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Journal of Clinical Microbiology, May 2006, p. 1836-1838, Vol. 44, No. 5
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.5.1836-1838.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rapid Differentiation between Members of the Anginosus Group and Streptococcus dysgalactiae subsp. equisimilis within Beta-Hemolytic Group C and G Streptococci by PCR

Liang-Chun Liu,1 Jui-Chang Tsai,2,4 Po-Ren Hsueh,3 and Lee-Jene Teng1,3*

Department of Clinical Laboratory Sciences and Medical Biotechnology,1 Center for Optoelectronic Biomedicine, National Taiwan University College of Medicine,2 Department of Laboratory Medicine,3 Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan4

Received 6 January 2006/ Accepted 14 February 2006


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ABSTRACT
 
Based on a pair of primers developed initially for differentiating the anginosus group from other viridans streptococci, the PCR reported here can also differentiate between members of the anginosus group and Streptococcus dysgalactiae subsp. equisimilis among beta-hemolytic group C and G streptococci. The resulting 742-bp PCR product was specific for members of the anginosus group, although a smaller, nonspecific product (361 bp) was generated from S. dysgalactiae subsp. equisimilis. Restriction digestion of the amplicon with XbaI and BsmI further differentiated Streptococcus anginosus from Streptococcus constellatus within the anginosus group.


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TEXT
 
The human isolates of beta-hemolytic group C (group C streptococci [GCS]) and group G streptococci (GGS) include the anginosus group (Streptococcus anginosus and/or Streptococcus constellatus) and Streptococcus dysgalactiae subsp. equisimilis. Among them, S. anginosus and S. dysgalactiae subsp. equisimilis are found in both GCS and GGS, but S. constellatus is found only in GCS. While these species are regarded as normal flora that colonize human skin, throat, intestine, and vaginal tract (6, 15), they can cause serious infections such as bacteremia, endocarditis, rheumatic fever, and streptococcal toxic shock syndrome (7, 9, 16).

Identification of the above-mentioned bacteria in the clinical laboratory setting usually depends on morphological observation and biochemical tests and serogrouping. Many laboratories report the identification of these bacteria to the group level but not the species level. However, differentiation to the precise species among these GCS and GGS should not be ignored, because it has been reported that large-colony GCS or GGS (referring to S. dysgalactiae subsp. equisimilis) may have virulence factors similar to those of Streptococcus pyogenes (9, 13) and are more invasive than the anginosus group among GCS or GGS (4).

Conventional methods of differentiation based on morphology and biochemical reactions were time-consuming, and the results were usually unsatisfactory (5). Molecular methods targeting the 16S rRNA gene, the 16S-23S rRNA intergenic spacer, or the groEL gene have recently been used as more accurate methods to identify species of the anginosus group or S. dysgalactiae subgroups (1, 2, 8, 16, 17). However, these methods usually need sequence determination and were not economical for clinical use.

We have previously determined the groESL sequences in viridans group streptococci (14). The groES and groEL genes (also known as cpn10/60) are evolutionarily conserved and are considered as an alternative for the identification of microorganisms. To differentiate the anginosus group from other viridans group streptococci, we designed a set of PCR primers specific for members of the anginosus group. In this study, we unexpectedly found that beta-hemolytic S. dysgalactiae subsp. equisimilis also generated a smaller, nonspecific PCR product.

Bacterial strains. The bacterial strains used in this study consisted of the following reference strains obtained from the American Type Culture Collection (ATCC) (Rockville, Md.) and 40 clinical isolates (23 GCS and 17 GGS) that were collected from the Bacteriology Laboratory, National Taiwan University Hospital, a 2,000-bed teaching hospital in northern Taiwan. The ATCC reference strains included S. pyogenes ATCC 19615, S. agalactiae ATCC 13813, S. anginosus ATCC 33397, S. constellatus ATCC 27823, S. intermedius ATCC 27335, S. sanguis ATCC 10556, S. gordonii ATCC 10558, S. bovis ATCC 43144, S. oralis ATCC 35037, S. mitis ATCC 49456, S. pneumoniae ATCC 49619, and S. dysgalactiae subspecies equisimilis ATCC 12388, ATCC 12394, and ATCC 35666.

