Identification of Arthrobacter oxydans, Arthrobacter luteolus sp. nov., and Arthrobacter albus sp. nov., isolated from human clinical specimens

Five Arthrobacter isolates from clinical specimens were studied by phenotypic, chemotaxonomic, and genetic characterization. Two strains had characteristics consistent with those of Arthrobacter oxydans. One strain was related to A. citreus; however, DNA-DNA hybridization and phenotypic characteristics indicated that this strain belongs to a new species, for which the name Arthrobacter luteolus sp. nov. is proposed. Two strains were closely related to A. cumminsii by 16S rRNA gene sequencing, but DNA-DNA hybridization, peptidoglycan type, and some phenotypic features indicated that they should be assigned to a new species, for which the name Arthrobacter albus sp. nov. is proposed. The type strain of A. luteolus is CF25 (DSM 13067). The type strain of A. albus is CF43 (DSM 13068).     

Facklamia languida sp. nov., isolated from human clinical specimens

Three strains of a gram-positive catalase-negative, facultatively anaerobic coccus-shaped organism originating from human clinical samples were characterized by phenotypic and molecular taxonomic methods. Sequencing of genes encoding 16S rRNA showed that the strains are phylogenetically closely related (99.9 to 100% sequence similarity) and represent a new subline within the genus Facklamia. The unknown bacterium was readily distinguished from all currently described species of the genus Facklamia (viz., Facklamia hominis, Facklamia ignava, and Facklamia sourekii) by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Facklamia languida sp. nov. The type strain of F. languida is CCUG 37842.     

Enterococcus gilvus sp. nov. and Enterococcus pallens sp. nov. isolated from human clinical specimens

Light yellow-pigmented (strain PQ1) and yellow-pigmented (strain PQ2), gram-positive, non-spore-forming, nonmotile bacteria consisting of pairs or chains of cocci were isolated from the bile of a patient with cholecystitis (PQ1) and the peritoneal dialysate of another patient with peritonitis (PQ2). Morphologically and biochemically, the organisms phenotypically belonged to the genus Eterococcus. Whole-cell protein (WCP) analysis and sequence analysis of a segment of the 16S rRNA gene suggested that they are new species within the genus Enterococcus. PQ1 and PQ2 displayed less than 70% identities to other enterococcal species by WCP analysis. Sequence analysis showed that PQ1 shared the highest level of sequence similarity with Enterococcus raffinosus and E. malodoratus (sequence similarities of 99.8% to these two species). Sequence analysis of PQ2 showed that it had the highest degrees of sequence identity with the group I enterococci E. malodoratus (98.7%), E. raffinosus (98.6%), E. avium (98.6%), and E. pseudoavium (98.6%). PQ1 and PQ2 can be differentiated from the other Enterococcus spp. in groups II, III, IV, and V by their phenotypic characteristics: PQ1 and PQ2 produce acid from mannitol and sorbose and do not hydrolyze arginine, placing them in group I. The yellow pigmentation differentiates these strains from the other group I enterococci. PQ1 and PQ2 can be differentiated from each other since PQ1 does not produce acid from arabinose, whereas PQ2 does. Also, PQ1 is Enterococcus Accuprobe assay positive and pyrrolidonyl-beta-naphthylamide hydrolysis positive, whereas PQ2 is negative by these assays. The name Enterococcus gilvus sp. nov. is proposed for strain PQ1, and the name Enterococcus pallens sp. nov. is proposed for strain PQ2. Type strains have been deposited in culture collections as E. gilvus ATCC BAA-350 (CCUG 45553) and E. pallens ATCC BAA-351 (CCUG 45554).     

Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov. isolated from clinical specimens of human origin

Three groups of previously unknown gram-positive, anaerobic, coccus-shaped bacteria were characterized using phenotypic and molecular taxonomic methods. Phenotypic and genotypic data demonstrate that these organisms are distinct, and each group represents a previously unknown subline within Clostridium cluster XIII. Two groups are most closely related to Peptoniphilus harei in the genus Peptoniphilus, and the other group is most closely related to Anaerococcus lactolyticus in the genus Anaerococcus. Based on the findings, three novel species, Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov., are proposed. The type strains of Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov. are WAL 10418(T) (= CCUG 53341(T) = ATCC BAA-1383(T)), WAL 12922(T) (= CCUG 53342(T) = ATCC BAA-1384(T)), and WAL 17230(T) (= CCUG 53340(T) = ATCC BAA-1385(T)), respectively.     

