Long-Chain Fatty Acids of Peptococci and Peptostreptococci

Transcription

1 JOURNAL OF CUNICAL MICROBIOLOGY, Dec. 1976, p Copyright American Society for Microbiology Vol. 4, No. 6 Printed in U.S.A. Long-Chain Fatty Acids of Peptococci and Peptostreptococci CAROL L. WELLS* AND CHARLES R. FIELD Wisconsin State Laboratory of Hygiene and Department of Medical Microbiology,* University of Wisconsin, Madison, Wisconsin Received for publication 23 July 1976 The long-chain fatty acids extracted from the whole cells of 12 clinically significant species of peptococci and peptostreptococci were characterized by gasliquid chromatography. The resulting methylated fatty acid profiles (and some unidentified compounds) of 82 strains allowed the 12 species to be separated into four groups. Fifteen strains of Peptostreptococcus anaerobius were placed in group I because they had a unique, prominent compound that occurred in the area where a 0, to C01 fatty acid would be expected. Group II, consisting of Peptostreptococcus intermedius, Peptostreptococcus micros, Peptostreptococcus parvulus, Peptococcus morbillorum, and Peptococcus constellatus, produced C14, C16:1, C18:1, and C18 fatty acids. Peptococcus prevotii, Peptococcus variabilus, Peptococcus magnus, Peptococcus asaccharolyticus, and Peptostreptococcus productus were placed in group III because they contained three to six additional, unidentified compounds that strikingly differentiated them from group II. Peptococcus saccharolyticus was the single species assigned to group IV because it yielded C14, C16, C18:1, C18, and C20 fatty acids and a prominent unidentified peak that occurred between C14 and C16 fatty acids. This study indicated that cellular long-chain fatty acids may be an important tool in clarifying the taxonomy of the peptococci and peptostreptococci. Peptococci and peptostreptococci are found as normal flora of the skin, upper respiratory tract, oral cavity, large intestine, and female genitalia (3); they are frequently associated with infectious diseases (13, 18), and their pathogenicity for man is now widely accepted. Accurate laboratory speciation of the medically important anaerobic gram-positive cocci is difficult. Different species of peptococci and peptostreptococci have similar biochemical reactions, and gas chromatography of short-chain acid metabolites (C1 to C8) often reveals identical fermentation products. The speciation ofthe peptococci and peptostreptococci is further complicated by the existence of many strains that do not fit the already described species of anaerobic cocci (3). In addition, four of the foremost authorities in clinical anaerobic bacteriology differ markedly in their guidelines for speciation of peptococci and peptostreptococci (5, 7, 14-16). This study presents an analysis of the longchain fatty acids (LCFAs) extracted from the whole cells of 12 clinically significant species (82 strains) of peptococci and peptostreptococci. The LCFAs (C8 to C20) of the anaerobic cocci have not yet been characterized, and such analyses have been useful in clarifying the taxonomy of other organisms (10, 12). MATERIALS AND METHODS Organisms and media. The organisms studied were obtained from stock cultures maintained at the Wisconsin State Laboratory of Hygiene, Madison, Wis. All cultures were clinical isolates sent to the State Laboratory for identification over a 4-year period from 1972 to The following identification tests were routinely performed: Gram stain from agar and peptone-yeast-glucose broth; fermentation tests on cellobiose, esculin, fructose, glucose, lactose, maltose, mannitol, and sucrose; esculin hydrolysis; gelatin liquefaction; indole production; nitrate reduction; gas-liquid chromatography of shortchain acid metabolites. In addition, other tests, such as stimulation by Tween 80, reactions in milk and meat, salicin and starch fermentation, starch hydrolysis, and hemolysis, were occasionally done. All tests were performed according to, and with results compatible with, the Virginia Polytechnic Institute (VPI) Anaerobe Laboratory Manual, 2nd ed. (7). Twelve species were studied: Peptostreptococcus anaerobius, Peptostreptococcus intermedius, Peptostreptococcus micros, Peptostreptococcus productus, Peptostreptococcus parvulus, Peptococcus asaccharolyticus, Peptococcus constellatus, Peptococcus magnus, Peptococcus morbillorum, Peptococcus prevotii, Peptococcus variabilus, and Peptococcus saccharolyticus. After identification, the cultures were stored at -70 C in chopped-meat-glucose broth. Before analysis, each culture was thawed and transferred to chopped-meat-glucose broth for 24 to 48 h at 370C. 515

