Christopher M. Rooney

Objectives This work aimed to resolve the conflicting reports on Prevotellaceae abundance in the development of rheumatoid arthritis (RA) and to observe structural, functional and temporal changes in the gut microbiome in RA progressors versus non-progressors. Methods Individuals at risk of RA were defined by the presence of anticyclic citrullinated protein (anti-CCP) antibodies and new musculoskeletal symptoms without clinical synovitis. Baseline sampling included 124 participants (30 progressed to RA), with longitudinal sampling of 19 participants (5 progressed to RA) over 15 months at five timepoints. Gut microbiome taxonomic alterations were investigated using 16S rRNA amplicon sequencing and confirmed with shotgun metagenomic DNA sequencing on 49 samples. Results At baseline, CCP+ at risk progressors showed significant differences in Prevotellaceae abundance compared with non-progressors, contingent on intrinsic RA risk factors and time to progression. Longitudinal sampling revealed gut microbiome instability in progressors 10 months before RA onset, a phenomenon absent in non-progressors. This may indicate a late microbial shift before RA onset, with Prevotellaceae contributing but not dominating these changes. Structural changes in the gut microbiome during arthritis development were associated with increased amino acid metabolism. Conclusion These data suggest conflicting reports on Prevotellaceae overabundance are likely due to sampling within a heterogeneous population along a dynamic disease spectrum, with certain Prevotellaceae strains/clades possibly contributing to the establishment and/or progression of RA. Gut microbiome changes in RA may appear at the transition to clinical arthritis as a late manifestation, and it remains unclear whether they represent a primary or secondary phenomenon.

Methicillin-resistant Staphylococcus aureus (MRSA) infections in neonates can result in significant morbidity and mortality. However, comparatively to adults, neonatal MRSA data remains relatively scarce. Additionally, while evidence-driven practices for adults have seen considerable progress, neonatal infection prevention strategies remain poorly described. The Leeds Newborn Service adopted a series of infection prevention and control (IPC) measures following a rise in MRSA cases in 2008–2009. This narrative review presents IPC measures for neonatal MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) infections and reflects upon local challenges and successes of these interventions. Our experience underscores the importance of an adaptive, evidence-based strategy, tailored to the neonatal population. Effectively addressing MRSA/MSSA requires continuous monitoring with sustained targeted interventions. Our key learning points highlight the intertwined difficulties of specific neonatal requirements and lack of definitive IPC guidance, suggesting a holistic approach is key for successful IPC outcomes in the neonatal intensive care unit setting.

Clostridioides difficile infection (CDI) remains a significant cause of morbidity and mortality worldwide. Historically, two antibiotics (metronidazole and vancomycin) and a recent third (fidaxomicin) have been used for CDI treatment; convincing data are now available showing that metronidazole is the least efficacious agent. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) management guidance for CDI were updated in 2021. This guidance document outlines the treatment options for a variety of CDI clinical scenarios and for non-antimicrobial management (e.g., faecal microbiota transplantation, FMT). One of the main changes is that metronidazole is no longer recommended as first-line CDI treatment. Rather, fidaxomicin is preferred on the basis of reduced recurrence rates with vancomycin as an acceptable alternative. Recommended options for recurrent CDI now include bezlotoxumab as well as FMT. A 2017 survey of 20 European countries highlighted variation internationally in CDI management strategies. A variety of restrictions were in place in 65% countries prior to use of new anti-CDI treatments, including committee/infection specialist approval or economic review/restrictions. This survey was repeated in November 2022 to assess the current landscape of CDI management practices in Europe. Of 64 respondents from 17 countries, national CDI guidelines existed in 14 countries, and 11 have already/plan to incorporate the ESCMID 2021 CDI guidance, though implementation has not been surveyed in 6. Vancomycin is the most commonly used first-line agent for the treatment of CDI (n = 42, 66%), followed by fidaxomicin (n = 30, 47%). Six (9%) respondents use metronidazole as first-line agent for CDI treatment, whereas 22 (34%) only in selected low-risk patient groups. Fidaxomicin is more likely to be used in high-risk patient groups. Availability of anti-CDI therapy influenced prescribing in six respondents (9%). Approval pre-prescription was required before vancomycin (n = 3, 5%), fidaxomicin (n = 10, 6%), bezlotoxumab (n = 11, 17%) and FMT (n = 10, 6%). Implementation of CDI guidelines is rarely audited. Novel anti-CDI agents are being evaluated; it is not yet clear what will be the roles of these agents. The treatment of recurrent CDI is particularly troublesome, and several different live biotherapeutics are being developed, in addition to FMT. Keywords C. difficile treatment Anti-CDI agents CDI guidelines Novel C. difficile agents C. difficile prophylaxis

