Permeability and Selective Toxicity of Nitrofurane Compounds

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1 Permeability and Selective Toxicity of Nitrofurane Compounds for Bacteria By Satoru OKA Food Industrial Experiment Station, Hiroshima Prefecture Received April 16, 1962 The bacterial growth is inhibited by nitrofurane compounds, although the yeast growth is hardly affected. In relation to the selective toxicity of nitrofuranes for bacteria, the interaction between microbes (Escherichia coli, Staphylococcus aureus and bakers' yeast) and nitrofurane compounds (5-nitro-2-furfural semicarbazone and 5-nitro-2-furylacryl amide) was examined. Apparently, in the bacterial suspension containing energy substrate, nitrofuranes are continuously reduced to corresponding aminofuranes, respectively. The velocity of the bacterial reduction at the growth inhibiting condition was evaluated as great as above 30 per cent of the limit of supplying velocity of coenzymes in the cell, the reduction velocity of such value is enough to inhibit the bacterial growth, because the electron transfer in the cell metabolism is disordered. On the other hand, in the yeast suspension, the reduction velocity was negligibly small. The difference of the reduction ability between bacteria and yeast was seemingly owing to the fact that the permeability of the nitrofuranes differs by the kind of microbe so that it was concluded that the antimicrobial effect of nitrofuranes is limited by the permeability for the microbe cell. The bacterial growth is inhibited by the nitrofurane compounds, whereas the yeast growth is hardly affected. In connection with the antibacterial action of the nitrofuranes, Brodie and Gots1) assumed that 5-nitro-2- furfural semicarbazone acts as the hydrogen acceptor against the reduced coenzyme in the cell, and it diverts the electron transfer in the cell metabolism from the normal course. Recently, Beckett and Robinson2) have proved that the reduction product of nitrofurane by bacteria is 5-amino-2-furfural semicarbazone. However, there is no information on the selective toxicity of nitrofuranes for bacteria. In the present work, the interaction between microbes and nitrofuranes was investigated, and the mechanism of the antibacterial effect and that of the selective toxicity of nitrofuranes were discussed. EXPERIMENTAL Test Organism. Escherichia coli HUT-8032, Staphylococcus aureus AM-1058 and bakers' yeast were used as the test organism. The cell of these microbes was prepared by the same procedure as described in the previous report3). Culture Condition. The growth of these microbes was observed at 30 Ž, koji extract (Ballg. 8, ph 5.6) for the yeast and bouillon (1% peptone and 1% beaf extract, ph 6.0) for the bacteria being used. 1) A.F. Brodie and J.S. Gots, Arch. Biochem. Biophys., 36, 165 (1952). Nitrofurane Compounds. 5-Nitro-2-furfural semi- 2) F.L. Beckett and A.E. Robinson, Chem. and Ind. (London) 1957, ) S. Oka, This Journal, 26, 515 (1962).

