Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation)

We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.     

Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis

Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium-related isolates, respectively. Within the Corynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01). Four isolates from the genera Arthrobacter, Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.     

Coryneform bacteria in infectious diseases: clinical and laboratory aspects.

Coryneform isolates from clinical specimens frequently cannot be identified by either reference laboratories or research laboratories. Many of these organisms are skin flora that belong to a large number of taxonomic groups, only 40% of which are in the genus Corynebacterium. This review provides an update on clinical presentations, microbiological features, and pathogenic mechanisms of infections with nondiphtheria Corynebacterium species and other pleomorphic gram-positive rods. The early literature is also reviewed for a few coryneforms, especially those whose roles as pathogens are controversial. Recognition of newly emerging opportunistic coryneforms is dependent on sound identification schemes which cannot be developed until cell wall analyses and nucleic acid studies have defined the taxonomic groups and all of the reference strains within each taxon have been shown by molecular methods to be authentic members. Only then can reliable batteries of biochemical tests be selected for distinguishing each taxon.     

Clinical and microbiologic characteristics of pediococci.

Over a 43-month period, 23 separate isolates of nonenterococcal alpha- and nonhemolytic streptococci were reported by our clinical microbiology laboratory to be resistant to vancomycin. This constituted 0.32% of nonenterococcal alpha- and nonhemolytic streptococci reported and 4.4% of such streptococci upon which susceptibility testing was performed. Of 13 isolates which were available for further study, all were highly resistant to vancomycin (MIC greater than or equal to 1,024 micrograms/ml), but none were actually streptococci. Three were clearly gram-positive rods by Gram stain and were found to be homofermentative lactobacilli. Two strains with elongated gram-positive cocci from colonies on agar showed small gram-positive rods when grown in thioglycolate broth and were physiologically identified as Lactobacillus confusus. Two isolates with lenticular gram-positive cocci appeared to be Leuconostoc mesenteroides subsp. mesenteroides. Six gram-positive isolates with round cells from growth on agar and from broth were arranged in tetrads in broth and closely resembled Pediococcus acidilactici. Twelve additional strains of pediococci that were not of human origin were also found to be highly resistant to vancomycin. These findings confirm published reports of clinical isolation of organisms resembling pediococci and suggest that clinically isolated, vancomycin-resistant bacteria which superficially resemble streptococci are probably other lactic acid bacteria.     

Two similar but atypical strains of coryneform group A-4 isolated from patients with endophthalmitis.

Corynebacterium species and other coryneform organisms isolated from clinical specimens are frequently considered contaminants. We isolated two strains of a gram-positive organism from the vitreous fluid of two patients with endophthalmitis who had previously received intraocular lens transplants. The biochemical characteristics and gas chromatographic patterns of both isolates were similar to those of coryneform group A-4 strains. Major differences included esculin hydrolysis, nitrate reduction, growth pigment, and lactic acid production. These two strains along with a limited number of strains collected at the Special Bacterial Pathogens Laboratory (Division of Bacterial Diseases, Centers for Disease Control, Atlanta, Ga.) may represent a subgroup of coryneform group A-4. Results of in vitro susceptibility testing performed with antimicrobial agents commonly used to treat patients with bacterial endophthalmitis underscore the importance of determining MBCs for slow-growing organisms. This report cautions microbiologists not to discard organisms frequently considered contaminants when isolated from body fluids that are normally sterile and from patients receiving local steroids.     

Vancomycin-resistant gram-positive bacteria isolated from human sources

Recent reports of infections with vancomycin-resistant gram-positive bacteria prompted us to study vancomycin-resistant isolates from human sources to characterize the types of bacteria displaying this phenotype. Thirty-six vancomycin-resistant gram-positive isolates, 14 from clinical specimens and 22 from stool samples, were identified. These isolates were tentatively identified as Lactobacillus spp. (25 strains), Leuconostoc spp. (6 strains), and Pediococcus spp. (3 strains) on the basis of morphology and physiological tests. Two isolates of indeterminate morphology could not be unambiguously assigned to a genus. Four isolates of vancomycin-resistant lactobacilli from normally sterile body sites were considered to be clinically significant. Vancomycin-resistant gram-positive bacteria may represent an emerging class of nosocomial pathogens. Better methods for distinguishing the various genera in the clinical microbiology laboratory are needed.     

Development of Amplified 16S Ribosomal DNA Restriction Analysis for Identification of Actinomyces Species and Comparison with Pyrolysis-Mass Spectrometry and Conventional Biochemical Tests

Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinical isolates (n = 36) of Actinomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination of HaeIII and HpaII endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification of Actinomyces spp. of clinical origin.     

