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Differentiation of cultured keratinocytes promotes the adherence of Streptococcus pyogenes.

Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections.

Bacterial Adhesion↗

Streptococcus pyogenes as a cause of nosocomial infection in a critical care unit.

A cluster of five cases of Streptococcus pyogenes infection or colonization was identified in an adult critical care unit following the admission of a patient with a severe cutaneous infection. This report deals with the nature of the outbreak and methods of control, and comments on infections of this nature in adult critical care areas.

Adult↗

PCR-enzyme-linked immunosorbent assay and sequencing as an alternative to serology for M-antigen typing of Streptococcus pyogenes.

A rapid PCR-enzyme-linked immunosorbent assay for identification of 10 important emm gene types of Streptococcus pyogenes was developed. The emm genotypes of a coded panel of strains of known M serotype were determined, and in 144 of 149 cases (97%) the results were congruous. Strains of types that were not included in the panel of capture probes were emm genotyped by sequencing.

Antigens, Bacterial↗

Rapid increase of resistance to erythromycin and clindamycin in Streptococcus pyogenes in Italy, 1993-1995. The Italian Surveillance Group for Antimicrobial Resistance.

A survey of antibiotic resistance in Streptococcus pyogenes in Italy showed a sharp increase in erythromycin resistance. In 1993, the incidence of erythromycin-resistant strains was on average 5.1%, with marked variations by geographic area. Two years later, the incidence of these strains had registered a 1.5- to roughly 20-fold increase, with a mean value of 25.9%, exceeding 40% in three centers out of 13 and 30% in another four. For all the strains studied, normal levels of susceptibility to penicillin were reported.

Anti-Bacterial Agents↗

Telithromycin and quinupristin-dalfopristin resistance in clinical isolates of Streptococcus pyogenes: SMART Program 2001 Data.

This study evaluated the current status of antimicrobial resistance in clinical isolates of Streptococcus pyogenes in Taiwan as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. In 2001, 419 different isolates of S. pyogenes, including 275 from respiratory secretions, 87 from wound pus, and 31 from blood, were collected from nine hospitals in different parts of Taiwan. MICs of 23 antimicrobial agents were determined at a central location by the agar dilution method. All of the isolates were susceptible to penicillin (MIC at which 90% of the isolates were inhibited [MIC(90)], moxifloxacin > ciprofloxacin = levofloxacin = gatifloxacin > gemifloxacin) demonstrated potent activity against nearly all of the isolates of S. pyogenes tested. Thirty-two isolates (8%) were not susceptible to quinupristin-dalfopristin. Seventeen percent of isolates had telithromycin MICs of >or=1 microg/ml, and all of these isolates exhibited erythromycin MICs of >or=32 microg/ml. The high prevalence of resistance to telithromycin (which is not available in Taiwan) limits its potential use in the treatment of S. pyogenes infections, particularly in areas with high rates of macrolide resistance.

Anti-Bacterial Agents↗

Purification and characterization of streptolysin O from Streptococcus pyogenes.

1. Streptolysin O, an exotoxin produced by group A beta-hemolytic streptococci, has been purified from Streptococcus pyogenes culture supernatants. 2. The isolation and purification procedure involved ammonium sulphate and polyethylene glycol precipitations, and ion-exchange chromatographies on CM-Sepharose and Mono Q. 3. The proposed procedure introduces two ion-exchange chromatography steps making the purification process simpler and, especially, more reproducible than other described protocols. 4. The purified streptolysin O was hemolytically active, had a specific activity of 415,000 HU/mg, an optimum pH of 7.0, a relative molecular mass of 60,100 and an isoelectric pH of 7.5.

Bacterial Proteins↗

Variation in sic gene encoding complement-inhibiting protein of Streptococcus pyogenes serotype M1 isolates in Japan.

DNA sequencing of the gene encoding a complement-inhibiting protein of Streptococcus pyogenes (streptococcal inhibitor of complement, Sic) was carried out on 49 strains of S. pyogenes serotype M1. Those strains were obtained from patients and asymptomatic carriers in Japan from 1969 to 1997 and had various pulsed-field gel electrophoresis (PFGE) patterns. Four identical polymorphic sites were found in the strains with the same PFGE pattern (Ia), but not in those giving the pattern IIa. The other identical sites were found in the strains with the PFGE pattern IIa, but not in those with the pattern Ia. These observations suggest that each of PFGE patterns was restricted to a set of variation in the sic gene.

Alleles↗

Immunochemical studies and complete amino acid sequence of the streptokinase from Streptococcus pyogenes (group A) M type 12 strain A374.

The complete amino acid sequence of the streptokinase (SKase) of Streptococcus pyogenes M type 12 strain A374, isolated from a patient with poststreptococcal glomerulonephritis (PSGN), was determined. The epitope domain for the monoclonal antibody N-59, which cross-reacts with SKases of both the PSGN-associated strain and S. equisimilis H46A (a non-PSGN-associated strain), was predicted to be localized in residues 370 to 374. The epitope domain specific for monoclonal antibody RU-1, which reacts only with the PSGN-associated SKase, was localized to residues 164 to 236.

