Search

A robust cell envelope is the first line of defense in infectious pathogens when encountering host immune defenses. In Gram-positive bacteria, LytR-CpsA-Psr (LCP) family proteins play a major role in the synthesis and assembly of the cell envelope. The Gram-positive bacteriumS. pyogenescauses a wide range of diseases from pharyngitis to septicemia, but the involvement of LytR in the synthesis and assembly of the cell wall envelope in this bacterium is not clear. Therefore, we investigated whether LytR ofS. pyogenes, like LytR of other streptococci, is involved in cell wall formation. ThelytRgene-deficient strains were used to investigate the bacteria's ability to form a chaining, drug sensitivity to penicillin G, an inhibitor of cell wall synthesis, bacterial biofilm formation, and the morphological structure of bacteria by transmission electron microscopy, and the expression ofpbp2b, which encodes a penicillin-binding protein, compared with the wild-type strain. Our results showed that LytR-deficient strains had reduced bacterial chaining compared to wild-type strains. Compared to wild-type strains, LytR-deficient strains were also more drug sensitive to the cell wall synthesis inhibitor penicillin G. This genetic study was accompanied by increased expression of thepbp2b gene in the LytR-deficient strains. In addition, the LytR-deficient strain showed a reduced ability to form biofilms, and thelytRgene-deficient strain showed morphological irregularities with abnormal bacterial septa. These results indicate that thelytRgene is involved in cell wall synthesis inS. pyognes.

Background. Scarlet fever, an infectious disease caused by Streptococcus pyogenes, largely disappeared in developed countries during the twentieth century. In recent years, scarlet fever is on the rise again, and there is a need for a better understanding of possible factors driving transmission. Methods. Using historical case notification data from the three largest cities in The Netherlands (Amsterdam, Rotterdam and The Hague) from 1906 to 1920, we inferred the transmission rate for scarlet fever using time-series susceptible-infected-recovered (TSIR) methods. Through additive regression modelling, we investigated the contributions of meteorological variables and school term times to transmission rates. Results. Estimated transmission rates varied by city, and were highest overall for Rotterdam, the most densely populated city at that time. High temperature, seasonal precipitation levels and school term timing were associated with transmission rates, but the roles of these factors were limited and not consistent over all three cities. Conclusions. While weather factors alone can only explain a small portion of the variability in transmission rates, these results help understand the historical dynamics of scarlet fever infection in an era with less advanced sanitation and no antibiotic treatment and may offer insights into the driving factors associated with its recent resurgence.

Surface-associated, coiled-coil M proteins ofStreptococcus pyogenes(Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, leading to its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic M protein variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule bound M87 in the crystal, but in solution recruited additional LL-37 molecules, consistent with a ‘protein trap’ neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain, and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

Background Streptococcus pyogenes (S. pyogenes) is a Gram positive bacterium which is a leading cause of pharyngitis, skin and soft tissue infection and post streptococcal syndromes. Due to lack of β-lactamase enzyme production, it was considered universally susceptible to penicillin group and later generation of β-lactam antibiotics. As such, empirical treatment was common which might leads to development of antibiotics resistance. Therefore, the aims of this study were to determine the prevalence, antibiotics susceptibility profile; and associated factors of S. pyogenes among pediatric patients with acute pharyngitis in Felege Hiwot Comprehensive Specialized Hospital (FHCSH), Northwest Ethiopia. Methods Hospital based cross-sectional study was carried out on 154 pediatric patients, whose age ranged from 0 to 18 years old using consecutive convenient sampling technique from 1st February to 19th June 2020 at FHCSH. S. pyogenes were identified by throat swab culture on 5% sheep blood agar with an overnight incubation at 37 °C in candle jar containing 5% CO2. Gram stain, catalase test and bacitracin test were used to identify S. pyogenes. Then,the data were entered into EpiData version 3.1 and analyzed by SPSS version 20 software. Finally, stepwise, bivariable and multivariable logistic regressions were carried out for identifyying factors having significant ssociation (p Results From the total throat swabs, 14 (9.1%) with (95% CI; 4.5–14.3) were culture positive for S. pyogenes. From these, all isolates were sensitive to penicillin and ampicillin. On the otherhand, 4 (35.7%), 4 (35.5%), 3 (21.4%), 2 (14.3%), 1 (7.1%), 7 (50.0%) and 1 (7.1%) isolates were resistant for ceftriaxone, vancomycin, erythromycin, tetracycline, chloramphenicol, clindamycin and levofloxacin, respectively. The presence of any smoker in home showed significant association with S. pyogenes acute pharyngitis. Furthermore, having tender lymphadenopathy and recurrence were clinical predictors for S. pyogenes acute pharyngitis (P Conclusion The prevalence of S. pyogenes was guaged at 9.1% which is considered as low prevalence. All S. pyogenes isolats remain sensitive to penicillin. However, resistance was reported to clindamycin 7 (50.0%), ceftriaxone 5 (35.7%) and erythromycin 3 (21.4%). The current practice of giving erythromycin, clindamycin instead of penicillin and ampicillin is againest the microbiology result. Therefore, current empirical treatment of acute pharyngitis shall take in to account the current evidences. Continuous surveillance of antibiotics resistance pattern of S. pyogenes for acute pharyngitis must be strengthen to improve the use of antibiotics in hospitals.

As a cause of vulvovaginitis in prepubertal girls, Streptococcus pyogenes (S. pyogenes) is commonly isolated from vaginal introitus swabs. Studies have identified several risk factors, but have not focused on the correlation between ambient air pollutants and S. pyogenes-induced vulvovaginitis in prepubertal girls. This study was conducted to determine whether ambient air pollutants were associated with S. pyogenes-induced vulvovaginitis in prepubertal girls. Daily data about S. pyogenes-induced vulvovaginitis in prepubertal girls from the outpatient department of Children’s Hospital at the Zhejiang University School of Medicine in Hangzhou City between January 1, 2015, and December 31, 2018, were retrospectively reviewed. Ambient air pollutants in Hangzhou were measured daily. A generalized additive model (GAM) was utilized to assess the associations between daily air pollutants and S. pyogenes isolates obtained from vaginal introitus swabs of prepubertal girls. The mean daily concentration of nitrogen dioxide (NO2) in Hangzhou City during the study period was 44.6 μg/m3 (25th–75th percentiles, 32.0–56.0 μg/m3). The GAM showed that the largest estimate effects in S. pyogenes-induced vulvovaginitis in prepubertal girls were found in NO2 with a moving (accumulative) average on day 3. The excess risk of NO2 in terms of the daily number of S. pyogenes isolates obtained from the vaginal introitus swabs was 14.91% (95% confidence interval [CI]: 4.85–25.94%) in the single-pollutant model. The multipollutant model revealed that an increase of 10 μg/m3 in NO2 exposure was associated with an 18.33% increased risk for acquiring S. pyogenes-induced vulvovaginitis in prepubertal girls (95% CI: 1.21–38.35%; P < 0.05). In conclusion, short-term NO2 exposure was strongly associated with the spread of S. pyogenes-induced vulvovaginitis in prepubertal girls.

