Antimicrobial and anti-biofilm activity of hexadentated macrocyclic complex of copper (II) derived from thiosemicarbazide against Staphylococcus aureus

Multidrug-resistant pathogens causing nosocomial and community acquired infections delineate a significant threat to public health. It had urged to identify new antimicrobials and thus, generated interest in studying macrocyclic metal complex, which has been studied in the past for their antimicrobial activity. Hence, in the present study, we have evaluated the antimicrobial activity of the hexadentated macrocyclic complex of copper (II) (Cu Complex) derived from thiosemicarbazide against Gram-positive and Gram-negative bacteria. We observed increased susceptibility against standard isolates of Staphylococcus aureus with a minimum inhibitory concentration (MIC) range of 6.25 to 12.5 μg/mL. Similar activity was also observed towards methicillin resistant and sensitive clinical isolates of S. aureus from human (n = 20) and animal (n = 20) infections. The compound has rapid bactericidal activity, and we did not observe any resistant mutant of S. aureus. The compound also exhibited antibiofilm activity and was able to disrupt pre-formed biofilms. Cu complex showed increased susceptibility towards intracellular S. aureus and was able to reduce more than 95% of the bacterial load at 10 μg/mL. Overall, our results suggest that Cu complex with its potent anti-microbial and anti-biofilm activity can be used to treat MRSA infections and evaluated further clinically.

Cu is one of the most investigated metal ions among other transition metals and hold importance due to their significant role in the various biological activity 22 . During intracellular bacterial infections, macrophages expose bacteria to increased Cu concentrations resulting in phagosomal killing 23 . Various reports of Cu complexes showing antibacterial and anticancer activity have been published earlier 21,22 . The hexadentated macrocyclic complex of copper (II) derived from thiosemicarbazide has shown potent antibacterial activity, which encouraged us to investigate its therapeutic potential 22 . The primary aim of our study is to determine the antibacterial activity of the Cu complex against clinically important bacterial pathogens and to investigate their mechanism of action, antibacterial and antibiofilm efficacy and capacity to kill intracellular bacteria.

Methods
Bacterial cultures. We used standard isolates of Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumanii, Clostridium difficile, Klebsiella pneumonia, and Escherichia coli in the study (Table 1). Antimicrobial susceptibility was determined in S. aureus clinical isolates from animal (n = 20) and human (n = 20) infections.
Synthesis of Cu complex. The Cu complex was synthesised by the condensation reaction between substituted carbohydrazone and thiosemicarbazide in the presence of Copper chloride as described earlier 22 . Briefly, a divalent Copper chloride (1 mmol) solution was added to a stirred hot methanolic solution (≈50 cm 3 ) of thiosemicarbazide (2 mmol) and substituted carbohydrazone. Further, the solution was refluxed for 8-10 h. The mixture was concentrated and kept overnight in desiccators. After overnight cooling, it formed a greenish coloured precipitate which was filtered, washed with methanol and dried in vacuum (Fig. 1). The compound obtained was assessed for its physical and analytical properties as described previously 22 . Antimicrobial susceptibility. Micro-broth dilution assay determined the minimum inhibitory concentrations (MICs) in 96 well plate format. The micro-broth dilution assay was performed as per guidelines of Clinical and Laboratory Standards Institute (CLSI) with a slight modification by using resazurin dye as described earlier 24   Resistance Detection Study. S. aureus (ATCC 29213 and ATCC 700699) cells were adjusted to the count of 10 10 CFU/mL and were plated on Mueller Hinton agar (MHA) plates containing 2×, 4× and 10× of the MICs value. The plates were then incubated for 48 h at 37 °C and observed for growth 27 . Serial passaging of the bacteria in the sub-inhibitory concentration of drug was done to evaluate the development of resistance as described earlier 1,27 . Briefly, MIC values of ATCC 29213 strain against Cu complex and ofloxacin were determined by micro-broth dilution assay. The bacterial cells growing at a sub-inhibitory concentration (0.5 × MIC) of the compounds (Cu complex and ofloxacin) were harvested and inoculated into fresh media. The inoculated bacterial cells were subjected to another MIC assay. After 18-24 h incubation, cells growing in the second highest concentration from the previous passage were again inoculated and used for the MIC determination assay. We repeated the procedure for 15 passages. The fold change in MIC was plotted against the number of passages. The experiment was performed in triplicates.
