Antibiotic resistance is a significant issue in modern infectious medicine and veterinary science, with animals, including horses, playing a crucial role in its dissemination. Antibiotics, used both for treatment and prevention of diseases in animals, influence the composition of the microbiota and promote the selection of resistant strains that can be transmitted to humans through direct or environmental contact. Horses, currently classified as companion animals, serve as a reservoir of commensal and pathogenic bacteria, including multidrug-resistant strains capable of horizontal gene transfer. Of particular importance are plasmids and integrons that carry genes encoding β-lactamases, which significantly reduce the effectiveness of therapy in both humans and animals. Analysis of the equine oral and gut microbiota highlights the potential of these environments as sources and vectors of resistance. Understanding the mechanisms underlying the transfer and persistence of resistance genes in horses is crucial for public health and helps define new directions for research within the One Health framework.

Streptococcus anginosus is considered an emerging opportunistic pathogen causing life-threatening infections, including abscesses and empyema. Noticeably, clinical data revealed that S. anginosus also constitutes an important component of polymicrobial infections. Here, we showed for the first time that S. anginosus inactivates the antibacterial potential of neutrophil extracellular traps (NETs). The process is determined by a cell wall-anchored nuclease referred to as SanA, which high expression dominates in clinical strains isolated from severe infections. Nuclease activity protects S. anginosus against the antibacterial activity of NETs, supporting at the same time the survival of coexisting highly pathogenic species of Enterobacteriales. Obtained data suggest that SanA nuclease should be recognized as a critical S. anginosus virulence factor determining severe monospecies purulent infections but also shielding other pathogens promoting the development of polymicrobial infections.

Background The antibiotic resistance determinants and associated mobile genetic elements (MGEs) were detected among Streptococcus pyogenes (group A streptococci [GAS]) clinical isolates of an M77 serotype collected in Poland between 2003 and 2017.Methods The genomes of 136 M77 GAS isolates were sequenced using short- and selected with long-read approach; whole genome sequences were analyzed to determine the genetic context of macrolide resistance determinants.Results The analysed strains were collected from in- and outpatients. Sequencing data analysis revealed that all strains carried the tet(O) gene. They were classified as a single sequence type, ST63. The unique erythromycin-resistance determinant, the erm(TR), was detected in 76.5% (n = 104) of isolates. It was found predominantly (n = 74) within a novel hybrid integrative conjugative element composed of the ICESp1108-like sequence and ICESp2906 variant, which was then named ICESp1109. However, in strains isolated before 2008, erm(TR) was located within ICESp2905 (n = 27) and in 3 strains - within stand-alone ICESp1108-like sequences.Conclusions Based on phylogenetic analysis results, the clonal dissemination of the macrolide-resistant S. pyogenes M77/ST63 strain with hybrid ICESp1109 was observed between 2008 and 2017. ICESp1109 is the novel hybrid ICE in gram-positive bacteria. A novel hybrid 103-kb integrative and conjugative element, ICESp1109, was identified in macrolide- and tetracycline-resistant Streptococcus pyogenes M77/ST63 strains collected in Poland between 2003 and 2017. Whole genome-based phylogenetic analysis revealed its clonal dissemination since 2008.

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.

For many years, Streptococcus anginosus has been considered a commensal colonizing the oral cavity, as well as the gastrointestinal and genitourinary tracts. However, recent epidemiological and clinical data designate this bacterium as an emerging opportunistic pathogen.

Three distinct streptococcal species: Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus, belonging to the Streptococcus anginosus group (SAG), also known as Streptococcus milleri group, have been attracting clinicians and microbiologists, not only as oral commensals but also as opportunistic pathogens. For years they have been simply classified as so called viridans streptococci, and distinct species were not associated with particular clinical manifestations. Therefore, description of SAG members are clearly underrepresented in the literature, compared to other medically relevant streptococci. However, the increasing number of reports of life-threatening infections caused by SAG indicates their emerging pathogenicity. The improved clinical data generated with the application of modern molecular diagnostic techniques allow for precise identification of individual species belonging to SAG. This review summarizes clinical reports on SAG infections and systematizes data on the occurrence of individual species at the site of infection. We also discuss the issue of proper microbiological diagnostics, which is crucial for further clinical treatment.

