Search

The aim of this study was to reinvestigate the relationships and the generic affiliations of the species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis. The nicotinamide phosphoribosyltransferase gene (nadV) conferring V factor-independent growth was identified in Haemophilus aphrophilus. The gene encodes a polypeptide of 462 amino acids that shows 74.5 % amino acid sequence identity to the corresponding enzyme from Actinobacillus actinomycetemcomitans. Ten isolates of Haemophilus paraphrophilus all carried a nadV pseudogene. DNA from Haemophilus aphrophilus was able to transform Haemophilus paraphrophilus into the NAD-independent phenotype. The transformants carried a full-length nadV inserted in the former locus of the pseudogene. The DNA-DNA relatedness between the type strains of Haemophilus aphrophilus and Haemophilus paraphrophilus was 77 %. We conclude that the division into two species Haemophilus aphrophilus and Haemophilus paraphrophilus is not justified and that Haemophilus paraphrophilus should be considered a later heterotypic synonym of Haemophilus aphrophilus. Forty strains of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis were investigated by multilocus sequence analysis. The 40 strains form a monophyletic group clearly separate from other evolutionary lineages of the family Pasteurellaceae. We propose the transfer of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis to a new genus Aggregatibacter gen. nov. as Aggregatibacter actinomycetemcomitans comb. nov. (the type species; type strain ATCC 33384(T)=CCUG 13227(T)=CIP 52.106(T)=DSM 8324(T)=NCTC 9710(T)), Aggregatibacter aphrophilus comb. nov. (type strain ATCC 33389(T)=CCUG 3715(T)=CIP 70.73(T)=NCTC 5906(T)) and Aggregatibacter segnis comb. nov. (type strain HK316(T)=ATCC 33393(T)=CCUG 10787(T)=CCUG 12838(T)=CIP 103292(T)=NCTC 10977(T)). The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro.

It is well established that infective endocarditis (IE) involving the HACEK (Hemophilus, Actinobacillus, Cardiobacter, Eikenella, Kingella) group of microbes occurs in patients with congenital heart defects (CHD) and in those with prosthetic grafts. Dental caries and gingival disease have been presumed to be the focus of microbial shedding. The purpose of this study was to determine if children with CHD had a more severe gingival inflammatory condition and harbored the HACEK group of microbes to a greater extent than normal children. Two groups of 12 age and sex matched children were selected for this study. The experimental group consisted of twelve children with CHD, 1-1/2 to 8 years of age. The control group consisted of 12 healthy children 2 to 8 years of age. Each child had a gingival index score recorded as described by Massler. Subgingival cultures were obtained. Gingival samples were cultured for HACEK microbes and total Streptococcus (spp) using standard techniques. Fisher's exact test was performed with significance defined at P < 0.05. Children with CHD had more severe gingival inflammatory index than the control group (P < 0.05). 8/12 CHD patient had Actinobacillus actinomycetemcomitans (A.a.) as compared with 2/12 controls (P < 0.05). Furthermore, all cyanotic CHD patients (4/4) had A.a. whereas, only 2/12 controls did (P < 0.05). 4/12 CHD patients harbored Eikenella corrodens (E.c.) compared to 1/12 controls (N.S.). There was no significant difference in colonization with E.c. or A.a. between cyanotic and acyanotic patients. No significant difference in total Streptococcus (spp) was found between the two groups. This study suggests that children with CHD have a more severe gingival inflammatory index and are colonized with specific HACEK microbes more so than normal children.

Twenty-four isolates of Actinobacillus seminis were typed by PCR ribotyping, repetitive extragenic palindromic element (REP)-based PCR, and enterobacterial repetitive intergenic consensus (ERIC)-based PCR. Five types were distinguished by REP-PCR, and nine types were distinguished by ERIC-PCR. PCR ribotyping produced the simplest pattern and could be useful for identification of A. seminis and for its differentiation from related species. REP- and ERIC-PCR could be used for strain differentiation in epidemiological studies of A. seminis.

