at –80°C for up to 6 months before use. Doripenem solutions

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were prepared on the day of use.

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2.3.

Media:

Tryptic soy broth supplemented with 1% glucose (TSB-glucose, Difco Laboratories,

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Franklin Lakes, NJ) was used for biofilm production, cation-adjusted Mueller-Hinton broth

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(CAMHB, Difco Laboratories) was used to determine the minimum inhibitory concentration

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(MIC) and minimum biofilm-eradication concentration (MBEC) values, and tryptic soy agar

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(TSA, Difco Laboratories) was used in colony counts to determine the minimum bactericidal

101 

concentrations (MBC) and MBEC values and in time-kill curve (TKC) analysis.

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2.4.

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MICs of antibiotics and AMPs were determined with a microbroth dilution technique as

MIC and MBC determinations:

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described by the Clinical and Laboratory Standards Institute (CLSI) [3]. The MIC was

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defined as the lowest concentration of antibiotic that completely inhibited visible growth.

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MBCs were determined at the end of the incubation period by removing two 10-µL

107 

samples from each well demonstrating no visible growth and plated onto TSA. Resultant

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colonies were counted after an overnight incubation at 37°C. The MBC was defined as the

109 

lowest concentration of antimicrobials that killed at least 99.9% of the initial inoculums [18].

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2.5.

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P. aeruginosa strains were cultured in 5 mL TSB-glucose for 24 h at 37°C with 360°

Biofilm formation:

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rotation (50 rpm) and diluted 1/50 in fresh TSB-glucose, yielding a final concentration of

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approximately 1×107 cfu/200 µL. This suspension was added to each well of a 96-well tissue

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culture microtitre plate (Greiner Bio-One, Kremsmuenster, Austria), and incubated at 37°C,

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24 h. The negative control was TSB-glucose. After the incubation, the waste media was

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aspirated gently and the wells of the plates were washed three times with 250 µl physiological

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buffered saline (PBS) solutions to remove unattached bacteria and air-dried. 200 µl of 99%

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methanol was added per well for 15 min for fixation and aspirated, and plates were allowed to

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dry. Wells were stained with 200 μl of 0.1% crystal violet (in water) for 5 min. Excess stain 4   

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was gently rinsed off with tap water, and plates were air-dried. Stain was resolubilized in 200

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μl of 95% ethanol, shaking in orbital shaker for 30 min. and measured the absorbance at 595

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nm [8]. 2.6. Biofilm attachment assay:

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Biofilm attachment assays were performed using a previously described method with

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some modifications [19]. The overnight cultures were diluted 1/50 to give 1×107 cfu/200 µL

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in TSB-glucose, and standard and clinical P. aeruginosa strains were added to each well of a

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96-well tissue culture microtitre plate with 1/10× MIC concentrations of AMPs and

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antibiotics. The plates were incubated for 1, 2, or 4 h at 37°C. Six wells were used for each

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AMP and antibiotic condition. The positive controls were P. aeruginosa strains in TSB-

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glucose without peptide or antibiotic. After incubation, the wells were washed twice with

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phosphate-buffered saline (PBS) solution and measured in PBS at 595 nm.

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2.6.

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Standard and clinical P. aeruginosa strains (1×105 cfu/200 µL) in TSB-glucose were

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Inhibition of biofilm formation:

incubated at 37°C, 24 h with AMPs or antibiotics at 1×, 1/10×, and 1/100× MIC

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concentrations in 96-well tissue culture microtitre plates. Six wells were used for each AMP

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or antibiotic. The positive controls were P. aeruginosa strains in TSB-glucose without peptide

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or antibiotic. After the incubation, wells were washed twice with PBS solutions and measured

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at 595 nm.

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2.7.

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Measurements of the antimicrobial susceptibilities of the P. aeruginosa biofilms were

Minimum biofilm eradication concentration (MBEC):

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assessed as previously described MBEC assay with the following modifications [2]. The 24-h

142 

biofilms in a 96 well tissue culture microtitre plates were washed three times with 250 µl PBS

143 

solutions and air-dried. Serial two fold dilutions ranging from 640 to 0.06 mg/L for AMPs

144 

and 5120 to 5 mg/L for antibiotics were prepared in CAMHB. 200 µl of each concentration

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was added to each corresponding well and plates were incubated 24 h at 37°C. After the

146 

incubation, antibiotics were aspirated gently and plates were washed two times with sterile

147 

PBS solutions, wells were scraped thoroughly, with particular attention to well edges. Well

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contents were removed, placed in 1 ml PBS solution, placed in a sonicating waterbath

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(Bandelin sonopuls HD 2200) for 5 min to disrupt the biofilm, and 100 µl samples were

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plated on TSA. Colonies were counted after 24 h at 37°C. The MBEC was defined as the

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lowest concentration of AMP or antibiotic that prevented bacterial regrowth.

