As Is Well Known, the Genus Lactobacillus Has Been Defined As Being Gram-Positive, Non-Motile, Non-Sporulating, Catalase-Negative and Micro- Aerophilic

J. Gen. Appl. Microbiol. Vol. 9, No. 1, 1963

SPOROLACTOBACILLUS NOV. SUBGEN.

KAKUO KITAHARA and JIRO SUZUKI Institute of Applied Microbiology, Uiniversity of Tokyo, Tokyo Received October 23, 1962

As is well known, the genus Lactobacillus has been defined as being Gram-positive, non-motile, non-sporulating, catalase-negative and microaerophilic. The organisms are rod-shaped bacteria producing lactic acid from glucose through either homo- or hetero-f ermentative pathways. One of the present authors (KITAHARA,(1)) pointed out, however, in 1940 that there are intermediate forms between the homo-f ermentative Lactobacillus and the genus Bacillus, and he named them wild lactobacilli. NAKAYAMA(2) made careful comparative studies on these organisms and classified the wild type into Bacillus coagulans calling them spore-forming lactic acid bacteria because they form spores in sugar-deficient media. This group includes Lactobacillus thermophilus, L. sporogenes, Bacillus dextrolacticus, B. thermo- acidurans, etc. The general characteristics of this group were described as catalase-positive, motile, spore-forming and (dextro-rotatory) L(+)-lactic acid forming. Recently, some investigators reported the presence of some strains that resemble normal homo-fermentative Lactobacillus in many respects except for their motility with peritrichous flagella in early growth phases. Re- presentative strains of these bacteria are L. plantarum var, mobile of HARRISONand HANSEN(3) and a variety of L. case2 of DEIBEL and NIVEN (4). The optical form of lactic acid produced by these bacteria varied with strain from L(+) to DL. During the course of studies on the distribution of micro-organisms in assorted chicken feed, the present authors isolated an unusal strain of lactic acid bacteria that is different from any of the species hitherto described, and tentatively named it strain EU. Taxonomic studies to be reported in this paper have revealed that this organisms cannot be classified into any of the known genera. The proposal was, therefore, made to class this strain as a representative of a new subgenus Sporolactobacillus.

METHODS

A medium of the following composition was used throughout this work unless otherwise noted : glucose, 2° ° ; yeast extract (Difco), 0.5%; peptone, 59 60 KITAHARA and SuzuKI VOL. 9

0.5% (GYP). Cultivation was carried out at 37°. Bacteriological determinative methods used were according to those described in Manual of Micro- biological Methods published by the American Society for Microbiology (5). Analyses of fermentation products were carried out as described earlier by KATAGIRIand KITAHARA(6). Heat resistance of vegetative cells and spores was examined by comparing the viable counts of suspension before and after heat treatments. The cells harvested from media were washed with and suspended in physiolgical saline. Heat treatment was applied by taking a small volume of cell suspension (ca. 1 ml) in a small test tube and immersing it in a water bath of desired temperature.

RESULTS

Morphology The cells of strain EU, cultured in the GYP medium, are rod-shaped with rounded ends, having an average width of 0.7'0.8 p and a length of 3'5 p. They are usually single but occasionally in pairs and rarely in short chains. The cells have granular appearance under the phase-contrast microscope. When young, they are remarkably motile with a small number of peritrichous flagella (Fig. 1). They retain their motility for more than 72 hr of incubation when the medium is buffered with sodium acetate or calcium carbonate. Strain EU forms endospores under suitable conditions (Fig. 2). The mature spores are cylindro-elliptical with dimension 0.8 x 1.0 p and occur in a terminal position in somewhat swollen sporangia. The number of spore- bearing cells is too small to be observed under the microscope when cultured in ordinary media such as the GYP-medium or B12 assay medium (Difco). Glu-

Fig. 1. A flagellated cell (x5,000) 1963 Sporolactobacillus Nov. Subgen. 61

Fig. 2. Spore-bearing cells (X 1, 500) (phase-contrast microscope) cose-yeast extract-CaCO3 medium containing a low concentration of glucose is favorable for the formation of spores. The presence of manganese ions at the concentration of 10-4 M promotes the sporulation (Table 1).