Background. Scarlet fever, an infectious disease caused by Streptococcus pyogenes , largely disappeared in developed countries during the twentieth century. In recent years, scarlet fever is on the rise again, and there is a need for a better understanding of possible factors driving transmission. Methods. Using historical case notification data from the three largest cities in The Netherlands (Amsterdam, Rotterdam and The Hague) from 1906 to 1920, we inferred the transmission rate for scarlet fever using time-series susceptible-infected-recovered (TSIR) methods. Through additive regression modelling, we investigated the contributions of meteorological variables and school term times to transmission rates. Results. Estimated transmission rates varied by city, and were highest overall for Rotterdam, the most densely populated city at that time. High temperature, seasonal precipitation levels and school term timing were associated with transmission rates, but the roles of these factors were limited and not consistent over all three cities. Conclusions. While weather factors alone can only explain a small portion of the variability in transmission rates, these results help understand the historical dynamics of scarlet fever infection in an era with less advanced sanitation and no antibiotic treatment and may offer insights into the driving factors associated with its recent resurgence.

Streptokoki su gram-pozitivne kokoidne bakterije koje mogu biti organizirane u lancima različitih duljina ili kao diplokoki. Rod uključuje neke od najčešćih patogenih bakterija kod ljudi, poput S. pyogenes, S. pneumoniae i S. agalactiae. Klasifikacija roda je složena, a identifikacija vrsta temelji se na kolonijalnoj morfologiji, tipu hemolize, biokemijskim reakcijama te serološkoj specifičnosti. Ovaj završni rad predstavlja osvrt na dosadašnju filogenetsku strukturu roda Streptococcus, mikromorfologiju i kolonijalnu morfologiju, metabolizam i fiziologiju, strukturu genoma, patogenost te rezistenciju streptokoka na antibiotike., Streptococci are gram-positive coccoid bacteria that can be organized in chains of different lengths or as diplococci. The genus includes some of the most common pathogenic bacteria in humans, such as S. pyogenes, S. pneumoniae and S. agalactiae. The classification of the genus is complex, and species identification is based on colonial morphology, type of hemolysis, biochemical reactions and serological specificity. This thesis presents a review of the current phylogenetic structure of the genus Streptococcus, micromorphology and colonial morphology, metabolism and physiology, genome structure, pathogenicity and resistance of streptococci to antibiotics.

Guava (Psidium guajava L.) leaf has long been the subject of diverse research initiatives as herbal medicine due to its antimicrobial activity against various bacteria. An Upper Respiratory Tract Infection (URTI) is a contagious infection of the upper respiratory tract, which can be caused by bacteria, i.e., Streptococcus pyogenes and Pseudomonas aeruginosa. In this research, guava leaves were extracted sequentially using solvents with different polarities. Furthermore, this research aimed to determine the antibacterial activity of guava leaf extract against URTI caused by bacteria (S. pyogenes and P. aeruginosa) and to formulate it into hard candy at various Minimum Inhibitory Concentrations (MIC), which were 2 MIC, 3 MIC, 4 MIC, and 5 MIC, to obtain hard candy with the highest antibacterial activity and organoleptic acceptability. To assist in the organoleptic acceptability aspect of the hard candy, lemongrass oil was used in varying concentrations (0%, 0.15%, 0.3%) as a flavoring agent. Results showed that ethanol guava leaf extract had the strongest antibacterial activity against both S. pyogenes (MIC=721.21 ppm) and P. aeruginosa (MIC=3085.91 ppm). Moreover, hard candy added with guava leaf extract at a concentration of 4 MIC and 0.15% lemongrass oil had the highest overall acceptability and antibacterial activity against S. pyogenes (inhibition diameter of 11.92 ± 0.56 mm) and P. aeruginosa (10.77 ± 0.69 mm).

SUMMARY Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function—annotated subsequently as S protein—in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory., In Brief Wierzbicki et al. show that S protein is a major group A Streptococcus (GAS) virulence factor that facilitates bacterial coating with lysed red blood cells to promote molecular mimicry, which increases virulence in vitro and in vivo. Removal of S protein reduces the abundance of multiple virulence factors and attenuates virulence., Graphical Abstract

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system’s function.

Previous studies have clearly demonstrated that the addition of lentisk oil (LO) to streptococcal cultures makes it possible to differentiate Streptococcus spp. into three categories with Streptococcus mitis and Streptococcus intermedius sensitive, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus mutans partially sensitive, and Streptococcus salivarius insensitive to the product. We have investigated here whether the winterization of LO, an easy and cheap procedure that removes some of the fatty substances contained within, resulted in a better antimicrobial effect on human pathogens affecting the pharyngeal mucosa and middle ear such as S. pyogenes, S. pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae, without affecting, or minimally affecting, S. salivarius strains, oral probiotics commonly used to reduce oral and middle ear infection recurrence, especially in children. Our results not only demonstrated a stronger antimicrobial action of winterized LO (WLO) on S. pyogenes, compared to what was seen with LO, but also demonstrated a strong antimicrobial action vs. S. pneumoniae and M. catarrhalis and a very limited effect on S. salivarius (strains K12 and M18). Moreover, WLO demonstrated a co-acting action when tested along with the antibiotics amoxicillin (A) and amoxicillin clavulanate (AC), effects clearly visible also on H. influenzae. Our results also showed that at least part of the antimicrobial effect observed was due to the presence of anacardic acids (AAs). Finally, WLO, when tested with human peripheral blood mononuclear cells (h-PBMCs), reduced the release of IL-6 and TNF-α and, in the case of cells stimulated by LPS, the release of IFN-γ. In conclusion, our study highlights an enhanced antimicrobial role for LO when winterized, suggests a co-acting effect of this when given with antibiotics, identifies AAs as possible active ingredients, and proposes a possible anti-inflammatory role for it.

Background Group A Streptococcal (GAS) infections cause the autoimmune disease acute rheumatic fever (ARF), which can progress to chronic rheumatic heart disease (RHD). Treating pharyngitis caused by GAS with antibiotics is important in preventing ARF. However, it is difficult to distinguish these infections from GAS carriers. There is growing evidence for GAS skin infections as a cause of ARF. This study will identify the incidence of true GAS pharyngitis and serological responses to GAS skin infections. The effectiveness of antibiotics for these conditions will be explored, and modifiable risk factors. Serum antibody titres indicating the upper limits of normal (ULN for ASO/ADB antibodies) will be established alongside carriage rates in asymptomatic children. Methods This is a prospective disease incidence study, with an associated case-control study. The study population includes 1000 children (5–14 years) from Auckland, New Zealand, 800 of whom have visited their healthcare professional, resulting in a throat or skin swab for GAS, and 200 who are asymptomatic. The conditions of interest are GAS throat swab positive pharyngitis (n = 200); GAS carriage (n = 200); GAS negative throat swab (n = 200); GAS skin infections (n = 200); and asymptomatic controls (n = 200). All participants, except asymptomatic controls, will have acute and convalescent serological testing for ASO/ADB titres (collected

The review is devoted to the analysis of molecular mechanisms of action ofS. pyogenesvirulence factors aimed at overcoming the protective functions of macrophages. The review describes in detail the main protective functions of macrophages and the mechanisms of their implementation in the course of streptococcal infection. The virulence factors ofS. pyogenes,which prevent the recruitment of macrophages to the site of infection, are examined. Particular attention is paid to the analysis of molecular effects that suppress the pathogen by the process of phagocytosis, intracellular bactericidal activity and the production of cytokines by macrophages. The analysis of molecular genetic mechanisms of regulation of the expression ofS. pyogenesvirulence factors that provide adaptation of the pathogen to changing conditions in the site of inflammation is carried out.

