In vitro antibacterial and time-kill evaluation of phosphanegold(I) dithiocarbamates, R3PAu[S2CN(iPr)CH2CH2OH] for R = Ph, Cy and Et, against a broad range of Gram-positive and Gram-negative bacteria

The in vitro antibacterial activity of a series of phosphanegold(I) dithiocarbamates, R3PAu[S2CN(iPr)CH2CH2OH] where R = Ph (2), Cy (3) and Et (4), against 25 strains of Gram-positive and Gram-negative bacteria were determined through the disk diffusion method, the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and by time-kill assay. Compounds 2 and 3 have been shown to be specifically active against the tested Gram-positive bacteria, with MIC values ranging from 7.81 to 125 μg/ml. Compound 4 has a broad-spectrum activity against 24 strains of Gram-positive and Gram-negative bacteria, with MIC values ranging from 0.98 to 1,000 μg/ml. Noteworthy was that 4, with a very low MIC value of 0.98 μg/ml, is particularly effective against methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus sp., as effective as the standard antibiotic ciprofloxacin. In time-kill studies, the bacteriostatic and bactericidal activities of the tested compounds towards susceptible strains were similar to their characteristics determined by MBC/MIC ratios. In the time-kill assay, 2 and 3 showed only bactericidal activity towards the susceptible strains tested, whereas 4 revealed varying degrees of bactericidal and bacteriostatic activities, results indicating different antibacterial mechanisms are involved.


Introduction
The emergence of resistance in pathogenic bacteria to multiple antibacterial agents has become a significant public health issue as there are fewer, or even sometimes no, effective antibiotic treatments available for these infectious diseases [1]; also see recent commentaries on this issue [2,3]. Apart from patients themselves, the threat of multidrug-resistant bacteria posed towards frontline health workers has also increased [4]. Due to their natural behaviour of rapid life cycle, fast reproduction and the ability to exchange genetic information with other strains of bacteria, the chances of bacteria to develop resistance to currently available antibiotics are fairly high [5]. Hence, over and above improving health care hygiene, there is a clear imperative to develop novel antimicrobial agents needed to meet the challenges posed by the rapid emergence of multidrug-resistant pathogens. As a case in point, over time, the original Gram-positive bacterium Staphylococcus aureus developed resistance towards a series of first-line, second-line and even third-line antibiotics [6] to evolve into methicillin-resistant S. aureus (MRSA). Now, as MRSA is able to resist the beta-lactam group of antibiotics, the treatment of this highly prevalent pathogen has become an urgent challenge. As a contribution to the development of new and effective antibacterial agents, herein, synthetic gold compounds derived from phosphanegold(I) dithiocarbamate ( Fig. 1) are demonstrated to exhibit convincing effects against a broad range of pathogens, including the particularly virulent bacterium, MRSA.
Since earliest civilization, gold and its compounds have been utilized by medical practitioners to treat various healthrelated problems, and amongst these are bacterial infections [7][8][9][10]. Despite this, antibacterial studies on gold compounds are still relatively limited [10][11][12][13][14][15] and more often than not focussing on gold(I) rather on gold(III) compounds. The primary focus of these studies is usually upon the inherent interest in the chemistry and consequently the accompanying antimicrobial work is often limited to the measurement of minimum inhibitory concentration (MIC) and sometimes minimum bactericidal concentration (MBC). One limitation of these studies is the inability of the method(s) to determine the kinetics of interaction between the putative antibacterial agents and the bacteria under investigation. In the present study, the MIC and MBC scores of some phosphanegold(I) dithiocarbamates (Fig. 1) against a wide range of Grampositive and Gram-negative bacteria are determined. This is augmented by an assessment of the killing kinetics determined by a time-kill assay, therefore enhancing the understanding of the pharmaco-dynamic relationships between the phosphanegold(I) dithiocarbamates and their effects on bacteria.
Reflecting the increasing interest in the antimicrobial activity of gold compounds, very recently, a comprehensive review of the developments in this field appeared [16]. While studies have been reported on phosphanegold(I) monofunctional thiolate compounds, none have yet appeared for bi-functional dithiolate analogues, such as dithiocarbamate. This is perhaps a little surprising owing to the substantial and ongoing efforts investigating the anti-tumour potential of gold dithiocarbamates. Thus, directly related to the compounds shown in Fig. 1, i.e. phosphanegold(I) dithiocarbamates, several studies focussing on cytotoxicity profiles and mechanisms of cell death have appeared [17][18][19]. Even more studies of gold(III) dithiocarbamates are available as these potent compounds exhibit in vivo potential, have limited nephrotoxicity and a different mechanism of action to the widely used anti-cancer drug cisplatin, (NH 3 ) 2 PtCl 2 [20][21][22][23][24]. Herein, we redress this shortcoming in the gold/antimicrobial literature by reporting the exciting antibacterial activity of 2-4, as outlined above.

