World J Microbiol Biotechnol (2014) 30:3015–3025 DOI 10.1007/s11274-014-1728-7
ORIGINAL PAPER
Antimicrobial activity of the imipenem/rifampicin combination against clinical isolates of Acinetobacter baumannii grown in planktonic and biofilm cultures Yang Wang • Wanguo Bao • Na Guo • Haiying Chen • Wei Cheng • Kunqi Jin • Fengge Shen • Jiancheng Xu • Qiaoli Zhang • Chao Wang Yanan An • Kaiyu Zhang • Feng Wang • Lu Yu
•
Received: 18 June 2014 / Accepted: 21 August 2014 / Published online: 9 October 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract To investigate the antimicrobial activity of imipenem and rifampicin alone and in combination against clinical isolates of Acinetobacter baumannii grown in planktonic and biofilm cultures. Minimum inhibitory concentrations were determined for each isolate grown in suspension and in biofilm using a microbroth dilution method. Chequerboard assays and the agar disk diffusion assay were used to determine synergistic, indifferent or antagonistic interactions between imipenem and rifampicin. We used the tissue culture plate method for A. baumannii biofilm formation to measure the percentage of biofilm inhibition and the amount of extracellular DNA after the treatment. To understand the synergistic mechanisms, we conducted hydroxyl radical formation assays. The results were verified by confocal laser scanning Y. Wang W. Bao N. Guo H. Chen W. Cheng K. Jin F. Shen Q. Zhang C. Wang Y. An K. Zhang F. Wang L. Yu (&) Department of Infectious Diseases, First Hospital of Jilin University, Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, China e-mail: [email protected] N. Guo Department of Food Quality and Safety, Jilin University, Changchun, China J. Xu Department of Clinical Laboratory, First Hospital of Jilin University, Changchun, China F. Wang (&) Department of Infectious Diseases, First Hospital of Jilin University, Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130021, China e-mail: [email protected]
microscopy. Imipenem and rifampicin showed effective antimicrobial activity against suspensions and biofilm cultures of A. baumannii, respectively. Synergistic antimicrobial effects between imipenem and rifampicin were observed in 13 and 17 of the 20 clinical isolates when in suspension and in biofilms, respectively. Imipenem and rifampicin alone and in combination generated hydroxyl radicals, which are highly reactive oxygen forms and the major components of bactericidal agents. Furthermore, treatment with imipenem and rifampicin individually or in combination has obvious antibiofilm effects. The synergistic activity of imipenem and rifampicin against clinical isolates of A. baumannii (in suspension and in biofilms) was observed in vitro. Therefore, we conclude that imipenem combined with rifampicin has the potential to be used as a combinatorial therapy for the treatment of infectious diseases caused by A. baumannii. Keywords Antimicrobial activity Biofilms Imipenem Rifampicin Synergy
Introduction Acinetobacter baumannii is a non-motile, gram-negative, opportunistic pathogen. A. baumannii has the ability to form biofilms, which may play a role in the process of colonization. A. baumannii has become increasingly important due to its resistance to a wide variety of antibiotics and its ability to survive in a hospital environment (Wilson et al. 2004). The emergence of the multi-drug resistant nosocomial A. baumannii infection has prompted the evaluation of newer antimicrobial agents and older warehoused agents as possible therapeutic candidates. Carbapenems (e.g.,
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imipenem) have been used as the antibiotics of choice for the treatment of A. baumannii infections, but increasing resistance to these antimicrobial agents mediated by blactamases is undermining this option. The resistance of A. baumannii to all major classes of antibiotics (except polymyxins) has substantially increased worldwide in the past decade (Talbot et al. 2006; Peleg et al. 2006). While researchers are looking for new antimicrobial agents to solve this problem, they are also trying to determine the interactions between different types of traditional antibiotics and identify those combinations of antibiotics with synergistic effects. Recent studies have noted promising results when testing rifampicin/imipenem combinations against A. baumannii in planktonic and animal models. It was found that some kind of bactericidal antibiotics could simulate hydroxyl radical formation in bacteria and this toxic chemical contributes to the killing efficiency of lethal antibiotics in 2007 (Kohanski et al. 2007). In some papers about different antibiotics combination therapy and the mechanisms of the synergistic effect, hydroxyl radical formation was detected to know if the introduction of hydroxyl radical formation was one of the synergistic mechanisms (Kim et al. 2011; Lee et al. 2012). But there were no reports about the hydroxyl radical formation level in the treatment with imipenem and rifampicin against A. baumannii. Inspired by this, we detected the hydroxyl radical production after treatment with imipenem and rifampicin against A. baumannii. In light of these results, we treated 20 clinical A. baumannii strains with a rifampicin/imipenem combination in both planktonic and in biofilm cultures. The data were collected prospectively to assess the efficacy of this combination and to discuss the mechanism of the effect of the combination.
Materials and methods Materials Unless stated otherwise, all reagents were purchased from Sigma Chemical Company (St. Louis, USA). The cell culture plates were polystyrene, flat-bottomed, sterile, tissue culture-treated 96-well microtiter plates with low evaporation lids purchased from Nest Biotech Ltd (Wuxi, China). Crystal Violet and DNase I were purchased from Sigma-Aldrich (Dorset, UK). Imipenem was from Kangmanlin Ltd (Nanjing, China). Rifampicin was from Dingguo Ltd (Beijing, China). DNase I (Sigma) stock solutions were prepared before each experiment in 0.15 M NaCl supplemented with 5 mM of MgCl2 (Qin et al. 2007). Working solutions of DNase I 20 lg ml-1 were prepared in RPMI medium.
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Micro-organisms Clinically isolated A. baumannii was obtained from four hospitals of Jilin University and five other hospitals of Jilin Province from blood and sputum samples of infected patients. The quality control (QC) strain ATCC 19606T was obtained from American Type Culture Collection (Manassas, USA). The A. baumannii strains were stored on MicroBank beads (Pro-Lab Diagnostics, Cheshire, UK) at -70 °C until required. Preparation of antimicrobial agents Imipenem were dissolved in deionized water to obtain a stock solution of 20.480 mg ml-1 under sterile conditions and stored at -70 °C until use. Rifampicin were dissolved in DMSO to obtain a stock solution of 20.480 mg ml-1 under sterile conditions and stored at -70 °C until use. Preparation of A. baumannii inoculum for suspension assay The strains were placed in LB agar and incubated at 37 °C overnight. Colonies from the plates were resuspended in MH broth and cultured at 37 °C for 16 h. With optical density (OD) at 660 nm, the concentration of each overnight cultured suspension was determined based on a previously established OD concentration standard curves The suspensions were further diluted with MH broth to obtain inocula containing 1 9 106 CFU ml-1 (Clock et al. 2013). Determination of MICs of imipenem and rifampicin for A. baumannii in suspension The MICs of imipenem and rifampicin were determined by the broth microdilution method adapted from previous studies and in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2012). Serial twofold dilutions of antimicrobial agents were prepared in MH broth to obtain the required concentrations (1.0 lg ml-1 to 1.024 mg ml-1). The assay was performed in 96-well microtiter plates, and a positive control, which contained inoculated broth without any drugs, was included in every plate. Each well contained 100 ll of the test antimicrobial and 100 ll of the A. baumannii suspension diluted in MH broth, containing 1 9 105 CFU. The plates were incubated in air for 24 h at 37 °C. The MIC was defined as the lowest concentration of the drug that inhibited the growth of the test micro-organism by [90 % visually (Oo et al. 2010). The assay was performed in triplicate.
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Assessment of the antimicrobial activity of imipenem in combination with rifampicin against A. baumannii in suspension via the chequerboard assay The antimicrobial activity of imipenem in combination with rifampicin was assessed in a suspension assay by the chequerboard method (Shin and Lim 2004). Serial twofold dilutions of the antimicrobial compounds were prepared in MH broth so that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for both imipenem and rifampicin. Fifty microliters of each imipenem solution was added to the rows of a 96-well microtiter plate in diminishing concentrations, and 50 ll of the rifampicin was also added to the columns in diminishing concentrations. The wells were then inoculated with 100 ll of A. baumannii suspension containing 1 9 105 CFU. The wells in column 12 served as controls containing MH broth either with or without the inoculum. After inoculation and agitation, the microplates were incubated in air at 37 °C for 24 h, and the MICs of the agents individually and in combination were determined as described previously. To assess the synergistic or antagonistic activity of antimicrobial combinations, the fractional inhibitory concentration (FIC) and FIC index (FICI) were determined using the following formulas (Oo et al. 2010): FIC = MIC of antimicrobial in combination/MIC of antimicrobial alone; FICI = FIC of imipenem ? FIC of rifampicin (Clock et al. 2013; Principe et al. 2013). In this study, the FICI, calculated as the sum of each FIC, was interpreted as follows: FICI B0.5 demonstrated synergy; 0.5 \ FICI B 4 indicated indifferent; and FICI [4 showed antagonism. Agar disk diffusion assay for imipenem combined with rifampicin in A. baumannii J9 A 100-ll sample of 106 CFU ml-1 A. baumannii J9, which exhibited the best synergistic effect, was spread onto an LB agar surface. Subsequently, 6-mm-diameter paper disks, impregnated with imipenem (4 lg ml-1), rifampicin (4 lg ml-1) and imipenem (4 lg ml-1) combined with rifampicin (4 lg ml-1), were placed onto the surface. The inhibition zones were measured using a dial caliper after a 48-h incubation at 37 °C. The tests were performed in duplicate (Kiraz et al. 2009).
