Streptococcus pyogenes (GAS) is a human pathogen that causes multiple infections worldwide. The pathogenic properties of GAS strains are often linked to the production of virulence factors such as toxins, proteases or DNases. Detection of virulence factors produced by GAS strains can be used to either determine pathogenic potential of the strain or as a rapid screening and typing method. We recently developed a method to detect simultaneously 20 GAS virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) in four low volume multiplex PCR reactions (Borek et al., 2011) and below we present a detailed protocol describing the method.

A b s t r a c tStreptococcus anginosus, Streptococcus intermedius and Streptococcus constellatus form a group of related streptococcal species, namely the Streptococcus Anginosus Group (SAG). The group, previously called "milleri" had been rarely described until 1980/1990 as source of infections. Nowadays SAG bacteria are often described as pathogens causing predominantly purulent infections. The number of infec tions is highly underestimated, as SAG strains are often classified in the microbiology laboratory as less virulent "viridans streptococci". Epidemiological situation regarding SAG infections in Poland has been unrecognized, therefore we performed a retrospective analysis of strains isolated between 1996 and 2012. Strains suspected of belonging to SAG were reidentified using an automated biochemical approach (Vitek2) and MALDITOF MS. We performed first analysis of antibiotic resistance among SAG strains isolated in Poland using automated methods (Vitek2), disk diffusion tests and ETests. We also performed PCR detection of resistance determinants in antibiotic resistant strains. Clonal structure of analyzed strains was evaluated with PFGE and MLVF methods. All three species are difficult to distinguish using automated diagnostic methods and the same is true for automated MIC evaluation. Our analysis revealed SAG strains are rarely isolated in Poland, predominantly from purulent infections. All isolates are very diverse on the genomic level as estimated by PFGE and MLVF analyses. All analyzed strains are sensitive to penicillin, a substantial group of strains is resistant to macrolides and the majority of strains are resistant to tetracycline. K e y w o r d s:

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The changes of perfusion caused by endovascular embolization of AVM can rely on both deterioration and improvement, and occur in the parenchyma surrounding the AVM and in the distant regions of the brain. Although deterioration of perfusion indicated that more frequent follow-up was necessary, it was not related with serious complications in our group of patients.

We developed a new method of typing for anginosus group streptococci (SAG). It is the first SAG-dedicated, PCR-based method, which allows to determine the relationship between strains. The method is based on the detection of tandem repeats among 9 genomic loci and is classified as multilocus variable number tandem repeats fingerprint (MLVF) type of analysis. Using the described method, it is possible to detect over half million MLVF patterns, which correlate with pulsed-field gel electrophoresis profiles. The other advantage of the method is relatively short time from "cell to data", low costs, and easy application for epidemiological and evolutionary studies.

Streptococcus pyogenes (GAS) is a human pathogen that causes millions of infections worldwide. Comparison between GAS strains isolated in different laboratories all over the world is often difficult. Three usually used methods have either low resolution (emm typing), are expensive (MLST) or time- and labor-consuming (PFGE). Our laboratory recently developed new, inexpensive methods of GAS typing—VF (virulence factor profiling) and PP (phage profiling). To improve the typing scheme developed for GAS, we recently proposed a new typing method. Here we present detailed protocol for MLVF analysis.

We recently developed a method that allows fast differentiation between Streptococcus pyogenes (GAS) strains. The method named phage profiling (PP) is based on a simple assumption that a regular PCR reaction with Taq polymerase and relatively short elongation time is not able to yield long DNA fragment, such as ~40–50 kb integrated prophage. Only fragments without any integrated DNA or short fragments inserted between integration sites can be efficiently amplified. We designed primers that anneal upstream and downstream prophage integration sites, so in simple PCR reaction we can test if any additional DNA is integrated into particular site. Profiling of integrated elements can be used as rapid, high resolution typing method, with the resolution as high as PFGE and is excellent predictor of PFGE type.