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-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. 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. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (less than 20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.

The taxonomic distinction between Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus and the taxonomic distinction between H. aphrophilus and Haemophilus paraphrophilus have been questioned. This study was done to determine whether multivariate statistical analyses of carbohydrate data from lipopolysaccharides could be used to distinguish between these closely related species. Lipopolysaccharides were extracted with phenol-water and purified. Carbohydrates were assessed by using gas chromatography and gas chromatography-mass spectrometry after methanolysis and derivatization with trifluoroacetic acid anhydride. The lipopolysaccharides from all of the species contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, and glucosamine plus galactosamine, but in varying amounts. A. actinomycetemcomitans and H. paraphrophilus also contained D-glycero-D-mannoheptose, while H. aphrophilus did not. Sample- and variable-oriented principal-component analyses of the carbohydrate data clearly distinguished among A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. Soft independent modelling of class analogy showed that no sample in the A. actinomycetemcomitans class fell within the 95% confidence limits of the H. aphrophilus class. H. paraphrophilus fell outside both classes.

DNA-DNA hybridization was used to identify clinical isolates as Haemophilus aphrophilus or Actinobacillus actinomycetemcomitans. Some of the isolates were naturally competent for genetic transformation and were also used as DNA recipients for identification of other isolates. The results obtained by hybridization were supported by interstrain-to-intrastrain transformation ratios. Distinction between the closely related species H. aphrophilus and A. actinomycetemcomitans was generally clear-cut by both methods. Distinction of H. aphrophilus and A. actinomycetemcomitans from type and reference strains of a diversity of species in the family Neisseriaceae and other gram-negative species was also demonstrated by both methods. This is the first description of the identification of clinical isolates of H. aphrophilus or A. actinomycetemcomitans by using them as recipients in genetic transformation. The results suggest that this is a reliable system for identification of new clinical isolates belonging to these taxonomic entities.

Soluble thermostable antigens prepared from Actinobacillus pleuropneumoniae, as commonly applied in the ring precipitation test, were used in rapid slide tests. This method was easier to perform than the ring precipitation test and showed the same specificity. This specificity was higher than that obtained in slide agglutination tests using whole bacterial cells.

The serological typing (by enzyme-linked immunosorbent assay) of 119 isolates of Actinobacillus (Haemophilus) pleuropneumoniae (representing in varying numbers the 12 serovars of this taxon) by monoclonal antibodies derived from the reference strains of serovars 1 to 5 in general correlated reasonably with the serotype previously established for these strains by conventional procedures employing polyclonal antisera. However, where there were reasonable numbers of isolates representing a given serovar to provide a decision, there was no instance where the correlation between the monoclonal and the polyclonal antibody was in complete accord. In addition, some of the differences between monoclonal and polyclonal antibody binding with some isolates suggest that the distribution of the serotype-specific antigens within the taxon may be even more complex than has previously been supposed.

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by means of SDS-PAGE patterns and reactions on immunoblots with rabbit antiserum against A. actinomycetemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefévre syndrome (PLS), from whom periodontal wild strains of A. actinomycetemcomitans had been isolated, were also studied. OMP were purified with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. actinomycetemcomitans and 4 reference strains of H. aphrophilus. OMP profiles from wild and reference strains of A. actinomycetemcomitans were similar while those from A. actinomycetemcomitans and H. aphrophilus differed. The most prominent difference was absence of a heat modifiable protein in H. aphrophilus strains. Immunoblotting revealed strong common antigens in most strains, including a heat modifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treatment with pronase and sodium periodate confirmed the protein nature of the major OMP antigens.

