Identification of 43 Streptococcus species by pyrosequencing analysis of the rnpB gene

Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.     

Ecology and nature of immunoglobulin A1 protease-producing streptococci in the human oral cavity and pharynx.

The identity and proportional distribution of immunoglobulin A1 (IgA1) protease-producing streptococci in the oral and pharyngeal microflora were studied. A collection of 459 streptococcal strains, including reference strains of Streptococcus species, and fresh isolates from human dental plaque and buccal and pharyngeal mucosa were identified by biochemical means and were examined for IgA1 protease production. IgA1 protease production was demonstrated in some, but not all, strains of Streptococcus sanguis and Streptococcus mitior and in a group of strains of uncertain taxonomic affiliation. The property was not associated with particular biotypes within the two species. Strains of S. sanguis and S. mitior isolated from Macaca fascicularis also cleaved human IgA1. A significantly different proportion of streptococcal populations in different ecosystems produced IgA1 protease. The enzyme was released by 62.7% of streptococcal isolates from buccal mucosa in contrast to only 7.8% from pharyngeal mucosa. In samples from initial and mature dental plaque 38 to 40% of streptococcal isolates produced IgA1 protease. This difference was largely a result of the frequency by which IgA1 protease activity was present in S. mitior, the predominant streptococcal species in all samples. Among otherwise identical isolates of S. mitior, 67.8% from buccal mucosa in contrast to only 5.9% from pharyngeal mucosa produced IgA1 protease. The results indicate that IgA1 protease may confer an ecological advantage to streptococci colonizing surfaces exposed to a secretory IgA-mediated selection pressure.     

Comparison of epidemic and endemic group G streptococci by restriction enzyme analysis.

Restriction enzyme profiles of group G beta-hemolytic streptococci associated with a point source outbreak and an outbreak of sporadic pharyngitis in two different communities were compared. To asses the epidemiologic utility of this approach for studying group G streptococci, DNA fingerprints of strains responsible for a point source outbreak of pharyngitis associated with the consumption of contaminated food were compared with DNA fingerprints of pharyngeal isolates from children with pharyngitis seen at a pediatric practice during a 6-month period. In each epidemiologic situation, a single strain characterized by a unique restriction enzyme pattern predominated. The results are compatible with the conclusion that human infections could be limited to a few strains of group G streptococci which have the capacity to spread through a given population. The restriction enzyme profiles proved to be a highly specific and precise means of evaluating strain relatedness and of providing further understanding of the epidemiology of group G streptococcal infections.     

New Actinomyces and Streptococcus coaggregation groups among human oral isolates from the same site.

The coaggregation properties of recent human oral streptococcal and actinomyces isolates from the same site were determined and compared with the coaggregation properties of well-characterized stock strains of these two kinds of bacteria. Streptococcus sanguis, Actinomyces viscosus, Actinomyces naeslundii, and phenotypically similar strains of actinomyces were isolated from subgingival samples from periodontally healthy older individuals, from persons participating in an experimental gingivitis study, and from young persons with localized (juvenile) and generalized (severe) periodontitis. All 34 of the actinomyces isolates coaggregated with reagent strains of S. sanguis that represented the four streptococcal coaggregation groups. Most of these actinomyces exhibited coaggregations identical to those of actinomyces stock strains. However, five isolates of an Actinomyces WVa-963 serovar exhibited a coaggregation pattern different from any previously described, which was used to define coaggregation group F. All coaggregations with members of this group were lactose inhibitable. Only 57% (8 of 14) of the recent S. sanguis isolates coaggregated with actinomyces reagent strains. But when the nonreactive streptococcal isolates were tested for their ability to coaggregate with actinomyces from the same patient, a new, highly specific coaggregation pattern (group 6) for S. sanguis was discovered. Coaggregation of these streptococci was observed only with certain isolates of A. naeslundii (members of coaggregation group D) from the same site, and none of these coaggregations were inhibited by lactose. Subsequent testing revealed that streptococci of group 6 coaggregated with group D actinomyces from other sources but not with actinomyces of other coaggregation groups. Only two strains of S. sanguis failed to coaggregate with any strain of actinomyces tested. These results indicate that nearly all fresh isolates of these species obtained from both diseased and healthy sites exhibit specific, nonrandom patterns of coaggregation and suggest the widespread occurrence of in vivo cell-to-cell recognition between oral actinomyces and streptococci.     

