β-Hemolytic streptococci are common pathogens that usually cause community-acquired infections.

In general, β-hemolytic streptococci isolated from human beings belong to Lancefield groups A, B, C, G, F, or more rarely, to L18. Until recently, group A streptococci (GAS, Streptococcus pyogenes)63 and group B streptococci (GBS, Streptococcus agalactiae)45,60 were considered the most important pathogens of this group of microorganisms in clinical settings.

GAS is responsible for a broad spectrum of human infections that result in significant morbidity and mortality, including pharyngitis, scarlet fever, skin and soft tissue infection (SSTI), streptococcal toxic shock syndrome (STSS), septicemia, pneumonia and rarely meningitis13. It is estimated that severe GAS diseases lead to more than 500,000 deaths each year via infections such as acute rheumatic fever, rheumatic heart disease, post streptococcal glomerulonephritis and invasive diseases8.

In contrast, group C and G Streptococcus dysgalactiae subsp. equisimilis (GCS and GGS) were long considered to be commensal organisms that only rarely cause invasive infections as opportunistic pathogens.

Since the mid-1980s there has been a marked increase in reported invasive group A infections, including cases of STSS in Europe and North America15,33,57.

These fulminant infections were characterized by acute hypotension, shock, multiorgan impairment and death. However, the factors underlying the worldwide resurgence of this pathogen remain unknown16.

In 1996, Vandamme et al62. proposed that a novel subspecies, S. dysgalactiae subsp. equisimilis (SDSE), was a clinically significant pathogen. This microorganism possesses group C or G antigens (rarely A or L), and exhibits strong β-hemolysis.

Beginning around the year 2000, invasive infections such as bacteremia caused by SDSE as well as those caused by GAS and GBS6,12,38,59 have been increasingly reported worldwide.

M proteins of GAS, which are encoded by emm genes and form elongated structures on the bacterial surface, play an important role in the pathogenesis of this microorganism. M proteins are known to be critical antiphagocytic constituents of GAS due to their role in resistance to opsonization4,14. GCS and GGS have shown a similar pathogenic pattern to S. pyogenes21, coincidently expressing homologs of the M virulence proteins of S. pyogenes7. Moreover, some strains contain superantigen genes firstly characterized in S. pyogenes25,29. As with the emm genes of S. pyogenes, GCS and GGS homologs have been used for sequence-based typing3,17,25.

We conducted a prospective study to assess the relative prevalence of SDSE versus GAS in invasive infections in Argentina during a twelve-month period. The objective of this study was to determine their epidemiological features, to explore the molecular characteristics of the infecting strains, to study their genetic diversity, virulence genes, antibiotic susceptibility and antibiotic resistance genes.

All S. pyogenes and SDSE isolated from invasive infections in 28 centers of 16 Argentinean cities from July 1, 2011 to June 30, 2012 were studied.

A microbiologist from each hospital was required to complete a data sheet of the isolates. During the study period, each laboratory collected one isolate per patient in accordance with the case definition. Isolates were appropriately sent to the reference center where they were preserved in 300μl of sheep blood at −80°C.

Invasive SDSE infections included sepsis with an unknown focus, cellulitis, septic arthritis, pneumonia, necrotizing fascitis, meningitis, infectious endocarditis, STSS, abscesses at sites other than skin, osteomyelitis, and others.

In our study most of the SDSE isolates were obtained from blood (n: 14). The rest of the isolates were obtained from bone, joint fluid or soft tissue (n: 7).

All our strains were correctly identified by both systems (manual and API 20 Strep) as SDSE. The correlation was excellent between the tests with ROSCO tablets (esculin, sorbitol, trehalose, α-galactosidase and β-glucuronidase) and the same biochemical tests in the API 20 Strep panel.

The frequency of invasive SDSE infection has increased in Asia5,38, in Europe19,34,36,37,48,53,54, and in America1,6,40 over the years. In particular, since 2003, the number of invasive SDSE infections, including STSS and severe soft tissue infection22,26,44, has gradually increased in Japan each year.