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2.8.

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When considering the large concentrations of AMPs and antibiotics required to treat

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biofilm-associated infections, we investigated the in vitro activities of the antibiotics with

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AMPs at subMBEC values. For this purpose, we added 64 mg/L AMP 1/10×MBEC) to each

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concentration of antibiotic and then performed the MBEC assay as explained above.

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MBECs of antibiotics in the presence of AMP:

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2.9.

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The modified TKC method, which we previously described, was used to determine the

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Time killing curve (TKC) studies:

dynamic bactericidal activities of AMPs, alone or in combination with antibiotics against 24-h

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biofilms [7]. For this purpose, 24-h biofilms were prepared in a 96-well tissue culture

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microtiter plate, washed three times with 250 µL PBS, and air-dried. AMPs and antibiotics

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were diluted in CAMHB and added to each corresponding well, yielding a final concentration

163 

of 1× MBEC, and the plates were incubated for 0, 2, 4, 7, or 24 h at 37°C. After incubation,

164 

the plates were washed two times with sterile PBS, scraped and the contents were incubated

165 

in a sonicating water-bath, as described above. Following the disruption, serial 1/10-fold

166 

dilutions were made and 100 µL samples were plated on TSA. Colonies were counted 24 h

167 

after incubation at 37°C. An antibiotic-free control of each strain was also included.

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TKCs were constructed by plotting mean colony counts (log10 cfu/mL) versus time. The

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lower limit of detection for the time-kill assays was 2 log10 cfu/mL. Bactericidal activity was

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defined as a ≥ 3-log10 cfu/mL decrease from the initial inoculum. The results of drug

171 

combinations were interpreted by comparing the effects of the various combinations and the

172 

effects of the most active agent alone. Synergy and antagonism were defined as a ≥ 2-log10

173 

decrease or increase, respectively, in the colony count after 24 h. If there was no 2-log10

174 

increase or decrease, the effect of combination was assumed as additive [18]. We also

175 

calculated the fractional inhibitory concentrations of bliss synergy coefficients to measure the

176 

degree of synergy/antagonism. For this purpose, we used the equation below, which

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mentioned in the model of Prichard et al [22].

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Z =X+ Y(1 -X)

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Z represents the total inhibition produced by the combination of drugs X and Y and X and Y

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represent the inhibition produced by drugs X and Y alone, respectively. Any peaks above this

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plane would be indicative of synergy. Similarly, any depressions below the plane would

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indicate antagonism.

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2.10. Statistical analysis:

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All experiments were performed in two independent assays. When the results were

185 

different for MIC, MBC, or MBEC determinations, a third test was performed. In biofilm

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attachment and inhibition of biofilm formation assays, results are presented as the mean ±

187 

standard deviations of two independent experiments performed with standard and four clinical

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strains. One-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison tests

189 

were used to compare differences between control and antimicrobial-treated biofilms. P

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values <0.001 were considered statistically significant.

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3. Results

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3.1.

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The in vitro activities of the studied antibiotics and AMPs against P. aeruginosa

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planktonic cells and biofilms are summarized in Table 1. The MIC values of the antibiotics

Susceptibility:

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against P. aeruginosa ATCC 27853 were within the CLSI accuracy range throughout the

196 

study [4]. As shown in Table 1, there were no major differences between bactericidal and

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inhibitory concentrations of the antibiotics and AMPs. The MBC values were generally equal

198 

to or two-fold greater than those of the MIC values.

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When we investigated the in vitro activities of the antibiotics that were active against

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biofilms, with the active AMPs at 1/10×MBEC values against biofilms, the MBEC values of

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those antibiotics were decreased by up to eight-fold (Table 2).

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3.2.

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When we incubated the 1/10 concentrations of AMPs and antibiotics with standard and

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Biofilm attachment assay:

clinical P. aeruginosa strains for 1, 2, or 4 h at 37°C to assess the adherence to the wells of

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tissue culture microtitre plates, all of the antimicrobial agents inhibited biofilm attachment in

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a time-dependent fashion (Figure 1).