Amino Acid Sequence↗

Cloning and characterization of the gene, speC, for pyrogenic exotoxin type C from Streptococcus pyogenes.

The structural gene of streptococcal pyrogenic exotoxin type C (SPE C) was cloned from the chromosome of Streptococcus pyogenes strain T18P into Escherichia coli using pBR328 as the vector plasmid. Subcloning enabled the localization of the gene (speC) to a 1.7 kb fragment. Partially purified E. coli-derived SPE C and purified streptococcal-derived SPE C, were shown to have the same molecular weight (23,800) and biological activities. A DNA probe, prepared from cloned speC, cross-hybridized with the structural genes of SPE A and SPE B indicating relatedness at the nucleotide level. The speC-derived probe also hybridized to a fragment of CS112 bacteriophage DNA containing the phage attachment site.

Bacterial Proteins↗

Streptococcus pyogenes infection in mouse skin leads to a time-dependent up-regulation of protein H expression.

Streptococcus pyogenes protein H (sph) is an immunoglobulin-binding protein present in the Mga regulon of certain M1 serotype isolates. Although sph is present in many strains, it is frequently not expressed. In this paper we show that protein H was highly expressed after bacteria were injected into the skin of mice and were recovered from the blood, kidney, or spleen at various times postinfection. The percentage of protein H-positive colonies increased with time, reaching 100% in the spleen and kidney within 24 to 72 h postinfection. The up-regulation of sph expression was also observed in a mga mutant.

Animals↗

A simplified method for purification of an antitumor acidic glycoprotein from Streptococcus pyogenes (Su strain) by immunoadsorbent chromatography.

A simplified method for purification of an antitumor acidic glycoprotein (SAGP) from Streptococcus pyogenes (Su strain) by immunoaffinity chromatography is described. A cell-free crude extract prepared from the cocci was applied to the anti-SAGP IgG coupled Sepharose column, and elution was conducted with an alkaline buffer. The material eluted was confirmed to be homogeneous and identical with SAGP as demonstrated by both relative mobility on the SDS-polyacrylamide gel column and the antigenicity on the double diffusion agar plate. The cell-growth inhibitory activity of SAGP prepared by the present method was almost the same as that of SAGP purified by the previous time-consuming method. Since this simplified method provides a higher yield of SAGP, it will be useful in further studies on the biological properties of SAGP.

Antineoplastic Agents↗

The characteristics of M proteins purified by column chromatography with hydroxyapatite from acid extracts of Streptococcus pyogenes of types 1, 3, 6, 12 and 17.

Purified M proteins were recovered from acid extracts of Streptococcus pyogenes, M-types 1, 3, 6, 12 and 17, by elution from columns of hydroxyapatite of the proteins precipitated with ammonium sulphate. M protein free from non-type-specific antigens was recovered only from M-type 12. Although similar fractions were not recovered from M-types 1, 3, 6 and 17, purified preparations containing a single cross-reactive antigen were obtained. In addition to the M proteins associated with cross-reactive antigens, type-specific antigens that did not stimulate opsonic antibodies were isolated from revealed molecular weights that ranged from 32,000 to 63,000 daltons, total amino acid compositions that were similar, and N-terminal amino acids that were variable.

Amino Acid Sequence↗

Pharmacoepidemiological analysis of provincial differences between consumption of macrolides and rates of erythromycin resistance among Streptococcus pyogenes isolates in Spain.

The M phenotype is by far the most common mechanism of erythromycin resistance among Streptococcus pyogenes isolates in Spain. A geographic analysis of the relationship between within-country differences in the prevalence of M-type resistance to erythromycin in S. pyogenes and the level of consumption of 14- and 15-membered macrolides within different provinces was carried out. From 1998 to 1999, a nationwide multicenter surveillance study yielded 2,039 consecutive pharyngeal isolates of S. pyogenes. Data on antibiotic consumption for the same period were gathered from IMS Health, and the corresponding daily defined doses per 1,000 inhabitants per day were calculated according to the Anatomic Therapeutic Classification index. Macrolide use was subdivided into dosages given three times a day (TID), twice a day (BID), or once a day (OD). Spearman nonparametric correlation coefficients (R) were calculated, and variables proving to be significantly associated (P < 0.1) were introduced into a linear regression model. The total consumption of macrolides presented a significant correlation with the prevalence of resistance (R = 0.527; P = 0.032). Neither TID nor BID macrolide consumption showed significant correlations. Only OD consumption had a significance below 0.1. These data are consistent with the hypothesis that only the total consumption of macrolides influences the local rates of M-type erythromycin resistance in S. pyogenes, and subgroups of macrolides seem to have an additive rather than a selective effect by contributing to increasing the final amount of macrolides used. Local variations in total consumption were associated only with BID consumption (R = 0.849; P = 0.004). The simple linear regression with total macrolide consumption showed a considerable determination coefficient (R(2) = 0.678; P = 0.006). The model explains up to 68% of the measured variation and is clearly better as a predictor of the prevalence of resistance than the mere mean is. By solving the regression equation, the resultant value of 2.2 defined doses per 1,000 inhabitants per day fits with the existence of a critical threshold of selective pressure.

