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Clinical significance of inhibition kinetics for Streptococcus pyogenes in response to penicillin.

OBJECTIVES: The antibiotic mode of action against clinical isolates of Streptococcus pyogenes and physiological factors involved in modifying the inhibitory response to the antibiotic were investigated. METHODS: We developed high-resolution respirometry for continuous monitoring of bacterial growth and inhibition kinetics. One hundred and ten clinical isolates from 90 paediatric patients were tested, including 48 isolates obtained from 28 patients with eradication failure. Respirometric inhibition curves were monitored at 4 mg/L penicillin G over a short 30 min period, corresponding to the drug's serum half-life. RESULTS: None of the clinical isolates exhibited penicillin tolerance. Latency in the respirometric response of S. pyogenes to penicillin increased significantly with decreasing strain-specific respirometric growth rate. No difference in inhibition kinetics was found in vitro for isolates from patients with or without bacteriological treatment failure. CONCLUSIONS: In streptococcal pharyngotonsillitis, tolerance is not a relevant concept to explain bacteriological treatment failure. Definitions of tolerance should be reconsidered in the framework of growth-dependent antibiotic susceptibility.

Humans↗

The epidemiology of severe Streptococcus pyogenes associated disease in Europe.

Several European countries reported outbreaks of severe disease caused by Streptococcus pyogenes in the late 1980s. This marked a departure from the previous decades, where very few such outbreaks were noted. These changes in disease occurrence formed part of a global phenomenon, the reasons for which have yet to be explained. Results of surveillance activities for invasive S. pyogenes infection within Europe over the past fifteen years identified further increases in many countries. However, variations in surveillance methods between countries preclude robust comparisons being made, illustrating the need for a unified surveillance strategy across Europe. This was finally embodied in the Strep-EURO programme, introduced in 2002.

Disease Outbreaks↗

Effect of streptolysin S from Streptococcus pyogenes on contact sensitivity in mice.

Streptolysin S, a hemolytic toxin produced by strains of Streptococcus pyogenes, was examined for its effect on cellular immune reaction in mice. The toxin given intraperitoneally for six consecutive days did not influence intensiveness of delayed hypersensitivity to oxazolone which has been used as a model of cellular immune reaction. Streptolysin S injected subcutaneously, closely to lymph nodes directly involved in immune response, markedly suppressed delayed hypersensitivity. Significant inhibition of lymphocyte proliferation by streptolysin S was observed both in vivo as well as in vitro experiments.

Animals↗

The post-antibiotic effects of rokitamycin (a 16-membered ring macrolide) on susceptible and erythromycin-resistant strains of Streptococcus pyogenes.

The post-antibiotic effects (PAEs) on susceptible and erythromycin-resistant strains of Streptococcus pyogenes (M phenotype and inducibly resistant) of rokitamycin and erythromycin were investigated in vitro using microbiological impedance measurement. Exposure of susceptible S. pyogenes strains to 1/4, 1/2, 1 and 2 MIC erythromycin and rokitamycin resulted in PAEs of rokitamycin in the same order of magnitude as those of erythromycin and that were dose dependent. The duration of rokitamycin PAEs in erythromycin-resistant S. pyogenes strains (M phenotype and those with inducible resistance) were comparable with those observed in susceptible strains. This was not the case for erythromycin. The investigation showed that a 16-membered ring macrolide such as rokitamycin has different PAEs from those of a 14-membered ring macrolide such as erythromycin. They also indicated that, as the PAEs of rokitamycin on the M phenotype and inducible resistant strains were comparable with those on susceptible strains, no re-evaluation of therapeutic dosing regimens was required.

Anti-Bacterial Agents↗

[Emergence of macrolide resistant Streptococcus pyogenes strains in pediatric patients in France].

A total of 206 recent throat isolates of Streptococcus pyogenes collected between 2002 and 2004 from children were tested for their susceptibility to penicillin, amoxycillin, erythromycin, clarythromycin and clindamycin. The erythromycin resistant isolates were further studied for their genetic mechanism of resistance by means of PCR. In all, 14.5% of the strains were erythromycin resistant and 13.5 and 1% expressed the constitutive MLS(B) and M resistance phenotypes and harbored the ermB and mef A genes respectively.