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3.3.

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All of the studied AMPs and antibiotics inhibited standard and clinical P. aeruginosa

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Inhibition of biofilm formation:

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biofilm formation at 24 h in a concentration-dependent manner (Figure 2). Significant biofilm

210 

inhibition was observed at all studied antibiotic concentration, while AMPs achieved this

211 

inhibition at 1× or 1/10×MIC values.

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3.4.

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According to the susceptibility test results, we performed the TKC studies with three

TKC studies:

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antibiotics (tobramycin, ciprofloxacin, and colistin) and two AMPs (LL-37, and CAMA) that

215 

were active against P. aeruginosa biofilms. The results of the TKC analyses show that 3-log10

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killing against P. aeruginosa biofilms was determined within 24 h using almost all studied

217 

antibiotics and AMPs alone. These analyses also demonstrate enhanced killing of biofilms

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when AMPs were used in combination with all of the examined antibiotics (Figure 3). As

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shown in Table 2, the synergistic interactions between the AMPs and antibiotics were 8   

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observed in most of the studied combinations against biofilms, especially in those that were

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treated with the CAMA/LL-37 + ciprofloxacin. According to the calculation of bliss

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synergism, synergistic interactions against P. aeruginosa biofilms were increased up to at

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least three strains with all of the antibiotic + AMP combinations (Data not shown). No

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antagonism was observed with any of the combinations.

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4. Discussion

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Biofilms are social communities of bacteria that involve several interactions, such as

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communication, cooperation, assistance, engagement, balance, and sharing. Generally,

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biofilm-associated bacteria causes chronic infections that can persist for decades, whereas

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planktonic forms cause acute infections. Bacteria on biofilm structures are protected from

230 

environmental conditions, antimicrobial agents, and host immune responses, and they also

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exhibit up to 1000-fold-increased antibiotic resistance to a wide range of antimicrobial agents

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[13].

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In this study, we investigated the in vitro activities of six clinically used antibiotics and

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five AMPs against biofilms of standard and clinical P. aeruginosa strains. When we examined

235 

the MIC values of antibiotics against planktonic P. aeruginosa cells, they ranged between

236 

0.25 - 128 mg/L. Only one strain showed resistance to tobramycin and piperacillin, while all

237 

of the other strains were susceptible or moderately susceptible to the studied antibiotics. On

238 

the other hand, the MIC values of AMPs were within the 8 - >128 mg/L range, and three

239 

cationic peptides (LL-37, CAMA, and melittin) were active against standard and clinical P.

240 

aeruginosa strains. When we considered the anti-biofilm activities of these antibiotics and

241 

AMPs, MBEC values ranged between 80 - >5120 mg/L and 640 - >640 mg/L, respectively.

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According to these results, three antibiotics (tobramycin, ciprofloxacin, and colistin) and two

243 

AMPs (LL-37 and CAMA) were active against P. aeruginosa biofilms. The MBEC/MIC

244 

ratios of these active antibiotics and AMPs were found to be between 320 - 2560 and 5 - 80 9   

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fold, respectively. These results are similar to our previous studies and other researchers’

246 

results [7,17,24,26]. Novel biofilm control strategies can be carried out in three main ways: first, reduction of

247 

the planktonic cells before biofilm formation; second, initial prevention of cell adhesion to

249 

surfaces; and third, removal or inhibition of mature biofilms. Because biofilm-associated

250 

bacteria are not affected by therapeutically achievable concentrations of antimicrobial agents,

251 

anti-biofilm therapies have generally focused on the inhibition of biofilm formation [14]. In

252 

some reports, peptides or antibiotics were able to inhibit the initial biofilm attachment (58-

253 

62%) at subMIC values. That was suggesting a potential interaction of these molecules with

254 

bacterial adhesins that mediates the intercellular adhesion of bacteria to the surfaces [5, 25].

255 

For this purpose, we investigated the inhibition of bacterial attachment to the surfaces, as well

256 

as the inhibition of biofilm production by MIC or subMIC values of antibiotics and AMPs.

257 

Almost all of the antibiotics and AMPs were able to inhibit the attachment of bacteria at

258 

1/10×MIC in 1-4 h (p<0.001). Ciprofloxacin and LL-37 significantly decreased the

259 

attachment (up to 70%) of P. aeruginosa to the surface in 1 h. We were also able to determine

260 

that antibiotics and AMPs significantly inhibited 24-h biofilm formation up to 80%, especially

261 

at 1× or 1/10×MIC (p<0.001). These results suggested that all of the studied antibiotics and

262 

AMPs were effective for inhibiting pre-formed biofilms, even if they were not active against

263 

formed biofilms. Even though inhibiting mature biofilm is very difficult, inhibition of biofilm

264 

formation in early critical stages seems to be more applicable.

