organisms isolated from fermenting substances no characters PLANTARUM (ORLA-JENSEN) BERGEY

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1 A STUDY OF THE SPECIES LACTOBACILLUS PLANTARUM (ORLA-JENSEN) BERGEY ET AL.1 CARL S. PEDERSON2 New York State Agricultural Experiment Station, Geneva, New York Received for publication, November 5, 1935 In systematic work it is essential that the characters used to define species shall be of such a nature that at least the majority of the strains can be properly and readily identified. In only relatively few cases have descriptions of species of bacteria been sufficiently comprehensive to include even known variations and, as a result, new strains or variants of species are frequently described as new species. For such reasons it was found difficult to arrange a key for the species of the genus Lactobacillus for Bergey's Manual of Determinative Bacteriology (1934). Many so-called species could not be separated from one another. This situation was particularly evident in those species of non-gasproducing organisms normally found in plant materials or in soil. In a study of over four hundred strains of non-gas-producing organisms isolated from fermenting substances no characters were observed which could satisfactorily be used to separate the cultures into groups that corresponded to described species. It seemed, therefore, that nearly all of the descriptions that have been given are not only too incomplete, but are also too restricted in nature and that more inclusive descriptions should be used. The description of the species Streptobacterium plantarum Orla- Jensen (1919) syn. Lactobacillus plantarum Bergey et al., is the 1 Approved by the Director, New York State Agricultural Experiment Station for publication as Journal Paper No The author wishes to acknowledge the many helpful criticisms and suggestions made by Drs. E. B. Fred and W. H. Peterson of the University of Wisconsin, Dr. J. M. Sherman, New York State College of Agriculture, and Dr. R. S, Breed, New York Agricultural Experiment Station. 217 JOBUNAL OF BACTZRIOLOGY, VOL. 31, NO. 3

2 218 CARL S. PEDERSON first one given in this group that is sufficiently general to be satisfactory. The name was applied by Orla-Jensen to a series of strains of bacteria isolated from butter, milk, cheese, fermenting potatoes, beets, cabbage and dough. Complete fermentation characters on forty-four strains were presented. Fermentation usually varied somewhat with the source of nitrogen. Inactive lactic acid was produced by the majority of strains, although a few produced an excess of dextro-lactic acid. Studies on changes produced in nitrogenous foods show that the strains do not split the proteins to any degree. More or less complete descriptions of other characters were given. The species differs but slightly from Streptobacterium case Orla-Jensen which is described, in contrast to Streptobacterium plantarum, as producing dextrolactic acid and as usually preferring lactose to maltose and sucrose. Variations in the sugar fermentations are the only characters of a sufficiently constant nature to raise the question of establishing more than one species in the group of cultures included under the name Streptobacterium plantarum. When the results secured by Orla-Jensen are summarized, it is found that if the group is split, it should be on the basis of the fermentation of the sugars xylose, arabinose, or raflinose. If these sugars are selected for use in separating the strains into species, there would be as many species as there are possible combinations of characters. Strains that produce a vigorous xylose fermentation can be separated into 67 per cent weak and 33 per cent strong arabinose fermenters, or into 17 per cent weak and 83 per cent strong raffinose fermenters. Strains of vigorous arabinose fermenters can be separated into 74 per cent weak and 26 per cent strong xylose fermenters, or into 24 per cent weak and 76 per cent strong raflinose fermenters. Variations are noted in the fermentation of these sugars. Over four hundred strains of non-gas-producing, short-rod forms from various fermenting substances have been used in our study. These studies have included morphology under different growth conditions, growth characters, resistance to heat and to

3 LACTOBACILLUS PLANTARUM (ORLA-JENSEN) BERGEY ET AL. 219 salt, hydrogen ion relationships, temperature relationships, products of fermentation, and ability to ferment carbon compounds. Morphology varied with conditions of growth and under like conditions the majority of strains were the same. Although other differences were noted, the greatest variation in characters was found to occur in ability to ferment sugars. Every gradation from a definite failure to ferment to a definite 60 s ~20 FIG. 1A. FREQUENCY OF ACID PRODUCTION FROM VARIOUS CARBON COMPOUNDS BY STRAINS OF LACTOBACILLUS PLANTARUM EXPRESSED AS PER CENT OF STRAINS PRODUCING FROM 0 TO 9 CC. OF ACID IN 10 CC. OF MEDIUM Ten spaces used for each sugar signify in the first block per cent of strains producing no acid, the second, per cent of strains producing 0.1 to 1 cc. of acid, the third space 1.1 cc. to 2.0, etc. The tenth space is used for percentage of strains producing 8.1 to 12.5 cc. positive fermentation was noted for nearly all of the carbon compounds (fig. 1, a and b). Almost all strains fermented glucose, levulose, mannose, galactose, sucrose, maltose, lactose, raffinose and salicin, and to a lesser extent, sorbitol, mannitol, dextrin and glycerol. The majority of strains did not ferment rhamnose, starch and inulin. A few strains had little or no action on arabinose, while a greater

