Several Bacteroides Strains

APPLIED MICROBIOLOGY, Nov., 1966 Vol. 14, No. 6 Copyright @ 1966 American Society for Microbiology Printed in U.S.A. Amino Acid and Vitamin Requirements of Several Bacteroides Strains GRACE QUINTO Cumberland College, Williamsburg, Kentucky Received for publication 20 July 1966 ABSTRACT QuINTo, GRACE (Cumberland College, Williamsburg, Ky.). Amino acid and vitamin requirements of several Bacteroides strains. Appl. Microbiol. 14:1022-1026. 1966.-Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required L-arginine hydrochloride, L-tryptophan, L-leucine, L-histidine hydrochloride, L-cysteine hydrochloride, DL-valine, DL-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, DL-lysine hydrochloride, DL-isoleucine, L-prohne, L-glutamic acid, DL-alanine, DL-phenylalanine, DL-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium D-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% L-tryptophan, 0.09% L-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in L-proline, glycine, L-glutamic acid, DL-serine, L-histidine hydrochloride, DL-alanine, or L-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% DL-serine but without vitamins. Initial attempts to characterize nutritionally several Bacteroides species were hindered by an unsuspected hemin requirement, and suboptimal growth responses to different defined media (1; Quinto, Ph.D. Thesis, Univ. Kentucky, Lexington, 1960). The present paper reports the amino acid and vitamin requirements of these species, determined by the methodology and media used for anaerobic rumen microbiology. MATERLALS AND METHODS Strains. Ristella perfoetens CCI, Zuberella clostridiformis Prevot 1957, and three hemin-requiring strains of R. pseudoinsolita, C4, C-7, and C-2795, previously described by Quinto (1) and Quinto and Sebald (2), were investigated. In addition, four non-heminrequiring strains of R. pseudoinsolita, 2808, 2929, 2937, and 2982, of the Pasteur Institute collection were included. Media. The maintenance medium was similar in salts and vitamins to the complete medium of Scott and Dehority (3), except for the following modifications. The concentration of casein hydrolysate was increased from 0.2 to 2.0%; all fatty acids except acetic acid were omitted; and cellulose was replaced 1022 with 0.5% glucose. The maintenance medium was adjusted to ph 7.0, tubed in 5.0-ml amounts in disposable glass test tubes (13 by 150 mm) with cotton plugs, and autoclaved at 15 psi for 15 min. The complete assay medium was similar to the maintenance medium, except that casein hydrolysate was replaced by 20 amino acids. The following 16 amino acids were added in 0.1% concentration: L-arginine hydrochloride, glycine, L-leucine, L-proline, hydroxy-l-proline, L-histidine hydrochloride, L- cysteine hydrochloride, DL-valine, DL-alanine, DLlysine hydrochloride, DL-threonine, DL-isoleucine, DL-tyrosine, DL-methionine, DL-serine, and DLphenylalanine. Because of solubility limitations, L-cystine was added in the concentration of 0.033%; L-tryptophan, 0.044%; L-glutamic acid, 0.09%; and DL-aspartic acid, 0.085%. Amino acid assay media were prepared in whichr a single amino acid was deleted at a time. Based upon results with single amino acid deletions, a composite medium A-23, containing salts, vitamins, and all the amino acids except threonine, cystine, hydroxyproline, and aspartic acid, was prepared for R. perfoetens. Another composite medium A-20 was prepared for Z. clostridiformis and the seven strains of R. pseudoinsolita. The medium contained: salts, vitamins,

