Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.     

Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain

We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.     

Invasive group A streptococcus infections.

The late 1980s have witnessed the emergence of severe group A streptococcus (GAS) infection; shock, bacteremia, and acute respiratory distress syndrome are common features, and death has been associated with this infection in 30% of patients. Such infections have now been described in all parts of the United States, Europe, and Australia and have occurred predominantly in otherwise healthy adolescents and adults. The characteristic clinical and laboratory features of the streptococcal toxic shock syndrome include deep-seated infection associated with shock and multiorgan failure. Strains of GAS isolated from patients with invasive disease have been predominantly M types 1 and 3, which produce pyrogenic exotoxin A or B or both. In this report, the clinical and demographic features of streptococcal bacteremia, myositis, and necrotizing fasciitis will be presented and compared with those of streptococcal toxic shock syndrome. Current concepts of the pathogenesis of invasive streptococcal infection will also be presented in terms of the interaction between virulence factors of GAS and host defense mechanisms. Finally, new concepts for future treatment of serious streptococcal infections will be proposed.     

Genome-wide molecular dissection of serotype M3 group A Streptococcus strains causing two epidemics of invasive infections

Molecular factors that contribute to the emergence of new virulent bacterial subclones and epidemics are poorly understood. We hypothesized that analysis of a population-based strain sample of serotype M3 group A Streptococcus (GAS) recovered from patients with invasive infection by using genome-wide investigative methods would provide new insight into this fundamental infectious disease problem. Serotype M3 GAS strains (n = 255) cultured from patients in Ontario, Canada, over 11 years and representing two distinct infection peaks were studied. Genetic diversity was indexed by pulsed-field gel electrophoresis, DNA-DNA microarray, whole-genome PCR scanning, prophage genotyping, targeted gene sequencing, and single-nucleotide polymorphism genotyping. All variation in gene content was attributable to acquisition or loss of prophages, a molecular process that generated unique combinations of proven or putative virulence genes. Distinct serotype M3 genotypes experienced rapid population expansion and caused infections that differed significantly in character and severity. Molecular genetic analysis, combined with immunologic studies, implicated a 4-aa duplication in the extreme N terminus of M protein as a factor contributing to an epidemic wave of serotype M3 invasive infections. This finding has implications for GAS vaccine research. Genome-wide analysis of population-based strain samples cultured from clinically well defined patients is crucial for understanding the molecular events underlying bacterial epidemics.     

Quantitative and qualitative comparison of virulence traits,including murine lethality,among different M types of group A streptococci

Epidemiological studies have proposed an association between group A streptococci (GAS) bearing a particular M serological type and pathologic conditions such as streptococcal toxic shock syndrome (STSS). M1 and M3 GAS are isolated from STSS cases more frequently, whereas M4 and M12 GAS are isolated from non-STSS cases more frequently. To investigate whether there is any difference contributing the M-type association among GAS, we compared various virulence traits, including the murine lethality of M4, M12, M1, and M3 GAS clinical isolates, which are not clonally related to one another. Murine lethality, the activities of superantigens, streptolysin O, and nicotinamide adenine dinucleotide glycohydrolase, and the presence of the speA and speC genes were closely associated with M type. These results indicate that M types may serve, in part, as markers for strains/clones with particular profiles of virulence traits and mouse lethality.     

Evolutionary origin and emergence of a highly successful clone of serotype M1 group a Streptococcus involved multiple horizontal gene transfer events

To better understand the molecular events involved in the origin of new pathogenic bacteria, we studied the evolution of a highly virulent clone of serotype M1 group A Streptococcus (GAS). Genomic, DNA-DNA microarray, and single-nucleotide polymorphism analyses indicated that this clone evolved through a series of horizontal gene transfer events that involved (1) the acquisition of prophages encoding streptococcal pyrogenic exotoxin A and extracellular DNases and (2) the reciprocal recombination of a 36-kb chromosomal region encoding the extracellular toxins NAD+-glycohydrolase (NADase) and streptolysin O (SLO). These gene transfer events were associated with significantly increased production of SLO and NADase. Virtual identity in the 36-kb region present in contemporary serotype M1 and M12 isolates suggests that a serotype M12 strain served as the donor of this region. Multiple horizontal gene transfer events were a crucial factor in the evolutionary origin and emergence of a very abundant contemporary clone of serotype M1 GAS.     