2 516 WELLS AND FIELD The purity of each culture was checked by Gram stain. All cultures were then transferred to 7 ml of peptone-yeast-glucose broth prepared according to the formula given in the VPI Anaerobe Laboratory Manual (7) (peptone made by GIBCO Laboratory, Madison, Wis.; yeast extract and glucose made by Difco Laboratories, Detroit, Mich.) for 48 h at 370C. The cultures were centrifuged, the supernatant was poured off, and the cell pellet was stored at -70 C until the fatty acids were extracted. LCFA extraction. Fatty acids were extracted from the whole cells according to the method of Moss et al. (11) with some modifications. The cells were thawed, suspended in 5 ml of 5% NaOH in 50% aqueous methanol and heated at 100 C for 15 min. The saponified material was cooled and acidified with 6 N HCl to a ph below 2. One milliliter of boron trifluoride methanol (BF3-CH30H, Applied Science Laboratories, State College, Pa.) was added to the saponified mixture and it was heated at 100 C for 5 min. The methylated solution was then added to 10 ml of saturated NaCl and the fatty acid methyl esters were extracted twice with an equal volume of 1:1 ether-hexane. The combined ether-hexane extracts were evaporated at room temperature to about 5 ml under a gentle stream of dry nitrogen and anhydrous sodium sulfate was then added to remove any water. A precipitate frequently formed during the evaporation procedure and was removed by centrifugation at 1,800 rpm for 5 min. Evaporation of the extract with dry nitrogen was continued until a final volume of 0.1 to 0.2 ml was achieved. The extract was stored at -70 C before fatty acid analysis Ġas chromatography. A Hewlett-Packard gas chromatograph (model 5710A) with automatic sampler (model 7671A) and reporting integrator (model 3380A) was used throughout this study. The gas chromatograph was equipped with a flame ionization detector. The flame was maintained by a mixture of hydrogen and air with flow rates of 30 ml/min and 250 ml/min, respectively. The reporting integrator was set for a slope sensitivity of 1 mv, an attenuation of 16 or 32, and a chart speed of 0.5 cm/ min. The integrator printed the retention time of each peak, calculated the area under each peak, or identified each peak relative to an external standard. The external standard contained caprylate (C8), caprate (C10), laurate (C12), myristate (C14), palmitoleate (C16:), palmitate (C16), oleate (C8:1), stearate (C18), and arachidate (C20) methyl esters (Applied Science Laboratories, State College, Pa.). The methyl esters of LCFAs were separated on a coiled-glass column 6 feet (ca. 183 cm) long with a 4- mm ID and a one-fourth-inch (ca cm) OD. The carrier gas was prepurified nitrogen (Badger Welding Supplies, Madison, Wis.), with a flow rate of 50 ml/min. Methylated fatty acid extracts were run on both a polar and a nonpolar column. The nonpolar column was packed with 3% SE-30 on 100/120 Gas- Chrom Q (Hewlett-Packard, Skokie, Ill.). The temperature of the nonpolar column was programmed from 110 to 240 C at 4 C/min, with a total analysis time of 45 min. For analysis on the nonpolar column, the sample size was 1,ul, the injection port tempera- J. CLIN. MICROBIOL. ture was 200 C, and the detector temperature was 350'C. The polar column, used primarily for confirmation of identified peaks, was packed with 15% EGSS-X on 80/100 Gas-Chrom P (Applied Science, State College, Pa.). The temperature of the polar column was maintained at 172 C for a total analysis time of 50 min. For analyses on the polar column, the sample size was 10 gl, the injection port temperature was 200 C, and the detector temperature was 4000C. All samples were first analyzed on the nonpolar column. The remaining extract was diluted 1:3 in ether-hexane to obtain a sufficient volume for analysis on the polar column. Consequently, a 10-,ul sample size was needed to obtain adequate peak size from the polar column, whereas a 1-,ul sample size gave adequate peak size from the nonpolar column. RESULTS Results of this study demonstrated that the peptococci and peptostreptococci, on the basis of gas chromatographic profiles of their methylated LCFAs, could be divided into four groups. Group I (Fig. 1) contained only one species, Peptostreptococcus anaerobius. Fifteen strains of this organism