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Antibacterial activity and time kill kinetics of Amlodipine, Thioridazine and Promethazine against pathogenic clinical bacterial isolates

Background: The emergence of multi-drug resistant bacterial strains worldwide has necessitated the scientific search for novel, potent, and affordable antimicrobial agents including medicinal plants and non-antibiotic drugs for therapy of infectious diseases. The objective of this study is to assess in vitro antibacterial activities and time kill kinetics of some non-antibiotic drugs against pathogenic clinical bacterial isolates. Methodology: In vitro antibacterial activities including minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill kinetics of Amlodipine (AML), Thioridazine (THI) and Promethazine (PRO) against Staphylococcus aureus, coagulase negative staphylococci (CoNS), Streptococcus spp, Escherichia coli, Enterobacter spp, Klebsiella pneumoniae and Pseudomonas aeruginosa clinical isolates were determined using disc diffusion, broth microdilution and plate count techniques. Results: The mean growth inhibition zones by the disc diffusion assay of AML, THI and PRO against the isolates were ≤15.1±1.0 mm with MIC and MBC values ranging from 12.5 to 50μg/ml and 25 to 100μg/ml respectively. The time-kill assay revealed bactericidal effect of AML, THI and PRO on Gram positive bacteria evidenced by mean log reductions in viable bacterial cell counts ranging from 0.13 Log10 to 2.41 Log10 CFU/ml for S. aureus, 0.88 Log10 to 2.08 Log10 CFU/ml for Streptococcus spp, and 0.26 Log10 to 2.34 Log10 CFU/ml for CoNS after ≤30hrs post inoculation at 1xMIC. The range of log reduction in viable cell counts of Gram-negative bacteria exposed to AML, THI and PRO were E. coli (0.11 to 3.23 Log10 CFU/ml), P. aeruginosa (0.52 to 2.56 Log10 CFU/ml), K. pneumoniae (0.85 to 3.0 Log10 CFU/ml) and Enterobacter spp (0.38 to 2.08 Log10 CFU/ml) after ≤30 hrs post inoculation at 1x MIC. Conclusion: These findings demonstrate in vitro antibacterial efficacies and time kill kinetics of AML, THI and PRO against pathogenic clinical bacterial isolates, which indicate that these non-antibiotic drugs may be useful therapeutic alternatives in the bid to reduce the burden of infectious diseases associated with antibiotic resistant pathogens.


Introduction:
Although the pharmaceutical industries are still in the business of producing new antibiotics for treatment of infectious diseases, the numbers of infections caused by resistant microorganisms continue to increase owing to combinations of microbial characteristics and selective pressure created by antimicrobial use (1,2). The mechanisms of microbial resistance to antimicrobial agents include the production of structure-inactivating enzymes, alteration of cell wall, cell membrane and enzyme (e.g. DNA gyrase) target sites and ribosomal modification (3). The emergence of multi-drug resistant (MDR) microbial strains has necessitated the scientific search for potent, novel and affordable antimicrobial agents including plants (4,5) and non-antibiotic drugs for the treatment of infectious diseases.
Promethazine is usually administered to attenuate inflammatory process in disease conditions such as allergic rhinitis, conjunctivitis, and urticaria (12,13). The use of antihistamines, especially the first-generation phenothiazine H1-inhibitors such as promethazine for patients with microbial infection may become inevitable and this has prompted the evaluation of their antimicrobial properties.
The antibacterial activity of amlodipine, a cardiovascular drug, was reported by Kumar et al., (8) on several clinical bacterial isolates including Staphylococcus aureus, Escherichia coli, Vibrio spp, Salmonella spp, Bacillus spp and Pseudomonas aeruginosa. Similarly, in addition to the antipsychotic properties of thioridazine, significant antimicrobial activities against intracellular microorganisms have been reported (14). The objective of this study is to determine the antibacterial activities and time kill kinetics of amlodipine, thioridazine and promethazine against selected pathogenic clinical bacterial isolates.

