emm typing of M nontypeable invasive group A streptococcal isolates in Israel

We performed emm typing of M nontypeable invasive group A streptococcal (GAS) isolates collected in a prospective population-based study in Israel. One hundred twenty of 131 isolates (92%) had emm sequences compatible with GAS, consisting of 51 different emm types. Eleven isolates were found to be group G streptococcus. Of the 120 isolates, 55 (46%) belonged to 32 types for which there were no typing sera available in the Streptococcal Reference Laboratory in Israel. The other 65 (64%) isolates, consisting of 19 types, had sera available and therefore could have been serotyped. Forty-three isolates had T and emm types which were not correlated according to standard M-typing protocols and were therefore missed. The principal effect of emm typing was the addition of 32 types not previously identified in Israel and the discovery of new associations between emm and T types. emm typing did not significantly change the proportion of M types; the five most common types were 3, 28, 2, 62, and 41. Twenty different types comprised 80% of all isolates. No new emm sequences were discovered. emm typing emphasized the unusually low incidence of M1 strains causing severe disease in Israel. As serological typing of GAS becomes more problematic due to lack of sera and the appearance of new emm types, reference laboratories should replace M typing with emm sequence typing. Development of a GAS vaccine relies on the emm type distributions in different geographical locations. In our study, 7% of isolates (types 41 and 62) are not included in a 26-valent vaccine that is being studied.     

Distribution of emm types among group A streptococcal isolates from Serbia

This is the first study concerning the molecular epidemiology of group A streptococcus in Serbia and includes 145 isolates from patients with various infections during the period 2001–2007. The emm types, superantigen profile and susceptibility pattern were determined. Among 31 emm types identified, the most prevalent were emm6, emm12, emm1, and emm58. All isolates showed uniform antimicrobial susceptibility to all tested antibiotics, with the exception of tetracycline and erythromycin (41% and 0.7% resistant strains, respectively). Significant heterogeneity of emm types was found, with a high frequency of emm6 and emm58, as well as a considerable prevalence of tetracycline resistance, and a low level of macrolide resistance.     

What is the size of the group A streptococcal vir regulon? The Mga regulator affects expression of secreted and surface virulence factors

The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3′ end of the core vir regulon. Utilizing the M49 wild type, a mga ^– mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga ^– mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome. Received: 2 May 1996     

Characterization of group C and G streptococcal strains that cause streptococcal toxic shock syndrome

Twelve strains (the largest number ever reported) of group C and G(1) streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as Streptococcus dysgalactiae subsp. equisimilis, and one strain was identified as Streptococcus equi subsp. zooepidemicus. We found that it was the first reported case of STSS caused by S. equi subsp. zooepidemicus. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the S. dysgalactiae strains belonged to clusters I and II, both of which were closely related. The emm types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular S. dysgalactiae strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the spegg gene was detected in seven S. dysgalactiae strains, with none of the other superantigen genes being detected in any of the strains. However, the sagA gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.     

Epidemiology, outcome and emm types of invasive group A streptococcal infections in Finland

In 2006, Finnish nationwide surveillance showed an increase of invasive group A streptococcal (iGAS) disease and clinicians were alarmed by severe disease manifestations, prompting the investigation of recent trends and outcome for iGAS. A case of iGAS was defined as Streptococcus pyogenes isolated from blood or cerebrospinal fluid. Cases during 1998–2007 and isolates during 2004–2007 were included. Case-patients’ 7-day outcome was available for 2004–2007. Isolates were emm typed. A total of 1,318 cases of iGAS were identified. The average annual incidence was 2.5/100,000 population. The rate was higher in males than females in persons aged 45–64 years, but lower in persons aged 25–34 years. The annual incidence was highest in 2007 (3.9/100,000). Occasional peaks occurred during midwinter and midsummer. The most common emm types were 28 (21%), 1 (16%), 84 (10%), 75 (7%) and 89 (6%). During 2004–2007, emm1 replaced emm28 as the most predominant type. The overall case fatality was 8%. Cases with emm1 were associated with high case fatality (14% vs. 8% in other types; p < 0.02); that of emm28 infections was 2% (p < 0.01). Changes in emm type prevalence influenced incidence and case fatality. Differences in age- and sex-specific incidence and seasonal patterns suggest variations in predisposing factors and underlying conditions.     

