Background:Culture-independent molecular techniques could potentially be used to measure microbiological efficacy in response to antibiotic treatment and improve understanding of the role of the airway microbiota in determining response in patients with chronic respiratory disease. Methods:Using molecular methods, we analysed changes in the sputum microbiota in samples from 107 participants with bronchiectasis recruited to the iBEST-1 study, and defined community endotypes based on response to tobramycin inhalation powder (TIP) treatment. The relationship between microbiota metrics in these endotypes and clinical and inflammatory biomarkers were also determined. Results:There was a significant reduction in Pseudomonas aeruginosa density, measured by quantitative polymerase chain reaction (qPCR), between Days 1 and 29 for participants in the TIP treatment (n=63; p<0.0001) but not placebo (n=20; p>0.05) group. Based on decrease in P. aeruginosa density (oprL copies·mL-1) over 28 days, two clusters of participants receiving TIP were observed and stratified as either responders (≥2Log10; n=26) or non-responders (<2Log10; n=37). In responders, a shift to a microbial community structure less dominated (p=0.018) by a pathogen was apparent and associated with a greater improvement in inflammatory and fewer participant exacerbations in the following 6 months (27% versus 49%; p=0.117) when compared to non-responders. Lung function was higher at Day 1 in responders (median=64.6% predicted) than non-responders (μ̃median=50.3% predicted) and independently predicted response to treatment with TIP (p=0.013). Conclusions:qPCR may be a useful, culture-independent microbiological efficacy end-point in clinical trials. Using qPCR, participants with bronchiectasis were stratified into endotpyes which predicted response to antimicrobial treatment, potentially allowing for a more personalised approach to therapy.

Background and Objective This study explored the relationship between total bacterial density, airway microbiota composition and clinical parameters in bronchiectasis. We determined changes with time during clinical stability and following antibiotic treatment of a pulmonary exacerbation. Methods We conducted a multicentre longitudinal cohort study of UK participants with CT confirmed bronchiectasis. Sputum samples and clinical parameters [FEV1% predicted, lung clearance index, C-reactive protein, white cell count and Quality of Life] were collected when participants were clinically stable and pre/post-antibiotic treatment of an exacerbation. Total bacterial density and microbiota community composition was measured by quantitative polymerase chain reaction and sequencing of the V4 region of bacterial 16S rRNA, respectively. Results Among 105 participants at baseline, 65 (62%) were female with a mean age of 65 years and FEV1 at 69% predicted. In participants who remained clinically stable (n=15), no significant changes were observed in bacterial density, microbiota diversity, richness, evenness, and dominance (p=0.30, 0.45, 0.54, 0.23 and 0.43; respectively) across four time points over a 1-year period. Similarly, for participants with paired pre/post-antibiotic treatment samples (n=19), no significant changes were observed (p=0.30, 0.46, 0.44, 0.71 and 0.58; respectively). However, considerable fluctuation in community composition between samples was apparent for most patients. Total bacterial density and microbiota composition did not correlate with clinical parameters at baseline (n=75). Conclusions Stability in bacterial density and microbiota diversity, richness, evenness and dominance was observed over time at a population level but considerable fluctuation was apparent in samples from individual patients.

Antimicrobial resistance poses a growing threat, especially in care homes where older residents are particularly vulnerable due to frequent antibiotic use and comorbidities. Following the COVID-19 pandemic, there has been a growing focus on wastewater surveillance for detecting and monitoring pathogens in healthcare settings. This study followed the Joanna Briggs Institute scoping review framework to map the extent of available literature on wastewater-based epidemiological studies addressing antimicrobial resistance in care homes for older adults. Six electronic databases (Medline, Embase, Scopus, Web of Science, ProQuest, and Google Scholar) were searched from date of inception until August 26th, 2024. The search strategy employed variations of the keywords: 'antimicrobial resistance', 'wastewater-based epidemiology', and 'care homes for older adults'. Studies were screened based on eligibility criteria, with data extracted by one researcher. Another researcher reviewed the charted data and resolved any queries. The search identified 83 studies, from which 11 studies, conducted between 2015 and 2024, were included. The studies used grab or composite sampling, combined with culture-based methods for bacterial identification, antimicrobial susceptibility testing, and molecular techniques such as polymerase chain reaction and whole-genome sequencing. Enterobacterales, including Escherichia coli and Klebsiella spp., were the most frequently detected, with high resistance rates, especially to some penicillins and cephalosporins. Despite the small sample sizes reported in this review, wastewater-based epidemiology shows promise in monitoring antibiotic-resistant bacteria in care home wastewaters, offering insights into trends and genetic diversity, with the potential to inform public health strategies and antibiotic stewardship programmes.

