Cellular fatty acids in Fusobacterium species as a tool for identification.

Identification of fusobacteria from clinical specimens currently requires analysis of metabolic end products by gas-liquid chromatography in addition to certain biochemical and enzymatic tests because of the relative biochemical inactivity of these bacteria. Even the finding of pointed, thin gram-negative cells on Gram-stained slides can no longer be relied on for identification of Fusobacterium nucleatum, since at least four other species of fusobacteria have been seen to exhibit similar morphology. We examined 46 clinical isolates and six American Type Culture Collection type strains of fusobacteria by conventional methods and by the Microbial ID Systems MIDI software package for analyzing cellular fatty acid patterns measured by capillary column gas-liquid chromatography. Distinctive patterns of major fatty acids could be used to reliably identify most clinical isolates to the species level. The MIDI system identified 89% of the isolates correctly and provides an alternative to conventional methods.     

Genotypic and phenotypic characterization of "Streptococcus milleri" group isolates from a Veterans Administration hospital population.

Because identification of the species within the "Streptococcus milleri" group is difficult for the clinical laboratory as the species share overlapping phenotypic characteristics, we wished to confirm biochemical identification with identification by 16S rRNA gene sequence analysis. Ninety-four clinical isolates previously identified as the "Streptococcus milleri" group were reclassified as S. anginosus, S. constellatus, or S. intermedius with the API 20 Strep system (bioMerieux Vikek, Hazelton, Mo.) and the Fluo-card (Key Scientific, Round Rock, Tex.). In addition, we determined the Lancefield group, hemolysis, colony size, colony texture, repetitive extragenic palindromic PCR (rep-PCR) pattern, and cellular fatty acid (CFA) profile (MIDI, Newark, Del.). 16S rRNA gene sequence analysis with 40 selected representative strains showed three distinct groups, with S. constellatus and S. intermedius found to be more closely related to each other than to S. anginosus, and further distinguished a biochemically distinct group of urogenital isolates within the S. anginosus group of isolates. Except for strains unreactive with the Fluo-card (8%), all S. anginosus and S. intermedius strains identified by sequencing were similarly identified by biochemical testing. However, 23% of the selected S. constellatus isolates identified by sequencing (9% of all S. constellatus isolates) would have been identified as S. anginosus or S. intermedius by biochemical tests. Although most S. anginosus strains formed one unique cluster by CFA analysis and most S. constellatus strains showed similar rep-PCR patterns, neither method was sufficiently dependable for identification. Whereas Lancefield group or lactose fermentation did not correspond to sequence or biochemical type, S. constellatus was most likely to be beta-hemolytic and S. intermedius was most likely to have a dry colony type. The most frequent isolate in our population was S. constellatus, followed by S. anginosus. There was an association of S. anginosus with a gastrointestinal or urogenital source, and there was an association of S. constellatus and S. intermedius with both the respiratory tract and upper-body abscesses.     

Evaluation of the Quantum II and Rapid E identification systems.

A total of 492 clinical isolates from the family Enterobacteriaceae were tested in the API 20E, Rapid E, and Quantum II identification systems. Discrepant identifications among these three systems were resolved by repeat testing in the identification systems or use of conventional biochemical tests. Of these isolates, 94.1% were correctly identified with the API 20E and Rapid E systems, and 97.0% were correctly identified with the Quantum II system. An additional 48 non-Enterobacteriaceae isolates were tested with the Quantum II system, and 83.3% were correctly identified. The majority of incorrect identifications with the Rapid E and Quantum II systems were caused by a single aberrant biochemical reaction. Reproducibility of the biochemical reactions obtained with these two systems was evaluated by testing 40 organisms in triplicate. Identical biocodes for all three tests were obtained for 10 organisms with the Quantum II system and for 19 organisms with the Rapid E system. Reproducibility of the Quantum II test results was improved with a subsequent modification of the photometer of this system. Both the Rapid E and Quantum II systems were inexpensive and were technically easy to inoculate and interpret.     