Abstract Streptococcus anginosus group (SAG), formerly known as Streptococcus milleri, consists of three distinct streptococcal species: Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius. SAG was considered as commensal bacteria of colon, oral cavity and vagina. Recent observation reported those bacteria as potent pathogens found in brain or liver abscess. Mechanism of SAG pathogenesis is still unknown, despite the strong set of clinical data. In our study, we determined the virulence of different SAG clinical isolates by examining the activity of hemolysins, DNAses and proteases. By using Dictyostelium discoideum as model for screening SAG virulence we selected a few high and low virulence strains out of 41 SAG isolates. Finally, we established and characterized the infection process of selected SAG strains by wax worm Galleria mellonella. Therefore, we aimed to characterize how those bacteria can interact with human innate immunity. In our experiments, using both human serum and whole blood we demonstrated, that these bacteria are resistant to killing by non-specific mechanisms of the immune response. Our studies have demonstrated that SAG have numerous virulence factors which differ among examined strains, and which allow bacteria to effectively colonize the human body and avoid elimination by the immune system. Supported by National Science Centre, Poland 2018/29/B/NZ6/00624

Streptococcus agalactiae (Group B Streptococcus, GBS) is a common commensal bacterium in adults but remains a leading source of invasive infections in newborns, pregnant women, and the elderly, and more recently, causes an increased incidence of invasive disease in nonpregnant adults. Reduced penicillin susceptibility and emerging resistance to non-β-lactams pose challenges for the development and implementation of novel, nonantimicrobial strategies to reduce the burden of GBS infections. Antimicrobial photodynamic inactivation (aPDI) via the production of singlet oxygen or other reactive oxygen species leads to the successful eradication of pathogenic bacteria, affecting numerous cellular targets of microbial pathogens and indicating a low risk of resistance development. Nevertheless, we have previously reported possible aPDI tolerance development upon repeated sublethal aPDI applications; thus, the current work was aimed at investigating whether aPDI tolerance could be observed for GBS and what mechanisms could cause it. To address this problem, 10 cycles of sublethal aPDI treatments employing rose bengal as a photosensitizer, were applied to the S. agalactiae ATCC 27956 reference strain and two clinical isolates (2306/02 and 2974/07, serotypes III and V, respectively). We demonstrated aPDI tolerance development and stability after 5 cycles of subculturing with no aPDI exposure. Though the treatment resulted in a stable phenotype, no increases in mutation rate or accumulated genetic alterations were observed (employing a RIF-, CIP-, STR-resistant mutant selection assay and cyl sequencing, respectively). qRT-PCR analysis demonstrated that 10 sublethal aPDI exposures led to increased expression of all tested major oxidative stress response elements; changes in sodA, ahpC, npx, cylE, tpx and recA expression indicate possible mechanisms of developed tolerance. Increased expression upon sublethal aPDI treatment was reported for all but two genes, namely, ahpC and cylE. aPDI targeting cylE was further supported by colony morphology changes induced with 10 cycles of aPDI (increased SCV population, increased hemolysis, increased numbers of dark- and unpigmented colonies). In oxidant killing assays, aPDI-tolerant strains demonstrated no increased tolerance to hypochlorite, superoxide (paraquat), singlet oxygen (new methylene blue) or oxidative stress induced by aPDI employing a structurally different photosensitizer, i.e., zinc phthalocyanine, indicating a lack of cross resistance. The results indicate that S. agalactiae may develop stable aPDI tolerance but not resistance when subjected to multiple sublethal phototreatments, and this risk should be considered significant when defining efficient anti-S. agalactiae aPDI protocols.

Streptococcus pyogenes encodes multiple virulence factors and their presence is often related to the severity of the disease. We designed the system of four low-volume multiplex PCR reactions to detect genes encoding 20 virulence factors: spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, speK, speM, speC, speI, speA, speH, speG, speJ, smeZ, and ssa. Classification of strains based on the virulence factors absence or presence correlates with PFGE MLST and emm typing results. The typing/detection system is fast and cost-effective, can be used to detect GAS virulence factors and as a rapid tool to effectively differentiate between strains.

BACKGROUND:Cholera is one of the most deadly diarrheal diseases that require new treatments. We investigated the neutralization of cholera toxin by five plant extracts obtained from the Rosaceae family that have been traditionally used in Poland to treat diarrhea (of unknown origin). METHODS:Hot water extracts were prepared from the dried plant materials and lyophilized before phytochemical analysis and assessment of antimicrobial activity using microdilution assays. The ability of the plant extracts to neutralize cholera toxin was analyzed by measurement of cAMP levels in cell cultures, enzyme-linked immunosorbent assay and electrophoresis, as well as flow cytometry and fluorescence microscopy studies of fluorescent-labeled cholera toxins with cultured human fibroblasts. RESULTS:The antimicrobial assays displayed modest bacteriostatic potentials. We found that the plant extracts modulate the effects of cholera toxin on intracellular cAMP levels. Three plant extracts (Agrimonia eupatoria L., Rubus fruticosus L., Fragaria vesca L.) suppressed the binding of subunit B of cholera toxin to the cell surface and immobilized ganglioside GM1 while two others (Rubus idaeus L., Rosa.canina L.) interfered with the toxin internalization process. CONCLUSIONS:The traditional application of the Rosaceae plant infusions for diarrhea appears relevant to cholera, slowing the growth of pathogenic bacteria and either inhibiting the binding of cholera toxin to receptors or blocking toxin internalization. The analyzed plant extracts are potential complements to standard antibiotic treatment and Oral Rehydration Therapy for the treatment of cholera.

