Effects of macrolides on pneumolysin of macrolide-resistant Streptococcus pneumoniae

Micro-organism

A clinical isolate of MRSP was obtained from Nagasaki University School of Medicine (Nagasaki, Japan). NU4471 (minimum inhibitory concentration (MIC) of penicillin G, 2 µg·mL⁻¹; serotype 19) was used in this experiment. The presence of resistance mechanisms for erm B and mef A genes was analysed using PCR 17. The bacterium was stored at −80°C until use.

Antimicrobial agents

CLR (Taisho Toyama Pharmaceutical, Tokyo, Japan) and AZM (Pfizer Japan Inc., Tokyo, Japan) were kindly supplied by the indicated companies. For the antimicrobial susceptibility test, each macrolide was dissolved in ethanol and diluted further with sterile water. For murine experiments, CLR powder was dissolved in 0.5 M HCl and diluted to a final concentration of 30 mg·mL⁻¹ with sterile water, then sonicated for 30 min. AZM powder was suspended to a final concentration of 30 mg·mL⁻¹ with 0.1% carboxymethyl cellulose. Both macrolides were prepared as recommended by the manufacturer. Macrolide stock solutions were diluted with sterile water immediately prior to use to yield the appropriate drug concentrations in a final administration volume of 0.3 mL. Drug solutions were stored at 4°C for no more than 72 h.

Antimicrobial susceptibility test

The MIC of each antibiotic was determined by the broth dilution method using Muller-Hinton broth (Difco Laboratories, Detroit, MI, USA) supplemented with 5% lysed horse blood (Nippon Biotest Laboratories Inc, Tokyo, Japan). Microtitre plates, containing 5.0×10⁵ colony-forming units (cfu)/well of NU4471 were incubated with antibiotics at 35°C for 18 h, and the lowest concentration of the drug that prevented visible growth was considered the MIC 18.

Western blot analysis

Suspensions of NU4471 (3.0×10⁶ cfu·mL⁻¹) in cell culture medium (D-MEM/F-12: Invitrogen Corp., Carlsbad, CA, USA) with 1, 2 and 4 µg·mL⁻¹ of CLR and AZM or sterile water (control) were incubated at 37°C for 8 h at late-log phase (n = 4) 19. Thereafter, pneumococci were lysed with protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan) with a sonicator (Ultrasonic Disruptor UD-201: Tomy Seiko, Tokyo, Japan) 20. The western blot procedure for PLY was then performed as described previously 21. Sample proteins (30 µg) of whole lysates were separated using SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and transferred onto nitrocellulose membranes. Blots were blocked and successively incubated with monoclonal murine antibodies to recombinant PLY (NCL-SPNm: Novocastra Laboratories, Newcastle-upon-Tyne, UK) and peroxidase-conjugated goat anti-mouse antibodies (Bio-Rad, Richmond, CA, USA). The reaction was developed with the ECL⁺ enhanced chemiluminescence western blotting detection reagents and exposed to Hyperfilm ECL (both from Amersham Biosciences UK, Buckinghamshire, UK). PLY was detected as a band at 53 kDa. Densitometry of the films was performed using a software program. Viable counts (cfu·mL⁻¹) were also determined after 8-h incubation by plating appropriate dilutions on blood agar plates and incubated at 37°C for 18 h. The lowest level of detectable cfu·mL⁻¹ was 50 cfu·ml⁻¹.

Haemolytic assay

Suspensions of NU4471 with 1, 2 and 4 µg·mL⁻¹ of CLR and AZM or sterile water (control) were incubated at 37°C for 8 h and lysed with a sonicator as described above (n = 4). The haemolytic activity was then determined using 1% (volume/volume) suspensions of fresh horse erythrocytes (washed three times) in PBS, as described previously 19.

Laboratory animals

For the study, 5-week-old female CBA/J specific-pathogen-free mice (body weight 16–20 g) were purchased from Charles River Japan (Yokohama, Japan). The CBA/J mouse model of S. pneumoniae has been described previously 22. All experiments were conducted taking into consideration the guidelines of the Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine.