Effective pathogen surveillance is critical for public health decision-making, with both individual and environmental monitoring playing essential roles. While wastewater (WW) and individual whole genome sequencing (WGS) have been used to monitor SARS-CoV-2 dynamics, their complementary potential for enhancing national-level genomic surveillance remains underexplored. This study aimed to evaluate the unique and combined contributions of WW and individual WGS to genomic surveillance. We conducted SARS-CoV-2 WGS on over 4000 WW samples and 23,000 individual samples across Northern Ireland (NI) between 2021 and 2023. SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 and Mini-XT protocols and sequenced on Illumina MiSeq. Variant compositions in WW data were analysed using Freyja and compared to individual data using time series analysis, correlation assessments, and volatility measurements via numerical derivatives, with mean absolute error (MAE) calculations used to assess concordance. Wastewater treatment plants (WWTPs) were ranked by concordance to individual WGS data. WW and individual WGS complementarity was quantified by mutation classification and overlap analysis. Temporal curve shifting was used to identify lags or leads in variant detection and to infer differences in geospatial spread between WW and individual sequencing data. We confirmed strong concordance between WW and individual variant compositions (mean MAE = 6.2 %). MAE was inversely correlated with sequencing rate (Pearson r=-0.37, p < 0.001) and increased during periods with more circulating variants, highlighting the value of increased sequencing efforts during volatile periods. The population size served by a WWTP was not a reliable indicator of how well its variant composition matched that of the national individual sequencing programme. Both individual and WW-based sequencing (WBS) detected unique, as well as common mutations. Patterns of variant spread within NI were consistent between both programmes (Pearson r = 0.63, p = 0.036), providing complementary insights into variant trends and geospatial spread. We demonstrate that integration of individual and WW WGS data offers more comprehensive SARS-CoV-2 genomic surveillance and improves confidence in predictions of variant composition and spread.

Background Influenza A viruses (IAVs) are significant pathogens of humans and other animals. Although endemic in humans and birds, novel IAV strains can emerge, jump species, and cause epidemics, like the latest variant of H5N1. Wastewater-based epidemiology (WBE) has been shown capable of detecting human IAVs. We aimed to assess whether whole-genome sequencing (WGS) of IAVs from wastewater is possible and can be used to discriminate between circulating strains of human and any non-human IAVs, such as those of avian origin. Methods Using a pan-IAV RT-quantitative PCR assay, six wastewater treatment works (WWTWs) across Northern Ireland were screened from Aug 1 to Dec 5, 2022. A nanopore WGS approach was used to sequence RT-qPCR-positive samples. Phylogenetic analysis of sequences relative to currently circulating human and non-human IAVs was performed. For comparative purposes, clinical data (PCR test results) were supplied by The Regional Virus Laboratory, Belfast Health and Social Care Trust (Belfast, Northern Ireland, UK). Findings We detected a dynamic IAV signal in wastewater from Sept 5, 2022, onwards across Northern Ireland, which did not show a clear positive relationship with the clinical data obtained for the region. Meta (mixed strain) whole-genome sequences were generated from wastewater samples displaying homology to only human and avian IAV strains. The relative proportion of IAV reads of human versus avian origin differed across time and sample site. A diversity in subtypes and lineages was detected (eg, H1N1, H3N2, and several avian). Avian segment 8 related to those found in recent H5N1 clade 2.3.4.4b was identified. Interpretation WBE affords a means to monitor circulating human and avian IAV strains and provide crucial genetic information. As such, WBE can provide rapid, cost-effective, year-round One Health surveillance to help control IAV epidemic and pandemic-related threats. However, optimisation of WBE protocols are necessary to ensure observed wastewater signals not only correlate with clinical case data, but yield information on the wider environmental pan-influenz-ome. Funding Department of Health for Northern Ireland.

Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limit the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of severe acute respiratory syndrome coronavirus 2 in the community, but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying reverse-transcription quantitative polymerase chain reaction and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and nonpharmaceutical interventions. The genomic wastewater-based epidemiology platform has the potential to complement ongoing global surveillance efforts and aid the management of respiratory syncytial virus by informing the timely deployment of pharmaceutical and nonpharmaceutical interventions.

