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.     

Superiority of molecular techniques for identification of gram-negative, oxidase-positive rods, including morphologically nontypical Pseudomonas aeruginosa, from patients with cystic fibrosis

Phenotypic identification of gram-negative bacteria from Cystic Fibrosis (CF) patients carries a high risk of misidentification. Therefore, we compared the results of biochemical identification by API 20NE with 16S rRNA gene sequencing in 88 gram-negative, oxidase-positive rods, other than morphologically and biochemically typical P. aeruginosa, from respiratory secretions of CF patients. The API 20NE allowed correct identification of the bacterial species in 15 out of 88 (17%) isolates investigated. Agreement between the API and the 16S rRNA gene sequencing results was high only in isolates with an API result classified as "excellent identification". Even API results classified as "very good identification" or "good identification" showed a high rate of misidentification (67% and 84%). Fifty-two isolates of morphological and biochemical nontypical Pseudomonas aeruginosa, representing 59% of all isolates investigated, were not identifiable or misidentified in the API 20NE. Therefore, rapid molecular diagnostic techniques like real-time PCR and fluorescence in situ hybridization (FISH) were evaluated in this particular group of bacteria for identification of the clinically most relevant pathogen, P. aeruginosa. The LightCycler PCR assay with a P. aeruginosa-specific probe showed a sensitivity and specificity of 98.1% and 100%, respectively. For FISH analysis, a newly designed P. aeruginosa-specific probe had a sensitivity and specificity of 100%. In conclusion, molecular methods are superior over biochemical tests for identification of gram-negative, oxidase-positive rods in CF patients. In addition, real-time PCR and FISH allowed identification of morphologically nontypical isolates of P. aeruginosa within a few hours.     

Use of 16S rRNA gene sequencing for identification of nonfermenting gram-negative bacilli recovered from patients attending a single cystic fibrosis center

During 1999, we used partial 16S rRNA gene sequencing for the prospective identification of atypical nonfermenting gram-negative bacilli isolated from patients attending our cystic fibrosis center. Of 1,093 isolates of nonfermenting gram-negative bacilli recovered from 148 patients, 46 (4.2%) gave problematic results with conventional phenotypic tests. These 46 isolates were genotypically identified as Pseudomonas aeruginosa (19 isolates, 12 patients), Achromobacter xylosoxidans (10 isolates, 8 patients), Stenotrophomonas maltophilia (9 isolates, 9 patients), Burkholderia cepacia genomovar I/III (3 isolates, 3 patients), Burkholderia vietnamiensis (1 isolate), Burkholderia gladioli (1 isolate), and Ralstonia mannitolilytica (3 isolates, 2 patients), a recently recognized species.     

Phenotypic and genotypic characteristics of non fermenting atypical strains recovered from cystic fibrosis patients

We used partial 16S rRNA gene (16S DNA) sequencing for the prospective identification of nonfermenting Gram-negative bacilli recovered from patients attending our cystic fibrosis center (h?pital Necker-Enfants malades), which gave problematic results with conventional phenotypic tests. During 1999, we recovered 1093 isolates of nonfermenting Gram-negative bacilli from 702 sputum sampled from 148 patients. Forty-six of these isolates (27 patients) were not identified satisfactorily in routine laboratory tests. These isolates were identified by 16S DNA sequencing as Pseudomonas aeruginosa (19 isolates, 12 patients), Achromobacter xylosoxidans (10 isolates, 8 patients), Stenotrophomonas maltophilia (9 isolates, 9 patients), Burkholderia cepacia genomovar I/III (3 isolates, 3 patients), Burkholderia vietnamiensis (1 isolate), Burkholderia gladioli (1 isolate) and Ralstonia mannitolilytica (3 isolates, 2 patients). Fifteen isolates (33%) were resistant to all antibiotics in routine testing. Sixteen isolates (39%) resistant to colistin were recovered on B. cepacia-selective medium: 2 P. aeruginosa, 3 A. xylosoxidans, 3 S. maltophilia and the 8 Burkholderia--Ralstonia isolates. The API 20NE system gave no identification for 35 isolates and misidentified 11 isolates (2 P. aeruginosa, 2 A. xylosoxidans and 1 S. maltophilia classified as B. cepacia ). Control measures and/or treatment were clearly improved as a result of 16S DNA sequencing in three of these cases. This study confirms the weakness of phenotypic methods for identification of atypical nonfermenting Gram-negative bacilli recovered from cystic fibrosis patients. The genotypic methods, such as 16S DNA sequencing which allows identification of strains in routine practice, appears to have a small, but significant impact on the clinical management of CF patients.     