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AMPs are one of the most promising groups of natural products for overcoming

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resistance problems in antimicrobial chemotherapy. Most AMPs exhibit broad spectrum

267 

antimicrobial activity against planktonic cells, independent from the antibiotic resistance

268 

patterns of bacteria, by pore-forming or non-pore-forming models. They also allow the entry

269 

of other substances like antibiotics inside the cell and serve as anti-resistance compounds of 10   

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classical antibiotics. However, the anti-biofilm activities of AMPs are not completely

271 

understood, a small number of studies have posited possible explanations including matrix

272 

disruption; binding of DNA; and altering the expression of biofilm-related genes, such as the

273 

production of pili and rhamnolipid, quorum sensing systems, and flagella assembly, and their

274 

dual capacity to act on both the cytoplasmic membrane and intracellular targets [1,12,14,28].

275 

Therefore, we investigated the in vitro activities of biofilm active antibiotics with AMPs at

276 

subMBEC values that can be tolerated by humans, and we found that the MBEC values of

277 

those antibiotics were decreased up to eight-fold. These results suggested that AMPs could be

278 

used to treat serious biofilm-related infections in combination with antibiotics.

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Although the MIC and MBEC values and inhibition of attachment or biofilm production

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demonstrated the efficacy of studied antibiotics and AMPs against P. aeruginosa biofilms,

281 

these techniques do not provide any information about the time course of antimicrobial

282 

activities. This limitation can be overcome with the use of TKC studies. The results of the

283 

TKC studies revealed that neither antibiotics nor AMPs showed at least 2 log10 (99 %) killing

284 

in 3 or 6 h, and 3 log10 (99.9 %) killing was achieved in 24 h at MBEC concentrations as

285 

expected.

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The synergistic effect achieved by using combinations of antimicrobial agents can be

286  287 

useful to rapidly enhance anti-biofilm activity and to help prevent or delay the emergence of

288 

resistance. TKC studies also indicated synergistic interactions against P. aeruginosa biofilms

289 

for at least two strains with all of the antibiotic + AMP combinations. When we calculated the

290 

results with bliss synergy method, the number of strains that showed synergism between

291 

antibiotic + AMP combinations, were increased. These results showed that the strains affected

292 

by this synergism were the same strains that expressed decreased MBEC values when AMP

293 

was applied at subMBEC concentrations. Especially ciprofloxacin + CAMA/LL-37

294 

combinations were synergistic against almost all P. aeruginosa biofilms. 11   

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Consequently, treatment of biofilm-related infections have become an important part of

295 

antimicrobial chemotherapy because biofilms are not affected by therapeutic concentrations

297 

of antibiotics. Using a combination of antimicrobial agents including AMPs or inhibiting pre-

298 

formed biofilms by blocking the attachment of bacteria to surfaces might be effective

299 

alternatives to combating biofilm-associated infections.

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Acknowledgement

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This work was supported by the Research Fund of Istanbul University (Project No: 29758).

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[10] Ghannoum M, O’Toole GA. Microbial Biofilms. ASM Pres, 2004, Washington, DC.

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[11] Gopal R, Kim YG, Lee JH, Lee SK, Chae JD, Son BK et al. Synergistic effects and anti-

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biofilm properties of chimeric peptides against MDR Acinetobacter baumannii strains.

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[12] Hancock RE, Sahl HG. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 2006;24:1551-7. [13] Haussler S, Fuqua C. Biofilms 2012: new discoveries and significant wrinkles in a

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[15] Kerr KG, Snelling AM. Pseudomonas aeruginosa: a formidable and ever present adversary. J Hosp Infect 2009;73:338-44.

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[16] Leid JG. Bacterial biofilms resist key host defenses. Microbe 2009;4:66-70.

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[17] Mataraci E, Dosler S. In vitro activities of antibiotics and antimicrobial cationic peptides

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alone and in combination against methicillin resistant Staphylococcus aureus biofilms.