Wastewater based epidemiology (WBE) is an integral component of the global COVID-19 surveillance strategy and has become a key public health tool. By detecting increasing incidence of SARS-CoV-2 in wastewater WBE can inform Public Health teams of community level infection rates. Levels of SARS-CoV-2 in wastewater are however influenced by the amount of water flowing through the treatment system. Daily variances in flow rates, due to rainfall, can lead to underestimation or overestimation of viral fragment prevalence. To account for this normalisation of the SARS-CoV-2 signal is required. Commonly normalisation is achieved by measuring the flow into a treatment works using a flow metering system. Yet such measured flow data is often not collated or reported until weeks after a sample has been taken limiting the functionality of WBE as a tool for warning of increasing incidence of infection. Other, biomarker-based systems, based on ammonia or crAssphage concentration have been developed. These are however costly and time consuming. Here we detail a new method for the normalisation of SARS-CoV-2 using localised rainfall data to estimate wastewater flow rates. Normalisation via rainfall was statistically similar to results obtained using flow metering for all treatment works tested and compares favourably to normalisation via ammonia flow estimation. We also present details of an optimised, moderately high-thoughput, SARS-CoV-2 wastewater detection workflow which utilises a concentrating pipette system (CP Select) and the Roche Classic Flow System for nucleic acid extraction and RT-qPCR. This methodology permits the quantitative detection of SARS-CoV-2 in wastewater in within 8 hours of sample receipt with a limit of detection of 1 x 102 gene copies. We demonstrate no significant difference between our methodology and those viral recovery protocols based on polyethylene glycol precipitation or centrifugal filtration. These methodologies have subsequently been integrated into Northern Ireland’s national SARS-CoV-2 wastewater surveillance program.

Introduction: This study determined changes in the antimicrobial resistome and airway microbiota following treatment with tobramycin inhalation powder (TIP) or placebo in the iBEST study. Methods: DNA was extracted from sputum collected at baseline and at day 29 (Day 1 vs Day 29) following treatment with TIP (224mg daily; n=11 patients) or placebo (n=5 patients). Shotgun-metagenomic sequencing (sgNGS) was performed on the Illumina Novaseq platform. P. aeruginosa density was determined by both total viable count (TVC) and qPCR of the oprL gene with total bacterial density measured by qPCR of the 16S rRNA gene. Resistome profiles were determined by mapping reads to the Comprehensive Antibiotic Resistance gene Database (CARD). Results: Using sgNGS sequences, reads mapped to 62 different resistance genes. Differences in resistance gene prevalence for multiple targets were observed pre- and post-treatment and were primarily associated with samples from patients receiving TIP. Limited or no difference was observed for those receiving placebo. Differences in resistome profiles were strongly aligned with a decrease in P. aeruginosa density determined by TVC (Day 1 vs. Day 29; TIP: p<0.001; Placebo: p=0.222) and qPCR (Day 1 vs. Day 29; TIP: p=0.001; Placebo: p=0.421). No difference was seen in total bacterial density (16S rRNA) for either treatment group (p=0.193 and p=0.841, respectively). Conclusion: Treatment with TIP reduced the number of resistance genes detected. This was mainly due to a decrease in P. aeruginosa density within the community. Supported by EU/EFPIA IMI iABC grant no° 115721

As of 8 July 2022, the World Health Organization (WHO) have reported 1010 probable cases of acute hepatitis of unknown aetiology in children worldwide, including approximately 250 cases in the United Kingdom (UK). Clinical presentations have often been severe, with liver transplantation a frequent clinical outcome. Human adenovirus F41 (HAdV-F41) has been detected in most children with acute hepatitis, but its role in the pathogenesis of this infection has yet to be established. Wastewater-based epidemiology (WBE) has become a well-established tool for monitoring the community spread of SARS-CoV-2, as well as other pathogens and chemicals. In this study, we adopted a WBE approach to monitoring levels of HAdV-F40/41 in wastewater before and during an acute hepatitis outbreak in Northern Ireland. We report increasing detection of HAdV-F40/41 in wastewater, concomitant with increasing numbers of clinical cases. Amplicon whole genome sequencing further classified the wastewater-derived HAdV as belonging to the F41 genotype which in turn was homologous to clinically derived sequences. We propose that WBE has the potential to inform community surveillance of HAdV-F41 and can further contribute to the ongoing global discussion supporting HAdV-F41 involvement in acute hepatitis cases.

BACKGROUND:Whether there is any benefit in integrating culture-independent molecular analysis of the lower airway microbiota of people with cystic fibrosis into clinical care is unclear. This study determined the longitudinal trajectory of the microbiota and if there were microbiota characteristics that corresponded with response to treatment or predicted a future pulmonary exacerbation. METHODS:At least one sputum sample was collected from 149 participants enrolled in this prospective longitudinal multi-centre study and total bacterial density and microbiota community measurements were determined and compared with clinical parameters. RESULTS:In 114 participants with paired samples when clinically stable, ∼8 months apart, the microbiota remained conserved between timepoints, regardless of whether participants received acute intravenous antibiotic treatment or not. In 62 participants, who presented with an acute exacerbation, a decrease in community richness correlated best with patient response to antibiotic treatment. Analysis of baseline samples from 30 participants who exacerbated within 4 months of their stable sample being collected and 72 participants who remained stable throughout the study showed that community characteristics such as lower richness at baseline may be predictive of an exacerbation in addition to several clinical parameters. However, lasso regression analysis indicated that only lung function (p = 0.014) was associated with a future exacerbation. CONCLUSIONS:The airway microbiota remains stable over periods <1 year with modest shifts related to treatment apparent which might provide some additional insights to patient-level measurements.

