Use of the Phoenix Automated System for Identification of Streptococcus and Enterococcus spp

Clinical isolates.Clinical isolates were obtained from routine clinical specimens at the Microbiology Laboratory of the Ospedale di Circolo, Varese, Italy. A total of 200 nonduplicated isolates of gram-positive, catalase-negative cocci were studied. The following strains were investigated: S. pneumoniae (n = 46), S. pyogenes (n = 15), S. agalactiae (n = 15), Streptococcus dysgalactiae subsp. equisimilis (n = 20), viridans group streptococci (n = 31), E. faecium (n = 36), E. faecalis (n = 25), other enterococcal species (n = 9), and other catalase-negative, gram-positive cocci (n = 3). Isolates were stored at −70°C in Todd-Hewitt broth containing 20% glycerol. Before ID assays were performed, all strains were passed twice on Mueller-Hinton agar containing 5% sheep blood (Oxoid SpA, Milan, Italy) to get them to an active-growth stage following metabolic inactivity while frozen.

Phoenix system procedures.The Phoenix system uses different panels for gram-positive cocci. The SMIC/ID-2 panel is dedicated to streptococci and the PMIC/ID-14 panel to enterococci and staphylococci. All panels include two separate sections: wells on the left contain ID substrates, and wells on the right side are dedicated to AST. Panel inoculation was performed according to the manufacturer's instructions. Both panel sections were inoculated, but only ID results have been taken into consideration for this study. After overnight culture, bacteria were suspended in the ID broth. Turbidity was adjusted to a 0.5 McFarland standard by using the CrystalSpec Nephelometer (Becton Dickinson). Panels were then sealed, logged, loaded into the instrument, and incubated at 35°C. Kinetic, colorimetric, and fluorescent signals were automatically collected by the instrument every 20 min until results were completed.

Comparator biochemical ID method.Two different API ID systems (bioMérieux, Marcy l'Étoile, France) were used to identify streptococcal and enterococcal isolates at the species level. The API 20 Strep system was used for beta-hemolytic streptococci. The rapid ID 32 Strep system was used for enterococci and non-beta-hemolytic streptococci. Inoculation, reading, and interpretation of panels were performed according to the manufacturer's instructions.

Data analysis and resolution of discrepancies.Isolates that were equally identified at the species level by both the API and the Phoenix systems were considered to be correctly identified and included in the “concordant ID” category. Due to the inability of the Phoenix system to discriminate between Enterococcus casseliflavus and Enterococcus gallinarum, the classification of an isolate as E. casseliflavus/E. gallinarum by the Phoenix system was considered concordant when the isolate was identified by the API system as either E. casseliflavus or E. gallinarum. Isolates with discordant species ID (i.e., an ID produced by the Phoenix system that differed from that obtained with the API system) were retested using both the Phoenix and the API systems. When discrepant results persisted, bacterial ID was investigated by molecular methods.

Amplification and sequencing of the 16S rRNA gene.Bacterial DNA was extracted from pure cultures by using a QIAamp DNA mini kit (QIAGEN, Basel, Switzerland). DNA eluted in Tris-EDTA was stored at −80°C. ABI 2400 thermal cyclers and PCR reagents were from Applied Biosystems (Foster City, CA). AmpliTaq Gold polymerase, PCR buffer II, standard deoxynucleoside triphosphate mixture, and the universal 16S rRNA gene primers 8f (5′-GAGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-TACGGCTACCTTGTTACGACT-3′) were used to produce a 1,498-bp amplicon (23). Amplification products were purified using a Mini Elut PCR purification kit (QIAGEN) and directly sequenced using an ABI 310 genetic analyzer (Applied Biosystems). Sequences for both DNA strands were determined, each by using the product of a different PCR as a template. Analysis and comparison of sequence data were carried out at the BLAST interface (http://www.ncbi.nlm.nih.gov/BLAST/ ) and ClustalW interface (http://www.ebi.ac.uk/clustalw/ ) websites.

Amplification and sequencing of housekeeping genes of viridans group streptococci.Samples were amplified with degenerate primers specific for the internal fragments of the zwf (encoding glucose-6-phosphate dehydrogenase) and gki (encoding glucose kinase) streptococcal genes (21). Two primer pairs were used: 5′-CCG(T/G)ATCGACCATTA(T/C)CTTGG(T/C)AAGG-3′ and 5′-TC(A/T)GTCAG(T/A)CGTTTACCTGT(A/G)CGGA-3′ for the zwf gene and 5′-GGCATTGGAATGGGATCACCAGG-3′ and 5′-CCGATAA(C/T)TCCAGCGTCATTTCC-3′ for the gki gene. Amplicons of 453 bp (zwf) and 624 bp (gki) were directly sequenced.

