Repurposing pinaverium bromide against Staphylococcus and its biofilms with new mechanisms

Abstract

Antibiotic resistance by methicillin-resistant Staphylococcus aureus (MRSA) is an urgent threat to human health. The biofilm and persister cells formation ability of MRSA and Staphylococcus epidermidis often companied with extremely high antimicrobial resistance. Pinaverium bromide (PVB) is an antispasmodic compound mainly used for irritable bowel syndrome. Here we demonstrate that PVB could rapidly kill MRSA and S. epidermidis planktonic cells and persister cells avoiding resistance occurrence. Moreover, by crystal violet staining, viable cells counting and SYTO9/PI staining, PVB exhibited strong biofilm inhibition and eradication activities on the 96-well plates, glass surface or titanium discs. And the synergistic antimicrobial effects were observed between PVB and conventional antibiotics (ampicillin, oxacillin, and cefazolin). Mechanism study demonstrated the antimicrobial and antibiofilm effects by PVB were mainly mediated by proton motive force disrupting as well as reactive oxygen species inducing. Although, relatively poor pharmacokinetics were observed by systemic use, PVB could significantly reduce the viable bacterial cell loads and inflammatory infiltration in abscess in vivo caused by the biofilm forming strain ATCC 43,300. In all, our results indicated that PVB could be an alternative antimicrobial reagent for the treatment of MRSA, S. epidermidis and its biofilm related skin and soft tissue infections.

Keywords: Staphylococcus; Biofilm; Drug repurposing; Persister; Proton motive force.

Fig. 1

Effective bactericidal activity of PVB against Staphylococcus avoiding resistance occurrence. (A) Bacterial killing dynamics of PVB against MRSA ATCC 43,300/USA300 and S. epidermidis RP62A. (B) Resistance inducing ability of PVB against MRSA and S. epidermidis. CIP was used as a positive control

Fig. 2

Combinational antimicrobial effects between PVB and conventional antibiotics against MRSA ATCC 43,300. Drug combinational activity between PVB and (A) tetracyclines, (B) macrolides, (C) levofloxacin (LEV), (D) daptomycin (DAP), (E) fosfomycin (FOS), (F) linezolid (LZD), (G) vancomycin (VAN), (H) β-lactams, and (I) aminoglycosides, respectively. (J) FICI values of the synergistical combinations. (K) Bacterial killing dynamics of the synergistical combinations. PVB and the conventional antibiotics were used at the concentration of 1/4×MIC. TET, tetracycline. DOX, doxycycline. E, erythromycin. AZI, azithromycin. P, penicillin. CAZ, ceftazidime. AMP, ampicillin. CEZ, cefazolin. CEF, cefotaxime. OXA, oxacillin. CRO, ceftriaxone. AMK, amikacin. GEN, gentamycin, KANA, kanamycin. TOB, tobramycin. SPC, spectinomycin

Fig. 3

Antibiofilm activity by PVB. (A) Biofilm inhibition activity of PVB determined by crystal violet staining. (B) Biofilm inhibition activity of PVB determined by CFU counting. (C) Biofilm inhibition activity of PVB observed by LCSM. (D) Biofilm eradication activity of PVB determined by crystal violet staining. (E) Biofilm eradication activity of PVB determined by CFU counting. (F) Biofilm eradication activity of PVB observed by LCSM. SYTO9 and PI were used for the live and dead bacterial cells tracing, respectively. Scale: 20 μm

Fig. 4

Antibiofilm effects of PVB against MRSA and S. epidermidis on Ti-discs. (A-B) Biofilm inhibitory (A) and eradicating (B) activity of PVB against USA300 determined by crystal violet staining and CFU counting, respectively. (C) Representative images of inhibition and eradication effects of PVB against USA300 biofilms on Ti-discs determined by crystal violet staining. (D-E) Biofilm inhibitory (D) and eradicating (E) activity of PVB against USA300 determined by crystal violet staining and CFU counting, respectively. (F) Representative images of inhibition and eradication effects of PVB against RP62A biofilms on Ti-discs determined by crystal violet staining

Fig. 5

PMF disruption and ROS inducing activities by PVB. (A) Microstructure observation of MRSA ATCC 43,300 by TEM and SEM, respectively. The bacterial cells were treated with 5×MIC of PVB for 1 h. The representative images were selected randomly. Scale: 250 nm for TEM and 1 μm for SEM. (B) Relative periplasmic space quantification of the S. aureus determined by the SEM. (C) Fluorescence unit quantification of DiSC3(5) probe after treated with PVB for 30 s. Melittin (2 µg/mL) was used as control. (D) MIC changes of PVB against MRSA ATCC 43,300 in the presence of varied proton concentrations. (E) PMF determination by BCECF-AM probe after treated with PVB for indicated time. Melittin (2 µg/mL) was used as control. (F-I) ROS (F) and its components of O2- (G), H2O2 (H), and OH (I) quantification after treated with 1×MIC of PVB for 30 min. (J) ROS tracing by DCFH-DA probe after treated with 1×MIC of PVB for 30 min. (K) Fold change of MIC values in the presence of GSH or anaerobic condition

Fig. 6

In vitro metabolic stability analysis. (A) Plasma protein bind rate of PVB. The binding rate of 200 µg/mL warfarin sodium was set as 100%. (B) Remaining rate of PVB in the presence of liver microsome. (C) Inhibitory effects of PVB against CYP450 sub-types. (D) IC50 values distribution by PVB against the CYP450 sub-types. (E) Catalytic activity determination of CYP450 sub-types after induced by PVB. (F) Pharmacokinetics analysis of PVB by i.p. injection

Fig. 7

Effective in vivo antimicrobial effects of PVB in an abscess model. (A) Representative images of abscesses after treated or untreated with PVB. (B) Viable cells quantification of the abscess (N = 6 mice per group). (C) Representative images of the viable cells counting in the abscess. (D) Pathological detection of the abscessed by H&E staining and immunohistochemistry (IL-1β, IL-6, and TNF-α), respectively