"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)).     

Aeromonas trota sp. nov., an ampicillin-susceptible species isolated from clinical specimens.

Previous DNA hybridization studies established 12 Aeromonas genospecies, from which nine phenotypic species have been proposed: Aeromonas hydrophila, A. sobria, A. caviae, A. media, A. veronii, A. schubertii, A. salmonicida, A. eucrenophila, and A. jandaei. We have delineated a new Aeromonas genospecies, A. trota, on the basis of 13 strains isolated primarily from fecal specimens from southern and southeastern Asia. All strains were highly related to the proposed type strain, AH2 (ATCC 49657T): 51 to 100% (60 degrees C) and 49 to 99% (75 degrees C), with 0.2 to 2.2 divergence. AH2 was only 16 to 41% (60 degrees C) related to all other Aeromonas type strains and DNA group definition strains. The unique profile of A. trota includes negative reactions for esculin hydrolysis, arabinose fermentation, and the Voges-Proskauer test, positive reactions for cellobiose fermentation, lysine decarboxylation, and citrate utilization, and susceptibility to ampicillin, as determined by the broth microdilution MIC method and the Bauer-Kirby disk diffusion method (10 micrograms). Nine of the A. trota strains were from a single study of 165 geographically diverse aeromonads. This finding questions the efficacy of screening fecal specimens for Aeromonas spp. with ampicillin-containing media and suggests a previously unrecognized prevalence of this new species.     

"Bacteroides nordii" sp. nov. and "Bacteroides salyersae" sp. nov. isolated from clinical specimens of human intestinal origin

Two groups of unknown bacteria, which phenotypically resemble members of the Bacteroides fragilis group but phylogenetically display >5% 16S rRNA gene sequence divergence from their nearest validly described species, Bacteroides thetaiotaomicron, were characterized by phenotypic and molecular taxonomic methods. Phylogenetically and phenotypically, the unidentified bacteria displayed a relatively close association with each other. However, a 16S rRNA gene sequence divergence of approximately 4% between the two unknown bacteria, as well as distinguishable biochemical characteristics, demonstrates that these organisms are genotypically and phenotypically distinct, and each group may represent a previously unknown subline within the Bacteroides phylogenetic cluster. Subsequent DNA-DNA hybridization studies confirmed that the two novel organisms were indeed distinct from each other. The previously described species closest to both of them is B. thetaiotaomicron (approximately 94% sequence similarity), but they can be differentiated easily from B. thetaiotaomicron by virtue of not utilizing trehalose. DNA-DNA pairing studies also documented the separateness of the unknown species and B. thetaiotaomicron. Based on the phenotypic and phylogenetic findings, two new species, "Bacteroides nordii" sp. nov. and "Bacteroides salyersae" sp. nov, are proposed. The G+C content of the DNA is 41.4 mol% for Bacteroides nordii and 42.0 mol% for Bacteroides salyersae. The type strains of Bacteroides nordii and Bacteroides salyersae are WAL 11050 (ATCC BAA-998 or CCUG 48943) and WAL 10018 (ATCC BAA-997 or CCUG 48945), respectively.     

Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. isolated from human clinical infections

One hundred eight isolates were previously identified in our laboratory as Clostridium clostridioforme by colonial and cellular morphology, as well as biochemical tests. Recent studies have indicated that there are actually three different species in this C. clostridioforme group: C. hathewayi, C. bolteae, and C. clostridioforme. Our isolates were reexamined using biochemical and enzymatic tests and molecular methods. Forty-six isolates were reidentified as C. hathewayi, 34 as C. bolteae, five as C. clostridioforme, and one as C. symbiosum. Twenty-two strains were identified only to the genus level by 16S rRNA gene sequencing, and although they are microscopically and morphologically indistinguishable from the above-mentioned three species, they are phenotypically different and only 96 to 98% similar by gene sequencing. Twenty of these 22 strains were indole positive and formed two novel species. We propose Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. as names for these new species. They are differentiated from each other by results for raffinose, rhamnose, alpha-galactosidase, and beta-galactosidase: positive, negative, positive, and positive, respectively, for the former species and negative, positive, negative, and negative, respectively, for the latter species. The type strain of C. aldenense is RMA 9741 (ATCC BAA-1318; CCUG 52204), and the type strain of C. citroniae is RMA 16102 (ATCC BAA-1317; CCUG 52203).     