Evaluation of CrystalTM Rapid Stool/Enteric ID System for Identification of Aerobic Gram-negative Bacilli

Objective: The evaluation of the new miniaturized Crystal^TM Rapid Stool/Enteric System (Becton-Dickinson, USA) for identification of aerobic gram-negative bacilli.
Methods: a total of 154 clinical organisms ( Enterobacteriaceae: 120 strains; oxidase-positive fermenters: 13 strains; non-fermenters: 21 strains) were tested. Results were compared with those obtained with the PASCO^R system (Difco, USA) and divergent identifications were evaluated by standard biochemical tests.
Results: without additional testing, correct identification was obtained for 146 strains ( Enterobacteriaceae: 95%; oxidase-positive fermenters 87%; non-fermenters 100%). For adequate identification of Yersinia enterocolitica strains, however, panels had to be incubated for 5 instead of 3 hours.
Conclusions: the Crystal^TM Rapid Stool/Enteric system offers a safe, accurate and rapid method for the identification of frequent isolates of the family Enterobacteriaceae and bacterial stool pathogens.     

Septic arthritis caused by a gram-negative bacterium representing a new species related to the Bordetella-Alcaligenes complex

A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species.     

Cellular fatty acid composition of Pseudomonas marginata and closely associated bacteria.

The cellular fatty acid compositions of the lectotype strain and four clinical isolates of Pseudomonas marginata were determined by gas-liquid chromatography and compared with 11 strains of the Centers for Disease Control Pseudomonas-like group 2, which are similar to P. marginata in a number of conventional biochemical tests. Isolates of P. marginata were readily distinguished from Pseudomonas-like group 2 by the presence of a C17:0 cyclopropane acid and hydroxy acids 3-OH-C14:0, 2-OH-C16:0, 3-OH-C16:0, and 2-OH-C18:1, whereas strains of Pseudomonas-like group 2 contained C16:1 delta 9 as a major acid with small amounts of 3-OH-C12:0 and 2-OH-C14:0 acids. Our data show that cellular fatty acid composition provides useful additional information that can be combined with selected conventional tests to provide a more reliable and rapid identification of P. marginata and related bacteria.     

Comparison of API Rapid Strep, Baxter MicroScan Rapid Pos ID Panel, BBL Minitek Differential Identification System, IDS RapID STR System, and Vitek GPI to conventional biochemical tests for identification of viridans streptococci

Viridans group streptococci (36 stock strains and 167 single patient blood culture isolates) were assessed using API Rapid Strep, Baxter MicroScan Rapid Pos ID Panel, BBL Minitek Differential Identification System, IDS RapID STR System, and Vitek GPI methods. Identification data obtained with these systems were compared with those indicated by conventional biochemical procedures. API, Baxter MicroScan, BBL, IDS, and Vitek corresponded with conventional biochemical identification in 74%, 66%, 65%, 50%, and 61% of the isolates, respectively; using recommended supplemental tests, agreement was augmented in 9%, 11%, 20%, 11%, and 21% of the isolates, respectively. Disagreement with conventional biochemical methods occurred in 14%, 17%, 14%, 32%, and 10% of the commercial techniques, respectively; no identification was possible in 2%, 5%, fewer than 1%, 6%, and 8% of specimens, respectively. BBL, API, and Baxter MicroScan systems provided the most reliable rapid identification, although supplemental testing often was required. Until a higher percentage of correct identification data can be obtained without supplemental procedures, conventional biochemical techniques will remain the methods of choice for identification of viridans streptococci.     

Subgrouping of Pseudomonas cepacia by cellular fatty acid composition.

The cellular fatty acid compositions were determined for 42 strains of Pseudomonas cepacia from five cystic fibrosis centers in North America. All isolates contained significant (20%) amounts of hexadecanoic (C16:0), and cis-9 hexadecenoic (C16:1 cis9) acids and an isomer of octadecenoic acid (C18:1). None had hydroxy acids containing fewer than 14 carbon atoms. The quantitative data from the fatty acid analysis were highly reproducible and provided a basis for numerical analysis. Five subgroups comprising all the strains were obtained by cluster analysis and further characterized by principal-component analysis. With minor exceptions, the predominant subgroup identified in each center was different from that identified in other centers and accounted for one-half of the isolates within each center. Cellular fatty acid composition is a useful adjunct to biochemical characterization for the identification of P. cepacia isolated from cystic fibrosis patients. Numerical analysis of the fatty acid data can separate P. cepacia into subgroups, which may provide useful epidemiologic information or a basis for further analysis by more complex techniques such as DNA probe analysis.     

Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens

A total of 430 strains of glucose-nonfermenting gram-negative bacteria representing 35 species were analyzed for their cellular fatty acid composition by gas-liquid chromatography (GLC). On the basis of qualitative differences in their cellular fatty acid composition, these bacteria could be divided into 19 distinct chromatographic groups. Eight Pseudomonas species, Achromobacter xylosoxidans, group Vd, and Agrobacterium radiobacter were identified from their fatty acid compositions alone. The other glucose-nonfermenting gram-negative bacterial species studied here, classified within nine distinct GLC groups, were easily recognized by using the GLC fatty acid analysis supplemented with a limited number of conventional biochemical tests. The results support the hypothesis that bacterial fatty acid composition is rather specific and that qualitative GLC fatty acid analysis can be adapted in the clinical laboratory either to provide additional criteria for differentiation of closely related groups or to serve as a rapid and highly reproducible method for their routine identification.     

Evaluation of the RapID-ANA system for identification of anaerobic bacteria of veterinary origin.

This study evaluated the ability of the RapID-ANA system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) to accurately identify a spectrum of freshly isolated veterinary anaerobes. A total of 183 isolates were tested and included 7 Actinomyces spp., 53 Bacteroides spp., 32 Clostridium spp., 2 Eubacterium spp., 65 Fusobacterium spp., 1 Peptococcus spp., 22 Peptostreptococcus spp., and 1 Propionibacterium spp. All isolates were initially identified by conventional biochemical testing and gas-liquid chromatography of short-chain fatty acid metabolites. Additional tests were performed as required by the RapID-ANA system. Of these isolates, 81.4% were correctly identified to the genus level, including 59.6% to the species level, 14.2% were incorrectly identified at the genus level, and 4.4% were not identified. Initially, 20.2% of the strains were not identified because the microcodes were not in the code book. The majority of the incorrect identifications were caused by the misidentification of Fusobacterium spp. as Bacteroides spp. Errors also occurred when veterinary anaerobes not included in the data base were assigned an identification from the existing data base. The RapID-ANA system appears to be a promising new method for rapid identification of veterinary anaerobes; however, further evaluation with an extended data base is needed before the system can accurately identify all clinically significant anaerobes.     

Evaluation of the Microbial Identification System for identification of clinically isolated yeasts.

The Microbial Identification System (MIS; Microbial ID, Inc., Newark, Del.) was evaluated for the identification of 550 clinically isolated yeasts. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C and conventional methods) and included Candida albicans (n = 294), C. glabrata (n = 145), C. tropicalis (n = 58), C. parapsilosis (n = 33), and other yeasts (n = 20). In preparation for fatty acid analysis, yeasts were inoculated onto Sabouraud dextrose agar and incubated at 28 degrees C for 24 h. Yeasts were harvested, saponified, derivatized, and extracted, and fatty acid analysis was performed according to the manufacturer's instructions. Fatty acid profiles were analyzed, and computer identifications were made with the Yeast Clinical Library (database version 3.8). Of the 550 isolates tested, 374 (68.0%) were correctly identified to the species level, with 87 (15.8%) being incorrectly identified and 89 (16.2%) giving no identification. Repeat testing of isolates giving no identification resulted in an additional 18 isolates being correctly identified. This gave the MIS an overall identification rate of 71.3%. The most frequently misidentified yeast was C. glabrata, which was identified as Saccharomyces cerevisiae 32.4% of the time. On the basis of these results, the MIS, with its current database, does not appear suitable for the routine identification of clinically important yeasts.     

Accuracy of four commercial systems for identification of Burkholderia cepacia and other gram-negative nonfermenting bacilli recovered from patients with cystic fibrosis.

Burkholderia cepacia has recently been recognized as an important pathogen in chronic lung disease in patients with cystic fibrosis (CF). Because of the social, psychological, and medical implications of the isolation of B. cepacia from CF patients, accurate identification of this organism is essential. We compared the accuracies of four commercial systems developed for the identification of nonfermenting, gram-negative bacilli with that of conventional biochemical testing for 150 nonfermenters including 58 isolates of B. cepacia recovered from respiratory secretions from CF patients. The accuracies of the four systems for identifying all nonfermenters ranged from 57 to 80%, with the RapID NF Plus system being most accurate. The accuracies of these systems for identifying B. cepacia ranged from 43 to 86%, with the Remel system being most accurate. Depending on the commercial system, from two to seven isolates were misidentified as B. cepacia. The relatively poor performance of the commercial systems requires that identification of certain nonfermenters be confirmed by conventional biochemical testing. These organisms include B. cepacia, Burkholderia sp. other than B. cepacia, and infrequently encountered environmental species (Pseudomonas and Flavobacterium species). In addition, conventional biochemical testing should be done if a commercial system fails to assign an identification to an organism. Confirmatory testing should preferably be performed by a reference laboratory with experience in working organisms isolated from CF patients.     

