CDC group O-3: phenotypic characteristics, fatty acid composition, isoprenoid quinone content, and in vitro antimicrobic susceptibilities of an unusual gram-negative bacterium isolated from clinical specimens.

Between 1983 and 1994, 13 phenotypically similar unidentified clinical isolates were received by the Special Bacteriology Reference Laboratory, Centers for Disease Control and Prevention (CDC). Sources included blood (four strains), lung (three strains), knee fluid and duodenal tissue (one strain each), bone, and lymph node tissue (two strains each). All were aerobic glucose-oxidizing, slender, long, curved gram-negative rods that utilized xylose, sucrose, and maltose; did not grow on MacConkey agar in 1 to 2 days; were oxidase positive; hydrolyzed esculin; and grew on Campylobacter selective medium. All were negative for urease, indole, nitrate reduction, and gelatin hydrolysis. All were motile by means of a single polar flagellum with a noticeably short wavelength; however, motility was sometimes difficult to demonstrate. The cellular fatty acid compositions of these strains, as analyzed by gas-liquid chromatography, were unique, characterized by relatively large amounts of 16:1omega7c, 16:0, and 18:1omega7c with smaller amounts of 12:0, 3-OH-12:1, 14:0, 15:0, 18:0, Br-19:1, and 19:0cyc11-12. High-performance liquid chromatography and mass spectrometry of the quinone extracts of three representative strains showed ubiquinone-10 as the major component. Based on the breakpoints for the family Enterobacteriaceae, all the strains were susceptible in vitro to aminoglycosides, sulfamethoxazole-trimethoprim, and chloramphenicol but were resistant to most beta-lactams except imipenem. The MICs of amoxicillin-clavulanate and ciprofloxacin for these strains clustered around the breakpoints, which makes it difficult to predict the strains' response in vivo to these agents. This group has been designated CDC oxidizer group 3 (O-3).

CDC group IIc: phenotypic characteristics, fatty acid composition, and isoprenoid quinone content.

Twenty strains of glucose-utilizing, small gram-negative slightly pleomorphic rods that grew well aerobically and that were isolated from clinical specimens formed a phenotypically similar group that was designated CDC group IIc. The phenotypic characteristics of CDC group IIc were most similar to those of CDC groups IIe and IIh, the major differences being that CDC group IIc produced acid from sucrose, hydrolyzed esculin, and usually reduced nitrate. The CDC group IIc strains were analyzed by gas-liquid chromatography for their cellular fatty acid compositions, and all contained relatively large amounts of isobranched hydroxy and nonhydroxy acids. High-performance liquid chromatography and mass spectrometry analysis of the quinone extract showed menaquinone-6 as the major component. Both the cellular fatty acid and isoprenoid quinone compositions were consistent with the profiles of CDC groups IIe and IIh. Thirty percent of the isolates were from human blood.

Characterization of ear fluid isolates of Alloiococcus otitidis from patients with recurrent otitis media.

Nineteen isolates of Alloiococcus otitidis from ear fluid samples collected by tympanostomy from patients at four geographic locations were identified by phenotypic characterization and genetic relatedness. Initial growth of A. otitidis isolates occurred after 3 days at 37 degrees C on brain heart infusion (BHI) agar with 5% rabbit blood. Heavy growth occurred in BHI broth supplemented with 0.07% lecithin and 0.5% Tween 80 after 4 days of incubation. The isolates were gram-positive cocci that divided on an irregular plane and produced metabolic lactic acid, pyrrolidonyl arylamidase, and leucine aminopeptidase. These cocci grew sparsely in 6.5% NaCl-BHI broth, were asaccharolytic on both fermentative and oxidative bases, and were cytochrome negative by the iron-porphyrin test. The cellular fatty acid profile of A. otitidis was distinguished from those of related genera and characterized by major amounts ( > or = 14%) of 16:0, 18:2, 18:1 omega 9c, and 18:0 and smaller amounts of 14:0, 16:1 omega 7c, 17:0, and 18:1 omega 7c. Fifteen isolates demonstrated > 69% relatedness by DNA-DNA hybridization. Four isolates plus the original 15 were confirmed as A. otitidis by dot blot hybridization with a digoxigenin-labeled nucleotide probe specific for this species. The intergenic space between the genes coding for the 16S and 23S rRNAs of alloiococci was amplified by PCR, analyzed by restriction fragment length polymorphism, and determined to consist of three different genetic types. Although beta-lactamase negative, A. otitidis demonstrated intermediate levels of resistance to beta-lactams, including expanded-spectrum cephalosporins, and were resistant to trimethoprim-sulfamethoxazole and erythromycin.

