Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa

Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa

International Journal of Antimicrobial Agents 27 (2006) 196–200 Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas ae...

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International Journal of Antimicrobial Agents 27 (2006) 196–200

Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa A. Abdi-Ali a,∗ , M. Mohammadi-Mehr b , Y. Agha Alaei b a

Department of Biology, Faculty of Science, Alzahra University, Tehran, Iran b Army University of Medical Sciences, Tehran, Iran Received 16 June 2005; accepted 6 October 2005

Abstract Clinical isolates of Pseudomonas aeruginosa were collected from hospitals in Tehran, Iran, and identified using biochemical tests. A modified microtitre plate test was used to determine the biofilm-forming capacity of the isolates, measured with an enzyme-linked immunosorbent assay (ELISA) reader. Results showed that P. aeruginosa strain 214 was the most efficient at producing biofilm compared with the other strains. Observation of the bacterial biofilm on Teflon sheets and on a catheter using a scanning electron microscope showed greater biofilm formation on the catheter than on Teflon sheets. In this study, we investigated the bactericidal activity of fluoroquinolones, ␤-lactams, macrolides and aminoglycoside. The results showed differences in the antibiotic susceptibility of planktonic and biofilm cell populations. Fluoroquinolones showed more potent activity than the other antibiotics, and biofilms were completely eradicated by treatment with 16 × the minimum inhibitory concentration (MIC) of ciprofloxacin and 64 × MIC of ofloxacin, whereas all biofilms survived 2560 ␮g/mL of imipenem and ceftazidime. Production of an exopolysaccharide matrix is one of the distinguishing characteristics of biofilms. It has been suggested that this matrix prevents access of antibiotics to the bacterial cells embedded in the community. In this study, we also evaluated the permeation of antibiotics through alginate of P. aeruginosa strain 214 using a sandwich cup method. Macrolides were most efficient, showing 100% penetration; fluoroquinolones and ␤-lactams had a high permeation rate >75%, whereas the rates for aminoglycosides were low (amikacin = 59%; gentamicin = 73%). © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Biofilms; Pseudomonas aeruginosa; Matrix penetration; Antibiotic efficacy

1. Introduction Pseudomonas aeruginosa is one of the most important opportunistic human pathogens [1]. It has emerged as a dominant pulmonary pathogen with biofilm-forming capability, resulting in progressive and intractable chronic pulmonary infections, especially in patients with cystic fibrosis [2]. There is also an increasing awareness of the important role of P. aeruginosa biofilms in the contamination of medical biomaterials such as catheters and prostheses [1]. Biofilm infections are difficult to eradicate with antimicrobial treatment, and in vitro susceptibility tests show considerable resistance of biofilm cells to killing. Biofilms have a role in up to 60% of human infections [3]. ∗

Corresponding author. E-mail address: [email protected] (A. Abdi-Ali).

There are two main reasons why biofilm bacteria are hard to eradicate using common antibiotic therapy. Alginate, which is the main constituent of P. aeruginosa biofilms, is an unbranched linear heteropolysaccharide consisting of polymannuronic–polyguluronic acid, which acts as a barrier and protects the infecting cells from humoral and cellular host defence systems as well as from the action of antibiotics. Another reason is that the biofilm bacteria are either slow-growing or non-growing [2,4]. The concentrations of antibiotics needed to kill bacteria in the sessile phase are often much higher than those required for bacteria in the planktonic phase [2]. In this study, using multiples of the minimum inhibitory concentration (MIC) of a test strain, we investigated the bactericidal activity of various antibiotics, including ceftazidime, imipenem, amikacin, gentamicin, ciprofloxacin, ofloxacin, azithromycin and erythromycin against

0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.10.007

A. Abdi-Ali et al. / International Journal of Antimicrobial Agents 27 (2006) 196–200

P. aeruginosa in the biofilm and planktonic phase of growth in vitro.

2. Materials and methods 2.1. Bacterial strain Forty-two isolates of P. aeruginosa were collected from different clinical sources, including blood, sputum, urine, stool, wound, ear, nose and burns. One isolate, P. aeruginosa 214, a mucoid clinical isolate, was selected for further study because this strain produced more biofilm compared with the other strains as assessed using a microtitre plate method. MICs of ceftazidime, imipenem, amikacin, gentamicin, ciprofloxacin, ofloxacin, azithromycin and erythromycin for P. aeruginosa 214, determined by the standard broth serial dilution method (microdilution method), were 1, 2, 2, 1, 0.25, 0.5, 4 and 4 ␮g/mL, respectively. Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 were used as controls. 2.2. Antimicrobial agents Ceftazidime, gentamicin, ciprofloxacin and ofloxacin were purchased from Fuzhou (China), imipenem from Merck Sharp and Dohma-chibert (France), amikacin from Zhejiang Younging (China), and azithromycin and erythromycin from Sandoz (Spain). 2.3. Modified microtitre plate test A modified microtitre plate test was used to determine biofilm formation [5]. Briefly, bacteria were grown overnight on Colombia agar plates and subcultured onto trypticase soy agar plus 5% glucose. Bacteria were then re-suspended in trypticase soy broth plus 5% glucose. The optical density at 650 nm (OD650 ) of the bacterial suspensions was determined and aliquots of 100 ␮L were inoculated in six parallel wells of a 96-well polystyrene plate. After incubation for 24 h at 37 ◦ C, the wells were rinsed with buffer A (0.01 M potassium phosphate buffer made isotonic with saline, pH 7.5) to remove detached cells and then fixed with 150 ␮L absolute methanol for 10 min. Attached bacterial material was then stained by adding 150 ␮L crystal violet (1% w/v) for 20 min. The plates were rinsed with tap water (to remove excess crystal violet dye) and the amount of attached material was measured by solubilisation of the crystal violet dye in 150 ␮L of 33% glacial acetic acid. The A570 was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

