One novel strategy is to utilize helper compounds in combination with traditional antibiotics. Helper compounds are drugs approved for other therapeutic purposes that also possess antibacterial activity (Dickey et al. 2017). Thioridazine (TZ) is primarily an antipsychotic drug and also functions as an efflux pump inhibitor, which can be used as a helper compound (Klitgaard et al. 2008; Pule et al. 2016). Several
The previous studies have shown that the innate immune response, including pattern recognition receptors (PRR), was activated upon infection with
Our study is the first to explore the combination of TZ, MCZ, and CXN in the treatment of mastitis using a mouse model to provide a basis for subsequent combined antibacterial therapy.
For the first clear well in each row of the microtitre plate containing both antimicrobial agents, the fractional inhibitory concentration (FIC) of each agent was calculated as follows:
FIC of drug A (FICA) = MIC of drug A in combination / MIC of drug A alone
FIC of drug B (FICB) = MIC of drug B in combination / MIC of drug B alone
The summation of both FICs (Σ FIC) in each well (FICI = Σ FIC = FICA + FICB) was used to classify the combination of antimicrobial agents at the given concentrations as synergistic (Σ FIC, ≤ 0.5), partially synergistic (Σ FIC, > 0.5 and ≤ 1.0), indifferent (Σ FIC, > 1 and ≤ 4), or antagonistic (Σ FIC, > 4) (Zore et al. 2011).
Using three-dimensional checkerboard microdilution with CXN, TZ, and MCZ the combined concentrations of each antibiotic showed synergy when their sum, FICI (Σ FICs) was lower than 1.0.
The MICs values of individual antimicrobial agents against
Antimicrobial agents | MIC (μg/ml) | ||
---|---|---|---|
Range | 50% | 90% | |
Cloxacillin | 4–512 | 16 | 512 |
Thioridazine | 16–64 | 32 | 64 |
Miconazole | 1–8 | 4 | 8 |
MIC – minimum inhibitory concentration
Summary of thioridazine, miconazole and cloxacillin activity in combination (expressed as the MIC value) against
Antimicrobial agents | MIC (μg/ml) | |||
---|---|---|---|---|
Range | 50% | 90% | ||
Cloxacillin | Thioridazine | 0.125–512 | 16 | 512 |
Miconazole | 0.25–512 | 4 | 512 | |
Thioridazine + Miconazole | 0.000972–16 | 0.5 | 8 |
MIC – minimum inhibitory concentration
The activity of the combination of cloxacillin and thioridazine against
Strain | FICI |
---|---|
MRSA14 | 0.38 |
MRSA15 | 0.63 |
MRSA16 | 0.75 |
MRSA20 | 0.25 |
MRSA21 | 0.50 |
MRSA22 | 0.63 |
MRSA25 | 0.31 |
MRSA29 | 0.19 |
MRSA30 | 1.06 |
MRSA64 | 0.25 |
MRSA65 | 0.25 |
MRSA75 | 1.13 |
MRSA76 | 1.13 |
MRSA92 | 0.31 |
MRSA94 | 0.19 |
MRSA97 | 0.25 |
MRSA98 | 0.19 |
MRSA125 | 0.27 |
MRSA126 | 0.50 |
MRSA134 | 0.16 |
MRSA135 | 0.16 |
MRSA142 | 0.63 |
MRSA162 | 0.38 |
ATCC 29213 | 0.28 |
MSSA10 | 0.31 |
MSSA13 | 0.56 |
MSSA14 | 0.63 |
MSSA31 | 0.63 |
MSSA36 | 0.38 |
MSSA41 | 0.63 |
MSSA42 | 0.56 |
MSSA44 | 0.38 |
MSSA50 | 0.38 |
MSSA51 | 0.14 |
MSSA54 | 0.38 |
MSSA56 | 0.63 |
MSSA62 | 0.63 |
MSSA65 | 0.63 |
MSSA66 | 1.13 |
MSSA67 | 1.13 |
MSSA68 | 1.13 |
MSSA70 | 1.13 |
MSSA72 | 1.06 |
MSSA73 | 0.63 |
MSSA78 | 1.25 |
MSSA79 | 1.25 |
MSSA80 | 0.25 |
See FICI criteria for details.
