Survey of emm-Like Gene Sequences from Pharyngeal Isolates of Group C and Group G Streptococci Collected in Spain

Abstract

We conducted a nationwide surveillance of the variable 5′ emm-like (M-like protein gene) sequences from 214 pharyngeal group C and group G streptococci. Almost 75% of the isolates exhibited emm or emm-like sequences previously described. We identified six new 5′ emm-like regions, and almost 23% of the isolates were nontypeable. Five emm-like sequences accounted for more than 50% of the isolates in group C and group G, suggesting horizontal gene transfer between strains of different species.

Group C beta-hemolytic streptococci (GCS) and group G beta-hemolytic streptococci (GGS) form a heterogeneous collection of microorganisms, which, unlike other beta-hemolytic streptococci, such as group A streptococci (GAS), can be isolated from either human or animal specimens (10). In humans, GCS and GGS may cause pharyngitis and skin and soft tissue infections, although not as frequently as GAS (6, 9, 18).

Human-associated strains of GGS and GCS may present virulence factors similar to those expressed by GAS, including the major virulence factor of these important pathogens, the M protein (5, 7, 12, 16).

M proteins of GAS exhibit a substantial polymorphism due to the high degree of heterogeneity among the N termini of diverse M proteins, leading to more than 80 different serological M or OF types. The variable sequences of surface-exposed amino termini of M proteins may be deduced by sequencing the 5′ M protein-encoding gene, emm. Thus, since the emm gene's possible polymorphism was first reported, in 1995 (4), its gene sequence analysis has proven quite useful for routine M typing of GAS and many epidemiological studies have applied this methodology for monitoring GAS strain diversity (2, 3, 8). M proteins of GAS, GCS, and GGS recovered from human infections have similar biological, immunochemical, and structural features (5-7, 12, 16). Collins et al. reported the first complete sequence of an M-like protein gene of a human GGS strain (7). Later, Smirnov et al. published the sequence of a GGS gene encoding an immunoglobulin G-binding protein (17). More recently, Bisno et al. reported that emm gene sequences from GCS pharyngeal isolates were highly homologous to those found in GGS (6). The proteins encoded by these genes exhibited features similar to those present in the GAS M proteins, but they differed significantly in the N terminal region. These data suggest that, like GAS, GGS and GCS can exhibit various M-like proteins encoded by emm-like genes (emmL). Based on these observations, Schnitzler et al. extended the earlier studies and demonstrated that the emmL gene from 38 epidemiologically unrelated GGS strains isolated from human specimens displayed a substantial diversity, suggesting that this polymorphism could provide the basis for a molecular typing of GGS (15).

In the present work, we analyzed a larger number of GCS and GGS isolates for the variability of the 5′ region of the emmL to study the M-like type distribution of these isolates in Spain.

Bacterial strains.

GCS and GGS isolates included in this study belong to the SAUCE II (November 1998 to October 1999) surveillance collection and were isolated from patients with acute pharyngitis in 17 different hospitals selected on the basis of population and geographic location in Spain (13). Among 2,292 beta-hemolytic streptococcus isolates collected during this period, 155 (6.8%) and 59 (2.6%) were GCS and GGS, respectively. Isolates were tested initially for beta-hemolysis by using 5% (vol:vol) defibrinated horse blood agar and serotyped by using specific C and G group antisera (Remel, Lenexa, Kans.). Colony size (smaller or larger than 0.5 mm in diameter), Voges-Proskauer test results (11), and production of β-d-glucuronidase (20) were determined to differentiate between Streptococcus equi subsp. zooepidemicus and Streptococcus dysgalactiae subsp. equisimilis from the “Streptococcus milleri” group (S. anginosus, S. intermedius, or S. constellatus).

Further testing to differentiate among species included acid production from trehalose and sorbitol to differentiate S. equi subsp. zooepidemicus from S. dysgalactiae subsp. equi and production of β-d-fucosidase, β-d-glucosidase, and/or α-d-glucosidase by using 4-methyllumbelliferyl-linked fluorogenic substrates in microtitration plates as previously described (20) to differentiate among S. anginosus, S. intermedius, and S. constellatus.

