DNA-DNA hybridization study of strains of Chryseobacterium, Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc, IIe, IIh and IIi, mostly from human clinical sources, and proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov.

The taxonomic classification of 182 phenotypically similar isolates was evaluated using DNA-DNA hybridization and 16S rRNA gene sequence analysis. These bacterial isolates were mainly derived from clinical sources; all were Gram-negative non-fermenters and most were indole-producing. Phenotypically, they resembled species from the genera Chryseobacterium, Elizabethkingia or Empedobacter or belonged to CDC groups IIc, IIe, IIh and IIi. Based on these analyses, four novel species are described: Chryseobacterium bernardetii sp. nov. (type strain NCTC 13530(T) = CCUG 60564(T) = CDC G229(T)), Chryseobacterium carnis sp. nov. (type strain NCTC 13525(T) = CCUG 60559(T) = CDC G81(T)), Chryseobacterium lactis sp. nov. (type strain NCTC 11390(T) = CCUG 60566(T) = CDC KC1864(T)) and Chryseobacterium nakagawai sp. nov. (type strain NCTC 13529(T) = CCUG 60563(T) = CDC G41(T)). The new combination Chryseobacterium taklimakanense comb. nov. (type strain NCTC 13490(T) = X-65(T) = CCTCC AB 208154(T) = NRRL B-51322(T)) is also proposed to accommodate the reclassified Planobacterium taklimakanense.

Characterization of human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi.

In this study, 16 human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi were evaluated using phenotypic methods, including traditional and commercial (API Coryne) biochemical tests, antimicrobial susceptibility testing, and 16S rRNA gene and gyrB gene sequencing. Positive results for both the hydrolysis of adenine and Christie-Atkins-Munch-Petersen (CAMP) reaction allowed for differentiation between the Dietzia isolates and the type strain of Rhodococcus equi; however, traditional and commercial phenotypic profiles could not be used to reliably identify Dietzia species. The analysis of 16S rRNA gene and gyrB gene sequences could discriminate all Dietzia strains from the type strain of R. equi. Most Dietzia species had distinct 16S rRNA gene and gyrB gene sequences; however, the 16S rRNA gene sequences of the type strains of D. schimae and D. cercidiphylli were identical to D. maris and D. natronolimnaea, respectively. Based on comparative sequence analysis, five clinical isolates clustered with D. maris/D. schimae and nine with D. natronolimnaea/D. cercidiphylli. The two remaining isolates were found to be most closely related to the D. cinnamea/D. papillomatosis clade. Even though molecular analyses were not sufficiently discriminative to accurately identify all Dietzia species, the method was able to reliably identify isolates that were previously misidentified by phenotypic methods to the genus level.

Aerococcus urinae: polyphasic characterization of the species.

A polyphasic characterization of Aerococcus urinae is presented. In this study the intraspecies relationships between 26 strains of varying geographical origin were examined by phenotypic tests, ribotyping and multilocus enzyme electrophoresis. The results demonstrated two main phenotypic patterns that could be distinguished in tests for hydrolysis of aesculin, and acid production from amygdalin and salicin. Strains were either negative (n=19) or positive (n=6) in these tests. One strain had a deviating pattern. Heterogeneity within the 19 pattern I strains was demonstrated especially by phenotypic tests (acid production from ribose, mannitol, sorbitol, sucrose and D-arabitol) and by multilocus enzyme electrophoresis. However, DNA sequence analysis of the 16S rRNA (n=7) and gyrB genes (n=3) from strains representing the two main patterns showed no variation in sequences among strains. Comparison of A. urinae and representatives of related taxa by 16S rDNA sequence analysis showed that the taxon is related to, but distinct from, other Aerococcus spp.

Reclassification of Leptospira parva Hovind-Hougen et al. 1982 as Turneriella parva gen. nov., comb. nov.

Analysis of the G+C content, DNA-DNA relatedness to other leptospires and 16S rRNA gene sequence of Leptospira parva showed that this species was not related to other Leptospira species. On the basis of these data, it is proposed that Leptospira parva should be transferred to the genus Turneriella as Turneriella parva gen. nov., comb. nov., with strain H(T) (=NCTC 11395(T)=ATCC BAA-1111(T)) as the type strain.

Genotyping of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease by Repetitive Elements-Based Polymerase Chain Reaction and DNA-DNA Hybridization.

