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Journal of Clinical Microbiology, May 2004, p. 2065-2073, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2065-2073.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Comparison of Conventional and Molecular Methods for Identification of Aerobic Catalase-Negative Gram-Positive Cocci in the Clinical Laboratory

P. P. Bosshard,* S. Abels, M. Altwegg, E. C. Böttger, and R. Zbinden

Institute of Medical Microbiology, University of Zürich, 8028 Zürich, Switzerland

Received 8 October 2003/ Returned for modification 2 December 2003/ Accepted 11 January 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In clinical laboratories the present means of identification of aerobic catalase-negative gram-positive cocci mainly rely on phenotypic tests. These tests have been miniaturized and semiautomated, leading to major progress in diagnostic accuracy (16). Among the commercially available test systems, the API 20 Strep system (bioMérieux sa, Marcy l'Etoile, France) is widely used and is generally accepted as a reliable identification system (2, 37). However, phenotypic tests are characterized by potential inherent problems; e.g., (i) not all strains within a given species may exhibit a common characteristic (3, 17), (ii) the same strain may give different results upon repeated testing (36), (iii) the corresponding database does not enclose newly or not yet described species, and (iv) the test result relies on individual interpretation and expertise. Moreover, small alterations in the execution of an assay may give false test results. Consequently, identification based on phenotypic tests does not always allow an unequivocal identification (24).

Small-subunit (16S) rRNA gene sequencing is a widely accepted tool for identifying bacterial isolates (4, 18, 21) and for diagnosing microbial infections (26, 27, 38, 40). rRNA molecules comprise several functionally different regions. Some of these are characterized by highly conserved sequences, i.e., sequences that can be found among a wide range of bacteria. Other regions show highly variable sequences, i.e., nucleic acid sequences that are specific for a species or a genus. Thus, the 16S rRNA sequence of a species is a genotypic feature which allows the identification of microbes at the genus or the species level (4). In addition, molecular identification offers the possibility of recognizing yet undescribed taxa, because ribosomal DNA (rDNA) similarity reflects phylogenetic relationships (41).

Despite the broad acceptance of 16S rDNA sequencing as a tool for identification of bacterial pathogens, few studies so far have systematically compared molecular and phenotypic identification procedures to determine their usefulness for the diagnostic laboratory (5, 8, 10, 11, 22, 32, 34, 35). The available studies focused on mycobacteria (8, 22, 32), gram-negative bacilli (11, 34), and gram-positive rods (35). In the prospective study described here, we have evaluated the suitability of 16S rDNA sequencing for the identification of aerobic catalase-negative gram-positive cocci under routine conditions in a clinical microbiology laboratory.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical isolates. From October 2000 to April 2002, a total of 171 isolates of gram-positive cocci were analyzed. Except for enterococci and beta-hemolytic streptococci, all clinically relevant aerobic catalase-negative gram-positive cocci were included in this study. For enterococci, only those isolates that were identified as unusual clinical species and those that were not clearly identified by the commercial API 20 Strep system (bioMérieux sa), i.e., isolates with only a genus-level identification or an equivocal species-level identification, were included. The isolates investigated were from cultures of blood or specimens from other normally sterile body sites.

Identification with the API 20 Strep system. Identification with the API 20 Strep was performed according to the instructions of the manufacturer (bioMérieux sa). Fermentations were read after 4 and 24 h. Identification was achieved after 24 h by using the corresponding identification software (version V6.0). According to these results, all strains were classified into one of the following three groups: (i) strains identified to the species level, (ii) strains identified to the genus level, and (iii) strains not identified (i.e., strains with a low level of discrimination). According to the manufacturer's instructions, strain identification to the species level was divided into four subgroups: (i) excellent species identification, %id of >=99.9% and a T value of >=0.75; (ii) very good species identification, %id of >=99.0% and a T value of >=0.5; (iii) good species identification, %id of >=90.0% and a T value of >=0.25; and (iv) acceptable species identification, %id of >=80.0% and a T value >=0.0 (with %id and T being manufacturer-defined variables).

Sequencing of 16S rDNA. DNA was extracted by enzymatic lysis and alkaline hydrolysis. A loopful of bacterial cells was lysed in 200 µl of lysis buffer (0.05 M Tris-HCl, 1 mM EDTA [pH 7.5]) containing 0.5 mg of lysozyme (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) by incubation for 1 h at 37°C. After addition of 10 µl each of 1 M NaOH and 10% sodium dodecyl sulfate, the mixture was incubated at 95°C for 10 min and neutralized with 10 µl of 1 M HCl. Nucleic acids were then purified with a QIAamp DNA blood mini kit (Qiagen AG, Basel, Switzerland), resulting in a sample volume of 100 µl.

