Identification ofCorynebacterium jeikeium andCorynebacterium CDC group D 2 with the API 20 strep system

A total of 170 strains ofCorynebacterium jeikeium and 23 strains ofCorynebacterium group D2 were examined in three British laboratories using the API 20 Strep identification system and three supplementary tests (catalase production, urease production and nitrate reduction). The isolates were collected from clinical specimens in various laboratories over a three-year period. The two species produced consistent reactions in these tests after 24 h. Two tests were highly discriminatory, with positive reactions for ribose fermentation seen forCorynebacterium jeikeium while urease production was observed withCorynebacterium group D2. This method allows routine clinical laboratories to rapidly identify these emerging pathogens.     

Proposal of Afipia gen. nov., with Afipia felis sp. nov. (formerly the cat scratch disease bacillus), Afipia clevelandensis sp. nov. (formerly the Cleveland Clinic Foundation strain), Afipia broomeae sp. nov., and three unnamed genospecies.

On the basis of phenotypic characterization and DNA relatedness determinations, the genus Afipia gen. nov., which contains six species, is described. The type species is Afipia felis sp. nov. (the cat scratch disease bacillus). Afipia clevelandensis sp. nov., Afipia broomeae sp. nov., and three unnamed not associated with cat-borne disease. All but one strain (Afipia genospecies 3) were isolated from human wound and respiratory sources. All Afipia species are gram-negative, oxidase-positive, nonfermentative rods in the alpha-2 subgroup of the class Proteobacteria. They are motile by means of a single flagellum. They grow on buffered charcoal-yeast extract agar and nutrient broth, but rarely on MacConkey agar, at 25 and 30 degrees C. They are urease positive; but they are negative in reactions for hemolysis, indole production, H2S production (triple sugar iron agar), gelatin hydrolysis, esculin hydrolysis, and peptonization of litmus milk. They do not produce acid oxidatively from D-glucose, lactose, maltose, or sucrose. The major cell wall fatty acids are 11-methyloctadec-12-enoic (CBr19:1), cis-octadec-11-enoic (C18:1omega7c), and generally, 9,10-methylenehexadecanote and 11,12-methyleneoctadecanoate; and there are only trace amounts of hydroxy acids. The guanineplus-cytosine content is 61.5 to 69 mol%. A. felis is positive for nitrate reduction and is delayed positive for acid production from D-xylose, but it is catalase negative. A. clevelandensis is negative in all of these tests. A. broomeae is weakly positive for catalase production and acid production from D-xylose, but it is negative for nitrate reduction.     

Isolation of sulfate-reducing bacteria from human thoracoabdominal pus

To evaluate the prevalence of sulfate-reducing bacteria in septic processes, we searched for these bacteria by culture in 100 consecutive abdominal and pleural pus specimens. Twelve isolates were obtained from abdominal samples and were identified by a multiplex PCR as Desulfovibrio piger (formerly Desulfomonas pigra) (seven strains), Desulfovibrio fairfieldensis (four strains), and Desulfovibrio desulfuricans (one strain).     

Evaluation of a New Assay for Vi Antibody in Chronic Carriers of Salmonella typhi

Biotyping of Haemophilus influenzae into five type and H. parainfluenzae into three types based on indole production, ornithine decarboxylase, and urease has been reported (M. Kilian, Acta Pathol. Microbiol. Scand. Sect. B 82:835--842, 1976). A commercially available test system designed for the 4-h identification of Enterobacteriaceae. Micro-ID, proved efficacious for the rapid biotyping of these two Haemophilus species. The nitrate reductase, indole production, ornithine decarboxylase, urease, and o-nitrophenyl-beta-D-galactopyranoside hydrolysis tests in Micro-ID correlated over 99% with conventional methodology. By utilizing the indole and o-nitrophenyl-beta-D-galactopyranoside tests it was possible, with 261 of 272 (96.1%) isolates, to distinguish H. influenzae from H. parainfluenzae. Cerebrospinal fluid isolates were over 90% H. influenzae biotype I, and conjunctival isolates were approximately 70% biotype II. Type b H. influenzae were predominantly biotypes I and II; these type b isolates were also overwhelmingly indole producers. Although over 90% of biotypes I and II have been reported to produce beta-lactamase, this was not confirmed by the small number of beta-lactamase producers encountered here. The 4-h Micro-ID should prove a useful mechanism, amenable to the routine clinical laboratory, for the further exploration of the association of Haemophilus with the site of isolation, antigenicity, and antibiotic resistance.     

Use of Presumpto Plates to identify anaerobic bacteria.

