Positive Rods Isolated from Frozen Vegetables1

Transcription

1 APPLED MCROBOLOGY, Jan., 1967, p Copyright 1967 American Society for Microbiology Vol. 15, No. 1 Printed in U.S.A. Numerical Taxonomy of Gram-Positive and Catalase Positive Rods solated from Frozen Vegetables1 D. F. SPLT7STOESSER, MARJORE WEXLER, JANET WHTE, AND R. R. COLWELL' Department offood Science and Technology, New York State Agricultural Experiment Station, Cornell University, Geneva, New York Received for publication 1 September 1966 ABSTRACT One hundred isolates from peas, beans, and corn were compared with cultures of Corynebacterium, Microbacterium, and Arthrobacter by use of numerical taxonomic procedures. Six groups (clusters), representing 75% of the isolates, resembled Corynebacteriaceae. There was some doubt regarding the genera represented because the groups were not closely related to many of the known strains. The relationship of the different groups to each other, as well as a number of their properties, is presented. n a study of the microbial contaminants of frozen vegetables, gram-positive, catalase-positive rods made up 1, 1, and 8% of the "total" counts on peas, snap beans, and corn, respectively (18). Because most of these organisms defied identification by traditional methods, even to the genus level, numerical taxonomic procedures were adopted to aid in their classification. This report describes the results obtained when 1 unknown isolates were compared with cultures of Corynebacterium, Microbacterium, and Arthrobacter. MATERALS AND METHODS Appropriate dilutions of blended vegetables were cultured on tryptone-glucose-yeast extract-agar (TGYE agar) (1). Colonies were picked after the plates had been incubated for days at 3 C. Five cultures were isolated from corn, from beans, and 73 from peas. Details with respect to the vegetables and isolation procedures have been published (18, 19). The following named cultures were studied along with the unknowns: C. flaccumfaciens CF-18, C. fascians CF-19 and CF-, C. insidiosum C-18, C. michiganense CM-9, C. poinsettiae CP-, A. tumescens ATCC 697, A. ureafaciens ATCC 756, A. globiformis ATCC 81, M. lacticum ATCC 818, and M. flavum ATCC 13. The corynebacteria were obtained from Robert Dickey, Department of Plant Pathology, Cornell University; the other cultures were purchased from the American Type Culture Collection. Approved by the Director of the New York State Agricultural Experiment Station for publication as Journal Paper 15. Present address: Department of Biology, Georgetown University, Washington, D.C. The cultures were subjected to a group of tests and observations that yielded 11 recordable characters (Table 1). Most of the methods are given by Skerman (15), Ewing (6), and the Committee on Bacteriological Technique (), and, thus, will not be described in detail. Observations on cell morphology were made on slides stained with methylene blue and crystal violet, as well as on wet mounts under a phase microscope. Gram-stained cells were used for size measurements. TGYE agar without the glucose served as the basal medium in carbon utilization tests. An alkaline reaction signified utilization of the organic acids. The medium and methods of Hugh and Leifson (8) were used to determine whether glucose was oxidized or fermented. The different inhibitory media were prepared and used as directed by their commercial sources. The tolerance tests were conducted by adding different concentrations of inhibitors to TGYE media. A medium containing 5% butterfat was used to detect lipolysis. The plates were flooded with a saturated solution of CUSO after 1 week of incubation. Kovacs' (11) oxidase test was performed, as well as the cytochrome oxidase test (7) that is listed. The data were processed on an nternational Business Machines (BM) 16, model, computer at Georgetown University. Similarity coefficients (per cent S) between each culture pair were calculated according to the formula of Sneath (16); thus, only positive matches were taken into account. The cultures were then grouped on the basis of single linkage, i.e., cultures were added to a given group at the highest per cent S shared by the entrant culture and any existing members of the group. The computer programs used in the analysis were GTP-1 and GTP- (Colwell and Troy, BM 16 Computer Users Library Programs). 158 Downloaded from on October 5, 18 by guest

