Fermentation of Polysaccharides by Klebsielleae and Other

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1980, p Vol. 39, No /80/ /05 $02.00/0 Fermentation of Polysaccharides by Klebsielleae and Other Facultative Bacilli GREG U. OCHUBAt AND V. LYLE VON RIESEN* Department of Medical Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska Fermentations of 10 polysaccharides by species of the family Enterobacteriaceae were examined. Algin, guar, karaya, xanthan, and xylan were not fermented by any of the strains tested. Most of the activity was found in the tribe Klebsielleae. Klebsiella oxytoca fermented amylopectin (97% of the strains studied), carrageenan (100%), inulin (68%), polypectate (100%), and tragacanth (100%). Klebsiella pneumoniae fermented amylopectin (91%), carrageenan (100%), and tragacanth (86%). Carrageenan was also fermented by Enterobacter aerogenes (100%), Enterobacter agglomerans (63%), Enterobacter cloacae (95%), and Pectobacterium (38%). Pectobacterium shared polypectate fermentation (100%) with K. oxytoca. With one exception, Serratia strains were negative on all polysaccharides. These results, along with other evidence, indicate that (i) the genus Klebsiella is biochemically the most versatile genus of the tribe, (ii) because of its distinct characteristics, K. oxytoca warrants species designation separate from K. pneumoniae, and (iii) some food additives generally considered indigestible can be metabolized by a few species of facultative bacilli, whereas others appear to be resistant. In 1976 von Riesen (39) stated that indolepositive strains of Klebsiella pneumoniae (hereinafter designated Klebsiella oxytoca) were able to digest polypectate (sodium polygalacturonate). This was confirmed subsequently by Starr et al. (34) and by Naemura and Seidler (28). In 1977 workers at the Center for Disease Control stated that, on the basis of the deoxyribonucleic acid relatedness shown by Jain et al. (19) and their own work (5), the indole-positive strains of K. pneumoniae are clearly different from the indole-negative strains of this species. They indicated (5) that as of 1 October 1977 the Center for Disease Control would report indolepositive strains as K. oxytoca. This species designation is in keeping with suggestions made by various workers (1, 3, 17, 19, 21, 24, 25, 35, 39) since as early as 1886 (16). A neotype strain for the species (K. oxytoca ATCC 13182) has been proposed by the International Committee on Systematic Bacteriology Subcommittee on Taxonomy of Enterobacteriaceae (4). Because deoxyribonucleic acid relatedness and several biochemical characteristics of K. oxytoca seem to set this species apart from the other species of Klebsiella and from other closely related genera in the tribe Klebsielleae, it seemed worthwhile to examine this group further to determine whether additional striking t Present address: Pathology Laboratory, University of Nebraska Medical Center, Omaha, NE differences could be detected. Since polypectate fits the definition of plant gums (40) and is used in foods (more properly, its methyl ester, pectin, is used) as a gelling or stiffening agent, we decided to study the metabolism of some additional, acidic and neutral, plant polysaccharides which are present in foods either as nutrients or as additives for the function which each serves in the food (i.e., gelling, stiffening, thickening, emulsifying, stabilizing). The polysaccharides selected for this study were algin, amylopectin, carrageenan, guar, inulin, karaya, polypectate, tragacanth, xanthan, and xylan. All but xylan are described in a book edited by Whistler and BeMiller (40). Spiller and Amen also describe many polysaccharides, including xylans (33). von Riesen (38, 39) observed essentially the loss of gel rigidity, resulting presumably from the breakdown of the glycosidic linkages of the polypectate molecules. Analytical studies (30) have shown that hydrolysis of polypectate can release galacturonate and unsaturated oligosaccharides. Since all of the organisms studied are active fermenters, we decided to examine the results in terms of fermentation; that is, we examined acid production with the polysaccharides as the fermentable (or nonfermentable) substrates. This method was one of several used recently by Talbot and Seidler (36) in their study of the utilization of cyclitols (cyclic hexahydric alcohols) by Klebsielleae. Although most of the 988