Experimental murine model of pneumococcal pneumonia

NU4471 was cultured on horse blood agar (Muller-Hinton ΙΙ agar: Becton, Dickinson and Company, Sparks, MD, USA, horse blood from same manufacturer) at 37°C for 24 h. It was then scraped and suspended in brain–heart infusion broth (Becton, Dickinson and Company) mixed with horse serum (Invitrogen Corp.) and cultured at 37°C for 6 h at mid-log phase. Bacteria were then harvested by centrifugation at 800×g for 3 min. The organisms were resuspended in normal saline and the final number of bacteria prepared was ∼10⁷ cfu·ml⁻¹, as determined by optical density. Infection was induced by intra-nasal inoculation of 0.05 mL bacterial suspension, containing about 5×10⁵ cfu·mouse⁻¹, into anesthetised mice (pentobarbital sodium, 40 mg·kg⁻¹ i.p.) 23, 24.

Treatment regimens for the survival study

Treatment was initiated at 12 h post-infection. The regimen included: CLR at 200 mg·kg⁻¹ and saline (control) administered 12, 24, 36, 48 and 60 h post-infection; AZM at 200 mg·kg⁻¹ administered 12, 36 and 60 h post-infection 25. Antibiotics were dispensed orally. Mice (four in each group) were killed by cervical dislocation at 72 h post-infection. The lungs were dissected out under aseptic conditions and suspended in saline (50 µL).

Bacteriological examination and immunoprecipitation of lungs for western blot analysis

For bacteriological examination, the lungs were homogenised just after death using a Polytron homogeniser (PT3000; Kinematica, Littau-Lucerne, Switzerland). The number of bacteria in the lungs was determined as described previously 23. Thereafter, 1 mL of lysis buffer was added to the lung lysates, incubated on ice for 10 min, and centrifuged at 12,000×g at 4°C as described previously 26. Lung extracts were isolated and 8 mg of proteins were rotated for 1 h at 4°C with 60 µg of monoclonal murine antibodies to recombinant PLY (NCL-SPNm) cross-linked to protein G beads (Amersham Biosciences AB, Uppsala, Sweden). The beads were washed four times with 1 mL of lysis buffer and boiled for 5 min in SDS-denaturing sample buffer. Finally, western blot analysis was performed as described above.

Treatment regimens for study of survival rates

Treatment was initiated at 12 h post-infection and continued for 7 days. The regimens included: CLR at 40 or 200 mg·kg⁻¹ twice daily; AZM at 40 or 200 mg·kg⁻¹ once daily; and saline (control) twice daily. Seven mice were included in each treatment group. Antibiotics were administered through oral gavage. The survival rates were determined daily over a 10-day post-infection period 25.

Statistical analysis

Data are expressed as mean±sem. Differences in the protein synthesis and haemolytic activities of PLY or the number of viable bacteria between macrolide-treated groups and the control group were evaluated using ANOVA. Survival analysis was performed using the log-rank test, and survival rates were calculated via the Kaplan–Meier method. A p-value <0.05 was considered statistically significantly.

Minimum inhibitory concentration of macrolides for Streptococcus pneumoniae NU4471

Both of the MICs of CLR and AZM were ≥256 µg·mL⁻¹. The presence of both erm B and mef E/A genes in NU4471 was confirmed by PCR (data not shown).

Effect of macrolides on pneumolycin protein synthesis

Figure 1a⇓ shows the representative results of western blot analysis of the suppressing effects of sub-MIC (1, 2 and 4 µg·ml⁻¹) of CLR and AZM on the protein synthesis of PLY by NU4471. After 8 h of incubation, the production of PLY was reduced in the presence of 1, 2 and 4 µg·mL⁻¹ of CLR and AZM. Moreover, the inhibitory effect of 4 µg·mL⁻¹ of CLR on the production of PLY was better than that of 4 µg·mL⁻¹ of AZM. There was no difference in the number of bacteria recovered in the presence or absence of macrolides after 8 h of incubation (fig. 1b⇓).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1—

a) The representative results of western blot analysis of the suppressing effects of sub-minimum inhibitory concentrations of clarithromycin (CLR) and azithromycin (AZM) on the protein synthesis of pneumolysin by NU4471 after 8 h of incubation. b) The number of colony-forming units (cfu) in media treated with saline (control), CLR at 1, 2 and 4 µg·mL⁻¹ and AZM at 1, 2 and 4 µg·mL⁻¹ were 8.33±0.03, 8.27±0.14, 8.33±0.11, 8.49±0.08, 8.20±0.12, 8.31±0.08 and 8.23±0.11 log10 cfu·mL⁻¹, respectively. Data present the mean±sem of four experiments.

