Pangenome evaluation of gene essentiality in Streptococcus pyogenes.

Bacterial species often consist of strains with variable gene content, collectively referred to as the pangenome. Variations in the genetic makeup of strains can alter bacterial physiology and fitness. To define biologically relevant genes of a genome, genome-wide transposon mutant libraries have been used to identify genes essential for survival or virulence in a given strain. Such phenotypic studies have been conducted in four different genotypes of the human pathogen Streptococcus pyogenes, yet challenges exist in comparing results across studies conducted in different genetic backgrounds and conditions. To advance genotype to phenotype inferences across different S. pyogenes strains, we built a pangenome database of 249 S. pyogenes reference genomes. We systematically re-analyzed publicly available transposon sequencing datasets from S. pyogenes using a transposon sequencing-specific analysis pipeline, Transit. Across four genetic backgrounds and nine phenotypic conditions, 355 genes were essential for survival, corresponding to ~24% of the core genome. Clusters of Orthologous Genes (COG) categories related to coenzyme and lipid transport and growth functions were overrepresented as essential. Finally, essential operons across S. pyogenes genotypes were defined, with an increased number of essential operons detected under in vivo conditions. This study provides an extendible database to which new studies can be added, and a searchable html-based resource to direct future investigations into S. pyogenes biology.IMPORTANCEStreptococcus pyogenes is a human-adapted pathogen occupying restricted ecological niches. Understanding the essentiality of genes across different strains and experimental conditions is important to direct research questions and efforts to prevent the large burden of disease caused by S. pyogenes. To this end we systematically reanalyzed transposon sequencing studies in S. pyogenes using transposon sequencing-specific methods, integrating them into an extendible meta-analysis framework. This provides a repository of gene essentiality in S. pyogenes which was used to highlight specific genes of interest and for the community to guide future phenotypic studies.

Glycan-Tailored Glycoproteomic Analysis Reveals Serine is the Sole Residue Subjected to O-Linked Glycosylation in Acinetobacter baumannii.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii, O-linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O-linked glycosylation.

UK guidelines for the management of soft tissue sarcomas.

Soft tissue sarcomas (STS) are rare tumours arising in mesenchymal tissues and can occur almost anywhere in the body. Their rarity, and the heterogeneity of subtype and location, means that developing evidence-based guidelines is complicated by the limitations of the data available. This makes it more important that STS are managed by expert multidisciplinary teams, to ensure consistent and optimal treatment, recruitment to clinical trials, and the ongoing accumulation of further data and knowledge. The development of appropriate guidance, by an experienced panel referring to the evidence available, is therefore a useful foundation on which to build progress in the field. These guidelines are an update of the previous versions published in 2010 and 2016 [1, 2]. The original guidelines were drawn up by a panel of UK sarcoma specialists convened under the auspices of the British Sarcoma Group (BSG) and were intended to provide a framework for the multidisciplinary care of patients with soft tissue sarcomas. This iteration of the guidance, as well as updating the general multidisciplinary management of soft tissue sarcoma, includes specific sections relating to the management of sarcomas at defined anatomical sites: gynaecological sarcomas, retroperitoneal sarcomas, breast sarcomas, and skin sarcomas. These are generally managed collaboratively by site specific multidisciplinary teams linked to the regional sarcoma specialist team, as stipulated in the recently published sarcoma service specification [3]. In the UK, any patient with a suspected soft tissue sarcoma should be referred to a specialist regional soft tissues sarcoma service, to be managed by a specialist sarcoma multidisciplinary team. Once the diagnosis has been confirmed using appropriate imaging and a tissue biopsy, the main modality of management is usually surgical excision performed by a specialist surgeon, combined with pre- or post-operative radiotherapy for tumours at higher risk for local recurrence. Systemic anti-cancer therapy (SACT) may be utilised in cases where the histological subtype is considered more sensitive to systemic treatment. Regular follow-up is recommended to assess local control, development of metastatic disease, and any late effects of treatment.

Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Technical considerations for isolated limb perfusion: A consensus paper.

BACKGROUND: Isolated limb perfusion (ILP) is a well-established surgical procedure for the administration of high dose chemotherapy to a limb for the treatment of advanced extremity malignancy. Although the technique of ILP was first described over 60 years ago, ILP is utilised in relatively few specialist centres, co-located with tertiary or quaternary cancer centres. The combination of high dose cytotoxic chemotherapy and the cytokine tumour necrosis factor alpha (TNFα), mandates leakage monitoring to prevent potentially serious systemic toxicity. Since the procedure is performed at relatively few specialist centres, an ILP working group was formed with the aim of producing technical consensus guidelines for the procedure to streamline practice and to provide guidance for new centres commencing the technique. METHODS: Between October 2021 and October 2023 a series of face to face online and hybrid meetings were held in which a modified Delphi process was used to develop a unified consensus document. After each meeting the document was modified and recirculated and then rediscussed at subsequent meeting until a greater than 90% consensus was achieved in all recommendations. RESULTS: The completed consensus document comprised 23 topics in which greater than 90% consensus was achieved, with 83% of recommendations having 100% consensus across all members of the working group. The consensus recommendations covered all areas of the surgical procedure including pre-operative assessment, drug dosing and administration, perfusion parameters, hyperthermia, leakage monitoring and theatre logistics, practical surgical strategies and also post-operative care, response evaluation and staff training. CONCLUSION: We present the first joint expert-based consensus statement with respect to the technical aspects of ILP that can serve as a reference point for both existing and new centres in providing ILP.

Surgical Management of Primary Anorectal Melanoma: Is Less More?

PURPOSE: Ano-uro-genital (AUG) Mucosal Melanoma UK guidelines recommended a less radical surgical strategy for anorectal melanoma (ARM) where possible. We report our experience of ARM consistent with that approach including clinical presentation, intervention undertaken and prognosis. METHODS: We present a retrospective study of 15 consecutive patients with ARM surgically treated between November 2014 and April 2023. Patients were divided into the two surgery types: wide local excision (WLE, n = 9) and abdominoperineal resection (APR, n = 6). Data on demographics, diagnosis, treatment and oncological outcomes were assessed between the groups. RESULTS: The mean age was 65.3 ± 17.4 years and 6 (40.0%) were female patients. Nine patients (60.0%) were diagnosed with stage I and six patients (40.0%) with stage II disease. R0 margins were achieved in all cases. The overall mean length of stay was lower following WLE compared to APR (2.6 ± 2.4 days versus 14.0 ± 9.8 days, p = 0.032). Two complications were observed in the WLE group compared to four complications after APR (p = 0.605). Five patients (55.5%) developed local/distant recurrence in the WLE group compared to three patients (50.0%) in the APR group (p = 0.707), with a median overall survival of 38.5 (12-83) months versus 26.5 (14-48) months, respectively. CONCLUSIONS: Achieving clear margins by the least radical fashion may have equivalent oncological outcomes to radical surgery, potentially reducing patient morbidity and preserving function. In our experience, the surgical management of ARM consistent with the 'less is more' approach adhering to AUG guidelines has acceptable outcomes.