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Streptococcus pyogenes (group A Streptococcus [GAS]) is an important pathogen whose virulence is related to the production of exotoxins and the presence of particular surface components. One hundred eighty-two GAS strains were collected in northwestern Italy between 1994 and 2002 and analyzed for phenotypic characteristics (opacity factor, proteolyic activity, and antimicrobial susceptibility) and by polymerase chain reaction for the presence of genes responsible for the production of exotoxins implicated in pathogenesis speA and speF and of prtF(1) (encoding fibronectin-binding protein F1). All strains were speF positive and 19.2% were speA positive and prtF(1) negative, whereas the prtF(1) gene was identified in 39.5% of the other strains. Of these, approximately half revealed the same pulse-field gel electrophoresis (PFGE) pattern but differed in both speA gene and macrolide resistance.

In this paper, a novel multiobjective evolutionary algorithm for optimal reactive power (VAR) dispatch problem is presented. The optimal VAR dispatch problem is formulated as a nonlinear constrained multiobjective optimization problem where the real power loss and the bus voltage deviations are to be minimized simultaneously. A new Strength Pareto Evolutionary Algorithm (SPEA) based approach is proposed to handle the problem as a true multiobjective optimization problem with competing and noncommensurable objectives. A hierarchical clustering algorithm is imposed to provide the decision maker with a representative and manageable Pareto-optimal set. The results demonstrate the capabilities of the proposed approach to generate true and well-distributed Pareto-optimal nondominated solutions in one single run. The results also show the superiority of the proposed approach and confirm its potential to solve the multiobjective VAR dispatch problem.

Recently, there arose some important multiobjective evolutionary algorithms (MOEAs), among these MOEAs, strength Pareto evolutionary algorithm (SPEA) seems the most effective technique for finding or approximating the Pareto-optimal set for multiobjective optimization problems with several characteristics. Unfortunately, there are always some basic and obvious characteristics or knowledge in pending problem, where the loss due to this negligence is sometimes considerable in dealing with complex problems. Based on these reasons, an improvement on SPEA with immunity is given to restrain degeneracy of the evolution process, where the immune operator is realized by vaccine extraction, vaccination and immune selection in turn. Simulations show the ISPEA is effective and feasible.

Toxic products, such as endotoxin from the gram-negative and exotoxin from the gram-positive bacteria, are the most important initiators of the inflammatory host response in sepsis. In addition to antibacterial treatment, numerous attempts have been made to interfere with the exaggerated proinflammatory cascade initiated by the toxins. As most antitoxic and anti-inflammatory agents have shown no clear efficacy, an attractive alternative has been to prevent or minimise their release. Therefore, it was of interest to further study the antibiotic-induced release of toxins after exposure to antibiotics used for the treatment of the most severe infections, especially if protein synthesis inhibitors could reduce the release induced by PBP 3-specific β-lactam antibiotics. There were significant reductions in endotoxin release from gram-negative bacteria when the combination of the PBP 3-specific β-lactam antibiotic, cefuroxime, and the protein synthesis inhibitor, tobramycin, was compared with cefuroxime alone. Increasing doses of tobramycin reduced endotoxin release and increased the killing rate. In a kinetic in vitro model the endotoxin release from E.coli was higher after the second dose of cefuroxime. Nevertheless, it was reduced after addition of tobramycin. No binding of tobramycin to endotoxin was observed, either in vivo or in vitro. In a porcine sepsis model, a possible anti-inflammatory effect of ceftazidime and tobramycin, expressed as late cytokine inhibition, was seen. The protein synthesis inhibitor, clindamycin, released less streptococcal pyrogenic exotoxin A (SpeA) from a group A streptococcus strain than penicillin, and addition of clindamycin to penicillin resulted in less toxin production than penicillin alone. The SpeA production was dependent on the bacterial number at the start of treatment. Higher doses of penicillin also led to less SpeA. The choice of antibiotic class and dose may be important in the severely ill septic patient in whom an additional toxin release could be deleterious. A combination of a β-lactam antibiotic and a protein synthesis inhibitor seems beneficial but further investigations are needed.