An 800-bp 16S rDNA fragment, corresponding to Escherichia coli positions 10 to 806 (7), was amplified with primers BAK11w [5'-AGTTTGATC(A/C)TGGCTCAG] and BAK2 [5'-GGACTAC(C/T/A)AGGGTATCTAAT] (6). Cycling parameters included an initial denaturation for 5 min at 95°C; 40 cycles of 1 min at 94°C, 1 min at 48°C, and 1 min at 72°C; and a final extension for 10 min at 72°C. Five microliters of the DNA extract was used for amplification in a total volume of 50 µl containing 1.25 U of AmpliTaq DNA polymerase LD (Applied Biosystems, Rotkreuz, Switzerland) and the appropriate buffer. Amplicons were purified with a QIAquick PCR purification kit (Qiagen AG) and were sequenced with forward primer BAK11w by use of the BigDye kit and an automatic DNA sequencer (ABI Prism 310 Genetic Analyzer; Applied Biosystems).

Sequence analysis. The 16S rDNA sequences were compared with those available in the GenBank, EMBL, and DDBJ databases by a two-step procedure. A first search was performed with the FASTA algorithm of the Wisconsin Genetics Computer Group program package (9). All positions showing differences from the best-scoring reference sequence were visually inspected in the electropherogram, and the sequence was corrected if adequate, i.e., when obvious sequencing software errors occurred, such as when false spacing occurred or when undetermined nucleotides in the sequence could be determined according to the electropherogram. Thereafter, a second search was done with the BLASTN algorithm. Undetermined nucleotides (designated by an N) in either the sequence determined or the reference sequence were counted as matches. The mean length of the sequences after manual editing was 429 ± 68 nucleotides, with 1.1 ± 1.7 undetermined (N) positions.

Criteria for identification. The following criteria were used for identification to the genus or species level: (i) when the comparison of the sequence determined with a reference sequence (i.e., a public database sequence) of a classified species yielded a similarity score >=99%, the unknown isolate was assigned to that species; (ii) when the score was <99% and >=95%, the unknown isolate was assigned to the corresponding genus; and (iii) when the score was <95%, the unknown isolate was not identified to any taxonomic level. If the unknown isolate was assigned to a species and the second classified species in the scoring list showed less than 0.5% additional sequence divergence, the unknown isolate was categorized as a "species with a low level of demarcation to the next species."

Discrepant analysis. If the results of sequencing were different from the results obtained with the API 20 Strep system or the species revealed was not in the database of the API 20 Strep system, testing with the API 20 Strep system was repeated with the isolate, which had been kept frozen at –70°C (except in the case in which API 20 Strep revealed Streptococcus acidominimus and sequencing resulted in Aerococcus urinae; see Results). In some cases, additional reactions, e.g., motility, were used for analysis.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolate identification with the API 20 Strep system. A total of 171 aerobic catalase-negative gram-positive isolates that included nine different genera comprising 29 different species were investigated. The API 20 Strep system identified 67 isolates to the species level and yielded excellent, very good, good, and acceptable species identifications for 6, 19, 30, and 12 isolates, respectively; identification to the genus level was achieved for 32 cases; and 72 isolates could not be identified (Table 1).


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  		TABLE 1. Molecular versus phenotypic identification for 171 isolates (unresolved data)

 
Isolate identification by rDNA sequencing. By use of the criteria defined for sequence analysis, 16S rDNA sequencing resulted in the identification of 138 isolates to the species level and 33 isolates to the genus level (Table 1). For 24 of the 138 isolates identified to the species level, comparisons of the sequences with those available in public databases resulted in the retrieval of two sequences for different species with identical similarity scores; thus, the isolate was not assigned to a single taxon but was reported to belong to either of the two species. Twenty-six of the 138 isolates identified to the species level were identified as a species with a low level of demarcation to the next species, i.e., less than 0.5% additional sequence difference from another sequence entry.

Sequencing of isolates identified to the species level with the API 20 Strep system. For 25 of the 67 strains identified to the species level with the API 20 Strep system, molecular identification assigned the isolate to the same species. Discrepant results were found for 42 isolates (Tables 2 and 3).


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  		TABLE 2. Molecular identification versus phenotypic identification for 67 isolates identified to the species level with the API 20 Strep system

 

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  		TABLE 3. Molecular versus phenotypic identification for 171 isolates (resolved data)

 
Analysis of discrepant results and assignment to different species. For 29 of 42 isolates with discrepant results (Table 2), 16S rDNA sequencing assigned the strains to a species different from that to which the strain was assigned by the API 20 Strep system. The results for 20 of the 29 isolates were regarded as major discrepancies; i.e., the isolate was assigned either to a different genus or to a different group within the streptococci (15).