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected.

Background Influenza A viruses (IAV) are significant pathogens of humans and other animals. Although endemic in humans and birds, novel IAV strains can emerge, jump species, and cause epidemics, like the latest variant of H5N1. Wastewater-based epidemiology (WBE) has very recently been shown to detect human IAV but whether it can detect avian-origin IAV, and if whole genome sequencing (WGS) can be used to discriminate circulating strains of IAV in wastewater remains unknown. Methods Using a pan-IAV RT-qPCR assay, six wastewater treatment works (WWTWs) across Northern Ireland (NI), were screened from August to December 2022. A WGS approach using Oxford Nanopore technology was employed to sequence positive samples. Phylogenetic analysis of sequences relative to currently circulating human and avian IAVs was performed. Findings We detected a dynamic IAV signal in wastewater from September 2022 onwards across NI. “Meta” whole genome sequences were generated displaying homology to both human and avian IAV strains. The relative proportion of human versus avian-origin IAV reads differed across time and sample site. A diversity in subtypes and lineages was detected (e.g. H1N1, H3N2, and several avian). Avian segment 8 related to those found in recent H5N1 clade 2.3.4.4b was identified. Interpretation WBE affords a means to monitor circulating human and avian IAV strains and provide crucial genetic information. As such WBE can provide rapid, cost-effective, year-round “one-health” IAV surveillance to help control epidemic and pandemic threats. Funding This study was funded by the Department of Health for Northern Ireland as part of the Northern Ireland Wastewater Surveillance Programme. Highlights Author Summary Influenza A virus (IAV) is a major pathogen of humans and other animals and causes regular epidemics and devastating pandemics. Recently, a novel variant of highly-pathogenic H5N1 avian influenza has emerged spreading across the world killing millions of birds and infecting mammals, enhancing its pandemic potential. Strengthening global surveillance systems for human and animal IAV is thus a major priority. Wastewater-based epidemiology (WBE) has been applied to track SARS-CoV-2 and IAV in humans but whether this approach could work for avian IAV is not known. Here, we develop a “one-health” method to survey pan-IAV levels and genetically characterise the viruses. Through this we highlight co-detection of human and avian IAVs in wastewater, with homology to recent H5N1 isolates. Our work demonstrates the potential for WBE to help defend against not only human infections but emerging, zoonotic IAVs of pandemic potential. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Department of Health for Northern Ireland as part of the Northern Ireland Wastewater Surveillance Programme. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. [1]: pending:yes

Introduction: qPCR can be useful for the detection and absolute quantification of target genes, whilst 16S rRNA MiSeq sequencing (NGS) provides data on the relative abundance of taxa within a community. Here we compare total, P. aeruginosa (PA) and H. influenzae (HI) bacterial density with ecological metrics obtained by NGS. Methods: Sputum samples (n=219) collected in the iBEST study at Days 1, 8 and Day 29 from patients receiving tobramycin inhalation powder (TIP; n=54) and placebo (n=19) were analysed by qPCR and NGS. The relationship between ecological metrics and total bacterial (16S rRNA), PA (oprL and ecfX) and HI (hpD and smpB) density was compared. Results: There was a significant reduction in total bacterial density between Day 1 and 29 in the TIP group (p< 0.001), with no difference in the placebo group (p=0.980). A significant decrease in PA density with TIP treatment was also apparent for both qPCR targets (oprL: p<0.001; ecfX: p<0.001), with no difference for the placebo group (p=0.740 and p=0.970, respectively). There was a significant negative correlation between Shannon-Wiener diversity and oprL and ecfX gene copy number (r=-0.427; p<0.001 and r=0.406; p<0.001), with a positive association between dominance and PA copy number (r=0.440; p<0.001 and r=0.430; p<0.001, respectively). No decrease in density or association with ecological markers was apparent in either treatment arm (p>0.05) for HI. Conclusion: Higher density of pathogens, such as PA, was associated with lower community diversity and higher dominance, highlighting that qPCR may be a useful end-point in clinical studies. Supported by EU/EFPIA IMI iABC grant n° 115721

