Electrochemical Classification of Gram-Negative and Gram-Positive Bacteria

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1985, p. 238-242 99-224/85/8238-5$2./ Copyright C) 1985, American Society for Microbiology Vol. 5, No. 2 Electrochemical Classification of Gram-Negative and Gram-Positive Bacteria TADASHI MATSUNAGA* AND TOSHIAKI NAKAJIMA Department of Applied Chemistry for Resources, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan Received 22 January 1985/Accepted 1 May 1985 Intestinal bacteria were classified as gram-positive or gram-negative by an electrode system with a basal plane pyrolytic graphite electrode and a porous nitrocellulose membrane filter to trap bacteria. When the potential of the graphite electrode was run in the range of to 1. V versus the saturated calomel electrode (SCE), gram-positive bacteria gave peak currents at.65 to.69 V versus the SCE. The peak potentials of gram-negative bacteria were.7 to.74 V versus the SCE. Gram-negative bacteria and gram-positive bacteria were also classified based on the ratio of the second peak current to the first peak current when the potential cycle was repeated twice. The numbers of cells on the membrane filter were determined from the peak currents. It was found that the peak currents result from the electrochemical oxidation of coenzyme A in the cells of Escherichia coli and Lactobacilus acidophilus. Various electrochemical methods have been developed to classify microbial cells (1, 3, 4, 9). For example, the impedance measurement of culture broth has been proposed for the determination of viable-cell numbers (1). The electrochemical method based on the detection of hydrogen molecules produced by bacteria has been used to estimate cell numbers of members of the family Enterobacteriaceae and other organisms (9). T. Matsunaga and co-workers have performed the amperometric determination of viable cells based on the analysis of microbial respiration (3) and lactic acid production (4). However, since the cell number is indirectly measured from bacterial metabolites and oxygen, the electric signal obtained does not reflect the true cell number. An electrode system has been developed for the continuous determination of cell numbers in fermentation media (2, 5). Although this system was shown to be convenient for the continuous determination of cell populations, the mechanism of current generation was unknown, and the classification of microorganisms was impossible. Recently, a novel method for detecting microbial cells has been developed based on cyclic voltammetry with a basal plane pyrolytic graphite electrode used alone (6) or modified with 4,4'-bipyridine (7). Electron transfer between cells and the electrodes is mediated by coenzyme A (CoA) present in the cell wall. As a result, the cell numbers were determined from the peak current of cyclic voltammograms. It was also suggested that the differences in the peak potentials may be used to classify some microbial cells. In this study, an electrode system using a basal plane pyrolytic graphite electrode and a porous nitrocellulose membrane filter to trap microorganisms is described and used to classify bacteria. The electrode system is applied to intestinal bacteria which are then classified into grampositive and gram-negative strains based on cyclic voltammograms. MATERIALS AND METHODS Materials. Tryptose and Trypticase were purchased from Oxoid Ltd., London, England, and yeast extract was obtained from Difco Laboratories, Detroit, Mich. Phos- * Corresponding author. 238 photransacetylase (EC 2.3.1.8) was purchased from P-L Biochemicals, Inc., Milwaukee, Wis. Acetyl phosphate was obtained from Boehringer GmbH, Mannheim, Federal Republic of Germany. Other reagents were commercially available analytical reagents or laboratory grade materials. Deionized water was used in all procedures. Microbial cells. The microbial cells used here for electrochemical classification were Lactobacillus fermentum ATCC 9338, L. casei ATCC 393, L. acidophilus ATCC 4356, Streptococcus bovis IID 676, Streptococcus durans IID 677, Streptococcus salivarius IID 5223, Streptococcus sanguis IID 5224, Streptococcus mitis IID 685, Streptococcus equinus IID 68, Staphylococcus aureus IID 671, Staphylococcus aureus ATCC 421, Staphylococcus epidermidis ATCC 12228, Escherichia coli K-12, and Proteus vulgaris. These microorganisms were cultured aerobically at 37 C for 16 to 18 h on Rogosa agar consisting of: Trypticase, 1%; tryptose,.3%; yeast extract,.5%; KH2PO4,.3%; (NH4)3- citrate,.2%; glucose, 