Evaluation of simplified DNA extraction methods for emm typing of group A streptococci

Simplified methods of DNA extraction for amplification and sequencing for emm typing of group A streptococci (GAS) can save valuable time and cost in resource crunch situations. To evaluate this, we compared two methods of DNA extraction directly from colonies with the standard CDC cell lysate method for emm typing of 50 GAS strains isolated from children with pharyngitis and impetigo. For this, GAS colonies were transferred into two sets of PCR tubes. One set was preheated at 94 degrees C for two minutes in the thermal cycler and cooled while the other set was frozen overnight at -20 degrees C and then thawed before adding the PCR mix. For the cell lysate method, cells were treated with mutanolysin and hyaluronidase before heating at 100 degrees C for 10 minutes and cooling immediately as recommended in the CDC method. All 50 strains could be typed by sequencing the hyper variable region of the emm gene after amplification. The quality of sequences and the emm types identified were also identical. Our study shows that the two simplified DNA extraction methods directly from colonies can conveniently be used for typing a large number of GAS strains easily in relatively short time.     

Polymorphism of the virulence regulon and allelic variations of the sic gene among the emm1 isolates of group A Streptococcus from western Norway

With the objective of finding genetic markers of invasiveness, 43 isolates of group A streptococcus, isolated in western Norway and from both severe invasive disease and superficial infections, were studied initially by restriction fragment length polymorphism of the virulence regulon (virR -RFLP). Polymorphism that seemed to be related to the severity of infection was observed within the emm1 sequence type, which included 11 invasive and seven non-invasive isolates. These emm1 isolates were further investigated by restriction mapping of the virR and sequence analysis of a polymorphic region, which revealed the presence of a hypervariable sic gene. Of the nine distinct sic alleles, seven were found in single isolates, of which only two were from patients with invasive disease. The other two alleles were shared among nine invasive and two non-invasive isolates. The presence of only two sic allotypes in nine of the 11 invasive isolates, as compared to a different allele in each of the five non-invasive, contemporary isolates supports the hypothesis that selection of the sic variants occurs at mucosal surfaces and implicates mainly two clones among the invasive emm1 isolates.     

Genetic diversity among type emm28 group A Streptococcus strains causing invasive infections and pharyngitis

Genome sequencing of group A Streptococcus (GAS) has revealed that prophages account for the vast majority of gene content differences between strains. Serotype M28 strains are a leading cause of pharyngitis and invasive infections, but little is known about genetic diversity present in natural populations of these organisms. To study this issue, population-based samples of 568 strains from Ontario, Canada; Finland; and Houston, Texas, were analyzed. Special attention was given to analysis of variation in prophage-encoded virulence gene content by a PCR-based method. Thirty and 29 distinct prophage-encoded virulence gene profiles were identified among pharyngitis and invasive infection isolates. Thirteen profiles, representing the majority of the strains, were shared between these two classes of isolates. Significant differences were observed in the frequency of occurrence of certain prophage toxin gene profiles and infection type. M28 strains are highly diverse in prophage-encoded virulence gene content and integration site, supporting the key concept that prophages are critical contributors to GAS genetic diversity and population biology. Nucleotide sequence variation in the emm gene (encodes M protein) was also examined. Only three allelic variants were identified in the hypervariable portion of the emm28 gene. All but one strain had the same inferred amino acid sequence in the first 100 amino acids of the mature M28 protein. In contrast, size differences in the emm28 gene and inferred protein due to variable numbers of C-terminal repeats were common. The presence of macrolide resistance genes (mefA, ermB, and ermTR) was analyzed by PCR, and less than 2% of the strains were positive.     

Distribution of emm types and subtypes among noninvasive group A, C and G streptococcal isolates in western Norway

