Studies on encapsulated lactobacilli—I

Arch.ora/ Biol. Vo1.9,pp.341-349,1964. Pergamon

Press Ltd. Printed in Gt. Britain.

STUDIES ON ENCAPSULATED UTILIZATION

OF CAPSULAR

MATERIAL

LACTOBACILLI-I BY LACTOBACZLLUS

CASEZ

B. F. HAMMOND and N. B. WILLIAMS University

of Pennsylvania, Microbiology Department, School of Dentistry, Philadelphia, Pennsylvania, U.S.A.

Summary-The role of capsular polysaccharides in the biochemical activities of mucoid (encapsulated) and non-mucoid (non-encapsulated) strains of Lactobacillus casei (L-324M) was investigated. Both the rate and extent of glucose oxidation were greater for non-encapsulated cells although the endogenous oxygen uptake was considerably less than that of encapsulated cells. A positive association was made between endogenous catabolism of capsular polysaccharide as measured by physicochemical means and the in vitro growth and maintenance of the organism under conditions of nutrient limitation. The taxonomic and serologic implications of the findings are discussed in relation to the oral ecosystem. INTRODUCTION ALTHOUGH considerable

information is now available concerning the chemical and biologic characterization of lactobacillus cell walls (IKAWA and SNELL, 1960) very little is known about their other surface structures. Descriptions of slime and capsule production in the genus Lactobacillus have been brief and generally limited to infrequent reports of the activities of heterofermentative strains (WARD, 1892; MAYER, 15139; SHIMWELL, 1949; WILLIAMSON, 1959). The only specific report of capsule formation among the homofermentative lactobacilli prior to 1961 (HAMMOND, 1961) was made in 1909 by BURRI and ALLEMANNin a communication concerning the of Lactobacihs casei. More recent reports (WILLIAMS and “schleimsubstanz” HAMMOND, 1962) have shown that this homofermenter produced up to 40 % of its cell dry weight as capsular polysaccharide and suggested that the material might be utilized under appropriate conditions for growth purposes. If the hypothesis of capsular utilization is true, there should be several measurable changes in the biochemical properties of the cell which accompany either removal or addition of the capsular material. Accordingly, the experimental approach consisted first of comparing the biochemical activities of encapsulated (mucoid) and non-encapsulated (non-mucoid) strains of the organism ; and secondly of assessing the importance of these biochemical changes in a context of in vitro growth and maintenance. MATERIALS AND METHODS Organism and Culture Conditions Lactobacillus casei, strain L-324M was used throughout these studies and maintained in agar deep tubes of Rogosa SL medium (Difco). Cultures were transferred at monthly intervals and examined microscopically for demonstration of contaminants. All incubations were done aerobically at 37°C unless otherwise noted. 341

342

B. F. HAMMONDAND N. B. WILLIAMS

Polysaccharide Production and Utilization The assay for capsule production and utilization was both morphological and chemical. Transverse capsule diameters were measured with an eyepiece micrometer using wet mounted India ink preparations and checked periodically with Alcian Blue 8GS stained sections. Chemical estimations of the capsule (a polysaccharide consisting of approximately equimolar amounts of glucose and rhamnose, HAMMOND, 1961) were performed using the anthrone procedure of SEIFTER et al. (1950). Control experiments with glucose and rhamnose indicated that the two sugars reacted in an additive manner in the anthrone test and confirmed the suitability of the method as a routine assay procedure for the polysaccharide. Residual glucose in culture filtrates was measured by the method of SOMOGYI (1945). The method of obtaining non-encapsulated cells was adapted from the procedure of JUNI and HEYM (1961) in which the capsular material was stripped from the cell by repeatedly forcing thick saline suspensions of the organism through the grid of a glass chromatogram sprayer with compressed air. Earlier attempts to grow the cells in media containing low concentrations of several uncoupling agents (NaN,, and 2,4 di-nitrophenol), or in media containing a low carbohydrate-nitrogen ratio, depressed capsule formation. Since there was no means of controlling other possible enzymatic changes which might result from exposure to uncoupling agents or to altered cultural conditions, the capsule stripping technique was used routinely for all endogenous studies. Because the capsule was resynthesized by stripped cells under growth conditions, a non-encapsulated variant isolated during the course of this investigation was used for all growth curve and survival studies. Measurements of oxygen consumption were made at 37°C by conventional Warburg procedures with air as the gas phase. Varying amounts of cells (5-25 mg cell dry weight) were suspended in 0.05 M phosphate buffer, final pH 6.8, containing additional components as noted in the text. Substrates were added from side-arms after temperature equilibration to give a final concentration of 0.05 M in a total volume of 3.2 ml. Centre wells normally contained 20% KOH; for measurements of cyanide inhibition this was replaced with 0.1 ml each of 0.2 M KOH and 2 M KCN and the desired final concentration added to the main chamber as KCN. The QO, values were calculated from periods of essentially linear activity after addition of the substrate and corrected for capsular weight when encapsulated cells were used. For measurements of endogenous catabolism a semi-macro procedure was used. Fifty millilitre suspensions of washed cells (Klett-Summerson readings of 400 with No. 42 filter) were placed in 500 ml Erlenmeyer flasks containing the same final concentration of buffer as in the Warburg flasks. The flasks containing a final volume of 100 ml were placed in 37°C water bath and air was bubbled through the system by means of three capillary pipettes connected to a water aspirator. The air was brought to the temperature of the water bath and saturated with water before it entered the reaction vessel. Air was passed through the suspension at a rate of approximately 250 cm”/hr. Less than 2 per cent volume change occurred during the 2 hr period. Samples, 2 ml each, were removed from the flask at 15 min intervals, chilled in an ice bath and separated from the suspending medium by centrifugation. After two washings in cold saline (O-85 %

