A study is made of the effect of experimental errors of microbiological tests upon similarity values used in numerical taxonomy. For the coefficient SSM approximations are given for the mean and standard error of similarity values when some test results are erroneous, S', if the true value of similarity (S), the average probability in I, o data of erroneous tests results ( p ) , and the number of tests (n) are known.Formulae are given for estimatingp from an analysis of variance, and for including or excluding chosen factors that affect test reproducibility.When p is over about 10 % the error of similarity values becomes unacceptably large. Test error also increases the existing sampling error. It is generally better to employ many tests even if they are not as reproducible as desired, rather than to use only a few extremely reproducible tests.Examples are given suggesting that within one laboratory p can usually be kept below 5 %, but the discrepancies between laboratories may often be much larger.It is advisable to replicate some of the strains in numerical taxonomic studies, so as to afford a check on test reproducibility.

No abstract

A numerical taxonomic study was made of strains belonging to the genera Bordetella, Alcaligenes, Brucella, Acinetobacter, Moraxella, Neisseria, Haemophilus, Actinobacillus, and Pasteurella, together with a few strains from allied genera. They were examined for 139 characters covering a wide range of tests and properties. The strains fell into two major groups. Group A contained Haemophilus, Actinobacillus, and Pasteurella. Group B contained eight main clusters, representing, respectively, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica combined with Alcaligenes faecalis, Brucella, Acinetobacter anitratus, Acinetobacter lwoffii, and two clusters containing Moraxella and Neisseria strains. Brucella and the three clusters of Bordetella (including Alcaligenes) were more similar to one another than t o the other clusters; it is suggested that the family Brucellaceae should comprise these three genera, and that the other genera should be placed elsewhere.Several families of bacteria listed in Bergey ' s ManuaI (13) represent aggregations of genera which at best can only be described as loose associations. Instead of encompassing organisms which overall are closely similar, they have been set up t o accommodate genera with one or two prominent characters in common. Such a group is Brucellaceae, or its predecessor Parvobacteriaceae (12,63). This family is the niche for those gram-negative, rod-shaped bacteria which require rich media for growth and are responsible for many human and animal infections. Their potential pathogenicity and the alleged difficulties of working with them account largely for their neglect by taxonomists. They tend to grow poorly or t o die out on the usual media, whereas the addition of blood t o increase growth often interferes with tests.Within Brucellaceae the genus Bordetella Moreno-L6pez 1952 has been one of the least studied. Three species are generally recognized: the type species B. pertussis (the whoopingcough bacillus), B. parapertussis, and B. bronchiseptica. Although these organisms have been placed in various genera (e-g., Haemophilus), in recent years their segregation as a separate genus has become generally accepted ( 1 3, 84). They do not require X factor (haematin) or V factor (nicotinamide adenine dinucleotide) as do species of Haemophilus. The only reason that B. pertussis requires blood is to remove toxic substances from growth media; the blood can be replaced by starch or charcoal (65) or by anionic-exchange resins (45).Bordetella-species are obligate aerobes, whereas species of Haernophilus are facultative anaerobes. The guanine plus cytosine (G + C ) ratios also differ, being 67 t o 70% for Bordetella and 38 to 42% for Haemophilus (20, 36, and findings reported below).The separation of Bordetella from Alcaligenes, Acinetobacter, and Brucella is more difficult to justify, and the main motive for the present work was to investigate this question. Many workers have noted points of similarity between Bordetella bronchiseptica and A k aligenes faecali...

A numerical taxonomic study of named strains of Haemophilus Winslow et al., A ctinobacillus Brumpt , and Pasteurella Trevisan was made using 134 characters covering a wide range of properties. The strains fell into four main clusters.Three of these represented Haemophilus. Cluster 1 contained strains requiring X factor and usually also V factor and corresponded t o H. influenzae (Lehmann and Neumann) Winslow et al. and closely allied organisms. Cluster 2 contained strains that required only V factor and corresponded t o H. parainfluenza Rivers and similar organisms. Cluster 3 contained strains with a requirement for a raised COz tension and contained H. aphrophilus Khairat and H. paraphrophilus Zinnemann. Cluster 4 contained strains of Actinobacillus and Pasteurella but not Yersinia van Loghem. The three genera Actinobacillus, Pasteurella, and Hae.mophilus have a close phenetic relationship and are facultative anaerobes that can acidify glucose fermentatively. They might best be separated from the other genera of the family Brucellaceae Breed et al. Winslow et al. 1917, which are similar in although of considerable medical importance, many ways to H. aphrophilus Khairat 1940. H. has received relatively little detai.led study, as agni Kennedy et al. 1958, which causes a shown by the paucity of comparative data in septicaemia of lambs, also appears t o resemble works such as the 7th edition of Bergey 's H. aphrophilus, and similar organisms have been Manual (7). Some species once included in the isolated from meningoencephalitis of cattle (25, genus have been segregated as a separate genus, 46). Bordetella; the remainder have been studied The genus HaemophilusHaemophilus pleuropneumoniae Shope 1963, mainly with respect t o rather few properties, a very pleomorphic, V-requiring organism causnotably growth requirements and hemolysis. ing pleuropneumonia of swine, is similar t o the The taxonomic structure within the genus, and strains of influenza bacilli from swine that its relation t o other genera, is uncertain, and we require V factor only (47), and thus it is therefore present this brief report in the hope probably similar t o H. parainfluenzae and to that it will stimulate further comparative work the recently described species H. paragallinarum on the group.