2%; Tween 8,.1%; cysteinehydrochloride,.2%; agar, 3%; and salt solution,.5% - (MgSO4 2H2, 11.5%; FeSO4 * 7H2,.68%; MnSO4 * 2H2, 2.4%). The ph was adjusted to 7., and the agar was autoclaved at 121 C for 15 min. Apparatus. The electrode system for the classification of microbial cells is depicted in Fig. 1. The electrode system consisted of a basal plane pyrolytic graphite electrode (surface area,.19 cm2; Union Carbide Corp., New York, N.Y.), a counter electrode (platinum wire), and a membrane filter for retaining microbial cells. Cyclic voltammograms were obtained by using a potentiostat (model HA31; Hokuto Denko), a function generator (model HB14; Hokuto Denko), and an X-Y recorder (F35; Riken Denshi). After each run, the graphite electrode was polished with emery paper (Nikken Kogyo Rodo, Tokyo, Japan). The measurement cell was an all-glass construction, approximately 25 ml in volume, incorporating a conventional threeelectrode system. The reference electrode was the saturated calomel electrode (SCE). It was separated from the main cell compartment by immersion in a glass tube terminated by a sintered glass frit. Procedure for classification of bacteria. Microbial cells were obtained from the Rogosa agar after incubation for 16 Downloaded from http://aem.asm.org/ on December 31, 218 by guest

VOL. 5, 1985 ELECTROCHEMICAL CLASSIFICATION OF BACTERIA 239 FIG. 1. (Left) A schematic diagram of the electrode system for detecting microbial cells. 1, Function generator; 2, potentiostat; 3, X-Y recorder; 4, counter electrode (platinum wire); 5, working electrode (basal plane pyrolytic graphite); 6, reference electrode (SCE); 7, microbial cells; 8, membrane filter; 9, holder. (Right) Photographs of working electrode and microbial cells on the membrane filter. Downloaded from http://aem.asm.org/ l- cu L.) 1 4) ci 3 on December 31, 218 by guest.5 1. v U.-, 1L.V A E (V vs S.C.E.) B E (V vs S.C.E.) FIG. 2. Cyclic voltammograms of (A) gram-positive bacteria (L. acidophilus) and (B) gram-negative bacteria (E. coli). The cell numbers of L. acidophilus and E. coli were 5. x 1O and 1. x 18 cells, respectively. The scan rates was 1 mv/s. The experiments were performed at ph 7. and at an ambient temperature (25 + 2 C).

24 MATSUNAGA AND NAKAJIMA APPL. ENVIRON. MICROBIOL. 1.5 LU LU- 2. LUJ LU) C--, R 1. ci~ 1-) wo. 5.67.66.65 C. al) ~ =31 4-), 1. c L) 3. 6. 1. 2. A Cell numbers (x18 cells ) B Cell numbers W(18 cells) FIG. 3. Relationship between peak current (), peak potential (), and cell numbers on the membrane filter for (A) L. acidophilus and (B) E. coli. to 18 h. Colonies on the agar were scraped off and suspended in 1 ml of.1 M phosphate buffer (ph 7.). The cell suspension was dropped onto the membrane filter (pore size,.45 j.m; Toyo Roshi Co.). Immediately, the cells were fixed on the membrane filter by filtration using an aspirator. The cells on the membrane filter were attached to the basal plane pyrolytic graphite. Cyclic voltammetry was run in the range of to 1. V versus the SCE. RESULTS AND DISCUSSION Cyclic voltammetry of gram-positive bacteria and gramnegative bacteria. Figure 2 shows the cyclic voltammograms of gram-positive bacteria (L. acidophilus) and gram-negative bacteria (E. coli). Anodic waves appeared at.68 V versus C,) -) 1.5 ~~~ ~.72.7 the SCE for L. acidophilus and.72 V versus the SCE for E. coli on the first scan in the positive direction. Upon scan reversal, no corresponding reduction peak was obtained. On the second scan, anodic waves appeared at the same potentials. However, the peak current of L. acidophilus decreased to 38% of that of the first scan, whereas that of E. coli was still 8%. Relationship between peak current and cell numbers on the membrane filter. Figure 3 shows the relationship between peak current, peak potential, and cell numbers on the membrane filter for L. acidophilus and E. coli. Linear relationships were obtained for cell concentrations below 6. x 18 for L. acidophiliis and below 2.5 x 18 for E. coli. The minimum detectable cell numbers were 1. x 18 and.5 x 18 for L. acidophiluls and E. coli, respectively. The peak LUJ LU 1- al) Cl.76 p. 72 -.68.1 / C) a1) ~ Downloaded from http://aem.asm.org/ on December 31, 218 by guest C) C) p-c- 15 2 25 Incubation time (hr) FIG. 4. Peak current per 18 cells when L. acidophilus () and E. coli () were incubated on Rogosa agar at 37 C for 8 to 25 h. ~.641-1 15 2 25 Incubation time (hr) FIG. 5. Peak potential per 18 cells when L. acidophilus () and E. coli () were incubated on Rogosa agar at 37 C for 8 to 25 h.