ABSTRACT The bacterium that causes cucurbit yellow vine disease (CYVD) has been placed in the species Serratia marcescens based on 16S rDNA and groE sequence analysis. However, phenotypic comparison of the organism with S. marcescens strains isolated from a variety of ecological niches showed significant heterogeneity. In this study, we compared the genomic DNA of S. marcescens strains from different niches as well as type strains of other Serratia spp. through repetitive elements-based polymerase chain reaction (rep-PCR) and DNA-DNA hybridization. With the former, CYVD strains showed identical banding patterns despite the fact that they were from different cucurbit hosts, geographic locations, and years of isolation. In the phylogenetic trees generated from rep-PCR banding patterns, CYVD strains clearly were differentiated from other strains but formed a loosely related group with S. marcescens strains from other niches. The homogeneity of CYVD strains was supported further by the DNA relatedness study, in that labeled DNA from the cantaloupe isolate, C01-A, showed an average relative binding ratio (RBR) of 99%, and 0.33% divergence to other CYVD strains. Used as a representative strain of CYVD, the labeled C01-A had a RBR of 76%, and a 4.5% divergence to the S. marcescens type strain. These data confirm the previous placement of CYVD strains in S. marcescens. Our investigations, including rep-PCR, DNA-DNA hybridization, and previous phenotyping experiments, have demonstrated that CYVD-associated strains of S. marcescens cluster together in a group significantly different from other strains of the species.

DNA relatedness, phenotypic characteristics, and antimicrobial susceptibilities of Globicatella sanguinis strains.

DNA-DNA reassociation was performed on 15 strains of Globicatella sanguinis to compare their taxonomic status with phenotypic characterization. All 15 strains selected for DNA-DNA reassociation readily met the criteria for species relatedness. The relative binding ratio was 81% or greater at the optimal temperature and 76% or greater at the stringent temperature, and the divergence was less than 3% for all strains hybridized with the type strain. These strains included nine strains from the Centers for Disease Control Streptococcus Laboratory culture collection that were previously included in comparative 16S rRNA gene sequencing studies as well as six additional phenotypically variant isolates. DNA-DNA relatedness was less than 18% at the optimal reassociation temperature to Aerococcus viridans, Enterococcus avium, and Streptococcus uberis, which are phenotypically similar to G. sanguinis. This study confirms these Globicatella strains were previously misidentified as S. uberis or S. uberis-like strains based on biochemical characteristics. The biochemical data from 28 strains was compiled to further define the phenotypic criteria for identification of this species. A revised description of the species should be variable reaction for pyrrolidonylarylamidase production (75% positive), positive reaction for the bile esculin test (100%), growth at 45 degrees C (96%), variable reaction for acid production from arabinose (45% positive), and negative starch hydrolysis (0% positive). We also evaluated four rapid identification systems, the Biomerieux rapid ID32 STREP (ID32), the Crystal rapid gram-positive identification (Cry4), the BBL Crystal gram-positive identification (Cry24), and the Remel IDS RapID STR (IDS) systems for their ability to identify these strains.

Enterococcus porcinus sp. nov. and Enterococcus ratti sp. nov., associated with enteric disorders in animals.

Recent insights have been brought to the taxonomy of the genus Enterococcus by studies applying whole-cell protein analysis and DNA-DNA reassociation experiments, in addition to conventional physiological tests. Using these techniques, a group of 10 strains resembling the physiological group III of enterococcal species was characterized. Five strains were recovered from pigs and five from rats with enteric disorders. On the basis of the results of conventional physiological tests, the most likely identity of these strains was Enterococcus durans or Enterococcus hirae. Analysis of the electrophoretic whole-cell protein profiles showed two distinct clusters of virtually indistinguishable profiles: one composed of strains isolated from pigs, and one composed of strains isolated from rats. These protein profiles were not similar to the profiles of any previously described Enterococcus species. The results of DNA-DNA relatedness experiments were consistent with the results of the protein-profile analysis. The high levels of DNA relatedness found for pig isolates demonstrated that they belong to a new enterococcal species, for which the designation Enterococcus porcinus sp. nov. is proposed (type strain =DS 1390-83T =ATCC 700913T =CCUG 43229T =NCIMB 13634T). Strains isolated from rats were found to comprise another new species, for which the designation Enterococcus ratti sp. nov. is proposed (type strain =DS 2705-87T =ATCC 700914T =CCUG 43228T =NCIMB 13635T). This report provides data on the phenotypic and genotypic characterization of these two new enterococcal species, which may represent diarrhoeagenic pathogens for animals.