No abstract

The taxonomic positions of several recently described species, Levinea malonatica, Levinea amalonatica, Citrobacter diversus, and Enterobacter agglomerans, were investigated by numerical analysis. A set of 141 strains, for which a total of 240 characters was recorded, was analyzed and also compared with representatives of a set of 384 strains of bacteria, examined in an earlier study, representing genera within the family Enterobacteriuceae . Three clusters of Citrobacter spp. were observed, Citrobacter freundii, Citrobacter spp. , and Levinea amalonutica, with strains received as Citrobacter diversus and Levinea malonatica clustering with the Citrobacter spp. Citrobacter intermedius was concluded to be synonymous with C. freundii. L . malonatica, from the results of this study, was included in the species C. diversus. Hydrogen sulfide-positive strains of Escherichia coli were not judged to warrant separate species status. Klebsiella aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, and Klebsiella edwardsii were found to be highly related (similarity values > 90%). It is proposed that these species be merged into a single species, Klebsiella pneumoniae.In a recently published study, 384 strains of bacteria representing genera within the family Enterobacteriaceae and the genus Yersinia were subjected to numerical analysis (38). Thirty-three phenetic clusters were distinguished, and these, for the most part, corresponded to the established species within the family Entero bacteriaceae . Useful information concerning taxonomic relationships among the E nterobacteriaceae was obtained. However, some members of the Enterobacteriaceae, including Erwinia, and the more recently described species Levinea malonatica, Levinea amalonatica, Citrobacter diversus, Enterobacter agglomerans, and Erwinia, were not represented. Therefore, an additional 141 strains of Enterobacteriuceae were examined, and the results were compared with those of the previous study.Morphology. Strains were subcultured onto Trypticase soy agar (BBL) and incubated at 30 C for 19 to 24 h. Cell form was examined by staining with Loeffler methylene blue. An India ink, wet-film method was used to detect capsules. Flagella were observed using Leifson's stain (11) and by electron microscopy. Gram reaction was determined using the Hucker modification of the Gram staining procedure (11). Colonial morphology was described from cultures grown on Trypticase soy agar (BBL) for 18 to 24 h at 30 C. Growth in nutrient broth was determined using heart infusion broth (Difco), followed by incubation at 30 C for up to 5 days.Pigmentation. Pigmentation was recorded from observation of growth on the media of King et al.(411, yeast extract-mannitol agar (27), and heart infusion agar (Difco) containing either 0.2% (wt/vol) DL-phenylalanine or 0.5% (wt/vol) tyrosine.Physiology and resistance. Tolerance to sodium chloride was detected in nutrient broth (Difco) containing 0, 0.5, 3.0, 5.0, 7.0, or 10% (wt/vol) NaCl. Temperature growth ranges (0 to 44.5 C) for cultures were d...

One hundred and five strains of gram-negative, fermentative bacteria, representing members of the Enterobacteriaceae together with strains of Vibrio and A erornonas, were subjected to numerical taxonomy and deoxyribonucleic acid (DNA)/DNA reassociation studies. The numerical taxonomy sorted the strains into major clusters corresponding t o Edwardsiella tarda, Escherichia coli, Citrobacter freundii, the tribe Klebsielleae, and Vibrio alginolyticus. Smaller clusters could be identified as Serratia marcescens, Hafnia ulvei, and Shigella species. Seven reference strains were used in the DNA/DNA reassociation studies. Klebsiella pneumoniae at 6 0 C shared reassociation values of 80 t o 100% with other strains of Klebsiella, 37 to 64% with Enterobacter spp., and lower values with Hafnia, Citrobacter, Serratiu, and Escherichia coli. Phenetically, K . pneurnoniae and Enterobacter aerogenes shared high relatedness, although they are only 41 t o 64% related in the reassociation studies. Hajnia, Salmonella typhirnurium, and Edwardsiella tarda showed little relatedness to any of the other strains included in this study. Both reference strains of Escherichia coli shared genetic resemblances greater than 65% at 60 C with Shigella and at least 70% with other strains of Escherichia coli. Overall, the agreement between numerical taxonomy and the DNA/DNA reassociation studies was satisfactory, with correlation coefficients ranging from 0.5 to 0.9. The choice of reference strains may well affect the correlation between the two techniques, and it is suggested that hypothetical median organisms be determined from the phenetic groupings and that actual strains that approximate t o the hypothetical median organisms be used as reference strains for DNA reassqciation.The taxonomy of the Enterobacteriaceae has been influenced strongly by clinical workers who require rapid and convenient identification methods and by tradition. Some of the inconsistencies in the taxonomy of the Enterobacteriaceae will be discussed in another paper (R. Johnson, R. Colwell, R. Sakazaki, and K. Tamura, submitted for publication), noteworthy among them being the nomenclature of the genus Salmonella and the taxonomy of Shigella. In addition, there is a direct contradiction between the molecular and traditional viewpoints of the species of Proteus, which are maintained by the latter in a single genus, based on intuitive, or alpha, classifications (2), whereas evidence from numerical taxonomy indicates the possible existence of three genera, with suggestion that at least two genera are supported by deoxyribonucleic acid (DNA) base composition data.Work has been underway in our laboratories on studies involving the application of numerical taxonomy and molecular genetic techniques t o the classification of the E n terobacteriaceae. Our initial approach was t o examine a wide range of strains representative of the Enterobacteriaceae. In this study, we attempted t o correlate the results obtained from numerical taxonomy with those obtained from DNA reassociation ex...

No abstract