Antibacterial and anti-biofilm activity of quinazolinone derived Schiff base and its Cu(II) complex

According to the last WHO report, the level of bacterial resistance is alarming all over the world. Drug resistant diseases cause at least 700 000 deaths a year [1]. The synthesis of new antibacterial compounds is an important step to overcome this problem.
Shiff bases are a privileged structure possessing a wide spectrum of pharmacological activities such as anticancer, antioxidant and antibacterial [2]. The synthesis of 3‑[(2‑hydroxy‑5‑nitrobenzylidene)amino]‑2‑(2‑hydroxy‑5‑nitrophenyl)‑2,3‑dihydroquinazolin‑4(1H)‑one was described, and its spectral analysis, antioxidant and cytotoxic activities were presented [3]. The aim of this work was to study the antibacterial activity of this compound and its Cu(II) complex [4] against a spectrum of bacterial strains as well as the eradication activity against S. aureus biofilm.
The compounds showed moderate antibacterial activity, which was slightly higher in the case of Cu(II) complex. The dynamics of activity against S. aureus was analyzed using the time-kill curve method, and the activity was considered as bacteriostatic. Minimum biofilm eradication activity (MBEC90) against staphylococcal biofilm was only twofold higher than MIC against planktonic cells, which shows a great potential of these derivatives as anti-biofilm active drugs.
 
This study was supported by the Ministry of Education of the Czech Republic (LO1305) and by project VEGA 2/0022/18.
 
References:

No time to wait–securing the future from drug-resistant infections. Report to the Secretary General of the Nations, 2019, https://www.who.int/docs/default-source/documents/no-time-to-wait-securing-the-future-from-drug-resistant-infections-en.pdf?sfvrsn=5b424d7_6.
 Sztanke, K.; Maziarka, A.; Osinka, A.; Sztanke, M. An insight into synthetic Schiff bases revealing antiproliferative activities in vitro. Med. Chem. 2013, 21(13):2648–3666.
Hricoviniova, Z. Hricovini, M.; Kozics, K. New series of quinazolinone derived Schiff’s bases: synthesis, spectroscopic properties and evaluation of their antioxidant and cytotoxic activity. Pap. 2018, 72(4): 1041–1053.
Hricoviniova, Z. et al. manuscript in preparation


Introduction
• Resistant bacteria are still an important global problem, which causes at least 700 000 deaths per year.
• Treating multi-resistant infections is not only more complicated, but also more expensive.
• Development of new active and safe antibacterial agents is necessary to overcome the risk of losing therapeutic perspectives for treating serious lifethreatening infections.
• Schiff bases are a group of compounds with wide spectrum of activity such as antibacterial, anticancer or antioxidant. Due to their structure they are able to serve as ligands in metal complexes, which usually have higher activity than ligands alone.

Methods
• Minimum inhibitory concentrations (MICs) were tested by broth dilution method in microtitration plates. After incubation, the lowest concentration which inhibited growing od bacteria was evaluated visually. MICs against M. tuberculosis were examined using resazurin • Following incubation, sub-cultivation of aliquots onto agar plates was used to assess minimum bactericidal concentration (MBCs) • Method of time-kill curves was used to study dynamics of antibacterial activity against S. aureus ATCC 29213.
• Formed biofilm cultivated for 48 hours in tryptic soy broth with 2% of glucose was treated by different concentration of tested compound for 24 hours. Minimum biofilm eradication concentration (MBEC 90 ) was counted as the lowest concentration of the compound which increased viability of the cells by 90% compared to the growth control. Viability was measured by MTT method. Antibacterial activity of tested compounds

Results and discussion
Antibacterial activity of tested compounds • The compounds showed good antibacterial activity against staphylococci including methicillin resistant isolates.
• Antimycobacterial activity was higher in the case of slowly growing mycobacteria as M. marinum and M. tuberculosis. Differences among the strains could be caused by differences in their cell wall components.
• The activity of Cu(II) complex was slightly higher than the activity of the ligand.
• Enterococcus was less sensitive than staphylococci, which can be caused its higher intrinsic resistance to antibacterial agents in general.

Dynamics of antibacterial activity
Graph 1: Dynamics of antibacterial activity of both tested compounds against S. aureus ATCC 29213.

Dynamics of antibacterial activity
• All of the compounds demonstrated only bacteriostatic activity, because the decrease of CFU/mL in all times and concentrations was < 3log compared to the time 0.
• Results were analysed using two-way ANOVA following Tukey test . At P = 0.05, the only statistically significant difference in activity was observed between ligand in concentration 1 MIC /24 hours and complex 2-4 MIC/24 hours.
• Thus overall, the difference in bactericidal activity among different concentration of ligands and complexes in different times is not statistically significant.

Results and discussion
• The compounds showed very good activity against pre-formed staphylococcal biofilm, when the MBEC 90 was only 2-fold higher than MIC against planktonic cells.
• In concentration equal to MICs the anti-biofilm activity of Cu(II) complex was only slightly higher than activity of the ligand ( 87.3 ± 4% vs. 81.7 ± 13.7%).
• In the concentration equal to ½ MIC, the Cu(II) complex reduced viability of the film by 66.4 ± 10.6%, but the ligand did not have any effect.
• Cu(II) complex is an interesting compound for deeper research in the field of antibiofilm active agents.
• Quinazolinone -based Schiff base and its Cu(II) complex were tested against a spectrum of bacterial pathogens, as well as against staphylococcal biofilm.
• The compounds showed good activity against staphylococci, which was defined as bacteriostatic using time-kill method.
• Anti-biofilm activity of these compounds is very promising, because the concentration needed to eradicate 90% of matured staphylococcal biofilm was only 2-fold higher than MICs against planktonic cells.
• The compounds are very perspective antibacterial agents and their features should be analysed deeper in following studies.