Site-specific manifestations of invasive group a streptococcal disease: type distribution and corresponding patterns of virulence determinants

As part of a national surveillance program on invasive group A streptococci (GAS), isolates that caused specific manifestations of invasive GAS disease in The Netherlands were collected between 1992 and 1996. These site-specific GAS infections involved meningitis, arthritis, necrotizing fasciitis, and puerperal sepsis. An evaluation was performed to determine whether GAS virulence factors correlate with these different disease manifestations. PCRs were developed to detect 9 genes encoding exotoxins and 12 genes encoding fibronectin binding proteins. The genetic backgrounds of all isolates were determined by M genotyping and pulsed-field gel electrophoresis (PFGE) analysis. The predominant M types included M1, M2, M3, M4, M6, M9, M12, and M28. Most M types were associated with all manifestations of GAS disease. However, M2 was found exclusively in patients with puerperal sepsis, M6 predominated in patients with meningitis, and M12 predominated in patients with GAS arthritis. While characteristic gene profiles were detected in most M types, the resolution of detection of different gene profiles within M genotypes was enhanced by PFGE analysis, which clearly demonstrated the existence of some clonal lineages among invasive GAS isolates in The Netherlands. M1 isolates comprised a single clone carrying highly mitogenic toxin genes (speA, smeZ) and were associated with toxic shock-like syndrome. Toxin profiles were highly conserved among the most virulent strains, such as M1 and M3.     

Natural Disruption of Two Regulatory Networks in Serotype M3 Group A Streptococcus Isolates Contributes to the Virulence Factor Profile of This Hypervirulent Serotype

Despite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α₂-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.     

Survey of emm gene sequences and T-antigen types from systemic Streptococcus pyogenes infection isolates collected in San Francisco, California; Atlanta, Georgia; and Connecticut in 1994 and 1995.

The variable 5' emm (M-protein gene) sequences and T-antigen types were determined from 340 systemic group A streptococcal (GAS) isolates taken from hospitalized patients in San Francisco, Calif.; Atlanta, Ga.; and Connecticut in 1994 and 1995. Eighty percent of these isolates had emm sequences and T-antigen types in agreement with previously recorded M- and T-antigen associations. Most of the remaining strains either were T nontypeable (11%) or contained emm genes encoding M proteins for which T-antigen associations have not been made (6%). One newly encountered emm gene, designated ST2974, from each of 13 isolates had the T type 8/25/Imp19. Another new emm gene, ST2967, from 8 of 11 isolates was T nontypeable. Six other unique emm gene sequences from seven isolates were encountered. Sequencing of the variable region of the emm gene of GAS isolates (emm typing) is effective for surveying the sequence variability of the M virulence protein, and combined with T typing, emm typing is useful for monitoring GAS strain diversity.     

Molecular analysis of group A Streptococcus type emm18 isolates temporally associated with acute rheumatic fever outbreaks in Salt Lake City, Utah

Acute rheumatic fever (ARF) and subsequent rheumatic heart disease are rare but serious sequelae of group A Streptococcus (GAS) infections in most western countries. Salt Lake City (SLC), Utah, and the surrounding intermountain region experienced a resurgence of ARF in 1985 which has persisted. The largest numbers of cases were encountered in 1985-1986 and in 1997-1998. Organisms with a mucoid colony phenotype when grown on blood agar plates were temporally associated with the higher incidence of ARF. To develop an understanding of the molecular population genetic structure of GAS strains associated with ARF in the SLC region, 964 mucoid and nonmucoid pharyngeal isolates recovered in SLC from 1984 to 1999 were studied by sequencing the emm gene. Isolates with an emm18 allele were further characterized by sequencing the spa, covR, and covS genes. Peak periods of ARF were associated with GAS isolates possessing an emm18 allele encoding the protein found in serotype M18 isolates. Among the serotype M18 isolates, the difference in the number of C repeats produced three size variants. Variation was limited in spa, a gene that encodes a streptococcal protective antigen, and covR and covS, genes that encode a two-component regulatory system that, when inactivated, results in a mucoid phenotype and enhanced virulence in mouse infection models. Pulsed-field gel electrophoresis showed a single restriction profile for serotype M18 organisms isolated during both peak periods of ARF. In SLC, the incidence of ARF coresurged with the occurrence of GAS serotype M18 isolates that have very restricted genetic variation.     