all yielded a similar chromatographic profile which contained a characteristic, prominent, unidentified peak with a retention time between C8 and C10. The average percent fatty acid composition of this unidentified compound was 20% (with a range of 2.4 to 29.0%). This compound was always present and could be used as a marker for group I anaerobic cocci since it was not produced by the other anaerobic cocci assayed. P. anaerobius was also unique among the other peptococci and peptostreptococci because each of 15 P. anaerobius strains analyzed contained from 14 to 25 different peaks (with an average of 19 peaks). All other species of peptococci and peptostreptococci studied never produced over 12 different peaks. After analysis of 15 P. anaerobius strains, the following statements could be made about group I: (i) a prominent, unidentified peak is located between C8 and C01; (ii) a total of 14 to 25 different peaks are produced; (iii) C14, C16, C18:1, and C18 are usually present; the latter fatty acids were present in 13 out of 15 strains studied. On the basis of their cellular LCFA content, Peptostreptococcus intermedius, Peptostreptococcus micros, Peptostreptococcus parvulus, Peptococcus morbillorum, and Peptococcus constellatus were all placed in group II. The average percentages of the LCFA composition of these organisms are summarized in Table 1. All strains within group II had C16 or C18:1 as the most prominent LCFAs on the chromatogram. Group II also produced smaller, but detectable amounts of C14, C16:1, and C18. A repre-

3 VOL. 4, 1976 LCFAs OF PEPTOCOCCI AND PEPTOSTREPTOCOCCI 517 GROUP I GROUP EI 18: llj (I) z 0 a. Uf) LLI wa: a: 0 wl a: ULAJ GROUP mf 14 C: 16 18: Downloaded from l 14 16: : MINUTES FIG. 1. Representative chromatograms of each of the four groups ofpeptococci and peptostreptococci based on long-chain fatty acid analysis of whole cells. sentative chromatogram for this group is shown in Fig. 1. Table 2 shows the average percentages of the fatty acid composition of group III organisms. Group m included Peptococcus variabilus, Peptococcus magnus, Peptococcus asaccharolyticus, Peptococcus prevotii, and Peptostreptococcus productus. The most prominent fatty acids in group III were identical to the fatty acids in group 11 (C16:1, C16, C18:1, and C18). Again, as in group II, the highest peak on the chromatogram was consistently C16 or Cj8:l. Group Ill differed from group II, however, by the presence of three to six additional compounds (Fig. 1). These peaks were all unidentified and occurred between C14 and C16:1, between C16 and C18:1, and between C18 and C20. Group IV contained one organism, Peptococcus saccharolyticus. The LCFAs extracted from P. saccharolyticus are shown in Table 3. Prominent peaks of C16, C08:1, and C18 were similar to those seen in groups II and III; however, C16:1 was conspicuously absent from group IV. P. saccharolyticus contained significant amounts on September 24, 2018 by guest

4 518 WELLS AND FIELD J. CLIN. MICROBIOL. TABLE 1. Average percentages of the long-chain fatty acids extracted from group II cocci Peptostreptococcus sp. Peptococcus sp. Fatty acids P. intermedius P. micros P. parvulus P. morbillorum P. constellatus (15 strains) (8 strains) (1 strain) (5 strains) (5 strains) C (0-3.7)a ( ) (0-6.2) (0-1.0) C ( ) (0-6.8) ( ) ( ) C16: ( ) ( ) ( ) ( ) C ( ) ( ) ( ) ( ) C18: ( ) ( ) ( ) ( ) C ( ) ( ) ( ) ( ) a The numbers in parentheses are the range (in percent) for all organisms tested. of C20 and an unidentified compound with a retention time between C14 and C16, which appeared to be unique characteristics of this species in group IV. Fig. 1 presents a representative chromatogram of group IV (P. saccharolyticus). DISCUSSION The taxonomy of the peptococci and the peptostreptococci is unclear. Four of the most notable publications in this area differ greatly in the speciation of these organisms. Publications of Rogosa (14, 15), Sutter et al. (16), Dowell and Hawkins (5), and Holdeman and Moore (7) describe 11 species of peptococci and six species of peptostreptococci, and they differ on the number and identity of species within each genus. Since all of the peptococci and peptostreptococci (except Peptococcus niger) are considered to be associated with infectious disease in humans, there should be reliable methods for identifying them in the clinical laboratory. Rogosa (14, 15) lists six species of peptococci and five species of peptostreptococci. Of the latter 11 