and Streptococcus Species

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1986, p /86/ $02.00/0 Copyright 1986, American Society for Microbiology Vol. 24, No. 4 New Test System for Identification of Aerococcus, Enterococcus, and Streptococcus Species MABEL S. YOU' AND RICHARD R. FACKLAM2* Emory University, Atlanta, Georgia 30322,1 and Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia Received 28 April 1986/Accepted 2 July 1986 A total of 244 strains of Aerococcus, Enterococcus, and Streptococcus species were tested by the RapID STR system (Innovative Diagnostic Systems Inc., Atlanta, Ga.) for identification. Strains were without additional tests or with additional conventional tests suggested by the IDS compendium manual. Our data indicate that the RapID STR system identifies 89% of the beta-hemolytic Streptococcus species if serological procedures are used in conjunction with the rapid physiological procedures. Of the group D streptococci, 98% of the Enterococcus species and 100% of the group D non-enterococcus species were correctly. Of the commonly occurring viridans group Streptococcus species, 93% were correctly, and 79% of the less frequently occurring viridans group Streptococcus species were correctly. All of the Streptococcus pneumoniae and Aerococcus strains tested were correctly. The identification of Streptococcus species is accomplished by a combination of serological and physiological tests. Convenient serological tests are available for the rapid identification of the beta-hemolytic Streptococcus species (10, 14). Rapid, convenient identification systems such as the API-20S (2, 9), the Rapid Strep system (API-20 Strep) (1, 11, 19), and the gram-positive identification system (GPI; Vitek Systems, Inc., Hazelwood, Mo.) (2, 9, 18) have been proposed as tools for identifying the group D and viridans group Streptococcus species. All of these systems are able to identify the group D Streptococcus species with 95% or better accuracy. However, none of these systems have more than 85% of the viridans group Streptococcus species even with additional testing. Identification of pneumococci has had mixed success by all previously evaluated identification systems. Generally, none of the rapid systems have accomplished a more convenient identification than the presumptive identification by the optochin test. We tested a recently marketed system, the RapID STR system, available from Innovative Diagnostic Systems, Inc. (IDS), Atlanta, Ga., for identification of aerococci, enterococci, and streptococci isolated from humans. MATERIALS AND METHODS The RapID STR system and inoculation fluid were obtained from IDS. The RapID STR system is packaged with 20 RapID STR panels, 1 dropper tube of RapID STR reagent, 20 report forms, and 1 package insert. The RapID inoculation fluid is not supplied with the RapID STR system but is available from IDS. The panel consists of 10 cavities of dehydrated substance for determining 14 physiological characteristics, of which 4 are bifunctional (two tests are contained in the same cavity). Five-digit profile numbers were obtained by adding the numerical values for the test results in the panel. The four bifunctional cavities were first scored without reagent addition for the initial test result, and then the same cavity was scored after reagent addition providing the second test result. The hemolysis reaction provided the 15th test result * Corresponding author. 607 for determining physiological characteristics. Beta-hemolysis is scored positive. Other types of hemolysis are scored negative. The identification and level of identification for each strain were obtained by matching the profile number to the profile identification number provided in the code compendium of the manufacturer. The current (March 1986) data base of the manufacturer was used to confirm all identifications generated by the IDS RapID STR system panels. Five levels of identification are described by IDS. Species level identification requires a probability level of 95% or greater as the first choice. These correct identification levels are given as implicit (I), satisfactory (S), and adequate (A), which do not require additional testing. If species level of identification is less than 95%, two levels of identification are possible; questionable (Q) and inadequate (IQ). When both the Q and IQ levels of identification are observed, RapID STR panels are repeated and the recommended additional tests included in the instructions are performed. A list of additional characteristics for beta-hemolytic and non-betahemolytic streptococci is provided in the RapID STR code compendium. Streptococcus strains. All of the strains tested were clinical isolates from humans and were selected from the culture collection of the Streptococcus Reference Laboratory, Bacterial Diseases Division, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Ga. All strains were gram-positive cocci arranged in chains or clusters. All streptococci were catalase negative when tested for effervescence of hydrogen peroxide (12). All strains were susceptible to vancomycin disks (30 mg) when tested by a previously described method (9). All Aerococcus strains were cytochrome negative when tested for cytochromes by the benzidene test (7). Hemolysis reaction and purity were determined under a dissecting microscope (x70). All strains were initially by the physiological and serological procedures described in the Manual of clinical microbiology (7). Reference specimens treated as quality control (QC) organisms for each possible species identification listed in the RapID STR system compendium were tested. The QC strains were taken from the stock culture collection of the Streptococcus Reference Laboratory. Results of QC tests are not included in the following tables.