For the last 40years, Sanger sequencing allowed to unveil crucial secrets of life. However, this method of sequencing has been time-consuming, laborious and remains expensive even today. Human Genome Project was a huge impulse to improve sequencing technologies, and unprecedented financial and human effort prompted the development of cheaper high-throughput technologies and strategies called next-generation sequencing (NGS) or whole genome sequencing (WGS). This review will discuss applications of high-throughput methods to study bacteria in a much broader context than simply their genomes. The major goal of next-generation sequencing for a microbiologist is not really resolving another circular genomic sequence. NGS started its infancy from basic structural and functional genomics, to mature into the molecular taxonomy, phylogenetic and advanced comparative genomics. Today, the use of NGS expended capabilities of diagnostic microbiology and epidemiology. The use of RNA sequencing techniques allows studying in detail the complex regulatory processes in the bacterial cells. Finally, NGS is a key technique to study the organization of the bacterial lifefrom complex communities to single cells. The major challenge in understanding genomic and transcriptomic data lies today in combining it with other sources of global data such as proteome and metabolome, which hopefully will lead to the reconstruction of regulatory networks within bacterial cells that allow communicating with the environment (signalome and interactome) and virtual cell reconstruction.

Staphylococcus aureus gene locus sdr is encoding proteins responsible for adhesion to bone tissue and therefore for the invasiveness of this bacteria. The aim of the study was to determine possible...

Streptococci are a diverse group of Gram-positive microorganisms sharing common virulence traits and similar strategies to escape the oral niche and establish an infection in other parts of the host organism. Invasive infection with oral streptococci is a perfect storm that requires the concerted action of multiple biotic and abiotic factors. Our understanding of streptococcal pathogenicity and infectivity should probably be less mechanistic and driven not only by the identification of novel virulence factors. The observed diversity of the genus, including the range of virulence and pathogenicity mechanisms, is most likely the result of interspecies interactions, a massive horizontal gene transfer between streptococci within a shared oral niche, recombination events, selection of specialized clones, and modification of regulatory circuits. Selective pressure by the host and bacterial communities is a driving force for the selection of virulence traits and shaping the streptococcal genome. Global regulatory events driving niche adaptation and interactions with bacterial communities and the host steer research interests towards attempts to define the oral interactome on the transcriptional level and define signal cross-feeding and co-expression and co-regulation of virulence genes.

Streptococcus anginosus group (SAG) is Gram-positive bacteria responsible for a number of purulent human infections such as brain and liver abscesses, which have been on the rise for last few decades. Although some virulence factors of SAG are described, they are mostly undefined and there are almost no methods for genetic manipulations of clinical SAG. Therefore, we presented various approaches to produce engineered strains of this poorly known group of streptococci. We developed a procedure of transformation characterized by transformation efficiency at the level of 104 per 1 μg DNA for certain strains. Moreover, mutagenesis for many SAG strain is possible based on the process of natural transformation. However, the usefulness of methods and their effectiveness are strain dependent.

Abstract Group A Streptococcus (GAS) is a Gram-positive human pathogen that causes a variety of diseases ranging from pharyngitis to life-threatening streptococcal toxic shock syndrome. Recently, several global gene expression analyses have yielded extensive new information regarding the regulation of genes encoding known and putative virulence factors in GAS. A microarray analysis found that transcription of the GAS gene M5005_Spy_1343 was significantly increased in response to interaction with human polymorphonuclear leukocytes. M5005_Spy_1343 is predicted to encode a member of the LysR family of transcriptional regulators and is located upstream of a putative operon containing six genes. Five of these genes have sequence similarity to genes involved in short-chain fatty acid metabolism, whereas the sixth gene (luxS) is found in many bacterial species and is involved in quorum sensing. Unexpectedly, inactivation of the M5005_Spy_1343 gene resulted in hypervirulence in an intraperitoneal mouse model of infection. Increased virulence was not due to changes in luxS gene expression. We postulate that short-chain fatty acid metabolism is involved in GAS pathogenesis.

Abstract Streptococcus 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 infections 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 re-identified using an automated biochemical approach (Vitek2) and MALDI-TOF MS. We performed first analysis of antibiotic resistance among SAG strains isolated in Poland using automated methods (Vitek2), disk diffusion tests and E-Tests. 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.

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.

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.