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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.

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.

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.

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).

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.

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.

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.

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.

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.

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).

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.

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).

Streptococcus pyogenes (group A streptococci, GAS) is an exclusive human bacterial pathogen. The virulence potential of this species is tremendous. Interactions with humans range from asymptomatic carriage over mild and superficial infections of skin and mucosal membranes up to systemic purulent toxic-invasive disease manifestations. Particularly the latter are a severe threat for predisposed patients and lead to significant death tolls worldwide. This places GAS among the most important Gram-positive bacterial pathogens. Many recent reviews have highlighted the GAS repertoire of virulence factors, regulators and regulatory circuits/networks that enable GAS to colonize the host and to deal with all levels of the host immune defense. This covers in vitro and in vivo studies, including animal infection studies based on mice and more relevant, macaque monkeys. It is now appreciated that GAS, like many other bacterial species, do not necessarily exclusively live in a planktonic lifestyle. GAS is capable of microcolony and biofilm formation on host cells and tissues. We are now beginning to understand that this feature significantly contributes to GAS pathogenesis. In this review we will discuss the current knowledge on GAS biofilm formation, the biofilm-phenotype associated virulence factors, regulatory aspects of biofilm formation, the clinical relevance, and finally contemporary treatment regimens and future treatment options.

Eine 23-jahrige Patientin stellte sich wegen seit einigen Tagen bestehenden linksthorakalen Schmerzen und Atemnot in unserer Notfallaufnahme vor. Tage zuvor wurde der hausarztliche Verdacht einer vertebrogenen Schmerzsymptomatik geausert, und der Patientin nichtsteroidale Antirheumatika (NSAR) verschrieben. Zur beschriebenen Symptomatik trat zusatzlich eine Tachykardie (HF: 100 bpm), sowie eine Hypotonie (90/50 mmHg). Bei Zustandverschlechterung musste die Patientin schlieslich intubiert, prolongiert reanimiert werden und war darauffolgend hochgradig katecholaminpflichtig. Ursachlich konnte ein linksseitiges Pleuraempyem gesichert werden, in welchem β-hamolysierende Streptokokken (S. pyogenes) nachweisbar waren. Eine Tonsillopharyngitis konnte klinisch ausgeschlossen werden. Der gynakologische Status war unauffallig. Beide Knie der Patientin hingegen waren nach einem Handballtraining wund erodiert, so dass wir diesen Befund als Eintrittspforte vermuteten. Aufgrund der fulminaten Zustandverschlechterung, bedingt durch einen septischen Schock und Multiorgandysfunktionssyndrom (MODS), einhergehend mit einer blasenbildenden Hautveranderungen und dem Nachweis von S. pyogenes wurde der klinische Verdacht auf ein Streptokokken- Toxic-Shock-Syndrom (STSS) geausert.

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).

Spider bites are a frequent complaint, often prompting patients to seek medical attention. 1 However, the diagnosis of a spider bite is frequently inferred by the patient, and a thorough evaluation subsequently reveals an alternate diagnosis. Gertsch et al demonstrated that of 600 consecutive “spider bite” cases, 80% were incorrectly diagnosed. 2 Alternative diagnoses included bites by arthropods other than spiders, 2 bacterial, viral or fungal infections 3 and malignant tumours. 3 In this study, we describe the case of a patient who was initially misdiagnosed as having been bitten by a spider. Later, the patient was demonstrated to have a spontaneous, soft tissue Staphylococcus aureus infection. We further describe the local epidemiology of cases presenting with spider bites and the subsequent microbiological findings.

Antimicrobial resistance is a growing problem among pathogens from respiratory tract infections. b-Lactam resistance rates are escalating among Streptococcus pneumoniae and Haemophilus influenzae. Macrolides are increasingly used for the treatment of respiratory tract infections, but their utility is compromised by intrinsic and acquired resistance. This article analyses macrolide-resistance mechanisms and their worldwide distributions in S pneumoniae, S pyogenes, and H influenzae.

Objective. Our objective was to confirm an observed temporal association of increased numbers of rheumatic fever cases concomitant with the appearance of an increased prevalence of mucoid strains of Streptococcus pyogenes. During the resurgence of rheumatic fever (RF) that has occurred in the Intermountain area surrounding Salt Lake City, Utah, since 1985, the largest number of cases occurred in 1985 and 1986 and 12 years later in 1997 and 1998. During the initial outbreak and the later exacerbation of the resurgence, an increased number of mucoid strains of S pyogenes were present in the community.Methods. The referred cases of RF that fulfilled Jones criteria have been systematically reviewed by the medical staff at Primary Children’s Medical Center yearly since 1985. Before the resurgence of RF, a program was initiated by the microbiology laboratory at Primary Children’s Medical Center to store frozen isolates of S pyogenes. All frozen specimens were randomly selected and entered into a log; the coded entry allowed for comments regarding the origin of the isolate and whether the isolate had a mucoid appearance on the blood agar culture plate. This log was reviewed in October 2002 to determine whether the percentage of frozen mucoid isolates stored during the resurgence of RF would support the clinical and epidemiologic suspicion that mucoid isolates seemed to be present with a higher frequency during the 2 periods of high incidence of RF. The percentage of mucoid isolates was compared with the yearly number of cases of RF. A Pearson r correlation analysis was completed to determine whether there was a significant association between the percentage of mucoid isolates and the number of cases of RF.Results. The highest number of cases of RF was temporally associated with the highest percentage of mucoid isolates. There was statistically significant correlation between percentage of mucoid strains and the number of RF cases.Conclusions. The Utah experience with the resurgence of RF in a civilian population during the last decade and a half of the 20th century confirmed the temporal association of mucoid strains of S pyogenes, primarily M-type 18, with a high incidence of RF.

