Cisplatin (cisplatinum, cis-diamminedichloroplatinum [II]), is one of the most important anticancer agents used in medicine. Its structure is shown in Figure 1. Owing to its ability to bind to DNA, cause the cross-linking of adjacent intrastrand purines, and interfere with DNA repair, cisplatin is an effective DNA-damaging and anticancer agent. Although cisplatin is nearly curative for testicular cancer and active against ovarian, head and neck cancer, the potential of this drug as a cure for many other types of cancer is limited because of cellular resistance to cisplatin1 and cisplatin's toxicity to humans; for example, renal toxicity, emesis, neurotoxicity, bone marrow suppression, anemia and hearing loss. Owing to the toxicity, cisplatin is administered intravenously in low dosage. The inhibition of Escherichia coli by cisplatin was discovered by Rosenberg et al.2, 3, 4 before it was known to be an effective antitumor agent. They made this discovery while performing an experiment to analyze the effect of an electric field on the growth of bacteria, the experiment involving the use of platinum electrodes.

Figure 1
figure 1

Molecular structure of cisplatin.

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Although Rosenberg et al.3 found that E. coli and other Gram-negative bacteria such as Aerobacter aerogenes, Alcaligenes faecalis, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Serratia marcescens were sensitive to cisplatin, it was unclear whether other bacteria were also inhibited. At a concentration 15-fold higher than that which inhibited cell elongation in E. coli, Gram-positive bacteria such as Streptococcus lactis, Streptococcus faecalis, Staphylococcus aureus, Sarcina lutea and Neisseria catarrhalis were not inhibited. Although other Gram-positive organisms were inhibited by this high concentration of cisplatin, they were much more resistant than E. coli. We felt it important to revisit this situation and determine the antimicrobial spectrum of cisplatin. Since those early days, three yeasts have been reported to be inhibited by cisplatin; that is, Saccharomyces cerevisiae,5 Schizosaccharomyces pombe6 and Candida albicans.7 One mold, Dictyostelium discoideum, has been reported to be sensitive to cisplatin.8 However, there has been very little screening effort focusing on the molds. Hence, we felt it important to expand our screening effort to include more filamentous fungi.

Inhibition of Gram-negative bacteria E. coli, A. aerogenes, A. faecalis, P. mirabilis, P. aeruginosa, K. pneumoniae and S. marcescens by cisplatin has been known for almost 45 years.2 In our initial tests, we confirmed the sensitivity of E. coli and S. marcescens to cisplatin. Table 1 shows such a test with E. coli strains 153zγ, ZK 650 and C600 R1 and with S. marcescens. Our further experiments showed inhibition of E. coli strains ZY and ESS as well as P. aeruginosa. The effect of increasing dosages of cisplatin from 16 to 40 ug per disc against E. coli C600 R1 is shown in Figure 2. It can be observed that the increase in zone size is proportional to the cisplatin dosage. We also studied the effect of cisplatin on growth of E. coli ZK650 in liquid medium and found inhibition (Table 2).

Table 1 Inhibition of bacteria by cisplatina
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Figure 2
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Inhibition of Escherichia coli C600 R1 by increasing doses of cisplatin. Inhibition was measured by the diameter of the clear zones in mm.

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Table 2 Cisplatin inhibition of Escherichia col i ZK650 in liquid culture in TSB medium
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Owing to the question in the literature regarding the sensitivity of Gram-positive bacteria to cisplatin, a number of such organisms were tested. Initial testing with Bacillus brevis 9999, Bacillus cereus 9139, Bacillus subtilis K and the actinomycete Streptomyces lividans B18 revealed inhibition by cisplatin at the lowest dose tested; that is, 16 ug per disc (Table 1). The results against B. subtilis K using different concentrations of cisplatin varying from 16 to 48 ug per disc, are shown in Figure 3. Later experiments showed inhibition of B. subtilis 168, B. subtilis JH642 and Staphylococcus aureus. The effect of cisplatin on growth of B. subtilis JH642 was also examined. Table 3 shows that cisplatin made up in dimethylformamide inhibited growth at levels of dimethylformamide that were not inhibitory (because of the disk evaporation technique used).