Multi-center evaluation of the VITEK® MS system for mass spectrometric identification of non-Enterobacteriaceae Gram-negative bacilli

Studies have demonstrated that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate method for the identification of clinically relevant bacteria. The purpose of this study was to evaluate the performance of the VITEK MS v2.0 system (bioMérieux) for the identification of the non-Enterobacteriaceae Gram-negative bacilli (NEGNB). This multi-center study tested 558 unique NEGNB clinical isolates, representing 18 genera and 33 species. Results obtained with the VITEK MS v2.0 were compared with reference 16S rRNA gene sequencing and when indicated recA sequencing and phenotypic analysis. VITEK MS v2.0 provided an identification for 92.5 % of the NEGNB isolates (516 out of 558). VITEK MS v2.0 correctly identified 90.9 % of NEGNB (507 out of 558), 77.8 % to species level and 13.1 % to genus level with multiple species. There were four isolates (0.7 %) incorrectly identified to genus level and five isolates (0.9 %), with one incorrect identification to species level. The remaining 42 isolates (7.5 %) were either reported as no identification (5.0 %) or called “mixed genera” (2.5 %) since two or more different genera were identified as possible identifications for the test organism. These findings demonstrate that the VITEK MS v2.0 system provides accurate results for the identification of a challenging and diverse group of Gram-negative bacteria.     

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.     

Revisited distribution of nonfermenting Gram-negative bacilli clinical isolates

Nonfermenting Gram-negative bacilli (NF-GNB) are ubiquitous environmental opportunistic bacteria frequently misidentified by conventional phenotypic methods. The aim of this study was to determine the distribution of NF-GNB species by 16?S rRNA gene sequencing (used as reference method) and to compare performances of biochemical tests and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). From nine French hospitals, 188 NF-GNB isolates (except P. aeruginosa and A. baumannii) were prospectively collected from 187 clinical samples between December 2008 and May 2009. By using the genotypic approach, 173 (92%) and 188 (100%) isolates were identified to the species and genus level, respectively. They covered 35 species and 20 genera, with a predominance of Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Pseudomonas putida group bacteria. Of the 173 species-level identified strains, concordant identification to the species-level was obtained for 75.1%, 83% and 88.9% of isolates with API 20 NE strip, the VITEK-2 (ID-GN card) system and MALDI-TOF-MS, respectively. By excluding S. maltophilia isolates accurately identified by the three methods, genus-level identification was much higher for MALDI-TOF-MS (92.9%), compared with API 20 NE and VITEK-2 (76.2% and 80.8%, respectively). In conclusion, MALDI-TOF-MS represents a rapid, inexpensive, and accurate tool for routine identification of NF-GNB in human clinical samples.     

Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available identification systems also evaluated, its database needs to be expanded for accurate identification of anaerobic Gram positive bacilli.     

