ABSTRACT
We evaluated three rapid identification systems—The Biomerieux rapid ID 32 STREP (ID32), the BBL Crystal rapid gram-positive identification (Crystal), and the Remel IDS RapID STR (IDS) systems—for their ability to identify 7 strains of Alloiococcus otitidis, 27 strains of Dolosigranulum pigrum, 3 strains of Ignavigranum ruoffiae, and 18 strains of 4 different Facklamia species. Since none of these six species of gram-positive cocci are included in the identification databases for these systems, the correct identification for the strains tested should be “unacceptable ID” for the ID32 and Crystal systems or “no choice” for the IDS system. The ID32 system identified all 27 strains of D. pigrum, 6 of 18 Facklamiaspecies, and 2 of 3 cultures of I. ruoffiae as “unacceptable ID.” The Crystal system identified 10 of 27 D. pigrum, 2 of 18 Facklamia species, and 2 of 3I. ruoffiae strains as “unacceptable ID.” The IDS system identified only 1 culture of D. pigrum as “no choice,” but it also identified 2 cultures of D. pigrumas a “questionable microcode” and 19 cultures of D. pigrum as an “inadequate ID, E. faecalis 90%,S. intermedius 9%.” A total of 2 of the 18 cultures ofFacklamia and all 3 of the I. ruoffiaecultures were correctly identified as “no choice.” The most common misidentifications of Facklamia species by the ID32 and IDS systems were as various Streptococcus species and asGemella species. In the Crystal system, the most common erroneous identification was Micrococcus luteus. These data indicate the need for the commercial manufacturers of these products to update their databases to include newly described species of gram-positive cocci.
Since the description of the firstFacklamia species in 1997, four additional species of this genus have been described (5-8, 17). The speciesFacklamia 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 pigrumwas 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 ofAlloiococcus otitidis for comparison purposes since the phenotypic characteristics of this organism resemble those ofDolosigranulum, Facklamia, andIgnavigranum 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 theA. 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
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 Tables1 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
Conventional tests.Strains were first identified as potentialDolosigranulum, Facklamia, andIgnavigranum 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-β-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 (Table1). 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.
Phenotypic characteristics of catalase-negative, gram-positive cocci
Facklamia species are differentiated from each other and from D. pigrum and I. ruoffiae by the deamination of arginine (Arg), by hydrolysis of hippurate (Hip) and esculin (Esc), and by acid formation in sucrose (Suc) and sorbitol broth (Sbl) (Table 2). D. pigrum is positive only for Esc; all other species, includingI. ruoffiae, are negative for Esc. I. ruoffiae and F. languida are both negative in all five tests; however, I. ruoffiae has a distinctive sauerkraut odor on blood agar, which helps to identify the species.F. hominis is positive for Arg and Hip and variable in Suc. F. ignava is positive only for Hip. F. sourekii is positive for Hip, Suc, and Sbl. The newest species,F. tabacinasalis, not yet documented from humans, is positive for Suc and Sbl only. This combination of tests identifies all of these strains to the species level.
Phenotypic characteristics of D. pigrum,I. ruoffiae, and Facklamia species as shown by conventional biochemical tests
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.
The ID32 system correctly identified 7 (39%) of the 18Facklamia species as “unacceptable ID.” Those that were correctly identified were 3 of 4 strains of F. hominis, 3 of 5 strains of F. ignava, none of 6 strains ofF. languida, and 1 of 3 strains of F. sourekii (Table 3). TheF. languida strains were most often misidentified asGemella morbillorum, as was one strain of F. ignava. This misidentification is somewhat understandable since there is little difference between the Gemella spp. andFacklamia spp.; however, the correct division of the two genera can be made with growth in 6.5% sodium chloride. Only theFacklamia species grow in broth containing 6.5% sodium chloride. Another common misidentification was Facklamiaspecies being identified as S. acidiminimus; this occurred with three different strains of Facklamia. This, too, may be understandable since the Centers for Disease Control and Prevention (CDC) Streptococcus Laboratory has distributed several strains previously identified as S. acidiminimus, which were reidentified as Facklamia species. In fact, we included these strains in previous studies of rapid identification kits (10, 12, 22). This points out a need to update the identification of strains obtained from the CDCStreptococcus Laboratory and to update the commercial databases as well.