4 220 CARL S. PEDERSON number had little or no action- on xylose. Melezitose was about the only carbohydrate in which an approach to a clear-cut positive or negative fermentation was obtained. The same range of variation occurred even where cultures were obtained from identical materials. If this group of cultures is divided into more than one species on the basis of fermentation characters, the carbon compounds so FIG. 1B. FREQUENCY OF ACID PRODUCTION FROM VARIOUS CARBON COMPOUNDS BY STRAINS OF LACTOBACILLUS PLANTARUM EXPRESSED AS PER CENT OF STRAINS PRODUrCINGE FROM 0 TO 9 CC. OF ACID IN 10 CC. OF MEDIUM1 Ten spaces used for each sugar signify in the first block per cent of strains producing no acid, the second, per cent of strains producing 0.1 to 1 cc. of acid, the third space 1.1 cc. to 2.0, etc. The tenth space is used forpercentageof strains producing 8.1 to 12.5 cc. which could be used would be melezitose and arabinose and possibly xylose and galactose; but when characters are studied carefully, little justification can be found for dividing the group. Even the fermentation of melezitose merely separates the less active strains from the more active strains. Since this sugar has not been used by previous workers and the separation has little importance, it seems more reasonable to consider this as a strain variation.

5 LACTOBACILLUS PLANTARUM (ORLA-JENSEN) BERGEY ET AL. 221 Among the strains studied, cultures have been found which agree in their characteristics with many of the closely related so-called species described. The name Lactobacillus plantarum (Orla-Jensen) Bergey et al. appears to be the most logical selection of a name for this species, even though several strains had previously been described and named. However, the name mentioned is the first one given to a well described and inclusive species. Described species which appear to be wholly or in part identical are as follows: Bacillus pabuli-acidi II Weiss3 is poorly described, and the name given is in the form of a trinomial. The species was considered to be related to Bacillus a of von Freudenreich now called Lactobacillus casei (Orla-Jensen) Holland. Possibly, strains described by Weiss were related to Lactobacillus casei rather than to Lactobacillus plantarum. Six of the so-called species described by Henneberg in 1903 appear to belong to Lactobacillus plantarum. The descriptions by Henneberg are very complete in certain respects, but incomplete in others. Descriptions differ but slightly, and variations may possibly be dependent upon growth conditions. Henneberg's species differ from one another primarily in respect to their fermentation of carbon substances. The first of these, to which he has applied a trinomial Bacillus cucumerisfermentati was later renamed Lactobacillus cucumeris by Bergey et al. in It is the only well known name and the only one of the group of which representative cultures are available. It is described as fermenting arabinose and dextrin vigorously. Two cultures received from Henneberg show no significant differences from cultures of Lactobacillus plantarum. Since the binomial Lactobacillus cucumeris was proposed later than the binomial Streptobacterium plantarum OrlaJensen 1919 (now Lactobacillus plantarum), the latter name is the valid one. Descriptions of the other species are similar. Bacillus Wortmanni is reported as fermenting alpha methyl glucoside. Bacillus Listeri shows a weak fermentation of arabinose. Bacillus Maerckeri shows a 3 Bibliographic references to all of these names will be found in Bergey (1934), pages

6 222 CARL S. PEDERSON weak action on arabinose and alpha methyl glucoside but a strong fermentation of dextrin. Bacillus Leichmanni II is an inulin fermenter, a weak arabinose fermenter but a high acid producer. Since it is described as granular, there may be some doubt whether or not this should be regarded as identical with the species Lactobacillus plantarum. Bacillus Leichmanni I and III either fail or slightly ferment arabinose, lactose and raffinose, and do not curdle milk. They seem to be more closely related to the species Lactobacillus Delbrueckii described by Leichmann as Bacillus Delbricki than to Lactobacillus plantarum. Possibly they are intermediate between Lactobacillus plantarum and L. Delbrueckii. Unfortunately the type of lactic acid formed was not determined for any of these types. Bacillus Beijerincki is a weak fermenter of arabinose and trehalose and has no action on lactose, mannitol and milk. It is a weak strain more like B. Leichmanni I and III and B. Delbricki in its sugar fermentations. From this brief summary, it may be noted that species differentations have been based primarily upon differences in sugar fermentations, although in many cases these are not definitely positive or negative. Two other species, Lactobacillus conglomeratus Beijerinck (1901) (1908) and Lactobacillus densus Beijerinck appear to be identical with Lactobacillus plantarum, but the descriptions given are very incomplete. Three other names have been given to organisms that apparently belong to this species subsequent to Orla-Jensen's description of Lactobacillus plantarum, namely Lactobacillus pentosus and Lactobacillus arabinosus as described by Fred, Peterson and Anderson and Bacterium busae-asiaticae as described by Tschekan. The latter is apparently identical with Lactobacillus plantarum. Lactobacillus pentosus and Lactobacillus arabinosus were described shortly after the publication of Orla-Jensen's work and are more properly regarded as varieties of this species, the differentiation being based on the fermentation of xylose. In an unpublished paper by Fred and Peterson (1935) complete biochemical characterization is given of non-gas producing rods from fermenting vegetables. In this manuscript, they have referred to the cul-

