Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

We report a Group A streptococcal (GAS) outbreak in a geriatric mental health in-patient unit. Communication with cognitively impaired patients, limitations in adherence to hygiene practices and communal dining may have facilitated transmission. Settle plates aided in identifying a colonized patient. Rapid access to whole genome sequencing facilitated assessment and management.

BACKGROUND: Silica-sprayed tubes (SSTs) are often used to transport synovial fluid samples in equine practice. They promote the coagulation of the sample. The objective of the study is to evaluate the effect of SST on bacterial culture.
METHODS: The study was divided into two parts: sterile saline (Part A) and synovial fluid (Part B). Four common bacteria associated with equine synovial sepsis were used: Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Three collection tubes were used: STT, plain (no-additives) and brain and heart infusion (BHI) broth. Bacteria were cultured in horse blood agar plates for 48 h. Outcome variables were negative culture, positive culture and total number of colony-forming units (CFUs). Statistical analysis was performed using Mann-Whitney U test, and significance was set at p < 0.05.
RESULTS: The total number of agar plates read was 1557 (779 saline; 778 synovial fluid). Total negative cultures were 25/779 on saline and 3/778 on synovial fluid. In broth, maximum growth CFU was achieved after 8 h for both saline and synovial fluid for all bacteria. S. pyogenesand E. coli produced a significantly lower number of CFU when in SST compared to plain or broth after 4 h, whereas S. aureus (American Type Culture Collection [ATCC] and MRSA) only after 24 h.
CONCLUSIONS: Silica-containing tubes reduced bacterial proliferation, whereas the use of a BHI broth provided the highest bacterial load in the sample. The use of SST may have a negative effect on bacterial proliferation in samples obtained from clinical cases.

Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

暂无摘要。

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP-binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP, and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to identification of amino acids that are crucial for C4BP-binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