2 Permeability and Selective Toxicity of Nitrofurane Compounds for Bacteria 521 carbazone (F) and 5-nitro-2-furylacryl amide (A) were used. Determination of Nitrofurane Compounds. The concentration of these nitrofuranes was polarographically determined. The polarographical procedure was essentially the same as that described in the previous report4). RESULTS Inhibiting Effect of Nitrofuranes on Microbe Growth. The influence of the nitrofuranes on the process of microbe growth was observed. The turbidimetric result is shown in Fig. 1, i.e., the nitrofuranes prolong the lag phase of the bacterial growth without producing any other effect, but the yeast growth is hardly affected. The concentration of nitrofuranes required to inhibit the microbe growth for 48 hours is illustrated in Table I. Germicidal Effect of Nitrofuranes. The death rate of microbes was evaluated in 0.5mM nitrofurane solutions after having been sus- As shown in Table II, the death rate of the microbes is very low, though in the case of bacteria, nitrofurane concentration is more than ten times the growth inhibiting concentration. This suggests that the growth inhibiting effect is almost independent of the germicidal action. TABLE II. GERMICIDAL EFFECT OF NITRO- FURANE COMPOUNDS FIG. 1. Influence of Nitrofurane Compounds on Microbe Growth. The cell of the yeast and E. coli was inoculated in the culture medium containing 5-nitro-2-furfural semicarbazone (F) or 5-nitro- 2-furylacryl amide (A), and the growth of microbes was turbidi- 1: Normal growth of the yeast and the growth in the presence of 0.5mM F or 0.8mM A, 2: Normal growth of E. coli, 3: E. coli in 0.02mM F, 4: E. coli in 0.01mM A, 5: E. coli in 0.02mM A. The behavior of St. aureus was essentially the same as that of E. coli. TABLE I. INHIBITING EFFECT OF NITROFURANE COMPOUNDS ON MICROBE GROWTH Concentration of nitrofuranes required for growth inhibition during 48 hrs. (mm) One thoufandth per cent of the microbe cell was inoculated into 0.5mM nitrofurane solution containing 0.05M phosphate (ph 6.0) and 1 per cent of glucose. Then, the death rate of microbe cell Stability of Nitrofuranes in Culture Medium. The change of nitrofurane concentration was followed in such culture medium as koji extract and bouillon. However, any change was not observed after 48 hours incubation at 30 Ž. Accordingly, it is considered that the toxicity of nitrofuranes is not affected by One ten-thousandth per cent of the microbe cell was inoculated into the culture medium, and the nitrofurane concentration required for the growth inhibition was determined after having been incubated 4) S. Oka, This Journal, 26, 387 (1962). any component of the culture medium. Behavior of Nitrofuranes in Suspension of Microbe Cell. The change of nitrofurane concentration was followed in the suspension of the

3 522 Satoru OKA FIG. 2. Reduction of Nitrofurane Compounds by Bacteria. One tenth per cent of the cell of E. coli was suspended in 0.1mM solution of 5-nitro-2-furfural semicarbazone (F) or 5- nitro-2-furylacryl amide (A) containing 0.05M phosphate (ph 6.0) and 2 per cent of glucose. Then, the change of the pol- 1: Initial state of 0.1mM F, 2: F after 9 `11min., 3: F after 19 `21min., 4: F after 39 `41min., 5: Initial state of 0.1mM A, 6: A after 9 `11min., 7: A after 19 `21min., 8: A after 39 `41min., The same behavior was observed in the case of St. aureus. microbe cell in the presence of glucose. The polarograms of F and A give reduction waves at and volt vs. S.C.E. in 0.05M phosphate buffer solution (ph 6.0) containing 2 per cent of glucose as seen at curves 1 and 5 in Fig. 2, respectively. By addition of 0.1 per cent of the cell of E. coli in nitrofurane solution, the wave height decreases with time as seen at curves 2 `4 and 6 `8 in Fig. 2. The same behavior is also observed in the suspension of St. aureus. However, in the case of the yeast, the decreasing velocity is negligibly small as seen at curves 1 and 6 in Fig. 3, and the time of half decay of nitrofurane is inversely proportional to the concentration of bacterial cell as seen at curves 2 `4 and 7 `9 in Fig. 3; and it is confirmed that the decreasing velocity of the

