Streptococcal erythrogenic toxin B abrogates fibronectin-dependent internalization of Streptococcus pyogenes by cultured mammalian cells

Streptococcus pyogenes secretes several proteins that influence host-pathogen interactions. A tissue-culture model was used to study the influence of the secreted cysteine protease streptococcal erythrogenic toxin B (SPE B) on the interaction between S. pyogenes strain NZ131 (serotype M49) and mammalian cells. Inactivation of the speB gene enhanced fibronectin-dependent uptake of the pathogen by Chinese hamster ovary (CHO-K1) cells compared to that in the isogenic wild-type strain. Preincubation of the NZ131 speB mutant with purified SPE B protease significantly inhibited fibronectin-dependent uptake by both CHO-K1 and CHO-pgs745 cells. The effect was attributed to an abrogation of fibronectin binding to the surface of the bacteria that did not involve either the M49 protein or the streptococcal fibronectin-binding protein SfbI. In contrast, pretreatment of the NZ131 speB mutant with SPE B did not influence sulfated polysaccharide-mediated uptake by CHO-pgs745 cells. The results indicate that the SPE B protease specifically alters bacterial cell surface proteins and thereby influences pathogen uptake.     

Augmented adherence and internalization of group A Streptococcus pyogenes to influenza A virus infected MDCK cells

Respiratory tract infections are one of the leading causes of morbidity and mortality. There is considerable epidemiologic evidence that infection with respiratory viruses increases the incidence and severity of secondary bacterial complications. However, very limited number of studies were concerned with the mechanism behind such synergy. In this context, our study aimed to explore the interaction between Group A Streptococcus pyogenes (GAS) and Influenza A virus (IAV). Our results revealed that the GAS adherence and internalization into Madin-Darby canine kidney (MDCK) cells markedly increased after IAV infection. When M6 protein defective mutant of GAS was used, the virus enhanced adherence and internalization was nearly abolished indicating the involvement of M protein binding sites on the MDCK cell surface. Interestingly, the modulation of some O-linked glycolproteins as well as sialic acid, mucin and fibrinogen-like residues on the surface of MDCK cells contributed to augmented bacterial adherence and/or internalization. In the same way, qRT-PCR experiments showed an overexpression of the membrane associated mucin (MUC1) on the surface of the MDCK cells after IAV infection. Altogether, the present study revealed that IAV infection augments the adherence and internalization of GAS to MDCK cells via modulation of membrane associated O-linked glycoproteins, fibrinogen, sialic acid residues and the mucin, MUC1 on the surface of MDCK cell.     

Biofilm formation or internalization into epithelial cells enable Streptococcus pyogenes to evade antibiotic eradication in patients with pharyngitis

Streptococcus pyogenes is the bacterium most frequently isolated from patients with pharyngitis. Although various antibiotics including penicillin are effective, antibiotic treatment failure in cases of streptococcal pharyngitis have been reported. Herein, we investigated mechanisms associated with recurrent streptococcal pharyngitis. Clinically isolated S.?pyogenes strains showed serotype-specific features, with emm12 strains most frequently detected and emm6 strains more likely to produce biofilm. The architectures of formed biofilms were observed using a fluorescence microscope with Live/Dead staining. Furthermore, various cationic antimicrobial peptides were tested to evaluate their inhibitory activities toward biofilms formed by S.?pyogenes. After treatments with high concentrations of antibiotics, S.?pyogenes survived in biofilm even when dead bacterial cells covered the surface. Other findings demonstrated that some antimicrobial peptides have inhibitory effects on forming and formed biofilm. Moreover, emm4, emm6, and emm75 strains showed significantly higher levels of invasion capacity into Detroit 562 cells than strains with other genotypes. Additionally, more than half of the strains temporarily escaped killing by penicillin alone by internalization into epithelial cells, even when the antibiotic concentration used was greater than the 10-fold minimum inhibitory concentration (MIC) for planktonic S.?pyogenes. Also, combined administrations of multiple antibiotics were more effective to eradicate strains more likely to be internalized. Finally, flow cytometric analyses demonstrated that emm12 strains with higher invasive capabilities expressed PrtF1 protein on the bacterial surface. These findings suggest that S.?pyogenes isolated from patients with recurrent streptococcal pharyngitis have emm type-specific features that allow escape from eradication by antibiotics.     

Invasive infections by Streptococcus pyogenes

Invasive infections by Streptococcus pyogenes continually increase both in France and in others industrialized countries. Because of the seriousness, the rapidity of the evolution and the epidemic potentialities, guidelines for managing these infections are requested by the Superior Council of Public Hygiene of France. The authors report herein a case of an adult stricken down by a violent evolution due to Streptococcus pyogenes. They point up how diagnosis, treatment and prophylaxis for family circle are difficult.     