The activity of the combination of cloxacillin and miconazole against
Strains | FICI |
---|---|
MRSA14 | 0.63 |
MRSA15 | 0.75 |
MRSA16 | 0.38 |
MRSA20 | 0.31 |
MRSA21 | 0.31 |
MRSA22 | 2.25 |
MRSA25 | 0.53 |
MRSA29 | 2.13 |
MRSA30 | 0.26 |
MRSA64 | 0.14 |
MRSA65 | 0.26 |
MRSA75 | 1.00 |
MRSA76 | 1.13 |
MRSA92 | 0.26 |
MRSA94 | 0.51 |
MRSA97 | 0.52 |
MRSA98 | 0.26 |
MRSA125 | 0.50 |
MRSA126 | 1.50 |
MRSA134 | 1.50 |
MRSA135 | 0.27 |
MRSA142 | 1.01 |
MRSA162 | 0.63 |
ATCC 29213 | 0.50 |
MSSA10 | 0.56 |
MSSA13 | 0.31 |
MSSA14 | 2.00 |
MSSA31 | 1.13 |
MSSA36 | 2.02 |
MSSA41 | 1.50 |
MSSA42 | 0.27 |
MSSA44 | 1.06 |
MSSA50 | 1.03 |
MSSA51 | 1.00 |
MSSA54 | 1.50 |
MSSA56 | 1.00 |
MSSA62 | 1.50 |
MSSA65 | 1.03 |
MSSA66 | 0.53 |
MSSA67 | 0.31 |
MSSA68 | 0.53 |
MSSA70 | 0.52 |
MSSA72 | 0.28 |
MSSA73 | 1.03 |
MSSA78 | 1.06 |
MSSA79 | 0.56 |
MSSA80 | 0.56 |
See FICI criteria for details.
The activity of the combination of cloxacillin, thioridazine, and miconazole against MRSA strains
Strain | CXN | TZ | MCZ | CXN | TZ | MCZ | FICI |
---|---|---|---|---|---|---|---|
MICs (Single) | MICs (Synergy) | ||||||
MRSA14 | 512 | 32 | 2 | 4 | 4 | 0.25 | 0.26 |
MRSA15 | 512 | 32 | 2 | 2 | 4 | 0.25 | 0.25 |
MRSA16 | 512 | 16 | 4 | 1 | 4 | 0.25 | 0.31 |
MRSA20 | 256 | 32 | 2 | 0.25 | 4 | 0.25 | 0.25 |
MRSA21 | 128 | 16 | 4 | 0.5 | 4 | 0.25 | 0.32 |
MRSA22 | 64 | 32 | 4 | 0.25 | 4 | 0.50 | 0.25 |
MRSA25 | 512 | 16 | 2 | 0.5 | 4 | 0.50 | 0.50 |
MRSA29 | 256 | 64 | 4 | 4 | 4 | 0.50 | 0.20 |
MRSA30 | 512 | 64 | 4 | 0.5 | 4 | 0.50 | 0.19 |
MRSA64 | 512 | 32 | 8 | 8 | 4 | 0.50 | 0.20 |
MRSA65 | 512 | 32 | 4 | 0.5 | 4 | 0.50 | 0.25 |
MRSA75 | 512 | 32 | 2 | 0.5 | 4 | 0.50 | 0.38 |
MRSA76 | 512 | 32 | 4 | 8 | 4 | 0.50 | 0.27 |
MRSA92 | 512 | 16 | 4 | 0.0078 | 4 | 0.50 | 0.38 |
MRSA94 | 512 | 32 | 2 | 0.0078 | 4 | 0.50 | 0.38 |
MRSA97 | 256 | 32 | 2 | 0.0156 | 4 | 0.50 | 0.38 |
MRSA98 | 512 | 32 | 4 | 0.0078 | 4 | 0.50 | 0.25 |
MRSA125 | 256 | 16 | 2 | 0.5 | 4 | 0.50 | 0.50 |
MRSA126 | 256 | 16 | 1 | 1 | 4 | 0.50 | 0.75 |
MRSA134 | 512 | 32 | 1 | 0.0078 | 4 | 0.50 | 0.63 |
MRSA135 | 256 | 32 | 4 | 0.25 | 4 | 0.50 | 0.25 |
MRSA142 | 512 | 32 | 1 | 2 | 4 | 0.50 | 0.63 |
MRSA162 | 512 | 32 | 4 | 16 | 4 | 0.50 | 0.28 |
ATCC 29213 | 4 | 16 | 4 | 0.0156 | 4 | 0.50 | 0.38 |
See FICI criteria for details.
MCZ – miconazole; TZ – thioridazine; CXN – cloxacillin;
FIC of drug A (FICA) = MIC of drug A in combination / MIC of drug A alone;
FIC of drug B (FICB) = MIC of drug B in combination / MIC of drug B alone;
Combination FIC (AB) = Σ FIC = FICA + FICB;
Synergistic (Σ FIC ≤ 0.5);
Partially synergistic (Σ FIC > 0.5 and ≤ 1.0);
Indifferent (Σ FIC > 1 and ≤ 4);
Antagonistic (Σ FIC > 4).