Lysogenic transfer of group A Streptococcus superantigen gene among Streptococci

A group A Streptococcus (GAS) isolate, serotype M12, recovered from a patient with streptococcal toxic shock syndrome was analyzed for superantigen-carrying prophages, revealing phi149, which encodes superantigen SSA. Sequence analysis of the att-L proximal region of phi149 showed that the phage had a mosaic nature. Remarkably, we successfully obtained lysogenic conversion of GAS clinical isolates of various M serotypes (M1, M3, M5, M12, M19, M28, and M94), as well as of group C Streptococcus equisimilis (GCSE) clinical isolates, via transfer of a recombinant phage phi149::Km(r). Phage phi149::Km(r) from selected lysogenized GAS and GCSE strains could be transferred back to M12 GAS strains. Our data indicate that horizontal transfer of lysogenic phages among GAS can occur across the M-type barrier; these data also provide further support for the hypothesis that toxigenic conversion can occur via lysogeny between species. Streptococci might employ this mechanism specifically to allow more efficient adaptation to changing host challenges, potentially leading to fitter and more virulent clones.     

Intrahost sequence variation in the streptococcal inhibitor of complement gene in patients with human pharyngitis

Selection of new variants of the streptococcal inhibitor of complement protein has been implicated in the perpetuation of epidemics caused by serotype M1 strains of group A Streptococcus (GAS). The frequency at which new streptococcal inhibitor of complement (Sic) variants arise in an infected individual is not known. To study this issue, the sic gene was sequenced in 100 isolates cultured from throat swabs of each of 20 patients with acute pharyngitis caused by serotype M1 GAS. Five patients were infected with GAS populations expressing 2 Sic variants characterized by deletion of a region of the protein. In contrast, no intrahost variation was detected in the number of a pentanucleotide repeat (CAAAA) that controls production of a bacterial cell-surface collagen-like protein by slipped-strand mispairing. Sic variation occurs at a sufficient frequency in vivo to result in mixed infections on the mucosal surface of human hosts, potentially contributing to pathogen survival.     

Polymorphisms in regulator of protease B (RopB) alter disease phenotype and strain virulence of serotype M3 group A Streptococcus

Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens.     

The flesh-eating bacterium: what's next?

Since the 1980s, there has been a marked increase in the recognition and reporting of highly invasive group A streptococcal (GAS) infections associated with shock and organ failure, with or without necrotizing fasciitis. Such dramatic cases have been defined as streptococcal toxic shock syndrome (StrepTSS). Strains of GAS isolated from patients with invasive disease have been predominantly M types 1 and 3, which produce either pyrogenic exotoxin A or B or both. The clinical and demographic features of streptococcal bacteremia, myositis, and necrotizing fasciitis are presented and compared with those of StrepTSS. Current concepts in the pathogenesis of invasive streptococcal infection will be presented, with emphasis on the interaction between GAS virulence factors and host defense mechanisms. Finally, new concepts in the treatment of StrepTSS will be discussed.     

Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2

The molecular basis of pathogen clone emergence is relatively poorly understood. Acquisition of a bacteriophage encoding a previously unknown secreted phospholipase A(2) (designated SlaA) has been implicated in the rapid emergence in the mid-1980s of a new hypervirulent clone of serotype M3 group A Streptococcus. Although several lines of circumstantial evidence suggest that SlaA is a virulence factor, this issue has not been addressed experimentally. We found that an isogenic DeltaslaA mutant strain was significantly impaired in ability to adhere to and kill human epithelial cells compared with the wild-type parental strain. The mutant strain was less virulent for mice than the wild-type strain, and immunization with purified SlaA significantly protected mice from invasive disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We conclude that transductional acquisition of the ability of a GAS strain to produce SlaA enhanced the spread and virulence of the serotype M3 precursor strain. Hence, these studies identified a crucial molecular event underlying the evolution, rapid emergence, and widespread dissemination of unusually severe human infections caused by a distinct bacterial clone.     