2 VOL. 39, 1980 species of the family Enterobacteriaceae were included in this study, we focused on the species in the tribe Klebsielleae and particularly on the activities of K. oxytoca and K. pneumoniae. MATERIALS AND METHODS POLYSACCHARIDE FERMENTATION BY KLEBSIELLEAE 989 Microorganisms. All of the strains used in this study (G. U. Ochuba, M.S. thesis, University of Nebraska Medical Center, Omaha, 1978) were collected by one of us (von Riesen) over a period of years from a variety of sources, but primarily from clinical specimens from humans. In addition to the genera of the family Enterobacteriaceae, a small number of Aeromonas and Vibrio strains were also included. All strains were tested for purity and were reidentified by conventional methods for the identification of enteric and related bacteria (11, 13-15, 32). The eight strains designated Pectobacterium were positive for polypectate digestion, as previously described for K. oxytoca (39) and Yersinia (38), but are not identifiable with any of these. Three of these strains may be polypectate-positive strains of Enterobacter cloacae, and four may be Enterobacter agglomerans strains. Cultures were maintained in cystine Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) at room temperature in the dark. Polysaccharides and media. The polysaccharides studied were algin, amylopectin, carrageenan, guar, inulin, karaya, polypectate, tragacanth, xanthan, and xylan. Amylopectin was obtained from Nutritional Biochemicals Corp., Cincinnati, Ohio, and inulin was from Difco Laboratories, Detroit, Mich. The other polysaccharides were purchased from Sigma Chemical Co., St. Louis, Mo. Control studies for the presence of free reducing sugar groups in the polysaccharides and for the hydrolyzing effects of autoclaving indicated that carrageenan at 1% contained a small amount of reducing sugar which was not increased by autoclaving. Inulin showed a trace of reducing sugar after autoclaving. None of the other eight polysaccharides contained free reducing sugar groups either before or after autoclaving. Further control studies with galactose (carrageenan) and fructose (inulin) established that the amounts of free reducing sugars in carrageenan and inulin were insufficient to produce falsepositive reactions under the conditions of this study. For this study the polysaccharides were incorporated into a standard fermentation base, phenol red broth base (Difco), at 0.5% (amylopectin, guar, karaya, and xanthan) or 1% (algin, carrageenan, inulin, polypectate, tragacanth, and xylan); the ph was adjusted for those media containing polysaccharides which produced sufficient changes in the ph of the medium to warrant readjustment. The media were dispensed in 6-nil volumes into tubes (16 by 150 ml) and were sterilized by autoclaving at 121 C for 15 min. The media were inoculated with cultures grown for 18 h on Trypticase soy agar (BBL Microbiology Systems), incubated at 37 C, and observed daily for 7 days. Results were considered positive when the color of the indicator (phenol red) showed that the medium was distinctly acid when compared with uninoculated complete medium and with inoculated phenol red broth base without polysaccharide. RESULTS Five of the polysaccharides (algin, guar, karaya, xanthan, and xylan) were not fermnented by any of the species tested. Table 1 shows the results for the five polysaccharides that were fermented by one or more species of the tribe Klebsielleae. These results indicate that K. oxytoca was the most active species (five of five polysaccharides fermented). K. pneumoniae was next (three of five) and, except for Enterobacter aerogenes, E. agglomerans, and E. cloacae on carrageenan, the remaining species of this group were relatively inactive in the fermentation of these polysaccharides. Table 1 also shows the results