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30 -(p-hydroxyphenyl) fluorescein (HPF; Invitrogen) was used at a concentration of 5 lM. ODs were measured with a spectrofluorophotometer (Infinite F200 Pro, TECAN, Switzerland) at a 492-nm excitation and a 525-nm emission wavelength (Price et al. 2009; Choi and Lee 2012). The increase in the percentage of hydroxyl radical was calculated using the following equation: [(OD490 of cells treated with agent - OD490 of non-treated control)/OD490 of nontreated control) 9 100]. Experiments were performed in triplicate, the data were averaged, and the standard deviation (SD) was calculated. Establishment of A. baumannii biofilms For each clinically isolated A. baumannii strain, the OD of the overnight suspension was determined at 660 nm and diluted in TSB to obtain the final concentration of 1 9 105 CFU ml-1. Bacterial biofilms were prepared by aliquoting 200 ll of the bacterial suspension diluted in 2 % glucose-TSB containing 1 9 105 CFU ml-1 into the wells of the 96-well microtiter plates. The microtiter plates were then incubated in air for 24 h at 37 °C (Rodrı´guez-Ban˜o et al. 2008). Determination of the MICs of imipenem and rifampicin for A. baumannii in biofilm Biofilms were grown in 96-well microtiter plates (1 9 105 CFU ml-1, 200 ll/well) for 24 h. After incubation, the microtiter plates containing A. baumannii biofilm were washed twice gently with sterile PBS to remove the unbound bacteria. Serial double dilutions of antimicrobial agents were prepared in TSB to obtain the required concentrations (both of imipenem and rifampicin 0.5–512 lg ml-1). To triplicate wells, 200 ll of each antimicrobial agent was added to each microtiter plate well in decreasing concentrations across the rows. Columns 12 served as controls containing the biofilm and saline. Following incubation in air at 37 °C for 24 h, the MIC was determined as the lowest concentration to show growth below or equal to that of the control visually (biofilm in saline).
Hydroxyl radical formation assay
Assessment of the antimicrobial activity of imipenem in combination with rifampicin against A. baumannii in biofilm via the Chequerboard assay
Bacterial cells (1 9 106 CFU ml-1) were treated with imipenem and rifampicin individually or in combinations, and the final concentration of the treated agents was the MIC for each agent or the combination treatments. The samples were incubated for 1 h at 37 °C. To detect hydroxyl radical formation, the fluorescent reporter dye
Biofilms were grown in 96-well microtiter plates (1 9 105 CFU ml-1, 200 ll/well) for 24 h and then gently washed once with PBS to remove the unbound bacteria. Serial twofold dilutions of the drugs were prepared in TSB so that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for both imipenem and rifampicin.
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One hundred microliters of each imipenem solution were added to the rows of a 96-well microtiter plate in diminishing concentrations, and 100 ll of the rifampicin solutions were also added to the columns in diminishing concentrations. Column 12 served as controls containing TSB both with and without the inoculum. Following incubation at 37 °C for 24 h, the antimicrobial supernatant was removed, and the wells were washed again with PBS twice. The MIC, FIC and FICI values were determined as described previously. The assay was performed in triplicate. Determination of biofilm formation by the tissue culture plate (TCP) method Quantitative biofilm assays of crystal violet-stained cells cultured in TSB were performed as described previously (Tomaras et al. 2003). Briefly, biofilms were grown in 96-well microtiter plates (1 9 105 CFU ml-1, 200 ll/well). After culturing for 24 h, the wells were washed softly with PBS twice to remove free-floating bacteria. Then, the solutions containing TSB and the drugs were added into the wells, and the plates were incubated for 24 h at 37 °C. After the incubation, the wells were washed twice with 200 ll of PBS. Biofilms formed by adherent ‘‘sessile’’ organisms in the plate were stained with crystal violet (1 %, w/v). Excess stain was rinsed off by washing with cold deionized water, and then the plates were dried. After drying, 95 % ethanol was added to the wells, the plates were vortexed, and the ODs were measured using a microtiter ELISA reader (Molecular Devices Emax, CA, USA) at 595 nm. These OD values were considered an index of bacterial adherence to the surface and the formation of biofilms. The percentage of biofilm inhibition was calculated using the following equation: [1 (OD595 of cells treated with agent/OD595 of non-treated control)] 9 100 (Wei et al. 2006; Rogers et al. 2010). Experiments were performed in triplicate, the data were averaged, and the SD was calculated. The results were expressed as percentage of biofilm inhibition. Determination of extracellular DNA (eDNA) of A. baumannii J9 in biofilm treated with imipenem combined with rifampicin
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were harvested by resuspension in 50 mM TrisHCl/10 mM ETDA/500 mM NaCl, pH 8.0, and transferred into prechilled tubes. After centrifugation for 5 min at 18,0009g at 4 °C, each supernatant was transferred to a tube containing threefold the volume of TE buffer (10 mM TrisHCl/1 mM EDTA, pH 8.0) and extracted once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). The aqueous phase of each sample was then mixed with threefold the volume of ice-cold 100 % (vol/vol) ethanol and 1/10 the volume of 3 M sodium acetate (pH 5.2) and stored at -20 °C overnight. Then, the ethanol-precipitated DNA was collected by centrifugation for 20 min at 18,0009g at 4 °C, washed with ice-cold 70 % (vol/vol) ethanol, air-dried, and dissolved in 40 ll of TE buffer. The concentration and purity of the DNA were determined spectrophotometrically by the absorbance ratio A260/A280 using a NanoDrop 2000 (Thermo Scientific; Steinberger and Holden 2005; Tetz et al. 2009). The assay was performed in triplicate. Confocal laser scanning microscopy (CLSM) Bacterial cells of A. baumannii strain J9 (1 9 105 CFU ml-1, 2 ml/well) were grown on coverslides in sixwell plates for 48 h to form the biofilms. After incubation, the biofilms were treated with imipenem and rifampicin individually or in combination. The coverslides were then washed gently with PBS, moved to a new plate and treated for 24 h with imipenem and rifampicin individually or in combination. The coverslides were washed again with PBS and stained with the LIVE/DEAD BacLight Bacterial Viability kit (Invitrogen Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. CLSM images were collected using an Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan) with a 60 9 objective lens. For the detection of SYTO 9 (green, alive), we used 488-nm excitation and 520-nm emission filter settings. For PI detection (red, dead), we used the 543-nm excitation and 572-nm emission filter settings. Image analyses and export were performed in a Fluoview, ver. 1.7.3.0 (Olympus; Sauer et al. 2009).
Results Bacterial cells of A. baumannii stain J9 (1 9 105 CFU ml-1, 2 ml/well) were grown in six-well plates for 24 h at 37 °C to form the biofilms and then were treated with imipenem and rifampicin alone or in combination for 24 h. Then, the plates were gently washed three times with PBS without disturbing the adherent film and chilled at 4 °C for 1 h, and 1 ll of 0.5 M EDTA was added to each well. The supernatants were removed, and unwashed biofilm
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The MICs of aqueous imipenem and rifampicin for A. baumannii in suspension and biofilm In our study, 20 clinical isolates of A. baumannii and one QC strain ATCC 19606T were selected. Imipenem and rifampicin demonstrated antimicrobial activity against suspensions and biofilms of A. baumannii (Table 1). The
World J Microbiol Biotechnol (2014) 30:3015–3025 Table 1 MICs of imipenem and rifampicin for suspension and biofilm cultures of A. baumannii (MICs in lg ml-1)
MIC minimum inhibitory concentrations
Strains
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Source
MIC for suspension
MIC for biofilm
Imipenem
Imipenem
Rifampicin
Rifampicin
A. baumannii J1
Blood
16
8
64
128
A. baumannii J2
Sputum
32
8
128
64
A. baumannii J3
Sputum
16
4
128
16
A. baumannii J4
Cerebrospinal fluid
8
8
128
128
A. baumannii J5
Sputum
16
16
256
128
A. baumannii J6
Blood
8
4
128
32
A. baumannii J7
Berebrospinal fluid
32
4
256
32
A. baumannii J8
Sputum
32
4
512
64
A. baumannii J9
Blood
64
16
512
128
A. baumannii J10
Sputum
32
4
512
32
A. baumannii S1 A. baumannii S2
Sputum Blood
16 16
4 4
128 128
32 64
A. baumannii S3
Sputum
32
8
256
64
A. baumannii S4
Sputum
32
4
256
32
A. baumannii S5
Sputum
16
4
128
32
A. baumannii S6
Sputum
2
4
32
64
A. baumannii S7
Sputum
64
16
256
128
A. baumannii S8
cerebrospinal fluid
64
4
512
64
A. baumannii S9
Blood
32
2
256
16
A. baumannii S10
Sputum
16
4
128
32
ATCC
1
2
4
16
T
ATCC 19606
MICs of imipenem and rifampicin against these clinical strains growing in suspension ranged from 2 to 64 and 2–16 lg ml-1, respectively. Among these tested isolates, the MICs of imipenem against biofilm ranged from 32 to 512 lg ml-1, while the MICs of rifampicin against biofilm ranged from 16 to 128 lg ml-1. The MIC values of imipenem and rifampicin were 4- to 16-fold higher for A. baumannii growing in biofilm compared with the same strain in suspension. The antimicrobial activity of imipenem in combination with rifampicin against A. baumannii in suspension and in biofilm To analyze the interaction of imipenem and rifampicin against A. baumannii in suspension and in biofilm in the chequerboard assay, we used the nonparametric FICI approach, based on the Loewe additivity theory. The antimicrobial activities of imipenem combined with rifampicin against A. baumannii in suspension and biofilm were shown in Tables 2 and 3, respectively. Using the FICI method, thirteen of the 20 A. baumannii clinical strains were determined to have synergistic interactions in suspension, with FICI values ranging from 0.25 to 0.5, and eight A. baumannii strains had indifferent interactions in suspension, with FICI values ranging from 0.5625 to 1.