Streptococcus pyogenes was the most common bacterial causes of tonsillitis. The detection of virulence factors of this pathogen can be used to determine pathogenic potential as a rapid screening method. A total of 109 isolates (46%) showed positive culture for S. pyogenes, these isolates recovered from 235 swabs were collected from tonsillitis patients in ENT department in Al–Habboby Teaching Hospital, Thi-Qar province, Iraq. S. pyogenes isolates exposed to detect the specific gene (spy1258)and one of the virulence factors were smeZ gene by PCR technique and DNA sequencing analysis. The PCR results recorded that 61% and 50% of isolates harbor spy1258 and smeZ genes, respectively. The sequencing of PCR products showed significant alignments identities (94-100%) to the S. pyogenes for both genes which are located in BLAST-NCBI Genbank. The four PCR products of both genes were registered in Genbank under the named as (ZKD1 Spy-like gene; ZKD2 Spy-like gene; ZKD3 SmeZ-like gene and ZKD4 SmeZ-like gene). The results of Multiple sequence alignment analysis recorded that C > T and T > C polymorphism for smeZ gene.

Previous studies have clearly demonstrated that the addition of lentisk oil (LO) to streptococcal cultures makes it possible to differentiate Streptococcus spp. into three categories with Streptococcus mitis and Streptococcus intermedius sensitive, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus mutans partially sensitive, and Streptococcus salivarius insensitive to the product. We have investigated here whether the winterization of LO, an easy and cheap procedure that removes some of the fatty substances contained within, resulted in a better antimicrobial effect on human pathogens affecting the pharyngeal mucosa and middle ear such as S. pyogenes, S. pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae, without affecting, or minimally affecting, S. salivarius strains, oral probiotics commonly used to reduce oral and middle ear infection recurrence, especially in children. Our results not only demonstrated a stronger antimicrobial action of winterized LO (WLO) on S. pyogenes, compared to what was seen with LO, but also demonstrated a strong antimicrobial action vs. S. pneumoniae and M. catarrhalis and a very limited effect on S. salivarius (strains K12 and M18). Moreover, WLO demonstrated a co-acting action when tested along with the antibiotics amoxicillin (A) and amoxicillin clavulanate (AC), effects clearly visible also on H. influenzae. Our results also showed that at least part of the antimicrobial effect observed was due to the presence of anacardic acids (AAs). Finally, WLO, when tested with human peripheral blood mononuclear cells (h-PBMCs), reduced the release of IL-6 and TNF-α and, in the case of cells stimulated by LPS, the release of IFN-γ. In conclusion, our study highlights an enhanced antimicrobial role for LO when winterized, suggests a co-acting effect of this when given with antibiotics, identifies AAs as possible active ingredients, and proposes a possible anti-inflammatory role for it.

Virulent strains of Streptococcus pyogenes (gram-positive group A Streptococcus pyogenes [GAS]) recruit host single-chain human plasminogen (hPg) to the cell surface—where in the case of Pattern D strains of GAS, hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM). The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R561-V562 peptide bond, resulting in the disulfide-linked two-chain protease, human plasmin (hPm). hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins. Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2hPg), but how this relates to the function of the full-length proteins is unclear. Herein, we use intact proteins to show that the lysine-binding site of K2hPg is a major determinant of the activation-resistant T-conformation of hPg. The binding of PAM to the lysine-binding site of K2hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b. Domain swapping between hPg and mouse Pg emphasizes the importance of the Pg latent heavy chain (residues 1–561) in PAM binding and shows that while SK2b binds to both hPg and mouse Pg, the activation properties of streptokinase are strictly attributed to the serine protease domain (residues 562–791) of hPg. Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface.

Infection of a surgical wound due to bacteria is a major problem for surgical patients. Cu-zeolite is a material that can suppress bacterial growth with reversible cation characteristics and adsorption to be developed into non-toxic disinfectants for humans. Packaging uses filter paper to keep disinfectant solutions or instruments that will be sterilized clean. This study aimed to observe the effects of contact time of natural Cu-zeolite on the growth of Streptococcus pyogenes (S. pyogenes) and Pseudomonas aeruginosa (P. aeruginosa) bacteria. An experimental research was simple randomized design. Cu-zeolite 10 grams were packaged in Whatman no 42 paper bags measuring 5 x 5 cm2, contacted for 15, 30 and 45 minutes in 99.5 ml of distilled water exposed to 0.5 x 108 CFU / ml of S. pyogenes and P. aeruginosa. Subsequently, bacterial growth was calculated using total plate count method. The average growth of S.pyogenes for 15, 30 and 45 minutes (1840 ± 571.236 CFU; 29 ± 16.33 CFU and 0 CFU) while P. aeruginosa was (2776 ± 725.277 CFU; 55 ± 23.214 CFU and 0 CFU) respectively. Based on the independent t-test on Cu-zeolite, the bacterial growth in the 15th and 30th minute contact between S. pyogenes and P. aeruginosa was significantly different (pth and 30th minute contact.