Characterization of the reservoir of beta-hemolytic streptococci in a community may shed light on the pathogenesis of severe infections caused by these bacteria. We used emm sequence typing to characterize group A streptococci (GAS), group C streptococci (GCS) and group G streptococci (GGS) in community isolates associated with noninvasive disease in western Norway. A total of 165 isolates during a 13-month period were examined. Skin and throat isolates accounted for 123 and 16, respectively, and the remaining 26 isolates were from other non-sterile sites. We identified 18 previously validated emm types and one novel subtype, emm11.7, among the 101 GAS isolates. The two predominant types, emm28 and 12, were found in 40.6% of the GAS isolates. Compared to other recent studies of noninvasive GAS infections from elsewhere in the world, we found a higher frequency of emm82 (5.9%) and emm87 (12.9%) and a lower frequency of emm1 (4.0%) and emm3 (4.0%). We found a different distribution of GAS emm types compared to a previous study from western Norway. Among the 64 isolates of GCS and GGS, 15 previously described emm types and four novel subtypes, stC1400.5, stCK401.3, stG6.3 and stG652.3, were found, stG6, stG643 and stG485 were the most prevalent types and accounted for 59.4% of the GCS and GGS isolates. The high proportion of skin isolates in the present study may indicate the existence of GAS, GCS and GGS strains with predominantly skin and soft tissue tropism in our community.     

National Department of Defense surveillance data for antibiotic resistance and emm gene types of clinical group A streptococcal isolates from eight basic training military sites

Antibiotic resistance and emm gene types were examined from 692 Group A streptococci isolates from eight United States military basic training sites between 1998 and 2001. Macrolide resistance was associated with geographic sites and emm type. These data are useful for vaccine development initiatives and antimicrobial treatment considerations.     

Characterization of a complement-binding protein, DRS, from strains of Streptococcus pyogenes containing the emm12 and emm55 genes

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity.     

Transfer of group A streptococcal pyrogenic exotoxin production to nontoxigenic strains of lysogenic conversion.

Production of group A streptococcal pyrogenic exotoxins (SPE) type A and C was transferred from toxigenic streptococcal strains to nontoxigenic strains by lysogeny. Lysogens were tested for SPE with Ouchterlony immunodiffusion on Todd-Hewitt agar plates; toxin diffusing from isolated colonies reacted with specific hyperimmune antisera to SPE. Phage prepared from strains T25(3) (T12gl) and 3GL16, both yielding SPE type A, formed plaques on T25(3) (NONLYSOGENIC) lawns. Over 90% of the colonies picked from the plaque centers yielded A toxin, suggesting SPE type A was transferred by lysogenic conversion. SPE type C formation was transferred to nontoxigenic strains T25(3) and K56 with supernatant fluids from mitomycin C-induced cultures of CS112, producing SPE types B and C. All lysogens tested were positive for SPE type C, indicating that C toxin induction also was transferred by lysogenic conversion. SPE type B formation was not transferable by lysogeny with the strains tested.     

Identification of group A streptococcal emm types commonly associated with invasive infections and antimicrobial resistance by the use of multiplex PCR and high-resolution melting analysis

M/emm typing, based either on serotyping of the M protein or on sequencing of the emm gene, is a major tool for epidemiological studies of group A streptococci (GAS). In order to simplify M/emm typing, we designed two multiplex polymerase chain reaction (PCR) formats capable of identifying the most frequent GAS M/emm types involved in invasive infections and antimicrobial resistance. A heptaplex PCR procedure was first developed in a conventional format coupled with gel electrophoresis to identify emm types 1, 3, 4, 6, 12, 28, and 89, based on the size of the amplification products. The other method, designed to identify the same seven emm types, together with emm11, was based on a real-time PCR format coupled with high-resolution melting (HRM) analysis, allowing the rapid typing of large strain collections.     

Characterization of group A streptococcal R-28 antigen purified by hydroxyapatite column chromatography.

Purified R-28 antigen from an M-protein-poor, R-antigen-rich strain of group A Streptococcus was prepared by sequential treatment of an acid extract of whole cells with ammonium sulfate fractionation and hydroxylapatite (HA) column chromatography. Purified R-28 antigen was eluted only with 0.10 M sodium phosphate, pH 6.7. Findings on quantitative amino acid composition, polyacrylamide gel electrophoresis pattern, and HA column elution pattern were similar but not identical to those previously reported for streptococcal M-proteins. Rabbits immunized with either HA-purified R-28 antigen or heat-killed cells developed two pepsin-sensitive, trypsin-resistant immunodiffusion lines of identity against HA-purified R-28 antigen but failed to form bactericidal antibody. One of these two lines formed a line of identity with R-28 antigen prepared by trypsinization of whole cells. The other line remained undefined, although it appeared not to be either streptococcal group A carbohydrate, M-protein, T-antigen, polyglycerophosphate, E4 antigen, or M-associated protein; by enzymatic criteria it is an R-antigen. Polyacrylamide gel electrophoresis of HA-purified R-28 antigen revealed multiple serologically active charge and size isomers. These findings suggest possible structural similarities between group A streptococcal M-proteins and R-antigens and also indicate that the same purification techniques may be utilized to study these protein antigens if the proper strain of Streptococcus is chosen.     