Effects of population density on layer performance.

Four experiments were conducted to study the effects of population density on layer performance. in each experiment, 20-wk--old White leghorn pullets were housed in 30.5 x 50.8 cm cages at the rate of one, two, three, and four birds per cage for Treatments 1, 2, 3, and 4, respectively. in Experiment 4, there were three additional treatments: for Treatments 5, 6, and 7, birds were placed into identical cages (30.5 x 50.8 cm) at the rate of three, two, and three birds per cage, respectively, at 20 wk of age. At 28 wk of age, one bird was removed from each cage in Treatment 5, and one bird was added to each cage in Treatments 6 and 7. In the first three experiments, all eggs that were collected for 2 consecutive d every 8 wk starting at 28 wk of age were used to measure egg traits. In Experiment 4, egg traits were determined once at 52 week of age. Birds at the highest population density had the lowest percentage hen-day egg production and had one of the poorest feed efficiencies. Egg production was negatively correlated (P < or = .01, Experiments 1, 2, 3; P < or = .05, Experiment 4) with population density. Feed required to produce a dozen eggs was positively correlated (P < or = .01, Experiments 1, 2; P < or = .05, Experiment 3) with population density in three out of the four experiments. The addition or removal of a cage mate to or from a multiple-bird cage in Experiment 4 did not (P > .05) affect egg production or feed efficiency. Final BW, mortality, and egg weight were not (P > .05) affected by population density. Only in one out of the four experiments was feed consumption (Experiment 1), eggshell thickness (Experiment 2), or albumen height (Experiment 3 lowered (P < or = .05) by having more than one bird per cage. This study showed that increasing population density decreased laying performance of the birds.

Effects of cage density on fear-related behavioral response and activity of layers.

Two experiments were conducted to study the effects of cage density on fear-related behavioral response and activity of White Leghorn layers. In Experiment 1, 20-wk old pullets were placed into 30.5 x 50.8 cm cages at the rate of one, two, three, and four birds per cage for Treatments 1, 2, 3, and 4, respectively. In Experiment 2, birds were placed into cages (30.5 x 50.8 cm) at the rate of one, two, three, four, three, two, and three birds per cage for Treatments 1, 2, 3, 4, 5, 6, and 7, respectively, at 20 wk of age. At 28 wk of age, one bird was removed from each cage in Treatment 5, and one bird was added to each cage in Treatments 6 and 7. Fear-related behavioral response of the birds, as determined by the duration of induced tonic immobility (TI), and bird activity, as determined by the head movement (HM) count, were measured at 52 wk of age in Experiment 1, and at 29, 36, and 52 wk of age in Experiment 2. Increasing cage density had no effect on fear-related response of the birds in both experiments. Cage density effect on bird activity was not consistent. In Experiment 1, HM count was not affected by cage density, whereas in Experiment 2, pullets in the multiple-bird cage had a lower (P < or = .05) HM count (three-period average) than the birds housed individually. There was no correlation between the duration of induced TI and HM count in both experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypic characteristics, fatty acid composition, and isoprenoid quinone content of CDC group IIg bacteria.

Eleven strains of eugonic, nonoxidative, gram-negative rods isolated from clinical specimens formed a distinct group that was designated CDC group IIg. Five of the 11 isolates were from wounds. The phenotypic characteristics of CDC group IIg were most similar to those of Weeksella species, with the major difference being that CDC group IIg strains grew on MacConkey agar in 1 to 2 days, did not hydrolyze gelatin, and did not produce urease. All 11 strains of CDC group IIg possessed a distinct fatty acid profile that was characterized by large amounts (19 to 29%) of 18:1 omega 7c, 16:0, and 16:1 omega 7c, moderate amounts (6 to 10%) of 3-OH-14:0 and 14:0, and smaller amounts (1 to 2%) of 18:2, 18:0, and 3-OH-16:0. This fatty acid profile differs from those of Weeksella species by the absence of branched-chain fatty acids. CDC group IIg contains ubiquinone-8, as opposed to menaquinone-6 in Weeksella species. The isolates were susceptible to a variety of antimicrobial agents, including the aminoglycosides, tetracyclines, quinolones, sulfonamides, and polymyxin B.

Chemotaxonomic analyses of Bacteroides gracilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter gracilis comb. nov.

