Investigating group A Streptococcus antibiotic tolerance in necrotizing fasciitis.

Group A Streptococcus (GAS) necrotizing fasciitis (NF) is a difficult-to-treat bacterial infection associated with high morbidity and mortality despite extensive surgery and targeted antibiotic treatment. Difficult-to-treat infections are often characterized by the presence of bacteria surviving prolonged antibiotic exposure without displaying genetic resistance, referred to as persisters. In the present study, we investigated the presence of GAS persisters in tissue freshly debrided from patients as well as in an in vivo mouse model of NF and examined the phenomenon of antibiotic tolerance. Time-lapse imaging of GAS plated directly upon isolation from NF debrided tissue and an antibiotic challenge-based persisters assay were used to assess the presence of persisters. We show for the first time that GAS recovered directly from freshly debrided NF tissue is characterized by heterogeneous and overall delayed colony appearance time, suggesting the presence of persisters. Acidic pH or nutrient stress exposure, mimicking the NF-like environment in vitro, led to a similar phenotypic heterogeneity and resulted in enhanced survival upon antibiotic challenge, confirming the presence of GAS persisters. GAS persisters might contribute to NF treatment failure, despite extensive surgery and adequate antibiotic treatment.IMPORTANCEDifficult-to-treat and recurrent infections are a global problem burdening society and the health care system alike. Unraveling the mechanisms by which bacteria can survive antibiotic treatment without developing genetic resistance is of utmost importance to lay the foundation for new, effective therapeutic approaches. For the first time, we describe the phenomenon of antibiotic tolerance in group A Streptococcus (GAS) isolated from necrotizing fasciitis (NF) patients. Dormant, non-replicating cells (persisters) are tolerant to antibiotics and their occurrence in vivo is reported in an increasing number of bacterial species. Tailored treatment options, including the use of persisters-targeting drugs, need to be developed to specifically target dormant bacteria causing difficult-to-treat and recurrent infections.

Dynamics of diversified A-to-I editing in Streptococcus pyogenes is governed by changes in mRNA stability.

Adenosine-to-inosine (A-to-I) RNA editing plays an important role in the post-transcriptional regulation of eukaryotic cell physiology. However, our understanding of the occurrence, function and regulation of A-to-I editing in bacteria remains limited. Bacterial mRNA editing is catalysed by the deaminase TadA, which was originally described to modify a single tRNA in Escherichia coli. Intriguingly, several bacterial species appear to perform A-to-I editing on more than one tRNA. Here, we provide evidence that in the human pathogen Streptococcus pyogenes, tRNA editing has expanded to an additional tRNA substrate. Using RNA sequencing, we identified more than 27 editing sites in the transcriptome of S. pyogenes SF370 and demonstrate that the adaptation of S. pyogenes TadA to a second tRNA substrate has also diversified the sequence context and recoding scope of mRNA editing. Based on the observation that editing is dynamically regulated in response to several infection-relevant stimuli, such as oxidative stress, we further investigated the underlying determinants of editing dynamics and identified mRNA stability as a key modulator of A-to-I editing. Overall, our findings reveal the presence and diversification of A-to-I editing in S. pyogenes and provide novel insights into the plasticity of the editome and its regulation in bacteria.

Developing small Cas9 hybrids using molecular modeling.

The contraction of CAG/CTG repeats is an attractive approach to correct the mutation that causes at least 15 neuromuscular and neurodegenerative diseases, including Huntington's disease and Myotonic Dystrophy type 1. Contractions can be achieved in vivo using the Cas9 D10A nickase from Streptococcus pyogenes (SpCas9) using a single guide RNA (sgRNA) against the repeat tract. One hurdle on the path to the clinic is that SpCas9 is too large to be packaged together with its sgRNA into a single adeno-associated virus. Here we aimed to circumvent this problem using the smaller Cas9 orthologue, SlugCas9, and the Cas9 ancestor OgeuIscB. We found them to be ineffective in inducing contractions, despite their advertised PAM sequences being compatible with CAG/CTG repeats. Thus, we further developed smaller Cas9 hybrids, made of the PAM interacting domain of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues. We also designed the cognate sgRNA hybrids using molecular dynamic simulations and binding energy calculations. We found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit their target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing larger scale complexes of Cas9/sgRNA hybrids.

Bioinformatics comparison of hemolysin in different bacteria and experimental evaluation of anti-cancer properties of extracts of some hemolysin-producing bacteria.