The Teflon sheets and catheter were fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS) (1 h), washed with PBS, transferred to 1% (w/v) tannic acid in PBS (1 h), then washed in PBS. They were dehydrated in a graded concentration of ethanol, frozen in a freezer (Snijder Scientific, The Netherlands) at −65 ◦ C and dried at the critical point of vacuum pressure in Christ instruments under the following temperature conditions: condenser temperature −53 ◦ C, shelf temperature 15 ◦ C. They were then covered with gold using a voltage of 15–16 kV and a coating time of ca. 30 s. Biofilms were then observed with a scanning electron microscope (LEO 440i; Electron Microscopy Ltd., Cambridge, UK). 2.5. Extraction of exopolysaccharide The procedure for production and extraction of exopolysaccharide has been described previously by May and Chacrabarty [7]. After incubation of the bacteria on MacConkey agar plus 2% glycerol for 10 days at 21 ◦ C, surface growth was collected in saline and vortexed vigorously until uniformly dispersed. After removal of the whole bacteria, alginate was precipitated by 95% ethanol, collected, washed twice with 95% ethanol and once with absolute alcohol and then dried. 2.6. Sandwich cup method for determination of the permeability of the alginate layer to antibiotics Alginate was isolated from P. aeruginosa mucoid strain 214. A log phase culture of P. aeruginosa 214 grown in nutrient broth at 37 ◦ C, adjusted to an OD600 of 0.6, was used as the indicator strain. Fig. 1 shows a diagram of the sandwich cup method with a few modification used in this study to assay the penetration of antimicrobial agents through pseudomonal alginate [8]. After inserting a culture plate with a 0.4 ␮m pore size filter (12 mm in diameter; Millipore Corp.

2.4. Electron microscopic study Bacterial biofilms on Teflon sheets and on a catheter were fixed by the method described previously by Ohgaki [6].

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Fig. 1. Diagram of the sandwich cup method.

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Bedford, MA) in the centre of a 100 mm diameter Petri dish, 20 mL of nutrient agar at 47 ◦ C was mixed with 50 ␮L of bacteria suspension and poured into the dish. After the agar had hardened, 200 ␮L of either heat solubilised 1% Noble agar or alginate preparation was placed into the insert and allowed to solidify. Finally, 50 ␮L of 64 ␮g/mL of different antibiotics was added to the insert. Plates were then incubated overnight at 37 ◦ C. Zone sizes representing inhibition of bacterial growth were measured and the radius (after the contribution of the insert was subtracted) of the zone that was observed with Noble agar was defined as representing 100% penetration of antibiotic [1]. Inhibition of antibiotic activity for each alginate concentration was determined as follows: Percentage of inhibition    radius with alginate = 100 × 1 − . radius with Noble agar

(1)

2.7. Bactericidal activity of antibiotics against biofilm-forming and planktonic cells To test the bactericidal activity of antibiotics against the biofilm cells, catheter pieces of 1.5 cm in length were incubated with the organism as described by Ishida et al. [2] and Ohgaki [6]. Briefly, bacteria were incubated in trypticase soy agar with 5% glucose for 16–20 h at 37 ◦ C and re-suspended in saline adjusted to 0.5 McFarland turbidity. Then, 200 ␮L of this suspension and the catheter pieces were added to 18.8 mL of trypticase soy broth with 1 mL of 5% glucose and incubated for 6 days at 37 ◦ C. The catheter pieces incubated with the organisms were washed gently with PBS and transferred to Mueller–Hinton broth (MHB) containing a given antibiotic for 6 h at 37 ◦ C. The bacteria recovered from the catheter and PBS were designated biofilm and planktonic cells, respectively. The catheter pieces were transferred to 1 mL of PBS and vortexed for 5 min. The suspensions were diluted and plated on nutrient agar plates and viable cells were counted after incubation for 24 h at 37 ◦ C. For planktonic cells, 1 mL of the PBS that the catheter pieces had been soaked in was transferred to MHB containing the desired antibiotic. The number of surviving bacteria was determined in the same way as for the biofilm cells.

3. Results 3.1. In vitro biofilm mode of bacterial growth on the Teflon and catheter surface The modified microtitre plate test was used to determine the biofilm-forming capacity of P. aeruginosa isolates measured using an ELISA reader at 570 nm. The results demonstrated that P. aeruginosa strain 214 produced more biofilm (OD = 2.89) compared with the other 41 strains.

