Guido Funke

Use of the BD PHOENIX Automated Microbiology System for Direct Identification and Susceptibility Testing of Gram-Negative Rods from Positive Blood Cultures in a Three-Phase Trial

ABSTRACT The present study describes the use of the automated BACTEC 9240 blood culture system, the Serum Separator Tube (SST), and the BD PHOENIX Automated Microbiology System in combination for the direct identification and antimicrobial susceptibility testing (AST) of gram-negative rods (GNRs) from positive blood cultures (BCs) without subculture. The study was conducted in three phases: (i) the recovery yield of Escherichia coli ATCC 25922 was determined with the SST between 0 and 8 h after spiked BC bottles turned positive; (ii) the identifications and susceptibility testing results obtained with the PHOENIX system for nine American Type Culture Collection strains of GNRs processed by the SST procedure and for colonies from agar medium were compared; and (iii) the procedure with the BACTEC system, SSTs, and the PHOENIX system was applied to positive cultures of blood from 309 patients during a 3-month period. The SST procedure with E. coli yielded sufficient numbers of cells to perform direct inoculation at any time between 0 and 8 h after a BC bottle turned positive. By using the identities obtained from pure cultures with the PHOENIX system and other biochemical identification systems as reference methods, the agreement between the reference methods and the PHOENIX system tested directly by using cultures of blood from patients was 92.9%. The 7.1% discrepant results were due to 6.5% incorrect identifications with the PHOENIX system with BC samples and 0.6% incorrect identifications with the PHOENIX system with samples from agar cultures. By AST the overall categorical accuracy was 99.0%, with 0.1% very major errors, 0.1% major errors, and 0.8% minor errors. In conclusion, use of the combination of the BACTEC system, SSTs, and the PHOENIX system has the potential to allow the agar isolation step to be skipped and the procedures for rapid direct identification and susceptibility testing of GNRs from positive BCs to be improved both in hospital-based and in central non-hospital-based laboratories.

Actinomyces europaeus sp. nov., isolated from human clinical specimens.

Ten strains of a hitherto undescribed catalase-negative, facultatively anaerobic, coryneform bacterium were isolated or collected by workers at three European clinical bacteriology laboratories or reference centers. These strains were isolated from humans, and most came from abscess material. Biochemical and chemotaxonomic characterization revealed that the strains belonged to the genus Actinomyces. The phenotypic features of the 10 strains were incompatible with the descriptions of the previously established Actinomyces species. A comparative 16S rRNA gene sequence analysis demonstrated that the previously undescribed strains constitute a new line in the genus Actinomyces. The name Actinomyces europaeus sp. nov. is proposed for these clinical isolates. The type strain is CCUG 32789A.

Turicella otitidis gen. nov., sp. nov., a Coryneform Bacterium Isolated from Patients with Otitis Media

The partial 16S rRNA gene sequences of some coryneform bacteria isolated from middle-ear fluids from patients with otitis media were determined. A comparative analysis performed with rRNA sequences of other high-G + C-content gram-positive bacteria showed that the coryneform bacteria isolated from otitis media patients were only remotely related to members of previously described genera. On the basis of the results of the phylogenetic analysis and previously published biochemical and chemotaxonomic data, we propose that the strains isolated from middle-ear infections should be classified in a new genus, Turicella, as Turicella otitidis gen. nov., sp. nov. The type strain of T. otitidis is strain DSM 8821.

Assignment of Actinomyces pyogenes-like (CDC coryneform group E) bacteria to the genus Actinomyces as Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov.

In a previous study the authors reported the characterization of some facultatively anaerobic, Gram‐positive, non‐sporeforming rods which were found in mixed cultures from various infectious processes, including patients with otitis, empyema, perianal abscesses and decubitus ulcers. Phenotypically these organisms closely resembled Actinomyces pyogenes although their precise taxonomic position remained unknown. In the present investigation the authors have determined the 16S rRNA gene sequences of some representative strains of the Actinomyces pyogenes‐like bacteria and report the results of a comparative sequence analysis. On the basis of the results of the present and earlier findings two new Actinomyces species, Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. are proposed. The type strains are DSM 9169T and DSM 9168T, respectively.

Identities of Microbacterium spp. Encountered in Human Clinical Specimens

ABSTRACT In the present study, 50 strains of yellow-pigmented gram-positive rods that had been isolated from human clinical specimens and collected over a 5-year period were further characterized by phenotypic and molecular genetic methods. All 50 strains belonged to the genus Microbacterium, and together they represented 18 different species. Microbacterium oxydans (n = 11), M. paraoxydans (n = 9), and M. foliorum (n = 7) represented more than half of the strains included in the present study. The isolation of strains belonging to M. hydrocarbonoxydans (n = 2), M. esteraromaticum (n = 1), M. oleivorans (n = 1), M. phyllosphaerae (n = 1), and M. thalassium (n = 1) from humans is reported for the first time. Microbacterium sp. strain VKM Ac-1389 (n = 1) and the previously uncultured Microbacterium sp. clone YJQ-29 (n = 1) probably represent new species. Comprehensive antimicrobial susceptibility data are given for the 50 Microbacterium isolates. This study is, so far, the largest on Microbacterium spp. encountered in human clinical specimens and outlines the heterogeneity of clinical Microbacterium strains.

