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INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1992, p. 12-18 Vol. 42, No. 1 0020-7713/921010012-07$02.OO/O Copyright 0 1992, International Union of Microbiological Societies

Application of Multivariate Analyses of Enzymic Data to Classification of Members of the Actinobacillus- Haemophilus-Pasteurella Group

VESLEMJbY MYHRVOLD,' ILIA BRONDZ,2t AND INGAR OLSEN'" Department of Microbiology, Dental Faculty, University of Oslo, Oslo, Norway,' and Research Department, National Institute of Occupational Health, UmeB, Sweden2

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-componentanalysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophiius. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. injluenzae, H. parainjluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.

Organisms belonging to the Actinobacillus-Haemophilus- The enzymic characterization data were treated statisti- Pasteurella group, which constitute the family Pasteurel- cally by using multivariate analyses. We have previously laceae, have assumed increasing clinical importance in med- used such methods as auxiliary techniques to define the icine and dentistry over the last few years (see references 30, following bacterial and yeast genera: Actinobacillus, Hae- 34, 38, 39). Unfortunately, generic separation in this family mophilus, and Pasteurella (6, 11); Porphyromonas (3, 8, 9); has been questioned, and with conventional biochemical Prevotella, Bacteroides, Campylobacter, and Wolinella (2, tests it can be difficult to distinguish among the sometimes 8, 9); Treponema (4); and Candida, Torulopsis, and Saccha- confusingly similar species of these genera. This particularly romyces (7, 10). applies to Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus para- MATERIALS AND METHODS phrophilus. Therefore, additional criteria should be sought to assist in the taxonomic separation of organisms belonging Bacteria. The bacterial species and strains which we used to the Actinobacillus-Haemophilus-Pasteurellagroup. and their sources are shown in Table 1. The organisms were Like a number of other gram-negative rod-shaped bacte- cultured in an atmosphere containing 80% N,, 10% H,, and ria, organisms belonging to the genera Actinobacillus, Hae- 10% CO, at 37°C for 3 days. Each cell culture was divided mophilus, and Pasteurella form budding extrusions (exten- into two parts; one part was used for isolation of vesicles and sions of the outer membrane) which can be either attached to fragments that were assessed enzymically, and the other part the bacterial cell surface or released to the environment was used for enzymic assessment of whole cells. In order to during growth (16, 19, 23, 25, 35). These structures are remove vesicles and fragments attached to whole-cell sur- referred to below as outer membrane vesicles or outer faces, a light ultrasonic treatment was performed before membrane fragments. They serve as vehicles for toxins and enzymic registration. enzymes (for a review, see reference 24), and their small Collection of outer membrane vesicles and fragments. The sizes (21 to 500 nm) permit them to cross epithelial barriers outer membrane vesicles and fragments were obtained after that are impermeable to whole cells. ammonium sulfate precipitation, differential centrifugation, The aim of this study was to examine whether the enzymic and dialysis (14). Briefly, cells from 0.5 liter of a 3-day contents of vesicles and fragments and the enzymic contents culture of bacteria were collected by centrifugation at 10,000 of whole cells, as determined by using the API ZYM X g for 15 min. Then, over a period of 2 h, 120 g of micromethod, can be used for taxonomic distinction among ammonium sulfate (May & Baker, Ltd., Dagenham, En- members of the Actinobacillus-Haemophilus-Pasteurella gland) was added to the culture supernatant (40% satura- group. Previously, the API ZYM method has been used to tion). After a second centrifugation at 20,000 x g for 40 min, distinguish whole cells of A. actinomycetemcomitans, Acti- the resulting pellet was suspended in 15.0 ml of 50 mM Tris nobacillus lignieresii, and H. aphrophilus (29). Another buffer (pH 9.5) containing 0.5 mM dithiothreitol (Sigma purpose of this study was to determine whether the enzyme Chemical Co., St. Louis, Mo.). This suspension was dia- contents of vesicles and fragments differed from the enzyme lyzed for 16 h at 4°C against 3 liters of the same buffer. The contents of whole cells within the same species. vesicles and fragments obtained were then collected by centrifugation at 27,000 x g for 40 min and resuspended in 5 ml of 50 mM Tris buffer (pH 7.2) containing 0.5 mM * Corresponding author. dithiothreitol. After a final centrifugation at 27,000 x g for 40 ? Present address: Department of Microbiology, Dental Faculty, min, the vesicles and fragments were resuspended in 1.0 ml University of Oslo, Oslo, Norway. of Tris buffer (pH 7.2) and kept at -20°C. The method

