Institute of Environmental Science and Research

ABSTRACTThe ecological impacts of plastics and their additives on marine microbiota remain unclear. We applied prokaryotic 16S rRNA gene and fungal ITS2 region amplicon sequencing, alongside shotgun metagenomic sequencing, to identify compositional and functional changes in microbial communities on marine plastic. Five common plastics, both non‐aged and artificially aged, were submerged in Auckland Harbour, Aotearoa‐New Zealand. Biofilms on linear low‐density polyethylene (LLDPE), nylon‐6 (PA), polyethylene terephthalate (PET), polylactic acid (PLA), oxo‐biodegradable LLDPE (OXO) and glass were sampled over 12 months. The taxonomy and functional potential of biofilm communities differed from surrounding seawater communities and varied with biofilm age. Younger biofilms were more diverse, with Proteobacteria, unknown fungi and unclassified Metazoa dominating prokaryotic, fungal and eukaryotic communities, respectively. Taxa related to previously reported plastic‐degraders were found in very low abundance across all substrates. Plastic type and UV‐ageing did not significantly shape biofilm communities over a year. Although some genes differed in relative abundance due to UV‐ageing, overall functional profiles remained consistent across plastics. Genes conferring reported plastic‐degrading traits were present regardless of plastic type, UV‐ageing and biofilm age. Nevertheless, nylon hydrolases were notably associated with PA, suggesting marine plastic impacts may be restricted to taxa or functions involved in its degradation.

Abstract Description There are millions of influenza infections annually, causing a range of illness severity. Although annual influenza vaccinations reduce infection and transmission, vaccine efficacy rates vary from 30-60%, highlighting the need for improved tools to predict influenza susceptibility and transmission. Household cohorts such as the Southern Hemisphere Influenza Vaccine Effectiveness and Response Study (SHIVERS IV) cohort help identify factors contributing to influenza infection and transmission. In this study, households are enrolled, and blood samples are taken before, during, and after transmission events. To capture all asymptomatic infections all household members are PCR tested every 3 days after a confirmed household infection. To identify immune correlates of protection and transmission, we performed high dimensional flow cytometry on the baseline samples of all household members examining both innate and adaptive immune markers. We hypothesize that those who transmit influenza to household contacts will have weaker post infection innate activation profiles compared to those who never spread influenza to their household contacts. Furthermore, those that are protected from influenza infection after exposure to a household contact, we predict will have higher rates of polyfunctional CD4+ T cells. Future plans include transcriptional and TCR repertoire profiling of transmitting and protected individuals. Funding Sources Supported by: St. Jude Center of Excellence for Influenza Research and Response (SJCEIRR) Human Immunology, Project 1 (NIAID HHSN27220140006C) awarded to Richard Webby, Paul G. Thomas? Topic Categories Immune Mechanisms of Human Disease (HUM)

Our laboratory began prospective genomic surveillance for healthcare-associated organisms in 2022 using Oxford Nanopore Technologies (ONT) as a standalone platform. While effective for early outbreak detection, the lower read accuracy compared to Illumina sequencing has limited single-nucleotide variant (SNV) analysis. This study aimed to determine whether Haplotype-aware ERRor cOrrection (HERRO) of ONT data could permit high-resolution comparison of outbreak isolates. ONT simplex reads were used from isolates involved in a recent outbreak of meticillin-resistant Staphylococcus aureus (MRSA) in our neonatal unit. Raw data were basecalled and adapter-trimmed using Dorado v0.7.0, followed by HERRO correction. Genome assemblies and phylogenies were compared with previous analyses (using Dorado v0.3.4 no HERRO correction, and data generated by Illumina sequencing). Five of nine outbreak isolates were analysed; four had insufficient read lengths (N50 values <10,000 bp) and did not achieve complete chromosome coverage post-HERRO correction. The average chromosome sequencing depth for nanopore data was 147× (range: 44-220×) with an average read N50 of 12,215 bp (interquartile range: 11,439-12,711 bp). The median pairwise SNV distance between outbreak isolates from the original investigation was 51 SNVs (range: 40-68), which decreased to 3 SNVs (range: 1-15) with HERRO correction. Illumina sequencing generated a median SNV distance of 2 (range: 0-13). The HERRO-corrected ONT phylogeny closely matched the Illumina-generated phylogeny. HERRO correction enabled high-resolution analysis of MRSA outbreak isolates comparable to Illumina sequencing. ONT sequencing with HERRO correction represents a viable standalone option for detailed genomic analysis of hospital outbreaks, provided sufficient read lengths are achieved.

ABSTRACT Macrophages play essential roles in generating a tolerogenic resident environment, but the interactions between bacteriophages and their action in macrophage tolerance memory remain unknown. Here, we find that gut bacteriophage exposure in vivo induces tolerance immunity via reprogrammed macrophages which significantly enhances protection against bacterial lipopolysaccharide (LPS). Bacteriophage-memory macrophages orchestrate LPS-challenge responses into tolerization or hyperresponsive gene expression clusters in a function-specific manner. The tolerized gene cluster encodes pro-inflammatory cytokines, while the induction cluster is a defense-specific response including anti-inflammatory cytokines, antiviral and antimicrobial effectors, and negative regulators of inflammation. Mechanistically, this augmented defense response is dependent on increased expression of IL-10, but not suppression of pro-inflammatory cytokines. Furthermore, bacteriophages suppressed LPS-induced pro-inflammatory genes by repressing histone acetylation target enhancers that coordinate chromatin accessibility to limit inflammation. Thus, our study identifies the function and mechanism of reprogramming actions for bacteriophage in moderating LPS immune responses.