The activity of the combination of cloxacillin, thioridazine, and miconazole against MSSA strains
Strain | CXN | TZ | MCZ | CXN | TZ | MCZ | FICI |
---|---|---|---|---|---|---|---|
MICs (Single) | MICs (Synergy) | ||||||
MSSA10 | 16 | 64 | 4 | 4 | 4 | 0.25 | 0.38 |
MSSA13 | 8 | 64 | 8 | 8 | 4 | 0.25 | 1.09 |
MSSA14 | 8 | 32 | 2 | 8 | 4 | 0.25 | 1.25 |
MSSA31 | 16 | 32 | 2 | 4 | 4 | 0.25 | 0.50 |
MSSA36 | 16 | 32 | 1 | 0.015625 | 4 | 0.25 | 0.38 |
MSSA41 | 8 | 32 | 1 | 4 | 4 | 0.25 | 0.88 |
MSSA42 | 16 | 64 | 8 | 0.125 | 4 | 0.25 | 0.10 |
MSSA44 | 16 | 32 | 1 | 0.5 | 4 | 0.25 | 0.41 |
MSSA50 | 16 | 32 | 1 | 0.125 | 4 | 0.25 | 0.38 |
MSSA51 | 16 | 32 | 2 | 0.001975 | 4 | 0.25 | 0.25 |
MSSA54 | 16 | 16 | 2 | 0.0625 | 4 | 0.25 | 0.38 |
MSSA56 | 16 | 32 | 2 | 0.03125 | 4 | 0.25 | 0.25 |
MSSA62 | 16 | 32 | 2 | 0.0009715 | 4 | 0.25 | 0.25 |
MSSA65 | 16 | 32 | 2 | 0.125 | 4 | 0.25 | 0.26 |
MSSA66 | 16 | 32 | 4 | 4 | 4 | 0.25 | 0.44 |
MSSA67 | 16 | 32 | 4 | 8 | 4 | 0.25 | 0.69 |
MSSA68 | 16 | 32 | 4 | 16 | 4 | 0.25 | 1.19 |
MSSA70 | 16 | 32 | 4 | 0.015625 | 4 | 0.25 | 0.19 |
MSSA72 | 16 | 64 | 8 | 0.5 | 4 | 0.25 | 0.13 |
MSSA73 | 16 | 32 | 2 | 0.0625 | 4 | 0.25 | 0.25 |
MSSA78 | 8 | 16 | 2 | 0.03125 | 4 | 0.25 | 0.38 |
MSSA79 | 8 | 16 | 4 | 0.03125 | 4 | 0.25 | 0.32 |
MSSA80 | 16 | 32 | 4 | 0.0078125 | 4 | 0.25 | 0.19 |
ATCC 29213 | 4 | 16 | 4 | 0.0156 | 4 | 0.50 | 0.38 |
See FICI criteria for details.
MCZ – miconazole; TZ – thioridazine; CXN – cloxacillin;
FIC of drug A (FICA) = MIC of drug A in combination / MIC of drug A alone;
FIC of drug B (FICB) = MIC of drug B in combination / MIC of drug B alone;
Combination FIC(AB) = Σ FIC = FICA + FICB;
Synergistic (Σ FIC ≤ 0.5);
Partially synergistic (Σ FIC > 0.5 and ≤ 1.0);
Indifferent (Σ FIC > 1 and ≤ 4);
Antagonistic (Σ FIC > 4).
Clinical observations of mammary tissue of mouse infected with
Clinical observations of mammary tissue of mouse infected with
Histopathology of mammary tissue by the hematoxylin and eosin stain (H&E) was observed (Fig. 2A, and 2B). No obvious pathological changes were found in the blank control group, which displayed normal acinar mammary glands and neat rows of acinar epithelial cells with prolactin. A large number of bacterial lumps and detached cells were seen in the infection control group, and both had red blood cell infiltrate observed in the interstitial space. The agglomerated cell mass was seen in the acinus, and large numbers of lymphocyte infiltrate were observed in the interstitium. In the MCZ or TZ monotherapy groups, epithelial cells were swollen, obvious bacterial masses were visible, and lymphocytes had infiltrated the stroma. Few lymphocytes and certain bacterial masses were observed in the CXN group. In the combination group, few lymphocytes and very few bacteria were visible, in addition to a lack of cell shedding. The results showed that, in addition to the known effect of CXN monotherapy, the combination of CXN, TZ, and MCZ has obvious therapeutic effects against infection by both strains of
Histopathological observations for each group of mice (control, infected, and treated with the drugs).
The culture of
MCZ – miconazole; TZ – thioridazine; CXN – cloxacillin; MCZ + TZ + CXN – the combination of miconazole, thioridazine, cloxacillin. There was no significant difference between the cloxacillin-treated and three-drugs-treated mice (
Drug effects on the cytokine level in mouse mastitis models.
(A) Serum IFN-γ level in mice infected with ATCC 29213; (B) Serum IFN-γ level in mice infected with MRSA135; (C) Serum IL-6 level in mice infected with ATCC 29213; (D) Serum IL-6 level in mice infected with MRSA135; (E) Serum TNF-α level in mice infected with ATCC 29213; (F) Serum TNF-α level in mice infected with MRSA135; MCZ – miconazole; TZ – thioridazine; CXN – cloxacillin; MCZ + TZ + CXN – combination of miconazole, thioridazine, cloxacillin. The * on the horizontal line indicates a significant difference analysis between the CXN group and the three-drug group. *
The levels of TNF-α, IL-6, and IFN-γ in mice treated with the three drugs together were significantly lower than those of the MRSA135-infected group (
A recent report indicated that developing of new natural compounds or combination therapies should be a focus on the fight against
Previous studies reported that TZ functions as an external pump inhibitor, and MCZ acts as an autolytic inducer against
In
In our study, we investigated the effects of TZ, MCZ, and CXN on