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MIC values of MFX and AZM against M. abscessus reference strain were determined as 2 μg/mL and 8 μg/mL, respectively; this suggests a moderate susceptibility to MFX and susceptibility to AZM (Supplementary Table S1 available in www.besjournal.com).
Table S1. Results of DST by Alamar blue 2-fold dilution method
Drug Culture time (d) MIC value (μg/mL) Susceptible breakpoint (μg/mL) Moderately susceptible breakpoint (μg/mL) Resistant breakpoint (μg/mL) Azithromycin 3 8 ≤ 1 2 ≥ 4 Moxifloxacin 3 2 ≤ 16 32 ≥ 64 -
Different bacteria concentrations and durations of observation were tested for establishing infected ZF model. High concentration would be expected to cause a rapid death. Low concentration would not generate enough fluorescence to generate permanent records of images when observed under the microscope. Three M. abscessus concentrations – 1.6 × 109 µg/mL, 2 × 109 µg/mL, and 5 × 109 µg/mL were chosen for testing. The following amounts of bacteria (in units, 1 unit means 1 M. abscessus) – 12,800, 6,400, 3,200, 1,600, and 800 were tested by injection and followed over the observation period from 3 to 7 d. For the final testing, M. abscessus concentration of 5 × 109 µg/mL containing 1,600 units of M. abscessus was injected into ZFs and observed over a period of 5 d.
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Several MTCs of the drugs were tested (Table 1), with the purpose that concentrations that did not affect ZF survivability would be selected for a follow up process. We established that MFX at ≤ 1,000 μg/mL and AZM at ≤ 250 μg/mL would not impact ZF survivability. Hence, MFX concentrations of 62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, 1,000 μg/mL, and AZM concentrations of 15.625 μg/mL, 31.25 L, 62.5 μg/mL, 125 μg/mL, and 250 μg/mL were chosen for the subsequent process.
Table 1. ZF survivability at different concentrations of MFX and AZM (n = 10 of in each group)
Group Concentration (μg/mL) Death number Mortality (%) Control group (Healthy ZF) − 0 0 Moxifloxacin 0 0 0 62.5 0 0 125 0 0 250 0 0 500 0 0 1,000 0 0 2,000 10 100 Azithromycin 0 0 0 15.625 0 0 31.25 0 0 62.5 0 0 125 0 0 250 0 0 500 7 70 -
We tested AZM at a wide range of concentrations from 15.625 μg/mL to 250 μg/mL, and MFX at concentrations ranging from 62.5 μg/mL to 1,000 μg/mL. Exposing ZF to aqueous solutions these drug concentrations did not show any indication of toxicity in our preliminary experiments.
When infected ZFs were exposed for more than 2 d to the above AZM concentrations, a significant increase in survival rate (P = 0.000) was observed depending on AZM concentration (Figure 1A); higher doses of AZM increased ZF survival. The treatment with low AZM doses failed to restrict mycobacterial growth. This result shows that AZM has a significant activity against M. abscessus in vivo in the M. abscessus-infected ZF test system. However, although some restriction to mycobacterial growth by MFX was observed, the association between the increased survival and the high dose of MFX was not found to be significant (Figure 1B). With the increasing MFX concentration, the survival curve did not show a corresponding significant increase in ZF survival (P = 0.061).
Figure 1. The survival analysis of AZM and MFX against M. abscessus infected ZF. (A) Increased survival was associated with a high dose of AZM. The treatment with low AZM doses failed to restrict mycobacterial growth. The survival curve showed significant difference between different AZM concentration group (P = 0.000). (B) Although some restriction to mycobacterial growth by MFX was observed, the association between increased survival and high dose of MFX is not significant (P = 0.061). Statistical comparison was tested between different drug concentration but without uninfected/untreated ZF control.
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The effect of MFX and AZM on bacterial burden was analyzed by quantifying CFU loads. Increased AZM concentration was associated with lower bacterial burdens as determined quantitatively by CFU plating (Figure 2A). Treatment with lower doses was correspondingly less effective in restricting mycobacterial growth. The same trend was observed with MFX. MFX concentration correlated with CFU loads (Figure 2B). In both AZM and MFX groups, no significant differences were observed between CFU loads at different concentrations (P > 0.05).