Among the 20 A. baumannii clinical strains, 17 strains were determined to have synergistic interactions in biofilm, with FICI values ranging from 0.25 to 0.5, and four A. baumannii strains had indifferent interactions in suspension, with FICI values ranging from 0.75 to 1. The synergism between imipenem and rifampicin was confirmed by agar diffusion tests The halo diameters produced by the combination were predominantly larger than ones produced by single-drug treatments. The sizes of the inhibition zones increased to 13.6 mm when 4 lg ml-1 imipenem (6.2 mm) was combined with 4 lg ml-1 of rifampicin (9.0 mm; Fig. 1). The hydroxyl radicals increase by treatment with imipenem and rifampicin for A. baumannii in suspension We tested whether A. baumannii strain J9 with imipenem and/or rifampicin generated hydroxyl radicals and examined how this influenced the synergistic effects. As seen in Fig. 2, both imipenem and rifampicin have the capability to generate hydroxyl radicals, and the bacterial cells treated with the MIC of imipenem and rifampicin in combination showed significantly increased hydroxyl radical formation.
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Table 2 Antimicrobial activities of imipenem combined with rifampicin against A. baumannii growing in suspension (MIC and FIC in lg ml-1) Strains
MIC of imipenem (in combination/alone)
FIC of imipenem (average)
MIC of rifampicin (in combination/alone)
FIC of rifampicin (average)
FICI (average)
Result
A. baumannii J1
1/16
0.0625
2/8
0.25
0.3125
Synergy
A. baumannii J2
8/32
0.25
4/8
0.5
0.75
Indifference
A. baumannii J3
4/16
0.25
2/4
0.5
0.75
Indifference
A. baumannii J4
4/8
0.5
2/8
0.25
0.75
Indifference
A. baumannii J5
2/16
0.125
2/16
0.125
0.25
Synergy
A. baumannii J6
0.5/8
0.0625
1/4
0.25
0.3125
Synergy
A. baumannii J7 A. baumannii J8
2/32 2/32
0.0625 0.0625
1/4 1/4
0.25 0.25
0.3125 0.3125
Synergy Synergy
A. baumannii J9
4/64
0.0625
4/16
0.25
0.3125
Synergy
A. baumannii J10
2/32
0.0625
1/4
0.25
0.3125
Synergy
A. baumannii S1
2/16
0.125
1/4
0.25
0.375
Synergy
A. baumannii S2
4/16
0.25
1/4
0.25
0.5
Synergy
A. baumannii S3
8/32
0.25
4/8
0.5
0.75
Indifference
A. baumannii S4
8/32
0.25
0.25/4
0.125
0.375
Synergy
A. baumannii S5
1/16
0.0625
1/4
0.25
0.3125
Synergy
A. baumannii S6
0.5/2
0.25
1/4
0.25
0.5
Synergy
A. baumannii S7
16/64
0.25
8/16
0.5
0.75
Indifference
A. baumannii S8
8/64
0.125
1/4
0.25
0.375
Synergy
A. baumannii S9
16/32
0.5
1/2
0.5
1
Indifference
A. baumannii S10
16
0.25
2/4
0.5
0.75
Indifference
ATCC 19606T
0.0625/1
0.0625
1/2
0.5
0.5625
Indifference
MIC minimum inhibitory concentrations, FIC fractional inhibitory concentration, FICI fractional inhibitory concentration index
The increase in the percentage of hydroxyl radicals when treated with the MIC of imipenem and rifampicin in combination was much higher than the corresponding value for treatment with the MIC of rifampicin in combination (p \ 0.05) and the MIC of rifampicin in combination (p \ 0.05), respectively. This result shows that hydroxyl radical formation might be an important factor in the synergism. Anti-biofilm formation effect of imipenem in combination with rifampicin against A. baumannii Treatment with 1/8 MIC of imipenem combined with 1/8 MIC of rifampicin had an inhibitory effect of 45.91 ± 6.47 % on pre-formed biofilms. The effect was much more significant than the inhibitory effect of treatment with 1/8 MIC of imipenem in combination (34.89 ± 2.59 %) and 1/8 MIC of rifampicin (5.88 ± 1.27 %; Fig. 3). The percentage of biofilm inhibition following combination treatment with imipenem and rifampicin was significantly different from the corresponding value for treatment with the MIC of rifampicin in combination (p \ 0.05). There was no significant difference between the percentage of biofilm inhibition when treated with 1/8 MIC of imipenem combined with 1/8 MIC of
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rifampicin and the corresponding value for treatment with the MIC of imipenem in combination (p = 0.08). The eDNA changes by treatment with imipenem and rifampicin in biofilm of A. baumannii strain J9 The amount of eDNA in biofilms was quantified by spectrophotometry and reported as eDNA per relative biomass in the biofilm treated with 1/8 MIC of imipenem and 1/8 MIC of rifampicin alone or 1/8 MIC of imipenem combined with 1/8 MIC of rifampicin while it was also treated with DNase I, which effectively degrades eDNA in biofilms. As shown in Fig. 4, treatment with imipenem or rifampicin resulted in a significant decrease in the amount of eDNA from the cell-free supernatants in A. baumannii strain J9 strain compared with the level in the cultures, and treatment with imipenem combined with rifampicin displayed a significant reduction in the amount of eDNA. The value of the quantity of eDNA treated with any agent was significantly different from the corresponding value of the untreated biofilms (control group; p \ 0.05). The value of the quantity of eDNA in biofilms treated with imipenem combined with rifampicin was significantly different from the corresponding value for biofilms treated with the imipenem (p \ 0.05) or rifampicin (p \ 0.05) individually.
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Table 3 Antimicrobial activities of imipenem combined with rifampicin against A. baumannii growing in biofilms (MIC and FIC in lg ml-1) Strains
MIC of imipenem (in combination/alone)
FIC of imipenem (average)
MIC of rifampicin (in combination/alone)
FIC of rifampicin (average)
FICI (average)
Result
A. baumannii J1
8/64
0.125
32/128
0.25
0.375
Synergy
A. baumannii J2
32/128
0.25
32/64
0.5
0.75
Indifference
A. baumannii J3
32/128
0.25
4/16
0.25
0.5
Synergy
A. baumannii J4
32/128
0.25
64/128
0.5
0.75
Indifference
A. baumannii J5
32/256
0.125
16/128
0.125
0.25
Synergy
A. baumannii J6
16/128
0.125
4/32
0.125
0.25
Synergy
A. baumannii J7 A. baumannii J8
32/256 128/512
0.125 0.25
4/32 8/64
0.125 0.125
0.25 0.375
Synergy Synergy
A. baumannii J9
64/512
0.125
16/128
0.125
0.25
Synergy
A. baumannii J10
32/512
0.0625
8/32
0.25
0.3125
Synergy
A. baumannii S1
16/128
0.125
8/32
0.25
0.375
Synergy
A. baumannii S2
32/128
0.25
16/64
0.25
0.5
Synergy
A. baumannii S3
64/256
0.25
16/64
0.25
0.5
Synergy
A. baumannii S4
64/256
0.25
8/32
0.25
0.5
Synergy
A. baumannii S5
8/128
0.0625
8/32
0.25
0.3125
Synergy
A. baumannii S6
8/32
0.25
16/64
0.25
0.5
Synergy
A. baumannii S7
128/256
0.5
64/128
0.5
1
Indifference
A. baumannii S8
128/512
0.25
16/64
0.25
0.5
Synergy
A. baumannii S9
64/256
0.25
4/16
0.25
0.5
Synergy
A. baumannii S10
32/128
0.25
8/32
0.25
0.5
Synergy
ATCC 19606T
1/4
0.25
8/16
0.5
0.75
Indifference
MIC minimum inhibitory concentrations, FIC fractional inhibitory concentration, FICI fractional inhibitory concentration index
CLSM of bacterial cell survival in biofilms exposed to imipenem and rifampicin individually and in combination The effects of imipenem and rifampicin individually and in combination on pre-existing 2d-biofilms was studied by using CLSM (Fig. 5). After treatment for 24 h, the control group was chiefly comprised of living bacterial cells (Fig. 5f). Compared with the control group, treatment with 1/8 MIC of imipenem or 1/8 MIC of rifampicin killed only a small portion of the bacterial population (Fig. 5c, d), while treatment with each of the MICs of imipenem and rifampicin killed a significant portion of the bacterial population and reduced the number of bacteria present in biofilm (Fig. 5a, b). In contrast, treatment with the 1/8 MIC of imipenem combined with 1/8 MIC of rifampicin killed the vast majority of the biofilm bacteria, with few survivors at the surface (Fig. 5e).
