From synthesis to biological impact of palladium bis(benzimidazol-2-ylidene) complexes: Preparation, characterization, and antimicrobial and scavenging activity

: Palladium-based complexes with the ligand N-heterocyclic carbene have long received attention as active catalysts for many catalytic reactions. Recently, the biological activities of these air-and moisture-stable complexes have also been investigated. In our work, bis(benzimi-dazol-2-ylidene)palladium complexes 3a – d were synthesized by reacting benzimidazolium salts 2a – d with PdCl 2 under re ﬂ ux in tetrahydrofuran for 24 h and analyzed by spectroscopy (FT-IR [Fourier transform infrared], 1 H NMR [proton nuclear magnetic resonance]) characterization, 13 C NMR [carbon-13 (C13) nuclear magnetic resonance]), and elemental analysis. The in vitro antibacterial and anti-fungal activities of these complexes were studied against Gram-positive and Gram-negative microorganisms, and two di ﬀ erent fungi showed their remarkable biological potential. In addition, the analysis of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals using spectrophotometry showed that they are an antioxidant. The potent antibacterial and antioxidant activities of the synthetic complexes suggest that they are more e ﬀ ective antibacterial agents. Our study extends the biological relevance of palladium bis(benzimi-dazol-2-ylidene) complexes with antibacterial and antioxidant activities. Furthermore, the main goal of the docking simulation is to provide a detailed analysis of the interaction between the complex and the protein of interest