Identification of major Streptococcal species by rrn-amplified ribosomal DNA restriction analysis

Amplified ribosomal DNA restriction analysis (rrn-ARDRA) is based on PCR amplification and restriction of a fragment of rRNA genes including 16S and 23S genes and the intergenic spacer. rrn-ARDRA was evaluated for the identification of species within the genus Streptococcus: A total of 148 type and reference strains of pyogenic, oral, and group D streptococci were examined in order to construct a database for identification of streptococci. The amplified product was a single band approximately 4500 bp long. This amplicon was digested separately with three (HhaI, MboII, and Sau3A) restriction endonucleases. Respectively, 27, 26, and 28 major patterns were observed after HhaI, MboII, and Sau3A restrictions. Streptococcal strains belonging to different species had different patterns or different combination of patterns. An identification system based upon a combination of the three restriction patterns in a single database was then proposed. rrn-ARDRA was successfully applied to 11 clinical isolates whose identification to the species level was difficult to obtain by phenotypic analysis. Using a database of well-characterized strains, rrn-ARDRA is a powerful method for the identification of streptococcal isolates.     

Identification of clinically relevant viridans group streptococci by phenotypic and genotypic analysis

Two phenotypic and three molecular methods were assessed for their ability to identify viridans group streptococci (VGS) to the species level. A panel of 23 clinical isolates, comprising strains isolated from infective endocarditis, blood cultures, pleural and peritoneal fluid, and 19 type/reference strains were analyzed. Identification was performed using two conventional phenotypic methods: API? rapid ID 32 Strep and the VITEK? 2 system, and genotypic analysis of the nucleotide sequence of the housekeeping gene sodA, restriction patterns generated by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene and multilocus sequence analysis (MLSA) of seven housekeeping genes. The API? rapid ID 32 Strep accurately speciated 79% of the strains assessed, while the VITEK? 2 generated a successful identification for 55%, presenting limitations particularly with regard to species belonging to the mitis group. RFLP of the 16S rRNA gene correctly speciated 24% of the strains, having failed to allocate a species for 36% of the isolates examined. In contrast, sequence analysis of the sodA gene provided a correct identification for 95% of the strains assessed, while identification using the MLSA technique was unsuccessful due to practical limitations. The results generated herein indicate that no single methodology can be used to provide an accurate identification to the species level of all VGS, although nucleotide sequence analysis of the sodA gene proved to be useful in providing reliable speciation.     

Evaluation of two matrix-assisted laser desorption ionization–time of flight mass spectrometry systems for identification of viridans group streptococci

In this study, the performances of two matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, MALDI Biotyper (Bruker Daltonics) and VITEK MS (bioMérieux), were evaluated in the identification of viridans group streptococci. Two collections of isolates were tested with both methods. From a panel of type collection strains (n?=?54), MALDI Biotyper gave correct species-level identification for 51/54 (94 %) strains and 37/54 (69 %) strains for the VITEK MS in vitro diagnostic (IVD) method. Additionally, a collection of blood cultures isolates which had been characterized earlier with partial sequencing of 16S rRNA (n?=?97) was analyzed. MALDI Biotyper classified 89 % and VITEK MS 93 % of these correctly to the group level. Comparison of species-level identification from the blood culture collection was possible for 36 strains. MALDI Biotyper identified 75 % and VITEK MS 97 % of these strains consistently. Among the clinical isolates, MALDI Biotyper misidentified 36 strains as Streptococcus pneumoniae. Nevertheless, our results suggest that the current MALDI-TOF methods are a good alternative for the identification of viridans streptococci and do perform as well as or better than commercial phenotypical methods.     