STUDIES ONENCAPSULATED LACTOBACILLI-I

343

NaCl), the cells were analysed for changes in total cell carbohydrate, capsular size and dry weight. Lactic acid was determined by the method of BARKER and SUMMERSON(1941). Estimation

of growth and total cell crop

For estimation of growth and growth rates, cells were grown in test tubes containing 10 ml of Rogosa SL broth (Difco) or in a nitrogendeficientmedium (HAMMOND, 1962). The inoculum consisted of 0.1 ml of a 12-hr broth culture originally taken from a stock agar deep. Growth was measured turbidometrically at 660 rnp with an Evelyn calorimeter using a tube of sterile medium as a blank. Differences in optical density due to the capsular material were corrected for by taking suitable aliquots from the growing culture, stripping the capsules from the cells, then taking another reading using the stripped cells and subtracting the difference from the original reading. I ,nonencapsulated

glucose

Minutes FIG.

of 15. casei

1. Oxygen uptake of encapsulated

and non-encapsulated

(stripped)

ceils

(L-324M).

RESULTS Figure 1 shows the differences in the rate and extent of glucose oxidation by encapsulated and non-encapsulated cells. Whereas non-encapsulated cells oxidized glucose much more rapidly and to a greater extent than encapsulated cells-the endogenous oxygen uptake was greater with encapsulated cells. The high values for oxidation of glucose by stripped cells indicated that the stripping procedure did not damage the oxidative enzymic machinery of the cell. Similar results (low endogenous and high exogenous oxygen uptake values) were also observed using a non-encapsulated variant also subjected to the stripping procedure. Thus, it appeared that the

344

B. F. HAMMONDAND N. B. WILLIAMS

1.

Minutes casei encapsulated

FIG. 2. The effect of uncoupling agents on the rate and extent of glucose oxidation by L. cusei(L-324M encapsulated). KCN conc.-OOl M, NaN, conc-O.01 M, glucose cont.--0.05 M.

FIG. 3. Polysaccharide production (“cell glucose”) and cell growth. The arrow represents the beginning of the diauxic phase of growth and the point at which the residual glucose in the medium is depleted (less than 2.0 pg/ml).

high endogenous rate of encapsulated cells depended upon the presence of a capsule. Figure 2 shows how the incomplete oxidation of substrate was changed by using NaN, and KCN. Using encapsulated cells oxidation was allowed to proceed toward completion in the presence of either assimilatory poison (QO, with KCN= 17.3;