Discussion Both imipenem and rifampicin are traditional and common antibiotics used to control bacterial infection with a wide antibacterial spectrum, and they have been used to treat A.
baumannii infection for a long time. However, the increasing emergence of drug-resistant A. baumannii strains has limited the application of these antibiotics in recent years. Synergistic activities have been previously observed between imipenem and rifampicin against A. baumannii in planktonic cultures, and the effects were also identified in an infected mice model recently (Wolff et al. 1999; Montero et al. 2004; Clock et al. 2013; Principe et al. 2013). Both of the two studies in vivo were conducted in a mouse pneumonia model. Majewski et al. (2014) identified the synergism or an additive interaction between rifampicin and imipenem most likely occurs in A. baumannii strains showing moderate resistance to imipenem in the latest paper. These studies only evaluated the effect by using the chequerboard method on planktonic bacteria or verified the effect in the animal model, but they did not demonstrate the mechanisms of the synergistic activity between imipenem and rifampicin and did not detect the activity of treatment with imipenem and rifampicin against A. baumannii in biofilms. Acinetobacter baumannii has the ability to form biofilms, which may play a role in the process of colonization. The ability to adhere to and form biofilms on the surface of inanimate objects might be the cause of its strong ability to survive in hospital environments. Our present study shows
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Fig. 1 Agar disk diffusion assay for imipenem combined with rifampicin in A. baumannii J9
Imipenem 4 µg ml-1
-1 rifampicin 4 µg ml-1 Imipenem 4 µg ml + rifampicin 4 µg ml-1
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Fig. 2 The percentage of hydroxyl radicals increased in A. baumannii strain J9 treated with imipenem, rifampicin and both antibiotics in combination, as verified by the HPF staining method. MIC of IMI: treatment with 64 lg ml-1 imipenem; MIC of RIF: treatment with 16 lg ml-1 rifampicin; 1/16 MIC of IMI: treatment with 4 lg ml-1 imipenem; 1/4 MIC of RIF: treatment with 4 lg ml-1 rifampicin; IMI ? RIF: treatment with 4 lg ml-1 imipenem and 4 lg ml-1 rifampicin. *p \ 0.05, compared with the group treated with imipenem combined with rifampicin
treatment with imipenem and rifampicin in combination against A. baumannii had a synergistic effect not only on the planktonic bacteria but also on the biofilms. The synergistic effect was observed both in imipenem-sensitive strains and in imipenem-resistant strains. Agar diffusion tests were conducted to confirm the synergistic effect. Biofilms are highly structured communities of bacteria attached to a surface and are recognized as a common cause of human infection. A biofilm is an aggregate of microorganisms in which the cells adhere to one another or to a surface in a self-produced matrix of eDNA, proteins, and polysaccharides. The cells forming the biofilms are morphologically, metabolically, and physiologically different from their planktonic counterparts. Biofilms help the
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Fig. 3 The percentage of biofilm inhibition by different antibiotics on 24-h-old biofilms of A. baumannii strain J9. 1/8 MIC of IMI: treatment with 64 lg ml-1 imipenem; 1/8 MIC of RIF: treatment with 16 lg ml-1 rifampicin; IMI ? RIF: treatment with 64 lg ml-1 imipenem and 16 lg ml-1 rifampicin. *p \ 0.05, compared with the group treated with imipenem combined with rifampicin
bacteria resist disinfection, while also allowing the participating cells to trade resistance genes, further facilitating the persistence of the pathogen (Stoodley et al. 2002). The present study shows that treatment with an imipenem/rifampicin combination against A. baumannii had synergistic effect on biofilm, which was greater than that on the planktonic bacteria. The results indicated that the combinations of imipenem and rifampicin had anti-biofilm properties on the pre-formed biofilms. One of very important component of biofilms is eDNA, and it has been demonstrated that the degradation of eDNA in biofilms of other pathogens could lead to inhibition of the formation of the biofilms (Mann et al. 2009; Martins et al. 2010). DNase I, which also can cause eDNA degradation, was used as a positive control agent for biofilms treatment in the present study (Mann et al. 2009; Martins et al. 2010). Though treatment with imipenem or
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Fig. 4 The quantity of eDNA in 24-h-old biofilms of A. baumannii strain J9 treated by imipenem, rifampicin and in combination. Control: without treatment; 1/8 MIC of IMI: treatment with 64 lg ml-1 imipenem; 1/8 MIC of RIF: treatment with 16 lg ml-1 rifampicin; IMI ? RIF: treatment with 64 lg ml-1 imipenem and 16 lg ml-1 rifampicin; DNase I: treatment with 20 lg ml-1 DNase I. *p \ 0.05, compared with the control group; #p \ 0.05, compared with the group treated with the MIC of imipenem and rifampicin in combination
rifampicin individually reduced the amount of eDNA in the biofilms, the combination treatment with imipenem and rifampicin reduced the amount of eDNA to an extraordinary level by comparison. The synergistic effect on eDNA by the combination treatment might improve the inhibitory
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effect on biofilm formations. Therefore, imipenem and rifampicin in combination have the potential to function as an anti-biofilm agent against bacterial biofilms. Kohanski et al. (2007) suggested an intriguing mechanism for how antibiotics kill bacteria. The authors showed that three major bactericidal antibiotics stimulate hydroxyl radical formation in bacteria, and this toxic chemical contributes to the killing efficiency of lethal antibiotics. This process was accompanied by the hyperactivation of NADH dehydrogenases and a depletion of NADH. Based on the results, they concluded that the generation of hydroxyl radicals is a common mechanism of cell death caused by antibiotics. Hydroxyl radical formation in bacteria was found to be one of the mechanisms of the synergistic effect of different antibiotics combination therapy (Kim et al. 2011; Lee et al. 2012). Consistent with this study, we tested whether the A. baumannii strains treated with imipenem and rifampicin individually and in combination generated hydroxyl radicals and examined how this influences the synergistic effects. As seen in Table 3, both imipenem and rifampicin have the capability to generate hydroxyl radicals. The strain cells treated with imipenem and rifampicin in combination showed increased hydroxyl radical formation. This result shows that hydroxyl radical formation might be an important factor in the synergism. Our study examined the activity of imipenem and rifampicin in combination against A. baumannii and our results showed significant differences in MIC between planktonic and biofilm growth of A. baumannii, which is
Fig. 5 Confocal laser scanning microscopy image of LIVE/ DEAD BacLight Bacterial Viability kit stained A. Baumannii J9 48-h-biofilm grown on coverslide discs: a Treatment of biofilms with the MIC of imipenem (512 lg ml-1). b Treatment of biofilms with the MIC of rifampicin (128 lg ml-1). c Treatment of biofilms with 1/8 MIC of imipenem (64 lg ml-1). d Treatment of biofilms with 1/8 MIC of rifampicin (16 lg ml-1). e Treatment of biofilms with 1/8 MIC of imipenem and 1/8 MIC of rifampicin. f Growth controls. Green viable cells, red dead cells
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consistent with the known behavior of biofilms resistant to antimicrobials. Synergistic effect was demonstrated more frequently against biofilms than against planktonic growth. But there are some limitations in our study and lots of work need to be done in further study. We would increase the clinical isolates numbers and analyze the delicate relationship between the effect of imipenem and rifampicin combination therapy. We could detect other components of biofilms of A. baumannii and to know the exact effect of combination therapy to each components of the biofilms, such as PNAG or bacteria in the biofilms. We would conduct a mouse model of A. baumannii catheter-associated biofilm infection (Heim et al. 2014) and treat the infected mice with imipenem and rifampicin, then identify the synergistic effect of imipenem and rifampicin combination therapy in vivo. In summary, imipenem combined with rifampicin might be a promising choice for the treatment of A. baumannii infection. Acknowledgments Financial support for this work came from the National Nature Science Foundation of China (Nos. 31172364, 31271951, 31000822); Program for New Century Excellent Talents in University (NCET-09-0434, NCET-13-0245); Fundamental Research Program of Shen Zhen (JCYJ20130401172016183; JCYJ20120616 142424467); and Shenzhen Promotion Plan Basic Research Laboratory in 2012 (ZDSY20120616141302982).