VoL. 14, 1966 NUTRITIONAL REQUIREMENTS OF BACTEROIDES 1.023 0.1% glycine, proline, arginine hydrochloride, glutamic acid, histidine hydrochloride, cysteine hydrochloride, and tyrosine; and 0.2% alanine, serine, and lysine hydrochloride. Cysteine assays were performed in medium A-23 containing 0.1% sodium thioglycolate in place of cysteine hydrochloride. Preliminary vitamin assays of R. perfoetens were made in maintenance medium prepared with single vitamin deletions and additions. These results were confirmed in medium A-23. Supplement. A carbonate-hemin-cysteine supplement was prepared with equal quantities of sterile 40.0% sodium carbonate, 10.0% cysteine hydrochloride, and 4.0% triethanolamine containing 100 my of hemin per ml. Just prior to inoculation, 0.15- ml amounts were added aseptically to 5.0 ml of medium or diluent. Anaerobiosis. Anaerobiosis was accomplished by deaerating the media in a steam bath, by including cysteine hydrochloride in the supplement, and by adding carbon dioxide gas-and sealing the tubes with sterile rubber stoppers. Preparation of the inoculum. Stock cultures in maintenance medium were prepared weekly, refrigerated, and subcultured to fresh maintenance medium with 0.1-ml inocula 48 hr before each experiment. The fresh stock cultures were incubated for 24 hr, and then subcultured to complete medium. Centrifugation of 15-hr cultures in complete medium was done at 3,000 X g for 6 min. The supernatant fluids were replaced with 5.0 ml of glucose-salts diluent, containing glucose, 0.1%; monopotassium phosphate, 0.2%; dipotassium phosphate, 0.7%; sodium citrate, 0.05%; ammonium sulfate, 0.1%; resazurin, 0.0001%; and carbonate-hemin-cysteine supplement. The sediments were mixed well by capillary pipet, incubated 7 to 8 hr at 37 C to starve the cells, and then recentrifuged. After the supernatant fluids were removed again, enough cells were pipetted to 5.0 ml of supplemented glucose-salts diluent to produce an optical density (OD) of 0.2 at 600 my in a Bausch & Lomb Spectronic-20 colorimeter. The starved, washed cells, adjusted to an OD of 0.2, were inoculated into test media in 0.1-ml amounts. Glucosesalts diluents were supplemented with carbonatehemin-cysteine mixture, except during the cysteine assay when cysteine was omitted. Assay procedure. All experiments were performed twice. Each strain was inoculated into duplicate tubes of each medium. Optical densities of duplicate cultures were recorded and averaged at 15 to 17 hr, 20 to 24 hr, 36 to 40 hr, 60 to 62 hr, 72 hr, and, if necessary, 4 days. Counts of viable cells were made according to the agar deep technique described previously (1). REsuLTs AND DIscussIoN Table 1 is a sunmation of all the single amino acid deletion experiments in which the OD values of cultures in deleted media were recorded at the time the control cultures in complete medium first showed a maximal OD of 1.0, TABLE 1. Effect of single amino acid deletions on growtha of nine Bacteroides strains Risldla pseudoinsoli*a strains erella Deletion clostridiformi-s R. perfocns 2808 2929 2937 2982 C-4 C-7 2795 Hydroxyproline... 1.40 1.50 1.40 1.40 1.50 1.50 1.30 1.35 1.20 Isoleucine... 1.40 1.25 1.05 1.25 1.30 1.45 1.10 1.45 0.30 Aspartic acid... 1.10 1.50 1.45 1.10 1.25 1.40 1.50b 1.40 1.10. Threonine...1.40 1.50 1.20 1. 50 1.45 1.25 1.50Jo 1.40 1.45b Tyrosine... 1.30 1.50 1.50 1.30 1.25 1.40 1.50 1.40 0.03c Glutamic acid... 1.50 1.45 1.10 1.50 1.45 1.25 1.25 0.32 0.13 Leucine... 1.20 1.40 1.05 1.45 1.20 1.75 1.30 1.30 0. 12c Glycine... 0.16 1.45 1.40 1.60 1.40 1.40 1.30 0.44 1.05 Alanine... 1.05 1.40 1.50 1.50 1.30 1.20 0.10 1.45 0.72 Phenylalanine... 1.15 1.35 1.20 1.40 1.10 1.30 1.00 1.45 0.30 Arginine... 1.25 1.40 1.00 1.00 1.00 1.30 1.20 1.40 Oc Lysine... 1.15 1.45 0.98 1.45 0.95 1.20 1.05 1.40 1.00 Valine... 1.25 1.00 0.90 1.10 1.20 1.00 0.98 1.30 0.02C Serine... 1.10 1.00 1.00 1.05 0.50 0.92 1.40 1.40 0.92 Methionine... 1.00 1.25 1.00 1.25 1.05 1.00 0.90 1.30 0.55 Proline... 0.88 0 0.11 1.20 1.30 1.25 1.60 0.40 0 Histidine... 1.20 1.25 0.22 1.45 1.10 1.25 1.15 1.35 oc Cystine and tryptophan.... 1.20 1.60 1.45 1.40 1.35 1.40 1.20 1.40 0.02. Cystine... 1.50 Tryptophantophn... 0. 17 a Optical densities were recorded at the time control cultures in complete medium first showed an OD of 1.0, usually between 20 and 24 hr. b Medium lacked aspartic acid, threonine, and hydroxyproline. c Optical densities were the same at 4 days. d Not done.