Description of human-derived Centers for Disease Control coryneform group 2 bacteria as Actinomyces bernardiae sp. nov.

Biochemical, chemotaxonomic, and molecular methods were used to establish the precise taxonomic position of the Centers for Disease Control (CDC) coryneform group 2 bacteria. The results of a comparative 16S rRNA sequence analysis demonstrated that the CDC coryneform group 2 bacteria constitute a distinct species within the genus Actinomyces. Actinomyces pyogenes was found to be the closest genealogical relative of the CDC coryneform group 2 bacteria, although these taxa were readily distinguished from each other and other Actinomyces spp. by using phenotypic criteria. On the basis of our findings we propose the name Actinomyces bernardiae sp. nov. for the CDC coryneform group 2 bacteria. The type strain is DSM 9152 (CCUG 33419).

Heterogeneity within human-derived centers for disease control and prevention (CDC) coryneform group ANF-1-like bacteria and description of Corynebacterium auris sp. nov.

Recently, Centers for Disease Control and Prevention coryneform group ANF-1 bacteria were described as Corynebacterium afermentans, and group ANF-1-like bacteria were described as Turicella otitidis. Over a 1.5-year period 10 strains of a previously undescribed, gram-positive, rod-shaped organism that was not partially acid fast and resembled ANF-1-like bacteria were isolated from different pediatric patients with ear infections. These previously undescribed coryneform bacteria exhibited a distinct colony morphology and consistency, had a carbon source utilization pattern distinct from the carbon source utilization patterns of C. afermentans and T. otitidis, had a cell wall based on meso-diaminopimelic acid, contained mycolic acids, and had DNA G+C contents of 68 to 74 mol%. A 16S rRNA gene sequence analysis revealed that these clinical isolates are members of the genus Corynbacterium and that they are distinct from C. afermentans and T. otitidis. On the basis of phenotypic and phylogenetic evidence we propose a new species, Corynebacterium auris, for these Centers for Disease Control and Prevention coryneform group ANF-1-like bacteria. The type strain is strain DSM 44122 (CCUG 33426).

Evaluation of the new VITEK 2 card for identification of clinically relevant gram-negative rods

ABSTRACT The VITEK 2 card for gram-negative bacteria (bioMérieux,Marcy-l′Etoile, France) has been redesigned to improve the identification of fermenting and nonfermenting bacilli. Forty-seven biochemical tests, including 19 enzymatic tests, are present in the new card and interpreted in a kinetic mode. Final identification results are available within 10 h. The database allows the identification of 159 different taxa. Six hundred fifty-five gram-negative rods (GNR; 511 fermenters and 144 nonfermenters), representing 54 taxa, were tested. Strains were taken from fresh routine primary isolation plates (n = 157), from stored routine plates (n = 301), and from stock cultures (n = 197). Six hundred thirty-seven strains (97.3%) were correctly identified to the species level, 14 strains (2.1%) gave low discrimination results requiring additional tests, and 4 strains (0.6%) gave discordant results; not a single strain remained unidentified. Nearly 92% of all isolates were correctly identified within 7 h of incubation. The robustness of the system was demonstrated by the fact that strains were grown on four different agar media before testing. The system may also have the potential to be applied directly to primary isolation plates, since in this instance 96.2% of 157 GNR were correctly identified and 3.8% gave low discrimination results. The new VITEK 2 card for gram-negative bacteria seems to be a promising new tool for routine, rapid identification of GNR.

First Comprehensively Documented Case of Paracoccus yeei Infection in a Human

ABSTRACT Paracoccus yeei was isolated in pure culture from an aerobic blood culture and bulla fluid from a 67-year-old male. The biochemical identification scheme for this recently described species is outlined. Because of its reaction pattern it is not unlikely that P. yeei is underdiagnosed.

Corynebacterium mucifaciens sp. nov., an Unusual Species from Human Clinical Material

Eight strains of a previously undescribed coryneform bacterium had been isolated from human clinical material over a 5-year period. Colonies of the unknown coryneform bacterium had an unusual appearance as they were slightly yellowish and very mucoid. Biochemical and chemotaxonomic characterization revealed that the unknown coryneform bacterium belonged to the genus Corynebacterium. It could be readily differentiated from all previously described Corynebacterium species. Electron microscopy demonstrated the production of an extracellular substance causing connecting filaments between cells as a morphological correlate to the mucoid colonies. Comparative 16S rRNA gene sequence analysis revealed that the unknown coryneform bacterium represented a new subline within the genus Corynebacterium, for which the name Corynebacterium mucifaciens sp. nov. is proposed. The type strain is CCUG 36878 (= DSM 44265 = CIP 105129).