Source of the non-antibiotic drugs (Amlodipine, Thioridazine and Promethazine)
Amlodipine (AML, MAF India Pharmaceutical, India), Thioridazine (THI, Indian Generic Company, India) and Promethazine (PRO, CSC Pharmaceuticals International, India) were purchased in tablet forms from registered Pharmacy stores in Uyo. Stock solution of each drug was prepared by dissolving 1g of each in 100ml of sterile distilled water to give a concentration of 10mg/ml. The stock solution was further diluted to produce 5mg/ml and 2.5 mg/ml concentrations for each of AML, THI and PRO.

Determination of antibacterial activity of the non-antibiotic drugs by disc diffusion assay
The antibacterial activities of AML, THI and PRO on the clinical bacterial isolates were determined by the disc diffusion method (16). Paper discs of AML, THI and PRO were prepared by punching sterile Whatman filter paper (No.1) with a 6 mm disc puncher and sterilizing them at 160°C for one hour. Each disc was then impregnated with 10μL of 2.5mg/ml and 5.0mg/ml concentrations of AML, THI and PRO to give disc contents of 25µg and 50µg respectively.
Mueller-Hinton agar (MHA) plates were aseptically prepared and 0.1ml of each bacterial inoculum, prepared directly from an overnight nutrient agar plate and adjusted to 0.5 McFarland standards, was inoculated using sterile pipette onto each of the MHA plate. The impregnated discs were carefully placed on the MHA plates and incubated aerobically at 37 o C for 24 hours followed by measurement of the diameters of zone of inhibition (in millimetres) using a calibrated ruler. The disc diffusion assays were performed in triplicates and the mean diameters of zones of inhibition were recorded.

Determination of minimum inhibitory (MIC) and minimum bactericidal concentration (MBC)
The MICs of AML, THI and PRO against the bacterial isolates were determined using macrobroth dilution technique in test tubes (5). One ml of the stock solution (10mg/ml) of AML, THI and PRO was serially diluted in test tubes to obtain concentrations of 5, 2.5, 1.25 and 0.625mg/ml for each of the non-antibiotic solution. To 0.1ml (100µL) of each of these concentrations was added 9.9ml nutrient broth (1 in 100 dilution) to give the final concentrations of AML, THI and PRO of 100, 50, 25, 12.5 and 6.25µg/ml for the MIC testing. A loopful of prepared inoculum of each bacterium was added to each of the non-antibiotic solution. A tube containing only nutrient broth was inoculated with bacterial isolate to serve as control. All culture tubes were incubated aerobically at 37 o C for 24 hours, after which the tubes were examined for microbial growth by observing for turbidity. The MIC was read as the least concentration of the non-antibiotic solution that visibly inhibited the growth of the test bacterial isolate after 24 hours incubation.
For the MBC, an aliquot of 1ml from each of the MIC broth tubes that showed no visible growth was streak-inoculated onto sterile nutrient agar plates using a sterile pipette. The inoculated plates were inverted and incubated at 37°C aerobically for 24 hours. The MBC values of AML, THI and PRO were considered as the concentration of each nonantibiotic solution that resulted in killing of the bacterial isolates, which showed as no visible colonies on the agar plates.

Time-kill analysis
The time-kill analysis was carried out using macrobroth dilution and pour plate techniques. An overnight nutrient broth culture of each bacterial isolate in test tube was adjusted to 0.5 McFarland turbidity standard to obtain a starting inoculum of between 10 5 and 10 6 CFU/ml (confirmed by quantitative plate counts). The tubes were incubated at 37 o C with shaken at 150 rpm for 90 mins to ensure that microbial growths were in the logarithmic (exponential) phase. One (1) millilitre of this exponentially growing bacterial cultures was added to 9ml of MHB containing MIC concentration (1ml) of AML, THI and PRO. Bacterial growth was quantified at time '0' hour and at 6,12,18,24, and 30 hours of incubation by aseptically taking 1ml of the aliquot, diluting serially (10-fold dilutions) in sterile normal saline and plating out 1ml of the final dilution onto nutrient agar plates. The plates were incubated aerobically for 24 hours at 37 o C after which the colonies on each plate were enumerated and viable cells expressed as CFU/ml. Inoculated medium without AML, THI and PRO was also set up and plated on nutrient agar as control. All experiments were performed in triplicates.
The percentage and logarithm reductions of the bacterial cells exposed to AML, THI and PRO were calculated for each of the time intervals. The Log10 CFU/ml of survived bacterial cells against exposure time (in hours) were plotted on a semi-logarithm graph for each bacterial pathogen to obtain the time-kill curve. Activity of the non-antibiotic drugs was considered bactericidal at the lowest concentration that reduced the initial inoculum by >3Log10 CFU/ml (99.9% killing). The percentage and logarithm reductions of the bacterial cells exposed to AML, THI and PRO were respectively calculated as; percentage reduction = (initial counts -counts at 'x' interval)/ initial counts (multiply by 100), while the logarithm reduction = Log10 (initial counts) -Log10 (counts at 'x' interval).

