Phenotypic and genotypic profiling of antimicrobial resistance genes and virulence factors in Pseudomonas aeruginosa isolates from keratitis patients
Abiye Tigabu, Mark D.P. Willcox, Fiona Stapleton
Abstract
BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is a major cause of ocular infections, exhibiting resistance to many antimicrobial agents and the ability to acquire further resistance through mutations and horizontal gene transfer. It employs a range of virulence factors to invade ocular tissues, leading to complications such as corneal scarring and perforation. Alarmingly, multidrug-resistant P. aeruginosa isolates are increasing worldwide. For instance, a recent outbreak in the United States of America (USA) involving an extensively drug-resistant P. aeruginosa PA1270 strain resulted in four deaths, four eye removals, and 14 cases of vision loss. A comprehensive understanding of the antibiotic resistance and virulence mechanisms of P. aeruginosa is essential for the effective management of corneal infections. METHODS: Whole-genome sequencing data from 70 P. aeruginosa isolates collected from corneal samples were retrieved from the National Center for Biotechnology Information (NCBI) database and annotated using Prokka 1.14.6. Unique genes identified in these datasets were analyzed against the Comprehensive Antibiotic Resistance Database (CARD) to determine antimicrobial resistance profiles. The presence of acquired resistance genes and virulence factors were assessed using ResFinder and the Virulence Factor Database (VFDB), respectively. Roary v3.13.0 was used for pangenome analysis, while Snippy v4.6.0 was employed for whole-genome variant analysis. Furthermore, mobile genetic elements (MGEs) and pathogenicity islands (PIs) were identified using MobileElementFinder v1.0.3 and IslandViewer 4, respectively. In parallel, phenotypic characterization, including determination of minimum inhibitory concentrations (MICs) for selected antimicrobial agents, was performed using the broth microdilution method. Additionally, a crystal violet assay was conducted to evaluate the biofilm-forming ability of P. aeruginosa. RESULTS: The corneal P. aeruginosa harboured numerous AMR genes against various classes of antibiotics. Notably, the most prevalent acquired resistance genes across all keratitis P. aeruginosa strains were β-lactams blaPAO, and blaOXA, aminoglycoside aph(3')-IIb, chloramphenicol catB7, and fosfomycin fosA. P. aeruginosa keratitis isolates harbored more core and cloud genes than environmental and cystic fibrosis (CF) strains. A significant difference in ciprofloxacin resistance gene crpP was observed between the keratitis and CF isolates. Additionally, a considerable number of insertion sequences (ISPa1, ISPa6, ISPa32) and transposons (Tn4661, Tn6082, Tn5563) were identified. Phenotypic characterization of antimicrobial resistance (AMR) revealed that gentamicin were the most effective antibiotics against corneal P. aeruginosa isolates. However, 68.9 % of the isolates exhibited resistance to imipenem. Surprisingly, 26.7 % of the P. aeruginosa strains were classified as MDR, all of them originating from India. Furthermore, 61.8 % of the corneal P. aeruginosa isolates were strong biofilm producers, with all MDR strains identified as strong biofilm formers. CONCLUSIONS: Genotypic analysis revealed key resistance mechanisms, including antibiotic efflux, inactivation, and target alteration, as well as several structural and secreted virulence factors. Whilst MDR and extensively drug-resistant (XDR) strains were identified among the keratitis P. aeruginosa isolates, most isolates in this study were susceptible to gentamicin. These findings offer valuable insights for developing targeted therapeutic strategies that, when used in combination with antibiotics, may improve treatment outcomes and help mitigate the emergence of resistance.