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.     

Platelet aggregation by Streptococcus pyogenes.

Heat-killed group A Streptococcus pyogenes induced platelet aggregation in platelet-rich plasma. Aggregation was dependent upon the ratio of platelets to bacteria, with maximal aggregation occurring at 0.8 platelets per bacterium (final concentration, 300,000 per microliter). Inhibition of the reaction by 3 mM EDTA indicated it was a true aggregation and not merely adhesion and agglutination. Lactic acid dehydrogenase assays indicated lysis of platelets did not occur during a 6-min incubation period. Aggregation was inhibited in a dose-dependent manner by acetylsalicylic acid (100 microM to 10 mM) and quinacrine (15.6 to 250 microM), with no decrease in aggregation at the lowest concentration of inhibitor tested. S. pyogenes induced the release of [14C]serotonin, which was maximal (50%) at 2.4 min, when aggregation was nearly complete. Gel-filtered platelets were not aggregated unless fibrinogen (final concentration, 1.8 mg/ml) was included in the reaction mixture. Staphylococcus aureus, a group B streptococcus, and Escherichia coli were unable to induce aggregation in platelet-rich plasma under the conditions used for S. pyogenes.     

Recurrent sepsis caused by Streptococcus pyogenes

I report that a 75-year-old man with severe atherosclerosis experienced two episodes of bacteremia with Streptococcus pyogenes of type emm87. Recurrent sepsis with S. pyogenes is extremely rare, and a foot ulcer was the suspected point of entry. The patient did not develop opsonizing antibodies to the isolate.     

Updated emm-typing protocol for Streptococcus pyogenes

ObjectivesPCR-based typing of the emm gene Streptococcus pyogenes often results in the amplification of multiple bands. This has resulted in the misclassification of strains into types based on non-emm gene sequences. We aimed to improve the specificity of the emm typing PCR reaction using a primer called CDC3, the sequence for which has been previously used to identify emm genes in silico.MethodsThe proposed primer CDC3 was validated in silico from a global database of 1688 GAS genomes and in vitro with 32 isolates. PCR reactions were performed on genomic DNA from each isolate, using the published CDC1 forward primer with the CDC2 reverse primer or the new CDC3 reverse primer. The products were examined by gel electrophoresis, and representative PCR products were sequenced.ResultsIn 1688 S. pyogenes genomes, the previous CDC2 reverse primer annealed in silico in 1671 emm genes and also in 2109 non emm genes in close proximity, whereas the new CDC3 primer annealed in 1669 emm genes only. The remaining 19 genes without a CDC3 binding site were chimeric emm genes. The PCR pair CDC1+CDC3 produced a single band at appropriate molecular weight in all 32 isolates tested, while the CDC1+CDC2 pair produced more than one band in 13 of 32 isolates (40%).ConclusionsThe new CDC3 primer is more specific for emm genes than the previous CDC2 primer and represents a simple solution to reduce the potential for mistyping S. pyogenes strains.     

Macrolide-resistance genes in clinical isolates of Streptococcus pyogenes

Macrolide-resistance genes were investigated in 103 macrolide-resistant strains of Streptococcus pyogenes, isolated from children with pharyngotonsillitis. The presence of mef(A), erm(B), and erm(TR) genes was detected by PCR. mef(A) was found in 48 out of 103 (46.6%) strains, whereas erm(B) was detected in 43 isolates (41.7%). All mef(A) strains showed a typical M phenotype (resistance to 14- and 15-membered macrolides, and sensitivity to lincosamides and streptogramin B), whereas erm(B) strains had the MLSB phenotype (resistance to macrolides, lincosamides, and streptogramin B antibiotics). erm(TR) was found in 10 strains, always together with other resistance genes. In seven cases erm(TR) was associated with erm(B), and three cases with mef(A). In two isolates with the M phenotype (1.9%), it was not possible to detect the presence of any of the three macrolide resistance genes tested. Inducible resistance to macrolides was shown for 24 out of the 53 MLSB strains. Analysis of macrorestriction fragment patterns by pulsed-field gel electrophoresis showed that erythromycin-resistant S. pyogenes are polyclonal, however each phenotype, MLSB and M, formed essentially homogeneous groups.     