Impetigo is the most common bacterial skin infection in children. The disease has two forms: nonbullous and bullous, of which nonbullous is the most common (70 %). Impetigo is caused by Staphylococcus aureus, Streptococcus pyogenes, or their combinations. In nonbullous impetigo, methicillin­resistant strains (MRSA) of S. aureus are most often detected. For the colonization of staphylococcus and streptococcus, the necessary conditions are damage to the integrity of the skin and violation of the composition of the usual microflora of the skin. Patients with inflammatory skin diseases are most often colonized by S. aureus and are at high risk for developing MRSA infections. In bullous impetigo, the rash elements are represented by bullae which quickly burst with the formation of surface erosion and a yellow crust. Nonbullous impetigo is manifested by vesicles, pustules, crusts. Typically, impetigo is clinically diagnosed. Suspicion of MRSA occurs in cases of spontaneous abscess or cellulitis, or if lesions are not eliminated by the recommended initial treatment with antibiotics. Topical therapy is the first line of treatment for uncomplicated nonbullous and bullous impetigo. If the lesion is limited to a small area (up to 6 cm2), the treatment involves soaking the crusts, exfoliating them and applying antimicrobial ointments (fusidic acid or a combination of neomycin and bacitracin) (EBM Guidelines, 2018). With more diffuse lesions, systemic antibiotic therapy is prescribed. In treatment of patients with eczema, topical and systemic antimicrobials with topical steroids are used. The combination of the aminoglycoside antibiotic neomycin and the polypeptide antibiotic bacitracin is presented at the Ukrainian pharmaceutical market as Baneocin drug. This combination has a pronounced synergistic effect on S. aureus, S. pyogenes and P. aeryginosa. An important advantage of Baneocin is its availability in two forms (powder and ointment), which allows the use of this drug at different stages of the infection process., Импетиго является наиболее общей бактериальной инфекцией кожи у детей. Заболевание имеет две формы: небуллезную и буллезную, из которых небуллезная является наиболее распространенной (70 %). Импетиго вызывают Staphylococcus aureus, Streptococcus pyogenes или их комбинации. При небуллезном импетиго чаще выявляют метициллино-резистентные штаммы (MRSA) S. aureus. Для колонизации стафилококка и стрептококка необходимыми условиями являются повреждение целости кожного покрова и нарушение состава обычной микрофлоры кожи. Пациенты с воспалительными кожными заболеваниями чаще других колонизированы S. aureus и находятся в зоне высокого риска в отношении развития инфекций, обусловленных MRSA. При буллезном импетиго элементы высыпаний представлены буллами, которые быстро разрываются с образованием поверхностной эрозии и желтой корочки. Небуллезное импетиго проявляется везикулами, пустулами, корочками. Как правило, диагноз импетиго устанавливают клинически. Подозрение на MRSA возникает в случаях спонтанного абсцесса или целлюлита либо если поражения не устраняются при рекомендованном начальном лечении антибиотиками. Топическая терапия является первой линией лечения неосложненных небуллезного и буллезного импетиго. Если поражение ограничено небольшой площадью (до 6 см2), то лечение предусматривает размачивание корочек, их отслоение и нанесение антимикробных мазей (фузидиевая кислота или комбинация неомицина с бацитрацином) (EBM Guidelines, 2018). При более распространенных поражениях назначают системную антибиотикотерапию. У пациентов с экземой в лечении используют топические и системные антимикробные препараты с топическими стероидами. Комбинация аминогликозидного антибиотика неомицина и полипептидного антибиотика бацитрацина представлена на фармрынке Украины препаратом «Банеоцин». Эта комбинация имеет выраженный эффект синергизма в отношении S. aureus, S. pyogenes и P. aeryginosa. Важным преимуществом «Банеоцина» является наличие двух лекарственных форм (порошок и мазь), что позволяет использовать этот препарат на разных стадиях инфекционного процесса., Імпетиго є найбільш загальною бактеріальною інфекцією шкіри у дітей. Захворювання має дві форми: небульозну та бульозну, з яких небульозна є найпоширенішою (70 %). Імпетиго спричиняють Staphylococcus aureus, Streptococcus pyogenes або їхні комбінації. При небульозному імпетиго частіше виявляють метицилінорезистентні штами (MRSA) S. aureus. Для колонізації стафілокока та стрептокока необхідними умовами є пошкодження цілості шкірного покриву та порушення складу звичайної мікрофлори шкіри. Пацієнти із запальними шкірними захворюваннями частіше за інших колонізовані S. aureus та перебувають у зоні високого ризику щодо розвитку інфекцій, спричинених MRSA. При бульозному імпетиго елементи висипу представлені булами, які швидко розриваються з утворенням поверхневої ерозії та жовтої кірочки. Небульозне імпетиго виявляється везикулами, пустулами, кірочками. Зазвичай діаг­ноз імпетиго встановлюють клінічно. Підозра на MRSA виникає у випадках спонтанного абсцесу чи целюліту або коли ураження не усуваються при рекомендованому початковому лікуванні антибіотиками. Топічна терапія є першою лінією лікування неускладнених небульозного та бульозного імпетиго. Якщо ураження обмежено невеликою площею (до 6 см2), то лікування передбачає розмочування кірочок, їхнє відшарування та нанесення антимік­робних мазей (фузидієва кислота або комбінація неоміцину з бацитрацином) (EBM Guidelines, 2018). При більш поширених ураженнях призначають системну антибіотикотерапію. У пацієнтів з екземою у лікуванні використовують топічні та системні антимікробні препарати із топічними стероїдами. Комбінація аміноглікозидного антибіотика неоміцину та поліпептидного антибіотика ацитрацину представлена на фармринку України препаратом «Банеоцин». Ця комбінація має виражений ефект синергізму по відношенню до S. aureus, S. pyogenes і P. aeryginosa. Важливою перевагою «Банеоцину» є наявність двох лікарських форм (порошок і мазь), що дає змогу використовувати цей препарат на різних стадіях інфекційного процесу.

The protective role of macrophages closely related to the production of bactericidal molecules, in which nitrogen monoxide (NO) play an important role. Arginine serves as a substrate for inducible NO synthase (iNOS) in course of NO production. Expression and activity of iNOS are regulated by the availability of the substrate (arginine) in the intercellular space. The bacterial enzyme arginine deiminase also uses arginine as a substrate, causing its deficiency for host cells. The aim of this study was to confirm the possible role of arginine deiminase from S. pyogenes in inhibiting NO synthesis by macrophages. For this purpose, a comparative study was made of the effect on the synthesis of NO by macrophages of the products of destruction of two strains: the initial S. pyogenes M49-16 and the isogenic mutant S. pyogenes M49-16 delArcA with the inactivated arginine deiminase gene (arcA). It has been shown that the ability of S. pyogenes M49-16 to inhibit production of NO by macrophages depends on its arginine deiminase activity because the isogenous mutant of S. pyogenes M49-16 delArcA with the inactivated gene arcA has lost its ability to inhibit NO synthesis. This allows us to consider the effects of S. pyogenes M49-16 as effects of arginine deiminase. An analysis of the inhibitory mechanisms of the enzyme showed that suppression of NO synthesis was not associated with the effect of destruction products of S. pyogenes M49-16 on the viability of macrophages. According to data of flow cytometry, incubation of cells in the presence of S. pyogenes destruction products of the original and mutant strains did not affect the level of iNOS expression, i.e. did not alter synthesis or stability of this enzyme. At the same time, the decrease in NO production under the influence of the original S. pyogenes strain M49-16 correlated with a decrease in the content of arginine in the culture medium. When exogenous arginine to the culture medium was added, the effect of the original strain of the suppression of NO production was declined. This confirms that the depletion of arginine is the main mechanism of the inhibitory effect of arginine deiminase on the production of NO by macrophages. The deficiency of NO production in the course of streptococcal infection can lead to a weakening of bactericidal activity of macrophages and to a decrease in the effectiveness of antimicrobial protection.

Streptococcal infection is characterized by a variety of manifestations from asymptomatic carriage of the pathogen to manifest forms. Recently, in addition to the bacteriological method for confirming streptococcal etiology, the rapid test for β-hemolytic streptococcus group A is increasingly being used. Isolation of streptococci does not always indicate their involvement in pathology, quite often a person is a healthy carrier of the pathogen. The share of carrier is 10—28% of cases. However, in the practical activities of a physician, positive tests for β -hemolytic streptococcus group A (rapid test or bacteriological culture) are often treated as acute streptococcal infection even in children without any clinical manifestations of acute tonsillopharyngitis and, as a result, antibiotic therapy is prescribed. For differential diagnosis, a correct evaluation of epidemiological and clinical data with a mandatory serological test — the determination of ASO in paired sera with an interval of 7—10 days is required. Absence of an increase in antibodies indicates carrier. b-hemolytic streptococcus group A carriers in most cases do not need antibiotic therapy. However, if there is a history or risk of developing rheumatic fever, acute poststreptococcal glomerulonephritis, antibiotic therapy is necessary. The authors proposed an algorithm for managing patients with the release of b-hemolytic streptococcus group A from the oropharynx.