To date, investigations of the microbiota in the lungs of people with Cystic Fibrosis (PWCF) have primarily focused on microbial community composition in luminal mucus, with fewer studies observing the microbiota in tissue samples from explanted lung tissue. Here, we analysed both tissue and airway luminal mucus samples extracted from whole explanted lungs of PWCF and unused donor lungs. We determined if the lung microbiota in end-stage CF varied within and between patients, was spatially heterogeneous and related to localized structural damage. Microbial community composition was determined by Illumina MiSeq sequencing and related to the CF-Computed Tomography (CT) score and features of end-stage lung disease on micro-CT. Ninety-eight CF tissue (n=11 patients), 20 CF luminal mucus (n=8 patients) and 33 donor tissue (n=4 patients) samples were analysed. Additionally, we compared 20 paired CF tissue and luminal mucus samples that enabled a direct "geographical" comparison of the microbiota in these two niches. Significant differences in microbial communities were apparent between the 3 groups. However, overlap between the three groups, particularly between CF and donor tissue and CF tissue and CF luminal mucus was also observed. Microbial diversity was lower in CF luminal mucus compared to CF tissue, with dominance higher in luminal mucus. For both CF and donor tissue, intra- and inter-patient variability in ecological parameters was observed. No relationships were observed between ecological parameters and CF-CT score, or features of end-stage lung disease. The end-stage CF lung is characterised by a low diversity microbiota, differing within and between individuals. No clear relationship was observed between regional microbiota variation and structural lung damage.

Abstract Background Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture. Methods Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94). Results Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC90 > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%). Conclusions This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing.

Bacterial vaginosis (BV) is a common dysbiosis of the human vagina in which commensal vaginal lactobacilli are displaced by mixed pathogenic bacterial populations. Current treatments by antibiotics result in short- term cure but often lead to recurrence. Lactobacilli are being actively developed as a probiotic treatment option for BV, given their ability to inhibit growth of pathogenic microorganisms and to maintain the health and stability of the vaginal tract microbiota. Therefore, incorporation of freeze-dried lactobacillus and metronidazole (MET) into matrix-type silicone elastomer vaginal rings (VR) is a long-effective novel treatment of BV which can also prevent recurrence. Here, MED-4870 matrix-type VRs containing four different lyoprotectants of three concentrations were manufactured by reaction injection molding, and Shore M Hardness test, compression test and twist test were applied to determine the impact of incorporation of lyoprotectants on mechanical characteristics of VRs. Results indicated that incorporation of lyoprotectants can increase the resistance force and decrease the rotational angles of the VRs. As the category and concentration of incorporated lyoprotectant also have impact on the releasing profile of MET and protecting ability of lactobacillus during freeze-drying process, more research such as in-vitro release tests and microbiology tests will be needed.

Background: Chronic methicillin resistant Staphylococcus aureus (MRSA) in CF is associated with worse outcomes compared to early or intermittent infection. This observation could be related to adaptive bacterial changes such as biofilm formation or anaerobic growth. Methods: MRSA isolates stored from incident and during chronic (>2 years) infection were included at two study sites. MRSA isolates were characterised by spa-typing, antimicrobial susceptibility testing, biofilm formation and haemolysis under aerobic and anaerobic culture conditions. Results: Paired MRSA isolates from 49 patients were included. Mean age at incident infection was 9.7 +/- 1.2 years with mild to moderate lung disease (FEV1 74 +/- 4% predicted). Twenty-five subjects showed progression of disease/symptoms after onset of MRSA with significantly increased use of antibiotics. Most isolates belonged to t002 (38%) and t008 (36%) spa-types and 8 patients had a change in spa-type over time. Antimicrobial susceptibility testing showed few differences between incident and late isolates but significantly lower MIC under anaerobic vs. aerobic conditions for vancomycin, fusidic acid, rifampin but higher MIC for trimethoprim-sulfamethoxazole. Biofilm formation and haemolysis did not differ by stage of infection or disease course but both were lower under anaerobic conditions (biofilm p=0.018; haemolysis p=0.002) in multi-variate analyses that included study site, growth condition and stage of infection. Conclusions: Persistent MRSA infection is frequently associated with clinical decline. Anaerobic growth conditions, which occur in CF airways, affect the expression of virulence factors and antibiotic susceptibility of MRSA more than duration of infection. (C) 2021 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society.