Figure 3
figure 3

Inhibition of Bacillus subtilis K by increasing doses of cisplatin. Inhibition was measured by the diameter of the clear zones in mm.

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Table 3 Cisplatin inhibition of Bacillus subtili s JH642 in liquid culture in TSB medium
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It is known that the yeasts S. cerevisiae,5 S. pombe6 and C. albicans7 are inhibited by cisplatin. Our initial studies, shown in Table 4, confirmed cisplatin inhibition of S. cerevisiae and S. pombe. Five strains of S. pombe; that is, WT, 972, NW158, NW214, NW240 and sp6 were tested and all were found to be inhibited. A later experiment showed inhibition of an additional strain of S. pombe; that is, Rad3Δ.

Table 4 Inhibition of fungi by cisplatina
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The molds were difficult to test because their growth in liquid culture was filamentous rather than as an evenly distributed turbid suspension. They often grew as pellets, sometimes very large, with or without the apparent presence of sporulation. To solve this problem, small glass beads (65 beads per flask) plus either 1.5% of carboxymethylcellulose or 0.3% carboxypolymethylene9 were added to the flasks. This resulted in a much more homogeneous type of growth. Our initial results showing cisplatin inhibition of Penicillium sp., Alternaria alternata and Aspergillus niger are shown in Table 4. Later experiments revealed cisplatin inhibition of additional molds such as Aspergillus fumigatus, Fusarium oxysporum, Pythium ultimum and Geotrichum candidum.

To summarize, the antitumor agent cisplatin has an extensive antimicrobial spectrum of activity. Growth of all 29 microbes, including seven Gram-negative bacterial strains, eight Gram-positive bacterial strains, seven yeast strains and seven mold strains was found to be inhibited. This agrees with the point made by Newman and Shapiro10 that most antitumor agents have antimicrobial activity. We are now using these cisplatin-inhibitable strains as prescreens to test the antimicrobial activity of novel cisplatin analogs chemically synthesized by Dr Baldwin King and his Drew University students. Those that are antibiotically active will be further tested for antitumor activity in the hope of identifying cisplatin analogs that do not have the toxicity problems associated with cisplatin.

Experimental Procedure

Organisms were preserved by storage in 30% glycerol solution at –80 °C. The cultures were transferred to liquid medium (Tryptic Soy broth for the bacteria and Sabouraud maltose broth for the fungi) at 40 ml per 250 ml flask and grown on the rotary shaker at 28 °C and 220 r.p.m. until heavy growth was observed. This usually required 2 to 3 days for the bacteria and 4 to 6 days for the fungi. The liquid cultures were then used to seed melted agar media at 500 μl per 100 ml of agar medium. The agar media used were as follows: LB agar and Tryptic Soy agar for unicellular bacteria; yeast extract agar for the actinomycete; Sabaroud's maltose agar, Tryptic Soy agar and yeast extract agar for yeasts; and Sabaroud maltose agar as well as Tryptic Soy agar for the molds. The inoculated agar was poured into Petri dishes at 10 ml per plate and allowed to solidify. Cisplatin was dissolved in water up to 5 mM concentration or in dimethylformamide at higher concentrations (10 and 20 mM). Cisplatin was then added to paper discs of 6 mm diameter, which were allowed to dry on an aluminum foil surface. Those discs that received the high concentration of cisplatin were incubated in a vacuum oven at 42 °C for 2–3 h to remove the dimethylformamide before placing them on the inoculated agar surface. Those that received the low concentration of cisplatin could be placed on the agar directly. Plates were incubated at 35–37 °C for bacteria and 26–28 °C for fungi until growth was observed. Activity was shown by inhibition zones, the diameters of which were measured.