Table 1. Con ditions for spore-formation

Cultural Features Agar plates : Grayish white pin-point colonies with a diameter of less than 1 mm are formed on the GYP-agar plate ; in the presence of calcium carbonate the colony appears more distinct, being surrounded by a transparent halo formed by the action of lactic acid produced. Semi-solid agar : Larger colonies with diameters of up to 3 mm appear on semi-solid agar (agar content 0.250) indicating the motility of the strain. Agar slant : Growth is almost invisible, 62 KITAHARA and SUZUKI VOL. 9

Agar stab : Filiform growth occurs uniformly along the stab-canal without noticeable sign of surface growth (Fig. 3). Agar tube : Small colonies are formed uniformly in agar tube except for the region near the surface of the medium (Fig. 4).

Fig. 3. Agar stab Fig. 4. Deep colonies

Gelatin stab : Almost the same as in agar stab. No sign of lique- faction. Liquid culture : Young cultures are slightly turbid giving a silky lustre when shaken while older cultures are heavily turbid, the cells precipitating gradually. Growth does not occur on the surface or in the absence of glucose or other fermentable sugars. These cultural features show that strain EU is microaerophilic in nature.

Physiology Strain EU is Gram-positive, catalase-negative and does not reduce nitrates to nitrites. Indole is not formed. Litmus milk is not changed. Lactic acid is produced actively without liberation of gas from glucose, fructose, mannose, sucrose, maltose, trehalose, raffinose, inulin, mannitol, sorbitol and a-methyl glucoside. No or only slight acid production takes place when the substrate given is galactose, pentoses (xylose, D- and L-arabi- 1963 Sporolactobacillus Nov. Subgen. 63 noses, ribose, rhamnose), lactose, cellobiose, melibiose, melezitose, dextrin, dextran, starch, glycogen, glycerol, erythritol, adonitol, dulcitol, inositol, salicin, aesculin or chitin (Table 2). Noteworthy is the fact that strain EU is a lactic acid bacterium capable of fermenting inulin but not pentoses

Table 2. Fermentability of sugars

Table 3. Fermentation products 64 KITAHARAand SuzuKI VOL. 9 and lactose. Determination of fermentation products (Table 3) showed that strain EU converts glucose and inulin quantitatively and vigorously to (leavo- rotatory) D(_)-lactic acid through typical homo-fermentative pathway. In the presence of CaC03 it consumes glucose completely even when the initial sugar concentration is as high as 20% or more (Table 3). Strain EU grows readily at temperatures between 25° and 40°. The optimal temperature for growth lies around 35°, while that for acid production appears to be somewhat lower. It shows slight growth at 15° after one week, but not at 45° (Table 4).

Table 4. Effect of temperature on growth and fermentation

The vegetative cells from a 20 hr culture in the GYP-CaC03 medium are killed rapidly when heated at 50°, while spores tolerate heating at 85° for ten min. (Fig. 5 a and b). Strain EU is not very exacting in respect to growth factors. It requires only pantothenic acid and biotin as essential growth factors; stimulation of growth is brought about by p-aminobenzoic acid and nicotinic acid. As for amino acids, valine and leucine are essential. None of purine- or pyrimidine-bases is required by strain EU. Summarizing the results described above, strain EU is characterized as follows : Gram-positive, spore-forming, motile rods ; catalase-negative, microaerophilic and mesophilic. A typical homo-fermenter producing n(-)-lactic acid. These properties correspond to those of typical Lactobacillus except for the two important points, motility and spore-forming nature. Moreover, the nutritional requirement of strain EU is not so complex as that of ordinary lactic acid bacteria. 1963 Sporolactobacillus Nov. Subgen. 65

Fig. 5. Heat-resistance of vegetative cells and spores 66 KITAHARA and SUZUKI VOL. 9