The cellular fatty acids, respiratory quinones, and proteins of the generically misnamed taxa Bacteroides gracilis and Bacteroides ureolyticus were analyzed and compared with the corresponding chemotaxonomic features of their closest relatives, the campylobacters. Our results and previously published data for genotypic and phenotypic characteristics were used in a polyphasic approach to reconsider the classification of these organisms. We transfer B. gracilis to the genus Campylobacter as Campylobacter gracilis comb. nov. B. ureolyticus can be considered a campylobacter on genotypic grounds; in contrast, the proteolytic metabolism and fatty acid components of this taxon exclude it from the genus Campylobacter. We prefer to consider this taxon a species incertae sedis pending the isolation and characterization of additional B. ureolyticus-like bacteria.

Bordetella holmesii sp. nov., a new gram-negative species associated with septicemia.

CDC nonoxidizer group 2 (NO-2) currently consists of 15 gram-negative, rod-shaped, oxidase-negative, asaccharolytic, brown soluble pigment-producing strains isolated from blood cultures, usually from young adults. On the basis of their cellular fatty acid profiles, NO-2 strains formed a single group that was identical with the profile of Bordetella avium. 16S rRNA sequencing of one NO-2 strain and the type strains of B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium showed a high degree of homology (> or = 98% over 1,525 bases). The NO-2 guanine-plus-cytosine content (61.5 to 62.3 mol%) and major ubiquinone analysis (ubiquinone-8) results were both consistent with those for the genus Bordetella. DNA relatedness studies (hydroxyapatite method) confirmed a close relatedness between NO-2 and Bordetella species and demonstrated that NO-2 strains were a single new species. The name B. holmesii sp. nov. is proposed for CDC group NO-2.

Characterization of Neisseria elongata subsp. glycolytica isolates obtained from human wound specimens and blood cultures.

Four slightly yellow-pigmented, alpha-hemolytic, gram-negative coccobacilli, three from wound specimens and one from multiple blood cultures of a patient with endocarditis, were identified as Neisseria elongata subsp. glycolytica on the basis of their overall biochemical and genetic similarities to this subspecies. These strains resembled N. elongata in their guanine-plus-cytosine contents (55.6 to 57.1 mol%) and in their overall cellular fatty acid profiles, which are characterized by large amounts of 16:0, 16:1 omega 7c, and 18:1 omega 7c fatty acids. Their identities were confirmed by species-level DNA relatedness (hydroxyapatite method) to the type strains of all three N. elongata subspecies. The biochemical profiles and cultural characteristics of these strains resembled those of the type strain of N. elongata subsp. glycolytica except for the production of a weak yellow growth pigment and alpha-hemolysis on sheep blood agar. They differed from N elongata subsp. elongata by the production of catalase, by the production of alpha-hemolysis on sheep blood agar, and by acid production from D-glucose. They differed from N. elongata subsp. nitroreducens by the production of catalase and an inability to reduce nitrate. These studies suggest a pathogenic potential for N. elongata subsp. glycolytica, usually considered to be a transient colonizer in humans.

Recognition of Dermabacter hominis, formerly CDC fermentative coryneform group 3 and group 5, as a potential human pathogen.

Thirty strains of fermentative coryneform-like bacteria designated CDC fermentative coryneform group 3 and coryneform group 5 were compared biochemically by cellular fatty acid analysis and by DNA relatedness with the type strain of Dermabacter hominis, ATCC 49369. DNA from 22 strains of both CDC groups showed 69 to 96% relatedness (hydroxyapatite method) to labeled DNA from ATCC 49369 and to DNA from CDC group 3 strain G4964, and the strains are considered to belong to D. hominis. The remaining eight strains were genetically but not phenotypically differentiable from D. hominis. They were genetically heterogeneous, but hybridization results indicated that they probably belong to the genus Dermabacter. Thirteen of the 22 D. hominis strains and all 8 of the other Dermabacter strains had been isolated from blood, which indicates the pathogenic potential of this species and genus.

Human infections caused by Brevibacterium casei, formerly CDC groups B-1 and B-3.