Purpose There is an immense concern in the international community about controlling the outburst of infectious diseases. An essential step towards diminishing it is the development of an adequate detection system. Among the huge plethora of microorganisms which may infect the human body, Streptococcus pyogenes is important one which infects the upper respiratory tract leading to sore throat, which eventually develops into rheumatic heart disease (RHD) in the absence of timely treatment. A major process in controlling the infection is to detect it at an early stage. Hence, there is a need to develop detection tools which are both rapid and reliable. Design/methodology/approach Different types of diagnostic methods are available for identification, but the most commonly used are culturing, staining and rapid antigen detection tests. For better sensitivity and specificity, this review describes the development of biosensor. Compared with the current available methods, which are usually cumbersome, time-consuming and expensive, this approach features sequence specificity, cost efficiency, rapid and ease of use. Findings This review outlines various sensors which are available for the detection of Streptococcus pyogenes which causes human RHD. The working scheme of the sensors, their sensitivity and limitation of detection has been described in the review. Originality/value The review fulfills an acknowledged the need to study various sensors that are available for the detection of Streptococcus pyogenes, causing human RHD.

Introduction: Sickle cell disease (SCD) is a red blood cell disorder caused by a single nucleotide mutation in the β-globin gene (HBB). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but is available to only a minority of patients and can be associated with high morbidity and mortality. CRISPR/Cas9 mediated genome editing may provide a permanent cure for SCD patients by correcting the sickle mutation in HBB in hematopoietic stem and progenitor cells (HSPCs). Previously, we achieved ~39% sickle mutation correction in SCD HSPCs by delivering S. pyogenes (Spy) Cas9/R-66S gRNA as ribonucleoprotein (RNP) and single-stranded oligodeoxynucleotides (ssODN) corrective donor template. S. aureus (Sau) Cas9 has potentially advantageous properties to improve therapeutic gene editing efficiency and safety, including smaller size allowing for efficient in vivo delivery and longer Protospacer Adjacent Motif (PAM) sequence for higher specificity. However, although in general, the cutting efficiency of SauCas9 is lower than SpyCas9, the differences in gene correction and other gene-editing outcomes between SpyCas9 and SauCas9 have not been well studied. Methods: With our R-66S gRNA sequence targeting the sickle mutation, the PAM sequence of SauCas9 (NGGRRT) is mutually permissive with that of SpyCas9 (NGG), allowing the same sequence to be targeted by both Cas9 nucleases. We delivered R-66S gRNA with SpyCas9 and SauCas9 respectively as RNP, along with corrective ssODN donor template into SCD HSPCs. We analyzed sickle mutation correction rate and small insertions and deletions (INDELs) profile by Next Generation Sequencing (NGS). Results/discussions: We found that although the INDEL rate of SpyCas9 is higher than SauCas9 at the same molar concentration of RNP, SauCas9 gave 43% sickle mutation correction, slightly higher than SpyCas9 (39%), demonstrating efficient homology-directed repair (HDR) mediated gene correction by SauCas9. To further investigate the potential for clinical translation, we will perform in-depth efficiency and safety characterization comparing SauCas9 and SpyCas9 mediated sickle mutation correction therapy in SCD HSPCs. Conclusion: In this work, we showed that, compared with the highly-optimized and widely-used SpyCas9, SauCas9 leads to a higher sickle mutation correction in SCD HSPCs, demonstrating the therapeutic potential of SauCas9 for treating SCD. We will further investigate the efficiency and safety of gene-edited therapy mediated by these two Cas9 orthologs, including in-depth characterization of off-target effects, chromosomal rearrangement and aberrations, and large genomic modifications. We will differentiate gene-corrected SCD HSPCs to study erythropoiesis and red cell phenotype, including normal hemoglobin production and reduced sickling under hypoxic conditions. Lastly, we will evaluate the engraftment efficiency of gene-edited cells in Nonirradiated NOD,B6.SCID Il2rγ -/- Kit (W41/W41) (NBSGW) mice that support the engraftment of human hematopoietic stem cells. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

There is a growing body of data about the cytopathic effect of bacterial arginine deiminase on human endothelial cells, but the precise mechanisms of endothelial dysfunction caused by the activity of the enzyme remain poorly understood. Activity of arginine deiminase causes arginine depletion in the microenvironment of the host organism cells. In view that arginylation of beta-actin regulates actin cytoskeleton structure and cell motility, we proposed that the cytopathic effect of arginine deiminase may be associated with disruption of actin in the cytoskeleton of endothelial cells. The aim of this study was to investigate the effect of arginine deiminase from S. pyogenes on migration and actin cytoskeleton structure of the human endothelial cells, line EA.hy926. The supernatant of sonicated S. pyogenes M49-16, its isogenic mutant with a deletion of the arginine deiminase gene (S. pyogenes M49-16delAD), supernatant of sonicated S. pyogenes M22, and arginine deiminase isolated from the latter strain were used. The effect of bacterial factors on migration activity of endothelial cells was studied in the model of "wound healing" in vitro. To analyze the influence of bacterial factors on the actin cytoskeleton structure, cells were stained with phalloidin-rhodamine. It was shown that supernatants of destroyed S. pyogenes, as well as arginine deiminase significantly reduced the migration activity of endothelial cells and altered the structure of their actin cytoskeleton. The supernatants of destroyed S. pyogenes M49-16delAD with deleted gene of arginine deiminase showed a significantly reduced ability to suppress cell migration as compared with the supernatant of sonicated S. pyogenes M49-16. No significant differences were revealed in the structure of actin filaments in cells cultured in the presence of supernatants of destroyed S. pyogenes M19-16, and cells cultured in the presence of isogenic mutant S. pyogenes M4916delAD. Adding exogenous arginine to the cells cultured with supernatants of destroyed S. pyogenes did not restore their migratory activity and the structure of their actin cytoskeleton. However, if arginine deficiency caused by the activity of arginine deiminase was compensated, endothelial cells migration activity was restored, and the structure of actin cytoskeleton was recovered. A decrease of migration activity of endothelial cells under the influence of streptococcal arginine deiminase was due to the disruption of actin cytoskeleton structure.

ObjectivesThe aim of this study was to determine the asymptomatic pharyngeal carriage rate ofS. pyogenes, antimicrobial pattern and related risk factors among school children in Hawassa, southern Ethiopia.ResultsOut of 287 school children’s screened, 35 (12.2%) were colonized withS. pyogenes. The carriage rate was significantly associated with factors such as sex (female p = 0.013) occupational status of mother (p = 0.002), lower income source (500–900 ETB, 1000–1500 ETB) (p = 0.001, and p = 0.042), history of hospitalization (p = 0.00) and residence of the children (p = 0.002). High level resistant to tetracycline and low level to vancomycin were observed, while penicillin, amoxicillin, erythromycin, chloramphenicol, and ceftriaxone were found to be effective.

CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli. The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola. We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes., eLife digest Viruses that attack bacteria are known as bacteriophages, or phages for short. Bacteria have developed an antiviral immune system called CRISPR-Cas that works by targeting particular genetic sequences, such as those of an invading phage, for destruction. To counteract this immune system, phages have evolved proteins that can block CRISPR-Cas known as anti-CRISPRs. Researchers have studied the CRISPR-Cas bacterial defense systems intensively over the past decade but much less is known about anti-CRISPRs. For example, the natural diversity and prevalence of anti-CRISPRs is still unknown, and identifying these proteins has proven difficult. To address this gap, Forsberg et al. developed a technique to identify new anti-CRISPRs based on their ability to inhibit CRISPR-Cas activity. The method relies on three elements. First, a piece of DNA that lets bacteria resist a specific antibiotic. Second, a test piece of DNA that might code for an anti-CRISPR. Third, a CRISPR-Cas system designed to target and destroy the antibiotic resistance DNA. The three elements are put into bacteria, and two things can happen. If the ‘test DNA’ does not code for an anti-CRISPR, then the CRISPR-Cas system destroys the antibiotic resistance DNA and the bacteria die when exposed to the antibiotic. On the other hand, if the test DNA does code for an anti-CRISPR, it will inhibit the CRISPR-Cas system and the antibiotic resistance DNA will remain intact. This means that the bacteria will survive when grown in the antibiotic, and new anti-CRISPRs can be found by examining the test DNA in those bacteria. Forsberg et al. employed this strategy to screen a huge library of DNA pieces, uncovering several new anti-CRISPRs. They then focused on an anti-CRISPR that was very common in the human gut called AcrIIA11. Biochemical characterization showed that AcrIIA11 inhibited CRISPR-Cas via a different mechanism from other known anti-CRISPRs. Moreover, it could inhibit CRISPR-Cas systems from many different bacteria. The potential to systematically identify anti-CRISPRs able to resist any bacterium’s CRISPR-Cas defense system could lead to the design of phages that can infect bacteria which are otherwise difficult to destroy. In the future, these phages could be used to clear antibiotic-resistant infections. Beyond its role as an antiviral system in bacteria, CRISPR-Cas is a widely used tool for genetic modification in biomedical research. Using anti-CRISPRs to regulate where, how, and when CRISPR-Cas systems act could make their many emerging applications safer and more effective.

Objective: To collect the most up‐to‐date information regarding pediatric osteoarticular infections, including the epidemiological and microbiological profiles, diagnosis, and treatment. Source of data: A non‐systematic review was performed on the search engines PubMed, SciELO, Lilacs, and Google Scholar, using the keywords “bone and joint infection”, “children”, “pediatric”, “osteomyelitis”, “septic arthritis” and “spondylodiscitis” over the last ten years. The most relevant articles were selected by the authors to constitute the database. Synthesis of data: Osteoarticular infections are still a major cause of morbidity in pediatrics. Their main etiology is Staphylococcus aureus, but there has been an increase in the detection of Kingella kingae, especially through molecular methods. Microbiological identification allows treatment direction, while evidence of inflammatory activity assists in treatment follow‐up. Imaging tests are especially useful in the initial diagnosis of infections. Empirical treatment should include coverage for the main microorganisms according to the age and clinical conditions of the patient, while considering the local resistance profile. Surgical procedures can be indicated for diagnosis, focus control, and function preservation. Acute complications include sepsis, deep venous thrombosis, and pulmonary embolism. Deaths are rare. Late complications are uncommon but may lead to deformities that compromise motor development. Conclusion: A correct and early diagnosis, prompt implementation of adequate antimicrobial therapy, and focus control, when indicated, are critical to a better prognosis. Resumo: Objetivo: Compilar as informações mais atuais referentes às infecções ostoarticulares em pediatria, inclusive perfil epidemiológico e microbiológico, diagnóstico e tratamento. Fonte dos dados: Feita revisão não sistemática nos mecanismos de busca Pubmed, Scielo, Lilacs e Google Scholar, com as palavras‐chave bone and joint infection, children, pediatric, osteomyelitis, septic arthritis e espondylodiscitis nos últimos 10 anos. Os artigos mais relevantes foram selecionados pelos autores para compor a base de dados. Síntese dos dados: As infecções osteoarticulares ainda são causa importante de morbidade na pediatria. A sua principal etiologia é o Staphylococcus aureus, porém há um aumento na detecção de Kingella kingae, especialmente através de métodos moleculares. A identificação microbiológica possibilita direcionamento de tratamento, enquanto que as provas de atividade inflamatória auxiliam no acompanhamento do tratamento. Exames de imagem são especialmente úteis no diagnóstico inicial das infecções. O tratamento empírico deve incluir cobertura para os principais microrganismos, de acordo com a faixa etária e as condições clínicas do paciente, considerando o perfil de resistência local. Procedimentos cirúrgicos podem ser indicados para diagnóstico, controle do foco e preservação da função. As complicações agudas incluem sepse, trombose venosa profunda e embolia pulmonar. Óbitos são raros. As complicações tardias são incomuns, mas podem levar a deformidades que comprometem o desenvolvimento motor. Conclusão: O diagnóstico correto e precoce, com pronta instituição de terapia antimicrobiana adequada e controle do foco, quando indicado, é fundamental para um melhor prognóstico. Keywords: Osteoarticular, Infections, Children, S. aureus, S. pyogenes, Palavras‐chave: Osteoarticular, Infecções, Crianças, S. aureus, S. pyogenes

We are writing to reply to the comment by Pouwels et al., 2019 about our recent study (Olesen et al., 2018) on antibiotic use and antibiotic resistance.

We are writing to reply to the comment by Pouwels et al., 2019 about our recent study (Olesen et al., 2018) on antibiotic use and antibiotic resistance.

This protocol outlines the steps to edit single point mutations into cells in a scarless manner. Uses single-stranded DNA repair templates and the S. pyogenes CRISPR/Cas9 system. Using a short single-stranded DNA repair template is advantageous because it is easy to order synthetically and it cannot be randomly integrated into other double-strand breaks in the cell. Furthermore, this protocol is "scarless" - that is, nothing is changed to the genome other than the desired changes. However, it may be easier to edit genes using a selectable marker, especially when knocking out a gene. This protocol is ideal when trying to make genetic changes with minimal perturbations to the gene locus and surroundings. Note that repair using a single-stranded short repair template (SSTR) uses a different DNA repair pathway than homology-directed repair from long DNA repair templates. Citations: image 1: synthego.com- verify your guide design tool

Antibiotic use is a primary driver of antibiotic resistance. However, antibiotic use can be distributed in different ways in a population, and the association between the distribution of use and antibiotic resistance has not been explored. Here we tested the hypothesis that repeated use of antibiotics has a stronger association with population-wide antibiotic resistance than broadly- distributed, low-intensity use. First, we characterized the distribution of outpatient antibiotic use across US states, finding that antibiotic use is uneven and that repeated use of antibiotics makes up a minority of antibiotic use. Second, we compared antibiotic use with resistance for 72 pathogen-antibiotic combinations across states. Finally, having partitioned total use into extensive and intensive margins, we found that intense use had a weaker association with resistance than extensive use. If the use-resistance relationship is causal, these results suggest that reducing total use and selection intensity will require reducing broadly-distributed, low- intensity use.

Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV–Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5.

