Legionella nagasakiensis sp. nov., isolated from water samples and from a patient with pneumonia.

A novel Legionella species was identified based on analysis of 16S rRNA and mip (macrophage infectivity potentiator) gene sequences, cellular fatty acids, isoprenoid quinones, biochemical reactions, antigens and quantitative DNA-DNA hybridization. Strain CDC-1796-JAP-E(T) was isolated from well water at the Nagasaki Municipal Medical Center, Japan. Two strains, CDC-3041-AUS-E and CDC-3558-AUS-E, were isolated from water samples during an outbreak of legionellosis in South Australia. Strain CDC-5427-OH-H was isolated from a 66-year-old female patient diagnosed with Legionnaires' disease in the US. Cells from these four strains were gram-negative, non-fluorescent, rod-shaped, and positive for alkaline phosphatase, esterase, leucine arylamidase, catalase, gelatinase, ß-lactamase and tyrosine browning assay. Phylogenetic analysis of 16S rRNA and mip genes revealed that the four strains formed a distinct cluster within the genus Legionella. The bacteria contained branched-chain fatty acids and quinones that are typical of members of the genus Legionella. Slide agglutination tests demonstrated no cross-reaction with 52 previously described members of the Legionellaceae. DNA-DNA hybridization studies indicated that DNAs from the four strains were highly related (78-84 %) but they showed 29 % relatedness to Legionella oakridgensis ATCC 33761(T) and less than 10 % to strains of other Legionella species tested. These characterizations suggest that the isolates represent a novel species, for which the name Legionella nagasakiensis sp. nov. is proposed; the type strain is CDC-1796-JAP-E(T) ( = ATCC BAA-1557(T) = JCM 15315(T)).

Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest.

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (>82 % relatedness at 55 degrees C and >76 % relatedness at 70 degrees C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a well-supported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were non-haemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120(T) (=ATCC BAA-1595(T) =BEIR NR 9579(T) =CCUG 56148(T)). L. marthii has not been associated with human or animal disease at this time.

Novel Brucella strain (BO1) associated with a prosthetic breast implant infection.

We report the microbiological, biochemical, and molecular characterization of an unusual Brucella strain (BO1) isolated from a breast implant wound in a 71-year-old woman with clinical symptoms consistent with brucellosis. Initial phenotypic analysis, including biochemical and antimicrobial susceptibility testing, cellular fatty acid analysis, and molecular analysis based on DNA-DNA reassociation and the presence of multiple copies of IS711 element suggested that the isolate was a Brucella-like organism, but species determination using microbiological algorithms was unsuccessful. Furthermore, molecular data based on 16S rRNA gene sequencing and multilocus sequence analysis demonstrated that BO1 was an unusual Brucella strain and not closely related to any currently described Brucella species. However, comparison with equivalent sequences in Ochrobactrum spp. confirms that the isolate is much more closely related to Brucella than to Ochrobactrum spp., and thus the isolate likely represents an atypical and novel strain within the genus Brucella.

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 clinical isolates previously identified as Oerskovia turbata: proposal of Cellulosimicrobium funkei sp. nov. and emended description of the genus Cellulosimicrobium.

Taxonomic studies were performed on 13 clinical isolates (ten of which were epidemiologically related) that had been previously identified as Oerskovia turbata. Comparative phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolates are closely related to Cellulosimicrobium cellulans with sequence similarity values ranging from 99.5 to 99.8 %. Chemotaxonomic results (fatty acid profiles and menaquinones) supported the inclusion of these isolates in the genus Cellulosimicrobium. The DNA G+C content was 74.5 mol%. The results of DNA-DNA reassociation, whole-cell sugars (with galactose as the characteristic whole sugar) and phenotypic properties, including antimicrobial resistance, indicated that these isolates are representatives of a novel species of the genus Cellulosimicrobium. The name Cellulosimicrobium funkei sp. nov. is proposed for the novel strains, with strain W6122T (=ATCC BAA-886T = DSM 16025T = CCUG 50705T) as the type strain. The definition of this novel Cellulosimicrobium species will assist in the understanding of the epidemiology and clinical significance of these micro-organisms.

Novel mycolic acid-containing bacteria in the family Segniliparaceae fam. nov., including the genus Segniliparus gen. nov., with descriptions of Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov.

