Stewart A. Koser

Aspartic Acid as a Partial Substitute for the Growth-Stimulating Effect of Biotin on Torula cremoris.

Conclusion The omission of biotin resulted in a pronounced delay of growth of a stock laboratory strain of Torula cremoris in an ammonium phosphate-glucose-inorganic salt medium. Under these conditions 1(—)- aspartic acid or dl-aspartic acid, when supplied in place of biotin but in much larger amounts, produced a distinct stimulation of growth. A less marked stimulation was caused by glutamic acid. Seventeen other amino acids did not produce this effect. Pimelic acid and cysteine did not stimulate growth in the absence of biotin under the conditions of these tests.

Nicotinic acid as an essential growth-substance for dysentery bacilli.

Recent evidence has shown that nicotinic acid is a compound of considerable biologic importance. Current interest in this compound can be attributed primarily to the work of Warburg, 1 Euler 2 and their associates, which demonstrated that the amide of nicotinic acid is a constituent of the coenzyme from horse blood and the cozymase of yeast. To those interested in the nutritive requirements of microörganisms this work has been of considerable significance as it supplied a clue to the chemical identity of one of the essential substances, or “growth-factors,” needed for development of certain of the more exacting bacteria. In studies of Staphylococcus aureus Knight 3 demonstrated that a combination of nicotinic acid and vitamin B1 (thiamin chloride) was effective in replacing a concentrate prepared from yeast. Neither substance would suffice in the absence of the other, but when both were supplied the staphylococcus developed in a culture-medium containing only known compounds. For growth of the diphtheria bacillus Mueller 4 found that nicotinic acid could replace one of several fractions obtained from liver. A combination of nicotinic acid, beta-alanine and, for some strains of the organism, pimelic acid was effective in promoting growth in the absence of tissue extract preparations. 5 Nicotinic acid without beta-alanine was relatively ineffective. 5 The work reported here deals with the growth-promoting effect of nicotinic acid upon dysentery bacilli. It is of interest for two reasons: first, through the use of nicotinic acid it is possible to cultivate dysentery bacilli in a solution of known chemical compounds and second, nicotinic acid alone is strikingly effective without the addition of any other “accessory” factor.

Comparative activity of nicotinic acid and nicotinamide as growth factors for microorganisms.

Summary Nicotinic acid and nicotinamide do not always produce equivalent growth-promoting effects upon microörganisms. A series of microörganisms is presented showing a ratio of activity of amide to acid varying from one-tenth to infinity. The last case is represented by certain organisms of the Pasteurella group whose growth and respiration is promoted by nicotinamide, but not by nicotinic acid. A further summary is presented of synthetic abilities of different microörganisms with respect to the entire enzyme-coenzyme complex.

Inhibition of Respiration of Dysentery Bacilli by Sulfapyridine.

Summary It has been found that sulfapyridine in concentrations of 30 mg % inhibits the stimulation of respiration due to nicotin-amide. This inhibition is apparently competitive since the sulfapyridine must be added prior to the addition of the nicotinamide to obtain this effect. While percentage inhibition has varied in different experiments, sufficient experimental data have been obtained so as to leave no doubt as to the validity of the inhibition.

The Amounts of Folic Acid and Vitamin B6 in Saliva

THE vitamin content of saliva is of interest from a number of standpoints. The present work is concerned with the amounts of two of the vitamins which influence growth of some oral lactobacilli. It was prompted by information now available concerning which of the vitamins are needed for growth of these microorganisms and the limitations imposed upon their growth and acid production by suboptimum levels of a required vitamin. Does saliva contain enough of each of the vitamins required by oral lactobacilli to support sufficient growth and acid production for attainment of a pH level in the 4.0 to 4.5 range? General experience with the several lactobacilli used for microbiologic assays and also information from a detailed study of some oral lactobacilli have shown that very small amounts of required vitamins often are sufficient to support rather extensive cell metabolism, growth, and acid production. Although these studies apply more particularly to pure cultures in laboratory media where ample supplies of other nutritive substances are available, the information is nevertheless of some interest from the standpoint of activity of these organisms in the mouth where numerous other factors may influence multiplication of microorganisms. Few attempts have been made to study the vitamin content of saliva, particularly with respect to those vitamins needed by lactobacilli. Kniesner, Mann, and Spies2 found that the pantothenic acid content of saliva of fifty-one persons averaged 0.088 [tg per milliliter. Glavind, Granados, Hansen, Schilling, Kruse, and Dam3 studied the saliva of eight young adults; the amounts of vitamins of the B group which they list, in terms of micrograms per milliliter of saliva, are: thiamine as the hydrochloride 0.007, riboflavin 0.05, nicotinic acid 0.03, pantothenate as the Ca salt 0.08, biotin 0.0008, folic acid 0.0001, and pyridoxine as the hydrochloride 0.6. In a later study, Granados, Glavind, Noer, and Dam4 reported the presence of vitamin B12 in two samples of human saliva; one contained 0.005 /ug per milliliter and the other 0.0015 jig. Since the amounts of folic acid and of pyridoxine reported for saliva seemed somewhat out of line with the relationship in amounts of the vitamins as reported for various tissues and blood, it was decided to investigate further the salivary content of these two vitamins. The results form the subject of ths report.