12 VOL.42, 1992 API ZYM PROFILES OF PASTEURELLACEAE 13

TABLE 1. Origins and sources of the strains of the incubated, and read according to the manufacturer’s direc- Actinobacillus-Haemophilus-Pasteurellagroup investigated tions. The enzymic tests were carried out aerobically after Strain Sample Site of isolation Sourcea inoculation with 65 r~.lof vesicles and fragments or whole bacterial cells emulsified in 2 ml of sterile, distilled water to A. actinomycetemcomitans a turbidity between McFarland no. 5 and 6 standards. All FDC Y4 1 Periodontitis FDC tests were performed three times by using material collected SUNY 75 2 Dental plaque SUNY from cultures obtained on different days. BK435 3 Pus Kilian Statistical analyses. The statistical analyses were based on FDC 511 4 Periodontitis FDC FDC 2097 5 Periodontitis FDC mean scores from three experiments. In this study the FDC 2112 6 Periodontitis FDC following two multivariate approaches were used: principal- ATCC 29523 7 Blood ATCC component analysis (18, 37) and soft independent modeling ATCC 29524 8 Chest aspirate ATCC of class analogy (SIMCA) (36). In the principal-component ATCC 33384T 9 Lung abscess ATCC method the original space for variable measurements is A. lignieresii projected down onto two low-dimension subspaces. One of ATCC 19393 17 Bovine lesion ATCC these is sample related, and the other is variable related. NCTC 4189T 25 Glands, cattle NCTC This projection also decides which of the variables contrib- A. ureae KWF 3520/59= 24 Ozaena KWF utes most to the sample-related projection. The complexity H. aphrophilus FDC 621 10 Periodontitis FDC of both models was determined by cross-validation (36, 37). FDC 626 11 Periodontitis FDC The approximate class borders for the SIMCA analysis were FDC 654 12 Periodontitis FDC constructed by using an F test. FDC 655 13 Periodontitis FDC ATCC 13252 14 ATCC ATCC 19415 15 Endocarditis ATCC ATCC 33389= 16 Endocarditis ATCC RESULTS H. paraphrophilus API ZYM data. The vesicles and fragments and the whole KWF IMoAI 27 Cerebellar abscess KWF bacterial cells obtained from 33 bacterial strains and as- KWF IIMoAI 28 Cerebellar abscess KWF KWFIIIRA43 29 Sputum KWF sessed in three independent experiments by using cultures H76 30 Oral cavity Sims produced on different days were negative for the following HK477 31 Dental plaque Kilian enzyme activities: lipase, trypsin, chymotrypsin, a-galac- ATCC 29240 32 Parietal abscess ATCC tosidase, P-galactosidase, P-glucuronidase, P-glucosidase, ATCC 29241T 33 Paronychia ATCC N-acetyl-P-glucosaminidase, a-mannosidase, and a-fucosi- H. infiuenzae dase. The results of the enzymic reactions for the bacterial ATCC 31441 21 Clinical isolate ATCC strains in the tests for the nine remaining enzymes are shown ATCC 33533 22 Blood ATCC in Table 2 (vesicles and fragments) and in Table 3 (whole ATCC 8143T 26 NCTC cells). H. aphrophilus and H. paraphrophilus produced H. parainfiuenzae NCTC 4101 19 Human tongue NCTC a-glucosidase, while A. actinomycetemcomitans, A. lignier- ATCC 7901 20 ATCC esii, Actinobacillus ureae, Haemophilus influenzae, Pas- P. haemolytica NCTC 9380T 23 NCTC teurella haemolytica, and Pasteurella multocida did not. P. multocida NCTC 10322T 18 NCTC Minor differences were occasionally found between the enzymic profiles of vesicles and fragments and the enzymic a ATCC, American