for the other species of facultative gram-negative bacilli in which one or more strains fermented any of the five polysaccharides. Carrageenan was fermented by the greatest number of species (15 species) and inulin was fermented by the smallest number (4 species). Polypectate was metabolized by the eight strains designated Pectobacterium on the basis of their ability to digest polypectate, by K. oxytoca, and by the three species of Yersinia, as shown previously by another method (38, 39). DISCUSSION Although it is not always possible to identify the currently recognized species of Klebsiella, Enterobacter, and Serratia in the early literature, the "aerogenes" type of Bacterium coli generally fits the nonmotile Klebsiella species and the "cloacae" type is closer to the motile Enterobacter species (6). Johnson and Levine (20) indicated that the aerogenes type of B. coli almost always fermented starch (97 to 100% of the strains), whereas the cloacae type was usually negative (4.5% positive). The results of other workers (10, 12, 22, 23) follow this same pattern. In our study 94% of the Klebsiella strains were positive on amylopectin (insoluble starch). Serratia, Pectobacterium, and Enterobacter were negative, or nearly so (E. cloacae and E. agglomerans). Thus, it appears that among the Klebsielleae fermentation of amylopectin (insoluble starch) is a characteristic of Klebsiella. In addition to K. oxytoca and K. pneumoniae, E. aerogenes (100% of the strains), E. agglomerans (63%), E. cloacae (95%), and Pectobacterjum (38%) fermented carrageenan. Table 1 shows that carrageenan was the polysaccharide fermented by the largest number of species and

3 990 OCHUBA AND VON RIESEN TABLE 1. Fermentation ofpolysaccharides by Klebsielleae and other gram-negative facultative bacillia Amylopectin Carrageenan Inulin Polypectate Tragacanth Organism' No. % No % No: % No: % No. % posi- posi Jposi- Posi- posi- Posi- pos- Posi- posi- Positive/no. tietive/no. tietive/no. tietive/no. tive tive/no. tive tested tested tested tested tested K. oxytoca 30/ / / / / K. pneumoniae 19/ / /21 5 0/ /21 86 K. ozaenae 1/ / /1 0 0/1 0 1/1 100 E. aerogenes 0/2 0 2/ /2 0 0/2 0 0/2 0 E. agglomerans 2/ / /16 0 0/16 0 0/16 0 E. cloacae 1/ / /19 0 0/19 0 0/19 0 E. hafniae 0/2 0 0/2 0 1/2 50 0/2 0 0/2 0 Pectobacterium sp. 0/8 0 3/8 38 1/8 13 8/ /8 0 Serratia liquefaciens 0/1 0 0/1 0 0/1 0 0/1 0 0/1 0 Serratia marcescens 0/16 0 0/16 0 0/16 0 0/16 0 0/16 0 Serratia rubidaea 0/1 0 1/ /1 0 0/1 0 0/1 0 Aeromonas hydrophila 2/ /2 50 0/2 0 0/2 0 2/2 100 Aeromonas shigelloides 0/1 0 1/ /1 0 0/1 0 0/1 0 Arizona hinshawii 0/3 0 2/3 67 0/3 0 0/3 0 2/3 67 Citrobacter diversus 0/3 0 1/3 33 0/3 0 0/3 0 0/3 0 C. freundii 0/ / /12 0 0/12 0 0/12 0 Escherichia coli 1/ / /16 0 0/16 0 0/16 0 P. vulgaris 0/4 0 0/4 0 0/4 0 0/4 0 2/4 50 P. mirabilis 0/12 0 0/12 0 0/12 0 0/12 0 8/12 67 Vibrio cholerae 1/ / /1 0 0/1 0 1/1 100 Vibrio parahaemolyticus 2/ /2 0 0/2 0 0/2 0 2/2 100 Yersinia enterocolitica 2/ /15 0 0/ / /15 53 Yersinia pestis 0/7 0 0/7 0 0/7 0 7/ /7 100 Yersinia pseudotuberculosis 0/8 0 0/8 0 0/8 0 8/ /8 0 a Algin, guar, karaya, xanthan, and xylan were not fermented within 7 days by any of the species tested. bone strain of Chromobacterium violaceum, 1 strain of Edwardsiella tarda, 10 strains of Proteus morganii, 3 strains of Proteus rettgeri, 8 strains of Providencia stuartii, 21 strains of Salmonella, and 7 strains of Shigella were also tested, and all were found to be unable to ferment any of the 10 polysaccharides tested. strains. The fermentation of this polysaccharide by Citrobacter freundii is noteworthy since it was the only polysaccharide fermented by this species. Our sample of carrageenan did contain small amounts of free reducing sugar groups (0.2% glucose reducing equivalents at a concentration of 1%). Control studies indicated that, if the free reducing sugar was galactose (substituted forms as the monosaccharide of carrageenan), almost all strains should have been positive on carrageenan (all but 4 of 255 strains fermented galactose). Such was not the case (Table 1). Nevertheless, these results should be checked