Forty-one clinical strains of CDC coryneform groups B-1 and B-3 were compared biochemically, by analysis of cell wall sugars, amino acids, and cellular fatty acids, and by DNA relatedness to the type strains of Brevibacterium casei, Brevibacterium epidermidis, and Brevibacterium linens. Twenty-two strains were shown to be B. casei, while five other strains formed a phenotypically inseparable genomospecies in the same genus. The remaining isolates were genetically heterogeneous, and most are probably members of the genus Brevibacterium. They were not further identified, but they were biochemically distinguishable from B. casei. Eleven of the clinical strains of B. casei were isolated from blood, and two each were isolated from cerebrospinal fluid and from pleural fluid. At least five isolates were from multiple blood or cerebrospinal fluid cultures. To our knowledge, these strains are the first described clinical isolates identified as B. casei, which was previously considered to be a nonpathogenic species.

Cellular fatty acid composition and phenotypic and cultural characterization of CDC fermentative coryneform groups 3 and 5.

Seventy strains of fermentative, asporogenous, gram-positive coccobacilli or short rods form two closely related groups which have been designated CDC fermentative coryneform groups 3 (32 strains, xylose fermenters) and 5 (38 strains, xylose nonfermenters). The two taxa are otherwise similar to each other phenotypically and culturally and by a distinctive Staphylococcus-like odor and by cellular fatty acid (CFA) composition. CDC group 3 and CDC group 5 strains have been isolated from clinical sources (blood, abscesses, and wounds but not urine or respiratory specimens) in Canada and the United States and among referrals from Belgium, Sweden, and Spain. Coryneform CDC group 3 strains were phenotypically similar to CDC coryneform group A-3 but were distinguishable by their inability to reduce nitrate and by their lack of motility. Coryneform CDC group 5 isolates were phenotypically somewhat similar to Actinomyces viscosus and Rothia dentocariosa, except that none of this group reduced nitrate. Both CDC groups could be differentiated from these similar bacteria by the ability to decarboxylate lysine and ornithine. The CFA compositions of CDC group 3 and 5 strains were similar to each other, were distinctive from those of other coryneforms, and were of the branched-chain type. API CORYNE codes were consistent for both CDC group 3 and CDC group 5 bacteria, suggesting that this method could be useful as an identification method.

Mycobacterium celatum sp. nov.

A new slowly growing nonphotochromogenic Mycobacterium species of clinical importance is described. The biochemical characteristics of this organism were similar to those of Mycobacterium xenopi and members of the Mycobacterium avium complex. However, none of the strains reacted with commercially available genetic probes for the M. avium complex. The strains were resistant to most antituberculosis drugs. Multilocus enzyme electrophoresis revealed two original electrophoretic types, which was suggestive of new species. The strains contained alpha-, keto-, and dicarboxylic mycolates, as determined by thin-layer chromatography. A mycolic acid analysis by high-performance liquid chromatography revealed a chromatographic pattern similar to that of M. xenopi, but distinct from the patterns of previously described Mycobacterium species. Hexadecanoic and tuberculostearic acids were identified as the major cell wall fatty acids by gas-liquid chromatographic analysis; hexacosanoic acid was the major mycolic acid cleavage product, and 2-eicosanol was the major alcohol. Evaluation of the 16S rRNA sequence confirmed the phylogenetic position of the organism among the slowly growing Mycobacterium species. Cultures representing this new species have been deposited in the American Type Culture Collection as strains ATCC 51130 and ATCC 51131T (T = type strain). The name Mycobacterium celatum is proposed.

Cellular fatty acid compositions of "Achromobacter groups B and E".

Strains of "Achromobacter groups B and E" were examined for cellular fatty acid (CFA) composition to evaluate their chemical relatedness to known bacterial species and groups. The CFAs were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. The CFA profiles of the two groups were identical and were distinct from CFA profiles of all other bacteria we have previously tested. These data provide support for results from whole-cell protein pattern analysis and DNA-DNA and rRNA-DNA hybridization studies, which show that "Achromobacter groups B and E" are biotypes of a single new genus and species.

Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis.

A Rochalimaea-like organism (strain F9251) was isolated from a patient with endocarditis after blood drawn for culture before antimicrobial therapy was subcultured onto blood and chocolate agars and incubated for 2 weeks in 5% CO2. The strain was phenotypically similar to known Rochalimaea species. The cellular fatty acid composition of strain F9251 was close to but distinct from those of the three known Rochalimaea species and was most similar to that of R. vinsonii. Labeled DNA from strain F9251 was 59 to 67% related to DNAs from type strains of the three described Rochalimaea species, and its 16S rRNA gene sequence was 98.9% or more homologous to their 16S rRNA gene sequences. These findings support classification of F9251 as a new Rochalimaea species, for which the name Rochalimaea elizabethae sp. nov. is proposed. The patient infected with the organism had large bacterial vegetations on his aortic valve and was cured with antibiotics and valve-replacement surgery. Recognition of the procedures required to identify this and other Rochalimaea species suggests that clinical laboratories should prolong the incubation times of cultures of blood and tissue from patients with suspected endocarditis, patients with fever of unknown origin, and immunocompromised patients with fever so that the full spectrum of disease caused by these organisms can be recognized.

