Epidemiology of Streptococcus pyogenes upper respiratory tract infections in Poland (2003-2017).

Streptococcus pyogenes (group A Streptococcus, GAS) is a major human pathogen and causes every year over 600 millions upper respiratory tract onfections worldwide. Untreated or repeated infections may lead to post-infectional sequelae such as rheumatic heart disease, a major cause of GAS-mediated mortality. There is no comprehensive, longitudinal analysis of the M type distribution of upper respiratory tract strains isolated in Poland. Single reports describe rather their antibiotic resistance patterns or focus on the invasive isolates. Our goal was to analyse the clonal structure of the upper respiratory tract GAS isolated over multiple years in Poland. Our analysis revealed a clonal structure similar to the ones observed in high-income countries, with M1, M12, M89, M28, and M77 serotypes constituting over 80% of GAS strains. The M77 serotype is a major carrier of erythromycin resistance and is more often correlated with upper respiratory tract infections than other serotypes.

Streptococcus sobrinus as a Predominant Oral Bacteria Related to the Occurrence of Dental Caries in Polish Children at 12 Years Old.

Dental caries is listed by the WHO as one of the major non-communicable diseases that need to be prevented and treated. The aim of the study was to evaluate the prevalence and severity of caries expressed as the Decayed, Missing and Filled Permanent Teeth (DMFT) index in 12-year-old Polish children and to verify bacterial species related to the occurrence of dental caries. Quantitative real-time PCR analysis of DNA isolated from saliva samples was performed to detect 8 cariogenic and periopathogenic bacterial strains. A total of 118 Polish children were enrolled in the study. They had low mean DMFT scores of 1.58 ± 1.98. The prevalence of dental caries in the children tested was low (53.4%), with a tendency to decrease compared to previous oral surveys. Bacterial abundance of other species in the dental caries and caries-free groups did not differ; however, periopathogenic Prevotella pallens, Fusobacterium nucleatum along with cariogenic Streptococcus mutans and Lactobacillus fermentum were significantly strongly correlated in the caries-active subjects. The prevalence of S. sobrinus was significantly higher in children with dental caries (p = 0.023) and correlated with higher DMFT. It may temporarily play an important role in the initiation of the cariogenic process or in its enhancement due to an ecological imbalance in dental microbiota.

New RAPMYCOI SensititreTM Antimicrobial Susceptibility Test for Atypical Rapidly Growing Mycobacteria (RGM).

Rapidly growing mycobacteria (RGM) cause an increasing international concern, mainly due to their natural resistance to many antibiotics. The aim of this study was to conduct species identification and determine the antimicrobial susceptibility profiles of RGM isolated in Poland. Antimicrobial susceptibility was tested using broth microdilution and the RAPMYCOI panel. A total of 60 strains were analysed, including the following species: M. fortuitum complex (30), M. abscessus subsp. abscessus (16), M. abscessus subsp. massiliense (7), M. chelonae (5), and M. mucogenicum (2). For 12 M. abscessus subsp. abscessus strains, the presence of the erm 41T28 genotype associated with inducible macrolide resistance and a functional erm gene was confirmed. A MUT2 mutation in the rrl gene (constitutive resistance) was identified for two strains from the subtype M. abscessus subsp. massiliense. Among the 15 tested antibiotics, amikacin and linezolid had the strongest antimycobacterial activity. Most of the tested strains were resistant to doxycycline and trimethoprim/sulfamethoxazole. Tigecycline MICs were low for all tested strains. Findings from our study highlight the importance of correct identification of clinical isolates and antimicrobial susceptibility testing.

Impact of Pneumococcal Vaccination on Nasopharyngeal Carriage of Streptococcus pneumoniae and Microbiota Profiles in Preschool Children in South East Poland.