by other methods for determining fermentation of carbohydrates. Inulin was fermented by K. oxytoca. Because the fermentation was a delayed-type fermentation, as previously noted by Kauffman (22), and the number of positive strains reached 21 of 31 strains by day 7 at 37 C, we decided to continue the incubation of all inulin cultures at room temperature for up to 21 days. Within this period eight additional strains of K. oxytoca (29 of 31) and two of K. pneumoniae (3 of 21) became positive. These results gave 68 and 94% positive at 7 and 21 days, respectively, for K. oxytoca APPL. ENVIRON. MICROBIOL. and 5 and 14% positive at 7 and 21 days, respectively, for K. pneumoniae. These results are simnilar to those of Malcolm (83% of K. oxytoca and 15% of K. pneumoniae positive at 14 days [27] and of Lautrop (76% of K. oxytoca and none of K. pneumoniae at 30 days [24]). Malcolm (27) showed that 19% of his E. aerogenes strains and 6% of his E. cloacae strains were positive at 14 days. In this study neither of these species was positive; however, one of two strains identified as Enterobacter hafniae was positive. Inulin was also fermented by one strain of Pectobacterium. This strain was positive for indole and polypectate, but because it was motile, citrate negative, arginine dihydrolase positive, and lysine decarboxylase negative, it was considered not to be K. oxytoca. All other species tested were negative for inulin. Thus, it appears that, except for a small percentage of strains of K. pneumoniae which ferment inulin slowly, inulin fermentation is a characteristic of K. oxytoca, as suggested by earlier workers (22, 24, 25, 27). In addition to searching for additional substrates metabolized by K. oxytoca and related species, this study served to determine whether the digestion of polypectate observed by one

4 VOL. 39, 1980 POLYSACCHARIDE FERMENTATION BY KLEBSIELLEAE 991 method (38, 39) could be confirmed by another method. Seidler (personal communication) indicated that he could detect pectin metabolism in an Analytab Products Inc. research device (Enteric 50 E-R), which based positive results on the detection of acid by a ph indicator. One of us (von Riesen, unpublished data) could not obtain results consistent with the results of Seidler. Naemura and Seidler (28) subsequently used the method of von Riesen (38, 39) to identify their pectin (polypectate)-positive strains of Klebsiella. In this study polypectate was studied at a concentration of 1% in phenol red broth base. All 31 strains of K. oxytoca (Table 1) showed fermentation of polypectate at 24 h. All of these strains were shown by the plate method (39) to digest polypectate. This agreement was also observed for strains of Pectobacterium and Yersinia. These results establish that polypectate digestion observed by one method (38, 39) was confirmed by another method (Table 1). The fermentation medium (1% polypectate in phenol red broth base) is less expensive and easier to prepare than the gel medium (3% polypectate) and might be preferred for these reasons. Recently, Macken and Pickaver (26) stated that a bacterium isolated and identified by Pickaver as E. cloacae and shown to be negative on polypectate gel media (29) could produce growth-associated polygalacturonase and polygalacturonate trans-eliminase, two enzymes associated with polypectate digestion. The activities of these enzymes were determined by photometric measurements (polygalacturonate trans-eliminase) and by release of reducing group products and decreased viscosity of polypectate solutions (polygalacturonase). Dias (9) described strains ofaerobacter aerogenes which oxidized polygalacturonate. None of our strains identified as E. cloacae or E. aerogenes fermented polypectate (Table 1) or showed digestion on a gel medium (von Riesen, unpublished data). Thus, metabolism of polypectate appears to be a characteristic of K. oxytoca, Yersinia, and Pectobacterium. The ecological relationships of these and the plant-pathogenic Erwinia species have been discussed by Starr and his coworkers (7, 34). Tragacanth was fermented by the nonmotile Klebsiella strains, but not