NONSPOREFORMING, ANAEROBIC BACTERIA'

THE FERMENTATION OF GLUCOSE BY CERTAIN GRAM-POSITIVE, NONSPOREFORMING, ANAEROBIC BACTERIA' New York State Agricultural Experiment Station, Geneva, N. Y. Received for publication June 30, 1945 The identity of the various nonsporeforming, gram-positive, anaerobic, and microaerophilic bacteria present in the human intestinal tract has puzzled bacteriologists for years. In general, it appears that a certain type may predominate, depending upon the particular condition existing at the time. Eggerth (1935) described eleven species of gram-positive rods from human feces and concluded that they differed sufficiently from the gram-negative species of the genus Bacteroides to justify classifying them either in the genus Lactobacillus or in a separate genus. Weiss and Rettger (1938) agreed that the genus name Bacteroides should be reserved for the gram-negative organisms and that some of the gram-positive types should be included in the genus Lactobacillus. Orla-Jensen, Orla-Jensen, and Winther (1936) observed that one group or type produced about half acetic acid and half dextro rotatory lactic acid. They suggested that this type was Bacterium bifidum (Bacillus bifidus Tissier). Still more recently, the generic name Butyribacterium, type species Butyribacterium rettgeri, has been suggested by Barker and Haas (1944) for those types that produce butyric and acetic acid. They base their new genus on a culture of Lactobacillus bifidus, type II, of Weiss and Rettger (1938) and Lewis and Rettger (1940), and think this culture possibly identical with Bacillus cadaveris butyricus of Buday (1898). However, Pederson (1937) pointed out that Eggerth's (1935) cultures of Bacteroides bifidus did not produce butyric acid and that other species described by him did produce higher volatile acids. Since this report on Eggerth's cultures appeared only in abstract form, it seems desirable to present more fully the data collected. METHOD OF STUDY Cultures were obtained from Eggerth which represented 10 of the 12 grampositive types described by him (1935). They were grown on a brain heart medium under anaerobic conditions. After a preliminary study of their characteristics, a broth consisting of 0.5 per cent peptone, 0.3 per cent yeast extract, 3.0 per cent glucose, and an excess of calcium carbonate was prepared. This broth was placed in Erlenmeyer flasks, sterilized at 15 pounds' pressure, and, when cooled, inoculated with 10 ml of a culture actively growing in the brain heart medium. The flasks of inoculated broth were incubated a month at 37 C with occasional stirring so that the calcium carbonate would neutralize any 'Approved by the director of the New York State Agricultural Experiment Station for publication as Journal Paper no. 642, June 21, 1945. 475

476 acids formed. After a month of incubation, the broths were analyzed for sugar, alcohol, and volatile and nonvolatile acid, using the methods described by Pederson, Petersen, and Fred (1926). Duclaux constants were determined for the volatile acids. RESULTS Eleven of the 12 cultures grew fairly well and produced lactic and volatile acids as fermentation end products. The total acid produced by the different cultures represented from 54 to 101 per cent of the sugar fermented (table 1). Four cultures produced a high percentage of volatile acid, but only one culture produced alcohol. It should be noted that Eggerth described several of these TABLE 1 The volatile and nonvolatile acid production by certain gram-positive, anaerobic bacteria of intestinal origin nonsporeforming VAOLATILE VOLAT ACMNONVOLATIL ACID ACID AS LACTIC WATZR 0F EGOERTH'S ALCO- AS ACETIC ACED ACID CRYSTALLI- EGGERTE'S SPZECII SPECIES DESIGNATION CUTURmZ ROL ATION OF ROTATION ZINC Grams Per Grams Per cent LACTATE grains cent per cola Bacteroides biformis Helen G 0.453 0.120 5.6 1.21 55.8 18.09 Bacteroides limosus... Barney 2 0.031 0.387 32.2 0.266 22.2 13.43 Bacteroides pseudoramosus Schafer 0.008 0.114 4.6 2.430 86.5 13.43-7.0 D Bacteroides avidus. John 2 0.008 0.602 40.0 0.219 14.4 Bacteroides bifidu. Larsen 6 0.017 1.120 43.9 1.328 62.1 13.25-7.2 D Bacteroides cornutus Frank 0.004 0.034 1.1 2.34 77.4 18.09 Bacteroides bifidus... Egg. 2 0.036 0.161 5.6 2.203 77.4 13.07-7.1 D Bacteroides bifidu. Egg. 2 0.027 0.155 5.0 2.310 75.0 12.96 Bacteroides aerofaciens... Lucille 4 0.028 0.148 5.2 2.112 74.1 13.17-7.3 D Bacteroides tortuosus. elen C 0.018 0.120 4.3 2.309 81.8 13.16-7.3 D Bacteroides catenaformis.. Conboy 5 0.041 0.116 7.7 0.836 55.3 13.48 +7.2 L Bacteroides bifidus. Lucille 1 0.003 0.611147.2 0.825 63.8 13.85-7.3 D cultures as gas producers, so that some of the sugar was probably converted to carbon dioxide. Inactive lactic acid was produced by the cultures of Bacteroides biformis and Bacteroides cornutus, levo rotatory lactic acid by those of Bacteroides catenaformis, and dextro rotatory lactic acid by other cultures. Four cultures produced a high percentage of volatile acid, and in two cases the volatile acid contained a high percentage of the higher fatty acids, presumably butyric acid (figure 1 and table 2). If it is assumed that acetic and butyric acids only were present, the proportions would be about 1 acetic to 3 butyric produced by Bacteroides limosus, and 1.5 to 1 by Bacteroides avidus, estimated from the Duclaux tables. The Duclaux constants in other cases indicated the presence of small amounts of volatile acids other than acetic acid. This is apparently somewhat variable since duplicate determinations in three cases gave slightly different constants.