2 VOL. 15,1967 MCROBAL CONTAMNANTS N FROZEN VEGETABLES 159 TABLE 1. Observations upon which the similarity indices were calculated Character No. coded Morphology: size, shape, stain reactions, capsules, spores, flagella, cell arrangement Culture: growth in broth and agar media; pigmentation Carbon metabolism: acid from glucose, fructose, mannose, galactose, arabinose, xylose, sucrose, maltose, lactose, melibiose, trehalose, melizitose, raffinose, mannitol, rhamnose, dextrin Utilization of lactate propionate, pyruvate, succinate, tartrate, benzoate 6 Hugh and Leifson test... Growth temperature: 3, 1, 3, 37, 5 C... 5 Growth in inhibitory media: EMB, telluriteglycine, M-Endo, azide dextrose, selenitecystine, ph.5 agar, brilliant green, deoxycholate, asparagine agar, glucose- NH-salts Tolerance to: Sodium chloride, potassium tellurite, ethyl violet, sodium azide, mercuric chloride, penicillin, streptomycin Thermal resistance: D values at 6 C... 1 Hydrolysis of: starch, gelatin, cellulose, sodium hippurate, esculin... 9 Reactions in litmus milk... Tests for: lipolysis, nitrate reduction, Voges Proskauer, methyl red, titratable acidity,,b-glucosidase, cytochrome oxidase, catalase, acid and alkaline phosphatase, NH3, HS, phenylalanine deamination... 1 RESULTS Similarity coefficients down to 5% were calculated for all culture pairs. The highest matches were at 89% S. At 5% S, all but two cultures, C. insidiosum and C. michiganense, had merged into one large group. At 66% S, six groups contained 75% of the vegetable isolates. Differences in the size and homogeneity of these groups are illustrated in the matrix of similarity coefficients (Fig. 1). Group V was the largest with cultures, and groups and V (not shown) were the smallest with 5 cultures each. The isolates that did not fit into these groups at this level consisted of a number of pairs and triplets having similarities as high as 78%. For example, three cultures that formed spores clustered at 7% S and finally merged with the other groups at 57% S. The intra- and interrelationships of the six main groups are shown in a dendrogram (Fig. ). The points represent the different subgroups and show the levels at which they were formed. t can be seen that groups to V originated at 8% S or above, whereas groups V and V formed at lower levels; thus, cultures within the latter two groups shared fewer properties. The data also show the similarity levels at which the different groups merged. At 63% S, only two groups remained, owing to the merger of groups to V; at 6% S, all six groups had joined. Only three of the named cultures united with the vegetable isolate groups at 65% S or above. A. ureafaciens joined group V at 66% S, and the two C. fascians strains merged with group V at 65% S. With the exception of C. insidiosum and C. michiganense, the other known cultures linked with the merged groups at levels ranging from 56 to 6% S. Most of the species within the three genera FG. 1. Similarity matrix showing the relationship between the 7 isolates that clustered into groups through V. 9F so8 7[ 6 FG.. Dendrogram illustrating the intra- and intergroup relationships of the coryneform isolates. The points show the per cent similarity at which the different subgroups formed. Downloaded from on October 5, 18 by guest

3 16 SPLTTSTOESSER ET AL. APPL. MCROBOL. differed as much from each other as they did from the vegetable isolate groups. Thus, C. insidiosum and C. michiganense paired at 57% S, the level at which C. poinsettiae and the two strains of C. fascians clustered. The three species of Arthrobacter joined at 6% S, and the two species of microbacteria formed a cluster at 56%. The frequency of occurrence of the positive characters in each of the six groups was measured. The approach was similar to that of the median organism of Liston et al. (1), with the exception that the characters shared by 75% rather than 5% of the cultures within a group were recorded. Some of the properties thought to be important for characterizing the different groups are presented in Table. Others will be discussed in the test. Certain properties were shared by the six groups. All were gram-positive, catalase-positive, nonsporeforming rods that oxidized glucose and Property TABLE. Morphology Size (,u)... Pleomorphic... Stained irregularly... Branched... Flagella... Capsules... TGYE agar pigment... Carbon metabolism Acid from hexoses... Acid from pentoses... Acid from disaccharides. Acid from trisaccharides. Organic acid utilization. Hugh and Leifson test... Growth in NaCl broth, 5%. Glucose-NH salts medium... Asparagine agar... Growth temperature range... Biochemistry Litmus milk... Gelatin liquefaction... Kovac oxidase... NH3 from peptone... HS production... Alkaline phosphatase... Reduced K-tellurite (.