A rapid microprocedure for isolating detergent (sodium N-lauroyl sarcosinate)-insoluble major outer membrane proteins from Haemophilus species produced results qualitatively identical to those obtained with a commonly used preparative isolation procedure. Proteins isolated by both procedures were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after staining with Coomassie brilliant blue R-250. The time for outer membrane protein isolation was substantially reduced with the rapid procedure, allowing a larger number of membrane preparations to be obtained rapidly for routine analysis.

Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and RPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates from 42 persons in towns where BPF cases occurred, and control strains from 32 persons in towns without BPF cases were characterized biochemically, genetically, and epidemiologically. Results indicated that a single clone was responsible for all BPF cases identified in six Brazilian towns from 1984 through 1986. All of 15 (100%) case strains were the same clone as was 1 of 32 (3%) control strains (P = <10-8). Isolates of the clone were preferentially intrarelated by DNA hybridization (99% relatedness, hydroxyapatite method at 60 and 75°C) and were separable from other H. influenzae biogroup aegyptius strains (approximately 90% relatedness at 60°C and 82% relatedness at 75°C). All isolates of the BPF clone and no other strains contained a 24-megadalton plasmid of restriction endonuclease type 3031, were of a single multilocus enzyme mobility type, were of a single sodium dodecyl sulfate-polyacrylamide gel electrophoresis type, and were in one of two ribosomal DNA restriction patterns. Ail BPF clone isolates reacted with monoclonal antibodies produced from a case strain; only 3 of 62 (5%) other strains reacted with this monoclonal antibody. Ninety percent of BPF clone strains and 27% of other strains were relatively resistant to sulfamethoxazole-trimethoprim.

Seventeen strains of Haemophilus ducreyi were isolated from genital lesions which were negative for syphilis by dark-field examination. Media used for primary isolation at various times during the study were enriched chocolate agar, chocolate agar plus vancomycin (3 microgram/ml), rabbit blood agar plus vancomycin (3 micrograms/ml), fetal bovine serum agar, and fetal bovine serum agar plus vancomycin (3 micrograms/ml). H. ducreyi was isolated on chocolate agar plus vancomycin from 10 of 14 patients found to be positive on one or more media, on rabbit blood agar plus vancomycin from 16 of 17 patients, and on fetal bovine serum agar plus vancomycin from 9 of 11 patients. Sera from six animal species were tested to determine if any would support the growth of H. ducreyi. Horse and rabbit sera supported light growth of some strains. Fetal bovine serum supported good growth of all strains included in the study. Biochemical and physiological tests were done on the 17 isolates, a reference strain of H. ducreyi, and two reference strains of Haemophilus haemoglobinophilus. The results agreed with those reported by Kilian, except that H. ducreyi produced alpha-hemolysis in stabs on rabbit blood agar and was oxidase positive, three strains were urease positive, and CO2 improved the growth of seven strains. All 17 isolates were beta-lactamase positive. The reference strains were beta-lactamase negative.

The relationship between nine Haemophilus species and Haemophilus influenzae was studied by DNA-DNA hybridization, by transformation of H. influenzae to streptomycin resistance with heterospecific DNA, by competition of heterospecific DNA for transformation by homospecific DNA and by the lethal effect of heterospecific DNA on competent H. influenzae. H. parainfluenzae, H. parasuis, and H. aegyptius DNA transformed at more than 10% efficiency when compared to homologous transformation, but only H. aegyptius demonstrated, by hybridization, a relative binding ratio of more than 80%. H. aphrophilus and H. paraphrophilus DNA demonstrated a relative binding ratio of less than 30% and transformed H. influenzae at only 10(-5) the efficiency of homologous DNA, but they competed for H. influenzae transformation as well as or better than homospecific DNA. The data indicated that in some of the species sharing the common ecological habitat of the mammalian respiratory tract, sequences necessary for competition and efficient uptake into H. influenzae are present in large numbers in their DNAs, which nevertheless have little overall homology with H. influenzae DNA.