2 608 YOU AND FACKLAM J. CLIN. MICROBIOL. TABLE 1. Levels of identification of beta-streptococci by RapID STR system Taxon No. of species Without adda tests With add tests incorrectly StAsteIQ Group Ab Group Bb Group C S. equi 2 2 S. equisimilis S. zooepidemicus 8 8 Group G Group F S. anginosus Group A Group C Group G Nongroupable 5 5 aadd, Additional. b Two nonhemolytic variants each for groups A and B are included. Inoculum. All strains were inoculated on Trypticase soy agar base (BBL Microbiology Systems, Cockeysville, Md.) containing 5% defibrinated sheep blood. The inoculated Trypticase soy agar base was incubated in a candle extinction jar at 35 C for 24 to 48 h. The instructions of the manufacturer recommended that Trypticase soy agar base be incubated anaerobically or in 5 to 7% CO2 for less than 50 h. Blood agar plates containing or supplemented with monoor disaccharides (i.e., Schaedler agar) are not recommended, since they may suppress glycolytic activity and reduce test selectivity. A bacterial suspension prepared in the RapID inoculation fluid (1 ml of a solution containing 0.75% KCl and 0.05% CaCl2) of a turbidity greater than that of a McFarland no. 1 standard was used to inoculate the RapID STR panels. A less turbid suspension may result in weak reactions. After the panels were incubated aerobically at 35 to 37 C in a non-co2 atmosphere, reactions were read at 4 h. All of the panels were read and interpreted by one individual with no knowledge of the identity of the strains. RESULTS Of the beta-hemolytic streptococci, the RapID STR system beta-hemolytic groups A, B, C, G, and F and Streptococcus anginosus. The RapID STR system is also capable of identifying nonhemolytic variants of group A and group B streptococci. All QC strains were to the correct species without additional tests. The results of testing 75 strains of beta-hemolytic streptococci are shown in Table 1. Serological testing was suggested for confirmatory identification. Of the 75 strains, 65 (87%) were at the I or S level. Two of three S. anginosus group G strains were correctly at the Q level. Five nongroupable S. anginosus strains and three group A S. anginosus strains were incorrectly by the same profile identification number as group C, G, or F streptococci. The testing of two strains of nonhemolytic group A and one strain of nonhemolytic group B resulted in S levels of identification. One strain of non-hemolytic group B was at an IQ level but was correctly with additional tests. The RapID STR system recognizes Enterococcus species according to recent classification proposals (6, 20). The QC strains of E. faecium, E. faecalis, and E. hirae were correctly to species level without additional testing; however, QC strains of E. durans, E. malodoratus, E. casseliflavus, and E. gallinarum were identifed to the IQ level and subsequently were correctly with additional tests. The results of testing 48 strains of Enterococcus species are shown in Table 2. Of the 48 strains, 43 (90%) were correctly to the I or S level. Two strains of E. faecium variant and E. avium variant and one strain of E. hirae required additional tests for correct identification (8%). One E. faecium variant was incorrectly with no profile identification number found in the code compendium. The results of testing 20 strains of group D nonenterococci, S. bovis, and S. bovis variants are also shown in Table 2. The QC strains for both species were to the I level. Of the 20 strains, 18 (90%) were correctly without additional tests. Two strains of S. bovis were correctly only to the IQ level with additional tests. The RapID STR system recognizes 10 species of viridans group streptococci (10). Of the 10 QC species, 7 were correctly to the S level with no additional test. The QC strains for S. intermedius and S. sanguis II were at an IQ level and required additional characteristic testing for correct identification. A S. uberis QC strain was mis as E. faecalis. The results of testing 81 strains of viridans group streptococci are shown in Table 3. Of the 81 strains, 50 (62%) were correctly without additional tests to the I, S, and A levels. The testing of 25 strains (31%) resulted in an IQ level of identification and required additional characteristic tests for correct identification. Of the 25 strains at the IQ level, 13 (52%) were S. mitis and S. intermedius. A total of six viridans group strains were incorrectly (7%). One strain each of S. mutans, S. intermedius, S. constellatus, and S. sanguis I were mis as S. bovis, S. salivarius, S. mitis, and S. sanguis II, respectively. Two strains of S. acidominimus generated profile numbers not included in the code compendium. Identification of Aerococcus sp. and S. pneumoniae is also possible with the RapID STR system. An S level of identification was achieved for both quality control strains. Of the 10 S. pneumoniae strains tested, 8 strains were correctly