Identification of haemolytic Haemophilus species isolated from human clinical specimens and description of Haemophilus sputorum sp. nov

Haemolytic Haemophilus strains with no requirement for X factor are regularly isolated from sputum and throat swabs and occasionally from invasive infections, but the classification of such strains is not clear. We characterized 56 strains with a phenotype concordant with Haemophilus parahaemolyticus (V, but not X factor-dependent; urease-positive; tryptophanase-negative; ornithine decarboxylase-negative) by extended phenotypic testing and 16S rRNA gene sequencing. In addition, 31 of the strains and representative type strains were investigated by multilocus sequence analysis based on 3 housekeeping gene fragments. Most strains could be assigned to H. parahaemolyticus and were characterized by expression of IgA1 protease and a negative test for β-galactosidase. Isolation of H. parahaemolyticus from various infections and its absence among more than 300 commensal Haemophilus isolates suggests a pathogenic potential of this organism. The majority of haemolytic strains with β-galactosidase activity did not cluster with the type strain of H. paraphrohaemolyticus, but constituted a distinct and coherent novel taxon. Ten strains of this new taxon proved to be genetically and phenotypically homogeneous. Few biochemical characters discriminate the new taxon from related Haemophilus species, but identification is easily accomplished by routine matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Genetic, biochemical, and spectrometry data show that the taxon merits recognition as a novel species of Haemophilus. The name Haemophilus sputorum is proposed, with CCUG 13788(T) (=DSM 24472(T)=NCTC 13537(T)) as the type strain.     

Neisseria bacilliformis sp. nov. isolated from human infections

Most Neisseria species are gram-negative cocci or diplococci; currently, N. elongata is the only species of human origin with a bacillary morphology. Here, we report isolation and characterization of eight strains of another bacillary Neisseria species from human infections. The organisms caused or contributed to either oral cavity-related or respiratory tract infections, and two strains were isolated from blood cultures. The 16S rRNA gene sequences of these organisms, being homogenous or nearly so (99.4 to 100% identity), matched at <96% known Neisseria species and formed a distinct group within the genus. Analysis of the cellular fatty acids showed broad similarity with a few Neisseria species. The organisms were gram negative and measured 0.6 mum by 1.3 to 3.0 mum. They grew well on chocolate agar and on sheep blood agar but did not grow on modified Thayer-Martin agar. They were positive for oxidase and negative for indole production. There was no acid production from dextrose, lactose, maltose, or sucrose. The tests for catalase reaction, nitrate reduction, and tributilin varied with the strains. These results suggest that these organisms represent a novel species within the genus Neisseria, for which the name Neisseria bacilliformis sp. nov. is proposed. The type strain is MDA2833 = ATCC BAA-1200(T) = CCUG50858(T). Distinction between N. bacilliformis and N. elongata can be made confidently by 16S rRNA gene sequencing or cellular fatty acid profiling but may be difficult by morphology or routine biochemical tests.     

Prevotella massiliensis sp. nov. isolated from human blood

We report a bacterial isolate (Marseille isolate) recovered from the blood of a patient hospitalized in an intensive care unit, presenting with severe trauma, fever and mechanical ventilation. Colonies appeared at 37 degrees C on blood agar after 72 h incubation. This isolate was a strictly anaerobic, Gram-negative rod phenotypically related to other Prevotella species described to date: non-motile, catalase-negative, oxidase-positive, non-glucose fermenting, resistant to vancomycin and susceptible to kanamycin. Cells exhibited a trilamellar membrane under electron microscopy. The fatty acid methyl ester profile was marginally related to that of Clostridium botulinum group A (distance: 26.27%) and Bifidobacterium bifidum GC subgroup B (distance: 26.38%). 16S rRNA gene sequence similarity was 90.0% with that of Prevotella oris and 89.1% with that of Prevotella melaninogenica. Partial rpoB gene sequence similarity was 84.5 and 86.4% with P. oris and P. melaninogenica, respectively. According to current standards, phenotypic traits, 16S rRNA and rpoB gene sequence analyses indicated that the Marseille isolate belonged to a previously unrecognized species of the genus Prevotella, and we propose classifying it in the new taxon "Prevotella massiliensis" sp. nov.     