Identification of anaerobic bacteria requires special media and growth conditions that contribute to a higher cost per identification than that for aerobic isolates. Newer rapid methods streamline the identification process, but confirmation to the species level is often difficult. The Presumpto Plate method for the identification of commonly encountered anaerobes consists of three quadrant plates, each containing four conventional media, that result in the generation of 21 test parameters: growth on Lombard-Dowell medium; production of indole, indole derivative, catalase, lecithinase, and lipase; proteolysis of milk, H2S, and esculin; growth on 20% bile; precipitate on bile; DNase, glucose, casein, starch, and gelatin hydrolysis; and fermentation of lactose, mannitol, and rhamnose. Identification charts were developed by using the results from 2,300 anaerobic isolates. Because conventional media were used, there was a high degree of agreement between the Presumpto Plate method and the reference method when testing commonly encountered anaerobes. The Presumpto Plate method is as accurate as commercially available enzyme systems for the identification of many anaerobic species but is less expensive to perform.     

Nature and identification of Exophiala werneckii.

PathoTec strips and spot biochemical tests were evaluated for the ability to biotype Haemophilus influenzae and Haemophilus parainfluenzae. Indole, urease, and ornithine decarboxylase reactions were tested. The results of PathoTec strips compared favorably with those conventional methods; the percent agreements were as follows: indole, 100; urease, 99.5; and ornithine, 95.5. Spot tests were simple and rapid, and the results also compared favorably with those of conventional tests; the percent agreements were as follows: indole, 99; urease, 100; and ornithine, 96.     

o-Nitrophenyl-beta-D-galactopyranoside-urease-indole broth, a new composite tube medium for Salmonella screening.

A new composite broth medium combining o-nitrophenyl-beta-D-galactopyranoside (ONPG) and urease and indole tests in a single tube is described. High-level agreement with individual conventional tests was recorded in comparative studies with 2,412 cultures of members of the family Enterobacteriaceae, i.e., 100% agreement with the exception of Hafnia spp. (96.3% agreement) for the ONPG test and Citrobacter, Enterobacter, and Hafnia spp. (75, 86.4, and 98.2% agreement, respectively) for the urease test. The new medium seems especially promising as a screen for Salmonella subgroup I which encompasses most pathogenic Salmonella species other than the Arizona subgroup.     

Capnocytophaga canimorsus sp. nov. (formerly CDC group DF-2), a cause of septicemia following dog bite, and C. cynodegmi sp. nov., a cause of localized wound infection following dog bite

CDC group DF-2 is the vernacular name given to a slow-growing gram-negative bacterium that causes septicemia and meningitis in humans. Infections frequently (one-third of cases) occur following dog bites or close contact with dogs or occasionally with cats. Splenectomy and alcoholism appear to be strong predisposing factors for DF-2 infection. In addition to 150 DF-2 strains received for identification, we received 9 DF-2-like strains; 6 were isolated from wound or eye infections, 3 of which were associated with dog bites and 1 of which was associated with a cat scratch, and 3 were isolated from dog mouths. The major characteristics of DF-2 include production of acid but no gas from lactose and maltose and usually D-glucose; positive reactions for oxidase, catalase, arginine dihydrolase, gliding motility, and o-nitrophenyl-beta-D-galactopyranoside; growth enhanced by serum and by incubation in a candle jar atmosphere; and negative reactions for sucrose, raffinose, inulin, melibiose, nitrate reduction, indole, and growth on MacConkey agar. DF-2-like strains had the same characteristics, except that acid was formed from sucrose, raffinose, inulin, and melibiose. By the hydroxyapatite method, DNAs from 12 DF-2 strains were 88% related in 60 degrees C reactions and 84% related in 75 degrees C reactions. Related sequences contained 0.5 to 1.5% unpaired bases (divergence). Three DF-2-like strains were 73 to 80% related at 60 degrees C (with 2.0 to 2.5% divergence) and 68 to 75% related at 75 degrees C. The relatedness of DF-2 and DF-2-like strains was 19 to 31% at 60 degrees Celsius and 13 to 19% at 75 degrees Celsius. The relatedness of DF-2 and DF-2-like strains to Capnocytophaga species was 4 to 7%. The DNA relatedness date indicate that eh DF-2 and the DF-2-like strains are separate, previously undescribed species. Both groups are phenotypically and genetically distinct from Capnocytophaga species, although they do share several characteristics with Capnocytophaga species, including cellular morphology, gliding motility, cellular fatty acid composition, enhancement of growth in a candle jar atmosphere, and G+C content. The new species differ from Capnocytophaga species by their positive oxidase and catalase reactions. We chose to avoid creating a new genus and proposed the names Capnocytophaga canimorsus sp. nov. for group DF-2 and C. cynodegmi sp. nov. for the DF-2-like strains.     