4 Permeability and Selective Toxicity of Nitrofurane Compounds for Bacteria 523 FIG. 3. Decreasing Process of Nitrofurane Compounds in Suspension of Microbes. The cell of microbes was suspended in 0.1mM solution of 5-nitro-2-furfural semicarbazone (F) or 5-nitro-2-furylacryl amide (A) containing 0.05M phosphate (ph 6.0) and 2 per cent of glucose. Then, the change of the nitrofurane coneentration was (F) 1: 5% yeast, 2: 0.05% E. coli, 3: 0.1% E. coli, 4: 0.2% E. coli, 5: 0.1% St. aureus, (A) 6: 5% yeast, 7: 0.05% St. aureus, 8: 0.1% St. aureus, 9: 0.2% St. aureus, 10: 0.1% E. coli, nitrofuranes is proportional to the cell concentration. From these facts, it is considered that the nitrofuranes are continuously reduced by the bacteria as shown by Beckett and Robinson2), although they are hardly reduced by the yeast. It is noteworthy that the bacteria which are sensitive to the nitrofuranes reduce the nitrofuranes, whereas the yeast which is tolerant hardly reduces the nitrofuranes. Nitrofurane Concentration and Reduction Velocity by Bacteria. The time of half decay of the nitrofuranes by bacterial reduction was determined in relation to the initial concentration, and the initial concentration was plotted versus the time of half decay at the definite concentration of the bacterial cell. As shown in Fig. 4, the time of half decay of these nitrofuranes linearly increases with the increase of the initial concentration, in which the reduction velocity at a given concentration of the nitrofuranes is expressed by Laidler and Shuler's equation concerning permeation velocity represented by equation (1)4 `7), 5) S. Oka, This Journal, 26, 393 (1962). 6) S. Oka, ibid., 26, 508 (1962). 7) K. J. Laidler and K.E. Shuler, J. Chem. Phys., 17, 851, 856 (1949). FIG. 4. Initial Concentration of Nitrofurane Compounds and Time of Half Decay by Bacterial One tenth per cent of the bacterial cell was suspended into the solution of 5-nitro-2-furfural semicarbazone (F) or 5-nitro-2-furylacryl amide (A) containing 0.05M phosphate (ph 6.0) and 2 per cent of glucose, and the time of half decay was determined in relation to the initial concentration. 1: A, E. coli, 2: A, St. aureus, 3: F. E. coli, 4: F, St. aureus. (1) where v is the reduction velocity (m-mol/ min/g-cell), V the maximum velocity of the reduction which is the limit of the diffusion

524 Satoru OKA velocity through the cell membrane (m-mol/ min/g-cell), Km the dissociation constant of the nitrofurane-cell surface complex and C the concentration of the nitrofuranes (mm).

5 524 Satoru OKA velocity through the cell membrane (m-mol/ min/g-cell), Km the dissociation constant of the nitrofurane-cell surface complex and C the concentration of the nitrofuranes (mm). The values of V and Km of each nitrofurane are illustrated in Table III. This relation is the same as that observed in the case of the reduction of quinone compounds below the critical concentration4 `6,8). However, the relation of equation (1) seems to be applicable in all range of the solubility of these nitrofuranes (below 1mM). yeast catalyzes the reduction of F by the reduced coenzyme. By assuming, form this TABLE III. REDUCTION VELOCITY OF NITRO- fact, that F permeated into the microbe cell is immediately reduced by the reduced co- The reduction velocity of a nitrofurane at a given concentration of nitrofurane is represented by the following equation, where v is the reduction velocity (m-mol./min./g-cell) C the nitrofurane concentration in the medium (mm) and V and Km the constants. In the case of yeast, the reduction velocity is so small that these constants can not be evaluated. DISCUSSION In the previous reports4 `6), it was observed that quinone compounds are reduced to corresponding hydroquinones by various microbes, and the author considered that when quinone concentration is below the definite critical concentration, the reduction velocity is limited by the premeation velocity of quinone into microbe cell, and that the hydrogen donor of the reduction-oxidation reaction is the reduced coenzymes such as di- or triphosphopyridine nucleotide in microbe cell. In the present work, it has been also observed that nitrofurane compounds are continuously reduced by the bacteria such as E. coli and St. aureus which are sensitive to nitrofuranes, though nitrofuranes are hardly reduced by yeast which is tolerant to nitrofuranes. In this case, the relation between the reduction velocity by the bacteria and the nitrofurane concentration follows Laidler and Shuler's equation in all range of the solubility of nitrofuranes, and the behavior of the nitrofuranes in the bacterial suspension seems to be essentially the same as that of quinones having concentration below the critical concentration. Formerly, Brodie and Gots1) have observed that the diaphorase prepared from bakers' enzymes in the cell, it is considered, as in the case of the reduction of quinones4-6), that the reduction velocity of F indicates the permeation velocity into the cell. On the other hand, yeast hardly reduces F which is considered to permeate hardly into the yeast cell, though the yeast diaphorase catalyses the reduction. For the case of A, the same explanation is applicable from the similarity of the chemical form and the behavior in microbe suspension. In connection with this, it has been observed that the permeability of quinones for the bacterial cell is much higher than that for the yeast cell6). This is in good agreement with the fact that the permeability of nitrofuranes for yeast cell is much lower than that for bacterial cell. Accordingly, nitrofuranes hardly affect yeast growth because of their extremely low permeability to the yeast cell. Brodie and Gots1) have assumed that F spends the reduced coenzyme in cell and it results in the bacteriostatic action, diverting the electron transfer in the cell metabolism from the normal course. In the case of qui. nones6), it has been also considered that when the reduction velocity approaches the limit of the supplying velocity of coenzymes in the