Chemotaxonomic data for strains of Actinobacillus, Haemophilus and Pasteurella spp. were analysed using three multivariate statistical strategies: principal components, partial least squares discriminant, and soft independent modelling of class analogy. The species comprised Actinobacillus actinomycetemcomitans. Haemophilus aphrophilus, H. paraphrophilus, H. influenzae, Pasteurella multocida, P. haemolytica and P. ureae. Strains were characterized by cell sugar and fatty acid composition, lysis kinetics during EDTA and EDTA plus lysozyme treatment, and methylene blue reduction. In total 23 quantitative variables were compiled from chemotaxonomic analyses of 25 strains. A. actinomycetemcomitans and H. aphrophilus formed distinct classes which differed from those of H. paraphrophilus, H. influenzae and Pasteurella spp. All characterization variables, except those describing fatty acid content, contributed significantly to inter-species discrimination.

Actinobacillus pleuropneumoniae strains of serotypes 1 and 9 were studied for their serological properties by means of agglutination, coagglutination (CoA), indirect hemagglutination (IHA), Co-IHA, ring precipitation (RP), and immunodiffusion (ID) tests. Particulate and soluble antigens of unheated and heat-treated bacterial cells were used in various serological tests. Agglutination, CoA, and RP tests demonstrated common antigens between strains of serotypes 1 and 9. Quantitative estimation of serotype-specific antigenic activity by CoA, RP, and ID tests proved useful in differentiating strains of serotypes 1 and 9. IHA and Co-IHA tests using sheep erythrocytes sensitized with unheated or heat-treated whole-cell saline extract and the ID test using boiled whole-cell saline extract as antigen distinguished the strains of serotypes 1 and 9. In studies of absorption of rabbit antisera with heterologous whole-cell antigens there was no absorption of antibodies in tube agglutination and IHA tests, suggesting that serotype 1 and 9 strains belong to two distinct serogroups. It appears that the cross-reactivity between serotype 1 and 9 strains could be due to common epitopes associated with cell wall antigens.

The pathogenicity of 2 isolates of each of serovars 7, 3, 1 and 2 of Actinobacillus pleuropneumoniae was tested by intranasal inoculation into 60, 6-week-old large white pigs. Four dose rates varying from 0.27 to 560 x 10(6) organisms per pig with 10-fold serial dilutions were used. Surviving pigs were necropsied 7 days after inoculation. The proportion of pigs dying and developing gross lesions following infection was significantly greater for pigs given serotype 1 than for each of the other 3 serotypes, which did not differ significantly from each other. Twelve of 16 pigs given either of the 2 isolates of serovar 1 died after acute illness and 1 of 44 pigs given either of the 2 isolates each of serovars 7, 3 and 2 died. Pigs given serovar 1 showed high temperatures, severe respiratory distress, frothy haemorrhagic nasal discharge and weight loss. Lung lesions were produced in all 16 pigs given serovar 1, in 7 of 14 pigs given serovar 7, 7 of 14 pigs receiving serovar 3 and in 5 of 16 pigs given serovar 2. The lethal infections were characterised by a severe acute fibrinohaemorrhagic necrotising pleuropneumonia, whereas non-lethal cases had lung lesions ranging from necrotising purulent pleuropneumonia to abscessation. Significant differences between isolates in proportions of tissues culture positive for A. pleuropneumoniae for serovars 7 and 2, but not for serovars 3 and 1 suggested that isolates may vary in virulence within serovars, but more detailed studies are needed to clarify this point.

Thirty Haemophilus strains and six Actinobacillus strains, all of porcine origin, were examined for their biochemical reactivity on API 20E and API ZYM test strips using dense cell suspensions (supplemented with NAD as appropriate) as strip inocula. When combined with a test for V-factor dependency, the use of both strips allowed adequate differentiation of closely related organisms. Numerical taxonomic analysis of the data demonstrated that the majority of the haemophili and actinobacilli studied could be placed in one of four major clusters; these clusters contained, respectively, the H. pleuropneumoniae--A. pleuropneumoniae strains, the H. parasuis strains, strains belonging to Haemophilus taxon "minor group," and strains belonging to an unusual group of mannitol-positive, urease-negative haemophili. A representative of Haemophilus species taxon C and an unusual Actinobacillus isolate appeared to be comparatively unrelated to organisms in the four major clusters. Although it may, on occasion, be difficult to place an unusual isolate in any one particular group, owing to the uncertain taxonomy of some of these organisms, it is concluded that API test strips can serve as useful tools for the characterization and differentiation of porcine haemophili and actinobacilli.

