Abstract
A total of 274 Streptococcus dysgalactiae subsp. equisimilis isolates was analyzed by emm typing and by determining the organization of their mgrC loci. Three of the most frequent emm types were stG485.0 (45/274, 16.4%), stG6.1 (43/274, 15.7%), and stC839.0 (32/274, 11.7%), in decreasing order. The cpdB-positive mgrC locus appears to be predominant in some emm types.
Streptococcus dysgalactiae subsp. equisimilis is a member of the beta-hemolytic group G streptococci (GGS) and is associated with pharyngitis, bacteremia, and streptococcal toxic shock syndrome (6, 12, 16). S. dysgalactiae subsp. equisimilis may present certain virulence factors, one of which is the M protein, which is encoded by emm genes (1, 7, 12). Two types of multigene regulator-like (mgrC) loci with an Mga (Emm protein)-like structure have been reported to occur in S. dysgalactiae subsp. equisimilis (5). The large-type mgrC locus contains five genes (nrd, mgc, emm, cpdB, and rel), whereas the small-type mgrC locus contains four genes (nrd, mgc, emm, and rel). However, little is known about the correlation of emm types with mgrC structure in the clinical isolates of S. dysgalactiae subsp. equisimilis. The aims of this study were to investigate the distribution of emm types in group G S. dysgalactiae subsp. equisimilis isolates and to determine emm's correlation with structure of the mgrC locus.
Species identification was reconfirmed by the method described previously (10) for 406 GGS collected from the National Taiwan University Hospital in northern Taiwan from 1998 to 2004. Among them, 274 were identified as S. dysgalactiae subsp. equisimilis, 130 as Streptococcus anginosus, and 2 as Streptococcus canis. The emm genetic type was determined and analyzed with the Streptococcus emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm) of the Centers for Disease Control and Prevention. For 274 (103 from blood and 171 from samples other than blood) S. dysgalactiae subsp. equisimilis isolates, the most frequent emm types were of sequence type G485.0 (stG485.0) (45/274, 16.4%), followed by stG6.1 (43/274, 15.7%), stC839.0 (32/274, 11.7%), stG652.0 (24/274, 8.8%), and stG652.1 (17/274, 6.2%), in decreasing order (Table 1). For 103 blood isolates, stG6.1 was the most common type (19/103, 18.4%), followed by stG485.0 (16/103, 15.5%), stC839.0 (11/103, 10.7%), stG840.0 (8/103, 7.8%), stG166b.0 (7/103, 6.8%), and stG5420.0 (7/103, 6.8%). Types stG485.0 and stG6.1 have been reported from patients with group G bacteremia in Jerusalem, S. dysgalactiae subsp. equisimilis infections in Portugal, and invasive beta-hemolytic streptococci in Argentina (4, 11, 14). It is noted that stC839.0 was reported to be a dominant emm type from group C S. dysgalactiae subsp. equisimilis isolates in Spain and Australia (2, 12). In our study, this type was also found in some (11.7%, 32/274) group G S. dysgalactiae subsp. equisimilis isolates.
TABLE 1.
Distribution of emm types in 274 Streptococcus dysgalactiae subsp. equisimilis isolates
| emm type | No. (%) of isolates |
||
|---|---|---|---|
| Blood | Other sites | Total | |
| stG485.0 | 16 | 29 | 45 (16.4) |
| stG6.1 | 19 | 24 | 43 (15.7) |
| stC839.0 | 11 | 21 | 32 (11.7) |
| stG652.0 | 6 | 18 | 24 (8.8) |
| stG652.1 | 3 | 14 | 17 (6.2) |
| stG840.0 | 8 | 9 | 17 (6.2) |
| stG166b.0 | 7 | 5 | 12 (4.4) |
| stG4222.0 | 4 | 8 | 12 (4.4) |
| stG5420.0 | 7 | 4 | 11 (4.0) |
| stG6.0 | 6 | 4 | 10 (3.6) |
| stGLP1.0 | 5 | 3 | 8 (2.9) |
| stG10.0 | 1 | 5 | 6 (2.2) |
| stG4831.0 | 2 | 3 | 5 (1.8) |
| stG480.0 | 1 | 3 | 4 (1.5) |
| stC46.0 | 1 | 2 | 3 (1.1) |
| stG6792.0 | 0 | 3 | 3 (1.1) |
| stC74a.0 | 2 | 1 | 3 (1.1) |
| stG653.0 | 1 | 2 | 3 (1.1) |
| stGrobn.0 | 0 | 2 | 2 (0.7) |
| stG245.0 | 1 | 1 | 2 (0.7) |
| stG643.0 | 0 | 2 | 2 (0.7) |
| stCK401.1 | 1 | 1 | 2 (0.7) |
| stG97.0 | 0 | 1 | 1 (0.4) |
| stG643.1 | 1 | 0 | 1 (0.4) |
| stG2078.0 | 0 | 1 | 1 (0.4) |
| stC5345.0 | 0 | 1 | 1 (0.4) |
| stC5344.1 | 0 | 1 | 1 (0.4) |
| stC5344.0 | 0 | 1 | 1 (0.4) |
| stC6979.0 | 0 | 1 | 1 (0.4) |
| stG211.0 | 0 | 1 | 1 (0.4) |
| Total | 103 | 171 | 274 |
In order to investigate the association of emm types with the structure of the mgrC locus, the sequence of the mgrC locus was determined from two clinical isolates, NTUH-5518 (stG840) and NTUH-7688 (stG6.1), representing large and small mgrC types, respectively. The large-type locus (GenBank accession number GQ392134, which is most similar to AJ133440) in NTUH-5518 contained five genes (nrd, mgc, emm, cpdB, and rel), and the small-type locus (GenBank accession number GQ438775, which is most similar to Y18363) in NTUH-7688 contained four genes (nrd, mgc, emm, and rel) and a truncated cpdB gene (Fig. 1). The gene contents in the above-named isolates were very similar to those reported previously (5).