Objectives: Intervention in the pre-arthritis phase of RA could prevent or delay the onset of disease. The primary aim of this study was to explore perspectives of being at risk and potential preventive interventions among individuals at risk of developing RA and to identify factors influencing their engagement with prevention. A secondary aim, established during the analytical process, was to understand and compare different approaches to health-related behaviours related to prevention of RA. Methods: Anti-CCP-positive (CCP+) at-risk individuals with musculoskeletal symptoms but no synovitis participated in semi-structured interviews. Data were analysed using reflexive thematic analysis, followed by a secondary ideal-type analysis. Results: Nineteen CCP+ at-risk individuals (10 women; age range 35–70 years) participated. Three overarching themes were identified: being CCP+ at risk; aiming to prevent RA; and influencers of engagement. Participants described distress related to symptoms and uncertainty about disease progression. Many participants had concerns about medication side effects. In contrast, most participants expressed willingness to make lifestyle changes with the aim of preventing RA. Engagement with preventive measures was influenced by symptom severity, personal risk level, co-morbidities, experiences of taking other medications/supplements, knowledge of RA, risk factors and medications, and perceived effort. Three types of participants were identified from the data: proactive preventers, change considerers and fearful avoiders. Overall orientation to health behaviours also impacted the attitude towards preventing RA. Conclusion: Findings could inform recruitment and retention in RA prevention research and promote uptake of preventive interventions in clinical practice.

Objective: Recent advances imply that early events triggering rheumatoid arthritis (RA) occur at mucosal surfaces. We aimed to evaluate whether intestinal permeability is altered in patients at increased risk of RA, and/or predicts the development of clinical arthritis, by measuring serum zonulin family peptides (ZFP) levels, which are shown to reflect intestinal barrier integrity.Methods: Two independent prospective observational cohorts were studied, including subjects with musculoskeletal symptoms and anticitrullinated protein antibodies (ACPA), but without clinical arthritis at baseline. In Sweden, 82 such at-risk patients were compared to 100 age-matched healthy blood donors. In the UK, 307 at-risk patients were compared to 100 ACPA-negative symptomatic controls. ZFP was measured in baseline sera by enzyme-linked immunoassays.Results: In the Swedish at-risk cohort, ZFP levels were significantly increased in patients compared to controls (mean 41.4 vs 33.6 ng/mL,P< 0.001) and Cox regression analysis showed prognostic value of ZFP for arthritis development (hazard ratio [HZ] 1.04 per ng/mL ZFP increase, 95% CI 1.01-1.07,P= 0.02). Elevated ZFP levels among ACPA-positive at-risk patients compared to symptomatic ACPA-negative controls were confirmed in the UK at-risk cohort (mean 69.7 vs 36.0 ng/mL,P< 0.001), but baseline ZFP were not associated with arthritis development (HR 1.00 per ng/mL ZFP increase, 95% CI 1.00-1.01,P= 0.30).Conclusion: Serum ZFP levels are elevated in ACPA-positive at-risk patients when compared to both healthy blood donors and symptomatic ACPA-negative controls. Thus, gut barrier function may be of importance in RA-associated autoimmunity. A possible prognostic value of serum ZFP merits further investigation, preferably in larger prospective cohorts.

Microbial taxonomic assignment based on 16S marker gene amplification requires multiple data transformations, often encompassing the use of a variety of computational platforms. Bioinformatics analysis may represent a bottleneck for researchers as many tools require programmatic access in order to implement the software. Here we describe a step-by-step approach for taxonomic assignment using QIIME2 and highlight the utility of graphical-based microbiome tools for further analysis and identification of biological relevant taxa with reference to an outcome of interest. Key words Gut microbiome 16S rRNA Bioinformatics QIIME 2 MEGAN LEfSe

We examine 3 different approaches to protecting the gut microbiome: highly targeted antibiotics, antibiotic destruction, and antibiotic binding. Each approach shows promise to prevent the off-target effects of antibiotics on the gut microbiome.