Chemistry
The R 3 PAu[S 2 CN(iPr)CH 2 CH 2 OH] compounds, where R = Ph (2), Cy (3) and Et (4), were prepared from the reaction of t h e r e s p e c t i v e R 3 PA u C l p r e c u r s o r w i t h t h e Na[S 2 CN(iPr)CH 2 CH 2 OH] salt as described in the literature [19]. The compounds exhibited the reported spectroscopic attributes (IR, 1 H and 13 C{1H} NMR). High-energy absorptions (CHCl 3 solution) ascribed to intraligand (IL) dithiocarbamate transitions [25] were noted in the UV-vis spectra run on an Agilent Cary 60 UV-vis spectrophotometer, see Supplementary Materials Table S1 for data. Photoluminescence (PL) measurements were performed on an Agilent Varian Cary Eclipse Fluorescence Spectrophotometer using a Xenon flash lamp as the excitation source at room temperature, also in CHCl 3 solution; see Supplementary Materials Table S1 for details. In the case of 3 and 4, the powder X-ray diffraction patterns recorded on a PANalytical Empyrean XRD system with Cu-Κα1 radiation (λ=1.54056 Å) in the 2θ range 5 to 40°with a step size of 0.026°were consistent with the simulated patterns calculated using the single crystal data using X'Pert HighScore Plus [ McFarland standard turbidity (corresponding to approximately 10 8 CFU/ml) by adding Mueller-Hinton broth. This suspension was then swabbed on the surface of Mueller-Hinton agar (MHA) plates using a sterile cotton swab. The test compounds were dissolved in DMSO to achieve a test concentration of 2 mg/ml. Sterile 6-mm filter paper discs were aseptically placed on MHA surfaces and 5 μl of the dissolved test compound was immediately added to the discs. Each plate contained one standard antibiotic paper disc, serving as the positive control, one disc served as negative control (5 μl broth) and one disc served as solvent control (5 μl DMSO). The plates were incubated at 37°C for 24 h. Antibacterial activity was evaluated by measuring the diameter of inhibition zone against each bacterial strains. Each experiment was performed in duplicate.

Determination of minimum inhibitory concentration and minimum bactericidal concentration
A broth micro-dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values according to the CLSI guidelines. The test compounds were serially twofold diluted in DMSO to achieve the range of test concentrations of 2,000-0.06 μg/ml and then placed into each well of a 96-well microplate. An inoculum suspension with density of 10 5 CFU/ml exponentially growing bacterial cells was added into each well. The 96-well microplates were incubated at The diameter of inhibition zones in millimetres (mm) were measured around the disc after 24 h incubation; −, no zone of inhibition     ; the salt was non-cytotoxic. The motivation for the present study arose from recent observations of remarkable antimicrobial activities, specifically against Gram-positive bacteria, exhibited by related phosphanegold(I) thiocarbamate compounds, i.e. Ph 3 PAu[SC(OR)=N(tol-p)], for R = Me, Et and iPr [29], and by the knowledge that other metal dithiocarbamates have been reported to exhibit antimicrobial activity [30][31][32]. Crystal structure analysis on 3 and 4 [19] proved linear P-Au-S coordination geometries with the second sulphur atom oriented towards gold as indicated in Fig. 1. While the structure of 2 remains unverified, literature precedents suggest a similar coordination arrangement [33,34]. 1 H NMR spectra run in DMSO solution after 24 h were unchanged compared with freshly prepared solutions proving the stability of 2-4 in the time-frame of the biological studies. The gold compounds are insoluble in water.

Anti-bacterial activity
The antibacterial properties of 2-4 along with the dithiocarbamate salt 1 were evaluated against both Gram-positive and Gram-negative bacteria using the Kirby-Bauer disk diffusion method. According to the results collected in Table 1, 2-4 were specifically effective against all tested Gram-positive bacteria but against not Gram-negative bacteria. By contrast, 4 was the most active compound with significant inhibitory activity towards all the tested Gram-positive and Gramnegative pathogens except P. aeruginosa. This finding indicates that 2 and 3 has a similar inhibitory mechanism of action towards Gram-positive bacteria only, whereas 4 possesses wider spectrum of inhibitory activity against both Grampositive and Gram-negative bacteria that is similar to that exhibited by the standard antibiotic ciprofloxacin.