Identification of mutans and other oral streptococci by random amplified polymorphic DNA analysis

The identification and classification of the non-haemolytic or viridans group of streptococci have long been recognised as difficult and unsatisfactory. Phenotypic and genotypic heterogeneity have resulted in ambiguous speciation, particularly with mutans streptococci and other oral streptococci. This study was done to determine whether random amplified polymorphic DNA (RAPD) analysis is useful to identify and even classify oral and other streptococci. DNA was prepared and purified from 25 strains of mutans streptococci including 11 reference strains of Streptococcus mutans, seven of S. sobrinus, three of S. rattus and one each of the four other species of the mutans group, together with 20 other reference species, mostly streptococci, and from 49 fresh isolates of mutans streptococci and of S. mutans from human saliva and dental plaque. DNA amplification was primed with each of three arbitrarily selected primers nine or 10 nucleotides in length. The amplified DNA fragments (amplicons) obtained were compared by agarose gel electrophoresis. Species- and strain-specific RAPD fingerprints were obtained not only from pure genomic DNA, but also from the supernates of crude cellular or colony extracts. Pending the analysis of numerous other strains, the data suggest that RAPD may be of value: (i) to distinguish the species S. mutans and S. sobrinus from each other and potentially from other species of oral streptococci, (ii) to differentiate and possibly classify oral streptococci and (iii) as a valuable tool in mutans streptococci epidemiology and transmission studies, by virtue of its rapidity, efficiency and reproducibility in generating genetic fingerprints of streptococcal isolates.     

PCR-Based Methods for Genotyping Viridans Group Streptococci

Standard repetitive extragenic palindromic (REP)-PCR, enterobacterial repetitive intergenic consensus-PCR, and Salmonella enteritidis repetitive element-PCR methods for bacterial strain typing were performed with DNA extracted by boiling members of each of the currently recognized species of human viridans group streptococci. Each of the methods was reproducible. The unique isolates (n = 72) from 15 species of viridans group streptococci were readily distinguishable, with no two isolates showing greater than 90% per cent similarity. The majority of strains exhibited much less than 90% similarity. Isolates identical by REP-PCR were also identical by the other two methods. These PCR-based typing methods, although they do not permit determination of the species of the isolates, are simple to perform and are suitable for clinical and ecological investigations of viridans group streptococci.     

Adherence of Streptococcus pyogenes M type 5 to pharyngeal and buccal cells of patients with rheumatic fever and rheumatic heart disease during a one-year follow-up.

In vitro adherence of Streptococcus pyogenes M type 5 to isolated pharyngeal and buccal epithelial cells was studied in patients with acute recurrent rheumatic fever (n = 21), chronic rheumatic heart disease (n = 33), streptococcal pharyngitis (n = 12), and in normal controls. Patients were investigated at admission and one, six and 12 months later. Streptococci adhered significantly more to the pharyngeal cells of patients with rheumatic fever and rheumatic heart disease than to the pharyngeal cells of controls. Adherence of streptococci to pharyngeal cells of patients with pharyngitis was not different from age-matched controls. The adherence of streptococci to the pharyngeal cells of patients with acute rheumatic fever fell during follow-up but even after one year remained significantly higher than in the control group. These findings suggest that host factor(s) controlling streptococcal adhesion and colonization at the pharyngeal mucosa may be important in the pathogenesis of acute rheumatic fever.     

Restricted distribution of Streptococcus milleri carbohydrate type antigens amongst other viridans streptococci.

The distribution of oral Streptococcus milleri carbohydrate type antigens in other viridans streptococcus species was examined. Rantz-Randall extracts of cells of the test strains grown in broth containing glucose were allowed to react with typing or grouping antisera for S. milleri serotypes a-k, or Lancefield groups A-G and K. Of 93 strains comprising more than 12 streptococcal species that included S. mutans and S. sanguis complexes, only 15 S. salivarius strains and one S. mitis strain were immunologically related to S. milleri serotype f. Unlike S. milleri strains, S. salivarius type f strains belonged to Lancefield group K, whereas the S. mitis strain was closely related to S. milleri serotype f but did not react with any of the Lancefield grouping antisera tested. Results suggest that oral S. milleri strains can be distinguished serologically from other oral viridans streptococci and that the typing antisera used in our researches might differentiate S. milleri isolates from the mouth from those associated with systemic purulent infections.     