Introduction
N-Heterocyclic carbenes (NHCs) are important supporting ligands (Froese et al., 2017) with their novel biological effects (Janssen-Müller et al., 2017).The steric and electronic properties of NHCs have helped them to be at the forefront of many developments (Azouzi et al., 2018).The NHC and phosphine ligands have played a crucial role in developing organometallic chemistry.These ligand classes, combining two-electron, neutral, and σ-donor, have developed the applications of transition metal complexes (Karthik et al., 2015).Diverse approaches have been made to add a phosphine group to the NHC skeleton in synthesizing organometallic compounds, and metalation has been successfully accomplished through both functionalities.The halide ligand and phosphine molecule used in such complexes are thought to hydrolyze and facilitate the in vitro stabilization of complex ions formed by the metal reduction in the metal's coordination sphere.Phosphine-based ligands have widespread pharmacological applications, including antibacterial antioxidant, anticarcinogenic, antiviral, antifungal, and antitumor.It has been recorded that especially phosphinebased nickel(II) and palladium(II) complexes have important bioactivities.These metal-based complexes are used as valuable anticancer drugs due to their effects such as inhibiting cell division and controlling gene expression.The problem with these complexes is that they decompose into highly reactive species in solution, so they do not reach their pharmacological targets such as deoxyribonucleic acid.However, this can be avoided by stabilizing the nickel(II) and palladium(II) complexes with bulky ligands such as triphenylphosphine.In 2006, Organ et al. reported a class of precatalysts based on Pd(II) containing pyridine-derived ligands (O'Brien et al., 2006).The complexes comprise one NHC, two anions (usually Cl or Br), and stabilizing throw-away pyridine derivative ligands (Bal et al., 2021;Erdemir et al., 2020;Lu et al., 2021).Recently, new works have been reported on the antimicrobial, anticancer, antileishmanial, antitoxoplasmal, and enzyme inhibition activities of the PEPPSI-type Pd(II)NHC complexes (Daşgın et al., 2021;Türker et al., 2020).Researchers were particularly synthesized palladium(II) nonplatinum metal complexes due to their strong biological activity and better lipophilicity or solubility than cisplatin (Díez-González and Nolan, 2007;Kumar et al., 2021).Due to its structural similarities to platinum and its potential in vitro cytotoxicity, palladium metal is an excellent metallodrug option (Dindar et al., 2022;Hahn et al., 2005).It has been researched and documented elsewhere that the antiviral, antibacterial, and antifungal activity of Pd(II) complexes with various types of ligands was extensively studied by Garoufis et al. (sulfur and nitrogen donor ligands, Schiff base ligands, and different drugs as ligands) (Scattolin et al., 2021;Zhang and Yu, 2020).Other interesting works showing different intensities of palladium complex activity on various species of bacteria and fungi were studied (Blettner et al., 1999;Crozet et al., 2006;Franzén and Xu, 2005;Jia et al., 2020;Leadbeater, 2005;Moore and Shaughnessy, 2004;Popović et al., 2021;Rufino-Felipe et al., 2021;Shaughnessy, 2006;Villemin et al., 2001;Wierenga et al., 2019).In our last studies, we have published bis(NHC)Pd(II) complexes that are effective catalysts for direct arylation reactions (Boubakri et al., 2019;Slimani et al., 2022).The purpose of this article is to synthesize new palladium(II) complexes and evaluate their impact on a variety of microbes in vitro.The primary focus of this investigation is on the effects of the newly synthesized Pd(II) complexes on various microorganisms, including Gram-positive and Gram-negative bacteria and different fungi.The goal is to overcome microbial infections to some extent.Thus, we synthesized novel Pd(II) complexes containing halo substitutions and analyzed their in vitro antimicrobial, antifungal, and antioxidant activities to achieve the abovementioned objectives.To optimize metal-NHC complexes, we chose the most famous hybrid density functional theory model (B3LYP) functional, which has been shown to be highly effective in our recent research (Hassen et al., 2022).In addition, we utilized Natural Bond Orbital (NBO) analysis (Glendening et al., 2012) to determine the charge populations at specific sites and quantify the charge transfer (CT) that occurred.
imidazole-(4-(tert-butyl)benzyl) (1a) was prepared by refluxing benzimidazole with 1-(bromomethyl)-4-(tert-butyl)benzene in EtOH for 16 h.The benzimidazolium salts (2a-d) were synthesized by quaternization of imidazole-(4-(tert-butyl) benzyl) (1a), with the corresponding benzylbromide derivatives for 48 h at 70°C in degassed dimethylformamide (DMF) to produce the respective benzimidazolium salts (2a-d) with yields in the range of 50-89%.The target salts were obtained as white solids.These salts are stable in solid state and solution against air and moisture.The salts are soluble in chlorinated solvents, alcohols, and water.The reaction has been monitored following thin-layer chromatography, and after this time, the formation of salts (2a-d) has been observed for every target compound.The benzimidazolium salts (2a-d) were air-and moisture-stable both in the solid state and in solution.The FTIR spectroscopy, 1 H NMR and 13 C{1H} NMR spectroscopy, and elemental analysis data of the title compounds confirm the proposed structures.The synthesis of NHC precursors and their respective ligands is described in Scheme 1.
Bis(benzimidazol-2-ylidene)palladium complexes (3a-d) were prepared by reaction of salts (2a-d) with PdCl 2 in high yields of 75-97% (Scheme 2).The reaction was carried out at reflux in anhydrous tetrahydrofuran (THF) in the presence of potassium carbonate (K 2 CO 3 ) as a base for 24 h.Complex 3 is an air-stable solid, soluble in acetone, chloroform, dichloromethane, DMF, dimethyl sulfoxide (DMSO), ethanol, and acetonitrile but insoluble in hexane, ether, and benzene.The structural characterization of the palladium complex was confirmed by NMR, FT-IR, and elemental analysis techniques.In the 1 H NMR spectra of complexes 3a-3d, the characteristic down-field signals for the acidic C(2)-H protons of the benzimidazolium salts 2a-d disappeared in the 1 H NMR spectra of palladium complex confirming the formation of the NHC-Pd bonds.In the 13 C NMR spectrum of complexes 3a-3d, characteristic signal of C(2)-carbon of imidazolinium salts 3a-3d between δ = 142.9-162.9ppm were completely disappeared, and the characteristic Pd-C (carbene) bond signals of the complexes 3a-3d were observed as singlet.In the 13 C NMR spectra, the carbene signals of the Pd-PEPPSI-NHC complexes 3a-3e were observed at δ = 180.58, 180.81, 180.87, and 180.83 ppm, respectively.The Fourier-transform infrared spectroscopy of the palladium complexes 3a-3d unveiled a characteristic ν(CN) band, which was observed at 1,608, 1,610, 1,606, and 1,612 cm −1 , respectively.The stretching frequency of v(CN) was lower compared to the salt, which can be attributed to the electron flow from the carbene ligand toward the palladium center, causing a weak C]N bond.Moreover, the data from the elemental analysis of the palladium complexes agreed with the suggested structure.
Numerous attempts were made to obtain a suitable crystal of palladium bis(benzimidazol-2-ylidene) complexes for single-crystal X-ray diffraction study in order to provide a more detailed explanation of the structures of our complexes.The solvent diffusion method was employed using various solvent systems, including dichloromethane/diethyl ether CH 2 Cl 2 /Et 2 O, ethanol/ether EtOH/Et 2 O, and others.However, despite all attempts, a suitable single crystal for X-ray analysis could not be obtained for the palladium bis(benzimidazol-2-ylidene) complexes.All spectroscopic and analytical data acquired were consistent with those of previously reported similar palladium complexes of the same type (Slimani et al., 2022).
Both optimized cis and trans isomer structures of the 3a complex are shown in Figure 1, taking into account the spin multiplicity of palladium(II).The obtained geometrical parameters are summarized in Table 1.Computed electronic energies relative to the lowest-energy spin multiplicity state S 2 , E Δ (in kcal•mol −1 ), ε LUMO , ε HOMO , and energy gap 1 multipli- cities at B3LYP/6-311 G(d,p)/LANL2DZ theory level are summarized in Table 2.
According to the data listed in Table 1, we can conclude that complex 3a crystallizes in square planar geometry.The trans isomer for a singlet spin multiplicity is more stable than the cis one with relative energy equal to 12.4 − − Br6 Pd4 C2, and − − C7 Pd4 Br5 bond angles in the case of trans isomer are equal to 90°.Whereas, in the cis isomer, a distorted square planar geometry was found (Table 1).This deformation may be explained by the steric hindrance of aromatic rings.