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[18] National Committee for Clinical Laboratory Standards. Methods for Determining

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[19] Overhage J, Campisano A, Bains M, Torfs EC, Rehm BH, Hancock RE. Human host defense peptide LL-37 prevents bacterial biofilm formation. Infect Immun 2008;76:4176-

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[20] Page MG, Heim J. Prospects for the next anti-Pseudomonas drug. Curr Opin Pharmacol

cr

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us

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Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and antibiofilm

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activity of natural and designed α-helical peptides against Staphylococcus aureus,

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Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. BMC Microbiol

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[22] Prichard MN, Prichard LE, Shipman C Jr.. Strategic design and three-dimensional

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analysis of antiviral drug combinations. Antimicrob Agents Chemother 1993;37:540-5.

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[23] Saising J, Dube L, Ziebandt AK, Voravutchikunchai SP, Nega M, Götz F. Activity of

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gallidermin on Staphylococcus aureus and Staphylococcus epidermidis biofilms.

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[24] Tang HJ, Chen CC, Cheng KC, Toh HS, Su BA, Chiang SR et al. In vitro efficacy of

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fosfomycin-containing regimens against methicillin-resistant Staphylococcus aureus in

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biofilms. J Antimicrob Chemother 2012;67:944-50.

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[25] Tchouaffi-Nana F, Ballard TE, Cary CH, Macdonald TL, Sifri CD, Hoffman PS.

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Nitazoxanide inhibits biofilm formation by Staphylococcus epidermidis by blocking

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accumulation on surfaces. Antimicrob Agents Chemother. 2010;54:2767-74.

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various antibiotics on matrix and viable mass of Staphylococcus aureus and

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Pseudomonas aeruginosa biofilms. Int. J Antimicrob Agants 2009;33:525-31

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[27] Valderrey AD, Pozuelo MJ, Jimenez PA, Macia MD, Oliver A, Rotger R. Chronic colonization by Pseudomonas aeruginosa of patients with obstructive lung diseases:

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[28] Yeaman MR, Yount NY. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 2003;55:27-55.

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[29] Yeung AT, Gellatly SL, Hancock RE. Multifunctional cationic host defence peptides and

an

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their clinical applications. Cell Mol Life Sci 2011;68:2161-76.

378 

M

379  380 

d

381 

Ac ce pt e

382  383  384  385  386  387  388  389  390  391  392 

15   

Page 16 of 22

393 

Table 1. In vitro antibacterial and anti-biofilm activities of antibiotics and AMPs against

394 

standard and clinical P. aeruginosa strains. Antimicrobial agents

MIC range (mg/L)

MBC range (mg/L)

MBEC range (mg/L)

Antibiotics 1-4

1-4

Tobramycin

0.25-16

0.25-32

Ciprofloxacin

0.25-2

0.25-8

Doripenem

0.25-4

0.5-8

Piperacillin

4-128

4-256

>5120

Colistin

0.25-2

0.5-2

160-2560

64-≥128

640

16-128

640

>128

≥640

8-64

Melittin

64->128

Ac ce pt e

395  396  397 

160-2560 80-1280

cr

us

an

CAMA

d

32-128

M

Cationic peptides LL-37

2560->5120

ip t

Ceftazidime

>5120

Defensin

>128

>128

>640

Magainin II

>128

>128

>640

398  399  400 

16   

Page 17 of 22

401 

Table 2. In vitro anti-biofilm activities of antibiotic and AMP combinations against biofilms

402 

of standard and clinical P. aeruginosa strains. Decrease in MBEC

Interactions in TKC

CI1

CI2

CI3

CI4

Std

CI1

CI2

+LL-37











A

A

+CAMA











A

A

+LL-37











+CAMA











+LL-37









+CAMA







CI3

CI4

Std



S

S

S

S

A

cr

A

A

S

S

S

S

S

S

S

S

an

S



S

A

S

A

A



A

A

S

S

A

M

Colistin

us

Ciprofloxacin

ip t

Tobramycin

CI: Clinical isolate, Std: Standard, A: additive, S: synergist, 1x: no significant decrease, 2-8x:

404 

2-8 fold decrease in MBEC. Synergist or additive effects were defined as a ≥2-log10 decrease,

405 

or no 2-Log10 increase/decrease, respectively, in the colony count after 24 h.

Ac ce pt e

d

403 

406 

17   

Page 18 of 22

406 

Highlights

407 

• MBEC values of all antibiotics&s were 80->5120 and 640->640 mg/L,

408 

respectively.

409 

• Tobramycin, ciprofloxacin, colistin, LL-37 and CAMA were active against biofilms.