Comparison of conventional and molecular methods for identification of aerobic catalase-negative gram-positive cocci in the clinical laboratory

Over a period of 18 months we have evaluated the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic catalase-negative gram-positive cocci in the clinical laboratory. A total of 171 clinically relevant strains were studied. The results of molecular analyses were compared with those obtained with a commercially available phenotypic identification system (API 20 Strep system; bioMérieux sa, Marcy l'Etoile, France). Phenotypic characterization identified 67 (39%) isolates to the species level and 32 (19%) to the genus level. Seventy-two (42%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 138 (81%) isolates to the species level and 33 (19%) to the genus level. For 42 of 67 isolates assigned to a species with the API 20 Strep system, molecular analyses yielded discrepant results. Upon further analysis it was concluded that among the 42 isolates with discrepant results, 16S rDNA sequencing was correct for 32 isolates, the phenotypic identification was correct for 2 isolates, and the results for 8 isolates remained unresolved. We conclude that 16S rDNA sequencing is an effective means for the identification of aerobic catalase-negative gram-positive cocci. With the exception of Streptococcus pneumoniae and beta-hemolytic streptococci, we propose the use of 16S rDNA sequence analysis if adequate species identification is of concern.     

Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.     

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.     

Comparative evaluation of RapID ANA and API 20A for identification of anaerobic bacteria

This study evaluated RapID ANA and API 20 A systems for identification of anaerobic gram-positive and gram-negative bacteria isolated from oral and non-oral infections using standard reference methods. A total of 480 isolates were tested in both systems. The RapID ANA system correctly identified 74 % of the strains to species level and 17.5% to genus level; 5 % were misidentified. The API 20 A system correctly identified 50 % of the strains to species level and 24.5 % to genus level; 8 % were misidentified and 17.5 % could not be identified by the API 20 A system.     

Characterisation of Eubacterium-like strains isolated from oral infections.

The genus Eubacterium currently includes a heterogeneous group of gram-positive, non-spore-forming anaerobic bacilli, many of which are slow growing, fastidious and generally unreactive in biochemical tests. As a consequence, cultivation and identification of isolates are difficult and the taxonomy of the group remains indifferent. In this study, 105 isolates from odontogenic infections, infections associated with dental implants or saliva from healthy subjects and provisionally assigned to the genus Eubacterium were subjected to phenotypic and genotypic analysis. Ninety-one of the isolates were identified as belonging to one of 14 previously described species: Atopobium parvulum (5 isolates), A. rimae (29), Bulleidia extructa (2), Cryptobacterium curtum (1), Dialister pneumosintes (1), Eubacterium saburreum (2), E. sulci (8), E. yurii subsp. yurii (1), Filifactor alocis (3), Lactobacillus uli (1), Mogibacterium timidum (13), M. vescum (6), Pseudoramibacter alactolyticus (6) and Slackia exigua (13). The remaining 14 isolates did not correspond to existing species. This study confirms the diversity of organisms provisionally assigned to the genus Eubacterium by conventional identification methods. This group of organisms is frequently isolated from oral infections but their role in the aetiology of these conditions has yet to be determined.     

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.     

Catheter-related Microbacterium bacteremia identified by 16S rRNA gene sequencing

We describe the application of 16S rRNA gene sequencing in defining two cases of catheter-related Microbacterium bacteremia. In the first case, a gram-positive bacillus was isolated from both the blood culture and central catheter tip of a 39-year-old woman with chronic myeloid leukemia. The API Coryne system identified the isolate as 98.9% Aureobacterium or Corynebacterium aquaticum. In the second case, a gram-positive bacillus was recovered from five sets of blood cultures from both central catheter and percutaneous venipuncture of a 5-year-old girl with acute myeloid leukemia. The isolate was identified by the API Coryne system as 99.7% Cellulomonas or Microbacterium species. Further phenotypic tests failed to identify the two isolates. 16S rRNA gene sequencing showed 99.4% similarity between the first isolate and Microbacterium oxydans and 98.7% similarity between the second isolate and Microbacterium trichotecenolyticum, indicating that both isolates were Microbacterium species. Microbacterium infections are rarely reported in the literature. Although the central venous catheter was previously proposed to be a source of bacteremia, the first case in this report represents the first culture-documented case of catheter-related Microbacterium bacteremia.     

Accuracy and reproducibility of the 4-hour ATB 32A method for anaerobe identification.

The ATB 32A system (API System SA, La Balme les Grottes, Montalieu-Vercieu, France) was evaluated for use in the identification of 214 anaerobes. Organisms included 73 isolates of the Bacteroides fragilis group, 24 Bacteroides spp., 10 fusobacteria, 43 clostridia, 28 cocci, and 36 gram-positive, nonsporeforming rods. With the concomitant use of Gram stain, pigmentation, catalase testing, and aerobic growth, the ATB 32A system correctly identified 97% of the B. fragilis group isolates, 88% of Bacteroides spp., 50% of fusobacteria, 74% of clostridia, 100% of cocci, and 86% of the gram-positive, nonsporeforming rods. Overall, 188 strains (88%) were correctly identified, with 18 (8%) requiring extra tests, other than the four mentioned above, for correct identification. Eight strains were misidentified, including one Bacteroides sp., three fusobacteria, one Clostridium sp., and three gram-positive, nonsporeforming rods. Reproducibility was very good, with 12 of 14 strains (86%) tested in triplicate yielding identical correct results on each of three occasions and 2 strains (14%) yielding identical correct results on two occasions. There was a low-probability identification (including the correct species) on the third testing. The ATB 32A system represents a worthwhile advance in systems used for the identification of clinically significant anaerobic bacteria.     