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3025 Wilson SJ, Knipe CJ, Zieger MJ, Gabehart KM, Goodman JE, Volk HM, Sood R (2004) Direct costs of multidrug-resistant Acinetobacter baumannii in the burn unit of a public teaching hospital. Am J Infect Control 32(6):342–344 Wolff M, Joly-Guillou ML, Farinotti R, Carbon C (1999) In vivo efficacies of combinations of beta-lactams, beta-lactamase inhibitors, and rifampin against Acinetobacter baumannii in a mouse pneumonia model. Antimicrob Agents Chemother 43(6):1406–1411
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ORIGINAL PAPER
Antimicrobial activity of the imipenem/rifampicin combination against clinical isolates of Acinetobacter baumannii grown in planktonic and biofilm cultures Yang Wang • Wanguo Bao • Na Guo • Haiying Chen • Wei Cheng • Kunqi Jin • Fengge Shen • Jiancheng Xu • Qiaoli Zhang • Chao Wang Yanan An • Kaiyu Zhang • Feng Wang • Lu Yu
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Received: 18 June 2014 / Accepted: 21 August 2014 / Published online: 9 October 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract To investigate the antimicrobial activity of imipenem and rifampicin alone and in combination against clinical isolates of Acinetobacter baumannii grown in planktonic and biofilm cultures. Minimum inhibitory concentrations were determined for each isolate grown in suspension and in biofilm using a microbroth dilution method. Chequerboard assays and the agar disk diffusion assay were used to determine synergistic, indifferent or antagonistic interactions between imipenem and rifampicin. We used the tissue culture plate method for A. baumannii biofilm formation to measure the percentage of biofilm inhibition and the amount of extracellular DNA after the treatment. To understand the synergistic mechanisms, we conducted hydroxyl radical formation assays. The results were verified by confocal laser scanning Y. Wang W. Bao N. Guo H. Chen W. Cheng K. Jin F. Shen Q. Zhang C. Wang Y. An K. Zhang F. Wang L. Yu (&) Department of Infectious Diseases, First Hospital of Jilin University, Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, China e-mail: [email protected] N. Guo Department of Food Quality and Safety, Jilin University, Changchun, China J. Xu Department of Clinical Laboratory, First Hospital of Jilin University, Changchun, China F. Wang (&) Department of Infectious Diseases, First Hospital of Jilin University, Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130021, China e-mail: [email protected]
microscopy. Imipenem and rifampicin showed effective antimicrobial activity against suspensions and biofilm cultures of A. baumannii, respectively. Synergistic antimicrobial effects between imipenem and rifampicin were observed in 13 and 17 of the 20 clinical isolates when in suspension and in biofilms, respectively. Imipenem and rifampicin alone and in combination generated hydroxyl radicals, which are highly reactive oxygen forms and the major components of bactericidal agents. Furthermore, treatment with imipenem and rifampicin individually or in combination has obvious antibiofilm effects. The synergistic activity of imipenem and rifampicin against clinical isolates of A. baumannii (in suspension and in biofilms) was observed in vitro. Therefore, we conclude that imipenem combined with rifampicin has the potential to be used as a combinatorial therapy for the treatment of infectious diseases caused by A. baumannii. Keywords Antimicrobial activity Biofilms Imipenem Rifampicin Synergy
Introduction Acinetobacter baumannii is a non-motile, gram-negative, opportunistic pathogen. A. baumannii has the ability to form biofilms, which may play a role in the process of colonization. A. baumannii has become increasingly important due to its resistance to a wide variety of antibiotics and its ability to survive in a hospital environment (Wilson et al. 2004). The emergence of the multi-drug resistant nosocomial A. baumannii infection has prompted the evaluation of newer antimicrobial agents and older warehoused agents as possible therapeutic candidates. Carbapenems (e.g.,
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imipenem) have been used as the antibiotics of choice for the treatment of A. baumannii infections, but increasing resistance to these antimicrobial agents mediated by blactamases is undermining this option. The resistance of A. baumannii to all major classes of antibiotics (except polymyxins) has substantially increased worldwide in the past decade (Talbot et al. 2006; Peleg et al. 2006). While researchers are looking for new antimicrobial agents to solve this problem, they are also trying to determine the interactions between different types of traditional antibiotics and identify those combinations of antibiotics with synergistic effects. Recent studies have noted promising results when testing rifampicin/imipenem combinations against A. baumannii in planktonic and animal models. It was found that some kind of bactericidal antibiotics could simulate hydroxyl radical formation in bacteria and this toxic chemical contributes to the killing efficiency of lethal antibiotics in 2007 (Kohanski et al. 2007). In some papers about different antibiotics combination therapy and the mechanisms of the synergistic effect, hydroxyl radical formation was detected to know if the introduction of hydroxyl radical formation was one of the synergistic mechanisms (Kim et al. 2011; Lee et al. 2012). But there were no reports about the hydroxyl radical formation level in the treatment with imipenem and rifampicin against A. baumannii. Inspired by this, we detected the hydroxyl radical production after treatment with imipenem and rifampicin against A. baumannii. In light of these results, we treated 20 clinical A. baumannii strains with a rifampicin/imipenem combination in both planktonic and in biofilm cultures. The data were collected prospectively to assess the efficacy of this combination and to discuss the mechanism of the effect of the combination.
Materials and methods Materials Unless stated otherwise, all reagents were purchased from Sigma Chemical Company (St. Louis, USA). The cell culture plates were polystyrene, flat-bottomed, sterile, tissue culture-treated 96-well microtiter plates with low evaporation lids purchased from Nest Biotech Ltd (Wuxi, China). Crystal Violet and DNase I were purchased from Sigma-Aldrich (Dorset, UK). Imipenem was from Kangmanlin Ltd (Nanjing, China). Rifampicin was from Dingguo Ltd (Beijing, China). DNase I (Sigma) stock solutions were prepared before each experiment in 0.15 M NaCl supplemented with 5 mM of MgCl2 (Qin et al. 2007). Working solutions of DNase I 20 lg ml-1 were prepared in RPMI medium.
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Micro-organisms Clinically isolated A. baumannii was obtained from four hospitals of Jilin University and five other hospitals of Jilin Province from blood and sputum samples of infected patients. The quality control (QC) strain ATCC 19606T was obtained from American Type Culture Collection (Manassas, USA). The A. baumannii strains were stored on MicroBank beads (Pro-Lab Diagnostics, Cheshire, UK) at -70 °C until required. Preparation of antimicrobial agents Imipenem were dissolved in deionized water to obtain a stock solution of 20.480 mg ml-1 under sterile conditions and stored at -70 °C until use. Rifampicin were dissolved in DMSO to obtain a stock solution of 20.480 mg ml-1 under sterile conditions and stored at -70 °C until use. Preparation of A. baumannii inoculum for suspension assay The strains were placed in LB agar and incubated at 37 °C overnight. Colonies from the plates were resuspended in MH broth and cultured at 37 °C for 16 h. With optical density (OD) at 660 nm, the concentration of each overnight cultured suspension was determined based on a previously established OD concentration standard curves The suspensions were further diluted with MH broth to obtain inocula containing 1 9 106 CFU ml-1 (Clock et al. 2013). Determination of MICs of imipenem and rifampicin for A. baumannii in suspension The MICs of imipenem and rifampicin were determined by the broth microdilution method adapted from previous studies and in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2012). Serial twofold dilutions of antimicrobial agents were prepared in MH broth to obtain the required concentrations (1.0 lg ml-1 to 1.024 mg ml-1). The assay was performed in 96-well microtiter plates, and a positive control, which contained inoculated broth without any drugs, was included in every plate. Each well contained 100 ll of the test antimicrobial and 100 ll of the A. baumannii suspension diluted in MH broth, containing 1 9 105 CFU. The plates were incubated in air for 24 h at 37 °C. The MIC was defined as the lowest concentration of the drug that inhibited the growth of the test micro-organism by [90 % visually (Oo et al. 2010). The assay was performed in triplicate.
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Assessment of the antimicrobial activity of imipenem in combination with rifampicin against A. baumannii in suspension via the chequerboard assay The antimicrobial activity of imipenem in combination with rifampicin was assessed in a suspension assay by the chequerboard method (Shin and Lim 2004). Serial twofold dilutions of the antimicrobial compounds were prepared in MH broth so that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for both imipenem and rifampicin. Fifty microliters of each imipenem solution was added to the rows of a 96-well microtiter plate in diminishing concentrations, and 50 ll of the rifampicin was also added to the columns in diminishing concentrations. The wells were then inoculated with 100 ll of A. baumannii suspension containing 1 9 105 CFU. The wells in column 12 served as controls containing MH broth either with or without the inoculum. After inoculation and agitation, the microplates were incubated in air at 37 °C for 24 h, and the MICs of the agents individually and in combination were determined as described previously. To assess the synergistic or antagonistic activity of antimicrobial combinations, the fractional inhibitory concentration (FIC) and FIC index (FICI) were determined using the following formulas (Oo et al. 2010): FIC = MIC of antimicrobial in combination/MIC of antimicrobial alone; FICI = FIC of imipenem ? FIC of rifampicin (Clock et al. 2013; Principe et al. 2013). In this study, the FICI, calculated as the sum of each FIC, was interpreted as follows: FICI B0.5 demonstrated synergy; 0.5 \ FICI B 4 indicated indifferent; and FICI [4 showed antagonism. Agar disk diffusion assay for imipenem combined with rifampicin in A. baumannii J9 A 100-ll sample of 106 CFU ml-1 A. baumannii J9, which exhibited the best synergistic effect, was spread onto an LB agar surface. Subsequently, 6-mm-diameter paper disks, impregnated with imipenem (4 lg ml-1), rifampicin (4 lg ml-1) and imipenem (4 lg ml-1) combined with rifampicin (4 lg ml-1), were placed onto the surface. The inhibition zones were measured using a dial caliper after a 48-h incubation at 37 °C. The tests were performed in duplicate (Kiraz et al. 2009).