Type Culture Collection, Rockville, Md.; NCTC, profiles of whole cells for specific organisms. In such cases National Collection of Type Cultures, London, England; FDC, Forsyth Dental Center, Boston, Mass.; SUNY, State University of New York at the activity tended to be highest in the whole-cell prepara- Buffalo, Buffalo, New York; Kilian, M. Kilian, Royal Dental College, Aarhus, tions. This was particularly observed with leucine arylami- Denmark; Sims, H. W. Sims, Royal Dental Hospital of London, London, dase, the activity of which was consistently high in the England; KWF, Kaptein Wilhelmsen og Frues Bakteriologiske Institutt, whole-cell preparations and usually absent in the vesicle and Rikshospitalet, University of Oslo, Oslo, Norway. fragment preparations. Statistical analyses. In the principal-component analysis, the enzymic data for membrane vesicles and fragments and described above provided membrane fragments (14); the the data for whole cells were combined. The two first generation of these fragments may have been related to the principal-component score vectors (tl and t2) were plotted natural shedding of cell wall components during bacterial against each other (Fig. 1). This sample-oriented (Lee,bac- growth or to successive treatments during preparation. The terial strain-oriented) projection described the two largest use of Tris buffer at pH 9.5 increased solubilization and thus variants of the data matrix. In this projection the A. actino- aided specific isolation of the vesicles by centrifugation (14). mycetemcomitans strains constituted a heterogeneous Enzymic assessment. We used the API ZYM semiquanti- group. While samples 2 to 4, 6, and 7 of A. actinomycetem- tative micromethod system (API System-La Balme, Les comitans formed a distinct cluster of organisms that was well Grottes, Montalieu, France), which allows rapid determi- separated from other organisms, including H. aphrophilus nation of 19 enzymic reactions in a complex, unpurified (samples 10 to 16), samples 1, 5, 8, and 9 of A. actinomyce- sample. Each enzyme strip was assayed, by using appropri- temcomitans were close to A. lignieresii (samples 17 and 25), ate chromogenic substrates, for alkaline phosphatase, es- H. influenzae (samples 21, 22, and 26), P. haemolytica terase, esterase-lipase, lipase, leucine arylamidase , valine (sample 23), and P. multocida (sample 18). Except for H. arylamidase, cystine arylamidase, trypsin, chymotrypsin, aphrophilus ATCC 33389 (sample 16), which was close to acid phosphatase, naphthol-AS-BI-phosphohydrolase,a-ga- Haemophilus parainfluenzae ATCC 7901 (sample 20), the H. lactosidase, P-galactosidase, P-glucuronidase, a-glucosidase, aphrophilus strains formed a distinct group, as did the H. P-glucosidase, N-acetyl-P-glucosaminidase, a-mannosidase , paraphrophilus strains (samples 27 to 33). The H. aphro- and a-fucosidase. The API ZYM strips were inoculated, philus and H. paraphrophilus clusters were well separated. 14 MYHRVOLD ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Results of enzymic characterization of outer membrane vesicles and fragments from bacteria with the API ZYM systema Activity of

Organism Alkaline Esterase- Leucine Valine Cystine Acid AS-BI- a-Glucosi- Sample phos- Esterase lipase arylami- arylami- arylami- phos- phospho- dase phatase dase dase dase phatase hydrolase