Antibiotics are important for the management of airway infection in Cystic Fibrosis (CF). Growth in lung microbiome research has expanded our awareness of the nature and significance of the polymicrobial communities harboured by people with CF (PWCF). They have illustrated that what is considered a healthy lung consists of a structurally complex, but finely balanced commensal ecosystem of microbes, perturbation of which provides opportunity for aggressive colonisation and dominance by opportunistic pathogens [1Dickson RP Huffnagle GB. The lung microbiome: new principles for respiratory bacteriology in health and disease.PLoS Pathogens. 2015; 11e1004923Crossref PubMed Scopus (292) Google Scholar, 2Malla MA Dubey A Kumar S et al.Exploring the human microbiome: the potential future role of next-generation sequencing in disease diagnosis and treatment.Front Immunol. 2019 9; (2868)Crossref Scopus (153) Google Scholar, 3Barcik W Boutin R Sokolowska M Finlay B. The role of lung and gut microbiota in the pathology of asthma.Immunity. 2020; 52: 241-255Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar]. Throughout their lifetime, PWCF are treated with multiple courses of broad-spectrum antibiotics that impact this ecosystem by applying selective pressures, accelerating antimicrobial resistance (AMR) development. A working group has published a series of papers addressing the state of our knowledge of AMR in CF [4Flume PA Waters VJ Bell SC et al.Antimicrobial resistance in cystic fibrosis: does it matter?.J Cyst Fibros. 2018; 17: 687-689Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 5Kidd TJ Canton R Ekkelenkamp M et al.Defining antimicrobial resistance in cystic fibrosis.J Cyst Fibros. 2018; 17: 696-704Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 6Somayaji R Parkins MD Shah A et al.Antimicrobial susceptibility testing (AST) and associated clinical outcomes in individuals with cystic fibrosis: a systematic review.J Cyst Fibros. 2019; 18: 233-240Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 7Waters V Kidd T Canton R et al.Reconciling antimicrobial susceptibility testing and clinical response in antimicrobial treatment of chronic cystic fibrosis lung infections.Clin Infect Dis. 2019; 69: 1812-1816Crossref PubMed Scopus (47) Google Scholar, 8Cogen JD Kahl BC Maples H et al.Finding the relevance of antimicrobial stewardship for cystic fibrosis.J Cyst Fibros. 2020; Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 9Zemanick E Burgel PR Taccetti G et al.Antimicrobial susceptibility testing in cystic fibrosis: a Delphi approach to defining best practices.J Cyst Fibros. 2020; 19: 370-375Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar]. The most recent addition of this group is published in this issue; Bullington et al [[10]Bullington W Hempstead S Smyth A et al.Antimicrobial resistance: concerns of healthcare providers and people with CF.J Cyst Fibrosis. 2021; Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar] describe the findings of a survey documenting the concerns of health care workers (HCWs) and PWCF regarding AMR. The survey examined five topics: knowledge of AMR; level of concern and perceived risks; potential consequences of AMR; desired information and preference with respect to information sources. Responses were unanimous and congruent between HCWs, PWCF and their carers, with all groups expressing worries regarding both resistance development and key pathogens in CF. In addition, a high proportion of both groups felt that AMR affected life expectancy and expressed concern with respect to person-to-person transmission as a source for AMR. While carriage of antibiotic resistant bacteria in the airways is common among people with CF [[11]Hahn A Burrell A Fanous H et al.Antibiotic multidrug resistance in the cystic fibrosis airway microbiome is associated with decreased diversity.Heliyon. 2018; 4: e00795Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar], the impact of AMR, even beyond the respiratory system, is of great interest. Aminoglycoside antibiotics, administered intravenously or by nebulization, are used widely in CF and consequently P. aeruginosa clinical isolates from CF patients commonly exhibit phenotypic resistance to this class of antimicrobials, but these are delivered systemically and may impact bacteria in other parts of the body. Also in this issue Taylor et al [[12]Taylor S Leong L Sims S et al.The cystic fibrosis gut as a potential source of multidrug resistant pathogens.J Cyst Fibrosis. 2021; Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar] utilised metagenomic next generation sequencing (NGS) in their comparison of the faecal microbiome of a cohort of adult CF patients to one of a healthy control group. Overall, they found no difference in total gut carriage of AMR genes between the groups. However, they discovered an increased proportion of AMR genes with potential for horizontal transmission among the CF cohort, notably plasmid-mediated aminoglycoside-modifying genes ant(6)-Ib, aac(6′)-Ip, and aph(3′)-IIIa. Culture-based stool analysis further confirmed the presence of aminoglycoside resistant Gram-negative bacteria in this group. Furthermore, the authors also isolated extended-spectrum beta-lactamase (ESBL) positive Enterobacterales representatives in some CF patients, that also displayed resistance to gentamicin and tobramycin. The resistance presented by these strains was associated with a further aminoglycoside modifying enzyme, aac(3)-IIa, present on the E. coli associated mobile genetic element pc15-1a. No resistant strains were isolated from any participants in the healthy control group. This study, although small, has clearly highlighted the possibility of a reservoir of transmissible resistance genes, and transmissible genetic elements implicated in spreading antimicrobial resistance, present in the gut microbiome of PWCF. This study provides a timely reminder of the importance of antimicrobial stewardship (AMS) in the context of CF, the threat posed by increasing AMR and the emergence and dissemination of multi-drug resistant (MDR) pathogens. The challenge of treating CF lung infection is targeting the principal pathogens while being cognisant of the broad impact on microbial flora. Though the emphasis is on treatment of respiratory pathogens, the potential for the development of AMR determinants and pathogens is clearly not limited to the lung. Repeated and prolonged antimicrobial exposure in CF, through cycles of maintenance and acute therapy, is associated with dysbiosis of the resident microbial communities in other regions such as the gut [[13]Budden KF Gellatly SL Wood DL et al.Emerging pathogenic links between microbiota and the gut-lung axis.Nat Rev Microbiol. 