Activity of moxifloxacin on biofilms produced in vitro by bacterial pathogens involved in acute exacerbations of chronic bronchitis

International Journal of Antimicrobial Agents 30 (2007) 415–421

Activity of moxifloxacin on biofilms produced in vitro by bacterial pathogens involved in acute exacerbations of chronic bronchitis S. Roveta, A.M. Schito, A. Marchese, G.C. Schito ∗ Sezione di Microbiologia, Di.S.C.A.T., University of Genoa, Largo R. Benzi 10, 16132 Genoa, Italy Received 16 May 2007; accepted 22 June 2007

Abstract The aim of this study was to assess whether moxifloxacin is able to inhibit the synthesis of and to disrupt biofilms produced in vitro by bacterial pathogens involved in acute bacterial exacerbations of chronic bronchitis. Three strains each of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus and Escherichia coli recently isolated from clinical respiratory specimens and capable of slime production were used. Biofilm formation on polystyrene plates was quantified spectrophotometrically by established methodologies. Moxifloxacin (0.5 mg/L) inhibited slime synthesis by >70% in S. aureus, H. influenzae and S. pneumoniae, 45–70% in E. coli and 35–70% in M. catarrhalis. Disruption of pre-formed structures was also promoted by moxifloxacin both for initial (5 h) and mature (48 h) biofilms. Drug concentrations reached during therapy (0.5–4 mg/L) resulted in a breakdown of initial biofilm of 60–80% in H. influenzae and S. pneumoniae, 48–86% in S. aureus, 37–69% in M. catarrhalis and 51–71% in E. coli. Mature biofilms were less susceptible to degradation. Moxifloxacin at concentrations that can be achieved in the bronchial mucosa during therapy therefore promotes a significant inhibition of biofilm synthesis and induces slime disruption, a feature that may be instrumental in reducing the exacerbations so frequently observed in this condition. © 2007 Published by Elsevier B.V. and the International Society of Chemotherapy. Keywords: Biofilm; Respiratory infection; Fluoroquinolone

1. Introduction Acute bacterial infections caused by specialised pathogens are caused by free-floating planktonic cells. These clinical conditions may be satisfactorily controlled in single patients with antibiotic therapy, chosen ideally after in vitro antimicrobial susceptibility testing, and in the general population with vaccines stimulating high levels of protective antibodies [1]. Possibly for these reasons, chronic ailments caused by less virulent environmental or human normal commensal flora are now the most prevalent infectious diseases [2]. These microorganisms make full use of the biofilm strategy. Biofilms represent a structured community of bacterial cells embedded in a self-produced polymeric matrix adherent to a natural or artificial surface and are therefore significantly protected from antimicrobial agents and host immune defences ∗

Corresponding author. Tel.: +39 010 353 7660; fax: +39 010 353 7698. E-mail address: [email protected] (G.C. Schito).

[3]. Antibiotic therapy typically overcomes the symptoms caused by waves of planktonic cells released from the biofilm during exacerbations of the disease, but fails to eradicate the infection as the sessile cells are inherently less affected by these drugs [4]. Cystic fibrosis, ventilation-associated pneumonia and bronchitis are prominently included among the impressive number of chronic and recurrent infections attributed to formation of bacterial biofilms [2–4]. Several antibiotic classes (from ␤-lactams to fluoroquinolones) are generally employed with varying clinical success in the treatment of acute bacterial exacerbations of chronic bronchitis (ABECB), especially the mild-to-severe forms [5]. Time to eradication of symptoms and time to relapse are now included among other essential parameters in order to rank the potency of compounds to be used in empirical therapy for these conditions. It may be safely hypothesised that compounds capable of inhibiting the synthesis of slime and of disrupting biofilms formed by respiratory pathogens common in causing ABECB (Haemophilus influenzae,

0924-8579/$ – see front matter © 2007 Published by Elsevier B.V. and the International Society of Chemotherapy. doi:10.1016/j.ijantimicag.2007.06.029