A method based on whole-cell methanolysis and trifluoroacetic acid anhydride derivatization was developed for routine laboratory differentiation between isolates from the Actinobacillus--Haemophilus--Pasteurella group. All species, except Haemophilus aphrophilus, contained D-glycero-D-mannoheptose, although in varying concentrations. The distribution of this sugar could be used to distinguish H. aphrophilus from Actinobacillus actinomycetemcomitans, H. paraphrophilus, H. influenzae type b, Pasteurella haemolytica, P. multocida and P. ureae, and also H. influenzae type b from Pasteurellae. The pattern of major sugars in P. ureae and P. haemolytica resembled that of A. actinomycetemcomitans. Major fatty acids of the whole-cell methanolysates provided no basis of interspecies differentiation.

In a comparative study, 63 collection cultures representing 38 nomenspecies of, or assigned to, the genera Actinobacillus, Haemophilus, or Pasteurella were characterized by phenotypical features and deoxyribonucleic acid base composition. The latter was calculated from the thermal denaturation point. Biochemical reactions were tested in differential media commonly used for Enterobacteriaceae, and two test procedures were compared: (i) pure cultures with haematin and nicotine adenine dinucleotide added, where necessary, and (ii) xenocultures with an asaccharolytic Acinetobacter strain (ST 661/60). Furthermore, the respiratory quinones, and the effect of fumarate on oxygen-limited growth were considered. On the basis of these and some additional physiological and morphological criteria, a definition of the Actinobacillus-Haemophilus-Pasteurella group as a whole was established which appears to rank as a family. Several misclassified species, i.e. the so-called Actinobacillus actinoides, Haemophilus piscium, Haemophilus vaginalis, Pasteurella anatipestifer, and the organisms of the Bovine Lymphangitis group were eliminated, and the position of so-called Pasteurella piscicida was questioned. Some principles of subdivision of the group, and some of the practical identification procedures were discussed.

The API ZYM system, a commercially-available technique that measures bacterial enzyme activity was used to test 43 isolates identified as H. somnus, H. ovis or A. seminis and 19 from related genera. The enzyme patterns resulting from the API ZYM differentiated H. somnus and H. ovis from A. seminis and related genera but not from each other. An identification scheme based on 9 of the enzymes in the API ZYM and a few simple biochemical tests is proposed for the rapid and reliable identification of these bacteria in a diagnostic bacteriology laboratory.

Instrumental analytical and bioenzymatic methods were used to differentiate between species of the Actinobacillus-Haemophilus-Pasteurella group. Long-chain fatty acids were analysed directly with gas chromatography (GC) without derivatization. GC of trifluoroacetylated whole-cell methanolysates was a rapid method for differentiation. Cellular sugars were more suitable for differentiation than fatty acids. D-Glycero-D-mannoheptose, the major localization of which was lipopolysaccharide, distinguished H. aphrophilus from A. actinomycetemcomitans, H. paraphrophilus, H. influenzae type b, P. haemolytica, P. multocida, and P. ureae. GC of single colonies, which is a new chemotaxonomic method, was preferable to GC of liquid-grown cells. Lysozyme-and EDTA-induced bacteriolysis and reduction of methylene blue by cellular hydrogenase served as additional criteria for differentiation.