Epidemiology of severe Streptococcus pyogenes disease in Europe

The past 2 decades have brought worrying increases in severe Streptococcus pyogenes diseases globally. To investigate and compare the epidemiological patterns of these diseases within Europe, data were collected through a European Union FP-5-funded program (Strep-EURO). Prospective population-based surveillance of severe S. pyogenes infection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe (Cyprus, the Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Romania, Sweden, and the United Kingdom) using a standardized case definition. A total of 5,522 cases were identified across the 11 countries during this period. Rates of reported infection varied, reaching 3/100,000 population in the northern European countries. Seasonal patterns of infection showed remarkable congruence between countries. The risk of infection was highest among the elderly, and rates were higher in males than in females in most countries. Skin lesions/wounds were the most common predisposing factor, reported in 25% of cases; 21% had no predisposing factors reported. Skin and soft tissue were the most common foci of infection, with 32% of patients having cellulitis and 8% necrotizing fasciitis. The overall 7-day case fatality rate was 19%; it was 44% among patients who developed streptococcal toxic shock syndrome. The findings from Strep-EURO confirm a high incidence of severe S. pyogenes disease in Europe. Furthermore, these results have identified targets for public health intervention, as well as raising awareness of severe S. pyogenes disease across Europe.     

Epidemiological markers of Streptococcus pyogenes strains in Tunisia

To further understand the epidemiology of Streptococcus pyogenes or group A streptococcus (GAS) infections in Tunisia, phenotypic and genomic markers of GAS isolates, including antibiotic susceptibility, biotypes, T and emm types and toxin gene profiles, have been characterized. A total of 103 isolates, collected between 2000 and 2006, were investigated; 47 were recovered from invasive infections, and 56 from non-invasive infections. Rates of tesistance to tetracycline, erythromycin, clindamycin and rifampin were 70.8%, 4.8%, 4.8% and 0.9%, respectively. High levels of resistance to streptomycin and kanamycin were observed in 1.9% and 4.8% of isolates, respectively. Biotype 3 was most common. Twenty different T patterns were observed, with a predominance of T3/13/B3264, and 38 different emm types. In both invasive and non-invasive isolates, emm118 (9.7%), emm42 (8.7%), emm1 (7.8%), st432 (6.8%), emm28 (5.8%) and emm76 (5.8%) were the most prevalent types; emm1, emm76 and emm18 were mainly observed among invasive infections, whereas emm118 (12.5%), emm42 (10.7%) and emm28 (8.9%) were predominant among non-invasive infections. The speB gene was detected in all isolates, but there were variable frequencies of speA, speC and ssa (20.3%, 32% and 25.2% respectively). Significant associations of emm1, emm18 and emm3 with speA and of emm4 and st432 with ssa were found. This first report from Tunisia revealed a unique emm distribution of GAS that differs from those of other regions. This information on the distribution of such emm types will be useful for the development of an appropriate vaccine in a country where the incidence of rheumatic fever remains high.     

The fibronectin-binding protein of Streptococcus pyogenes, SfbI, is involved in the internalization of group A streptococci by epithelial cells.

Streptococcus pyogenes organisms (group A streptococci) are considered to be highly adhesive extracellular pathogens. However, it has recently been reported that S. pyogenes has the capacity to efficiently invade eukaryotic cells. In this study, we demonstrate that the interaction of S. pyogenes fibronectin-binding protein (SfbI) with fibronectin on nonphagocytic HEp-2 cells triggers bacterial internalization. Blocking of the SfbI adhesin by either antibodies against the whole protein or antibodies against the fibronectin-binding domains of SfbI, as well as pretreatment of HEp-2 cells with purified SfbI protein, prevents both S. pyogenes attachment and internalization. Inert latex beads precoated with the purified SfbI protein are ingested by eukaryotic cells, demonstrating that SfbI is per se enough to trigger the internalization process. Experiments performed with a recombinant SfbI domain encompassing the two fibronectin-binding regions of the SfbI molecule demonstrated that these binding regions are essential and sufficient to activate uptake by HEp-2 cells. These results demonstrate that the fibronectin-binding protein SfbI is involved in both S. pyogenes' attachment to and ingestion by HEp-2 cells and contribute to elucidation of the underlying molecular events leading to eukaryotic cell invasion by S. pyogenes.     

Severe Soft Tissue Infection Caused by a Non-Beta-Hemolytic Streptococcus pyogenes Strain Harboring a Premature Stop Mutation in the sagC Gene

We recovered a non-beta-hemolytic Streptococcus pyogenes strain from a severe soft tissue infection. In this isolate, we detected a premature stop codon within the sagC gene of the streptolysin S (SLS) biosynthetic operon. Reintroduction of full-length sagC gene on a plasmid vector restored the beta-hemolytic phenotype to our clinical isolate, indicating that the point mutation in sagC accounted for loss of hemolytic activity. To the best of our knowledge, this is the first report to demonstrate that a severe soft tissue infection can be caused by a non-beta-hemolytic S. pyogenes strain lacking a functional SagC.