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30 -(p-hydroxyphenyl) fluorescein (HPF; Invitrogen) was used at a concentration of 5 lM. ODs were measured with a spectrofluorophotometer (Infinite F200 Pro, TECAN, Switzerland) at a 492-nm excitation and a 525-nm emission wavelength (Price et al. 2009; Choi and Lee 2012). The increase in the percentage of hydroxyl radical was calculated using the following equation: [(OD490 of cells treated with agent - OD490 of non-treated control)/OD490 of nontreated control) 9 100]. Experiments were performed in triplicate, the data were averaged, and the standard deviation (SD) was calculated. Establishment of A. baumannii biofilms For each clinically isolated A. baumannii strain, the OD of the overnight suspension was determined at 660 nm and diluted in TSB to obtain the final concentration of 1 9 105 CFU ml-1. Bacterial biofilms were prepared by aliquoting 200 ll of the bacterial suspension diluted in 2 % glucose-TSB containing 1 9 105 CFU ml-1 into the wells of the 96-well microtiter plates. The microtiter plates were then incubated in air for 24 h at 37 °C (Rodrı´guez-Ban˜o et al. 2008). Determination of the MICs of imipenem and rifampicin for A. baumannii in biofilm Biofilms were grown in 96-well microtiter plates (1 9 105 CFU ml-1, 200 ll/well) for 24 h. After incubation, the microtiter plates containing A. baumannii biofilm were washed twice gently with sterile PBS to remove the unbound bacteria. Serial double dilutions of antimicrobial agents were prepared in TSB to obtain the required concentrations (both of imipenem and rifampicin 0.5–512 lg ml-1). To triplicate wells, 200 ll of each antimicrobial agent was added to each microtiter plate well in decreasing concentrations across the rows. Columns 12 served as controls containing the biofilm and saline. Following incubation in air at 37 °C for 24 h, the MIC was determined as the lowest concentration to show growth below or equal to that of the control visually (biofilm in saline).
Hydroxyl radical formation assay
Assessment of the antimicrobial activity of imipenem in combination with rifampicin against A. baumannii in biofilm via the Chequerboard assay
Bacterial cells (1 9 106 CFU ml-1) were treated with imipenem and rifampicin individually or in combinations, and the final concentration of the treated agents was the MIC for each agent or the combination treatments. The samples were incubated for 1 h at 37 °C. To detect hydroxyl radical formation, the fluorescent reporter dye
Biofilms were grown in 96-well microtiter plates (1 9 105 CFU ml-1, 200 ll/well) for 24 h and then gently washed once with PBS to remove the unbound bacteria. Serial twofold dilutions of the drugs were prepared in TSB so that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for both imipenem and rifampicin.
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One hundred microliters of each imipenem solution were added to the rows of a 96-well microtiter plate in diminishing concentrations, and 100 ll of the rifampicin solutions were also added to the columns in diminishing concentrations. Column 12 served as controls containing TSB both with and without the inoculum. Following incubation at 37 °C for 24 h, the antimicrobial supernatant was removed, and the wells were washed again with PBS twice. The MIC, FIC and FICI values were determined as described previously. The assay was performed in triplicate. Determination of biofilm formation by the tissue culture plate (TCP) method Quantitative biofilm assays of crystal violet-stained cells cultured in TSB were performed as described previously (Tomaras et al. 2003). Briefly, biofilms were grown in 96-well microtiter plates (1 9 105 CFU ml-1, 200 ll/well). After culturing for 24 h, the wells were washed softly with PBS twice to remove free-floating bacteria. Then, the solutions containing TSB and the drugs were added into the wells, and the plates were incubated for 24 h at 37 °C. After the incubation, the wells were washed twice with 200 ll of PBS. Biofilms formed by adherent ‘‘sessile’’ organisms in the plate were stained with crystal violet (1 %, w/v). Excess stain was rinsed off by washing with cold deionized water, and then the plates were dried. After drying, 95 % ethanol was added to the wells, the plates were vortexed, and the ODs were measured using a microtiter ELISA reader (Molecular Devices Emax, CA, USA) at 595 nm. These OD values were considered an index of bacterial adherence to the surface and the formation of biofilms. The percentage of biofilm inhibition was calculated using the following equation: [1 (OD595 of cells treated with agent/OD595 of non-treated control)] 9 100 (Wei et al. 2006; Rogers et al. 2010). Experiments were performed in triplicate, the data were averaged, and the SD was calculated. The results were expressed as percentage of biofilm inhibition. Determination of extracellular DNA (eDNA) of A. baumannii J9 in biofilm treated with imipenem combined with rifampicin
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were harvested by resuspension in 50 mM TrisHCl/10 mM ETDA/500 mM NaCl, pH 8.0, and transferred into prechilled tubes. After centrifugation for 5 min at 18,0009g at 4 °C, each supernatant was transferred to a tube containing threefold the volume of TE buffer (10 mM TrisHCl/1 mM EDTA, pH 8.0) and extracted once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). The aqueous phase of each sample was then mixed with threefold the volume of ice-cold 100 % (vol/vol) ethanol and 1/10 the volume of 3 M sodium acetate (pH 5.2) and stored at -20 °C overnight. Then, the ethanol-precipitated DNA was collected by centrifugation for 20 min at 18,0009g at 4 °C, washed with ice-cold 70 % (vol/vol) ethanol, air-dried, and dissolved in 40 ll of TE buffer. The concentration and purity of the DNA were determined spectrophotometrically by the absorbance ratio A260/A280 using a NanoDrop 2000 (Thermo Scientific; Steinberger and Holden 2005; Tetz et al. 2009). The assay was performed in triplicate. Confocal laser scanning microscopy (CLSM) Bacterial cells of A. baumannii strain J9 (1 9 105 CFU ml-1, 2 ml/well) were grown on coverslides in sixwell plates for 48 h to form the biofilms. After incubation, the biofilms were treated with imipenem and rifampicin individually or in combination. The coverslides were then washed gently with PBS, moved to a new plate and treated for 24 h with imipenem and rifampicin individually or in combination. The coverslides were washed again with PBS and stained with the LIVE/DEAD BacLight Bacterial Viability kit (Invitrogen Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. CLSM images were collected using an Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan) with a 60 9 objective lens. For the detection of SYTO 9 (green, alive), we used 488-nm excitation and 520-nm emission filter settings. For PI detection (red, dead), we used the 543-nm excitation and 572-nm emission filter settings. Image analyses and export were performed in a Fluoview, ver. 1.7.3.0 (Olympus; Sauer et al. 2009).
Results Bacterial cells of A. baumannii stain J9 (1 9 105 CFU ml-1, 2 ml/well) were grown in six-well plates for 24 h at 37 °C to form the biofilms and then were treated with imipenem and rifampicin alone or in combination for 24 h. Then, the plates were gently washed three times with PBS without disturbing the adherent film and chilled at 4 °C for 1 h, and 1 ll of 0.5 M EDTA was added to each well. The supernatants were removed, and unwashed biofilm
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The MICs of aqueous imipenem and rifampicin for A. baumannii in suspension and biofilm In our study, 20 clinical isolates of A. baumannii and one QC strain ATCC 19606T were selected. Imipenem and rifampicin demonstrated antimicrobial activity against suspensions and biofilms of A. baumannii (Table 1). The
World J Microbiol Biotechnol (2014) 30:3015–3025 Table 1 MICs of imipenem and rifampicin for suspension and biofilm cultures of A. baumannii (MICs in lg ml-1)
MIC minimum inhibitory concentrations
Strains
3019
Source
MIC for suspension
MIC for biofilm
Imipenem
Imipenem
Rifampicin
Rifampicin
A. baumannii J1
Blood
16
8
64
128
A. baumannii J2
Sputum
32
8
128
64
A. baumannii J3
Sputum
16
4
128
16
A. baumannii J4
Cerebrospinal fluid
8
8
128
128
A. baumannii J5
Sputum
16
16
256
128
A. baumannii J6
Blood
8
4
128
32
A. baumannii J7
Berebrospinal fluid
32
4
256
32
A. baumannii J8
Sputum
32
4
512
64
A. baumannii J9
Blood
64
16
512
128
A. baumannii J10
Sputum
32
4
512
32
A. baumannii S1 A. baumannii S2
Sputum Blood
16 16
4 4
128 128
32 64
A. baumannii S3
Sputum
32
8
256
64
A. baumannii S4
Sputum
32
4
256
32
A. baumannii S5
Sputum
16
4
128
32
A. baumannii S6
Sputum
2
4
32
64
A. baumannii S7
Sputum
64
16
256
128
A. baumannii S8
cerebrospinal fluid
64
4
512
64
A. baumannii S9
Blood
32
2
256
16
A. baumannii S10
Sputum
16
4
128
32
ATCC
1
2
4
16
T
ATCC 19606
MICs of imipenem and rifampicin against these clinical strains growing in suspension ranged from 2 to 64 and 2–16 lg ml-1, respectively. Among these tested isolates, the MICs of imipenem against biofilm ranged from 32 to 512 lg ml-1, while the MICs of rifampicin against biofilm ranged from 16 to 128 lg ml-1. The MIC values of imipenem and rifampicin were 4- to 16-fold higher for A. baumannii growing in biofilm compared with the same strain in suspension. The antimicrobial activity of imipenem in combination with rifampicin against A. baumannii in suspension and in biofilm To analyze the interaction of imipenem and rifampicin against A. baumannii in suspension and in biofilm in the chequerboard assay, we used the nonparametric FICI approach, based on the Loewe additivity theory. The antimicrobial activities of imipenem combined with rifampicin against A. baumannii in suspension and biofilm were shown in Tables 2 and 3, respectively. Using the FICI method, thirteen of the 20 A. baumannii clinical strains were determined to have synergistic interactions in suspension, with FICI values ranging from 0.25 to 0.5, and eight A. baumannii strains had indifferent interactions in suspension, with FICI values ranging from 0.5625 to 1.