STUDIES

ON ENCAPSULATED

LACTOBACILLI--I

345

without KCN, QOe= 8.1) whereas neither poison materially affected the oxidation of glucose when a non-encapsulated variant was used (& 2 per cent). Neither agent affected the rate or extent of endogenous systems. Again the high endogenous rate of encapsulated cells was observed; in this case the values were roughly half of those observed for glucose oxidation in the absence of KCN or NaN,. These results are typical of the process of oxidative assimilation where the substrate, instead of being oxidized to completion is incorporated in part into the cell proper as a polymeric product, which, in turn, may be disposed of according to the physiologic capacities and needs of the cell. These data, however suggestive of endogenous breakdown of assimilated material, did not allow us to equate a high endogenous QO, with utilization of capsular polysaccharide. Also, even though the endogenous RQ (respiratory quotient) was found to be unity (a value classically associated with the catabolism of carbohydrate), several workers have pointed out that manometric data on assimilating organisms indicate only a general range of possibilities of endogenous substrates-so that what might appear as a classical RQ for carbohydrates in reality may be a summation of several endogenous reactions in which protein or other lipid substrates may be involved (LAMANNA and MALLETTE, 1959). For a more direct approach it was decided to study growing cells and compare the changes in the growth cycle with changes in the total amounts of capsular polysaccharide. Figure 3 shows a typical growth curve of this organism in the nitrogendeficient medium. It will be observed that polysaccharide production (represented as “cell glucose”) roughly paralleled cell crop where the organisms were in the log phase of growth-but in both the lag and stationary phases cell polysaccharide decreased rapidly although cell crop either increased slightly or remained constant. It should be pointed out also that the secondary increase in cell crop, after the stationary phase as shown in Fig. 3 was a constant and repeatable finding even when readings were made at l- and 2-hr intervals. (Asamatter of fact shorter intervals accentuated the diphasic or diauxic character of the growth curve). It was also discovered that the diphasic part of the optical density curve (note arrow on Fig. 3) never began until all of the residual glucose in the medium was depleted (i.e. less than 2.0 pg/ml). It may be inferred that in the absence of exogenous nutrient these cells utilize their own cellular reserve materials for limited growth and maintenance, a finding consonant with the high endogenous QO, of encapsulated cells. Because the diphasic growth curve was never observed during growth of the non-encapsulated variant it appeared probable that the causal factor for this change was the capsular polysaccharide. Since the absence of a diphasic growth curve in the non-encapsulated variant might be attributable to strain-associated differences, information of a more direct nature using a single strain was indicated. Data on capsule size, cell dry weight and total cell carbohydrate in a semimacro endogenous system are shown in Fig. 4. It will be observed that all three parameters of measurement decreased with time; the transverse capsule diameters decreased from 3.5 to 2~ (change of 35 per cent) ; cell dry weight decreased by 15 per cent; and cell polysaccharide decreased by 18 per cent. Under anaerobic conditions (95 % N,-5 % CO,) it appeared that lactate was not the only product of endogenous

B. F.

346

HAMMOND AND N. B. WILLIAMS

activity as it accounted for only 30 per cent of the 18 per cent decrease in cellular polysaccharide on a wt/wt basis. The end products of aerobic endogenous metabolism have not yet been defined. Stripped cells or non-encapsulated variants subjected to similar endogenous study showed less than 1 per cent decrease in cell dry weight, cell diameter and less than 5 per cent change in cellular polysaccharide.

Z.capsuie 3.cell

t

diameter “glucose’

: ‘1

I ‘2

’

I ’

Hours FIG.

4.

Endogenous

semi-macro

determination

of capsular

polysaccharide

utilization.

Viability changes of encapsulated and non-encapsulated cells in similar endogenous systems were compared over a 36-hr period. Samples were plated in triplicate at 0, 3, 6, 12, 18, 24 and 36 hr. The results presented in Table 1 show that 15-20 per cent of the encapsulated cells survived after 24 hr whereas less than 5 per cent of the non-encapsulated cells survived in the same period. TABLE 1. SURVIVAL OF ENCAPSULATED AND NON-ENCAPSULATED L. CUSCiIN A NON-NUTRIENT MEDIUM

The organisms were grown aerobically in Rogosa SL broth medium (Difco) for 20 hr, washed three times and incubated in a saline-buffer (PH 5%) at 37°C aerobically. Percentage viability figures obtained from viable plate counts done in triplicate on Rogosa, SL agar medium Viable cells (%) Time (hr) 0 3 6 12 18 24 36