By the year 2011 the Argentine population was 40.73 million inhabitants, therefore, the annual prevalence of SDSE invasive infections (n: 23) was at least 0.06/100,000 in Argentina during the 2011–2012 period. This prevalence seems to be lower than that recorded in a previous multicenter study conducted in 1998–1999, in which 27 invasive SDSE infections were reported during six-months; however, more health institutions participated in that study40.

As for age distribution, a population-based surveillance carried out by Broyles et al. (n: 212)6 found that most patients suffering from invasive diseases due to SDSE (59.0%) were adults under 65 years old. Moreover, Takahashi et al. analyzed 286 SDSE infections from August 2006 to December 200960 and all patients with invasive infections were adults, often elderly (74.0%), and most of them with underlying diseases. Another investigation58 also indicated that invasive SDSE infection (n=42) mostly occurred in elderly adults (60–80 years old). Severe underlying conditions (i.e., diabetes mellitus, liver or renal dysfunction, and others) were associated with 85.7% of these invasive SDSE infections.

In our study, the age range was 21–86 years; 57.1% of the patients were adults older than 50 years old (12 out of 21 patients).

With regard to underlying diseases, in U.S. surveillance reports, 96.2% of patients with invasive infections possessed underlying medical conditions13.

Similarly to the study by Sunaoshi et al.58, 69.9% of SDSE-infected patients in Argentina presented at least one underlying disease and 17.4% had more than one, being diabetes the most frequent one.

Concerning SDSE disease outcomes, the mortality rate in a Japanese study (12.7%) was similar to that previously described in Hong Kong and the United States (12 and 15%)6,60,64. In our series, the mortality rate due to SDSE invasive infections was 8.7% (2 cases) and both were adults. The same percentage corresponded to STSS cases. Three STSS cases and 3 deaths were recorded in a previous six-month Argentinean study40.

Neither fatalities nor SDSE-mediated STSS were detected in children. SDSE-mediated STSS was a consequence of infections due to types stG840 and stG62647 and fatal infections were associated with stC74.a and stG62647.

The dominant emm types in Japan (stG6792 and stG485) were different from those in the United States (stG6, stG245, stG2078, and stG643). The geographical factor may not be the only cause accounting for such difference since both study periods differed in these studies (2002–2004 vs. 2006–2009)6,60. The emm type stG6792 was the most frequently found in SDSE Japanese isolates (n: 65; 22.7%) and was more strongly related to poor outcome of SDSE diseases than other SDSE emm types60. Furthermore, those isolates displayed similar DNA profiles with PFGE suggesting the clonal expansion of a specific subpopulation of strains rather than the spread of distinct strains.

In another study58, 3 emm types, stG6792, stG485, and stG2078, predominated among the 42 invasive strains; strains having the same emm type showed uniform DNA profiles by PFGE. Moreover, previous reports described emm types and the DNA profiles by PFGE as variable among SDSE strains22,26. In two other SDSE bacteremia studies carried out in the U.S. and Israel, stG485, stG6, stG245 and stG2078 types were the most frequently found6,12.

In our study, 19 distinct DNA profiles by PFGE were detected, not having significant relationship among them. These results were expected because strains were not isolated in the context of an epidemic outbreak and were obtained from different geographic places. Predominant emm-types were stG62647 (n: 4), stG840 (n: 3) and stG653 (n: 3). stG6792 (n: 1), stG485 (n: 2) and stG2078 (n: 0) types were less represented in the present study, in contrast to the study conducted by Sunaoshi et al58. The types in this study were also different from those reported in the previous Argentinean multicenter study (1998), in which stG6.0 (n: 6), stG480 (n: 5) and stG10.0 (n: 4) types40 predominated.

Therefore, emm typing is an excellent comparative epidemiological tool for the analysis of SDSE strains belonging to different geographical regions or from the same region but isolated at different times.

The complete genomic sequence of SDSE GGS_124 (stG480.0) isolated from patients with STSS (GenBank accession No. AP010935) have been recently determined. The genome size was 2.1 Mbp, and sequence coverage with GAS genomes was 61–63% identity20. Interestingly, many genes encoding virulence factors in GAS were identified in SDSE.