2017; 15: 55-63Crossref PubMed Scopus (715) Google Scholar], which may, in turn, potentially impact the progression of airway disease in CF [[14]Marsland BJ Trompette A Gollwitzer ES. The gut–lung axis in respiratory disease.Ann Am Thorac Soc. 2015; 12: S150-S156Crossref PubMed Scopus (329) Google Scholar]. While we are aware of and seek to mitigate against the potential for AMR transmission between CF patients in the clinical environment, a more holistic view of AMR needs to be taken. A "One Health" approach has highlighted that we inhabit a complex interconnected microbial ecosystem, where overuse of antimicrobials in one niche can drive development of MDR pathogens and spread of AMR in another [[15]Mulani MS Kamble EE Kumkar SN et al.Emerging strategies to combat ESKAPE pathogens in the era of antimicrobial resistance: a review.Front Microbiol. 2019 Apr 1; 10 (PMID: 30988669; PMCID: PMC6452778): 539https://doi.org/10.3389/fmicb.2019.00539Crossref PubMed Scopus (632) Google Scholar]. Molecular diagnostics, such as NGS, have a role to play in generating timely and informative data around AMR, offering an opportunity to describe its presence and persistence, understand its evolution, and determine how and where it is transmitted [[16]Yadav S Kapley A. Antibiotic resistance: global health crisis and metagenomics.Biotechnol Rep (Amst). 2021 Feb 23; 29 (PMID: 33732632; PMCID: PMC7937537): e00604https://doi.org/10.1016/j.btre.2021.e00604Crossref PubMed Scopus (32) Google Scholar]. Utilised correctly, contemporary NGS workflows can provide species level identification and sufficient data for the classification of antibiotic resistance genes, all in less than 48 hours, offering time saving alternatives to current antimicrobial susceptibility testing (AST) and, if needed, more accurate and rapid pathogen identification [17Charalampous T Kay GL Richardson H et al.Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection.Nat Biotechnol. 2019; 37 (Epub 2019 Jun 24. PMID: 31235920): 783-792https://doi.org/10.1038/s41587-019-0156-5Crossref PubMed Scopus (259) Google Scholar, 18Dmitrijeva M Kahlert CR Feigelman R et al.Strain-resolved dynamics of the lung microbiome in patients with cystic fibrosis.mBio. 2021; 12 (e02863-20PMID: 33688005)https://doi.org/10.1128/mBio.02863-20Crossref PubMed Scopus (7) Google Scholar, 19Gu W Deng X Lee M et al.Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids.Nat Med. 2021; 27 (Epub 2020 Nov 9. PMID: 33169017): 115-124https://doi.org/10.1038/s41591-020-1105-zCrossref PubMed Scopus (193) Google Scholar]. The desire to improve our understanding of the effect of antibiotics on the lung microbiome has seen NGS driven culture independent methods applied in the study of targeted antibacterial therapy (CFMATTERs, [[20]Einarsson G Flanagan E Lee A et al.Longitudinal airway microbiota profiling in cystic fibrosis patients enrolled in the CFMATTERS clinical trial.J Cyst Fibrosis. 2017; 16 (WS03 Jun 1): S4Abstract Full Text PDF Google Scholar]) or as novel exploratory endpoints (iBEST, [[21]Loebinger MR Polverino E Blasi F et al.iBEST-1 Trial Team. Efficacy and safety of tobramycin inhalation powder in bronchiectasis patients with P. aeruginosa infection: Design of a dose-finding study (iBEST-1).Pulm Pharmacol Ther. 2019; 58 (Epub 2019 Aug 18. PMID: 31433997)101834https://doi.org/10.1016/j.pupt.2019.101834Crossref PubMed Scopus (8) Google Scholar]). Research such as this represents a move away from traditional monoculture enrichment-based laboratory techniques, the results of which do not often show a relationship to clinical response [[6]Somayaji R Parkins MD Shah A et al.Antimicrobial susceptibility testing (AST) and associated clinical outcomes in individuals with cystic fibrosis: a systematic review.J Cyst Fibros. 2019; 18: 233-240Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar]. Microbiome-directed methodologies may lead to more targeted, patient-specific, and effective antibacterial therapies compared to empirical treatments, a research area, noted by Bullington et al., in which HCWs, PWCF and their parents have expressed a keen interest. However, there is still an important role for culture. Enhanced culture methods promise to bridge gaps in our knowledge by identifying unassigned sequences and uncovering new bacterial strains missed or overlooked by NGS, conceivably providing more comprehensive data than sequencing can alone [[22]Whelan FJ Waddell B Syed SA et al.Culture-enriched metagenomic sequencing enables in-depth profiling of the cystic fibrosis lung microbiota.Nat Microbiol. 2020; 5: 379-390Crossref PubMed Scopus (39) Google Scholar]. Furthermore, continued advances in the analysis of metabolites, proteins and transcripts have meant that combinational approaches which integrate data from these multiple omics' sources could offer new avenues for monitoring infections in CF or help inform decisions regarding disease management [[23]Güemes AGC Lim YW Quinn RA et al.Cystic fibrosis rapid response: translating multi-omics data into clinically relevant information.mBio. 2019; 10 (e00431-19PMID: 30992350; PMCID: PMC6469968)https://doi.org/10.1128/mBio.00431-19Crossref Google Scholar]. Combatting AMR will require unavoidable policy and societal changes. It will necessitate investment in and expansion of AST and AMR surveillance programmes, strengthening and reforming aspects of our health-care services, incentivising drug development and promoting education. Lastly, ensuring everyone has fair access to diagnostics and current and future antimicrobials will be essential. The COVID-19 pandemic has graphically illustrated the need for immediate, coordinated action across all sectors when tackling global health threats. Leveraging this hard-won experience into sustained and effective action on AMR will undoubtedly benefit both CF and non-CF populations alike. The authors have no conflicts of interest to declare.