Vitamin Requirements of Oral Lactobacilli

IN CONSIDERING the relationship of oral bacteria to conditions arising in the mouth, the advantage of a thorough knowledge of metabolism of the microorganisms often has been apparent. One important phase of microbic metabolism is the vitamin requirement. It is now well established that certain bacteria are unable to synthesize some of the vitamins needed in their metabolic processes. This is important from the standpoint of development of the organism, for if a needed vitamin or a satisfactory substitute (of which there are few known) is lacking, cell metabolism and multiplication stop even when other environmental conditions are appropriate. When the vitamin is supplied these processes are carried on readily. Thus it happens that growth of such bacteria is dependent upon a supply of the preformed vitamin, and also that the extent of growth and of resultant metabolic products, such as acid produced from sugars, often is proportional within certain limits to the concentration of the vitamin. Since the oral lactobacilli are associated prominently with the various process, it was deemed desirable to know more of their nutritive requirements and particularly of the vitamin requirement. Previous studies of other workers have given some insight into this problem. Hill and Kniesner1 reported that 9 strains of oral lactobacilli all needed pantothenic acid and that the amount of panthothenic acid in human saliva is usually sufficient for optimal growth of these organisms. Weisberger and Johnson2 found that thiamine, pantothenic acid, nictotinic acid, and tryptophane were needed by an oral lactobacillus. When these substances were supplied the organism grew readily and produced maximal acid in a hydrolyzed casein digest-glucose-salts medium. Riboflavin, biotin, pyridoxine, folic acid, and some purines and pyrimidines were nonessential for this organism. Coolidge, Williams, Ebisch, and Hodges3 noted that nicotinic acid, pantothenic acid, biotin, thiamine, and riboflavin were important in the nutrition of several strains of oral lactobacilli which were used in a study of variation in fermentation and other metabolic properties. The current study of classification of lactobacilli reported by Rogosa, Mitchell, and Fitzgerald4 promises to supply much needed information. These workers used many oral strains and included the vitamin needs of the organisms along with other characteristics used for separating different types or species. From their preliminary report it is evident that pantothenic acid, nicotinic acid, biotin, folic acid, thiamine, riboflavin, vitamin B6, and vitamin B12

Quantitative response of the dysentery bacillus to nicotinamide and related compounds.

Summary By means of a titration method for estimation of bacterial growth it was found that growth is proportional to the quantity of nicotinamide present. Nicotinamide is more active than an equivalent amount of either pyridine-containing coenzyme. Hydrolysis increases the activity of the latter, indicating that the function of nicotinamide is not based entirely on synthesis to either of the known coenzymes. A method has been developed for determining nicotinamide and related substances in blood. The values obtained are higher if autoclaved blood is used.

Growth and acid production by oral lactobacilli in the presence of varying amounts of required vitamins.

ECENT studies 2 of vitamin requirements of oral lactobacilli have shown that the needs of many cultures can be met by supplying from 4 to 6 of the B vitamins. In a series of 26 such cultures studied in this laboratory it was found all required biotin, nicotinic acid, pantothenic acid, and either riboflavin or thiamine; some also required pteroylglutamic acid (folic acid), vitamin B6, or both vitamin B6 and pteroylglutamic acid. These last 2 compounds at times accelerated growth when not an absolute requirement. In the series of 26 cultures examined, an absolute need for vitamin B,,, thymidine, phosphorylated vitamin B6, or unidentified factors was not encountered. However, it is known that some oral lactobacilli need one or more of these substances,2 as do occasional lactobacilli obtained from other sources.3 Since a suboptimum amount of one required vitamin limits both growth o f the microorganism and the products of cell metabolism, even when all other vitamin and nutritive needs are fully met, the question arises as to how much acid would be produced by oral lactobacilli when any one of the required vitamins happens to be present in only small quantities. The usual studies of vitamin requirements do not supply much information on this point since often the vitamin is added somewhat in excess in the endeavor to secure optimum nutritive conditions for abundant growth. However, the use of microorganisms for vitamin assay procedures and especially the use of certain strains of lactobacilli of the L. case and L. arabinosus types has supplied some information of the metabolic response in relation to the amount of needed vitamin in the environment, at least for the cultures which have now become more or less standard for use in microbiological assay procedures. In the present work particular attention was devoted to the amounts of each vitamin capable of supporting acid production at a level which might be significant with respect to dental caries. The lactobacilli employed were from the collection of cultures previously used in the study of vitamin requirements and had been isolated originally either from saliva of persons with active caries, from plaques, or from cavities.

Vitamin Requirements of Torula crmoris.

Summary Nicotinamide, biotin, calcium pantothenate, and thiamin must be supplied for prompt and abundant growth of Torula cremoris in a basal medium of inorganic salts, glucose and ammonium phosphate or amino acids. The combination of nicotinamide and biotin supports slow growth and under certain conditions nicotinamide alone may suffice. Tests with the 4 vitamins singly and in various combinations show that the response to the vitamins depends to some extent upon the basal medium used in the tests. The results emphasize the importance of the composition of the basal medium in studies of vitamin requirements.

Pyridine Derivatives and Other Compounds as Growth-Promoting Substances for Dysentery Bacilli.∗

Summary Vitamin B6, nicotinamide methiodide, pyrazine monocarboxylic acid and pyrazine 2,3-dicarboxylic acid showed no growth-promoting effect for dysentery bacilli when added to a basal synthetic medium. Thiazole 5-carboxylic acid and its amide exerted a low order of growth-promoting activity. The use of purified quinolinic acid in 1 × 10-4 molar concentration was uniformly followed by culture development. This may have been due to gradual conversion to nicotinic acid. Heating the quinolinic acid solution greatly increased its activity. The efficacy of quinolinic acid in curing pellagra may be due to contamination with small amounts of nicotinic acid.