VOL. 5, 1985.4 ELECTROCHEMICAL CLASSIFICATION OF BACTERIA 241 1. cn -) -'.3 C: 1-4-J.2 3 CL 1. - _ w C1 ) "%, -4 ' ;2 C _< C-).5,_ I4- =s 1 2 3 4 5 1 2 3 4 5 A Sonicating time (min) B Sonicoting (min) time FIG. 6. Relationship between peak current and amount of CoA eluted in the solution when whole cells of (A) L. acidophilus and (B) E. coli were sonicated. After the cell suspension was sonicated, it was dropped on the membrane filter. The cells on the membrane filter were attached to the electrode for cyclic voltammetry, and the peak current was obtained. The CoA concentration in the filtrate was determined by the method of Stadtman et al. (8). current was reproducible with an average relative error of 4% when microbial cells from 2 Rogosa agar plates were used for the experiments. These results indicate that cell numbers on the membrane filter can be determined from the peak current of cyclic voltammetry in the.range of 1. x 18 to 6. 5< 18 cells for L. acidophilus and.5 x 18 to 2.5 x 18 cells for E. coli. The oxidation peak current increased linearly with the square root of the scan rate as expected for a diffusion controlled electrode reaction of a totally irreversible system using L. acidophilus and E. coli. The slopes of the line were.11 pua/mv112 *s-112 for E. coli in the range over 1 to 5 mv112 * S-112 and.37 jxaimv"12 S-1/2 for E. coli in the range over 1 to 5 mv1"2 S-1/2. Figure 4 shows the peak current per 18 cells when L. acidophilus and E. coli were incubated on Rogosa agar for 8 to 25 h. The peak current of E. coli was higher than that of L. acidophilus. The peak currents obtained from cells on the membrane filter were almost constant for 13 to 25 h. TABLE 1. Cl),.5, ~ Classification of gram-positive and gram-negative bacteria. As shown in Fig. 2, gram-positive (L. acidophilus) and gram-negative (E. coli) bacteria gave different peak potentials when incubated for 16 to 18 h. Figure.5 shows the relationship between peak potentials and incubation timne when L. acidophilus and E. coli were cultured on Rogosa agar for 8 to 24 h. The peak potentials of L. acidophilus were in the range of.67 to.69 V versus the SCE. On the other hand, E. coli gave peak potentials at.71 to.73 V versus the SCE. The peak potentials and the first and second peak current values of various intestinal bacteria are given in Table 1. Gram-positive bacteria such as L. fermentum, L. acidophilus, L. casei, Streptococcus equinus, Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguis, Staphylococcus aureus, and Staphylococcus epidermidis gave peak currents at.65 to.69 V versus the SCE. The peak potentials of gram-negative bacteria such as E. coli and Peak potentials and first and second peak currents of various intestinal bacteria Peak current (j±a/18 cells) Peak Strain pvs the SCE) Scan 1 (A) Scan 2 (B) C - 5. 5 2= - w acoo C.-) 2.5 4o.E C_ B/A (%) Downloaded from http://aem.asm.org/ on December 31, 218 by guest Gram-stain positive Lactobacillus fermentum.66 ±.2.27.12 44 Lactobacillus acidophilus.66 +.2.21.8 38 Lactobacillus casei.66 ±.1.21.8 38 Streptococcus equinus.66 ±.1.19.4 47 Streptococcus mitis.65 ±.1.27.9 33 Streptococcus salivarius.66 +.1.42.15 36 Streptococcus sanguis.68 +.1.45.12 27 Staphylococcus aureus.67 ±.1.33.13 38 Staphylococcus epidermidis.68 ±.1.47.19 4 Gram-stain negative Escherichia coli.71 ±.1 1.25.99 8 Proteus vulgaris.72 ±.2.81.74 91