Inverse relation between disease severity and expression of the streptococcal cysteine protease, SpeB, among clonal M1T1 isolates recovered from invasive group A streptococcal infection cases

The streptococcal cysteine protease (SpeB) is one of the major virulence factors produced by group A streptococci (GAS). In this study we investigated if differences exist in SpeB production by clonally related M1T1 clinical isolates derived from patients with invasive infections. Twenty-nine of these isolates were from nonsevere cases and 48 were from severe cases, including streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) cases. The expression and amount of the 28-kDa SpeB protein produced were determined by quantitative Western blotting, and protease activity was measured by a fluorescent enzymatic assay. A high degree of variation in SpeB expression was seen among the isolates, and this variation seemed to correlate with the severity and/or clinical manifestation of the invasive infection. The mean amount of 28-kDa SpeB protein and cysteine protease activity produced by isolates from nonsevere cases was significantly higher than that from STSS cases (P = 0.001). This difference was partly due to the fact that 41% of STSS isolates produced little or no SpeB compared to only 14% of isolates recovered in nonsevere cases. Moreover, the cysteine protease activity among those isolates that expressed SpeB was significantly lower for STSS isolates than for isolates from nonsevere cases (P = 0.001). Increased SpeB production was also inversely correlated with intact M protein expression, and inhibition of cysteine protease activity blocked the cleavage of the surface M protein. Together, the data support the existence of both an "on-off" and a posttranslational regulatory mechanism(s) controlling SpeB production, and they suggest that isolates with the speB gene in the "off" state are more likely to spare the surface M protein and to be isolated from cases of severe rather than nonsevere invasive infection. These findings may have important implications for the role of SpeB in host-pathogen interactions via regulation of the expression of GAS virulence genes and the severity of invasive disease.     

EMM types of Streptococcus pyogenes in Chennai

The M protein of group A Streptococcus (GAS) is the major virulence factor and is coded by the emm gene. The current serologic M typing methods are now being replaced by alternate means of M type deduction such as emm gene sequencing. This is the first report of emm types of GAS which are prevalent in south India. We found no marked preponderance of any single emm sequence among our clinical isolates with 11 emm sequences being present in 34 isolates.     

Cytotoxic effects of streptolysin o and streptolysin s enhance the virulence of poorly encapsulated group a streptococci

Although the toxicity of streptolysin O (SLO) and streptolysin S (SLS) in purified group A streptococci (GAS) has been established, the effect of these molecules in natural infection is not well understood. To identify whether biologically relevant concentrations of SLO and SLS were cytotoxic to epithelial and phagocytic cells that the bacteria would typically encounter during human infection and to characterize the influence of cell injury on bacterial pathogenesis, we derived GAS strains deficient in SLO or SLS in the background of an invasive GAS M3 isolate and determined their virulence in in vitro and in vivo models of human disease. Whereas bacterial production of SLO resulted in lysis of both human keratinocytes and polymorphonuclear leukocytes, GAS expression of SLS was associated only with keratinocyte injury. Expression of SLO but not SLS impaired polymorphonuclear leukocyte killing of GAS in vitro, but this effect could only be demonstrated in the background of acapsular organisms. In mouse invasive soft-tissue infection, neither SLO or SLS expression significantly influenced mouse survival. By contrast, in a mouse model of bacterial sepsis after intraperitoneal inoculation of GAS, SLO expression enhanced the virulence of both encapsulated and acapsular GAS, whereas SLS expression increased the virulence only of acapsular GAS. We conclude that the cytotoxic effects of SLO protect GAS from phagocytic killing and enhance bacterial virulence, particularly of strains that may be relatively deficient in hyaluronic acid capsule. Compared to SLO, SLS in this strain background has a more modest influence on GAS pathogenicity and the effect does not appear to involve bacterial resistance to phagocytosis.     