7 LACTOBACILLUS PLANTARUM (ORLA-JENSEN) BERGEY ET AL. 223 tures by the names Lactobacillus plantarum and Lactobacillus cucumeris. CONCLUSION It appears logical to retain the name Lactobacillus plantarum as the species name for bacteria having the following characteristics: Lactobacillus plantarum (Orla-Jensen) Bergey et al. Synonyms in whole or in part: Bacillus pabuli-acidi II Weiss, Bacillus cucumeris fermentati Henneberg, Bacillus Wortmanni Henneberg, Bacillus Listern Henneberg, Bacillus Maerckeri Henneberg, Bacillus Leichmanni II Henneberg, Bacillus Beijerincki Henneberg, Lactobacillus pentosus Fred, Peterson and Anderson, Lactobacillus arabinosus, Fred, Peterson and Anderson and Bacterium busae-asiaticae Tschekan and probably Lactobacillus densus Beijerinck and Lactobacillus conglomeratus Beijerinck. Morphology: Non-motile, Gram-positive rods, ordinarily 0.7 to 1.0 by 3.0 to 8.0 microns, occurring singly or in short chains with rounded ends. Under favorable growth conditions these organisms tend to be short rods. Under adverse conditions they tend to be longer; for example, in tomato juice agar at 450C. (Pederson, 1929). In fermenting vegetables, the organisms tend to become longer as the acidity becomes greater. The organisms are usually longer in milk than in broths. Differences in morphology are well illustrated by Orla-Jensen.4 Gelatin colonies: No growth. Gelatin-yeast extract-glucose stab.: Filiform growth. No liquefaction. Agar slant: Growth, if any, is very faint. Broth: Turbid, clearing after a few days. A few strains flocculate. Litmus milk: Acid, usually coagulated. Potato: No growth. Indol not formed. Nitrates not reduced. The majority of strains form acid in glucose, levulose, mannose, 4 See plates 41 and 45, Orla Jensen, 1919.

8 224 CARL S. PEDERSON galactose, arabinose, sucrose, maltose, lactose, raffinose, and salicin, and to a lesser extent, in sorbitol, mannitol, dextrin, glycerol, and xylose. Rhamnose, starch and inulin are usually not fermented. Lactic acid, usually inactive, with only small quantities of acetic acid and carbon dioxide, are formed in the fermentation of hexose sugars. Acetic and lactic acid are produced from the pentoses. Forms up to 1.2 per cent acid in broth. Salt tolerance: Usually grow in salt up to 5.5 per cent. Microaerophilic. Optimum temperature 30-35TC. Maximum 40TC. Minimum 100C. Thermal death temperature: 650 to 75TC. for 15 minutes. Sources from which isolated: milk, cheese, butter, kefir, feces, fermenting potatoes, beets, corn, chard, bread dough, sauerkraut, cucumber pickles, tomato pickles, cauliflower pickles and spoiled tomato products. Habitat: Widely distributed in nature, particularly in fermenting plant and animal products. REFERENCES BEIJERINCK, M. W Archives N6erlandaises de Sciences Exactes et Naturelles Haarlem, S6rie II, Tome VII, Also in Verzamelde Geschriften van M. W. Beijerinck, Delft, 4, 54. BEIJERINCK, M. W Centr. fur Bakt., II Abt., 20, 1908, BERGEY, D. H Manual of Determinative Bacteriology. 4th Ed. 664 pp. Baltimore. FRED, E. B., AND PETERSON, W. H Manuscript. ORLA-JENSEN, S MWm. de l'acad. Roy. d. Sci. et d. Lettres de Danemark, Copenhague. Sect. d. Sci., 8 s6r., 5, No. 2. PEDERSON, C. S Tech. Bulls. 150 and 151, N. Y. State Agr. Exp. Station.