FIG. 1.
Genetic organization of mgrC loci in S. dysgalactiae subsp. equisimilis NTUH-5518 (large type) and NTUH-7688 (small type). Genes are shown as arrows indicating the direction of transcription. The sequence of the 8-nucleotide repeat (GCAACTAA) in NTUH-7688 (one copy) and NTUH-5518 (two copies) is indicated. The dotted arrows indicate undetermined sequences of nrd and rel. The start (ATG) and stop (TAA) codons of cpdB are underlined.
The major difference between the large and small mgrC loci is the length of the cpdB gene, which is 2,382 bp in the large type and 86 bp in the small type. A duplication of an 8-nucleotide sequence (GCAACTAA) was found in the cpdB gene (Fig. 1). The 8-nucleotide sequence was also found in the truncated cpdB gene in the small-type locus (accession number GQ438775). By searching the database, we could not find this 8-bp repeat in other species. Comparison of the cpdB genes from NTUH-5518 and NTUH-7688 revealed two conserved regions. The first one, located from the start codon to the 8-nucleotide sequences, was 47-bp long, whereas the other one, located from the 8-nucleotide sequence site to the stop codon, was 31-bp long (Fig. 1).
The homolog of CpdB has been identified in Haemophilus parasuis, Escherichia coli, Yersinia enterocolitica, and Streptococcus suis. The apparent deletion of 1,100 bp from the middle of the cpdB gene has been found in some serovars of H. parasuis (13). In E. coli and Yersinia enterocolitica, the CpdB protein was shown to participate in RNA degradation and enable bacteria to grow on 2′,3′-cAMP as a sole source of carbon and energy (9, 15). Furthermore, the cpdB and relA genes were upregulated under iron restriction conditions in S. suis (8), indicating that cpdB responded to a stringent environment and was regulated by carbon source availability. However, it is not clear if CpdB in S. dysgalactiae subsp. equisimilis works in a similar manner.
To clarify the types of mgrC loci in the remaining S. dysgalactiae subsp. equisimilis isolates, two sets of PCR were designed from accession numbers GQ392134 and GQ438775. One set of primers, emmF (5′-TATTG/CGCTTAGAAAATTAA-3′, corresponding to nucleotide positions 28 to 46 of the emm gene) and cpdBR5 (5′-GGTTACAAGTCAAAAAGCGCC-3′, corresponding to nucleotide positions 13 to 33 of the cpdB gene), is supposed to generate a 1.6-kb amplicon in the small-type or a 4.2-kb amplicon in the large-type mgrC locus. The other set of primers, emmF and relR (5′-CAACTTCTTCTCCTGTTAAATTG-3′, corresponding to nucleotide positions 15 to 37 of the relR gene), generates a 1.9-kb (small-type) or a 4.5-kb (large-type) amplicon. Among 274 isolates, 91 large- and 183 small-type mgrC loci were detected (Table 2). Our results also indicated that the mgrC types have significant correlation with emm types by two-way analysis of variance (ANOVA) (P < 0.001) but were not associated with blood or sites other than blood. These results were different from those for group A streptococci (GAS), in which the emm patterns were correlated with tissue tropism and disease presentation (3, 7). The small mgrC type was predominant in emm types stG6.1 (100%, 43/43 isolates), stG485.0 (96%, 43/45), and stG652.0 (84%, 21/24), whereas the large type (cpdB-positive) was predominant in stG840.0 (100%, 17/17 isolates), stG166b.0 (100%, 12/12), and stG4222.0 (92%, 11/12).
TABLE 2.