3 VOL. 24, 1986 RapID STR SYSTEM 609 TABLE 2. Levels of identification of group D series by RapID STR system Taxon ntested No. of species Without adda tests With add tests incorrectly Enterococcus faecalis E. faecium E. faecium var E. avium E. avium var E. durans 4 4 E. hirae Streptococcus bovis S. bovis var a add, Additional. to the I or S level without additional tests, and 2 (10, 13). Group A S. anginosus strains do not possess the strains were inadequately and required additional same virulence factors possessed by group A S. pyogenes tests. Of the 9 Aerococcus sp. tested, 8 strains were cor- strains (10). This system could be used to accurately identify rectly without additional tests and 1 strain required S. pyogenes and S. agalactiae. However, the group C additional tests for correct identification. species (S. equisimilis, S. equi, and S. zooepidemicus) as well as S. anginosus were not as accurately by DISCUSSION RapID STR as they are by the other rapid identification Most of the strains used in our evaluation of the RapID methods we have evaluated (9, 10). STR system were the streptococci commonly encountered in Our results are comparable to those reported by Apthe clinical laboratory. However, we also included a few plebaum et al. (3) with group A, B, and G streptococci. atypical strains of streptococci not frequently encountered in However, Applebaum et al. did not test the diversity of other clinical specimens. Users of this system should be aware of beta-hemolytic streptococci we tested; hence, the results the nomenclature changes suggested by several authors, cannot be compared. which this system has adopted (6, 20). S. anginosus is a The RapID STR identification of the common Enterococminute colony-forming beta-hemolytic Streptococcus sp. cus species was similar to the identification of these species that can be further divided into serological group A, C, or G, as members of the Streptococcus genus by other or nongroupable (8). The RapID STR system offers an systems (API-20S, GPI, or Rapid STREP system). All alternative nomenclature of calling these strains "S. mil- strains of E. faecalis, E. faecium, E. durans, and E. avium leri." This system recognizes that different Streptococcus were to the species level with 100% accuracy. species mnay possess common group antigens (group A, S. Variant strains of E. faecium and E. avium included in this pyogenes and S. anginosus; and group C, S. equisimilis, S. study were not as well as were the typical strains. anginosus, and S. zooepidemicus). Like mnost other investi- The variant E. faecium strains differ from typical strains by gators, we agree that serogrouping is a more rapid, efficient, forming acid in raffinose broth. Four of the five variant E. and useful technique for identifying most clinical isolates of faecium tested formed acid raffinose in the RapID STR beta-hemolytic streptococci than are rapid biochemical iden- panel. This indicates that the identification of these variant tification procedures. However, serogrouping does not cor- strains could be accomplished easily in the RapID STR relate with accurate species identification and in some cases system. Variant E. avium strains differ from typical E. avium may lead to erroneous assumptions concerning the pathoge- strains by forming acid in melibiose and raffinose broths, nicity of the strain. Beta-hemolytic strains of S. while the typical strains do not. The variant E. avium strains anginosus with group A antigen are occasionally isolated differ from E. malodoratus by forming acid in arabinose TABLE 3. Levels of identification of viridans group streptococci by RapID STR system Taxon No. tested of species Without adda tests With add tests incorrectly S. mutans S. mitis S. intermedius S. constellatus S. morbillorum S. sanguis I S. sanguis II S. salivarius S. acidominimus S. uberis a add, Additional.