In this paper, we propose a high performance multi-objective evolutionary algorithm (HPMOEA) based on the principles of the minimal free energy in thermodynamics. The main innovations of HPMOEA are : (1) providing of a new fitness assignment strategy by combining Pareto dominance relation and Gibbs entropy, (2) the provision of a new criterion for selection of new individuals to maintain the diversity of the population. We use convergence and diversity to measure the performance of the proposed HPMOEA, and compare it with the other four well-known multi-objective evolutionary algorithms (MOEAs): NSGA II, SPEA, PAES, TDGA for a number of test problems. Simulation results show that the HPMOEA is able to find much better spread of solutions and has better convergence near the true Pareto-optimal front on most problems.

This chapter describes a quantitative assay for the streptococcal superantigen, streptococcal pyrogenic exotoxin A (SPEA), which can be used in broth, tissue-culture media, and certain sera. The protocol can be adapted to allow measurement of any bacterial superantigen or protein toxin, using different oligonucleotides to amplify the coding sequences from bacterial DNA. Previously, measurement of bacterial toxins such as SPEA was limited by poor test sensitivity and test samples could only be analyzed if concentrated. Techniques included Ouchterlony immunodiffusion or enzyme-linked immuno-sorbent assay (ELISA), although the lower limit of detection was 100-fold greater than that achievable in this system. (1-3) Recent methods described have achieved improved sensitivities, some using sandwich ELISA techniques, others using competitive ELISA techniques. (4-8).

The influence of mutationally induced changes in protein folding on development of effective neutralizing antibodies during vaccination remains largely unexplored. In this study, we probed how mutational substitutions of streptococcal pyrogenic exotoxin A (SPEA), a model bacterial superantigen, affect native conformational stability and antigenicity. Stability changes for the toxin variants were determined using circular dichroism and fluorescence measurements, and scanning calorimetry. Self-association was assayed by dynamic light scattering. Inactivated SPEA proteins containing particular combinations of mutations elicited antibodies in HLA-DQ8 transgenic mice that neutralized SPEA superantigenicity in vitro, and protected animals from lethal toxin challenge. However, a highly destabilized cysteine-free mutant of SPEA did not provide effective immunity, nor did an irreversibly denatured version of an otherwise effective mutant protein. These results suggest that protein conformation plays a significant role in generating effective neutralizing antibodies to this toxin, and may be an important factor to consider in vaccine design.

Recognising the multiobjective nature of the decision process for rehabilitation of water supply distribution systems, this paper presents a comparative study of two multiobjective evolutionary methods, namely, multiobjective genetic algorithm (MOGA) and strength Pareto evolutionary algorithm (SPEA). The analyses were conducted on a simple hypothetical network for cost minimisation and minimum pressure requirement, treated as a two-objective problem. For the application example studied, SPEA outperforms MOGA in terms of the Pareto fronts produced and processing time required.

This paper examines the effect of mating restriction on the search ability of EMO algorithms. First we propose a simple but flexible mating restriction scheme where a pair of similar (or dissimilar) individuals is selected as parents. In the proposed scheme, one parent is selected from the current population by the standard binary tournament selection. Candidates for a mate of the selected parent are winners of multiple standard binary tournaments. The selection of the mate among multiple candidates is based on the similarity (or dissimilarity) to the first parent. The strength of mating restriction is controlled by the number of candidates (i.e., the number of tournaments used for choosing candidates from the current population). Next we examine the effect of mating restriction on the search ability of EMO algorithms to find all Pareto-optimal solutions through computational experiments on small test problems using the SPEA and the NSGA-II. It is shown that the choice of dissimilar parents improves the search ability of the NSGA-II on small test problems. Then we further examine the effect of mating restriction using large test problems. It is shown that the choice of similar parents improves the search ability of the SPEA and the NSGA-II to efficiently find near Pareto-optimal solutions of large test problems. Empirical results reported in this paper suggest that the proposed mating restriction scheme can improve the performance of EMO algorithms for many test problems while its effect is problem-dependent and algorithm-dependent.