Distribution of two types of mgrC loci in 274 Streptococcus dysgalactiae subsp. equisimilis isolates
| emm type | No. (%) of isolates from indicated site(s) with small or large mgrC locus |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Blood |
Other sites |
Blood and other sites |
|||||||
| Small | Large | Total | Small | Large | Total | Small | Large | Total | |
| stG485.0 | 16 (100) | 0 (0) | 16 | 27 (93) | 2 (7) | 29 | 43 (96) | 2 (4) | 45 |
| stG6.1 | 19 (100) | 0 (0) | 19 | 24 (100) | 0 (0) | 24 | 43 (100) | 0 (0) | 43 |
| stC839.0 | 8 (73) | 3 (27) | 11 | 13 (62) | 8 (38) | 21 | 21 (66) | 11 (34) | 32 |
| stG652.0 | 5 (83) | 1 (17) | 6 | 16 (89) | 2 (11) | 18 | 21 (88) | 3 (12) | 24 |
| stG652.1 | 2 (67) | 1 (33) | 3 | 11 (79) | 3 (21) | 14 | 13 (76) | 4 (24) | 17 |
| stG840.0 | 0 (0) | 8 (100) | 8 | 0 (0) | 9 (100) | 9 | 0 (0) | 17 (100) | 17 |
| stG166b.0 | 0 (0) | 7 (100) | 7 | 0 (0) | 5 (100) | 5 | 0 (0) | 12 (100) | 12 |
| stG4222.0 | 1 (25) | 3 (75) | 4 | 0 (0) | 8 (100) | 8 | 1 (8) | 11 (92) | 12 |
| stG5420.0 | 7 (100) | 0 (0) | 7 | 4 (100) | 0 (0) | 4 | 11 (100) | 0 (0) | 11 |
| stG6.0 | 4 (67) | 2 (33) | 6 | 1 (25) | 3 (75) | 4 | 5 (50) | 5 (50) | 10 |
| stGLP1.0 | 0 (0) | 5 (100) | 5 | 1 (33) | 2 (67) | 3 | 1 (12) | 7 (88) | 8 |
| stG10.0 | 1 (100) | 0 (0) | 1 | 5 (100) | 0 (0) | 5 | 6 (100) | 0 (0) | 6 |
| stG4831.0 | 2 (100) | 0 (0) | 2 | 3 (100) | 0 (0) | 3 | 5 (100) | 0 (0) | 5 |
| stG480.0 | 0 (0) | 1 (100) | 1 | 0 (0) | 3 (100) | 3 | 0 (0) | 4 (100) | 4 |
| stC46.0 | 0 (0) | 1 (100) | 1 | 1 (50) | 1 (50) | 2 | 1 (33) | 2 (67) | 3 |
| stG6792.0 | 0 (0) | 0 (0) | 0 | 0 (0) | 3 (100) | 3 | 0 (0) | 3 (100) | 3 |
| stC74a.0 | 0 (0) | 2 (100) | 2 | 0 (0) | 1 (100) | 1 | 0 (0) | 3 (100) | 3 |
| stG653.0 | 1 (100) | 0 (0) | 1 | 2 (100) | 0 (0) | 2 | 3 (100) | 0 (0) | 3 |
| stGrobn.0 | 0 (0) | 0 (0) | 0 | 0 (0) | 2 (100) | 2 | 0 (0) | 2 (100) | 2 |
| stG245.0 | 1 (100) | 0 (0) | 1 | 1 (100) | 0 (0) | 1 | 2 (100) | 0 (0) | 2 |
| stG643.0 | 0 (0) | 0 (0) | 0 | 1 (50) | 1 (50) | 2 | 1 (50) | 1 (50) | 2 |
| stCK401.1 | 1 (100) | 0 (0) | 1 | 1 (100) | 0 (0) | 1 | 2 (100) | 0 (0) | 2 |
| stG97.0 | 0 (0) | 0 (0) | 0 | 1 (100) | 0 (0) | 1 | 1 (100) | 0 (0) | 1 |
| stG643.1 | 0 (0) | 1 (100) | 1 | 0 (0) | 0 (0) | 0 | 0 (0) | 1 (100) | 1 |
| stG2078.0 | 0 (0) | 0 (0) | 0 | 0 (0) | 1 (100) | 1 | 0 (0) | 1 (100) | 1 |
| stC5345.0 | 0 (0) | 0 (0) | 0 | 0 (0) | 1 (100) | 1 | 0 (0) | 1 (100) | 1 |
| stC5344.1 | 0 (0) | 0 (0) | 0 | 1 (100) | 0 (0) | 1 | 1 (100) | 0 (0) | 1 |
| stC5344.0 | 0 (0) | 0 (0) | 0 | 1 (100) | 0 (0) | 1 | 1 (100) | 0 (0) | 1 |
| stC6979.0 | 0 (0) | 0 (0) | 0 | 0 (0) | 1 (100) | 1 | 0 (0) | 1 (100) | 1 |
| stG211.0 | 0 (0) | 0 (0) | 0 | 1 (100) | 0 (0) | 1 | 1 (100) | 0 (0) | 1 |
| Total | 68 (66) | 35 (34) | 103 | 115 (67) | 56 (33) | 171 | 183 (67) | 91 (33) | 274 |
In conclusion, based on our data and those of others, the distribution of emm types in S. dysgalactiae subsp. equisimilis isolates may vary in different countries or regions. The cpdB-positive (large) mgrC locus appears to be predominant in some emm types, suggesting that cpdB may play a role in the clinical significance of isolates.
Footnotes
Published ahead of print on 23 June 2010.
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