Cancer is one of the main causes of death in the world. Resistance to anticancer treatments in patients with advanced solid tumors leads to new treatments. Therefore, more alternative anticancer methods have been found over time with greater specificity against tumor cells and with less or no adverse effects on normal cells. Bacterial spores of obligate anaerobes exclusively germinate in the hypoxic/necrotic areas and not in the well oxygenated areas of the body. This unique phenomenon has been exploited in using bacterial spores as a remedy for cancer. Bacterial toxins also play a significant role in either directly killing tumor cells or altering the cellular processes of the tumor cells which ultimately leads to the inhibition and regression of the solid tumor. In the microbial environment, pathogens such as Staphylococcus aureus, Bacillus cereus, or Streptococcus pyogenes produce hemolysin. This protein is used as an anti-cancer protein. To identify the production of hemolysin by bacteria, which can destroy cancer cells more effectively, different bacterial strains were first cultured in blood agar culture medium. The Strains that completely lysed red blood cells, creating transparent zones, were selected for further investigation. Then, to find out which strains have more ability to lyse red blood cells, the qualitative method of halo diameter measurement was used. Also, using quantitative methods, hemolysin strength in microtubes was determined compared to control samples. The results of the hemolysis in the microtube and the qualitative test results showed similar results. In the next step, the cell viability test was performed with the partially purified proteins. Then, bioinformatics studies such as secondary structure investigation, physicochemical properties, pseudo amino acid composition, and molecular docking were performed. The results of molecular docking showed that the hemolysin protein has the highest affinity for the cholesterol of the cytoplasmic membrane, respectively, of Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus bacteria which play a significant role in either directly killing tumor cells or altering the cellular processes of the tumor cells which ultimately leads to the inhibition and regression of the solid tumor.

PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

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.

Emergent emm4 group A Streptococcus evidences a survival strategy during interaction with immune effector cells.

The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence among emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. By creating and analyzing isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates than the historic strains. Via the creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found a high ratio of mucosal (i.e., pharyngeal) relative to invasive infections among emm4 GAS. Since ever-increasing virulence is unlikely to be evolutionarily advantageous for a microbial pathogen, our data further understanding of the well-described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.

Structural basis for pegRNA-guided reverse transcription by a prime editor.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

Capsule-deficient group A Streptococcus evades autophagy-mediated killing in macrophages.

The hyaluronic acid capsule is crucial in protecting group A Streptococcus (GAS) against phagocytic killing. However, there have been reported outbreaks caused by capsule-deficient GAS strains, and the mechanisms underlying their evasion of immune clearance remain unclear. This study demonstrated that the capsule-deficient mutant [Cap(-)] of the emm1 strain increased survival within phagocytic cells compared to the wild-type strain [Cap(+)]. Although both Cap(+) and Cap(-) strains exhibited similar abilities to disrupt the phagosome, only the Cap(+) strain was colocalized with lysosomes and acidified compartments in phagocytic cells, indicating its susceptibility to autophagosome elimination. In contrast, the Cap(-) mutant evaded the recognition of galectin-8 and ubiquitin, impairing selective autophagy-mediated elimination. These findings suggest that a deficiency in the capsule could impair the intracellular elimination of GAS in macrophages, revealing previously unknown aspects of the host's recognition of the GAS capsule in macrophages. IMPORTANCE: Group A Streptococcus (GAS) is a Gram-positive bacterium that causes diseases ranging from mild pharyngitis to severe necrotizing fasciitis. Phagocytic cells serve as the primary defense against bacterial infections, exhibiting remarkable efficiency in eliminating intracellular pathogens. The hyaluronic acid capsule is a critical virulence factor that contributes to the resistance of phagocytosis in GAS. Nevertheless, the outbreaks caused by GAS strains that lack the hyaluronic acid capsule have been reported, and the selective advantage of capsule-deficient strains during infection is not fully understood. This study showed that the autophagic adaptor proteins recognize the capsulated GAS strain but not the capsule-deficient mutant, indicating that the hyaluronic acid capsule could be the autophagic target in macrophages. These findings imply that the hyaluronic acid capsule of GAS actually enhances its elimination within phagocytic cells, subverting the understanding of the capsule in GAS pathogenesis.

Outbreak of Streptococcus pyogenes emm89 ST646 in a head and neck surgical oncology ward.