Anti-biofilm activity. The inhibitory effect of Cu complex on biofilm formation of S. aureus isolate was determined by 96-well plate-based Crystal violet (CV) assay 1,28 . An overnight culture of S. aureus (ATCC 33592) was diluted 1:200 in Tryptic Soy Broth (TSB, containing 0.25% glucose and 0.5% NaCl) and dispensed into 96-well plate (200 µL/well). The plate was incubated at 37 °C for 24 h and washed gently three times with PBS to remove planktonic bacteria. After washing, Cu complex and vancomycin was added ranging from 0 to 125 μg/mL, and the plate was incubated for another 16 h at 37 °C. All the wells were then again washed with PBS three times and fixed with methanol for 15 minutes. The plate was air dried for 30 minutes and 0.1% CV solution was added to each well and incubated at room temperature for 20 minutes. The picture of the wells was taken using a digital camera. After washing with distilled water, 33% acetic acid was added to each well and absorbance was taken at 590 nm. Mean absorbance values of each sample was calculated and compared with the mean values of controls.
Similarly, the anti-biofilm activity was also assessed using resazurin. The protocol used was same as mentioned above with slight modifications 29,30 . Briefly, the anti-biofilm effect of Cu complex was determined on 24 h and 72 h old biofilms. The pre-formed biofilms (24 h and 72 h) were treated for 24 h with different dilutions of Cu complex (0 to 125 μg/ml). After treatment, the wells were washed with PBS, and 100 μL of resazurin was added to each well and incubated for 30 min at 37 °C. The fluorescence was measured by using a multimode reader (Perkin Elmer, excitation wavelength = 550 nm and emission wavelength = 590 nm). The results were expressed as percent cell viability in treated wells as compared with untreated control. All experiments were repeated thrice in quadruplicates.

Determination of the anti-biofilm activity by confocal microscopy. An overnight grown bacterial
culture (diluted 1:200) was added to the chambered slide and incubated at 37 °C for 24 h 1 . The planktonic cells were carefully removed and washed with 1 × PBS. The Cu complex (50 µg/mL) was added to the slide and incubated for 4 h. Wells containing only medium were treated as control. After incubation, the wells were washed with 1 × PBS and stained with SYTO-9 (3 µM) and Propidium Iodide (PI, 15 µM) for 20 minutes and images were taken using a confocal microscope.
Cell membrane permeability. A cell membrane permeability assay was performed using propidium iodide (PI). The PI uptake was determined by flow cytometry (FACs). Bacterial cells of S. aureus (ATCC 29213) was grown to exponential phase in TSB and incubated with 50 μg/mL of Cu Complex at 37 °C for 60 min. PI was added to the treated cells (10 6 CFU/mL) at final concentrations of 5 μM. The cells were incubated in the dark at 25 °C for 5 min, and the sample was run on a flow cytometer. 10,000 events were recorded for control and treated samples.
Scanning Electron Microscopy. ATCC 29213 cells were grown till the exponential phase and resuspended at a concentration of 10 8 CFU/mL 31 . Cells were incubated with Cu Complex (50 µg/mL) for 60 min at 37 °C and cells without drug were used as a control. Further, the cells were pelleted and fixed with 2% glutaraldehyde solution for 2 h at 4 °C as described earlier 31 . The cells were dehydrated in a graded series of alcohols. The samples were then coated by sputter coater and were observed with Zeiss ULTRA 55 scanning electron microscope (SEM).
