Introduction  

Antimicrobial resistance (AMR) is now a global threat as a consequence of the excessive use of antimicrobials in many applications [1-3]. Extensive and inappropriate uses of antimicrobial agents have promoted microbial evolution and adaptation, leading to the existence of multi-drug resistant (MDR) strains that cause lethal infections [4]. Also, the development of resistance is attributed to the loss of power of anti-microbial drugs as a consequence of long-term and improper use [5-7].

High levels of antimicrobial resistance of E. coli and S. aureus from food-producing animals have been reported in various countries [1-7]. Antimicrobials such as ampicillin, sulfamethoxazole, and tetracycline have been widely used for many years in the treatment of animals to treat infections [8]. Resistant S. aureus against ampicillin is related to the ability of this microorganism to produce penicillinase [9, 10]. Ampicillin-resistant E. coli has been reported worldwide, suggesting the need to consider a new approach to combat this type of bacteria [11, 12]. In an attempt to develop new antibacterial agents, research on organometallic compounds for medicinal purposes continues to grow [13], inspired by successful of organometallic complexes (cisplatin) as a chemotherapy agent for the treatment of testicular and ovarian cancer [14].

In previous studies [15, 16], it was reported that metal complexes with organic ligands exhibit significant biological activity, contributed by the ligands and the metal in the compounds. The organic ligands contribute to biological activity, increase efficiency, reduce toxic effects or side effects, reduce therapeutic doses, protect and transport the metal, avoid side reactions on the route to potential targets, and overcome the mechanism of drug resistance, while the metal acts as a carrier and or stabilizer of the drug to reach the target [15, 16].

The tin(II) compound with a Schiff bases derived from 2-hydroxy-1- naphth-aldehyde has a significant influence on the inhibition of S. aureus and E. coli [17]. Organotin(IV) derivatives based on 2-((2-methoxyphenyl)carbamoyl)benzoic acid compounds are known to have good activity as antibacterial compounds [18]. Antibacterial testing against bacterium Bacillus sp by the diffusion method with the best results obtained using diphenyltin(IV) di-4-nitrobenzoate with a concentration of 200 ppm, whereas in the dilution test, diphenyltin(IV) di-4-nitrobenzoate) was effective at concentration of 0.4 mg/2mL [19]. Diphenyltin(IV) and triphenyltin(IV) 3-chlobenzoate have been reported to exhibit activity against Pseudomonas aeruginosa and Bacillus subtilis [17]. The antibacterial activity using the diffusion method on diphenyltin(IV) dibenzoate and triphenyltin(IV) benzoate compounds on B. substilis and P. aeruginosa gave maximum growth inhibition at a concentration of 200 ppm [20]. In recent years, studies on organometallic chemistry revealed that derivatives of organotin(IV) posses activity as anticancer [16, 18, 21, 22], antifungal [23, 24], antimalarial [25-27], corrosion inhibitor [28, 29] and antimicrobial [17-20].

Based on the interesting results reported in the literature on the activity of organotin(IV) derivatives, this work aims to investigate the potential of diphenyltin(IV) o-, m-, p-dichlorobenzoate and triphenyltinIV) o-, m-, p-chlorobenzoate as antimicrobials against S. aureus and E. Coli.

Materials and Methods

Materials

The materials used in this work were diphenyltin(IV) dihydroxide [(C₆H₅)₂Sn(OH)₂], triphenyltin (IV) hydroxide [(C₆H₅)₃SnOH], o-, m-, p-chlorobenzoic acids [o,- m-, p-(C₆H₄(Cl)COOH)], chloramphenicol, nutrient Agar (NA) media, and sodium chloride (NaCl) were obtained from Sigma-Aldrich, methanol (CH₃OH) was obtained from J.T Baker. All of these chemicals were used as received, double distilled water was purchased from Ikapharmindo, Indonesia, Gram-negative bacteria E. coli and Gram-positive bacteria S. aureus were obtained from the Lampung Provincial Health Laboratory.

Preparation of solution test

Test solutions consisting of: Diphenyltin(IV) dihydroxide (1), triphenyltin(IV) hydroxide (2), diphenyltin(IV) di-o-dichlorobenzoate (3), diphenyltin(IV) di-m-dichlorobenzoate (4), diphenyltin(IV) p-dichlorobenzoate (5) , triphenyltin(IV) o-chlorobenzoate (6), triphenyltin(IV) m-chlorobenzoate (7) and triphenyltin(IV) p-chlorobenzoate (8). The compounds 3-8 were prepared based on the procedure previously described [19-29]. The antibacterial activity test was carried using the procedure available in the literatures [19, 20, 29-32].