In the present study, the closely related facultative, Gram-negative rods, Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus, were distinguished taxonomically by means of their carbohydrate composition in phenol-extracted lipopolysaccharide. Both A. actinomycetemcomitans and H. aphrophilus lipopolysaccharide contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, galactosamine, and glucosamine. The content of galactose was approximately twice as high in lipopolysaccharide from H. aphrophilus as in lipopolysaccharide from A. actinomycetemcomitans. D-Glycero-D-mannoheptose was detected exclusively in lipopolysaccharide from A. actinomycetemcomitans where it constituted 11.8-16.7% of the sugar content. This aldoheptose may therefore serve as a marker for chemotaxonomic differentiation between A. actinomycetemcomitans and H. aphrophilus. The present study also describes fragmentation of methylheptoside derivatives of trifluoroacetic acid (D-glycero- and L-glycero-D-mannoheptose) from A. actinomycetemcomitans as suggested by mass spectrometry.

The fatty acid composition of 35 Haemophilus influenzae strains was found to be grossly similar and characterized by relatively large amounts of 14:0, 3-OH-14:0, 16:1 and 16:0. The three C18 fatty acids 18:2, 18:1 and 18:0 were also present, but in much lower concentrations. This general pattern was also found for most of the other species of Haemophilus examined (H. aegyptius, H. aphrophilus, H. canis, H. gallinarum, H. haemolyticus, and H. parainfluenzae). Small but distinct quantitative discrepancies were detected for H. ducreyi and the haemin-independent species H. paraphrohaemolyticus, H. paraphrophilus and H. suis. Actinobacillus actinomycetemcomitans was found to be indistinguishable from H. influenzae. Pasteurella multocida also exhibited a fatty acid pattern closely related to that of Haemophilus, but could be distinguished by its higher concentration levels of the C18 fatty acids. The fatty acid pattern of H. vaginalis was considerably different from those of the other species examined. This species lacked 3-OH-14:0 and 18:2 and contained small amounts of 14:0 and 16:0, whereas 18:1 and 18:0 were the major constituents.

The current classification of recognized actinobacilli and pasteurellas does not allow differentiation of the two genera by their phenotypic features. Recent investigations of their genetic relationships have shown that several species hitherto assigned to the genus Pasteurella are more closely related to the actinobacilli. Moreover, some recently described taxa were located by DNA-DNA hybridization in one or the other of the two genera. On the basis of the genetic system, improved identification keys have been devised which separate the taxonomic groups on the genus and species levels according to an appropriate set of biochemical characteristics.

Reference strains of serotypes 1 to 12 of Actinobacillus (Haemophilus) pleuropneumoniae were cultured in Eagle minimal essential medium with 10% Serum Plus. Culture supernatants were examined for cytotoxicity to alveolar macrophages and for the ability to hemolyze sheep erythrocytes. All strains except the reference strain of serotype 6 produced cytotoxin, whereas only serotypes 1, 5, 9, 10, and 11 produced hemolysin. Both cytotoxin and hemolysin appeared to be heat labile. Antisera were raised against cytotoxin- and hemolysin-containing culture supernatants of serotypes 1 to 11. Cross-neutralization studies revealed that the hemolysins were serologically homogeneous. In contrast, four serologically different cytotoxins were distinguished. One cytotoxin was produced by serotypes 1, 5, 9, and 11, and a second was produced by serotypes 2, 3, 4, and 8. A third cytotoxin was produced by serotypes 7 and 12; this cytotoxin was related to the cytotoxins of serotypes 1, 2, 4, 5, 9, and 11. A fourth cytotoxin, produced by serotype 10, was related to the cytotoxin of serotypes 1, 5, 9, and 11. Seventy field strains belonging to serotypes 2, 3, 7, 8, 9, and 11 were also tested for production of cytotoxin and hemolysin. All strains belonging to serotypes 9 and 11 produced hemolysin and cytotoxin, whereas all strains of serotypes 2, 3, 7, and 8 produced only cytotoxin. Hemolysins and cytotoxins of both the field strains and the corresponding serotype reference strains were comparably neutralized. These findings strongly suggest that the observed differences in production and type of hemolysin and cytotoxin were related to serotype and not to strain.