Permeability and Selective Toxicity of Nitrofurane Compounds for Bacteria 525 cell, quinones inhibit the microbe growth, disordering the electron transfer in the cell metabolism.

6 Permeability and Selective Toxicity of Nitrofurane Compounds for Bacteria 525 cell, quinones inhibit the microbe growth, disordering the electron transfer in the cell metabolism. The limit of the supplying velocity of the reduced coenzymes was evaluated as great as 0.04m-mol /min/g-cell with the same bacteria used in the present work. In connection with the bacterial reduction of nitrofuranes, Beckett and Robinson2) have proved that the reduction product of F in the bacterial suspension is 5-amino-2-furfural semicrabazone, though the aminofurane is easily transformed to glyoxylopropionitryl semicarbazone automatically. Thus, it is considered that three molecules of the reduced coenzymes are spent for the bacterial reduction of one molecule of nitrofurane, though one molecule is spent for the case of quinones. In order to compare the disordering effect on the electron transfer in the cell metabolism, it is convenient to express the reduction velocity with the spending velocity of the coenzymes, the reduction velocity of nitrofuranes being multiplied by three. In Fig. 5, the spending velocity thus evaluated is plotted versus nitrofurane concentration. As seen in Fig. 5, at the bacteriostatic con- FIG. 5. Nitrofurane Concentration and Spending Velocity of Coenzymes in Cell by Bacterial Reduction of Nitrofuranes. 1: A, E. coli, 2: A, St. aurens, 3: F. E. coli, F, St. aureus. centration of nitrofuranes, the spending velocity of coenzymes by the bacterial reduction is held above 30 per cent of the limit of the supplying velocity of coenzymes of cell. The spending velocity of such value is considered, as well as in the case of the quinones, to be enough to disorder the electron transfer in the cell metabolism. From the view point of reduction velocity, the mechanism of the bacteriostatic action of nitrofuranes proposed by Brodie and Gots1) is supported. In this case, the permeability is not so high that the permeation velocity does not exceed. the limit of the supplying velocity of coenzymes in any concentration of nitrofuranes. This indicates that the concentration of nitrofuranes in bacterial cell is held practically zero in any condition, and that nitrofuranes can not kill the bacteria, even if they have the activity to react with the component of of cell. In fact, the germicidal activity of nitrofuranes is very weak, so that, the lag time prolonged by nitrofuranes is determined by the concentration of nitrofuranes and that of the bacterial cell inoculated. Because the bacteria begins to grow at the normal rate, when the nitrofuranes in the medium is almost reduced by the bacteria. This mechanism of the bacteriostatic effect is essentially the same as that of microbe-static quinone such as 2-methyl-1,4-naphthoquinone6). From above mentioned considerations, it is concluded that the antimicrobial action of nitrofuranes is limited by the permeability for the microbe cell, though the mechanism of the antimicrobial effect is essentially the same as that of the microbe-static quinone. Acknowledgements. The author wishes to express his sincere thanks to Prof. Y. Sakurai of Tokyo University for his interest shown in this work. Thanks are also due to Dr. M. Fujimaki, Dr. S. Okimasu, Dr. R. Nomi and Mr. K. Shimizu for their valuable advices.

Biochemical Studies on the Mineral Components in Sake Yeast. Part V. The Relationship of the Mineral Composition of Yeast to Fermentation

[Agr, Biol. Chem. Vol. 30, No. 9, p. 925 `930, 1966] Biochemical Studies on the Mineral Components in Sake Yeast Part V. The Relationship of the Mineral Composition of Yeast to Fermentation By Tsuyoshi