Streptococcus pyogenes causes a variety of human infections, and hospital outbreaks with this pathogen have also been reported. The purpose of this study is to describe the clinical characteristics of an outbreak of S. pyogenes involving 15 patients and four healthcare workers (HCWs), as well as the molecular characteristics of the causative isolates. The course and response to the outbreak were reviewed, and information on the characteristics of the patients was extracted retrospectively from the medical records. Whole-genome sequencing of the 16 causative isolates (14 from patients and two from HCWs) was also performed. All 15 patients were postoperative of head and neck cancer with tracheotomy, and 12 had invasive infections, primarily surgical site infections, all of which resolved without causing serious illness. All but the first case was detected more than 7 days after admission. S. pyogenes was detected in two patients after empiric antimicrobial administration was performed on all inpatients and HCWs, and the outbreak was finally contained in approximately 2 months. All isolates detected in patients and HCWs belonged to emm89/clade 3, a hypervirulent clone that has emerged worldwide and was classified as sequence type 646. These isolates had single nucleotide polymorphism (SNP) differences of zero to one, indicating clonal transmission. This study demonstrated an outbreak of S. pyogenes emm89/clade 3 in a ward of patients with head and neck cancer. The global emergence of hypervirulent isolates may increase the risk of outbreaks among high-risk patients. IMPORTANCE: This study describes an outbreak of Streptococcus pyogenes that occurred in a ward caring for patients with head and neck cancer and tracheostomies. Many cases of invasive infections occurred in a short period, and extensive empiric antimicrobial administration on patients and healthcare workers was performed to control the outbreak. Whole-genome sequencing analysis of the causative strains confirmed that it was a monoclonal transmission of strains belonging to emm89/clade 3. The epidemiology and clinical characteristics of S. pyogenes infections have changed with the replacement of the prevalent clones worldwide. In the 1980s, there was a reemergence of S. pyogenes infections in high-income countries due to the spread of hypervirulent emm1 strains. emm89/clade 3 has recently been spreading worldwide and shares common features with emm1, including increased production of two toxins, NADase, and streptolysin O. The outbreak reported here may reflect the high spreading potential and virulence of emm89/clade 3.

An adenine base editor variant expands context compatibility.

Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. Existing ABEs function most effectively when the target A is in a TA context. Here we evolve the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to generate TadA8r, which extends potent deoxyadenosine deamination to RA (R = A or G) and is faster in processing GA than TadA8.20 and TadA8e, the two most active TadA variants reported so far. ABE8r, comprising TadA8r and a Streptococcus pyogenes Cas9 nickase, expands the editing window at the protospacer adjacent motif-distal end and outperforms ABE7.10, ABE8.20 and ABE8e in correcting disease-associated G:C-to-A:T transitions in the human genome, with a controlled off-target profile. We show ABE8r-mediated editing of clinically relevant sites that are poorly accessed by existing editors, including sites in PCSK9, whose disruption reduces low-density lipoprotein cholesterol, and ABCA4-p.Gly1961Glu, the most frequent mutation in Stargardt disease.

Molecular Genetic Characterization of Streptococcus pyogenes Strains Isolated from Patients with Various Manifestations of Streptococcal Infection.

In 82 clinical strains of Streptococcus pyogenes (group A streptococci) isolated from patients with various manifestations of streptococcal infection, emm-typing revealed 27 emm-types (n=77) with a predominance of emm-89 (n=15; 18%), emm-75 (n=9; 11%), and emm-1 (n=6; 7%); types emm-3, emm-12, and emm-58 (n=4; 5% each) were found with almost equal frequency; other types were less common. The superantigen genes speC, speG, speH, speI, speJ, speK, speL, speM, smeZ, and SSA were identified in S. pyogenes strains using multiprimer PCR; the genes of the superantigen SpeA and cysteine proteinase SpeB were detected using real-time PCR. All the studied S. pyogenes strains contained superantigen genes, and 98% of the strains had several (from 2 to 7) genes. The number of variants of these sets reached 37; 2% of the strains contained only one superantigen gene. The distribution frequencies of superantigen genes in the studied strains were: speA - 43%; speC - 38%; speG - 93%; speH - 13%; speI - 6%; speJ - 24%; speK - 13%; speL and speM - 11% each; smeZ - 98%; SSA - 15%. All studied S. pyogenes strains contained the speB gene. Our studies have demonstrated that the sets of superantigen genes of group A streptococci are characterized by pronounced diversity to some extent associated with emm-type.

Sustained increase of paediatric invasive Streptococcus pyogenes infections dominated by M1UK and diverse emm12 isolates, Portugal, September 2022 to May 2023.

