Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

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.     

Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, Haemophilus and Pasteurella spp

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.     

Differentiation between Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus paraphrophilus by multilocus enzyme electrophoresis

Genetic relationships among isolates assigned to Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and H. paraphrophilus were determined by analysis of electrophoretically demonstrable allelic variation in 14 structural genes encoding metabolic enzymes. Among the 51 isolates analysed there were 25 electrophoretic types (ETs), among which mean genetic diversity per locus was 0.753. Cluster analysis of ETs demonstrated one well-defined group of 11 ETs representing solely the genotypes of all 17 isolates assigned to A. actinomycetemcomitans. The remaining 14 ETs represented the genotypes of the 34 isolates of H. aphrophilus and H. paraphrophilus. With the exception of ATCC 13252, all strains of H. aphrophilus were closely related, whereas strains assigned to H. paraphrophilus included distantly related lineages, some of which were similar to those of H. aphrophilus and should be assigned to this species. Thus, the study showed that there is no significant overall genetic similarity between A. actinomycetemcomitans and the two Haemophilus spp.     

Cellular fatty acid composition of Haemophilus species, Pasteurella multocida, Actinobacillus Actinomycetemcomitans and Haemophilus vaginalis (Corynebacterium vaginale)

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.     

Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.     

Synthetic oligodeoxynucleotide probes for the rapid detection of bacteria associated with human periodontitis

Analysis of the subgingival microflora has recently implicated Actinobacillus (Haemophilus) actinomycetemcomitans and several black Bacteroides species in the aetiology of juvenile, adult and rapidly progressing periodontitis. Rapid bacteriological diagnosis has been hampered by the slow growth and fastidious nature of these bacteria. To construct diagnostic probes, dideoxy sequencing of the 16S rRNA molecules from A. (H.) actinomycetemcomitans, Haemophilus aphrophilus, Bacteroides gingivalis, Bacteroides intermedius subgroup II, Bacteroides asaccharolyticus and several closely related species was performed. Next, oligodeoxynucleotides, complementary to defined regions of the 16S rRNA exhibiting considerable evolutionary divergence, were synthesized for use as molecular probes. In a dot-blot hybridization assay, all strains from each of the species for which probes were constructed were correctly identified, with a detection limit of less than 5 x 10(3) organisms. No cross-hybridization to closely related species (except for H. aphrophilus and Haemophilus paraphrophilus) or contaminating bacteria was observed. Using a modified DNA/RNA hybridization technique, the detection could be performed in less than 12 h, as compared to 2-3 weeks using conventional bacteriological procedures.     

Distribution of diaminopropane, putrescine and cadaverine in Haemophilus and Actinobacillus

Cellular levels of diaminopropane, putrescine and cadaverine, and decarboxylase activities to produce these diamines in six species (16 strains) of Haemophilus and four species (5 strains) of Actinobacillus belonging to the family Pasteurellaceae of the gamma subclass of the class Proteobacteria, were determined by high performance liquid chromatography (HPLC). Diaminopropane was ubiquitously distributed within all Haemophilus and Actinobacillus species, and L-2,4-diaminobutyric acid decarboxylase activity was detected in them. Putrescine and ornithine decarboxylase activity were found in H. aphrophilus, H. parainfluenzae and H. influenzae (type a, b, d, e and f except for type c) but not detected in H. aegyptius, H. parahaemolyticus, H. ducreyi and Actinobacillus species. Cadaverine occurred in H. aphrophilus, H. aegyptius, H. influenzae, H. parainfluenzae, A. actinomycetemcomitans, A. equuli and A. lignieresii, whereas their lysine decarboxylase activity was scarcely detected. Cadaverine was not found in H. parahaemolyticus, H. ducreyi and A. suis. The diamine profile serves as a phenotypic marker for the chemotaxonomic classification of the family Pasteurellaceae.     