Anti-Bacterial Agents↗

Activity of telithromycin compared with seven other agents against 1039 Streptococcus pyogenes pediatric isolates from ten centers in central and eastern Europe.

In total, 1039 pediatric Streptococcus pyogenes isolates from Bulgaria, Croatia, the Czech Republic, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia and Slovenia were studied. All strains were susceptible to penicillin G, levofloxacin, and quinupristin-dalfopristin, 91-100% to telithromycin, and 82-100% to erythromycin, azithromycin, and clarithromycin, and 90-100% to clindamycin. Macrolide resistance occurred mainly in Slovakia (25%), the Czech Republic (17.3%), and Croatia (15.8%). Overall, 9.7% of S. pyogenes isolates were erythromycin resistant due to erm(B)- or erm(A)-encoded methylases (72.3%) or to a mef(A)-encoded efflux pump (25.7%). One strain had alterations of both 23S rRNA (A2058G Escherichia coli numbering) and ribosomal protein L22 (G95D).

Adolescent↗

Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes.

We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PCR). Genomic DNA from 22 isolates representing 14 M serotypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and ennX were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which correspond to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein.

Antigens, Bacterial↗

Increased expression of the ska gene in emm49-genotyped Streptococcus pyogenes strains isolated from patients with severe invasive streptococcal infections.

Since 2000, emm49-genotyped Streptococcus pyogenes strains have been isolated from patients with severe invasive group A streptococcal infections in Japan, although they had not been isolated before 1999. We compare the characteristics of these strains with those of strains isolated from patients with non-invasive infections. SmaI-digested pulsed-field gel electrophoresis profiles of the isolates were almost indistinguishable between these groups, however, we found that ska (streptokinase gene) transcriptional levels in the strains isolated from patients with severe invasive infections were significantly higher than those in non-invasive infections.

Antigens, Bacterial↗

Recognition of Streptococcus pyogenes and skin autoantigens in guttate psoriasis.

BACKGROUND: Guttate psoriasis is associated with infections by Streptococcus pyogenes and cross-reactions between skin and streptococcal antigens have been reported, suggesting an autoimmune component in the disease. METHODS: In this work, the authors looked for antibodies against S. pyogenes M-5 antigens by immunoblot in 52 sera of psoriasis patients and in 52 sera of normal individuals. Histological and immunohistochemical analysis in skin biopsies from lesions of another group of 16 clinically diagnosed guttate psoriasis patients and four healthy controls were also carried out. RESULTS: All guttate psoriasis patients studied (11) had IgG antibodies that intensively recognized three different proteins of 70, 60 and 14 kDa, as compared to sera from patients with other forms of psoriasis or from healthy controls. The diagnosis of psoriasis was confirmed in 14 of the patients by hematoxylineosin staining. Of the other two patients, one was diagnosed as parapsoriasis and the other as liquen. By indirect immunofluorescence (IFI), all 14 psoriatic patients had autoantibodies against their own lesional skin that did not recognize normal skin from control subjects or from the two non-psoriatic patients. The parapsoriatic and the liquen patients did not have autoantibodies. A rabbit immune serum against S. pyogenes antigens reacted with lesional skin from the 14 guttate psoriatic patients, but not with normal skin from controls or with lesional skin from the 2 non-psoriatic patients. CONCLUSIONS: The recognition by immunoblot of streptococcal antigens by serum of guttate psoriasis patients, the presence of autoantibodies against their own skin, and recognition of the same skin antigens by anti-streptococcal rabbit antibodies confirm the participation of the immune system and of streptococcal infections in guttate psoriasis.

Adult↗

Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370.

The mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes.

Attachment Sites, Microbiological↗

Crystallization and preliminary X-ray diffraction studies of Streptococcus pyogenes plasmid pSM19035-encoded omega transcriptional repressor.

The transcriptional repressor, omega protein, from the Streptococcus pyogenes broad-host-range plasmid pSM19035 was crystallized at pH 7. 5 and 8.5 by the vapour-diffusion method using PEG 4000 as precipitant. Two crystal forms were obtained; the first belongs to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 and the second to the hexagonal space group P6(1) or P6(5). The crystals are most likely to contain one omega protein in the asymmetric unit, with V(m) values of 3.2 and 3.5 A(3) Da(-1), respectively. The crystals diffract X-rays to 2.4 and 2.9 A resolution for the tetragonal and hexagonal systems, -respectively.