Background: Treatment with Ivacaftor provides a significant clinical benefit in people with cystic fibrosis (PWCF) with the class III G551D-CFTR mutation. This study determined the effect of CFTR modulation with ivacaftor on the lung microbiota in PWCF. Methods: Using both extended-culture and culture-independent molecular methods, we analysed the lower airway microbiota of 14 PWCF, prior to commencing ivacaftor treatment and at the last available visit within the following year. We determined total bacterial and Pseudomonas aeruginosa densities by both culture and qPCR, assessed ecological parameters and community structure and compared these with biomarkers of inflammation and clinical outcomes. Results: Significant improvement in FEV1, BMI, sweat chloride and levels of circulating inflammatory biomarkers were observed POST-ivacaftor treatment. Extended-culture demonstrated a higher density of strict anaerobic bacteria (p = 0.024), richness (p = 1.59* 10(-4)) and diversity (p = 0.003) POST-treatment. No significant difference in fold change was observed by qPCR for either total bacterial 16S rRNA copy number or P. aeruginosa density for oprL copy number with treatment. Culture-independent (MiSeq) analysis revealed a significant increase in richness (p = 0.03) and a trend towards increased diversity (p = 0.07). Moreover, improvement in lung function, richness and diversity displayed an inverse correlation with the main markers of inflammation (p < 0.05). Conclusions: Following treatment with ivacaftor, significant improvements in clinical parameters were seen. Despite modest changes in overall microbial community composition, there was a shift towards a bacterial ecology associated with less severe CF lung disease. Furthermore, a significant correlation was observed between richness and diversity and levels of circulating inflammatory markers. (C) 2021 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Introduction: We investigated (i) changes in total bacterial density in sputum during clinical stability and PEx and (ii) the relationship between total bacterial density, inflammation and FEV1. Methods: Two subgroups from the BRONCH-UK cohort study (n=74) were analysed; A) clinically stable patients (n=54) who provided sputum at baseline and one further visit within 12 months; B) PEx patients (n=20) who provided sputum at baseline, pre- and post-Abx treatment of an PEx and one further visit within 12 months when clinically stable. Total bacterial density (16S rRNA) was determined by qPCR and patients were stratified as having moderate (105- <107 copies/ml) or high bacterial (≥107 copies/ml) density. Non-parametric tests were used for analysis. Results: Of 74 patients, 58% female, mean (SD) age 66 (8.7) years, FEV1 67 (20.5) %. At baseline, 23/54 patients (43%) in the stable group had high bacterial density vs. 7/20 patients (35%) in the PEx group. No difference was observed in age, FEV1 or CRP in patients with moderate vs. high bacterial density (p>0.05). Between baseline and final stable visit, there was no difference in bacterial density (median Log10 16S rRNA 6.8 [4.9-7.9] vs. 6.7 [5.2-8.0] copies/ml, respectively; p= 0.58) and no significant change in the number of patients with high or moderate bacterial density. No difference was observed in bacterial density pre and post Abx treatment (median Log10 16S rRNA 6.9 [5.4-8.0] vs 7.1 [5.7-8.1] copies/ml, respectively; p=0.28). Bacterial density did not correlate with FEV1 (r=0.11; p=0.37) or CRP (r=0.10; p=0.40) at baseline. Conclusion: Bacterial density was stable during clinical stability and did not change following Abx therapy of a PEx.