Reciprocal, Temporal Expression of SpeA and SpeB by Invasive M1T1 Group A Streptococcal Isolates In Vivo

The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including speA, speB, speF, speG, and smeZ. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of the speA-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that speA gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of the speA gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced speA gene expression and suppressed speB gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of streptococcal virulence factors in vivo. The model described here should facilitate such studies.     

Epidemiological analysis of non-M-typeable group A Streptococcus isolates from a Thai population in northern Thailand

Infection with group A streptococci (GAS) can lead to the development of severe postinfectious sequelae such as rheumatic fever (RF). In Thailand, RF and rheumatic heart disease (RHD) remain important health problems. More than 80% of GAS circulating in this population are non-M antigen typeable by conventional M serotyping methods. In this study, we determine the M protein sequence types of GAS isolates found in northern Thailand. The emm genes from 53 GAS isolates, collected between 1985 and 1995 from individuals with pharyngitis, impetigo, acute RF (ARF), RHD, or meningitis as well as from individuals without infections, were amplified by PCR and sequenced. Thirteen new sequence types that did not show homology to previously published sequences were characterized. Six of these sequence types could be isolated from both skin and throat sites of impetigo and pharyngitis/ARF patients, respectively. In many cases we could not specifically differentiate skin strains or throat strains that could be associated with ARF or acute glomerulonephritis. Antigenic variations in the emm gene of the isolates investigated, compared to published M protein sequences, were predominantly due to point mutations, small deletions, and insertions in the hypervariable region. One group of isolates with homology to M44 exhibited corrected frameshift mutations. A new M type isolated from an RHD patient exhibited nucleotide sequence corresponding to the N terminus of M58 and the C terminus of M25, suggesting that recombination between the two types may have occurred. This study provided epidemiological data relating to GAS endemic to northern Thailand which could be useful for identification of vaccine candidates in a specific region of endemicity.     

中国儿童A族链球菌感染菌株emm分型及超抗原基因谱分布

目的 研究我国儿科A族β溶血性链球菌感染菌株emm分型及其超抗原在我国的分布情况。方法 用聚合酶链反应（PCR）联合测序对来自中国儿科2005年-2006年222株A族β溶血性链球菌感染的临床菌株进行emm分型;PCR检测speA, speC, speH, speI, speG, speJ, ssa和 SMEZ超抗原基因的分布情况。结果 共检测到9个emm型,emm12为主占55.86%（124/222）,其次emm1占39.64%（88/222）;78.39%的GAS分离株携带有6个或6个以上的超抗原基因,emm型和超抗原基因谱之间存在密切联系。结论 中国儿科GAS感染流行菌株具有emm型分布集中和超抗原基因携带率高的特点,不同的emm分型有不同的超抗原基因谱,这将为我国GAS疫苗的研究提供重要的分子流行病学依据。     

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.     

M1T1 group A streptococcal pili promote epithelial colonization but diminish systemic virulence through neutrophil extracellular entrapment

Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures.     

Molecular characterization of a strain of group a streptococcus isolated from a patient with a psoas abscess

We report the first case of a primary group A streptococcus (GAS) psoas abscess in a 31-year-old woman. The psoas abscess was preceded by an episode of acute pharyngitis. The M-protein gene (emm) and streptolysin S structural gene (sagA) were present in the isolate, with no significant amino acid differences from previously described sequences of M1 GAS isolates. Multilocus sequence typing (MLST) showed that the isolate belonged to MLST sequence type (MLST-ST) 28, the predominant MLST-ST associated with invasive disease caused by M1 isolates.     