species, five species (Peptococcus niger, Peptococcus aerogenes, Peptococcus activus, Peptococcus anaerobius, and Peptostreptococcus lanceolatus) are not recognized by either Sutter et al. (16), Dowell and Hawkins (5), or Holdeman and Moore (7). Table 4 presents a summary of the current classification of the 12 most generally recognized species of peptococci and peptostreptococci and includes the grouping based on LCFA profiles. Of the 12 species listed in Table 4, there is general agreement on the taxonomic status of only two species, Peptostreptococcus anaerobius and Peptostreptococcus intermedius. It is evident that much work needs to be done on the taxonomy of the peptococci and the peptostreptococci. This study, for the first time, characterized the LCFAs of the 12 most uniformly recognized species of peptococci and peptostreptococci. On the basis of their LCFA chromatographic patterns, each species of peptococcus and peptostreptococcus assayed could be placed into one of four different groups. As a result of LCFA profiles, Peptostreptococcus anaerobius was placed in a group by itself. One distinguishing feature of P. anaerobius was the large number (14 to 25) of peaks on the chromatograms. No other species studied produced over 12 peaks. The most characteristic feature of the profile of P. anaerobius was a unique, prominent peak between C5 and C00. Unfortunately, the latter peak has not yet been identified and may be an iso-acid. Group II cocci contained Peptostreptococcus intermedius, Peptostreptococcus micros, Peptostreptococcus parvulus, Peptococcus morbillorum, and Peptococcus constellatus. The species in group II produced characteristic amounts of C14, C16:i, C16, Cl8:1, and Cl5 fatty acids. The latter profile is similar to the profile reported for fatty acids in some of the facultative streptococci (2, 9). Some of the organisms in this group are now considered to be members of the genus Streptococcus since they produce lactic acid as a major metabolic product. On the latter basis, P. intermedius is now generally recognized as belonging to the genus Streptococcus (4, 8, 14-16). Similarly, P. morbillorum and P. constellatus are now widely considered to be members of the streptococci (4, 8, 16). Peptostreptococcus micros, which produces lactic acid as a minor product, is still generally

5 VOL. 4, 1976 LCFAs OF PEPTOCOCCI AND PEPTOSTREPTOCOCCI 519 TABLE 2. Average percentages of the long-chain fatty acids extracted from group III cocci Peptococcus sp. Peptostreptococcus Fatty acids P. variabilus P. magnus P. asaccharolyti- P. prevotii productus (1 strain) (5 strains) (8 strains) cus (9 strains) (6 strains) C (0-1.8)a ( ) (0-3.4) C (0-3.3) ( ) (0-9.0) (0-1.0) (0-1.6) (0-4.1) (0-2.9) (0-3.6) (0-2.8) C16: ( ) ( ) ( ) (2)2-13.0) C ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (0-5.6) (0-12.2) ( ) C18: ( ) ( ) ( ) ( ) C ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (0-4.7) ( ) (0-2.2) (0-1.1) a The numbers in parentheses are the range (in percent) for all organisms tested. TABLE 3. Average percentages ofthe long-chain fatty acids extracted from group IV cocci Fatty Fatty acids Peptococcus saccharolyticus C14 C16 C18:1 C.8 C20 acids ~~(4 strains) 2.9 ( )a 26.5 ( ) 12.8 ( ) 2.5 (0-9.8) 14.3 ( ) 18.3 ( ) 2.5 ( ) 5.7 (0-8.8) 13.2 ( ) a The numbers in parentheses are the range (in percent) for all organisms tested. considered to be a member of the peptostreptococci. The fatty acids in P. parvulus also placed this organism in group II; however, only one strain was analyzed and this should not be considered a definitive characterization. The VPI Anaerobe Laboratory Manual also describes P. parvulus on the basis of a single isolate and the Manual also notes that this organism produces lactic acid as a major metabolic product (7). Thus, the most significant interpretations of the LCFA profiles of group II organisms are: (i) these LCFA profiles are consistent with the profiles of the facultative streptococci; and (ii) all species now being considered for classification with the streptococci have all been placed in group II (Table 4) on the basis of LCFA analysis. Group III cocci, consisting ofpeptococcus variabilus, Peptococcus magnus, Peptococcus asaccharolyticus, Peptococcus prevotii, and Peptostreptococcus productus, appeared to be a miscellaneous assortment of organisms that produce similar LCFA profiles that consist of C16:1, C16, C08:1, and C1l fatty acids in amounts similar to those seen in group II. The chromatograms of the organisms in group m contained from three to six additional, unidentified peaks not seen in group H organisms, and thus warranted the formation of a separate group. The organisms in group III possessed some associations that were consistent with the published literature on anaerobic cocci. Holdeman and