Fig. 2. Scanning electron micrographs of Pseudomonas aeruginosa strain 214 on the surface of Teflon sheets incubated for (a) 1 day or (b) 7 days.

Figs 2 and 3 show scanning electron micrographs of sessile cells on the surface of Teflon sheets and a catheter, respectively, that had been incubated with P. aeruginosa 214 for 1 day or 7 days. 3.2. Permeation of antibacterials through the alginate layer Table 1 shows the rate of drug diffusion through the agar layer containing 1% alginate. The value for permeation of drugs through an agar layer without alginate was assigned a value of 100%. When alginate was

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Table 2 Bactericidal activities of antibiotics against biofilm and planktonic cells Antibiotics

Biofilm cells

Planktonic cells

Gentamicin Amikacin Ofloxacin Ciprofloxacin Azithromycin Erythromycin Imipenem Ceftazidime

512 × MIC 256 × MIC 64 × MIC 16 × MIC 2560 ␮g/mL 2560 ␮g/mL >2560 ␮g/mL >2560 ␮g/mL

4 × MIC 4 × MIC 1 × MIC 4 × MIC 64 × MIC 128 × MIC 64 × MIC 128 × MIC

MIC, minimum inhibitory concentration.

3.3. Bactericidal activities of antimicrobials against biofilm-forming sessile cells and floating (planktonic) cells Several different bactericidal antimicrobials (ceftazidime, gentamicin, ciprofloxacin, ofloxacin, imipenem, amikacin, azithromycin and erythromycin) were chosen to test the relative resistance to killing of planktonic and biofilm cells. Table 2 shows the bactericidal action of the various antibiotics on sessile and planktonic cells of P. aeruginosa 214.

4. Discussion

Fig. 3. Scanning electron micrographs of Pseudomonas aeruginosa strain 214 on the surface of a catheter incubated for (a) 1 day or (b) 7 days.

added at 1% to the agar, the diffusion rates of fluoroquinolones, ␤-lactams and aminoglycosides decreased but remained above 50%; macrolides showed a permeation rate of 100%. Table 1 Rates of diffusion through the agar layer containing 1% alginate Antibiotics

Penetration rate (%)

Inhibition rate (%)

Gentamicin Amikacin Ofloxacin Ciprofloxacin Azithromycin Erythromycin Imipenem Ceftazidime

73 59 78 90 100 100 86 95

27 41 22 10 – – 14 5

Scanning electron microscopy observation of the bacterial biofilm on the catheter and Teflon sheets showed that biofilm formation on the catheter was greater than on Teflon sheets. Moreover, Ishida et al. [2] reported data that support our conclusions. It is commonly accepted that biofilms are more resistant to antibiotics than planktonic cells. Several factors have been suggested to account for biofilm tolerance, for example slow growth and the presence of an exopolysaccharide matrix that can slow the diffusion of antibiotics, as well as the presence of unknown resistance mechanisms [3]. The ␤-lactam antibiotics imipenem and ceftazidime, and the aminoglycosides gentamicin and amikacin were hardly effective. There are several reasons why these antibacterial agents are not as effective on biofilm cells as they are on planktonic cells. Some antibiotics, such as ␤-lactams, require rapid bacterial growth to kill cells [3]. Also, a reduction in antibacterial penetration through the biofilm layer owing to alginate, the main constituent of the biofilm, acts as a barrier [2]. Fluoroquinolones showed a strong bactericidal activity against the biofilm-forming cells compared with the other antibiotics, and biofilms were completely eradicated by treatment with 16 × MIC of ciprofloxacin and 64 × MIC of ofloxacin. Planktonic cells were killed by 4 × MIC of ciprofloxacin and 1 × MIC of ofloxacin. For other antibiotics in this study, persistence of biofilm cells was 10–1000 times more than planktonic cells. The results are supported by some investigators [9–11]. Our results demonstrated a macrolide penetration rate

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through alginate of 100%; fluoroquinolones and ␤-lactams showed a relatively high penetration rate >75%, whereas aminoglycosides showed low penetration (gentamicin 73% and amikacin 59%). In this respect, some of our results were in contrast to those of Ishida et al. [2]. The results of the bactericidal activity of antibiotics and the permeability study of the alginate layer show that alginate is not the only resistance factor for biofilm cells. Results for ␤-lactams and macrolides showed high permeation but low bactericidal activity (>2560 ng/mL), whereas aminoglycosides with low penetration showed greater bactericidal activity than the ␤-lactams and macrolides. The results indicated that the fluoroquinolones show higher permeability in alginate as well as higher bactericidal activity and consequently can be considered a first-choice antibiotic in the treatment of P. aeruginosa biofilms.

[2]

[3]

[4]

[5]

[6] [7]

Acknowledgments This work was supported by a research grant from the Army University of Medical Sciences, Tehran, Iran. We would like to thank Dr M.H. Heidari for his assistance in electron microscopy.

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[9]

[10]

References [11] [1] Hatch RA, Schiller NL. Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid

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