Figure 2. The analysis of AZM and MFX efficacy against M. abscessus infected ZF by CFU loads. (A) From day 1 to day 3, 5 ZF from each tested concentration were collected and were lysed and plated on 7H10. Increased AZM concentration was associated with lower bacterial burdens as determined quantitatively by CFU plating. Treatment with lower doses had less effect on mycobacterial growth. No significant difference was observed between different AZM concentrations (P > 0.05). (B) The same trend was observed with MFX. MFX concentrations correlated with CFU loads. No significant difference was observed among different MFX concentrations (P > 0.05).
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3 days after infection, 5 ZFs in each concentration group that generated images of adequate quality were collected and analyzed. Exposure to AZM was associated with a significant reduction in the number of abscesses (Figure 3A). With increasing AZM concentration (15.625 μg/mL, 31.25 μg/mL, 62.5 μg/mL, 125 μg/mL), bacterial fluorescence intensity in ZF showed significant decrease (161,828 ± 6,605, 157,329 ± 5,356, 142,300 ± 13,715, 132,942 ± 11,243) (Figure 3A). This decrease in fluorescence intensity was consistent with the inhibition rate. AZM inhibition rates at 15.625 μg/mL, 31.25 μg/mL, 62.5 μg/mL, and 125 μg/mL concentration were 13%, 15%, 24%, and 29%. The inhibition rate also showed significant difference when compared with no-drug group (P < 0.05) indicating that AZM possesses good inhibition efficacy (Figure 4A). However, exposure of infected ZF to MFX showed no significant decrease in the frequency of abscesses (Figure 3B), although increased MFX concentrations did decrease fluorescence intensity slightly when observed under fluorescence microscope. At MFX concentrations of 62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, and 1,000 μg/mL, fluorescence intensities in ZF were 247,306, 243,523, 229,586, 221,573, and 219,640 pixels (Figure 3B), and the inhibition rates were 0%, 1%, 7%, 10%, and 11%, respectively, with all P value > 0.05 indicating statistical insignificance when comparing with the control group (Figure 4B).
Figure 3. The analysis of AZM and MFX efficacy against M. abscessus infected ZF by fluorescence intensity (by pixel). Three days after infection, 5 ZFs from each concentration were collected and imaged. The fluorescence intensity (by pixel) of M. abscessus treated with different concentrations of AZM (A) and MFX (B) were compared with that each of M. abscessus treated without drug. n = 20 for each group. *P < 0.05; **P < 0.01.
Figure 4. The analysis of AZM and MFX efficacy against M. abscessus infected ZF by inhibition rate(%). Three days after infection, 5 ZFs at each concentration were imaged, and the inhibition rate for each concentration was calculated. Inhibition rates at different concentrations of AZM (A) and (B) were compared with that each of M. abscessus treated without drug. One-way analysis of variance and t test were performed. *P < 0.05.
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The effect of AZM and MFX on bacterial fluorescence dissemination was examined. In the AZM control group (without drug), M. abscessus disseminated in the heart, brain, and veins. The transfer-occurrence rate was 50%. In 15.625 μg/mL AZM group, M. abscessus disseminated in the brain and veins, with transfer-occurrence rate of 30%. In 31.25 μg/mL, 62.5 μg/mL, and 125 μg/mL AZM, M. abscessus disseminated only in the vein, with transfer-occurrence rate of 20%. All the transfer rates at different concentrations were compared with those in the control group with P > 0.05. In the MFX control group (without drug), M. abscessus disseminated in the liver, heart, brain, and veins; the transfer-occurrence rate was 70%. In 62.5 μg/mL MFX, M. abscessus disseminated in the heart and veins, with transfer-occurrence rate of 60%. In 125 μg/mL MFX, M. abscessus disseminated in the brain and veins, with transfer-occurrence rate of 50%. In 250 μg/mL, 500 μg/mL, and 1,000 μg/mL MFX, M. abscessus disseminated in the brain and veins, with transfer-occurrence rate of 40%. All the transferring rates at different concentrations compared with those in the control group showed P > 0.05. Therefore, although both two groups showed some inhibition of M. abscessus dissemination, no significant differences were observed for AZM and MFX groups when compared with the control group.