by the motile Enterobacter and Serratia strains. This adds another difference to the biochemical characteristics which aid in the separation of strains within this group. Table 1 indicates that tragacanth can also be fermented by other less related genera of facultative bacilli. The fermentation by two species of Yersinia is interesting since this genus shared polypectate fermentation with K. oxytoca and Pectobacterium. We could find no previous studies on the fermentation of tragacanth by enteric and similar bacteria. Although the fermentation of some of the negative substrates by anaerobes has been reported (31), we could find very little information concerning the fermentation of these substrates (algin, guar, karaya, xanthan, and xylan) by facultative gram-negative bacilli. Davis and Ewing (8) reported that strains of K. pneumoniae (including indole-positive strains) and Klebsiella ozaenae could use algin as a source of carbon. Inoue and Ando (18) reported the formation of an alginase by A. aerogenes, and Boyd and Turvey (2) isolated an enzyme from Klebsiella aerogenes which was specific for the alpha-lguluronosyl linkages in algin. None of our strains fermented algin. Williams and Doetsch (41) found no activity for guar in some nonrumen bacteria, including Proteus vulgaris, Proteus mirabilis, E. coli, and A. aerogenes. None of our strains fermented guar. We have no information concerning the other three polysaccharides. In conclusion, we believe that several things can be derived from this study. First, it establishes further that K. oxytoca is different from K. pneumoniae. As Lautrop (24) said, "no other group [oxytocum] in the Enterobacteriaceae family ferments so many different sugars, including inulin." We now confirm amylopectin, inulin, and polypectate and add carrageenan and tragacanth to the growing list. On a different tack, Taylor and others (37) recently pointed out that a histamine-producing strain of K. pneumoniae which was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning was indole positive. Thus, all studies, past and present, whether of deoxyribonucleic acid relatedness, immunological relationships, or biochemical characteristics, add to the justification for describing K. oxytoca as a separate species. Additionally, our results and the recent report of Talbot and Seidler (36) describing utilization and fermentation of cyclitols by Klebsielleae show that the genus Klebsiella is the most active of the tribe Klebsielleae and probably of the family Enterobacteriaceae. Second, this study shows that as food additives, some of these polysaccharides (algin, guar, karaya, xanthan, and xylan) seem safe from destruction by facultative fermenters (but not from some anaerobes [31]), whereas others (amylopectin, carrageenan, polypectate, and tragacanth) may lose their additive properties when exposed in various ways to enteric organisms. If this digestion occurs in vivo, it may add a few uncounted calories; however, if, perhaps after contamination, the digestion occurs in vitro, the food could lose some of its esthetic value.

5 992 OCHUBA AND VON RIESEN ACKNOWLEDGMENTS We gratefully acknowledge the help of S. Grzywa, L. Karnish, and D. Burgin in the preparation of the media and manuscript. LITERATURE CITED 1. Bascomb, S., S. P. Lapage, W. R. Willcox, and M. A. Curtis Numerical classification of the tribe Klebsielkae. J. Gen. Microbiol. 66: Boyd, J., and J. R. Turvey Isolation of a poly-a- L-guluronate lyase from Klebsiella aerogenes. Carbohydr. Res. 57: Brenner, D. J Characterization and clinical identification of Enterobacteriaceae by DNA hybridization. Prog. Clin. Pathol. 7: Brenner, D. J Some taxonomic recommendations and a proposal of neotype strains for nineteen species of Enterobacteriaceae. Int. J. Syst. Bacteriol. 29: Brenner, D. J., J. J. Farmer, F. W. Hickman, M. A. Asbury, and A. G. Steigerwalt Taxonomic and nomenclature changes in Enterobacteriaceae. U. S. Department of Health, Education, and Welfare Publication (CDC) U. S. Department of Health, Education, and Welfare, Washington, D.C. 6. 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