4 610 YOU AND FACKLAM broth, whereas the prototype strain of E. malodoratus does not. Of the three differential tests required to identify E. avium and E. avium var. from E. malodoratus, only raffinose and arabinose are included in the RapID STR panel. Two of the four variant E. avium strains tested formed acid in the raffinose cavity in the RapID STR system, indicating a less than ideal correlation between conventional and rapid tests. However, all four variant E. avium strains and none of the nine typical E. avium strains tested positively for alphaglucosidase in the RapID STR system. The prototype strains of both E. avium and E. malodoratus do not produce alpha-glucosidase. This indicates that these variant strains could be accounted for easily in the RapID STR system, even though we are not sure of the specific classification. Users of this system should be aware that a supplemental test must be performed to differentiate between E. durans and E. hirae strains. The RapID STR system will differentiate some strains of E. hirae from E. durans by the raffinose reaction (E. hirae is positive, whereas E. durans is negative). However, only two of six E. hirae strains tested were raffinose positive in the RapID STR system. We suggest that the user perform the conventional test for acid formation in raffinose or sucrose broths for a more sensitive result to differentiate between E. durans (negative) and E. hirae (positive) strains. Each reference strain of E. gallinarum, E. casseliflavus, and E. malodoratus was at the IQ level. The testing of type strain of E. gallinarum profile identification number resulted in probability overlap with E. casseliflavus, which was not resolved with the provided IDS additional test chart. This reflects the changes in the physiological description of the two species. Recently, Collins et al. (5) redescribed E. gallinarum and E. casseliflavus in the acid formation of sorbitol and raffinose broths. In the same study, they discussed the differential tests of these two species from a newly characterized species, E. mundtii. Among the reported differential tests, pigmentation and motility are used to differentiate E. casseliflavus from E. gallinarum and E. mundtii. The testing of E. malodoratus profile identification number resulted in probability overlap with E. faecalis. This was resolved by performing the suggested test of acid formation in sorbose broth, positive for E. malodoratus and negative for E. faecalis. The probability overlap that occurred between these organisms may be due to a very small number of strains in the IDS data bank. Applebaum et al. (3) reported similar identification for the Enterococcus species. Strains of E. faecalis, E. faecium, and E. durans were correctly, but strains of E. casseliflavus were mis by the RapID STR system. It is important that any system used to differentiate Streptococcus species provides an accurate identification of S. bovis strains, since the isolation of S. bovis strains from the bloodstream is a potential indicator of colonic cancer (4, 16, 17). The identification of S. bovis by the RapID STR system is similar to that of other rapid identification systems (1, 2, 9, 10, 18, 19). A total of 9 of 11 strains of S. bovis were by the RapID STR system without additional testing. The remaining two strains were correctly by performing the conventional test for glucan production. All nine variant strains of S. bovis were with no additional tests required. When compared with the three other systems, RapID STR showed a higher rate of correct identification for the S. bovis variant strains. This is especially important now that S. bovis variant strains have been associated with colonic neoplasms similar to those in the typical S. bovis strains (4). Ten species of viridans group streptococci are included in J. CLIN. MICROBIOL. the RapID STR system. This system recognizes the taxon "S. milleri" as an alternative for S. intermedius and S. constellatus taxons. However, DNA homology studies have not resolved the taxonomic or physiological differences between these