Statistical analysis
All experiments were performed in triplicates and data analysis was done with the Statistical Package for the Social Sciences (SPSS) version 20.0. Data were presented as mean ± SD and comparison between mean values was done using the Duncan multiple range test, with significance level at p<0.05.
The AML, THI and PRO inhibited the growth of all the 14 isolates tested with MIC values in the range of 12.5 -50 µg/ml, 12.5 -50µg/ml and 12.5 -˃50µg/ml respectively. The MIC values of THI were lowest for S. aureus, E. coli, K. pneumoniae, Enterobacter spp, P. aeruginosa and CoNS. The MBC values of AML, THI and PRO were in the range of 25 -100 µg/ ml. The MBC of AML for 78.6% of the bacterial isolates tested was 50 µg/ml, the MBC of THI for 35.7% of bacterial isolates tested was 25 µg/ml while the MBC of PRO for 7.1% of the bacterial isolates tested was 100 µg/ml ( Table  2). The results also indicated that both MIC and MBC end points obtained by visual reading for AML on CoNS and K. pneumoniae were equal.
The bactericidal activity was deemed to be present if there was a ≥ 99.9% reduction in survival from the original inoculum (≥99.9% killing). Table 3 showed the percentage and log reductions in GPB cells exposed to AML, THI and PRO at 6 hours intervals after incubation. The percentage and log reduction in viable cell counts of S. aureus exposed to AML ranged from 33.3 to ≥ 99.9% and 0.18 to 2.41 Log10 CFU/ml after 30 hrs of interaction respectively while the percentage and log reduction in viable cell counts of Streptococcus spp exposed to AML ranged from 86.96 to ≥ 99.9% and 0.88 to 2.08 Log10 CFU/ml after 30 hours of interaction respectively.
The lowest percentage and logarithm reduction in viable cell count of CoNS exposed to AML was ≥ 58.6% and 0.38 Log10 CFU/ml respectively. The log reduction in viable cell counts of S. aureus, Streptococcus spp and CoNS exposed to PRO for 30 hrs ranged from 0.13 to 1.54 Log10 CFU/ml, 0.26 to 2.08 Log10 CFU/ml and 0.26 to 2.20 Log10 CFU/ml respectively (Table 3). At 1xMIC, THI achieved bactericidal effects on S. aureus, Streptococcus spp, E. coli, K. pneumoniae and P. aeruginosa at 24 hours post inoculation while ≥ 99.9% reduction in survival from the original inoculum was achieved for CoNS and Enterobacter spp at 30 hours post inoculation (Tables 3 and 4). The ranges of log reduction in viable cell counts of GNB exposed to AML for 30 hrs were E. coli (0.7 to 2.30 Log10 CFU/ml), Enterobacter spp (0.38 to 2.0 Log10 CFU/ml), K. pneumoniae (0.85 to 2.23 Log10 CFU/ml) and P. aeruginosa 0.56 to 2.3 Log10 CFU/ml) ( Table 4). The bactericidal activity of PRO on K. pneumoniae and P. aeruginosa was achieved at 18-and 30-hours post inoculation respectively whereas 1.0 x 10 2 CFU/ml Enterobacter spp were still viable at 30 hours post inoculation ( Table 4) The increase in viable cell counts of the GPB and GNB not exposed to AML, THI and PRO within the 30 hours of incubation were observed and presented in Tables 5 and 6. The viable cell count of S. aureus increased from 5.57 to 7.79 Log10 CFU/ml, those of Streptococcus spp cells increased from 5.61 to 7.85 Log10 CFU/ml while those of CoNS increased from 5.61 to 7.85 Log10 CFU/ml ( Table 5). The increase in the viable cell count of GNB within the 30 hours incubation period was 5.60 to 7.84 Log10 CFU/ml for E coli, 5.41 to 7.60 Log10 CFU/ml for Enterobacter spp, 5.58 to 7.72 Log10 CFU/ml for K. pneumoniae and 5.76 to 7.86 Log10 CFU/ml for P. aeruginosa (Table  6). The global emergence of multi-drug resistant pathogens as well as the continuing challenge of infectious diseases have necessitated the scientific exploration of novel, potent and affordable antimicrobial agents such as medicinal plants (5) and non-antibiotic drugs (6) in the management of infectious diseases. In this study, amlodipine (AML) demonstrated higher growth inhibitory activities at 5mg/ml concentration (50µg disk) against the GPB (S. aureus, CoNS, Streptococcus spp) and GNB (E. coli, Enterobacter spp, K. pneumoniae and P. aeruginosa) than at 2.5mg/ml concentration (25µg), indicating a concentration-dependent inhibition of bacterial growth.