Assignment of CDC weak oxidizer group 2 (WO-2) to the genus Pandoraea and characterization of three new Pandoraea genomospecies.

CDC weak oxidizer group 2 (WO-2) consists of nine phenotypically similar human clinical isolates received by the Centers for Disease Control and Prevention between 1989 and 1998. Four of the isolates were from blood, three were from sputum, and one each was from bronchial fluid and maxillary sinus. All are aerobic nonfermentative, motile gram-negative rods with one to eight polar flagella per cell. All grew at 25 and 35 degrees C and were positive for catalase, urease (usually delayed 3 to 7 days), citrate, alkalinization of litmus milk, oxidization of glycerol (weakly), and growth on MacConkey agar and in nutrient broth without NaCl. All except one strain were oxidase positive with the Kovács method, and all except one isolate weakly oxidized D-glucose. All were negative for oxidation of D-xylose, D-mannitol, lactose, sucrose, maltose, and 20 other carbohydrates, esculin hydrolysis, indole production, arginine dihydrolase, and lysine and ornithine decarboxylase. Only two of nine isolates reduced nitrate. Broth microdilution susceptibilities were determined for all strains against 13 antimicrobial agents. Most of the strains were resistant to ampicillin, extended-spectrum cephalosporins, and aminoglycosides, including gentamicin, tobramycin, and amikacin, but they varied in their susceptibility to fluoroquinolones. High-performance liquid chromatographic and mass spectrometric analyses of the WO-2 group identified ubiquinone-8 as the major quinone component. The percent G+C of the WO-2 strains ranged from 65.2 to 70.7% (thermal denaturation method). All shared a common cellular fatty acid (CFA) profile, which was characterized by relatively large amounts (7 to 22%) of 16:1omega7c, 16:0, 17:0cyc, 18:1omega7c, and 19:0cyc(11-12); small amounts (1 to 3%) of 12:0 and 14:0; and eight hydroxy acids, 2-OH-12:0 (4%), 2-OH-14:0 (trace), 3-OH-14:0 (12%), 2-OH-16:1 (1%), 2-OH-16:0 (3%), 3-OH-16:0 (4%), 2-OH-18:1 (2%), and 2-OH-19:0cyc (3%). This profile is similar to the CFA profile of Pandoraea, a recently described genus associated with respiratory infections in cystic fibrosis patients (T. Coenye et al., Int. J. Syst. Evol. Microbiol., 50:887-899, 2000). Sequencing of the 16S rRNA gene (1,300 bp) for all nine strains indicated a high level (> or =98.8%) of homogeneity with Pandoraea spp. type strains. DNA-DNA hybridization analysis (hydroxyapatite method; 70 degrees C) confirmed the identity of WO-2 with the genus Pandoraea and assigned three strains to Pandoraea apista and three to Pandoraea pnomenusa, and identified three additional new genomospecies containing one strain each (ATCC BAA-108, ATCC BAA-109, ATCC BAA-110). This study also shows that Pandoraea isolates may be encountered in blood cultures from patients without cystic fibrosis.

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France.

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.

Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C. rodentium and mouse-pathogenic Escherichia coli.

Citrobacter rodentium (formerly Citrobacter freundii biotype 4280 and Citrobacter genomospecies 9) was described on the basis of biochemical characterization and DNA-DNA hybridization data and is the only Citrobacter species known to possess virulence factors homologous to those of the human pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli. These virulence factors are encoded on the locus of enterocyte effacement (LEE), a pathogenicity island required for the characteristic attaching and effacing (AE) pathology seen in infection with these three pathogens. C. rodentium, which apparently infects only mice, provides a useful animal model for studying the molecular basis of AE pathology. No work has been done to assess differences in pathogenicity between C. rodentium isolates from diverse sources. Here, we report the examination of 15 C. rodentium isolates using a battery of genetic and biochemical approaches. No differences were observed between the isolates by repetitive-element sequence-based PCR analysis, biochemical analysis, and possession of LEE-specific virulence factors. These data suggest that members of the species are clonal. We further characterized an atypical E. coli strain from Japan called mouse-pathogenic E. coli (MPEC) that, in our hands, caused the same disease as C. rodentium. Applying the same battery of tests, we found that MPEC possesses LEE-encoded virulence factors and is indistinguishable from the previously characterized C. rodentium isolate DBS100. These results demonstrate that MPEC is a misclassified C. rodentium isolate and that members of this species are clonal and represent the only known attaching and effacing bacterial pathogen of mice.