En los últimos años se ha producido un aumento significativo en la prevalencia de resistencias de los principales patógenos respiratorios a la mayoría de los antibióticos utilizados en el ámbito extrahospitalario que ha comprometido en muchos casos el tratamiento empírico de algunas infecciones respiratorias, siendo de especial interés el progresivo aumento de las tasas de resistencia deStreptococcus pneumoniae a macrólidos y penicilina y deS. pyogenes a macrólidosVarios estudios han confirmado la asociación entre consumo de antibióticos y desarrollo de resistencia en S. pneumoniae y S. pyogenes. Sin embargo, ni todos los antibióticos tienen la misma capacidad de selección de resistencias, ni todos se ven afectados de la misma maneraEl objetivo del tratamiento antimicrobiano en las infecciones del tracto respiratorio es la erradicación bacteriológica. Los parámetros farmacodinámicos pueden utilizarse para predecir la erradicación bacteriológica de los antimicrobianos : During the last few years, increasing antibiotic resistance amongst the major respiratory pathogens in the community has compromised the choice of empirical therapy for some respiratory tract infections. Of special interest has been the progressive increase in the resistance rates of Streptococcus pneumoniaeto macrolides and penicillin, and of S. pyogenes to macrolidesSeveral studies have confirmed the association between community use of certain antibiotics and the development of resistance inS. pneumoniae and S. pyogenes. Nevertheless, not all the antibiotics have the same ability to select resistance and not all microorganisms are affected in the same wayThe aim of antimicrobial therapy in respiratory tract infections is bacterial eradication. Pharmacokinetic and pharmacodynamics can be used to predict bacteriological eradication with antimicrobial therapy

A Streptococcus pyogenes clade that emerged in Portugal during the past decade quickly outcompeted several other locally circulating lineages of this pathogen, according to Mario Ramirez and his collaborators at the University of Lisbon in Portugal. What happened in Portugal also apparently took place elsewhere in Northern Europe and in the United States, they note. “Taken together, these observations suggest that, in spite of the differences in the structures of the S. pyogenes emm89 populations previously circulating in different countries, the same clade disseminated and outcompeted other emm89 lineages over the first decade of the 21st century.” These findings carry implications for the design of diagnostic tests and vaccines aimed at detecting and controlling these pathogens. Details appeared 1 December 2015 in mBio (doi:10.1128/mBio.01780–15).

The objective of this study was to consider the invasive properties of Streptococcus pyogenes in human pharyngeal epithelial cells, and to correlate these with their clinical significance. Clinical isolates of S. pyogenes obtained from blood cultures over a period of 10 years, and throat and skin isolates from a community-based study, were used in this investigation. The S. pyogenes isolates were inoculated in HEp-2 cells and subsequently treated with antibiotics to kill the extracellular bacteria. The cells were then lyzed, and a colony count was carried out to check for invasion. The throat and skin isolates had 45.7%, 25.7% and 28.5% of low, intermediate and high invasion efficiencies, respectively, while 80%, 8.6% and 11.4% of the blood isolates had low, intermediate and high invasion efficiencies. We concluded that the throat and the skin isolates from superficial infections were more invasive than the blood isolates, which is an interesting and paradoxical feature.

Resume Objectif : Apprecier l’activite de la levofloxacine sur les streptocoques et pneumocoques Methode : Mille deux cent cinquante cinq souches de pneumocoques (1015) et streptocoques (240) ont ete isolees (octobre 1999 a mars 2000), dans 46 laboratoires (35 hospitaliers, 11 liberaux). Un antibiogramme (diffusion en gelose) a ete realise pour chacune ainsi que des CMI (Etests) pour penicilline G, amoxicilline, cefotaxime, cefpirome, cefpodoxime et cefuroxime. Les CMI de la levofloxacine ont ete determinees sur un echantillon representatif de souches de Streptococcus pneumoniae sensibles et sur toutes les souches resistantes a l’antibiogramme. Resultats : 31,1 % et 41,3 % des souches de S. pneumoniae etaient sensibles a la penicilline G, 57,4 % et 34 % intermediaires, 11,5 % et 24,7 % resistantes, respectivement dans les laboratoires liberaux et hospitaliers. La sensibilite a la levofloxacine atteignait 100 % (liberaux) et 98,2 % (hopitaux), et a l’erythromycine 21,6 % (ville) et 41,2 % (hopital). Les plus forts taux de souches non sensibles a l’erythromycine retrouves en ville, 64,2 % versus 26,5 %, refletaient la predominance de souches isolees chez des enfants, en ville. L’activite de la penicilline G sur Streptococcus pyogenes etait de 100 %, celle des macrolides de 93,3 %. La sensibilite a la levofloxacine de S. pyogenes etait de 97,8 %. Les streptocoques non groupables etaient moins sensibles puisque seuls 73,5 % et 47 % etaient respectivement sensibles a la penicilline G et aux macrolides ; l’activite de la levofloxacine atteingnait 95,4 %. Conclusion : Cette etude montre la bonne activite de la levofloxacine vis-a-vis de S. pneumoniae a l’hopital et en ville.