Conventional identification. Colony size, hemolytic reaction, Voges-Proskauer (VP) reaction, and ß-glucuronidase (ß-GUR) activity were tested for 40 clinical isolates that were initially identified as Lancefield group C or G streptococci by an agglutination kit (Streptex; Murex Biotech Ltd., Dartford, United Kingdom). The colony size of each isolate was observed after 24 h of incubation at 37°C in 5% CO2 on a 5% sheep blood agar plate and was recorded as small, intermediate, or large. Small colonies were defined as having "sand-like" morphology, with a colony diameter of less than 0.5 mm. Large colonies were defined as having a diameter of ≥1.0 mm, and intermediate-sized colonies were defined as having a diameter between 0.6 and 0.9 mm. Two distinct types of hemolysis were observed, vague (Fig. 1A) and clear (Fig. 1B).


Figure 1
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  		FIG. 1. Colony morphologies of GCS and GGS. (A) S. anginosus clinical isolate GCS 927. (B) S. anginosus clinical isolate GCS 1577. (C) S. anginosus ATCC 33397. A, B, and C showed different colony sizes and hemolytic characteristics. (D) S. dysgalactiae subsp. equisimilis ATCC 12388. (E) S. dysgalactiae subsp. equisimilis clinical isolate GGS 4115. The colony sizes of E were larger than that of the corresponding reference strain (D). Colony sizes of B and D were similar. The line is a scale that represents 10 mm.

Among 23 isolates of GCS, 12 isolates had small colonies, 11 of which showed vague hemolysis (two of them were VP-positive S. anginosus isolates, eight were VP-positive S. constellatus isolates, and one was a VP-negative S. constellatus isolate) and one of which showed clear hemolysis (VP-positive S. anginosus). Five isolates had intermediate colony size, three of which had vague hemolysis (one was a VP-negative S. anginosus isolate, one was a VP-negative S. constellatus isolate, and one was a VP-positive S. constellatus isolate) and two of which had clear hemolysis (one VP-positive S. anginosus isolate and one VP-negative S. anginosus isolate). The remaining six isolates had large, clear hemolytic colonies, which were VP negative, and were identified as S. dysgalactiae subsp. equisimilis by 16S rRNA gene sequencing.

Three types of colony morphologies were found among the 17 GGS clinical isolates. Three isolates formed small, vaguely hemolytic colonies (two VP-positive isolates and one VP-negative isolate). Three isolates had intermediate, clearly hemolytic colonies (one VP-positive and two VP-negative isolates). The six isolates described above were identified as S. anginosus by 16S rRNA gene sequencing. The remaining 11 isolates had large, clearly hemolytic colonies that were VP negative and were identified as S. dysgalactiae subsp. equisimilis by 16S rRNA gene sequencing.

The ß-GUR test was performed as described previously by Fox et al. (6). It has been reported that the ß-GUR test may provide better differentiation between S. dysgalactiae subsp. equisimilis and S. anginosis. In the present study, the results from 23 isolates of GCS were in agreement with results reported previously by Fox et al. (6), which showed that all anginosus group isolates displayed negative ß-GUR activity, while S. dysgalactiae subsp. equisimilis had positive reactions. However, in GGS, one exception occurred. One isolate that was identified as S. dysgalactiae subsp. equisimilis by 16S rRNA gene sequencing displayed negative ß-GUR activity. A similar result was reported previously by Lawrence et al. (12). The negative ß-GUR activity may lead to misidentification.

Anginosus group-specific PCR. On the basis of known streptococcal groESL sequences (14), we designed a pair of primers to differentiate members of the anginosus group from other members of viridans group streptococci. The primers are milleri-EL-F (5'-ACTCTTGTGTTAAATAAAATCC-3'), corresponding to nucleotide positions 775 to 796, and milleri-EL-2R (5'-ACGCAGCATTTTGAAGRGCA-3') (where R = A + G), corresponding to nucleotide positions 1516 to 1497 of the groEL gene. PCR was performed for 30 cycles, with each cycle consisting of 1 min at 94°C, 30 s at 53°C, and 1 min at 72°C, followed by a final extension step at 72°C for 7 min. PCR products were subjected to 1.5% agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light.