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Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Enterobacteriaceae and, more rarely, Pseudomonas aeruginosa) may offer advantages over molecules not endowed with these abilities. The aim of this study was to assess whether moxifloxacin, a fourth-generation oral fluoroquinolone whose antimicrobial spectrum encompasses almost all the microorganisms involved in ABECB, is able to interfere with the ability of a bacteria to produce biofilms and is capable of causing disruption of slimes independent of the degree of maturation. 2. Materials and methods 2.1. Microorganisms Three strains each of H. influenzae, S. pneumoniae, M. catarrhalis, S. aureus and Escherichia coli recently isolated from respiratory clinical specimens, identified according to standard procedures [6] and shown to be capable of slime production, were employed in this study. 2.2. Drug Preparation of sterile stock solutions of moxifloxacin (Bayer, Milan, Italy) was performed in accordance with the manufacturer’s instructions. 2.3. Susceptibility tests Minimal inhibitory concentrations (MICs) of moxifloxacin were determined following the microdilution procedure of the Clinical and Laboratory Standards Institute [7] using as test medium cation-adjusted Mueller–Hinton (MH) broth (Biolife, Milan, Italy) for M. catarrhalis, S. aureus and E. coli, MH broth supplemented with 5% lysed horse blood for S. pneumoniae and Haemophilus Test Medium for H. influenzae. Overnight cultures of bacteria were diluted to give a final concentration of ca. 5 × 105 cells/mL. Samples were then added to equivalent volumes of the various concentration of antibiotic distributed on a microplate and prepared from serial two-fold dilutions, ranging from 0.015 to 32 ␮g/mL. After 16–20 h of incubation at 35 ◦ C (20–24 h for H. influenzae, S. pneumoniae and M. catarrhalis), the concentration of drug that prevented visible growth was recorded as the MIC.

2.5. Biofilm production The presence and extent of biofilm structures were quantified spectrophotometrically using a method based on that reported by Cramton et al. [11] as previously reported [12]. To produce biofilms, 25 ␮L of stationary-phase bacterial cultures were added aseptically to a well of a 96-well polystyrene tissue culture plate (Corning, Milan, Italy) containing 175 ␮L of appropriate growth medium (the same as that employed in the screening procedure for each microorganism, respectively). Biofilms were obtained (at 37 ◦ C and in a 5% CO2 atmosphere for H. influenzae, S. pneumoniae and M. catarrhalis) at 5–6 h or 48 h. The growth medium was discarded and replaced every 12 h. To evaluate the effect of moxifloxacin on biofilm synthesis, the same procedure was followed except that the drug was added to the growth medium. Each well was washed three times with phosphate-buffered saline (PBS) under aseptic conditions to eliminate unbound bacteria, and a suitable concentration of moxifloxacin in appropriate medium was added. After 24 h of exposure, drug solutions were discarded and each well was washed three times with PBS. Adherent microorganisms were fixed with Bouin’s solution and stained with crystal violet. Excess stain was rinsed off with running tap water and the plates were dried. Adherent bacterial films were quantified spectrophotometrically by determining the optical density at 570 nm (OD570 nm ). The results were derived from three separate experiments and OD570 nm values were expressed as mean ± standard deviation. The OD570 nm value obtained for each strain without drug was used as the control. The percentages of biofilm formed in the presence of different concentrations of drug were calculated employing the ratio between the values of OD570 nm with and without drug, adopting the following formula: [(OD570 nm with drug/OD570 nm without drug) × 100]. 2.6. Statistical analysis Student’s t-test was employed to evaluate any significant differences between the OD570 nm values obtained without the drug (controls) and those observed in the presence of different drug concentrations. Paired t-test was used to compare the mean between each control/drug-treated group. Differences were considered statistically significant at P < 0.05. Analyses were done using SPSS statistical software (SPSS Inc., Chicago, IL). 2.7. Counting of viable cells

2.4. Screening for slime production Strains of S. aureus and E. coli were screened for qualitative slime production using Congo red agar plate test as described by Freeman et al. [8]. Biofilm production in H. influenzae, S. pneumoniae and M. catarrhalis was evaluated spectrophotometrically using the protocol described by Wen et al. [9,10].

