Comparison of Antimicrobial and Antibiofilm Activity of Proflavine Co-crystallized with Silver, Copper, Zinc, and Gallium Salts

Abstract

Here, we exploit our mechanochemical synthesis for co-crystallization of an organic antiseptic, proflavine, with metal-based antimicrobials (silver, copper, zinc, and gallium). Our previous studies have looked for general antimicrobial activity for the co-crystals: proflavine·AgNO₃, proflavine·CuCl, ZnCl₃[Proflavinium], [Proflavinium]₂[ZnCl₄]·H₂O, and [Proflavinium]₃[Ga(oxalate)₃]·4H₂O. Here, we explore and compare more precisely the bacteriostatic (minimal inhibitory concentrations) and antibiofilm (prevention of cell attachment and propagation) activities of the co-crystals. For this, we choose three prominent "ESKAPE" bacterial pathogens of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. The antimicrobial behavior of the co-crystals was compared to that of the separate components of the polycrystalline samples to ascertain whether the proflavine-metal complex association in the solid state provided effective antimicrobial performance. We were particularly interested to see if the co-crystals were effective in preventing bacteria from initiating and propagating the biofilm mode of growth, as this growth form provides high antimicrobial resistance properties. We found that for the planktonic lifestyle of growth of the three bacterial strains, different co-crystal formulations gave selectivity for best performance. For the biofilm state of growth, we see that the silver proflavine co-crystal has the best overall antibiofilm activity against all three organisms. However, other proflavine-metal co-crystals also show practical antimicrobial efficacy against E. coli and S. aureus. While not all proflavine-metal co-crystals demonstrated enhanced antimicrobial efficacy over their constituents alone, all possessed acceptable antimicrobial properties while trapped in the co-crystal form. We also demonstrate that the metal-proflavine crystals retain antimicrobial activity in storage. This work defines that co-crystallization of metal compounds and organic antimicrobials has a potential role in the quest for antimicrobials/antiseptics in the defense against bacteria in our antimicrobial resistance era.

Keywords: antimicrobials; co-crystallization; copper; gallium; quaternary-ammonium compound; silver; zinc.

Scheme 1. Neutral PF and Its Monoprotonated Proflavinium Cation (HPF⁺)

Figure 1

Relevant packing features in crystalline PF·CuCl, showing the herring-bone arrangement of the PF molecules (a) and the 1D (CuCl···CuCl···)_n chains (b); H atoms omitted for clarity.

Figure 2

(a) Projection along the a-axis of crystalline ZnCl₃(HPF), showing the stackings of proflavinium ligands along the c-axis direction. (b) Space-filling representation. H atoms omitted for clarity.

Figure 3

(a) Projection down the crystallographic b-axis of crystalline [HPF]₂[ZnCl₄]·H₂O, showing the herring-bone pattern of HPF⁺ cationic pairs. (b) Space-filling representation. Water oxygens in blue; H atoms omitted for clarity.

Figure 4

Packing in crystalline [HPF]₃[Ga(ox)₃]·4H₂O viewed along the crystallographic b-axis (a). Proflavinium cations envelop around the [Ga(ox)₃]^3– anion (b). O_W in blue; H atoms omitted for clarity.

Figure 5

Bacteriostatic MIC efficacy of the metal salt and PF starter compounds and their co-crystallization products. Bars with no visible errors indicate that there was no variation in the values obtained between trials. Note y-axis scale is log-2.

Figure 6

MBIC efficacy of the metal salt and PF starter compounds and their co-crystallization products. These values represent the ability of the compounds to inhibit cell attachment and proliferation of biofilm biomass. Bars with no visible error bars indicate that there was no variation in the values obtained between trials. Note the y-axis scale is log-2.

Figure 7

Comparison of efficacy from storage of co-crystals CC-1, CC-2, CC-3, and CC-4.