A novel ruthenium based coordination compound against pathogenic bacteria

International Journal of Molecular Sciences Article A Novel Ruthenium Based Coordination Compound Against Pathogenic Bacteria Vishma Pratap Sur 1,2,* , Aninda Mazumdar 1,2, Pavel Kopel 3, Soumajit Mukherjee 1, Petr Vítek 4, Hana Michalkova 1, Markéta Vaculoviˇcová1,2 and Amitava Moulick 1,2,* 1Department of Chemistry and Biochemistry, Faculty of AgriSciences, Mendel University in Brno, CZ-613 00 Brno, Czech Republic; [email protected] (A.M.); [email protected] (S.M.); [email protected] (H.M.); [email protected] (M.V.) 2Central European Institute of Technology, Brno University of Technology, CZ-61200 Brno, Czech Republic 3Department of Inorganic Chemistry, Faculty of Science, Palacky University, CZ-771 46 Olomouc, Czech Republic; [email protected] 4Global Change Research Institute of the Czech Academy of Sciences, CZ603 00 Brno, Czech Republic; [email protected] *Correspondence: [email protected] (V.P.S.); [email protected] (A.M.) Received: 10 March 2020; Accepted: 7 April 2020; Published: 10 April 2020   Abstract: The current epidemic of antibiotic-resistant infections urges to develop alternatives to less-effective antibiotics. To assess anti-bacterial potential, a novel coordinate compound (RU-S4) was synthesized using ruthenium-Schiffbase-benzimidazole ligand, where ruthenium chloride was used as the central atom. RU-S4 was characterized by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), and Raman spectroscopy. Antibacterial effect of RU-S4 was studied against Staphylococcus aureus (NCTC 8511), vancomycin-resistant Staphylococcus aureus (VRSA) (CCM 1767), methicillin-resistant Staphylococcus aureus (MRSA) (ST239: SCCmecIIIA), and hospital isolate Staphylococcus epidermidis. The antibacterial activity of RU-S4 was checked by growth curve analysis and the outcome was supported by optical microscopy imaging and fluorescence LIVE/DEAD cell imaging. In vivo (balb/c mice) infection model prepared with VRSA (CCM 1767) and treated with RU-S4. In our experimental conditions, all infected mice were cured. The interaction of coordination compound with bacterial cells were further confirmed by cryo-scanning electron microscope (Cryo-SEM). RU-S4 was completely non-toxic against mammalian cells and in mice and subsequently treated with synthesized RU-S4. Keywords: coordination compound; antimicrobial compound; ruthenium; benzimidazole; SEM; EDS 1. Introduction The appearance of antibiotic-resistant pathogenic bacteria during mid-nineteenth century created an urge to develop a treatment against these life-threatening pathogens, alongside the existing drug molecules known as antibiotic. The present scenario in terms of the effectivity of novel antimicrobials is quite complex due to the rapid uprising of antibiotic-resistance in bacteria. Their ability to evade the effect of antibiotics by using various mechanisms depends on their evolving nature, which makes these pathogens smarter and better. Antibiotic resistance is an emerging problem in society and a crucial challenge to medical biology. The epidemiology of Staphylococcus aureus and the antibiotic-resistant S. aureus strains are changing constantly. S. aureus causes a range of mild to life-threatening community-associated infections and currently is more significant with the evolution of antibiotic-resistant species like vancomycin-resistant S. aureus (VRSA), and, methicillin-resistant S. aureus (MRSA) [ 1 ]. VRSA and MRSA are some of the primary causes of nosocomial infections associated commonly with increased morbidity and increases treatment Int. J. Mol. Sci. 2020,21, 2656; doi:10.3390/ijms21072656 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020,21, 2656 2 of 18 duration and medical cost [ 2 ]. The VRSA strain is thought to acquire the vancomycin-resistance gene from the antibiotic-resistant vancomycin-resistant Enterococci (VRE), another predominantly concerning pathogen that causes infections similar to VRSA and MRSA [ 3 ]. Initially, penicillin used to be