Together, these results suggest that AZM exerts a therapeutic effect, whereas MFX exerts a limit therapeutic effect, by preventing the development of abscesses and protecting ZF by killing bacteria.
doi: 10.3967/bes2020.047
Efficacy of Moxifloxacin against Mycobacterium abscessus in Zebrafish Model in vivo
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Abstract:
Objective Moxifloxacin (MFX) shows good in vitro activity against Mycobacterium abscessus and can be a possible antibiotic therapy to treat M. abscessus infection; however, other studies have shown a lower or no activity. We aimed to evaluate MFX activity against M. abscessus using zebrafish (ZF) model in vivo. Methods A formulation of M. abscessus labeled with CM-Dil was micro-injected into ZF. Survival curves were determined by recording dead ZF every day. ZF were lysed, and colony-forming units (CFUs) were enumerated. Bacteria dissemination and fluorescence intensity in ZF were analyzed. Inhibition rates of MFX and azithromycin (AZM, positive control) were determined and compared. Results Significantly increased survival rate was observed with different AZM concentrations. However, increasing MFX concentration did not result in a significant decrease in ZF survival curve. No significant differences in bacterial burdens by CFU loads were observed between AZM and MFX groups at various concentrations. Bacterial fluorescence intensity in ZF was significantly correlated with AZM concentration. However, with increasing MFX concentration, fluorescence intensity decreased slightly when observed under fluorescence microscope. Transferring rates at various concentrations were comparable between the MFX and AZM groups, with no significant difference. Conclusion MFX showed limited efficacy against M. abscessus in vivo using ZF model. Its activity in vivo needs to be confirmed. -
Key words:
- Mycobacterium abscessus /
- Moxifloxacin /
- Azithromycin /
- Zebrafish
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Figure 1. The survival analysis of AZM and MFX against M. abscessus infected ZF. (A) Increased survival was associated with a high dose of AZM. The treatment with low AZM doses failed to restrict mycobacterial growth. The survival curve showed significant difference between different AZM concentration group (P = 0.000). (B) Although some restriction to mycobacterial growth by MFX was observed, the association between increased survival and high dose of MFX is not significant (P = 0.061). Statistical comparison was tested between different drug concentration but without uninfected/untreated ZF control.
Figure 2. The analysis of AZM and MFX efficacy against M. abscessus infected ZF by CFU loads. (A) From day 1 to day 3, 5 ZF from each tested concentration were collected and were lysed and plated on 7H10. Increased AZM concentration was associated with lower bacterial burdens as determined quantitatively by CFU plating. Treatment with lower doses had less effect on mycobacterial growth. No significant difference was observed between different AZM concentrations (P > 0.05). (B) The same trend was observed with MFX. MFX concentrations correlated with CFU loads. No significant difference was observed among different MFX concentrations (P > 0.05).
Figure 3. The analysis of AZM and MFX efficacy against M. abscessus infected ZF by fluorescence intensity (by pixel). Three days after infection, 5 ZFs from each concentration were collected and imaged. The fluorescence intensity (by pixel) of M. abscessus treated with different concentrations of AZM (A) and MFX (B) were compared with that each of M. abscessus treated without drug. n = 20 for each group. *P < 0.05; **P < 0.01.
Figure 4. The analysis of AZM and MFX efficacy against M. abscessus infected ZF by inhibition rate(%). Three days after infection, 5 ZFs at each concentration were imaged, and the inhibition rate for each concentration was calculated. Inhibition rates at different concentrations of AZM (A) and (B) were compared with that each of M. abscessus treated without drug. One-way analysis of variance and t test were performed. *P < 0.05.
S1. Results of DST by Alamar blue 2-fold dilution method
Drug Culture time (d) MIC value (μg/mL) Susceptible breakpoint (μg/mL) Moderately susceptible breakpoint (μg/mL) Resistant breakpoint (μg/mL) Azithromycin 3 8 ≤ 1 2 ≥ 4 Moxifloxacin 3 2 ≤ 16 32 ≥ 64 Table 1. ZF survivability at different concentrations of MFX and AZM (n = 10 of in each group)
Group Concentration (μg/mL) Death number Mortality (%) Control group (Healthy ZF) − 0 0 Moxifloxacin 0 0 0 62.5 0 0 125 0 0 250 0 0 500 0 0 1,000 0 0 2,000 10 100 Azithromycin 0 0 0 15.625 0 0 31.25 0 0 62.5 0 0 125 0 0 250 0 0 500 7 70 -
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