Role of Adherence in the Pathogenesis of Neonatal Group B Streptococcal Infection

The ability of group B streptococci to attach to buccal epithelial cells from adult volunteers, healthy neonates, and infants with invasive group B streptococcal infection was assessed by using (3)H-labeled bacteria incubated at a bacteria-to-cells ratio of 1,000:1. Type III group B streptococcal clinical isolates adhered significantly better to the epithelial cells of healthy neonates than to those of adults (mean bacteria per cell of 31 versus 7, respectively; P < 0.005). In contrast, no statistically significant differences in adherence of type Ia or type II strains to cells of neonates and adults were noted. The adherence of strains isolated from 15 infants with invasive group B streptococcal infection was significantly greater to the cells of infected infants than to those of age-matched controls (mean bacteria per cell of 39 versus 18, respectively; P < 0.005). In contrast, no significant difference was noted in the adherence of a usually adherent type Ia strain and a nonadherent type III strain to the cells of infected infants compared with control infants. These results indicate that the serotype of group B streptococci with the greatest virulence for neonates (type III) adheres better to neonatal than to adult epithelial cells. Infants who develop invasive infection may have an increased number of epithelial cell surface receptor sites for attachment of group B streptococci, the bacteria may elaborate products which unmask receptor sites, or both.     

Use of the VITEK 2 system for rapid identification of clinical isolates of Staphylococci from bloodstream infections

Staphylococci are an increasing cause of bloodstream infections. Rapid reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment. We evaluated the ability of the VITEK 2 system (bioMérieux, Inc, Hazelwood, Mo.) to identify these organisms rapidly and accurately. A total of 405 clinically relevant nonduplicate staphylococcal isolates (Staphylococcus aureus, n = 130; coagulase-negative staphylococci, n = 275) collected from blood cultures were tested. VITEK 2 results were considered correct when they were identical to those furnished by the comparison method based on the ID 32 STAPH system (bioMérieux, Marcy l'Etoile, France) plus supplementary manual testing. When discrepancies occurred, isolate identity was verified by molecular typing. The VITEK 2 correctly identified 387 (95.6%) isolates at the species level: 379 (including all but one [99.2%] of 130 S. aureus isolates and 249 of 275 [90.5%] coagulase-negative isolates) were identified by the automated reading; for the other eight, supplemental tests suggested by the manufacturer had to be used. Only one strain (0.2%) was misidentified (Staphylococcus hominis as Staphylococcus epidermidis), and four (1%), all S. epidermidis, were not identified. For the remaining 13 strains (including 10 S. hominis), the VITEK 2 system was unable to discriminate among two species, and no supplemental tests were suggested for conclusive identification. Over 90% of results were obtained within 4 h. These results suggest that the VITEK 2 system can provide rapid, accurate, and reliable species-level identification of staphylococci responsible for bloodstream infections, although there is room for improvement in the identification of certain coagulase-negative species, especially S. hominis.     

Virulence properties of erysipelas-associated group A streptococci

Group A streptococcal isolates (n=53) recovered from 38 erysipelas patients in 1988 and 1990 in Sweden were analysed with respect to serotype, erythrogenic toxin production and polymorphism in theemm gene region. Serotype determination showed a dominance of type T1M1 (28.6 % of the strains), but T type 8 was also prevalent (14.3 %). In the majority of the strains only a low production of erythrogenic toxin A was demonstrated, while both toxin B and C production were high. Polymorphism was detected in theemm gene region of T1M1 strains at a frequency of 64 %. These erysipelas associated group A streptococci were more heterogenic with respect to serotype distribution and polymorphism in theemm gene region compared to previously studied group A streptococci isolated during an outbreak of serious streptococcal infections in Sweden in 1988/1989. The material included isolates from two cases of recurrence, and typing of the isolates indicated that the patients had been infected by the same serotype as in the primary infection.     

Use of the Phoenix automated system for identification of Streptococcus and Enterococcus spp