Fifty-seven clinical isolates of previously unidentified gram-positive, fermentative, nonsporeforming rods were studied and compared to the type strains of Oerskouia turbata and 0 . xanthineolytica. Thirty-five of the isolates were identified as Oerskouia species: 9 were identified as 0. turbata, and 26 were identified as 0. xanthineolytica. The Oerskouia cultures could be differentiated from the other isolates on the basis of the development of filamentous colonies. The genus Oerskouia, described by Prauser et al. (€9, Sukapure et al. (ll), and emended by Lechevalier (61, consists of yellow-pigmented organisms with branched hyphae. These hyphae break up into motile, rodlike elements that "appear bacterial" in smears. These rods are gram positive and non-acid fast. Previously described sources of the organisms are soil, aluminum hydroxide gel antacid, and dry grass cuttings (6). During the last 20 years, the Special Bacteriology Section (SBS), Bacteriology Division, Center for Disease Control (CDC), has received for identification a number of motile, gram-positive, nonsporeforming, yellow pigment-producing organisms isolated from clinical sources. These organisms were not recognized as belonging to any established species and were arbitrarily designated as group A Corynebacterium sp. Later, this group was divided into five subgroups, primarily on the basis of carbohydrate reactions. When these isolates were observed to have characteristics similar to those reported for the genus Oerskovia, a collaborative study between the Actinomycete Laboratory of CDC's Mycology Division and the SBS was initiated to further characterize and classify them. Listeria denitrificans was included in this study because two of our colleagues, D. Hollis and G. Wiggins, had noted during earlier work on the genus Listeria that many biochemical characteristics of this species were similar to those of the group A strains.

Genetic transformation studies were used to determine relatedness within the family Pasteurelluceae. Among strains with <60 % relatedness to Haemophilus influenzae based on deoxyribonucleic acid hybridization, two groups were identified; one, showing competition for homospecific transformation with H. influenzae, contained Haemophilus parainfluenzae, Haemophilus parasuis, Haemophilus aphrophilus, Haemophilus paraphrophilus, Pasteurellu pneumotropica, Pasteurella multocida, and ActinobaciElus actinomycetemcomitans, and the other, showing little or no competition for homospecific transformation with H. influenme, contained Haemophilus ducreyi, Haemophilus parahaemolyticus, Actinobacillus ligniermii, Actinobacillw equuli, Actinobacillus pleuropneumoniae, and Pasteurellu ureae. Such groupings support existing studies which have used only deoxyribonucleic acid hybridization or numerical analysis. Currently there is much interest in the taxonomy of the family Pasteurellaceae, particularly the genus Haemophilus, and it is recognized that traditional genus designation based on limited phenetic traits may be inadequate (15). Despite requirements for hemin (X factor) or nicotinamide adenine dinucleotide (V factor), deoxyribonucleic acid (DNA) hy-bridization studies have suggested that Haemophilus pleuropneumoniae is more closely related to members of the genus Actinobacillus ; that Haemophilus ducreyi is a mono-specific genus unrelated to the type species Haemophilus influenzae; that avian species of Haemophilus are related to Pasteurella species; and that Haemophilus somnus, Haemo-philus agni and Histophilus ovis are related and belong to a single distinct species (4, 13, 15, 16, 25). Overall DNA sequence relationships as determined by DNA hybridization studies have become the standard parameter for determining taxonomic relationships, and the concept of the genospecies is thought to be of little value (11). Recent studies, however, hqve demonstrated regional differences within the genome for both presumed rates and mechanisms of evolution (17). We think, therefore, that taxonomic methods based on characteristics other than overall sequence relatedness will be necessary for completeness. Not long after Alexander and Leidy recognized transformation in H. influenzae in 1950, this genetic method was used in classification studies for members of the genus Haemophilus (2, 12). Since that time, much work has been done on the molecular events that accompany transformation , and several steps in transformation that are of potential taxonomic significance have been identified: (i) development of competence, (ii) recognition and uptake of transforming DNA, (iii) integration of DNA in the recipient genome, and (iv) expression of phenotype in the recipient cell (6, 9, 11, 18-20, 22). To avoid problems of expression of phenotype, we have chosen markers thought to be conserved, such as ribosomal subunits or (in this study) a subunit of the DNA-dependent ribonucleic acid polymerase (7). To avoid problems of variability in development o...

Haemophilus aegyptius and Haemophilus influenzae biotype III are morphologically and biochemically similar; however, their outer membrane protein (Sarkosyl insoluble) profiles are distinct. Of 18 strains of H. aegyptius examined, 15 had a type 1 protein profile, and 3 had a type 2 profile, whereas the 5 strains of H. influenzae biotype III examined had three other protein profile types. All Haemophilus strains examined had 31-and 76-kilodalton (kDa) proteins and minor proteins with molecular masses between 20 and 100 kDa. H. aegyptius, with a type 1 protein profile, had major outer membrane proteins with apparient molecular masses of 27, 35.5, and 41.5 kDa, and H. aegyptius, with a type 2 protein profile, had 26-, 29-, 39.5-, and 41-kDa proteins. The type strain of H. influenzae biotype III had three major outer membrane proteins with apparent molecular masses of 29, 38.5 and 40 kDa. Four other strains designated as H. influenzae biotype III had major outer membrane proteins between 27 and 41.5 kDa representing two additional protein profiles.

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