Epidemiology and molecular characterization of Streptococcus pyogenes recovered from scarlet fever patients in central Taiwan from 1996 to 1999

Abstract

One hundred seventy-nine Streptococcus pyogenes isolates recovered from scarlet fever patients from 1996 to 1999 in central Taiwan were characterized by emm, Vir, and pulsed-field gel electrophoresis (PFGE) typing methods. The protocols for Vir and PFGE typing were standardized. A database of the DNA fingerprints for the isolates was established. Nine emm or emm-like genes, 19 Vir patterns, and 26 SmaI PFGE patterns were detected among the isolates. Among the three typing methods, PFGE was the most discriminatory. However, it could not completely replace Vir typing because some isolates with identical PFGE patterns could be further differentiated into several Vir patterns. The prevalent emm types were emm4 (n = 81 isolates [45%]), emm12 (n = 64 [36%]), emm1 (n = 14 [8%]), and emm22 (n = 13 [7%]). Some emm type isolates could be further differentiated into several emm-Vir-PFGE genotypes; however, only one genotype in each emm group was usually predominant. DNA from nine isolates was resistant to SmaI digestion. Further PFGE analysis with SgrAI showed that the SmaI digestion-resistant strains could be derived from indigenous strains by horizontal transfer of exogenous genetic material. The emergence of the new strains could have resulted in an increase in scarlet fever cases in central Taiwan since 2000. The emm sequences, Vir, and PFGE pattern database will serve as a basis for information for the long-term evolutionary study of local S. pyogenes strains.

FIG. 1.

Representative Vir patterns of genomes of 16 S. pyogenes isolates digested with HaeIII. Vir typing was performed by the standard protocol described in Materials and Methods. Lanes M, 100-bp ladders used as reference size markers; lanes 1 to 16, S. pyogenes isolates Sp07233 (VT1, emm1), Sp15088 (VT1.2, emm1), Sp11029 (VT2, emm1), Sp10837 (VT3, emm4), Sp15066 (VT4, emm4), Sp15580 (VT4.2, emm4), Sp15581 (VT4.4, emm4), Sp15735 (VT14, emm6), Sp15459 (VT6, emm12), Sp18217 (VT6.2, emm12), Sp09459 (VT7, emm12), Sp04348 (VT8, emm12), Sp08850 (VT9, emm22), Sp11433 (VT10, emm33), Sp11792 (VT11, emm74), and Sp11014 (VT12, st11014), respectively.

FIG. 2.

Dendrogram and Vir patterns of S. pyogenes isolates and the association with emm types and the number of isolates belonging to the genotype. The dendrogram was constructed with BioNumerics software, with 3% optimization and 0.85% position tolerance, by using the UPGMA algorithm with Dice similarity coefficients.

FIG. 3.

Restriction profiles of 10 rarely cutting restriction enzymes for S. pyogenes strain MGAS315 and the number of restriction sites in the genomes of strains MGAS315, MGAS8232, SF370, and SSI-1. The prediction was done with the whole genome sequences of the indicated strains by use of the Restriction Digest Tool provided on The Institute for Genome Research website (http://www.tigr.org/). Fragments above the dashed line (>21 kb) are effective for analysis with the reference size markers, XbaI-digested DNA of S. enterica serovar Braenderup strain H9812. The numbers at the bottom indicate the numbers of restriction sites on the genome of the indicated strains.

FIG. 4.

Representative PFGE patterns of 11 S. pyogenes isolates obtained by the standard PFGE protocol with SmaI digestion described in Materials and Methods. Lanes M, chromosomal DNA of S. enterica serovar Braenderup H9812 digested with XbaI as reference size markers (size range, 20.5 to 1,135 kb). Lane 1, S. pyogenes isolate Sp15601 (SPYS16.0006, emm4); lane 2, Sp16314 (SPYS16.0026, emm4); lane 3, Sp15090 (SPYS16.0006, emm4); lane 4, Sp15581 (SPYS16.0006, emm4); lane 5, Sp15735 (SPYS16.0020, emm6); lane 6, Sp18297 (SPYS16.0021, emm6); lane 7, Sp04853 (SPYS16.0019, emm12); lane 8, Sp05265 (SPYS16.0019, emm12); lane 9, Sp7286 (SPYS16.0019, emm12); lane 10, Sp08504 (SPYS16.0013, emm12); lane 11, Sp8536 (SPYS16.0015, emm12). The chromosomal DNA of strain Sp16314 was resistant to SmaI digestion.

FIG. 5.

Dendrogram and PFGE patterns of SmaI-digested chromosomal DNA of S. pyogenes isolates and association with emm types, Vir patterns, and the number of isolates belonging to these genotypes. The dendrogram was constructed with BioNumerics software, with 4% optimization and 1% position tolerance, by using the UPGMA algorithm and Dice similarity coefficients. The clusters (clusters A, B, C, D, E, and F) contained isolates with similarity coefficients greater than 70% (indicated by the dashed line).

FIG. 6.

Representative PFGE patterns of genomes of 10 S. pyogenes isolates digested with SgrAI. The chromosomal DNA of the S. pyogenes isolates was resistant to SmaI digestion. Lanes M, chromosomal DNA of S. enterica serovar Braenderup H9812 digested with XbaI as reference size markers (size range, 20.5 to 1,135 kb); lane 1, S. pyogenes isolate Sp11433 (SPYS15.0006, emm33); lane 2, Sp15006 (SPYS15.0016, emm12); lane 3, Sp15066 (SPYS15.0001, emm4); lane 4, Sp15504 (SPYS15.0016, emm12); lane 5, Sp15685 (SPYS15.0014, emm12); lane 6, Sp15782 (SPYS15.0016, emm12); lane 7, Sp18278 (SPYS15.0016, emm12); lane 8, Sp18471 (SPYS15.0014, emm12); lanes 9 and 10, Sp16314 (SPYS15.0003, emm4).

FIG. 7.

Dendrogram and PFGE patterns of chromosomal DNA of S. pyogenes isolates digested with SgrAI and the association with SmaI PFGE patterns, emm types, and the numbers of isolates belonging to these genotypes. The dendrogram was constructed with BioNumerics software, with 3% optimization and 1% position tolerance, by using the UPGMA algorithm and Dice similarity coefficients. A 220-kb DNA fragment (shown by the arrow) appeared with the six strains (Sp15685, Sp18471, Sp15006, Sp15504, Sp15782, and Sp18278) resistant to digestion with SmaI.