242 MATSUNAGA AND NAKAJIMA P. vulgaris were.7 to.74 V versus the SCE. Gramnegative and gram-positive bacteria can be classified from the peak currents in intestinal bacteria. The first peak current per 18 cells of gram-positive bacteria was.19 to.47 p.a, which was lower than that of gram-negative bacteria (.74 to.99 p.a). Moreover, the peak current of grampositive bacteria decreased to 27 to 47% of the first peak current on the second scan. On the other hand, the second peak current of gram-negative bacteria retained 8 to 91% of the first peak current. It is also possible to classify gramnegative and gram-positive bacteria by using the ratio of the second peak current to the first peak current. Mechanism of electrochemical classification. Recently, it was found that an electron transfer between cells and the graphite electrode is mediated by CoA present in the cell wall of Saccharomyces cerevisiae (6, 7). Therefore, the relationship between peak current and the amount of CoA eluted in the solution was studied when whole cells of L. acidophilus and E. coli were sonicated (Fig. 6). CoA was enzymatically detected in the exudate solution by the method of Stadtman et al. (8). The concentration of CoA in the exudate solution increased as the peak current decreased. The amount of CoA in the eluent from L. acidophilius increased from.3 to.9 nmol/18 cells, and that from E. coli increased from 1.4 to 5.7 nmol/18 cells. The decrease in CoA content in the cell was also determined after sonication of cells. The CoA content of L. acidophilus decreased from 2.3 to 1.7 nmol/18 cells, and that of E. coli decreased from 7. to 2.7 nmol/18 cells. These results support the idea that CoA present in the cell wall also mediates an electron transfer between the graphite electrode and the L. acidophilus and E. coli cells. Further developmental studies are in progress in our laboratories to determine and classify various species of microorganisms by electrochemical techniques. ACKNOWLEDGMENTS APPL. ENVIRON. MICROBIOL. This work was partially supported by grant-in-aid for scientific research no. 5985134 from the Ministry of Science and Culture. We thank Y. Kawai and N. Suegara, Advance Research and Development Co., for supplying intestinal bacteria and helpful suggestions. LITERATURE CITED 1. Hadley, W. K., and G. Senyk. 1975. Early detection of microbial metabolism and growth by measurement of electrical impedance, p. 12-21. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. 2. Matsunaga, T., I. Karube, T. Nakahara, and S. Suzuki. 1981. Amperometric determination of viable cell numbers based on sensing microbial respiration. Eur. J. AppI. Microbiol. Biotechnol. 12:97-11. 3. Matsunaga, T., I. Karube, and S. Suzuki. 1979. Electrode system for the determination of microbial populations. Appl. Environ. Microbiol. 37:117-121. 4. Matsunaga, T., I. Karube, and S. Suzuki. 198. Electrochemical determination of cell populations. Eur. J. Appl. Microbiol. Biotechnol. 1:125-131. 5. Matsunaga, T., I. Karube, N. Teraoka, and S. Suzuki. 1982. Determination of cell numbers of lactic acid producing bacteria by lactate sensor. Eur. J. Appl. Microbiol. Biotechnol. 16: 157-16. 6. Matsunaga, T., and Y. Namba. 1984. Detection of microbial cells by cyclic voltammetry. Anal. Chem. 56:798-81. 7. Matsunaga, T., and Y. Namba. 1984. Selective determination of microbial cells by graphite electrode modified with adsorbed 4,4'-bipyridine. Anal. Chim. Acta 159:87-94. 8. Stadtman, E. R., G. D. Novelli, and F. Lipman. 1951. Coenzyme A function in and acetyl transfer by the phosphotransacetylase system. J. Biol. Chem. 191:365-376. 9. Wilkins, J. R., R. N. Young, and E. H. Boykin. 1977. Multichannel electrochemical microbial detection unit. Appl. Environ. Microbiol. 35:214-215. Downloaded from http://aem.asm.org/ on December 31, 218 by guest