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological relationship of some V-factor dependent Pasteurellaceae (Haemophilus sp.) from guineapigs and rabbits

Culture of guinea pig and rabbit respiratory tracts for bacteria using X- (haemin) and V- (NAD) factor in agar media detected infection by V-factor dependent Pasteurellaceae (Haemophilus sp.) in three colonies of guinea pigs and a group of rabbits. The 12 Haemophilus strains comprised three API NH codes classed as Haemophilus parainfluenzae and two codes classed as Haemophilus aphrophilus/paraphrophilus. Six cell wall lipid profiles were detected, but these were not related to API NH codes. Both bacteriological properties were used to select strains for serological studies but any relationship between bacteriological and serological properties of the Haemophilus strains was not evident. Varying serological relationships occurred between the newly isolated Haemophilus strains, [Pasteurella] pneumotropica NCTC 8284 and Haemophilus strains previously isolated from rats.     

Utilization of transferrin-bound iron by Haemophilus species of human and porcine origins

Haemophilus influenzae and H. haemolyticus acquired iron bound to human transferrin but not to human lactoferrin, ovo- or porcine transferrins. Conversely the swine pathogens H. pleuropneumoniae and H. parasuis used iron bound only to porcine transferrin. Growth under conditions of iron deprivation induced the production of siderophores and iron-repressible outer membrane proteins in H. parainfluenzae, H. paraphrophilus and H. parasuis but not in H. influenzae, H. haemolyticus or H. pleuropneumoniae. The latter 3 Haemophilus species appear to sequester transferrin bound iron via a siderophore-independent mechanism. However, the ability to produce iron chelating compounds did not enable H. parainfluenzae or H. paraphrophilus to utilize transferrin bound iron.     

Vertebral osteomyelitis due to coccobacilli of the HB group.

Three cases of pyogenic vertebral osteomyelitis occurred in which unusual, fastidious, Gram negative coccobacilli belonging to the "HB" group were isolated. The organisms were Haemophilus aphrophilus in case 1, intermediate between H aphrophilus and Actinobacillus actinomycetemcomitans in case 2, and Eikenella corrodens in case 3. All HB bacteria are sensitive to a wide range of antibiotics.     

Anomalous but helpful findings from the BBL Crystal ID kit with Haemophilus spp

Fourteen strains of Haemophilus (12 H. influenzae , 1 H. parainfluenzae and 1 H. aphrophilus ) were processed in BBL Crystal ID Enteric/Nonfermenter, API 20E and API 20NE kits, to determine whether the BBL kit misidentifies, as API kits may do, Haemophilus spp. as Pasteurella spp. The 13 H. influenzae and H. parainfluenzae strains produced uninterpretable colour reactions in the Crystal kit, thus signalling that an inappropriate species had been tested. On the other hand, the API kits (especially 20NE) often confidently 'identified' Haemophilus spp. as Pasteurella spp., giving no warning that this was a misidentification.     

Binding of human hemoglobin by Haemophilus influenzae

Abstract Binding of biotinylated human hemoglobin to Haemophilus influenzae was detected when organisms were grown in heme-deplete, but not heme-replete, conditions. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled human hemoglobin or human hemoglobin complexed with human haptoglobin. Binding was only partially inhibited by rat hemoglobin, bovine hemoglobin, human globin, and bovine globin, and not at all by heme, human serum albumin, bovine serum albumin, human transferrin, or myoglobin. Hemoglobin binding was saturable at 16–20 ng of hemoglobin per 10⁹ cfu. Binding of human hemoglobin was detected in serotypes a-f and serologically non-typable strains of H. influenzae , as well as Haemophilus haemolyticus but not Haemophilus parainfluenzae, Haemophilus aphrophilus, Haemophilus parahaemolyticus , or Escherichia coli .     

A numerical taxonomic study of Actinobacillus, Pasteurella and Yersinia

A numerical taxonomic study of strains of Actinobacillus, Pasteurella and Yersinia, with some allied bacteria, showed 23 reasonably distinct groups. These fell into three major areas. Area A contained species of Actinobacillus and Pasteurella: A. suis, A. equuli, A. lignieresii, P. haemolytica biovar A, P. haemolytica biovar T, P. multocida, A. actinomycetemcomitans, 'P. bettii', 'A. seminis', P. ureae and P. aerogenes. Also included in A was a composite group of Pasteurella pneumotropica and P. gallinarum, together with unnamed groups referred to as 'BLG', 'Mair', 'Ross' and 'aer-2'. Area B contained species of Yersinia: Y. enterocolitica, Y. pseudotuberculosis, Y. pestis and a group 'ent-b' similar to Y. enterocolitica. Area C contained non-fermenting strains: Y. philomiragia, Moraxella anatipestifer and a miscellaneous group 'past-b'. There were also a small number of unnamed single strains.     