0.614 g (0.002 moles) diphenyltin(IV) dihydroxide [(C₆H₅)₂Sn(OH)₂] was reacted with 0.626 g (0.004 moles) o-, m-, p-chlorobenzoic acids in 30 mL of methanol solvent. The reaction mixtures were refluxed for 4 h at 60 ^oC. Upon completion of the reaction, the solvent was evaporated and the products obtained were dried in a desiccator until the compounds were completely dried. The same procedure was used in the preparation of triphenyltin(IV) derivatives, in this reaction, 0.7336 gram (0.002 moles) triphenyltin(IV) hydroxide was reacted with 0.313 g -chlorobenzoic acid in 30 mL of methanol. The results obtained diphenyltin(IV) di-o-, m-, p-chlorobenzoate were white solid compound, 1.0781 g (92.30%); for triphenyltin(IV) o-, m-, p-chlorobenzoate 0.9236 g (91.36%)

The nutrient agar (NA) media were made by 2.8 g of NA was dissolved in 100 mL double distilled water, then heated and sterilized in an autoclave at 121 °C and 1 atm for 15 min. The warm NA media were placed into a test tube-tilted, let stand until solidified, and then stored in the refrigerator. 1 ose of each bacterium was diluted with 2 mL of saline water (NaCl 0.85%) and then used as a bacterial suspension. A 1 ml bacterial suspension was inoculated into a sloping NA test media previously prepared and flattened on the surface of the media using a spreader.

Antibacterial activity test with diffusion method

Each sterile petri dish containing 15 mL of NA media was added with 1 mL of a bacterial suspension before solidifying, homogenized for 5 min, and allowed to solidify. After the media has solidified, four wells are made using the blue tip. The diffusion method test uses the procedure available in the literature [19, 20, 30-32]. In petri dish 1, the first well was filled with positive control K (+) chloramphenicol, the second well was filled with negative control K (-) (containing methanol p.a.), the third well was filled with a solution of compound 1, diphenyltin(IV) dihydroxide and the fourth well with diphenyltin(IV) di-o-chlorobenzoate 100 ppm. Petri dish 2, four wells were filled with diphenyltin(IV) di-o-chlorobenzoate test solution with concentrations of 200, 300, 400, and 800 ppm. Each experiment was performed three times. Then they were incubated for 1-2 days at 37 °C, then the inhibition zone was observed and measured using calipers. Data on the inhibition zone were calculated as average from the three repetitions. Similar procedures were carried out for compounds 2, and 4-8.

Antibacterial activity test with dilution method

Based on the results of the diffusion test, the most effective inhibitory concentration from compounds 1-8 was obtained and each compound was dissolved in methanol. The dilution test was performed using the procedure as in the literature [19, 20, 31] by preparing volume variations for the compounds having inhibitory properties to bacteria. 15 mL of liquid NA media were prepared and kept at 55 °C, followed by placing the compounds tested into the liquid NA media. Homogenized with vortex, then the mixture was poured into a petri dish, let stand until solidified. The suspension of E. coli and S. aureus inoculated in the NA media were incubated at 37 oC for 2-3 days. The growth of bacteria was observed daily. The most effective compound tested was the compound having the smallest concentration but having the highest inhibitory effect on bacterial growth [19, 20, 30-32].

Results and Discussion

The results of the antibacterial activity test on compounds 1-8 by diffusion and dilution methods against S. aureus and E. coli are shown in Table 1.

The starting materials tested both diphenyltin(IV) dihydroxy and triphenyltin(IV) hydroxy gave inhibition against S. aureus but not against the E. coli. Out of the diphenyltin(IV) di-chlorobenzoates (compounds 3-5), diphenyltin(IV) di-o-chlorobenzoate (3) produced no inhibition, while compound 4 and 5 showed activity against S aureus. However, compounds (3-5) did not give inhibition against E. coli. The same observations were also found for the derivative of triphenyltin(IV) chlorobenzoate as shown in Table 1. The results reported here for these compounds were surprisingly weaker than similar compounds reported by others [17] for bacterial activity against E. coli although the compounds reported also showed very weak activity [17].

Based on Table 1, the weak antibacterial activity against S. aureus i.e. inhibition zone diameter <10 mm was observed for compound 4 (100) ppm, compound 6 (100) ppm, compound 7 (100 and 200 ppm), and compound 8 (100) ppm. Medium antibacterial activity i.e. inhibition zone diameter of 10-16 mm against S. aureus were observed for compound 4 (200, 300, 400, and 800 ppm), compound 5 (800 ppm), compound 6 (200 ppm), compound 8 (200 and 300 ppm). Strong antibacterial activity i.e. inhibition zone diameter > 16 mm against S. aureus were observed for compound 6 (300, 400, and 800 ppm), compound 7 (300, 400, and 800 ppm), and compound 8 (400 and 800 ppm). Unfortunately, no compound was found to exhibit activity against E coli.

The effectiveness of compounds against bacteria was also calculated based on the ratio of the diameter of the inhibition zone (in cm) to the concentration of the compound (in ppm). Based on Table 2, of six organotin(IV) chlorobenzoate derivative compounds prepared, compound 4 (100 ppm) is the most effective to inhibit the growth of S. aureus with a ratio of 0.0096 and it was the highest inhibition ratio among all of the compounds tested comparable to other compounds reported [17, 19, 20].

The dilution method is used to determine the minimum inhibitory concentration (MIC). The MIC for compound 4 (100 ppm), 5 (600 ppm), 6-8 (60 ppm) against S. aureus is shown in Table 3.

The differences in antibacterial activity of a compound against S. aureus and E coli are due to the differences in the structure and composition of the cells of the two bacteria [17, 19, 20]. It is also observed that the small difference in the structure of the compound produces different antibacterial activities [17, 19, 20].

Note:

+++++ = very high bacterial growth

++++ = high bacterial growth

+++ = medium bacterial growth

++ = little bacterial growth

+ = very little bacterial growth

 - = no bacterial growth