Use of the MicroSeq 500 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly

Reliable automated identification and susceptibility testing of clinically relevant bacteria is an essential routine for microbiology laboratories, thus improving patient care. Examples of automated identification systems include the Phoenix (Becton Dickinson) and the VITEK 2 (bioMerieux). However, more and more frequently, microbiologists must isolate "difficult" strains that automated systems often fail to identify. An alternative approach could be the genetic identification of isolates; this is based on 16S rRNA gene sequencing and analysis. The aim of the present study was to evaluate the possible use of MicroSeq 500 (Applera) for sequencing the 16S rRNA gene to identify isolates whose identification is unobtainable by conventional systems. We analyzed 83 "difficult" clinical isolates: 25 gram-positive and 58 gram-negative strains that were contemporaneously identified by both systems--VITEK 2 and Phoenix--while genetic identification was performed by using the MicroSeq 500 system. The results showed that phenotypic identifications by VITEK 2 and Phoenix were remarkably similar: 74% for gram-negative strains (43 of 58) and 80% for gram-positive strains were concordant by both systems and also concordant with genetic characterization. The exceptions were the 15 gram-negative and 9 gram-positive isolates whose phenotypic identifications were contrasting or inconclusive. For these, the use of MicroSeq 500 was fundamental to achieving species identification. In clinical microbiology the use of MicroSeq 500, particularly for strains with ambiguous biochemical profiles (including slow-growing strains), identifies strains more easily than do conventional systems. Moreover, MicroSeq 500 is easy to use and cost-effective, making it applicable also in the clinical laboratory.     

Evaluation of the new Vitek 2 GN card for the identification of gram-negative bacilli frequently encountered in clinical laboratories

The new Vitek 2 GN card (bioMérieux, Marcy-l'Etoile, France) was developed for better identification of fermenting and nonfermenting bacilli. This new card allows the identification of 159 taxa. A total of 426 isolates (331 fermenting and 95 nonfermenting gram-negative bacilli) belonging to 70 taxa covered by the database were evaluated. All isolates were identified in parallel with the ID 32 GN, the API 20E, and the API 20NE methods. The system correctly identified 97.4% (n=415) of the strains. Only 2.1% (n=9) needed additional testing. One strain (0.25%) was misidentified (Klebsiella pneumoniae subsp. pneumoniae), and another one (0.25%) was not identified (Morganella morganii subsp. morganii). The new GN card gives more accurate identifications overall for gram-negative bacilli when compared to the systems described in other similar studies. This study was presented in part at the 104th General Meeting of the American Society for Microbiology, New Orleans, Louisiana, 2004, Abstract C-180.     

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.     

Usefulness of the MicroSeq 500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles

Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the "gold standard," we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification.     

Bacterial identification, clinical significance, and antimicrobial susceptibilities of Acinetobacter ursingii and Acinetobacter schindleri, two frequently misidentified opportunistic pathogens

The species belonging to the Acinetobacter genus are currently reported as opportunistic pathogens in hospitalized patients with underlying predispositions. However, except for the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, the identification of other species is frequently unreliable, especially for Acinetobacter ursingii and Acinetobacter schindleri, newly described in 2001. Thus, the clinical significance, phenotypic features, and antimicrobial susceptibilities of these two misidentified species remain unclear. Of 456 Acinetobacter sp. clinical strains isolated from 2002 to 2005 in Henri Mondor Hospital, 15 isolates (10 A. ursingii and 5 A. schindleri isolates) were studied. They were characterized using a phenotypic approach (API 20 NE and VITEK 2 systems), 16S rRNA gene sequencing, and susceptibility to antimicrobial agents with evaluation of impact in clinical relevance. The two corresponding type strains were also included for comparison. All isolates were identified to the species level using molecular tools, whereas the phenotypic methods remained unreliable due to the absence of these two species in the manufacturers' databases. However, the API 20 NE system appeared to be a reasonably reliable phenotypic alternative for the identification of A. ursingii when the numerical code 0000071 was found. Conversely, no discriminative phenotypic alternative existed for A. schindleri isolates. Concerning antimicrobial susceptibility, A. ursingii strains appeared to be more resistant to antibiotics than A. schindleri strains, which could imply therapeutic consequences. Finally, the prevalence of infections caused by A. ursingii and A. schindleri (representing 9.7% and 4.8% of non-A. calcoaceticus-A. baumannii complex strains, respectively) seems to be underestimated.     