the major effective treatment for S. aureus infections until the efficacy of the antibiotic reduced. It is due to notable evidence of acquiring the penicillin-resistance gene and consequently the development of resistant mechanism toward prominent broad-range antibiotics, such as methicillin and vancomycin [4]. Contrary to efficacy, both incomplete dosage and over-usage of antibiotics cause side effects which further complicates the well-being of the patient. The use of broad-range antibiotics such as vancomycin was considered as one of the last resorts against significantly life-threatening, multidrug-resistant infections causedbygram-positive bacteria[ 5 , 6 ].However, theincreasing complexityof differentantibiotic resistance mechanisms in S. aureus strains such as VRSA and MRSA resulted in a significant negative impact on health and clinical settings. With the increasing mortality rate causing by antibiotic-resistant bacterial strains and with fewer appropriate treatment options for multi-drug resistant infections, currently, there is a necessity to investigate new candidates as potential alternative to antibiotics. In pursuit of new effective and non-cytotoxic antimicrobials, recent reports witnessed a rise in the usage of novel metal-based coordination compounds, nanoparticles (NPs) [ 7 – 11 ] or antimicrobial peptides as alternatives to existing antibiotics. It has been previously reported that transition metal-based coordination compounds and ligand-based coordination compounds were used as potential antimicrobial agents [7,9–12]. Thus, in this study, we have aimed to develop a novel metal-based coordination compound to treat life-threatening infections caused by S. aureus, VRSA, MRSA, and Staphylococcus epidermidis (isolated and obtained from hospital sample). In this study ruthenium-based coordination compound synthesized with benzimidazole and Schiff base ligands (ruthenium-Schiff base–benzimidazole coordination compound). Benzimidazole derivatives are heterocyclic molecules that have a wide range of biological activities and reported to have antibacterial, antimicrobial, anthelmintic, analgesic, antitumor, and anti-inflammatory properties [ 7 , 13 ]. In other studies, it has been shown to inhibit DNA gyrase and to exhibit DNA binding affinity [ 14 , 15 ]. Thus, benzimidazole derivatives can be used as the potential candidates for developing new biologically active compounds [ 14 ]. Schiff bases are versatile biologically active molecules with antimicrobial activity and being used for drug molecule designing and industrial purposes [ 12 ]. Ruthenium-based compounds are well-known antibacterial compounds, where ruthenium played a key role [ 9 – 11 ]. Whereas, ruthenium provides the metal ion background and the complexes formed by them have biological importance. Thus, the ruthenium-Schiff base benzimidazole coordination complex with a unique combination was synthesized as new novel compounds to study its antimicrobial efficacy. Further, the chemical characterization of ruthenium, Schiffbase and benzimidazole were carried out using biophysical techniques like Fourier-transform infrared spectroscopy, scanning electron microscope and Raman microscopy. Energy-dispersive X-ray spectroscopy (EDS) elemental analyses were performed to confirm the presence of all the individual components. Further, the effect of this ruthenium-based coordination compound was used to study the antibacterial efficacy against S. aureus, VRSA MRSA, and S. epidermidis. Thereafter, the MIC was calculated and the lowest concentration with the highest effectivity against bacterial growth was used for the toxicity test. The toxicity test was performed by several assays including MTT assay and hemolytic assay. In vivo study of the RU-S4 was also carried out to better understand its biocompatibility and activity towards S. aureus and its resistant strains. Int. J. Mol. Sci. 2020,21, 2656 3 of 18 2. Results 2.1. Chemical Synthesis and Electron Microscopy and Energy-Dispersive X-ray Spectroscopy (EDS) Confirmation The