Determination of the relationship between group A streptococcal genome content, M type, and toxic shock syndrome by a mixed genome microarray

Group A streptococci (GAS), or Streptococcus pyogenes, are associated with a remarkable variety of diseases, ranging from superficial infections to life-threatening diseases such as toxic-shock-like syndrome (TSS). GAS strains belonging to M types M1 and M3 are associated with TSS. This study aims to obtain insight into the gene profiles underlying different M types and disease manifestations. Genomic differences between 76 clinically well characterized GAS strains collected in The Netherlands were examined using a mixed-genome microarray. Inter-M-type genomic differences clearly outweighed intra-M-type genome variation. Phages were major contributors to observed genome diversification. We identified four novel genes, including two genes encoding fibronectin-binding-like proteins, which are highly specific to a subset of M types and thus may contribute to M-type-associated disease manifestations. All M12 strains were characterized by the unique absence of the citrate lyase complex and reduced growth under hypoxic, nutrient-deprived conditions. Furthermore, six virulence factors, including genes encoding a complement-inhibiting protein (sic), an exotoxin (speA), iron(III) binding factor, collagen binding factor (cpa), and fibrinogen binding factor (prt2-like), were unique to M1 and/or M3 strains. These virulence factors may contribute to the potential of these strains to cause TSS. Finally, in contrast to M-type-specific virulence profiles, we did not identify a common virulence profile among strains associated with TSS irrespective of their M type.     

Role for Streptococcal Collagen-Like Protein 1 in M1T1 Group A Streptococcus Resistance to Neutrophil Extracellular Traps

Streptococcal collagen-like protein 1 (Scl-1) is one of the most highly expressed proteins in the invasive M1T1 serotype group A Streptococcus (GAS), a globally disseminated clone associated with higher risk of severe invasive infections. Previous studies using recombinant Scl-1 protein suggested a role in cell attachment and binding and inhibition of serum proteins. Here, we studied the contribution of Scl-1 to the virulence of the M1T1 clone in the physiological context of the live bacterium by generating an isogenic strain lacking the scl-1 gene. Upon subcutaneous infection in mice, wild-type bacteria induced larger lesions than the Δscl mutant. However, loss of Scl-1 did not alter bacterial adherence to or invasion of skin keratinocytes. We found instead that Scl-1 plays a critical role in GAS resistance to human and murine phagocytic cells, allowing the bacteria to persist at the site of infection. Phenotypic analyses demonstrated that Scl-1 mediates bacterial survival in neutrophil extracellular traps (NETs) and protects GAS from antimicrobial peptides found within the NETs. Additionally, Scl-1 interferes with myeloperoxidase (MPO) release, a prerequisite for NET production, thereby suppressing NET formation. We conclude that Scl-1 is a virulence determinant in the M1T1 GAS clone, allowing GAS to subvert innate immune functions that are critical in clearing bacterial infections.     

Characterization of group A streptococcal strains Sv and Su: determination of emm gene typing and presence of small vir regulon

The emm gene typing of GAS (group A streptococcus) strains Sv and Su and the molecular structure of the vir regulon were decided. An emm(-like) gene from the chromosomal DNA of GAS strain Sv was amplified with forward and reverse primers, which were selected from the best conserved portion in leader sequences of different strains and the C-terminal conserved portion, respectively, for determination of the M protein gene type. Strain Sv was defined as serotype M23, because deduced N-terminal amino acid positions of the products are identical to those of the M type 23 (emml) gene derived from GAS strain M23-MEMPHIS (M serotype 23, GenBank accession number U11953). When the vir regulon of strain Sv was examined by polymerase chain reaction mapping and compared with that of GAS strain Su, they had a similar size in length. In addition, when sequencing analysis of the DNA fragment of 4791 base pairs (bp) encoding three open reading frames (orf, mga, and emm) and the upstream region of scpA from genomic DNAs of both strains was performed, the sequence of the DNA from strain Sv was, except for 1 bp (T for C at position 4124), identical to that of the DNA from strain Su. These data show that both strains possess the genes in the order of mga (virR or mry) -emm -scpA designated as the small vir regulon. The effect of the formation of alternative pathway C3 convertase of complement on the GAS strains Sv and Su was also examined. When GAS strains Sv and Su were incubated in NHS containing radiolabeled C3 in the presence of Mg-EGTA, binding of C3 to Su bacteria was dose-dependent, whereas less binding of C3 to Sv bacteria was seen. Taken together, the data suggest that M protein could be expressed on the surface of the Sv bacteria, but not on the Su bacteria.