The antibacterial activity of 1-4 were quantitatively assessed by determining their minimum inhibitory concentration (MIC) values and the results are tabulated in Table 2; a lower MIC value indicates a better antimicrobial agent as less compound is required to inhibit growth of the bacteria. The MIC values of compounds 2-4 were in the range 0.98-2,000.00 μg/ml, whereas ciprofloxacin was active in the range of 0.06-125.00 μg/ml, tetracycline in the range of 1.95-250.00 μg/ml and chloramphenicol in the range of 62.50-250.00 μg/ml towards susceptible tested bacteria. Salt 1 exhibited low activity and only shows inhibition against B. subtilis, L. monocytogenes and S. pyogenes, with high MIC values of 2,000.00, 2,000.00 and 500.00 μg/ml, respectively. This result points to the importance of phosphine gold in imparting antibacterial activity.
Compounds 2 and 3 were effective in inhibiting the growth of all tested Gram-positive bacteria with MIC values in the range of 7.81-62.5 and 31.25-125.00 μg/ml, respectively. As shown in Table 2, the antibiotic ciprofloxacin used as standard drug was more potent than the tested compounds 2 and 3 against Gram-positive bacteria at low concentration (MIC= 0.98-125.00 μg/ml) with the exception against S. pyogenes where it was less active compared with 2. However, standard drugs tetracycline and chloramphenicol were less potent than 2 and 3 against Gram-positive bacteria at higher concentration (MIC = 125.00-250.00 μg/ml) with exception against B. subtilis and S. saprophyticus. Interestingly, 4 displayed excellent inhibitory activity towards Gram-positive bacteria, with lower MIC values in the range 0.98-3.91 μg/ml, compared with 2 and 3, and the standard drugs ciprofloxacin, tetracycline and chloramphenicol,. In addition, 4, with MIC values in the range of 15.63-1,000.00 μg/ml, also showed moderate antibacterial activity against Gram-negative bacteria. The standard drugs ciprofloxacin, tetracycline and chloramphenicol showed remarkable high activity against Gramnegative bacteria (MIC=0.06-250.00 μg/ml) compared with 4 with the exception of chloramphenicol against P. vulgaris.
The bactericidal properties of 2-4 against susceptible strains (i.e. excluding P. aeruginosa) were analysed by the minimum bactericidal concentration (MBC) assay and summarized as MBC/MIC ratios in Table 2. An antimicrobial agent is considered bactericidal if the MBC is not more than  [40]. Compounds 1-3 were shown to be bactericidal (MBC/MIC≤2) towards the susceptible Gram-positive strains with exception of 3, against E. faecium, with the MBC being eightfold higher than the MIC indicating bacteriostatic character. For 4, bactericidal activity was observed positive against B. cereus, B. subtilis, L. monocytogenes, S. saprophyticus, S. pyogenes, A. hydrophilla, S. paratyphi A, S. flexneri, S. Sonnei, P. mirabilis and V. parahaemolyticus, whereas bacteriostatic activity on E. faecalis, E. faecium, MRSA, S. aureus, A. baumannii, C. freundii, E. aerogenes, E. cloacae, E. coli, K. pneumonia, S. typhimurium, S. maltophilia and P. vulgaris was indicated. These results suggest that the bacteriostatic and bactericidal activities of 2-4 are dependent on the bacterial strain. This behaviour is similar to standard antibiotic ciprofloxacin, which is classified primarily as a bactericidal drug, so that the MBC/MIC≤4 might have been predicted. Ciprofloxacin has been shown to kill bacteria by binding their DNA Gyrase subunit which causes inhibition of bacteria DNA replication [41]. However, the MBC values of ciprofloxacin towards E. faecium (MBC/MIC = 8), E. aerogenes (32), S. paratyphi A (8), S. maltophilia (64) and P. mirabilis (8) were fourfold, or more, higher than the MIC indicating bacteriostatic activity. The present findings confirm the conclusions of earlier studies [42,43], where it was shown that the antibacterial agent vancomycin is generally bactericidal against S. aureus and pneumococci, but bacteriostatic against enterococci.
Time-kill assays have been widely used for in vitro investigations of new antimicrobial agents as these provide descriptive (qualitative) information on the pharmacodynamics of antimicrobial agents [44]. In the present study, only gold compounds with high activity towards susceptible bacteria strains, i.e. with MIC<100 μg/ml, were selected for time-kill studies. The kinetic interaction between susceptible bacteria and 2-4 were examined at concentrations of two times the MIC (2x MIC), MIC and one-half of the MIC (½x MIC).