1024 QUINTO APPL. MICROBIOL. usually between 20 and 24 hr after inoculation. It was concluded that R. perfoetens required arginine, valine, tryptophan, leucine, histidine, and tyrosine, since only scant turbidity occurred in 4 days in media without one of these amino acids. Isoleucine, proline, glutamic acid, alanine, phenylalanine, and methionine stimulated R. perfoetens, as cultures in these deleted media developed maximal turbidity within 37 to 72 hr, that is, more slowly than control cultures in complete medium. All of the other strains shown in Table 1 grew to maximal turbidity in all single amino acid deletion media within 4 days, but the deletion of glutamic acid, glycine, or proline delayed the attainment of maximal turbidity of Z. clostridiformis. Similarly, for the R. pseudoinsolita strains, glycine deletion delayed the attainment of maximal turbidity of strain 2808; proline, of strains 2929 and 2937; histidine, of strain 2937; alanine, of strain 2795; and serine, of strain C-4. The 20th amino acid in each case hastened the appearance of maximal turbidity in the complete medium as compared with deleted media. Based on these results, a composite medium A-23 was designed for R. perfoetens, and a second composite medium A-20, for the other eight strains. Medium A-23 contained salts, vitamins, the 12 amino acids found to be essential or stimulating for R. perfoetens, cysteine hydrochloride for anaerobiosis, and serine, lysine hydrochloride, and glycine. Medium A-20 contained salts, vitamins, the six amino acids found to be stimulating for one or more of the other eight strains, cysteine hydrochloride, lysine hydrochloride, alanine, and tyrosine. The con-. centrations of the DL-amino acids, serine, lysine hydrochloride, and alanine in mediuml A-20 were doubled to compensate for the loss in organic nitrogen when 10 amino acids were deleted simultaneously. Although R. perfoetens grew well in medium TABLE 2. Effect of single amino acid additions to medium A-20a on the growthb ofeight Bacteroides strains Ristdla pseudoinsolita strains Additionc _l-_ ostridiformis 2808 2929 2937 2982 C4 C-7 2795 None... 0.34 1.25 0.18 0.95 0.78 0.58 0.66 0.95 Valine... 0.22 1.20 0.27 0.74 0.43 0.31 0.14 0.33 Methionine...... 1.30 1.00 0.29 0.80 0.88 0.44 0.88 0.92 Leucine....88 1.25 0.63 1.20 1.00 0.64 0.94 0.92 Phenylalanine... 0.14 0.76 0.14 0.60 0.12 0.42 0.19 0.21 Tryptophan... 0.12 0.90 0.09 0.16 0.18 0.13 0 0.22 Valine, methionine, leucine, phenylalanine, tryptophan... 0.88 1.10 0.12 1.10 1.05 0.43 0.69 1.05 Complete medium...... 1.00 1.25 0.72 1.15 0.82 0.72 0.88 0.98 amedium A-20 contained: salts; vitamins; 0.1% glycine, L-glutamic acid, L-proline, L-histidine hydrochloride, L-arginine hydrochloride, L-cysteine hydrochloride, and DL-tyrosine; and 0.2% DLlysine hydrochloride, DL-alanine, and DL-serine. b Optical densities were recorded at the time control cultures in complete medium first showed maximal turbidity. DL-Valine, DL-methionine, DL-phenylalanine, and L-leucine were added in 0.1% concentration, and L-tryptophan, in 0.044% concentration. TABLE 3. Effect of multiple amino acid additions to medium A-20a on growthb of eight Bacteroides strains Additions' Ristella pseudoinsolita strains 2808 2929 2937 2982 C4 C-7 2795 dostridiformis Methionine and leucine... 1.40 1.35 0.07 0.95 0.81 0.50 0.73 0.95 Methionine and isoleucine... 1.45 1.35 0.05 0.75 1.35 0.54 0.33 1.05 Methionine, leucine, isoleucine... 1.15 1.30 0 1.00 1.30 1.25 0.55 1.40 Complete medium...... 1.45 1.30 1.30 1.00 1.15 1.20 0.88 1.05 a For composition of medium A-20, see first footnote to Table 2. boptical densities were recorded at the time control cultures in complete medium first showed maximal turbidity. c DL-Methionine, DL-isoleucine, and L-leucine were added in 0.1% concentration.