Cell Cytotoxicity Assay. Cell cytotoxicity was determined using mouse macrophage adherent cell line, RAW 264.7. 5 × 10 3 cells were seeded per well in 96 well tissue culture plate and allowed to adhere in DMEM medium containing 10% FBS for 24 h at 37 °C, 5% CO 2 . Cu Complex was added at different concentration (0, 1, 10, 50 & 100 μg/mL) in the cells in DMEM medium containing 10% FBS. After 24 h of incubation, resazurin dye was added to each well and incubated for 6-8 h at 37 °C, 5% CO 2 . The fluorescence was measured (excitation wavelength, 550 nm; emission wavelength, 590 nm) using the multimode reader. The results were expressed as percent cell viability, compared with untreated cells. Intracellular activity of Cu complex. RAW cell line 264.7 was seeded at a density of 50,000 cells per well in a 24 well tissue culture plate at 37 °C, 5% CO 2 . Macrophage cells were then infected with ATCC 29213 for 2 h at a 1:10 multiplicity of infection (macrophage to bacteria ratio). Cells were then washed with 1 × PBS twice, and gentamycin 50 μg/mL was added to each well to kill extracellular bacteria for 1 h. Cells were washed with 1 × PBS and incubated again for 24 h with different concentrations (0, 0.1 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL) of each antibiotic (oxacillin, vancomycin, linezolid and Cu Complex) at 37 °C, 5% CO 2 . After 24 h, the cells were washed twice with 1 × PBS and lysed using 0.1% saponin. The cell lysates were diluted and plated on tryptic soy agar plates, and colony-forming units (CFU) was counted. The results were expressed as % bacterial survival at different drug concentrations in comparison to control cells.  (Table 1). Antimicrobial activity was observed against methicillin resistant and sensitive (MRSA & MSSA) isolates of S. aureus, which showed MICs ranged from 6.25 to 12.5 μg/mL) (Fig. 2). Further, reduced susceptibility with a MIC of 50 μg/mL was seen against E. faecalis. However, no antibacterial activity against Gram-negative bacteria was found up to 100 μg/mL. No resistance against Cu complex was observed during continuous serial passaging with sub-inhibitory concentration for 15 days (Fig. 3C). However, resistance was developed against ofloxacin within few days of exposure. We did not find any mutant lines of S. aureus resistant to Cu complex, plated on MHA plates containing a variable concentration of Cu complex (2 × MIC, 4 × MIC & 10 × MIC).

Susceptibility profile of Clinical isolates of S. aureus and no detectable resistance against Cu
Anti-biofilm activity. Cu complex was significantly effective in disrupting the 24 h pre-formed biofilm and killing the bacterial cells in comparison with vancomycin (p < 0.05) which was revealed by a reduction in biomass percentage (Fig. 4A). Confocal imaging using SYTO-9 and PI showed the killing of biofilm cells treated with Cu complex in comparison to control (Fig. 4B). The resazurin viability assay showed 50% inhibition (Mean IC 50 ) of 24 h and 72 h old biofilms at 9.18 ± 0.16 μg/mL and 8.51 ± 0.68 μg/mL of Cu complex, respectively (Fig. 4C). There was no significant difference (p > 0.05) between Cu complex activity to kill 24 h or 72 h old biofilm.
Cell membrane permeability. Membrane permeability of S. aureus was determined by the uptake of PI using FACs. Cells treated with Cu complex and control cells with no treatment were incubated with PI and run on FACs. In treated cells, there was increased uptake of PI showing 41.7% of the cells positive for the stain as compared to 8.3% in control cells. Addition of Cu complex to S. aureus cells caused an increase in PI fluorescence, indicating that Cu complex permeabilizes the bacterial membrane (Fig. 5A). SEM images revealed prominent morphological alteration in the cells treated with Cu complex in comparison to control (Fig. 5B). In control, the cell membranes were found intact. However, Cu complex treated cells were showing numerous protrusion on its surface, damage to the cell membrane and cell lysis was observed. Intracellular Anti-S. aureus activity and cell cytotoxicity. We also assessed the antibacterial activity of Cu complex against intracellular S. aureus in RAW 264.7 cell line and compared its efficacy with different antibiotics (Oxacillin, Vancomycin, and Linezolid). Cu Complex showed a maximum bacterial reduction of 96.9% at a concentration of 10 μg/mL; however, none of the other antibiotics had shown a reduction in bacterial load of more than 77.3%. We have also observed bacterial clearance activity at 0, 0.1, 1, 5 and 10 μg/mL of Cu complex and compared with other antibiotics (Fig. 6A). Cu complex cytotoxicity was assessed using RAW 264.7 cells at variable concentrations ranging from 0 μg/mL to 100 μg/mL for 24 h. The inhibitory concentration at which 50% toxicity of RAW 264.7 cells occurred was 62.99 ± 1.48 μg/mL (Fig. 6B).