Anti-Bacterial Agents↗

Molecular population genetic analysis of a Streptococcus pyogenes bacteriophage-encoded hyaluronidase gene: recombination contributes to allelic variation.

Many strains of the human pathogenic bacterium Streptococcus pyogenes produce hyaluronidase, an enzyme that degrades hyaluronic acid, a major component of the extracellular matrix. Degradation of hyaluronic acid is thought to aid in host tissue invasion and dissemination of S. pyogenes. The molecular population genetics of the bacteriophage-encoded hyaluronidase gene (hyl) was analysed by sequencing the gene from 13 streptococcal strains representing seven well-differentiated multilocus enzyme electrophoretic types and eight M or T protein serotypes. Substantial levels of allelic polymorphism were identified, and the analysis found strong statistical evidence that recombinational processes have contributed to the generation of molecular variation in this gene. A 111 base pair segment of hyl encoding a collagenous motif, that may bind collagen, was absent in a serotype M14 isolate and 13 serotype M18 multilocus enzyme electrophoretic type 20 strains examined. The analysis provides a molecular population genetics framework for studies examining the role of naturally occurring hyaluronidase variation in host-pathogen interactions.

Alleles↗

[Increasing methylase-mediated resistance to macrolides in Streptococcus pyogenes in a children's hospital in Barcelona (Spain)].

INTRODUCTION: The genetic mechanisms and changes in resistance of Streptococcus pyogenes to clarithromycin and clindamycin were studied in 480 strains from a children's hospital in Barcelona (1996-2003). RESULTS: There was a progressive increase of strains with the MLSB phenotype (55.6% of resistant strains in 2002) and a relative decrease in the M phenotype. The overall rate of macrolide resistance was 29.8%, with an increase from 27.4% in the 1996-2001 period to 35.8% in the 2002-2003 period. The MefA gene was detected in M phenotype strains, the ermB gene in constitutive MLSB strains and the ermTR gene in some inducible MLSB strains. CONCLUSION: The increase of the resistance and the changes in the implied mechanisms reduce the effectiveness of macrolides and clindamycin as an alternative treatment.

Adolescent↗

Effect of lipoteichoic acid on the uptake of Streptococcus pyogenes by HEp-2 cells.

Lipoteichoic acid (LTA) is thought to play a role in the interactions between Streptococcus pyogenes and host cells. We have examined the effect of exogenous LTA on the adherence and entry of S. pyogenes JRS4 strain into HEp-2 epithelial cells. LTA markedly inhibited bacterial entry in a concentration-dependent manner, up to 250 microg ml(-1). In contrast, LTA had only a slight inhibitory effect on adherence. LTA also inhibited the entry but not adherence of Salmonella typhimurium strain into HEp-2 cells. Binding experiments showed a dose-dependent binding of LTA to cells up to 10 microg ml(-1). Confocal laser microscopy imaging and analysis revealed that LTA was internalized by the epithelial cells and colocalized with F-actin. These results might imply that, following binding, exogenous LTA enters HEp-2 cells and exerts a cytotoxic effect that interferes with bacterial internalization. A possible target for LTA activity might be the actin cytoskeleton, which is known to be essential for bacterial uptake.

Bacterial Adhesion↗

Strains of Streptococcus pyogenes from severe invasive infections bind HEp2 and HaCaT cells more avidly than strains from uncomplicated infections.

Epidemiologically unrelated Streptococcus pyogenes strains isolated from blood, throat, and skin were assayed for adherence to HEp2 and HaCaT cells. Invasive isolates showed significantly higher avidity for these cell lines than isolates from skin and throat. In general, S. pyogenes showed greater binding to HaCaT cells than to HEp2 cells.

Bacterial Adhesion↗

Typing of Streptococcus pyogenes strains isolated from throat infections in the region of Aachen, Germany.

BACKGROUND: Changes in the epidemiology of Streptococcus pyogenes infections may be associated with the introduction and reappearance of individual serotypes within a population. MATERIALS AND METHODS: Typing of 216 consecutive isolates of S. pyogenes from patients with pharyngitis in the region of Aachen, Germany, was performed by sequencing the emm gene, slide-agglutination of the T-antigen and determining the serum opacity reaction (SOR). RESULTS: All 216 isolates were unequivocally emm-typable. emm1 was most common (18.5%), foLlowed by emm12 (15.7%), emm3 (14.4%) and emm28 (13.9%). Only four isolates contained newly validated emm types: emm89 or emm94 were harbored by two isolates each. In one isolate, the sequence type s104 was found. CONCLUSION: Despite an anticipated selective pressure, the prevalence of emm1 among isolates from throat infections in northwestern Germany remains high, but does not reflect the predominance of emm1 among invasive isolates in Germany.

Adolescent↗