For 22 of the 29 isolates, the 16S rDNA sequence determined exhibited less than 97% similarity to the 16S rDNA sequence of the species to which it was assigned by the API 20 Strep system (for 21 isolates the sequence similarity was even less than 93%). According to Stackebrandt and Goebel (33), 16S rDNA similarities of less than 97% indicate that isolates belong to different species. Although only partial sequences were used here, it was thus concluded that these isolates do not belong to the species identified by the API system. For example, 12 strains were identified as Streptococcus acidominimus with the API 20 Strep system, whereas sequencing resulted in 99.7 to 100.0% similarity with Aerococcus urinae and less than 85% similarity with S. acidominimus. These isolates clearly do not belong to S. acidominimus but belong to A. urinae. Of note, A. urinae is not included in the API 20 Strep system database. It has been shown previously that an unknown isolate that shows a profile for S. acidominimus in the API 20 Strep system and that is positive for ß-glucuronidase and leucine arylamidase should be reported as A. urinae (42). If this rule is applied (which would result in the assignment of 12 isolates to A. urinae on the basis of the results obtained with the API 20 Strep system), molecular identification and phenotypic identification would assign an isolate to the same species for 37 of the 67 isolates for which species assignment was achieved with the API 20 Strep system (5 of 6, 13 of 19, 14 of 30, and 5 of 12 isolates with excellent, very good, good, and acceptable species identifications by the API 20 Strep system, respectively).

For 7 of the 29 isolates with discrepant results, the 16S rDNA sequence of the isolate showed >=97% sequence similarity to the 16S rDNA sequence of the species to which the isolate was assigned by the API 20 Strep system. It thus cannot be excluded that the strains belong to the species identified by the API 20 Strep system. For three of these seven isolates, however, a repeat of the test with the API 20 Strep system did not confirm the primary result obtained with the system. For these isolates it is thus assumed that the molecular approach correctly identified the species (99.5% sequence similarity with Streptococcus oralis [two isolates] and 99.1% sequence similarity with Streptococcus parasanguis, which is not included in the API 20 Strep system database). The results for four isolates remained unresolved.

Analysis of discrepant results and assignment to the genus level by molecular analysis. For 13 of 42 isolates with discrepant results (Table 2), the isolates were identified to the genus level by sequencing; i.e., these isolates showed less than 99.0% similarity (our defined threshold value for species-level identification) to the best-scoring reference sequence. For 7 of these 13 isolates, the similarity of the sequence to that of the species identified by the API 20 Strep system was below 97%, leading us to conclude that these isolates do not belong to the species identified by the API 20 Strep system. For example, phenotypic identification resulted in Aerococcus viridans II (five isolates); the 16S rDNA sequences determined showed, however, that the isolates had between 95.5 and 95.8% sequence similarity with A. urinae and between 93.3 and 93.7% sequence similarity with A. viridans. It is likely that these five isolates represent an Aerococcus species that has yet not been described.

For 6 of the 13 isolates, the nucleic acid sequences determined showed 97% or more similarity with the sequences of the species to which the isolates were assigned by the API 20 Strep system. For two of these isolates, the species determined with the API 20 Strep system was identical to the best-scoring species, as determined by sequence analysis. It is thus assumed that the biochemical system correctly assigned the two isolates. For four of the six isolates, the API 20 Strep system assigned the isolate to a species different from the best-scoring species from the molecular investigation. For example, for three isolates identified as Streptococcus sanguis by the API 20 Strep system, the 16S rDNA sequences determined showed between 98.7 and 98.8% sequence similarity with Streptococcus gordonii and between 97.3 and 97.7% sequence similarity with S. sanguis; the three isolates were reported to belonging to the genus Streptococcus. Thus, for these four isolates, the species identity could not be determined conclusively (unresolved data).

Sequencing of isolates identified to the genus level with the API 20 Strep system. With the API 20 Strep system, 32 of 171 gram-positive cocci investigated were identified to the genus level. For 23 of them, 16S rDNA sequencing allowed assignment to a species (Tables 3 and 4). For all but two isolates, the species assignment did not contradict the genus assignment determined conventionally: for one strain, the strain was identified as a Streptococcus sp. with the API 20 Strep system, whereas molecular methods resulted in a sequence that was identical to that of A. urinae; in the other case, the strain was identified as a Gemella sp. with the API 20 Strep system, whereas sequence analysis resulted in Streptococcus mitis or S. pneumoniae.