After 24 h of exposure, moxifloxacin-containing solutions were discarded and the wells were washed three times and filled with PBS. The surfaces of the wells were vigorously scraped with a sterile bacteriological loop as previously described [12]. Plates wrapped in plastic were placed in a sonicator bath for 15–30 min to aid dissolution of bacterial clumps. The number of viable cells was estimated by plate

S. Roveta et al. / International Journal of Antimicrobial Agents 30 (2007) 415–421

count after transferring 100 ␮L of the mixture and further dilutions to selected agar plates containing the appropriate media for each pathogen species. The results have been derived from three separate experiments.

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0.06–0.125 mg/L for M. catarrhalis and 0.06 mg/L and 0.03 mg/L for all three strains of S. aureus and E. coli, respectively. The results of susceptibility testing are reported in Table 1. 3.2. Inhibition of slime synthesis

3. Results 3.1. MICs The MICs of moxifloxacin were 0.06–0.25 mg/L for S. pneumoniae, 0.015–0.03 mg/L for H. influenzae,

Moxifloxacin at a concentration of 0.5 mg/L inhibited slime synthesis by >70% in all S. aureus, H. influenzae and S. pneumoniae strains. At the same concentration, reduction of biofilm synthesis was 45–70% in E. coli and 35–70% in M. catarrhalis strains (Fig. 1).

Table 1 Minimal inhibitory concentrations (MICs) of moxifloxacin and reduction of pre-formed initial (5 h) biofilm in the different respiratory pathogens Microorganism

Strain

MIC (mg/L)

Concentration of drug (mg/L)

OD570 nm ± S.D.

Haemophilus influenzae

206

0.03

480

0.03

521

0.015

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

0.55 0.17 0.13 0.76 0.21 0.17 0.63 0.16 0.13

± ± ± ± ± ± ± ± ±

PD16

0.25

PD26

0.125

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

0.49 0.20 0.17 0.53 0.19 0.15 0.61 0.19 0.12

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

Streptococcus pneumoniae

Moraxella catarrhalis

Staphylococcus aureus

Escherichia coli

PD37

0.06

UD5

0.06

VA6

0.06

396b

0.125

1876

0.06

1880

0.06

1890

0.06

1263

0.03

1293

0.03

169

0.03

P-value

Biofilm reduction (%)

0.041 0.015 0.011 0.044 0.023 0.013 0.042 0.013 0.015

– 0.011 0.0064 – 0.0072 0.0058 – 0.0067 0.0049

– 70 77 – 72 78 – 74 80

± ± ± ± ± ± ± ± ±

0.035 0.016 0.012 0.049 0.025 0.012 0.052 0.017 0.010

– 0.018 0.015 0.014 0.0083 – 0.012 0.0048

– 60 65 – 64 72 – 69 80

0.56 0.27 0.20 0.63 0.32 0.19 0.45 0.28 0.24

± ± ± ± ± ± ± ± ±

0.081 0.033 0.026 0.094 0.045 0.016 0.058 0.018 0.027

– 0.026 0.013 – 0.038 0.012 – 0.049 0.038

– 52 65 – 49 69 – 37 46

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

0.83 0.17 0.12 0.91 0.47 0.41 0.77 0.20 0.15

± ± ± ± ± ± ± ± ±

0.055 0.015 0.011 0.070 0.032 0.029 0.068 0.043 0.013

– 0.0067 0.0054 – 0.041 0.039 – 0.0080 0.0071

– 80 86 – 48 55 – 74 81

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

0.87 0.43 0.38 0.80 0.34 0.25 0.74 0.28 0.21

± ± ± ± ± ± ± ± ±

0.091 0.037 0.025 0.083 0.046 0.038 0.095 0.036 0.019

– 0.036 0.032 – 0.038 0.010

– 51 56 – 57 69 – 62 71

CTR, control without drug; OD570 nm , optical density at 570 nm; S.D., standard deviation.