Four strains of novel, rapidly growing, acid-alcohol-fast-staining bacteria were characterized with a polyphasic approach. Isolates were received by the Centers for Disease Control and Prevention from domestic health department laboratories for reference testing as unidentifiable, clinical mycobacteria. Bacteria were rod-shaped and produced non-pigmented (white to beige), non-photochromogenic, smooth or wrinkled-rough colonies on Middlebrook 7H10 and 7H11 media at 33 degrees C. The smooth and wrinkled colony forms were representative of two species with 68.0 and 72.0 mol% DNA G+C content. The cell wall contained meso-diaminopimelic acid and mycolic acids. Species were characterized by cellular fatty acids of C10:0, C14:0, C16:1omega9t, C16:0, C18:1omega9c and 10-methyl C18:0 (tuberculostearic acid). HPLC analysis of mycolic acids produced a novel late-emerging, genus-specific mycolate pattern. TLC analysis demonstrated a novel alpha(+)-mycolate. Species were 98.9% similar by comparison of 16S rRNA gene sequences; however, the DNA-DNA association was <28 %. Phylogenetic analysis of 16S rRNA gene sequences demonstrated an association with Rhodococcus equi, although a DNA-DNA relatedness value of 2% did not support a close relationship. PCR analysis of a proposed, selected actinomycete-specific 439 bp fragment of the 65 kDa heat-shock protein was negative for three of the four isolates. The creation of Segniliparaceae fam. nov. is proposed to encompass the genus Segniliparus gen. nov., including two novel species, the type species Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov., with the respective type strains CDC 1076(T) (=ATCC BAA-972(T)=CIP 108378(T)) and CDC 945(T) (=ATCC BAA-974(T)=CIP 108380(T)).

Phenotypic and genetic characterization of clinical isolates of CDC coryneform group A-3: proposal of a new species of Cellulomonas, Cellulomonas denverensis sp. nov.

CDC coryneform group A-3 bacteria are rare human pathogens. In this study, six group A-3 isolates (two from blood, one from cerebrospinal fluid, and one each from homograft valve, lip wound, and pilonidal cyst) were compared to the type strains of phenotypically related organisms, Cellulomonas fimi, Cellulomonas hominis, Oerskovia turbata, and Sanguibacter suarezii, and characterized by phenotypic, chemotaxonomic, and genotypic studies. DNA-DNA reassociation analysis identified two genomic groups, and phylogenetic analysis of the 16S rRNA gene sequence identified the taxonomic positions of these groups to genus level. Two groups were defined, and both were more closely related to Cellulomonas species: one group of three strains, for which we propose the new species Cellulomonas denverensis sp. nov., with the type strain W6929 (ATCC BAA-788(T) or DSM 15764(T)), was related to C. hominis ATCC 51964(T) (98.5% 16S rRNA gene sequence similarity), and the second group of three strains was related to C. hominis ATCC 51964(T) (99.8 to 99.9% 16S rRNA gene sequence similarity). The definition of this new Cellulomonas species and the confirmation of three strains as C. hominis serve to further clarify the complex taxonomy of CDC coryneform group A-3 bacteria and will assist in our understanding of the epidemiology and clinical significance of these microorganisms.

Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. nov., Mycobacterium houstonense sp. nov., Mycobacterium neworleansense sp. nov. and Mycobacterium brisbanense sp. nov. and recognition of Mycobacterium porcinum from human clinical isolates.

The Mycobacterium fortuitum third biovariant complex (sorbitol-negative and sorbitol-positive) contains unnamed taxa first characterized in 1991. These organisms can cause respiratory infections, a spectrum of soft tissue and skeletal infections, bacteraemia and disseminated disease. To evaluate this group of organisms, clinical reference isolates and the type strains of M. fortuitum third biovariant complex sorbitol-negative (n = 21), M. fortuitum third biovariant complex sorbitol-positive (n = 3), M. fortuitum (n = 3), Mycobacterium peregrinum (pipemidic acid-susceptible) (n = 1), Mycobacterium porcinum (n = 1), Mycobacterium senegalense (n = 2) and Mycobacterium septicum (n = 1) were characterized by using conventional phenotypic (morphological, physiological and antimicrobial susceptibilities), chemotaxonomic (HPLC and cellular fatty acids) and genotypic [RFLP of the rRNA gene (ribotyping), PCR-RFLP of a 439 bp segment of the 65 kDa hsp gene (PCR restriction analysis) and 16S rRNA gene sequence] analysis, DNA G + C content and DNA-DNA relatedness analyses. The results of these studies indicated that the strains comprised M. porcinum (n = 13), M. septicum (n = 1) and four novel closely related genetic groups within the M. fortuitum third biovariant complex: Mycobacterium boenickei sp. nov. (n = 6), Mycobacterium houstonense sp. nov. (n = 2), Mycobacterium neworleansense sp. nov. (n = 1) and Mycobacterium brisbanense sp. nov. (n = 1), with type strains ATCC 49935T (= W5998T = DSM 44677T), ATCC 49403T (= W5198T = DSM 44676T) ATCC 49404T (= W6705T = DSM 44679T) and ATCC 49938T (= W6743T = DSM 44680T), respectively.