6 520 WELLS AND FIELD J. CLIN. MICROBIOL. TABLE 4. Current classification of the species of peptococci and peptostreptococci a Organism Rogosa (14, 15)8 Sutter et al. Dowell and Holdeman and Long-chain fatty (16) Hawkins (4, 5) Moore (6-8) acid grouping Ps. anaerobius R R Rc R 1 Ps. intermedius S S S S 2 Pc. morbillorum U S S S 2 Pc. constellatus R R S S 2 Ps. micros R R Q R 2 Ps. parvulus R R Q R 2 Pc. magnus Ud Rd Q Re 3 Pc. variabilus Ud Rd Q Re 3 Pc. asaccharolyticus R R Rf R 3 Pc. prevotii Ug R Rh R 3 Ps. productus R R Q R 3 Pc. saccharolyticus U U RI R 4 a Abbreviations: Pc., Peptococcus; Ps., Peptostreptococcus; R, recognized species; U, unrecognized species; Q, questionable status; S, Streptococcus species. b Numbers in parentheses indicate literature reference. c Designated Peptostreptococcus Center for Disease Control (CDC) group 3. d Considered the equivalent of Peptococcus anaerobius. e Considered equivalent organisms. ' Designated Peptostreptococcus CDC group 1. 9 Considered the equivalent of Peptococcus asaccharolyticus. h Designated Peptostreptococcus CDC group 2. i Designated Peptococcus CDC group 2. Moore (6) consider P. magnus and P. variabilus to be the same organism since their biochemical profiles are identical if one adds Tween 80 to their growth medium. Rogosa (14) claims that P. variabilus and P. magnus are the equivalent of a Peptococcus anaerobius. As a solution to this obvious confusion, West and Holdeman (17) propose that Peptococcus anaerobius should be rejected since it is a nomen confusum due to the existence of Peptostreptococcus anaerobius. An example of two other organisms that may not be separate species are P. prevotii and P. asaccharolyticus. Rogosa (14) states that P. prevotii and P. asaccharolyticus are indistinguishable since they differ only by an indole reaction out of over 100 other characteristics. The placement of P. productus in group III must not be considered a reliable classification since only one strain was available for analysis. Thus, as seen in Table 4, group Ill contains five organisms and any two organisms considered equivalent by any taxonomist were always placed in the same LCFA group. On the basis of the LCFA profiles of whole cells, a single species, P. saccharolyticus, was placed in group IV. P. saccharolyticus contained fatty acids C14, C16, Ci8:1, and C18 that appeared to be typical of the other peptococci and peptostreptococci. This organism, however, had two unique characteristics not seen in the other peptococci and peptostreptococci: (i) significant amounts of C20 were produced; and (ii) a prominent, unidentified peak with a retention time between C14 and C16 was present. This peak may be a branched-chain 15-carbon fatty acid similar to that reported in cell extracts of the genus Ruminococcus (1). Some of the literature on the peptococci and peptostreptococci support placing P. saccharolyticus in a separate group. Dowell and Hawkins (5), in the Center for Disease Control Laboratory Manual, listed P. saccharolyticus as the only species in the peptococci. Rogosa (14) claimed that P. saccharolyticus was frankly saccharoclastic and should be excluded from the genus Peptococcus, without, however, mentioning where the organism should be placed. On the basis of LCFA analysis, these studies agree with Rogosa (14, 15) and with Dowell and Hawkins (5), that P. saccharolyticus is significantly different from the other peptococci and peptostreptococci and should be placed in a group by itself. This study showed that LCFA analysis can be used as an aid in taxonomy, but cannot be relied on as a definitive test for species designation. Further work must be done if the speciation of the peptococci and the peptostreptococci is to be clarified. Additional biochemical tests, serological studies, guanine-cytosine ratios, or deoxyribonucleic acid homology studies might prove helpful for speciation. The grouping of species obtained from the LCFA profiles may be an indication that these species do overlap each other a great deal. Perhaps our goal should not be to describe new species of these organisms, but to consolidate the species known to exist.