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to 16S rRNA gene sequencing for species identification of nonfermenting bacteria

Nonfermenting bacteria are ubiquitous environmental opportunists that cause infections in humans, especially compromised patients. Due to their limited biochemical reactivity and different morphotypes, misidentification by classical phenotypic means occurs frequently. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for species identification. By using 248 nonfermenting culture collection strains composed of 37 genera most relevant to human infections, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurement and MALDI BioTyper software (Bruker Daltonik GmbH, Leipzig, Germany), i.e., by using a mass range of 2,000 to 20,000 Da and a new pattern-matching algorithm. To evaluate the database, 80 blind-coded clinical nonfermenting bacterial strains were analyzed. As a reference method for species designation, partial 16S rRNA gene sequencing was applied. By 16S rRNA gene sequencing, 57 of the 80 isolates produced a unique species identification (>or=99% sequence similarity); 11 further isolates gave ambiguous results at this threshold and were rated as identified to the genus level only. Ten isolates were identified to the genus level (>or=97% similarity); and two isolates had similarity values below this threshold, were counted as not identified, and were excluded from further analysis. MALDI-TOF MS identified 67 of the 78 isolates (85.9%) included, in agreement with the results of the reference method; 9 were misidentified and 2 were unidentified. The identities of 10 randomly selected strains were 100% correct when three different mass spectrometers and four different cultivation media were used. Thus, MALDI-TOF MS-based species identification of nonfermenting bacteria provided accurate and reproducible results within 10 min without any substantial costs for consumables.     

Evaluation of 16S rRNA sequencing and reevaluation of a short biochemical scheme for identification of clinically significant Bacteroides species

Sequence analysis of the 16S rRNA gene represents a highly accurate and versatile method for bacterial classification and identification, even when the species in question is notoriously difficult to identify by phenotypic means. In this study, we evaluated the utility of 16S rRNA gene sequencing as a means of identifying clinically important Bacteroides species. We sequenced 231 clinical isolates that had been identified by a short biochemical scheme. Based on the sequence analysis, 192 clinical isolates were assigned to an established species, with the other 39 clinical strains revealing five unique sequences that may represent five novel species. This is in contrast to identification obtained from a short biochemical scheme, by which only 73.5% (172 of 231) of isolates were correctly identified to species level. Based on the solid identification obtained from 16S rRNA gene sequencing, the short biochemical scheme was modified and improved to provide clinical laboratories with an inexpensive and simple alternative for the identification of isolates of clinically significant Bacteroides species.     

Re-analysis of 178 previously unidentifiable Mycobacterium isolates in the Netherlands in 1999–2007

Nontuberculous mycobacteria (NTM) that cannot be identified to the species level by reverse line blot hybridization assays and sequencing of the 16S rRNA gene comprise a challenge for reference laboratories. However, the number of 16S rRNA gene sequences added to online public databases is growing rapidly, as is the number of Mycobacterium species. Therefore, we re-analysed 178 Mycobacterium isolates with 53 previously unmatched 16S rRNA gene sequences, submitted to our national reference laboratory in 1999–2007. All sequences were again compared with the GenBank database sequences and the isolates were re-identified using two commercially available identification kits, targeting separate genetic loci. Ninety-three out of 178 isolates (52%) with 20 different 16S rRNA gene sequences could be assigned to validly published species. The two reverse line blot assays provided false identifications for three recently described species and 22 discrepancies were recorded in the identification results between the two reverse line blot assays. Identification by reverse line blot assays underestimates the genetic heterogeneity among NTM. This heterogeneity can be clinically relevant because particular sub-groupings of species can cause specific disease types. Therefore, sequence-based identification is preferable, at least at the reference laboratory level, although the exact targets needed for clinically useful results remain to be established. The number of NTM species in the environment is probably so high that unidentifiable clinical isolates should be given a separate species status only if this is clinically meaningful.     

Evaluation of two rapid methods for identification of commonly encountered nonfermenting or oxidase-positive, Gram-negative rods.