Lipopolysaccharide composition of three strains of Haemophilus influenzae

The lipopolysaccharides of three strains of Haemophilus influenzae with varying beta-lactam susceptibility were examined. All three strains contained galactose, glucose, galactosamine, glucosamine, heptose, phosphate, and a trace of mannose. None contained fucose, rhamnose, or mannosamine. Levels of 2-keto-3-deoxy-octulosonic acid were consistently detected in all three strains at levels similar to that of Salmonella typhimurium LT2, but only following hydrolysis with 4 N hydrochloric acid.     

Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence

Members of the family Pasteurellaceae are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene, nadV, whose presence allows NAD-independent growth of Haemophilus influenzae and Actinobacillus pleuropneumoniae. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in Haemophilus ducreyi. When transformed into A. pleuropneumoniae, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida, and Actinobacillus actinomycetemcomitans. P. multocida and A. actinomycetemcomitans are among the NAD-independent members of the Pasteurellaceae. Homologues of NadV were not found in the sequenced genome of H. influenzae, an NAD-dependent member of the Pasteurellaceae, or in species known to utilize a different pathway for synthesis of NAD, such as Escherichia coli. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the Pasteurellaceae should help to elucidate the role of its homologues in other species.     

Delineation of the Species Haemophilus influenzae by Phenotype,Multilocus Sequence Phylogeny,and Detection of Marker Genes

