Biology subjects

Iszatt, J. J.

Publications and source records attributed to Iszatt, J. J..

4 recordsLinked to original sources

bioRxiv2026

Preclinical safety assessment of Koomba kaat 1 and Biyabeda mokiny 1 phages for respiratory application against Staphylococcus aureus

Therapeutic bacteriophages are promising alternatives to antibiotics for treating antimicrobial-resistant bacterial infections. For pulmonary infections, direct respiratory delivery offers therapeutic advantages; however, preclinical evaluation of respiratory safety remains underdeveloped. Bacteriophages exhibit genomic and biological diversity, and safety cannot be assumed for individual candidates. Here, we evaluated the respiratory safety of lytic Staphylococcus aureus bacteriophages, Koomba kaat 1 and Biyabeda mokiny 1, using human and murine preclinical models. Differentiated primary airway epithelial cells derived from six healthy paediatric donors were exposed apically to purified phage (1 x 109 PFU/mL) for 24 hours. Barrier integrity, epithelial morphology, mucus production, cytotoxicity, and interleukin-8 release were assessed. Safety was further investigated in adult C57BL/6J mice receiving intranasal phage administration (1 x 109 PFU) twice daily (14 days). Clinical observations, body weight, organ pathology, blood biochemistry, bronchoalveolar lavage cellularity, protein concentration, and inflammatory mediators were evaluated. Neither phage altered epithelial morphology, barrier integrity, mucus production, cytotoxicity, nor inflammatory responses in differentiated cultures. Repeated intranasal administration was well tolerated in vivo, with no adverse clinical signs, weight loss, macroscopic pathology, or treatment-associated changes in pulmonary cellularity or tissue histopathology. Differences in blood biochemistry and inflammatory mediators were small and not accompanied by epithelial injury or pulmonary inflammation. Collectively, these findings demonstrate that Koomba kaat 1 and Biyabeda mokiny 1 exhibit favourable safety profiles following repeated airway administration. This study establishes a comprehensive framework for the preclinical respiratory safety assessment of bacteriophages and provides support for the clinical development of inhaled phage for S. aureus respiratory infections.

microbiology↗

bioRxiv2026

Long read and short read whole genome sequencing are equivalent for genomic characterisation of bacteriophage: considerations for high throughput analysis

BackgroundAntimicrobial resistance (AMR) is a global health crisis, necessitating alternative antibacterial strategies. Bacteriophages (phages) offer a promising solution, and their use as a therapeutic agent relies on stringent bioinformatic characterisation using whole genome sequencing (WGS) technologies. However, phages are highly diverse, with no clear consensus on best practices concerning phage DNA extraction or sequencing platform. Efficient and repeatable DNA extraction, sequencing, and bioinformatics processes are critical for safety assessments but remain poorly defined. Additionally, the impact of sequencing platform choice and DNA extraction methods on downstream genomic analyses is not well understood. MethodsWe evaluated multiple DNA extraction, library preparation, and sequencing approaches using a diverse collection of Pseudomonas phages from the PhageWA biobank. Column-based and precipitation-based DNA extraction methods were compared for DNA yield and recovery efficiency. Genome sequencing was performed using short-read (Illumina) and long-read (Oxford Nanopore Technologies) platforms, incorporating multiple library preparation kits and Nanopore basecalling models. Assemblies were assessed for completeness, quality, and sequence concordance using standardised bioinformatics pipelines, with hybrid Illumina-Nanopore assemblies used as references for comparison. ResultsDNA extraction efficiency varied substantially between protocols, with the Puregene precipitation-based method yielding significantly higher DNA recovery than column-based approaches when normalised to phage titre. Illumina sequencing consistently generated complete genome assemblies, although assembly fragmentation was observed for several jumbo phages when using the SeqWell ExpressPlex 2.0 library preparation method. For Nanopore sequencing, ligation-based native barcoding libraries produced longer reads than rapid barcoding libraries, while selection of the Dorado v5.0.0 basecalling model significantly improved read quality. Genome assembly success was dependent on phage genus; native Nanopore sequencing failed to assemble several Pbunavirus genomes, likely due to modified DNA bases, but an amplification-based library preparation successfully resolved these genomes. Across successfully assembled samples, Illumina and Nanopore platforms produced highly concordant genomes with comparable completeness scores, and hybrid polishing identified only minor sequence differences. ConclusionsDNA extraction methodology, sequencing chemistry, and basecalling model selection significantly influence phage WGS outcomes. Precipitation-based DNA extraction improved DNA recovery, while both Illumina and Nanopore sequencing generated high-quality phage genomes suitable for therapeutic characterisation. Nanopore sequencing provided assemblies comparable to Illumina with minimal benefit from hybrid polishing, supporting its routine use for phage genomics. These findings provide practical guidance for phage genome characterisation workflows and contribute to the development of standardised, regulatory-grade approaches for therapeutic phage assessment.

microbiology↗

bioRxiv2026

Genomic Determinants of Phage Activity Against Pseudomonas aeruginosa: Roles of Receptors, Defence Systems, and Anti-Defences

Abstract/SummaryPseudomonas aeruginosa is a priority pathogen in chronic and multidrug-resistant infections, yet therapeutic phages targeting this organism often exhibit variable and unpredictable efficacy. A mechanistic understanding of the genomic determinants governing phage-host interactions is therefore critical for the rational design of robust phage therapeutics. Here, we systematically dissected the genetic featrures underlying lytic outcomes across a diverse panel of P. aeruginosa-infecting phages. We comprehensively annotated receptor-binding proteins (RBPs), bacterial defence systems and phage-encoded anti-defence genes, and experimentally defined host receptor usage for each phage, linking receptor specificity to cognate RBPs. We identified multiple anti-defence systems--including vcrx089, acrIIA24, atd1, gnarl1, klcA, darA and nmna (NARP2-associated)--and RBPs targeting lipopolysaccharide, type IV pili and flagella that are associated with enhanced lytic activity and represent tractable engineering targets. Across 174 genomic features analysed, 110 significantly influenced phage activity, with heterogeneous and context-dependent effects. Leveraging these features, we trained a machine-learning classifier that accurately predicted phage-host outcomes (AUC-ROC = 0.875), demonstrating that interaction phenotypes are encoded in definable genomic signatures. Together, our findings reveal the quantitative contribution of phage anti-defence systems to infectivity in P. aeruginosa and define a genomic framework for predicting and engineering lytic success. These results establish a foundation for the rational design of synthetic phages with enhanced host ranges.

microbiology↗

bioRxiv2021

Development and validation of a miniaturized host range screening assay for bacteriophages

Antimicrobial resistance is a global health crisis, partly contributed by inappropriate use of antibiotics. The increasing emergence of multidrug resistant infections has led to the resurgent interest in bacteriophages as an alternative treatment. Current procedures assessing susceptibility and breadth of host range to bacteriophage are conducted using large-scale manual processes that are labor-intensive. The aim here was to establish and validate a scaled down methodology for high-throughput screening in order to reduce procedural footprint. Bacteriophages were isolated from wastewater samples and screened for specificity against 29 clinical Pseudomonas aeruginosa isolates and PA01 using a spot test (2 L/ drop). Host range assessment was performed on four representative P. aeruginosa isolates using both double agar overlay assay on petri dishes and 24-well culture plates. The breadth of host range of bacteriophages that exhibited lytic activity on P. aeruginosa isolates were corroborated between the current standard practice of whole plate phage assay and 24-well phage assay. The high correlation achieved in this study confirms miniaturization as the first step in future automation that could test phage diversity and efficacy as antimicrobials.

microbiology↗