Streptococcus sinensis sp. nov., a novel species isolated from a patient with infective endocarditis

A bacterium was isolated from the blood culture of a patient with infective endocarditis. The cells were facultative anaerobic, nonsporulating, gram-positive cocci arranged in chains. The bacterium grows on sheep blood agar as alpha-hemolytic, gray colonies of 0.5 to 1 mm in diameter after 24 h of incubation at 37 degrees C in ambient air. Growth also occurs in 10 or 40% bile and on bile esculin agar but not in 6% NaCl. No enhancement of growth is observed in 5% CO(2). It is nongroupable with Lancefield groups A, B, C, D, F, or G antisera and is resistant to optochin and bacitracin. The organism is aflagellated and is nonmotile at both 25 and 37 degrees C. It is Voges-Proskauer test positive. It produces leucine arylamidase and beta-glucosidase but not catalase, urease, lysine decarboxylase, or ornithine decarboxylase. It hydrolyzes esculin and arginine. It utilizes glucose, lactose, salicin, sucrose, pullulan, trehalose, cellobiose, hemicellulase, mannose, maltose, and starch. 16S rRNA gene sequencing showed that there were 3.6, 3.7, 4.3, 4.7, and 5.9% differences between the 16S rRNA gene sequence of the bacterium and those of Streptococcus gordonii, Streptococcus intermedius, Streptococcus constellatus, Streptococcus sanguis, and Streptococcus anginosus, respectively. The G+C content of it (mean plus minus standard deviation) was 53.0% plus minus 2.9%. Based on phylogenetic affiliation, it belongs to the mitis or anginosus group of Streptococcus. For these reasons a new species, Streptococcus sinensis sp. nov., is proposed, for which HKU4 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging cause of infective endocarditis.     

Evaluation of rapid tests for the identification of mycobacteria

The use of eight rapid tests for the identification of 1307 strains of mycobacteria belonging to 18 species was evaluated. The standard niacin, nitrate-reductase and catalase tests were supplemented by new tests for the detection of beta glucosidase, urease, penicillinase, trehalase and cephalosporinase. This combination of eight rapid tests was not able to replace more conventional procedures but in some cases was of value in discriminating between closely related species.     

Short prereduced anaerobically sterilized (PRAS) biochemical scheme for identification of clinical isolates of bile-resistant Bacteroides species.

The rapid identification of isolates of bile-resistant Bacteroides species has clinical and therapeutic relevance because of differences in their patterns of susceptibility and virulence. Five hundred twenty-one strains of bile-resistant Bacteroides species that were previously identified by conventional biochemical methods were reexamined to determine the minimum essential parameters necessary for correct identification. Rapid tests for bile resistance, indole production, and catalase were combined with a novel scheme for biochemical determination of saccharolytic activity on arabinose, trehalose, rhamnose, and/or xylan that included the postincubation addition of bromthymol blue for visual pH determination. Organisms were inoculated into prereduced anaerobically sterilized (PRAS) carbohydrates directly from plates, and identification was complete within 24 h of obtaining a pure culture. Ninety-three percent of bile-resistant Bacteroides species from routine clinical specimens were identified correctly by this scheme; a small number of other indole-positive strains, B. splanchnicus, B. eggerthii, and B. stercoris, were misidentified as B. uniformis.     

Laribacter hongkongensis gen. nov., sp. nov., a novel gram-negative bacterium isolated from a cirrhotic patient with bacteremia and empyema.

A bacterium was isolated from the blood and empyema of a cirrhotic patient. The cells were facultatively anaerobic, nonsporulating, gram-negative, seagull shaped or spiral rods. The bacterium grows on sheep blood agar as nonhemolytic, gray colonies 1 mm in diameter after 24 h of incubation at 37 degrees C in ambient air. Growth also occurs on MacConkey agar and at 25 and 42 degrees C but not at 4, 44, and 50 degrees C. The bacterium can grow in 1 or 2% but not 3, 4, or 5% NaCl. No enhancement of growth is observed with 5% CO(2). The organism is aflagellated and nonmotile at both 25 and 37 degrees C. It is oxidase, catalase, urease, and arginine dihydrolase positive, and it reduces nitrate. It does not ferment, oxidize, or assimilate any sugar tested. 16S rRNA gene sequencing showed that there are 91 base differences (6.2%), 112 base differences (7.7%), and 116 base differences (8.2%) between the bacterium and Microvirgula aerodenitrificans, Vogesella indigofera, and Chromobacterium species, respectively. The G+C content (mean and standard deviation) is 68.0% +/- 2.43%, and the genomic size is about 3 Mb. Based on phylogenetic affiliation, the bacterium belongs to the Neisseriaceae family of the beta-subclass of Proteobacteria. For these reasons, a new genus and species, Laribacter hongkongensis gen. nov., sp. nov., is proposed, for which HKU1 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging pathogen.     

