INTRODUCTION

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Since the description of the first Facklamia species in 1997, four additional species of this genus have been described (5-8, 17). The species Facklamia hominis, F. ignava, F. sourekii, and F. tabacinasalis are most often arranged in chains, whereas F. languida is most often arranged in clusters with very little chaining. Dolosigranulum pigrum was described in 1993 (2). This bacterium is arranged in pairs, tetrads, and clusters. Ignavigranum ruoffiae, recently described (9), is arranged primarily in chains. Information on the identification of these newly described bacteria is minimal, and in most cases the authors initially describing these species have used a combination of molecular characterization, conventional tests, and miniaturized rapid tests such as the API 50CH (Biomerieux, Inc., Hazelwood, Mo.). The identification of these bacteria is problematic since none of the rapid testing systems have them in their databases. We also included seven strains of Alloiococcus otitidis for comparison purposes since the phenotypic characteristics of this organism resemble those of Dolosigranulum, Facklamia, and Ignavigranum strains at the genus level.

In a previous study examining 120 strains of unidentified gram-positive cocci with phenotypic characteristics that eliminated them from the known genera of gram-positive bacterial genera, e.g., Aerococcus, Enterococcus, Gemella, Lactococcus, and Streptococcus, we identified 18 strains as Facklamia species, 27 strains of D. pigrum, and 3 strains of I. ruoffiae by using conventional test methods (11). Rapid identification test systems for gram-positive cocci have been used for nearly 20 years. We evaluated three systems for their capability to correctly identify the A. otitidis, D. pigrum, I. ruoffiae, and Facklamia species: the rapid ID 32 STREP (ID32; Biomerieux, Inc., Hazelwood, Mo.) (15), the BBL Crystal Rapid Gram-Positive ID Kit (Crystal; BD Bioscience, Cockeysville, Md.) (20), and the RapID STR (Remel, Inc., Lenexa, Kans.) (3, 16, 19). The ID32 system is a modification of the API 20S system (16, 19). The Remel RapID STR system is the same test kit previously distributed by Innovative Diagnostics, Inc., as the IDS RapID STR system and is referred to here simply as IDS.

	MATERIALS AND METHODS

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The 55 strains tested were taken from the culture collection of the Streptococcus Laboratory at the Centers for Disease Control and Prevention. All strains were identified to the genus level by previously described conventional tests (11) (see Tables 1 and 2). Seventeen of the strains were additionally identified by 16S rRNA sequencing. The sources of these gram-positive cocci were similar to those of the viridans streptococci; 24 cultures were isolated from blood cultures, 6 were isolated from cultures of the eye, 4 were isolated from nasopharyngeal swabs, 2 cultures each were from cerebrospinal fluid and abscesses, and one each was from cultures of bone, ear, gall bladder, gastric contents, sinus, sputum, urine, vagina, wound, and unknown. All seven cultures of A. otitidis were isolated from inner ear cultures. Antimicrobial susceptibilities, sources, clinical diagnosis, and other demographic information on these organisms will be reported elsewhere (L. L. LaClaire and R. R. Facklam, submitted for publication).

The ID32 kit and reagents, the Crystal kit, and the IDS system were all used according to the manufacturer's instructions provided. Most strains were tested at least two different times. Some strains were tested four times.

	RESULTS AND DISCUSSION

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Conventional tests. Strains were first identified as potential Dolosigranulum, Facklamia, and Ignavigranum spp. by determining unique phenotypic characteristics in conventional tests used to identify the different genera of catalase-negative, gram-positive cocci. These three genera are characterized as susceptible to vancomycin, with negative gas production, positive L-pyrrolidonyl--naphthylamide (PYR) reactions, positive leucine aminopeptidase (LAP) reaction, growth in 6.5% NaCl broth, negative bile esculin reaction, and negative growth at 10°C and 45°C, are nonmotile, and are either alpha-hemolytic or gamma-hemolytic on 5% sheep blood agar (Table 1). Since the arrangement of cells into chains or clusters is not reliable and depends on the medium from which the cells were taken for the Gram strain, identification to the genus level must rely on the combination of tests listed in Table 1. The combination of reactions listed above is not unique to the genus level; Alloiococcus otitidis, D. pigrum, Facklamia spp., and I. ruoffiae all have the same phenotypic characteristics. A. otitidis does not grow anaerobically, which differentiates it from the other three genera (1, 13, 18). The cultures we described as "Alloiococcus-like" in a previous study (18) have since been identified as D. pigrum.

ID32. Since A. otitidis gave the same genus identification as the Dolosigranulum, Facklamia, and Ignavigranum cultures, we included seven strains in this study for comparison purposes (Table 1). None of the A. otitidis strains were correctly identified by the ID32, Crystal, or IDS systems. A. otitidis strains presented difficulty in preparing satisfactory inocula for the test systems because of poor growth and adherence properties. These strains must be grown on heart infusion rabbit blood agar for at least 48 to 72 h.

Crystal. As stated above, A. otitidis was included in this study because of the similar genus identification. Six of the seven strains tested in the Crystal system were identified as Micrococcus species. The seventh was identified as Streptococcus pneumoniae (Table 3). The identification of A. otitidis may have been compromised because of the difficulties in preparing the inoculum as previously stated; however, its identification may point out a more serious problem for this system. Micrococcus species are catalase positive, whereas most of the cultures we included in this study were catalase negative. Some of the A. otitidis cultures are catalase positive, but we have found that this is not true in every case. Most of the A. otitidis cultures we have examined have been very weak-to-negative catalase producers.

IDS. None of the A. otitidis strains were correctly identified in the IDS system. Four strains were identified as a probable overlap between G. morbillorum and Aerococcus viridans, two strains were identified as implicit identification for G. morbillorum, one was identified as an implicit identification of S. mitis, and one was identified as a probable S. mitis. Only 3 of 18 (17%) Facklamia strains were identified as "no choice" (Table 3). The identification of F. hominis, F. ignava, and F. sourekii as S. acidiminimus with high levels of confidence with the IDS kit was not surprising. As explained above for the ID32 kit, strains originally identified as S. acidiminimus have been reidentified as F. hominis. It is probable that some of the strains that the CDC Streptococcus Laboratory had identified and distributed as S. acidiminimus were used to generate databases for the IDS system (22). Also, the identifications of the various Facklamia species as G. morbillorum and as various Streptococcus species may be interpreted as poorly growing Streptococcus species or misidentified strains as Gemella that have been used to generate databases. Gemella species do not grow in 6.5% NaCl broth, so strains with profiles identical to the ones described here should be retested for growth in 6.5% sodium chloride. We observed only one "no choice" profile in 27 strains of D. pigrum tested. The majority of D. pigrum strains tested (19 of 27 [70%]), generated one profile number: "inadequate ID, Enterococcus faecalis, 90%." This is an erroneous identification, even though the confidence level is quite low (Table 3). Other misidentifications of Streptococcus, including group A streptococci and Enterococcus species, are inexplicable.