VOL. 14, 1966 NUTRITIONAL REQUIREMENTS OF BACTEROIDES 1025 A-23, some of the other eight strains grew more slowly in medium A-20 than in complete medium. Only four strains produced maximal turbidity in medium A-20 as quickly as in complete medium (Table 2). The addition of 0.1% DL-methionine or L-leucine stimulated several strains. However, the addition of 0.044% L-tryptophan or 0.1% DL-phenylalanine delayed the development of maximal turbidity of four and six strains, respectively. When medium A-20 was prepared with TABLE 4. Effect of vitamins on the growtha of Ristella perfoetens in medium A-23b Medium Optical density A-23 without vitamins... OC A-23 with calcium-d-pantothenate and nicotinamide... 0.85 A-23 with calcium-d-pantothenate and p-aminobenzoic acid.. 0 A-23 with nicotinamide and p- aminobenzoic acid... 0.08c A-23 with calcium-d-pantothenate, nicotinamide, and p-aminobenzoic acid... 1.40 Complete medium... 1.05 a Optical densities were recorded at 24 hr when control cultures in complete medium first showed maximal turbidity. b Medium A-23 was similar to the complete medium, except that it lacked aspartic acid, hydroxyproline, threonine, and cystine. Calcium-Dpantothenate and nicotinamide were added in 0.0002% concentration. p-aminobenzoic acid was added in 0.00001% concentration. c No additional turbidity developed in 4 days. added 0.1% methionine, leucine, and isoleucine, six of the eight strains shown in Table 3 produced maximal turbidity as quickly as in complete medium. Preliminary single vitamin addition and deletion studies with R. perfoetens in maintenance medium yielded no growth without calcium-dpantothenate or nicotinamide, and only partial growth without p-aminobenzoic acid in 48 hr. R. perfoetens produced maximal turbidity in 24 hr in complete medium and in medium A-23 containing these three vitamins, but required 40 hr to produce maximal turbidity in medium A-23 containing calcium-d-pantothenate and either nicotinamide or p-aminobenzoic acid (Table 4). R. perfoetens failed to grow in 4 days in medium A-23 containing nicotinamide and p-aminoben- TABLE 5. Effect of single amino acid deletions from medium A-23a on growth6 of Ristella perfoetens Medium Optical density per Cels ml A-23 without cysteine... 0. 16c A-23 without lysine... 0.68 A-23 without glycine... 0.08 A-23 without serine... 1.25 2.2 X 109 Complete medium... 1.25 2.5 X 109 a For composition of medium A-23, see second footnote to Table 4. b Optical densities and cell counts were recorded at 15 hr when control cultures in complete medium first showed maximal turbidity. c No additional turbidity occurred in 4 days. d Not done. TABLE 6. Effect of vitamin and cysteine deletion from medium A-23a on the growth of eight Bacteroides strains Cysteine and vi.tamisn Time OD and OD when deleted Cmltemdu Co m Organism cell counts vitamins are recorded deleted OD Cells per ml OD Cells per ml Ristella pseudoinsolita Strain 2808... 15 1.50 1.25 3 X 109 1.50 1.3 X 109 Strain 2929... 15 1.50 1.35 3 X 10' 1.15 1.3 X 109 Strain 2937... 15 1.35 1.15 9 X 10' 0.94 1.2 X 109 Strain 2982... 20 1.20 1.45 9.7 X 10' 1.40 1.6 X 109 Strain C-4...... 15 1.40 1.30 1.3 X 109 1.30 3.1 X 10' Strain C-7... 15 1.45 0.456 -c 1.0 6.5 X 109' Strain 2795...... 15 0.90 1.05 2.6 X 108 0.36d 1.8 X 108 Zuberella clostridiformis.20 1.40 1.40 1.3 X 109 1.30 1.8 X 108 a Medium A-23 was similar to the complete medium, except proline, threonine, and cystine. b This OD became 1.30 at 20 hr. c Not done. d This OD became 0.81 at 40 hr. that it lacked aspartic acid, hydroxy-