Discussion
The ability of the bacterial pathogens to develop resistance against major classes of antibiotics has placed human and animal life at risk. S. aureus is one such pathogen, which infects broad host range and causes mild to chronic infections 13,32-34 . The additional worrisome factor associated with S. aureus infection is their ability to form a biofilm, which makes them highly resistant to antimicrobials and host attack 3,13,35,36 . There is an urgent need for antibiotics, which can not only kill planktonic cells but can also eradicate biofilm. In light of the current status, preliminary studies with Cu complex showed potent antibacterial and anticancer activity as reported previously, which encouraged us to explore its activity further 22 . In this study, we have evaluated Cu complex efficacy against different forms of S. aureus infections: planktonic cells, biofilm cells, and intracellular cells.
Cu complex showed increased susceptibility against standard isolates of S. aureus ATCC 29213, ATCC 33592 and ATCC 700699. We have also determined the efficacy of Cu complex in clinical isolates of S. aureus from human (MRSA = 10, MSSA = 10) and animal (MRSA = 10, MSSA = 10) infections. The planktonic growth of all isolates was inhibited and showed a MIC ranged from 6.25 to 12.5 μg/mL.
S. aureus isolates are notorious for developing resistance against various classes of antibiotics 37 . Therefore, to be a valid treatment option, it is crucial to determine the ability of S. aureus to develop resistance against Cu complex. We were unable to find mutant of S. aureus resistant to Cu complex when plated on media containing the compound (4 × MIC and 10 × MIC). Additionally, serial passage of S. aureus for 15 days in the presence of Cu complex also failed to detect resistant mutant. These results highlight the potential of Cu complex as a treatment option.
Biofilm infections are of much bigger concern in comparison to infections caused by planktonic cells 1,[8][9][10][11][12][13]28 . In case of biofilm-associated infections, higher concentrations of drugs are required, which makes such infections extremely difficult to cure 1,7,10,13,28 . The Cu complex was also able to kill bacterial cells in 24 and 72 h pre-formed biofilms with no significant difference, which enhances its probability to be an effective antimicrobial agent. These results emphasise that Cu complex could be developed as antimicrobial agents for treating planktonic and biofilm-associated infections.
Further, S. aureus can invade and survive inside the host cells causing chronic infections. At the intracellular stage, the treatment becomes very challenging due to the difficulty of antibiotics passage through the cellular membranes. Therefore, in addition to extracellular antimicrobial activity, intracellular antimicrobial activity is also required. We evaluated the intracellular antimicrobial activity of Cu complex. We found that Cu complex was able to reduce the bacterial load to more than 95% at an intracellular level as compared to Vancomycin (75.67%), Linezolid (82.34%), and Oxacillin (74.67%) at 10 μg/mL concentration of each. Cell cytotoxicity analyses revealed 50% toxicity observed at 62.99 ± 1.48 μg/mL. However, this value is quite higher than the concentration at which   MRSA growth can be inhibited. Owing to the cytotoxicity caused by Cu complex we are also trying to modify the complex to reduce its cell toxic effect.
Overall, the Cu complex exhibit substantial antimicrobial activity against both MRSA and MSSA clinical isolates of human and animal origin. It is not only effective in killing planktonic cells but also has antimicrobial activity against biofilm and intracellular bacterial cells. Thus, these studies warrant further in-depth investigation for the development of Cu complex as an effective agent against biofilm-associated and chronic intracellular infections.