Effect of macrolides on haemolytic activities of pneumolysin

The effects of sub-MIC (1, 2 and 4 µg·mL⁻¹) of CLR and AZM on the haemolytic activities of PLY by NU4471 are shown in figure 2⇓. Data reflect absorbance at 541 nm. After 8 h of incubation, the haemolytic activities were significantly lower in the presence of CLR (2 and 4 µg·mL⁻¹) and AZM (4 µg·mL⁻¹) than control (p<0.0001). The inhibitory effect of 4 µg·mL⁻¹ of CLR on haemolytic activity was better than that of 4 µg·mL⁻¹ of AZM (p<0.05).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2—

Effects of sub-minimum inhibitory concentrations of clarithromycin (CLR) and azithromycin (AZM) on the haemolytic activities of pneumolysin (PLY) by NU4471. After 8 h of incubation, the haemolytic activity of PLY treated with saline (control), CLR at 1, 2 and 4 µg·mL⁻¹ and AZM at 1, 2 and 4 µg·mL⁻¹ were 1.07±0.01, 0.95±0.04, 0.50±0.08, 0.24±0.10, 1.03±0.03, 1.00±0.03 and 0.54±0.03, respectively. Data present the mean±sem of four experiments. ^#: p<0.0001 versus control; *: p<0.05 versus AZM at 4 µg·mL⁻¹.

Effect of macrolides on the production of pneumolysin in murine lungs

Figure 3⇓ shows western blot analysis of the inhibitory effects of CLR at 200 mg·kg⁻¹ twice daily, AZM at 200 mg·kg⁻¹ once daily and saline (control) twice daily on the production of PLY by NU4471 in murine lungs. Treatment with CLR at 200 mg·kg⁻¹ and AZM at 200 mg·kg⁻¹ significantly reduced the production of PLY in murine lungs compared with the control (p<0.05).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3—

a) Representative results of western blot analysis of the inhibitory effects of clarithromycin (CLR) at 200 mg·kg⁻¹ twice daily, azithromycin (AZM) at 200 mg·kg⁻¹ once daily and saline (control) twice daily on the production of pneumolycin by NU4471 in murine lungs. b) Signal intensity of each band treated with CLR at 200 mg·kg⁻¹, AZM at 200 mg·kg⁻¹ and control. Data present mean±sem of four mice. *: p<0.05 versus control.

The numbers of viable bacteria in mice lungs treated with CLR, AZM and saline were 7.21±0.27, 7.68±0.48 and 7.66±0.18 log10 CFU·lung⁻¹, respectively. There was no difference in the total CFU of viable bacteria in the murine lungs recovered after treatment with CLR, AZM and saline.

Survival rates

The survival rates of mice infected with NU4471 and treated with CLR at 40 or 200 mg·kg⁻¹ twice daily, AZM at 40 or 200 mg·kg⁻¹ once daily and saline (control) twice daily are shown in figure 4⇓. Treatment with CLR at 40 and 200 mg·kg⁻¹ and AZM at 200 mg·kg⁻¹ significantly improved the survival rates compared with the control (p<0.05).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4—

Kaplan–Meier survival curves of mice (seven in each group) infected with NU4471 and treated with clarithromycin (CLR; two different doses), azithromycin (AZM; two different doses) or control (saline) for 7 days. •: mice treated with saline; □: mice treated with AZM at 40 mg·kg⁻¹; ▵: mice treated with AZM at 200 mg·kg⁻¹; ▪: CLR at 40 mg·kg⁻¹; ▴: CLR at 200 mg·kg⁻¹. *: p<0.05 versus control.