The PCR was found to be specific to the anginosus group, with the exception of S. dysgalactiae subsp. equisimilis. The isolates that were identified as belonging to the anginosus group (including S. anginosus and S. constellatus) by 16S rRNA gene sequencing always produced the 742-bp products. S. dysgalactiae subsp. equisimilis isolates can also generate the 361-bp nonspecific products (Fig. 2). The sequence of this nonspecific 361-bp amplicon was determined and compared to published sequences in the GenBank database by using BlastN algorithm. The closest match was obtained with the DNA gyrase subunit b of S. pyogenes MGAS315, section 11 of 37 of the complete genome (GenBank accession number AE014146) (88% nucleotide sequence identity and 99.8% amino acid identity).


Figure 2
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  		FIG. 2. Gel electrophoresis of anginosus group-specific PCR products from group C and group G streptococcal clinical isolates. Lane M, molecular size marker (100-bp ladder; Gibco BRL, Gaithersburg, Md.); lanes 1 to 3, group C streptococcus S. constellatus; lanes 4 to 6, group C streptococcus S. anginosus; lanes 7 and 8, group C streptococcus S. dysgalactiae subsp. equisimilis; lanes 9 to 11, group G streptococcus S. dysgalactiae subsp. equisimilis; lanes 12 to 15, group G streptococcus S. anginosus; lane C, negative control (no DNA). All S. anginosus and S. constellatus isolates generated 742-bp products. All S. dysgalactiae subsp. equisimilis isolates (lanes 7 to 11) generated 361-bp products.

PCR-RFLP. In order to further differentiate the species (S. anginosus and S. constellatus) among the anginosus group of GCS, the 742-bp amplification product was subsequently digested with the restriction enzymes XbaI (37°C, 3 h) and BsmI (65°C, 3 h) (Gibco BRL, Gaithersburg, Md.). XbaI digested the 742-bp PCR product of S. constellatus into 138-bp and 604-bp fragments but did not do so for S. anginosus (Fig. 3A). BsmI digested 742-bp PCR product of S. anginosus into 151-bp and 591-bp fragments but did not do so for S. constellatus (Fig. 3B). Therefore, PCR restriction fragment length polymorphism (RFLP) could be used as a confirmatory test for the identification of S. anginosus in GGS and differentiation between S. anginosus and S. constellatus in GCS. Among clinical isolates, we discovered that regardless of the morphology and biochemical results, those isolates that generated the 742-bp PCR products and that were digested by XbaI into two fragments were all identified as S. constellatus. Those that generated the 742-bp PCR products and that were digested by BsmI into two fragments were all identified by 16S rRNA gene sequencing to be S. anginosus. Isolates that generated small (361-bp) amplicons were identified as S. dysgalactiae subsp. equisimilis.


Figure 3
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  		FIG. 3. RFLP of PCR-amplified 742-bp products. (A) XbaI digestion. (B) BsmI digestion. Lane M, molecular size marker (100-bp ladder; Gibco BRL, Gaithersburg, Md.); lane 1, S. constellatus ATCC 27823; lanes 2 to 5, S. constellatus clinical isolates; lane 6, S. anginosus ATCC 33397; lanes 7 to 10, S. anginosus clinical isolates.

It was previously reported that some S. dysgalactiae subsp. equisimilis and S. anginosus display the group A antigen, which can lead to their misidentification as S. pyogenes (5, 11, 17). S. dysgalactiae subsp. equisimilis and S. pyogenes have been reported to cause similar diseases such as streptococcal toxic shock syndrome, pharyngitis, and rheumatic fever; they also share some virulence factors such as emm, scpA, ska, slo, speM, ssa, smeZ, and sag genes (3, 4, 9, 10, 13). The PCR that we developed in this study can help differentiate between S. dysgalactiae subsp. equisimilis and S. pyogenes among those isolates with the group A streptococcal antigen.

In conclusion, the anginosus group-specific PCR provided rapid and reliable differentiation among members of the anginosus group, S. dysgalactiae subsp. equisimilis, and S. pyogenes. PCR amplification coupled with RFLP allowed further differentiation between S. anginosus and S. constellatus. This PCR test provides an alternative identification method for use in clinical laboratories.


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ACKNOWLEDGMENTS
 
This work was supported by grant NSC 92-2314-B-002-353 from the National Science Council of Taiwan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, 1 Chang-Te Street, Taipei 100, Taiwan. Phone: 886-2-23123456, ext. 6918. Fax: 886-2-23711574. E-mail: ljteng{at}ha.mc.ntu.edu.tw. Back


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Journal of Clinical Microbiology, May 2006, p. 1836-1838, Vol. 44, No. 5
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.5.1836-1838.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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