Innovative compounds to battle multiresistence to antibiotics: use of PVA-Tannic acid nanoparticles to inibit staphylococcus pseudointermedius growth

45 Sinergias en la investigación en STEM INNOVATIVE COMPOUNDS TO BATTLE MULTIRESISTANCE TO ANTIBIOTICS: USE OF PVA-TANNIC ACID NANOPARTICLES TO INHIBIT STAPHYLOCOCCUS PSEUDOINTERMEDIUS GROWTH Guillermo Martínez1, María Pérez-Aranda1,2, Manuel Merinero1,2, Belén Begines1, Tamara Ortiz3, Salvadora Navarro2, Raúl Aguilera-Velázquez4, María-Jesús Martín5, Ignacio Rodríguez2, Eloisa Pajuelo2, Ana-Paula Zaderenko4, Ana Alcudia1 1 Departamento de Química Orgánica Y Farmacéutica. Facultad de Farmacia. Universidad de Sevilla. C/ Profesor García González, s/n cp.: 41012 (Sevilla). 2 Departamento de Microbiología y Parasitología. Facultad de Farmacia. Universidad de Sevilla. C/ Profesor García González, s/n cp.: 41012 (Sevilla). 3 Departamento de Citología e Histología Natural y Patológica. Facultad de Medicina. Universidad de Sevilla. Avda. Doctor Fedriani, s/n cp.:41009 (Sevilla). 4 Departamento de Sistemas Físicos, Químicos y Naturales. Universidad Pablo de Olavide. ES-41013 (Sevilla). 5 Departamento de Química Analítica. Facultad de Química. Universidad de Sevilla. C/ Profesor García González, s/n cp.: 41012 (Sevilla). E-mail de correspondencia: mtne[email protected] ABSTRACT Antibiotic resistance is an increasing public health problem that affects numerous pathogens, including Staphylococcus pseudintermedius, which has a high prevalence of methicillin resistance and can be easily transmitted to humans. Nowadays, the scientific community is developing new compounds that not only improve classic therapies in fighting antibiotic resistances but also prevent its appearance, essential to maintain health protection. The main objective of the present study is to synthesize tannic acid and polyvinyl alcohol nanoparticles and to determine their potential as growth inhibitors for S. pseudintermedius, to be considered a potential alternative therapy. The study includes diameter and Z-potential measurements for nanoparticles characterization and antimicrobial effect assays with different nanoparticles concentrations. MIC90 is determined as 112 μg/mL. Nonetheless, further studies to identify the underlying action mechanisms of these nanoparticles are going on in our group. INTRODUCTION AND OBJECTIVES OF THE STUDY S. pseudintermedius is an opportunistic pathogen commonly associated with skin and soft tissue infections (SSTIs) in dogs. The high prevalence of antibiotic resistance, particularly to methicillin, along with its zoonotic potential could lead to treatment implications in human infections (Somayaji et al., 2016). In this sense, searching 46 Sinergias en la investigación en STEM for new bactericide molecules that could prevent the appearance of resistance has become a critical issue for WHO priority plan. Biocompatible polymers, such as polyvinyl alcohol (PVA), have become crucial materials in the development of novel drug delivery systems due to their low toxicity and high biocompatibility (Begines et al., 2015). Tannic acid (TA), existing in plant tissues, has been reported to possess natural antioxidant, antibacterial and antiviral activity (Ivanova et al., 2018). Thus, the presentation of both compounds in the form of nanoparticles, that have shown to be very useful structures in biomedicine, may offer new therapeutic possibilities such as controlled delivery of the TA, increased toxicity to bacteria, etc. (Wu et al., 2017). Furthermore, polyvinyl alcohol - tannic acid nanoparticles (PVA-TA NPs) have been described as non-toxic and biodegradable nanomaterials, which makes them an ideal candidate for developing new alternative therapies (Velázquez et al., 2016). The purpose of the present study is to describe an easily executable synthesis protocol for PVA-TA NPs and to determine their potential capacity to inhibit S. pseudintermedius growth in vitro to be considered a potential alternative drug compared to classic therapies. MATERIALS AND METHODS