Trichophyton eboreum sp. nov. isolated from human skin

An unusual dermatophyte was isolated from the plantar scales of a human immunodeficiency virus-positive man with tinea pedis. Morphology, physiology, and molecular data provided evidence to support the new species Trichophyton eboreum. This dermatophyte is characterized by rapid growth on common mycological media, a flat powdery off-white colony, formation of clavate microconidia, smooth- and thin-walled cylindrical or club-shaped macroconidia with two to nine cells, the presence of hook-shaped hyphae, the production of cleistothecium-like structures and spiral hyphae in older cultures, positive hair perforation, the absence of pigmentation on potato glucose agar, the absence of a requirement for vitamins, a weak positive urease reaction, no growth at 37 degrees C, resistance to 5% NaCl, resistance to fluconazole, good growth on human epidermal keratin, and the production of various enzymes on different media by the API-ZYM test. More than 5% divergence from any known species of dermatophyte was revealed by sequence analysis of the internal transcribed spacer of the rRNA gene.     

Characterization of three new enterococcal species, Enterococcus sp. nov. CDC PNS-E1, Enterococcus sp. nov. CDC PNS-E2, and Enterococcus sp. nov. CDC PNS-E3, isolated from human clinical specimens

As a reference laboratory, the Streptococcus Laboratory at the Centers for Disease Control and Prevention (CDC) is frequently asked to confirm the identity of unusual or difficult-to-identify catalase-negative, gram-positive cocci. In order to accomplish the precise identification of these microorganisms, we have systematically applied analysis of whole-cell protein profiles (WCPP) and DNA-DNA reassociation experiments, in conjunction with conventional physiological tests. Using this approach, we recently focused on the characterization of three strains resembling the physiological groups I (strain SS-1730), II (strain SS-1729), and IV (strain SS-1728) of enterococcal species. Two strains were isolated from human blood, and one was isolated from human brain tissue. The results of physiological testing were not consistent enough to allow confident inclusion of the strains in any of the known enterococcal species. Resistance to vancomycin was detected in one of the strains (SS-1729). Analysis of WCPP showed unique profiles for each strain, which were not similar to the profiles of any previously described Enterococcus species. 16S ribosomal DNA (rDNA) sequencing results revealed three new taxa within the genus ENTEROCOCCUS: The results of DNA-DNA relatedness experiments were consistent with the results of WCPP analysis and 16S rDNA sequencing, since the percentages of homology with all 25 known species of Enterococcus were lower than 70%. Overall, the results indicate that these three strains constitute three new species of Enterococcus identified from human clinical sources, including one that harbors the vanA gene. The isolates were provisionally designated Enterococcus sp. nov. CDC Proposed New Species of Enterococcus 1 (CDC PNS-E1), type strain SS-1728(T) (= ATCC BAA-780(T) = CCUG 47860(T)); Enterococcus sp. nov. CDC PNS-E2, type strain SS-1729(T) (= ATCC BAA-781(T) = CCUG 47861(T)); and Enterococcus sp. nov. CDC PNS-E3, type strain SS-1730(T) (= ATCC BAA-782(T) = CCUG 47862(T)).     

Actinomyces cardiffensis sp. nov. from human clinical sources

Eight strains of a previously undescribed catalase-negative Actinomyces-like bacterium were recovered from human clinical specimens. The morphological and biochemical characteristics of the isolates were consistent with their assignment to the genus Actinomyces, but they did not appear to correspond to any recognized species. 16S rRNA gene sequence analysis showed the organisms represent a hitherto unknown species within the genus Actinomyces related to, albeit distinct from, a group of species which includes Actinomyces turicensis and close relatives. Based on biochemical and molecular genetic evidence, it is proposed that the unknown isolates from human clinical sources be classified as a new species, Actinomyces cardiffensis sp. nov. The type strain of Actinomyces cardiffensis is CCUG 44997(T).     

Nocardia mexicana sp. nov., a new pathogen isolated from human mycetomas

Three isolates collected from human mycetomas and showing an unusual brownish purple pigmentation on Bennett agar plates were analyzed by a polyphasic taxonomic approach, including morphological, biochemical, physiological, and chemotaxonomic properties coupled with genomic and phylogenetic analysis. It clearly appeared that these microorganisms were distinct from their closest phenotypic and genetic match, the most related species according to 16S rRNA gene sequence analysis being Nocardia pseudobrasiliensis. The data obtained indicated that the three clinical strains should be recognized as a new species for which the name Nocardia mexicana sp. nov. is proposed.     