Francesco Di Pierro,1 Maria Colombo,2 Alberto Zanvit,3 Paolo Risso,4 Amilcare S Rottoli5 1Scientific Department, Velleja Research, Milan, 2Pediatric Department, University of Parma, Parma, 3Stomatology Institute, Milan, 4Laboratory of Epidemiology and Social Psychiatry, Mario Negri Institute, Milan, 5Pediatric Department, Uboldo Hospital, Cernusco sul Naviglio, Italy Background: Streptococcus salivarius K12 is an oral probiotic strain releasing two lantibiotics (salivaricin A2 and salivaricin B) that antagonize the growth of S. pyogenes, the most important bacterial cause of pharyngeal infections in humans also affected by episodes of acute otitis media. S. salivarius K12 successfully colonizes the oral cavity, and is endowed with an excellent safety profile. We tested its preventive role in reducing the incidence of both streptococcal and viral pharyngitis and/or tonsillitis in children. Materials and methods: We enrolled 61 children with a diagnosis of recurrent oral streptococcal disorders. Thirty-one of them were enrolled to be treated daily for 90 days with a slow-release tablet for oral use, containing no less than 1 billion colony-forming units/tablet of S. salivarius K12 (Bactoblis®), and the remaining 30 served as the untreated control group. During treatment, they were all examined for streptococcal infection. Twenty children (ten per group) were also assessed in terms of viral infection. Secondary end points in both groups were the number of days under antibiotic and antipyretic therapy and the number of days off school (children) and off work (parents). Results: The 30 children who completed the 90-day trial with Bactoblis® showed a significant reduction in their episodes of streptococcal pharyngeal infection (>90%), as calculated by comparing the infection rates of the previous year. No difference was observed in the control group. The treated group showed a significant decrease in the incidence (80%) of oral viral infections. Again, there was no difference in the control group. With regard to secondary end points, the number of days under antibiotic treatment of the treated and control groups were 30 and 900 respectively, days under antipyretic treatment 16 and 228, days of absence from school 16 and 228, and days of absence from work 16 and 228. The product was well tolerated by the subjects, with no side effects, and only one individual reported bad product palatability and dropped out. Conclusion: Prophylactic administration of S. salivarius K12 to children with a history of recurrent oral streptococcal disease resulted in a considerable reduction of episodes of both streptococcal and viral infections and reduced the number of days under antibiotic and/or antipyretic therapy and days of absence from school or work. Keywords: Blis K12, pediatric trial, Bactoblis, S. pyogenes, antibiotic therapy

This interactive small-group session integrates microbiology of the respiratory tract with pharmacology (antimicrobials). It is intended as a review following lectures or interactive sessions in basic microbiology, respiratory microbiology, and antimicrobials. This integrated case-based microbiology and pharmacology session reinforces the basic concepts of respiratory microbiology with basic concepts of antimicrobial therapy. The cases require students to recall clinical presentations of various microbes, microbiology laboratory tests, and appropriate antimicrobials for treatment of specific pathogens. During the inaugural year of our curriculum, the students scored lower than anticipated on the microbiology and pharmacology sections of the respiratory course final exam. During the second year of our curriculum we aimed to remedy this and strengthen both disciplines with additional microbiology and pharmacology sessions, such as this. A comparison of final exam scores from the inaugural year and the second year of the respiratory course demonstrated a rise in scores for microbiology- and antimicrobial (pharmacology)-related questions.

Resume Lors d'un essai clinique randomise ouvert, nous avons etudie la cinetique d'eradication de Streptococcus pyogenes chez 48 patients presentant une angine aigue traitee per os par 10 jours de penicilline V ou 5 jours de josamycine. Des prelevements oropharynges ont ete pratiques quotidiennement chez chaque patient. Les deux antibiotiques ont permis une reduction de 3 ou 4 log du taux initial de S. pyogenes apres 48 heures de traitement, la plupart des patients presentant alors une culture negative. Les prelevements realises 48 heures apres la fin du traitement ont confirme l'eradication de S. pyogenes . Des controles realises a 30 jours puis a 3 ou 4 mois ont montre que certains patients s'etaient a nouveau colonises, certains S. pyogenes isoles ne presentant pas le meme serotype M que celui retrouve lors de l'episode initial aigu. Cette etude souleve le probleme du role de l'entourage dans la recolonisation des patients ainsi que la valeur des prelevements a distance, 3 a 6 semaines apres la fin du traitement antibiotique.