Among the 20 A. baumannii clinical strains, 17 strains were determined to have synergistic interactions in biofilm, with FICI values ranging from 0.25 to 0.5, and four A. baumannii strains had indifferent interactions in suspension, with FICI values ranging from 0.75 to 1. The synergism between imipenem and rifampicin was confirmed by agar diffusion tests The halo diameters produced by the combination were predominantly larger than ones produced by single-drug treatments. The sizes of the inhibition zones increased to 13.6 mm when 4 lg ml-1 imipenem (6.2 mm) was combined with 4 lg ml-1 of rifampicin (9.0 mm; Fig. 1). The hydroxyl radicals increase by treatment with imipenem and rifampicin for A. baumannii in suspension We tested whether A. baumannii strain J9 with imipenem and/or rifampicin generated hydroxyl radicals and examined how this influenced the synergistic effects. As seen in Fig. 2, both imipenem and rifampicin have the capability to generate hydroxyl radicals, and the bacterial cells treated with the MIC of imipenem and rifampicin in combination showed significantly increased hydroxyl radical formation.
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Table 2 Antimicrobial activities of imipenem combined with rifampicin against A. baumannii growing in suspension (MIC and FIC in lg ml-1) Strains
MIC of imipenem (in combination/alone)
FIC of imipenem (average)
MIC of rifampicin (in combination/alone)
FIC of rifampicin (average)
FICI (average)
Result
A. baumannii J1
1/16
0.0625
2/8
0.25
0.3125
Synergy
A. baumannii J2
8/32
0.25
4/8
0.5
0.75
Indifference
A. baumannii J3
4/16
0.25
2/4
0.5
0.75
Indifference
A. baumannii J4
4/8
0.5
2/8
0.25
0.75
Indifference
A. baumannii J5
2/16
0.125
2/16
0.125
0.25
Synergy
A. baumannii J6
0.5/8
0.0625
1/4
0.25
0.3125
Synergy
A. baumannii J7 A. baumannii J8
2/32 2/32
0.0625 0.0625
1/4 1/4
0.25 0.25
0.3125 0.3125
Synergy Synergy
A. baumannii J9
4/64
0.0625
4/16
0.25
0.3125
Synergy
A. baumannii J10
2/32
0.0625
1/4
0.25
0.3125
Synergy
A. baumannii S1
2/16
0.125
1/4
0.25
0.375
Synergy
A. baumannii S2
4/16
0.25
1/4
0.25
0.5
Synergy
A. baumannii S3
8/32
0.25
4/8
0.5
0.75
Indifference
A. baumannii S4
8/32
0.25
0.25/4
0.125
0.375
Synergy
A. baumannii S5
1/16
0.0625
1/4
0.25
0.3125
Synergy
A. baumannii S6
0.5/2
0.25
1/4
0.25
0.5
Synergy
A. baumannii S7
16/64
0.25
8/16
0.5
0.75
Indifference
A. baumannii S8
8/64
0.125
1/4
0.25
0.375
Synergy
A. baumannii S9
16/32
0.5
1/2
0.5
1
Indifference
A. baumannii S10
16
0.25
2/4
0.5
0.75
Indifference
ATCC 19606T
0.0625/1
0.0625
1/2
0.5
0.5625
Indifference
MIC minimum inhibitory concentrations, FIC fractional inhibitory concentration, FICI fractional inhibitory concentration index
The increase in the percentage of hydroxyl radicals when treated with the MIC of imipenem and rifampicin in combination was much higher than the corresponding value for treatment with the MIC of rifampicin in combination (p \ 0.05) and the MIC of rifampicin in combination (p \ 0.05), respectively. This result shows that hydroxyl radical formation might be an important factor in the synergism. Anti-biofilm formation effect of imipenem in combination with rifampicin against A. baumannii Treatment with 1/8 MIC of imipenem combined with 1/8 MIC of rifampicin had an inhibitory effect of 45.91 ± 6.47 % on pre-formed biofilms. The effect was much more significant than the inhibitory effect of treatment with 1/8 MIC of imipenem in combination (34.89 ± 2.59 %) and 1/8 MIC of rifampicin (5.88 ± 1.27 %; Fig. 3). The percentage of biofilm inhibition following combination treatment with imipenem and rifampicin was significantly different from the corresponding value for treatment with the MIC of rifampicin in combination (p \ 0.05). There was no significant difference between the percentage of biofilm inhibition when treated with 1/8 MIC of imipenem combined with 1/8 MIC of
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rifampicin and the corresponding value for treatment with the MIC of imipenem in combination (p = 0.08). The eDNA changes by treatment with imipenem and rifampicin in biofilm of A. baumannii strain J9 The amount of eDNA in biofilms was quantified by spectrophotometry and reported as eDNA per relative biomass in the biofilm treated with 1/8 MIC of imipenem and 1/8 MIC of rifampicin alone or 1/8 MIC of imipenem combined with 1/8 MIC of rifampicin while it was also treated with DNase I, which effectively degrades eDNA in biofilms. As shown in Fig. 4, treatment with imipenem or rifampicin resulted in a significant decrease in the amount of eDNA from the cell-free supernatants in A. baumannii strain J9 strain compared with the level in the cultures, and treatment with imipenem combined with rifampicin displayed a significant reduction in the amount of eDNA. The value of the quantity of eDNA treated with any agent was significantly different from the corresponding value of the untreated biofilms (control group; p \ 0.05). The value of the quantity of eDNA in biofilms treated with imipenem combined with rifampicin was significantly different from the corresponding value for biofilms treated with the imipenem (p \ 0.05) or rifampicin (p \ 0.05) individually.
World J Microbiol Biotechnol (2014) 30:3015–3025
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Table 3 Antimicrobial activities of imipenem combined with rifampicin against A. baumannii growing in biofilms (MIC and FIC in lg ml-1) Strains
MIC of imipenem (in combination/alone)
FIC of imipenem (average)
MIC of rifampicin (in combination/alone)
FIC of rifampicin (average)
FICI (average)
Result
A. baumannii J1
8/64
0.125
32/128
0.25
0.375
Synergy
A. baumannii J2
32/128
0.25
32/64
0.5
0.75
Indifference
A. baumannii J3
32/128
0.25
4/16
0.25
0.5
Synergy
A. baumannii J4
32/128
0.25
64/128
0.5
0.75
Indifference
A. baumannii J5
32/256
0.125
16/128
0.125
0.25
Synergy
A. baumannii J6
16/128
0.125
4/32
0.125
0.25
Synergy
A. baumannii J7 A. baumannii J8
32/256 128/512
0.125 0.25
4/32 8/64
0.125 0.125
0.25 0.375
Synergy Synergy
A. baumannii J9
64/512
0.125
16/128
0.125
0.25
Synergy
A. baumannii J10
32/512
0.0625
8/32
0.25
0.3125
Synergy
A. baumannii S1
16/128
0.125
8/32
0.25
0.375
Synergy
A. baumannii S2
32/128
0.25
16/64
0.25
0.5
Synergy
A. baumannii S3
64/256
0.25
16/64
0.25
0.5
Synergy
A. baumannii S4
64/256
0.25
8/32
0.25
0.5
Synergy
A. baumannii S5
8/128
0.0625
8/32
0.25
0.3125
Synergy
A. baumannii S6
8/32
0.25
16/64
0.25
0.5
Synergy
A. baumannii S7
128/256
0.5
64/128
0.5
1
Indifference
A. baumannii S8
128/512
0.25
16/64
0.25
0.5
Synergy
A. baumannii S9
64/256
0.25
4/16
0.25
0.5
Synergy
A. baumannii S10
32/128
0.25
8/32
0.25
0.5
Synergy
ATCC 19606T
1/4
0.25
8/16
0.5
0.75
Indifference
MIC minimum inhibitory concentrations, FIC fractional inhibitory concentration, FICI fractional inhibitory concentration index
CLSM of bacterial cell survival in biofilms exposed to imipenem and rifampicin individually and in combination The effects of imipenem and rifampicin individually and in combination on pre-existing 2d-biofilms was studied by using CLSM (Fig. 5). After treatment for 24 h, the control group was chiefly comprised of living bacterial cells (Fig. 5f). Compared with the control group, treatment with 1/8 MIC of imipenem or 1/8 MIC of rifampicin killed only a small portion of the bacterial population (Fig. 5c, d), while treatment with each of the MICs of imipenem and rifampicin killed a significant portion of the bacterial population and reduced the number of bacteria present in biofilm (Fig. 5a, b). In contrast, treatment with the 1/8 MIC of imipenem combined with 1/8 MIC of rifampicin killed the vast majority of the biofilm bacteria, with few survivors at the surface (Fig. 5e).