Antimicrobial sensitivity and characterization of Streptococcus pyogenes strains isoleated from a scarlatina outbreak

OBJECTIVE: To evaluate the in vitro activities of 13 antimicrobial agents against 47 group A Streptococcus pyogenes (GAS) strains, and to determine the presence of genes encoding streptococcal pyrogenic exotoxin A (SpeA) and the M-protein serotypes. MATERIALS AND METHODS: A cross-sectional study was conducted at Centro de Salud Dr. José Castro Villagrana, during a scarlet fever outbreak occurring between December 1999 and January 2000, among 137 children at Colegio Espíritu de América. Minimum Inhibitory Concentrations (MICs) were obtained by the semiautomated microdilution method. Automated DNA sequencing was used for analysis of sequence variation in genes encoding the M protein, and SpeA. RESULTS: All strains were sensitive to betalactams and clindamycin. Six (12.7%) were resistant to erythromycin. The M2 type was the most frequently isolated GAS (27); almost all (96%) bacteria with the SpeA gene had the gene encoding the M2 protein. CONCLUSIONS: The recent resurgence of GAS infections calls for molecular epidemiology research and studies on the sensitivity to macrolides and beta-lactams.     

Toxin-gene profile heterogeneity among endemic invasive European group A streptococcal isolates

We determined the toxin-gene profiles of 239 endemic, invasive group A streptococcal (GAS) isolates that circulated, within a 5-year period, in European university hospitals. Profiling was performed by use of multiplex polymerase chain reaction that screened for 9 streptococcal pyrogenic exotoxins (speA, speB, speC, speF, speG, speH, speJ, ssa, and smeZ). Analysis revealed that invasive GAS isolates do not share a common toxin-gene profile. Although all emm types were characterized by several different toxin-gene profiles, a predominance of 1 or 2 toxin-gene profiles could be observed, reflecting that a few invasive clones have spread successfully throughout the world. Remarkably, statistical pair-wise analysis of individual toxin genes revealed that strains that did not share the predominant profile still showed a nonrandom distribution of key toxin genes characteristic of the specific emm type. This could indicate that M proteins function, directly or indirectly, as barriers for horizontal gene exchange.     

Epidemiologic analysis of group A streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis.

More than 1100 group A streptococcal isolates collected in the United States (1988-1990) were examined to document an association of individual serotypes with specific clinical infections during the recent resurgence of group A infections and their sequelae. The most commonly isolated strains from patients with only uncomplicated streptococcal pharyngitis ("control" strains) were M serotypes 1, 2, 4, and 12. M1, M3, and M18 were statistically significantly more frequently isolated from patients with serious invasive infections and M3 and M18 from patients with rheumatic fever compared with the distribution of serotypes from the 866 control strains. An unexpected and important finding indicated that isolation rates of M1 streptococci varied geographically within the United States by year. The propensity for M1 streptococci to be statistically associated with severe systemic infections appeared unrelated to the M1 isolation rates from patients with only uncomplicated pharyngitis, thus offering additional support for the concept of strain-associated virulence rather than virulence broadly related to a given serotype.     

Group A beta-hemolytic streptococcus meningitis: clinical and microbiological features of nine cases.

Group A beta-hemolytic streptococcus (GAS) meningitis is a rare disease in adults. We conducted a retrospective study to describe clinical and microbiological features of nine cases of GAS meningitis in Switzerland. Of nine patients, six had neurosurgical conditions, and five had upper respiratory tract infections. Eight cases were community-acquired. The outcome of GAS meningitis was favorable; only one patient died of neurosurgical complications. No patient presented with toxic shock syndrome. Serotyping failed to reveal a dominant strain, and genotyping revealed that two strains carried the gene encoding the streptococcal pyrogenic exotoxin C and that one strain carried the gene encoding the streptococcal pyrogenic exotoxin A. Our observations suggest that GAS meningitis in adults is associated with neurosurgical conditions and/or an upper respiratory tract infection.     