Arginine deiminase is a bacterial enzyme that hydrolyses arginine with citrulline and ammonia formation. In recent years, increasing evidence is reported about in vitro and in vivo anti-angiogenic action of arginine deiminase from Mycoplasma spp. Our studies have shown that arginine deiminase from Streptococcus pyogenes M22 exerts similar effects, i.e., inhibits proliferation and other endothelial cell functions related to angiogenesis. To confirm a leading role of arginine deiminase, as a factor responsible for the anti-proliferative effect, we have constructed an isogenic S. pyogenes M49-16 mutant unable to express arginine deiminase. A comparative analysis of anti-proliferative activity of original S. pyogenes M49-16 strain and its isogenic mutant with arginine deiminase gene deletion (M49-16delAD) was performed, using an endothelial EA.hy926 cell line. The bacterial supernatantes obtained by sonication of S. pyogenes M49-16 and M49-16delAD were tested. The ability of S. pyogenes M49-16 and M49-16delAD supernatantes to hydrolyze arginine was assessed. Moreover, we compared effects of the Streptococcus supernatantes upon proliferative activity of endothelial cells and their distribution through the cell cycle phases.Supernatantes from original S. pyogenes 49-16 strain were shown to inhibit endothelial cell proliferation to a significant degree (down to 50% of controls). This effect was due to its arginine hydrolyzing activity, i. e. addition of exogenous arginine to the medium resulted into recovery of the cell proliferation levels. The supernatante from S. pyogenes M49-16delAD showed a lower ability to hydrolyze arginine as compared to the supernatante of original strain. Culturing of endothelial cells supplied with S. pyogenes M49-16delAD supernatantes resulted into reduction of their proliferative activity by 10% of control values. Analysis of the cell cycle distribution was concordant with these results. S. pyogenes M49-16 supernatante caused a decrease in S-phase cell fraction by 20% against controls. With a supernatants from S. pyogenes M49-16delAD, such drop in DNA-synthesizing cell ratio was significantly weaker (by only 5% of the control). These results reveal new pathogenetic mechanisms of endothelial dysfunction during streptococcal infection and suggest anti-angiogenic potential of streptococcal arginine deiminase.

Cas9 Nuclease,S. pyogenes, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNAin vitrousing Cas9 and a single guide RNA (sgRNA).

A protocol to in vitro transcribe sgRNAs from oligos for S. pyogenes Cas9.

Group A Streptococcus (GAS) remains one of the top 10 deadliest human pathogens worldwide despite its sensitivity to penicillin. Although the most common GAS infection is pharyngitis (strep throat), it also causes life-threatening systemic infections. A series of complex networks between host and pathogen drive invasive infections, which have not been comprehensively mapped. Attempting to map these interactions, we examined organ-level protein dynamics using a mouse model of systemic GAS infection. We quantified over 11,000 proteins, defining organ-specific markers for all analyzed tissues. From this analysis, an atlas of dynamically regulated proteins and pathways was constructed. Through statistical methods, we narrowed organ-specific markers of infection to 34 from the defined atlas. We show these markers are trackable in blood of infected mice, and a subset has been observed in plasma samples from GAS-infected clinical patients. This proteomics-based strategy provides insight into host defense responses, establishes potentially useful targets for therapeutic intervention, and presents biomarkers for determining affected organs during bacterial infection.

Although mef(A) and its subclass mef(E) genes have long been considered to play a central role in macrolide efflux-based resistance, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A) gene plays a predominant role in Streptococcus pyogenes macrolide resistance. The mef(A) and mef(E) genes are carried by different genetic elements and the resistance associated with mef(A) was reported to be higher than that associated with mef(E); therefore, we further investigated the functional relevance of mef(A)/mef(E) and its associated msr(D). We established additional mef(A)-, mef(E)-, and their associated msr(D)-knockout strains and confirmed the predominance of msr(D) over mef(A)/mef(E). In addition, we performed experiments introducing mef(A), mef(E), and their associated msr(D) genes into mef(A)/mef(E)-msr(D) double-knockout and mef(A)/mef(E) negative strains. Neither mef(A) nor mef(E) genes had effects on erythromycin resistance. However, both associated msr(D) showed significant effects, and the mef(A)-associated msr(D) exhibited more effect than the mef(E)-associated one. These results suggest that an overall functional predominance of msr(D) over mef(A)/mef(E) is conceivable in efflux-based macrolide resistance in at least some S. pyogenes strains. Furthermore, the higher resistance of mef(A) system over mef(E) system could be derived at least in part from functional differences of mef(A)- and mef(E)-associated msr(D).

Yanlış veya gereksiz antibiyotik kullanımı nedeni ile antibiyotik dirençliliği kazanmış patojen bakteriler tüm dünya için bir tehdit oluşturmaktadır. S. pyogenes, penisiline duyarlıdır ve günümüzde S. pyogenes enfeksiyonlarının tedavisinde ilk aşamada kullanılmaktadır. Penisiline direnç rapor edilmemiştir, ancak penisilin alerjisi olan kişilerde kullanılan makrolid antibiyotiklere dirençli izolatların sayısı giderek artmaktadır. Bu sorunun çözümü için potansiyel ilaç moleküllerinin elde edilmesi amacı ile S. pyogenes’in virulans mekanizmasında rol oynayan SpyCEP enzimi hedef olarak seçildi. DNA aptamerlerinin geliştirilmesi amacı ile SELEX yöntemi kullanılarak SpyCEP enzimine bağlanan ve inhibe eden DNA aptamerleri geliştirildi., Pathogenic bacteria that have acquired antibiotic resistance due to incorrect or unnecessary use of antibiotics pose a threat to the whole world. S. pyogenes is penicillin sensitive and penicillin is currently used in the first step in the treatment of S. pyogenes infections. Penicillin resistance has not been reported, but the number of macrolide antibiotic resistant isolates used in people with penicillin allergy is increasing. To solve this problem, SpyCEP enzyme, which plays a role in the virulence mechanism of S. pyogenes, was selected as a target for obtaining potential drug molecules. DNA aptamers were developed using the SELEX method to develop DNA aptamers that bind to and inhibit the enzyme SpyCEP.

The secreted Streptococcus pyogenes cysteine protease SpeB is implicated in host immune system evasion and bacterial virulence. We present a small molecule inhibitor of SpeB 2477 identified from a high-throughput screen based on the hydrolysis of a fluorogenic peptide substrate Ac-AIK-AMC. 2477 inhibits other SpeB-related proteases but not human caspase-3, suggesting that the molecule targets proteases with the papain-like structural fold. A 1.59 Å X-ray crystal structure of 2477 bound to the SpeB active site reveals the mechanism of inhibition and the essential constituents of 2477 necessary for binding. An assessment against a panel of 2477 derivatives confirms our structural findings and shows that a carbamate and nitrile on 2477 are required for SpeB inhibition, as these moieties provide an extensive network of electrostatic and hydrogen-bonding interactions with SpeB active site residues. Surprisingly, despite 2477 having a reduced inhibitory potential against papain, the majority of 2477-related compounds inhibit papain to a much greater and broader extent than SpeB. These findings indicate that SpeB is more stringently selective than papain for this panel of small molecule inhibitors. On the basis of our structural and biochemical characterization, we propose modifications to 2477 for subsequent rounds of inhibitor design that will impart specificity to SpeB over other papain-like proteases, including alterations of the compound to exploit the differences in CA protease active site pocket sizes and electrostatics.

article

In this review two aspects dealt with Streptococcus pyogenes — one of the leading agent responsible for infectious diseases and another related to their complications in humans worldwide — are given. In the first part of the review the comparative evaluation of laboratory diagnostic approaches and methods used in the second half of the twentieth century and molecular technologies developed during last twenty years are described. In the second part the role of the main microbial pathogenic factors as well as the data on intra- and interspecies genetic exchange with extrachromosomal genetic elements and their influence on biological properties of the pathogen are discussed. Essential for today possibilities for molecular epidemiology of streptococcal pathology approaches must be introduces in diagnostic laboratories within the country.