Classification of Proteus vulgaris biogroup 3 with recognition of Proteus hauseri sp. nov., nom. rev. and unnamed Proteus genomospecies 4, 5 and 6.

Strains traditionally identified as Proteus vulgaris formed three biogroups. Biogroup 1, characterized by negative reactions for indole production, salicin fermentation and aesculin hydrolysis, is now known as Proteus penneri. Biogroup 2, characterized by positive reactions for indole, salicin and aesculin, was shown by DNA hybridization (hydroxyapatite method) to be a genetic species separate from biogroup 1 and from biogroup 3 which is positive for indole production and negative for salicin and aesculin. In this study, 52 strains were examined, of which 36 strains were Proteus vulgaris biogroup 3, which included the current type strain of the species P. vulgaris (ATCC 29905T), and compared to seven strains of Proteus vulgaris biogroup 2 and nine type strains of other species in the genera Proteus, Providencia and Morganella. By DNA hybridization, these 36 strains were separated into four distinct groups, designated as Proteus genomospecies 3, 4, 5 and 6. DNAs within each separate Proteus genomospecies were 74-99% related to each other in 60 degrees C hybridization reactions with < or = 4.5% divergence between related sequences. Proteus genomospecies 3 contained the former P. vulgaris type strain and one other strain and was negative in reactions for salicin fermentation, aesculin hydrolysis and deoxyribonuclease, unlike the reactions associated with strains considered as typical P. vulgaris which are positive in reactions for salicin, aesculin and DNase. Genomospecies 3 can be distinguished from Proteus genomospecies 4, 5 and 6 because it is negative for Jordan's tartrate. Proteus genomospecies 4, containing five strains, was differentiated from Proteus penneri, genomospecies 3 and 6 and most, but not all, strains of genomospecies 5, by its ability to ferment L-rhamnose. Proteus genomospecies 5 and 6, containing 18 and 11 strains, respectively, could not be separated from each other by traditional biochemical tests, by carbon source utilization tests or SDS-PAGE of whole-cell proteins. In an earlier publication, a request was made to the Judicial Commission that the former type strain of P. vulgaris (ATCC 13315) be replaced by P. vulgaris biogroup 2 strain ATCC 29905T, a strain considered more biochemically typical of P. vulgaris strains. This would have the effect of assigning the name P. vulgaris to P. vulgaris biogroup 2. Since this request has been acceded to, the name Proteus hauseri is herein proposed for Proteus vulgaris genomospecies 3. Its type strain is ATCC 700826T. Proteus genomospecies 4, 5 and 6 will remain unnamed until better phenotypic differentiation can be accomplished. All Proteus genomospecies were similar in their antimicrobial susceptibility patterns. Nineteen strains were isolated from urine, four from faeces, two from wounds, nine from other human sources and two from animals.

Phenotypic and genotypic characterization of Vibrio viscosus sp. nov. and Vibrio wodanis sp. nov. isolated from Atlantic salmon (Salmo salar) with 'winter ulcer'.

Two groups of Vibrio strains isolated from Atlantic salmon with 'winter ulcer' were characterized phenotypically and genotypically. The data obtained indicated that each of the two groups represented a new species in the genus Vibrio. The names Vibrio viscosus sp. nov. [type strain NVI 88/478T (= NCIMB 13584T)] and Vibrio wodanis sp. nov. [type strain NVI 88/441T (= NCIMB 13582T)] are proposed for the new species. V. viscosus strains exhibited a similar total DNA RFLP pattern and a similar plasmid DNA profile. DNA relatedness (hydroxyapatite method) of the V. viscosus type strain to nine other V. viscosus strains was 81-93% at 60 degrees C. Divergence within related sequences was 0.0-1.5% and relatedness at 75 degrees C was 74-100%. V. wodanis strains exhibited marked heterogeneity on the basis of RFLP analysis and plasmid profiles. DNA relatedness of the V. wodanis type strain to 10 other V. wodanis strains was 66-94% at 60 degrees C. Divergence within related sequences was 0.0-1.5% and relatedness at 75 degrees C was 55-97%. Relatedness between V. viscosus and V. wodanis type strains was approximately 20%. Among other Vibrio species, the closest relative of V. viscosus was Vibrio marinus (ATCC 15381T) (43% relatedness at 60 degrees C) and that of V. wodanis was Vibrio logei (ATCC 15382) (57% relatedness at 60 degrees C). These same pairs were the closest phenotypic relatives. DNA sequence analysis of the 16S rRNA gene of V. viscosus indicated an intimate relationship to V. marinus. A total evaluation of the results, however, supports V. viscosus to be a separate species in the genus Vibrio. The analysis of the sequence of the 16S rRNA gene of V. wodanis supports that V. logei (ATCC 15382) was the most related species. Ability to degrade casein, oxidative production of acid from trehalose and production of lysine decarboxylase are important biochemical tests that will differentiate between V. viscosus, V. wodanis, V. marinus (ATCC 15381T) and V. logei (ATCC 15382).