In 2017, Poland introduced the 10-valent pneumococcal conjugate vaccine (PCV) into its national immunization schedule. This prospective study was conducted between March and June 2020 to determine the impact of vaccination on prevalence of the nasopharyngeal carriage of S. pneumoniae in 176 healthy children and to determine how conjugate vaccines indirectly affect colonization of nasopharyngeal microbiota. Pneumococcal isolates were analyzed by serotyping and antimicrobial resistance tests. Nasopharyngeal microbiota were detected and identified using the culture method and real-time PCR amplification primers and hydrolysis-probe detection with the 16S rRNA gene as the target. In the vaccinated group of children, colonization was in 24.2% of children, compared to 21.4% in the unvaccinated group. Serotypes 23A and 23B constituted 41.5% of the isolates. Serotypes belonging to PCV10 and PCV13 constituted 4.9% and 17.1% of the isolates, respectively. S. pneumoniae isolates were resistant to penicillin (34.1%), erythromycin (31.7%), and co-trimoxazole (26.8%). Microbial DNA qPCR array correlated to increased amounts of Streptococcus mitis and S. sanguinis in vaccinated children, with reduced amounts of C. pseudodiphtericum, S. aureus, and M. catarrhalis. Introduction of PCV for routine infant immunization was associated with significant reductions in nasopharyngeal carriage of PCV serotypes and resistant strains amongst vaccine serotypes, yet carriage of non-PCV serotypes increased modestly, particularly serotype 23B.

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia.

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004-2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes--emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies.

Multiple locus VNTR fingerprinting (MLVF) of Streptococcus pyogenes.

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.

Typing of Streptococcus pyogenes strains using the phage profiling method.

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.

Detection of Streptococcus pyogenes virulence factors by multiplex PCR.

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.

Antimicrobial susceptibilities of Listeria monocytogenes strains isolated from food and food processing environment in Poland.

A total of 471 Listeria monocytogenes isolates from different types of food and food-related sources in Poland during 2004-2010 were examined. This number includes 200 isolates from fish, 144 from fresh and frozen vegetables, 43 ready-to-eat products (deli foods, cold cuts), 13 from dairy products, 16 from raw meats, 15 from confectionery products and 40 directly from processing plants. All isolates were subjected to serotyping and lineage assays using PCR, and antimicrobial susceptibility using E-test and a broth microdilution method. Of all isolates, 256 (54.4%), 120 (25.5%), 59 (12.5%), 36 (7.6%) were identified as serotypes 1/2a (or 3a), 1/2c (or 3c), 1/2b (or 3b or 7), and 4b (or 4d or 4e), respectively. A direct correlation between the most common serotypes and three L. monocytogenes lineages was also observed. All L. monocytogenes isolates belonged to lineages I (20.2%) and II (79.8%). All strains were sensitive to ampicillin, amoxicillin, gentamicin, erythromycin, trimethoprim, rifampicin, vancomycin, chloramphenicol and sulfamethoxazol. Two of the L. monocytogenes strains (0.42%) showed phenotypic resistance. One strain was resistant to tetracycline and minocycline due to the presence of tet(M). It did not carry gene int, which may indicate that the tet(M) gene in this strain was not integrated in the transposon Tn916-Tn1545 family. The resistance of the second strain to ciprofloxacin and norfloxacin was attributed to active efflux associated with overexpression of gene lde. Our data indicate the low prevalence of antimicrobial resistance among L. monocytogenes isolates from food and food-related sources in Poland.

A new rapid and cost-effective method for detection of phages, ICEs and virulence factors encoded by Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.

Multilocus variable number tandem repeat analysis (MLVA) of Streptococcus pyogenes.

We tested 21 polymorphic loci encoded by the genome of Streptococcus pyogenes (group A Streptococcus, GAS). Seven of them were chosen for the MLVA scheme. The primer pairs, designed for selected loci, detect from few to several alleles, and the method has a Simpson's Index of diversity of 0.957. To test the overall performance of the method, multiplex PCR reactions were carried out for over 700 GAS strains. Using the method we were able to detect differences between highly clonal strains that share the same emm, MLST and PFGE types. The most diverse strains were M4, M2, M3 and M28. We developed a typing method that can be employed to differentiate between GAS strains. The method has high resolution and measures diversity of the GAS core chromosome, on the contrary to methods such as PFGE.