SDSE possesses many virulence factors shared with GAS28, such as M protein, streptolysin O, streptolysin S, streptokinase, fibronectin binding protein, collagen binding protein and DNase. They also exhibit homology in streptococcal inhibitor of complement (SIC). However, they do not possess certain virulence factors, such as some superantigens, cysteine protease (SPE-B) and the ABC operon52. However, it was reported that some strains of SDSE may contain superantigen genes, which were firstly characterized in S. pyogenes25,29.

It has been suggested that these factors were transmitted from GAS to SDSE species30.

We analyzed speA, speB, speC, speF, speG, speH, speJ, smeZ and ssa genes. All the strains harbored only speJ and ssa genes simultaneously.

In the study by Ikebe et al. from Japan, all isolates were negative to speA, speB, speC, speH, speI, speJ, speL and speM, however, 12 out 16 isolates had the speG gene26.

As shown in the present study and in previous reports, SDSE secretes only a few previously known superantigen exotoxins23,35,46.

The clinical manifestations of STSS caused by SDSE are similar to EGA. speA, speB, and speC genes were not observed in our study although they are the most frequent detected exotoxins in S. pyogenes. Therefore, we suspect that not yet identified superantigen exotoxins or exoenzimes may exist, which could play an important role in the development of STSS caused by SDSE.

Although 60 years have passed since the introduction of PEN, β-hemolytic streptococci still continue being susceptible to this antibiotic, even though some S. agalactiae strains showed reduced susceptibility32.

Macrolide, CLI and TET resistance has been a matter of real concern because of the development of ERY-resistant S. pyogenes outbreaks in Japan, Australia and several Europeans countries since the 70s42,51,56. Macrolide and TET resistance has been observed both in GAS and SDSE31,43.

SDSE isolates (n=212) collected in a multicenter surveillance study by Broyles et al.6 in the United States showed resistance rates of 28.8% to ERY, 4.2% to CLI, and 0.9% to fluoroquinolones.

In Korea39, high frequency of the TET resistance-mediating tet(S) gene was demonstrated (68.8%), while ERY, CLI, and chloramphenicol resistance rates were low (9.4, 3.1 and 9.4%, respectively)61.

Of 231 SDSE isolates in the study by Takahashi et al.60, four harbored the mef(A) gene, and 13 and six isolates carried the erm(A) and erm(B) genes, respectively.

In our study, 19.0% were non-susceptible to CLI and 26.1% to ERY. Only one strain was resistant to ERY (iMLSB phenotype, ermTR positive) and no CLI constitutive resistance was detected in the previous multicenter study40.

In our study, TET resistance was 30.4%. Two tetracycline-resistant strains only carried the tet(M) gen while five others carried both the tet(M) and tet(O) genes.

TET resistance was also common in SDSE during the 1998–1999 period in Argentina (33.3% in group C SDSE and 40% in group G SDSE)40. In such study, TET resistance was 40.7% and all TET resistant strains carried only the tet(M) gen.

In Portugal, a high rate of fluoroquinolone resistance (12%) was detected between 1998 and 200547, although a previous study from Europe and the United States detected less than 1% resistance toward this group of antibiotics2.

All our isolates were in vitro susceptible to LEV. Only one isolate (strain 9) was found to have reduced susceptibility to NOR, PEF and OFL.

Fluoroquinolones act by inhibition of bacterial DNA gyrase and DNA polymerase. The alterations of these enzymes were the prevalent mechanism of resistance in the Streptococcus genus24. The mutation in the gyrA subunits (gyrase DNA) and parC (DNA topoisomerase) are found in the so-called quinolone resistance determining regions (QRDR). The mechanisms involved in the small diameters observed by the disk diffusion test with strain 9 deserve to be further studied.

In the future, we should continue to survey invasive SDSE infections to further clarify the prevalence of infection, the study of new virulence factors, the distribution of emm types and to monitor antibiotic susceptibility rates in the Argentinean population.

The authors declare that they have no conflicts of interest.