Morphology, biochemical reactions, pigmentation, antigens, and cell envelope proteins were examined in 12 strains of Haemophilus somnus, Haemophilus agni, Histophilus ovis, and Actinobacillus seminis. All of the strains except A. seminis are related and are considered as a single Haemophilus-Histophilus (HH) group. In immunodiffusion tests, HH group bacteria had at least two antigens common to all members of the group, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that they have similar cell envelope protein profiles. A quantitatively variable yellow pigment with absorption maxima of 430 to 435 nm was present in strains of H. somnus and H. agni. The HH group did not produce catalase and grew only in air containing 10% CO2. Of 10 HH group bacteria, 9 required thiamine monophosphate for growth. A. seminis was distinguished from the HH group by its lack of yellow pigment, production of catalase, growth in air, lack of a thiamine monophosphate requirement, and different cell envelope protein profile. In gel immunodiffusion tests, A. seminis antigens produced two lines of partial identity with the HH group when antiserum against H. somnus was used. Reference strains of Haemophilus influenzae, Actinobacillus lignieresii, and Haemophilus haemoglobinophilus were compared with the test strains. In immunodiffusion tests, a single antigen was found to be common to H. haemoglobinophilus, A. seminis, and the HH group. No similarities between any of the test strains and H. influenzae or A. lignieresii were noted. The close relationship of H. somnus, H. agni, and Histophilus ovis suggests that these unofficially named bacteria may belong to a single taxon.

In order to assess the relationships among strains of the genera Actinobacillus and Haemophilus, DNAs from 50 strains of these genera were isolated and purified. The guanine plus cytosine (G + C) content of DNAs from strains of Haemophilus segnis and Haemophilus parainfluenzae were determined by thermal denaturation. DNA-DNA homologies were measured using labelled probes from one strain representing Haemophilus segnis (strain ATCC 10977), and two strains representing Haemophilus parainfluenzae (strains ATCC 9796 and ATCC 7901). Strains isolated as H. segnis had a G + C content of 39.0 to 42.9% and were 49-92% homologous with the ATCC 10977 DNA probe. All of the strains freshly isolated as H. parainfluenzae were 70-81% homologous with the ATCC 9796 DNA probe and had a G + C content of 34.9 to 38.3%. Strain ATCC 7901 was 11% homologous with the ATCC 9796 DNA probe, had a G + C content of 42.4%, and was 65-78% homologous to DNA from strains identified as Haemophilus aphrophilus and Haemophilus paraphrophilus. From these results we conclude that strain ATCC 7901 is a mislabelled strain of H. paraphrophilus. The results of multiple DNA-DNA hybridizations indicated that separate species designations were appropriate for H. segnis, H. parainfluenzae, Actinobacillus actinomycetemcomitans ("Haemophilus actinomycetemcomitans"), and H. aphrophilus. H. aphrophilus and H. paraphrophilus were closely related organisms and did not fulfill the generally accepted criteria for designation as separate species.

The taxonomy of the family Pasteurellaceae has remained controversial despite investigations of biochemistry, serology, and nucleic acid relatedness. In an attempt to resolve some of this confusion, we have partially sequenced the 16S rRNAs of seven members of the family, representing all three genera. The sequences were aligned, similarity scores calculated, and single, average and complete linkage cluster analysis of the resulting distance matrix performed. In this way, an evolutionary branching pattern of these closely related species was reconstructed, and the approximate phylogenetic position of the family determined. Actinobacillus (Haemophilus) actinomycetemcomitans clustered with Haemophilus instead of Actinobacillus, supporting transfer of this species to the genus Haemophilus. Thus cluster analysis of phylogenetic relatedness was found to be particularly useful for studying closely related organisms, and could be performed using a microcomputer.

Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.