Differentiation criteria are summarized as follows. S. equi subsp. zooepidemicus or S. dysgalactiae subsp. equisimilis were presumed for those isolates forming minute colonies (less than 0.5 mm in diameter) and confirmed only when positive β-d-glucuronidase and negative Voges-Proskauer test results were obtained. Further differentiation between both species was based on acid production from trehalose and/or sorbitol (S. equi subsp. zooepidemicus is trehalose negative or variable and sorbitol positive, while S. dysgalactiae subsp. equisimilis is trehalose positive and sorbitol negative). The S. anginosus, S. intermedius, and S. constellatus group was presumed when isolates formed colonies larger than 0.5 mm in diameter and confirmed by negative β-d-glucuronidase and positive Voges-Proskauer tests. Further species differentiation among these three species was done according to the production of β-d-fucosidase, β-d-glucosidase, and/or α-d-glucosidase (S. anginosus, β-d-glucosidase positive and β-d-fucosidase negative; S. intermedius, β-d-fucosidase and α-d-glucosidase positive; S. constellatus, β-d-fucosidase negative and α-d-glucosidase positive).

Among the GCS isolates, we identified 29 as S. constellatus, 70 as S. dysgalactiae (subsp. equisimilis), 7 as S. intermedius, and 49 as S. anginosus. Almost 82% (48) of the GGS isolates were S. dysgalactiae (subsp. equisimilis), and only 8 and 3 isolates were identified as S. anginosus and S. constellatus, respectively.

emm-like gene typing.

The emmL gene types of GCS and GGS isolates were determined by amplification and sequencing of the emmL gene with primers G1F and G1R (15). Lysates of the GCS and GGS isolates were prepared with mutanolysin as we previously described for GAS (1). The sizes of the amplicons ranged from about 0.8 kb to approximately 1.6 kb, a size range similar to that found for GAS emm genes (2). PCR products were sequenced with primer G1F and a dye terminator mix (Perkin-Elmer, Applied Biosystems, Madrid, Spain) and were subjected to automated sequence analysis on a 377 DNA sequencer (Perkin-Elmer, Applied Biosystems). DNA sequences were subjected to homology searches against the bacterial DNA database with BLASTN. Sequences were given the GenBank emm or emmL designation following the criteria previously described (4, 15).

Prevalent emmL genes.

Results of the survey of the emmL gene sequences are shown in Table 1. Overall, 155 (72.43%) of 214 GCS and GGS pharyngeal isolates included in our study had 5′ emm sequences that were ≥95% identical to the first 120 bases of one of the emm or emm-like genes deposited in GenBank. For most of these sequences, this high level of identity actually extended to 200 to 450 bases without diminishing. For some sequences, we found more than one emm or emm-like gene in the GenBank with a homology of between 95 and 100% in the 5′ emm region. In these cases, we gave the emm designation with the highest homology. Nine of the 214 isolates had an undocumented emm or emmL gene sequence. All of the sequences were less than 90% identical over the first 160 bases to sequences deposited in GenBank. The sequences were designated emmLC1900, emmLC1901, emmLC1902, emmLC1903, emmLC1904, and emmLC1905 and have been deposited in the EMBL data library.

TABLE 1.

emm or emm-like sequences of pharyngeal GCS and GGS isolated in Spain in 1998-1999^a