A. actinomycetemcomitans FDC Y4 1 SUNY 75 2 HK435 3 FDC 511 4 FDC 2097 5 FDC 2112 6 ATCC 29523 7 ATCC 29524 8 ATCC 33384T 9 A. lignieresii ATCC 19393 17 NCTC 4189= 25 A. ureae KWF 3520/59T 24 H. aphrophilus FDC 621 10 FDC 626 11 FDC 654 12 FDC 655 13 ATCC 13252 14 ATCC 19415 15 ATCC 33389T 16 H. paraphrophilus KWF IMoAI 27 KWF IIMoAI 28 KWF IIRA43 29 H76 30 HK477 31 ATCC 29240 32 ATCC 29241T 33 H. injluenzae ATCC 31441 21 ATCC 33531 22 ATCC 8143T 26 H. parainjuenzae NCTC 4101 19 ATCC 7901 20 P. haemolytica NCTC 9380T 23 P. multocida NCTC 10322T 18

a Enzymic activity was scored as negative (01, weak (l),or strong (2). The results of three experiments run on different days are shown.

P. haemolytica and P. multocida were close to each other, 21, 22, and 26), H. parainfluenzae (samples 19 and 20), P. and A. ureae (sample 24) was more distant. While the H. muftocida (sample 18), P. haemolytica (sample 23), and A. influenzae samples fell almost together, the samples from H. ureae (sample 24) fell outside both the A. actinomycetem- parainfluenzae strains (samples 19 and 20) were more dis- comitans class and the H. aphrophifus class, The type strain tinct. of A. fignieresii (sample 25) fell outside the A. actinomyce- In the sample-oriented projection shown in Fig. 2 only temcomitans class, while A. lignieresii ATCC 19393 (sample samples from A. actinomycetemcornitans strains (samples 1 17) fell on the borderline of this class. to 9) and H. aphrophilus strains (samples 10 to 16) were In the SIMCA analysis shown in Fig. 4, none of the included. The heterogeneity of A. actinomycetemcornitans samples in the H. aphrophilus class (class 1, samples 10 to was again apparent, with samples 2 to 4, 6, and 7 clustering 16) fell within the 95% confidence interval of the H. para- at a distance from samples 1, 5, 8, and 9, which were fairly phrophilus class (class 2, samples 27 to 33). A. actinomyce- close to the type strain of H. aphrophifus (sample 16). The temcomitans (samples 1 to 9), A. lignieresii (samples 17 and other H. aphrophilus samples were more distant from A. 25), H. influenzae (samples 21, 22, and 26), H. parainflu- actinomycetemcomitans. enzae (samples 19 and 20), P. haemolytica (sample 23), P. The SIMCA analysis demonstrated that none of the sam- muftocida (sample 18), and A. ureae (sample 24) fell outside ples in the A. actinomycetemcomitans class (class 1, sam- both the H. aphrophilus class and the H. paraphrophilus ples 1 to 9) fell within the 95% confidence limits of the H. class. While H. parainfluenzae was relatively close to both aphrophilus class (class 2, samples 10 to 16) (Fig. 3). H. of the Haemophilus classes, A. actinomycetemcornitans was paraphrophilus (samples 27 to 33), H. influenzae (samples remote from them. VOL.42, 1992 API ZYM PROFILES OF PASTEURELLACEAE 15

TABLE 3. Results of enzymic characterization of whole bacterial cells with the API ZYM systema

Activity of

Organism Alkaline Leucine Valine Cystine Acid Sample phos- Esterase Esterase-lipase arylami- arylami- arylami- phos- AS-BI- a-Glucosi- phatase dase dase dase phatase phospho- dase hydrolase A. actinomycetemcomitans FDC Y4 1 SUNY 75 2 HK435 3 FDC 511 4 FDC 2097 5 FDC 2112 6 ATCC 29523 7 ATCC 29524 8 ATCC 33384T 9 A. lignieresii ATCC 19393 17 NCTC 4189T 25 A. ureae KWF 3520/59T 24 H. aphrophilus FDC 621 10 FDC 626 11 FDC 654 12 FDC 655 13 ATCC 13252 14 ATCC 19415 15 ATCC 33389T 16 H. parap h rop hilus KWF IMoAI 27 KWF IIMoAI 28 KWF IIRA43 29 H76 30 HK477 31 ATCC 29240 32 ATCC 29241T 33 H. influenzae ATCC 31441 21 ATCC 33531 22 ATCC 8143T 26 H. parainfluenzae NCTC 4101 19 ATCC 7901 20 P. haemolytica NCTC 9380T 23 P. multocida NCTC 10322= 18

a Enzymic activity was scored as negative (0). weak (l),or strong (2). The results of three experiments run on different days are shown.