1026 QUINTO zoic acid but no calcium-d-pantothenate. R. perfoetens has an absolute requirement for calcium-d-pantothenate, and is stimulated to grow more quickly by nicotinamide and p-aminobenzoic acid. The cysteine assay and confirmatory assays of serine, lysine, and glycine were performed with R. perfoetens in medium A-23 containng calcium-d-pantothenate, nicotinamide, andp-aminobenzoic acid. R. perfoetens produced only scant turbidity during 4 days of incubation in medium A-23 in which 0.1% sodium thioglycolate replaced cysteine hydrochloride (Table 5). R. perfoetens produced maximal turbidity in medium A-23 without glycine or lysine in 40 hr, compared with 15 hr in complete medium or medium A-23 without serine. R. perfoetens produced the same number of viable cells in medium A-23 without serine as in complete medium at 15 hr. These data indicate that R. perfoetens requires cysteine but not serine, and is stimulated to grow more quickly by glycine and lysine. Because two strains failed to produce rapid maximal turbidity in medium A-20 plus methionine, leucine, and isoleucine, all strains were tested in medium A-23 in which they grew optimally. Table 6 shows that eight Bacteroides strains produced maximal turbidity in 15 to 20 hr in medium A-23 without vitamins, as well as in complete medium. In medium A-23 in which 0.1% sodium thioglycolate replaced cysteine hydrochloride, all strains except C-7 produced maximal turbidity and viable cells as quickly as in complete medium. Therefore, seven Bacteroides strains are not stimulated by cysteine, and none of the eight strains requires vitamins. In summar, several defined media, supplemented with a carbonate-hemin-cysteine mixture and incubated under carbon dioxide gas, support rapid maximal growth of nine Bacteroides strains. The complete medium contains the salts and vitamins included in the complete medium of Scott and Dehority (3), as well as 0.1% L-argmine hydrochloride, glycine, L-leucine, L-proHne, hydroxy-l-proline, L-histidine hydrochloride, L- cysteine hydrochloride, DL-valine, DL-alanme, DL-lysine hydrochloride, DL-threonine, DL-iSOleucine, DL-serine, DL-tyrosine, DL-methionine, APPL. MIcROBIOL. and DL-phenylalanine; 0.033% L-cystine; 0.09% L-glutamic acid; 0.085% DL-aspartic acid; and 0.044% L-tryptophan. All nine strains of Bacteroides tested in this medium grew to maximal turbidity within 20 hr. Medium A-23, developed for R. perfoetens, is similar to the complete medium, except that it contains only three vitamins, calcium-d-pantothenate, nicotmiamide, and p-aminobenzoic acid, and all amino acids except aspartic acid, threonine, hydroxyproline, serine, and cystne. Modified medium A-23, without vitamins but containing 0.1% DL-serine in addition to the 15 amino acids mentioned above, supports optiml growth of Z. clostridiformis and seven strains of R. pseudoinsolita. Medium A-20, which supports optimal growth of Z. clostridiformis and five strains of R. pseudoinsolita, is siminla to the complete medium, except the concentrations of lysine, alanine, and serine are doubled, and valine, phenylalanine, tryptophan, aspartic acid, cystine, threonine, snd hydroxyproline are omitted. Throughout this report, emphasis has been placed on the rapid development of maximal turbidity which is of paramount importance in clinical anaerobic microbiology. In this area, a delayed or marginal growth response prevents a timely diagnosis. One of the problems encountered with fastidious nonsporulating anaerobes is loss of viability, or the inability of these anaerobes to grow in serial culture, so that they can be studied and identified. The application of the methodology of anaerobic rumen microbiology, which has facilitated the serial trnsfer of nine Bacteroides strains for 5 months, may help to solve two underlying difficulties intmhe cultivation of anaerobes, effective anaerobiosis and adequate nutrients. LrrwTuix CADm 1. QUINTo, G. 1962. Nutrition of five Bacteroides strains. J. Bacteriol. 84:559-562. 2. Quinro, G., Am M. SEBALD. 1964. Identification of three hmin-requiing Bacteroides stains. Am. J. Med. Technol. 30:381-384. 3. ScoTr, H. W., AND B. A. DEHoRrTy. 1965. Vitamin requirements of several cellulolytic rumen bacteria. J. Bacteriol. 89:1169-1175.