Identification of A. otitidis, D. pigrum, Facklamia species, and I. ruoffiae by three commercial rapid identification systems
Although 46 profiles were generated from testing 27 strains of D. pigrum in the ID32 test system, all 46 profiles resulted in an “unacceptable ID” (Table 3). These results were very encouraging since the product's manufacturer would only have to include profiles we have generated for D. pigrum in its database to accurately identify this species.
Two of the three strains of I. ruoffiae were correctly identified by the ID32 system. The third strain tested had a doubtful profile. Not enough numbers of this species have been tested to conclude that the system can be adjusted to identify the species accurately; however, these results are encouraging.
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 asMicrococcus species. The seventh was identified asStreptococcus pneumoniae (Table 3). The identification ofA. 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.
Like the ID32 system, the Crystal system identified seven (39%) strains of Facklamia species as “unacceptable ID” (Table3). One of four strains of F. hominis, three of five strains of F. ignava, none of six strains ofF. languida, and three of three strains ofF. sourekii were correctly identified. Of the 18 strains of Facklamia tested, 13 were identified as variousMicrococcus species. These misidentifications indicate the need to include catalase reactions in the identification schemes for gram-positive cocci.
Among the 27 strains of D. pigrum, 17 “unacceptable ID” profiles were observed with 14 different strains (Table 3). This indicates that half of the cultures could be accurately identified by incorporating these newly generated profile numbers into the Crystal database. As with the Facklamia species (indicating a problem that arises by not including the catalase test into the identification process), eight strains of D. pigrumgenerated eight profile numbers that were identified as variousMicrococcus species. Eleven different profile numbers identifying strains as various streptococcal species were observed with 11 different strains of D. pigrum. The majority of these profiles had low percentages of confidence for identification (Table3).
We observed three profiles with the three strains of I. ruoffiae: one was an “unacceptable ID” and two were low-level confidence M. luteus. These misidentifications again indicate the need to include catalase in the identification scheme.
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%) Facklamiastrains 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 asF. 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 (Table3). Other misidentifications of Streptococcus, including group A streptococci and Enterococcus species, are inexplicable.
A discouraging point for all three test kits is the incidence of generated profile numbers shared among different species and genera. The ID32 had one instance in which a single strain of F. hominis and F. ignava had the same profile. This phenomenon occurred twice with the Crystal system among two different strains of F. ignava and among three different strains of F. hominis. The Crystal system also generated shared profiles between different genera: F. ignava andF. sourekii with Alloiococcus sp. The IDS system had three instances in which F. sourekii andF. ignava shared profiles, as well as two instances in which three different strains of F. hominis andF. ignava had the same profiles.
Previous investigators have used a combination of conventional tests and reactions from one or more of the rapid identification systems to describe and differentiate the newly identified genera and species (2, 4-9, 17). We explored the possibility that each one of the three systems we evaluated could be used to identify the species ofAlloiococcus, Dolosigranulum,Facklamia, and Ignavagranulum. When we used conventional tests first to identify these four genera with common phenotypic traits, we then tested these rapid kits to determine whether they could identify all the species of these genera. Although we have included Alloiococcus strains in these investigations, it is unlikely that microbiologists would confuse this bacterium with the other genera in Tables 4,5, and 6.A. otitidis is a strict aerobe and, to our knowledge, has been isolated only from inner ear cultures from patients with otitis. The results of using the ID32 for differentiation of D. pigrum and I. ruoffiae from theFacklamia species are presented in Table 4. Although not perfect, combinations of reactions can identify most of the species.D. pigrum can be identified by positive reactions in arginine and β-galactosidase (β-Gar), by acidification of trehalose and maltose, and by negative reaction in alanine-phenylalanine-proline aryalamidase (APPA). F. hominis can be identified by positive reactions in β-Gar, APPA, β-galactosidase (β-Gal), and glycyl-tryptophane arylamidase (GTA) and by negative reactions in trehalose. F. ignava is problematic because there are too many variable reactions to the majority of tests in the ID32 system. F. languida is easily identified because it is positive in only 2 of the 14 tests: acid formation in trehalose and GTA. F. sourekii is identified by acidification of mannitol, sorbitol, trehalose, maltose, d-arabitol, and saccharose and by hydrolysis of Hip. Only a single nonhuman isolate ofF. tabacinasalis is represented in Table 4, so these results should be cautiously interpreted. I. ruoffiaeis identified by positive reactions in Arg, acidification of mannitol, trehalose, and saccharose, and hydrolysis of hippurate and by negative reactions in β-Gar, acidification of sorbitol, APPA, β-Gal, and GTA.
