924 Inhibitory Action of Metabolites of Pseudomonas aeruginosa against Gram-negative Bacteria Zhongxing LI, Xiuhua WANG, Yuezhu GUO and Jianhong ZHAO Bacteriological Laboratory, The Second Affiliated Hospital of Hebei Medical College (Received: March 20, 1995) (Accepted: June 13, 1995) Key words: Pseudomonas aeruginosa, inhibitory action, Gram-negative bacteria Abstract Fifty clinical isolates of Pseudomonas aeruginosa were tested for inhibition of growth of clinical isolates of Escherichiacoli, Salmonella infantis, Klebsiella pneumoniae and other Gram-negative bacteria in the authors' laboratory. Pseudomonas aeruginosa was strongly active against both E. coli and Enterobacter cloacae, with 89.4% and 94.7% inhibition respectively, but weakly active against S. infantis, K. pneumoniae and Proteus mirabilis with 56.3%, 48.8% and 23.8% inhibition, respectively. The pigmented strains were found to have stronger antimicrobial activity than the unpigmented strains. Pyocyanin, the major metabolite of Pseudomonas aeruginosa, has been shown to inhibit Escheri- chia coli, Proteus spp. and other Gram-negative bacteria, by research with a few strains of P . aeruginosa and a single inhibited strains'. However, little attempt has been made to determine the inhibitory action of many strains of P. aeruginosa against a large number of clinical isolates such as Escherichia spp., Klebsiella spp., and Salmonella spp., up to now. For this reason, in this study we examined 50 randomly selected clinical isolates of P. aeruginosa for inhibition of growth of a wide range of Gram-negative bacteria, including 30 strains of E. coli, 30 of K. pneumoniae, 30 of S. infantis, 6 of Enterobacter cloacae and 9 of Proteus mirabilis. Materials and Methods Bacterial strains: All 50 isolates of P. aeruginosa were obtained from clinical samples in our hospital (25 strains from pus, 13 from sputum, 10 from blood, 2 from feces), including E. coli, K. pneumoniae, S. infantis, E. cloacea and Proteus mirabilis. Medium: Mueller-Hinton agar manufactured by Hangzhou Microbiogical Reagent Factory was used for all agar plates. Bacterial preparation: Cultures of P. aeruginosa and other organisms to be tested on Mueller- Hinton agar plates at 35C for 18-24 h were adjusted to suspensions of about 108 CFU/ml with Mueller-Hinton broth. Methods: A loopful of a suspension of P. aeruginosa was streaked across the middle of an Mueller-Hinton agar plate in a straight line. After overnight incubation at 35C , the colonies were scraped off the plate with a sterile slide. The agar plate was placed in an inverted dish and exposed to chloroform vapor (a cotton globe soaked with chloroform on the lid of the dish) for 15 minutes at 35C to kill the residual P. aeruginosa. The cotton globe was removed and the surface of the plate was air-dried for 15 minutes by leaving the plate partially open to eliminate residual chloroform vapor. Correspondence to: Zhongxing LI, M.D. Bacteriological Laboratory, The Second Affiliated Hospital of Hebei Medical College , Shijiazhuang, China 050000 感染症学雑誌 第69巻 第8号 Inhibitory Action of Metabolites of Pseudornonas aeruginosa 925 Then the streaked plate was cross-streaked horizontally with a loopful of a suspension of the test organisms (about 6 cross-streaks on a plate) prepared as described above and incubated at 35•Ž for 24 h. The plates were then examined for an inhibition zone along P. aeruginosa streak. The strain was scored as resistant if no inhibition zone appeared or sensitive if there was an inhibition zone of > 10 mm. Results Of all the isolates tested, E. coli was the most sensitive to P. aeruginosa and only 23.8% of the strains of Proteus mirabilis were inhibited. Pseudomonas aeruginosa also strongly inhibited E. cloacae though only 6 strains were tested. Furthermore, zone of inhibition of E. coli was the widest (21.2 mm) and that of P. mirabilis was the narrowest (14 mm). The results are shown in Table 1. The inhibitory activity of P. aeruginosa containing different pigments against Gram-negative bacteria is shown in Table 2. In order to investigate the inhibitory powers of the different strains of P. aeruginosa against the Gram-negative bacteria, 10 randomly selected P. aeruginosa strains were tested against the five Gram-negative bacteria described above. The results showed that the inhibitory power differed Table 1 Inhibitory action of the metabolite of 50 strains of P. aeruginosa against Gram- negative bacteria + Average width of inhibition zone (mm), *Number showing sensitivity, **Number showing resistance Table 2 Inhibitory action of P. aeruginosa containing various pigments against Gram-negative bacteria For explanations of column 2, see footnotes to Table. 