CT analysis
A detailed analysis of the CTs that occur between the palladium and NHC fragment was reported since the UV absorption spectrum showed a MLCT transition.We have considered two main fragments, as shown in Figure 2, and the results are displayed in Table 4.An energy diagram showing the molecular orbital interactions and localizations of frontier molecular orbitals is presented in Figure 3.
To evaluate the CT taking place, an analysis of selected sites was performed using NBO analysis (Esrafili and Hadipour, 2011;Karthikeyan et al., 2011;Kepler et al., 2019).This analysis allowed for the determination of the electronic transitions and the extent of charge redistribution between these specific sites.By utilizing NBO analysis, insights into the nature and magnitude of the CT occurring in the system were obtained, enabling a better understanding of the electronic interactions.
According to the data summarized in Table 2, for carbine.This unit is the most efficient donor fragment because it has the highest charge transfer q CT value.Accordingly, the organic moiety (C 26 H 28 N 2 ) is the donor, while the PdCl 2 moiety is the acceptor.An important CT amount occurred from carbene to Pd(II) was obtained.This transferred charge amount was mostly attracted by both chlorides since the latters exhibit strong electron affinity.The CT was confirmed by second-order perturbation theory analysis of the Fock matrix on an NBO basis, as shown in Table 5.The larger the E(2) value, the more intensive the interaction between electron donors and electron acceptors, i.e., the more the donating tendency from electron donors to electron acceptors, the greater the extent of conjugation of the whole system (Sebastian and Sundaraganesan, 2010).The significantly higher stabilization energy value E(2) is observed in the case of intramolecular interaction for the ), and fluorescence quantum yield ) )  Table 4: Computed charge population (before the complexation q 1 , after the complexation q 2 ) and the amount of the transferred charge (q CT ) in (e) of selected sites within complex 3a at B 3   Depending on the type of ligand, the complexes exhibited different levels of antibacterial activity.Palladium bis (benzimidazol-2-ylidene) complexes show increased activity compared to benzimidazolium salts.The complexes showed increased antibacterial activity, which could be explained by the synergistic effect of the complexes to increase lipophilicity.The observed antibacterial activity of these complexes was comparable to our previous palladium bis (benzimidazol-2-ylidene) complexes (Achar et al., 2018).
The results obtained are shown in Table 6 and Figure 4.
Table 7 shows that palladium complexes 3a and 3b are the most active against M. luteus and have a zone of inhibition of 37 mm and a zone of inhibition of 29 mm.Furthermore, Table 6 shows that complex 3a show the high activity against L. monocytogenes with a zone of inhibition of 26 mm.Compound 2a was also shown to be effective against M. luteus LB 141107 with a zone of inhibition of 35 mm.Tetracycline was used as a benchmark when comparing the results obtained (Kirby and Schmidt, 1997;Sellem et al., 2016).Table 7 shows that the minimum inhibitory concentration (MIC) values of L. monocytogenes ATCC 1911 range from 0.1552 to 0.1963 μg•mL −1 , and that of Staphylococcus aureus ATCC 6538 range from 0.1526 to 1.942 μg•mL −1 .From 0.1561 to 0. 4136 μg•mL −1 , the values correspond to S. typhimurium ATCC 14028.