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  		TABLE 4. Molecular identification versus phenotypic identification for 32 isolates identified to the genus level with the API 20 Strep system

 
For 9 of 32 strains identified to the genus level with the API 20 Strep system, 16S rDNA sequencing did not yield more discriminative results; i.e., the isolate was assigned to the same genus without further species assignment.

Sequencing of isolates not identified with the API 20 Strep system. Molecular methods allowed identification of all 72 strains which could not be assigned to a genus by the API 20 Strep system identification procedure (Tables 3 and 5); 63 strains were identified to the species level, and 9 strains were identified to the genus level.


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  		TABLE 5. Molecular identification versus conventional methods for 72 isolates not identified by the API 20 Strep system

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This prospective study was performed under routine diagnostic conditions. A collection of clinically relevant strains (n = 171) of aerobic catalase-negative gram-positive cocci isolated in the diagnostic laboratory was investigated over a period of 18 months. Accurate identification of these strains, mostly obtained from normally sterile body sites, was attempted with the commercially available API 20 Strep system. rDNA sequencing was performed in parallel.

We demonstrate that 16S rDNA sequence analysis has an improved ability to identify aerobic gram-positive cocci compared to that of the API 20 Strep system: (i) 81% (138 of 171) of isolates were identified to the species level by sequence analysis, whereas 39% (67 of 171) were identified to the species level with the API 20 Strep system; (ii) for 72% (23 of 32) of the isolates which could be identified only to the genus level with API 20 Strep system, sequence analysis allowed identification to the species level; and (iii) among the strains that could not be discriminated at any taxonomic level biochemically (72 of 171), all of the isolates could be assigned to a species (89%) or a genus (11%) level by molecular analysis.

Molecular analysis yielded discrepant results for 42 of the 67 strains which were assigned to the species level by the API 20 Strep system. For 32 of the 42 isolates with discrepant results, it was concluded that 16S rDNA sequencing correctly identified the isolates (or at least had more discriminative power, as sequence analysis revealed that the isolate did not belong to a classified species; e.g., the sequence similarity to a reference sequence was less than 97% [33]). For two isolates with discrepant results, it was assumed that the API 20 Strep system yielded a correct species assignment. For eight isolates with discrepant results, further investigations such as DNA-DNA hybridization or sequencing of other targets (e.g., the manganese-dependent superoxide dismutase [24]) would be necessary to resolve the discrepancies. For 12 isolates with major discrepancies, the phenotypic system misidentified A. urinae as S. acidominimus, a finding that has been reported previously (42). In the future, gram-positive cocci in tetrads that are identified as S. acidominimus with the API 20 Strep system (and which are positive for ß-glucuronidase and leucine arylamidase) should be reported to probably be A. urinae.

It is concluded that under routine conditions in a clinical laboratory the API 20 Strep system frequently does not provide accurate identifications. The possible reasons for misidentifications are that (i) the species is not included in the API 20 Strep system database (e.g., A. urinae, Aerococcus sanguinicola, and S. gordonii); (ii) the strain presumably belongs to a new, not yet described species (sequence similarity to a classified species, <97%); (iii) the reactions of the API 20 Strep system are misinterpreted; and (iv) biochemical variability exists within a species. It has been shown previously that commercial phenotypic identification systems, such as the API 20 Strep system or the Rapid ID 32 Strep system, are not entirely satisfactory for accurate identification of a strain to the species level (13, 14, 16, 29, 39). Supplementary manual tests are often needed, which somewhat impairs the usefulness of commercial kits.

It has been proposed that molecular methods such as PCR-restriction fragment length polymorphism analysis (20, 28, 31), DNA sequencing (1, 15, 23, 24), and other PCR-based protocols (12, 25) accurately identify aerobic catalase-negative gram-positive cocci. However, those studies exhibited several drawbacks that limit the routine use of these methods in a clinical laboratory: (i) they were restricted to certain groups of bacteria and did not cover the whole range of aerobic catalase-negative gram-positive cocci (1, 12, 14-16, 20, 23, 24, 25, 28, 31, 39); (ii) they cannot be applied to other bacteria unless the corresponding databases (i.e., restriction patterns and sequences of genes other than 16S rDNA) are enlarged (12, 15, 20, 24, 28, 31); (iii) they have not been tested under routine conditions (12, 15, 23, 24, 25, 28, 31); and (iv) their use is limited to reference laboratories (20, 28, 31).