Evaluation of DNA given activity propositions (EGALP) is a widely discussed topic at this time, particularly in the United States. There is concern about opinions given in testimony that are not properly founded. Guidance on evaluative reporting given alleged activities can be found in published papers, official documents, and specialized textbooks. In this work, we aim to align and compare recommendations on evaluative reporting of DNA results. Some of these recommendations are explicitly stated while others are woven into the text. All documents pertain to evaluative reporting; they agree on the use of likelihood ratios and the need to avoid the transposed conditional. There is some disagreement such as whether a quantitative or qualitative LR should be reported. However, the majority situation is that one topic is covered explicitly by one document but only implicitly covered or not mentioned in the others. We identify 19 consensus recommendations and highlight five gaps or areas of disagreement for which we offer suggestions. It is our hope that this will encourage conversations that will lead to a more uniform set of guidelines, perhaps during a periodic updating of existing documents.

<h2>TO THE EDITOR</h2><p dir="ltr">Song <i>et al</i> recently reported an evaluation of the feasibility of Epstein-Barr virus (EBV) testing on the cobas 6800 (Roche Diagnostics) using whole blood, comparing it against the EBV RG PCR kit (Qiagen). We also recently evaluated the cobas EBV assay using whole blood on the Roche 6800 instrument against our existing, not fully automated, Roche LightMix EBV kit (TIB MolBiol). The existing assay runs on Roche’s LightCycler 480 instrument and was already validated for whole blood samples. <p dir="ltr">The cobas EBV assay is approved by the US Food and Drug Administration and is Conformite Europeenne (CE) in vitro diagnostics (IVD) marked only for EDTA plasma but not whole blood samples.2 <p dir="ltr">Most commercially available systems use plasma as the preferred sample type. However, analyzing whole blood and peripheral blood mononuclear cells enables a more sensitive diagnosis due to the detection of clonal expansion of B cells for EBV-associated infections, such as posttransplant lymphoproliferative disorder.3-5 Additionally, the effects of cell lysis on plasma viral load measurements are mitigated, providing a more stable measurement over time.3

Wastewater pathogen surveillance was rapidly implemented across Australia and New Zealand as a public health tool during the COVID-19 pandemic. To assess method consistency and identify opportunities for harmonization, we surveyed all Australian and New Zealand Laboratories conducting government-funded wastewater pathogen surveillance. The survey demonstrated alignment of some method choices, particularly municipal wastewater treatment plant (WWTP) sampling and the use of electromagnetic membrane filtration followed by RT-qPCR. However, key differences were observed in the wastewater sample volumes; nucleic acid purification methods; validation approaches; and sequencing, analysis, and reporting methods for SARS-CoV-2 lineages. A lack of consensus on best-practice methods was evident, highlighting the need for interlaboratory sample and data exchanges to support validation and comparability. Following the pandemic period, several jurisdictional programs were discontinued despite the mounting international evidence for the utility of wastewater-based epidemiology for a range of pathogens. Subsequently, a nationally funded program was announced in Australia, necessitating the re-establishment of laboratory capacity for some jurisdictions and expansion of target pathogens for other centers. The results of this survey are intended to inform the re-establishment and enhancement of regional capacity and to provide a foundation for best-practice knowledge sharing and approach harmonization.

Despite the rapid growth in aptamer-based biosensor research, there remains a significant demand for aptasensors that operate without the need for sample preparation and calibration, to better facilitate real-world applications. Electrochemical aptamer-based (EAB) sensors, particularly those utilizing a dual-frequency, calibration-free approach, have shown promising advances toward commercialization. Single-use, disposable sensors represent a cost-effective solution for at-home and on-site point-of-care (POC) diagnostics. However, the development of these sensors presents unique challenges compared to in vivo monitoring and reusable platforms, with pronounced variations across sensors and batches. Motivated by these challenges, we have comprehensively investigated the dual-frequency, calibration-free approach, focusing on sensor-to-sensor and batch-to-batch variations. Our research explored the use of a nonresponsive frequency-based ratiometric method for detecting cocaine with laser-ablated, disposable EAB sensors. Additionally, to overcome the absence of nonresponsive frequencies in some aptasensors, we developed strategies to modify the aptamer structure and optimize operational conditions, effectively tailoring nonresponsive frequencies to allow for rapid result turnover. Moreover, we assessed the effects of various filter types on saliva pretreatment using liquid chromatography with tandem mass spectrometry (LCMS/MS) and developed a saliva collection workflow using an oral swab. This workflow and the disposable aptasensors developed herein achieved low μM sensitivity in saliva, with results obtainable in under 5 min, including saliva collection and processing. Furthermore, our findings indicate that certain food and drink residues in saliva can compromise sensor accuracy, highlighting an area for future refinement.