FERMENTATION OF GLUCOSE BY CERTAIN GRAM-POSITIVE BACTERIA 477 1a 8 1. AceticAcid 2. Butyric Acid 3. Bacteroides biformis (Helen G) 4. Bacteroides limosus (Barney 2) 5. Bacteroides avidus (John 2) 6. Bacteroides bifidus (Larsen 6) 50 ml of Aistilate FIG. 1 Duclaux Constants For Acid From: 7. Bacteroides bifidw (Egg. 2) 8. Bacteroides pseudoramosus (Schafer) 9. Bacteroides tortuosus (Helen C) 10. Bacteroides aerofaciens (Lucille 4) 11. Bacteroides cornutus (Frank) 12. Bacteroides bifidus (Lucille 1) DISCUSSION Considering the characteristics presented by Eggerth (1935) and the results of this analysis of the end products produced from the fermentation of glucose, there seems little doubt that Eggerth's culture of Bacteroides bifidus, strain E2, might be considered as belonging to the genus Lactobacillus. Weiss and Rettger (1934) considered their Lactobacillus bifidus type I almost identical with Lacto-

478 bacillus bifidus, and later (1938) showed the close relationship of their type to the Thermobacterium intestinale of Orla-Jensen, Orla-Jensen, and Winther (1936) and to Eggerth's Bacteroides bifidus group I. However, culture E2, reported upon here, was one of Eggerth's group 2 cultures rather than a group 1 culture. This confusion is apparently due to the fact that Eggerth's grouping was made on the basis of sugar fermentations rather than upon the more important physiological character of fermentation end products. Eggerth's cultures Larsen 6 and Lucille 1 both produced dextro rotatory lactic acid and a high percentage of: volatile acid. However, this volatile acid is primarily acetic acid, and therefore these cultures should not be considered as strains of the genus Butyribacterium. Rather they may be similar to the type described by Orla-Jensen, Orla- TABLE 2 Duclaux constants for volatile acid produced by various strains of gram-positive, nonspore- _ forming bacteria RACTION 1 2 3 4 5 6 7 8 9 10 Acetic acid.......7.4 15.3 23.6 32.3 41.4 61.2 61.7 72.9 85.4 100.0 Butyric acid. 17.6 33.6 47.5 60.0 70.6 79.5 86.5 92.5 97.0 100.0 Bacteroidesbiformis (Helen G). 7.2 14.7 22.4 30.9 39.7 49.2 59.3 70.7 83.8 100.0 Bacteroidesbiformis(Helen G). 7.5 15.3 23.5 32.0 41.0 50.7 61.0 72.2 84.9 100.0 Bacteroideslimosus (Barney 2).. 14.5 28.3 41.1 52.7 64.4 72.1 80.9 88.2 94.5 100.0 Bacteroides avidus (John 2). 11.3 22.5 33.4 44.1 54.4 64.5 74.2 83.4 92.2 100.0 Bacteroides bifidu8 (Larsen 6)... 7.7 15.0 24.5 33.4 42.8 52.5 63.0 74.2 86.8 100.0 Bacteroides bifidus (Egg. 2)... 7.3 14.9 22.8 31.2 39.8 49.0 58.9 70.1 83.2 100.0 Bacteroides bifidus (Egg. 2)... 7.8 15.8 23.9 32.5 41.4 50.5 60.7 71.8 83.9 100.0 Bacteroides pseudoramosus (Schafer)... 8.4 16.0 23.5 31.1 39.5 48.0 57.8 68.5 81.7 100.0 Bacteroides pseudoramosus (Schafer)... 8.1 16.2 24.4 33.1 42.3 51.7 62.0 73.0 85.3 100.0 Bacteroides tortuosus (Helen C). 7.8 15.4 23.3 31.4 39.8 48.7 58.5 69'.7 82.7 100.0 Bacteroides aerofaciens (Lucille 4)... 6.8 14.1 21.7 30.0 38.7 49.0 58.4 70.1 83.6 100.0 Bacteroides cornutus (Frank)... 7.9 15.6 23.7 32.1 41.0 50.4 60.2 71.4 83.9 100.0 Bacteroides bifidus (Lucille 1)... 7.3 15.0 23.2 31.7 40.8 50.5 60.9 72.2 85.4 100.0 Jensen, and Winther (1936) as Bacterium bifidum. It seems unlikely that these cultures are similar to the strains of Lactobacillus bifidus type II of Weiss and Rettger, which were studied by Barker and Haas. Neither Orla-Jensen, Orla- Jensen, and Winther nor Weiss and Rettger reported Duclaux constants for the volatile acids produced by their cultures, but the former stated that half the acid was acetic acid and that the lactic acid was dextro rotatory. Cultures Barney 2 of Bacteroides limosus and John 2 of Bacteroides avidus of Eggerth appear to be closely related to Butyribacterium rettgeri. They ferment several sugars and produce gas and volatile acid of which butyric acid is a main component. It is to be noted that the amount of lactic acid recovered is very low, in fact, too low to obtain a specific rotation. The relationships of the other strains studied are not clear. None of them