Bacterial Proteins↗

Isolation and immunochemical characterization of carbohydrate antigens prepared from group A Streptococcus pyogenes.

Group A carbohydrate antigens were prepared from isolated cell walls or whole cells of Streptococcus pyogenes strain Sv (group A, M type 3). Cell walls were lysate by the enzymatic action of M1 endo-N-acetylmuramidase. The cell wall lysate was concentrated and chromatographed on a Sephadex G-100 column. Two fractions reactive with group A antisera were obtained. These were composed of N-acetylglucosamine, rhamnose, and peptidoglycan components such as glutamic acid, lysine, alanine, N-acetylmuramic acid and N-acetylglucosamine. For comparison, hot TCA extract of the cell walls (TCAgA) or Rantz and Randall extract of the whole cells (RRgA) was purified chromatographically. The latter two also contained rhamnose and glucosamine, and were reactive with group A antisera. The molecular weights of M1gA antigens were larger than those of TCAgA and RRgA. Hapten inhibition study indicated that N-acetylglucosamine was the most potent inhibitor.

Antigens, Bacterial↗

[Streptococcus pyogenes: in vitro susceptibility to several antimicrobials in two date periods].

BACKGROUND: The frequency of Streptococcus pyogenes infections with deep tissue invasion and toxic shock syndrome has increased in the last decade throughout the world. AIM: To compare antimicrobial susceptibility of S. pyogenes strains isolated during 1986 and during 1994-95. MATERIAL AND METHOD: Eighty two S. pyogenes strains isolated in 1986 and 67 strains isolated in 1994-95, were studied. MIC 50 and 90 were determined by and agar dilution method for penicillin, ampicillin, cefazolin, cefuroxime, erythromycin, roxithromycin and miocamycin. RESULTS: Eighty eight strains came from skin of soft tissues, 19 from surgical wounds, 18 from invasive infections, 15 from pharyngeal swabs and 9 from other locations. All strains were susceptible to penicillin, ampicillin, cefazolin, cefuroxime, roxithromycin and miocamycin. Ninety nine percent of strains were susceptible to erythromycin. Strains isolated in 1995-95 had a higher MIC 50 and 90 for erythromycin than those isolated in 1986. CONCLUSIONS: The changes in susceptibility to erythromycin of recently isolated strains could be due to the widespread use of macrolides in Chile.

Adolescent↗

Flow cytometric evaluation of adhesion of Streptococcus pyogenes to epithelial cells.

The precise roles of various surface molecules in the attachment of Streptococcus pyogenes to host epithelia are currently unclear. A flow cytometry assay that facilitates the analysis of the kinetics of S. pyogenes adhesion to epithelial cells was developed. Dose- and time-dependent adhesion isotherms with both buccal epithelial cells (BECs) and Hep-2 cells as substrata were obtained. Although binding equilibrium is reached within 2 h on both cell types, saturation of binding sites on BECs is not achieved within a wide range of experimental conditions. This indicates a high degree of non-specific attachment to that cell type. Since no rinsing step is necessary when using flow cytometry to analyze adhesion, low-affinity associations were observable. This was confirmed by determining bacterial desorption rates early and late in the adsorption process. Binding irregularities were also easily detected since the cytometer records and displays data for up to 10,000 epithelial cells per time point. It is proposed to use this methodology to assign roles to particular surface molecules/characteristics during distinct phases of adhesion.

Bacterial Adhesion↗

The influence of penicillin on growth and morphology of Streptococcus pyogenes in vivo.

The influence of penicillin (pc) on the growth, phagocytosis and killing of Streptococcus pyogenes was studied for an M protein positive (M+) and an M protein negative (M-) strain in vivo as well as in vitro. In vivo studies were based on a tissue cage model and the analyses were performed by CFU determinations and electron microscopic investigations. The M- strain was easily phagocytized with and without pc, but killing only occurred after pc treatment and thus the number of viable bacteria rapidly decreased under the influence of pc. M+ streptococci were not reduced in numbers by pc-treatment in vivo, but morphological changes and at high pc concentrations, phagocytosis could be seen. When this strain (M+) was cultivated in the absence of pc, the phagocytic cells were totally destroyed - a reaction that was prevented by penicillin. Variations in surface morphology of the two strains seem to influence the differences in sensitivity to penicillin, phagocytosis and killing.