%O)... gave positive tests for acid phosphatase and f3- glucosidase. Most of the organisms gave negative tests for starch hydrolysis, cytochrome oxidase, and phenylalanine deamination. Only one isolate, a culture in group V, decomposed cellulose after a -week incubation period. A number of observations on morphology were recorded (Table ). Club-shaped forms, as well as other pleomorphic forms, predominated in four of the six groups. The irregular staining reported for groups and refers to the fact that certain sections of the cells were gram-positive, whereas other areas were gram-negative. None of the isolates was acid-fast. Branched forms, which predominated in the group V isolates, also were observed in a few of the cultures included in group V. The possession of flagella was a characteristic common only to the organisms that clustered in group V; 1 of the 18 cultures exhibited them when stained by the Leifson (13) Properties possessed by 75% of the cultures within each group a Refers to the number of compounds utilized (see Table 1). 1.5 X.5 3a Alkaline 1. X.5 Orange 1S 1 Facultative 3-37 Reduced Group 1.5 X.5 ± Proteolysis V 1. X Acid v V.8 X.551~ X Ring White 1 5 i 3-37 Alkaline Downloaded from on October 5, 18 by guest

4 VOL. 15, 1967 MCROBAL CONTAMNANTS N FROZEN VEGETABLES procedure. The number of cultures and observed flagellum types were as follows: five polar-monotrichous, three polar-lophotrichous, three lateralmonotrichous, two peritrichous, and one in doubt. Of the 7 cultures in groups to V, 6 produced a nondiffusible, yellow or orange pigment when grown on TGYE or nutrient agar. t would appear that this pigmentation correlated with a number of other features, since only of the 3 vegetable isolates that did not fall into these groups were pigmented. The organisms in groups, ll, and V produced acid from a greater number of sugars than those in the other groups (Table ). Groups V and V, which attacked few sugars, utilized all of the organic acids that were tested. Although group was the only one in which 75% or more of the organisms fermented, as well as oxidized, glucose, 8 of 13 organisms in group and 1 of in group V also utilized this sugar anaerobically. Facultative organisms, therefore, were present but did not predominate in the other groups. Growth in the glucose-nh salts medium of Ayers et al. () was characteristic of groups to V when the synthetic medium was inoculated (by needle) with cells grown on TGYE agar or broth. However, only the organisms in group V grew after three serial transfers in the synthetic medium. These results suggest that, although NH3 served as a nitrogen source for the four groups, growth factors were required by all but group V. The group V organisms tolerated the greater number of inhibitors. They grew in telluriteglycine, selenite-cystine, azide dextrose, and brilliant green media, as well as in the presence of Ag/ml of HgC1, units of streptomycin per ml., 1.5 units of penicillin per ml, or.83 mg of ethyl violet per liter. The other groups were inhibited by most of these media and compounds. For example, group V cultures were inhibited by all but the selenite-cystine medium, whereas the group organisms tolerated only the penicillin. The heat resistance of the isolates was measured to determine whether any of them resembled heat-resistant strains of M. lacticum. After heating for 5, 1, and 15 min at 6 C, cells were plated in 1 mm sodium phosphate solution (ph 7). The results were expressed as D values; i.e., the number of minutes required to reduce the viable count by 9%. These studies showed that the characteristic D6 for all groups, except group, was in the 5- to 1-min range. Group was less resistant, in that four of the five cultures yielded values under 5 min. Thirteen of the isolates, primarily in groups V and V, exhibited a D6 of 1 to 15 min. Our single M. lacticum culture was in the 5- to min range and, thus, in our experiments, failed to exhibit special heat resistance. DSCUSSON The predominant gram-positive bacilli in frozen peas, beans, and corn resembled members of the family Corynebacteriaceae, in that they were catalase-positive, nonsporeforming organisms that showed the diversity of morphology characteristic for this group. t had been hoped that, by use of numerical taxonomic principles, a comparison of these organisms with representatives of different groups within the Corynebacteriaceae would indicate which genera they resembled most closely. This approach was not completely successful, however, because the species within the three genera