ruthenium-Schiff base–benzimidazole complex (chemical structure of benzimidazole ligand, Schiff base ligand and probable structure of RU-S4, Figure 1) and the Energy-dispersive X-ray spectroscopy (EDS) method confirms that in the composed complex have 39% ( ± 2) ruthenium (Figure S2). The presence of nitrogen 41% ( ± 2) proves that this compound contains nitrogenous benzimidazole and Schiff base and ruthenium (Figures S1 and S2). Visualization of the prepared complex on scanning electron microscope (SEM) shows partial particle character of the sample. The average diameter of the greater particles is about 500 nm, but the smaller entities have a size of about 100 nm and less. The formation of bigger particles was assumed as a result of sample drying for SEM visualization. In solution, we suppose the average size of entities is about 100 nm (Figure 2). Figure 1. ( a ) benzimidazole ligand structure, ( b ) Schiffbase ligand structure, ( c ) possible structure of RU-S4. Figure 2. Scanning electron microscopy image for RU-S4 in different scales: ( a ) scale 500 nm, ( b ) scale 1µm, (c) scale 2 µm. 2.2. Characterization of the Ru Complexes by Raman Spectroscopy Raman spectra of Ru-S4 complex provide a spectral pattern, with bands assignable to benzimidazole located at 1006, 1048, 1276, 1318, and 1378 cm −1 (Wang et al., 2004, Suwaiyan et al., 1990) [ 16 , 17 ]. Features attributed to Schiff base are located at 1582 and 1618 cm−1(Figure 3). Int. J. Mol. Sci. 2020,21, 2656 4 of 18 Figure 3. Raman spectra and listed bands of Ru-S4 complex as obtained by the 514.5 nm excitation. 2.3. Growth Curve Snalysis The effect of ruthenium-Schiffbase − benzimidazole (Ru-S4) complex compound was investigated against VRSA (CCM 1767), MRSA (ST239: SCCmecIIIA), and the hospital sample Staphylococcus epidermidis also. The effect of the RU-S4 compound and the MIC were determined by bacterial growth curve analysis [ 18 ]. Antibacterial activity of the RU-S4 compound was studied by turbidimetry technique, the absorbance was measured at 600 nm for 24 h against MRSA (ST239: SCCmecIIIA), VRSA (CCM 1767), S. aureus (NCTC 8511), and hospital sample Staphylococcus epidermidis [ 19 – 24 ]. The bacterial viability was calculated as mentioned in our previous work [ 25 ]. It was evaluated from the growth curve and viability curve that the ruthenium compound has the most adverse effects on all bacterial species used in this experiment. Different concentration was used for application, but 25 µ l/mL and 50 µ l/mL concentration show most adverse effects against all bacterial populations in both commercial bacteria and hospital samples also. For all bacterial populations MIC value was 25 µ g/mL (except 25 µ g/mL and 50 µ g/mL no other concentrations showed relevance effect) Figure 4a–d. The percentage of viability for VRSA (CCM 1767) resulted >8 and >4 with a concentration of Ru-S4 of 25 µ g/mL and 50 µ g/mL, respectively. The percentage of viability for MRSA (ST239: SCCmecIIIA) was >4% with a concentration of Ru-S4 of 25 µ g/mL and >2% with a concentration of 50 µ g/mL. The percentage of viability for (NCTC 8511) resulted >4 and >3 with a concentration of Ru-S4 of 25 µ g/mL and 50 µ g/mL respectively. The percentage of viability for S. epidermidis was >3% with a concentration of Ru-S4 of 25 µg/mL and >5% with a concentration of 50 µg/mL Int. J. Mol. Sci. 2020,21, 2656 5 of 18 Figure 4. Bacterial growth curve and viability curve ( a – d ); The growth curve of VRSA (CCM 1767), MRSA (ST239: SCCmecIIIA), S. aureus (NCTC 8511), Staphylococcus epidermidis respectively ( e – h ); Viability percentage of VRSA (CCM 1767), MRSA (ST239: SCCmecIIIA), S. aureus (NCTC 8511), hospital sample Staphylococcus epidermidis respectively. Data represent the mean ±SD, n=3. The cytotoxicity of Ru-S4 was estimated by using normal breast cell line HBL 100 and Cancer Breast cell line MDA MB-468 and prostate epithelial cell line PNT1A. RU-S4 showed negligible toxicity towards the all cell lines (Figure 5, Figure 6, Figure S9). The end conclusion is RU-S4 is completely nontoxic. Int. J. Mol. Sci. 2020,21, 2656 6 of 18 Figure 5. Cytotoxicity for RU-S4 against the human cell line. Figure 6. Percentage of hemolysis of blood cells treated with RU-S4 in a blood sample. 