Application of SmartGene IDNS software to partial 16S rRNA gene sequences for a diverse group of bacteria in a clinical laboratory

Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bacterial clinical isolates was compared with gene sequences interrogated by the SmartGene and MicroSeq databases. Of 300 isolates, SmartGene identified 295 (98%) to the genus level and 262 (87%) to the species level, with 5 (2%) being inconclusive. MicroSeq identified 271 (90%) to the genus level and 223 (74%) to the species level, with 29 (10%) being inconclusive. SmartGene and MicroSeq agreed on the genus for 233 (78%) isolates and the species for 212 (71%) isolates. Conventional methods identified 291 (97%) isolates to the genus level and 208 (69%) to the species level, with 9 (3%) being inconclusive. SmartGene, MicroSeq, and conventional identifications agreed for 193 (64%) of the results. Twenty-seven microorganisms were not represented in MicroSeq, compared to only 2 not represented in SmartGene. Overall, SmartGene IDNS provides comprehensive and accurate identification of a diverse group of bacteria and has the added benefit of being a user-friendly program that can be modified to meet the unique needs of clinical laboratories.     

Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting gram-negative bacilli from patients with cystic fibrosis

Pseudomonas aeruginosa and other gram-negative isolates from patients with cystic fibrosis (CF) may be difficult to identify because of their marked phenotypic diversity. We examined 200 gram-negative clinical isolates from CF respiratory tract specimens and compared identification by biochemical testing and real-time PCR with multiple different target sequences using a standardized combination of biochemical testing and molecular identification, including 16S rRNA partial sequencing and gyrB PCR and sequencing as a "gold standard." Of 50 isolates easily identified phenotypically as P. aeruginosa, all were positive with PCR primers for gyrB or oprI, 98% were positive with exotoxin A primers, and 90% were positive with algD primers. Of 50 P. aeruginosa isolates that could be identified by basic biochemical testing, 100% were positive by real-time PCR with gyrB or oprI primers, 96% were positive with exotoxin A primers, and 92% were positive with algD primers. For isolates requiring more-extensive biochemical evaluation, 13 isolates were identified as P. aeruginosa; all 13 were positive with gyrB primers, 12 of 13 were positive with oprI primers, 11 of 13 were positive with exotoxin A primers, and 10 of 13 were positive with algD primers. A single false-positive P. aeruginosa result was seen with oprI primers. The best-performing commercial biochemical testing was in exact agreement with molecular identification only 60% of the time for this most difficult group. Real-time PCR had costs similar to those of commercial biochemical testing but a much shorter turnaround time. Given the diversity of these CF isolates, real-time PCR with a combination of two target sequences appears to be the optimum choice for identification of atypical P. aeruginosa and for non-P. aeruginosa gram-negative isolates.     

Internal transcribed spacer region sequence analysis using SmartGene IDNS software for the identification of unusual clinical yeast isolates

Rapid and accurate identification of clinically important yeasts is essential given their inherent differences in antifungal susceptibility. We implemented nucleic acid sequencing for those species that could not be identified by phenotypic methods. Internal Transcribed Spacer region 1 and 2 (ITS1 and ITS2) sequences were investigated using SmartGene IDNS software, an rDNA sequence database and analysis program for microbial identification (ID). Over a 2.5-year period, 2,938 specimens were evaluated. Most (94%) isolates were fully identified by conventional methods, with Candida species accounting for the majority of them. Of the 169 organisms that required molecular analysis, 79% were identified to species level, 19% to genus and 2% remained unresolved. Sequenced isolates encompassed 33 unique species of which approximately half (52%) were common pathogens with atypical biochemical profiles and the remainder were rarer yeast species. A significant proportion (33%) of sequenced organisms displayed elevated MICs to fluconazole. Our experience supports the use of molecular techniques as an adjunct to conventional methods for the identification of medically important yeasts. Susceptibility testing alone may provide valuable treatment information in situations where phenotypic assessments are inconclusive and molecular or proteomic testing is not readily available.     