UNASSIGNED: Scarlet fever is an infectious disease caused by Streptococcus pyogenes. However, there is limited data regarding the disease in the Arab World, including the United Arab Emirates.
UNASSIGNED: To analyse a scarlet fever outbreak in United Arab Emirates.
UNASSIGNED: This retrospective cross-sectional study included scarlet fever cases diagnosed at the Kanad Hospital, Al Ain, United Arab Emirates in 2022 and 2023. Data were retrieved from the hospital records and analysed using SPSS version 23.0. Chi-Square, Mann-Whitney, and Monte Carlo tests were applied.
UNASSIGNED: Two hundred and twenty-two cases (13.5% in 2022 and 86.5% in 2023) were confirmed (P<0.001). Majority (67.1%) of the patients were aged 3-6 years, with a mean age of 4.56 ± 1.99 years. Rash, fever and sore throat were observed in 100%, 99.1%, and 82.0% of cases, respectively. The majority (85.1%) were managed as outpatients and 77.0% responded to oral penicillin. Patients\' age was not significantly associated with nonresponse to penicillin and in-hospital admission. The outbreak had winter and summer peaks, with the highest incidence occurring during January and February 2023.
UNASSIGNED: This study serves as a valuable reference for other studies, which should include antimicrobial susceptibility testing and the prevailing genetic variance of Streptococcus pyogenes.
تحليل وصفي لفاشية الحمى القرمزية في الإمارات العربية المتحدة.
إيمان خليفة صبح، ثياجاراج أديمولام كوماراسامي، زهراء خليفة صبح.
UNASSIGNED: ان الحُمَّى القرمزية مرض مُعدٍ تسببه المكورات العقدية المقيحة. ولكن توجد بيانات محدودة عن هذا المرض في العالم العربي، ومنه الإمارات العربية المتحدة.
UNASSIGNED: هدفت هذه الدراسة الى تحليل فاشية الحُمَّى القرمزية في الإمارات العربية المتحدة.
UNASSIGNED: شملت هذه الدراسة المقطعية الاسترجاعية حالات الحمى القرمزية التي شُخِّصت في مستشفى كند في مدينة العين بالإمارات العربية المتحدة في عامَي 2022 و2023. واستُُرجعت البيانات من سجلات المستشفى، وحُلِّلت ببرنامج SPSS، الإصدار 23.0. وطُبِّقت اختبارات مربع كاي، ومان ويتني، ومونت كارلو.
UNASSIGNED: رُصدت 222 حالة مؤكدة (13.5٪ منها في عام 2022 و86.5٪ في عام 2023) (القيمة الاحتمالية: P<0.001). وتراوحت أعمار غالبية المرضى (67.1٪) بين 3 و6 سنوات، وبلغ متوسط أعمارهم 4.56 ± 1.99 سنوات. ولُوحظت أعراض الطفح الجلدي والحمى والتهاب الحلق في 100٪ و99.1٪ و82.0٪ من الحالات على الترتيب. وقُدِّم العلاج لغالبية المرضى (85.1٪) في العيادات الخارجية، واستجاب 77.0٪ منهم للبنسلين الفموي. ولم ترتبط أعمار المرضى ارتباطًا ملحوظًا بعدم الاستجابة للبنسلين والحاجة إلى دخول المستشفى. وبلغت الفاشية ذروتها في الشتاء وفي الصيف، وحدثت أعلى معدلات الإصابة خلال شهرَي يناير/ كانون الثاني وفبراير/ شباط 2023.
UNASSIGNED: تعد هذه الدراسة مرجع قيِّم لدراسات أخرى، وهي الدراسات التي ينبغي أن تشمل اختبارات بشأن الحساسية لمضادات الميكروبات والتباين الجيني السائد للمكورات العقدية المقيحة.
Analyse rétrospective d’une flambée de scarlatine aux Émirats arabes unis.
UNASSIGNED: La scarlatine est une maladie infectieuse causée par Streptococcus pyogenes. Cependant, il existe peu de données à ce sujet dans le monde arabe, et notamment aux Émirats arabes unis.
UNASSIGNED: Analyser une flambée de scarlatine survenue aux Émirats arabes unis.
UNASSIGNED: La présente étude transversale rétrospective a inclus des cas de scarlatine diagnostiqués à l\'hôpital Kanad, à Al Ain, aux Émirats arabes unis en 2022 et 2023. Les données ont été extraites des dossiers d\'hôpital et analysées à l\'aide du logiciel SPSS version 23.0. Les tests de khi carré, de Mann-Whitney et de Monte-Carlo ont été appliqués.
UNASSIGNED: Deux cent vingt-deux cas (13,5 % en 2022 et 86,5 % en 2023) ont été confirmés (p<0,001). La majorité des patients (61,7 %) étaient âgés entre trois et six ans, l\'âge moyen étant de 4,56 ± 1,99 ans. Des éruptions cutanées, de la fièvre et des maux de gorge ont été observés dans 100 %, 99,1 % et 82 % des cas respectivement. La majorité des personnes touchées (85,1 %) ont été prises en charge en ambulatoire et 77,0 % ont répondu à la pénicilline par voie orale. L\'âge des patients n\'était pas significativement associé à la non-réponse à la pénicilline et à l\'hospitalisation. La flambée a connu des pics en hiver et en été, l\'incidence la plus élevée s\'étant produite en janvier et février 2023.
UNASSIGNED: Cette étude sert de référence précieuse pour d\'autres études, qui devraient inclure des tests de sensibilité aux antimicrobiens et la variance génétique prévalente de Streptococcus pyogenes.

Necrotizing soft tissue infections (NSTIs), characterized by extensive soft tissue destruction, are rare but life-threatening. We present a case of a NSTI in a healthy 65-year-old woman following a closed distal radius fracture. The patient presented with severe pain, fever, and lethargy 4 days after her index injury, with physical examination of the right upper limb revealing erythema and swelling to the mid-humeral level and blisters of the fingers and hand. Multiple surgical debridements were required to control the infection, which was caused by Streptococcus pyogenes. This case highlights the rapid progression and devastating consequences of NSTI, which can occur even in the setting of closed injuries in patients without comorbidities. Prompt diagnosis, early surgical intervention, and appropriate antimicrobial therapy are crucial in managing this pathology.Level of Evidence: Level 5.