Table 5. Vitamin and base requirements

Table 6. Amino acid requirements 1963 Sporolactobacillus Nov. Subgen. 67

DISCUSSION

Homo-f ermentative rod-shaped lactic acid bacteria had been classified by ORLA-JENSEN(7) into two sub-genera, namely Streptobacterium and Ther- mobacterium. According to ORLA-JENSEN, Streptobacterium and Thermo- bacterium differ not only in their temperature tolerance but also in the optical properties of lactic acid produced. The former generally produces L(+)-lactic acid or its racemized form, while the latter produces n(-)- or its racemized form. Although the opinion of ORLA-JENSENis generally valid especially as far as the thermophilic Thermobacterium is concerned, his concept about the mesophilic Streptobacterium is vulnerable to some criticisms. For example, L. leichmannii is described as a mesophilic n(-)-lactic acid former in Bergey's Manual of Determinative Bacteriology (8). However, the fact that L. leichmanii ATCC 4797 and 7380 can grow at 48° and 50° (9) makes their distinction from L. delbrueckii obscure. Similar opinion was recently published by ROGOSAand SHARPE(10). Bacillus leichmanii I of HENNEBERG(11) and L. homohiochii of KITAHARAet al. (9) produce n(-)-lactic acid, although they are mesophilic. One of the present authors demonstrated that some mesophilic DL-f ormers such as L. plantarum and L. japonics are originally n-f ormers, n-lactic acid produced being racemized by the action of lactate racemase contained in these species (12). The biochemical properties of strain EU, which is mesophilic and a strong n(-)-lactic acid former, resemble those of L. leichmanii described in Bergey's Manual, except for minor differences in their ability of fermenting mannitol, sorbitol, raffinose and salicin. L, leichmanii requires for growth various vitamins including vitamin B12, whereas strain EU requires only pantothenic acid and biotin. Motility of strain EU is observed invariably in several different media, and the cells retain the motility as long as 72 hr or more when media are buffered by sodium acetate or calcium carbonate. Strain EU differs, therefore, from hitherto described motile homo-f ermentative lactic acid bacteria which are motile only in the early growth phase or within only 10 to 20 hr of incubation. The so-called spore-forming lactic acid bacteria are as a rule thermophilic, but are different from Thermobacterium of ORLA-JENSEN. These spore-f ormers are catalase-positive, more or less aerophilic and produce invariably L(+)-lactic acid. Therefore, they should be considered to be affiliated to the genus Bacillus rather than to Lactobacillus, or to belong, as suggested by NAKAYAMA(2), to an intermediate form between Lactobacillus and Bacillus. Strain EU resmbles these bacteria in its motility and spore- forming ability, but differs from the latter in other important points such as catalase production, oxygen-requirement and the optical form of lactic acid produced (Table 7). 68 KITAHARA and SUZUKI VOL. 9

Clostridium is the only genus thus far known to include catalase- negative and spore-forming bacteria. Fermentative clostridia are anaerobic (in most cases strictly anaerobic, rarely aerotolerant) and produce from carbohydrates a large amount of gas which is composed of CO2 and H2 in addition to volatile acids. Since Embden-Meyerhof's glycolytic enzyme system is widely distributed in living organisms, it is not surprising that a certain amount of lactic acid may be found in the fermentation broth of clostridia. OSBURN,BROWN and WERKMAN(13) pointed out that Cl. but ??Jlieum produces DL-lactic acid besides its normal fermentation products when sodium

Table 7. Comparison of features of strain EU with those of the so-called spore-forming lactic acid bacteria and fermentative Clostridium 1963 Sporolactobacillus Nov. Subgen. 69 bicarbonate is added to the medium. KuBowITZ (14) and recently KATAGIRIet al. (15) reported that Cl. butylicum, which is a butyl-fermenter under normal conditions, ferments carbohydrates according to homo-lactic fermentation when hydrogen cyanide or carbon monoxide is added to the medium. Lactic acid obtained by KuBowITZ was DL+L(+)-form, while that of KATAGIRI et al. was always DL-form. In spite of these interesting reports, there exists a large gap between clostridia and strain EU which