To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of Haemophilus influenzae by comparative analysis of H. influenzae reference strains with closely related bacteria including strains assigned to Haemophilus haemolyticus, cryptic genospecies biotype IV, and the never formally validated species “Haemophilus intermedius”. Multilocus sequence phylogeny based on six housekeeping genes separated a cluster encompassing the type and the reference strains of H. influenzae from 31 more distantly related strains. Comparison of 16S rRNA gene sequences supported this delineation but was obscured by a conspicuously high number of polymorphic sites in many of the strains that did not belong to the core group of H. influenzae strains. The division was corroborated by the differential presence of genes encoding H. influenzae adhesion and penetration protein, fuculokinase, and Cu,Zn-superoxide dismutase, whereas immunoglobulin A1 protease activity or the presence of the iga gene was of limited discriminatory value. The existence of porphyrin-synthesizing strains (“H. intermedius”) closely related to H. influenzae was confirmed. Several chromosomally encoded hemin biosynthesis genes were identified, and sequence analysis showed these genes to represent an ancestral genotype rather than recent transfers from, e.g., Haemophilus parainfluenzae. Strains previously assigned to H. haemolyticus formed several separate lineages within a distinct but deeply branching cluster, intermingled with strains of “H. intermedius” and cryptic genospecies biotype IV. Although H. influenzae is phenotypically more homogenous than some other Haemophilus species, the genetic diversity and multicluster structure of strains traditionally associated with H. influenzae make it difficult to define the natural borders of that species.Bacterial diversity is usually expected to be organized into phenotypic and genetic clusters recognized as species. The diversity within species is thought to be confined by natural forces, although uncertainty exists as to the nature of the cohesion that constrains this diversity. There is increasing evidence that recombination occurs between distantly related bacteria and that barriers to this process, which could be used to define species naturally, are not always apparent (7, 11). As the bacterial species concept is central to taxonomy and evolutionary biology and has consequences for other disciplines such as the discrimination of pathogens from related bacteria with no pathogenic potential, there is a need to reevaluate to what extent accepted species are indeed coherent units.The gram-negative, nonmotile, facultatively anaerobic bacterium Haemophilus influenzae is a commensal organism of the pharynx frequently involved in localized infections of the respiratory tract, middle ear, and conjunctiva and sometimes in invasive infections such as meningitis or bacteremia. Presumptive identification is based on inability to synthesize NAD (V factor dependence) and porphyrin/hemin (X factor dependence). The latter characteristic is particularly notable as it is shared with few other microorganisms. Further metabolic characteristics of H. influenzae include fermentation of d-xylose, d-ribose, and d-galactose but not sucrose or d-mannose (17). By DNA analysis the X-factor-dependent species H. influenzae, Haemophilus aegyptius, and Haemophilus haemolyticus constitute a closely related group (4, 5, 14, 35), and housekeeping gene nucleotide dissimilarities between these species are exceeded by intraspecific nucleotide dissimilarities within the commensal species Haemophilus parainfluenzae (33).H. aegyptius was conceived as a species distinct from H. influenzae with a particular propensity to cause conjunctivitis (37). However, the existence of H. aegyptius as a separate species is controversial (5), and the designation H. influenzae biogroup aegyptius has been proposed to include traditional H. aegyptius and the closely related clones responsible for the invasive infection known as Brazilian purpuric fever (3), although formally the epithet aegyptius has priority. H. haemolyticus is distinguished from H. influenzae primarily by its hemolytic action on erythrocytes. A recent characterization of a large number of presumptive H. influenzae isolates from the respiratory tract of patients with chronic obstructive airway disease demonstrated that 102 of 258 strains clustered with the type strain of H. haemolyticus by 16S rRNA gene sequence, although many of the isolates were nonhemolytic. In contrast to H. influenzae, the atypical isolates were not associated with exacerbations of the disease (31), emphasizing the need to discriminate between the two species.Genetic methods have identified unnamed taxa of Haemophilus related to H. influenzae. Variant strains isolated from the genitourinary tract were first reported from Canada (1) and have been further studied by Quentin and coworkers (13, 39). These “cryptic genospecies biotype IV” strains (negative for tryptophanase/indole production and positive for urease and ornithine decarboxylase) are distinguishable from typical strains of H. influenzae by multilocus enzyme electrophoresis, 16S rRNA gene sequence, and DNA hybridization. However, only some biotype IV strains belong to the cryptic genospecies, and the lack of phenotypic characters enabling discrimination from H. influenzae has been an obstacle to the formal validation as a species. Another taxon, “Haemophilus intermedius,” was suggested (but never validly published) by Burbach (4). By DNA hybridization and by selected phenotypic traits, these strains take up an intermediate position between H. influenzae and H. parainfluenzae. Two subspecies were described, “Haemophilus intermedius subsp. intermedius,” capable of synthesizing porphyrin from δ-aminolevulinic acid and of fermenting sucrose, and “Haemophilus intermedius subsp. gazogenes,” characterized by the fermentation of mannose and the production of gas from glucose.A number of investigations have addressed the population structure of H. influenzae with assessment of particular virulence traits associated with specific subgroups or clades (9, 26, 32, 41). In the present study we attempted to define the borders of the species by performing phenotypic characterization, 16S rRNA sequence comparison, multilocus housekeeping gene sequence phylogeny, and detection of putative virulence and marker genes on a collection of 42 strains of H. influenzae and representatives of the above-mentioned closely related bacteria.     

Relationships among isolates of oral haemophili as determined by DNA-DNA hybridization

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 para-influenzae 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.     

Numerical taxonomic analysis of imperfect yeast species in Candida and Torulopsis shows no basis for generic separation

A numerical taxonomic analysis was performed on 79 phenotypic characters of 147 imperfect yeast species currently assigned to the genus Candida. The characters used were drawn from two monographs on yeast taxonomy. The analysis revealed 10 clusters of three or more species that were similar at the level of 75% or more, and seven clusters containing only one or two species. None of the 10 major clusters contained exclusively species that were traditionally assigned to the genus Torulopsis, while the 12 Candida species of basidiomycetous affinity fell into three clusters with only one species of ascomycetous affinity included. Statistical determination of the five most important differential characters for each cluster failed to show the property of pseudomycelium/mycelium formation as significant for any cluster. The study provides no evidence to support a distinction between taxa that were formerly divided between the genera Candida and Torulopsis and supports previous proposals that these genera should be fused.