0.015 0.0091

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Fig. 1. Effect of moxifloxacin on biofilm synthesis of (A) Haemophilus influenzae, (B) Streptococcus pneumoniae, (C) Moraxella catarrhalis, (D) Staphylococcus aureus and (E) Escherichia coli.

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3.3. Disruption of pre-formed initial and mature biofilm

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niae, 37–69% in M. catarrhalis and 51–71% in E. coli. In S. aureus, the extent of disruption was more variable (48–86%) and was strongly strain-dependent (Table 1). Remarkable results were also obtained when attempting to disrupt more mature biofilms. Moxifloxacin used at concentrations of 0.5–4 mg/L reduced pre-formed mature slimes by ≥60% in all H. influenzae strains, 58–70% in S. pneumoniae and 45–70% in S. aureus. At the same moxifloxacin concentrations, however, reductions were less significant in M. catarrhalis (35–50%) and E. coli (45–55%) (Table 2).

Not only biofilm synthesis was inhibited by moxifloxacin, but slime disruption was also promoted with a potency directly related to the antibiotic concentration (except with the lowest doses tested) both for initial and mature biofilm. Using moxifloxacin at concentrations achievable during therapy (0.5–4 mg/L), slime disruption of pre-formed initial biofilm was 70–80% in H. influenzae, 60–80% in S. pneumo-

Table 2 Minimal inhibitory concentrations (MICs) of moxifloxacin and reduction of pre-formed mature (48 h) biofilm in the different respiratory pathogens Microorganism

Strain

MIC (mg/L)

Concentration of drug (mg/L)

OD570 nm ± S.D.

Haemophilus influenzae

206

0.03

480

0.03

521

0.015

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

0.78 0.30 0.29 0.85 0.34 0.30 0.69 0.26 0.21

± ± ± ± ± ± ± ± ±

PD16

0.25

PD26

0.125

PD37

0.06

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

0.58 0.24 0.21 0.65 0.26 0.22 0.71 0.25 0.21

UD5

0.06

VA6

0.06

396b

0.125

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

1876

0.06

1880

0.06

1890

0.06

1263

0.03

1293

0.03

169

0.03

Streptococcus pneumoniae

Moraxella catarrhalis

Staphylococcus aureus

Escherichia coli

P-value

Biofilm reduction (%)

0.096 0.045 0.051 0.10 0.059 0.068 0.090 0.027 0.035

– 0.018 0.015 – 0.020 0.013 – 0.016 0.0095

– 61 63 – 60 65 – 62 70

± ± ± ± ± ± ± ± ±

0.083 0.018 0.015 0.052 0.029 0.026 0.095 0.037 0.019

– 0.026 0.014 – 0.019 0.011 – 0.014 0.0097

– 58 63 – 60 66 – 65 70

0.62 0.40 0.33 0.67 0.42 0.34 0.50 0.33 0.29

± ± ± ± ± ± ± ± ±

0.085 0.048 0.056 0.094 0.051 0.045 0.063 0.054 0.037

– 0.048 0.039 – 0.046 0.030 – 0.049 0.038

– 35 46 – 38 50 – 35 42

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

1.25 0.55 0.38 1.53 0.84 0.49 0.98 0.48 0.37

± ± ± ± ± ± ± ± ±

0.16 0.073 0.045 0.18 0.093 0.056 0.13 0.072 0.025

– 0.023 0.010 – 0.037 0.012 – 0.026 0.018

– 56 70 – 45 68 – 51 62

CTR 0.5 4 CTR 0.5 4 CTR 0.5 4

1.65 0.91 0.78 1.27 0.69 0.61 1.09 0.57 0.49

± ± ± ± ± ± ± ± ±

0.19 0.098 0.085 0.15 0.74 0.096 0.11 0.083 0.067

– 0.041 0.031 – 0.039 0.028 – 0.034 0.025

– 45 53 – 46 52 – 48 55

CTR, control without drug; OD570 nm , optical density at 570 nm; S.D., standard deviation.