The trade-off between obtaining a good distribution of Pareto-optimal solutions and obtaining them in a small computational time is an important issue in evolutionary multi-objective optimization (EMO). It has been well established in the EMO literature that although SPEA produces a better distribution compared to NSGA-II, the computational time needed to run SPEA is much larger. In this paper, we suggest a clustered NSGA-II which uses an identical clustering technique to that used in SPEA for obtaining a better distribution. Moreover, we propose a steady-state MOEA based on ɛ-dominance concept and efficient parent and archive update strategies. Based on a comparative study on a number of two and three objective test problems, it is observed that the steady-state MOEA achieves a comparable distribution to the clustered NSGA-II with a much less computational time.

12 páginas 1 figura, 2 tables et al.., Pelobatoidea is a clade of ancient anurans with obscure relationships to the remaining clades of frogs.We used partial sequences of two mitochondrial genes (cytochrome b and 16S RNA) from all Pelobatoidea subclades, including all species of Pelobatidae and Pelodytidae and four outgroup taxa (Xenopus, Ascaphus, Discoglossus, and Rana), to propose a phylogenetic hypothesis for relationships within Pelobatoidea.Maximum likelihood and Bayesian analyses support the monophyly of Pelobatoidea, but our hypothesis of internal relationships differs substantially from all previous hypotheses.Megophryidae is sister to Pelobates, and this clade is sister to Pelodytes.The most basal clade within Pelobatoidea is formed by Scaphiopus and Spea.The family Pelobatidae, as previously defined is not monophyletic, and it is split into Eurasian spadefoot toads Pelobates which retain the name Pelobatidae and North American spadefoot toads Scaphiopus and Spea which comprise the revived taxon Scaphiopodidae.Our analysis uncovers the existence of morphologically cryptic taxa within previously recognized species of the genus Spea and reveals marked genetic differentiation within Iberian Pelodytes.We discuss biogeographic implications and the evolution of fossoriality in the light of the new phylogenetic hypothesis., Tissue collection was supported in part by NSF Grant #9800886 to D.R.B., and the DNA analyses have been partially funded by the project REN2000-1541/GLO (Ministerio de Ciencia y Tecnolog ıa, Spain).

Why do closely related, coexisting species typically differ in phenotypic features associated with resource use? One answer to this question is that such differences might generally evolve in allopatry, as different species adapt to divergent environmental conditions, and any differences that thereby accumulate might subsequently enable coexistence in sympatry. Alternatively, coexisting species might generally diverge in sympatry, because of selection to reduce competition for food (character displacement). Here we evaluated these two causes of character divergence by asking which hypothesis better explains differences in feeding morphology between tadpoles of two species of spadefoot toads, Spea bombifrons and S. multiplicata. We found that, in natural ponds containing both species, S. multiplicata almost always developed into a smaller, round-bodied tadpole with normal sized jaw muscles used for feeding on detritus at the pond bottom (the omnivore morph), whereas S. bombifrons almost always developed into a larger, flat-headed tadpole with greatly enlarged jaw muscles used for feeding on crustaceans in open water (the carnivore morph). By contrast, in all but one of 18 similar ponds containing a single species, both species expressed both phenotypes. Divergence between species in morph production appears to have evolved in sympatry: when we compared population means for each of four key trophic characters, we found that no allopatric population of S. bombifrons was as carnivore-like as the sympatric S. bombifrons, and, for three of four characters, no allopatric population of S. multiplicata was as omnivore-like as the sympatric S. multiplicata. In contrast to significant differences in trophic characters, we found no divergence between allopatric and sympatric populations in a character not directly involved in feeding on detritus or crustaceans (overall body size). These data, together with our earlier experimental work, reveal that coexisting S. bombifrons and S. multiplicata have diverged from one another in resource use and in phenotypic features associated with resource use because of selection to reduce competition for food (i.e., character displacement). Spea tadpoles, therefore, are one of few systems for which both experimental and observational evidence link phenotypic divergence to resource competition, thereby providing a model for understanding why coexisting species often differ phenotypically.