Clinical evaluation of the RapID-ANA II panel for identification of anaerobic bacteria.

The accuracy of the RapID-ANA II system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) was evaluated by comparing the results obtained with that system with results obtained by the methods described by the Virginia Polytechnic Institute and State University. Three hundred anaerobic bacteria were tested, including 259 clinical isolates and 41 stock strains of anaerobic microorganisms representing 16 genera and 48 species. When identifications to the genus level only were included, 96% of the anaerobic gram-negative bacilli, 94% of the Clostridium species, 83% of the anaerobic, nonsporeforming, gram-positive bacilli, and 97% of the anaerobic cocci were correctly identified. When correct identifications to the genus and species levels were compared, 86% of 152 anaerobic gram-negative bacilli, 76% of 34 Clostridium species, 81% of 41 anaerobic, nonsporeforming, gram-positive bacilli, and 97% of 73 anaerobic cocci were correctly identified. Eight isolates (3%) produced inadequate identification in which the correct identification was listed with one or two other possible choices and extra tests were required for separation. A total of 9 isolates (3%) were misidentified by the RapID-ANA II panel. Overall, the system was able to correctly identify 94% of all the isolates to the genus level and 87% of the isolates to the species level in 4 h by using aerobic incubation.     

Characteristics of CDC group 3 and group 5 coryneform bacteria isolated from clinical specimens and assignment to the genus Dermabacter.

Over a 1-year period, 11 isolates (including 5 from blood cultures) of the recently described CDC group 3 and group 5 coryneform bacteria were derived from clinical specimens and compared with reference strains. Biochemical characteristics indicated a very close relationship between CDC group 3 and group 5 coryneform bacteria. The ability of CDC group 3 and the inability of CDC group 5 coryneform bacteria to ferment xylose were the only reactions that were different for the two taxa. Chemotaxonomic features of the two groups included the presence of meso-diaminopimelic acid, a lack of mycolic acids, and the presence of predominantly branched cellular fatty acids, a combination found among gram-positive rods only in Brevibacterium spp., Brachybacterium faecium, and Dermabacter hominis. 16S rRNA gene sequence analysis revealed that CDC group 3 and group 5 coryneform bacteria are members of the genus Dermabacter, which to date has been isolated exclusively from human skin.     

Premarket evaluation of IDS RapID SS/u system for identification of urine isolates.

A total of 170 fresh clinical urine isolates were tested with a premarket configuration of the RapID SS/u system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.), a qualitative micromethod for the identification of selected organisms commonly isolated from urine specimens. Results were compared with those obtained with conventional methods of identifying gram-positive isolates and with the AutoMicrobic system (Vitek Systems, Inc., Hazelwood, Mo.), utilizing Gram-Negative Identification cards for the identification of gram-negative rods. Organisms representing 12 taxa were included in the study. Of the 170 isolates, 163 (95.9%) were correctly identified. A total of 144 strains (84.7%) were correctly identified without additional testing, whereas 19 isolates (11.2%) required further testing. Seven isolates (4.1%) were incorrectly identified. The SS/u system required minimal hands-on time inoculate and interpret reactions. Discrepancies most often occurred with regard to misinterpretation of Escherichia coli and Enterobacter sp. as Citrobacter sp. The IDS RapID SS/u system may indeed prove valuable for the rapid manual identification of urine isolates.     

Comparison of PRAS II, RapID ANA, and API 20A systems for identification of anaerobic bacteria.

This study evaluated the PRAS II, RapID ANA, and API 20A systems for the identification of anaerobic bacteria. A total of 80 isolates (68 fresh clinical isolates and 12 stock cultures) were examined and included 25 Bacteriodes spp., 7 Fusobacterium spp., 12 Clostridium spp., 2 Veillonella spp., 16 gram-positive cocci, and 18 gram-positive nonsporeforming bacilli. All isolates were initially identified by the procedures outlined in Holdeman et al. (ed.), Anaerobe Laboratory Manual, Virginia Polytechnic Institute and State University, Blacksburg, Va., 1977; identifications from the PRAS II, RapID ANA, and API 20A systems were compared with these initial identifications. If no supplemental tests were required, the RapID ANA and API 20A systems had incubation times of 4 and 24 h, respectively; the PRAS II system generally required 2 to 5 days of incubation, depending on the growth rate of the isolate. PRAS II identified 74% correct to species level, 14% correct to genus only, and 6% incorrect; 6% could not be identified. PRAS II data were reevaluated according to a revised data base that was provided after completion of the study; PRAS II (revised) identified 82% correct to species, 12% correct to genus only, and 6% incorrect. RapID ANA identified 62% correct to the species level, 28% correct to genus only, and 10% incorrect. API 20A identified 71% correct to the species level, 10% correct to genus only, and 3% incorrect; 16% could not identified. The API 20A is a more established system for identification of anaerobic bacteria; PRAS II and RapID ANA appear to be promising new methods for the identification of anaerobic bacteria.