Encapsulated 100 55 40 30 20 15-20 10

Non-encapsulated 100 50 30 25 5 2-4 24

STUDIES ON ENCAPSULATED LACTOBACILLI-I

347

DISCUSSION

Although numerous investigators have consistently found L. casei to be the most frequently occurring lactobacillus in the human mouth (GRUBB and KRASSE,1953; ROGOSA et al., 1953; DAVIS, 1955; HAYWARD,1957) the reasons for this fact are unknown. The point is often made that this organism is the “hardiest of the oral lactobacilli” (ROGOSAet al., 1953) and it is tacitly assumed that some hardiness factor (e.g. resistance to environmentally caused deviations) associated with the organism is responsible for its high incidence in the oral cavity and the ease with which it is maintained in stock culture. A comparison of its reported biochemical properties and nutritional requirements with those of other oral lactobacilli does not reveal any outstanding or fundamental differences which would give it a selective advantage under in vitro or in vivo conditions. The present investigation indicates that the presence of a capsule is correlated with several physiologic properties of the organism which are of importance in its in vitro growth and maintenance. Encapsulated cells but not unencapsulated cells show a diphasic growth curve; have high endogenous oxidative rates (with an RQ indicative of carbohydrate catabolism); and show simultaneous decreases in cell dry weight, total cell carbohydrate and capsule diameter under conditions of nutrient limitation. Although such functions for capsules had been suspected for some time it was not until FABER and ROSENDAL(1954) showed that Str. pyogenes could depolymerize its own hyaluronic acid capsules that serious interest was given the idea that extracellular polysaccharides might serve as reserve energy sources (OGINSKY and UMBREIT,1959). The mere depolymerization of a material, however, need not be equated with beneficial utilization of the material nor causally related to survival. The association of a diphasic growth curve with decreases in capsular polysaccharide might be interpreted to mean that cells in a partially exhausted growth medium could derive benefit from utilization of the material, but our failure to observe critical differences in the survival rate and viability patterns of encapsulated and nonencapsulated cells in the semi-macro endogenous system make it difficult to arrive at a definite conclusion concerning the role of capsules in the survival process. It is tempting, nonetheless, on the basis of the more positive data presented to speculate that the enzymic degradation of this polysaccharide under conditions of nutrient limitation might explain the hardiness phenomenon of this organism and aid in the regulation of the microbial ecology of the oral cavity. Apart from its implications for oral ecology, the concept of an encapsulated Lactobacillus casei is an extremely interesting one and bears directly on many basic biological problems of the organism itself and of the lactobacillus group as a whole. The ability of the organism to synthesize a capsular polysaccharide of the same general nature as the glucose-rhamnose polysaccharide found in its cell wall (IKAWA and SNELL, 1960) and to utilize the material under appropriate conditions of nutrient limitation suggest that this newly recognized surface structure may be of taxonomic importance. None of the definitions or descriptions of the genus Lactobacillus including the one formulated by BREED, MURRAY and SMITH in the seventh edition of Bergey’s manual (1957) make any mention of capsule formation or the ability to

B. F. HAMMONDAND N. 8. WILLIAMS

348

assimilate glucose and other simple sugars into cellular polysaccharide. The production of levans, dextrans and other extracellular polysaccharides is a well known means of identification of other lactic acid bacteria, viz., Str. salivarius, Leuconostoc spp., and it seems reasonable that similar properties among the lactobacilli might be used to advantage. The serological implications of a capsule may be of real importance in understanding many unexplained cross reactions and anomalies reported in serologic studies of the lactobacilli. For example, there are two antigenic groups of L. casei, and the serological specificity of each group depends upon the molar ratios of glucose and rhamnose in the cell wall polysaccharides (KNOX, 1963). In addition to the fact that the purified capsular material of this organism contains both sugars, it should also be borne in mind that antigenic analysis can be masked or confused by steric interference of non-reactive complexes at the surfaces of bacterial cells. Preliminary studies along these lines using specific capsular (Quellung) reactions and tube agglutination tests have indicated the presence of specific haptenic groupings associated with the surfaces of encapsulated cells but absent from non-encapsulated cell surfaces. Thus, with further experimental evidence, it seems entirely possible that capsular material may determine major serologic as well as biochemical properties of this lactobacillus. Acknowledgement-This work was supported in part by a grant, DE-00175, from the National Institute of Dental Research, United States Public Health Service. R&urn&Le

r6ie des polysaccharides capsulaires dam I’activiti biochimique de souches mucoides (encapsulees) et non-mucoldes (non encapsulees) de Lactobacillus cusei (L-324 M) est Ctudie. A la fois la proportion et l’importance de l’oxydation du glucose sont plus tlev&es pour des cellules non encapsulees bien que leur consommation d’oxygtne endogene est nettement moins elevee que celle des cellules encapsulees. Une association positive est trouv&e entre le catabolisme endogene des polysaccharides capsulaires, mesure par des methodes physico-chimiques, et la croissance in vitro et le maintien en vie de la bacterie dans des conditions limites de nutrition. Les consequences taxonomiques et serologiques de ces rcsultats sont discutees en fonction de leur rapport avec I’ecologie buccale. Zwmmenfassung-Es wurde die Rolle der kapsularen Polysaccharide fur die biochemische Aktivitat mukoidet (eingekapselter) und nicht-mukoider (nichteingekapselter) Stamme des Lactobacihs casei (L-324 M) untersucht. Sowohl Rate als such Ausmass der Glukoseverbrennung waren bei nichteingekapselten Z&en grosser, obwohi der endogene Sauerstoffverbrauch betrtichtlich geringer war als der jenige der eingekapselten Zellen. Ein positiver Zusammenhang wurde zwischen der endogenen Dissimilation der kapsullren Polysaccharide, wie mit physikalisch-chemischen Mitteln gemessen, und dem in vitro-Wachstum wie such der Erhaltung der Organismen unter Bedingungen begrenzter Emahrung darstellt. Die taxonomische und serologische Bedeutung der Ergebnisse wird in Beziehung zum System des Mundhohlenbiotops diskutiert.