Objective: Individuals with newly diagnosed RA have a distinct microbiome when compared with healthy controls. However, little is known as to when these microbiome perturbations begin. Using a prospective at-risk cohort of individuals positive for anti-citrullinated protein (anti-CCP) antibody with new onset musculoskeletal symptoms, but without clinical arthritis, we investigated for the presence of a gut dysbiosis before the onset of RA. Methods: The gut microbiota of 25 anti-CCP positive individuals without clinical synovitis were sequenced targeting the V4 region of the 16S rRNA gene. Using a publicly available database, a control population of 44 individuals, approximately matched in age, gender, diet and ethnicity was selected for comparison, using the same sequencing methodology. Median interval between sample collection and progression to RA was 188 days. Taxonomic analysis was performed using QIIME and MEGAN, and statistical analysis using R software. Results: There were significant differences (P =0.01) at family level in gut microbiomes of anti-CCP positive individuals vs controls. The anti-CCP positive population had an overabundance of Lachnospiraceae, Helicobacteraceae, Ruminococcaceae, Erysipelotrichaceae and Bifidobacteriaceae, among others. Five individuals progressed to RA between sample collection and analysis. Clustering of the progressor population was observed on a phylogenetic network created using a probabilistic similarity index (Goodall’s index). Conclusions: Anti-CCP positive at-risk individuals without clinical synovitis appear to have a distinct gut microbiome compared with healthy controls. Phylogenetic clustering was observed in individuals who progressed to RA, suggesting that distinct taxa are associated with the development of RA many months before its onset.

Background: Significant nosocomial transmission of SARS-CoV-2 has been demonstrated. Understanding the prevalence of SARS-CoV-2 carriage amongst HCWs at work is necessary to inform the development of HCW screening programmes to control nosocomial spread. Methods: Cross-sectional ‘snapshot’ survey from April-May 2020; HCWs recruited from six UK hospitals. Participants self-completed a health questionnaire and underwent a combined viral nose and throat swab, tested by Polymerase Chain Reaction (PCR) for SARS-CoV-2 with viral culture on majority of positive samples. Findings: Point prevalence of SARS-CoV-2 carriage across the sites was 2·0% (23/1152 participants), median cycle threshold value 35·70 (IQR:32·42-37·57). 17 were previously symptomatic, two currently symptomatic (isolated anosmia and sore throat); the remainder declared no prior or current symptoms. Symptoms in the past month were associated with threefold increased odds of testing positive (aOR 3·46, 95%CI 1·38-8·67; p=0·008). SARS-CoV-2 virus was isolated from only one (5%) of nineteen cultured samples. A large proportion (39%) of participants reported symptoms in the past month. Interpretation: The point-prevalence is similar to previous estimates for HCWs in April 2020, though a magnitude higher than in the general population. Based upon interpretation of symptom history and testing results including viral culture, the majority of those testing positive were unlikely to be infectious at time of sampling. Development of screening programmes must balance the potential to identify additional cases based upon likely prevalence, expanding the symptoms list to encourage HCW testing, with resource implications and risks of excluding those unlikely to be infectious with positive tests. Funding: Public Health England. Word Count: Research in context: Evidence before this study: A search of PubMed was performed on 29 ^th April 2020 to identify other major works in this field, using the search terms (“novel coronavirus” OR “SARS-CoV-2” OR “COVID-19” OR “coronavirus”) AND (“workers” OR “staff”) AND (“testing” OR “screening”) from 31 ^st December 2019 onwards with no other limits. This search was updated on 10 ^th May 2020, and in addition reference lists were checked and pre-print papers were shared with us through professional networks. We found three papers commenting on prevalence of asymptomatic/pauci-symptomatic SARS-CoV-2 infection in healthcare workers, with prevalence estimates ranging from 1·1 to 8%. One of these studies explored previous symptoms in depth, though this was based upon a retrospective questionnaire and thus subject to recall bias. None of these studies explored exposures to the SARS-CoV-2 virus, commented on whether participants had been tested prior to the start of the study, or broke down results by staff role. Only one reported on estimated viral load (as inferred from cycle threshold [Ct] value), and none reported attempting viral culture. Added value of this study: This is the first published study of which we are aware that has been conducted across multiple sites in England and is therefore potentially more representative of the overall prevalence of SARS-CoV-2 infectivity amongst HCWs in the workplace. We explored symptoms in the preceding month in more depth than previous studies and in addition asked about previous test results and various exposures, also not commented on in other studies. Additionally, we attempted to isolate virus from some PCR-positive samples to look for evidence of infectious virus. Implications of all the available evidence: Authors of previous studies have proposed that screening asymptomatic HCWs for SARS-CoV-2 RNA may be beneficial, in addition to screening symptomatic HCWs. Our findings suggest that when prevalence of COVID-19 is very low, routine and repeated screening would be unlikely to have significant value, especially given the majority of participants testing positive in this study were unlikely to be infectious. However, in situations where prevalence levels are high in a particular population or setting, for example in a hospital outbreak, widening the case definition, or screening all HCWs irrespective of symptoms, may be of benefit.

Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden. To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon. Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing. CPE populations increased during inoculation, plateauing at ∼105 log10 cfu/mL in both models and persisting throughout the experiments (>65 days), with no evidence of CPE 'washout'. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742 bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (∼170 kb IncFIB/IncFII plasmid), and CPE populations rose from 45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model). CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials.