Research Article

Integrated Genomic, Virulence, and Immunoinformatic Characterization of Clinical Staphylococcus aureus Isolates for Identification and Prioritization of Conserved Vaccine Candidate Proteins

Abstract

Background: Staphylococcus aureus is a major opportunistic pathogen associated with a wide range of clinical infections, while the increasing prevalence of methicillin-resistant S. aureus (MRSA), multidrug resistance, and diverse virulence determinants represents a significant therapeutic and public-health challenge. The genetic diversity of S. aureus further complicates the development of broadly applicable vaccine strategies. This study aimed to integrate phenotypic, molecular, genomic, and immunoinformatic approaches to characterize clinical S. aureus isolates and identify conserved proteins with potential as vaccine candidate antigens. Methods: A total  of 150 clinical specimens were collected from different clinical sources in Babil Governorate, Iraq. Confirmed S. aureus isolates were subjected to antimicrobial susceptibility testing and PCR detection of antimicrobial resistance and virulence-associated genes. Representative isolates were confirmed by 16S rRNA gene sequencing, and 24 genetically and clinically representative isolates were selected for whole-genome sequencing (WGS). Genome assemblies were subjected to quality assessment, annotation, antimicrobial resistance and virulence profiling, multilocus sequence typing, phylogenomic analysis, and pan-genome characterization. Conserved surface-associated and secreted proteins were subsequently screened using a reverse-vaccinology pipeline based on conservation, biological relevance, human-homology exclusion, predicted antigenicity, allergenicity, and toxicity. The prioritized proteins were further evaluated using immunoinformatic analyses of B-cell and T-cell epitopes, MHC class I and II interactions, and predicted population coverage. Results: Of the 150 clinical specimens, 112 (74.7%) were confirmed as S. aureus. Penicillin resistance was the most prevalent phenotype (81.3%), followed by erythromycin (46.4%) and cefoxitin (42.9%), with 48 isolates (42.9%) classified as MRSA and 43 (38.4%) exhibiting multidrug-resistant profiles. Among the investigated resistance determinants, blaZ was the most prevalent (83.0%), followed by mecA (41.1%). Virulence-associated genes were widely distributed, particularly spa (97.3%), clfA (94.6%), clfB (92.0%), hla (90.2%), and icaD (83.9%). The 24 WGS isolates represented multiple sequence types and clonal complexes, demonstrating substantial genomic diversity. A pan-genome analysis identified approximately 3,800 gene clusters: 2,250 core, 900 accessory, and 650 unique. Among the approximately 2,700 predicted proteins, 285 were classified as surface-associated or secreted; a tiered screening and prioritization process for these proteins resulted in the selection of 94 conserved candidates. Conclusion: An integrated genomic and immunoinformatic analysis identified conserved Staphylococcus aureus proteins with computational characteristics that support further vaccine research. Because clinical isolates show high antimicrobial resistance, virulence, and genetic diversity, continued molecular surveillance is necessary. Of the four candidates, Protein C5 had the most favorable computational profile. Experimental work is still needed to verify its expression, immunogenicity, safety, and protective efficacy before it can be considered a validated vaccine candidate.

Authors affiliation:

Ameer Hayder Razzaq Kaiwan  (ORCID): College of Health and Medical Techniques, University of Alkafeel, Najaf, Iraq.

How To Cite: A.H.R. Kaiwan. Integrated Genomic, Virulence, and Immunoinformatic Characterization of Clinical Staphylococcus aureus Isolates for Identification and Prioritization of Conserved Vaccine Candidate Proteins. Current Research in Health Sciences, 4(2):37–47, 2026. https://doi.org/10.58613/crhs426