Molecular docking
The docking study was performed using ezCADD (Tao et al., 2019) Smina (Koes et al., 2013) software (https://www.dxulab.org/software).In order to visualize the results, BIOVIA discovery studio 2021 was used (BIOVIA Product Portfolio -BIOVIA -Dassault Systèmes®, n.d.).The salt 2a is an antibacterial activity and can act against Yersinia pestis.The simulation system was built on the crystal structure of PDB ID: 5JQ9 and 2X4M (Zhao et al., 2016), which was downloaded using the Protein Data Bank as a source (https://www.rcsb.org).The protein molecules were purified using pymol (PyMOL|Pymol.Org, n.d.).The downloaded construct is Y. pestis, a determinant of viability or virulence in every bacterial cell tested.Automatic pocket detection was performed using fpocket3 (Schmidtke et al., 2010).Minimum binding affinity (E b ) values (kcal•mol −1 ) were calculated to evaluate the resulting binding complexes.Molecular docking simulations were performed to predict the best binding configuration of a ligand for its  iiπ anion with GLU, ( ) iii − π σ with PHE, (iv) π-π stacked with TYR, (v) alkyl with ILE, and (vi)π alkyl with ALA.The obtained results allowed us to conclude that the methyl-substituted aromatic ring organic molecule electronic could play a significant role in the biological activity indicated.To investigate the impact of the metal fragment on the biological activity of complex 3a, we utilized the H-Dock server (Yan et al., 2017).The quality of the biological activity was evaluated using the LGscore, which categorizes it as follows: Correct (LGscore > 1.5), Good (3 < LGscore < 5), and Very Good (LGscore > 5).Upon analyzing Table 8, it was observed that complex 3a displayed a very good biological activity with a high LGscore.
Moreover, the data presented in Figure 8 led to the conclusion that the PdCl 2 fragment did not interact with the amino acids of Y. pestis.Similar to 2a, complex 3a exhibited conventional interactions with Y. pestis amino acids, including π-donor, π-alkyl, and π-anion interactions.
To investigate the impact of the metal fragment on the biological activity of complex 3a, we utilized the H-Dock  server (Yan et al., 2017).The quality of the biological activity was evaluated using the LGscore, which categorizes it as follows: Correct (LGscore > 1.5), Good (3 < LGscore < 5), and Very Good (LGscore > 5).Upon analyzing Table 7, it was observed that complex 3a displayed a very good biological activity with a high LGscore.
Moreover, the data presented in 8 led to the conclusion that the PdCl 2 fragment did not interact with the amino acids of Y. pestis.Similar to 2a, complex 3a exhibited conventional interactions with Y. pestis amino acids, including π-donor, π-alkyl, and π-anion interactions.

Antioxidant activity
The scavenging activities have been investigated to support the biological potential of the palladium bis(benzimidazol-2-ylidene) complexes 3a-d (Jomova et al., 2012;Meyerstein, 2013).The palladium bis(benzimidazol-2-ylidene) complexes 3a-d were used to examine the decrease in absorbance or scavenging action of a stable-free DPPH (Taha et al., 2011;Trivedi et al., 2012).Comparing the DPPH absorption at 517 nm to the Pd(II) complexes' percentage scavenging activity was done in a concentration-dependent manner (Li et al., 2006;Suh and Chaires, 1995).For Pd(II) complexes, the absorbance of the DPPH free radical at 517 nm with DMSO was 0.906.Complexes have expressed a decrease in absorption (Figure 8).