Since autumn 2022, observed numbers of paediatric invasive group A Streptococcus infections in Portugal (n = 89) were higher than in pre-COVID-19 seasons. Between September 2022 and May 2023, the dominant diagnoses were pneumonia (25/79), mostly with empyema (20/25), and sepsis (22/79). A number of cases required admission to intensive care (27/79) and surgery (35/79), and the case fatality rate was 5.1% (4/79). Genomic sequencing (n = 55) revealed multiple genetic lineages, dominated by the M1UK sublineage (26/55) and more diverse emm12 isolates (12/55).

RopB-regulated SpeB cysteine protease degrades extracellular vesicles-associated streptolysin O and bacterial proteins from group A Streptococcus.

Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. Streptococcus pyogenes (group A Streptococcus, GAS) is one of the most common pathogens of monomicrobial necrotizing fasciitis. Spontaneous inactivating mutation in the CovR/CovS two-component regulatory system is related to the increase of EVs production via an unknown mechanism. This study aimed to investigate whether the CovR/CovS-regulated RopB, the transcriptional regulator of GAS exoproteins, would participate in regulating EVs production. Results showed that the size, morphology, and number of EVs released from the wild-type strain and the ropB mutant were similar, suggesting RopB is not involved in controlling EVs production. Nonetheless, RopB-regulated SpeB protease degrades streptolysin O and bacterial proteins in EVs. Although SpeB has crucial roles in modulating protein composition in EVs, the SpeB-positive EVs failed to trigger HaCaT keratinocytes pyroptosis, suggesting that EVs did not deliver SpeB into keratinocytes or the amount of SpeB in EVs was not sufficient to trigger cell pyroptosis. Finally, we identified that EV-associated enolase was resistant to SpeB degradation, and therefore could be utilized as the internal control protein for verifying SLO degradation. This study revealed that RopB would participate in modulating protein composition in EVs via SpeB-dependent protein degradation and suggested that enolase is a potential internal marker for studying GAS EVs.

Interplay between human STING genotype and bacterial NADase activity regulates inter-individual disease variability.

Variability in disease severity caused by a microbial pathogen is impacted by each infection representing a unique combination of host and pathogen genomes. Here, we show that the outcome of invasive Streptococcus pyogenes infection is regulated by an interplay between human STING genotype and bacterial NADase activity. S. pyogenes-derived c-di-AMP diffuses via streptolysin O pores into macrophages where it activates STING and the ensuing type I IFN response. However, the enzymatic activity of the NADase variants expressed by invasive strains suppresses STING-mediated type I IFN production. Analysis of patients with necrotizing S. pyogenes soft tissue infection indicates that a STING genotype associated with reduced c-di-AMP-binding capacity combined with high bacterial NADase activity promotes a 'perfect storm' manifested in poor outcome, whereas proficient and uninhibited STING-mediated type I IFN production correlates with protection against host-detrimental inflammation. These results reveal an immune-regulating function for bacterial NADase and provide insight regarding the host-pathogen genotype interplay underlying invasive infection and interindividual disease variability.

Acyl Homoserine Lactone Sensitised Streptococcus Pyogenes Differentially Regulates the Transcriptional Expression of Early Growth Response 1 (EGR1) in Epithelial and Macrophage Cells.

The host transcriptional activator Early growth response 1 (EGR1) plays a vital role in cell cycle and differentiation, cell proliferation, and regulation of cytokines and several growth factors. It is an immediate-early gene that is expressed as an initial response to various environmental stimuli. Bacterial infection is one such factor that can trigger the expression of EGR1 in host. Therefore, it is imperative to understand expression of EGR1 during early stages of host-pathogen interaction. Streptococcus pyogenes is an opportunistic bacteria causing skin and respiratory tract infections in humans. The quorum-sensing molecule, N-(3-oxododecanoyl)-l-homoserine lactone (Oxo-C12), not synthesised by S. pyogenes, can be sensed by S. pyogenes leading to molecular changes in the pathogen. In this study, we investigated the role of Oxo-C12 on EGR1 regulation in lung epithelial and murine macrophage cell line upon S. pyogenes infection. We report that Oxo-C12 sensitised S. pyogenes upregulates the transcriptional expression of EGR1 through ERK1/2 pathway. It was observed that EGR1 was not involved in the intial attachment of S. pyogenes to A549 cells. However, inhibition of EGR1 in macrophage cell line, J774A.1, through the ERK1/2 pathway resulted in decreased adhesion of S. pyogenes. The EGR1 upregulation by Oxo-C12 sensitised S. pyogenes plays a vital role in enhancing the survival of S. pyogenes in murine macrophages, leading to persistent infection. Thus, understanding the molecular modulation in the host during bacterial infection will further help develop therapeutics to target specific sites.