Identification of some charcoal-black-pigmented CDC fermentative coryneform group 4 isolates as Rothia dentocariosa and some as Corynebacterium aurimucosum: proposal of Rothia dentocariosa emend. Georg and Brown 1967, Corynebacterium aurimucosum emend. Yassin et al. 2002, and Corynebacterium nigricans Shukla et al. 2003 pro synon. Corynebacterium aurimucosum.

Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4). Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses. Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains). The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains of Rothia dentocariosa and Rothia mucilaginosa, while the FCG4b strains were similar to Corynebacterium species. DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant of R. dentocariosa. Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species, Corynebacterium aurimucosum and Corynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain of R. dentocariosa; therefore, the Corynebacterium-like strains were classified as a charcoal-black-pigmented variant of C. aurimucosum, because this name has nomenclatural priority over C. nigricans. These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of both R. dentocariosa and C. aurimucosum; hence, the descriptions of both R. dentocariosa and C. aurimucosum have been amended to include charcoal-black-pigmented variants, and C. nigricans is a pro synonym of C. aurimucosum.

Bordetella holmesii bacteremia: a newly recognized clinical entity among asplenic patients.

Bordetella holmesii is a recently identified gram-negative bacterial species associated with bacteremia, endocarditis, and respiratory illness, mainly in immunocompromised patients. From isolates submitted to the Centers for Disease Control and Prevention from 1983 through 2000 for further identification, we identified 30 patients with B. holmesii bacteremia. Of the 26 patients for whom data were available, 22 (85%) were anatomically or functionally asplenic. In 25 (96%) of the 26 patients, B. holmesii was the only organism isolated from blood samples, and 14 patients (54%) had B. holmesii recovered from > or =2 blood cultures. The clinical course of the infection was generally characterized by a nonspecific febrile illness. Twenty-one patients (81%) were treated with various antimicrobial agents, and 20 (77%) were admitted to the hospital. There were no deaths. Our findings support evidence that B. holmesii may be a true pathogen associated with bacteremia among asplenic patients.

Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry.

Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) is a species-common, immunogenic surface lipoprotein. In this study, the psaA gene was expressed as a nonfusion acylated protein in an Escherichia coli expression system. Yields of pure recombinant PsaA (rPsaA) were 8-10 mg/liter of fermentation culture. Analysis of rPsaA tryptic digests by HPLC-electrospray mass spectrometry (MS) confirmed 98% of the expected protein sequence. GC/MS data demonstrated very similar acylation of native and rPsaA by C12:0-C22:0 fatty acids, with C16 and C18 predominating. Negative ion electrospray MS/MS analysis of the rPsaA lipid anchor released by Pronase-E confirmed that the structure was based on an N-terminal palmitoylcysteine (Pam(3)Cys). Electrospray MS heterogeneity analysis of intact rPsaA indicated that all of the observed heterogeneity could be accounted for by the fatty acid distributions. The availability of well-characterized rPsaA will facilitate the continued research and development of protein-based vaccines for the prevention of pneumococcal disease.

Paracoccus yeeii sp. nov. (formerly CDC group EO-2), a novel bacterial species associated with human infection.

CDC eugonic oxidizer group 2 (EO-2) is a group of unclassified gram-negative bacterial strains isolated from various human sources. As determined by biochemical tests and analyses of fatty acid compositions, these organisms form a homogeneous group that appears to be distinct from but related to other Paracoccus species. Molecular studies were performed on a set of 13 EO-2 strains from various clinical sources and geographic locations in the United States and Canada to determine their relationship to the Paracoccus genus. Control strains were Paracoccus denitrificans ATCC 17741(T), P. versutus ATCC 25364(T), P. aminophilus ATCC 49673(T), P. solventivorans ATCC 700252(T), and Psychrobacter immobilis ATCC 43116(T), which are phenotypically similar to EO-2. Nearly complete (1,500-base) 16S rRNA gene sequencing of eight EO-2 strains showed a high level of sequence similarity (>99.3%) within the group, and a BLAST search of GenBank placed the EO-2 cluster in close proximity to Paracoccus species (95 to 97% similarity). DNA-DNA hybridization studies of 13 of the EO-2 strains showed all to be related at the species level, with >70% relatedness under stringent conditions and a divergence within the group of less than 2%. None of the Paracoccus control strains hybridized at >54% with any of the EO-2 strains. These results indicate that EO-2 represents a new Paracoccus species, the first isolated from human clinical specimens. A new species, Paracoccus yeeii, is proposed for the EO-2 strains. The type strain of P. yeeii is CDCG1212 (ATCC BAA-599 and CCUG 46822), isolated in Pennsylvania from dialysate of a 77-year-old male with peritonitis.