Nanoparticles synthesis and characterization 32 mg of TA were dissolved in 10 mL deionized water and 100 mg of PVA in 10 mL deionized water using different flasks. Then, TA solution was added dropwise to the PVA solution at room temperature. The solution turned white and opaque and it was left stirring for 15 minutes. Nanoparticles’ characteristics were measured using Dynamic Light Scattering. BACTERIAL GROWTH INHIBITION ASSAYS Different concentrations of PVA-TA NPs in TSB – sterile water liquid culture medium was prepared. TSB was prepared at triple concentration (3x TSB) to be at normal concentration when mixed with the other compounds. “Stock solutions” were prepared with the following proportions: 1. 600 μL of 3xTSB, 1110 μL of sterile H20 and 90 μL of PVA-TA NPs. 2. 600 μL of 3xTSB, 1020 μL of sterile H20 and 180 μL of PVA-TA NPs. 3. 600 μL of 3xTSB, 930 μL of sterile H20 and 270 μL of PVA-TA NPs. 47 Sinergias en la investigación en STEM 4. 600 μL of 3xTSB, 840 μL of sterile H20 and 360 μL of PVA-TA NPs. 5. 600 μL of 3xTSB, 750 μL of sterile H2O and 450 μL of PVA-TA NPs. 6. 600 μL of 3xTSB, 660 μL of sterile H2O and 540 μL of PVA-TA NPs. Once prepared, a volume of 300 μL of each was poured in eppendorf tubes. Finally, 5 μL of an overnight grown S. pseudintermedius liquid culture were inoculated per tube, except the first of each (negative control). Once the dilutions were prepared, they were incubated for 24 hours at 37 oC. 200 μL of each eppendorf were transferred to a microtiter plate for determining bacterial growth as a measurement of the absorbance at 600 nm (turbidity). RESULTS AND DISCUSSION Nanoparticles synthesis and characterization The synthesis protocol was proven to be effective and nanoparticles always presented identical characteristics. The final solution was a white, non-aggregated and completely opaque colloid. Three samples of three different batches of nanoparticles were characterized at different times (Table 1). The average NP size was 164 nm, with PDI of 0.16 and a Z-potential of -20 mV. As standard deviations showed, values obtained were almost identical between the three samples. Table 1. Hydrodynamic diameter (size, nm), Z-potential (Z-pot, mV) and polydispersity index (PDI) obtained from three samples, A, B and C, of PVA-TA NPs. Sample A Sample B Sample C Average St.Dev Size (nm) 164,3 165,5 163,0 164,3 1,2 PDI 0,155 0,168 0,176 0,16 0,01 Z-pot (mV) -19,1 -20,7 -20,1 -20,0 0,8 Source: own elaboration. Low PDI values obtained indicated that nanoparticles size distribution was homogeneous, which is an ideal characteristic for nanoparticles to be considered as therapeutic agents (Clogston & Patri, 2011). Also, the negative values in Z-potential could mean a reduction in possible toxic effects in humans, but further studies are needed to determine this feature. Bacterial growth inhibition assays The results of bacterial growth (Table 2) showed a great reduction in the absorbance rate from dilution D (40 μL of nanoparticles), suggesting that the bacterial growth 48 Sinergias en la investigación en STEM was inhibited at this concentration. MIC90 is determined as 112 μg/mL, a promising result to take them into account as an alternative therapy or combined with regular antibiotic drugs. Table 2. Absorbance at 600nm of cultures of S. pseudintermedius incubated in the presence of increasing concentrations (0-60 μL) of PVA-TA NPs. Stock solutions Control 1 2 3 Average A (10μL NPs) 0.26 0.77 0.748 0.718 0.624 B (20μL NPs) 0.291 0.55 0.73 0.625 0.549 C (30μL NPs) 0.227 0.341 0.392 0.452 0.353 D (40μL NPs) 0.223 0.264 0.347 0.276 0.278 E (50μL NPs) 0.217 0.220 0.247 0.221 0.226 F (60μL NPs) 0.241 0.211 0.294 0.214 0.24 Source: own elaboration. CONCLUSIONS These nanoparticles present an outstanding balance among their properties. PDI values obtained indicates that nanoparticles size is very homogeneous, meaning that, along with negative Z-potential values, their potential toxicity for animals and humans is low. 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