emm or emmL type	No. of isolates of:								Total (%)
	S. constellatus		S. anginosus		S. dysgalactiae subsp. equisimilis		S. intermedius
	C	G	C	G	C	G	C	G
emm12	1								1 (0.47)
emm28D	4		5		10	1	1		21 (9.81)
emm3							1		1 (0.47)
emm9			1		2				3 (1.40)
emmC1139	3		4		7	1	4		19 (8.87)
emmC839	2		10	1	19	3			35 (16.35)
emmLG480	1				1	5			7 (3.27)
emmLG485		1			2	5			8 (3.74)
emmLG494			1			2			3 (1.40)
emmLG643						1			1 (0.47)
emmLG935		1				4			5 (2.34)
fcrV	3	1	3	3	4	17			31 (14.48)
C74a					1	1			2 (0.93)
emm73					1				1 (0.47)
mlc36	3		3		9	1			16 (7.48)
emmLC1900					2	1			3 (1.40)
emmLC1901					1				1 (0.47)
emmLC1902					1				1 (0.47)
emmLC1903					1				1 (0.47)
emmLC1904	2								2 (0.93)
emmLC1905						1			1 (0.47)
st1400c	1								1 (0.47)
NT	9		22	4	9	5	1		50 (23.36)
Subtotal	29	3	49	8	70	48	7	0
Total for species		32		57		118		7	214

^aC, GCS; G, GGS; NT, nontypeable.

We were not able to amplify the emm or emmL gene from 50 (23.36%) isolates (nontypeable isolates) with primer pair G1F-G1R or GASM1-GASM2 (4). Although it has been reported that all human-associated GGS strains harbor emmL genes, it may be possible that nontypeable isolates do not contain this gene, as occurs with those strains recovered from animal sources (16). In fact, Podbielski et al. (14), utilizing PCR assays and genomic fingerprinting, detected emm gene in 75% of 28 GCS strains isolated from human specimens. The author's findings are in agreement with our results, since 76.63% of our isolates harbor emm or emmL sequences that were amplified by PCR. Interestingly, more than half of the nontypeable isolates were S. anginosus, and almost half of the isolates of this species were nontypeable. These results suggest that the nontypeable isolates express another protein(s) or use a different mechanism to replace the M protein since this protein is a critical virulence factor causing pharyngitis and resists opsonophagocytosis in GAS and in many GCS and GGS strains. Alternatively, it may be possible that the G1F-G1R and GASM1-GASM2 annealing regions are not conserved in the nontypeable isolates and as a consequence we failed to amplify these genes.

emmC839, fcrV, emm28D, emmC1139, and mlc36 were the most prevalent emm sequences among the GCS and GGS isolated in Spain. They accounted for 16.35, 14.48, 9.81, 8.87, and 7.48%, respectively, of the isolates. These results indicate that there are ongoing horizontal gene transfers between the species of these two groups of beta-hemolytic streptococci. The remaining isolates represented less than 50% of the total and were associated with 17 different emm or emmL types. As occurred in the survey of emm gene sequences from pharyngeal GAS isolates conducted in Spain (2), only five emm types account for the majority of the isolates, suggesting that the M-like proteins encoded by these genes are more advantageous for the colonization of the human pharynx by GCS and GGS. However, the most prevalent GAS emm types (emm1, emm3, emm12, and emm9) were not represented among the most frequently observed emm or emmL types for GCS and GGS. By contrast, two GCS and five GGS strains exhibited emm gene sequences (emm12, emm3, emm9, and st1400c) that were identical over the first 160 bases to those reported for GAS, suggesting a horizontal gene transfer between strains of these species of beta-hemolytic streptococci. This transference could be responsible for the development of gene mosaics and the evolution of emm genes in beta-hemolytic streptococci (19).

In conclusion, using a large number of unrelated GCS and GGS pharyngeal isolates, we have confirmed the existence of an emmL gene polymorphism in these isolates and observed that this emmL gene diversity provides the basis for an emmL typing technique. The application of this typing technique to nationwide multicenter surveillance has revealed the existence of five prevalent types in Spain and has suggested that there is a horizontal transfer of emm genes between beta-hemolytic streptococci.

Nucleotide sequence accession numbers.

The sequences described in this study were designated emmLC1900, emmLC1901, emmLC1902, emmLC1903, emmLC1904, and emmLC1905 and were deposited in the EMBL data library with the accession numbers AY686726, AY686727, AY686728, AY686729, AY686730, and AY686731, respectively.