Molecular epidemiology of nga and NAD glycohydrolase/ADP-ribosyltransferase activity among Streptococcus pyogenes causing streptococcal toxic shock syndrome

Severe invasive group A streptococcal (GAS) infections emerged in the late 1980s, yet no single virulence factor has been common to all isolates from infected patients. A strong association was recently found between isolates of such cases (regardless of M type) and the production of NAD glycohydrolase (NADase). Of interest, all M-1 strains isolated after 1988 were positive for NADase, whereas virtually all M-1 GAS were previously negative for NADase. Genetic analysis demonstrated that GAS isolates were >96% identical in nga and >99% identical in their upstream regulatory sequences. Furthermore, because NADase-negative strains did not produce immunoreactive NADase, we concluded that additional regulatory element(s) control NADase production. NADase purified from GAS altered neutrophil-directed migration and chemiluminescence responses and had potent ADP-ribosyltransferase activity. In summary, the temporal relationship of NADase expression, alone or with other streptococcal virulence factors, may contribute to the pathogenesis of invasive GAS infections.     

Group A streptococcus clones causing repeated epidemics and endemic disease in intravenous drug users

Clones of Group A streptococcus (GAS) may spread epidemically and may be associated with enhanced virulence. Sociodemographic and clinical characteristics, together with bacterial isolates, of 79 patients with GAS infection in the Berne region between January 1993 and February 1997 were analysed retrospectively. Using pulsed-field gel electrophoresis, most strains (71%) were found to belong to one of 12 clones. Clonal strains caused significantly more frequent skin abscesses and more severe invasive disease than non-clonal strains. The largest clone (M serotype 1) occurred endemically in non-IVDU patients and caused severe disease in most. Three clones occurred almost exclusively among IVDUs: an M serotype 11 was associated with severe, endemic disease; the other 2 clones, both of M serotype 25, caused epidemics of needle abscesses. Epidemic and endemic spread of GAS clones among IVDUs may be more frequent than previously assumed.     

Hypervariability generated by natural selection in an extracellular complement-inhibiting protein of serotype M1 strains of group A Streptococcus

In many countries, M1 strains of the human pathogenic bacterium group A Streptococcus are the most common serotype recovered from patients with invasive disease episodes. Strains of this serotype express an extracellular protein that inhibits complement [streptococcal inhibitor of complement (Sic)] and is therefore believed to be a virulence factor. Comparative sequence analysis of the 915-bp sic gene in 165 M1 organisms recovered from diverse localities and infection types identified 62 alleles. Inasmuch as multilocus enzyme electrophoresis and pulsed-field gel electrophoresis previously showed that most M1 organisms represent a distinct streptococcal clone, the extent of sic gene polymorphism was unexpected. The level of polymorphism greatly exceeds that recorded for all other genes examined in serotype M1 strains. All insertions and deletions are in frame, and virtually all nucleotide substitutions alter the amino acid sequence of the Sic protein. These molecular features indicate that structural change in Sic is mediated by natural selection. Study of 70 strains recovered from two temporally distinct epidemics of streptococcal infections in the former East Germany found little sharing of Sic variants among strains recovered in the different time periods. Taken together, the data indicate that sic is a uniquely variable gene and provide insight into a potential molecular mechanism contributing to fluctuations in streptococcal disease frequency and severity.     

Molecular basis of group A streptococcal virulence

The group A streptococcus (GAS) (Streptococcus pyogenes) is among the most common and versatile of human pathogens. It is responsible for a wide spectrum of human diseases, ranging from trivial to lethal. The advent of modern techniques of molecular biology has taught much about the organism's virulence, and the genomes of several GAS types have now been deciphered. Surface structures of GAS including a family of M proteins, the hyaluronic acid capsule, and fibronectin-binding proteins, allow the organism to adhere to, colonise, and invade human skin and mucus membranes under varying environmental conditions. M protein binds to complement control factors and other host proteins to prevent activation of the alternate complement pathway and thus evade phagocytosis and killing by polymorphonuclear leucocytes. Extracellular toxins, including superantigenic streptococcal pyrogenic exotoxins, contribute to tissue invasion and initiate the cytokine storm felt responsible for illnesses such as necrotising fasciitis and the highly lethal streptococcal toxic shock syndrome. Progress has been made in understanding the molecular epidemiology of acute rheumatic fever but less is understood about its basic pathogenesis. The improved understanding of GAS genetic regulation, structure, and function has opened exciting possibilities for developing safe and effective GAS vaccines. Studies directed towards achieving this long-sought goal are being aggressively pursued.