Amplification of housekeeping genes in enterococci.The ID of E. faecium and E. faecalis isolates was confirmed by amplification of a fragment internal to the ddl gene encoding a d-Ala-d-Ala ligase (4, 6). The reaction mixture contained 250 ng of DNA as a template, 50 pmol of each primer, 200 pmol per liter of each deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP), 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl₂, 50 mM KCl, and 2 U of AmpliTaq Gold. Upon electrophoresis on a 2% agarose gel, ethidium bromide-stained DNA fragments were visualized under UV light with a Kodak CF440 camera (NEN Life Science Products, Boston, MA).

Quality controls.The following strains were included in each run: S. pneumoniae ATCC 49619, S. agalactiae ATCC 13813, and E. faecalis ATCC 29212. The identification results obtained with the above-mentioned reference strains were consistently satisfactory.

Biochemical identification of clinical isolates.Compared with the API system, the Phoenix system correctly identified 180/200 (90.0%) test organisms at the species level: 116/129 (89.9%) streptococci, 63/70 (90%) enterococci, and 1/1 Aerococcus viridans isolate. Results are summarized in Table 1.

Concordant ID for S. pneumoniae was obtained in 45/46 cases, since one isolate (identified as S. pneumoniae by the API system) was classified as a “not identified organism” by the Phoenix system. After resolution of discrepancies, however, this isolate was ultimately defined as Streptococcus mitis by sequencing the 16S rRNA gene. Fourteen out of 15 (93.3%) S. pyogenes and 21/31 (67.7%) viridans streptococci isolates were correctly identified by the Phoenix system. Among the latter, 8/8 members of the Streptococcus anginosus group, one Streptococcus mutans isolate, 3/4 members of the Streptococcus sanguinis group, and 9/17 members of S. mitis group were correctly identified. Correct species ID was obtained for 90% of the enterococci. Four E. faecium and three E. faecalis isolates were misidentified as E. casseliflavus/E. gallinarum or Enterococcus durans. Thus, these isolates were identified only at the genus level. Overall, the Phoenix system correctly identified all isolates of the following species: S. pneumoniae, S. agalactiae, S. dysgalactiae subsp. equisimilis, and Streptococcus bovis.

Analysis of discrepancies.Species ID of 20 discordant isolates was investigated by molecular methods. As shown in Table 2, sequencing of the 16S rRNA gene assessed that one S. mitis isolate was not identified by either the API or the Phoenix system. One S. pyogenes isolate (not identified by the Phoenix system) was correctly identified by the API system. Five isolates (which had been assigned to the S. mitis or Streptococcus salivarius group by the API system) did belong to the S. sanguinis group (Streptococcus parasanguinis, n = 2; S. sanguinis, n = 2; and Streptococcus gordonii, n = 1). The above-mentioned five isolates were correctly classified at the species level by the Phoenix system, not by the API system. One discordant isolate (resolved by 16S rRNA gene sequencing as S. gordonii) was not identified by the Phoenix system and was identified only at the group level by the API. Sequencing of the 16S rRNA and the housekeeping zwf and gki genes failed to resolve five additional isolates at the species level. The most probable molecular ID for those five isolates appeared to be S. mitis, Streptococcus oralis, or S. pneumoniae (species belonging to the S. mitis group) (26).

Concerning seven enterococci identified by the Phoenix system as E. casseliflavus/E. gallinarum (n = 5) or Enterococcus durans (n = 2), PCR analysis of the ddl gene confirmed the IDs given by the API system (four E. faecium and three E. faecalis isolates).

The performance of the Phoenix and API systems with regard to discordant isolates is summarized in Table 3. Of 13 streptococci, 9 were correctly identified at the species or group level by the Phoenix system and 5 by the API system. Three isolates could not be identified by either system. The correct IDs for seven discordant enterococci were given by the API system, not by the Phoenix system. Of the streptococci that could not be resolved at the species level by molecular methods, 4/5 were identified at the group level by both the API and the Phoenix systems. Overall, taking into consideration species IDs given by molecular methods, the accuracy of the Phoenix system in identifying streptococci rose from 89.9% to 93.8%. For enterococci, accuracy of ID at the species level remained at 90%.