1. 平成7年8月20日 926 Zhongxing LI et al Table 3 Inhibitory power of 10 strains of P. aeruginosa against Gram-negative bacteria For explanations of column 3, see footnotes to Table 1. among the strains; the strains with a higher inhibition rate produced a wider inhibitory zone, and vice versa. The detailed results are showen in Table 3. Discussion Pseudomonas aeruginosa strongly inhibits the growth of Staphylococcus aureus (both MRSA and MSSA) and Staphylococcus epidermidis2m. However, the inhibitory activity of P. aeruginosa against Gram-negative bacteria is not very clear, although there have been a few reports involving a single experimental strain". During this study designed to examine the inhibition of Gram-negative bacteria, including 30 strains each of E. coli, K. pneumoniae and S. infantis, by using 50 P. aeruginosa strains produced in our laboratory, we showed that P. aeruginosa can inhibit the growth of all of those Gram-negative bacteria. Of all the isolates tested, E. coli showed the highest inhibition rate (89.4%), and that of K. pneumoniae was 48.8%. However, the test for inhibition of E. cloacae showed that the frequency of resistance was only 12/300 tests and the inhibition rate was as high as 94.7%, though only 6 strains of E. cloacae were used, and showed that most P. aeruginosa strains can inhibit the growth of E. cloacae, while most of the 9 strains of P. mirabilis failed to exhibit inhibition (inhibition rate, 23.8%). The major antibiotic substance produced by P. aeruginosa is pyocyanin, a kind of phenazine pigment (C j 3H 1o N 2O)5) which is soluble in water, chloroform, and n-butanol. It appears deep red in acid solution and blue in alkaline solution7). Its inhibitory action depends upon the superoxid free radical (021, a high oxidation radical, which is produced during an oxidation-reduction reaction. The product has strong toxicity for a wide range of organisms. However, the toxicity can be eliminated. Hassan et al. demanstrated this by a adding superoxide dismutase (SOD) to the cultures". The pyocyanin produced by P. aeruginosa lost its activity against E. coli due to SOD, a cleaner for 02-, 感 染 症学 雑 誌 第69巻 第8号 Inhibitory Action of Metabolites of Pseudornonas aeruginosa 927 and reduced the 02- in the cultures. The difference in inhibition among the five Gram-negative bacteria by P. aeruginosa may result from the difference in the quantity of SOD produced by the different organism5). In addition to P. aeruginosa, another pseudomonad, P. fluorescens can also produce an antibitic material, pseudomonic acid, which has been used to treat human skin infection7'8). For this reason, the antimicrobial agent from P. aeruginosa should be studied further for its possible use for the health of human being5). References 1) Hassan, H.M. & Fridovich, I.: Mechanism of the antibiotic action of pyocyanine, J Bacteriol. 141: 156-163, 1980. 2) Ogino, J., Yamada, T., Goto, R., Kikushima, K. & Fujimori, I.: Growth inhibitory activity of clinical isolates of Pseudornonas aeruginosa against Staphylococcus aureus (MRSA and MSSA). J.J.A. Inf. D., 66: 909-913, 1992. 3) Ogino, J., Yamada, T., Kozeni, T., Ito, M., Kikushima, K., Goto, R., Fujimori, I., Hisamatsu, K. & Murakami, Y.: Effect of erythromycin on and staphylococcal activity and dye producion of Pseudornonas aeruginosa isolated from clinical materials. J.J.A. Inf. D., 67: 18-23, 1993. 4) Knight, M., Hartman, P.E., Hartman, Z. & Young, V.M.: A new method of preparation of Pyocyanine and demonstration of an unusual bacterial sensitivity. Anal Biochem, 95: 19-23, 1979. 5) Morrison, M.M. & Sawyer, D.T.: Flavin model systems 2. Pyocyanine complexes of divalent manganese, iron, nickel, copper, and zinc in dimethyl sulforxide. J Am Chem Soc., 100: 211, 1978. 6) Frank, L.H. & Demoss, R.D.: On the biosynthesis of pyocyanine, J Bacteriol 77: 776-782, 1959. 7) Hughes, J. & DeMoss, R.D.: On the biosynthesis of pyocyanine, J Bacteriol. 77: 776, 1959. 8) Wuite, J., Davies, B.I., Lamber, J., Jackson, D. & Mellows, G.: Pseudomonic acid: A new topical antimicrobial agent, Lancet, 13: 394, 1983. 緑 膿 菌 の産生 物質 の グラ ム陰性 桿菌 に対 す る発 育 阻止 力 中国河北医学院第二医院細菌室 李 仲興 王 秀隼 郭 月珠 趙 建宏 50株 の臨床分離 Pseudomonas aeruginosa を用 89.4%, E. cloacae の94.7%の 発 育 を 阻 止 し た が, い て本 菌 の 産 生 物 質 の30株 の Escherichia coli, S. infantis, K. pneumoniae お よ び P. mirabilis Klebsiella pneumoniae お よび Salmonella infantis に 対 し て は 各 々56.3%,48.8%お よ び23.8%の 株 な らびに9株 の Proteus mirabilis, 6株 の Enter- の 発 育 を 阻 止 す る の に と ど ま っ た.ま た,Paer- obacter cloacae に対 す る発育 阻止力 を検討 した. ugznosa の他菌種 に対する発育阻止力は色素産生 その結果 P. aeruginosa の産生物質 は E. coli の 株 の方が非産生株 よ り強力 で あった. 平 成7年8月20日.