The Phoenix system (Becton Dickinson Diagnostic Systems, Sparks, MD) was evaluated for identification (ID) to the species level of streptococci and enterococci. Two hundred clinical isolates were investigated: beta-hemolytic streptococci (n = 50), Streptococcus pneumoniae organisms (n = 46), viridans group streptococci (n = 31), Enterococcus faecium (n = 36), Enterococcus faecalis (n = 25), and other catalase-negative cocci (n = 12). The API system (bioMérieux, Marcy l'Etoile, France) was used as a comparator. Molecular methods (sequencing of 16S rRNA and zwf and gki genes and ddl gene amplification) were used to investigate discordant results. Upon resolution of discrepancies, correct species ID was achieved by the Phoenix system for 121/129 (93.8%) streptococci and 63/70 (90.0%) enterococci. Excellent results were obtained for S. pneumoniae (45/45) and beta-hemolytic streptococci (49/50). With regard to viridans streptococci, the accuracy of the Phoenix system was 83.9%. Among the latter organisms, the best performance was obtained with isolates of the Streptococcus sanguinis group and Streptococcus anginosus group; problems were instead encountered with the Streptococcus mitis group. Four E. faecium and three E. faecalis isolates were misidentified as Enterococcus casseliflavus/Enterococcus gallinarum or Enterococcus durans. Thus, these isolates were identified only at the genus level. Compared with commercially available systems, the Phoenix system appears a reliable diagnostic tool for identifying clinically relevant streptococci and enterococci. The SMIC/ID-2 panel proved particularly effective for beta-hemolytic streptococci and pneumococci.     

Identification of clinically relevant viridans group streptococci by sequence analysis of the 16S-23S ribosomal DNA spacer region

The feasibility of sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (ITS) for the identification of clinically relevant viridans group streptococci (VS) was evaluated. The ITS regions of 29 reference strains (11 species) of VS were amplified by PCR and sequenced. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The ITS lengths (246 to 391 bp) and sequences were highly conserved among strains within a species. The intraspecies similarity scores for the ITS sequences ranged from 0.98 to 1.0, except for the score for S. gordonii strains. The interspecies similarity scores for the ITS sequences varied from 0.31 to 0.93. Phylogenetic analysis of the ITS regions revealed that evolution of the regions of some species of VS is not parallel to that of the 16S rRNA genes. One hundred six clinical isolates of VS were identified by the Rapid ID 32 STREP system (bioMérieux Vitek, Marcy l'Etoile, France) and by ITS sequencing, and the level of disagreement between the two methods was 18% (19 isolates). Most isolates producing discrepant results could be unambiguously assigned to a specific species by their ITS sequences. The accuracy of using ITS sequencing for identification of VS was verified by 16S rDNA sequencing for all strains except strains of S. oralis and S. mitis, which were difficult to differentiate by their 16S rDNA sequences. In conclusion, identification of species of VS by ITS sequencing is reliable and could be used as an alternative accurate method for identification of VS.     

A new alkaline pH-adjusted medium enhances detection of beta-hemolytic streptococci by minimizing bacterial interference due to Streptococcus salivarius

A new selective medium (CNA-P) that reduces or eliminates the inhibitory activity of bacteriocin-producing Streptococcus salivarius against beta-hemolytic streptococci has been developed and compared with sheep blood agar (SBA) for the sensitive detection of small numbers of beta-hemolytic streptococci in clinical specimens. CNA-P has as its basis a commercial medium (Difco Columbia CNA agar) supplemented with 5% (vol/vol) sheep blood, and the CNA is further modified by addition of 100 mM PIPES buffer [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 7.5) to maintain cultures at an alkaline pH during incubation. CNA-P was shown to inhibit the production and/or release of four different types of S. salivarius bacteriocins or bacteriocin-like inhibitory molecules. The efficacies of CNA-P and SBA for detection of beta-hemolytic streptococci in 1,352 pharyngeal samples from 376 children were compared. The beta-hemolytic streptococcal isolates recovered from the samples included 314 group A (S. pyogenes), 61 group G, 33 group B, and 5 group C streptococci. Of 314 samples that yielded S. pyogenes, 300 were positive on CNA-P (96%) and 264 (86%) were positive on SBA. A significantly greater number of S. pyogenes isolates from these samples were recovered only on CNA-P (50 of 314) compared with the number of isolates recovered only on SBA (14 of 314). In addition, the degree of positivity, a measure of the total numbers of S. pyogenes isolates on the plate, was significantly higher on CNA-P than on SBA (2.40 versus 2.07; P < 0.001). Interestingly, CNA-P was also found to enhance the hemolytic activity of streptolysin O, allowing detection of streptolysin S-deficient S. pyogenes strains which might otherwise go undetected on SBA and other isolation media.     