Table 8. Descriptions of the new subgenus and the new species Sporolactobacillus nov. subgen. sporo: spore-forming, lactobacillus: genus Lactobacillus Gram-positive rods. Motile with peritrichous flagella. Endospores formed. Catalase negative, nitrates not reduced to nitrites. Micro- aerophilic. Ferments glucose by typical homofermentation, producing exclusively lactic acid. Type species Sporolactobacillus (Lactobacillus) inulinus nove, sp. Sporolactobacillus (Lactobacillus) inulinus nov. sp. inulinus: inulin fermenting. Gram-positive rods, 0.7'-0. 8 X 3'-'5 p in size, with granular appearance, occurring singly, occasionally in pairs and rarely in short chains. Motile with a small number of long peritrichous flagella. Cylindro-elliptical endospore, 0.8 X 1.0 p in size, formed in terminal position where the sporangium swells with the matura- tion of endosore. Can tolerate heating for 10 min at 85°. Some spores survive after 10 min', heating at 95°. Catalase negative, nitrates not reduced to nitrites. Indole not formed. Gelatin not liquefied. Litmus milk not changed. Grows well in glucose-yeast extract-peptone (GYP) or B12assay medium. Growth in GYP medium first silky then turbid with gradual formation of precipitate; no growth on surface. GYP agar colonies: small pin-head colonies; larger colonies appearing on or in semi-solid agar show the motility of the strain. When CaCO3is present, colonies become distinct being surrounded by transparent halos. GYP agar slant: growth very faint. GYP agar stab : filiform growth along the stab canal, no surface growth. GYP agar deep colonies: small colonies distribute equally except for surface layer. Strictly homo-lactic fermentation producing exclusively D(_)-lactic acid; in the presence of CaCO3i glucose given at the initial concentration of more than 15% can be completely fermented. Produces abundantly acid but no gas from glucose, fructose, mannose, maltose, sucrose, trehalose, raffinose, inulin, mannitol, sorbitol and a-methyl glucoside. Slight or no acid from galactose, pentoses, lactose, melibiose, cellobiose, melezitose, dextrin, starch, glycogen, dextran, glycerol, erythritol, adonitol, dulcitol, salicin and aesculin. Can grow between 15° and 40°, with the optimum temperature at around 35°; no growth at temperatures below 10° or above 45°. Aerophily: microaerophilic Isolated from an assorted chicken feed. 70 KITAHARAand SuzuKI VOL. 9

produces a large amount of lactic acid exclusively by homo-fermentation. Furthermore, strain EU is not strictly anaerobic or aerotolerant but microaerophilic. It forms colonies on agar plates, while Clostridium can form colonies only under anaerobic conditions. Important properties of f ermentative clostridia and strain EU are summarized for comparison in Table 7. The characteristics of strain EU described above indicate that it can hardly be excluded from the category of lactic acid bacteria, since its description agrees completely with that of Lactobacillaceae except for only two points, motility and spore formation. In its property of being Gram- positive and forming spores, strain EU is similar to the organisms belonging to Bacillaceae ; however, its biochemical properties are entirely different from those described for the two known genera of Bacillaceae, Bacillus and Clostridium. The authors' opinion is, therefore, that strain EU should be considered as an intermediate form between Lactobacillus and Clost- ridium. In 1909 ORLA-JENSEN(16) found a motile and spore-forming strain in Sarcina and proposed a subgenus Sporosarcina, which was later elevated to a tribe by KLUYVERand VANNIEL (17) in 1936. BREED et al. (18) described Sporosarcina as an only spore-forming genus not belonging to Bacillaceae. Following ORLA-JENSEN'sexample, the authors would like to propose a new subgenus Sporolactobacillus for strain EU and to give it a protruded position in the genus Lactobacillus as in the case of Sarcina ureae in Bergey's Manual. With emphasis of its strong inulin-fermenting property, the authors propose to name strain EU Sporolactobacillus (Lactobacillus) inulinus nov. sp. Summarized descriptions of the features of the new subgenus and new species are given in Table 8. Since it was suggested by METCHNIKOFF(19) that lactic acid bacteria are effective in suppressing the proliferation of undesirable intestinal micro- flora, many kinds of dried preparations of lactic acid bacteria have been placed on the market. An important problem for the manufacturers has been to preserve the viability of the bacteria in dried conditions, a problem, which seems to be easily solvable by utilizing the spore-forming property of the organism of this new subgenus.

SUMMARY

A motile, spore-forming and rod-shaped bacterium was isolated from a chiken feed. The strain is Gram-positive, catalase-negative and is a typical homo-lactic acid f ermenter producing leavo-rotatory lactic acid. Taxonomic position of this unusual strain was discussed and it was suggested that the organism represents an intermediate form between Lactobacillus and Clostridium, for which erection of a new subgenus Sporolactobacillus (in Lactobacillaceae) was proposed. The species was named Sporolactobacillus inulinus nov. sp. taking into account its powerful inulin fermenting activity. 1963 Sporolactobacillus Nov. Subgen. 71

The authors wish to express their heartful thanks to Miss T. Ogihara and Mr. T. Miyoshi of this laboratory for their assistance, and also to Mr. S. Kitahara of Noda Central Laboratory for taking electron micrographs. This work was supported in part by the Scientific Research Grant from the Ministry of Education.

REFERENCES

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