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Table 3 Drug concentrations producing a reduction of viable cells in pre-formed initial (5 h) and mature (48 h) biofilm in the different respiratory pathogens Microorganism

Strain

Concentration of drug (mg/L) producing % reduction of viable cells Initial biofilm

Mature biofilm

99%

99.9%

99%

99.9%

Haemophilus influenzae

206 480 521

0.125 0.125 0.125

0.25 0.25 0.25

0.125 0.125 0.125

0.25 0.5 0.25

Streptococcus pneumoniae

PD16 PD26 PD37

1 0.5 0.25

2 1 0.5

1 0.5 0.25

2 1 0.5

Moraxella catarrhalis

UD5 VA6 396b

0.5 0.5 1

2 2 4

1 1 1

4 4 4

Staphylococcus aureus

1876 1880 1890

0.25 0.25 0.25

1 1 1

0.25 0.25 0.25

1 1 1

Escherichia coli

1263 1293 169

0.25 0.25 0.25

1 1 0.5

0.5 0.25 0.25

1 1 1

3.4. Reduction of viable cell counts Moxifloxacin (≤4 mg/L) reduced viable cell counts by >99.9% of the initial concentration in pre-formed initial and mature biofilms of all strains considered in this study (Table 3). When 1 mg/L of moxifloxacin was employed, the reduction in viable cells was less evident in M. catarrhalis (99%) than in the other test microorganisms, in which a diminution of the viable cell count of 99.9% was observed both at 1 mg/L and at 4 mg/L.

4. Discussion One of the major goals in the field of modern clinical microbiology is the development of new strategies capable of reducing the incidence of biofilm infections and of effectively curing chronic conditions related to the establishment of these difficult to eradicate bacterial structures [13]. Moxifloxacin, a fourth-generation oral fluoroquinolone, has a broad spectrum of antimicrobial activity, including typical respiratory pathogens, atypical and intracellular respiratory organisms, Gram-negative pathogens and a vast range of anaerobes. This broad and appropriate activity makes the drug particularly suitable for the therapy of respiratory tract infections, including ABECB, overwhelmingly caused by S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus and Enterobacteriaceae [14]. Moxifloxacin is easily absorbed, reaching a peak serum concentration (3.1–4.5 mg/L) in 1–2 h; target tissues are reached by passive diffusion and the drug achieves an adequate concentration in the interstitial fluid [14–18]. Previous studies [19] investigated moxifloxacin concentrations

in serum and pulmonary tissue after a single or repeated oral or intravenous administration of a 400 mg once-daily dose. The concentrations observed in plasma, epithelial lining fluid and bronchial mucosa reached bactericidal levels for common pathogens found in respiratory tract infections. After a dose of 400 mg, the drug (0.5–4 mg/L) rapidly appears in bronchial secretions, reaching maximum concentrations within 1–2 h [20]. In previous studies, the ability of moxifloxacin to reduce biofilms has been demonstrated against slimes synthesised by different Gram-negative and Gram-positive microorganisms. The drug has decreased the density of biofilms formed in vitro by clinical isolates of Stenotrophomonas maltophilia, S. aureus, coagulase-negative staphylococci and viridans streptococci [21–25]. Moxifloxacin was the most effective antibiotic even when tested against biofilms produced by periodontopathic bacteria such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Streptococcus constellatus [26]. In a rat model of implant-related S. aureus osteomyelitis, moxifloxacin monotherapy significantly reduced the number of organisms recovered from bone, soft tissue and the surface of the implanted device [27]. In the present study, the efficacy of moxifloxacin in reducing biofilm synthesis was confirmed with all respiratory pathogens assayed. The drug at concentrations that can be easily reached in the bronchial mucosa during therapy promotes a sizable degree of slime disruption both in initial and mature biofilms. A remarkable reduction in the cell viability of sessile cells contained in these slimes was also observed. The heterogeneity of the response displayed by individual isolates may be attributed to chemical and physical heterogeneity of the specific biofilms formed or may represent the result of differential gene regulation patterns utilised by the strains involved.

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