The kill kinetic profiles of 2 and 3 (Figs. 2 and 3) displayed rapid bactericidal activity towards all susceptible strains, showing a ≥3log 10 reduction in viable cell count relative to the initial inoculum at all tested concentrations after 1 h exposure ( Table 3). As expected from the determined MBC/ MIC ratios, the time-kill assays for 2 towards B. cereus, B. subtilis, E. faecalis, E. faecium, L. monocytogenes, MRSA, S. aureus, S. saprophyticus and S. pyogenes were consistent with bactericidal characteristic. A similar conclusion is apparent for 3 towards B. cereus, B. subtilis, L. monocytogenes, MRSA and S. pyogenes.
The kill kinetic profiles of 4, shown in Fig. 4, exhibited varying degrees of bactericidal and bacteriostatic activities depending on the tested strains and concentrations. After 1 h and at all concentrations tested, Table 3, 4 had a similar killing rate as 2 and 3 against B. cereus, B. subtilis and S. pyogenes.
The killing rate of 4 was slower than 2 and 3 against E. faecalis, E. faecium, L. monocytogenes and MRSA in which bactericidal activities were only seen after 3 h interaction at 2x MIC. Compared to 2, 4 exhibited a slower killing rate against S. aureus and S. saprophyticus, showing bactericidal activity only after 3 h (2x MIC) and 4 h (½x MIC), respectively. In summary and consistent with the MBC/MIC ratios (Table 2), at its MIC value, 4 was found to be bactericidal towards B. cereus, B. subtilis, L. monocytogenes, S. saprophyticus and S. pyogenes after 24 h exposure. On the other hand, at its MIC value 4 is bacteriostatic towards E. faecalis, E. faecium, MRSA and S. aureus, as well as the Gram-negative bacteria E. coli and P. vulgaris.
Aggressive bactericidal activities for 4 can be achieved at concentrations higher than MIC, e.g. 2x MIC, over 24 h with E. faecalis, E. faecium, MRSA, S. aureus and E. coli. At its MIC value, 4 showed bacteriostatic activity against P. vulgaris (Fig. 4k) after 4 h contact but the strain regrew to the same level as the control inoculum after 24 h. Similarly, regrowth occurred in E. coli after 4 h of exposure to 4 at MIC and ½x MIC. This regrowth incidence was not found with the other strains tested, but is common in studies of bacterial killing rate with antimicrobial agents in time-kill assays [45]. The regrowth phenomenon was attributed to two distinct subpopulations with different susceptibility in which the selective growing of resistant sub-population take over the preferential killing of the susceptible sub-population at a specified time of interaction [44].
In order to place the time-kill assays determined for 2-4 in context, some observations from the literature are made. Vidaillac et al. [46] demonstrated that oritavancin exhibited rapid bactericidal activity against MRSA after 9 h exposure. In the present study, the kill kinetic profiles of 2 and 3 displayed much more rapid bactericidal activity, i.e. within 1 h, toward MRSA and other susceptible pathogens compared to oritavancin. Furthermore, the kill kinetic profiles of 4 exhibited both bactericidal and bacteriostatic activities depending on the tested strains and concentrations. The behaviour is similar to that exhibited by various oxazolidinone derivatives which demonstrated bacteriostatic effects toward Staphylococcus spp. and Enterococcus spp. but a bactericidal effect toward Streptococcus spp. [47].
In conclusion, three active gold compounds possess potent and differential activity against Gram-positive and Gramnegative bacteria pathogens, including the MRSA strain, which is often multi-resistant to several classes of antibiotics and can cause severe hospital-acquired and communityacquired infections. With rapid bactericidal activity against Gram-positive bacteria, 2 and 3 could provide clinical benefits over bacteriostatic therapy in neutropenia by rapid elimination of a bacterial pathogen and thereby reduce the likelihood of the spread of infection. With low MIC values, 4 could serve as a potential broad-spectrum antibacterial agent against Gram-positive and Gram-negative bacterial infections. The time-kill studies have provided valuable information on the rate, concentration and potential action of antibacterial agents in vitro. As the antibacterial activities and bacterial killing rates of 2-4 were different from each other, it is likely that different mechanisms are involved. Further investigation is needed to determine the mechanism(s) of action of these compounds in order to strengthen their potential as therapeutic antibiotics. Further derivatives, i.e. by varying both phosphane-and dithiocarbamate-substituents, will also be developed in a structure-activity study. In particular, of the present series, 4, with its potent and specific antibacterial profile, is deserving of further investigation and in vivo studies are planned.