7 VOL. 4, 1976 This approach seems more logical, at least until definitive pathogenicity studies have been completed. ACKNOWLEDGMENTS This study was supported by the Wisconsin Alumni Research Federation. We are grateful for the cooperation of the members of the General Bacteriology Laboratory at the Wisconsin State Laboratory of Hygiene. We especially appreciate assistance given by A. Helstad. We also wish to acknowledge E. Balish for his advice in the preparation of this manuscript. LITERATURE CITED 1. Allison, M. J., M. P. Bryant, I. Katz, and M. Ke_a_y Metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. II. Biosynthesis of higher branched-chain fatty acids and aldehydes. J. Bacteriol. 83: Amatein, C. F., and P. A. Hartman Differentiation of some enterococci by gas chromatography. J. Bacteriol. 113: Balows, A., R. M. DeHaan, V. R. Dowell, Jr., and L. B. Guze (ed.) Anaerobic bacteria: role in disease. Charles C Thomas, Springfield, ill. 4. Dowell, V. R Anaerobic cocci. Newsletter of November, Center for Disease Control, Atlanta, Ga. 5. Dowell, V. R., Jr., and T. M. Hawkins Laboratory methods in anaerobic bacteriology. CDC laboratory manual. DHEW publication no. (CDC) U.S. Government Printing Office, Washington, D.C. 6. Holdeman, L. V Anaerobe newsletter, no. 2. Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. 7. Holdeman, L. V., and W. E. C. Moore (ed.) Anaerobe laboratory manual, 2nd ed. Anaerobe Laboratory, Virginia Polytechnic Institute and State LCFAs OF PEPTOCOCCI AND PEPTOSTREPTOCOCCI 521 University, Blacksburg, Va. 8. Holdeman, L. V., and W. E. C. Moore New genus, Coprococcus, twelve new species, and emended descriptions of four previously described species of bacteria from human feces. Int. J. Syst. Bacteriol. 24: Lambert, M. A., and C. W. Moss Cellular fatty acid composition of Streptococcus mutans and related streptococci. J. Dent. Res. Special Issue A 55:A96- A Mos, C. W., D. S. Kellogg, D. C. Farshy, M. A. Lambert, and J. D. Thayer Cellular fatty acids of pathogenic Neisseria. J. Bacteriol. 104: Moss, C. W., M. A. Lambert, and W. H. Kerwin Comparison of rapid methods for analysis of bacterial fatty acids. Appl. Microbiol. 28: Moss, C. W., S. B. Samuel, and R. E. Weaver Cellular fatty acid composition of selected Pseudomonas species. Appl. Microbiol. 24: Pien, F. D., R. L. Thompson, and W. J. Martin Clinical and bacteriological studies of anaerobic gram positive cocci. Mayo Clin. Proc. 47: Rogosa, M Genus I. Peptococcus, p In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore, Md. 15. Rogosa, M Genus II. Peptostreptococcus, p In R. E. Buchannan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams and Williams Co., Baltimore, Md. 16. Sutter, V. L., V. L. Vargo, and S. M. Finegold Wadsworth anaerobic bacteriology manual, 2nd ed. Anaerobic Bacteriology Laboratory, Wadsworth Hospital Center, Los Angeles, Calif. 17. West, S. E. H., and L. V. Holdeman Placement of the name Peptococcus anaerobius (Hamm) Douglas on the list of nomina rejicienda. Int. J. Syst. Bacteriol. 23: Zabranski, R. J Isolation of anaerobic bacteria from clinical specimens. Mayo Clin. Proc. 45: