Bacteriophage Susceptibility Testing Using Community Associated Staphylococcus aureus Clinical Isolates
Department
Biomedical and Translational Sciences, Macon & Joan Brock Virginia Health Sciences Eastern Virginia Medical School at Old Dominion University
Graduate Level
Master’s
Presentation Type
Poster Presentation
Abstract
INTRODUCTION: Staphylococcus aureus (SA) is a significant health threat, capable of establishing persistent infections using virulence factors (VFs) that promote antibiotic resistance and immune evasion. Through targeted gene deletion, the ability to investigate the contribution of individual VFs to SA pathogenesis is possible. This methodology requires successful bacteriophage transduction; however, strong restriction-modification (R-M) and phage-resistance mechanisms often impede this process. To mitigate this obstacle, gene-deletion studies often rely on well-characterized, lab-adapted strains of a single clonal complex (CC) group containing highly related SA, despite these strains potentially being no longer clinically relevant. Previously, our lab generated a gene deletion in a SA clinical isolate using the generalized staphylococcal transduction phage φ11. As our lab maintains a clinical isolate library with SA strains of various infection type (IT) and genomic profile, this study aimed to investigate phage susceptibility differences in IT and CC among diverse clinical strains.
METHODS: Plaque assays with various SA clinical isolates were used to assess susceptibility of φ11 propagated on restriction-deficient SA RN4220. Briefly, bacteria grown overnight were mixed with diluted phage, CaCl2, and 0.5% molten agar, then overlaid on TSA plates. Plates were incubated at 37°C overnight, and plaques were enumerated. Whole-genome-sequencing was previously performed using the Illumina iSeq 100 System. Low-quality ends and reads were filtered and trimmed with Trim-Galore! (v0.6.10), assembled using SPAdes (v4.0.0), then polished for error-correction with Pilon (v1.24). BLAST and MEGA were used to identify and compare genes associated with phage adsorption (tarM, tarP, tarS) and the R-M gene hsdS1.
RESULTS: Plaque assays suggest significant differences in susceptibility of SA clinical isolates based on CC. Interestingly, isolates with similar genomic backgrounds (USA300) that differed in IT also showed differences in phage susceptibility. The sequence-specificity R-M gene, hsdS1, was present in all isolates assessed, with expected variation across CC. Carriage of genes in the tar family was more variable across isolates.
CONCLUSION: Overall, these data highlight the impact of diverse genomic profiles in SA on φ11 susceptibility and contribute to an increased understanding of the ability of bacteriophages to infect clinical isolates, encouraging their use in future virulence studies.
Keywords
Staphylococcus aureus, Bacteriophage
Bacteriophage Susceptibility Testing Using Community Associated Staphylococcus aureus Clinical Isolates
INTRODUCTION: Staphylococcus aureus (SA) is a significant health threat, capable of establishing persistent infections using virulence factors (VFs) that promote antibiotic resistance and immune evasion. Through targeted gene deletion, the ability to investigate the contribution of individual VFs to SA pathogenesis is possible. This methodology requires successful bacteriophage transduction; however, strong restriction-modification (R-M) and phage-resistance mechanisms often impede this process. To mitigate this obstacle, gene-deletion studies often rely on well-characterized, lab-adapted strains of a single clonal complex (CC) group containing highly related SA, despite these strains potentially being no longer clinically relevant. Previously, our lab generated a gene deletion in a SA clinical isolate using the generalized staphylococcal transduction phage φ11. As our lab maintains a clinical isolate library with SA strains of various infection type (IT) and genomic profile, this study aimed to investigate phage susceptibility differences in IT and CC among diverse clinical strains.
METHODS: Plaque assays with various SA clinical isolates were used to assess susceptibility of φ11 propagated on restriction-deficient SA RN4220. Briefly, bacteria grown overnight were mixed with diluted phage, CaCl2, and 0.5% molten agar, then overlaid on TSA plates. Plates were incubated at 37°C overnight, and plaques were enumerated. Whole-genome-sequencing was previously performed using the Illumina iSeq 100 System. Low-quality ends and reads were filtered and trimmed with Trim-Galore! (v0.6.10), assembled using SPAdes (v4.0.0), then polished for error-correction with Pilon (v1.24). BLAST and MEGA were used to identify and compare genes associated with phage adsorption (tarM, tarP, tarS) and the R-M gene hsdS1.
RESULTS: Plaque assays suggest significant differences in susceptibility of SA clinical isolates based on CC. Interestingly, isolates with similar genomic backgrounds (USA300) that differed in IT also showed differences in phage susceptibility. The sequence-specificity R-M gene, hsdS1, was present in all isolates assessed, with expected variation across CC. Carriage of genes in the tar family was more variable across isolates.
CONCLUSION: Overall, these data highlight the impact of diverse genomic profiles in SA on φ11 susceptibility and contribute to an increased understanding of the ability of bacteriophages to infect clinical isolates, encouraging their use in future virulence studies.