This paper proposes a new mating scheme for evolutionary multiobjective optimization (EMO), which simultaneously improves the convergence speed to the Pareto-front and the diversity of solutions. The proposed mating scheme is a two-stage selection mechanism. In the first stage, standard fitness-based selection is iterated for selecting a pre-specified number of candidate solutions from the current population. In the second stage, similarity-based tournament selection is used for choosing a pair of parents among the candidate solutions selected in the first stage. For maintaining the diversity of solutions, selection probabilities of parents are biased toward extreme solutions that are different from prototypical (i.e., average) solutions. At the same time, our mating scheme uses a mechanism where similar parents are more likely to be chosen for improving the convergence speed to the Pareto-front. Through computational experiments on multi-objective knapsack problems, it is shown that the performance of recently proposed well-known EMO algorithms (SPEA and NSGA-II) can be improved by our mating scheme.

In the present study, 37 group A Streptococcus (GAS) strains belonging to 13 new emm sequence types identified among GAS strains randomly isolated in Brazil were characterized by using phenotypic and genotypic methods. The new types were designated st204 , st211 , st213 , st809 , st833 , st854 , st2904 , st2911 , st2917 , st2926 , st3757 , st3765 , and st6735 . All isolates were susceptible to the antimicrobial agents tested, except to tetracycline. They all carried the speB gene, and 94.6% produced detectable SpeB. Most strains belonging to a given emm type had similar or highly related pulsed-field gel electrophoresis profiles that were distinct from profiles of strains of another type. The other characteristics were variable from isolate to isolate, although some associations were consistently found within some emm types. Unlike the other isolates, all type st213 isolates were speA positive and produced SpeA. Strains belonging to st3765 were T6 and opacity factor (OF) negative. Individual isolates within OF-positive emm types were associated with unique sof gene sequence types, while OF-negative isolates were sof negative by PCR. This report provides information on new emm sequence types first detected in GAS isolates from a geographic area not extensively surveyed. Such data can contribute to a better understanding of the local and global dynamics of GAS populations and of the epidemiological aspects of GAS infections occurring in tropical regions.

We have shown previously that an isogenic SPEA-negative Streptococcus pyogenes strain did not attenuate virulence in a murine model of necrotizing fasciitis. The aim of this study was to confirm that streptococcal pyrogenic exotoxin A (SPEA) is not crucial for streptococcal invasiveness in murine invasive infection. The SPEA-negative S. pyogenes (H326) was complemented with speA extra-chromosomally to create strain H361 which produced 2.2-fold more SPEA compared with the parental speA(+)wild-type (H305). The growth phase-regulated expression of SPEA in vitro was unaffected in this strain. Complementation with speA resulted in reduced virulence and bacterial counts in invasive murine infection. SPEA production was quantitated from muscle tissue of infected mice. However, H361 did not produce more SPEA than H305 in vivo. We conclude that SPEA does not play a key role in invasive murine streptococcal infection.

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.

The streptococcal pyrogenic toxins A, B, and C (SPEA, SPEB, and SPEC) are responsible for the fever, rash, and other toxicities associated with scarlet fever and streptococcal toxic shock syndrome. This role, together with the ubiquity of diseases caused by Streptococcus pyogenes, have prompted structural analyses of SPEA by several groups. Papageorgiou et al. (1999) have recently reported the structure of SPEA crystallized in the absence of zinc. Zinc has been shown to be important in the ability of some staphylococcal and streptococcal toxins to stimulate proliferation of CD4+ T-cells. Since cadmium is more electron dense than zinc and typically binds interchangeably, we grew crystals in the presence of 10 mM CdCl2. Crystals have been obtained in three space groups, and the structure in the P2(1)2(1)2(1) crystal form has been refined to 1.9 A resolution. The structural analysis revealed an identical tetramer as well as a novel tetrahedral cluster of cadmium in all three crystal forms on a disulfide loop encompassing residues 87-98. No cadmium was bound at the site homologous to the zinc site in staphylococcal enterotoxins C (SECs) despite the high structural homology between SPEA and SECs. Subsequent soaking of crystals grown in the presence of cadmium in 10 mM ZnCl2 showed that zinc binds in this site (indicating it can discriminate between zinc and cadmium ions) using the three ligands (Asp77, His106, and His110) homologous to the SECs plus a fourth ligand (Glu33).