The ability of the expanded API-20E and the Oxi-Ferm System to identify 176 isolates of nonfermenting or oxidase-positive, gram-negative rods from 17 species or groups was studied. The expanded API-20E, without referral to a computer reference center, was able to identify 61.4% of the isolates to the species level. If reference to the computer center was utilized, an additional 25% could be identified. Of the isolates examined, 13.6% were misidentified, partially identified, or assigned no identification. Those assigned to the incorrect genus constituted 0.5% of the isolates tested; those assigned to the correct genus, but the wrong species, constituted 0.5%; 4.5% were placed in the correct genus with no species given; and 7.9% were assigned no identification. The Oxi-Ferm System was able to assign 75% of the isolates to the correct species without further testing, and an additional 19.3% required additional testing for correct identification. Those assigned to the incorrect genus represented 3.4% of the isolates tested; 1.1% were assinged to the correct genus, but the wrong species; and 1.1% were assigned to the correct genus, with no species indicated.     

Evaluation of the Sensititre system for identification of Enterobacteriaceae.

The Sensititre identification system (Seward Laboratory/GIBCO Laboratories) consists of a microplate containing a pattern of 24 biochemicals repeated four times together with an automatic inoculation device and a microcomputer-assisted data interpretation component. A total of 1,415 isolates of Enterobacteriaceae plus 6 isolates of other glucose-fermenting gram-negative bacilli were tested in three hospital laboratories in parallel with API 20E (Analytab Products). Discrepancies were resolved by conventional biochemical testing. Sensititre yielded correct identifications at the species level with 94.6% of the isolates and at the genus level with an additional 1.9%. API 20E yielded correct species identification with 91.1% and genus only identification with an additional 6.7% of the isolates. For the routine identification of clinical Enterobacteriaceae isolates, the Sensititre system compares favorably with API 20E and offers clinical laboratories the economy of a microtiter plate system as well as the benefit of a microcomputer capable of other microbiological and data management applications.     

Identification of clinically relevant viridans group streptococci by phenotypic and genotypic analysis

Two phenotypic and three molecular methods were assessed for their ability to identify viridans group streptococci (VGS) to the species level. A panel of 23 clinical isolates, comprising strains isolated from infective endocarditis, blood cultures, pleural and peritoneal fluid, and 19 type/reference strains were analyzed. Identification was performed using two conventional phenotypic methods: API? rapid ID 32 Strep and the VITEK? 2 system, and genotypic analysis of the nucleotide sequence of the housekeeping gene sodA, restriction patterns generated by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene and multilocus sequence analysis (MLSA) of seven housekeeping genes. The API? rapid ID 32 Strep accurately speciated 79% of the strains assessed, while the VITEK? 2 generated a successful identification for 55%, presenting limitations particularly with regard to species belonging to the mitis group. RFLP of the 16S rRNA gene correctly speciated 24% of the strains, having failed to allocate a species for 36% of the isolates examined. In contrast, sequence analysis of the sodA gene provided a correct identification for 95% of the strains assessed, while identification using the MLSA technique was unsuccessful due to practical limitations. The results generated herein indicate that no single methodology can be used to provide an accurate identification to the species level of all VGS, although nucleotide sequence analysis of the sodA gene proved to be useful in providing reliable speciation.     

Use of a genus- and species-specific multiplex PCR for identification of enterococci

The 16S rRNA gene has previously been used to develop genus-specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (sodA) has been identified as a potential target for species differentiation of enterococci. In this study, Enterococcus genus-specific primers developed by Deasy et al. (E1/E2) were incorporated with species-specific primers based upon the superoxide dismutase (sodA) gene for development of a multiplex PCR. This assay provides simultaneous genus and species identification of 23 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to standard biochemical testing, the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources, and retail food were evaluated and, overall, results for 90% of the isolates tested by PCR agreed with results obtained using standard biochemical testing and VITEK. Eighty-five percent and 82% of PCR results agreed with results from the API Rapid ID 32 Strep and BBL Crystal tests, respectively. With the exception of concurrence between identification using standard biochemical testing and VITEK (85%) and between BBL Crystal and VITEK (83%), the percent agreement for PCR was higher than or equal to any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.