streptococci (15). Our study with the RapID STR system included 81 strains of 10 different viridans group streptococcus species. The overall identification rates were different for different species and numbers of each species tested. When we analyzed our data from the evaluation of the other three Streptococcus identification systems for the seven most commonly occurring viridans group species (S. constellatus, S. intermedius, S. mitis, S. mutans, S. salivarius, S. sanguis I, and S. sanguis II), we found that all three systems correctly 85% of the strains, whereas the RapID STR system 93%. Overall, 62% of the 81 strains were correctly without additional tests, and 31% required additional testing. Fiftysix percent of the seven most commonly occurring species could be correctly without additional tests, and 37% required additional testing. These percentages for correct identification of viridans group streptococci are similar to those reported by Applebaum et al., who evaluated the IDS RapID STR system (3). Applebaum et al. reported that correct identification of the seven most commonly occurring viridans group streptococci was 52% without additional testing and 99% with additional testing. This indicates that in some cases supplemental testing is required by this system for correct identification. For example, in our study 60% of 10 S. mitis strains required additional testing to resolve probability overlap with S. pneumoniae and S. constellatus. Four S. mitis strains were correctly by demonstrating that they were optochin negative, differentiating them from S. pneumoniae, and two S. mitis strains formed acid in lactose broth, differentiating them from S. constellatus. The QC strain of S. intermedius and 88% of 8 S. intermedius strains tested in our study required an additional lactose broth (positive) to differentiate them from S. constellatus. A lactose fermentation test is not included in the RapID STR panel. Unlike the other Streptococcus identification systems, the RapID STR system exceeded the rate of accuracy for identifying the three less commonly occurring species (S. acidominimus, S. uberis, and S. morbillorum). Only 10% were by the other systems (5, 6), whereas 79% were by the IDS system. The majority of successes in identification were S. morbillorum strains. For 10 strains tested, the identification rate was 100% without additional tests. When we excluded S. morbillorum strains, S. acidominimus and S. uberis were correctly 55% of the time, with and without additional tests. Thus, under our test conditions, when supplemental testing was performed, 93% of the commonly occurring strains were correctly. However, without supplemental testing for the common occurring viridans group streptococci, only 62% were correctly. Supplemental testing was not required for S. mutans. Very few strains of S. sanguis II and S. constellatus required additional tests for correct identification. Overall, the rate of error in our study for the 81 strains of viridans group streptococci was 7%. Identification of pneumococci by the RapID STR system requires an optochin test for confirmation. Therefore, we suggest that for simplification and cost effectiveness, a presumptive optochin test should be used to screen for S. pneumoniae before attempting to use this system. Generally, none of the rapid systems have accomplished a more convenient identification than the presumptive optochin test. The accuracy of the RapID STR system in identifying