Background: The virulence of Streptococcus pyogenes is determined by a variety of structural molecules, toxins and complex enzymes. Pyrogenic exotoxins cause fever, erythematous reactions, cytotoxic and immunological effects. Aim: To assess the frequency of speA, speB and speC genes in Chilean Streptococcus pyogenes strains and their association with the invasiveness of infections. Material and methods: The genes for pyrogenic exotoxins SpeA, SpeB and SpeC were determined by polymerase chain reactions in 114 strains of group A Streptococcus pyogenes isolated from Chilean patients with invasive or non invasive infections. Results: The gene for SpeA was present in 30.7% of isolates, the gene for SpeB was present in 69.3% and the gen for SpeC in 44.7% of isolates. The gene for SpeA was present in 20 of 33 invasive infections and in 15 of 81 non invasive infections (p

Epidemiological studies strongly implicate the bacterial superantigen, streptococcal pyrogenic exotoxin A (SPEA), in the pathogenesis of necrotizing soft-tissue infection and toxic shock syndrome resulting from Streptococcus pyogenes. SPEA can act as a superantigen and cellular toxin ex vivo, but its role during invasive streptococcal infection is unclear. We have disrupted the wild-type spea gene in an M1 streptococcal isolate. Supernatants from toxin-negative mutant bacteria demonstrated a 50% reduction in pro-mitogenic activity in HLA DQ-positive murine splenocyte culture, and up to 20% reduction in activity in human PBMC culture. Mutant and wild-type bacteria were then compared in mouse models of bacteraemia and streptococcal muscle infection. Disruption of spea was not associated with attenuation of virulence in either model. Indeed, a paradoxical increase in mutant strain-induced mortality was seen after intravenous infection. Intramuscular infection with the SPEA-negative mutant led to increased bacteraemia at 24 h and a reduction in neutrophils at the site of primary muscle infection. Purified SPEA led to a dose-dependent increase in peritoneal neutrophils 6 h after administration. SPEA is not a critical virulence factor in invasive soft-tissue infection or bacteraemia caused by S. pyogenes, and it could have a protective role in murine immunity to pyogenic infection. The role of this toxin may be different in hosts with augmented superantigen responsiveness.

Bacteriophage T12, the prototypic bacteriophage of Streptococcus pyogenes carrying the erythrogenic toxin A gene (speA), integrates into the bacterial chromosome at a gene for a serine tRNA (W. M. McShan, Y.-F. Tang, and J. J. Ferretti, Mol. Microbiol. 23:719-728, 1997). This phage is a member of a group of related temperate phages, and we show here that not all speA-carrying phages in this group use the same attachment site for integration into the bacterial chromosome. Additionally, other phages in the group use the same serine tRNA gene attachment site as phage T12 and yet do not carry speA. The evidence suggests that recombination between phage genomes has been an important means of generating diversity and disseminating virulence-associated genes like speA.

More than 100 cases have been identified as streptococcal toxic shock-like syndrome (TSLS) in Japan, since the first case was reported in 1993. Of 26 S. pyogenes isolates associated with TSLS, 69% were M-types 1 and 3. This study focused on speA alleles which were carried by all M-type 1 and 3 isolates and isolates of some other M-types, irrespective of sources. We sequenced the nucleotides of the relevant region of the speA allele in all of these isolates. Consistent with other reports, these M-type 1 and 3 isolates carried the speA2 and speA3 alleles, respectively. Our results indicate that these two speA alleles, which were prevalent worldwidely have been disseminating in Japan.

Erythrogenic toxins are also known as streptococcal pyrogenic exotoxins (spe) which cause a skin rash in scarlet fever (3). They also appear to be involved in the pathogenesis of severe infection (1). We investigated the detection of speA, speB, speC genes in group A streptococci (GAS) isolated from clinical specimens and in throat of school children.