Role of serotype and virulence determinants of Streptococcus pyogenes biofilm bacteria in internalization and persistence in epithelial cells in vitro.

Streptococcus pyogenes causes a multitude of local and systemic infections, the most common being pharyngitis in children. Recurrent pharyngeal infections are common and are thought to be due to the re-emergence of intracellular GAS upon completion of antibiotic treatment. The role of colonizing biofilm bacteria in this process is not fully clear. Here, live respiratory epithelial cells were inoculated with broth-grown or biofilm bacteria of different M-types, as well as with isogenic mutants lacking common virulence factors. All M-types tested adhered to and were internalized into epithelial cells. Interestingly, internalization and persistence of planktonic bacteria varied significantly between strains, whereas biofilm bacteria were internalized in similar and higher numbers, and all strains persisted beyond 44 hours, showing a more homogenous phenotype. The M3 protein, but not the M1 or M5 proteins, was required for optimal uptake and persistence of both planktonic and biofilm bacteria inside cells. Moreover, the high expression of capsule and SLO inhibited cellular uptake and capsule expression was required for intracellular survival. Streptolysin S was required for optimal uptake and persistence of M3 planktonic bacteria, whereas SpeB improved intracellular survival of biofilm bacteria. Microscopy of internalized bacteria showed that planktonic bacteria were internalized in lower numbers as individual or small clumps of bacteria in the cytoplasm, whereas GAS biofilm bacteria displayed a pattern of perinuclear localization of bacterial aggregates that affected actin structure. Using inhibitors targeting cellular uptake pathways, we confirmed that planktonic GAS mainly uses a clathrin-mediated uptake pathway that also required actin and dynamin. Clathrin was not involved in biofilm internalization, but internalization required actin rearrangement and PI3 kinase activity, possibly suggesting macropinocytosis. Together these results provide a better understanding of the potential mechanisms of uptake and survival of various phenotypes of GAS bacteria relevant for colonization and recurrent infection.

An optimized SpCas9 high-fidelity variant for direct protein delivery.

Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.

Engineered CRISPR prime editors with compact, untethered reverse transcriptases.

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.

The Effect of Amoxicillin with Clavulanate on the Microbiota of Tonsillar Tissue in Disease: a Randomized Control Trial.

Despite antibiotics being the primary medical treatment for recurrent tonsillitis, the impact of antibiotics on the tonsillar microbiome is not well understood. This study aimed to determine the effect of amoxicillin with clavulanate on the composition and quantity of bacteria in the tonsils of children with recurrent tonsillitis. A multicenter randomized clinical trial in Auckland, New Zealand was undertaken between August 1, 2017, and June 30, 2018. Sixty children undergoing tonsillectomy for the indication of recurrent tonsillitis were recruited for this study. Following random allocation, 30 participants were prescribed amoxicillin with clavulanate for the week before surgery. The remaining 30 received no antibiotics. Immediately following surgery, the crypts of the right and left tonsils were swabbed. Bacterial 16S rRNA gene-targeted amplicon sequencing and histological techniques were utilized. In the control group, there were significantly higher relative abundances of Haemophilus, Streptococcus, Neisseria, and Porphyromonas. Members from the genera Fusobacterium and Treponema were found to be significantly more abundant in the antibiotic group. There were no significant differences in the absolute quantities of bacteria between the groups. Microscopic examination found fewer bacterial microcolonies present in the tonsillar crypts of participants in the antibiotic group. Streptococcus pyogenes was not present in these bacterial microcolonies. These results suggest that a single course of antibiotics has a significant impact on the tonsil microbiota composition. The duration of this effect and the effect that the altered microbiome has on the course of the condition need to be determined. IMPORTANCE Several studies have identified the presence of multiple pathogenic bacteria in hyperplastic adenoids and palatine tonsils. However, there are currently no studies that utilize this technology to investigate the effect of oral antibiotics in children with recurrent tonsillitis on the tonsillar microbiome. This is the first study to investigate the effect of antibiotics on the microbiome of tonsillar tissue in children with recurrent tonsillitis using molecular techniques. This study has shown that participants who received amoxicillin with clavulanate immediately before tonsillectomy had a significantly reduced number of bacterial taxa commonly associated with recurrent tonsillitis, as well as the number of bacterial microcolonies observed in the tonsillar crypts. This novel finding suggests that either the effect of antibiotics is not sustained or that they are not an effective treatment for recurrent tonsillitis.