Conclusions
This study reports the synthesis of benzimidazolium salts 2a-d and their palladium bis(benzimidazol-2-ylidene) complexes 3a-d.The structures of these compounds were unambiguously characterized by standard NMR and IR spectroscopy techniques as well as elemental analysis.Palladium bis(NHC) combinations 3(a-d) and benzimidazolium salts 2a-d showed selectivity and moderate activity against a variety of microorganisms.Of particular interest are the results for Gram-positive species that are widespread in the environment.Additionally, these compounds have been shown to scavenge free radicals, suggesting they may have medicinal uses.This has enhanced the penetration of the complexes into the lipid membrane and inhibited the growth of the tested Gram-positive and Gram-negative bacteria.The latter phenomenon demonstrates the widerange activities of the complexes.

Experimental General methods
All manipulations were carried out under argon using standard Schlenk line techniques in accordance with our earlier work (Slimani et al., 2021).Chemicals and solvents were purchased from Sigma-Aldrich Co. (Poole, Dorset, UK).The solvents used were purified by distillation over the drying agents indicated and were transferred under argon.Melting points were measured in open capillary tubes using an Electrothermal-9200 melting points apparatus.IR spectra were recorded on an ATR unit in the range of 400-4,000 cm −1 with a Perkin Elmer Spectrum 100 spectrophotometer.

Synthesis of benzimidazolium salts (2a-2d)
The synthesis is carried out by reacting 1.0 g (3.42 mmol) of 1-(4-tert-butylbenzyl) benzimidazole (1a) with an equivalent molar amount of the appropriate aryl bromide or chloride and then dissolving in 2 mL of dried N,N-DMF.The solution was stirred at 70°C for 24-48 h.As a result, a product as a white powder was obtained.

Synthesis of palladium-bis(NHC) complexes 3(a-d)
The complexes considered in this work are prepared by mixing a solution of 5,6-dimethylbenzimidazolium salts (1 mmol), PdCl 2 (0.5 mmol; 0.09 g), and K 2 CO 3 (0.6 g, 4.3 mmol).Next, the anhydrous THF (25 mL) was added, and the mixture was heated at reflux for 24 h at a temperature of about 100°C.The obtained solid was solved in DCM (5 mL) and then purified by flash column chromatography.

Computational details
The ground state S 0 of the complex was optimized using DFT with hybrid B3LYP (Becke, 1993;Stephens et al., 1994) functional, combined with the 6-311G(d) basis set for the non-metal atoms and a double-ζ quality basis set LANL2DZ (Chiodo et al., 2006) for palladium.Singlet and triplet spin multiplicities were taken into account.Vibrational frequency analyses were performed to confirm that the optimized structures were a true minimum.The electronic populations were analyzed by the NBO population in order to investigate the CT.The Gaussian16A program package (Gaussian 16, Revision B.01 et al., 2016) was used to perform all calculations.We opted for the B3LYP functional due to its demonstrated effectiveness in optimizing metal-NHC complexes, as established in our recent research (Hassen et al., 2022).NBO analysis (Glendening et al., 2012) was utilized to determine the charge populations at specific sites and subsequently quantify the CT that took place between the relevant fragments in the complex.
The second-order Fock matrix was carried out to evaluate the donor-acceptor interactions in the NBO analysis.The interaction result is a loss of occupancy from the localized NBO of the idealized Lewis structure into an empty non-Lewis orbital.For each donor (i) and acceptor (j), the stabilization energy E(2) associated with the delocalization ij is estimated as follows (Dindar et al., 2022): UV-Vis electronic absorption spectrum and fluorescence were recorded on a UV-Vis Shimadzu 1650 spectrophotometer, as shown in Figure 9. Quartz cells of 10 mm in length were used.Absorption was measured in the spectrum range of 200-800 nm.The fluorescence spectrum was taken on a Jazco, FP-8200 spectrofluorometer, excitation bandwidth 5 nm, emission bandwidth 5 nm, with an Xe lamp Light source.
Fluorescence quantum yield in liquid was determined using the optically dilute solution relative method with either 9,10-diphenyleanthracene or quinine sulfate solutions, depending on the emission wavelength range.Light intensity was determined using ferrioxalate actinometry (Guven et al., 2017).Eq. 1 was applied to calculate the fluorescence quantum yields: The integrals denote the corrected fluorescence peak areas, A denotes the absorbance at the excitation wavelength, and n denotes the solvent's refractive index.The subscripts s and r indicate sample and reference, respectively.