Discussion Both imipenem and rifampicin are traditional and common antibiotics used to control bacterial infection with a wide antibacterial spectrum, and they have been used to treat A.
baumannii infection for a long time. However, the increasing emergence of drug-resistant A. baumannii strains has limited the application of these antibiotics in recent years. Synergistic activities have been previously observed between imipenem and rifampicin against A. baumannii in planktonic cultures, and the effects were also identified in an infected mice model recently (Wolff et al. 1999; Montero et al. 2004; Clock et al. 2013; Principe et al. 2013). Both of the two studies in vivo were conducted in a mouse pneumonia model. Majewski et al. (2014) identified the synergism or an additive interaction between rifampicin and imipenem most likely occurs in A. baumannii strains showing moderate resistance to imipenem in the latest paper. These studies only evaluated the effect by using the chequerboard method on planktonic bacteria or verified the effect in the animal model, but they did not demonstrate the mechanisms of the synergistic activity between imipenem and rifampicin and did not detect the activity of treatment with imipenem and rifampicin against A. baumannii in biofilms. Acinetobacter baumannii has the ability to form biofilms, which may play a role in the process of colonization. The ability to adhere to and form biofilms on the surface of inanimate objects might be the cause of its strong ability to survive in hospital environments. Our present study shows
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Fig. 1 Agar disk diffusion assay for imipenem combined with rifampicin in A. baumannii J9
Imipenem 4 µg ml-1
-1 rifampicin 4 µg ml-1 Imipenem 4 µg ml + rifampicin 4 µg ml-1
a
Fig. 2 The percentage of hydroxyl radicals increased in A. baumannii strain J9 treated with imipenem, rifampicin and both antibiotics in combination, as verified by the HPF staining method. MIC of IMI: treatment with 64 lg ml-1 imipenem; MIC of RIF: treatment with 16 lg ml-1 rifampicin; 1/16 MIC of IMI: treatment with 4 lg ml-1 imipenem; 1/4 MIC of RIF: treatment with 4 lg ml-1 rifampicin; IMI ? RIF: treatment with 4 lg ml-1 imipenem and 4 lg ml-1 rifampicin. *p \ 0.05, compared with the group treated with imipenem combined with rifampicin
treatment with imipenem and rifampicin in combination against A. baumannii had a synergistic effect not only on the planktonic bacteria but also on the biofilms. The synergistic effect was observed both in imipenem-sensitive strains and in imipenem-resistant strains. Agar diffusion tests were conducted to confirm the synergistic effect. Biofilms are highly structured communities of bacteria attached to a surface and are recognized as a common cause of human infection. A biofilm is an aggregate of microorganisms in which the cells adhere to one another or to a surface in a self-produced matrix of eDNA, proteins, and polysaccharides. The cells forming the biofilms are morphologically, metabolically, and physiologically different from their planktonic counterparts. Biofilms help the
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b
Fig. 3 The percentage of biofilm inhibition by different antibiotics on 24-h-old biofilms of A. baumannii strain J9. 1/8 MIC of IMI: treatment with 64 lg ml-1 imipenem; 1/8 MIC of RIF: treatment with 16 lg ml-1 rifampicin; IMI ? RIF: treatment with 64 lg ml-1 imipenem and 16 lg ml-1 rifampicin. *p \ 0.05, compared with the group treated with imipenem combined with rifampicin
bacteria resist disinfection, while also allowing the participating cells to trade resistance genes, further facilitating the persistence of the pathogen (Stoodley et al. 2002). The present study shows that treatment with an imipenem/rifampicin combination against A. baumannii had synergistic effect on biofilm, which was greater than that on the planktonic bacteria. The results indicated that the combinations of imipenem and rifampicin had anti-biofilm properties on the pre-formed biofilms. One of very important component of biofilms is eDNA, and it has been demonstrated that the degradation of eDNA in biofilms of other pathogens could lead to inhibition of the formation of the biofilms (Mann et al. 2009; Martins et al. 2010). DNase I, which also can cause eDNA degradation, was used as a positive control agent for biofilms treatment in the present study (Mann et al. 2009; Martins et al. 2010). Though treatment with imipenem or
World J Microbiol Biotechnol (2014) 30:3015–3025
Fig. 4 The quantity of eDNA in 24-h-old biofilms of A. baumannii strain J9 treated by imipenem, rifampicin and in combination. Control: without treatment; 1/8 MIC of IMI: treatment with 64 lg ml-1 imipenem; 1/8 MIC of RIF: treatment with 16 lg ml-1 rifampicin; IMI ? RIF: treatment with 64 lg ml-1 imipenem and 16 lg ml-1 rifampicin; DNase I: treatment with 20 lg ml-1 DNase I. *p \ 0.05, compared with the control group; #p \ 0.05, compared with the group treated with the MIC of imipenem and rifampicin in combination
rifampicin individually reduced the amount of eDNA in the biofilms, the combination treatment with imipenem and rifampicin reduced the amount of eDNA to an extraordinary level by comparison. The synergistic effect on eDNA by the combination treatment might improve the inhibitory
3023
effect on biofilm formations. Therefore, imipenem and rifampicin in combination have the potential to function as an anti-biofilm agent against bacterial biofilms. Kohanski et al. (2007) suggested an intriguing mechanism for how antibiotics kill bacteria. The authors showed that three major bactericidal antibiotics stimulate hydroxyl radical formation in bacteria, and this toxic chemical contributes to the killing efficiency of lethal antibiotics. This process was accompanied by the hyperactivation of NADH dehydrogenases and a depletion of NADH. Based on the results, they concluded that the generation of hydroxyl radicals is a common mechanism of cell death caused by antibiotics. Hydroxyl radical formation in bacteria was found to be one of the mechanisms of the synergistic effect of different antibiotics combination therapy (Kim et al. 2011; Lee et al. 2012). Consistent with this study, we tested whether the A. baumannii strains treated with imipenem and rifampicin individually and in combination generated hydroxyl radicals and examined how this influences the synergistic effects. As seen in Table 3, both imipenem and rifampicin have the capability to generate hydroxyl radicals. The strain cells treated with imipenem and rifampicin in combination showed increased hydroxyl radical formation. This result shows that hydroxyl radical formation might be an important factor in the synergism. Our study examined the activity of imipenem and rifampicin in combination against A. baumannii and our results showed significant differences in MIC between planktonic and biofilm growth of A. baumannii, which is
Fig. 5 Confocal laser scanning microscopy image of LIVE/ DEAD BacLight Bacterial Viability kit stained A. Baumannii J9 48-h-biofilm grown on coverslide discs: a Treatment of biofilms with the MIC of imipenem (512 lg ml-1). b Treatment of biofilms with the MIC of rifampicin (128 lg ml-1). c Treatment of biofilms with 1/8 MIC of imipenem (64 lg ml-1). d Treatment of biofilms with 1/8 MIC of rifampicin (16 lg ml-1). e Treatment of biofilms with 1/8 MIC of imipenem and 1/8 MIC of rifampicin. f Growth controls. Green viable cells, red dead cells
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consistent with the known behavior of biofilms resistant to antimicrobials. Synergistic effect was demonstrated more frequently against biofilms than against planktonic growth. But there are some limitations in our study and lots of work need to be done in further study. We would increase the clinical isolates numbers and analyze the delicate relationship between the effect of imipenem and rifampicin combination therapy. We could detect other components of biofilms of A. baumannii and to know the exact effect of combination therapy to each components of the biofilms, such as PNAG or bacteria in the biofilms. We would conduct a mouse model of A. baumannii catheter-associated biofilm infection (Heim et al. 2014) and treat the infected mice with imipenem and rifampicin, then identify the synergistic effect of imipenem and rifampicin combination therapy in vivo. In summary, imipenem combined with rifampicin might be a promising choice for the treatment of A. baumannii infection. Acknowledgments Financial support for this work came from the National Nature Science Foundation of China (Nos. 31172364, 31271951, 31000822); Program for New Century Excellent Talents in University (NCET-09-0434, NCET-13-0245); Fundamental Research Program of Shen Zhen (JCYJ20130401172016183; JCYJ20120616 142424467); and Shenzhen Promotion Plan Basic Research Laboratory in 2012 (ZDSY20120616141302982).
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