2.4. Hemolytic Assay Drug-induced hemolysis is a major toxicity problem. High drug concentrations can also be a cause of hemolysis inpatient. US FDA recommended hemolysis screening for drug molecules is a very important requirement of drug development [26]. It is clear from this Figure 6that RU-S4 exhibited no hemolysis, where 0.1% TritonX exhibits 100% hemolysis. 2.5. Optical Microscopy Optical microscopy showed that bacterial cells were disrupted after treatment with the RU-S4 complex (Figure 7and Figure S13). Int. J. Mol. Sci. 2020,21, 2656 7 of 18 Figure 7. Optical microscopy image for RU-S4 treatment against S. aureus (NCTC 8511), MRSA (ST239: SCCmecIIIA), VRSA (CCM 1767), and untreated cells. No morphological changes were seen in untreated cells whereas the treated bacterial cells were ruptured and almost no visible structured cells can be seen. Scale bar is 5 µm. 2.6. LIVE/DEAD Cell Imaging by Fluorescence Microscopy The viability of S. aureus, VRSA (CCM 1767), and MRSA (ST239: SCCmecIIIA) (Figure 8) and S. epidermidis (Figure S14) after treatment with RU-S4 was observed by the LIVE/DEAD cell imaging assay [ 8 , 25 , 27 ]. The bacterial samples treated with RU-S4 showed considerably decreased cell count with green fluorescent, but a significant increase in number of dead cells with red fluorescence, whereas in the case of control bacterial cells, the number of green fluorescent cells were high with red fluorescence (Figure 8and Figure S14). Figure 8. LIVE/DEAD cell imaging for RU-S4 treatment against S. aureus (NCTC 8511), MRSA (ST239: SCCmecIIIA), and VRSA (CCM 1767). Green cells define living cells whether red cells stand for dead cells. Scale bar is 10 µm. Int. J. Mol. Sci. 2020,21, 2656 8 of 18 2.7. Cryo-SEM Microscopy Imaging for Bacterial Cells Treatment with RU-S4 VRSA (CCM 1767) cells treated with RU-S4 and imaged under scanning electron microscope, where untreated bacterial cells appeared as intact cocci shape with no cell wall rupture or collapse (Figure 9). On the other hand, bacterial cells treated with RU-S4 showed cell outer shape bubbling rough surface and clumped and deformed which can be seen in Figure 9[28]. Figure 9. Scanning electron microscope (SEM) image: ( a ) left panel shows untreated coccus cells (VRSA) (CCM 1767), (b) right panel shows treated coccus (VRSA) (CCM 1767). 2.8. In Vivo Animal Model After the infection started spreading in the mouse near the dermal part near neck swells and wound started to form. After swelling and wound formation their treatment was started with RU-S4 and day by day infection and wound started to heal and their skin started to recover from the traumatic infection situation which can be seen in Figure 10. Here we use uninfected control (Figure S10). The control animal was maintained throughout the experiment for monitoring, wherever no changes were found. Whereas infected untreated control animals were monitored (Figure S11). During the experimental condition the morbidity of the infected untreated animals were 100% within 8 days, where dermal infection growth and simultaneously internal infection growth were observed. MMPSense was also gave fluorescence (against infection) until 9 days. From day 12 of observation there was no fluorescence for MMPSense (Figure 11). The decreased MMP (Matrix metallopeptidases) fluorescence intensity was also decreased (Figure S12). Int. J. Mol. Sci. 2020,21, 2656 9 of 18 Figure 10. In vivo infection model preparation, treatment, and recovery: ( a ) after bacterial infection dose, ( b ) day 1 Infection initiation, ( c ) day 2 Infection and wound growth, ( d ) day 3 inflammation and swelling, ( e ) day 6 no significant changes were observed, ( f ) day 9 wound started to heal, ( g ) day 12 wound started to heal, (h) day 15 fully recovered. Figure 11. 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