Mycobacterium septicum sp. nov., a new rapidly growing species associated with catheter-related bacteraemia.

Rapidly growing mycobacteria are capable of causing several clinical diseases in both immunosuppressed and immunocompetent individuals. A previously unidentified, rapidly growing mycobacterium was determined to be the causative agent of central line sepsis in a child with underlying metastatic hepatoblastoma. Four isolates of this mycobacterium, three from blood and one from the central venous catheter tip, were studied. Phenotypic characterization, HPLC and genetic analysis revealed that while this organism most closely resembled members of the Mycobacterium fortuitum complex and Mycobacterium senegalense, it differed from all previously described species. Phenotypic tests useful in differentiating this species from similar rapidly growing mycobacteria included: growth at 42 degrees C, hydrolysis of acetamide, utilization of citrate, production of arylsulfatase (3-d), acidification of D-mannitol and i-myo-inositol, and susceptibility to erythromycin, vancomycin and tobramycin. The name Mycobacterium septicum is proposed for this new species. The type strain has been deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen as DSM 44393T and in the American Type Culture Collection as strain ATCC 700731T.

Specific primer for AP-PCR identification of Actinobacillus actinomycetemcomitans.

We used arbitrarily-primed polymerase chain reaction (AP-PCR) to design and construct a specific primer pair for the identification of Actinobacillus actinomycetemcomitans. We analyzed 25 DNA samples of A. actinomycetemcomitans isolated from patients with localized-juvenile periodontitis. From 90 AP-PCR primers screened, one amplification product was selected, cloned in pCR II vector, and sequenced. The sequence was used to design a single pair of specific primers. The sequence was compared with GenBank entries using BLAST and showed no significant matches. PCR amplification using the new primer pair AA1416 produced a characteristic 3.5-Kb band in all A. actinomycetemcomitans DNAs tested. Primer pair AA16S produced no or different amplicon profiles using DNA samples from bacterial species other than A. actinomycetemcomitans. Our results show that this single primer pair AA1416 can be used in PCR to identify A. actinomycetemcomitans isolates and differentiate them from other periodontal bacteria. These approaches appear promising in facilitating laboratory identification and taxonomy of putative periodontopathogens.

Arbitrarily primed-polymerase chain reaction for identification and epidemiologic subtyping of oral isolates of Fusobacterium nucleatum.

BACKGROUND: Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F. nucleatum. Subtypes may differ in virulence and, hence, are important as periodontal pathogens. Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment. METHODS: We analyzed 70 DNA samples of F. nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30). From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced. These sequences were used to design new pairs of specific primers. Sequences were compared to GenBank entries with BLAST and showed no significant matches. RESULTS: Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nucleatum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls. Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001). All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F. nucleatum. CONCLUSIONS: Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F. nucleatum isolates and distinguish them from other bacteria. The primer pair M8171 could also be used to differentiate F. nucleatum isolated from periodontal patients or healthy individuals. These specific primers can be used in PCR analysis for specific identification of F. nucleatum and to distinguish it from other bacteria associated with human periodontitis. These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens.

Isolation of Providencia heimbachae from human feces.

Providencia heimbachae was first described in 1986. It has been isolated from penguin feces and an aborted bovine fetus. To date, there has been no reported isolation of this organism from human specimens. We now report the isolation of P. heimbachae from the stool of a 23-year-old woman with idiopathic diarrhea. The identity of the human strain was determined biochemically and by DNA relatedness to the type strain of P. heimbachae.

Isolation of a new subspecies, Bartonella vinsonii subsp. arupensis, from a cattle rancher: identity with isolates found in conjunction with Borrelia burgdorferi and Babesia microti among naturally infected mice.