WOS:000330925100006 PubMed: 23988167 The aim of this study is to evaluate antibiotic susceptibilities, emm gene types, toxin gene profiles and clonal relatedness of group A streptococci (GAS) isolates obtained from patients and carriers. A total of 79 clinical isolates from patients and 60 isolates from carriers were included in the study. Emm typing, toxin gene detection for speA, speB, speC, speG and smeZ genes and pulsed-field gel electrophoresis (PFGE) was performed. Twenty-one distinct emm types were detected; the most common types were emm12, emm89, emm1, emm77, emm4 and emm3. The. detection rates of both emm types and the toxin genes didn't differ significantly between patients and carriers. The presence of speA and smeZ was significantly higher in emm1 and speG was significantly lower in emm4 when compared to the other emm types. The rate of clustering obtained with PFGE wasn't significantly different in patients and carriers. As a result, twelve of the 21 emm types detected in this study were covered by the 26-valent vaccine, constituting 77.7% of the emm typeable isolates; however the emm4 type which is one of the most common types in the present study is not among this coverage. (C) 2013 Association of Basic Medical Sciences of FB&H. All rights reserved

In this study, we develop a bi-objective programming approach for due-date setting in make-to-order manufacturing with different classes of customers. We formulate the problem of quoting due-dates under the assumption that demand is dependent on lead-time and price is determined by the length of the delivery time. In addition, other parameters such as production policy, inventory holding, delivery system and capacity utilisation should be considered in due-date decisions. To this purpose, we consider additional objective function in traditional due-date management problem. So, the proposed bi-objective model attempts to maximise total profit and minimise rates of changes in capacity utilisation simultaneously. To obtain a set of Pareto solutions efficiently, we propose an algorithm based on multi-objective ant colony optimisation. The proposed algorithm is compared with a noticeable multi-objective genetic algorithm, i.e. SPEA, based on some comparison metrics with random instances.

The gene encoding the bacterial superantigen streptococcal pyrogenic exotoxin A is often found in streptococcal strains associated with the recently described streptococcal toxic shock syndrome. Here we demonstrate that this gene is expressed at approximate fourfold higher levels in cells grown at 37 degrees C when compared to cells grown at 26 degrees C. This suggests there is increased production of this toxin when Streptococcus pyogenes is found in infections of the soft tissues and bloodstream, as opposed to S. pyogenes that have not breached the epithelial layers and are living on the surface of the skin.

Recent studies have used muscle denervation experiments to examine the function of muscles during feeding in frogs. By comparing the results of denervation experiments among taxa, it is possible to identify evolutionary changes in muscle function. The purpose of this study was to examine the function of jaw and tongue muscles during prey capture in Spea multiplicata, a representative of the superorder Mesobatrachia. All members of this group possess a disjunct hyoid apparatus. We predicted that Spea would possess a novel mechanism of tongue protraction on the basis of its hyoid morphology. High-speed video motion analysis and muscle denervation were used to study the feeding behavior and mechanism of tongue protraction in Spea. Although Spea possesses a relatively long tongue, its feeding behavior is similar to that of short-tongued frogs of similar body size. Denervation of the m. submentalis had no effect on feeding behavior. When the m. geniohyoideus was denervated, the tongue pad was raised and moved forward slightly, but did not leave the mouth. When the m. genioglossus was denervated, the tongue pad was raised slightly, but no forward movement of the tongue occurred. A similar result was obtained after the mm. genioglossus and geniohyoideus were denervated simultaneously. Thus, both the mm. genioglossus and geniohyoideus are necessary for normal tongue protraction in Spea. In contrast, only the m. genioglossus is necessary for normal tongue protraction in archaeobatrachians and neobatrachians. We hypothesize that the disjunct hyoid is responsible for the greater role of hyoid movement during feeding in mesobatrachians.