Therefore, we decided to evaluate the use of 16S rDNA sequencing for the identification of aerobic catalase-negative gram-positive cocci under routine conditions. 16S rDNA sequencing for identification is not restricted to a specific group of bacteria and can readily be implemented in the laboratory. The procedure for sequence analysis (i.e., database search and manual editing of the sequence) in combination with the criteria for species and genus assignment (i.e., >=99% sequence similarity for species assignment and >=95% sequence similarity for genus assignment) proved to be helpful for the accurate identification of the isolates. If the sequence can be assigned to a species but the second-scoring reference species shows less than 0.5% additional sequence divergence, this should be noted (as was noted in our category of species with a low level of demarcation to the next species). It has been shown previously that this approach allows accurate species identification for gram-positive rods (5).

The part of the 16S rRNA gene chosen for analysis covers the most discriminating regions within the 16S rDNA and is therefore suitable for identification purposes (19). In general, 16S rDNA analysis has low phylogenetic resolving power at levels of close relatedness (above 97% similarity [33]); in the extreme, two species may share identical 16S rDNA gene sequences. It has been shown previously that S. mitis, S. pneumoniae, and S. oralis exhibit more than 99% sequence homology to each other (15). Similar findings have been reported for some enterococci (23). In the present study, the 16S rDNA sequences of some isolates (n = 24) were identical to those of different species. This was true in particular for S. mitis and S. pneumoniae, S. gordonii and S. mitis; Enterococcus faecium and Enterococcus durans (and in some cases, additionally, Enterococcus faecalis), Enterococcus raffinosus and Enterococcus malodoratus, and Enterococcus gallinarum and Enterococcus casseliflavus. These organisms can readily be distinguished by additional phenotypic tests, such as the bile solubility test (which differentiates S. pneumoniae from S. mitis) and standard biochemical tests (which are also part of the API 20 Strep system). For 21 of these 24 isolates with equivocal results, a definite species assignment was achieved by additional phenotypic tests.

Another problem arises from the quality of the public databases, such as the GenBank, EMBL, and DDBJ databases. Sequences can be deposited in these databases largely independently of their quality, e.g., regardless of the number of ambiguous nucleotides, the length of the sequence, or the correct assignment of the strain investigated. However, such situations should normally not lead to false identifications but, rather, should lead to problems assigning a strain to a particular species (a low level of demarcation), at least if the correct species is also contained in the database. This in turn would induce further investigations (e.g., biochemical tests or phylogenetic analysis of the sequences).

In this study, sequence-based identification was compared to the identification based on the widely used commercial API 20 Strep system. As a limitation, we did not consider other commercially available identification systems, such as the Rapid ID 32 Strep system (bioMérieux), the VITEK 2 system (bioMérieux), the BD Phoenix automated microbiology system (BD diagnostic systems), the BBL Crystal system (BD Diagnostic Systems), or the MicroLog system (Biolog Inc.). However, as discussed for the API 20 Strep system, most phenotypic systems have general drawbacks, such as the quality and the quantity of the underlying database and phenotypic variability within a species. This demonstrates that identification by molecular analysis is superior to that with the API 20 Strep system and is ready to be implemented in the clinical laboratory.

In our study, the majority (96%) of strains were not reliably identified to the species level by the API 20 Strep system or the species assignment was doubtful (6 of 19, 15 of 30, and 6 of 12 isolates with very good, good, and acceptable qualities of identification were falsely identified). A species assignment in the API 20 Strep system may be considered reliable only when an excellent species identification according to the criteria of the system is achieved. However, this was the case for only 6 of 171 isolates. We thus conclude that the API 20 Strep system is not an effective system for the identification of gram-positive catalase-negative cocci. Consequently, corresponding isolates, with the exception of S. pneumoniae and beta-hemolytic streptococci, should be subjected to 16S rDNA sequence analysis if adequate species identification is of concern (see the algorithm in Fig. 1). Phenotypic tests may be used for definite species assignment only for those few strains for which the sequencing result is equivocal.



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  		FIG. 1. Algorithm for the identification of aerobic catalase-negative gram-positive cocci. Pos, positive; Neg, negative; VP, Voges-Proskauer test.

 


   ACKNOWLEDGMENTS
 
We thank the technicians of the Institute of Medical Microbiology for excellent technical assistance.

This study was supported by the University of Zürich.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30, CH-8028 Zürich, Switzerland. Phone: 41 1 634 27 00. Fax: 41 1 634 49 06. E-mail: philboss{at}immv.unizh.ch. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Clinical Microbiology, May 2004, p. 2065-2073, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2065-2073.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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