FERMENTATION OF GLUCOSE BY CERTAIN GRAM-POSITIVE BACTERIA 479 produced a marked change in hydrogen ion concentration, but four of the strains produced a high percentage of lactic acid on the basis of sugar fermented. Unlike the lactobacilli, Bacteroides pseudoramosus forms indole, reduces nitrate, and liquefies gelatin. Bacteroides cornutus is slightly hemolytic. Possibly these six types are forms intermediate between the true, nonproteolytic, sugar-fermenting lactobacilli of the normal intestine and the more or less proteolytic and parasitic types of gram-positive, nonmotile, nonsporeforming rods. SUMMARY The end products of fermentation produced by eleven cultures of gram-positive, anaerobic, nonsporeforming species of the genus Bacteroides described by Eggerth have been determined. One of these cultures may be regarded as belonging in the genus Lactobacillus, two are related to Butyribacterium rettgeri Barker and Haas, and two others are related to Bacterium bifidum of Orla-Jensen, Orla-Jensen, and Winther. The six remaining cultures of Eggerth are types intermediate between the true, sugar-fermenting, nonproteolytic lactobacilli and the more or less proteolytic types found in pathologic conditions. REFERENCES BARKER, H. A., AND HAAS, V. 1944 Butyribacterium, a new genus of gram-positive, nonsporulating anaerobic bacteria of intestinal origin. J. Bact., 47, 301-305. BUDAY, K. 1898 Zur Kenntnis der abnormen postmortalen Gasbildung. Zentr. Bakt. Parasitenk., I, 24, 369-375. EGGERTE, A. H. 1935 The gram-positive, non-spore-bearing anaerobic bacilli of human feces. J. Bact., 30, 277-299. LEwIs, K. H., AND RETTGER, L. F. 1940 Non-sporulating anaerobic bacteria of the intestinal tract. I. Occurrence and taxonomic relationships. J. Bact., 40, 287-307. ORLA-JENSEN, S., ORLA-JENSEN, A. D.,'AND WINTHER, 0. 1936 Bacterium bifidum und Thermobacterium intestinale. Zentr. Bakt. Parasitenk., II, 93, 321-343. PEDERSON, C. S., PETERSON, W. H., AND FRED, E. B. 1926 The forms of lactic acid produced by pure and mixed cultures of bacteria. J. Biol. Chem., 68, 151-164. PEDERSON, C. S. 1937 The fermentation of glucose by certain gram-positive Bacteroides. J. Bact., 33, 450. WEIss, J. E., AND RETTGER, L. F. 1934 Lactobacillus bifidus. J. Bact., 28, 501-518. WEIss, J. E., AND RETTGER, L. F. 1938 Taxonomic relationships of Lactobacillus bifidus (B. bifidus Tissier) and Bacteroides bifidus. J. Infectious Diseases, 62, 115-120.