Potentiation effect of mallotojaponin B on chloramphenicol and mode of action of combinations against Methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus, the causative agent of many infectious diseases has developed resistance to many antibiotics, even chloramphenicol which was the essential antibiotic recommended for the treatment of bacterial infection. Thus, other alternatives to fight against S. aureus infections are necessary; and combinatory therapy of antibiotics with natural compounds is one of the approaches. This study evaluated the activity of the combination of mallotojaponin B and chloramphenicol against Methicillin-resistant Staphylococcus aureus (MRSA). Antibacterial activities were evaluated by broth microdilution and the checkerboard methods. Modes of action as time-kill kinetic, Nucleotide leakage, inhibition and eradication of biofilm, and loss of salt tolerance were evaluated. Cytotoxicity was evaluated on Vero and Raw cell lines. Mallotojaponin B showed good activity against MRSA with a MIC value of 12.5 μg/mL. MRSA showed high resistance to chloramphenicol (MIC = 250 μg/mL). The combination produced a synergistic effect with a mean FICI of 0.393. This combination was bactericidal, inducing nucleotide leakage, inhibiting biofilm formation, and eradicating biofilm formed by MRSA. The synergic combination was non-cytotoxic to Vero and Raw cell lines. Thus, the combination of mallotojaponin B and chloramphenicol could be a potential alternative to design a new drug against MRSA infections

Variation in the number of <i>S</i>. <i>aureus</i> colonies as a function of extract and NaCl concentration.

MIC: Minimum Inhibitory Concentration; PC: positive control; NC: negative control; Comb: Combination; Comb 1: 0.781 μg/ml of mallotojaponin B and 1.95 μg/mL of chloramphenicol; Comb 2: 0.781 μg/mL of mallotojaponin B and 7.812 μg/mL of chloramphenicol; Comb 3: 1.562 μg/mL of mallotojaponin B and 3.9 μg/mL of chloramphenicol; For each salt concentration, histograms carrying the same letters are not significantly different (p˃0.05); while for each test sample, histograms with same Greek alphabets are not significantly different, Waller Duncan test.</p

Growth curves of MRSA following exposure to various concentrations of mallotojaponin B and synergistic combinations.

MIC: Minimum Inhibitory Concentration; PC: positive control (Ciprofloxacin); NC: negative control; Comb: combination; Comb 1: 0.781 μg/mL of mallotojaponin B and 1.95 μg/mL of chloramphenicol; Comb 2: 0.781 μg/mL of mallotojaponin B and 7.812 μg/mL of chloramphenicol; Comb 3: 1.562 μg/mL of mallotojaponin B and 3.9 μg/mL of chloramphenicol.</p

Percentage of inhibition of synergistic combinations and chloramphenicol on Vero and Raw cells.

CPL: chloramphenicol; Comb: combination; Comb 1: 0.781 μg/mL of mallotojaponin B and 1.95 μg/mL of chloramphenicol; Comb 2: 0.781 μg/mL of mallotojaponin B and 7.812 μg/mL of chloramphenicol; Comb 3: 1.562 μg/mL of mallotojaponin B and 3.9 μg/mL of chloramphenicol; Comb 4: 0.781 μg/mL of mallotojaponin B and 15.625 μg/mL of chloramphenicol; Comb 5: 0.781 μg/mL of mallotojaponin B and 31.25 μg/mL of chloramphenicol; Comb 6: 3.125 μg/mL of mallotojaponin B and 3.9 μg/mL of chloramphenicol; PODO: Podophyllotoxin; For the same cell lines, bars with the same letter show that there is no significant difference between the different treatments at p ≤0.05.</p

The diagonal lines for the interpretation of isobolograms in the interaction study between two bioactive substances.

A: Synergy, B: Additivity, C: Indifference, D: Antagonism.</p

Fig 7 -

(A and B). Biomass of biofilms produced by MRSA ATCC 33591 in different cultures medium after 24 and 48hrs of incubation. MHB: Muller Hinton Broth; MHB 1%: Muller Hinton Broth with Glucose 1%; MHB 2%: Muller Hinton Broth with Glucose 2%; TSG 1%: Tryptic Soy with Glucose 1%; TSG2%: Tryptic Soy with Glucose 2%; BHI: Brain Heart Infusion; BHI 1%: Brain Heart Infusion with Glucose 1%; BHI 2%: Brain Heart Infusion with Glucose 2%. Histograms with the same letters are not significantly different (p ˃0.05), while those carrying the same Greek alphabet are not significantly different for the same medium.</p

Percentage of inhibition of biofilm of MRSA ATCC 33591 in MHB 2%.

CP: Ciprofloxacin; Comb: Combination; Comb 1: 0.781 μg/mL of mallotojaponin B and 1.95 μg/mL of chloramphenicol; Comb 2: 0.781 μg/mL of mallotojaponin B and 7.812 μg/mL of chloramphenicol; Comb 3: 1.562 μg/mL of mallotojaponin B and 3.9 μg/mL of chloramphenicol; MIC: Minimum Inhibitory Concentration; NC: Negative Control. Histograms with the same letters are not significantly different (p˃0.05).</p

Fractional Minimum inhibitory concentration indices (FICI) of different combinations of mallotojaponin B and chloramphenicol vis-à-vis MRSA ATCC 33591.

Fractional Minimum inhibitory concentration indices (FICI) of different combinations of mallotojaponin B and chloramphenicol vis-à-vis MRSA ATCC 33591.</p

Effect of the mallotojaponin B and the different combinations on nucleotide leakage MRSA ATCC 33591.

MIC: Minimum Inhibitory Concentration; PC: positive control (TritonX); NC: negative control; Comb: combination; Comb 1: 0.781 μg/mL of mallotojaponin B and 1.95 μg/mL of chloramphenicol; Comb 2: 0.781 μg/mL of mallotojaponin B and 7.812 μg/mL of chloramphenicol; Comb 3: 1.562 μg/mL of mallotojaponin B and 3.9 μg/mL of chloramphenicol.</p

Biofilms eradication percentage of mallotojaponin B at different concentrations and combinations.

PC: Ciprofloxacin; Comb: Combination; Comb 1: 0.781 μg/mL of mallotojaponin B and 1.95 μg/mL of chloramphenicol; Comb 2: 0.781 μg/mL of mallotojaponin B and 7.812 μg/mL of chloramphenicol; Comb 3: 1.562 μg/mL of mallotojaponin B and 3.9 μg/mL of chloramphenicol; MIC: Minimum Inhibitory Concentration; NC: Negative Control. Histograms with the same letters are not significantly different at p ≤0.05.</p