DISCUSSION paraphrophilus fell outside both of these classes. A. actino- In this study, in which we used multivariate statistical mycetemcomitans also fell outside the confidence limits of analyses of enzymic data obtained by using the API ZYM the H. aphrophilus and H. paraphrophilus classes. We micromethod and outer membrane vesicles and fragments, studied too few samples of A. lignieresii, A. ureae, H. as well as whole cells, we distinguished the closely related injluenzae, H. parainfluenzae, P. haemolytica, and P. mul- species A. actinomycetemcomitans, H. aphrophilus, and H. tocida for these organisms to be used as bases for separate paraphrophilus. In previous studies performed with whole class models. However, all of the samples of these bacteria cells the authors concluded that the API ZYM method is a fell outside the A. actinomycetemcomitans, H. aphrophilus, simple, rapid, and reproducible micromethod which may and H. paraphrophilus classes. Previously, workers have serve as a valuable adjunct to traditional biochemical testing demonstrated differences between H. uphrophilus and H. in identifying bacteria (15, 20, 29). Our findings support this paraphrophilus in NAD dependence, biochemical reactions contention. However, both the accuracy and the predictabil- (21), and cellular sugar contents (6, 11). When multilocus ity of the API ZYM method were strengthened by subjecting enzyme electrophoresis was used to assess genetic diversity, the enzymic data obtained to multivariate statistical analy- this technique distinguished between A. actinomycetemcom- ses. Previously, we have used multivariate analyses as itans and H. aphrophilus but not between H. aphrophilus auxiliary techniques for the chemotaxonomy of bacteria and and H. paraphrophilus (13). In other studies it was not fungi (24, 611). possible to differentiate between H. aphrophilus and H. A. actinomycetemcomitans and i?. aphrophilus formed paraphrophilus on the basis of biochemical characteristics, two classes of bacteria that were separated by 95% confi- DNA-DNA homology data (32), DNA-DNA hybddization dence limits, as demonstrated with the SIMCA test, and H. data, or genetic transformation data (33). 16 MYHRVOLD ET AL. INT. J. SYST.BACTERIOL.

33 . SD t 1 32 7. 7. 6i 3. 2.''1 -.9A.+*

3. 70 6. 33 250 22:26 2. 4. . 0 170 08. 9.1. 24*** 19 A 21~5f*~~ 1. 28 18* I 27. 32. . 20Al60 012 150 31 .

5. 21r I 29 . I 19A 30.

I 20A - -I------

0 I I * 0 SD2

FIG. 1. Sample-oriented (i.e., bacterial strain-oriented) princi- FIG. 3. Residual standard deviation for each sample when the pal-component projection (tllt2). Principal components t, and t, samples were fitted to the A. actinomycetemcomitans (0)(samples describe the largest variance and the second largest variance in the 1 to 9) class model (SD1) and the H. aphrophilus (0)(samples 10 to enzymic data in Table 2 and 3. The positions of strains of A. 16) class model (SD2). The dotted lines indicate the approximate actinomycetemcomitans (0)(samples 1 to 9), A. lignieresii (0) class borders based on the results of F tests (P = 0.05). Samples 27 (samples 17 and 25), H. aphrophilus (0)(samples 10 to 16), H. to 33 (I)are H. paraphrophilus, samples 17 and 25 (0)are A. paraphrophilus (W) (samples 27 to 33), H. influenzae (A) (samples 21 lignieresii, samples 21 and 22 (A) are H. influenzae, samples 19 and and 22), H. parainfluenzae (A) (samples 19 and 20), P. haemolytica 20 (A) are H. parainfluenzae, sample 23 (**) is P. haemolytica, (**) (sample 23), P. multocida (*) (sample 18), and A. ureae (***) sample 18 (*) is P. multocida, and sample 24 (***) is A. ureae. (sample 24) are shown. For sample identification, see Table 1.