represented by named strains from culture collections differed as much from each other as they did from the vegetable isolates. The difficulty might have been predicted, since a number of reports point to marked heterogeneity within the coryneform group. Thus, organisms within a given genus may differ considerably in morphology (9), pathways of carbohydrate metabolism (1), cell wall composition (), and nutritional requirements (1). Because of such variation, recent numerical taxonomic analyses have indicated that the plant pathogens should be removed from the genus Corynebacterium (5). nspection of the properties of the six groups (Table ) suggests that groups through V resembled the organisms found in a number of foods, including eggs, meats, and dairy products (1, 17, ). They probably have been most commonly identified as species of Corynebacterium or Microbacterium. The isolates in groups V and V, on the other hand, resembled more closely the soil organisms described by Conn and Dimmick (3), the Arthrobacter. Many were branched, their nutritional requirements were relatively simple, and acid was produced from only a few sugars. Although the characteristic transition from gramnegative cocci to rod forms was not commonly observed, this failure may merely reflect an insufficient number of microscopic examinations during the first hours of growth. The heterogeneity of group V was further evidenced by the mixed flagella types represented. Airborne contamination of the surfaces of processing equipment was probably the source of these nonsporeforming organisms, since they do not survive the blanch (18). Once introduced, they undoubtedly became established as a part of the processing-line microflora, since the counts of thousands per gram found on vegetables (19) are indicative of actual growth of the organisms on equipment surfaces or the product, or both. Downloaded from on October 5, 18 by guest

5 16 SPLTTSTOESSER ET AL. APPL. MCROBOL. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant EF-55 from the Division of Environmental Engineering and Food Protection, and by grant GB 3363 from the National Science Foundation. LTERATURE CTED 1. AMERcAN PUBLC HEALTH AssoCATON Recommended methods for the microbiological examination of foods, p. 7. American Public Health Association, New York.. COMMTTEE ON BACTEROLoGCAL TECHNQUE Manual of microbiological methods, p McGraw-Hill Book Co., nc., New York. 3. CONN, H. J., AND. DMMCK Soil bacteria similar in morphology to Mycobacterium and Corynebacterium. J. Bacteriol. 5: CUMMNS, C. S Chemical composition and antigenic structure of cell walls of Corynebacterium, Mycobacterium, Nocardia, Actinomyces, and Arthrobacter. J. Gen. Microbiol. 8: DA SLVA, G. A. N., AND J. G. HOLT Numerical taxonomy of certain coryneform bacteria. J. Bacteriol- 9: EwNG, W. H Enterobacteriaceae. U.S. Public Health Serv. Publ. 73, p GABY, W. L., AND E. FREE Differential diagnosis of Pseudomonas-like microorganisms in the clinical laboratory. J. Bacteriol. 76:-. 8. HUGH, R., AND E. LEFSON The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J. Bacteriol. 66: JENSEN, H. L The coryneform bacteria. Ann. Rev. Microbiol. 6: KEDDE, R. M., B. G. S. LEASK, AND J. M. GRANGER A comparison of coryneform bacteria from soil and herbage: cell wall composition and nutrition. J. Appl. Bacteriol. 9: KovAcs, N dentification of Pseudomonas pyocyanea by the oxidase reaction. Nature 178: KRAFT, A. A., J. C. AYRES, G. S. TORREY, R. H. SALZER, AND G. A. N. DA SLVA Coryneform bacteria in poultry, eggs, and meat. J. Appl. Bacteriol. 9: LEFSON, E Staining, shape, and arrangement of bacterial flagella. J. Bacteriol. 6: LsrON, J., W. WEBE, AND R. R. COLWELL Quantitative approach to the study of bacterial species. J. Bacteriol. 85: SKERMAN, V. B. D A guide to the identification of the genera of bacteria, p. 17. The Williams & Wilkins Co., Baltimore. 16. SNEATH, P. H. A The application of computers to taxonomy. J. Gen. Microbiol. 17: SPECK, M. L A study of the genus Microbacterium. J. Dairy Sci. 6: SPLTrSTOESSER, D. F., AND. GADJO The groups of microorganisms composing the "total" count population in frozen vegetables. J. Food Sci. 31: SPLirrsToEssER, D. F., W. P. WETTERGREEN, AND C. S. PEDERSON Control of microorganisms during preparation of vegetables for freezing.. Green beans. Food Technol. 15: SULZBACHER, W. L., AND R. A. McLEAN The bacterial flora of fresh pork sausage. Food Technol. 5: ZAGALLO, A. C., AN C. H. WANG Comparative carbohydrate metabolism in Arthrobacter. J. Gen. Microbiol. 9: Downloaded from on October 5, 18 by guest