Identification of "Haematobacter," a new genus of aerobic Gram-negative rods isolated from clinical specimens, and reclassification of Rhodobacter massiliensis as "Haematobacter massiliensis comb. nov."

Twelve strains of gram-negative, nonfermenting rods recovered mainly from septicemic patients were studied using conventional and molecular methods. The phenotypic profiles of these strains most closely resembled Psychrobacter phenylpyruvicus. They produced catalase, oxidase, urease, and H(2)S (lead acetate paper) but did not produce indole, reduce nitrate or nitrite, or hydrolyze gelatin or esculin. No acid production was observed in a King's oxidation-fermentation base containing d-glucose, d-xylose, d-mannitol, sucrose, lactose, or maltose. All strains were nonmotile and nonpigmented. Most strains produced green discoloration on blood agar. All strains grew at 25 degrees C and 35 degrees C and most grew on MacConkey agar. They shared a common cellular fatty acid (CFA) profile characterized by large amounts (56% to 90%) of 18:1omega7c and the presence of 3-OH-10:0, 16:1omega7c, 16:0, and 19:0cycomega8c that overall was most similar to that of Rhodobacter species but was quite distinct from that of P. phenylpyruvicus. The MICs for most beta-lactams, fluoroquinolones, aminoglycosides, and carbapenems were low. MICs for aztreonam and piperacillin were higher, with MICs for some strains of > 64 mg/liter and > 128 mg/liter, respectively. Polyphasic analysis of these strains, including morphological, biochemical, CFA composition, DNA-DNA hybridization, 16S rRNA gene sequencing, and percent guanine-plus-cytosine (G+C) content analysis, demonstrated that these strains and Rhodobacter massiliensis represent a new genus, "Haematobacter" (proposed name), with the species H. missouriensis (type strain H1892(T) = CCUG 52307(T) = CIP 109176(T)) and H. massiliensis comb. nov. (type strain Framboise(T) = CCUG 47968(T) = CIP 107725(T)) and an unnamed genomospecies.     

Characterization of a Haemophilus paracuniculus isolated from gastrointestinal tracts of rabbits with mucoid enteritis.

The isolation, characterization, and identification of a microorganism isolated from gastrointestinal tracts of rabbits with mucoid enteritis are described. The isolated organism did not grow on standard media. This organism grew around colonies of Staphylococcus aureus and Lactobacillus desidiosus and around disks saturated with diphosphopyridin nucleotide (factor V) on brain heart infusion agar. The growth of this organism was also observed on media supplemented with beta-nicotinamide adenine dinucleotide. The organism appeared as gram-negative, pleomorphic rods or coccobacilli. It was positive for urease, oxidase, catalase, glycosidases, porphyrin, and indole, and it fermented glucose and sucrose. All of these characteristics suggest that the organism is a member of the genus Haemophilus. Because of its isolation from rabbits and differences in some characteristics from other species of this genus, the name Haemophilus paracuniculus is proposed for this organism.     

"Bacteroides goldsteinii sp. nov." isolated from clinical specimens of human intestinal origin

Phenotypic and phylogenetic studies were performed on an unknown gram-negative, strictly anaerobic, rod-shaped bacterium isolated from human clinical specimens. This organism was indole negative, resistant to 20% bile, produced acetic and a lesser amount of succinic acids as the major end products of glucose metabolism, and possessed a G+C content of approximately 43 mol%. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was a member of the Cytophaga-Flavobacter-Bacteroides phylum of gram-negative bacteria and formed a close association (with an average sequence similarity of 93.6%) with the second subcluster of the Porphyromonas cluster in the Bacteroides subgroup. Phylogenetically and phenotypically it resembled Bacteroides merdae; however, a 16S rRNA gene sequence divergence of approximately 5.5% between the unknown bacterium and B. merdae, as well as distinguishable biochemical characteristics, demonstrate that the unknown bacterium is genotypically and phenotypically distinct and represents a previously unknown subline within the Porphyromonas phylogenetic cluster. Furthermore, a DNA-DNA reassociation value of 17.8% between isolates WAL 12034(T) (the type strain of this novel taxon) and ATCC 43184(T) (B. merdae type strain) also documented the separateness of the unknown species and B. merdae. Based on the phenotypic and phylogenetic findings, a new species, "Bacteroides goldsteinii sp. nov," is proposed. The G+C content of the DNA is 43 mol% for Bacteroides. The type strain of "B. goldsteinii" is WAL 12034(T) (= CCUG 48944(T) = ATCC BAA-1180(T)).     