Biological activities Antibacterial activity Microorganism Test
The biological potential of the synthesized palladium(II) complex was tested against six microorganisms.They studied their antibacterial activity against human pathogenic bacteria, both Gram-negative (E.coli; NCIM 2109 and P. aeruginosa; NCIM 2036) and Gram-positive (S. aureus; NCIM 2079 and B. subtilis; NCIM 2250) and two strains of the fungus (Candida albicans; NCIM 3471 and Aspergillus niger; NCIM 545) by the Kirby-Beurs disc diffusion method using DMSO as solvent on Mueller Hinton agar medium, the concentration is 200 µg•mL −1 .Inhibition zones were measured in millimeters (mm) after incubation for 24 h at 37°C, pH 7.4.The area of inhibition was compared with the standard drugs chloramphenicol (10 μg) and ciprofloxacin (10 μg).Discs containing only DMSO were used as positive controls.

Antioxidant Activities
The antioxidant activity of the stabilized 1-2,5-diphenyl-2-trinitrophenylhydrazine (DPPH˙) was investigated for its free radical scavenging ability.For this, the complex was mixed with a stock solution of the Pd(II) complex of DPPH( 0.002%) in DMSO + water (1:1).To prepare samples, mix DPPH˙solution with complex solution in a 1:1 ratio, shake vigorously, and incubate for 30 min in the dark.Measure the UV absorbance at 517 nm using a UV/Vis spectrophotometer and observe a decrease in DPPH˙absorbance, indicating radical scavenging activity, calculated using the following formula: AS is the absorbance of DPPH˙with the test compound, and A0 is the absorbance of DPPH˙without the test compound.Absorbance data are expressed as the mean ± standard error of triplicate determinations Acknowledgments: The authors extended their appreciation to the Researchers Supporting Project number (RSP2023R75), King Saud University, Riyadh, Saudi Arabia.

Figure 1 :
Figure 1: Structures of trans and cis isomers of complex 3a.

Figure 3 :
Figure 3: An interaction diagram for complex 3a of symmetry C 1 .

Figure 4 :
Figure 4: Inhibition zone of ligands and complexes against Gram(+) (M.luteus LB 141107, L. monocytogenes ATCC 1911, S. aureus ATCC 6538, and B. cereus ATCC 14579 and Gram(−) (S. typhimurium ATCC 14028).Antibacterial activities of salts 2a-d and their complexes 3a-d against M. luteus LB 141107.Antibacterial activities of salts 2a-d and their complexes 3a-d against L. monocytogenes ATCC 1911.Antibacterial activities of salts 2a-d and their complexes 3a-d against S. aureus ATCC 6538.Antibacterial activities of salts 2a-d and their complexes 3a-d against Salmonella typhimurium.6c 6a Antibacterial activities of salts 2a-d and their complexes 3a-d against B. cereus ATCC 14579.6a Method for determining the MIC.

Figure 7 :
Figure 7: Best position and the interactions of the 3a complex within the 5JQ9 (Yersinia pestis).

1H
NMR and 13 C NMR spectra were recorded using Bruker Avance AMX and Bruker Avance III spectrometer operating at 400 MHz ( 1 H NMR) and 100 MHz ( 13 C NMR) in CDCl 3 with TMS added.NMR multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, hept = heptet, and m = multiplet signal.The NMR studies were carried out in highquality 5 mm NMR tubes.The chemical shifts (d) are reported in ppm relative to CDCl 3 .Coupling constants (J values) are given in hertz.Elemental analyses were performed by the LECO CHNS-932 elementary chemical analyzer.The chemical shifts (d) are reported in ppm relative to CDCl 3 .Coupling constants (J values) are given in hertz.

Figure 8 :
Figure 8: The antioxidative activity of compounds 2a-d and 3a-d synthesized was assessed by DPPH and ABTS techniques and expressed as IC 50 in g•mL −1 .The BHT was used as a control.

Table 1 :
Computed a Before annihilation.b After annihilation.c When there are two lines, the first corresponds to α molecular orbital and the second to β molecular orbital.

Table 5 :
Second-order perturbation theory analysis of fock matrix in NBO basis of 3a complex

Table 6 :
Zones of bacterial inhibition measured in mm for salts 2a-d and their palladium bis(benzimidazol-2-ylidene) complexes 3a-d a Zone of bacterial inhibition measured in mm.