Potentially useful tests included in the ID32 system for differentiating unusual gram-positive cocci
Potentially useful tests included in the Crystal system for identifying unusual gram-positive cocci
Potentially useful tests included in the IDS system for identification of unusual gram-positive cocci
The same process can be used for the Crystal system (Table 5). The reactions of A. otitidis overlap somewhat with the reactions of some of the D. pigrum and Facklamia species.D. pigrum is FPY (l-pyroglutamic acid-AMC), FTR (l-trytophan–7-amino-4-methylcoumarin [AMC]) and BGL (p-n-p-β-d-glucoside) positive. F. hominis is FPR (l-proline-AMC), FTR, and PPG (p-n-p-β-galactoside andp-n-α-d-galactoside) positive. F. ignava is problematic because of the number of variable reactions.F. languida is identified by a positive reaction in FPY and negative reactions in FPR, MTT (acid formation in maltotriose), BGL, and PPG. F. sourekii is identified by positive reactions in FPY and BGL and negative reactions in MTT and PPG. Again, only a single strain of F. tabacinasalis (nonhuman) was tested, so these reaction percentages should be interpreted cautiously.I. ruoffiae is identified by positive reactions in FPY and FTR and by negative reactions in BGL and PPG.
Attempting to apply the same interpretation to the IDS system is more difficult because of the limited number of tests with positive reactions (Table 6). Cultures of F. hominis andI. ruoffiae appear to have unique profiles, but the cultures of D. pigrum and the remainder of theFacklamia species have a common profile.
We have previously compared various reactions included in rapid identification tests with conventional tests and have indeed found differences (14). Comparing the individual tests in these systems to the conventional tests resulted in several interesting observations. In the conventional disk test we use for the PYR test (l-pyrrolidonyl-β-naphthylamide) all of the D. pigrum, Facklamia species, and I. ruoffiae cultures were positive. However, in the ID32 test, two strains of D. pigrum, one strain of F. hominis, four strains of F. ignava, two strains ofF. languida, and one strain I. ruoffiaewere negative. These numbers were similar to those for the Crystal and IDS PYR tests. In the conventional test for deamination of Arg, only the F. hominis and I. ruoffiae strains were positive. But in the ID32, Crystal, and IDS systems, 88, 79, and 79% of the D. pigrum cultures, respectively, were positive. Of the F. ignava cultures, three of five, four of five, and three of five strains were positive for Arg in the ID32, Crystal, and IDS systems, respectively; none of these cultures were positive in the conventional Moeller's decarboxylase tests. These results were similar to those of West et al. (21), who also reported major differences in Arg hydrolysis by viridans streptococci, depending on the rapid test used.
It is apparent that the manufacturers of these rapid identification kits need to update their databases by including the identification of newly described genera and species of gram-positive cocci, including the Dolosigranulum, Facklamia andIgnavigranum species.
FOOTNOTES
- Received 22 December 1999.
- Returned for modification 11 February 2000.
- Accepted 8 March 2000.
- Copyright © 2000 American Society for Microbiology