REFERENCES S. B. and SUMMERSON, W.H. 1941. The calorimetric determination of lacticacid in biological material. J. biol. Chem. 138, 535-554. BREED, R. S., MURRAY, E. G. and SMITH,N. R. 1957. Bergey’s Manualof Determinative Eacteriology (7th ed.), p. 541. Williams and Wilkins, Baltimore, Md.

BARKER,

STUDIESON ENCAPSULATED LACTOBACILLI-I

349

BURRI, R. and ALLEMANN,0. 1909. Chemische-biologische Untersuchungen iiber schleimbildende Milchsaurebakterien. Z. Vnrersuch. Nahr.- u. Genussm. 18, 449-460. DAVIS, G. H. G. 1955. The classification of lactobacilli from the human mouth. J. gen. Microbial. 13,481&493.

FABER, V. and ROSENDAL,K. 1954. Studies on the production of hyaluronidase and hyaluronic acid by representatives of all types of hemolytic streptococci belonging to group A. Acfa path. microbial. stand. 35, 159-164. GRUBB, R. and KRASSE,B. 1953. Classification of oral strains of lactobacilli. Acta path. microbial stand. 32, 539-548.

HAMMOND,B. F.

1961. Isolation

and characterization

of a capsular polysaccharide

of L. casei.

Bact. Proc., p. 95.

HAMMOND,B. F. 1962. Studies on capsule formation in L. casei. Ph.D. Thesis, University of Pennsylvania, Philadelphia, Pennsylvania. HAYWARD,A. C. 1957. A comparison of Lactobacillus species from human saliva with those from other natural sources. &it. dent. J. 102, 450-451. IKAWA, M. and SNELL, E. E. 1960. Cell wall composition of lactic acid bacteria. J. biol. Chem. 235, 1376-1382. JUNI, E. and HEYM, G. 1961. Capsule resynthesis by decapsulated resting cell suspensions. Bacf. hoc., p. 193. KNOX, K. W. 1963. Isolation of group specific products from L. casei and L. casei var. rhamnosus. J. Gen. Microbial. 31, 59-72.

KOSER, S. A. and THOMAS,J. L. 1955. Amino acid requirements of lactobacilli. J. infecf. Dis. 97, 287-298. LAMANNA, C. and MALLETTE,M. F. 1959. Basic Bacteriology (2nd ed.), p. 621. Williams and Wilkins, Baltimore, Md. MAYER, H. D. 1939. Das “Tibi-” konsortium nebst einem beitrag sur kenntnis der bakteriendissoziation, pp. l-188. In: Druk naamlooze venootschap. Neineme, Delft. OGINSKY, E. L. and UMBREIT,W. W. 1959. An Introduction to Bacterial Physiology (2nd ed.), pp. 26-27. Freeman, San Francisco. ROGOSA, M., WISEMAN,R. F., MITCHELL,J. A., DISRAELY,M. N. and BEAMAN,A. J. 1953. Species differentiation of oral lactobacilli from man including descriptions of Lactobacillus salivarius nov. spec. and L. ceilobiosus nov. spec. J. Bact. 65, 681-699. SEIFTER,S., DAYTON, S., NOVIC, B. and MUNTWYLER,E. 1950. The estimation of glycogen with the anthrone reagent. Arch. Biochem. 25, 191-200. SHIMWELL,J. L. 1949. A study of ropiness in beer. J. Ins,. Brew. 46, 26-33. SOMOGYI,M. 1945. A new reagent for the determination of sugar. J. biol. Chem. 160, 61-68. WARD, H. M. 1892. The ginger-beer plant and the organisms composing it: a contribution to the study of the fermentation yeasts and bacteria. Roy. Sot. Lond. Phil. Trans. B. 183, 125-197. WILLIAMS,N. B. and HAMMOND,B. F. 1962. Proc. int. Ass. dent. Res. 40th General Meeting. WILLIAMSON,D. H. 1959. Studies on lactobacilli causing ropiness in beer. J. appl. Bacf. 22, 39242.