411 

• All studied antibiotics and AMPs were effective for inhibiting pre-formed biofilms.

412 

• The MBEC values of antibiotics, with AMPs at subMBEC, were decreased up to 8-

us

cr

ip t

410 

fold.

413 

an

• Especially ciprofloxacin+CAMA/LL-37 combinations were synergistic against

414 

biofilms.

M

415 

Ac ce pt e

d

416 

18   

Page 19 of 22

*

*

100 90 80 70 60 50 40 30 20 10 0

*

** *

*

*

**

* *

an

M

1h 2h 4h

M

ag

*

D ef

el

-M

C

37

LL -

* * **

C

Ac

on

tr

ol

ce pt

* *

*

**

ed

* *

*

M

100 90 80 70 60 50 40 30 20 10 0

B * *

cr

us

C ol

Pi p

D or

C ip

b To

tr C on

C ef

ip t

*

Antibiotics

Attachment %

1h 2h 4h

*

* *

* *

ol

Attachment %

A

Antimicrobic peptides

Figure 1. Inhibition of P. aeruginosa surface attachment in wells containing (A) antibiotics and (B) AMPs. Each well in the 96-well plates contained 1/10 the MIC of antibiotics or AMPs and an inoculum of 1×107 cfu/200 μl P. aeruginosa in TSB-glucose. The plates were incubated for 1, 2, or 4 h at 37°C. Six wells were used for each antibiotic and AMP. Each experiment is representative of at least two independent tests with standard and four clinical isolates of P. aeruginosa; the error bars indicate the standard deviations between strains. Control bars indicate P. aeruginosa biofilms without any antimicrobial, set as 100%. Results were expressed as percentage of biofilm formed with respect to control. *P < 0.001 vs control, ANOVA test followed by Bonferroni’s multiple comparison test. Page 20 of 22

*

*

*

*

*

*

* *

*

C

l C

*

*

ef

b To

C

*

*

ip

D

*

or

p Pi

*

ed

*

tr on

ol

C

L

37 L-

*

*

* *

1xMIC 1/10xMIC 1/100xMIC

*

ce pt

*

ol

*

M

*

Ac

Biofilm production %

B *

C

an

Antibiotics

100 90 80 70 60 50 40 30 20 10 0

*

*

ip t

*

o tr n o

1xMIC 1/10xMIC 1/100xMIC

*

cr

100 90 80 70 60 50 40 30 20 10 0

us

Biofilm production %

A

C

-M

el

M

D

ef

ag M

Antimicrobic peptides

Figure 2. Inhibition of P. aeruginosa biofilm formation by (A) antibiotics and (B) AMPs. Each well of the 96-well plates contained 1×, 1/10×, or 1/100×MIC of antibiotic or AMP, and an inoculum of 5×105 cfu/200 μl P. aeruginosa in TSB-glucose. The plates were incubated for 24 h at 37°C. Six wells were used for each antibiotic and AMP condition. Each experiment is representative of at least two independent tests with standard and four clinical isolates of P. aeruginosa; the error bars indicate the standard deviations between strains. Control bars indicate P. aeruginosa biofilms without any antimicrobial, set as 100%. Results were expressed as percentage of biofilm formed with respect to control. *P < 0.001 vs control, ANOVA test followed by Bonferroni’s multiple comparison tests. Page 21 of 22

A 8

6 5 4

CAMA Tob+LL-37

3

Tob+CAMA

2

0

6 9

3

24

Hours

us

B 8

an

7 6

Control

5 4

M

Log cfu/ml

ip t

Control Tob LL-37

cr

Log cfu/ml

7

2

0

3

ed

3

6 9

Cip LL-37 CAMA Cip+LL-37 Cip+CAMA

24

Hours

C

ce pt

8

6 5

Control Col LL-37

Ac

Log cfu/ml

7

CAMA

4

Col+LL-37

3

2

0

Col+CAMA 6 9

3

24

Hours

Figure 3. (A) Tob: tobramycin, (B) Cip: ciprofloxacin, (C) and Col: colistin + LL-37 or CAMA combinations observed by time-kill determinations against biofilms of standard and clinical P. aeruginosa strains at 1× MBEC. The X- and Y-axis represents time, and logarithmic P. aeruginosa survival in biofilm, respectively. Error bars indicate the standard deviations between strains. cfu: colony-forming unit, Control: P. aeruginosa biofilms without any antimicrobial treatment. Page 22 of 22