A sulfate-reducing bacterium that can detoxify U(VI) and obtain energy via nitrate reduction

Bacterial strain UFZ B 490, which was isolated from a uranium dump and is closely related to Desulfovibrio vulgaris oxamicus(T) (DSM 1925(T)), is able to detoxify U(VI) in aqueous media. In experiments reported here, U(VI) was used as an electron acceptor and lactate as electron donor. The reduction of soluble U(VI) to solid U(IV) (uraninite) did not provide energy for growth of strain UFZ B 490. However, the isolate is able to grow when supplied with nitrate as sole electron acceptor and nitrogen source, using lactate as a source of carbon and energy. In comparative studies, the strains Desulfovibrio vulgaris oxamicus(T) (DSM 1925(T)) and Desulfovibrio vulgaris vulgaris(T) (DSM 644(T)), as well as the isolate, all utilized 0.6 mol lactate per mol U(VI) reduced. Strains UFZ B 490 and Desulfovibrio vulgaris oxamicus(T) (DSM 1925(T)) were found to consume 2.4 mol lactate per mol nitrate reduced, but Desulfovibrio vulgaris vulgaris(T) (DSM 644(T)) did not display dissimilatory nitrate reduction. In further experiments it was found that strain UFZ B 490 preferred sulfate as electron acceptor in the presence of both sulfate and nitrate, irrespective of whether it had been precultivated on sulfate or nitrate.     

Atypical campylobacters associated with gastroenteritis.

Nine strains of Campylobacter species other than Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis were isolated from patients with acute diarrhea. All nine strains showed preferred growth at 37 degrees C under microaerophilic conditions. Conventional microbiological tests and DNA-DNA dot blotting were used to identify these strains. Three of the nine Campylobacter strains hydrolyzed hippurate, reduced nitrate, produced catalase, were resistant to cephalothin, and were shown to be highly related to C. jejuni type strains. Two strains had negative or weak catalase activity and were hippurate negative. Three other strains had characteristics similar to those of Campylobacter cinaedi. The ninth strain, isolated from a homosexual man with antibodies to human immunodeficiency virus (human T-cell lymphotropic virus type III), showed unique features different from those of all the known campylobacters used in this study. This strain grew well at 25 and 37 degrees C and was catalase and nitrate positive, hippurate negative, and resistant to cephalothin.     

Enterobacteriaceae: genera and species in 1988

Numerical taxonomy, G + C content determination, DNA-DNA reassociation studies and divergence values delta tm) elicit considerable proliferation in the number of genera and species in recent years. Until 1988 Enterobacteriaceae contain 30 genera and 99 species which can be identified by biochemical tests. Only 20-25 species of Enterobacteriaceae family are of real importance in medical microbiology.     

Isolation, characterization, and mapping of Tn5 insertions into the 140-megadalton invasion plasmid defective in the mouse Sereny test in Shigella flexneri 2a

Using Shigella flexneri 2a YSH6000, we isolated 304 independent Tn5 insertion mutants in the 230-kilobase invasion plasmid, pMYSH6000. The site of each Tn5 insertion was assigned to 23 SalI fragments on the previously made SalI cleavage map of pMYSH6000. Among the 304 insertions, 150 were negative in expression of four phenotypes examined (mouse Sereny test [Ser], invasion into epithelial cells [Inv], Congo red binding [Pcr], and inhibition of bacterial growth [Igr] ): 12 were Ser- Inv+ Pcr+ Igr+, and 142 were positive in all four phenotypes. Tn5 insertions in the avirulent mutants were distributed in two separate SalI fragments, F and G, and in four contiguous SalI fragments, B, P, H, and D. Fragment G contains a novel class of determinant(s) which is required only for Ser+ but not for Inv+, Pcr+, and Igr+. Fragment F contains the previously characterized virF locus. B, P, H, and D each contained both virulent and avirulent Tn5 insertions. This indicates that more than two gene clusters exist within this region. Both are required for expression of all four virulence phenotypes.