Characterization of group A streptococcal T-12 protein purified by ion-exchange column chromatography.

The aim of the present study was to describe the physicochemical characteristics of streptococcal T antigen. T protein isolated from Streptococcus pyogenes type 12 (R53/1077, Colindale) and purified by ion-exchange column chromatography resulted in a preparation that was homogeneous when tested electrophoretically (in two systems, in presence and in absence of sodium dodecyl sulfate) and by gel filtration on Sephadex G-100. The purified T antigen was resistant to enzymatic degradation by trypsin and pepsin. It formed a single precipitin line with standard T-12 antiserum and was not contaminated with group A carbohydrate and M protein. The molecular weight of protein T, determined by means of polyacrylamide gel electrophoresis and calculated from its amino acid composition, was about 39,000. The molecular weight of this protein, determined by means of high-speed sedimentation equilibrium, ranged between 80,000 and 120,000. Glutamic and asparatic acids, lysine, alanine, and leucine were the predominant amino acids.     

Arrangement and number of clustered regularly interspaced short palindromic repeat spacers are associated with erythromycin susceptibility in emm12, emm75 and emm 92 of group A streptococcus

Clustered regularly interspaced short palindromic repeats (CRISPR) are composed of numerous repeat-spacer units and are considered a prokaryotic defence system against foreign nucleic acids. Since antibiotic-resistant genes are frequently encoded in foreign nucleic acids, the aim of this study was to test whether erythromycin susceptibility in group A streptococcus (<Streptococcus pyogenes) is associated with characteristics of CRISPR elements. Erythromycin susceptibility of 330 isolates collected between 1997 and 2003 was analysed. Among 29 emm types, emm12, emm75 and emm92 showed significant changes in erythromycin-resistance rates. By sequencing the spacers from two CRISPR loci, spacer contents in emm12, emm75 and emm92 strains were associated with erythromycin susceptibility. Strains with fewer spacers were more resistant to erythromycin. Moreover, in emm4 strains, which showed no significant change in their annual erythromycin-resistance rate, CRISPR type and number of spacers were not correlated with erythromycin susceptibility. These results highlight a novel association between CRISPR spacer content and erythromycin susceptibility in group A streptococcus.     

M protein gene (emm type) analysis of group A beta-hemolytic streptococci from Ethiopia reveals unique patterns

The genetic diversity of group A streptococcal (GAS) isolates obtained in 1990 from Ethiopian children with various streptococcal diseases was studied by using emm gene sequence analysis. A total of 217 GAS isolates were included: 155 and 62 isolates from throat and skin, respectively. A total of 78 different emm/st types were detected among the 217 isolates. Of these, 166 (76.5%) belonged to 52 validated reference emm types, 26 (11.9%) belonged to 16 already recognized sequence types (st types) and 25 (11.5%) belonged to 10 undocumented new sequence types. Resistance to tetracycline (148 of 217) was not correlated to emm type. Isolation rate of the classical rheumatogenic and nephritogenic strains was low from cases of acute rheumatic fever (ARF) and acute glomerulonephritis (AGN), respectively. Instead, the recently discovered st types were overrepresented among isolates from patients with ARF (3 of 7) and AGN (9 of 16) (P < 0.01) compared to isolates from subjects with tonsillitis and from healthy carriers (10 of 57 and 16 of 90, respectively). In contrast to rheumatogenic strains from the temperate regions, more than half of the isolates from ARF (four of seven) carried the genetic marker for skin preference, emm pattern D, although most of them (six of seven) were isolated from throat. Of 57 tonsillitis-associated isolates, 16 (28%) belonged to emm pattern D compared to <1% in temperate regions. As in other reports emm patterns A to C were strongly associated with throat, whereas emm pattern D did not correlate to skin. This first large-scale emm typing report from Africa has demonstrated a heterogeneous GAS population and contrasting nature of GAS epidemiology in the region.