Isoniazid potentiates tigecycline to kill methicillin-resistant Staphylococcus aureus

Abstract

Therapeutic option for treating methicillin-resistant Staphylococcus aureus (MRSA) infection is urgently required since its resistance to a broad spectrum of currently available antibiotics. Here, we report that isoniazid is able to potentiate the killing efficacy of tigecycline to MRSA. The combination of isoniazid and tigecycline reduces the minimal inhibitory concentration of clinic MRSA strains to tigecycline. The killing activity of tigecycline is further confirmed by killing experiments and murine infection model. We further demonstrate the mechanism that isoniazid increases intracellular accumulation of tigecycline by promoting the influx but limiting the efflux of tigecycline through proton motive force. We also show that isoniazid and tigecycline synergize to increase the abundance of isoniazid-NAD adduct, which in turn damage cell membrane, possibly contributing to the disruption of PMF. Whereas phosphatidylethanolamine and cardiolipin are able to abrogate the synergistic effect of isoniazid plus tigecycline. Thus our study provides a new perspective that antibiotics, e.g. isoniazid, once recognized only to target Mycobacterium tuberculosis, can be repurposed as antibiotic adjuvant to tigecycline, expanding our choice of antibiotic-antibiotic combinations in treating bacterial infectious diseases.

Keywords: Tigecycline; antibiotic influx; isoniazid; methicillin-resistant Staphylococcus aureus; proton motive force.

Figure 1.

INA potentiates TIG to eliminate MRSA. (A) The FICI of INA combined with different types of antibiotics. (B) The FICI of INA combined with TIG to different MRSA strains. (C) The MIC of MRSA to TIG in the presence of 1/2 MIC of INA. INA, isoniazid; GEN, gentamycin; KAN, kanamycin; AMP, ampicillin; CFT, ceftaxime; CIP, ciprofloxacin; OFX, ofloxacin; TET, tetracycline; TIG, tigecycline; LIN, lincomycin; CM, chloramphenicol.

Figure 2.

INA and TIG eliminate MRSA infection in vitro and in vivo. (A) Percent survival of MRSA11 in the presence of the increasing concentration of INA plus 0.04 μg/mL TIG. (B) Percent survival of MRSA11 in the presence of the increasing concentration of TIG plus 8 mM INA. (C) Percent survival of MRSA11 in the presence of 8 mM INA plus 0.04 μg/mL TIG at indicated incubation time points. (D) Percent survival of persister in the presence of INA plus TIG. (E) Percent survival of biofilm in the presence of INA plus TIG. (F) Bacterial loads of mice infected with MRSA in the presence or absence of INA, TIG or both. Results are displayed as mean ± standard errors of the means (SEM) (N ≥ 3 technical replicates per sample), and statistically significant differences are identified by Student’s t test. *, p < 0.05, **, p < 0.01. Each experiment was repeated independently at least three times.

Figure 3.

INA enhances TIG bacteriostatic activity. (A) MIC and MBC of MRSA11 in the presence or absence of INA. (B) Percent of cells having PI of MRSA11 in the absence or presence of INA plus TIG. (C) Percent of cells having calcein of MRSA11 in the absence or presence of INA plus TIG. (D) Scanning electron micrographs of MRSA11 being treated with INA, TIG, or both (Scalar bar = 1, 2, 10 μm, respectively). Results are displayed as mean ± standard errors of the means (SEM) (N ≥ 3 technical replicates per sample), and statistically significant differences are identified by Student’s t test. *, p < 0.05, **, p < 0.01. Each experiment was repeated independently at least three times.

Figure 4.

INA enhances intracellular accumulation of TIG. (A) NPN fluorescence intensity of MRSA11 in the presence or absence of 8 mM INA plus 0.04 μg/mL TIG. (B) Intracellular TIG of MRSA11 being treated with indicated concentrations of 8 mM INA plus 0.04 μg/mL TIG. (C) Intracellular TIG of MRSA11 being treated with INA plus the increasing concentration of TIG. (D) Intracellular TIG of MRSA11 being treated with INA plus TIG at the indicated incubation periods. (E) pH fluorescence intensity of MRSA11 in the presence or absence of 8 mM INA plus 0.04 μg/mL TIG. (F) Percent survival of MRSA11 in the presence of the increasing concentration of AS with INA plus TIG. (G) Membrane potential of MRSA11 in the presence or absence of 8 mM INA plus 0.04 μg/mL TIG. (H, I) Percent survival of MRSA11 in the presence of the increasing concentration of CCCP with INA plus TIG. (J) qRT-PCR for expression of mepA of MRSA11 in the absence and presence of INA plus TIG. Results are displayed as mean ± standard errors of the means (SEM) (N ≥ 3 technical replicates per sample), and statistically significant differences are identified by student’s t test. *, p < 0.05, **, p < 0.01. Each experiment was repeated independently at least three times.

Figure 5.

INA plus TIG destabilize cellular membrane to promote TIG accumulation. (A) INA-NAD adduct of MRSA11 in the presence or absence of 8 mM INA plus 0.04 μg/mL TIG. (B) Percent of cells having PI of MRSA11 in the absence or presence of PE or CL with 8 mM INA plus 0.04 μg/mL TIG. (C) NPN fluorescence intensity of MRSA11 in the absence or presence of PE or CL with 8 mM INA plus 0.04 μg/mL TIG. (D, E) Percent survival of MRSA11 in the absence or presence of PE or CL with 8 mM INA plus 0.04 μg/mL TIG. (F) Intracellular TIG of MRSA11 in the absence or presence of PE or CL with INA plus TIG. Results are displayed as mean ± standard errors of the means (SEM) (N ≥ 3 technical replicates per sample), and statistically significant differences are identified by student’s t test. *, p < 0.05, **, p < 0.01. Each experiment was repeated independently at least three times.

Figure 6.

Proposed model. INA enhances the TIG influx to enhance intracellular TIG accumulation to combat MRSA infection.