Applications of bacteriocin-like inhibitory substance (BLIS) typing in a longitudinal study of the oral carriage of beta-haemolytic streptococci by a group of Dunedin schoolchildren

A longitudinal study of the distribution of oral beta-haemolytic streptococci in 103 children (ages 5-6 y) attending three Dunedin schools was initiated in February 1987. A total of 858 paired pharyngeal and saliva specimens were obtained in nine principal sampling sessions, the last being in May 1989. The detection rate of Lancefield group A streptococci in directly plated pharyngeal cultures (19.8%) was considerably higher than in the corresponding saliva cultures (5.1%). By contrast, Lancefield group F and serologically non-groupable beta-haemolytic Streptococcus anginosus tended to be more prevalent in saliva (10.9% positive) than in pharyngeal specimens (7.1%). Group A streptococci were recovered at least once from 59 (57.3%) of the subjects. Representative beta-haemolytic streptococcus isolates were typed according to their production of bacteriocin-like inhibitory substances (BLIS) and by traditional serological techniques. The epidemiological value of BLIS typing was particularly evident in that both BLIS-positive and BLIS-negative strains could be differentiated within serotypes M1, M12 and M22 and also the serologically not typable group A streptococcus isolates. Ten children consistently had small numbers of group A streptococci present in their pharyngeal cultures, for periods of at least 12 months. In six of these subjects the carried strain was M-type 4/BLIS-type 655. This study has shown that in young schoolchildren long-term carriage of a variety of species of beta-haemolytic streptococci with little associated clinical evidence of infection may be common.     

Identification of group D streptococci by SeroSTAT.

Clinical isolates of group D streptococci presumptively identified by biochemical methods were grouped by latex agglutination using a commercially prepared reagent specifically sensitized with group D antiserum (SeroSTAT; Scott Laboratories, Inc., Fiskeville, R.I.). Streptococcus species tested included S. faecalis, S. faecium, S. durans, S. avium, S. bovis, and S. equinus. Colonies of the organism to be tested were picked from agar plates, emulsified in a drop of glycine-buffered saline on a slide, and mixed with a drop of the latex reagent. Macroscopic agglutination occurred within 60 s. A total of 115 isolates of group D streptococci were tested; 103 (89.6%) gave positive reactions with SeroSTAT. Twelve strains failed to react with the latex reagent; these 12 strains also gave negative results with group D antiserum when tested by the Lancefield method. Two of 14 group A streptococci also reacted with the SeroSTAT group D reagent; after trypsinization, the cross-reaction was eliminated. Group B streptococci, viridans group streptococci, anaerobic streptococci, and staphylococci all gave negative reactions with the SeroSTAT reagent. The SeroSTAT reagent is a useful diagnostic tool for the prompt identification of enterococcal and non-enterococcal group D streptococci.     

Rapid and accurate identification of Staphylococcus species by tRNA intergenic spacer length polymorphism analysis.

PCR-amplified tRNA gene (tDNA) intergenic spacer length polymorphism (tDNA-ILP) was analyzed for its ability to identify to the species level type strains (n = 18) and clinical isolates (n = 163) of staphylococci. Amplified products obtained by PCR with outwardly directed consensus tDNA primers were separated by agarose and polyacrylamide gel electrophoreses. The results were compared with those obtained by biochemical identification and ribotyping. Each type strain presented a specific tDNA-ILP pattern. PCR with fluorescent primers allowed for the detection of labelled DNA fragments on polyacrylamide gels by using an automated laser fluorescence sequencer and provided enhanced pattern resolution in comparison with that by analysis on agarose gels. tDNA patterns indistinguishable from those of the type strains were produced by clinical isolates of all tested species except for some isolates of S. aureus (n = 3) and S. haemolyticus (n = 1), which showed variant patterns. Strains of S. saprophyticus and S. xylosus showed very closely related profiles, and S. cohnii subspecies were indistinguishable. The identities obtained by tDNA-ILP analysis agreed with those obtained by the biochemical method to the species level for 99% (162 of 163) of the strains tested and to the subspecies level for 96% (156 of 163) of the strains tested. These results indicate that fluorescence-labelled PCR analysis of tDNA-ILP provides an accurate and rapid molecular method for the identification of human staphylococci.