Biochemical characteristics and fatty acid composition of Gilardi rod group 1 bacteria.

Fifteen strains of eugonic, nonoxidative, gram-negative rods isolated primarily from human wounds of the extremities and blood formed a distinct group which was designated Gilardi rod group 1. The phenotypic characteristics of Gilardi rod group 1 were most similar to those of CDC group M-5, with the major difference that nitrite reduction was observed with CDC group M-5. All 15 strains of Gilardi rod group 1 possessed a distinct fatty acid profile which was characterized by large amounts (> 15%) of cis-vaccenic (18:1 omega 7c), palmitic (16:0), myristic (14:0), and lactobacillic (19:0 cyc11,12) acids and moderate amounts (3 to 5%) of lauric (12:0), 3-hydroxylauric (3-OH-12:0), and palmitoleic (16:1 omega 7c) acids. This fatty acid profile is unique compared with the profiles of CDC group M-5 and other bacteria we have tested and is useful for the rapid identification of Gilardi rod group 1 isolates.

Characterization of Centers for Disease Control group NO-1, a fastidious, nonoxidative, gram-negative organism associated with dog and cat bites.

Seventeen strains of fastidious, nonoxidative, gram-negative rods, isolated from human wounds resulting primarily from dog or cat bites, formed a distinct group, which was designated Centers for Disease Control (CDC) group nonoxidizer 1 (NO-1). The phenotypic characteristics of CDC group NO-1 were most similar to non-acid-producing Acinetobacter species, with the major difference being a negative reaction in the transformation assay test for Acinetobacter spp. The cellular fatty acid composition of CDC group NO-1 was different from those of Acinetobacter species and all other bacteria tested to date. The isolates were susceptible to a variety of antimicrobial agents including the aminoglycosides, beta-lactam antibiotics, tetracyclines, quinolones, and sulfonamides. Fifty percent of the isolates were resistant to trimethoprim. Ubiquinone-8 was present as the major isoprenoid quinone in CDC group NO-1.

Legionella lansingensis sp. nov. isolated from a patient with pneumonia and underlying chronic lymphocytic leukemia.

A Legionella-like organism, strain 1677-MI-H, was isolated from the bronchoscopy washings of a patient with pneumonia who had a 2-year history of progressive, chronic lymphocytic leukemia. The growth characteristics, cellular fatty acids, and ubiquinone content of the isolate were consistent with those for Legionella spp. The isolate was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization studies showed that strain 1677-MI-H (ATCC 49751) represents a new Legionella species which is named Legionella lansingensis.

Identification of some uncommon monounsaturated fatty acids of bacteria.

Location of the double-bond position of monounsaturated fatty acids of various bacteria was accomplished with combined gas chromatography-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary gas chromatography-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Twenty-one relatively novel monoenoic fatty acids were identified among the bacteria studied. All Flavobacterium species contained i17:1 omega 8c, Bacillus alvei contained i16:1 omega 11c and i17:1 omega 12c, and Psychrobacter immobilis contained 12:1 omega 9c. Resolution of cis and trans isomers with capillary gas chromatography and subsequent mass spectrometry permitted positive identification of 16:1 omega 7c and 16:1 omega 7t in Arcobacter (Campylobacter) cryaerophila and 16:1 omega 9c and 16:1 omega 9t in Aerococcus viridans.

Legionella shakespearei sp. nov., isolated from cooling tower water.

A Legionella-like organism (strain 214T [T = type strain]) was isolated from a cooling tower in Stratford-upon-Avon, England. This strain required L-cysteine and contained cellular branched-chain fatty acids that are typical of the genus Legionella. Strain 214T produced pink colonies on buffered charcoal-yeast extract agar. Ubiquinone Q-12 was the major quinone. Strain 214T was serologically distinct from other legionellae as determined by a slide agglutination test. The results of DNA hybridization studies showed that strain 214T (= ATCC 49655T) is a member of a new Legionella species, Legionella shakespearei.