The antibacterial activity of AML in this study substantiates the findings of Kumar et al., (8) that AML, a calcium channel blocker and cardiovascular drug, exhibited antibacterial activities against S. aureus, E. coli and P. aeruginosa. The antibacterial activities of AML on both GPB and GNB indicated its broadspectrum activity and these findings concurs with the reports of Mazumdar et al., (17) and Pereira et al., (18), which reported broadspectrum antibacterial activities of AML. The pattern of time-kill kinetics, investigated by the microbroth kinetic growth assay, against the GPB and GNB showed that AML exhibited bactericidal activities (≥ 99.9% reduction in survival from the original inoculum) within 30 hours of exposure against the bacteria. These bactericidal activities of AML against the isolates in this study confirmed the findings previously reported by Kumar et al., (8) and Mazumdar et al., (17).
The MBC values of THI for the isolates ranged between 25 and 100µg/ml with bactericidal effects on S. aureus, Streptococcus spp, E. coli, K. pneumoniae and P. aeruginosa at ≤ 24 hours post inoculation. The antibacterial activities of THI on GPB and GNB in this study corroborates the findings of Radhakrishnan et al., (19) on the potentiality of THI as an effective antibacterial agent. Our findings on antibacterial efficacy of THI on P. aeruginosa was also in conformity with a previous study by Mukherjee et al., (20), who reported that THI, an anti-psychotic drug, was a highly promising agent in the treatment of acute infections caused by P. aeruginosa. Studies have shown that THI exerts a bactericidal effect on bacteria by damaging the cell wall and causing major changes in expression of many genes involved in peptidoglycan biosynthesis (21). THI has also been reported to reverse antibiotic resistance by facilitating the elimination of resistance (R) plasmid from MDR bacteria (14,19).
In this study, PRO also exhibited antibacterial activities on both GPB and GNB, with log reduction in viable cell counts of GPB exposed for 30 hours ranging from 0.13 to 2.20 Log10 CFU/ml. Our finding substantiates the reports of Dasgupta et al., (22) on antibacterial activity of PRO against antibiotic resistant bacterial isolates. Bactericidal activity of PRO against S. aureus in our study also agrees with the findings of Dastidar et al., (23). PRO, a phenothiazine, acts by inhibiting the efflux pump that protects bacterial cell against harmful chemical substances (24), and also affects energy sources, adenosine triphosphatase (ATPase), and genes that regulate permeability in bacteria (25). The increased permeability ensures that PRO molecules get to the DNA sites, intercalate between the bases, and consequently hinder the DNA transcription and translation processes (26). It has also been reported that PRO can reverse the phenotypes of MDR bacteria (14).

Conclusion:
Our study demonstrated in vitro antimicrobial efficacies and time kill kinetics of amlodipine, thioridazine and promethazine against pathogenic clinical bacterial isolates, which indicates that these non-antibiotic drugs may be useful therapeutic alternatives in the bid to reduce the burden of infectious diseases associated with antibiotic resistant pathogens. Consequent upon these findings, in vivo antibacterial studies of these non-antibiotic drugs are required.