A. lignieresii ATCC 19393 fell on the borderline of the A. closely related to A. lignieresii than to H. aphrophilus and actinomycetemcomitans class, while the type strain of A. H. paraphrophilus. lignieresii, strain NCTC 4189, fell outside this class. There- In this study we demonstrated that there is heterogeneity fore, we could not conclude that A. actinomyceterncomitans among organisms belonging to A. actinomycetemcomitans, is more closely related to H. aphrophilus than to A. lignier- which consists of two groups of strains. One group contains esii. Interestingly, when Pohl (28) assessed DNA relatedness, he found that A. actinamycetemcomitans is more

SD1

1. 3. 7. 06 04 tl 2.

0 0 8. 9. 1. 5. 8. 5. 170 181 9. 7. 3. 4. 2. 21 A 2223.. r025 A26

10 0 FIG. 4. Residual standard deviation for each sample when the * samples were fitted to the H. aphrophilus (0)(samples 10 to 16) 0 f2 class model (SD1) and the H. paraphrophilus (W) (samples 27 to 33) FIG. 2. Sample-oriented principal-component projection of the class model (SD2). For the symbols for A. actinomycetemcomitans, A. actinomycetemcomitans (0)and H. aphrophilus (0) samples A. lignieresii, A. ureae, H. influenzae, H. parainfluenzae, P. hae- shown in Fig. 1. molytica, and P. multocida, see the legend to Fig. 3. VOL. 42, 1992 API ZYM PROFILES OF PASTEURELLACEAE 17 strains ATCC 29523, HK435, FDC 511, FDC 2112, and 2. Brondz, I., J. Carlsson, and M. Sjostrom. Submitted for publi- SUNY 75, while the other group comprises strains ATCC cation. 33384= (T = type strain), ATCC 29524, FDC 2097, and FDC 3. Brondz, I., J. Carlsson, M. Sjostrom, and G. Sundqvist. 1989. Y4. Previously, we demonstrated that there is heterogeneity Significance of cellular fatty acids and sugars in defining the among A. actinomycetemcomitans strains on the basis of genus Porphyromonas. Int. J. Syst. Bacteriol. 39:314-318. 4. Brondz, I., N.-E. Fiehn, I. Olsen, and M. Sjostrom. 1991. data obtained after bacteriolysis induced by EDTA and Multivariate analyses of cellular fatty acids and carbohydrates lysozyme (26) and on the basis of free cellular fatty acid of 1:2:1 and 2:4:2 spirochetes. APMIS 99567-575. contents (5). Subgroups of A. actinomycetemcomitans have 5. Brondz, I., and I. Olsen. 1983. Differentiation of Actinobacillus also been established after cluster and multidimensional actinomycetemcomitans from Haemophilus aphrophilus by gas scaling analyses of phenotypic features (32). Furthermore, chromatography of hexane extracts from whole cells. J. Chro- different biotypes, serotypes, leukotoxic groups, and protein matogr. 278: 13-23. profile groups have been described for this species (1,12,17, 6. Brondz, I., and I. Olsen. 1990. Multivariate analyses of carbo- 22, 27, 31). hydrate data from lipopolysaccharides of Actinobacillus (Hae- mophilus) actinomycetemcomitans, Haemophilus aphrophilus, We did not detect P-galactosidase activity in any of the and Haemophilus paraphrophilus. Int. J. Syst. Bacteriol. 40: preparations derived from the H. aphrophilus, H. paraphro- 405408. philus, or A. Eignieresii strains. This finding deviated from 7. Brondz, I., and I. Olsen. 1990. Multivariate analyses of cellular previous results (29). All of these species are considered carbohydrates and fatty acids of Candida albicans, Torulopsis lactose fermenters, although A. lignieresii ferments lactose glabrata, and Saccharomyces cerevisiae. J. Clin. Microbiol. slowly. The enzyme profiles were the same whether the cells 28: 1854-1857. were sonicated or not. Quality control of our media, strips, 8. Brondz, I., and I. Olsen. 