Bartonella koehlerae sp. nov., isolated from cats

Two of the 25 Bartonella isolates recovered during a prevalence study of Bartonella henselae bacteremia in domestic cats from the greater San Francisco Bay region were found to differ phenotypically and genotypically from all prior B. henselae isolates. These isolates, C-29 and C-30, which were recovered from the blood of two pet cats belonging to the same household, grew on chocolate agar as pinpoint colonies following 14 days of incubation at 35 degrees C in a candle jar but failed to grow on heart infusion agar supplemented with 5% rabbit blood. Additional phenotypic characteristics distinguished the isolates C-29 and C-30 from other feline B. henselae isolates. The restriction patterns obtained for C-29 and C-30 by citrate synthase PCR-restriction fragment length polymorphism (RFLP) analysis as well as by genomic RFLP could not be distinguished from each other but were distinctly different from that of the B. henselae type strain. In reciprocal reactions, DNAs from strains C-29 and C-30 were 97 to 100% related under optimal and stringent DNA reassociation conditions, with 0 to 0.5% divergence within related sequences. Labeled DNA from the type strain of B. henselae was 61 to 65% related to unlabeled DNAs from strains C-29 and C-30 in 55 degrees C reactions, with 5.0 to 5.5% divergence within the related sequences, and 31 to 41% related in stringent, 70 degrees C reactions. In reciprocal reactions, labeled DNAs from strains C-29 and C-30 were 68 to 92% related to those of the B. henselae type strain and other B. henselae strains, with 5 to 7% divergence. The 16S rRNA gene sequence of strain C-29 was 99.54% homologous to that of the type strain of B. henselae. On the basis of these findings, the two isolates C-29 and C-30 are designated a new species of Bartonella, for which we propose the name Bartonella koehlerae. The type strain of Bartonella koehlerae is strain C-29 (ATCC 700693).     

First description of Curtobacterium spp. isolated from human clinical specimens

During a 4-year period, five strains (three of which were doubtless clinically significant) of yellow- or orange-pigmented, oxidative, slowly acid-producing coryneform bacteria were recovered from human clinical specimens in two reference laboratories or referred to them. The strains were motile, catalase positive, nitrate reductase negative, and urease negative, but strongly hydrolyzed esculin. In all reference and clinical strains described in the present study, anteisopentadecanoic (C(15:0ai)) and anteisoheptadecanoic (C(17:0ai)) acids represented more than 75% of all cellular fatty acids except in one clinical strain and in Curtobacterium pusillum, in which both the unusual omega-cyclohexyl fatty acid (identified as C(18:1omega7cis/omega9cis/omega12trans) by the Sherlock system) represented more than 50% of all cellular fatty acids. In all clinical strains, ornithine was the diamino acid of the cell wall, the interpeptide bridge consisted of ornithine, and acetyl was the acyl type of the peptidoglycan. Therefore, the five clinical strains were unambiguously identified as Curtobacterium spp. Analyses of the complete 16S rRNA genes of the five clinical strains with homologies to the established Curtobacterium species ranging from 99.2 to 100% confirmed the identifications as Curtobacterium spp. Data on the antimicrobial susceptibility pattern of curtobacteria are reported, with macrolides and rifampin showing very low MICs for all strains tested. This report is the first on the isolation of Curtobacterium strains from human clinical specimens.     

Legionella gratiana sp. nov. isolated from French spa water

During an epidemiologic survey, an unidentified strain of Legionella was isolated from water of a thermal spa in France. The strain (Lyon 8420412) had the cultural and biochemical characteristics typical of the genus Legionella. In direct immunofluorescence tests, the strain reacted weakly with fluorescein-conjugated antisera prepared against L. bozemanii serogroups 1 and 2, L. longbeachae serogroups 1 and 2 and L. anisa, and failed to react with sera prepared against 36 other species or serogroups. A fluorescein-conjugated antiserum prepared against strain Lyon 8420412 reacted strongly with the homologous strain and only weakly with the above-mentioned species. The cell-wall fatty acid profile, with a predominance of hexadecenoic (16:1) and hexadecanoic (16:0) acids, ubiquinone Q10 as the major quinone and a characteristic protein electrophoresis profile suggested that the isolate was different from other Legionella species. In DNA-DNA hybridization experiments, the strain was distinct from all named Legionella species, and from all unnamed species currently under study at the Centers for Disease Control. The name Legionella gratiana is proposed for the new species (type strain Lyon 8420412; CDC 1242). A serologic survey of antibodies reacting against L. gratiana indicated that personnel or patients at the spa therapy centre where the organism was isolated had higher antibody titres than a control population.