The distribution of the genes of streptococcal pyrogenic exotoxin (speA, B and C) among the 400 clinical isolates of Streptococcus pyogenes from Japanese pharnyx was detected by the PCR method (Nihonrinsho 50: 326, 1992). Sixty, 399 and 303 isolates were positive for the speA, B and C genes, respectively. However, only one isolate had none of these genes. Several isolates possessed two or three genes, i.e., both speB & C, speA & B or speA, B & C were found in 258, 30 and 30 isolates, respectively. No isolate possessing both speA & C without speB was found in this study. We concluded that the PCR method is much more useful for the epidemiological study on streptococcal pyrogenic exotoxins (SPE) of S. pyogenes isolates because of specificity, rapidity and sensitivity of this method compared with the conventional SPE identification method.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of „superantigens” produced by Staphylococcus aureus and Streptococcus pyogenes and its T lymphocyte stimulating activity is involved into the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have produced and characterized recombinant SPEA molecules in Escherichia coli. These molecules are indistinguishable from natural SPEA in both T cell stimulatory and HLA class II binding activities. Human class II molecules are more efficient than mouse class II molecules in presenting SPEA to T cells. In binding tests to major histocompatibility complex class II-positive cells SPEA competes with staphylococcal enterotoxin B and A but not with toxic shock syndrome toxin-1.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of 'superantigens' produced by Staphylococcus aureus and Streptococcus pyogenes, and its T lymphocyte stimulating activity is involved in the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have generated nine mutant SPEA molecules by substituting amino acids in the regions of homology between different streptococcal and staphylococcal superantigens. An additional mutant was created by deletion of the 10 N-terminal amino acids. The mutants were expressed as fusion proteins. Several mutations led to a loss of function due to a loss of class II-binding activity. Such loss mutations did not cluster to a certain region of the SPEA molecule. Rather, even a substitution of neighboring amino acids had opposite effects. None of the loss mutations affected the binding of neutralizing mAb and all loss mutants could be precipitated in Ouchterlony tests by a polyclonal anti-SPEA serum. We conclude that the functional activities of SPEA, and probably of other superantigens as well, cannot be attributed to a defined region of the molecule.

Summary Erythrogenic toxin type A (ETA) is assumed to play a causative role in both scarlet fever and the streptococcal toxic shock-like syndrome (TSLS). For a molecular epidemiological analysis of the gene of erythrogenic toxin type A (speA) we used altogether 497 clinical isolates of Streptococcus pyogenes belonging to three groups: a) isolates from patients with scarlet fever b) isolates from cases with TSLS c) isolates from patients with other streptococcal infections (like otitis media, tonsillitis, impetigo) (general group) We found that less than 50% of the scarlet fever-associated strains possessed the speA gene as compared to 25% of the general group. Only 5 to 30% of these strains secreted the toxin under experimental conditions in very low quantities. Among strains isolated from TSLS, 67% appeared to contain the speA gene. The amount of ETA secreted into the medium was also extremely low. Southern hybridization patterns proved to be the same with an speAspecific probe in all three groups of streptococcal isolates ( Hae III, Hind III). Increased occurrence of the speA gene among scarlet fever and TSLS-associated strains does not seem to be sufficient to support the hypothesis that ETA may have a causative role in both diseases since a considerable number of strains in these groups did not possess the speA gene.

SUMMARYClosely related species often specialize on different types of prey, but little is known about the fitness consequences of making an evolutionary transition to a novel diet. Spadefoot toad larvae provide a unique opportunity to reconstruct these evolutionary events. Although most anuran larvae feed on detritus or plankton, Spea larvae have also evolved the ability to consume large anostracan fairy shrimp. To investigate the changes that may have accompanied the shift to shrimp prey, we compared shrimp-induced physiological responses of Spea larvae with those of its sister genus, Scaphiopus, that has not made this transition. Although Spea larvae performed equally well on either diet, shrimp-fed Scaphiopus larvae experienced reduced growth and developmental rates,as well as elevated levels of the stress hormone corticosterone when compared with those that ate the ancestral detritus diet. These results suggest that ancestral Spea likely experienced reduced fitness when they first adopted a carnivorous feeding strategy.

A new type or mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein an SDS-PAGE, The molecular weight of the purified mitogenic factor was determined to be 25.370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gin-Thr-Gin-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu-Asn-Glu-Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.

The distribution of the Plains Spadefoot, Spea bombifrons, in southwestern Manitoba appears to be determined by soil type. Preference is shown for coarse textured to moderately textured soils, followed by medium textured soils. Drainage appeared to be less important in regard to distribution.

Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25(3)c and is found only following induction of a T25(3)c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.