Bacteremia with fever due to a novel subspecies of Bartonella vinsonii was found in a cattle rancher. The subspecies shared major characteristics of the genus Bartonella in terms of most biochemical features and cellular fatty acid profile, but it was distinguishable from other subspecies of B. vinsonii by good growth on heart infusion agar supplemented with X factor and by its pattern of enzymatic hydrolysis of peptide substrates. DNA relatedness studies verified that the isolate belonged to the genus Bartonella and that it was genotypically related to B. vinsonii. The highest level of relatedness was observed with recently characterized strains from naturally infected mice that were coinfected with Borrelia burgdorferi and Babesia microti. We propose the name Bartonella vinsonii subsp. arupensis subsp. nov. as the new subspecies to accommodate these human and murine isolates.

Biochemical identification of Citrobacter species defined by DNA hybridization and description of Citrobacter gillenii sp. nov. (formerly Citrobacter genomospecies 10) and Citrobacter murliniae sp. nov. (formerly Citrobacter genomospecies 11).

Recent work describing six named species and two unnamed genomospecies within Citrobacter has enlarged the genus to 11 species. DNA relatedness and phenotypic tests were used to determine how well these species can be identified. One hundred thirty-six strains were identified to species level by DNA relatedness and then identified phenotypically in a blinded fashion. By using conventional tests, 119 of the 136 strains (88%) were correctly identified to species level. Three additional strains (2%) were identified as citrobacteria but were not identified to species level, and 14 strains (10%) were misidentified as other Citrobacter species. Carbon source utilization tests were used to identify 86 of the strains. Eighty-four strains (98%) were correctly identified, and two strains (2%) were misidentified as other Citrobacter species. Additional strains of Citrobacter genomospecies 10 and Citrobacter genomospecies 11 were identified, allowing these species to be formally named as Citrobacter gillenii sp. nov. and Citrobacter murliniae sp. nov., respectively.

DNA relatedness of Leptospira strains isolated from beef cattle in Zimbabwe.

The DNA relatedness of 17 Leptospira strains isolated from beef cattle in Zimbabwe was determined using the hydroxyapatite method. Similarly to previously speciated African strains, all Zimbabwe isolates belonged to either Leptospira borgpetersenii or Leptospira kirschneri. All serovars within serogroups Pyrogenes (kwale, mombe and a strain closely related to serovar nigeria), Hebdomadis (marondera and mhou), Tarassovi (ngavi) and Sejroe (balcanica and hardjo) were L. borgpetersenii. L. kirschneri contained all strains in serovars of serogroups Icterohaemorrhagiae (zimbabwe), Australis (fugis), Bataviae (paidjan) and Pomona (a strain closely related to mozdok). The species designations of the Zimbabwe fugis and paidjan strains were different from those of the reference strains of these two serovars, both of which belong to Leptospira interrogans.

Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies.

DNA relatedness was determined among 303 strains of Leptospira and Leptonema. Included in the analysis were reference strains from 228 well-characterized and recognized serovars. The study included 268 serovars from 29 named and one or more unnamed serogroups. The strains clustered into 17 DNA hybridization groups, representing 12 previously described species (292 strains) and five new genomospecies (11 strains). The largest groups included Leptospira interrogans (91 strains from 82 serovars), Leptospira santarosai (65 strains from 59 serovars), Leptospira borgpetersenii (49 strains from 43 serovars), Leptospira kirschneri (29 strains from 26 serovars) and Leptospira noguchii (20 strains from 20 serovars). The new genomospecies include Leptospira genomospecies 1 (two strains, serovars pinagchang and sichuan), Leptospira genomospecies 2 (six strains, serovars lushui, manhao 3, manzhuang, nanding, mengla and yunnan), Leptospira genomospecies 3 (one strain, serovar holland), Leptospira genomospecies 4 (one strain, serovar hualin) and Leptospira genomospecies 5 (one strain, serovar saopaulo). With the exception of Ballum, all serogroups with greater than one serovar studied were genetically heterogeneous. Phenotypic tests, including optimal growth temperature, lipase activity and growth inhibition by copper sulfate or 2,6-diaminopurine, were of little use in differentiating DNA relatedness groups. The name Leptospira alexanderi sp. nov. is proposed for Leptospira genomospecies 2 (type strain L 60T = ATCC 700520T, serovar manhao 3).