Evaluation of the Rapid ID 32A system for identification of anaerobic Gram-negative bacilli, excluding the Bacteroides fragilis group

Objective: To evaluate the Rapid ID 32A system (bioMérieux, Marcy-l'Etoile, France) for the identification of anaerobic Gram-negative bacilli, excluding the Bacteroides fragilis group.
Methods: Five hundred and twenty-eight identified clinical isolates of non- B. fragilis group anaerobic Gram-negative bacilli were tested in the Rapid ID 32A system, and identifications were compared with those obtained with conventional biochemical tests and gas-liquid chromatography.
Results: The Rapid ID 32A system correctly identified 280 (60.9%) of the 460 isolates tested for which taxa were included in the database, without the need for additional testing. A further 97 (21.1%) isolates were correctly identified to species level following the performance of complementary tests recommended by the manufacturer. Fifty-nine (12.8%) isolates were identified at the genus level only, and 21 (4.6%) were misidentified at the species level. Three isolates of Prevotella were not identified by the system. Of the 68 isolates belonging to taxa not included in the database, no identification was obtained for 33 (48.5%), while 35 (51.5%) were misidentified.
Conclusions: The Rapid ID 32A system provided a rapid and reliable method for the identification of non- B. fragilis group, anaerobic Gram-negative bacilli to the genus level, while the success of species-level identification varied with different taxa. There was poor discrimination between Fusobacterium nucleatum and F. necrophorum , between Porphyromonas asaccharolytica and Porphyromonas endodontalis , and between Prevotella buccalis, Prevotella denticola, Prevotella loescheii, Prevotella melaninogenica and Prevotella oralis . The need to perform conventional complementary tests on 149 (32.4%) of the 460 isolates compromised the usefulness of the system for rapid species identification.     

Evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of nonfermenting gram-negative bacilli isolated from cultures from cystic fibrosis patients

The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P = 0.099).     

Identification of clinical isolates of Mycobacterium spp. by sequence analysis of the 16S ribosomal RNA gene. Experience from a clinical laboratory

Twenty-one mycobacterial type strains and 334 clinical isolates of mycobacteria were identified by standardized sequence analysis using part of the gene encoding 16S rRNA. Apart from two clinical isolates, the resulting sequences corresponded to previously published sequences. The results of the molecular determinations of the type strains completely overlapped the identities obtained using conventional techniques (cultural characteristics, biochemical tests, commercial DNA probes, and gas chromatographic lipid profiles). Of 323 isolates conventionally identified as slow-growing mycobacteria, 318 (98.5%) were identified to the same species or group level by 16S rDNA sequence analysis, while 6 of the 11 strains of rapid growers obtained a corresponding identity with the two approaches. The sequencing protocol combined with a few cultural characteristics (i.e. growth rate, pigmentation and susceptibility testing) offers a rapid, reliable and usually definite identification of mycobacterial isolates.     

Evaluation of API Coryne in comparison with conventional methods for identifying coryneform bacteria.

A study was performed to evaluate a new manual miniaturized system, API Coryne (API-bioMérieux, Inc., La Balme les Grottes, France), in which conventional biochemical methods were used to identify 240 isolates of coryneform and related bacteria. A total of 40% of the isolates were excluded from the study because they could not be identified by conventional methods. Identifications of the 240 isolates obtained with API Coryne showed a 97.6% concordance with conventional methods (79% after 24 h of incubation, 21% after 48 h of incubation): 158 (65.8%) isolates were identified with no further testing, and extra testing was required for 76 (31.8%) isolates. In three (1.2%) cases, the organisms did not correspond to any key in the code book and could not be identified by the computer service of the manufacturer. Only three (1.2%) strains were misidentified. The system was shown to be reliable and rapid when compared with standard identification methods.     

Gas-liquid chromatographic analysis of cellular fatty acids for identification of gram-negative anaerobic bacilli.

A commercially available, computer-assisted microbial identification system (MIS) employs gas-liquid chromatographic analysis of cellular fatty acids for bacterial identification. MIS was compared with conventional identification systems. Of 225 gram-negative anaerobes tested, MIS identified 72.4% of the strains to the species level, 88.9% to the appropriate group, and 93.3% to the correct genus.