1991. Multivariate analyses of cellular and reagents based on the use of Bacteroides uniformis fatty acids in Bacteroides, Prevotella, Porphyromonas, Wo- (Bacteroides thetaiotaomicron) ATCC 8492, P-glucosidase linella, and Campylobacter spp. J. Clin. Microbiol. 29:183-189. 9. Brondz, I., I. Olsen, M. Haapasalo, and A. J. Van Winkelhoff. (Sigma), and a-chymotrypsin (Sigma), as recommended by 1991. Multivariate analyses of fatty acid data from Prevotella, the manufacturer, gave the expected results. Furthermore, Bacteroides and Porphyromonas spp. J. Gen. Microbiol. 137: the type strain of H. aphrophilus, strain ATCC 33389, and A. 1445-1452. lignieresii ATCC 19393 were reordered from the American 10. Brondz, I., I. Olsen, and M. Sjiistrom. 1989. Gas chromatographic Type Culture Collection and retested for enzymic activity by assessment of alcoholyzed fatty acids from yeasts: a new chemo- using the API ZYM system, and the results were similar to taxonomic method. J. Clin. Microbiol. 27:2815-2819. the results obtained in our first series of experiments. 11. Brondz, I., I. Olsen, and M. Sjostrom. 1990. Multivariate Although the substrate which we used, 2-naphthyl-P-~- analysis of quantitative chemical and enzymic characterization galactopyranoside, and o-nitrophenyl-P-D-galactopyrano- data in classification of Actinobacillus, Haemophilus and Pas- side are different, they may both be split by a P-galactosi- teurella spp. J. Gen. Microbiol. 136507-513. 12. Calhoon, D. A., W. R. Mayberry, and J. Slots. 1981. Cellular dase. It is also known that a given p-galactosidase may split fatty acid and soluble protein composition of Actinobacillus only one of these substrates. Since the results of the P-ga- actinomycetemcomitans and related organisms. J. Clin. Micro- lactosidase test did not conform with the expected reactions biol. 14:376-382. for H. aphrophilus, H. paraphrophilus, and A. lignieresii, 13. Caugant, A,, R. K. Selander, and I. Olsen. 1990. Differentiation the API ZYM test should probably be supplemented with the bet ween Act ino ba cil1 us (Hae mop h il us) ac tinomy ce tem com i- o-nitrophenyl-P-D-galactopyranosidetest or by a conven- tans, Haemophilus aphrophilus and Haemophilus paraphro- tional lactose fermentation test for more reliable diagnosis of philus by multilocus enzyme electrophoresis. J. Gen. Microbiol. lactose fermenters. 136:2135-2141. Since the semiquantitative API ZYM method does not 14. Grenier, D., and D. Mayrand. 1987. Functional characterization of extracellular vesicles produced by Bacteroides gingivalis. require standardized inocula according to the protein con- Infect. Immun. 55111-117. tents of the microbial suspensions to be assessed, care 15. Hofstad, T. 1980. Evaluation of the API ZYM system for should be taken in the interpretation of the relative differ- identification of Fusobacterium species. Med. Microbiol, Im- ences between the enzyme activities of membrane vesicles munol. 168:173-177. and fragments and the enzyme activities of whole cells from 16. Holt, S. C., A. C. R. Tanner, and S. S. Socransky. 1980. the same bacterial strain. Nevertheless, it was noteworthy Morphology and ultrastructure of oral strains of Actinobacillus that the activity of leucine arylamidase was consistently high actinomycetemcomitans and Haemophilus aphrophilus. Infect. in whole cells and usually absent in vesicles and fragments. Immun. 30:588-600. For comparison, membrane vesicles of Porphyromonas 17. Jellum, E., V. Tingelstad, and I. Olsen. 1984. 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