5 VOL. 24, 1986 Aerococcu,s species was comparable to that of other Streptococcus systems. None of these species was to an accuracy rate of more than 92%. This difficulty in identifying aerococci is reflected by their diverse physiological characteristics. Therefore, any system that would correctly identify these species would be of significant value (10). In conclusion, the results from our comparative study indicate very few differences between the overall Streptococcus identification rates with RapID STR system and other rapid test systems. Each system has advantages and disadvantages that must be assessed by the potential user. The RapID STR system identifies more Enterococcus species. The system includes several tests that were useful in recognizing variant species. However, the RapID STR system did not clearly distinguish between group C Streptococcus species and S. anginosus. The overall rate of accuracy without additional tests was 79% and with additional testing the accuracy rate was 94% with a 6% error rate. LITERATURE CITED 1. Applebaum, P. C., P. S. Chaurushiya, M. R. Jacobs, and A. Duffett Evaluation of the Rapid Strep system for species identification of streptococci. J. Clin. Microbiol. 19: Applebaum, P. C., M. R. Jacobs, J. I. Heald, W. A. Palko, A. Duffett, R. Crist, and P. A. Naugle Comparative evaluation of the API 20S System and the AutoMicrobic System Gram-Positive Identification Card for species identification of streptococci. J. Clin. Microbiol. 19: Applebaum, P. C., M. R. Jacobs, W. M. Palko, E. E. Frauenhoffer, and A. Duffett Accuracy and reproducibility of the IDS RapID STR system for species identification of streptococci. J. Clin. Microbiol. 23: Beeching, N. J., T. I. Christmas, R. B. Ellis-Pegler, and G. I. Nicholson Streptococcus bovis bacteraemia requires rigorous excludes of colonic neoplasia and endocarditis. Q. J. Med. 56: Collins, M. D., J. A. E. Farrow, and D. Jones Enterococcus mundtii sp. nov. Int. J. Syst. Bacteriol. 36: Collins, M. D., D. Jones, J. A. E. Farrow, R. Kilpper-Balz, and K. H. Schleifer Enterococcus avium nom. rev., comb. nov.; E. casseliflavus nom. rev., comb. nov.; E. durans nom. rev., comb. nov.; E. gallinarum comb. nov.; and E. malodoratus sp. nov. Int. J. Syst. Bacteriol. 34: Deibel, R. H., and J. B. Evans Modified benzidene test for the detection of cytochrome-containing respiratory systems in microorganisms. J. Bacteriol. 79: Facklam, R. R The major differences in the American and RapID STR SYSTEM 611 British streptococcus taxonomy schemes with special reference to Streptococcus milleri. Eur. J. Clin. Microbiol. 3: Facklam, R., G. S. Bosley, D. Rhoden, A. R. Franklin, N. Weaver, and R. Schulman Comparative evaluation of the API 20S and AutoMicrobic Gram-Positive Identification systems for non-beta-hemolytic streptococci and aerococci. J. Clin. Microbiol. 21: Facklam, R. R., and R. B. Carey Streptococci and aerococci, p In E. H. Lennette, A. Balows, W. J. Hausler, and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 11. Facklam, R. R., D. L. Rhoden, and P. B. Smith Evaluation of the Rapid Strep system for the identification of clinical isolates of Streptococcus species. J. Clin. Microbiol. 20: Hendrickson, D. A Reagents and stains, p In E. H. Lennette, A. Balows, W. J. Hausler, and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 13. Jablon, J. M., B. Brust, and M. S. Saslaw P-Hemolytic streptococci with group A and type II carbohydrate antigens. J. Bacteriol. 89: Keville, M. W., and G. V. Doern Comparison of the API 20S Streptococcus identification system with an immunorheophoresis procedure and two commercial latex agglutination tests for identifying beta-hemolytic streptococci. J. Clin. Microbiol. 16: Kilpper-Balz, R., B. L. Williams, R. Lutticken, and K. H. Schleifer Relatedness of "Streptococcus milleri" with Streptococcus anginosus and Streptococcus constellatus. System. Appl. Microbiol. 5: Klein, R. S., R. A. Recco, M. T. Catalana, S. C. Edberg, J. I. Casey, and N. H. Steigbigel Association of Streptococcus bovis with carcinoma of the colon. N. Engl. J. Med. 297: Reynolds, J. G., E. Silva, and W. M. McCormack Association of Streptococcus bovis bacteremia with bowel disease. J. Clin. Microbiol. 17: Ruoff, K. L., M. J. Ferraro, M. E. Jerz, and J. Kissling Automated identification of gram-positive bacteria. J. Clin. Microbiol. 16: Ruoff, K. L., and L. J. Kunz Use of the Rapid STREP system for identification of viridans streptococcal species. J. Clin. Microbiol. 18: Schleifer, K. H., and R. Kilpper-Bfilz Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nov. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int. J. Syst. Bacteriol. 34:31-34.