How do species that interbreed become reproductively isolated? If hybrids are less fit than parental types, natural selection should promote reproductive isolation by favoring the evolution of premating mechanisms that prevent hybridization (a process termed reinforcement). Although reinforcement should generate a decline in hybridization over time, countervailing forces of gene flow and recombination are thought to preclude natural selection from enhancing and finalizing reproductive isolation. Here, I present recent estimates of hybridization frequency between two species of spadefoot toad, Spea multiplicata and S. bombifrons. I compare these recent measures of hybrid frequency with previously published estimates and show that hybridization between these species has declined precipitously over the past 27 years. Although previous studies suggest that reinforcement possibly accounts for this decline in hybrids over time, three alternative hypotheses also can explain the observed decrease in hybridization. First, if one of the two interacting species becomes rare, opportunities for and incidence of hybridization may decrease. Second, if one of the two interacting species is initially rare, hybridization may be initially common if the rare species has difficulty locating conspecific mates. Third, if hybrids are produced only in particular environments, hybrid frequency may decline if habitat changes result in loss of those environments that promote hybrid formation. I found no support for these three alternative explanations of the decline in hybrids. Instead, reinforcement appears to best account for the evolution of enhanced reproductive isolation between these species. Moreover, the finding that hybridization declined precipitously in only 27 years suggests that many systems that have undergone reinforcement may be overlooked because reproductive isolation between the interacting populations or species may already be complete.

The fitness consequences of hybridization critically affect the speciation process. When hybridization is costly, selection favors the evolution of prezygotic isolating mechanisms (e.g., mating behaviors) that reduce heterospecific matings and, consequently, enhance reproductive isolation between species (a process termed reinforcement). If, however, selection to avoid hybridization differs between species, reinforcement may be impeded. Here, we examined both the frequency and fitness effects of hybridization between plains spadefoot toads (Spea bombifrons) and New Mexico spadefoot toads (S. multiplicata). Hybridization was most frequent in smaller breeding ponds that tend to be ephemeral, and heterospecific pairs consisted almost entirely of S. bombifrons females and S. multiplicata males. Moreover, in controlled experimental crosses, hybrid offspring from crosses in which S. multiplicata was maternal had significantly lower survival and longer development time than pure S. multiplicata offspring. By contrast, hybrid offspring from crosses in which S. bombifrons was maternal outperformed pure S. bombifrons offspring by reaching metamorphosis faster. These data suggest that, although S. multiplicata females are under selection to avoid hybridization, selection might favor those S. bombifrons females that hybridize with S. multiplicata if their breeding pond is highly ephemeral. Generally, the strength of selection to avoid hybridization may differ for hybridizing species, possibly impeding reinforcement.

The virulence of Group A Streptococcus (GAS) is due to multiple factors including the production of superantigens such as Streptococcus Pyrogenic Exotoxin A (SPEA). Superantigens activate the T-lymphocyte through a non-specific MHC-antigen presenting cell complex of which intercellular adhesion molecule-1 (ICAM-1) is a necessary component. We sought to determine the contribution of ICAM-1 in the host response to SPEA-producing and non-SPEA producing GAS.

Several outbreaks of scarlet fever caused by Streptococcus pyogenes were recently reported. Scarlet fever is historically considered a toxin-mediated disease, dependent on the production of the exotoxins SpeA and SpeC, but a strict association between scarlet fever and these exotoxins is not always detected. The aims of this study were to characterize the scarlet fever bacterial isolates recovered from patients in a Lisbon hospital and to identify any distinctive characteristics of such isolates.We characterized a collection of 303 pharyngeal S. pyogenes collected between 2002 and 2008. One-hundred and one were isolated from scarlet fever patients and 202 were associated to a diagnosis of tonsillo-pharyngitis. Isolates were characterized by T and emm typing, pulsed field gel electrophoresis profiling and superantigen gene profiling.The diversity of the scarlet fever isolates was lower than that of the pharyngitis isolates. Specific lineages of emm87, emm4 and emm3 were overrepresented in scarlet fever isolates but only 1 pulsed field gel electrophoresis major lineage was significantly associated with scarlet fever. Multivariate analysis indicated associations of ssa, speA and speC with scarlet fever.In nonoutbreak conditions, scarlet fever is caused by a number of distinct genetic lineages. The lower diversity of these isolates and the association with specific exotoxin genes indicates that some lineages are more prone to cause this presentation than others even in nonoutbreak conditions.