The Streptococcus pyogenes phospholipase A2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes, which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the ΔslaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.

RNA-guided endonucleases (RGENs) have invigorated the field of site-specific nucleases. The success of Streptococcus pyogenes Cas9 (SpCas9) has led to the discovery of several other CRISPR-associated RGENs. As more RGENs become available, it will be necessary to refine their activity before they can be translated into the clinic. With this in mind, we sought to demonstrate how deep mutational scanning (DMS) could provide details about important functional regions in SpCas9 and speed engineering efforts. Consequently, we developed a nuclease screening platform which could distinguish active Cas9 mutants. We screened a library of 1.9 × 107 with over 8500 possible non-synonymous mutations and inferred the effects of each mutation using DMS. We demonstrate that the RuvC and HNH domains are the least tolerant regions to mutation. In contrast, the Rec2 and PI domains tolerate mutation better than other regions. The mutation information defined in this work provides a foundation for further SpCas9 engineering. Together, our results demonstrate how DMS can be a powerful tool to uncover features important to RGEN function. Application of this approach to emerging RGENs should enhance their engineering and optimization for therapeutic and other applications.

Cas9 Nuclease, S. pyogenes (Cas9), is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA).

The RNA degradosome is a highly structured protein complex responsible for bulk RNA decay in bacteria. The main components of the complex, ribonucleases, an RNA helicase, and glycolytic enzymes are well-conserved in bacteria. Some components of the degradosome are essential for growth and the disruption of degradosome formation causes slower growth, indicating that this complex is required for proper cellular function. The study of the Escherichia coli degradosome has been performed extensively for the last several decades and has revealed detailed information on its structure and function. On the contrary, the Gram-positive bacterial degradosome, which contains ribonucleases different from the E. coli one, has been studied only recently. Studies on the Gram-positive degradosome revealed that its major component RNase Y was necessary for the full virulence of medically important Gram-positive bacterial pathogens, suggesting that it could be a target of antimicrobial therapy. This review describes the structures and function of Gram-positive bacterial RNA degradosomes, especially those of a Gram-positive model organism Bacillus subtilis, and two important Gram-positive pathogens, Staphylococcus aureus and Streptococcus pyogenes.

Bacteria of the genus Streptococcus are common asymptomatic colonisers of humans and animals. As opportunistic pathogens they can however, depending on their host’s immune status and other circumstances, cause mild to very severe infections. Streptococci are highly intertwined with specific host species, but can also cause zoonosis or anthroponosis in more uncommon hosts. Prolonged and reoccurring infections require immune evasion strategies to circumvent detection and eradication by the host’s immune defence. A substantial part of the immune defence against bacterial pathogens is mediated by immunoglobulins. This thesis is based on work to identify and characterise immunoglobulin degrading enzymes secreted by different Streptococcus species as a means to sabotage and evade antibody-mediated immune responses. Stoichiometric and kinetic analysis of the IgG degrading enzyme IdeS from the important human pathogen S. pyogenes revealed that IdeS cleaves IgG, opposed to previous publications, as a monomer following classical Michaelis-Menten kinetics. The IdeS homologue of S. suis, IdeSsuis, did however not cleave IgG, but was highly specific fo rporcine IgM. S. suis was found to possess yet another protease, IgdE, capable of cleaving porcine IgG. Both of these proteases were shown to promote increased bacterial survival in porcine blood during certain conditions. IgdE is the founding member of a novel cysteine protease family (C113). Novel streptococcal members of this protease family were shown to specifically degrade certain IgG subtypes of the respective Streptococcus species’ main host. The observed substrate specificity of IgdE family proteases reflects the host tropism of these Streptococcus species, thereby giving insights into host-pathogen co-evolution. The abundance of immunoglobulin degrading enzymes among Streptococcus species indicates the importance of evasion from the antibody mediated immune responses for streptococci. These novel identified immunoglobulin degrading enzymes of the IdeS and IgdE protease families are potential valid vaccine targets and could also be of biotechnological use.

International audience; A newly developed MLVA seven-loci scheme for Streptococcus pyogenes is described. The method can be successfully applied by using both agarose gel with visual inspections of bands and Lab on Chip technology. The potential of the present MLVA has been tested on a collection of 100 clinical GAS strains representing the most common emm types found in high-income countries plus 18 published gap-free genomes, in comparison to PFGE and MLST. The MLVA analysis defined 30 MLVA types with ten out of the considered 15 emm types exhibiting multiple and specific MLVA types. In only one occasion the same MLVA profile was shared between isolates belonging to two different emm types. A robust congruency between the methods was observed, with MLVA discriminating within clonal complexes as defined by PFGE or MLST. This new MLVA scheme can be adopted as a quick, low-cost and reliable typing method to track the short-term diffusion of GAS clones in inter-laboratory-based surveillance.

PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001, eLife digest Streptococcus pyogenes is the bacterium that causes throat infections and other serious infections in humans. Antibiotics such as penicillin are used to treat active infections, but so-called “strep throat infections” often return after treatment. This is because S. pyogenes can enter the cells that line the throat and hide from the antibiotics, which cannot enter the throat cells. Endolysins are enzymes produced by viruses that attack bacteria, and these enzymes target and destroy the bacterial cell wall. A previous study revealed that an endolysin known as PlyC could destroy S. pyogenes bacteria on contact. PlyC and other endolysins have the potential to act as alternatives to common antibiotics, but before these enzymes can be developed as therapeutics, it is important to understand how they interact with human host cells. Like antibiotics, the PlyC endolysin was not expected to enter throat cells. However, Shen, Barros et al. have now discovered that not only can PlyC enter throat cells, it can essentially chase down and kill S. pyogenes that are hiding inside. Other similar enzymes could not act in this way, and further studies confirmed that PlyC could move around inside a throat cell without causing it damage. Shen, Barros et al. also determined that PlyC has a pocket on its surface that binds with a specific component of the throat cell membrane, a molecule called phosphatidylserine. This interaction – which is a bit like a lock and key – grants PlyC access into the cell. While it is clear that PlyC eventually kills S. pyogenes hiding inside throat cells, future experiments will aim to determine how PlyC moves around once inside an infected throat cell. Together, an understanding of how an endolysin enters cells and destroys hiding S. pyogenes will contribute to the development of endolysins with broader activity, which can be used as alternatives to common antibiotics. DOI: http://dx.doi.org/10.7554/eLife.13152.002