Animals↗

Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system.

Macrolide-resistant Streptococcus pyogenes isolates from Finland, Australia, and the United Kingdom and, more recently, Streptococcus pneumoniae and S. pyogenes strains from the United States were shown to have an unusual resistance pattern to macrolides, lincosamides, and streptogramin B antibiotics. This pattern, referred to as M resistance, consists of susceptibility to clindamycin and streptogramin B antibiotics but resistance to 14- and 15-membered macrolides. An evaluation of the macrolide-lincosamide-streptogramin B resistance phenotypes among our streptococcal strains collected from 1993 to 1995 suggested that this unusual resistance pattern is not rare. Eighty-five percent (n = 66) of the S. pneumoniae and 75% (n = 28) of the S. pyogenes strains in our collection had an M phenotype. The mechanism of M resistance was not mediated by target modification, as isolated ribosomes from a pneumococcal strain bearing the M phenotype were fully sensitive to erythromycin. Further, the presence of an erm methylase was excluded with primers specific for an erm consensus sequence. However, results of studies that determined the uptake and incorporation of radiolabeled erythromycin into cells were consistent with the presence of a macrolide efflux determinant. The putative efflux determinant in streptococci seems to be distinct from the multicomponent macrolide efflux system in coagulase-negative staphylococci. The recognition of the prevalence of the M phenotype in streptococci has implications for sensitivity testing and may have an impact on the choice of antibiotic therapy in clinical practice.

Anti-Bacterial Agents↗

Resistance to macrolides in Streptococcus pyogenes in France in pediatric patients.

A total of 1,500 recent throat isolates of Streptococcus pyogenes collected between 1996 and 1999 from children throughout France were tested for their susceptibility to erythromycin, azithromycin, josamycin, clindamycin, and streptogramin B. The erythromycin-resistant isolates were further studied for their genetic mechanism of resistance, by means of PCR. The clonality of these strains was also investigated by means of serotyping and ribotyping. In all, 6.2% of the strains were erythromycin resistant, and 3.4 and 2.8% expressed the constitutive MLS(B) and M resistance phenotypes and harbored the ermB and mefA genes, respectively; ermTR was recovered from one isolate which also harbored the ermB gene. Ten serotypes and 8 ribotypes were identified, but we identified 17 strains by combining serotyping with ribotyping. Among the eight ribotypes, the mefA gene was recovered from six clusters, one being predominant, while the ermB gene was recovered from four clusters, of which two were predominant.

Anti-Bacterial Agents↗

High-frequency invasion of epithelial cells by Streptococcus pyogenes can be activated by fibrinogen and peptides containing the sequence RGD.

The ability of Streptococcus pyogenes to invade human epithelial cells has been suggested to be an important contributing factor to the bacterium's ability to cause severe, invasive infections. We know little, however, of the mechanism underlying intracellular invasion by this organism. In this study, we demonstrate that the invasion of cultured human epithelial cells by a serotype M1 strain of S. pyogenes (strain 90-226) is stimulated over 50-fold by the addition of fetal calf serum (FCS) to the cell culture medium (RPMI medium). Purified human fibrinogen and peptides containing the sequence Arg-Gly-Asp (RGD) were also found to promote bacterial invasion of cultured cells. Experiments that demonstrate that the agonists stimulate invasion by interacting with bacterial cells are described. Invasion stimulation did not appear to involve de novo synthesis of a bacterial protein, as FCS and fibrinogen stimulated invasion in the presence of chloramphenicol. Although the agonists stimulated adherence by up to threefold, strain 90-226 efficiently adhered to cultured cells in unsupplemented RPMI medium. The invasion index (the number of internalized CFU/the number of adherent CFU) of strain 90-226 was increased 10- to 25-fold by the addition of the agonists. Postinternalization survival of bacteria was unaffected by fibrinogen or FCS. Thus, the agonistic factors affect the efficiency by which adherent bacteria are internalized by epithelial cells.

Amino Acid Sequence↗